Peek

Resistance to anticoccidial drugs: alternative
strategies to control coccidiosis in broilers
Herman Peek
Utrecht
2010
Resistance to anticoccidial drugs: alternative strategies to control

coccidiosis in broilers
Herman Peek
ISBN
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: 978-90-393-5272-4
: Marije Brouwer
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: Animal Health Service (GD), Deventer
Resistance to anticoccidial drugs: alternative

strategies to control coccidiosis in broilers
Resistentie tegen anticoccidiosemiddelen: alternatieve strategien

voor de controle van coccidiose bij vleeskuikens
(met een samenvatting in het Nederlands)
Proefschrift
ter verkrijging van de graad van doctor aan de Universiteit Utrecht
op gezag van de rector magnificus, prof.dr. J.C. Stoof,
ingevolge het besluit van het college voor promoties
in het openbaar te verdedigen
op donderdag 18 februari 2010
des middags te 2.30 uur
door
Hermanus Wilhelmus Peek

geboren op 2 december 1952 te Harmelen
Promotor:
Prof. dr. J.A. Stegeman
Co-promotor:
Dr. W.J.M. Landman
The research was financially supported by the Commodity Board for Poultry and Eggs
(PPE), the Commodity Board for Animal Feed (PDV) and the Animal Health Service
(GD).
Paranimfs:
Brenda Vermeulen
Gerda Peek
CONTENTS
Chapter 1
Chapter 2
Chapter 3
Chapter 4
Chapter 5
Chapter 6
Introduction
General introduction and aim of the thesis
Coccidiosis in poultry: anticoccidial products and
alternative prevention strategies (a review)
Anticoccidial drug resistance in the field
Resistance to anticoccidial drugs of Dutch avian
Eimeria spp. field isolates originating from 1996, 1999 and 2001
(Avian Pathology, 32, (2003), pp. 392-401)
Anticoccidial drug sensitivity profiles of German, Spanish and
Dutch Eimeria spp. field isolates
(Tijdschrift voor Diergeneeskunde, 129, (2004), pp. 210-214)
Influence of ibuprofen, a mucolytic enzyme and a
mannanoligosaccharide preparation (MOS) on a
coccidiosis infection in broilers
Effect of ibuprofen on coccidiosis in broiler chickens
(Avian Diseases, 48, (2004), pp. 68-76)
Dietary protease can alleviate negative effects of a coccidiosis
infection on production performance in broiler chickens
(Animal Feed Science and Technology, 150, (2009), pp. 151-159)
The effect of an in-feed mannanoligosaccharide preparation
(MOS) on a coccidiosis infection in broilers
(Animal Feed Science and Technology, 134, (2007), pp. 347-354)
Influence of live coccidiosis vaccination on the occurrence
of anticoccidial drug resistance
Higher incidence of Eimeria spp. field isolates sensitive for
diclazuril and monensin associated with the use of live
coccidiosis vaccination with Paracox-5 in broiler farms
(Avian Diseases, 50, (2006), pp. 434-439)
1
2
5
105
106
125
139
140
157
171
181
182
Improvement of live anticoccidial vaccines

Cross protection studies between E. acervulina and
E. maxima, and E. tenella (submitted)
198
Summarizing discussion
215
Samenvatting
Dankwoord
Curriculum vitae
List of publications
197
227
239
241
243
Chapter 1
Introduction
General introduction
Introduction
Coccidiosis is a parasitic disease affecting mainly the intestinal tract. It is caused by
members of the genera Eimeria and Isospora belonging to the phylum Apicomplexa, and
characterized by a complex life cycle. It affects many species of mammals and birds, and
is of great economic significance in farm animals, especially poultry. Most animals are
parasitized by more than one species of Eimeria, which usually differ in pathogenicity and
tissue tropism. Although multiple species of coccidia have been identified, new
operational taxonomic units are being discovered regularly.
Most knowledge on coccidiosis has been obtained from poultry, in which the
disease has been studied most intensively as in this species the parasite causes the most
devastating losses due to the large number of birds per flock and high stock densities The
economic importance of coccidiosis can be related to decreased profit caused by higher
feed conversion, growth depression, increased mortality and the costs of prevention and
treatment. Worldwide the annual costs inflicted by coccidiosis to commercial poultry have
been estimated at 2 billion, stressing the urgent need for more efficient strategies to
control the parasite (Williams, 1999; Shirley et al., 2004; Dalloul & Lillehoj, 2006).
The traditional control of coccidiosis mainly relies on chemoprophylaxis which
appeared to be effective for the last decades. However the increased occurrence of
resistance against all anticoccidial drugs has left the poultry industry with a renewed
challenge for coccidiosis prevention and control and propelled the search for alternative
strategies amongst which vaccination is of major importance.
Aim of the thesis

Manuscripts documenting the occurrence of resistance against all commonly used
anticoccidial drugs abroad, together with the high incidence of clinical coccidiosis in the
field (60-90% of flocks) in the Netherlands, were the reasons to start investigations on the
occurrence of resistance of Dutch and other European Eimeria spp. field isolates (Chapter
2). This was preceded by an extensive literature review focusing on anticoccidial products
and alternative prevention strategies (Chapter 1). In this review, first, a general overview
of coccidial infections with Eimeria spp. in farm and game birds including history,
classification, life cycle and the determination and diagnosis of the disease is given. This is
followed by an outline on the host and site specificity of Eimeria spp., different
commercialized anticoccidial products (categories, mode of action, the occurrence and
management of resistance) and the influence of feed (structure, composition, ingredients,
toxins and administration) on the course of coccidiosis infections. Additionally, alternative
anticoccidial strategies such as biosecurity, management, homeopathy, phytotherapy,
aromatherapy, pre- and probiotics and vaccination are presented. The review is finalized
with conclusions and future perspectives.
CHAPTER 1
Based on the literature study one alternative coccidiosis control strategy, namely a
mannanoligosaccharide (a prebiotic) was chosen for further experimental studies in broiler
chickens. Additionally, two other products i.e. ibuprofen (a non-steroidal antiinflammatory drug- NSAID) and a mucolytic enzyme (protease) were also used in
experimental research aiming at assessing their anticoccidial potential. Ibuprofen was
chosen because as cyclooxygenase inhibitor it was expected to reduce the pathology of
coccidiosis lesions through impairment of the production of prostaglandins, and because it
was reported that another NSAID (indometacin) was able to reduce oocyst shedding
(Allen, 2000). The mucolytic enzyme was studied as it was speculated that a protease
might impair the attachment of Eimeria to the mucus layer through its degradation
(Chapter 3).
In view of the poor anticoccidial effect found in the studies performed with
mannanoligosaccharide, ibuprofen and the mucolytic enzyme, further research focused on
vaccination against chicken Eimeria, which despite some drawbacks was shown to be
highly effective against outbreaks of clinical coccidiosis (Williams, 2002). Another reason
to focus on vaccination was the inefficacy of rotation and shuttle programs to solve the
coccidiosis resistance problem, which is explained by the fact that resistance is stable even
in the absence of drug selection pressure (Gardiner & McLoughlin, 1963; Ball, 1968;
Williams, 1969; McLoughlin, 1970; Chapman, 1986). A welcome side effect of
vaccination is the improved sensitivity of Eimeria spp. field isolates for anticoccidial
drugs reported by some researchers. This phenomenon may play a key role in reducing the
anticoccidial drug resistance problem. However, large-scale field studies documenting this
were lacking. Therefore, in Chapter 4, the relation between the coccidiosis prevention
program (vaccination with a live attenuated anticoccidial vaccine or anticoccidial drugs in
feed) and anticoccidial drug sensitivity profiles of Eimeria spp. field isolates for diclazuril
and monensin was studied.
Major disadvantages of live anticoccidial vaccines are: 1/ propagation has to be
performed in the natural host, 2/ yield of oocysts is lowered for attenuated precocious
vaccines due to precocity characteristics and 3/ all relevant Eimeria spp. must be included
in the vaccine further increasing production costs. Coccidiosis vaccines could be produced
more efficiently if cross protection between Eimeria spp. would be relevant enough to
reduce the number of species included in the vaccine and/or reduce the vaccination doses.
Therefore, cross protection studies between an E. acervulina vaccine line and E. tenella,
and E. maxima were performed in Chapter 5.
The thesis is finalized with a summarizing discussion with conclusions and
perspectives for future research.
GENERAL INTRODUCTION
References
Allen, P.C. (2000). Effects of treatment with cyclooxygenase inhibitors on chickens infected with Eimeria
acervulina. Poultry Science, 79, pp. 1251-1258.
Ball, S.J. (1968). The stability of resistance to glycarbylamide and 2-chloro-4-nitrobenzamide in Eimeria
tenella in chicks. Research in Veterinary Medicine, 9, pp. 149-151.
Chapman, H.D. (1986). Eimeria tenella: stability of resistance to halofuginone, decoquinate and aprinocid
in the chicken. Research Veterinary Science, 40, pp. 139-140.
Dalloul, R.A. & Lillehoj, H.S. (2006). Poultry coccidiosis: recent advancements in control measures and
vaccine development. Expert Review of Vaccines, 5, pp.143-163.
Gardiner, J.L. & McLoughlin, D.K. (1963). Drug resistance in Eimeria tenella. III. Stability of resistance
to glycarbylamide. Journal of Parasitology, 49, pp. 657-659.
McLoughlin, D.K. (1970). Coccidiosis: experimental analysis of drug resistance. Experimental
Parasitology, 28, pp. 129-136.
Shirley, M.W., Ivens, A., Gruber, A., Madeira, A.M.B.N., Wan, K.L., Dear, P.H. & Tomley, F.M. (2004).
The Eimeria genome projects: a sequence of events. Trends on Parasitology, 20, pp. 199-201.
Williams, R.B. (1969). The persistence of drug resistance in strains of Eimeria species in broiler chickens
following a change in coccidiostat. Research Veterinary Science, 10, pp. 490-492.
Williams, R.B. (1999). A compartmentalized model for the estimation of the costs of coccidiosis to the
worlds chicken production industry. International Journal for Parasitology, 28, pp. 1209-1229.
Williams, R.B. (2002). Anticoccidial vaccines for broilers chickens: pathway to success. Avian
Pathology, 31, pp. 317-353.
Coccidiosis in poultry: anticoccidial products and

alternative prevention strategies (a review)
H.W. Peek and W.J.M. Landman
Animal Health Service (GD), P.O. Box 9, 7400 AA Deventer, the Netherlands
Summary
Coccidiosis in poultry is a parasitic disease with great economic significance which has
been controlled succesfully for decades using mainly anticoccidial products. However, the
increasing occurrence of resistance against all anticoccidial drugs and a possible upcoming
ban restricting their use prompted us to review the existing literature on anticoccidial
products and alternative coccidiosis prevention and control strategies. In the present
review the following subjects are discussed: (1) coccidiosis in poultry: history,
classification, Eimeria spp. life cycle, determination and diagnosis; (2) host and site
specificty of the coccidia; (3) anticoccidial products: mode of action, the occurrence of
resistance and efficious use; (4) the influence of feed structure, composition, toxins and
administration on the course of a coccidiosis infection; (5) other preventive strategies
against coccidiosis and (6) conclusions and perspectives. In chickens, seven Eimeria spp.
are considered of which E. acervulina, E. maxima and E. tenella are of crucial importance
as they most frequently affect broilers and these birds form a major part of the poultry
industry. The diagnosis of coccidiosis is generally performed at postmortem by identifying
the localization, morphology and extent of the intestinal lesions of Eimeria spp.
Shortcomings in the traditional diagnosis system are being dealt with by developing new
laboratory techniques such as computational analysis of microscopic images of oocysts
and various PCR-coupled and other molecular techniques. Particular attention has been
given to the usefulness of ITS-1 and -2 markers for the diagnosis and the analysis of
genetic variation of Eimeria. Host specificity of coccidia was thought to be very strict;
however, research showed that this is only true as far as completion of their life cycle is
concerned. Eimeria spp. are able to infect other species of hosts, restrictions being less
pronounced the closer host species are genetically related. Regarding site specificity,
coccidia invade narrowly defined cell types within a tissue or organ. In chickens, the
parasite develops in specifc defined regions of the intestine and this is also the case in
foreign hosts. Most anticoccidial products have more than one biochemical effect on a
specific stage of the coccidia and despite the lack of detailed knowledge on the selective
action, a broad categorization of their mode of action has been performed. Products
affecting cofactor synthesis are ethopabate, sulphonamides, pyrimethamines and thiamine
analogues like amprolium; products affecting mitochondrial functions are quinolones
(buquinolate, decoquinate and nequinate), pyridinols (meticlorpindol), nicarbizin,
robenidine and toltrazuril; and products that affect cell membrane function are polyether
antibiotics or ionophores (monensin, semduramycin, narasin, salinomycin, lasalocid and
COCCIDIOSIS IN POULTRY
maduramicin). Products with unknown mode of action are diclazuril and halofuginone.
The worldwide use of anticoccidial drugs has led to the development of resistance against
all these drugs. In order to minimize the occurrence of resistance, a rotation of various
anticoccidial drugs in single and/or shuttle programmes are used. Recently, live
anticoccidial vaccines have been incorporated into rotation programmes resulting in
increased incidence of anticoccidial drug sensitive Eimeria spp. field isolates. Various
feed components seem to affect Eimeria parasites, either directly by distributing the
development and multiplication of coccidia or indirectly by stimulating the immune
system, influencing the intestinal flora and mucosa, changing the physiology of the
digestive tract, etc. Feed components which negatively affect Eimeria parasites directly are
-3 fatty acids. Development of coccidia is negatively influenced in an indirect fashion by
-glucans, vitamin E, selenium, feed restriction, -3 fatty acids, vitamin A, C, K, medium
chain fatty acids and NaHCO3. Feed components that have been shown to favour the
recovery from a coccidiosis infection are: proteins, vitamin A, C and K. Management and
biosecurity measures should aim at avoiding the introduction of the parasite to the poultry
premises and infected poultry houses are best sanitated by using a lot of cleaning water
and disinfection with a solution of ammonia (5-10%). Phytotherapy has become
increasingly popular during the last decade due to a renewed interest in natural medicine.
Products showing a direct inhibitory effect on the development of the parasite are
arteminsin, citric extracts and some different herb extracts. Herbal products showing an
indirect effect on the development of coccidia are oregano, Echinacea, mushrooms
(lectin), turmeric and betain. Prebiotic mannanoligosaccharide (MOS) have been reported
to act directly on coccidia and either inhibit the development of the parasite or show no
effect. Probiotics have an indirect hindering effect on Eimeria parasites by stimulating the
humoral immune response. Both, attenuated and non-attenuated live anticoccidial vaccines
are becoming increasingly popular as an alternative control strategy against coccidiosis.
Nevertheless, high production costs, loss of infectivity with time affecting their expiry and
management shortcomings during application have limited their use in practice. Subunit
vaccines may circumvent most shortcomings of live vaccines; however, at present these
products underperform due to the lack of immunogenicity. It can be concluded that future
coccidiosis control is unlikely to be achieved solely by using anticoccidial products as feed
additives and/or through feed composition and management. The occurrence of resistance
against anticoccidial products will most probably further increase and although some
dietary compounds seem capable to reduce directly or indirectly a coccidiosis infection,
named compounds alone will not be sufficient to control the disease. In contrast,
anticoccidial vaccines offer a much better perspectieve despite a number of drawbacks
associated with their production and use. If with time the immunogenicity of subunit
vaccines can be improved, they could represent the next generation highly efficient and
low cost anticoccidial strategy.
CHAPTER 1
General introduction
Introduction
Coccidiosis is a disease caused by parasites of the genus Eimeria and Isopsora belonging
to the phylum Apicomplexa with a complex life cycle, affecting mainly the intestinal tract
of many species of mammals and birds. It is of great economic significance in farm
animals, especially chickens. Most knowledge on coccidiosis has been obtained from
chickens, where the disease has been studied most intensively as it is in commercial
poultry that this parasite causes the most damage due to the fact that birds are reared
together in large numbers and high densities. The economic significance of coccidiosis is
attributed to decreased animal production (higher feed conversion, growth depression and
increased mortality) and the costs involved in treatment and prevention. Worldwide the
annual costs inflicted by coccidiosis to commercial poultry have been estimated at 2
billion , stressing the urgent need for more efficient strategies to control this parasite.
Traditional coccidiosis control in poultry mainly relies on chemoprophylaxis;
however, the increasing occurrence of resistance against all anticoccidial drugs and a
possible upcoming ban on the use of these products as feed additives have propelled the
search for alternative control strategies for coccidiosis of which vaccination is of major
importance. The need for cost-efficient alternative anticoccidial strategies, which lays at
the basis of this thesis prompt us to perform an extensive literature review exploring the
alternative approaches to control Eimeria in poultry.
In the present review the following subjects are discussed:
1. History, classification, Eimeria spp. life cycle, determination and diagnosis
History and classification of the genus Eimeria
Eimeria spp. in farm and game birds
Life cycle of avian Eimeria spp.
Eimeria spp. determination and diagnosis
2. Host and site specificity of the coccidia

Host and site specificity of Eimeria spp.
3. Anticoccidial products: mode of action, the occurrence of resistance and efficious use
Anticoccidial products, categories and mode of action
Anticoccidial product resistance and management of resistance
4. The influence of feed structure, composition, toxins and administration on the course
of a coccidiosis infection
Feed structure, Feed composition (ingredients), Toxins and Feed management
5. Other preventive strategies against coccidiosis

Management and biosecurity
Alternative coccidiosis control (plant (herb) extracts, pre- and probiotics)
Vaccines
6. Conclusions and perspectives

7. References
7
1. Coccidiosis in poultry: history, classification, Eimeria spp. life

cycle, determination and diagnosis
1.1. Introduction
Coccidiosis is a parasitic disease with a complex life cycle, affecting many species of farm
and game birds and is of great economic significance, especially in chickens.
Most birds are parasitized by more than one species of Eimeria, which usually differ
in pathogenicity. Many species have been identified, although new operational taxonomic
units are being discovered regularly.
Traditionally, the diagnosis of coccidiosis is based on postmortem (assessment of
macroscopic lesions) and histopathological analysis. Moreover, morphological
identification of oocysts in native scrapings is performed using light microscopy. The
advent of computer aided oocyst morphology identification systems and molecular
techniques are greatly improving the shortcomings of classical diagnostic systems.
Especially techniques based on molecular biology may, in a near future, contribute
significantly to the understanding of the epidemiology of coccidiosis.
1.2. History and classification of the genus Eimeria

The first protozoan seen under the microscope by Antoine van Leeuwenhoek in 1674 was
probably an eimerian. At the time what he described as pus globules and hepatic lesions
due to tuberculosis, was identified 150 years later as unsporulated oocysts of Eimeria
stiedae in the bile duct of a rabbit (Hake, 1839). Eimer described in 1870 the endogen
cycles of Gregarina falciformis in the mouse, which was renamed later by Schneider as E.
falciformis and proposed as a new genus (Schneider, 1875). The class Sporozoa was
established by Leuckart in 1879 and the name Coccidium was introduced as a generic term
for the rabbit parasite.
Since then and up till now, the taxonomy of coccidia has been further defined and
underwent many changes. In the traditional general classification scheme (e.g. Btschli
1880-1882) the phylum Protozoa was divided into four classes (Mastigophora
(flagellates), Sarcodina (amoeboid organism), Infusoria (ciliates) and Sporozoa (a parasitic
group)), Coccidia being a subclass of the class Sporozoa. This structure dominated mainly
the systematic protozoology from the 1880s until the 1980s and during this era only a
few trends modified this Btschlian view. One trend was the process of a hierarchical
increase in the number and rank of taxa. In the early schemes only a single phylum of
protozoa was documented and this view was maintained until the 1960s (Honigberg et al.,
1964). Later on, some lifted the protozoa to the Kingdom status because of the increase of
number of phyla (Levine et al., 1980; Cavalier-Smith, 1998; Corliss, 1998). However, this
increase did not go together with important new information and eventually led to
widespread taxonomic dismissal. The end of the protozoa as a phylum was favoured by
8
CHAPTER 1
the gradual recognition that the popular Btschlian view was artificial and out of date,
moreover electron microscopy, serial endosymbiosis theory of cell evolution, 16S rRNA
sequence data and protein gene sequences further triggered changes in evolutionary
protistology, finally coming to a classification in which the four classes of protozoa are no
longer present (Patterson, 2002). More recently, integrating ultrastructural data with
molecular phylogenetic studies a scheme based on nameless ranked systematics was
proposed, in which six clusters of eukaryotes (similar to traditional kingdoms) are
recognized. The former genus Eimeria has been allocated within the Coccidiasina
(Leuckart, 1879), Conoidasida (Levine, 1988) and Apicomplexa (Levine et al., 1980; Adl
et al., 2005), which in turn belongs to the first rank of Alveolata (Cavalier-Smith, 1991).
The latter is part of the super-group Chromalveolata of the Eukaryotes (Adl et al., 2005).
1.2.1. Characteristics of Coccidiasina (Adl et al., 2005)
Mature gametes develop intracellularly; microgamont typically produces numerous
microgametes; syzygy absent; zygote rarely motile; sporocysts usually formed within
oocysts. Including the genus Cryptosporidium, Cyclospora, Eimeria and Hepatozoon.
1.3. Eimeria spp. in farm and game birds

Great pioneering work on the life cycle of coccidia in different hosts, parasite morphology,
studies on host-specificity was performed during the 1920s and 1930s amongst others by
Edward Tyzzer (Tyzzer, 1929; Tyzzer et al., 1932). He is considered the father of modern
coccidiology and produced a series of outstanding papers on monospecific Eimeria
infections in chicken, turkey and some game birds.
Generally coccidia are highly host organ and tissue-specific. Under normal circumstances,
most animals shed small amounts of oocysts in their faeces without clinical signs of
coccidiosis. However, coccidiosis can become clinically manifest in case chickens are
exposed to large numbers of oocysts instead of low doses typical for trickle infections. The
former situation is favoured particularly by husbandry systems with high farm animal
density.
1.3.1. Farm birds
Eimeria spp. are found in the domestic fowl, turkeys, waterfowl (geese and ducks) and
pigeons.
1.3.1.1. Domestic fowl

In chickens nine Eimeria spp. have been described: i.e. E. acervulina (Tyzzer, 1929), E.
brunetti (Levine, 1942), E. maxima (Tyzzer, 1929), E. mitis (Tyzzer, 1929), E. mivati
(Edgar & Siebold, 1964), E. necatrix (Johnson, 1930), E. praecox (Johnson, 1930), E.
tenella (Raillet & Lucet, 1891) and E. hagani (Levine, 1938). However, as the existence of
E. hagani and E. mivati is questioned, in practice only seven species are considered. If, no
convincing evidence for the existence of E. hagani and E. mivati is delivered in the future,
both will be eliminated from the reference list with time (McDougald, 2003; Shirley,
1986).
The diagnostic characteristics of Eimeria spp. in chickens have been outlined in Table 1.
1.3.1.2. Turkeys
Seven different Eimeria spp. of turkeys have been described but only a few species are
considered to be economically important. The most important pathogenic species are E.
meleagrimitis, E. adenoeides, E. gallopavonis and E. dispersa. Recently, Matsler and
Chapman (2006) have investigated the potential pathogenicity of E. meleagridis and they
suggested that the significance of this species as a pathogen should be reconsidered. In
another article they also described the selection of a precocious line of E. meleagridis
(Matsler & Chapman, 2007).
The detection of oocysts (even large numbers) in faeces can only yield a tentative
diagnosis for coccidiosis since differentiating the oocysts of the pathogenic species from
the less pathogenic or milder species is very difficult. Therefore, a satisfactory diagnosis
can only be obtained after postmortem examination, although the lesions of some species
are poorly described. E. innocua and E. subrotunda have been found so rarely that their
existence is questioned. The diagnostic characteristics of Eimeria species in turkeys can be
found in Table 2 (McDougald, 2003).
More recently a comprehensive review on coccidiosis in turkeys is published by Chapman
H.D. (2008) where the biology of the Eimeria spp. of turkeys, host specificity and
resistance, immunity, pathology, pathogenicity, diagnosis, prevalence, treatment and
prevention are described.
1.3.1.3. Geese
Five species of coccidia have been described in domestic and wild geese. Some of which
are associated with severe disease and even death. The most prevailing and harmful in
commercial flocks are E. truncata, which causes renal coccidiosis, and E. anseris which
causes intestinal coccidiosis (Betke & Wilhelm, 1976). E. truncata occurs in the kidney of
young goslings and may cause high mortality due to a blockage of the kidney function
(Sezen et al., 1981).
1.3.1.4. Ducks
Outbreaks of coccidiosis in ducks are sporadic but occur frequently enough in order to
draw the attention of veterinarians. Until now, five Eimeria species have been described in
ducks (Levine, 1985). Cases of coccidiosis from moderate to severe appear to be very
10
CHAPTER 1
common worldwide. These infections can cause high morbidity and mortality as well as
poor technical performance. E. danailovi is considered to be one of the most pathogenic
species (Grfner et al., 1965).
1.3.1.5. Pigeons
To date two species of coccidia have been reported in pigeons: E. labbeana and E.
columbarum. However, it is well possible that it concerns only one species as the sizes of
their oocysts overlap and the morphology of both species is similar. E. labbeana is
considered to produce the smallest oocysts for those believing that both species exist,
moreover it is considered to be the most pathogenic species.
All Eimeria species found to date in farm birds are outlined in Table 3.
1.3.2. Game birds (pheasants, partridges, quails and grouse)
Coccidiosis occurs in all species of game birds (Norton, 1986). It is mainly a disease of
young animals and is characterised by signs of weakness and watery droppings frequently
containing blood. Affected birds are listless and show decreased water and feed intake. As
the illness progresses moderate to high mortality can occur.
1.4. Life cycle of avian Eimeria spp.

Fantham (1910) was the first to describe the entire life cycle of an eimerian parasite in an
avian host. Later on, Tyzzer (1929) published detailed descriptions of the life cycle stages
of various Eimeria spp. (E. acervulina, E. mitis, E. maxima and E. tenella) in sections of
intestines. In 1932, he also described details of the life cycle of E. praecox and E. necatrix
(Tyzzer et al., 1932).
More recently, the life cycle of avian Eimeria spp. has been well documented by various
other authors (Long & Reid, 1982; Fernando, 1990; McDougald, 2003).
Although numerous drawings on the life cycle of avian Eimeria spp. have been published,
the website of the book Encyclopedic Reference of Parasitology by Heinz Mehlhorn
(2001) shows a clear and detailed scheme (Figure 1), which can be easily accessed online.
11
Table 1. Diagnostic characteristics of Eimeria spp. in the chicken
Table 2. Diagnostic characteristics of Eimeria spp. in the turkey
McDougald, L.R. (2003). Coccidiosis. In: Saif, Y.M., Barnes, H.J., Glisson, J.R., Fadly, A.M., McDougald, L.R., & Swayne, D.E. (eds.). Diseases of Poultry 11th
edn. (pp. 986). Ames: Iowa State University Press. 2003
Table 3. Oocyst habitat, morphology and pathogenicity of Eimeria spp. in farm birds
(Levine, N.D., 1985)
Species
Oocyst size (m)

Length Width
Shape
Pathogenicity
Reference
12-23
14-34
9-17
12-26
Ovoid
Ovoid
Low
Moderate
Tyzzer, 1929
Levine, 1942
16-21
21-42
10-21
11-20
14-19
16-30
9-18
12-17
Low
Low to moderate
Low
Low to moderate
12-29
11-24
Ovoid
Ovoid
Subspheric
Ellipsoid
or ovoid
Ovoid
Levine, 1942
Tyzzer, 1929
Tyzzer, 1929
Edgar & Siebold,
1964
Johnson, 1930
20-25
14-31
16-20
9-25
Ovoid
Ovoid
No
High
Johnson, 1930
Raillet & Lucet,
1891
19-31
13-21
High
22-31
18-24
Ellipsoid
or ovoid
Ovoid
Low
Moore & Brown,

1951
Tyzzer, 1929
22-33
15-19
Ellipsoid
Moderate
Hawkins, 1952
19-26
17-25
Subspheric
No
19-31
14-23
Ellipsoid
No
16-27
16-26
13-22
14-24
Subspheric
Subspheric
Moderate
No
Tyzzer, 1929
Moore et al., 1954
E. anseris
E. kotlani
Small intestine,
caeca, rectum
Small intestine
Small intestine
Geese
Small intestine
Large intestine
Moore & Brown,

1952
Tyzzer, 1927
16-24
29-33
13-19
23-25
Ovoid
Moderate
Low
E. nocens
E. stigmosa
E. truncate
Small intestine
Small intestine
Kidneys
25-33
23-16
14-27
7-24
23-17
12-22
Ovoid
Ovoid
Ovoid
Moderate
No
High
Kotlan, 1932
Grfner &
Graubman, 1964
Kotlan, 1933
Klimes, 1963
Raillet & Lucet,
1891
E. anatis
E. battakhi
Ducks
Small intestine
?
14-19
19-24
11-16
16-21
19-23
17-21
16-26
11-15
13-15
12-20
E. columbarum
Small intestine
Small intestine
?
Pigeons
Intestine
Ovoid
Ovoid to
subspheric
Ovoid
Ovoid
Ovoid
19-21
17-20
Ellipsoid
E. labbeana
Intestine
15-18
14-16
Subspheric
to spheric
E. acervulina
E. brunetti
E. hagani
E. maxima
E. mitis
E. mivati
E. necatrix
E. praecox
E. tenella
E. adenoeides
E. dispersa
E. gallopavonis
E. innocua
E. meleagridis
E. meleagrimitis
E. subrotunda
E. danailovi
E. saitamae
E. schachdagica
14
Host/Habitat
Chickens
Small intestine
Small intestine,
rectum, caeca,
cloaca
Small intestine
Small intestine
Small intestine
Small, large
intestine
Small intestine,
caeca
Small intestine
Caeca
Turkey
Small intestine,
caeca, colon
Small, large
intestine,
Large intestine,
caeca
Small intestine
High
High
High
?
Scholtyseck, 1955
Dubey & Pande,
1963
Grfner et al., 1965
Inoue, 1967
Musaev et al., 1966
Low to mild
Mitra & Das Gupta,

1937
Pinto, 1928
CHAPTER 1
Figure 1. Life cycle of Eimeria spp. in chickens

1 After oral uptake of sporulated oocysts the sporozoites hatch in the small intestine from the sporocysts.
26 After penetration, multinucleate schizonts are formed (3) inside a parasitophorous vacuole (PV). The
schizonts produce motile merozoites (DM, M), which may initiate another generation of schizonts in other
intestinal cells (25) or become gamonts of different sex (7, 8). 7 Formation of multinucleate
microgamonts, which develop many flagellated microgametes (7.1-7.2). 8 Formation of uninucleate
macrogamonts, which grow to be macrogametes (8.1) that are characterized by the occurrence of two
types of wall-forming bodies (WF1, WF2). 9 After fertilization the young zygote forms the oocyst wall by
consecutive fusion of both types of wall-forming bodies (FW). 10 Unsporulated oocysts are set free via
faeces. 1113 Sporulation (outside the host) is temperature dependent and leads to formation of four
sporocysts, each containing two sporozoites (SP), which are released when the oocyst is ingested by the
next host.
DG, developing microgametes; DM, developing merozoite; DW, developing wall-forming bodies; FW,
fusion of WF1 to form the outer layer of OW; M, merozoite; N, nucleus; NH, nucleus of host cell; OW,
oocyst wall; PB, polar body (granule); PV, parasitophorous vacuole; R, refractile (= reserve) body; SB,
sporoblast; SP, sporozoite; SPC, sporocyst; SPO, sporont; WF1, wall-forming bodies I; WF2, wall-forming
bodies II; Z, cytoplasm of zygote (= young oocyst) (Mehlhorn, 2001).
Reproduced with kind permission of Springer Science and Business Media.
15
1.5. Eimeria spp. determination and diagnosis

All Eimeria spp. of the chicken parasitize the intestine and can be differentiated by their
morphology, cell tropism and pathogenicity (Long et al., 1976; Long & Reid, 1982).
These criteria are adequate enough to identify the five most pathogenic and prevailing
species: E. acervulina, E. brunetti, E. maxima, E. necatrix and E. tenella. Their
pathogenicity varies from moderate to very severe. The other two species E. praecox and
E. mitis do not induce very clear characteristic identifiable lesions and are considered to be
benign. However, in experiments performed by Williams (1998), these species caused
intestinal (mucous membrane) inflammations, diarrhea and reduced feed conversion. It
suggested that they can actually cause economic damage and ought to be controlled as
well.
Traditionally, the diagnosis of coccidiosis in poultry is performed most accurately during
postmortem and histopathology. Macroscopically, this is done by thoroughly examining
the location and extents of lesions in the intestines and is complemented by analyzing
native intestinal scrapings with light microscopy in order to morphologically identify
schizonts and oocysts. Assessment of coccidiosis lesions is performed standard following
the scoring system introduced by Johnson and Reid (1970). E. acervulina lesion score 1
features ladder-like white streaks in de duodenal loop ( 5/cm2); in lesion score 2 the
lesion density is higher but not coalescent; in lesion score 3 the lesions are more numerous
and coalesce, while thickening of the intestinal wall is visible, and finally in lesion score 4
the mucosal wall is greyish with lesions completely coalesced. Lesion score 1 of E.
maxima is characterised by few small red petechiae in the mid-intestine, in score 2 more
numerous petechiae are found and the intestinal content may be orange, in score 3 the
intestinal wall might be ballooned and thickened with pinpoint blood clots and mucous
filling the intestinal contents. Finally, in lesion score 4 the intestinal wall is thickened and
ballooned over most of its length, containing numerous blood clots and digested red blood
cells. Lesion score 1 of E. tenella shows few scattered petechiae on the caecal wall, in
score 2 noticeable blood can be found in the caecal contents, while the caecal wall is
thickened. In lesion score 3 large amounts of blood or caecal cores are present, caecal
walls are greatly thickened, and in lesion score 4 caecal walls are enormously distended
with blood or large caseous cores.
The standard McMaster oocyst counting technique is a widely used laboratory method to
monitor coccidiosis infection pressure in chicken houses (Hodgson, 1970). This technique
can also be used to determine individual oocyst shedding patterns and to study the effect
of anticoccidial drugs. A commonly used modification of the standard oocyst counting
technique per gram faeces (OPG) is performed by making a faeces suspension in a salt
solution of which a small volume is pipetted into saturated sodium chloride. A portion of
the latter suspension is subsequently run into a McMaster chamber and oocysts are
counted using a microscope (Peek & Landman, 2003). More recently, other modifications
of the standard counting technique have been described (Haug et al., 2006).
16
CHAPTER 1
1.5.1. Advances in diagnosis

1.5.1.1. Species identification based on morphology
Due to the overlap of morphological characteristics of Eimeria spp. oocysts, visual
identification of species is far from accurate. Recently, identification of Eimeria oocysts
was greatly improved by classification through computational analysis of microscope
digital images. Images are pre-processed and the parametric contour of oocyst wall is
defined, then, using shape and textural characteristics (oocyst length and width, perimeter,
area, curvature, texture and symmetry) they are classified after automated feature
extraction. Images can be easily uploaded and the user can easily obtain a real-time
electronic
diagnosis
of
the
Eimeria
spp.
concerned
at
http://puma.icb.usp.br/coccimorph/requirements.html (Castanon et al., 2007).
1.5.1.2. Molecular techniques
The first molecular technique used for the identification of Eimeria spp. was the
characterization of isoenzyme patterns of oocysts by starch block electrophoresis (Shirley,
1975). However, this method requires large number of parasites, is time-consuming and
difficult to perform. Moreover, the low and limited number of variable enzymes and the
low level of polymorphism restricted the application of this method (Johnston &
Fernando, 1997).
In the early 1990s, it was demonstrated that rRNA and rDNA probes could be used to
identify Eimeria spp. through characteristics restriction fragment patterns (Ellis &
Bumstead, 1990). Shortly thereafter, the random amplified polymorphic DNA (RAPD)
technique was developed, which is based on the amplification of DNA using arbitrary
primers (Welsh & McClelland, 1990, Williams et al., 1990). However, despite its broad
use, the RAPD technique had a lack of reliability and reproducibility (MacPherson et al.,
1993; Schierwater & Ender, 1993).
A specific PCR diagnostic assay based on an amplification of the Internal Transcribed
Spacer-1 (ITS-1), was then developed (Schnitzler et al., 1998; 1999). This test became a
standard for the molecular diagnosis of seven Eimeria spp. and was also used to study the
intra-strain variation of Eimeria in chickens (Lew et al., 2003; Su et al., 2003). However,
due to the occurrence of variability in the amplification of E. maxima strains due to the
polymorphic nature of ITS-1, new markers were sought. A novel RAPD-derived marker,
the (Sequence-Characterised Amplified Region) SCAR, that could be amplified under
stringent conditions was found (Paran & Michelmore, 1993). These SCAR markers made
it even possible to develop a simple multiplex PCR test, which can differentiate in one test
between seven Eimeria spp. (Fernandez et al., 2003a, 2003b) and a public relational
database was constructed, the Eimeria SCARdb, a free-access database (available at the
address: http://puma.fmvz.usp.br/eimeriaScardb) that represents a complete and important
source of SCAR markers (Fernandez et al., 2004).
Recently, a PCR-coupled capillary electrophoretic approach using genetic markers of the
ITS-2 region of nuclear ribosomal DNA has been developed and investigated for its use
for the identification and epidemiology of seven Eimeria spp. (Gasser et al., 2005; Morris
17
& Gasser, 2006). This technique may, in a near future, contribute significantly to the
understanding of the epidemiology of each Eimeria spp. This knowledge may be used in
formulating and implementing new prevention and control programmes (Morris et al.,
2007).
18
CHAPTER 1
2. Host and site specificity of the coccidia

2.1. Introduction
Historically it was assumed that host specificity of coccidia was very strict. The term
refers to the restriction of a species of parasite to one or limited species of hosts (Levine,
1973; Noble & Noble, 1976). However, further research on basic characteristics of
parasite biology brought up evidence questioning the strictness of host specificity, which
seems strict only as far as completion of their lifecycle with the production of oocysts is
concerned (Duszynski, 1981; Marquardt, 1981; Fayer, 1980; Fernando, 1990). The
certainty that Eimeria spp. exclusively invade the intestinal tract has been revoked by the
demonstrations of parenteral (extraintestinal) development of some eimerians in mammals
and birds (Table 4) and may even be more prevalent than we appreciate (Desser, 1978;
Overstreet, 1981; Ball et al., 1989; Novilla & Carpenter, 2004).
2.2. Host specificity of Eimeria spp.

Eimeria species include some of the most common and best known protozoan parasites
with over 900 different species found in a wide range of animals including annelids,
insects, reptiles, amphibians, birds and mammals (Levine, 1973; Noble & Noble, 1976;
Pellerdy, 1974). Some hosts have more than one Eimeria spp., but one species rarely
occurs in more than one host species although there are some exceptions (Tsunoda &
Muraki, 1971; Norton & Pierce, 1971; Tsutsumi, 1972; Marquardt, 1981; Joyner, 1982;
Ruff et al., 1984; Ruff, 1985).
Host specificity of Eimeria spp. in chicken was probably first demonstrated by Johnson
(1923). He was unable to infect turkeys with Eimeria from chickens indicating host
specificity. Later on host specificity of Eimeria spp. of various groups was investigated by
performing cross-transmission experiments with rodents (Levine & Ivens, 1965), domestic
chicken and turkeys (McLoughlin, 1969), lagomorphs (Duszynski & Marquardt, 1969)
and ruminants (Levine & Ivens, 1970).
A detailed description on the results of cross transmission experiments with Eimeria spp.
of chickens and turkeys is outlined in Table 5, which shows some remarkable
inconsistencies (McLoughlin, 1969). Discrepancies also occurred in some transmission
experiments of Doran (1978) with E. dispersa from the turkey. He was able to induce
patent infections in Leghorn chickens, the Chukar partridge, ring-necked pheasant and
bobwhite quail and these results were in agreement with previous work of Long & Millard
(1979), Tyzzer (1929) and Norton (1979) whereas others have not been able to infect
chickens with E. dispersa (Kogut & Long, 1981).
Later on, in a survey, concerning the host specificity of the Eimeria spp. of rodents,
Levine & Ivens (1988) found that 14 out of 112 attempts were successful in transmitting
the Eimeria spp. from one genus of rodents to another and that within the same genus 39
of 49 transmissions of Eimeria spp. between species of hosts were successful.
19
Norton (1967) demonstrated that E. colchici, an Eimeria spp. from the pheasant, could
produce coccidial infections in turkeys if large numbers of oocysts was inoculated and
Marberry & Marquardt (1973) succeeded in transmissing E. separata from the rat to the
mouse.
Most species of Eimeria are still considered to be monoxenous (i.e. they complete their life
cycle in one host) (strictly host specific) and stenoxenous (each species parasitizes a single
host) these characteristics can however no longer be regarded as absolute (Fayer, 1980;
Marquardt, 1981; Joyner, 1982) considering the results of cross transmission studies,
which strongly suggest that closely related species or subspecies may serve as the most
adequate host for transmitting Eimeria spp. In this regard E. chinchillae, a coccidian
species which parasitizes the chinchilla (de Vos & van der Westhuizen, 1968) is an
exception as it showed a total lack of the normal host specificity characteristics of Eimeria
spp. crossing families of hosts (de Vos, 1970).
Table 5. Summary of the results of cross transmission experiments with Eimeria spp. of
chickens and turkeys (McLoughin, D.K., 1969)
Speciesa
Eimeria spp.
E. acervulina
E. maxima
E. mitis
E. tenellab
E. avium
(= E. tenella ?)
E. adenoeides
20
From (host)
Turkey
Duck
Chicken
"
"
"
Quail
"
Chicken
Chicken
"
Quail (?)
Quail
"
Chicken
"
"
"
"
"
"
"
To (receiver)
Chicken
"
Quail
"
Pheasant
Turkey
Chicken
"
Quail
Quail
Turkey
Chicken
Chicken
"
Quail
Turkey
Duck
Pheasant
"
Pigeon
"
"
Transmission
Yes
No
No
No
No
No
Yes
No
No
No
No
Yes
Yes
Yes
No
No
No
No
Yes
Yes
Yes
Yes
Grouse
Chicken
"
Turkey
"
Chicken
Turkey
Duck
Chicken
Guinea
Yes
No
No
No
No
Author
Henry, 1931
Tiboldy, 1933
Patterson, 1933
Tyzzer, 1929
Tyzzer, 1929
Steward, 1947
Henry, 1931
Venard, 1933
Patterson, 1933
Patterson, 1933
Tyzzer, 1929
Henry, 1931
Henry, 1931
Venard, 1933
Patterson, 1933
Patterson, 1933
Patterson, 1933
Patterson, 1933
Haase, 1939
Hofkampd
Nieschulz, 1921
Yakimoff & IwanoffGobzem, 1931
Fantham, 1910
Johnson, 1923
Johnson, 1923
Moore & Brown, 1951
Moore & Brown, 1951
CHAPTER 1
E. gallopavonis
E. meleagridis
E. meleagrimitis
E. innocua
E. subrotunda
"
"
"
Turkey
"
"
"
Turkey
"
"
"
"
"
"
"
"
Turkey
"
"
Turkey
"
"
"
Turkey
"
"
"
Pheasant
Quail
Chicken
Partridge
Pheasant
Quail
Chicken
Chicken
"
"
"
"
Pheasant
Partridge
Quail
"
Quail
Partridge
Chicken
Chicken
Guinea
Pheasant
Quail
Chicken
Guinea
Pheasant
Quail
No
No
No
Yes
No
No
Yes
Yes
Yes
No
No
No
No
No
No
No
No
No
Yes
No
No
No
No
No
No
No
No
Moore & Brown, 1951

Moore & Brown, 1951
Clarkson, 1959a, b
Hawkins, 1952
Hawkins, 1952
Hawkins, 1952
Gill, 1954
Steward, 1947
Gill, 1954
Tyzzer, 1929
Moore et al., 1954c
Clarkson, 1959a, b
Tyzzer, 1929
Hawkins, 1952
Tyzzer, 1929
Hawkins, 1952
Hawkins, 1952
Hawlins, 1952
Gill, 1954
Moore & Brown, 1952
Moore & Brown, 1952
Moore & Brown, 1952
Moore & Brown, 1952
Moore et al., 1954
Moore et al., 1954
Moore et al., 1954
Moore et al., 1954
Natural infections of E. dispersa in a number of hosts have been reported, therefore studies with this
species are not included.
b
Haase, 1939, also reported E. tenella from quail.
c
Moore, Brown and Carter, cited by Moore, 1954.
d
Quoted by Yakimoff & Iwanoff-Gobzem, 1931.
The mechanisms responsible for inhibiting the reproduction/multiplication of Eimeria spp.

in foreign hosts are currently unknown, undeniably very complex and probably diverse
regarding the differences in the degree of host specificity. However, mentioned
mechanisms are extremely effective because sporozoites from various Eimeria spp. are
able to invade the intestinal mucosa of foreign hosts, indicating that excystation of
sporozoites is a non-specific event in most foreign hosts (Joyner, 1982; Kogut & Long,
1984; Sundermann et al., 1987;), but are not able to complete their lifecycle (Marquardt,
1966; Todd et al., 1971; Vetterling, 1976; Long & Millard, 1979; Marquardt, 1981;
Joyner, 1982; Mayberry et al., 1982; Rose & Millard, 1985; Naciri, 1986).
The basis of the mechanisms, which are responsible for this host specificity, probably lays
in many biotic interactions e.g. the parasite biochemistry and nutrition, the host genome
and in particular the host immune system.
21
2.2.1. Parasite biochemistry and nutrition

After invading the host cells, coccidia are retained within a parasitophorous vacuole
membrane (PVM) for the duration of their intracellular phase. Therefore, nutrients and
wastes have to cross the parasite membrane, the PVM and the host cell membrane.
However, there is still a lack of knowledge on the mechanisms involved in the transport of
nutrients through the different membranes, despite the fact that they are interesting drug
targets. The PVM is permeable to low molecular solutes by high-capacity channels
(Werner-Meier & Entzeroth, 1997). Although not completely characterized to the
molecular level, uptake of nutrients by the parasite seems to occur through a combined
action of transporters and channels in the parasite membrane and endocytosis (Saliba &
Kirk, 2001).
A list of nutrients necessary for the development of E. tenella through the second
generation merogony was obtained by studying the parasite in vitro. However, the
requirements in vivo may be quite different, which is suggested by the lack of host
specificity in vitro, indicating that the parasite will only develop in those cases where the
nutrient requirements are satisfied. Named nutrients are: p-aminobenzoic acid (PABA),
thiamine, glutamine, pyridoxal, biotin, folic acid, ascorbic acid, riboflavin, nicotinamide,
pyridoxone, vitamin A, menadione, choline chloride, vitamin B12, calciferol, -tocopherol
and inositol (Warren, 1968; Ryley & Wilson, 1972; Sofield & Strout, 1974; Doran &
Augustine, 1978; Latter & Holmes, 1979;).
Studying the biochemistry of Eimeria may be of help in understanding their further
nutrient requirements. Eimeria spp. are obligate intracellular parasites, which has made
biochemical research on their nutritional requirements very difficult. Despite this
limitation gathered data indicate that although the coccidia go through complicated life
cycles the basic pattern of metabolic activities have not significantly changed (Naciri,
1986). These metabolic activities mainly include consumption of polysaccharides (Wagner
& Foerster, 1966), cell division (Hammond, 1973; Ryley, 1973; LaFon & Nelson, 1985),
amplification or continuation of polysaccharide storage (Wagner & Foerster, 1966; Fry et
al., 1984; Smith & Lee, 1986) and the process of active excitation and invading new host
cells.
The biochemistry of coccidia has significant differences with that of vertebrates providing
a wealth of possibilities for drug-designers. Biochemistry pathways that could be used as
anticoccidial drug targets are: plastid-associated activities, energy metabolism,
pyrophosphate metabolism and acidocalcisomes, electron transport chain and the
acquisition of pyrimidines and purines, polyamine metabolism, proteases and host cell
invasion, anti-oxidants, the mannitol cycle, folate metabolism and the newly discovered
protein kinases (Coombs & Mller, 2002). So far, knowledge on the biochemistry of
Eimeria spp. has yielded a few examples of drugs with proven or potential interest to
control coccidiosis infections. Nitrophenide a drug inhibiting the mannitol cycle,
effectively reduced oocyst excretion (Schmatz, 1997). Aprinocid was reported to interfere
with the hypoxanthine and guanine acquisition by Eimeria (Wang et al., 1979). The
enzyme -difluoromethylornithine (inhibitor of ornithine decarboxilase) has shown
22
CHAPTER 1
activity against E. tenella in chickens and some proteinase inhibitors have also shown to
affect coccidia parasites (Coombs et al., 1997).
2.2.2. Genetic makeup of the host
Host specificity is believed to be connected to genetic nearness, as phylogenetic closely
related hosts are more likely to sustain a familiar parasite (Doran, 1953; Vetterling, 1976).
Later studies further supported this view and indicated that resistance to coccidiosis is
under genetic control of the host (Mayberry et al., 1982; Bumstead & Millard, 1987;
Mathis & McDougald, 1987; Bumstead & Millard, 1992; Bumstead et al., 1995). In
resistant hosts the Eimeria infection is probably controlled by an efficient immune
response, which in turn is dictated by the genetic makeup.
2.2.3. Host immune system
Eimeria parasites have a complicated life cycle with asexual and sexual stages as well as
intra- and extracellular phases resulting in an equally complex immune response. Although
humoral and cell-mediated immunity are activated after an Eimeria infection besides the
participation of non-specific immunity, understanding of Eimeria immunology is still
subject of ongoing research.
The role of antibodies in controlling coccidiosis infections seems limited as hormonal and
chemical bursectomy does not abrogate an efficient host response (Lillehoj, 1987; Rose &
Long, 1970; Yun et al., 2000) and further indicates the importance of cell-mediated
immunity. This was further evidenced by the fact that abrogation of the T cell function
impaired protective immunity against coccidiosis. Extensive research has shown that the
antigen-specific and non-specific activation of T lymphocytes and macrophages are of
main importance in protective immunity against Eimeria. More specifically, Th 1
responses elicited by IFN- seem dominant in the immune response induced by Eimeria
(Lowenthal et al., 1997; Lillehoj & Choi, 1998; Lowenthal et al., 1998; Lillehoj et al.,
2000).
Attempts to identify the genes participating in coccidiosis immunity are ongoing and will
in near future enable a better understanding of the immune responses to coccidiosis and
the host-parasite interaction. QTL (quantitative trait loci) associated with resistance to
coccidiosis were recently mapped with DNA marker technology (Zhu et al., 2003), while
using DNA array with EST (Expressed Sequence Tags) from activated T cell library
several genes associated with immune responses to E. maxima and E. tenella were
identified; most clearly IL-15 and IFN- being upregulated (Min et al., 2003).
2.3. Site specificity of Eimeria spp.

Although Eimeria spp. parasitize a great diversity of hosts, individual species exhibit a
high degree of host specificity, as far as the ability to complete their life cycle with oocyst
production is concerned (Levine, 1973; Joyner, 1982), and site specificity. Coccidia
23
invade narrowly defined cell types within a tissue or organ (Table 4, Novilla & Carpenter,
2004). Site specificity for invasion is even so precise that intravenous, intramuscular, or
intraperitoneal injections of mammalian or avian Eimeria spp. results in infection in the
same area of the intestine as if the animals were infected with the parasite orally. This
happens in the natural host but it also occurs in foreign host (Davies & Joyner, 1962;
Sharma & Reid, 1962; Long & Rose, 1965; Long & Millard, 1976; Joyner, 1982;
Augustine & Danforth, 1986; Augustine & Danforth, 1990; Augustine, 2001). Thus, site
specificity for invasion of sporozoites in the intestine may be a result of some
characteristics of the intestines that are shared by a number of hosts rather than a unique
peculiarity of the natural host.
The invasion of host cells by Eimeria sporozoites is, similar to other members of the
Apicomplexa, likely largely regulated by the apical complex. Although exact mechanisms
have not been elucidated yet, there is evidence that proteinases play a key role in the
invasion process: i.e. penetration of the mucus layer as well as attachment and entry to the
host cell. Proteinases of other parasites have been characterised and the activity of
proteinase inhibitors against them has been demonstrated, suggesting that this is an area
that may hold promise for an alternative therapeutic approach for coccidiosis (Coombs &
Mller, 2002). Besides proteinases, there is evidence from in vitro studies that specific
molecules on intestinal cells act as receptor molecules for attachment and invasion. Some
host cell molecules involved in invasion are 22, 31 and 37 kDa antigens, surface
membrane glyconjugates and common epitopes. Especially host cell membrane
glyconjugates are regarded as potential receptors for parasite lectins (lectin-ligand binding)
but also microneme proteins like thrombospondin. However, treatments altering
mentioned putative receptors were unable to completely block parasite invasion
(Augustine, 2000; 2001), suggesting that other mechanisms may be operational as well.
Table 4. Eimeria spp. found in extraintestinal tissues of birds and mammals (Novilla et al.,
2004)
Species
E. stiedai
E. tenella
E. deblieki
E. hiepei
E. adenoeides
E. arloingi, christenseni,
crandalis
E. arloingi, faurei,
ninakohlyakimovae
E. neitzi
E. gaviae
E. truncate
E. boschadis,
christiansensi, somateria
E. reichenowi, gruis
24
Host(s)
Rabbit
Chicken embryo
Pig, chamois
Mink
Turkey
Goat
Sheep and goat
Impala
Common loon
Goose
Mallard duck, mute
swan, eider
Sandhill crane,
whooping crane
Tissue
Liver
Liver
Liver
Bile ducts
Gall bladder
Mesenteric
lymph node
Mesenteric
lymph node
Uterus
Kidney
Kidney
Kidney
Reference
Pellerdy, 1969
Long, 1971
Desser, 1978
Ball et al., 1989
Critchley et al., 1986
Lima, 1979
McCully et al., 1970

Montgomery et al., 1978
Gajadhar et al., 1983
Ball et al., 1989
Widespread
Novilla et al., 1981
Lotze et al., 1964
CHAPTER 1
3. Anticoccidial products: mode of action, the occurrence of

resistance and efficacious use
3.1. Introduction
Before the pioneering work on the antibacterial and anticoccidial properties of
sulphonamides (Levine, 1939; 1940), which led to the development of numerous other
anticoccidial drugs, early treatment of coccidiosis was attempted with flowers of sulphur
or other compounds, like dry or liquid skim milk and buttermilk (Beach & Corl, 1925;
Beach & Freeborn, 1927; Herrick & Holmes, 1936). Later on, the realization that
treatment of clinical diseased birds was less efficient than preventing coccidiosis
outbreaks, favoured the emergence of preventive in feed medication, which has been of
great significance in the upscaling of todays poultry industry. Up till now, commercial
poultry production still largely relies on preventive in feed medication with anticoccidial
drugs in order to avoid disease outbreaks. If despite in feed medication clinical disease
occurs, treatment of coccidiosis is used as last resource.
3.2. Anticoccidial products

The terms coccidiostat and coccidiocidal (coccidiocide), which have been used extensively
and characterize the mode of action of drugs used against coccidia, are frequently
confused. Drugs with a coccidiostatic mode of action arrest the development of certain
parasite stages in a reversible way and their withdrawal will still lead to the completion of
the coccidiosis life cycle. In contrast, coccidiocidal drugs kill or irreversibly damage most
parasite stages with no signs of disease relapse after drug withdrawal. On the other hand,
some products can have coccidiostatic and coccidiocidal properties at the same time
depending on the dose used and exposure time. In order to avoid misuse of terminology
and due to the fact that, as said, some products may present both modes of action, the term
anticoccidial, which conceals both terms, was introduced by Reid (Reid, 1975).
Numerous anticoccidial drugs have been introduced since 1948 (Table 6), when
sulfaquinoxaline and nitrofurazone were first approved by the American Food and Drug
Administration (FDA) (Chapman, 1997; McDougald, 2003). Most of the anticoccidial
products currently approved in different regions of the world for prevention of coccidiosis
in birds are listed in Table 7 and an overview of the anticoccidial products recommended
for therapeutic treatment is given in Table 10 (Conway & McKenzie, 2007). Some of the
drugs mentioned in both tables may not be available in specific countries. Moreover,
national regulatory limitations may apply to some products. A number of anticoccidials
have been withdrawn overtime because of safety or efficacy issues, nevertheless many
drugs are still available and used until today.
25
Table 6. Historical overview of anticoccidial products, reports on the resistance of Eimeria spp. field isolates and withdrawal periods
(Chapman, 1997; McDougald, 2003; Conway & McKenzie, 2007)
Generic or chemical name
(Manufacturer)
Tradename
Year of
introduction
Resistance described
(Country)
Year
Eimeria spp.
Sulfaquinoxaline, 0.015-0.025%
(Merck)
Nitrofurazone, 0.0055%
(Hess & Clark, Smith-Kline)
Arsanilic acid or sodium arsanilate, 0.04%
(Abott)
Butynorate, 0.0375% for turkeys
(Solvay)
Nicarbazin, 0.0125%
(Merck, Sharp and Dohme; Merial)
Furazolidone 0.0055-0.011%
(Hess & Clark)
Nitromide, 0.025% + sulfanitran, 0.03% +
roxarsone, 0.005
(Solvay)
Oxytetracycline, 0.022%
(Pfizer)
Amprolium, 0.0125-0.025%
(MSD; Agvet; Merial)
Chlortetracycline, 0.022% (American
Cyanamid)
Zoalene, 0.004-0.015%
(Solvay)
Amprolium, 0.0125% + ethopabate,
0.0004/0.004%
(Merck)
Aviochina, Quinatrol, SQ,

Sulquin
Nfz, Amifur, Furacin,
Furazol, Nitrofural, Coxistat
Pro-Gen,
Roxarsone
Tinostat
1948
Waletsky et al. (USA)
1954
E. tenella
Withdrawal
period
(days)
10
1948
1955
Not given
1949
Cuckler & Malanga

(USA)
Unknown
1954
Unknown
Nicarb, Nicoxin, Nicrazin
1955
Hemsley (GBR)
NF-180, Furovag, Furoxane,

Furoxone, Giarlam
Unistat-3, Tristat, Unistat
1957
Unknown
1958
Unknown
Terramycin
1959
Unknown
Amprol
1960
Hemsley (GBR)
Aureomycin
1960
Unknown
Zoamix, DOT, DNOT
1960
Hemsley (GBR)
Amprol Plus, Amprol Hi-E
1963
Unknown
5
28
1964
1964
E. tenella
E. brunetti
0
?
1964
E. tenella
E. necatrix
5
5
0
Clopidol or meticlorpindol, 0.0125-0.025%

(A.L. Laboratories)
Coyden
1966
Williams (GBR)
1969
Buquinolate, 0.00825%
(Norwich-Eaton)
Nequinate (methyl benzoquate )
Decoquinate, 0.003%
(Rhone Poulenc; Rorer)
Sulfamethoxine, 0.0125% + ormetoprim,
0.0075%
(Hoffmann-La Roche)
Monensin, 0.01-0.0121%
(Elanco)
Robenidine, 0.0033%
(American Cyanamid)
Halofuginone 0.0003%
(Hoechst-Roussell Agri-Vet)
Lasalocid, 0.0075-0.0125%
(Hoffmann-La Roche)
Aprinocid, 0.006%
(Merial)
Salinomycine, 0.004-0.0066%
(Hoechst, Pfizer, Roche, Agri-Bio)
Bonaid
1967
McManus (USA)
1968
E. acervulina
E. maxima
E. tenella
Not given
Statyl
Deccox
1967
1967
Millard (GBR)
Millard (GBR)
1970
1970
E. tenella
E. tenella
Agribon, Albon, Rofenaid
1970
Unknown
Elancoban, Coban,
Coxidin
Robenz, Cycostat
1971
Jeffers (USA)
1974b
E. maxima
1972
Jeffers (USA)
1974b
E. maxima
Stenorol
1975
Hamet (FRA)
1986
Avatec
1976
1977
Arpocox
1980
Weppelman et al.
(USA)
Chapman (GBR)
E. acervulina
E. tenella
E. acervulina
1982a
E. tenella
Sacox,
Bio-Cox, Coxistac, Salgain,
Salocin
Baycox
1983
Jeffers (USA)
1984
Various species
1986
1993
Not given
18
Monteban
1988
Vertommen & Peek

(Netherlands)
Weppelman et al.
(USA)
1977
Cygro
1989
1987a
Maxiban
1989
McDougald et al.
(USA)
Chapman (GBR)
E. acervulina
E. maxima
E. tenella
Various
1989.
E. tenella
Clinacox
1990
Kawazoe & Fabio

(Brazil)
1994
E. acervulina
E. maxima
E. tenella
Aviax
1995
Totrazuril, 7 mg/kg bodyweight

(Bayer)
Narasin, 0.0054-0.0072%
(Elanco)
Maduramicin, 0.0005-0.0006%
(American Cyanamid)
Narasin + nicarbazin, 0.0054-0.0090%
(Elanco)
Diclazuril, 0.0001%
(Janssen Pharmaceutica B.V.)
Semduramycin, 0.0025%
(Pfizer)
0 at 0,0125%
5 at 0,025%
0
0
5
3.2.1. Categories
The anticoccidial products can be classified in three categories according to their origin
(Chapman, 1999a, 1999b; Allen & Fetterer, 2002c).
1. Synthetic compounds.
These compounds are produced by chemical synthesis and are often referred to as
chemicals. Synthetic drugs have a specific mode of action against the parasite
metabolism. For example, amprolium competes for the absorption of thiamine
(vitamin B1) by the parasite.
2. Polyether antibiotics or ionophores.
These products are produced by the fermentation of Streptomyces spp. or
Actinomadura spp. and destroy coccidia by interfering with the balance of
important ions like sodium and potassium. The following groups of ionophores
exist:
a. Monovalent ionophores (monensin, narasin, salinomycin)
b. Monovalent glycosidic ionophores (maduramicin, semduramycin)
c. Divalent ionophores (lasalocid)
3. Mixed products.
A few drug mixtures, consisting of either a synthetic compound and ionophore
or
two
synthetic
compounds
(nicarbazin/narasin
(Maxiban))
(meticlorpindol/methylbenzoquate (Lerbek)), are also used against coccidiosis.
28
CHAPTER 1
Table 7. Contemporary anticoccidial products and recommended doses for prophylactic

treatment of coccidiosis in chickens (Conway & McKenzie, 2007)
Chemical name
Chemicals
Amprolium
Amprolium + ethopabate
Aprinocid
Clopidol
Decoquinate
Diclazuril
Dinitolmide (zoalene)
Halofuginone
Nequinate (methyl benzoquate)
Nicarbazin
Robenidine
Polyether ionophores
Lasalocid
Maduramicin
Monensin
Narasin
Narasin + nicarbazin
Salinomycin
Semduramycin
Bird type
Concentration in feed (ppm)
Broiler, rearing
Broiler, rearing
Broiler
Broiler, rearing
Broiler
Broiler, rearing
Broiler, rearing
Broiler
Broiler, rearing
Broiler
Broiler
125-250
125-250 + 4
60
125
30
1
125
3
20
125
33
Broiler
Broiler
Broiler, rearing
Broiler
Broiler
Broiler
Broiler
75-125
5-6
100-120
60-80
54-90 (of both drugs)
44-66
25
Products listed are known to be used with some frequency in Europe, Latin America, Asia/Pacific region
and/or North America.
3.2.2. Mode of action

Most anticoccidial products frequently have more than one biochemical effect upon a
specific developmental stage of coccidia. Unfortunately, detailed knowledge on the
selective action of these compounds against specific stages of the parasite is frequently
lacking (Wang, 1982). Nevertheless, despite these shortcomings, a broad categorization of
the mode of action of anticoccidials on the parasite metabolism has been undertaken
(Chapman, 1997).
A summarizing overview of mode of action of various anticoccidial products is given in
Table 8.
3.2.2.1. Products affecting cofactor synthesis
Several anticoccidial products influence essential biochemical pathways of the parasitic
cell by affecting an important cofactor of named pathway (Greif et al., 2001).
Ethopabate, which is often used in combination with amprolium to improve the spectrum
of efficacy, is a folate antagonist and blocks a step in the synthesis of PABA and prevents
29
the formation of nuclei acids and the construction of vitamins (Rogers et al., 1964).
Ethopabate affects the second generation of schizonts (Reid, 1975) and is most active
against E. maxima and E. brunetti.
Sulphonamides were the first drugs to be extensively used for the control of coccidiosis
and also inhibit the folic acid pathway. Similar to ethopabate, the synthesis of
dyhydrofolate is prevented by interfering with the dihydropteroate synthetase reaction,
blocking the conjugation of pteridine and PABA. Dihydropteroate synthetase is only
present in the parasite. Sulphonamides mainly affect the second generation of schizonts
(Reid, 1975). They are very effective against the intestinal species E. brunetti, E. maxima
and E. acervulina and to a much lower degree against both caecal species E. tenella and E.
necatrix (Ryley & Betts, 1973). A major drawback of sulphonamides is their small safety
margin, which easily leads to intoxications especially if they are used as treatment for
coccidiosis outbreaks. Sulphonamide toxicity is characterized by decrease in egg
production, loss of eggshell pigmentation, immune suppression, bone marrow depression
and thrombocytopenia frequently leading to multiple hemorrhages (hemorrhagic
syndrome).
A third product affecting the folate pathway of coccidia is pyrimethamine by preventing
the reduction of dihydrofolate to tetrahydrofolate by inhibiting the enzyme dihydrofolate
reductase. Pyrimethamine has a clear synergistic effect with sulphonamides (Kendall &
Joyner 1956). Similarly to sulphonamides, they affect the second generation of schizonts
(Reid, 1973).
Thiamine analogues, like amprolium block the absorption of thiamine completely and
have probably an antagonistic effect on the vitamin B1 supply. In vivo thiamine is
transformed into thiamine pyrophosphate, an important coenzyme in the carbohydrate
metabolism. Amprolium, competitively inhibits the uptake of thiamine, however it can not
be transformed into vitamin B1 as it lacks a hydroxyl group necessary for the translation
into thiamine pyrophosphate (Rogers, 1962). Amprolium seems especially efficacious
during schizogony as then the demand of thiamine is at its highest (James, 1980). There is
a difference in sensitivity of the thiamine transport system of host and parasite (more
sensitive) to amprolium, rendering the product efficacious against coccidiosis. It affects
the first generation of schizonts and to a lesser extent the gametogony (Reid, 1973)
allowing immune response to develop. It is most effective against the caecal species E.
tenella and E. necatrix and to a lesser degree against E. acervulina, E. maxima and E.
brunetti (Kaemmerer, 1965; Ryley & Betts, 1973).
3.2.2.2. Products affecting mitochondrial function
Quinolone drugs amongst which buquinolate, decoquinate and nequinate (methyl
benzoquate) are listed, show anticoccidial activity at very low concentrations. These
products inhibit the respiration of coccidia by blocking the electron transport in their
mitochondria (Wang, 1975). Although they are relatively safe and do not influence the egg
production, egg quality and feed conversion, resistance is rapidly induced (McManus et
al., 1968). This has significantly limited their use in the field. Quinolones arrest the
development in the sporozoite stage of the coccidiosis life cycle, impairing the
30
CHAPTER 1
development of an adequate immune response (Reid, 1973; Yvor, 1968). They were
found to be effective against E. acervulina, E. brunetti, E. maxima and E. mivati and to a
lesser degree to E. necatrix and E. tenella (Ryley & Betts, 1973).
Meticlorpindol is the most important compound of the pyridone group. Similar to the
quinolones it inhibits electron transport of mitochondria, however possibly at another level
as cross resistance with quinolones does not occur. A synergistic effect between
meticlorpindol and 4-hydroxiquinolones has been described (Challey & Jeffers, 1973). A
widely used pyridone-quinolone combination drug is Lerbek consisting of meticlorpindol
and methyl benzoquate. Pyridones, similar to quinolones, affect early stages of the
coccidiosis life cycle of all Eimeria spp. (Reid, 1973; Ryley & Wilson, 1975) and inhibit
development of immunity (Bennejean et al., 1970).
The true mode of action of nicarbazin (4, 4-dinitrocarbanilide) is unknown. The product
has been shown to inhibit both, the succinate-linked NAD reduction in mitochondria of
beef hearts and the energy dependent transhydrogenase and accumulation of Ca2+ ions by
rat lever mitochondria (Dougherty, 1974). It is not suitable for layers as it affects
negatively the egg production and egg quality. It may cause yolk mottling and eggshell
depigmentation. Nicarbazin mainly affects the second generation of schizonts and to a
lesser extent the other parasite stages (McLoughlin & Wehr, 1960). It is most effective
against E. tenella, E. necatrix and E. acervulina and less to E. maxima and E. brunetti.
Development of immunity is not inhibited.
Similar to nicarbazin, the exact anticoccidial mechanism of robenidine (a guanidine
derivative) is still unknown, however from studies in mammals, where the product has
shown to inhibit the oxidative phosphorylation of mitochondria at high concentrations, it is
assumed that it also affects this process in the parasite (Wong et al., 1972). It targets the
first and second generation of schizonts and allows the development of immune response.
Robenidine is highly effective against all Eimeria spp. (Kantor et al., 1970).
Another anticoccidial drug possibly affecting mitochondrial function is the triazinetrione
compound toltrazuril, which is applied in drinking water for preventive and therapeutic
treatment. Harder and Haberkorn (1989), showed that activities of some enzymes of the
respiratory chain, such as succinate-cytochrome C reductase, nicotinamide adenine
dinucleotide (NADH) oxidase and succinate oxidase from mouse liver, were reduced in
the presence of toltrazuril. They also showed an inhibitory effect on the dihydroorotatecytochrome C reductase from mouse liver. More recently, it has been suggested that
toltrazuril might affect plastid-like organelles (Hackstein et al., 1995). Toltrazuril is
efficacious against all intracellular stages (schizogony and gametogony) of all important
Eimeria spp. in the chicken (Mehlhorn et al., 1984; 1988). It induces cidal changes in the
organelles of the parasite at multiple levels, and it does not seem to impair the
development of natural immunity (Greif & Haberkorn, 1997; Greif, 2000).
3.2.2.3. Products affecting cell membrane function
Polyether antibiotics, commonly known as ionophores, with anticoccidial activity
influence the transport of mono- or divalent cations (Na+, K+ en Ca++) across cell
membranes inducing osmotic damage (Berger, 1951; Shumard & Callender, 1967). These
31
drugs are accumulated by extracellular stages (like sporozoites and merozoites) of the
parasite in the lumen of the intestine (Long & Jeffers, 1982, Mehlhorn et al., 1983). An
overview of all ionophores can be found in Table 7.
Monensin has a good efficacy against all Eimeria spp. although the efficacy seems to be
less against E. tenella (Reid et al., 1972; Folz et al., 1988).
Semduramycin showed a good efficacy against all Eimeria spp. and an excellent
protection against E. maxima (Logan et al., 1993; Conway et al., 1995).
Narasin has a good efficacy against all Eimeria spp. more or less comparatively to
monensin (Ruff et al., 1979; Ruff et al., 1980; Walters et al., 1981).
Salinomycin has a broad anticoccidial spectrum against all Eimeria spp. comparable to or
more effective than monensin and lasalocid (Migaki et al., 1979; Bedrnik, et al., 1980;
Greuel & Raether, 1980; McDougald et al., 1981). Fetotoxicity has been described for
salinomycin, robenidine, and arprinocid (Atef et al., 1989).
Lasalocid shows good efficacy against all Eimeria spp. In efficacy tests it showed a better
performance against E. brunetti and E. maxima and lower effect against E. acervulina, E.
necatrix and E. tenella in comparison to salinomycin and monensin (Mitrovic &
Schildknecht, 1974; Reid et al., 1975; Migaki et al 1979).
Maduramicin shows good efficacy against all Eimeria spp. In animal experiments,
maduramicin showed a better effect on E. acervulina, E. maxima and E. tenella than
monensin and narasin, but comparable to salinomycin (Kantor & Schenkel, 1984; Kantor
et al., 1984; McDougald et al., 1984; Schenkel et al., 1984; Wang et al., 1986). In another
comparative study with halofuginone, lasalocid, monensin and salinomycin, maduramicin
was the least effective against an E. maxima infection (Folz et al, 1988).
In general, treatment with ionophore anticoccidials allows some level of infection and
consequently the development of immunity against coccidia, however this depends upon
the dosage used, the Eimeria spp. and the infection level (Jeffers, 1989; Chapman &
Hacker, 1993; Chapman, 1999b).
3.2.2.4. Products with unknown mode of action: diclazuril and halofuginone
Diclazuril shows a coccidicidal effect towards all Eimeria spp. of the chicken; however its
exact mode of action has not been unraveled yet. It is a nucleoside analogue thought to be
involved in acid nucleic synthesis, possibly affecting later phases of coccidia
differentiation (Verheyen et al., 1988). It has been shown to affect parasite wall synthesis
resulting in the formation of an abnormally thickened, incomplete oocyst wall and zygote
necrosis in both, E. brunetti and E. maxima (Verheyen et al., 1989).
Halofuginone is a quinazolinone derivative with unknown mode of action effective against
all six pathogenic Eimeria spp. of chicken. It affects the sexual stages, particularly the first
generation of the schizogony.
32
CHAPTER 1
Table 8. Metabolic process affected by anticoccidial products, their mode of action and
speed of resistance (Chapman, 1997)
Anticoccidial drug
Ionophores (monensin,
lasalocid, narasin,
salinomycin,
maduramicin &
semduramycin)
Amprolium
(Sulfonamides +
dihydrofolate
reductase (DHFR)
inhibitor)
Quinolones
(buquinolate,
decoquinate &
nequinate)
Pyridones (clopidol)
(clopindol &
mehylclorpindol)
Nicarbazin
Robenidine
Halofuginone
Diclazuril
Metabolic process
Membrane
permeability
Mode of action
Cation transport
Speed of resistancea
Slow
Cofactor synthesis
Inhibit thiamine
uptake
Blocks folate
synthesis
Slow
Slow
Mitochondrial
function
Electron transport
Fast
Mitochondrial
function
Electron transport
Moderate
Mitochondrial
function
Mitochondrial
function
Unknown
Unknown
Slow
Moderate
?
?
Moderate
Moderate
Resistance has been described for all anticoccidial drugs introduced.

? = Unknown.
3.2.3. Antimicrobial & growth promoting properties of anticoccidial products

The antimicrobial and growth promoting properties of ionophores have been recognized
previously. Besides being active on parasites, they have been found to inhibit grampositive organisms and mycoplasmas (Shumard & Callender, 1967; Dutta & Devriese,
1984; Stipkovits et al., 1987). Monensin and narasin were shown to inhibit Clostridium
perfringens (types A and C) in chickens and turkeys (Elwinger et al., 1992; Vissiennon et
al., 2000). Ionophores may therefore have contributed in some cases to the control of
necrotizing enteritis (Martel et al., 2004) similar to traditional antibiotic growth promoters
(AGPs) like virginiamycin, zinc bacitracin, avoparcin and avilamycin (Elwinger et al.,
1998). Due to the occurrence of drug resistant C. perfringens strains (Devriese et al., 1993;
Benno et al., 1988), it is not possible to blindly rely on AGPs including ionophores for the
control of necrotizing enteritis.
33
As necrotizing enteritis is a multifactorial disease in which coccidiosis may be involved,

but also many other factors such as environmental and management conditions, stress and
immune suppression, the diet, etc., to conclude that the ban on AGPs will invariably lead
to increased outbreaks of necrotizing enteritis is an oversimplification of reality (Van
Immerseel et al., 2004, Williams, 2005). On the other hand until recently (before the
introduction of Clostridium perfringens vaccines) antibiotic treatment has been found to
be the only reliable way to control necrotizing enteritis. Antibiotic candidate replacement
treatments including probiotics, prebiotics, organic acids, enzymes, plant extracts, hen egg
antibodies, bacteriophages, vaccinations and diet composition (increase feed particle size,
corn-based diets, reducing non-starch polysaccharides and reducing proteins of animal
origin) might be effective to some extent in controlling necrotizing enteritis although no
single non-antibiotic treatment has proved satisfactory (Dahiya et al., 2006).
No effect of monensin and nicarbazin on the caecal colonization of Salmonella could be
demonstrated experimentally in chickens (Manning et al., 1994). Salinomycin has been
shown to reduce the number of resistant coliforms (sulfadiazine) and streptococci
(erythromycin and lincomycin) (George et al., 1982). It was also shown that salinomycin
seemed to reduce the number of resistant Salmonella Typhimurium bacteria (Ford et al.,
1981).
3.2.4. Incompatibilities of anticoccidial products
Ionophores are incompatible with some therapeutic antibiotics like tiamulin,
chlorampheniol, erythromycin, oleandromycin and certain sulfonamides. Ionophores are
also incompatible with some antioxidants (XAX-M, Duokvin, TD) (Umemura et al., 1984;
Prohaszka et al., 1987; Dowling, 1992; Von Wendt et al., 1997).
3.2.5. Regulations of anticoccidial products within the European Union (EU)
In the EU, the regulation of anticoccidial drugs, which are considered as feed additives, is
stipulated in Directive 70/524 since 1970. More recently, Directive 96/51 and the
Regulation (EC) 1831/2003 have been added. The main changes put forward by
Regulation 1831/2003 are the implication of the European Food Safety Authority (EFSA)
in the scientific review of the dossier of products and the call for maximum residue limits.
The future status of anticoccidial drugs is uncertain at this moment: their current status as
feed additives may be maintained or new legislation and phasing-out of these drugs as feed
additives could be proposed. Article 11 of Regulation 1831/2003 called for a report of the
Commission on this subject, which should have been made public in January 2008.
Actual and forthcoming information of the obtained regulations can be found on the
website of the European commission animal nutrition feed additives
(http://ec.europa.eu/food/food/animalnutrition/feedadditives/legisl_en.htm).
34
CHAPTER 1
3.2.5.1. Anticoccidial products currently available in the Netherlands

In Table 9 a complete list of all authorized anticoccidial products used in the Netherlands
is given.
Table 9. Authorized anticoccidial products in the Netherlands with registration number,
end of authorization, target species, dose and withdrawal period
Additive
Decoquinate
Monensin
Registration
number
E 756
E 757
Robenidine
E 758
Lasalocid
E 763
Halofuginone
Narasin
Salinomycin
Maduramicin
E 764
E 765
E 766
E 770
Diclazuril
E 771
Narasin/
Nicarbazin
Semduramycin
E 772
End of
authorization
17/07/2014
30/07/2014
30/07/2014
30/07/2014
29/10/2014
29/10/2014
20/08/2014
20/08/2014
30/09/2009
30/09/2009
21/08/2014
21/08/2014
30/09/2009
15/12/2011
30/09/2009
20/01/2013
28/02/2011
30/09/2009
E 773
30/09/2016
Animal
species
Broiler
Broiler
Chicken
Turkey
Broiler
Turkey
Broiler
Chicken
Turkey
Chicken
Broiler
Broiler
Broiler
Turkey
Broiler
Chicken
Turkey
Broiler
Broiler
Dose (ppm)
Minimum Maximum
20
40
100
125
100
120
60
100
30
36
30
36
75
125
75
125
90
125
2
3
60
70
60
70
5
5
5
5
1
1
1
1
1
1
80
100
20
25
Withdrawal
period (d)
3
1
1
1
5
5
5
5
5
5
1
1
5
5
5
5
5
5
5
3.3. Anticoccidial product resistance

The World Health Organization defines drug resistance in antimalarial chemotherapy,
which can also be applied to coccidiology, as "the ability of a parasite strain to survive
and/or multiply despite the administration and absorption of a drug in doses equal to or
higher than those usually recommended but within the limits of tolerance of the subject"
(World Health Organization, 1965).
Generally, drug resistance in coccidia can be complete, in which case increasing doses up
the maximum tolerated by the host is ineffective (i.e. diclazuril, nicarbazin). In contrast,
relative resistance to anticoccidial drugs is characterized by the fact that increasing doses
tolerated by the host still will show efficacy (i.e. ionophores).
The worldwide intensive use of anticoccidial drugs to prevent coccidiosis, has inevitably
led to the development of resistance to all anticoccidial drugs as long term exposure to any
drug will result in loss of sensitivity. The widespread occurrence of resistance has been
described in the United States, South America, Europe and China (Jeffers, 1974a, 1974b,
35
1989; Chapman, 1978, 1982b, 1984, 1997; Ryley, 1980; Hamet, 1986; Litjens, 1986;
McDougald et al., 1986; 1987b; Zeng & Hu, 1996; Zhou et al., 2000; Peek & Landman,
2003, 2004). In some cases resistance is induced very quickly, as in the case of quinolones
and pyridinols, which led to a decline in their use; while in other instances it may take
several years, as in the case of the ionophores.
Despite the widespread occurrence of resistance, at least in Europe, coccidiosis outbreaks
seem to have had limited impact so far. This is explained by the fact that resistance in
many cases will have allowed the occurrence of trickle infections, which are essential in
the built-up of immunity (Jeffers, 1989; Chapman, 1998; Peek & Landman, 2003).
The occurrence of anticoccidial drug resistance in the field and corresponding first reports
has been described in Table 6.
3.3.1. Resistance and cross resistance: contributing factors and genetic basis
An important contributing factor to the occurrence of resistance is the high reproductive
potential of coccidia and subsequent forthcoming genetic variation, which increases the
chances for resistant strains to be selected from the parasite population. Also, long-term
exposure to anticoccidial drugs will increase the frequency of resistance genes by selection
of resistant mutants that will become the dominant phenotype. Additionally, the
occurrence of large numbers of asexual haploid stages against which most anticoccidials
are effective, will efficiently allow the selection of resistant phenotypes because genetic
complexities as found in diploid organisms are not interfering (Jeffers, 1989; Chapman,
1997).
Drug resistance of coccidia is based on the occurrence of single mutations or mutations at
several loci with subsequent selection of resistant phenotypes. Multiple resistance is
believed amongst others to be the result of genetic recombination.
Cross resistance between anticoccidial drugs with similar mode of action is likely to occur.
This is an important motivation for the alternation of different drugs and drug programmes
when it comes to sustainable coccidiosis control in poultry.
3.3.2. Management of resistance
The development of resistance to anticoccidial drugs, which is widespread and has been
described for all products introduced so far (Chapman, 1986, 1997; Peek & Landman,
2003, 2004), is a major drawback in coccidiosis control. To minimize the occurrence of
resistance, rotation (a given anticoccidial product is used during a maximum of two
months or two fattening periods) of various anticoccidial drugs or shuttle programmes
(two or more anticoccidial drugs are used within a fattening period) are used. The rationale
behind this is the fact that, as said previously, the loss of sensitivity is correlated to the
length of drug exposure, which should be kept short if possible. Due to the occurrence of
cross resistance between anticoccidial drugs, anticoccidial drugs with distinct mode of
action should be used within rotation and shuttle programmes.
36
CHAPTER 1
In order to optimize the use of prophylactic medication in the field, scientific information
on the drug-sensitivity profiles of Eimeria spp. field isolates concerned, is vital. This
information can only be obtained by performing an in vivo Anticoccidial Sensitivity Test
(AST) in battery cages. Despite the fact that the AST is the only accurate tool to detect
anticoccidial drug resistance, the procedure is slow and expensive, isolates frequently
originate from disease outbreaks (and may not always be representative for the field) and
need to be propagated first in specified pathogen free (SPF) chickens for multiplication
(which may result in selection of non-relevant coccidia). Nevertheless, it is generally
accepted by the scientific community that ASTs provide valuable information and should
be given more attention in coccidiosis prevention programs in order to maintain the
efficacy of the steadily decreasing number of available anticoccidial drugs.
A more recent development in managing anticoccidial drug resistance is the rotation of
anticoccidial drugs with live Eimeria spp. vaccines. Several studies have documented a
higher incidence of sensitive Eimeria spp. field isolates when live anticoccidial vaccines
and anticoccidial products are rotated. The exact mechanism resulting in an increase of
sensitivity of Eimeria spp. field isolates is currently unknown, but it is explained by the
fact that chicken houses are seeded with drug-sensitive vaccine oocysts (Jeffers, 1976;
Mathis & McDougald, 1989; Chapman, 1994, 1996; Newman & Danforth, 2000; Mathis
& Broussard, 2006; Peek & Landman, 2006). This may lead to outgrow of resistant strains
by reproductively more advantageous drug-sensitive coccidia, interbreeding between field
and vaccine parasites resulting in (more) sensitive interbreeds or a combination of both.
Moreover, in case live attenuated coccidiosis vaccines are used, less virulent interbreed
field isolates may be produced (Shimura & Isobe, 1994; Williams, 2002a).
3.3.2.1. Anticoccidial programs
In order to minimize the occurrence of resistance it is crucial to shorten the exposure time
to anticoccidial drugs as much as possible and to alternate compounds with a different
mode of action. This has lead to a great variety of coccidiosis control programs. The
design of the various programs will depend on the goal of named program. In replacement
layer and breeder birds full coccidiosis control is not desired, instead it should be
monitored, for instance by regularly determining the mean lesion score in birds, number of
oocysts per gram faeces, etc., as the desired development of immunity takes place (Long
& Jeffers, 1986). In contrast, in broilers maximal growth and optimum feed conversion
with minimum disease of the birds is the main concern.
In general the efficacy of anticoccidial drug programs depends on nutritional factors (see
section 4) and adequate mixing procedures. Regarding the latter, it is crucial that the levels
of anticoccidial drugs meet the required dose. Assessment of mixing procedures by means
of chemical analysis of feed samples originating from the field should be performed
regularly, although in practice this is rarely done.
In single drug programs, a single anticoccidial product is continuously supplied to the
birds during successive grow-out periods until its efficiency declines and coccidiosis
outbreaks occur.
37
Shuttle programs are characterized by the alternation of anticoccidial compounds within a

single grow-out period: i.e. the drug used in the starter feed is different to the one used in
grower feed. Frequently, a chemical compound is used in the starter, followed by an
ionophore in the grower feed, although sometimes ionophores with distinct mode of action
are used. The choice of drugs may depend on a number of factors amongst which the past
experience and the season of the year (nicarbazin given in summer increases the chances
of heat stress) (Buys & Rasmussen, 1978; McDougald & McQuistion, 1980).
Single drug and shuttle programs can be used within rotation programs, which are
characterized by the use of distinct anticoccidial drugs between successive grow-out
periods. The use of live attenuated coccidiosis vaccines can be included within rotation
programs (Chapman et al., 2002; Williams, 2002a). In these cases, a chemical
anticoccidial drug is preferably used in the last grow-out period before vaccination
especially if during earlier fattening periods ionophores have been used. The chemical
drug will then more efficiently control the coccidiosis population including ionophore
resistant parasites and favour the repopulation of the poultry house with drug sensitive
vaccine oocysts, subsequently increasing the sensitivity of Eimeria spp. field isolates
(Jeffers, 1976; Mathis & McDougald, 1989; Chapman, 1994, 1996; Newman & Danforth,
2000; Mathis & Broussard, 2006; Peek & Landman, 2006). Also in rotation programs
coccidiosis control can be performed using toltrazuril applied in drinking water during two
days (Mathis et al., 2004).
Table 10. Anticoccidial products and doses for therapeutic treatment of coccidiosis in
chickens (Conway & McKenzie, 2007)
Chemical name
Amprolium
Sulfadimetoxine
Sulfaguanidine
Sulfamethazine
Sulfaquinoxaline
Sulfaquinoxaline +
pyrimethamine
Furazolidone
Nitrofurazone
Toltrazuril
38
Application form
Feed
Water
Water
Water
Feed
Feed
Water
Water
Feed
Used concentration
250 ppm
0.006%
0.012-0.24%
0,05%
10,000-15,000 ppm
4,000 ppm
0.1%
0.05%
1,000 ppm
Feed
Water
500 ppm
0.04%
Water
0.005%
+ 0.0015%
110 ppm
110 ppm
0.0082%
0.0025%
0.0075%
Feed
Feed
Water
Water
Water
Treatment schedule
2 wks
1-2 weeks
3-5 days
6 days
5-7 days
3-5 days
2 days
4 days
2-3 days on,3 days off; then 500
ppm for 2 days on
3 days on, 3 days off, 3 days on
2-3 days on, 3 days plain water
and then 2 days 0.025%
2-3 days on, 3 off and 2 days on
5-7 days on, 2 weeks 55 ppm on
5 days
5 days
2 days continuous
6-8 h/day for 2 days
CHAPTER 1
4. The influence of feed structure, composition, toxins and

administration on the course of a coccidiosis infection
4.1. Introduction
Optimal animal health is amongst other factors directly dependent on an adequate supply
of dietary nutrients. Nutritional deficiencies can be primary (deficiency of one or more
essential nutrients) or secondary (suboptimal use of nutrients by the animal) in nature and
have been described extensively by various authors. Besides many reports on specific
nutritional deficiencies or diseases resulting from ingestion of toxic of nutrients, there is a
wealth on scientific literature describing the direct influence of nutrients and diet
composition on the immune competence of animals and humans. Inadequate nutrient
supplies, even marginal, frequently result in impaired resistance to infectious diseases, but
can also result in reduced growth, deficient egg production and hatchability.
According to the National Research Council (1994), diets of poultry are mostly a
mixture of ingredients like cereal grains, soybean meal, animal by-product meal, fats,
vitamins and mineral premixes. Together with water these compounds deliver proteins,
amino acids, carbohydrates, fats, minerals, vitamins and energy that the birds need to
grow, to reproduce and to stay healthy.
Deficiencies of nutrients or energy levels can cause problems with the biochemical
processes which depend on the involved nutrient and may result in inadequate
performances and even disease. In the field, nutrient deficiencies are often erroneously
attributed to management problems and infectious diseases (NRC, 1994).
Changes in feed and feed ingredient quality can often result in health problems of
the animals. It is therefore highly desirable to provide diets being stable in nutrient
composition, energy content, moisture content, physical structure and texture.
In view of the importance of feed in disease resistance, almost all macronutrients
(carbohydrates, proteins and polyunsaturated fatty acids) and micronutrients (minerals,
oligo-elements and vitamins) have been investigated for their potential use as dietary
supplements for coccidiosis control. Some of these nutrients have a direct beneficial effect
on the development of the parasite within the host and are in fact coccidiosis promoting
compounds, while others actively enhance the hosts resistance against Eimeria by
stimulating the immune system, thus decreasing the pathological effects of a coccidiosis
infection. Moreover, nutrients that improve the recovery after an Eimeria infection have
also been described (Allen et al., 1998; Crvieu-Gabriel & Naciri, 2001).
In the present section scientific research on the influence of feed structure, feed
composition (macro and micro ingredients, special additives) and feed management on
coccidiosis is reviewed. The main conclusions obtained in the field of nutrition and
coccidiosis control have been summarized in Table 11 and Figure 2 for convenience.
39
4.2. Feed structure

Feed structure or texture is partly determined by the feed particle size, which in turn is
largely dependent on the presence of cereals and the extent, to which they have been
grounded. Processing of feed into pellet or crumb at the factory may further decrease the
average particle size of poultry feedstuffs. Describing the particle size of ground feed
ingredients involves using a sieve set of specific designation to separate a representative
sample of feed into eight size categories. The Modulus of Uniformity (MU) is then used to
indicate the percentage of the various filter fractions (coarse, medium and fine), while the
Modulus of Fineness is used to reflect the coarseness or fineness of the analyzed feed
(http://www.asft.ttu.edu/ansc5001/). The average particle size of a sample is frequently
expressed as geometric mean diameter (GMD in mm or m; <600 m is fine, 700-900 m
medium and >1000 m is coarse). Particle size uniformity is described by geometric
standard deviation (GSD), a small GSD representing higher uniformity.
The physical structure of the feed influences the development of the intestinal tract, the
secretion of digestive enzymes and the development of the gastrointestinal flora. Feed
mainly consisting of fine particles has a strong inhibiting effect on the contraction and
reflux activity of the intestine, and thus on the development of the intestinal tract, in
comparison to coarse feeds based on larger particles. This was shown in pigs fed with
larger maize grains resulting in higher villi and lamina propria (Healy et al., 1994). Feed
particle size also plays a major role in the development of the gizzard: greater gizzards
were found in chicks fed medium or coarse particle size diets compared to fine particulate
feeds.
Feed based on whole cereals improved the motility of the intestinal tract and the health
status of the birds; however, studies on the influence of such feeds on the development of
a coccidiosis infection have yielded ambivalent results.
Birds housed in cages supplied with whole cereals up to 60-70% and high protein
concentrations (40%) in their diet, showed reduced oocyst shedding and mortality most
prominently for E. tenella but also E. acervulina and E. maxima in a mixed coccidiosis
infection model (Cumming, 1987). These results were in agreement with those obtained in
floor pen trials (Cumming, 1989; 1992a, b; 1994). In these studies heavier gizzards as well
as lower pH in the intestines were found. It was hypothesized that the mechanical function
of the better developed gizzards will have destroyed more oocysts, while the lower pH will
probably have resulted in a more hostile environment for excysted sporozoites, thus
tempering the coccidiosis infection.
The promising results presented by Cumming could not be repeated by other European
research groups except by Langhout (1999), who reported in a non-peer-reviewed journal
lower coccidiosis lesion scores for E. acervulina in broilers fed coarse wheat compared to
birds fed fine wheat. However, the coccidiosis inhibiting effect of coarse wheat could not
be demonstrated in case of an E. tenella infection.
Feed supplemented with whole wheat up to 30%, increased the weight of the gizzard 1.2
to 1.5% of the bodyweight (less than found by Cumming in 1992b), however it had no
significant effect on a subclinical (low infection doses) infection with E. tenella or E.
40
CHAPTER 1
maxima regarding lesion scores, oocyst shedding and performance (Waldenstedt et al.,
1998).
In another study, no changes were found in oocyst excretion during an infection with E.
acervulina when birds were given feed containing 20% whole wheat compared with feeds
containing 20% ground wheat (Banfield et al., 1998). Banfield & Forbes (2001) also failed
to demonstrate that neither dilution nor substitution of feeds with whole wheat did
influence the E. acervulina infection as measured by oocyst excretion, although whole
wheat significantly increased the size of gizzards.
Previously, it was shown that the administration of whole wheat may be detrimental
instead of beneficial: substitution of 20 and 40% of ground wheat with whole wheat,
showed that the oocyst excretion was significantly increased and that significant poorer
feed conversion efficiency was achieved in case of an E. acervulina infection. Increased
viscosity of the diet did not influence the coccidiosis infection either (Banfield et al., 1999;
Waldenstedt et al., 2000a). A detrimental effect of whole wheat supplementation on the
performance of broiler chickens during the course of an E. acervulina infection was also
shown by Banfield et al. (2002). More recently, a detrimental effect of feeding whole
wheat compared to ground wheat on an experimental coccidiosis infection was shown by
Gabriel et al. (2006). The whole wheat diet led to lower weight gain in E. acervulina, E.
maxima or E. tenella infected birds during the acute phase of infection, although in
uninfected birds it lead to a more efficient feed conversion and more beneficial microflora.
Further, the oocyst excretion was higher in whole wheat fed birds (x 1.5 for E. maxima
and x 10 for E. tenella). These results confirmed previous studies (Gabriel et al., 2003)
showing that feeding whole wheat increases the more detrimental effect of Eimeria
parasites on the birds during the acute phase of a coccidiosis infection compared to a diet
with ground wheat.
The conflicting results between the work of Cumming and other research groups was
explained by the much higher concentrations of whole wheat used by the former (60-70%
compared to 20-40%), which resulted in the largest diet-induced gizzard development. The
bigger gizzards will probably have had a bigger mechanical impact on the oocysts,
sporocysts and sporozoites resulting in their destruction. In contrast, the limited gizzard
development induced by moderate whole wheat diets will have yielded optimal
mechanical forces for the breakage of oocysts, resulting in increased coccidiosis infection.
Moreover, low-to-moderate whole wheat diets (20-40%) have shown to increase pancreas
weight (Banfield et al., 2002; Wu et al., 2004), which may lead to increase production of
pancreatic enzymes favourable for excystation (Farr & Doran, 1962) and thus aggravating
the clinical signs of a coccidiosis infection.
Summarizing, it can be concluded that feed structure and degree of fineness of cereal
grains in particular may influence the course of a coccidiosis infection. As cereals form an
important basis for todays poultry diets in Western Europe, these effects and the
underlying mechanisms deserve to be further explored.
41
4.3. Feed composition

Besides texture, specific nutrients or feed ingredients may have a direct effect on the
Eimeria parasite by influencing certain stages of its life cycle, while other components
may modulate indirectly a coccidiosis infection by enhancing the immune response or
improving the recovery after infection.
4.3.1. Basic ingredients of feed
Poultry feeds are most frequently based on wheat or corn as main energy source. Relative
few research data on the influence of these ingredients in poultry diets on a coccidiosis
infection are available.
The effect of natural feedstuffs added to a semi-purified diet on E. tenella, was studied by
Colnago et al. (1984b), who showed that corn and corn fermentation solubles contain
factors that enhance a clinical E. tenella infection, however soybean meal did not. In
contrast, in a later study, where a corn diet was compared to wheat, lower mortality was
found in the birds infected with E. tenella given the first diet (Williams, 1992a). More
evidence suggesting a beneficial effect of a corn based diet on a coccidiosis infection, was
published by Morgan and Catchpole (1996), showing superior growth in E. tenella and E.
acervulina infected broilers fed corn compared to wheat.
The different effect of corn and wheat on a coccidiosis infection is possibly related to
differences in their micronutritive components as suggested by Williams (1992a): while
corn is rich in vitamin A (x 1.8) and E (x 1.7) that may boost the immune response, wheat
contains about twice as much niacin and x 1.3 riboflavin, which are beneficial to the
parasite (Warren, 1968). Moreover, a wheat-based diet may also alter the intestinal flora
favouring the development of the parasite. Compared to corn, such a diet has been shown
to increase mortality by C. perfringens (Branton et al., 1987).
4.3.2. Macronutrients
4.3.2.1. Carbohydrates
Carbohydrates make up the largest portion of a poultry diet, are the primary source of
metabolizable energy and influence the intestinal microbial activity. Modulation of the
intestinal flora mainly depends upon the type of carbohydrate. In animal feed
carbohydrates are usually classified into sugars (mono- (glucose, fructose, galactose) and
disaccharides (sucrose, lactose and maltose), oligosaccharides (raffinose, stachyose and
verbascose), starch (amylose and amylopectin) and non-starch polysaccharides (NSP)
(cellulose, hemicellulose, fructans, galactans, mannans, pectic substances, and gumsand
substances)). Except NSP, which need previous fermentation by the intestinal microflora,
most sugar fractions are readily digested in the small intestine with the aid of pancreatic
enzymes (Trowell et al., 1976).
Carbohydrates have been investigated frequently for their influence on a coccidiosis
infection; most attention has been given to the non-digestible carbohydrates (i.e. NSP).
42
CHAPTER 1
Different types of saccharides

The influence of various carbohydrate compounds mainly mono- and disaccharides on E.
tenella coccidiosis was studied after their in feed administration. The following results
were obtained: mannose slightly favours growth and reduces lesions, the latter is also
induced by lactose, however does not modify oocyst shedding. Fructose, maltose,
saccharose, amylopectin and glucose favour oocyst shedding without enhancing
coccidiosis lesions except fructose. Potato starch increased coccidial lesions, while
amylose did not influence their occurrence (Matsuzawa, 1979).
In general, if the saccharide content exceeds 50% of the diet, the growth of birds is
influenced negatively, while the oocyst excretion is enhanced. These effects are explained
by the modification of the intestinal flora by the high saccharide content of the diet.
NSP (non-starch polysaccharides) or dietary fibre
NSP are polysaccharides that cannot be degraded by endogenous enzymes and therefore
reach the colon almost indigested where they are fermented by microbes. There are two
kinds of NSP: insoluble (cellulose and hemicellulose) and soluble (fructans, galactans,
mannans, pectic substances (protopectin, pectin and pectic acid) and gumsand mucilages
(-glucans, xyloglucans and mannoglucans)). Insoluble NSP do not dissolve in water and
do not swell in water to form a gel; soluble NSP do not dissolve in water completely but
swell to form a gel. The anti-nutritional effects of soluble NSP are related to their viscous
properties contributing to a higher viscosity of the aqueous fraction of the intestinal
content impairing the efficient transport of nutrients. Also, the diffusion of digestive
enzymes may be affected by the increased viscosity. Insoluble NSP do not seem to affect
nutrient digestibility, however, they may change the anti-nutritional effect of soluble NSP
due to their laxative properties. Most plant foods contain both types NSP although
proportions vary. Insoluble NSP can be found in wheat, corn, rice, vegetables and pulses,
while soluble NSP are found in apples, citrus fruits, beans, barley, rye and oats.
Studies on the effect of the dietary fibre content in poultry diets on coccidiosis infections
have yielded conflicting results. Feed with high level of crude fibres (10% instead of
6.5%) aggravated clinical coccidiosis with E. tenella resulting in higher mortality and
caecal hemorrhages (Mann, 1947). However, in another study no effect of almost 10%
cellulose on E. tenella infection was observed (Koltveit, 1969). More recently, increased
fibre content (9%) was shown to reduce oocyst excretion of E. tenella and E. acervulina
although lesions remained unaffected (Muir & Bryden, 1992). In contrast, Cumming
showed previously (1987) that increased levels of fibre decreased oocysts yields related to
a better gizzard development and subsequent oocyst destruction.
The increased intestinal viscosity entrained by NSP (through increase of pentosans) could
favour the development of coccidiosis. The coccidiosis promoting effect may be lowered
by enzymes that reduce viscosity like pentosanases. In chickens infected with E.
acervulina and E. tenella, they seemed to diminish the growth retardation (Morgan &
Catchpole, 1996). In agreement with previous research, experimental increase of intestinal
viscosity with carboxymethylcellulose diminished the feed efficiency of E. acervulina
infected chickens, however oocyst shedding was not affected (Banfield et al., 1999).
43
Another study on the influence of intestinal digesta viscosity showed that the performance,
lesion scores and C. perfringens counts of E. acervulina and E. praecox infected birds was
not affected (Waldenstedt et al., 2000a).
NSP derived from barley (containing -glucans) could have an indirect effect on a
coccidiosis infection by their immunoprotective properties. Nevertheless, although
zymosan (a product which contains -glucan a.o.) enhanced the immune responsiveness in
mammals (Vadiveloo, 1999; Volman et al., 2005) and it promoted oocyst shedding in E.
tenella challenged chickens (Smith & Ovington, 1996).
Although NSP seemed to have little or no effect on a coccidiosis infection, more
complementary research is needed to determine the effect of these ingredients.
4.3.2.2. Proteins
Proteins are essential dietary components vital for growth, maintenance and repair of
muscle tissue and energy supply.
A protein concentration dependent effect on coccidiosis has been described by various
authors (Mann, 1947; Britton et al., 1964; Sharma et al., 1973). In general, low levels of
dietary protein ( 13%), which are mostly detrimental for growth performance, seem to
diminish coccidiosis mortality, oocyst shedding and coccidiosis lesions. This is attributed
to a reduction of trypsin activity in the small intestine, thereby limiting sporozoite
excystation and subsequent parasite invasion (Britton et al., 1964). High dietary protein
may also favour bacterial growth in the intestines and enhances the development of
coccidiosis lesions due to E. tenella (Mann, 1947). On the other hand, increased dietary
protein ( 16%) protects against weight reduction during clinical coccidiosis and
stimulates compensatory growth (Sharma et al., 1973).
Raw soybeans as protein source have a protective effect against coccidiosis-induced
growth retardation and lesions scores by several Eimeria spp., attributed to protease
inhibitors limiting excystation. This effect was not seen when continuously feeding raw
soybeans as it induced pancreas hypertrophy and hyperfunction (Mathis et al., 1995). In
practice, raw soybeans are not used due to the high level of antinutritional factors and the
low protein digestibility.
4.3.2.3. Lipids
In response to the growing number of lipids expected to be discovered through lipidomics
and advances in lipid research, recently a new classification scheme for lipids has been
proposed. Lipids are divided into eight primary categories: fatty acyls, glycerolipids,
glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids and
polyketides. These categories are based on the functional backbone of the lipid molecule
from a chemical standpoint. The categories are further subdivided into classes and
subclasses to handle the existing and emerging arrays of lipid structures
(http://www.lipidmaps.org/).
Lipids are important in poultry feed as source of energy, they are essential components of
cellular and subcellular membranes, serve as biological carriers for the absorption of fatsoluble vitamins and yield essential fatty acids.
44
CHAPTER 1
Fatty acyls
Unsaturated fatty acids seem to promote clinical and pathological signs of coccidiosis and
pathology. Essential fatty acids are required for the development of Eimeria parasites.
Chickens fed diets enriched with coconut oil, composed of medium chain saturated fatty
acids, showed better performance after an E. acervulina infection than those birds given
soy oil (unsaturated fatty acids) (Adams et al., 1996). Similarly, the use of coconut oil in
essential fatty acids deficient purified diet reduced the lesions and mortality as a result of a
coccidiosis infection (E. tenella and E. mivati) compared to a diet supplemented with corn
and soya or corn oil (unsaturated essential fatty acid) (Charney et al., 1971).
Regarding performance loss due to coccidiosis, medium chain fatty acids seem
advantageous compared to long chain fatty acids. Coconut oil significantly improved fat
digestion and performance values during a coccidiosis infection with E. acervulina in
comparison with natural long chain saturated fatty acids of animal fat. This is explained by
the better digestibility of medium chain fatty acids and their more efficient absorption by
the diseased mucosal surface (Babayan, 1987; Adams et al., 1996).
In case the intestinal health is compromised, as during coccidiosis infection, the
digestibility of lipids (especially long fatty acids) in feed is decreased first. It is probably
the consequence of the inactivation of bile salts due to microbiological activity. Bilesalts
are essential in the process of lipid emulgation, which is particularly important during the
digestion of long chain fatty acids. This provides an explanation for the fact that young
chickens suffering from clinical coccidiosis show better performance whenever they are
supplied with medium chain fatty acids. Moreover, these type of fatty acids also have
antimicrobial properties.
Essential fatty acids
Essential fatty acids (EFAs) must be obtained from the diet as they cannot be synthesized
from other components by any known chemical pathway by land animals except the
landsnail. There are two families of EFAs: -3 (or omega-3 or n-3 or -linolenic acid) and
-6 (omega-6, n-6 or linoleic acid). Both acids form the starting point for the creation of
longer and more unsaturated fatty acids, which are also referred to as long chain
polyunsaturated fatty acids.
Examples of -3 fatty acids are eicosapentaenoic acid and docosahexaenoic acid, and of
-6 fatty acids are gamma-linolenic acid, dihomogamma-linolenic acid and arachidonic
acid.
EFA -3 belonging to the family of polyunsaturated fatty acids can be found abundantly
in fish oil, flaxseed oil and whole flaxseed and can be easily supplemented to the feed.
In E. tenella infection experiments, lesion scores and growth retardation were significantly
reduced in birds supplemented with 2.5, 5 and 10% fish oil and 10% flaxseed oil
compared to unsupplemented diets (Allen et al., 1996; 1997a; Korver et al., 1997). The
beneficial effect of -3 fatty acids was explained by the retarded development of E.
tenella (Allen et al., 1996) and ultrastructural changes of both asexual and sexual stages
induced by these compounds (Danforth et al., 1997). These results are consistent with
reports on the influence of -3 fatty acids on other parasites and suggest that a state of
45
oxidative stress is created that is harmful to the parasite (Allen et al., 1998). In agreement
herewith, high vitamin E concentrations (35 IU/kg in stead of 4.2-21 IU/kg) added to feed
in order to limit the oxidation of unsaturated fatty acids seemed to nullify the positive
effect of -3 fatty acids on an E. tenella infection (Allen et al., 2000).
In contrast to the positive results obtained for E. tenella, dietary flaxseed supplementation
showed to be ineffective against moderate and severe E. maxima infections (Allen et al.,
1997a). This difference was attributed to differences in intestinal environment of both
species (caeca versus less anaerobic mid intestine) making E. maxima less vulnerable to
oxidative stress (Allen et al., 1998).
Although the immunomodulating and anti-inflammatory effects of -3 fatty acids are well
documented and these substances are able to dampen the growth depressing effect of
coccidiosis, a relation between their anti-inflammatory properties and effect on growth has
not been found yet (Korver et al., 1997).
4.3.3. Micronutrients
Micronutrients are generally necessary in small amounts for living and include minerals
and vitamins.
4.3.3.1. Minerals
Dietary minerals form the inorganic part of poultry feed and are needed in order to
maintain the osmotic balance, as co-factors for enzymes, for cellular activity and skeleton
physiology. They are available for immediate use after absorption and do not require
digestion. Given in excess quantities some minerals can be toxic. Minerals that are needed
in relatively large quantities such as calcium, salt (sodium and chloride), magnesium and
potassium (along with phosphorus and sulfur) are also referred to as macrominerals. In
contrast, those consistently needed in small amounts like iron, cobalt, chromium, copper,
iodine, manganese, selenium, zinc and molybdenum are designated as microminerals or
trace elements.
4.3.3.1.1. Macrominerals
Calcium
In contrast to a slight (1.5%) calcium overdose (Watkins et al., 1989), high dietary calcium
concentrations (approx. 2% or higher) exceeding the optimum required in a poultry diet,
consistently had a negative influence on the outcome of coccidiosis infections with both E.
tenella or E. acervulina (Holmes et al., 1937; Zucker et al., 1967; Bafundo et al., 1984b).
The activation of trypsine (important in excystation) by calcium, may explain the
coccidiosis enhancing effect of high dietary calcium. A higher calcium supplementation
(even slighty increased (1.5%)) had a negative effect on healthy birds in comparison with
coccidiosis infected birds. This effect is probably caused by a reduced absorption of
calcium in the coccidiosis infected birds (Watkins et al., 1989).
46
CHAPTER 1
Dietary buffers (NaHCO3, Al(OH)3, Al(OH)2NaCO3, kaolin, CaCO3, MgO).

Fox and co-workers (1987) were the first to test the effect of various dietary buffers
(NaHCO3, Al(OH)3, Al(OH)2NaCO3, kaolin, CaCO3, MgO) on an E. acervulina infection.
Only NaHCO3 (1%) added to diet at the rate of 1% resulted in an improvement of the
weight gain and feed conversion in infected chicks in comparison to uninfected controls.
The positive effect of NaHCO3 was attributed to the electrolyte status of the diet and
partial alleviation of the coccidiosis-induced acidosis.
The effect of dietary NaHCO3 on coccidial challenge with E. acervulina, E. maxima and
E. tenella and ionophore coccidiostats was studied by Hooge and others (1999). NaHCO3
levels of 0.2-0.4% yielded improvement in body weight, feed efficiency, coccidial lesion
scores, liveability, carcass yield, breast yield and occasionally abdominal fat pad. NaHCO3
potentiated the effect of the ionophores salinomycin and monensin possibly by rapid early
induction of immunity triggered by enhanced coccidial invasion of the intestinal wall as
found for NaHCO3 and monensin by Augustine in 1997 (Hooge et al., 1999).
Magnesium
The effect of magnesium on a coccidiosis infection varies according to the form in which
it is presented in the feed. An overexposure in the form as carbonate (0.6 or 1.2%) or
sulfate (0.3%) did not have an effect on an E. acervulina infection; however, if presented
as magnesium oxide (0.3%) it induced a reduction in performance possibly due to its
laxative effect (Giraldo et al., 1987; Zucker et al., 1967).
4.3.3.1.2. Microminerals or trace elements
Except zinc, which has a positive effect on weight gain of E. acervulina infected chickens
(Bafundo et al., 1984b) and copper, that reduced E. tenella-induced mortality (Czarnecki
& Baker, 1984; Zucker et al., 1967), most of the trace elements (like, cadmium, cobalt,
copper, iron, lead, manganese) aggravate the effect of an E. acervulina infection by an
increased absorption resulting in intoxications (Czarnecki & Baker, 1982; Southern &
Baker, 1982, 1983; Bafundo et al., 1984a).
Immunization of chickens against E. tenella coccidiosis is enhanced by selenium;
moreover it reduces mortality and increases body weight in challenged birds (Colnago et
al., 1984a). Jensen and co-workers (1978) previously observed similar positive effects of
selenium on mortality after infection with six Eimeria spp. except E. necatrix.
4.3.3.2. Vitamins
A vitamin is an organic molecule that is required in minute quantities for essential
metabolic functions to preserve normal growth and health. Vitamins are bio-molecules,
which are involved in chemical reactions as catalysts or as substrates. As catalysts they
bind to enzymes as cofactor enabling specific chemical reactions. As coenzymes vitamins
carry chemical groups between enzymes acting as substrates for these enzymes without
forming permanent part of the enzyme structure.
Based on their solubility, vitamins are classified in two groups: fat soluble and water
soluble vitamins.
47
The fat soluble vitamins only dissolve in lipids and are found in association with fat in
feed. Their intestinal absorption into the lymphatic system and blood circulation is
promoted by the presence of fat. In chickens these vitamins can also be absorbed directly
into the blood circulation due to the poor development of the lymphatic system. These
vitamins can be stored in the liver and to a lesser extent in other tissues. The fat soluble
vitamins are: vitamin A, D, E and K.
The water soluble vitamins are readily dissolved in water and directly absorbed from the
intestinal tract into the blood stream in order to reach the organs and tissues. They are
available in every cell where they participate in metabolic functions by releasing energy
from the nutrients. They are not stored in the organs or tissues and in excess they are easily
excreted from the body. The water soluble vitamins are: vitamin B complex (B1, B2, B6,
B12, niacin, pantothenic acid, folic acid, and biotin) and vitamin C.
A number of vitamins like, biotin, thiamine, nicotinic acid, folium acid and riboflavin are
known to be necessary for the complete development of the parasite within the host
(Warren, 1968). Although the idea of using vitamin deficient diets to control coccidiosis
seemed appealing, it proved unattainable as many vitamins are generated by the intestinal
flora or the host (Banfield & Forbes, 1999).
Vitamins as a strategy against coccidiosis infections were mainly studied in the period
1960-70.
4.3.3.2.1. Fat-soluble vitamins (A, D, E and K)
Vitamin A has a positive effect on the growth performance, the reduction in mortality and
oocyst excretion in E. acervulina or E. tenella infected chickens (Erasmus et al., 1960;
Coles et al., 1970; Singh & Donovan, 1973). In agreement herewith, vitamin A
deficiencies render chickens more susceptible to E. acervulina coccidiosis (Dalloul et al.,
2002). This was explained by a reduction in gut immunity due to a decrease in
intraepithelial lymphocyte (IEL) subpopulations, especially CD4+ cells, but also a
reduction in the systemic immune response in vitamin A deficient birds (Dalloul, et al.,
2002). Furthermore, although vitamin A is known to be essential for maintenance of the
mucosal integrity as it plays an important role in the differentiations of epithelial cells
(Chew & Park, 2004), a correlation between vitamin A levels in feed, intestinal epithelium
changes and numbers of E. acervulina parasites, could not be found (Coles et al., 1970).
High doses of vitamin D enhance E. tenella coccidiosis due to its immune suppressive
properties (Sherkov & Denovski, 1964; Jiropongsananuruk et al., 2000; Becker et al.,
2002).
Vitamin E is the collective name of eight antioxidants, four tocopherols and four
tocotrienols. -Tocopherol is the most important one and is frequently found in nature.
High doses of -tocopherol (100 IU/kg instead of the 10 IU/kg recommended by the NRC,
1994) reduced the growth retardation of immunized birds and mortality in non-immunized
chickens after a challenge with E. tenella (Colnago et al., 1984a). Mortality in nonimmunized birds was also reduced in vitamin E supplemented animals after challenge with
E. tenella (Jensen et al., 1978). An explanation for the obtained results is that vitamin E
stimulates the innate and acquired immune response. In contrast to the previous results,
48
CHAPTER 1
diet supplementation with 25 or 225 mg/kg -tocopherol did not affect the lesion scores
and the amount of oocysts shed during an E. maxima infection (Allen & Fetterer, 2002a).
These results are in accordance with earlier studies (Allen & Fetterer, 2002b) indicating
that -tocopherol supplemented in diets is not effective against an E. maxima infection.
Adding -tocopherol to the diet at a concentration of 8 ppm and given to chickens infected
with E. maxima improved the weight gain and reduced the amount of lesions, and oocyst
excretion, but in case of an E. tenella infection no improvements were noticed (Allen et
al., 1998) suggesting that -tocopherol is more effective against E. tenella. The higher
capability of -tocopherol to inactivate nitrogen dioxide in the intestine may provide an
explanation for its superior activity against E. maxima (Cooney et al., 1993; Christen et
al., 1997).
Vitamin K is characterized by its coagulation promoting properties and has therefore been
used to treat hemorrhages associated with coccidiosis and other diseases. Its
supplementation reduced mortality induced by an E. necatrix and E. tenella infection,
which are associated with blood loss, but no effect was found on growth, blood losses in
faeces and hematocryt. Vitamin K had no effect on E. acervulina, E. brunetti or E. maxima
infections (Baldwin et al., 1941; Ryley & Hardman, 1978).
4.3.3.2.2. Water-soluble vitamins (B and C)
The B-vitamins are essential for the development of Eimeria parasites (Warren, 1968) and
explains the coccidiosis enhancing effect of some of them. Hence, many anticoccidial
drugs are antagonists of analogues of one or more representatives of the vitamin complex.
Vitamin B1 increases E. tenella-induced mortality and oocyst shedding (Sherkov, 1976). In
contrast, increasing the concentrations of PABA, a member of the vitamin B complex, had
a coccidiosis impairing effect after a single (E. tenella) or a mixed species (E. tenella and
E. maxima) infection (Warren, 1968; Waldenstedt et al., 2000b).
Vitamin C is an antioxidant, which stabilizes membranes and could therefore be beneficial
in coccidiosis. It has also been reported to stimulate cellular immunity (McKee &
Harrison, 1995). Vitamin C is synthesized by chickens but under certain circumstances not
in sufficient quantities. Animal model studies on the effect of vitamin C have yielded
conflicting results. No effect on growth and mortality was found after E. maxima, E.
necatrix, E. brunetti or E. tenella infection, however in a mixed infection model (E.
acervulina, E. maxima, E. necatrix, E. brunetti and E. tenella) better growth was found
after vitamin C supplementation, although mortality was enhanced (Little & Edgar, 1971).
In layers, vitamin C supplementation reduced mortality and improved weight gain in E.
tenella infected chickens (Attia et al., 1978). Using the same Eimeria spp. in broilers no
effect was found on growth, although feed intake increased (McKee & Harrison, 1995).
49
4.3.4. Special additives

Milk products
Early treatment of coccidiosis was performed with milk products such as dry or liquid
skim milk, buttermilk and condensed whey (Beach & Corl, 1925; Beach & Freeborn,
1927; Herrick & Holmes, 1936), which likely affect the intestinal flora. Although these
products seemed to have an inhibitory effect against E. tenella (Beach & Corl, 1925), later
on the effect of dried buttermilk could not be demonstrated unambiguously by Becker &
Wilcke (1938).
Egg albumen
The value of egg albumen as a feed additive to control coccidiosis needs further research
in view of the conflicting results obtained in a few experimental studies. The addition of
egg albumen and thiamin (vitamin B1) to the feed of E. tenella infected chickens seems to
have a coccidiosis promoting effect, most probably due to the presence of thiamin
(Sherkov, 1976). However, in other studies, after adding egg albumin to the feed of E.
acervulina, E. tenella and E. maxima infected chickens lower oocyst shedding was
observed (Prasad, 1963; Warren & Ball, 1967) possibly due to the biotin (vitamin B7
essential for the coccidiosis parasite) binding properties of the protein avidin, which is
available at high levels in egg albumin.
Semipurified feed
Considering the origin of basic feed ingredients three types of poultry feeds exist: natural,
semisynthetic (semipurified) and synthetic. In the semisynthetic feed, a part of the natural
basic ingredients has been substituted by purified ingredients and (chemically synthesized)
nutrients, whereas in synthetic diets all basic ingredients are of purified origin and single
nutrients are included. The greater the synthesized part of the feed is, the more constant its
composition is, however its costs increase parallelly.
Compared to a corn-soybean feed, a semipurified diet appeared to ameliorate an infection
with E. tenella in chickens but not mixed infection with E. acervulina, E. maxima and E.
brunetti) (Colnago et al., 1984c). In agreement with previous study, Koltveit (1969)
obtained similar results for E. tenella earlier. Named scientist showed a spectacular
reduction in E. tenella mortality in birds given a semipurified diet compared to a ration of
natural feedstuffs, although clinical signs (onset and duration of bloody diarrhea, anorexia
and depression) did not differ between both diets. It suggested that natural feedstuffs
contain an unidentified ingredient(s) that enhances E. tenella lethality.
50
Table 11. Overview of the influence of feed components including, the active compound, dose and mode of action, on a coccidiosis
infection in poultry
Feed component
Cereals
Maize/corn
Maize
Wheat
Maize/wheat
Wheat
Wheat
Macronutrients
Carbohydrates
Active compound & dose
50, 100 and 200 g/kg
Mode of action
Coccidiosis model
Effect on clinical
parameters
Eimeria spp.
Infection dose
(# oocysts)
E. tenella
2 x 105
E. tenella
5 x 10
E. tenella
5 x 104
Mixed infect.
E. tenella
E. acervulina
E. acervulina
5 x 103
5 x 104
1.5 x 105
Increased digestive
efficiency by modification
of intestinal physiology
(improved hydrolyses,
absorption due to more
beneficial microflora)
E. acervulina
E. maxima
E. tenella
2.5 x 105
5 x 103
2 x 104
Changing intestinal flora
E. tenella
Severe
Increased intestinal flora
E. tenella
Unknown
Vitamin A (x 1.8)
Vitamin E (x 1.7)
Niacin (x 2)
Riboflavin (x 1.3)
+ soluble arabinoxynol
(different levels enzymes)
Stimulating intestinal
health & immunity
Enhancing parasite
development
Reduced viscosity
Whole wheat (200-400

g/kg)
Whole wheat (400 g/kg)
Increased viscosity
E. tenella
2 x 105
NSP
Mono- and disaccharides

( 500 g/kg)
Fibres, 100 g/kg (crude
fibre content)
Fibres 33, 66 and 99 g/kg
(cellulose)
Fibres 90 g/kg
NSP
Zymosan (-glucans)
Immune stimulation
E. tenella
E. acervulina
E. tenella
Proteins
Proteins
130 g/kg (low)

0-50 g/kg (very low)
Decreased trypsin activity

Decreased trypsin activity
E. tenella
E. tenella
?
?
NSP
NSP
Global
effect*
Reference
Mort**
BWG**
Mort
Colnago et al., 1984b
Mort
LS**
BWG
Maize (-)
Wheat (+)
Williams, 1992a
Warren, 1968
Williams, 1992a
Warren, 1968
Morgan & Catchpole, 1996
FCE**
OPG** =
BWG
OPG
+/-
Banfield et al., 2002
Gabriel et al., 2003

Gabriel et al., 2006
Matsuzawa, 1979
Mann, 1947
Koltveit, 1969
Muir & Bryden, 1992
Smith & Ovington, 1996
Mann, 1947
Britton et al., 1964
BWG
OPG
Mort
Haemorrhages
No difference
OPG
LS =
OPG
LS
LS
Mort
Protein
160, 200 and 240 g/kg
Increased trypsin activity
E. acervulina
E. tenella
105
104
Both species
BWG and FCE
E. ac OPG
E. ten Mort
+/-
Sharma et al., 1973
Lipids
(corn oil)
Unsaturated fatty acids
E. mivati
E. tenella
106
105
LS
Mort
Charney et al., 1971
Lipids
(coconuts oil)
Medium chain saturated

fatty acids (saturated fatty
acids)
-3 fa (25, 50 and 100
g/kg)
Enhanced parasite
development
(reproduction) &
influencing intestinal
mucosa for the expression
of pathology
Influencing intestinal
mucosa for the expression
of pathology
Immune stimulation &
reducing parasite
development by oxidative
stress
E. mivati
E. tenella
E. acervulina
E. tenella
106
105
6 x 105
5 x 104
LS
Mort
BWG
LS
BWG
Charney et al., 1971
E. maxima
5 x 104
LS =
Adams et al., 1996

Allen et al., 1996
Allen et al., 1997a
Korver et al., 1997
Allen et al., 1998
Lipids (fish and

flaxseed oil)
Macrominerals
Calcium
Buffers
Buffers
Microminerals
Selenium
Vitamins
Vitamin A
Vitamin D
Ca (10, 15, 18, 20 or 27

g/kg)
Increased trypsin activity
E. acervulina
E. tenella
Different
doses
Mort
BWG
NaHCO3 (10 g/kg)
Decreased coccidiosisinduced acidosis

Potentiation ionophores &
immune stimulation
E. acervulina
106, 5 x 105
E. acervulina
E. maxima
E. tenella
10
5 x 104
104
BWG
FCE
BWG
FCE
Mort
LS
Holmes et al., 1937

Zucker et al., 1967
Bafundo et al., 1984b
Fox et al., 1987
Hooge et al., 1999
Se (0.00025 to 0.001
g/kg)
Immune stimulation by
protection leucocytes
during phagocytic activity
Six Eimeria
spp. except E.
necatrix
BWG
Mort
Colnago et al., 1984a

Jensen et al., 1978
A (various from 4400 to

8000 IU/kg)
Stimulation intestinal
health & immunity
E. acervulina
E. tenella
Various
BWG
Mort
OPG
D (high)
Immune suppression
E. tenella
Erasmus et al., 1960

Coles et al., 1970
Singh & Donovan, 1973
Dalloul et al., 2002
Sherkov & Denovski, 1964
NaHCO3 (2 to 4 g/kg)
Vitamin E
E (100 IU/kg)
Vitamin E
E (0.025 or 0.225 g/kg tocopherol)

E (0.008 g/kg tocopherol)
Vitamin E
reduction in synthesis of
prostaglandins
Immune stimulation***
E. tenella
1.5 x 105
BWG
Mort
Colnago et al., 1984a
E. maxima
Mild & severe
Inactivation of reactive
nitrogen radicals
E. maxima
Allen & Fetterer, 2002a

Allen & Fetterer, 2002b
Allen et al., 1998
E. tenella
E. tenella
E. necatrix
Eimeria spp.
?
?
BWG
LS =
BWG
LS
OPG
No effect
Mort
=
-
Ryley & Hardman, 1978
Conflicting
results
+/-
Mort
OPG
Little & Edgar, 1971

Attia et al., 1978
McKee & Harrison, 1995
Sherkov, 1976
Mort
Beach & Corl, 1925
Mort
Becker & Wilcke, 1938
Vitamin K
Vitamin C
Vitamin B
B (0.1 g/kg)
Enhanced parasite
development (schizogony
(E. acervulina) &
(gametogony (E. tenella)
E. tenella
Dry or liquid skim and

buttermilk, condensed
whey (as drinks or dry in
the feed (100 and 400
g/kg)
Changed intestinal flora
E. tenella
Special additives
Milk products
Enhanced coagulation
intestinal mucosa
Stimulation immunity &
healing intestinal mucosa
E. tenella
10
* - sign indicates that the component has an inhibiting effect on coccidiosis, + sign indicates that the component promotes coccidiosis and = indicates no effect on
coccidiosis.
** BWG = body weight gain, FCE = feed conversion efficiency (g weight gain/g food intake), LS = lesion scores, Mort = mortality, OPG = oocysts per gram
faeces.
*** malabsorption (caused by the coccidiosis infection) prevented access of dietary vitamin E and thus immunity stimulation does not occur.
4.4. Toxins in feed

Mycotoxins
Mycotoxins are poisonous secondary metabolites produced by various species of moulds
and yeasts developing in the feeds and bedding of animals. A great variety of mycotoxins
(about 300 to 400) have been described and they may affect animal health and production
differently. Due to their great resistance to decomposition, digestion and various
treatments (high temperatures and cooking) mycotoxins can remain in the food chain. The
major mycotoxins are: aflatoxins, citrinin, ergot alkaloids, fumonisins, ochratoxins, patulin
and trichothecenes.
Various feed components (e.g. corn, wheat, oats) are susceptible to moulds and should be
tested regularly for the occurrence of mycotoxins. Even very low concentrations of
mycotoxins in feed can result in a reduction of feed intake, but also in immune suppressed
birds (Conway, 1996).
Aflatoxins are produced by many species of Aspergillus. They are toxic and carcinogenic,
and have been shown to enhance coccidiosis, especially E. tenella. Their influence on
blood clothing explains the increased mortality associated with greater caecal bleedings
found when aflatoxins are given to E. tenella infected birds (Edds et al., 1973; Wyatt et
al., 1975; Witlock & Wyatt, 1978). In E. acervulina infection, aflatoxins provoked strong
growth retardation, bad feed conversion and lesser pigmentation (Ruff, 1978; Southern et
al., 1984). A negative influence of dietary aflatoxin on coccidiosis infected quails has also
been described: aflatoxin supplemented and coccidiosis infected quails had higher
mortality, coccidial lesion scores and oocyst shedding (Awadalla, 1998).
Ochratoxins are produced by fungi of the genus Aspergillus and Penicillium and are
mainly nephrotoxic. Most common is ochratoxin A, which has been shown to enhance an
E. tenella or E. acervulina infection in chicks characterized by greater kidney function
impairment, histopathological changes of organs and growth depression (Stoev et al.,
2002; Koynarski et al., 2007).
T-2 fusariotoxin, which belongs to the type A trichothecenes, is produced by various
species of the genus Fusarium and may cause damage of cell membranes, structural lipids
and inhibit protein (inhibition of peptidyl-transferase), DNA and RNA synthesis. T-2
fusariotoxin has been reported to negatively influence the efficacy of monensin in
coccidiosis infected chickens (Vnyi et al., 1989) and of lasalocid in E. tenella and E.
mitis infected cockerels (Varga & Vnyi, 1992).
Mycotoxins have also been shown to impair the efficacy of some anticoccidial products.
Aflatoxins reduced the effect of monensin on an E. tenella infection, although the efficacy
of amprolium was not altered (Edds et al., 1973, Wyatt et al., 1975). In case of an E.
acervulina infection the action of monensin was not influenced either (Southern et al.,
1984).
54
CHAPTER 1
4.5. Feed management

4.5.1. Feed restriction
Feed restriction is frequently applied to commercial poultry, especially reproduction
broilers in order to control growth. During the past decades broilers have been genetically
selected for increased growth rate and lower feed conversion. In order to allow
reproduction broilers to survive until egg production and warrant longevity by avoiding
health disorders associated with obesity (skeletal disorders, heart failure, thermal
discomfort, reduced disease resistance, multiple ovulations, lower fertility of males, etc.),
these birds need to be fed restricted. It is nevertheless believed to compromise seriously
the welfare of chickens and ought to be discouraged. The use of slower-growing genotypes
would render feed restriction unnecessary (SCAHAW, 2000).
Feed restriction has been shown to have an inhibitory effect on E. tenella infections. It is
thought to improve the immune response after the initial stress induced by restricted
feeding has vanished. Indeed, White Plymouth Rock chickens that had been feed restricted
three weeks before an infection with E. tenella showed less coccidiosis lesions compared
to their ad libitum fed counterparts (Zulkifli et al., 1993). Another explanation for the
greater resistance of feed restricted birds is the lower production of trypsin, which
contributes to excystation of sporozoites in these chickens (Britton et al., 1964; Nir et al.,
1987). In contrast to the results obtained with E. tenella during feed restriction, increased
coccidiosis lesions were seen in feed restricted broilers infected with E. acervulina, E.
maxima and E. tenella and supplemented with anticoccidial drugs (salinomycin or
monensin) suggesting that higher levels of anticoccidial drugs are necessary during feed
restriction (McNaughton et al., 1990). In model studies using E. maxima, Newcombe and
others (1992) also found significantly higher coccidiosis lesion scores in male broilers
subjected to feed restriction. More recently in another study, changing the protein
concentration (15 or 19% CP) to restrict-fed broiler breeder pullets, did not influence mild
coccidial infections induced via the administration of commercial anticoccidial vaccines
(Yaissle et al., 1999).
55
Intestinal lumen
corn
wheat
whole cereals
+
EFA
viscosity
proteins
calcium
intestinal flora & mucosa
fibers (NSP)
milk products
vitamin K
medium chain FA
NaHCO3
feed restriction
+
-
aflatoxins
ionophores
+
-3 FA
Extraintestinal environment
vitamin A, C
trypsine activity
-glucans
vitamin E
-tocopherol
selenium
vitamin D
vitamin B
+
-
immunity
Figure 2. Vitamin B, essential fatty acids (EFA) and aflatoxins seem to influence the development of the
Eimeria parasite positively. Moreover, aflatoxins also have a negative effect on the activity of ionophores.
Therefore, the first two products should be avoided in excess and aflatoxins completely. Ionophores and
-3 fatty acids (-3 FA) influence the development of the Eimeria spp. negatively. Sodium bicarbonate
(NaHCO3) has a positive effect on the ionophores resulting in a negative effect on the development of the
coccidiosis infection. Proteins and calcium may have, depending on their concentration in the feed, an
indirect positive or negative effect on the development of the parasite. The viscosity, the intestinal flora &
mucosa of the digestive tract may have a variable influence on the development of the parasite. The
viscosity of the intestine content can be influenced by the presence of corn, wheat and whole cereals.
These components can together with fibers (NSP) and milk products influence the intestinal flora.
Additionally, the availability of whole cereals instead of ground cereals may influence the physiology of
the digestive tract. The Eimeria parasite may induce lesions and hemorrhages in the intestinal mucosa.
Components like vitamin K and medium chain fatty acids (FA) have a positive effect on the intestinal
mucosa and coccidiosis-induced lesions by promoting coagulation and healing. Vitamin A and C have a
positive influence on the intestinal mucosa and the immune system. Furthermore, -glucans, vitamin E, tocopherol, selenium and -3 FA also stimulate the immune system. Feed restriction showed conflicting
results on the development of Eimeria.
56
CHAPTER 1
5. Other preventive strategies against coccidiosis

5.1. Introduction
The prevention and control of coccidiosis in commercial poultry and mainly broilers is
largely based on the administration of anticoccidial drugs in feed, although in some cases
an anticoccidial drug administered shortly via drinking water is applied. Moreover,
biosecurity measures aiming at preventing the introduction of the parasite to the farm can
be of additional strategic value against clinical coccidiosis. During the last decade, live
coccidiosis vaccination has become increasingly popular as an alternative strategy to
control Eimeria. In feed products with supposed anticoccidial activity like herbs, essential
oils, probiotics and prebiotics represent additional alternative strategies. Control of
coccidiosis by means of feed composition and structure, which could also have been
considered here, has been discussed previously (section 4).
The quest for alternative strategies for the control and prevention of coccidiosis has
been propelled by increasing concerns of consumers on food safety. This has resulted in a
loud call for residue-free poultry products, which in Europe has first been translated into a
re-evaluation of all existing anticoccidial products and regulations safeguarding lower
residues. It has also favoured politic debates on the use of in feed medication in general.
Currently, the future status of anticoccidial drugs is uncertain: their status as feed additives
may be maintained or new legislation and phasing-out of these drugs as feed additives
could be proposed. Another contributing factor to the search for alternative control and
prevention strategies has been the development of resistance against all in feed
anticoccidial drugs used to date.
5.2. Management and biosecurity

Good health and hence optimal immune response is essential in order to minimize the
impact of infectious diseases. General management measures safeguarding the basic
requirements of poultry should therefore always be implemented: birds should be provided
with proper feed and drinking water intake, adequate bedding, temperature, humidity,
lighting and ventilation.
Management and biosecurity measures for the control of coccidiosis should focus on
preventing the introduction of the parasite to the premises, which is almost impossible
under practical circumstances, and control its multiplication and spread in case flocks have
been infected. In the early days of coccidiosis research Johnson and Tyzzer (1920) already
stressed that management and the environmental factors are very important in the
incidence and epizootiology of coccidiosis (Chapman, 2003). This has become common
knowledge and was more recently confirmed by Graat and others (1998) who quantified
risk factors of coccidiosis in broilers using on-farm data based on a veterinary practice and
statistical modeling following a case-control design. They found an enhanced risk of
57
coccidiosis due to environmental and management factors that increase the risk of
introducing contamination or that are related to hygienic measures.
5.2.1. Avoiding the introduction of the parasite
Eimeria parasites are ubiquitous and have enormous reproduction capabilities leading to
high contamination levels of infected poultry houses and their surroundings. Moreover, the
oocyst wall protects the parasite from desiccation and chemical disinfectants ensuring
long-term survival in the environment from which it may be introduced to a farm through
a variety of ways such as personnel, equipment, rodents, insects, etc. (Belli et al., 2006).
Also contaminated water, feed and bedding can be a source of infection. On the other
hand, oocysts may already be present in the farm house if cleaning and disinfection was
not adequate. It is very difficult for infected premises to achieve freedom from coccidiosis
as one oocyst, containing four sporocysts with each two sporozoites, is sufficient to start
infection (see section 1.4 Life cycle of avian Eimeria spp.).
Biosecurity measures aiming to prevent the introduction of Eimeria parasites to the farm
are similar to those applied for the prevention of other infectious poultry diseases and
should focus on:
1. Isolation. Birds should be separated from the environment by fencing and
other animals including rodents and insects should be kept out.
2. Traffic control should not only be performed at the farm, but also traffic
between farms should be restricted.
3. Sanitation includes disinfection of materials, people and equipment entering
the farm and poultry house.
5.2.2. Control of coccidiosis on infected premises
If performed adequately, cleaning and disinfection between flocks and maximizing the
downtime period should reduce significantly the number of parasites in contaminated
chicken houses, while biosecurity measures should prevent additional introductions.
However, in practice it is almost impossible to keep poultry free of coccidiosis unless
birds are housed under SPF-like housing conditions.
5.2.2.1. Environmental factors
In case coccidiosis infections occur, their multiplication can not be restricted by
influencing the climate environment of the chicken house as Eimeria thrives in
atmospheric conditions that are beneficial to the birds also.
In order for Eimeria to become infective, it must sporulate after excretion in the faeces.
The degree and rate of sporulation of excreted oocysts determine the infection pressure in
a chicken flock. Sporulation of the oocyst depends mainly on the following basic factors:
temperature, humidity and aeration (access to oxygen) (Kheysin, 1972).
The best sporulation temperature is approx. 24-28 C (Edgar, 1955), while a temperature
above 35 C is lethal for oocysts (Schneider et al., 1979). Due to the fact that the ideal
58
CHAPTER 1
sporulation temperature is within the range of temperatures frequently encountered in the

poultry environment and that high temperatures are detrimental to the birds, temperature is
not a factor that can be used to control parasite multiplication.
A general misconception, regarding humidity is the belief that the drier the climate, the
less coccidiosis occurs. Research data have shown that in the field sporulation in wet litter
is suboptimal possibly due to the occurrence of ammonia and bacteria (Williams, 1995); in
fact, dry litter showed better sporulation rates (Graat et al., 1994; Waldenstedt et al.,
2001). However, increasing the litter humidity as a control measure to reduce sporulation
does not seem feasible as it might cause footpad lesions and skin burns in the birds.
Adequate ventilation of the poultry house is essential for good performance and health of
the birds although proper aeration will also favour sporulation.
Theoretically, a higher bird density will result in greater oocyst accumulation in the litter
and increase the chances of clinical coccidiosis (Williams et al., 2000). Thus, reducing
bird density could help to control Eimeria infections.
In order to prevent clinical coccidiosis it is essential that the birds have adequate access to
the medicated feed. It is also vital that Eimeria spp. field strains concerned are sensitive to
the anticoccidial drugs used, which underlines the necessity to perform AST on a regular
basis. Moreover, rotation and shuttle programs eventually alternated with vaccination
should be used to minimize the occurrence of resistance (see section 3).
5.2.2.2. Cleaning and disinfection
Cleaning and disinfection between flocks and maximizing the downtime period is
important in order to reduce significantly the number of parasites in contaminated chicken
houses. However, there are mixed points of view regarding the use of cleaning and
disinfection for the control of coccidiosis. Some consider that the presence of oocysts in
the poultry environment enabling early establishment of immunity in order to avoid
outbreaks at later age, beneficial. In case coccidiosis vaccines are used to repopulate the
houses with drug sensitive strains, survival of vaccine parasites between vaccinated and
non-vaccinated flocks is desired and therefore cleaning and disinfection should be skipped.
Nevertheless, in case of severe clinical coccidiosis outbreaks especially due to
anticoccidial drug resistant strains, it is customary in Europe, where birds are not housed
on deep litter, to clean and disinfect the chicken houses in order to reduce infection
pressure.
The oocyst wall makes the parasite resistant to many disinfectants and environmental
conditions. They are nevertheless sensitive for desiccation and high temperatures (Ryley,
1973). Despite the protecting capabilities of the oocyst wall, a limited number of chemical
products is able to penetrate the oocyst through small pores, which are crucial for the
access of oxygen. Lipid-soluble substances and small molecules, including ammonia,
methyl bromide and carbon disulphide have been reported to penetrate the oocyst
(Kheysin, 1935; Monn & Hnig, 1954; Ryley, 1973; Kuticic & Wikerhauser, 1996). Due
to their toxicity, methyl bromide and carbon disulphide can not be used as disinfectants
against Eimeria.
59
Ammonium hydroxide has been reported as a highly effective disinfectant against

sporulated and non-sporulated oocysts. It has been shown to be effective at a concentration
of 5% in both fluid and vapour form (Chroustova & Pinka, 1987). Also products that
generate ammonia after mixing two components (ammonium salt and sodium hydroxide)
have shown to be oocidal (Oocide, Antec International Ltd, UK). More recently a cresolbased product (Neopredisan 135-1, Menno Chemie, Norderstedt, Germany) has been
marketed in Germany for the control of coccidiosis by chemical disinfection: a
concentration of 4% and a contact time of 2 hours is advised. It has been shown to be
efficient against E. tenella (Daugschies et al., 2002) and other Eimeria spp. (Houdek et al.,
2002). In a recent study the efficacy of eight disinfectants against E. tenella unsporulated
oocysts isolated from broilers was studied in vitro. The best disinfection efficacy was
observed for the combination formol 37% and sodium dodecylbenzene sulfonate 12%;
orthodichlorobenzene 60% and xylene 30%, and sodium hypochloride 2%, being 79.49%,
75.60%, 63.56%, respectively (Gumares et al., 2007).
There are no disinfectants specifically registered against coccidiosis in Netherlands.
However, in practice, poultry houses are disinfected using calcium hydroxide in
combination with ammonium sulphate (per 500 m2 40 kg calcium hydroxide is spread and
subsequently moisturized with approximately 500 l water, after which 80 kg ammonium
sulphate is added). Ammonium, which is lethal to the oocysts, is generated at combining
the afore mentioned products.
5.3. Alternative coccidiosis control

Various alternative methods like homeopathy, phytotherapy and aromatherapy have been
used during the past decennia for the treatment of various poultry diseases. Homeopathy is
a system for treating disease based on the administration of minute doses of a drug that in
massive amounts produces clinical signs in healthy individuals similar to those of the
disease itself. The treatment is thus based on law of similars, i.e. similia similibus curentur
= the most similar remedy will cure (Saine, 2000). It is thought to enhance the bodys
natural defenses. Homeopathy is often confused with herbalism also known as botanical
medicine, medicinal botany, medical herbalism, herbal medicine, herbology and
phytotherapy. It is based on the use of plant and plant extracts for the treatment and
prevention of disease. Its scope is sometimes extended to include fungi, bee products,
minerals, shells and certain animal parts. Aromatherapy, which is closely related to
phytotherapy, is the treatment or prevention of disease by means of essential oils, and
other scented compounds from plants. It involves the use of distilled plant volatiles, a
twentieth century innovation. Essential oils differ in chemical composition from other
herbal products because the distillation process only recovers lighter phytomolecules.
Although many papers have been published on the efficacy of homeopathic treatment in
humans, its effectiveness has remained a matter of debate to the scientific community.
Studies assessing the methodological quality of a large number of human homeopathic
experiments report a lack of evidence for efficacy of homeopathic treatments because of
60
CHAPTER 1
frequent low methodological quality and publication bias (Kleynen et al., 1991; Linde et
al., 2001). According to Velkers and others (2005) Efficacy of medicines should be
beyond placebo effect and observation and interpretation should be without subjective
elements. Therefore, all medicines, including homeopathic remedies, should be tested in a
double blind randomized clinical trial. Published studies on homeopathy in poultry are
rare and similar to human studies are frequently affected by low methodological quality
(Velkers et al., 2005). There are no scientific reports on the homeopathic treatment of
coccidiosis.
Some plant products or derived pharmaceutical drugs have been incorporated into
mainstream medicine; nevertheless most herbal treatments have been developed without
modern scientific assessment. Although later on many herbs have been found efficacious
in in vitro, animal models and/or clinical studies (Srinivasan, 2005), there are also many
studies showing negative results (Pittler et al., 2000). However, evaluation of the literature
on complementary/alternative therapies is difficult due to the occurrence of location bias
in the corresponding controlled clinical trials. More positive than negative trials are
published, except in high-impact mainstream medical journals. Further, in
complementary/alternative medicine journals positive studies are of poorer quality than
corresponding negative studies. The latter was not the case in mainstream medical journals
publishing on a wider range of therapies.
Pre- and probiotics have also been included within alternative coccidiosis controls
strategies although they are quite distinct from phyto- and aromatherapy. Probiotics
consist of in feed administered live bacteria or yeasts, which are supposed to be beneficial
for the individuals health. In contrast, prebiotics are non digestible food ingredients,
however, they also have a beneficial effect on the host health.
5.3.1. Plant (herb) extracts
Many different herbal compounds have been investigated for their potential use as a
dietary supplement to control coccidiosis and are reported next in alphabetical order. The
main conclusions obtained in the research of alternative coccidiosis control have been
summarized in Table 12 and Figure 3.
5.3.1.1. Artemisinin
Artemisinin is a herbal extract, which can be obtained from Artemisia annua (annual
wormwood) and A. sieberi. Its antimalarial activity in humans has been attributed to the
ability to induce oxidative stress through the production of free radicals and to alkylate
proteins (Klayman, 1985; Krungkrai & Yuthavong, 1987; Levander et al., 1989; Meshnick
et al., 1989; Yang et al., 1994).
To date only three manuscripts on the effect of artemisinin on coccidiosis in poultry have
been published. The first study was reported by Oh and others (1995), who described
improved weight gain and feed conversion as well as a reduction in lesion scores after an
E. tenella infection in chickens supplemented with A. annua extracts. Later on, Allen and
co-workers (1997b) showed that dried leaves of this plant at a dietary concentration of 5%
61
(equivalent to 17 ppm pure artemisinin) during three weeks provided a significant

protection against lesions of E. tenella but not against lesions of E. acervulina and E.
maxima. However, when pure artemisinin was fed for a period of 4 weeks at levels of 2,
8.5 and 17 ppm it significantly reduced oocyst output from single and dual species
infection of E. tenella and E. acervulina. No reduction in oocyst excretion was notified in
case of an E. maxima infection. Thus, artemisinin proved to be effective against at least
two Eimeria spp. (Allen et al., 1997b).
In another study it was shown that artemisinin isolated from A. sieberi was also active
against E. tenella and E. acervulina but not against E. maxima (Arab et al., 2006), which is
in agreement with the other studies mentioned above.
5.3.1.2. Betaine
Betaine is a sweet crystalline alkaloid (trimethylglycine C5H11NO2), choline analogue and
methyl donor occurring in sugar beets and other plants, which is used in the treatment of
certain metabolic disorders (muscular weakness and degeneration). It has been shown to
protect cells against osmotic stress by stabilizing cell membranes enabling the
maintenance of osmotic pressure in the cells and ensuring normal metabolic activity
(Rudolph et al., 1986). The osmoprotective effect of betaine is not restricted to the
intestinal cells, but also affects developing stages of the coccidia. It protects different cell
types against chemical and environmental stress (Kunin & Rudy, 1991), including the
asexual stages (sporozoites) of E. acervulina against the destructive action of salinomycin
(Augustine & Danforth, 1999). Nevertheless, feed efficacy and weight gain was
significantly improved in birds infected with a mixture of E. acervulina, E. maxima and E.
tenella if betaine (0.15%) and salinomycin (44 or 66 ppm) were supplemented separately,
but much more if both components were given together suggesting a palliative effect of
betaine on the coccidiosis infection. Betaine alone did not influence the severity of lesions
scores and mortality (Augustine et al., 1997). Both products alone or in combination
inhibited the invasion of E. acervulina and E. tenella (sporozoites) and the development of
E. acervulina (second generation schizonts).
The ionophore amplifying effect of betaine was only observed once and could not be
found with other anticoccidial ionophores drugs like monensin and narasin in experiments
performed by other scientists (Matthews et al., 1997; Waldenstedt et al., 1999).
Waldenstedt and others proved that betaine, used as a single feed supplement also
improved the weight gain of chickens infected with a mixture of Eimeria spp., but given in
combination with narasin no positive effect could be achieved. Improvement of weight
gain was also found in betaine supplemented birds infected with E. maxima, but not in E.
acervulina or E. tenella infected birds (Fetterer et al., 2003), which seems in contradiction
with the results obtained earlier.
The weight gain and feed conversion promoting capabilities found in some studies may be
explained by betaines osmoprotectant properties ensuring normal metabolism of intestinal
cells. This may warrant normal development of the chicks despite the fact that the
coccidiosis infection is not fully halted. Another contributing factor to the positive effect
of betaine on coccidiosis infection may be the fact that it increases intraepithelial
62
CHAPTER 1
lymphocytes in the duodenum and the functional properties of phagocytes of Eimeria

infected chickens (Klasing et al., 2002).
5.3.1.3. Citric Extracts
A product based on citric extracts and organic acids amongst others supplemented to
broilers showed a moderate efficacy against a challenge with various Eimeria spp. (E.
acervulina, E. maxima, E. tenella, E. brunetti and E. mivati) considering coccidiosis lesion
scores and oocyst production (Tamasaukas et al., 1997). In a previous study by the same
group, extracts obtained from seeds of citric fruits also showed anticoccidial activity but
not against E. tenella (Tamasaukas et al., 1996).
5.3.1.4. Echinacea purpurea
The anticoccidial effect of Echinacea purpurea has been attributed to its
immunomodulating properties, which have been widely documented (Stimple et al., 1984;
Burger et al., 1997; See et al., 1997; Sun et al., 1999; Currier & Miller, 2001; Goel et al.,
2002). In veterinary medicine Echinacea therapy has been reported in horses (ONeill et
al., 2002), swine (Stahl et al., 1990) and cattle (Schuberth et al., 2002), and more recently
in chickens against coccidiosis. Ground root preparations of E. purpurea (0.1% - 0.5%)
supplemented to broilers during two weeks reduced weight gain retardation and coccidial
lesions after a mixed challenge infection at the age of 28 days with E. acervulina, E.
maxima, E. tenella and E. necatrix (Allen, 2003).
5.3.1.5. Gentian violet
Gentian violet also named crystal violet, methyl Violet and hexamethyl pararosaniline
chloride is known for its antifungal and antibacterial properties, but also for its use in
diagnostic bacteriology as part of the Gram stain test. It is not derived from gentians, but
got its name since it is pink-violet like some gentians in the genera of Centaurium,
Gentiana and Gentianella. Gentian violet is derived from coal tar.
Gentian violet has been shown to reduce coccidiosis lesion scores in the duodenum and
improve weight gain in Eimeria spp. challenged birds. In combination with anticoccidial
drugs it improved feed conversion (Sharkey, 1978).
5.3.1.6. Mushrooms and their extracts
Mushrooms and their extracts have gained interest in medicine and as dietary supplement
due to their immune enhancing and antitumor properties. However, they should be used
cautiously as mushrooms may harbour toxic levels of metals (arsenic, lead, cadmium and
mercury) and radioactive contamination with 137Cs (Borchers et al., 2004).
Polysaccharide extracts originating from Lentinus edodes and Tremella fuciformes as well
as the herb Astragalus membranaceus showed a positive effect on the cellular and humoral
immunity of E. tenella infected female broilers (Guo et al., 2004).
In another study, the same extracts yielded better growth during immunization in
comparison with the non-treated vaccinated birds. During challenge with E. tenella,
63
broilers supplemented with these mushroom extracts showed similar lesion scores as the
vaccinated only birds, however they had lower oocyst countings (Guo et al., 2005).
Extracted lectin from the mushroom Fomitella fraxinea injected in 18 day-old embryos,
protected broilers inoculated with E. acervulina at one week post-hatch against weight loss
and was associated with a significant reduction in oocyst shedding compared to untreated
embryos. The mushroom lectins showed potent mitogenic activity on chicken splenic
lymphocytes, they induced NO secretion in HD11 macrophages and suppressed tumor cell
growth. This study suggested that the used lectines act as immunopotentiators of the innate
immune response and could be used as an alternative anticoccidiosis strategy (Dalloul et
al., 2006).
5.3.1.7. Oregano
The essential oils of Origanum vulgare are known for their antibacterial activity (Hammer
et al., 1999) and effect against some parasites (Milhau et al., 1997). Furthermore, some
essential oils of oregano, mainly carvacrol and thymol, have an anticoccidial effect against
E. tenella although lower than lasalocid (Giannenas et al., 2003). However, in
subsequently performed studies with diets supplemented with a mixture of the essential
oils, oregano thymol, eugenol, curcumin and piperin a beneficial effect of these oils on a
coccidiosis vaccination was not found (Oviedo-Rondn et al., 2006).
5.3.1.8. Turmeric (Curcuma longa)
Turmeric is a spice and colorant made from the rhizomes of the plant Curcuma longa, a
leafy plant belonging to the ginger family. The rhizomes are boiled for several hours and
then dried in ovens, after which they are ground into a deep orange yellow powder
commonly used as a spice in curry. Turmeric is also known for having medical properties.
Its medicinally active compound is the phenolic compound curcumin, which has been
shown to have antioxidative, anti-inflammatory and antitumour properties
(Mukhopadhyay et al., 1982; Conney et al., 1991; Ammon et al., 1993; Brouet &
Ohshima, 1995).
E. maxima infected chicks fed diets supplemented with 1% curcumin showed an improved
weight gain and a reduction in the lesion scores and oocyst excretion. Nevertheless, the
activity was only shown against E. maxima and not against E. tenella. A significant
reduction of plasma NO2 and NO3 was only found in E. maxima infected and curcumin
treated birds and provide a possible explanation for the difference in anticoccidial activity
found for both Eimeria spp. (Allen et al., 1998). A similar effect on lesion scores, oocyst
shedding, growth and plasma NO2 and NO3 was found for -tocopherol. The
antioxidative properties of curcumin by inhibiting NOS induction by macrophages
stimulated with lipopolysaccharide and interferon-, has been shown previously (Brouet &
Ohshima, 1995). Although NO is an important defence mechanism against the invasion of
different Apicomplexa parasites (Adams et al., 1990; Mellouk et al., 1991) it was
suggested more recently that NO might promote the development of coccidial lesions
(Allen, 1997a, b).
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CHAPTER 1
5.3.1.9. Incidental reports on the anticoccidial activity of other herb(s) extracts

A product prepared from Holarrhea pubescens, Berneris aristata, Embelia ribe and
Acorus calamus was reported to be active against E. necatrix when supplemented to the
diet at a concentration of 0.6% (Mandal et al., 1994).
Preparations of Persian lilac (Melia azedarach) and the bitter melon (Momordica
charantia) were shown to improve the weight gain retardation and reduce the oocyst
excretion after an infection with different Eimeria spp. (Hayat et al., 1996).
Fifteen different Asian plants were screened for their anticoccidial activity against E.
tenella. A number of these extracts seemed to have a beneficial effect. The mortality,
bloody diarrhea, clinical signs, lesion scores, body weight gains and oocyst excretions
were mostly influenced positively by the extract of the rhizomes of Sophora flavescens, a
small species of tree. Extracts of the seeds and barks of the elm (Ulmus macrocarpa) and
the rhizomes of the Korean anemone (Pulsatilla koreana) reduced only the mortality and
the coccidial lesions. The nuts of the Chinese honeysuckle (Quisqualis indica) improved
the weight gain and extracts of the trunks and the roots of the deciduous climber plant
Sinomenium acutum decreased the bloody diarrhoea. Both extracts of Quisqualis indica
and Sinomenium acutum delayed the oocyst excretion period with one or two days (Youn
& Noh, 2001).
The anticoccidial activity of the fruits of the neem tree (Azadirachta indica) was
investigated in comparison with salinomycin against an E. tenella infection. The results
showed that neem fruits at a concentration of 150 g/50 kg feed had excellent performance
in reducing the oocyst excretion and mortality as compared to the salinomycin treated
group (Tipu et al., 2002).
Using a herbal complex consisting of eight different herbs (Uncariae Ramulus cum Uncis,
Agrimoniae Herba, Sanguisorbae Radix, Eclipta Prostrate Herba, Pulsatillae Radix,
Sophorae Flavescentis Radix, Rehmanniae Radix and Glycyrrhizae Radix), the
anticoccidial activity against an E. tenella infection was assessed. The complex
significantly decreased the coccidiosis lesion scores and increased the bodyweight gain of
the birds after infection. The herbs of the complex that mainly contributed to the
anticoccidial effect remain to be identified (Du & Hu, 2004).
In another study the dietary supplementation of Apacox, a commercial preparation of a
mixture of four different herbal extracts, was investigated for its anticoccidial activity
against E. tenella in comparison with lasalocid. Results showed that Apacox has an
anticoccidial effect against E. tenella but this effect was significantly lower than the effect
of lasalocid. Similar to the study of Du and Hu (2004), further research is required to
identify the active components of Apacox (Christaki et al., 2004).
More recently, in a study performed in Korea the anticoccidial effect against an E. maxima
infection of green tea supplemented to diets was investigated. The parameters used to
assess anticoccidial activity were oocyst excretion and body weight gain. The birds
supplemented with green tea had significantly reduced oocyst excretion compared to E.
maxima infected birds fed the standard diet. However, the body weight gain was not
improved in the birds feed the green tea-based diet (Jang et al., 2007).
65
5.3.2. Pre- and probiotics

The term prebiotic was introduced by Gibson and Roberfroid (1995), who defined it as a
non digestible food ingredient that beneficially affects the host by selectively stimulating
the growth and/or activity of one or a limited number of bacteria in the colon, and thus
improves host health. The positive influence of prebiotics on the intestinal flora has been
confirmed by a number of studies (Van Loo et al., 1999). Recently, the definition of the
prebiotics was narrowed with the introduction of a prebiotic index by Roberfroid (2005),
who stated that a preparation might be called prebiotic if it is capable to produce at least 4
x 108 colony forming units of Bifidobacteria/gram faeces per daily dose (gram) ingested.
Only three large groups meet this criterion: inulin and oligofructose,
galactoseoligosaccharides and xylooligosaccharides.
Probiotics consist of beneficial live bacteria or yeasts supplemented to the diet. According
to the Food and Agriculture Organization (FAO) and World Health Organization (WHO)
probiotics are live microorganisms, which when administrated in adequate amounts
confer a health benefit on the host (FAO/WHO, 2002). The most frequently used
probiotics in humans are species of the genera Lactobacillus and Bifidobacteria whereas
species of Bacillus, Enterococcus and Saccharomyces yeasts have been the most
commonly used organisms in livestock. In poultry production, probiotics are known for
their capacity to restore the intestinal microflora after being disrupted by antibiotic
treatment or enteric infections (Rada & Rychly, 1995; Line et al., 1998; Pascual et al.,
1999). They are also known for their ability to boost the immune system and used against
allergies and other immune diseases (Zulkifli et al., 2000; Dalloul et al., 2003b; Kabir et
al., 2004; Koenen et al., 2004).
A treatment with prebiotics can be easily combined with a probiotic, in a so called
synbiotic approach (Gibson and Roberfroid, 1995). An advantage of this combination is
the improved survival of probiotics when given in a medium of prebiotics. The use of preand probiotics in poultry has been recently reviewed by Patterson and Burkholder (2003).
5.3.2.1. Prebiotics
Mannanoligosaccharides (MOS) are derived from the cell wall of the yeast Saccharomyces
cerevisae and are widely used in animal feed to promote gastrointestinal health and
performance. MOS have been described as a prebiotic but the mode of action may be
different; they are thought to block the binding of pathogens to mannan receptors on the
mucosal surface and stimulate the immune response (Spring et al., 2000). Some brand
names are: BioMos, SAF-Mannen, Y-Mos and Celmana.
In poultry MOS enhances the development of Bifidobacteria spp. and Lactobacillus spp.
in the intestinal tract of young chickens and suppresses the number of enterobacteriacea
(Fernandez et al., 2002). In an experiment performed with coccidia, dietary MOS (1 g/kg
feed) was able to reduce the severity of a single E. tenella infection with 3500 or 5000
sporulated oocysts (Elmusharaf et al., 2006). In another experiment a dietary
supplementation of MOS, at a concentration of 10 g/kg feed, reduced the oocyst excretion
and diminished the severity of E. acervulina lesions of birds infected with a mixture of E.
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CHAPTER 1
acervulina, E. maxima and E. tenella at subclinical doses of 900, 570 and 170 sporulated
oocysts, respectively (Elmusharaf et al., 2007).
In a study performed by McCann and others (2006) supplementation of MOS (0.5 g/kg
feed) or tannin (0.5 g/kg feed) either individually or in combination did not reduce the
severity of a mixed coccidiosis infection of E. acervulina, E. maxima and E. tenella given
at clinical doses of 50,000, 15,000 and 15,000 sporulated oocysts, respectively.
These contradictory results are possibly explained by differences in MOS concentrations
in feed and the magnitude of Eimeria spp. inoculation doses. Further research is necessary
to confirm whether MOS has anticoccidial activity when used at higher concentrations in
feed in combination with higher challenge doses.
5.3.2.2. Probiotics
Lower intestinal invasion and development of coccidia and lower oocyst production,
explained by enhanced local cell-mediated immunity, was achieved with a Lactobacillusbased probiotic supplemented diet in E. acervulina infected broilers (Dalloul et al., 2003a;
2003b; 2005).
More recently, in a study performed with a Pediococcus-based commercial probiotic
(MitoGrow) given to birds infected with an E. acervulina or E. tenella infection,
increased resistance of birds against coccidiosis and a partial protection against growth
retardation was demonstrated (Lee et al., 2007a). In another study performed with a
Pediococcus- and Saccharomyces-based probiotic (MitoMax) given to birds challenged
with 5000 oocysts of either E. acervulina or E. tenella, less oocyst shedding and a better
antibody response was found in probiotic fed birds compared to non-probiotic controls.
These results suggest that MitoMax may improve the resistance against coccidiosis by
enhancing the humoral immune response when included in the diet (Lee et al., 2007b).
67
Table 12. Overview of the influence of alternative feed additives including, the active compound, dose and mode of action, on a coccidiosis
infection in poultry
Alternative feed
additive
Active compound &

dose
Mode of action
Coccidiosis model
Eimeria spp.
Effect on
clinical
parameters
Global
effect*
Reference
Artemisia
annua
Artemisinin extracts
Induction oxidative
stress (free radicals)
E. tenella
Infection dose
(# oocysts)
?
Artemisia
annua
Dried leaves at 5, 10 &

50 g/kg 3 wks
Pure artemisinin 0.002,
0.0085 & 0.017 g/kg 4
wks
Polysaccharide extracts
(1 g/kg)
Lectin (FFrL) injected
into amniotic cavity
(100 g in 100 L/egg)
Essential oil, carvacrol
& thymol (0.3 g/kg)
Induction oxidative
stress (free radicals)
E. acervulina
E. tenella
105
5 x 104
BWG**
FCE**
LS**
OPG**
LS
E. maxima
5 x 103
No effect
Immune stimulation
E. tenella
BWG
Immune stimulation
E. acervulina
6 x 104
9 x 104
104
BWG
OPG
Stimulation mucosal
immunity (antimicrobial
effect)
Stimulation mucosal
immunity (antimicrobial
effect)
E. tenella
5 x 104
BWG
Giannenas et al., 2003
No beneficial
effect on
vaccination
Oviedo-Rondn et al.,
2006
LS
Mandal et al., 1994
BWG
OPG
Hayat et al., 1996
Mushrooms
extracts
Mushrooms
extracts
Oregano
Oregano
Herb(s) extracts
Herb(s) extracts
Essential oil, thymol,

eugenol, curcumin &
piperin (0.1 g/kg)
Various Indian herb

extracts (6 g/kg)
Preparation of Persian
lilac (Melia azedarach)
0.3 g/kg & bitter melon
(Momordica charantia)
30 g/kg
Unknown
Unknown
Advent
(day 1)
Challenged
(day 19)
E. acervulina
E. maxima
E. tenella
E. necatrix
Mixed infect.
Eimeria spp.
2 x 102
102
5 x 103
?
5 x 104
E. tenella E. acervulina
E. maxima =
-
Oh et al., 1995
Allen et al., 1997b
Guo et al., 2004; 2005
Herb(s) extracts
Herb extract
Herb(s) extracts
Fifteen Asian herb(s)

extracts (6-30 g/l
drinkingwater)
Neem fruit (Azadirachta
indica) (1, 2 & 3 g/kg)
Herb extract
Complex of eight herbs

(2 g/ml & 10 g/kg)
Complex of four herbs
(Apacox) (0.5 & 1.0
g/kg)
Green tea (5 & 20 g/kg))
Betaine (sugar
beet solids)
Trymethylglycine (0.5,
0.75, 1, 1.5 & 5 g/kg)
Citric fruits
Extracts & organic acids

(2, 3, & 4 ml/10 l
drinkingwater)
Herb(s) extracts
Echinacea
purpurea
Turmeric
(Curcuma
longa)
Prebiotics
Probiotics
Dried ground root

preparation including
glycoproteins, chiroric
acid & alkamides
(1 to 5 g/kg first 2 wks)
Diferuleolymethane
(phenolic compound,
curcumin 10 g/kg)
Mannanoligosaccharides
1, 0.5 & 10 g/kg (BioMos)
Lactobacillus
(Primalac, 1 g/kg)
Unknown
E. tenella
105
Variable results
different herbs
Unknown
E. tenella
3 x 104
Unknown
E. tenella
105
Unknown
E. tenella
6 x 104
Unknown
E. maxima
104
Stabilization cell
membranes and immune
stimulation
(osmoprotectant)
?
Mixed infect.
E. acervulina
E. tenella
E. maxima
Mixed infect.
E. acervulina
E. maxima
E. tenella
E. brunetti
E. mivati
ImmucoxC1
(day 1)
Challenged
(day 28)
Various
(3 g/kg)
Mort
OPG
LS
BWG
BWG
FCE
OPG
BWG =
OPG
BWG
OPG =
LS =
Immune stimulation,
enhancement
macrophage activity
inactivation of reactive
nitrogen radicals (tocopherol like)
Immune stimulation and
blocking binding to
mucosal surface
Stimulation local cell
immunity
E. maxima
E. tenella
E. acervulina
E. tenella
E. maxima
E. acervulina
+/-
Youn & Noh, 2001
Tipu et al., 2002
Du & Hu, 2004
Christaki et al., 2004
Jang et al., 2007
Augustine et al., 1997

Mathews et al., 1997
Waldenstedt et al., 1999
Klasing et al., 2002
Tamasaukas et al., 1996
Tamasaukas et al., 1997
4.5 x 104
LS
OPG
(E. tenella 5 x
103)
Except for E.
tenella
115 (0.5 x
dose)
BWG
LS
Allen, 2003
Allen et al., 1998
?
Various
BWG
LS
OPG
No effect
Variable results
104
OPG
2.3 x 105 (1000

x vaccine dose
?
Mixed infect.
E. tenella =
=
+/-
Elmusharaf et al., 2006

Elmusharaf et al., 2007
McCann et al., 2006
Dalloul et al., 2003a
Dalloul et al., 2003b
Probiotics
Probiotics
Pediococcus (MitoGrow, 1 & 2 g/kg)
Pediococcus and
Sacchromyces
(MitoMax 0.1, 1 & 10
g/kg)
Immune stimulation
(enhanced humoral
response)
Immune stimulation
(enhanced humoral
response)
E. acervulina
5 x 103 or 104
BWG
OPG
E. tenella
5 x 103
E. acervulina
E. tenella
5 x 103
5 x 103
BWG =
OPG =
BWG =
OPG
Lee et al., 2007a
Lee et al., 2007b
*- sign indicates that the substrate has an inhibiting effect on coccidiosis, + sign indicates that the substrate promotes coccidiosis and = indicates no effect on
coccidiosis.
** BWG = body weight gain FCE = feed conversion efficiency (g weight gain/g food intake). LS = lesion scores. Mort = mortality. OPG = oocysts per gram
faeces.
CHAPTER 1
artemisinin extracts
citric fruits
different herb extracts
intestinal flora & mucosa
betaine
prebiotica
Extraintestinal environment
Intestinal lumen
oregano extracts
(carvacrol, thymol)
Echinacea purpurea
mushroom extracts
turmeric
probiotics
+
immunity
Figure 3. Artemisinin extracts, citric fruits, and different herb extracts seem to have an inhibitory effect
on the development of Eimeria. Prebiotics and oregano have an indirect inhibitory effect on the
development of Eimeria parasite. Betaine has an indirect effect on the development of the parasite
through its osmoprotective properties on the intestinal mucosa and stimulation of the intraepithelial
lymphocytes. Echinacea purpurea, mushrooms extracts, turmeric and probiotics have an indirect
inhibitory effect on the development of Eimeria through stimulation of the immune system.
5.4. Vaccines
Immunity to coccidiosis, which can be induced by passive or active immune responses, is
generally defined as the occurrence of resistance to a challenge infection with an Eimeria
spp. and can be determined by a reduction of the pathogenic effects of coccidiosis: less
macroscopically visible lesions and/or a decrease in oocyst production, and increased
performance of birds.
The first study showing that chickens infected with E. tenella were resistant to
homologous challenge was reported by Beach and Corl (1925) and formed the basis of
modern coccidiosis vaccinology. However, it would take another 27 years before the first
commercial live coccidiosis vaccine CocciVac was registered in the USA (Edgar & King,
1952).
71
During the last twenty years various reports describing coccidiosis vaccines and their use
in poultry have been published (Shirley, 1988; Williams, 1992b, 1996, 1998, 1999,
Chapman, 2000; Williams, 2002a, 2002b; Dalloul & Lillehoj, 2006; Shirley et al., 2007).
In Table 13 an overview showing the available coccidiosis vaccines and their usage is
given (Shirley et al., 2005; Williams, 2002a).
Vaccination can be alternated with the anticoccidial drugs in feed within rotation
programmes in combination with biosecurity.
5.4.1. Subunit vaccines
Subunit vaccines are composed of a purified antigenic determinant that is separated from
the virulent organism. Such vaccines can be obtained by different technologies and may
consist of native antigens or of recombinant proteins expressed from DNA of various
developmental stages (sporozoites, merozoites, gametes) of the Eimeria parasite.
Despite an increasing number on manuscripts exploring the feasibility of subunit vaccines
against coccidiosis, no commercial products, except CoxAbic, have been marketed to
date (Brother et al., 1988; Jenkins et al., 1989; Miller, et al., 1989; Jenkins et al., 1990;
Crane, et al., 1991; Bhogal et al., 1992; Jenkins, 1998; Vermeulen, 1998; Jenkins, 2001;
Vermeulen et al., 2001; Dalloul & Lillehoj, 2006). A major limiting factor has been the
fact that until now none of the antigens responsible for potent protective immune response
against Eimeria has been isolated. Systematic and detailed analysis of host-parasite
interactions at the molecular and cellular levels including studies of basic immunology
need to be completed before successful subunit vaccine products will be made available.
In this regard, the E. tenella genome project may help to further understand how protective
immune responses against Eimeria spp. are developed and help to identify antigens of vital
importance in coccidial immunology (Shirley et al., 2007).
5.4.1.1. Maternal immunisation, transmission blocking immunity
CoxAbic is a vaccine against coccidiosis, which induces maternally derived antibodies to
protect broiler chickens (Michael, 2003, 2007; Finger & Michael, 2005; Ziomko et al.,
2005). It is an inactivated, subunit vaccine (oil emulsion) containing affinity purified
proteins (230 kDa, 82 kDa and 56 kDa) from the oocyst wall-forming bodies of E. maxima
gametocytes. Cross protection resulting in lower oocyst shedding of E. maxima, E.
acervulina and E. tenella has been described after the administration of low dose challenge
inocula (Wallach et al., 1995; Wallach, 1997). This is remarkable because after natural
Eimeria infections cross immunity has not been described (Rose & Long, 1962). However,
Crane and co-workers (1991) found cross protection against four Eimeria spp. (E.
acervulina, E. maxima, E. necatrix and E. tenella) after administration of a single
recombinant antigen.
Offspring originating from vaccinated parent birds are fed an anticoccidial drug free diet
in order to ensure natural exposure to the parasites and subsequent development active
immunity after maternal antibodies have disappeared.
72
CHAPTER 1
5.4.2. Live vaccines

Live Eimeria vaccines consist of sporulated oocysts and are either non-attenuated (wild
type strains of Eimeria spp.) or attenuated. Further differentiation is based on the Eimeria
spp. included, their anticoccidial drug sensitivity profile and application. Protective
immunity can be achieved if chickens are infected with either a single high dose or
multiple low doses (trickle infections) of Eimeria (vaccine) parasites (Joyner & Norton,
1973, 1976; Long et al., 1986). It is crucial that anticoccidial drugs are withdrawn from
feed in case chicken flocks are vaccinated with drug sensitive live vaccine parasites in
order to avoid vaccination failures.
A beneficial side effect of the use of drug sensitive live coccidiosis vaccines is their
association with increased sensitivity to anticoccidial drugs (Jeffers, 1976; Mathis &
McDougald, 1989; Chapman, 1994, 1996; Newman & Danforth, 2000; Mathis &
Broussard, 2006; Peek & Landman, 2006). See subject 3.3.2. Management of resistance.
5.4.2.1. Non-attenuated (or wild-type strains of Eimeria spp.) vaccines
Non-attenuated vaccines consist of Eimeria parasites which have not been modified in any
way to change their pathogenicity and originate from laboratory or field strains. Examples
of such vaccines are: CocciVac, Immucox, VAC M, Nobilis COX ATM, Inovocox
and ADVENT. Many of these vaccines are used worldwide covering the extensive
coccidiosis market. Between the year 2001 and 2004 about 2500 million doses CocciVac
and Immucox were sold (Shirley et al., 2005).
CocciVac is available as two different products CocciVac B, containing E. acervulina,
E. maxima, E. mivati and E. tenella, which is applied to broilers; CocciVac D, containing
E. acervulina, E. brunetti, E. hagani, E. maxima, E. mivati, E. necatrix, E. praecox and E.
tenella, which is applied to breeders and layers.
Likewise for Immucox two different products exist. Immucox C1 containing E.
acervulina, E. maxima, E. necatrix, and E. tenella, is used in broilers; Immucox C2
containing E. acervulina, E. brunetti, E. maxima, E. necatrix, and E. tenella, is used in
breeders and layers.
VAC M was used in the USA for a defined period of time in areas where coccidiosis
outbreaks with E. maxima strains resistant to ionophores occurred frequently. It consisted
of a non-attenuated E. maxima strain. According to Williams (2002b) the E. maxima strain
included in VAC M was ionophore resistant and not sensitive as cited in the literature.
Nobilis COX ATM is composed of three Eimeria spp. E. acervulina, E. maxima (2
antigenic different strains) and E. tenella which are all ionophore tolerant. This enables the
use of this vaccine in flocks exposed to anticoccidial drugs in feed. The advantage of this
vaccine is that it allows the use of ionophores during the first 3-4 weeks when immunity is
not complete and coccidiosis field strains could interfere (Schetters et al., 1999).
ADVENT is a non-attenuated coccidiosis vaccine harboring E. acervulina, E. maxima
and E. tenella. It is used to immunize broilers against Eimeria parasites. ADVENT
enables in vitro assessment of parasite viability using ViacystSM (non-viable sporocysts
73
stain with ethidium bromide), an innovative aspect in coccidiosis vaccinology, allowing a

better control of the viability of the vaccine dose administrated (Dibner et al., 2003).
Inovocox is a live coccidiosis vaccine consisting of four non-attenuated strains and three
species (E. acervulina, E. maxima (2 antigenic different strains) and E. tenella). In
contrast to all other vaccines it is applied to eggs using Inovoject technology (Weber &
Evans, 2003, Weber et al., 2004).
During the usage of non-attenuated coccidiosis vaccines it is crucial that all birds are given
the required dose and the occurrence of clinical coccidiosis is avoided. Therefore,
application protocols should be followed strictly (Chapman et al., 2002). In order to
diminish such risks attenuated vaccines have been developed.
5.4.2.2. Attenuated vaccines
Attenuated vaccines consist of Eimeria spp. strains, which have been manipulated in the
laboratory in order to decrease their virulence. Reduced virulence has been performed by
serial passages of the parasite in chicken embryos; e.g. E. tenella in Livacox vaccines.
Selection for precocity is the second described method for attenuation; e.g. remaining
Eimeria spp. in Livacox and all lines in Paracox vaccines (Shirley & Bedrnik, 1997;
Shirley et al., 1995; McDonald & Shirley, 1984; Shirley & Miljard, 1986). Precocity is
characterized by a shortened endogenous life cycle due to the fact that the number of
generations of schizogony is decreased because last generations of schizogony disappear
by selecting early oocysts. A consequence is that the number of oocysts produced during
infection is reduced; however, the immunizing potential is maintained (Jeffers, 1975,
1986; McDonald et al., 1986; Shirley & Miljard, 1986).
According to the poultry type targeted, Paracox and Livacox vaccine types are
composed of different Eimeria spp. I.e. Paracox-8 for breeder birds and layers has 8
Eimeria spp. (E. acervulina, E. brunetti, E. maxima (2 antigenic different strains), E. mitis,
E. necatrix, E. praecox and E. tenella), while Paracox-5, which was developed for
broilers only has five Eimeria spp. (E. acervulina, E. maxima (2 antigenic different
strains), E. mitis and E. tenella). Livacox Q is composed of oocysts of E. acervulina, E.
maxima, E. necatrix and E. tenella and is intended for the administration to breeder birds
and layers. In contrast, Livacox T, which only has E. acervulina, E. maxima and E.
tenella, is applied to broilers.
Eimeriavax 4m is composed of attenuated lines of E. acervulina, E. maxima, E. necatrix
and E. tenella and has been developed in Australia. It is used in breeders, layers and
broilers by eye-drop application.
Eimerivac plus, which is composed of attenuated lines of E. acervulina, E. maxima and
E. tenella has been developed in China, but registration of the vaccine has not been
obtained yet. This vaccine is intended for use in breeders, layers and broilers through oral
administration.
Another Chinese coccidiosis vaccine is Supercox, which is composed of wild strains of
E. acervulina and E. maxima, and a precocious line of E. tenella (Suo et al., 2006). This
vaccine is applied orally to broilers.
74
CHAPTER 1
A South American (Argentina) vaccine named Inmuner Gel-Coc consists of four

attenuated Eimeria spp.: E. acervulina, E. brunetti, E. maxima and E. tenella. This product
is given orally to breeders, layers and broilers.
Hipracox Broilers is another coccidiosis vaccine that recently was introduced in Europe
and is intended for use in broilers. It is composed of attenuated lines of E. acervulina, E.
maxima, E. mitis, E. praecox and E. tenella and is applied in the drinking water.
In the Netherlands up till now only both Paracox vaccines (Paracox-5 and Paracox-8)
have been registered and are currently used in the field.
A major drawback of live coccidiosis vaccines is their loss of infectivity with time
affecting their expiry (Jeston et al., 2002). Other concerns are their high production costs
and management shortcomings during their application such as dosage errors that may
result in insufficient immune response or clinical coccidiosis in case non-attenuated
vaccines are used, erroneously adding anticoccidial drugs to the feed frustrating effective
vaccination of drug sensitive strains, vaccination of sick birds, etc. Further, reversal of
virulence of live vaccines may be another point of concern. Subunit vaccines offer
possibilities to circumvent mentioned drawbacks and could provide a sustainable solution
for the coccidiosis problem in commercial poultry if with the help of new technologies
their immunogenicity can be increased.
75
Table 13. Overview of anticoccidial vaccines that are being used or being registered for use in chickens (Shirley et al., 2005; Williams,
2002a; manufacturers technical information bulletins and websites)
Vaccine (Manufacturer)
ADVENT
(Novus International)
CocciVac-B
(Shering Plough Animal Health)
CocciVac-D
(Shering Plough Animal Health)
CoxAbic
(Abic Biological Laboratories)
Eimerivac Plus
(Guangdong academy of
Agricultural Sciences)
Eimeriavax 4m
(Bioproperties Pty)
Hipracox Broilers
(Laboratorios Hipra, SA)
ImmucoxC1
(Vetech Laboratories)
ImmucoxC2
(Vetech Laboratories)
Inmuner Gel-Coc
(Vacunas Inmuner)
Inovocox
(Embrex Inc. and Pfizer)
Livacox Q
(Biopharm)
Eimeria speciesa
Eac, Emax, Eten
Attenuation
Non-attenuated
Bird type
Broilers
Eac, Emax, Emiv,

Eten
Eac, Ebr, Eha,
Emax, Emiv,
Enec, Epra, Eten
Non-attenuated
Broilers
Non-attenuated
Breeders/layers
Killed antigen of Emax

gametocytes
Attenuated
Eac, Emax, Eten

Eac, Emax, Enec,
Eten
Eac, Emax, Emit,
Epra, Eten
Eac, Emax, Enec,
Eten
Eac, Ebr, Emax,
Enec, Eten
Eac, Ebr, Emax,
Eten
Eac, Emax x 2,
Eten
Eac, Emax, Enec,
Eten
Livacox T
(Biopharm)
Eac, Emax, Eten
Nobilis COX-ATM
(Intervet international)
Eac, Emax x 2,
Eten
Administration route
Hatchery spray, water or
feed spray
Ocular, hatchery spray,
water or feed spray
Ocular, hatchery spray,
water or feed spray
First registration
2002 (USA)
Breeders (to protect

hatchlings)
Breeders/layers/broilers
Killed antigen, one species,

intramuscular
Oral
2002 (Israel)
Attenuated (precocious)
Eye-drop application
2003 (Australia)
Attenuated
Broilers
Drinking water
2007 (Spain)
Non-attenuated
Broilers
Water or gel
1985 (Canada)
Non-attenuated
Breeders/layers
Water or gel
1985 (Canada)
Attenuated
Oral
2005 (Argentina)
Non-attenuated
Broilers
2006 (USA)
Attenuated (precocious,
except Eten (embryoadapted)
Attenuated (precocious,
except Eten (embryoadapted)
Non-attenuated (all
ionophore tolerant)
Breeders/layers
In ovo injection with the

inovoject system
feed spray
1952 (USA)
1951 (USA)
expected (China)
1992 (Czech Republic)
Broilers

feed spray
1992 (Czech Republic)
Broilers
Water or feed spray
2001 (Netherlands)
Paracox-8
(Schering Plough Animal Health)
Paracox-5
(Schering Plough Animal Health)
Supercox
(Qilu Animal Pharmaceutical
Company)
VAC M
(Elanco)
Eac, Ebr, Emax x

2, Emit, Enec,
Epra, Eten
Eac, Emax x 2,
Emit, Eten
Eac, Emax, Eten
Emax
Breeders/layers
Water or feed spray
1989 (UK)
Broilers
1989 (UK)
Attenuated (precocious:
Eten) non-attenuated (Eac
and Emax)
Non-attenuated
(ionophore resistantb)
Broilers

feed spray
Oral
Broilers
Beak-o-Vac machine
1989 (USA)
2005 (China)
a
Eac = E. acervulina, Ebr = E. brunetti, Eha = E. hagani, Emax = E. maxima, Emit = E. mitis, Emiv = E. mivati, Enec = E. necatrix, Epra = E. praecox, Eten = E.
tenella and Emax x2 = two antigenically different lines of E. maxima.
b
According to personal communication of Dr. T.K. Jeffers (Williams, 2002a).
6. Conclusions and perspectives

Coccidiosis is a major parasitic disease of poultry with great economic impact, which
mainly affects the intestinal tract of birds. The clinical and economic importance of
coccidiosis is likely to remain unchanged during the coming decades as long as
commercial poultry is reared in large numbers at high densities, which seems necessary to
make the poultry industry profitable.
Many studies concerning the effects of diet composition (ingredient and nutrient
composition), feed structure and use of alternative feed additives (phyto- and
aromatherapy) on Eimeria parasites have been published to date. The results of these
studies are frequently ambivalent, while the number of papers describing clear direct
anticoccidial activity against various Eimeria spp. is relatively scarce. More research is
required on the mode of action and effects of nutrients, dietary constituents and additives
in alleviating the negative effects of a coccidiosis infection.
Comparing the mode of action of anticoccidial drugs with that of feed components and
feed additives no similar mechanisms have been described in literature, except that of
vitamin B (thiamine).
From this literature review it can be concluded that future coccidiosis control is unlikely to
be achieved solely through feed composition and management. However, some dietary
compounds seem capable to reduce the effects of an Eimeria infection either by a direct
effect on the parasite or indirectly by stimulating immune responses and recovery of the
birds. In this regard, natural feed additives also seem promising.
Considering the immune stimulating and intestinal health promoting effect of some feed
constituents and a few natural feed additives, these products could also play a significant
role in ameliorating the efficiency of anticoccidial vaccines, which at present seems the
most solid anticoccidiosis strategy especially in case anticoccidial drugs are banned as
feed additives. This is an area of research that also deserves future attention.
To date coccidiosis control has relied mainly on chemoprophylaxis, however, the
occurrence of resistance and the increasing regulations, and possible upcoming bans on the
use of anticoccidial drugs as feed additives, have been the principal motivators in the quest
for alternative control strategies, amongst which the application of live vaccines has
proved most successful so far. Although there are a number of drawbacks associated with
the production and use of live coccidiosis vaccines, their efficacy and the fact that they
have been associated with increased sensitivity of Eimeria spp. isolates to anticoccidial
drugs, have further stimulated their use. If the immunogenicity of subunit vaccines can be
improved they could represent the next generation highly efficient and low cost
anticoccidial strategy.
78
CHAPTER 1
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104
Chapter 2
Anticoccidial drug resistance in the field
Resistance to anticoccidial drugs of Dutch avian

Eimeria spp. field isolates originating from 1996, 1999
and 2001
Animal Health Service (GD), Poultry Health Centre, P.O. Box 9, 7400 AA, Deventer, the
Netherlands
Avian Pathology (August 2003), 32(4), 391-/401
Summary
Fifteen Eimeria spp. field isolates, sampled on Dutch broiler farms were subjected to an
Anticoccidial Sensitivity Test (AST) in a battery cage study. Four isolates dated from
1996, another four from 1999 and the last seven isolates from 2001. The selected
anticoccidial drugs were monensin, narasin, salinomycin, lasalocid, nicarbazin, diclazuril,
halofuginone, maduramicin and meticlorpindol/methylbenzoquate.
Maduramicin and halofuginone were not included in the AST of 1999 and 2001, while
meticlorpindol/methylbenzoquate was not tested in 1996 and 1999. E. acervulina present
in each of the four 1996 field isolates showed resistance for almost all products tested
except maduramicin (1/4) and salinomycin (1/4) which appeared to be reduced sensitive.
In 1999 the same species presented a similar resistance pattern for most products although
reduced sensitivity occurred for salinomycin (1/4), and sensitivity was found for diclazuril
(2/4), monensin (1/4) and narasin (1/4). In the year 2001 increased sensitivity to various
products was found. Higher sensitivity was found for meticlorpindol/methylbenzoquate
(7/7) and salinomycin and narasin (both 4/7), followed by nicarbazin (3/7) and monensin
(2/7). Reduced sensitivity was found for monensin (3/7), lasalocid (2/7), salinomycin and
narasin (1/7). E. maxima was only found in one field isolate per year. The E. maxima from
1996 was resistant to all products except narasin (sensitive) and halofuginone (reduced
sensitive). In 1999 this species was reduced sensitive to narasin and lasalocid, showing
resistance for the other products. The strain originating from the 2001 isolate was reduced
sensitive to most products except monensin and narasin (resistant). Full sensitivity was
found for meticlorpindol/methylbenzoquate. E. tenella was present in one isolate of 1996,
two of 1999 and four of 2001. The AST of 1996 showed reduced sensitivity for
nicarbazin, and sensitive to narasin, maduramicin and halofuginone. All other products
showed resistance. In 1999 both strains show resistance to all products tested. For the year
2001 full sensitivity was found to meticlorpindol/methylbenzoquate. Sensitivity was also
found for salinomycin (2/4), nicarbazin (2/4), diclazuril (2/4) and lasalocid (2/4),
monensin (1/4) and narasin (1/4). Reduced sensitivity was found for nicarbazin (1/4),
lasalocid (1/4) and narasin (1/4). The different resistance patterns of Dutch coccidiosis
isolates and resistance of coccidia in general is discussed.
CHAPTER 2
Introduction
A protozoan parasite belonging to the subclass Coccidia and family Eimeriidae causes
coccidiosis in poultry. It is still a major health problem with great economic impact on
poultry production. The economic costs are estimated at $ 1.5 billion world-wide (Weber,
1997).
Penetration of the intestinal villous and crypt epithelial cells by asexual (schizogony
or merogony) and/or sexual (gametogony) stadia may result in clinical disease. The major
pathologic manifestations of clinical coccidiosis are intestinal haemorrhage,
malabsorption, diarrhoea and reduction of body weight gain (Lillehoj & Lillehoj, 2000).
Control of coccidiosis has focused on management, vaccines, natural feed additives
and prophylaxis with anticoccidial drugs. Management practices may contribute to the
control of parasitic disease and prove especially useful in cases that no other measures are
effective or available. Mentioned practices should focus on good sanitation, cleaning out
contaminated litter, the use of adequate ventilation and watering systems that safeguard
good litter condition avoiding sporulation, not rising birds of different ages together, etc.
In view of the immunogenetic properties of the various Eimeria spp., vaccination
represents another possible strategy to prevent coccidiosis. Live vaccines consisting of
either attenuated or non-attenuated coccidial strains are becoming increasingly popular in
the poultry industry. Attenuated vaccines which contain parasites of reduced virulence,
can be obtained by passages through embryonated eggs or selection of precocious strains
(Shirley, 1993). Although no recombinant vaccines are commercially available yet,
considerable developmental work has been performed over the past ten years. The major
impeding factors are the identification of protective antigens and the lack of crossimmunity between Eimeria spp. (Jenkins, 1998).
Natural feed additives have been investigated in search of alternative methods to
control coccidiosis in view of increasing resistance development to anticoccidial drugs.
Moreover, consumers are becoming more aware of the detrimental effects of residues in
poultry products. A number of natural feed additives have shown anticoccidial activity.
Amongst them fish oils, flaxseed oil, and whole flaxseed containing high concentrations of
-3 fatty acids (docosahexaenoic acid, eicosapentaenoic acid and linolenic acid) (Allen et
al., 1996), artemisinin isolated from the Chinese herb Artimisia annua (Allen et al., 1997),
extracts from Sophora flavescens Aiton, Pulstilla koreana, Sinomenium acutum, Ulmus
macrocarpa and Quisqualis indica (Young & Noh, 2001) and feed supplements such as tocopherol, the spice tumeric and curcumin (Allen et al., 1996). However, large-scale
application of these products has not been carried out yet.
Prophylactic medication with anticoccidial drugs in the feed has proven to be
successful in the control of coccidiosis over the past half century and until now has been
the most widely practised strategy to prevent outbreaks of clinical coccidiosis. To
minimise the occurrence of drug resistance, rotation of various anticoccidial drugs,
combining chemical and ionophore treatments or shuttle programs have been used. In spite
of the use of such programmes resistance against in-feed medication against coccidiosis is
becoming increasingly important. Development of resistance has been described for most
107
ANTICOCCIDIAL DRUG RESISTANCE IN THE FIELD
anticoccidial drugs, chemical and ionophore compounds alike (Chapman, 1986, 1997). To
optimise the use of prophylactic medication in the field, knowledge of the drug-sensitivity
profile of the parasites concerned is vital. The only accurate method available to obtain
such information is the in vivo anticoccidial sensitivity test (AST) (Chapman, 1998). In the
present manuscript we present the results of the AST of Dutch avian Eimeria spp. field
isolates dating from 1996, 1999 and 2001.
Material and methods

Experimental design
Three AST were performed on Dutch Eimeria spp. field isolates. The first was carried out
in 1996 on four isolates against eight anticoccidial products. The second test took place in
1999 analysing another four field isolates against six anticoccidial products, and the last in
2001 comprising seven isolates tested against seven anticoccidial drugs. As reference
strain an E. acervulina Weybridge isolate was tested in 1999 against five products (Table
1). The drug concentrations per kg feed tested were: diclazuril (Clinacox) 1 mg,
halofuginone (Stenorol) 3 mg, lasalocid (Avatec) 90 mg, maduramicin (Cygro) 5 mg,
meticlorpindol/methylbenzoquate (Lerbek) 100 mg, monensin (Elancoban) 100 mg,
narasin (Monteban) 70 mg, nicarbazin (Nicarb) 125 mg and salinomycin (Sacox) 60
mg (Fowler, 1995).
At the age of eight days (D 0) chicks fed with anticoccidial drugs were inoculated in the
crop with a defined number of sporulated oocysts (see inocula). An Infected Untreated
Control (IUC) group (positive control) was given the same inoculum, while the Uninfected
Untreated Control (UUC) group was used as negative control. Access to the medicated
feeds was enabled two days prior to inoculation of oocysts (D-2) until termination of the
experiment. At six days post-inoculation a postmortem examination on five birds per
experimental group was performed and coccidial lesions were determined.
In 1996 and 1999 five birds were used per experimental group, while in 2001 nine chicks
per group were used. The E. acervulina reference strain was tested in fivefold using three
birds per group.
Experimental animals, housing and (medicated) feed
In each trial one-day-old male commercial broilers chicks were reared coccidiosis free on
a large stain-less-steel wire cage (height x width x depth = 30 cm x 200 cm x 100 cm) in a
separated room until the age of six days, during which period they received a feed without
anticoccidial products. At the age of six days, two days prior to infection (D-2) the birds
were, ad random, placed on battery stain-less-steel wire cages (height x width x depth =
30 cm x 40 cm x 50 cm) in the experimental room. The number of birds per experimental
group, i.e. per anticoccidial drug and corresponding controls, has been mentioned in the
experimental design.
108
CHAPTER 2
After transfer, the chicks were supplied with in-feed medication until termination of the
experiment. Twenty five kg of a complete formulated broiler starter mash (Apparent
Metabolizable Energy (AME) 12.0 MJ/kg)), free of anticoccidial products, was mixed
with commercial anticoccidial drug premixes to produce the desired concentrations in the
medicated rations. The required quantity of premix was first mixed with approximately 2.5
kg of mash to safeguard adequate mixing. Thereafter, it was further mixed in a blender
(Naturamix, machine nr. m19091, Naturamix B.V., Haarlem, the Netherlands) with the
remainder of the feed. Feed and water were offered ad libitum and the chicks were
subjected to a lighting scheme of 22 hours of light.
Table 1. Overview of the anticoccidial drugs analysed per Anticoccidial Sensitivity Test
(AST)
1996
1999
2001
Diclazuril: Clinacox (DIC)

Halofuginone: Stenorol (HAL)
Lasalocid: Avatec (LAS)
Maduramicin: Cygro (MAD)
DIC
DIC
LAS
LAS
MON
NAR
NIC
SAC
Meticlorpindol/methylbenzoquate:
Lerbek (METI/METH)
MON
NAR
NIC
SAC
Monensin: Elancoban (MON)

Narasin: Monteban (NAR)
Nicarbazin: Nicarb (NIC)
Salinomycin: Sacox (SAC)
1999
E. acervulina
DIC
MAD
MON
NIC
SAC
Assessment of the mixing procedure of anticoccidial drugs in feed

In order to asses the mixing procedure of anticoccidial drugs in feed, medicated feed
samples were analysed prior to the start of two of the experiments (1999 and 2001). We
also report the results of chemical analyses on anticoccidial drug concentrations in feed
done in 1988, 1989 and 1990.
For the chemical analysis of feed samples the following general procedure was performed:
feed samples were grinded into particles of 1 mm3 and extracted in mixtures of organic
solvents (e.g. methanol or acetonitrile depending on the product subjected to the analysis)
and water. The extraction procedure lasted for one hour, during which time the mixture
contained in an erlenmeyer of 300 ml was placed on a shaking device (Edmund Buhler
SM25, Salm & Kipp B.V. Breukelen, the Netherlands). After filtration through paper
(Schleicher & Schuell Faltenfilter, ref. no. 10311645, diameter 150 mm, Omnilabo, Breda,
the Netherlands) the extracts were injected onto HPLC system, (autosampler Waters 717
plus, Etten-Leur, the Netherlands) equipped with a Reversed Phase C18 column (5 m)
and methanol/water or acetonitrile/water mixture were used as mobile phase. The elutes
109
were analysed with a UV spectrophotometric detector (Photodiode Array Detector, Waters

966, Etten-Leur, the Netherlands) and computersystem (Waters millenium 32, Etten-Leur,
the Netherlands). For some anticoccidial drugs (narasin, monensin and salinomycin) the
elute was mixed with a solution of 4-methoxybenzaldehyde in a heated mixing coil before
examination with the detector.
Data on the proportions and concentrations of the extraction mixtures, which vary
according to the product to be analysed, can be found in the corresponding references of
lasalocid, monensin, salinomycin and narasin (Dusi & Gamba, 1999); clopidol (Dusi et al.,
2000); halofuginone (Tiller et al., 1988); diclazuril (De Kock et al., 1992); maduramicin
(Gliddon et al., 1988) and nicarbazin (Hurlbut et al., 1985).
Origin of isolates
The Eimeria spp. field isolates were obtained from samples of faeces/litter originating
from Dutch broiler farms participating in the coccidiosis broiler monitoring program in the
Netherlands (see below). The isolates from 1996 were selected from farms where clinical
coccidiosis problems emerged, and those of 1999 and 2001 originated from farms with
subclinical disease.
The E. acervulina reference laboratory strain (Weybridge W119), was obtained from the
Central Veterinary Laboratory (CVL), Weybridge, UK. This strain was kept at the Animal
Health Service since 1990 and rejuvenated frequently.
Parasitology and preparation of inocula
Oocysts were purified from the faeces/litter samples by means of a saturated sodium salt
flotation technique, and sporulated with modification of the method of Ryley et al. (1976).
Briefly, approximately 450 g faeces were suspended in a saturated salt (NaCl) solution (3
l). The suspension was homogenised during 15 to 30 min in an erlenmeyer with a
magnetic stirrer. Subsequently, the suspension passaged through a 1000-m sieve to
separate feathers and other coarse debris. The filtered fluid was transferred to 750 ml
bottles and centrifuged (5 min at 250 x g) three times. After each centrifugation, the oocyst
containing scum (approximately 20 ml) was removed with a pipette connected to a
vacuum pump. The three crops of oocysts were diluted in water (700 ml) and centrifuged
(15 min at 2750 x g). The supernatant was discarded and the sediment was resuspended in
2.5% (w/v) potassium dichromate (1 l). The suspension was aerated at 29 oC during three
days for sporulation. Oocyst counting of the Eimeria inocula was performed with the aid
of a Fuchs-Rosenthal haemocytometer counting chamber. The oocyst cultures were
refrigerated at 2-8 C until application.
Each Eimeria spp. field isolate was passaged through male Specific Pathogen Free (SPF)
broilers for multiplication and identification. The Eimeria species were identified by the
location and appearance of the gross lesions in the intestine and by microscopical
examination and measurement of the oocysts (Long et al., 1976; McDougald & Reid,
1997). From day 4 until day 7 post-infection, faeces samples were collected and subjected
110
CHAPTER 2
to oocysts purification as described above. The inoculation dose was adjusted to obtain a
coccidial lesion score of 2 to 3 in the IUC group on the scale of Johnson & Reid (1970)
and to avoid mortality. Adjustment was done based on data from literature (Conway et al.,
1993), previously performed AST (non-published results) and, on the lesion scores and
mortality recorded during the multiplication in SPF broilers. The inocula were prepared
and the potassium dichromate was removed by means of centrifugation. The birds were
inoculated individually in the crop with a calibrated syringe without needle in a volume of
approximately 1 ml tap water.
Further procedures, postmortem examination, coccidial lesion scores, Oocysts Per Gram
faeces (OPG) and evaluation of resistance
The birds were observed daily and deceased birds were subjected to postmortem
examination. At six days post-inoculation (D+6) necropsy was performed on five birds per
experimental group.
The individual coccidial lesion scores were determined according to the method of
Johnson & Reid (1970). Briefly, E. acervulina lesion score 1 features ladder-like white
streaks in de duodenal loop (5/cm2); in lesion score 2 the lesion density is higher but not
coalescent; in lesion score 3 the lesions are more numerous and coalesce, while thickening
of the intestinal wall is visible, and finally in lesion score 4 the mucosal wall is greyish
with lesions completely coalesced. Lesion score 1 of E. maxima is characterised by few
small red petechiae in the mid-intestine, in score 2 more numerous petechiae are found and
the intestinal content may be orange, in score 3 the intestinal wall might be ballooned and
thickened with pinpoint blood clots and mucous filling the intestinal contents. Finally in
lesion score 4 the intestinal wall is thickened and ballooned over most of its length,
containing numerous blood clots and digested red blood cells. Lesion score 1 of E. tenella
shows few scattered petechiae on the caecal wall, in score 2 noticeable blood can be found
in the caecal contents, while the caecal wall is thickened. In lesion score 3 large amounts
of blood or caecal cores are present, caecal walls are greatly thickened, and in lesion score
4 caecal walls are enormously distended with blood or large caseous cores.
Thereafter, the mean lesion score per Eimeria spp. and experimental group was calculated.
In fresh-collected faeces material (of 1996 and 1999 from D+4 till D+6 and of 2001 from
D+4 till D+8) the OPG-countings were performed by means of a modification of the
McMaster counting chamber technique of Hodgson (1970). Briefly, a 10% (w/v) faeces
suspension in a salt solution (151 g/l) was made. After shaking vigorously, 1 ml of the
suspension was pipetted into 9 ml of saturated sodium chloride (311 g/l). After mixing
again, a proportion of the latter suspension was run into the McMaster counting chamber
and the oocysts were count using 100 and 200 x magnification.
The anticoccidial-sensitivity profile of each Eimeria spp. present in the field isolates and
of the control isolate was based on the reduction of the mean lesion score of the medicated
group compared to the IUC group using the formula: 100% - (mean lesion score of treated
group/mean lesion score of the IUC group x 100%). A reduction percentage in the
medicated birds compared with the IUC birds of 0-30% indicates coccidial resistance; 31111
49% reduction indicates reduced sensitivity or partial resistance and 50% or more
indicates full sensitivity to the tested anticoccidial drug (McDougald et al., 1986).
Statistical analysis
Statistical analyses were performed on the individual lesion scores of E. acervulina, E.
maxima and E. tenella by means of the non-parametric Kruskal-Wallis test, testing
differences in lesion score Rank sums between groups treated with different anticoccidial
drugs and the IUC group (SAS, 1989). Differences were considered significant when P
0.05. Statistical analysis of the E. acervulina reference strain (1999) with respect to
differences in lesion scores between treatments was performed by the Mantel-Haensel
Chi-Square test which was corrected for differences in lesion scores between repetitions of
the experiment. Differences were considered significant when P 0.05.
Coccidiosis broiler monitoring program in the Netherlands
In 1996 a coccidiosis broiler-monitoring program was started in the Netherlands. Broiler
chickens from participating farms were sent to the Animal Health Service twice (between
22-25 days of age and between 30-32 days of age) during the fattening period. Each time
five birds were sent in for postmortem analysis. The number of participating farms was
238 in 1996, 329 in 1997, 244 in 1998, 172 in 1999, 112 in 2000 and 99 in 2001. The
number of broiler flocks analysed from 1996 until 2001 was 362, 510, 387, 267, 165 and
137 respectively. The participating farms belonged to 12 different integrators in 1996, 10
in 1997, five in 1998, four in 1999 and two in 2000 and 2001. The total Dutch broiler
population varied from 44,142,000 in 1996 to 50,937,000 in 2000 (LEI & CBS, 2001).
Results
Inocula
Half of the field isolates consisted of mixtures of two or three Eimeria species. All isolates
contained E. acervulina. E. maxima was found in one isolate per year. This species
occurred in combination with E. tenella in 1999 and 2001. Five other isolates also
contained E. tenella (one in 1996, another in 1999 and three in 2001).
The Eimeria isolates tested and the species involved as well as the inoculation doses are
presented in Table 2.
112
CHAPTER 2
Table 2. Eimeria spp. present in the field isolates and inoculation dose (number of
sporulated oocysts) per chick
Eimeria spp. field isolates
A (1996)
B (1996)
C (1996)
D (1996)
E (1999)
F (1999)
G (1999)
H (1999)
I (2001)
J (2001)
K (2001)
L (2001)
M (2001)
N (2001)
O (2001)
Reference isolate Weybridge (W119)
a
Eimeria spp. involveda

E. ac
E. ac/E. max
E. ac
E. ac/E. ten
E. ac
E. ac/E. ten
E. ac
E. ac/E. max/E. ten
E. ac/E. ten
E. ac
E. ac/E. ten
E. ac
E. ac/E. ten
E. ac
E. ac/E. max/E. ten
E. ac
Inoculation dose (x 103)

75
150
100
50
41
20
25
50
20
50
35
50
50
50
35
50
E.ac = E. acervulina; E. max = E. maxima; E. ten = E. tenella.
Assessment of the mixing procedure of anticoccidial drugs in feed

The results of the chemical analyses show anticoccidial drug concentrations close to the
desired dose for each compound. The average concentration (mg/kg standard deviation)
found were 103.1 3.7 for clopidol (n = 8), 0.94 0.05 for diclazuril (n = 5), 2.53 0.78
for halofuginone (n = 6), 101.8 9.0 for lasalocid (n = 7), 106.0 10.3 for monensin (n =
8), 70.5 5.7 for narasin (n = 8), 127.2 8.9 for nicarbazin (n = 9) and 70.0 4.0 for
salinomycin (n = 9 ).
AST of 1996
E. acervulina from field isolates collected in 1996 showed resistance against all
anticoccidial drugs tested except salinomycin and maduramicin which presented reduced
sensitivity against two different isolates. The single E. maxima isolate was resistant for all
products except narasin. Regarding E. tenella it was sensitive to narasin, maduramicin and
halofuginone and reduced sensitive for nicarbazin. This strain was resistant for the
remaining products.
Three birds died during the AST in 1996. The birds were infected with isolate A receiving
salinomycin, isolate B receiving diclazuril or isolate D receiving monensin. The latter bird
succumbed due to the E. tenella infection. In the other cases the mortality was regarded as
non-specific (Table 3).
113
Table 3. Individual lesion scores (ILS), mortality (Mort) and oocysts per pram faeces
(OPG) of the anticoccidial sensitivity test (AST) of 1996
Isolate nr/anticoccidial drug
Isolate A
DIC
HAL
LAS
MAD
MON
NAR
NIC
SAC
IUC
Isolate B
DIC
HAL
LAS
MAD
MON
NAR
NIC
SAC
IUC
Isolate C
DIC
HAL
LAS
MAD
MON
NAR
NIC
SAC
IUC
Isolate D
DIC
HAL
LAS
MAD
MON
NAR
NIC
SAC
IUC
UUC
a,b
E. acervulina
ILS
E. maxima
ILS
E. tenella
ILS
4 3 3 4 4a
4 4 3 4 4a
4 4 4 4 4a
3 4 4 4 3a
3 4 4 3 4a
3 2 3 3 3a
3 3 4 4 4a
1 2 2 2 xb
3 3 3 4 3a
3 3 3 1 xa
3 4 3 3 3a
3 4 2 3 3a
1 1 2 2 1b
2 2 3 3 4a
2 3 3 3 3a
3 3 2 3 1a
2 2 2 3 2a
3 2 2 3 2a
2 1 0 1 xa
1 1 1 0 1a
2 1 1 1 1a
1 1 1 1 1a
1 1 1 1 1a
1 0 0 0 1b
1 1 1 1 1a
1 1 1 1 1a
2 1 1 1 1a
4 4 4 3 3a
3 2 3 4 4a
3 3 3 4 4a
3 3 4 3 3a
4 3 4 4 4a
3 2 1 3 3a
4 4 3 4 4a
2 3 2 3 3a
4 3 3 4 3a
3 2 3 2 3a
2 2 1 1 1a
4 4 4 4 3a
3 3 3 3 3a
3 3 3 4 xa
4 3 2 2 3a
3 3 4 4 4a
1 1 1 1 3a
1 2 2 1 3a
00000
1 1 2 2 1a
0 0 0 0 0b
3 1 1 2 3a
0 1 0 0 0b
0 1 1 3 4a
1 1 0 1 1b
1 1 2 1 1a
1 2 1 3 1a
1 1 2 3 2a
00000
Mort
(n = 5)
OPG
(x 103)
0/5
0/5
0/5
0/5
0/5
0/5
0/5
1/5
0/5
790
860
970
450
890
1,400
460
580
640
1/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
410
310
700
260
190
370
910
970
880
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
300
520
420
1,200
49
330
490
160
320
0/5
0/5
0/5
0/5
1/5
0/5
0/5
0/5
0/5
0/5
640
640
620
150
310
310
650
440
610
0
Treatment groups with no common superscript within columns differ significantly from the IUC group
(P 0.05) with respect to lesion score Rank sums (non-parametric Kruskal-Wallis test).
114
CHAPTER 2
AST of 1999
E. acervulina in isolate H was sensitive for diclazuril, monensin, narasin and reduced
sensitive to salinomycin. Another E. acervulina belonging to isolate F was sensitive to
diclazuril. The remaining isolates were resistant. The only E. maxima isolate was reduced
sensitive lasalocid and narasin. It was resistant to the other compounds. E. tenella present
in two isolates was resistant to all drugs. No mortality was recorded (Table 4).
Table 4. Individual lesion scores (ILS), mortality (Mort) and oocysts per gram faeces
Isolate nr/anticoccidial drug
Isolate E
DIC
LAS
MON
NAR
NIC
SAC
IUC
Isolate F
DIC
LAS
MON
NAR
NIC
SAC
IUC
Isolate G
DIC
LAS
MON
NAR
NIC
SAC
IUC
Isolate H
DIC
LAS
MON
NAR
NIC
SAC
IUC
UUC
E. acervulina
ILS
E. maxima
ILS
E. tenella
ILS
2 3 3 3 3a
3 3 4 3 3a
2 2 3 3 3a
4 3 3 2 2a
3 2 3 2 3a
3 3 2 3 3a
3 3 3 3 3a
2 1 2 1 1b
2 2 3 2 2a
3 3 3 3 3a
2 2 3 2 2a
2 3 2 3 2a
3 2 2 3 2a
3 3 3 3 3a
2 2 2 3 3a
2 2 2 2 2b
2 2 4 4 4a
2 2 3 3 3a
3 3 3 2 3a
3 3 2 3 2a
3 3 3 2 2a
3 3 3 2 3a
3 2 3 3 3a
2 3 2 3 2b
3 3 3 2 3a
3 2 2 3 3a
3 3 2 3 3a
3 3 3 3 3a
2 1 2 1 1b
3 2 3 2 3a
1 2 2 1 1b
1 2 1 2 1b
2 2 3 2 2a
2 2 1 2 1b
2 3 3 3 3a
00000
2 2 1 1 1a
1 1 1 1 1b
3 2 1 1 1a
2 1 1 1 1a
1 2 2 1 1a
2 2 2 1 1a
2 3 2 1 1a
00000
2 2 2 4 1a
1 3 2 2 2a
2 2 2 3 1a
2 2 3 2 2a
2 3 2 3 3a
2 3 2 2 2a
2 3 3 3 3a
00000
Mort
(n = 5)
OPG
(x 103)
0/5
0/5
0/5
0/5
0/5
0/5
0/5
2,500
4,600
1,500
3,800
9,100
3,700
4,400
0/5
0/5
0/5
0/5
0/5
0/5
0/5
5,800
1,500
1,400
1,900
1,400
1,100
7,900
0/5
0/5
0/5
0/5
0/5
0/5
0/5
3,700
7,500
2,900
13,000
3,300
4,400
15,000
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
2,600
1,100
1,100
850
1,600
1,400
2,200
0
a,b
115
AST of 2001
Seven E. acervulina isolates were tested in 2001. All seven were resistant for diclazuril,
five for lasalocid, four for nicarbazin, two for salinomycin, monensin and narasin.
Reduced sensitivity was recorded for three isolates regarding monensin, two for lasalocid
and
one
for
salinomycin
and
narasin.
Sensitivity
was
found
for
meticlorpindol/methylbenzoquate (seven isolates), salinomycin and narasin (four isolates),
nicarbazin (three isolates) and monensin (two isolates). E. maxima was sensitive for
meticlorpindol/methylbenzoquate, resistant to monensin and narasin, and reduced sensitive
to the remaining drugs. Three out of the four E. tenella isolates were resistant to monensin,
three for salinomycin, two for diclazuril and narasin, and one for nicarbazin and lasalocid.
Reduced sensitivity was found in one isolate for nicarbazin, lasalocid and narasin. All
isolates were sensitive for meticlorpindol/methylbenzoquate, two for nicarbazin, diclazuril
and lasalocid, one isolate was sensitive for salinomycin, monensin and narasin.
Some mortality was found in isolate I due to the severity of the E. tenella infection. In
total seven out of 72 birds (eight groups of nine birds) died (Table 5).
Table 5. Individual lesion scores (ILS), mortality (Mort) and oocyst per gram faeces
Isolate nr/anticoccidial drug E. acervulina
ILS
Isolate I
DIC
3 3 3 2 2a
LAS
3 3 2 2 3a
METI/METH
0 0 0 0 0b
MON
1 2 2 3 1a
NAR
1 1 2 1 2b
NIC
2 3 2 3 2a
SAC
1 1 1 2 1b
IUC
3 2 3 3 3a
Isolate J
DIC
3 2 2 3 3b
LAS
3 3 3 3 3b
METI/METH
0 1 1 0 0b
MON
3 2 2 1 2b
NAR
1 1 2 2 1b
NIC
1 1 2 2 3b
SAC
1 1 1 1 1b
IUC
3 4 4 3 4a
Isolate K
DIC
2 2 2 2 2a
LAS
2 2 1 2 xa
METI/METH
1 1 0 0 1b
MON
1 1 1 0 1b
NAR
2 2 1 1 2a
116
E. maxima
ILS
E. tenella
ILS
Mort
(n = 9)
OPG
(x 103)
2 3 3 3 3a
3 3 3 3 3a
0 0 0 0 0b
3 3 3 3 3a
3 2 3 3 3a
3 3 3 3 3a
3 3 3 3 3a
3 3 3 3 3a
1/9
0/9
0/9
2/9
1/9
0/9
1/9
2/9
1,100
3,700
0.330
1,800
2,200
1,400
500
3,800
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
750
1,100
0.330
250
440
650
150
920
0/9
0/9
0/9
0/9
0/9
96
260
5.9
380
310
0 0 0 0 0a
0 0 0 0 xa
0 0 0 0 0a
0 1 0 0 0a
0 0 0 0 0a
CHAPTER 2
NIC
SAC
IUC
Isolate L
DIC
LAS
METI/METH
MON
NAR
NIC
SAC
IUC
Isolate M
DIC
LAS
METI/METH
MON
NAR
NIC
SAC
IUC
Isolate N
DIC
LAS
METI/METH
MON
NAR
NIC
SAC
IUC
Isolate O
DIC
LAS
METI/METH
MON
NAR
NIC
SAC
IUC
UUC
2 1 1 1 1b
2 3 1 2 2a
2 3 2 3 3a
0 0 0 0 0a
0 1 0 0 1a
0 0 0 0 1a
2 3 3 3 3a
2 3 2 3 3a
0 0 0 0 0b
1 1 1 1 1b
1 1 1 1 1b
2 2 3 2 2a
1 2 1 1 1b
3 3 2 3 3a
2 2 2 3 2a
2 1 3 2 1a
0 0 0 1 0b
1 1 2 1 2b
1 1 1 1 2b
1 1 1 1 1b
1 1 1 0 1b
3 3 3 2 2a
0 0 0 0 0b
0 0 0 0 0b
0 0 0 0 0b
0 1 0 2 0a
0 1 0 1 2a
0 0 0 0 0b
1 1 0 0 0b
2 1 1 1 1a
3 3 3 3 3a
3 4 4 3 3a
1 1 1 0 1b
3 3 3 3 3b
3 3 4 3 3a
3 4 4 3 4a
3 2 2 3 3b
3 4 4 4 3a
2 3 3 3 2a
3 3 2 3 3a
0 0 0 0 0b
3 2 2 3 2a
2 2 2 2 3a
3 3 2 1 2a
1 2 1 2 2a
2 2 3 2 3a
00000
2 1 2 1 1b
1 1 2 2 2b
0 0 0 0 0b
2 3 2 2 1a
2 2 2 2 3a
1 1 1 2 3a
1 1 2 2 1b
2 3 2 3 3a
00000
2 2 3 2 2b
1 2 1 1 3b
0 0 0 0 0b
3 3 2 3 3a
2 2 3 3 3a
2 2 2 2 2b
3 2 3 2 3a
3 3 3 3 3a
00000
0/9
0/9
0/9
68
340
830
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
520
940
0.660
210
540
980
710
1,700
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
1,500
420
36
240
300
810
170
800
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
290
3,100
12
240
950
1,000
1,100
1,300
0/9
0/9
0/9
0/9
0/9
1/9
0/9
0/9
0/9
940
700
0.660
270
1,000
490
1,300
550
0
a,b
AST of the E. acervulina reference strain

The E. acervulina reference strain was sensitive to all anticoccidial drugs tested.
The individual lesion scores and OPG per anticoccidial product of the AST of the E.
acervulina reference strain that was performed in fivefold in 1999 are outlined in Table 6.
117
Table 6. Individual lesion scores (ILS) (n = 3) and oocysts per gram faeces (OPG x 103)
of the AST of the E. acervulina reference strain performed fivefold in 1999
Anticoccidial
drug
DIC
MAD
MON
NIC
SAC
IUC
Group 1
ILS
0 0 1b
0 0 0b
0.0 1b
0 0.0b
0 1 1b
1 2 2a
OPG
0
78
150
4.3
3.6
720
Group 2
ILS
1 0 1b
0 0 0b
1 1 0b
0 0 0b
1 0 0b
1 2 2a
Group 3
OPG
0
34
32
11
19
1,100
ILS
1 0 1b
0 0 0b
1 1 1b
0 0 0b
1 1 0b
1 2 2a
OPG
0
40
93
1.0
16
1,700
Group 4
ILS
1 0 1b
0 0 0b
0 0 1b
0 0 0b
0 1 0b
1 2 2a
OPG
0
16
25
11
4,3
1,100
Group 5
ILS
1 0 0b
0 0 0b
1 1 0b
0 0 0b
1 1 0b
1 2 2a
OPG
0
16
140
3.3
0.7
1,000
a,b
Evaluation of resistance
In Table 7, the results of the interpretation of the calculated percent reduction of lesion
score according to the criteria of McDougald et al. (1986) are presented.
Table 7. Summarising classification of the AST per Eimeria spp. found in the field
isolates*
Anticoccidial drug
DIC
HAL
LAS
MAD
METI/METH
MON
NAR
NIC
SAC
R
4/2/7
4/-/4/4/5
3/-/-/-/0
4/3/2
4/3/2
4/4/4
3/3/2
E. acervulina
RS
S
0/0/0
0/2/0
0/-/0/-/0/0/2
0/0/0
1/-/0/-/-/-/0
-/-/7
0/0/3
0/1/2
0/0/1
0/1/4
0/0/0
0/0/3
1/1/1
0/0/4
R
1/1/0
0/-/1/0/0
1/-/-/-/0
1/1/1
0/0/1
1/1/0
1/1/0
* Data presented as 1996/1999/2001.

- = not done.
R = resistant; RS = reduced sensitive; S = sensitive.
118
E. maxima
RS
0/0/1
1/-/0/1/1
0/-/-/-/0
0/0/0
0/1/0
0/0/1
0/0/1
S
0/0/0
0/-/0/0/0
0/-/-/-/1
0/0/0
1/0/0/
0/0/0
0/0/0
R
1/2/2
0/-/1/2/1
0/-/-/-/0
1/2/3
0/2/2
0/2/1
1/2/3
E. tenella
RS
0/0/0
0/-/0/0/1
0/-/-/-/0
0/0/0
0/0/1
1/0/1
0/0/0
S
0/0/2
1/-/0/0/2
1/-/-/-/4
0/0/1
1/0/1
0/0/2
0/0/1
CHAPTER 2
Coccidiosis broiler monitoring program in the Netherlands

The results of the Dutch coccidiosis monitoring program in broiler shows an increase in
the incidence of coccidiosis positive broiler flocks from approximately 70% in 1996 to
91% in 2000. In 2001 the percentage decreased being approximately 73%.
The percentage of E. acervulina infected flocks ranged from 68 in 1996 to 84 in 2000. In
2001 67% of flocks showed E. acervulina coccidiosis. The percentage of E. maxima
infected flocks ranged from 6 in 1996 to 33 in 2000. In 2001 25% of flocks showed E.
maxima coccidiosis. Finally, the percentage of E. tenella infected flocks ranged from 21 in
1996 to 26 in 2000. In 2001 20% of flocks showed E. tenella coccidiosis. The percentages
of coccidial infections mentioned above concern single species or mixed infections.
The results of the Dutch broiler coccidiosis-monitoring program are presented in Figure 1.
Total coccidiosis
E. acervulina
E. tenella
E. maxima
100
90
% positive flocks
80
70
60
50
40
30
20
10
2001-IV
2001-III
2001-I
2001-II
2000-IV
2000-II
2000-III
1999-IV
2000-I
1999-II
1999-III
1999-I
1998-II
1998-III
1998-IV
1998-I
1997-IV
1997-I
1997-II
1997-III
1996-III
1996-IV
1996-I
1996-II
Time in quarter of year

Figure 1. Incidence of coccidiosis infections in Dutch broiler flocks participating in the coccidiosis
monitoring program expressed as total percentage positive flocks and per Eimeria spp. per quarter of
year.
119
Discussion
Although the AST is the only accurate tool to detect resistance against anticoccidial drugs,
it has some drawbacks. The test is slow and expensive. Moreover, the isolates tested
frequently originate from farms with disease outbreaks, therefore the anticoccidial profile
found may not be representative of what is occurring in the average broiler population in
the field. Further, the resistance pattern and pathogenicity of field isolates may be
influenced by the need to propagate them first in SPF broiler chickens for multiplication.
During this procedure which can also affect the proportion of species in the isolate, nonrelevant coccidia may be selected.
The field isolates analysed in the first AST originated from clinical cases of
coccidiosis suggesting the occurrence of anticoccidial drug resistance. Indeed, the results
of the first sensitivity study (1996) show a higher degree of anticoccidial drug resistance
compared to subsequent studies (1999 and 2001) where the isolates originated from
subclinical cases of coccidiosis. However, the fact that in the first study a higher
inoculation dose was used, may have at least in part, further compromised the effectivity
of the anticoccidial drugs tested. Conway et al. (1999) have shown a dose effect on the
lesion score of unmedicated and salinomycin-medicated chickens; higher lesion scores
occurred in both categories if a higher inoculation dose was used.
Resistance for all products tested has been reported previously i.e. for nicarbazin
(McLoughlin & Gardiner, 1967), clopidol (McLoughlin & Chute, 1973), monensin
(Jeffers, 1974), maduramicin (McDougald et al., 1987), salinomycin (Bedrnik et al.,
1987), diclazuril (Chapman, 1989; Vertommen & Peek, 1993), narasin (Weppelman et al.,
1977), halofuginone (Mathis & McDougald, 1982) and lasalocid (Weppelman et al.,
1977).
The results of OPG are difficult to correlate with the outcome of the coccidiosis
lesion scores in some cases. This might be due to the occurrence of a crowding effect
(Williams, 2001), mixed infections and different sensitivity patterns of species in these
mixed infections (Reid, 1975). Nevertheless, it is important to assess whether oocyst
shedding takes place in view of its importance in the development of immunity. According
to Tyzzer (1929) and Tyzzer et al. (1932) both E. maxima and E. praecox (and E.
acervulina as well) develop in the upper/mid-small intestine but they differ in the sites of
the intestinal villus where they invade. The zone of maximal invasion for E. maxima is the
crypt region which has the youngest enterocytes ready to migrate to the apical region (Uni
et al., 1998), while E. praecox and similarly E. acervulina target the mid and apical zone.
In case of a mixed infection where E. maxima is controlled efficiently by an anticoccidial
drug and E. acervulina is not, lesions due to the latter parasite may appear more severe as
more target cells become available.
The E. acervulina reference strain (Weybridge 119) included in the AST of 1999
showed full sensitivity towards all anticoccidial drugs tested. However, clear differences
were found in oocyst shedding between the various products analysed. The highest
numbers of oocysts per gram faeces were found in the groups treated with the ionophores
maduramicin and monensin. These drugs perform well due to their chemotherapeutic
120
CHAPTER 2
effect and the allowance of the development of an adequate immune response (Jeffers,
1989). Oocyst shedding in the presence of ionophores occurs even if parasites are not drug
resistant (Chapman, 1976). This will nevertheless favour the development of immunity
due to the occurrence of multiple small (trickle) infections. In the group treated with
diclazuril oocysts shedding was absent, while at postmortem lesions containing oocysts
were found. A similar effect of diclazuril on E. maxima and E. brunetti has been described
previously (Verheyen et al., 1989). The normal pattern of oocyst wall establishment was
completely disturbed, resulting in the formation of an abnormally thickened, incomplete
oocyst wall and the necrosis of the zygote in all fertilised macrogamonts of both species.
The different effects on oocyst shedding will have an impact on the development of
immunity.
Despite the occurrence of resistance, other positive properties of anticoccidial
products should be considered. Most anticoccidial drugs (mainly ionophores) have growth
promoting properties, which may enhance the flock performance (Radu et al., 1987;
Jeffers et al., 1988). Moreover, due to the occurrence of resistance, trickle infections that
allow build up of immunity take place. This principle has formed the basis for the
development of new vaccines consisting of Eimeria spp. resistant to ionophores. In fact a
form of spontaneous vaccination might be occurring in the field as broiler flocks are being
treated with anticoccidial drugs that show resistance against the prevailing Eimeria spp.
This suggestion is evidenced by the occurrence of a high prevalence of coccidiosis
infections in the field in the Netherlands during the past years (Figure 1), which did not
result in an increase of clinical problems.
Acknowledgements
The authors thank A.R.W. Elbers for performing the statistical analysis and E. Kamps for
his contribution to the Dutch broiler coccidiosis monitoring program.
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tenella. Veterinary Parasitology, 96, pp. 257-263.
123
Anticoccidial drug sensitivity profiles of German,

Spanish and Dutch Eimeria spp. field isolates
Netherlands
Tijdschrift voor Diergeneeskunde (April 2004), 127(7), 210-214
Summary
Anticoccidial drug sensitivity profiles of twenty-four Eimeria spp. field isolates
originating from Dutch, German and Spanish broiler farms were determined. The tested
anticoccidial
products
were
diclazuril,
halofuginone,
lasalocid,
meticlorpindol/methylbenzoquate, monensin, narasin, narasin/nicarbazin, nicarbazin,
robenidine and salinomycin. The sensitivity tests were performed in cage trials. Almost all
Eimeria spp. found in the field isolates showed resistance. Exceptions were some Eimeria
acervulina strains, which were sensitive for meticlorpindol/methylbenzoquate (10/12),
salinomycine (7/21), robenidine (3/6) and the combination narasin/nicarbazin (1/16). A
number of E. tenella strains were sensitive for halofuginone (2/2),
meticlorpindol/methylbenzoquate (3/3), diclazuril (2/4) and robenidine (1/1). E. maxima
strains showed sensitivity to halofuginone (4/4), meticlorpindol/methylbenzoquate (5/8),
the combination narasin/nicarbazine (5/8), nicarbazin (4/10) salinomycin (3/11) narasin
(1/5) and robenidine (1/1).
Introduction
Coccidiosis is a disease caused by intracellular parasites belonging to the genera Eimeria
and Isospora (phylum Apicomplexa). In the vertebrate (Chordata), 575 different species of
which 362 species belong to mammals have been described. These are the Eimeria spp. of
approximately 1.7% of the Chordata and 10% of the mammals. If the Chordata were
thoroughly investigated, the number of Eimeria spp. could probably reach 35,000, of
which 3,400 in mammals (Levine, 1973).
In chickens (Gallus gallus domesticus) nine different Eimeria spp. have been
described: Eimeria acervulina, E. brunetti, E. maxima, E. mitis, E. mivati, E. necatrix, E.
praecox, E. tenella and E. hagani (McDougald & Reid, 1997). All mentioned species
parasitize the intestinal epithelial cells and can be differentiated from each other by their
morphology, tissue tropism, pathogenicity and clinical signs (Johnson & Reid, 1970). In
broilers, E. acervulina, E. maxima and E. tenella are most frequently diagnosed (Peek &
Landman, 2003). E. mitis and E. praecox are diagnosed to a lesser extent and their
pathogenicity has been subject of debate (Williams, 1998).
The lifecycle of the parasite (McDougald & Reid, 1997) starts with the ingestion of
sporulated oocysts, which can be found in litter, water, feed, contaminated boots and/or
equipment. During the life cycle intestinal epithelial cells and the mucosal surface may be
destroyed by the intracellular replication of coccidia. In severe cases this may disrupt the
uptake of nutrients and cause mortality.
The traditional control of coccidiosis is mainly based on the use of anticoccidial
products in feed, while more recently vaccines are being used also. In broilers, these
vaccines are not yet intensively used due to their relative high price and the unfounded
fear for their negative influence on chicken performance and insufficient immunity build
up with time (Williams, 2002).
A major drawback related to the use of anticoccidial products is the occurrence of
resistance, which has been described for all products worldwide (Chapman, 1976; 1997;
Braunius, 1980). The speed at which the onset of resistance occurs is directly related to the
time the parasite is exposed to the anticoccidial product. Therefore, this period should be
as short as possible. To realize this, shuttle and rotation programmes have been
implemented. In case of a shuttle programme at least two different anticoccidial
products, with a likely different mode of action, within one grow-out period, are
alternated. In case of a rotation programme anticoccidial products are used during a preestablished period. Regularly a period of four months or two grow-outs are used before
another product is applied. An alternative for a rotation programme is a continuous
programme in which anticoccidial product(s) are used until a coccidiosis problem occurs
or a new product is introduced on the market. Shuttle programmes can be built-into
rotation programmes.
If full resistance occurs, exchange of anticoccidial products is often not enough to control
the coccidiosis. In these cases vaccination of birds may represent a good and efficacious
alternative. A welcome side effect of vaccination is its ability to increase the sensitivity of
Eimeria spp. field isolates by seeding the vaccinated houses with drug sensitive vaccine
126
CHAPTER 2
Eimeria spp. and therefore restoring the efficiency of anticoccidial products (Vertommen,
1996; Chapman, 2000; Chapman et al., 2002).
Knowledge on the anticoccidial sensitivity patterns of the Eimeria spp. field isolates
is crucial if the use of the steadily decreasing number of anticoccidial products is to be
optimised. The only reliable method to obtain anticoccidial drug sensitivity profiles is by
means of an in vivo anticoccidial sensitivity test (AST).
In the present study the anticoccidial sensitivity profiles of twenty-four Eimeria spp.
field isolates recently obtained from German, Dutch and Spanish broiler farms have been
assessed and the problem of resistance in general is discussed.
Material and Method

Experimental design
In the period of 2000-2002 four ASTs were conducted. In Table 1, an overview of the
investigated anticoccidial products per trial and participating country is given.
At the age of eight days (D 0), chicks in groups fed medicated diets and in one group fed
an unmedicated diet (infected unmedicated control (IUC)) were infected with the Eimeria
spp. field isolates. At six days post-inoculation a postmortem examination was performed
and coccidial lesion scores were determined. In fresh collected faeces the number of
oocyst per gram was determined (see lesion scores and oocysts per gram faeces).
In the ASTs with the German and Dutch field isolates, ten chicks per group were used
whereas in the trial with the Spanish isolates 5.
The birds were observed daily and deceased birds were collected for postmortem
examination.
Experimental animals and housing
One-day-old Hybro male broiler chicks (Nutreco, Putten, the Netherlands) were reared
coccidiosis free on a large stain-less-steel wire cage (height x width x depth = 30 cm x 200
cm x 100 cm) in a separated room until the age of 6 days. At the age of 6 days, two days
prior to infection (D-2) the birds (n = 5) were, ad random, placed on battery stain-lesssteel wire cages (height x width x depth = 30 cm x 40 cm x 50 cm) in the experimental
room. After transfer, the chicks in the medicated groups were supplied with in-feed
medication while the chicks in the unmedicated groups received a feed without
medication. Feed and water were offered ad libitum and the chicks were subjected to a
lighting scheme of 22 h light per twenty-four hours.
127
Table 1. Overview of the anticoccidial products analysed per anticoccidial sensitivity test
(AST) and country
Anticoccidial product
Diclazuril (Clinacox)(DIC)
Halofuginone (Stenorol)(HAL)
Lasalocid (Avatec)(LAS)
Meticlorpindol/methylbenzoquate (Lerbek)
(METI/METH)
Monensin (Elancoban)(MON)
Narasin (Monteban)(NAR)
Narasin/nicarbazin (Maxiban)NAR/NIC
Nicarbazin (Nicarb)NIC
Robenidine (Robenz)(ROB)
Salinomycin (Sacox)(SAL)
Spain
(2000)
Country (year)
Germany
Netherlands
(2001)
(2001)
X
X
X
X
X
X
X
X
Netherlands
(2002)
X
X
X*
X*
X
X
X
X
X
* = with four field isolates, lasalocid was tested and with the remainder two isolates
meticlorpindol/methylbenzoquate.
Feed and anticoccidial products

Twenty-five kilograms of a complete formulated broiler starter mash (Apparent
Metabolizable Energy (AME) 12.0 MJ/kg), free of anticoccidial products, were mixed
with commercial anticoccidial drug premixes. The concentrations of the anticoccidial
products per kilogram feed were: diclazuril (Clinacox) 1 mg, halofuginone (Stenorol) 3
mg, lasalocid (Avatec) 90 mg, meticlorpindol/methylbenzoquate (Lerbek) 100 mg,
monensin (Elancoban) 100 mg, narasin (Monteban) 70 mg, narasin/nicarbazin
(Maxiban) 40/40 mg, nicarbazin (Nicarb) 125 mg, robenidine (Robenz) 33 mg and
salinomycin (Sacox) 60 mg. To warrant a homogenous mixture, the required quantity
was first mixed with approximately 2.5 kg mash. Thereafter, it was further mixed in a
blender (Naturamix, machine nr. m19091, Naturamix B.V., Haarlem, the Netherlands)
with the remainder feed. The concentrations of the anticoccidial products in the diets were
controlled as described earlier by Peek & Landman (2003).
Preparation of the inocula
Oocysts of the Eimeria spp. field isolates were purified from the fresh faeces/litter samples
and were passaged through Specified Pathogen Free (SPF) broilers for multiplication and
identification (Ryley et al., 1976). The oocyst cultures were suspended in 2.5% (w/v)
potassium dichromate and refrigerated at 2-8 C until application. The Eimeria spp.
present in the field isolates were identified by the location and appearance of the gross
lesions in the intestine and by microscopical examination and measurement of the oocysts
128
CHAPTER 2
(Long et al., 1976; McDougald & Reid, 1997). The inoculation dose was adjusted to
obtain a coccidial lesion score of 2 to 3 in the IUC on the scale of Johnson & Reid (1970).
The inocula were prepared and the potassium dichromate was removed by means of
centrifugation and the oocysts were suspended in tap water. Oocyst counting of the inocula
was performed with the aid of a Fuchs-Rosenthal haemocytometer counting chamber and
the chicks were inoculated individually in the crop with a calibrated syringe without
needle in a volume of 1 ml.
Coccidial lesion scores and oocyst per gram faeces (OPG)
At six days after infection (D+6) necropsy was performed on five birds per experimental
group and the individual lesion scores were determined according to the method of
Johnson & Reid (1970). In fresh collected faeces material from D+4 till D+8 (of the
Spanish isolates from D+4 till D+6) the OPG-counting were performed by means of a
modification of the McMaster counting chamber saturated salt flotation technique
described by Peek & Landman (2003).
Determination and evaluation of the sensitivity profiles
The anticoccidial sensitivity profile of each Eimeria spp. present in the field isolates was
based on the reduction of the mean lesion scores of the medicated group compared to the
IUC group. The following formula was used: 100% - (mean lesion score of medicated
group/mean lesion score of the IUC group x 100%). A reduction percentage of 0-30%
indicates coccidial resistance, 31-49% reduction indicates reduced sensitivity or partial
resistance and a reduction of 50% or more indicates full sensitivity to the tested
anticoccidial product (McDougald et al., 1986; 1987).
Results
Inocula
The twenty-four Eimeria spp. field isolates consisted of different Eimeria spp. E.
acervulina was found in twenty-two isolates of which nine as single species, two in
combination with E. tenella, nine in combination with E. maxima and two in a mixture
with E. tenella and E. maxima. E. tenella and E. maxima were, beside the previous
mentioned combinations, only once found as single species.
E. acervulina was most frequently seen and was identified in 92% (22/24) of the field
isolates. E. maxima was observed in 50% (12/24) field isolates and E. tenella 21% (5/24).
The Eimeria spp. field isolates tested and the involved species as well as the inoculation
dose (number of sporulated oocysts per chick) are given in Table 2.
129
Lesion scores
The mean lesion score standard deviation (SD) of E. acervulina (n = 105) in the infected
unmedicated control was 2.1 0.8, of E. tenella (n = 15) 2.3 1.1 and of E. maxima (n =
55) 1.7 0.9. The lesion scores of the Eimeria spp. present in the field isolates 2001-4 and
2001-11 are not included, as mortality occurred in these groups (see later on).
Table 2. Eimeria spp. present in the field isolates and inoculation dose (number of
sporulated oocysts) per chick
Eimeria spp. field isolate
2000-1 (ES)
2000-2 (ES)
2000-3 (ES)
2000-4 (ES)
2000-5 (ES)
2001-1 (DEU)
2001-2 (DEU)
2001-3 (DEU)
2001-4 (DEU)
2001-5 (DEU)
2001-6 (DEU)
2001-7 (DEU)
2001-8 (NL)
2001-9 (NL)
2001-10 (NL)
2001-11 (NL)
2001-12 (NL)
2001-13 (NL)
2002-1 (NL)
2002-2 (NL)
2002-3 (NL)
2002-4 (NL)
2002-5 (NL)
2002-6 (NL)
Eimeria spp. involveda

E. ac/E. max
E. ac
E. ac/E. max
E. ac/E. max
E. ac/E. ten
E. ac
E. ac
E. ac/E. max
E. ac/E. ten/E. max
E. ac/E. max
E. ac
E. ac/E. max
E. max
E. ac/E. max
E. ac
E. ten
E. ac/E. max
E. ac/E. ten/E. max
E. ac
E. ac
E. ac
E. ac/E. max
E. ac
E. ac/E. ten
Inoculation dose (x 103)

50
35
10
50
10
50
40
50
15
30
50
30
36
41
53
10
40
21
50
50
50
50
50
10
E. ac = E. acervulina; E. max = E. maxima; E. ten = E. tenella.

(ES) = Spain; (DEU) = Germany; (NL) = the Netherlands.
Anticoccidial sensitivity test (AST)

An overview of the mean coccidial lesion scores and the OPG per anticoccidial product
and Eimeria spp. field isolate is given in Table 3.
The mortality of the chicks in the IUC group of field isolate 2001-4 was 80%. This
mortality was caused by a pathogenic E. tenella strain present in this field isolate. Due to
130
CHAPTER 2
this high rate of mortality it was impossible to determine the sensitivity of E. acervulina
and E. maxima also present in this field isolate. In the IUC group and the group medicated
with narasin of isolate 2001-11, mortality which was caused by E. tenella was 40 and
60%, respectively.
In Table 4 all anticoccidial drug sensitivity profiles per Eimeria spp. and field isolate
according to the criteria of McDougald et al., 1986; 1987 are given.
131
Table 3. Overview of the mean lesion scores and number of oocysts per gram faeces (x
106) per Eimeria spp. field isolate and anticoccidial product
2000-1 (ES)
2000-2 (ES)
2000-3 (ES)
2000-4 (ES)
2000-5 (ES)
2001-1 (DEU)
2001-2 (DEU)
2001-3 (DEU)
2001-4 (DEU)
2001-5 (DEU)
2001-6 (DEU)
2001-7 (DEU)
2001-8 (NL)
2001-9 (NL)
2001-10 (NL)
2001-11 (NL)
2001-12 (NL)
2001-13 (NL)
2002-1 (NL)
2002-2 (NL)
2002-3 (NL)
2002-4 (NL)
2002-5 (NL)
2002-6 (NL)
132
ac
2.4
2.8
1.4
3.0
0.8
--1.2
1.4
--1.6
1.8
2.2
2.4
2.6
3.0
2.0
0.8
DIC
te ma
--- 2.0
--- ----- 1.4
--- 1.2
0.0 ---
------0.0
--2.0
----------2.6
2.6
0.4
----1.0
1.2
------2.4
-----
opg ac
4.20
1.30
0.19
0.68
0.28
3.0
2.0
2.0
0.4
1.0
2.2
2.4
0.01
2.00
4.70
0.00
4.00
3.50
2.10 1.4
2.20 3.0
3.37 1.8
0.78 1.8
4.50 2.4
0.54 1.0
te
HAL
ma opg ac
------0.8
-------
----0.0
0.4
0.6
--0.2
1.30
0.68
0.11
0.26
0.25
3.40
1.40
----------1.0
------0.0
-----
2.23
3.10
1.69
0.68
1.70
0.07
2.2
2.4
2.4
1.0
LAS
METI/METH
ma opg ac te ma opg ac
1.6
3.0
1.0
2.0
1.0
0.4 --- --- 0.03
0.6 --- --- 0.15
0.0 --- 0.0 0.01
0.0 0.0 1.2 0.03
0.2 --- 1.6 0.08
2.0 --- --- 0.28
1.6 --- 0.6 0.15
--- --- 2.6 0.08
0.0 --- 0.0 0.00
0.0 --- --- 0.00
--- 0.0 --- 0.00
0.4 --- 0.2 0.01
0.2 0.0 0.0 0.01
--- --- 3.80
--- --- 3.99
0.0 --- --- 0.00
0.0 --- 2.0 0.14
--- --- 6.19
2.0 --- 0.31
te
MON
te ma
--- 1.0
--- ----- 1.0
--- 1.0
0.6 ---
opg
0.48
1.20
0.18
0.70
0.36
CHAPTER 2
DIC = diclazuril; HAL = halofuginone; LAS = lasalocid; METI/METH =

meticlorpindol/methylbenzoquate; MON = monensin; NAR = narasin; NIC = nicarbazin;
ROB = robenidine; SAL = salinomycin; IUC = infected unmedicated control.
ac = Eimeria acervulina; te = E. tenella; ma = E. maxima.
ES = Spain; DEU = Germany; NL = the Netherlands.
ac
--1.6
1.4
--1.8
1.8
1.4
1.6
2.8
2.4
2.2
1.0
NAR
NAR/NIC
ma opg ac te ma opg ac
2.4
2.8
1.8
2.6
1.0
2,.2 --- --- 0.73 2.4
2.2 --- --- 1.30 2.8
2.4 --- 0.2 0.40 1.8
0.8 3.0 1.0 0.26 0.8
1.0 --- 1.4 0.17 1.4
2.6 --- --- 3.0 2.6
1.6 --- 0.6 1.30 1.0
--- 2.4 0.05 --- --- 1.2 0.02 ----- 0.2 2.20 1.4 --- 0.0 1.60 1.8
--- --- 1.70 1.6 --- --- 2.50 1.6
3.6 --- 0.02 --- 3.0 --- 0.09 ----- 0.8 2.10 2.2 --- 0.6 2.10 2.4
2.4 1.4 3.30 1.0 2.6 0.8 1.80 1.2
--- --- 2.89 1.0 --- --- 2.00
--- --- 2.90 2.0 --- --- 1.47
--- --- 1.69 1.6 --- --- 2.29
--- 1.6 0.96 1.8 --- 2.2 1.05
--- --- 3.10 2.4 --- --- 3.80
2.8 --- 0.74 1.0 2.8 --- 0.49
te
NIC
te ma
--- 1.0
--- ----- 1.4
--- 0.0
0.8 ----- ----- ----- 1.0
3.2 0.8
--- 1.8
--- ----- 1.0
--- 2.6
--- 0.0
--- --3.2 ----- 0.4
2.8 1.4
opg ac
0.52
2.00
0.59
0.32
0.12
1.50
1.50
0.20
0.10
0.17
2.60
1.00
0.07
2.30
1.00
0.01
3.00
2.60
1.6
2.6
1.0
0.8
0.2
0.8
ROB
te ma opg ac
0.6
1.0
0.4
1.4
0.0
2.2
2.4
1.2
1.0
1.0
2.6
1.6
--1.2
1.2
--1.0
1.0
--- --- 1.18 1.4
--- --- 2.59 2.6
--- --- 0.70 1.2
--- 0.0 0.20 2.2
--- --- 0.02 2.0
0.0 --- 0.11 1.0
SAL
te ma
--- 1.4
--- ----- 0.8
--- 0.0
0,8. ----- ----- ----- 1.2
2.6 1.2
--- 1.8
--- ----- 1.4
--- 1.6
--- 0.6
--- --3.2 ----- 1.2
2.8 0.8
--- ----- ----- ----- 1.2
--- --3.0 ---
opg
0.19
0.37
0.10
0.30
0.18
1.70
0.97
0.64
0.29
0.23
3.90
1.20
0.05
0.90
1.40
0.22
0.14
1.40
1.05
1.13
1.44
0.52
2.90
0.34
ac
2.2
2.6
1.8
2.4
1.0
2.6
3.0
3.0
0.0
1.4
3.4
1.4
--1.2
2.2
--2.0
1.6
2.2
3.0
2.6
2.8
1.6
1.0
IUC
te ma
--- 1.6
--- ----- 1.2
--- 1.2
0.8 ---- ----- ----- 1.2
4.0 0.0
--- 2.2
--- ----- 2.2
--- 3.2
--- 1.0
--- --3.6 ----- 0.8
3.0 1.6
--- ----- ----- ----- 2.1
--- --3.0 ---
opg
5.10
1.20
0.71
0.07
0.11
2.40
2.40
1.40
0.44
0.35
3.80
1.10
0.04
2.20
4.10
0.03
3.40
4.00
7.59
6.79
6.19
2.70
2.99
0.84
133
Table 4. Anticoccidial sensitivity profiles per Eimeria spp. found in the field isolates
according to the criteria of McDougald et al. (1986; 1987)
Anticoccidial
product
DIC
HAL
LAS
METI/METH
MON
NAR
NAR/NIC
NIC
ROB
SAL
E. acervulina
R
5/-/3/6*
-/3-/3
-/-/-/4
-/1/0/0
4/-/-/-/-/3/4
-/6/3/2
5/5/4/-/-/-/3
0/5/1/4
E. tenella
E. maxima
RS
S
R
RS
S
R
RS
S
0/-/1/0 0/-/0/0 0/-/0/1 0/-/1/0 1/-/1/0 3/-/3/1 0/-/0/0 0/-/1/0
-/3/-/3 -/0/-/0 -/0/-/0 -/0/-/0 -/1/-/1 -/0/-/0 -/0/-/0 -/3/-/1
-/-/-/0 -/-/-/0 -/-/-/0 -/-/-/1 -/-/-/0
-/-/-/-/-/-/-/-/-/-/1/0/0 -/4/4/2 -/0/0/- -/0/0/- -/1/2/- -/1/1/1 -/0/0/- -/2/3/0
1/-/-/- 0/-/-/- 1/-/-/- 0/-/-/- 0/-/-/- 2/-/-/- 1/-/-/- 0/-/-/-/-/1/2 -/-0/0 -/-/2/1 -/-/0/0 -/-/0/0 -/-/3/1 -/-/0/0 -/-/1/0
-/0/1/3 -/0/0/1 -/1/2/1 -/0/0/0 -/0/0/0 -/0/1/1 -/1/0/0 -/2/3/0
0/1/0/- 0/0/0/- 1/1/2/- 0/0/0/- 0/0/0/- 1/2/2/- 1/0/0/- 1/1/2/-/-/-/0 -/-/-/3 -/-/-/0 -/-/-/0 -/-/-/1 -/-/-/0 -/-/-/0 -/-/-/1
1/0/2/1 4/1/1/1 1/0/2/1 0/1/0/0 0/0/0/0 1/2/1/0 1/1/1/1 1/0/2/0
* Data represent successively the number of isolates per category of Spain (2000)/Germany (2001)/the
Netherlands (2001)/the Netherlands (2002).
DIC = diclazuril; HAL = halofuginone; LAS = lasalocid; METI/METH =
meticlorpindol/methylbenzoquate; MON = monensin; NAR = narasin; NIC = nicarbazin; ROB =
robenidine; SAL = salinomycin.
- = not done.
Discussion
The infection doses of the inocula were chosen aiming at lesion scores of 2-3 on the scale
of Johnson & Reid (1970) and no mortality in the IUC groups. The obtained mean lesion
score of E. acervulina was 2.1, of E. tenella 2.3 and of E. maxima 1.7. Globally, the target
lesion scores pursued, were obtained with the inoculations doses used. Exceptions were
isolates 2001-4 and 2001-11, in which mortality was caused by a severe clinical E. tenella
infection and E. maxima, with lesion scores below 2-3. The lower lesion score of E.
maxima may have been the result of a small amount of E. maxima oocysts present in the
inoculum. However, it is also possible that the infection of E. maxima was influenced by
the E. acervulina infection, since this species has a shorter prepatent period in comparison
with E. maxima. Moreover, a severe E. acervulina infection can affect large parts of the
intestine beside the duodenum destroying intestinal epithelial cells used for the
development of E. maxima (Williams, 2001).
All Eimeria spp. field isolates from this study were collected from broiler farms
where no severe clinical coccidiosis problems had been diagnosed. However, the tested
Eimeria spp. field isolates showed resistance against most anticoccidial products analysed.
Regarding the evaluation of sensitivity of E. maxima against diclazuril an important
consideration should be made. Diclazuril is mainly active against the stages of the parasite
found at the end of the lifecycle resulting in the formation of an abnormally thickened,
134
CHAPTER 2
incomplete oocyst wall and ending in necrosis of the produced zygote (Verheyen et al.,
1989). Therefore, despite a fully sensitive anticoccidial profile of E. maxima against
diclazuril, lesions can be observed in the intestine but then oocyst excretion is limited or
even nullified. In contrast, if resistance occurs the oocyst excretion will not be limited or
nullified.
The number of oocysts excreted after a coccidiosis infection with different Eimeria
spp. field isolates varies greatly and may be difficult to interpret. This is explained by the
fact that many field isolates consist of Eimeria spp. differing in their pathogenicity and
oocyst production. Moreover, Eimeria spp. often have different anticoccidial sensitivity
profiles (Reid, 1975), while the so called crowding effect may additionally also influence
oocyst production. This phenomenon occurs when many host cells are lost during the
schizogony (asexual phase) and no cells to accomplish the gametogony (sexual phase) and
hence the production of oocysts (Williams, 2001) remain. Another explanation can be the
individual variation of the coccidiosis sensitivity (resistance) of the laboratory animals
used, even if they are closely related. In this regard, as the trials were performed in
different periods, dissimilarities between batches of laboratory animals may have played a
role.
The OPG determined in groups inoculated with the same field isolate are often good
interpretable. Especially, if the isolate is sensitive to the tested anticoccidial product. An
example for this is the anticoccidial profile obtained for meticlorpindol/methylbenzoquate.
The full sensitivity profile based on the reduction of coccidiosis lesion scores was
confined by the OPG results.
Resistance against all existing anticoccidial products is common and has been
widely described (Chapman, 1986; 1990; 1997; McDougald et al., 1986; 1987;
Vertommen & Peek, 1993). However, susceptibility to anticoccidial products may be
regained after long periods of withdrawal from feed (Chapman, 1990). Evidence for this
was also found in the ASTs performed with the product meticlorpindol/methylbenzoquate,
which showed high anticoccidial activity likely due to its infrequent use (the product had
almost not been used since 1999 according to the participating farms).
Products with high anticoccidial activity could be used to effectively control coccidiosis at
farms with resistance associated problems, while rotation with a live sensitive vaccine
could increase the sensitivity of the existing Eimeria spp. field population to the
anticoccidial products. The exact number of grow-outs that must be vaccinated
successively in order to induce higher incidence of sensitive Eimeria spp. field isolates
will depend upon the vaccine used and the pathogenicity of the resistant Eimeria spp.
(Chapman, 2000; Chapman et al., 2002). By regularly performing ASTs, which currently
is the only reliable method to measure the anticoccidial drug sensitivity, changes and
evolution in anticoccidial drug profiles can be monitored.
Although, in recently performed ASTs anticoccidial drug resistance was
extensively found in the Netherlands, the number of outbreaks of clinical coccidiosis
was not increased (Peek & Landman, 2003). Possibly as a result of the (partial)
inefficiency of the anticoccidial products due to resistance, immunisation of the birds
caused by subclinical trickle infections, as has been described during vaccination
135
(Chapman, 1976; Jeffers, 1989), occurred. This may also explain the absence of severe
clinical problems at the farms where the Eimeria spp. field isolates for this study were
collected.
In general, ASTs are not performed on a regular basis, which is the cause for a
paucity on actual and a continuous flow of data on anticoccidial drug resistance
(Chapman, 1998). They are nevertheless of major significance for the optimal use of
the anticoccidial products and the design of efficient coccidiosis prevention programs.
The latter are crucial to prevent a further increase in the prevalence of multi drug
resistant Eimeria spp. field isolates.
References
Braunius, W.W. (1980). Coccidiosis bij slachtkuikens, Tijdschrift voor Diergeneeskunde, 105, pp. 11-20.
Chapman, H.D. (1976). Eimeria tenella in chickens: studies on resistance to the anticoccidial drugs
monensin and lasalocid. Veterinary Parasitology, 2, pp. 187-196.
Chapman, H.D. (1986). Isolates of Eimeria tenella: studies on resistance to ionophorous anticoccidial
drugs. Research in Veterinary Science, 41, pp. 281-282.
Chapman, H.D. (1990). Drug resistance in coccidia of the fowl. In Boray J.C., Martin, P.J. & Roush, R.T.
eds. Resistance of parasites to antiparasitic drugs. Rahway N.J: MSD Agvet, Merck & Co.,
Incorporate, pp. 33-41.
Chapman, H.D. (2000). Practical use of vaccines for the control of coccidiosis in the chicken. World
Chapman, H.D., Cherry, T.E., Danforth, H.D., Richards, G., Shirly, M.W. & Williams, R.B. (2002).
In: Yvore, P. (Ed.). Proceedings of the Vth International Coccidiosis Conference, (pp. 295-308).
Tours, France.
Levine, N.D. (1973). Introduction, history and taxonomy. In: The Coccidia 1. University Park Press,
Baltimore.
Long, P.L., Millard, B.J., Joyner, L.P. & Norton, C.C. (1976). A guide to laboratory techniques used in
the study and diagnosis of avian coccidiosis. Folia Veterinaria Latina, 6, pp. 201-217.
McDougald, L.R., DaSilva, J.M., Solis, J. & Braga, M. (1987). A survey of sensitivity to anticoccidial
287-292.
McDougald, L.R. & Reid, W.M. (1997), Coccidiosis. In: Calnek, B.W., Barnes, H.J., Beard, C.W.,
McDougald, L.R. & Saif, Y.M. (eds.). Diseases of Poultry 10th edn,. (pp. 865-883). Ames: Iowa State
University Press.
136
CHAPTER 2
Reid, W.M. (1975). Relative value of oocysts counts in evaluating activity. Avian Diseases, 19, pp. 802811.
Verheyen, A., Maes, L., Coussement, W., Vanparijs, O., Lauwers, F., Vlaminckx, E. & Marsboom, R.
(1989). Ultrastructural evaluation of the effects of diclazuril on the endogenous stages of Eimeria
maxima and E. brunetti in experimentally inoculated chickens. Parasitology Research, 75, pp. 604610.
sensitivity tests; resistance development; cross resistance to Baycox. In: Barta, J.R. & Fernando,
M.A. (eds.). Proceedings of the VIth International Coccidiosis Conference (pp. 154-155). Guelph,
Canada.
Vertommen, M. (1996). Coccidiosis control methods. In: Proceedings of the Paracox European
Symposium (pp. 11-20). Annecy, France.
Williams, R.B. (1998). Epidemiological aspects of the use of live anticoccidial vaccines for chickens.
species of the domesticated fowl: its importance for experimental design and the production of oocysts
Williams, R.B. (2002). Anticoccidial vaccines for broiler chickens: pathways to success. Avian
137
Chapter 3
Influence of ibuprofen, a mucolytic enzyme and a

mannanoligosaccharide preparation (MOS) on a
coccidiosis infection in broilers
Effect of ibuprofen on coccidiosis in broiler chickens

B. Vermeulena, H.W. Peekb, J.P. Remona and W.J.M. Landmanb
a
Laboratory of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Ghent

University, Belgium
b
Animal Health Service (GD), Poultry Health Centre, P.O. Box 9, 7400 AA Deventer, the
Netherlands
Avian Diseases (January 2004), 48(1), 68-76
Summary
Ibuprofen (IBU), a non-steroidal anti-inflammatory drug (NSAID), inhibits the
biosynthesis of prostaglandins (PG) with pro-inflammatory and immunosuppressive
properties and is therefore proposed as a candidate molecule for the treatment of
coccidiosis in broiler chickens. In all experiments, IBU was administered via drinking
water. In a first experiment, chickens were infected at 10 or 21 days of age with oocysts of
Eimeria acervulina (5 x 104), E. maxima (3 x 104) and E. tenella (7.5 x 103) and
medicated with IBU at a dose of 15 mg/kg body weight (BW). In a second experiment,
chickens were infected at six days of age with 104 oocysts of E. acervulina and medicated
with IBU at a dose of 100 mg/kg BW. In the third experiment an inoculum consisting of 5
x 104 or 105 E. acervulina oocysts was administered at six days of age to chickens
medicated with IBU at a dose of 100 mg/kg BW. In a fourth experiment, the effect of IBU
on sporulation and infectivity of E. acervulina oocysts was studied. Coccidial lesion
scores (CLSs), oocyst shedding and weight gain were used as evaluation parameters in all
experiments, except the weight gain which was not taken into account in the fourth
experiment. In addition, the sporulation percentage was determined in the last experiment.
No influence of IBU on the indicated parameters was observed after providing the drug at
a dose of 15 mg/kg BW, while CLSs and oocyst shedding were reduced when IBU was
provided in a dose of 100 mg/kg BW. However, IBU did not significantly show any effect
on the degree of sporulation and infectivity of E. acervulina oocysts at a dose of 100
mg/kg BW.
Keywords: coccidiosis, prostaglandins, ibuprofen, drinking water, broiler chickens
CHAPTER 3
Introduction
Coccidiosis is a widespread parasitic poultry disease with great economic relevance
(Weber, 1997) and is caused by protozoa of the genus Eimeria (Phylum Apicomplexa). Of
the seven Eimeria species described in chickens, E. acervulina, E. maxima and E. tenella
are the most significant in broiler chickens. These different species can be distinguished
by the morphology of the lesions and their location in the intestine, and the size of oocysts
produced (McDougald & Reid, 1991).
Control of coccidiosis in broiler chickens is still problematic due to the large scale
occurrence of resistance against most anticoccidial drugs (Chapman, 1997; Peek &
Landman 2003) and the limited use of vaccines for economic reasons, non-founded
supposed adverse effects on chick growth as well as fears for insufficiently developed
immunity (Williams, 2002).
The previous mentioned shortcomings have prompted to a search for alternatives in
the combat against this parasitic disease. A possible alternative such as non-steroidal antiinflammatory drugs (NSAIDs) like ibuprofen (IBU) was therefore investigated.
Host immune responses against coccidial infections are very complex. Similar to
the immune responses to many other parasitic infections of the gastrointestinal tract, the
role of humoral factors such as antibodies is not completely understood. In contrast, the
role of T lymphocytes, which are involved in the development of cellular immunity, is
well-documented (Lillehoj, 1998; Lillehoj & Lillehoj, 2000; Yun et al., 2000): T
lymphocytes are involved in the production of different cytokines that participate in
immune responses against coccidia. The five most important cytokines are interferon-
(IFN-), tumour necrosis factor- (TNF-), interleukin-2 (IL-2), interleukin-15 (IL-15)
and transforming growth factor- (TGF-) (Lillehoj & Lillehoj, 2000; Yun et al., 2000).
IFN- and TNF- are involved in the generation of nitric oxide (NO) from L-arginine
(ARG) (Lillehoj & Lillehoj, 2000; Ovington et al., 1995) by activation of inducible nitric
oxide synthase (iNOS). NO is an important defense mechanism against the invasion of
different Apicomplexa parasites (Adams et al., 1990; Mellouk et al., 1991) however,
Allen (1997a; 1997b) suggested that NO might promote the development of coccidial
lesions and thereby contribute to the pathology of coccidiosis. Increased NO
concentrations activate inducible cyclooxygenase (iCOX), which in turn is involved in the
production of prostaglandins (PG) with pro-inflammatory and immunosuppressive
properties (Vane et al., 1994). NSAID - known as inhibitors of COX - inhibit the
biosynthesis of these PG.
Oral administration of another NSAID namely indomethacin had no effect on
weight gain or coccidial lesions but reduced oocyst shedding in chickens infected with E.
acervulina oocysts (Allen, 2000). Moreover, indomethacin also reduced oocyst shedding
after infection of chickens with Cryptosporidium bailey (Hornok et al., 1999).
In the present manuscript, the effect of IBU on coccidiosis in broilers was assessed
after administration of different doses of the drug via drinking water. In addition, the
effect of IBU on the sporulation and infectivity of E. acervulina oocysts was studied.
141
EFFECT OF IBUPROFEN ON COCCIDIOSIS IN BROILER CHICKENS

Experimental design
Three experiments were performed to study the effect of the drinking water administration
of IBU on coccidiosis in broiler chickens. Another experiment (experiment 4) was carried
out to study the effect of IBU on sporulation and infectivity of E. acervulina oocysts.
Experiment 1
One medicated group (n = 20) treated with IBU at a dose of 15 mg/kg body weight (BW)
was made. Medication was given from one day of age until seven days post-infection
(p.i.) (17 or 28 days of age). Another group served as infected unmedicated control (n =
20). Ten chickens per group were infected at ten days of age with an inoculum consisting
of 5 x 104 E. acervulina, 3 x 104 E. maxima and 7.5 x 103 E. tenella oocysts. Another ten
chickens per group were infected with the same Eimeria species at 21 days of age, except
the E. acervulina inoculum, which was administered at 23 days of age. In addition, a third
group served as uninfected unmedicated control (n = 20). All birds were euthanased seven
days p.i. to assess coccidial lesion scores (CLSs) according to the method of Johnson &
Reid (1970). Oocyst shedding, expressed as the number of oocysts per gram faeces
(OPG), was determined by collecting faeces 4-7 days p.i. in each cage. Oocyst shedding
was performed on one sample of homogenized faeces material deposited in each cage.
After homogenization of the faeces, oocysts were counted by means of a modification of
the McMaster counting chamber technique of Hodgson (1970) as described by Peek &
Landman (2003). Birds monitored for oocyst shedding were weighted before infection
and three and six days p.i. to determine weight gain.
Experiment 2
One medicated group consisting of 15 chickens was given IBU at a dose of 100 mg/kg
BW from one day before infection (five days of age) until five days p.i. (eleven days of
age). A second group served as infected unmedicated control (n = 15). At six days of age,
the birds of both groups were infected with an inoculum of 104 E. acervulina oocysts. A
third group served as uninfected unmedicated control (n = 15). Ten chickens per group
were euthanased five days p.i. to determine CLSs as described in experiment 1. The
remaining birds were used to determine oocyst shedding from 4-9 days p.i. The collected
homogenized faeces were divided in two portions and two samples from each portion
were prepared for oocyst counting. The number of oocysts per gram faeces and the weight
gain were determined as described in experiment 1.
142
CHAPTER 3
Experiment 3
A group consisting of 20 chickens was medicated with IBU at a dose of 100 mg/kg BW
and medication was provided from one day prior to infection (five days of age) until five
days p.i. (eleven days of age). An uninfected unmedicated control and infected
unmedicated control group each consisting of 20 birds also formed part of the experiment.
An inoculum consisting of 5 x 104 E. acervulina oocysts was administered to ten birds of
the medicated and unmedicated infected group at 6 days of age. The remaining birds of
both groups (ten chickens per group) were infected with a dose of 105 E. acervulina
oocysts at the same age. Intestinal lesions were assessed on five birds per group five days
p.i., while oocyst shedding and weight gain were determined on the remaining birds as
described in experiment 2.
Experiment 4
One group of chickens (n = 15) was medicated with IBU at a dose of 100 mg/kg BW. The
medication was provided one day prior to infection (five days of age) until five days p.i.
(eleven days of age). Another group with the same number of birds served as infected
unmedicated control. All birds were infected at six days of age with 5 x 104 E. acervulina
oocysts. Eight birds per group were euthanased six days later (twelve days of age) to
assess coccidial lesions as described before. From the remaining birds, oocyst shedding
was determined on daily basis from 3-9 nine days p.i. (nine to fifteen days of age). After
dividing the faeces as described in experiment 2, oocysts were counted as mentioned in
experiment 1. Faeces excreted four days p.i. were collected to study the effect of IBU on
sporulation and to prepare the inocula for the second part of the experiment in which the
infectivity of oocysts originating from faeces of IBU medicated and unmedicated chickens
was studied. Two groups of unmedicated chickens each of fifteen birds were infected at
six days of age with E. acervulina oocysts either from unmedicated (A) or medicated
chickens (B). Eight chickens per group were euthanased 6 days p.i. to determine CLSs as
described above. Additionally, oocyst shedding was determined daily in droppings
collected from 3-9 days p.i.
Chickens and housing
In experiment 1, commercial male Hybro (Nutreco, Putten, the Netherlands) chickens
were obtained at one day of age and housed in three HEPA isolators (Beyer and Eggelaar,
Utrecht, the Netherlands) with a volume of 1.3 m3 (140 cm length, 75 cm width, 125 cm
height) each. After infection, chickens were transferred to stainless steel cages (200 cm
length, 100 cm width and 30 cm height). A lighting scheme of 23 h light and 1 h dark was
used in both, the isolators and the cages. In the other experiments, commercial male Ross
(Belgabroed, Merksplas, Belgium) chicks were obtained at one day of age and housed in a
large steel cage (150 cm length, 60 cm width and 40 cm height). One day prior to
infection (five days of age), chickens were transferred to separate steel cages (75 cm
143
length, 60 cm width and 40 cm height) and provided with constant lighting. In all
experiments, drinking water and a complete formulated broiler starter mash (ArkervaartTwente, Nijkerk, the Netherlands) containing 12.0 megajoules/kg Apparent Metabolizable
Energy (AME), were available ad libitum.
Drinking water medication with ibuprofen
IBU (Knoll Pharma Chemicals, Nottingham, England) was mixed with ARG (Orffa,
Londerzeel, Belgium) to improve its solubility and enable drinking water medication.
Equal weight amounts of IBU (24.5 g) and ARG were mixed and 2% (w/w) citric acid
(Federa, Brussels, Belgium) was added to this mixture. The powders were moistened with
2 ml of an aqueous solution of 25% (w/w) Tween 60 (Federa). Next the wet mass was
passed through a 750 m sieve and dried at room temperature (20 2 C) until constant
weight. The drinking water medication was prepared daily and the required amount of the
drug formulation was calculated based on daily average body weight and water
consumption per group of chickens.
Inocula
Sporulated oocysts of E. acervulina (W119, Weybridge), E. maxima (Weybridge) and E.
tenella (Houghton) laboratory strains were used to prepare the inocula. These strains were
obtained from the Central Veterinary Laboratory (Weybridge, UK) and maintained alive
by regular passage in chickens at the Animal Health Service - Poultry Health Centre
(Deventer, the Netherlands). Sporulated oocysts were conserved in a 2.5% (w/v) K2Cr2O7
(Vel, Leuven, Belgium) solution to prevent bacterial and fungal contamination. The
number of oocysts per ml suspension was counted with a haemocytometer (FuchsRosenthal counting chamber). A volume containing the required number of sporulated
oocysts was transferred into a scaled plastic tube and K2Cr2O7 was washed out by
centrifugation. The chickens were individually inoculated in the crop with a scaled 1ml
syringe containing the required number of sporulated oocysts in 0.5 ml tap water.
Sporulation assessment
In experiment 4, oocysts were purified from faeces collected four days p.i. (ten days of
age) within 3 h after defecation by means of a saturated sodium salt flotation technique
based on the method of Ryley et al. (1976) and modified by Peek & Landman (2003).
Sporulation of oocysts occurred by aerating the oocyst suspensions at 29 C during 72 h.
Four samples of one ml of each suspension were removed 0, 3, 6, 9, 12, 18, 24, 30, 36, 48,
60 and 72 h after initiation of sporulation. The sporulation percentage was determined by
means of an haemocytometer (Fuchs-Rosenthal counting chamber) and oocysts were
counted as sporulated when four sporocysts could be seen at a 400 x magnification.
144
CHAPTER 3
The results of CLSs were analyzed non-parametrically using the Kruskal-Wallis test and
were considered significantly different at P 0.05. Data on weight gain and oocyst
shedding were analyzed using a one-way ANOVA after verifying normality and
homogeneity and were considered significantly different at P 0.05. Normality of data
was verified using the Kolmogorov-Smirnov test and the Levene test was used to verify
homogeneity of variances. For all statistical analyses, SPSS for Windows (version 10.0,
2001) was used.
Results
Drinking water medication with ibuprofen
In experiment 1, a theoretical IBU dose of 15 mg/kg BW was attempted. The daily doses
obtained varied from 9.8 to 18.2 mg/kg. The mean administered dose in the birds infected
at ten days of age was 14.3 2.9 mg/kg, while in chickens infected at 21 days of age a
mean dose of 15.2 2.3 mg/kg BW was found. In experiment 2 (IBU 100 mg/kg BW),
the daily doses varied between 87 and 99 mg/kg BW, while a mean administered dose of
95 5 mg/kg BW was found. Doses administered in experiment 3 (IBU 100 mg/kg BW)
varied from 70 to 119 mg/kg BW while mean administered doses of 92 19 and 96 18
mg/kg BW were obtained after infection with 5 x 104 and 105 E. acervulina oocysts,
respectively. In experiment 4 (IBU 100 mg/kg BW) the daily doses varied between 84 and
116 mg/kg BW. A mean administered dose of 102 14 mg/kg BW was found.
Experiment 1
The results of experiment 1 are shown in Table 1. CLSs due to E. acervulina were
minimal after infection at ten days of age. In contrast, CLSs of 2.8 0.4 and 2.4 0.5
were obtained for the unmedicated and medicated group, respectively after infection at 23
days of age. E. maxima lesions were more severe after infection at 21 days of age (2.7
0.7 and 2.5 1.1 for the unmedicated and medicated group, respectively) compared with
those after infection at ten days of age (1.7 0.5 and 1.5 0.5 for the unmedicated and
medicated group, respectively). E. tenella lesion scores of 2.8 0.4 (unmedicated group)
and 2.8 0.4 (medicated group) were obtained after infection at ten days of age, while
scores of 3.1 0.3 (unmedicated group) and 3.2 0.4 (medicated group) were determined
after infection at 21 days of age. CLSs of the medicated group did not differ significantly
from the unmedicated group after infections at ten and 21 days of age. The number of
oocysts counted per gram faeces, defined as oocyst shedding, was higher in the groups
infected at ten days of age (2.32 x 106 and 2.44 x 106 for the unmedicated and medicated
group, respectively) compared with chickens infected at 21 days of age (0.61 x 106 and
0.66 x 106 for the unmedicated and medicated group, respectively). Oocyst shedding was
145
not influenced by administration of IBU at any time. Compared with the uninfected
unmedicated control chickens, the average weight gain was significantly (P 0.05) lower
3-6 days p.i. in the infected groups after infection at ten and 21 days of age.
Experiment 2
The results of experiment 2 are outlined in Table 2. Significant (P 0.05) reduced CLSs
were obtained in the medicated group (0.3 0.5) compared with the unmedicated birds
(0.9 0.7). The number of oocysts per gram faeces was significantly (P 0.05) reduced
in the medicated chickens (3.89 0.09 x 104) compared with the unmedicated birds (6.28
0.10 x 104). The average weight gain was not significantly reduced in the infected
groups compared with the uninfected birds.
Experiment 3
The results of experiment 3 are summarized in Table 3. CLSs were lower in the
medicated group (2.0 0.7) compared with the unmedicated group (2.8 0.4) after
infection with a dose of 5 x 104 E. acervulina oocysts. After infection with 105 E.
acervulina oocysts, lesion scores were also lower in the medicated group (2.4 0.5)
compared with the unmedicated group (3.0 0.7). Although lesion scores were less
severe in medicated birds, this difference was not significant. In the medicated group, 1.00
0.03 x 106 and 1.49 0.04 x 106 oocysts per gram faeces were counted after infection
with 5 x 104 and 105 E. acervulina oocysts, respectively. They were significantly (P
0.05) lower than in the unmedicated group. The numbers of oocysts counted per gram
faeces in the unmedicated group were 1.99 0.04 x 106 and 4.58 0.09 x 106 after
infection with 5 x 104 and 105 E. acervulina oocysts, respectively. A significant (P 0.05)
lower weight gain was noted 3-6 days p.i. in the infected groups compared with the
uninfected group after infection with 105 E. acervulina oocysts.
146
Table 1. Coccidial lesion scores (CLSs) (7 days p.i.), number of oocysts per gram faeces (OPG) (4-7 days p.i.) and weight gain (g) (0-3
(P1) and 3-6 days (P2 ) p.i ) of experiment 1
GroupC
UUC
IUC
IM
UUC
IUC
IM
Medication
E. ac
0
5 x 104
5 x 104
0
5 x 104
5 x 104
None
None
IBU
None
None
IBU
Inoculum
E. max
0
3 x 104
3 x 104
E. ten
0
7.5 x 103
7.5 x 103
3 x 104
3 x 104
7.5 x 103
7.5 x 103
Age at
InfectionA
10
10
21 & 23
21 & 23
CLSs SD (n = 10)
E. ac
E. max
E. ten
0
0
0
0.2 0.4
1.7 0.5
2.8 0.4
0.1 0.3
1.5 0.5
2.8 0.4
0
0
0
2.8 0.4
2.7 0.7
3.1 0.3
2.4 0.5
2.5 1.1
3.2 0.4
OPGB
(x 106)
0
2.32
2.44
0
0.61
0.66
Weight gain SD (g)

P1
P2
128 7
152 11a
129 16
42 21b
128 22
44 23b
146 23
225 34a
183 18
69 54b
174 27
83 50b
E. acervulina oocysts were administrated at 10 and 23 days of age, while other Eimeria spp. were administrated at 10 and 21 days of age.
Faeces collected from 10 chickens per experimental group were used for the determination of oocyst shedding.
C
UUC = uninfected unmedicated control; IUC = infected unmedicated control; IM = infected medicated with IBU 15 mg/kg body weight.
a,b
Values of groups with no common superscript within columns differ significantly (P 0.05).
B
Table 2. Coccidial lesion scores (CLSs) (5 days p.i.), number of oocysts per gram faeces (OPG SD) (4-9 days p.i.) and weight gain (g)
(0-3 (P1) and 3-6 days (P2) p.i.) of experiment 2
GroupA
Medication
UUC
IUC
IM
None
None
IBU
Inoculum
(E. acervulina)
0
104
104
Age at infection
(Days)
6
6
CLSs SD
(n = 10)
0
0.9 0.7a
0.3 0.5b
OPGB SD
(x 104)
0
6.28 0.10a
3.89 0.09b
UUC = uninfected unmedicated control, IUC = infected unmedicated control, IM = infected medicated with IBU 100 mg/kg BW.
Faeces collected from 5 chickens per experimental group were used for determination of oocyst shedding.
a, b
B
Weight gain SD
P1
P2
57 14
86 9
70 5
87 6
56 12
85 7
Table 3. Coccidial lesion scores (CLSs) (5 days p.i.), number of oocysts per gram faeces (OPG) (4-9 days p.i.) and weight gain (g) (0-3
(P1) and 3-6 days (P2) p.i.) of experiment 3
GroupA
Medication
UUC
IUC
IM
UUC
IUC
IM
None
None
IBU
None
None
IBU
Inoculum
(E. acervulina)
0
5 x 104
5 x 104
0
105
105
Age at
Infection
6
6
6
6
CLSs SD
(n = 5)
0
2.8 0.4
2.0 0.7
0
3.0 0.7
2.4 0.5
OPGB
(x 106)
0
1.99 0.04a
1.00 0.03b
0
4.58 0.09a
1.49 0.04b
UUC = uninfected unmedicated control; IUC = infected unmedicated control; IM = infected medicated with IBU 100 mg/kg BW.
Faeces collected from 5 chickens per experimental group were used for determination of oocyst shedding.
a, b
B
Weight gain SD (g)

P1
P2
86 13
106 26
81 15
79 19
92 7
86 16
89 6
74 7a
96 6
44 15b
79 12
42 16b
CHAPTER 3
Experiment 4
Data on CLSs are outlined in Table 4. Lesions in IBU medicated chickens (1.8 0.7)
were significantly (P 0.05) lower compared with those in the unmedicated group (2.5
0.5). CLSs of birds infected with E. acervulina oocysts originating from IBU medicated
birds (Group B) (1.6 1.1) were not significantly different from those of birds infected
with unmedicated oocysts (Group A) (1.8 0.7). The daily OPG for both groups are
shown in Figure 1. In the first part of the experiment, the maximal oocyst shedding
occurred 5-6 days p.i. and was reduced by 20% in the medicated group. Maximal oocyst
shedding in the second part of the experiment was registered 3-4 and 4-5 days p.i. after
infection with oocysts from unmedicated chickens and medicated birds, respectively. The
percentage of sporulation is shown in Figure 2. It was 0% in both suspensions shortly
after starting the sporulation process. After 12 h, 90% of oocysts isolated from the faeces
of unmedicated birds were sporulated. The sporulation was decreased by 10% in the
suspension containing oocysts from faeces of IBU medicated chickens. During the rest of
the sporulation process these percentages remained unchanged.
# Ooc/g faeces (x 10 5)
unmedicated group
IBU medicated group
Group A
Group B
0
3-4
4-5
5-6
6-7
7-8
8-9
Days PI
Figure 1. Daily oocyst shedding (expressed as the number of oocysts per gram faeces) (x 105) in the first
( unmedicated group, IBU medicated group) and second part ( Group A, Group B) of experiment 4.
149
Sporulation percentage
100
90
80
70
60
50
40
30
20
10
0
unmedicated group
IBU medicated group
0
12 18 24 30 36 48 60 72
Time (h)
Figure 2. Sporulation percentage in function of time. Closed symbols represent sporulation percentages
of oocysts originating from unmedicated broilers and open symbols those of oocysts collected from
faeces of IBU medicated birds.
Table 4. Coccidial lesion scores (CLSs) (6 days p.i.) of experiment 4

GroupA
Part 1 of experiment 4
IUC
IM
Part 2 of experiment 4
Group A
Group B
A
Medication
None
IBU
None
None
Inoculum and origin

5 x 104 (CVL)B
5 x 104 (CVL
CLSs SD (n = 8)
2.5 0.5a
1.8 0.7b
5 x 104 (IUC, part 1)

5 x 104 (IM, part 1)
1.8 0.7
1.6 1.1
IUC = infected unmedicated control; IM = infected medicated with IBU 100 mg/kg BW.
CVL = Central Veterinary Laboratory (Weybridge, UK).
a, b
Mean values SD. Per part of the experiment, values within columns having a different superscript
are significantly (P 0,05) different. Columns without superscript are not significantly different.
B
150
CHAPTER 3
Discussion
IBU, a NSAID, inhibits COX, which is involved in the biosynthesis of PG from
arachidonic acid. COX exists in two isoforms: a constitutive enzyme (cCOX or COX-1)
involved in different physiological functions and an inducible enzyme (iCOX or COX-2)
activated in macrophages and other cells or tissues by inflammatory cytokines. Inducible
COX is involved in the synthesis of PG with pro-inflammatory and immunosuppressive
properties (Hornok et al., 1999). In coccidial infections, an increased PG biosynthesis
might be expected after a NO mediated increased activity of iCOX (Vane et al., 1994).
When a COX inhibitor such as IBU is administered, the biosynthesis of proinflammatory PG is inhibited. Therefore, it was postulated that IBU could reduce lesions,
oocyst shedding and weight gain suppression in case of coccidiosis. IBU was
administered as a salt with a basic amino acid such as ARG to improve its solubility in
water and enable drinking water medication. Although ARG is known as a substrate for
the biosynthesis of NO, an increased ARG concentration could theoretically promote the
formation of PG and subsequently aggravate the lesions caused by Eimeria oocysts.
However, experimental data on supplemental administration of ARG to chickens did not
shown such effect on the course of coccidiosis (Allen, 1999). IBU was administered via
drinking water and doses close to the theoretical doses of 15 or 100 mg/kg BW were
provided. Based on the registered water uptake, 15 to 20% deviations were found in
experiments 1, 3 and 4. This was explained by a decrease in water consumption due to the
infection. The decrease of water consumption was however difficult to predict. A smaller
standard deviation was reached in experiment 2 due to a less pronounced decrease in
water uptake as these birds were infected with a lower dose (104) of E. acervulina oocysts.
Nevertheless, based on the obtained results it can be concluded that a reproducible dose of
IBU can be provided when daily body weight and drinking water consumption are taken
into account.
In experiment 1 (Table 1), E. acervulina lesions were not observed seven days p.i.
in the groups infected at ten days of age. The absence of lesions can be explained by the
shorter prepatent period (96 h) of this species compared with E. maxima (121 h) and E.
tenella (115 h) (McDougald & Reid, 1991). Lesions caused by E. acervulina oocysts peak
five to six days p.i. and epithelial cells occupied by this species are already partially or
completely regenerated at seven days p.i. When chickens were infected with E. acervulina
two days later than E. maxima and E. tenella as done in the second part of experiment 1,
lesions were observed as expected. More severe E. maxima lesions were observed in
chickens infected at 21 days of age compared with birds infected at ten days of age. Older
chickens seem to be more sensitive for E. maxima infections. No influence of age of birds
on E. tenella lesions was remarked.
The number of oocysts counted per gram faeces was lower in birds infected at 21
days of age compared with those infected at 10 days of age. This was explained by the
fact that oocyst shedding is mainly determined by E. acervulina because, compared with
E. maxima and E. tenella oocysts, this species has the best oocyst productive potential
(Brackett & Bliznick, 1952). As birds infected at 21 days of age received the E.
151
acervulina inoculum two days later, oocyst shedding to the full extent was missed
compared with infection at 10 days of age. No significant differences in growth were
obtained 0-3 days p.i., while weight gain was significantly (P 0.05) reduced in all
infected groups compared with the uninfected group 3-6 six days p.i. From the results
shown in Table 1, it can be concluded that the administration of IBU at a dose of 15
mg/kg BW has no influence on coccidial lesions, oocyst shedding and weight gain. Given
the fact that IBU has a low oral bioavailability (Roder et al., 1996; Vermeulen & Remon,
2001), a dose of 15 mg/kg BW may be insufficient to affect the severity of coccidial
lesions, the oocyst shedding and the weight loss. Therefore in subsequent studies IBU was
administered in a dose of 100 mg/kg BW.
Indeed, medication with IBU at a dose of 100 mg/kg BW significantly (P 0.05)
reduced CLSs after infection with 104 oocysts of E. acervulina (Table 2). Although after
infection with 5 x 104 or 105 E. acervulina oocysts coccidial lesions were reduced in the
medicated group, they were not significantly different from the unmedicated group (Table
3). A previous study published by Allen (2000) reported no effect of COX inhibitors such
as indomethacin and nimesulide on coccidial lesions induced by 105, 5 x 105 and 106 E.
acervulina oocysts. In the mentioned study the COX inhibitors were supplied via feed
(solid form) or as a solution once or twice daily. Both ways of administration (solid form
via feed and solution once or twice daily) are in contrast with the continuous supply of a
dissolved formulation via drinking water as given in this study and this difference in drug
administration might explain the discrepancy found. Moreover, oocyst shedding was
significantly (P 0.05) reduced in the medicated group for each infection dose (104, 5 x
104 and 105 E. acervulina oocysts). The reduced intestinal lesions and oocyst shedding
after medication with IBU at 100 mg/kg BW is most probably due to the inhibition of
iCOX. No significant difference (P 0.05) in weight gain was reported in the infected
groups compared with the uninfected group from 0-3 days p.i. for all infection doses.
However, weight gain from 3-6 days p.i. was significantly reduced in the groups infected
with 105 E. acervulina oocysts whether drinking water medication was provided or not
(Experiment 3). From the observations obtained in experiments 2 and 3, it can be
concluded that IBU administered at 100 mg/kg BW significantly (P 0.05) reduces
oocyst shedding after infection with different doses (104, 5 x 104 and 105) of E. acervulina
oocysts when compared with unmedicated chickens. Also, intestinal coccidial lesions are
significantly (P 0.05) lower in medicated birds after infection with 104 E. acervulina
oocysts compared with unmedicated chicks.
Because many anticoccidials have proved to reduce the sporulation rate of oocysts
(Joyner & Norton, 1977; Ruff et al., 1978; Von Lwenstein & Kutzer, 1989 Arakawa et
al., 1991) and to lower the infectivity of sporulated oocysts (Ruff et al., 1978), the effect
of IBU on sporulation and infectivity of shedded oocysts was regarded. Oocysts obtained
from both unmedicated and medicated chickens started sporulation at the same time
(Figure 2). The maximum percentage of sporulation in oocyst suspensions of both groups
was reached after 18 h. These results are in accordance with experimental data described
elsewhere (Graat et al., 1994). E. acervulina oocysts sporulate within 17 hours with a
maximum percentage oscillating between 85 and 95% if maintained under optimal
152
CHAPTER 3
conditions (29 1C, continuous aeration, 2.5% (w/v) K2Cr2O7. The maximum
sporulation percentage was 80% and 90% in suspensions with oocysts obtained from
medicated and unmedicated chickens, respectively. Unfortunately, this difference in
sporulation will likely not be a significant contribution to the reduction of coccidiosis
infection pressure in the poultry environment. Therefore, it can be concluded that IBU has
no effect on the sporulation of excreted oocysts.
The CLSs obtained in the second part of experiment 4 were lower than those
induced in the unmedicated group in the first part (Table 4). In the first part of the
experiment, the oocysts derived from faeces sampled between four and seven days p.i.,
while in the second part of the experiment oocysts collected at four days p.i. were taken.
Oocysts that are excreted earlier during a coccidiosis infection are less pathogenic than
oocysts shedded later because they have completed their life cycle in the host faster
(precocious lines) (Jeffers, 1975; Shirley, 1993). Inocula with the latter will induce less
severe lesions.
There were no significant differences in CLSs between birds infected with oocysts from
unmedicated and medicated chickens. These results indicated that IBU medication does
not decrease the infectivity of excreted oocysts.
It can be concluded that IBU administered via drinking water at a dose of 100
mg/kg BW significantly (P 0.05) decreased oocyst shedding after infection with doses
of E. acervulina oocysts ranging between 104 and 105. However, CLSs were reduced for
all those infection doses; they were significantly lower only after infection with 104 E.
acervulina oocysts. In addition, IBU seems not to have effect on sporulation and
infectivity of shedded oocysts.
Acknowledgements
The authors wish to thank D. Tensy, J. Welner and M. Esmann for their practical
assistance during the experiments.
References
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and course of sporulation of oocysts of Eimeria acervulina under different environmental conditions.
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Johnson, J. & Reid, M.R. (1970). Anticoccidial drugs: lesion scoring techniques in battery and floor pen
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against Eimeria maxima, E. brunetti and E. acervulina with particular reference to oocyst
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Lillehoj, H.S. (1998). Role of T lymphocytes and cytokines in coccidiosis. International Journal for
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155
Dietary protease can alleviate negative effects of a

coccidiosis infection on production performance in
broiler chickens
H.W. Peeka, J.D. van der Klisb, B. Vermeulenc and W.J.M. Landmana
a
Netherlands
b
Division Nutrition and Food, Animal Sciences Group of Wageningen UR, Lelystad, the
Netherlands Current address: Schothorst Feed Research, P.O. Box 533, 8200 AM,
Lelystad, the Netherlands
c
Orotech N.V., Dijkstraat 30, 9140 Temse, Belgium
Animal Feed Science and Technology (March 2009), 150(1-2), 151-159
Summary
Two experiments were conducted to determine the effect of dietary protease on coccidiosis
infection, production performance, the intestinal mucus layer thickness, and brush border
enzyme activity using broilers challenged with Eimeria spp. laboratory isolates (Eimeria
acervulina, E. maxima and E. tenella). In the first study the protease was supplied at a
concentration of 2.5 x 104 amylase-DU per kg feed. Broiler chickens were housed in cages
and were infected at ten days of age with 104.0 E. acervulina, 103.8 E. maxima or 103.3 E.
tenella sporulated oocysts. Coccidial lesion scores, oocysts shedding, sporulation
assessment and daily weight gain were used as parameters to quantify the effect of the
protease. In the second study the effects of the protease (supplied at a concentration of 2.5
x 104 amylase-DU per kg feed) on the thickness of the mucus adherent layer and sucraseisomaltase activity (SIA) of three regions (duodenum, jejunum and caecum) of the
intestinal tract were determined. In experiment 1, no significant interaction between
dietary enzyme supplementation and single Eimeria spp. challenge was observed on body
weight gain. However, protease addition to the diet resulted in a significant (P = 0.046)
higher weight gain after comparing all supplemented and non-supplemented groups. E.
maxima infected chickens showed a significant lower body weight gain in comparison
with the other Eimeria infected groups. Coccidial lesions were not significantly affected
by the dietary protease supplementation, despite the slight tendency to a higher lesion
score for E. acervulina on the enzyme supplemented diets. In Experiment 2, it was shown
that the adherent mucus layer of the duodenum (P < 0.001), jejunum (P < 0.001) and caeca
(P = 0.005) was significantly thicker in birds fed the enzyme supplemented diet. The
sucrase-isomaltase activity (SIA) in mucosal scrapings of the jejunum was significantly
lower (P = 0.005) in birds fed the enzyme supplemented diet, indicating a higher villus
turnover rate. In conclusion, our data suggest that dietary supplementation with a protease
reduced the negative impact of a coccidiosis infection on body weight gain of broilers,
although the coccidial lesions and oocyst excretion remained unaffected.
EFFECT OF DIETARY PROTEASE ON COCCIDIOSIS IN BROILER CHICKENS
Introduction
Coccidiosis in poultry is caused by a protozoan parasite belonging to the genus Eimeria of
the family Eimeriidae (Phylum Apicomplexa). It is considered as the disease with greatest
economic impact in the poultry industry. Worldwide the annual costs have been estimated
at 2 billion (Williams, 1999; Shirley et al., 2004; Dalloul & Lillehoj, 2006). Eimeria
species are highly host specific (Levine, 1973; Joyner, 1982). Furthermore, it is well
recognized that Eimeria parasites only infect particular cell types, tissues and organs. The
underlying mechanisms are not yet fully understood and suggest distinct intraspecies and
interspecies antigenic diversity among Eimeria parasites.
Infections with Eimeria spp. induce various pathological and immunological
responses, which stimulate the hosts defense mechanisms and acquired immunity.
However, prior to the development of an acquired immune response, non-specific immune
pathways such as native microbial flora, mucus, lysozymes, increased gastric and bile salts
secretions, and increased peristalsis are considered to be important defense routes (Lillehoj
& Lillehoj, 2000; Yun et al., 2000). It has been suggested that these innate defense
mechanisms may modulate the start of the Eimeria life cycle and hence determine the
course of an acquired antigen specific immune response (Lillehoj and Trout, 1993).
The intestinal enterocytes are covered by a mucus layer, which is secreted by goblet
cells throughout the gastrointestinal tract. Its thickness and fluidity affect nutrient
digestion, absorption, intestinal barrier function and also the mucosa functionality
(Smirnov et al., 2004).
It is well-known that nutrient digestibility will be adversely affected by coccidiosis.
This might be due to: 1) the aforementioned non-specific defense mechanisms such as
altered gastric and bile secretions, increased peristalsis, etc.; 2) decreased activity of
important mucosal enzymes (Adams et al., 1996), which are anchored at the brush border
membrane such as sucrase-isomaltase, peptidases and phosphatases (Semenza, 1986) and
are involved in the final stages of hydrolysis; 3) increased thickness of the mucus layer
due to the gastrointestinal defense mechanisms, which might reduce the transport rate of
nutrients.
Proteases might counteract the negative effects of a coccidiosis infection on the
nutrient digestion and absorption via partial degradation of the mucus layer and hence the
attachment of Eimeria parasites (sporozoites-merozoites) to the mucus. Subsequently,
coccidia may be excreted as passants instead of binding to and invading epithelial cells. In
addition, the protease could have direct effect on the dietary protein by digesting it and
subsequently mitigating the detrimental consequences of the coccidiosis infection.
In the present study the effect of a protease supplied via feed on E. acervulina, E.
maxima or E. tenella challenged broilers was studied.
158
CHAPTER 3

Experimental design
In experiment 1, eight experimental groups consisting of ten birds each were used (Table
2). Four groups received a diet supplemented with the protease 1 while the other four
groups were fed the control diet without the protease. Within the groups fed the protease
supplemented diet, three groups were infected at day ten with E. acervulina, E. maxima or
E. tenella, while the fourth group acted as non-inoculated uninfected control group. The
four groups fed the control feed received exactly the same treatments. Birds were orally
inoculated and six days post-inoculation (p.i.) five birds per group were sacrificed for
postmortem examination and assessment of coccidial lesion scores. The number of oocysts
per gram faeces (OPG) was determined in droppings collected from day four till day eight
p.i. Bodyweight gain was calculated between ten and sixteen days of age for each group.
In experiment 2, the effect of the protease on the thickness of the adherent mucus layer
and the sucrase-isomaltase activity (SIA) of three intestinal regions (duodenum, jejunum
and caeca) of sixteen day-old uninfected broilers was analyzed.
Chickens and housing
In experiment 1, eighty day-old male Hybro broilers (Nutreco, Putten the Netherlands)
were reared coccidiosis free in a large stainless-steel cages with a wired floor (height x
width x depth = 30 cm x 200 cm x 100 cm) and received a diet without anticoccidial
products. At six days of age eight groups of ten randomly chosen birds were placed in
stainless steel battery cages (height x width x depth = 30 x 40 x 50 cm). Each bird was
tagged in the neck with a unique number (Swiftack). After transfer, four experimental
groups were provided a diet supplemented with protease, while the other four received a
control diet without the protease. Birds were fed per group.
In experiment 2, fifteen day-old Hybro broilers were reared coccidiosis free and at the age
of six days transferred to the stainless steel battery cages. Directly after transfer, one group
of ten birds received the diet supplemented with protease while the other group, consisting
of five birds, was given the diet without the protease.
During both trials a lighting scheme of 23 h of light was used. The birds were observed
daily and deceased birds were collected for postmortem examination.
The study was performed with approval of the Animal Experimental Committee in
accordance with the Dutch law on experimental animals.
Supplied by DSM Food Specialities Agri Ingredients, Delft, the Netherlands
159
Table 1. Ingredients and composition of the feed

Ingredient
Maize
Wheat
Soybean meal
Peas
Soybean oil
Animal fat
Chalk stones
Monocalcium phosphate
Sodium bicarbonate
Ronozyme wx (CT)A
PremixB
SporavitC
Natuphos (phytase)
Avilamycin
Lysine
Threonine
g/kg
291.0
299.9
275.0
50.0
15.2
20.2
11.2
10.4
2.3
0.3
10.0
5.0
2.0
2.0
5.0
0.5
Total
1000
Calculated chemical composition

AME (MJ/kg)
Crude protein
Calcium
Phophorus
Sodium
Chloride
12.0
207.7
9.4
6.4
1.6
2.1
Coated non-dusty light brown granulate with a minimum xylanase acitivity of 1000 FXU/g.
The premix consists per kg of: Vitamin A (1,000,000 IU), Vitamin D3 (200,000 IU), Vitamin E (2,500
IU), Vitamin B1 (50 mg), Vitamin B2 (500 mg), pantothenic acid (800 mg), nicotinic acid (4,000 mg),
Vitamin B6 (300 mg), folic acid (100 mg), Vitamin B12 (1.5 mg), biotin (10 mg), Vitamin K3 (125 mg),
choline (20,000 mg), methionine (200 g), CuSO45H2O (1,200 mg), FeSO4H2O (4,500 mg), MnO (7,000
mg), ZnSO4H2O (3,700), Ca(IO3)2 (100 mg) and Na2SeO35H2O (15 mg).
C
Sporavit consists per kg of: Vitamin A (1,000,000 IU), Vitamin D3 (100,000 IU), Vitamin E (5,000 IU),
Vitamin B1 (400 mg), Vitamin B2 (800 mg), pantothenic acid (2,000 mg), nicotinic acid (4,000 mg),
Vitamin B6 (400 mg), folic acid (200 mg), Vitamin B12 (3 mg), Vitamin C (10,000 mg), biotin (10 mg),
Vitamin K3 (200 mg), choline (40,000 mg), Fe (12,000 mg), Mn (10,000 mg), Co (40 mg) and I (40 mg).
B
Protease and feed

A protease produced by DSM Food Specialties, Agri Ingredients, Delft, the Netherlands,
was used in this experiment. The protease was obtained from Bacillus licheniformis and
160
CHAPTER 3
contained 558,700 DU 2 /ml. In both experiments the enzyme was mixed in a concentration
of 25,000 DU/kg feed in a broiler starter mash (Arkervaart-Twente, Nijkerk, the
Netherlands) free of anticoccidial products (Table 1). Feed and water were provided ad
libitum.
Origin of Eimeria spp. laboratory isolates and inocula
Three Eimeria spp. laboratory strains provided by the Central Veterinary Laboratory
(CVL, Weybridge, UK) were used. All strains have been rejuvenated approximately every
half year and were conserved as a suspension of sporulated oocysts at 2-8 C in a 2.5%
(w/v) potassium dichromate (K2Cr2O7, Vel, Leuven, Belgium) solution until application.
The number of oocysts per ml suspension was counted with a haemocytometer (FuchsRosenthal counting chamber). The inoculation doses were adjusted to induce lesion scores
of 2-3 for each Eimeria spp. on the scale of Johnson & Reid (1970). The amount of
volume containing the required number of sporulated oocysts was taken from the
suspensions and K2Cr2O7 was washed out by centrifugation. Inocula containing 104.0, 103.8
and 103.3 sporulated oocysts were obtained for E. acervulina, E. maxima and E. tenella,
respectively. The birds were inoculated individually in the crop with a calibrated syringe
without needle using a volume of 1 ml tap water.
Bodyweight gain, coccidial lesion scores and oocyst shedding
Individual bodyweight (BW) was measured daily between six and sixteen days of age. The
mean BW gain ( SEM) per experimental group was calculated from ten until sixteen days
of age (zero to six days p.i.). At sixteen days of age five birds per group were sacrificed
for postmortem examination. Parasitological examination of the entire gut was carried out
and the coccidial lesion scores (CLS) were determined according to the method of Johnson
and Reid (1970). Briefly, E. acervulina lesion score 1 features ladder-like white streaks in
the duodenal loop ( 5/cm2); in lesion score 2 the lesion density is higher but not
coalescent; in lesion score 3 they are more numerous and coalesce, while thickening of the
intestinal wall is visible; and finally in lesion score 4 the mucosal wall is greyish with
lesions completely coalesced. Lesion score of 1 of E. maxima is characterized by few
small red petechiae in the mid-intestine, in score 2 more numerous petechiae are found and
the intestinal content may be orange, and in score 3 the intestinal wall might be ballooned
and thickened with pinpoint blood clots and mucous filling the intestinal contents. Finally,
in lesion score 4 the intestinal wall is thickened and ballooned over most of its length,
containing numerous blood cloths and digested red blood cells. Lesion score 1 of E.
tenella shows a few scattered petechiae on the caecal wall, and in score 2 noticeable blood
can be found in the caecal contents, while the caecal wall is thickened. In lesion score 3
large amounts of blood or caecal cores are present and in the caecal walls are greatly
2
One DU is defined as the quantity of enzyme that liberates 7 micromoles of paranitroaniline from
casein per minute.
161
thickened, and in lesion score 4 the caecal walls are enormly distended with blood or large
caseous cores.
The mean coccidial lesion score for each experimental group was subsequently calculated.
Oocyst shedding expressed as the number of oocysts per gram faeces (OPG) was
determined in one homogenized sample of collected faeces droppings (4-8 days p.i.) per
cage. Countings were performed by means of a modification of the McMaster counting
chamber technique as described by Peek & Landman (2003, 2004).
Assessment of sporulation
Faeces collected within 4 hr after defecation at sixteen days of age (six days p.i.) were
suspended (6%, w/v) in a solution of 2.5% (w/v) K2Cr2O7. Sporulation was induced by
aeration of the suspensions at 29 C during 36 hr. Samples of each suspension were
removed 0, 12, 24, and 36 hr after initiating sporulation and the sporulation percentage
was determined by using a light microscope (400 x magnification). Oocysts were counted
as sporulated when four sporocysts were observed (Tyzzer, 1929; Wagenbach & Burns,
1969).
Histopathology and sucrase-isomaltase activity
In experiment 2, the influence of the protease on the thickness of the mucous adherent
layer of three regions of the intestine tissue (duodenum, jejunum and caeca) of sixteen
days-old uninfected broilers was measured by the modified method of Jordan et al. (1998).
Briefly, a 1 cm fragment of the duodenum, jejunum and caeca was longitudinally cut
inside out, snap frozen in liquid nitrogen and stored at 70 C. Cryostat cross-sections of
15-20 m of the whole frozen tissue were made and stained according to the modified
procedure of the Periodic Acid Schiff/Alcian Blue (PAS/AB) technique described by
Jordan. The mucus thickness was measured using a light microscope (400 x
magnification) fitted with an ocular eyepiece graticule with divisions of 10 m.
Approximately seven measurements were made on each slide of the mucus layer from the
duodenum, jejunum and caeca.
Sucrase-isomaltase activity of mucosal scrapings, isolated from the intestinal sections
(duodenum, jejunum and caeca) was determined according to the method of Messer &
Dahlqvist (1966). Briefly, the mucosal scrapings were snap frozen and stored in liquid
nitrogen. For analysis the scrapings were transferred to plastic tubes and milliQ water was
added to a final concentration of 5% (w/v) and homogenised with the Ultra-Turrax at full
speed (2 x 104 rpm) twice during 30 s separated by an interval of 30 s. Then, the
homogenate was diluted 5 times and sonicated twice for 15 s separated by a 15 s interval
at an amplitude of 30 m with a MSE Soniprep 150. The protein content of the samples
was determined by the Pierce BCA assay kit (Smith et al., 1985) in microtitreplates.
Subsequently, the specific enzyme activity (SEA) of sucrase (EC 3.2.1.48) -isomaltase
(EC 3.2.1.10) was measured by adding saccharose as substrate, incubating for 60 min at 37
C and measuring the extinction at 405 nm spectrophotometrically. The SEA activity is
162
CHAPTER 3
expressed in units/g protein (U/g). One unit is the amount of enzyme that produces 1 mol
disaccharide per minute.
Statistical analyses
The individual bird was used as experimental unit for comparing the effects of diets (+
protease vs. protease) on weight gains and coccidiosis infections.
The influence of the protease on weight gain after coccidial infection was analyzed by a
two-way ANOVA. Tests of homogeneity of variances were performed by the Levine test.
Residuals were checked on normality by inspecting their normality plots. Pairwise
comparison were done using the Bonferroni test (SPSS, 2008).
The influence of the enzyme on the coccidial lesion scores was analyzed with the nonparametric Wilcoxon Rank Sum Test (Statistix, 2003).
The influence of the protease on the thickness of the mucus layer in the duodenum,
jejunum and caeca was assessed using GEE model (Stata, 2008), which corrects for
correlation within animals. The influence of protease on the mucosal SIA was analyzed by
means of the Kruskal-Wallis test (Stata, 2008). Differences were considered significant if
P < 0.05.
Results
Bodyweight gain, coccidial lesion scores and oocyst shedding
Mean CLS as determined six days p.i., the OPG per experimental group and the mean BW
gain between 10 and 16 days of age are presented in Table 2 and Table 3.
There was no significant interaction on BW gain (P 0.05) between the protease
supplementation and single species coccidiosis infections, therefore the main effects were
further investigated and showed a significantly (P = 0.046) higher average growth in all
groups receiving the protease supplemented diet. The weight gain of birds infected with E.
maxima was significantly lower in comparison with the remaining groups (Table 3).
No significant influence of dietary protease supplementation on the severeness of CLS was
found after statistical analysis. Only E. acervulina showed a tendency towards higher
lesion scores in birds receiving the diet supplemented with the protease (Table 2).
The obtained oocyst shedding was in agreement with the achieved lesions scores in all
groups.
163
Table 2. Experimental groups, mean coccidial lesion scores (CLS)(n = 5) and number of
oocysts per gram faeces (OPG)
Group nr.
1
2
3
4
5
6
7
8
Experimental groups
Infection Eimeria spp.
(# oocysts/bird)
E. acervulina (104.0)
E. acervulina (104.0)
E. maxima (103.8)
E. maxima (103.8)
E. tenella (103.3)
E. tenella (103.3)
Uninfected
Uninfected
Protease
Yes
No
Yes
No
Yes
No
Yes
No
Experimental results
Mean CLS
OPG
SEM
2.6 0.25a
106.5
a
2.0 0.00
106.3
b
2.2 0.37
104.9
b
2.8 0.20
104.8
c
0.0 0.00
105.0
c
0.2 0.20
105.4
0
0
0
0
a-c
Groups with different superscript per Eimeria spp. infection (E. acervulina, E. maxima or E. tenella)
are significantly different from each other (P < 0.05).
Table 3. Mean body weight gain SEM (g) (n = 10) measured between 10 and 16 days of
age and statistical analyses with the Bonferroni test
Diet type
Uninfected
E. acervulina
E. maxima
E. tenella
- Protease
+ Protease
Main effect
coccidiosis
273 8.6
277 14.2
275 8.1a
272 7.0
284 9.0
278 5.7a
191 9.7
224 8.0
208 7.2b
254 14.0
264 11.9
259 9.0a
Mean
difference
67.5
70.2
51.2
15.1
Lower
bound
37.3
40.0
21.0
0.2
Difference between
(Bonferroni test)
E. maxima
Uninfected
E. maxima
E. acervulina
E. maxima
E. tenella
- protease
+ protease
0.001
0.001
0.001
0.046
Main effect
protease
247 7.3a
262 6.4b
Upper bound
97.7
100.4
81.4
30.0
a, b
Groups with different superscript within the row main effect coccidiosis or the column mean effect
protease are significantly different from each other (P < 0.05, Bonferroni test).
Assessment of sporulation
There was no sporulation shortly after initiating the sporulation process (0 h) while after
12 h sporulation started for all Eimeria spp. At this time the sporulation percentages of
oocysts isolated from the faeces of E. acervulina was 80 and 86%, of E. maxima 42 and
164
CHAPTER 3
50% and of E. tenella 75 and 70% in the groups receiving diets supplemented with or
without the protease, respectively. After 24 hr, 90 and 96% of E. acervulina and 90%
(both groups) of E. tenella oocysts were sporulated as for E. maxima oocysts these high
percentages (both groups 90%) were reached after 36 hr. These percentages remained
unchanged for the rest of the sporulation process.
Histopathology and sucrase-isomaltase activity (SIA)
The mean ( SEM) thickness and results of statistical analyses of the adherent mucus layer
of the duodenum, jejunum and caeca are shown in Table 4. A significant thicker mucus
layer was measured in the duodenum (P < 0.001), jejunum (P < 0.001) and caeca (P =
0.005) of birds with dietary protease enzyme supplementation.
The SIA ( SEM) of the mucosal scrapings per gram of intestinal protein (U/g protein) is
presented in Table 4. The SIA was significantly lower (P = 0.005) in mucosal scrapings
from the jejunum of chickens receiving the diet supplemented with the protease. No
significant influence of the supplementation of the protease on the SIA of the scrapings
obtained from the duodenum and caeca samples was noted.
Table 4. Mean thickness of adherent mucus layer (AML) (m) and mean sucraseisomaltase activity (SIA) (U/g protein) in mucus of duodenum, jejunum and caeca
Intestinal region
Duodenum
Duodenum
Jejunum
Jejunum
Caeca
Caeca
Protease
Yes
No
Yes
No
Yes
No
AML SEM
8.8 0.29a
6.3 0.39b
8.6 0.36a
5.4 0.20b
6.4 0.36a
4.9 0.31b
(n)
(61)
(35)
(64)
(35)
(45)
(23)
SIA SEM
29 3.2a
35 2.9a
78 6.9a
124 5.7b
-2 3.6a
7 3.5a
(n)
(10)
(5)
(10)
(5)
(10)
(5)
a, b
Groups with different superscript per intestinal region (duodenum, jejunum and caeca) are significantly
different from each other (P < 0.05).
Discussion
The mean CLS ( SEM) of birds given the non-supplemented diets and infected with E.
acervulina, E. maxima or E. tenella were 2.0 0.0, 2.8 0.2 and 0.2 0.2, respectively,
while for the supplemented birds the mean CLS were 2.6 0.25, 2.2 0.37 and 0.0 0.0,
respectively. The results of both treatment categories were not significantly different from
each other. Therefore, it seems reasonable to conclude that dietary supplementation of
protease did not influence the severity of coccidial lesions. This was also the case for the
oocyst shedding. The fact that the OPG levels for E. maxima and E. tenella were almost
165
similar while their lesion scores varied greatly can be explained by differences in oocyst
yield between these species (Williams, 2001).
The low mean CLS obtained with E. tenella was explained by the low inoculation dose
administrated as the isolate was known to be very pathogenic in previously performed
experiments (data not shown).
Only sporulated oocysts are infectious and it is known from literature that some
anticoccidial drugs diminish the infective potential of the excreted oocysts by reducing the
sporulation rate (Joyner & Norton, 1977; Ruff et al., 1978; Von Lwenstein & Kutzer,
1989; Arakawa et al., 1991). Nevertheless, it was shown that dietary protease
supplementation had no influence on the rate and onset of sporulation of all Eimeria
species studied, suggesting that in feed administration of protease will not reduce the
coccidiosis infection pressure in the poultry environment.
Significantly increased thickness of the mucus layer was measured in the
duodenum, jejunum and caeca regions compared to the same regions of non-protease
supplemented birds (Table 4). A possible explanation for this increase in mucus thickness
is a higher mucus production due to the activity of the protease. This can lead to a partial
immature layer of different composition and increased thickness. The variations in mucus
composition may result in local pH differences and subsequent differences in Alcian blue
staining as confirmed by the variation in coloration observed during the thickness
measurements.
The SIA measured in the jejunum of protease supplemented and non-supplemented
birds was greater than that of duodenum, which in turn was greater than that of caeca,
being in agreement with the work of Major & Ruff (1978) and Adams et al. (1996). The
SIA was lower in all mucosal scrapings of birds fed the protease supplemented diet. A
significant decrease was found in mucosal scrapings of the jejunum, whereas it was not
affected in the duodenum and caeca (Table 4). These measurements suggest a higher
mucosal turnover rate in the jejunum, which might represent a lower mucosal protection
against an Eimeria infection.
The effect of the protease treatment on BW gain was least in the uninfected group
(4 g), while a higher BW gain of 12 (4%), 33 (15%) and 10 g (4%) was observed in E.
acervulina, E. maxima and E. tenella infected birds, respectively.
Compared to the uninfected chickens, E. acervulina or E. tenella infected broilers with or
without protease did not show significant growth retardation, whereas the reduced body
weight gain in chickens infected with E. maxima with or without protease was shown to be
statistically significant. These observations agree earlier studies (Conway et al., 1993)
showing that impairment of body weight development is dependent on the numbers of
oocysts in the inoculum, which will also determine the severeness of the clinical
coccidiosis. In the present study, only the oocysts doses used for E. maxima were close to
the growth retardation-inducing doses described by Conway et al. (1993).
There was no significant interaction on BW gain (P 0.05) between the protease
supplementation and single species coccidiosis infections, therefore the main effects were
further investigated and showed a significantly (P = 0.046) higher average growth in all
groups receiving the supplemented diet.
166
CHAPTER 3
During a coccidiosis infection, BW gain suppression is generally caused by a

reduced feed intake, a disturbed digestibility and decreased nutrient utilization due to a
diminished digestive absorptive surface caused by damaged, shortened or flattened villi of
the intestinal cells (Fernando & McCraw, 1970). The protease can possibly have
influenced this process by initiating a higher rate of mucus production and jejunal mucosal
turnover resulting in an increased mucus layer thickness, which may have had a protective
effect against the parasite although in the present study it could not be measured using
CLS. The protease could also have stimulated feed intake resulting in better BW
development. Another explanation is that it may have prevented/reduced the leakage of
plasma proteins caused by parasite stages damaging the intestinal mucosa through
increased mucus thickness (Rose & Long, 1969; Schalm et al., 1975; Shane et al., 1985).
Also changes in the mucus layer induced by protease treatment may have favored
digestibility of nutrients. Alternatively a combination of these factors may have occurred.
In conclusion, the results of this study suggest that dietary protease supplementation
stimulates BW gain, which seems more prominent in coccidiosis infected broilers.
This experiment did not support the suggestion that an increased thickness of the mucus
layer reduced the absorbability of nutrients as the protease did stimulate mucus production
as well as BW gain. Differences in feed intake between treatments might have been a
confounding factor and need further research.
Acknowledgements
We acknowledge Peter C.J. Tooten for his assistance in the sucrase-isomaltase detection
assay, Dr. R.M. Dwars for performing the histopathology and Prof. Dr. J.A. Stegeman for
critically reading the manuscript.
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169
The effect of an in-feed mannanoligosaccharide

preparation (MOS) on a coccidiosis infection in broilers
M.A. Elmusharafa, H.W. Peekb, L. Nolletc and A.C. Beynena
a
Department of Nutrition, Faculty of Veterinary Medicine, Utrecht University, the

Netherlands
b
Animal Health Service (GD), Poultry Health Centre, Deventer, the Netherlands
c
Alltech Biotechnology Centre, Dunboyne, Ireland
Animal Feed Science and Technology (April 2007), 134(3-4), 347-354
Summary
In the current study with broiler chickens, the objective was to assess whether a
mannanoligosaccharide (MOS) preparation would have anticoccidial activity. One-day-old
birds were given a single, mild coccidiosis infection with a mixture of three Eimeria
species: Eimeria acervulina, Eimeria maxima and Eimeria tenella. Infected and noninfected birds were fed a diet without or with 10 g MOS/kg. Growth performance, feed
intake and feed conversion were not different (P > 0.05) among the treatment groups. The
feeding of the MOS preparation reduced oocyst excretion and diminished the severity of
coccidiosis lesions induced by E. acervulina (P < 0.05) but did not affect the lesions
mediated by E. maxima and E. tenella (P = 0.69 and 0.84, respectively). Further research
is necessary to explain the observed effects in terms of mechanisms and to assess their
practical relevance.
Keywords: broilers; coccidia; mannanoligosaccharide
EFFECT OF MANNANOLIGOSACCHARIDE (MOS) ON COCCIDIOSIS IN BROILERS
Introduction
The protozoan parasite of the genus Eimeria may cause coccidiosis in poultry, an
infectious disease causing major economic losses through increased mortality and reduced
growth (McDougald, 2003). In order to control coccidiosis in poultry, coccidial
vaccination, in drinking water and in-feed supplementation of anticoccidial products, are
commonly used. However, it is expected that within 5 years from now (2012), the
anticoccidial products currently used will be banned (van den Ban et al., 2005). Thus,
there is an urgent need for alternative agents to control coccidiosis in poultry. In this
context, mannanoligosaccharide (MOS) preparations, which are derived from the cell wall
of the yeast Saccharomyces cerevisiae, can be useful. In broiler chickens infected with
Eimeria tenella, the feeding of a MOS preparation was shown to reduce the number of the
asexual stage schizonts in the lamina propria of the caecum (Elmusharaf et al., 2006). The
outcome of that study was interpreted as evidence for a protective effect of MOS against
coccidiosis infection and enhanced immunity in broilers. Jeurissen et al. (1996) found in
immune chickens that significantly fewer sporozoites reached the crypt epithelium and
that the formation of schizonts was inhibited.
In a previous study (Elmusharaf et al., 2006), the chickens were infected with only
one species of Eimeria. In the present study, the possible anticoccidial activity of MOS
was further investigated in broiler chickens infected mildly with a mixture of three
Eimeria species: Eimeria acervulina, E. maxima and E. tenella. Infected and non-infected
birds were fed a diet without or with MOS. Growth performance, intestinal lesions and
oocyst excretion were measured.
Materials and methods

Animals and diets
The experiment was of a factorial design, with/without MOS and with/without Eimeria
mixture. Two hundred and fifty-six one-day-old male broiler chickens (Ross 308) were
purchased from a local hatchery. On the day of arrival (day one), the birds were wing
banded, weighed and randomly allocated to four treatment groups of 64 birds each. Each
group was further divided into eight replicates of eight birds each. All replicates were
housed in 32 separate wire-suspended cages equipped with plastic sides and bottoms
covered with clean wood shavings. Continuous lighting was provided. The temperature in
the cages was 32 C on arrival of the chickens and from day 8 of the experiment the
temperature was decreased gradually by 2 C every day until it reached 20 C by day
fourteen.
Two groups received the control diet and the other two groups were fed the basal diet
supplemented with MOS. The diets did not contain growth promoters or anticoccidial
products (Table 1). The experimental diet was prepared by adding a MOS preparation
(Bio-Mos, Alltech) at a level of 10 g/kg diet at the expense of an identical amount of
172
CHAPTER 3
cornstarch. The diets were in a pelleted form (2.5 mm in diameter). Throughout the
experiment, the birds had free access to their diet and tap water.
Body weights were measured on days 1, 7, 14 and 19. Amounts of feed provided and
leftovers were weighed per cage. Feed intakes were determined per week per cage and
expressed as g/bird/day. Feed conversion ratio is calculated as feed intake per cage divided
by weight gain of birds in the cage.
Table 1. Ingredients and the composition of the basal diet
Ingredient
Wheat (+ xylanase)
Maize
Peas
Soybean meal (467 g CP/kg)
Sunflower (320 g CP/kg)
Fish meal (720 g CP/kg)
Potato protein
Soybean oil
PremixA
Salt
Limestone
Sodium chloride
Calcium carbonate
Monocalcium phosphate
L-Lysine HCL
DL-Methionine
L-Threonine
Natuphos 5000G (phytase)
g/kg
250
321
50
225
40
25
15
40
5
1.7
13.5
5
1.7
4
0.8
1.7
0.5
0.1
Total
1000
Calculated chemical composition

AMEn (MJ/kg)
Crude protein (g/kg)
11.7
215.6
10 g premix consists of 24.0 mg Vitamin A (500,000 IU/g), 6.0 mg Vitamin D3 (100,000 IU/g), 60.0
mg Vitamin E (500 IU/g), 6.6 mg Vitamin K3 (purity, 22.7%), 100.0 mg Vitamin B12 (purity, 0.1%),
2000.0 mg biotin (purity, 0.01%), 1100.0 mg choline chloride (purity, 50%), 1.1 mg folic acid (purity,
90%), 65.2 mg nicotinic acid (purity, 100%), 16.3 mg d-pantothenate (purity, 92%), 4.5 mg Vitamin B6
(purity, 100%), 12.5 mg riboflavin (purity, 80%), 2.5 mg Vitamin B1 (purity, 100%), 32.00 mg
CuSO45H2O, 333.20 mg FeSO4H2O, 166.80 mg MnO, 1.00 mg Na2SeO35H2O, 220.00 mg
ZnSO4H2O, 4.80 mg CoSO47H2O, 0.56 mg KI, 100.00 mg ethoxyquin and 5742.94 mg corn meal as
carrier.
173
Coccidiosis infection
On day one, one group fed the control diet and one group fed the experimental diet were
challenged with a mixture of Eimeria containing 900 sporulated oocysts of E. acervulina
(Weybridge strain), 570 sporulated oocysts of E. maxima (Weybridge strain) and 170
sporulated oocysts of E. tenella (Houghton strain). The oocysts were obtained from the
Animal Health Service Ltd., Deventer, the Netherlands. The oocysts were laboratory
strains and the dose and the species used simulated commercially available live vaccines
(Williams, 2002).
The sporulated oocysts were administered with 1ml of tap water directly into the crop via
a scaled 1-ml syringe without needle. The non-infected groups were given 1 ml of oocystfree water into the crop. In order to avoid cross-contamination, the cages were equipped
with plastic sides, and the non-infected groups were always taken care of first. On day
fourteen, one bird per cage per treatment was randomly selected and killed by cervical
dislocation and used for lesion scoring. On day nineteen, three birds per cage and
treatment were killed by cervical dislocation, dissected and the different coccidial lesions
were scored.
Oocyst counting
Fresh excreta samples were collected from the four corners and the middle of each cage on
days 5, 7, 9, 12, 14, 16 and 19 of the experiment for oocyst counting. Excreta collection
was done in the evening and the samples were stored overnight in a refrigerator. The
amount of oocysts per gram faeces of each cage (eight samples/treatment) were counted
the next day and expressed as the number of oocysts per gram of faeces. For oocyst
counting, a modified McMaster counting chamber technique of Hodgson (1970) was used.
A 10% (w/v) faeces suspension in a salt solution (151 g NaCl mixed into 1 l of water) was
prepared. After shaking thoroughly, 1 ml of the suspension was mixed with 9 ml of a salt
solution (311 g of NaCl mixed into 1 l of water). Then, the suspension was put into the
McMaster chamber using a micropipette and the number of oocysts was counted (Peek &
Landman, 2003).
Lesion scoring
On days fourteen and nineteen of the experiment, eight and twenty-four birds per
treatment, respectively, were randomly selected and coccidial intestinal lesion scored. The
04 lesion scoring system of Johnson & Reid (1970) was used. The areas scored were the
upper, middle and the caecal regions of the intestine which are the natural infected sites for
E. acervulina, E. maxima and E. tenella, respectively. Based upon severity of the lesions, a
score of 0 (no lesions), 1 (mild lesions), 2 (moderate lesions), 3 (severe lesions) or 4
(extremely severe lesions) is recorded for each chicken. The severity of coccidial lesions
was scored while the investigator was blinded to treatment modality.
174
CHAPTER 3
All data for each variable were subjected to univariate analysis of variance using SPSS
(2006) (SPSS Inc., Chicago, USA). The oocyst values were logarithmically transformed
(log10 (X + 1)) to create a normal distribution before being analyzed, and lesion scores
were transformed using multinomial transformation. When significant treatment effects
were disclosed, differences between the four treatments were evaluated by the post hoc
multiple comparison least significant difference (LSD) test. Lesion scores were compared
using the non-parametric MannWhitney test. The level of statistical significance was preset at P 0.05.
Results
Chick performance in the various intervals of the experiment showed no significant
differences between all groups (P > 0.05) (Table 2). Oocysts were not detected in the
excreta obtained from non-infected groups. The pattern of oocyst shedding shows peaking
on days 5, 12 and 16. The two earlier peaks were most pronounced in the infected
controls. The feeding of MOS significantly lowered (P < 0.05) the number of oocysts per
gram of faeces on days five and twelve of the experiment (Figure 1). In the non-infected
birds, no lesions were found for either day fourteen or nineteen. Lesion scores for day
fourteen of the experiment showed no significant difference (P > 0.05) between the
infected birds fed either the diet without or with MOS (data not presented). For day
nineteen, there were twenty-four dissected birds per treatment and the results showed a
significant reduction (P 0.05) in E. acervulina lesions in the infected group fed the diet
with MOS (Table 3). The lesions induced by E. maxima and E. tenella showed no
difference (P > 0.05) between the two infected groups.
Table 2. Body weight gain (g), feed conversion (feed/gain) of uninfected and infected
broiler chickens fed diets without (Control) or with a mannanoligosaccharide preparation
(MOS)
Variable
Body weight gain
Days 1-7
Days 7-14
Days 14-19
Feed conversion
Days 1-7
Days 7-14
Days 14-19
Control
MOS
Pooled
SEM
P-value
Uninfected
Infected
Uninfected
Infected
86
241
264
97
247
264
85
253
265
93
229
262
4.9
9.2
11.9
0.293
0.319
0.999
1.26
1.28
1.54
1.20
1.46
1.45
1.22
1.37
1.44
1.22
1.48
1.43
0.04
0.06
0.09
0.73
0.48
0.81
175
Infected MOS
Infected Control
6.00
5.00
OPG
(x 105)
4.00
3.00
2.00
1.00
0.00
0
12
14
16
19
Day
Figure 1. Oocyst excretion pattern (number of oocysts per gram of faeces) of infected birds fed diets
without (control) or with a mannanoligosaccharide preparation (MOS).
* Statistically different P < 0.05, n = 8 samples/treatment.
Table 3. Mean lesion scores SEM and frequencies of lesions scores at day 19 in infected
birds fed diets without (control) or with a mannanoligosaccharide preparation (MOS)
Variable
Mean lesion scores
E. acervulina
E. maxima
E. tenella
Infected control
Infected MOS
0.46a 0.1
0.71 0.14
0.42 0.13
0.13b 0.09
0.63 0.13
0.33 0.1
Frequencies of lesion scoresA

Score
0
1
E. acervulina
13
11
E. maxima
10
11
E. tenella
16
6
a, b
2
0
3
2
3
0
0
0
0
22
11
16
1
1
11
8
2
1
2
0
P-value
< 0.05
0.690
0.841
3
0
0
0
Mean SEM values within the same row with different superscript letters are significantly different (P
< 0.05).
A
Number of birds dissected 24/treatment.
176
CHAPTER 3
Discussion
In the current experiment, the infection of broiler chickens with coccidiosis was successful
based on the intestinal lesions and oocyst shedding. No effect of the infection on
performance was seen. The lack of effect of infection on growth performance may relate
to the mildness of the infection. Under conditions of more severe infection with Eimeria,
weight gain is generally reduced (Johnson & Reid, 1970; Conway et al., 1993;
McDougald, 2003; Chapman et al., 2004).
The pattern of oocyst excretion as found in this experiment was fairly predictable
(Figure 1). This was because the initial infection dose was low and more or less equal to
the dose of a non-attenuated vaccine. Williams (2002) reported that in vaccinated birds the
oocyst stage of Eimeria that follow excystation initiate the vaccinal infection, hence
stimulating immunity, and during subsequent recycling of infection maintains this
immunity. The phenomenon establishes trickle infections that have been shown to be
very effective in stimulating protective immunity (Joyner & Norton, 1973; 1976). On days
five and twelve, the infected birds fed the diet with MOS showed significant lower peak
excretions of oocysts compared to the infected control (Figure 1). The MOS-induced
reduction in oocysts production indicates that it might have anticoccidial activities. Thus,
it can be concluded that the MOS preparation has the potential to lower the severity and
the pressure of the infection and at the same time maintain the oocysts production, which
is crucial for the re-infection and the maintenance of the immunity stimulated by the initial
infection. It has been shown that the feeding of a MOS preparation reduced the number of
the asexual stage schizonts in the lamina propria of the caecum broiler chickens infected
with E. tenella (Elmusharaf et al., 2006). The protective effect of MOS may be due to the
increase in the villi length and improved integrity and uniformity of the gut as observed in
chickens fed a diet supplemented with MOS (Loddi et al., 2002). Moreover, Ferket et al.
(2002) observed modulation of the systemic immunity in birds fed a diet fortified with
MOS. Williams (1995) has reported that there is reciprocity between the immune status of
chickens and their excretion of oocysts.
Intestinal lesion scores on day nineteen of the experiment showed that E. acervulina
lesions in the infected birds fed MOS were significantly reduced (P < 0.05), whereas the
severity of lesions produced by the two other species of Eimeria were reduced but not
significantly. The absence of the lesions due to E. maxima and E. tenella on day fourteen
is probably due to the very low initial dose and the low oocyst productive potential of both
species compared to E. acervulina (Brackett & Bliznick, 1952). The lesions seen on days
fourteen and nineteen should be considered as secondary and third lesions (Williams &
Andrews, 2001). Thus, it appears that the MOS preparation had anticoccidial activity
which counteracted the secondary and third (due to the recycling of the infection after the
initial infective dose) attack by E. acervulina and not the secondary and third attack by E.
maxima and E. tenella.
In conclusion, the results of the current experiment show that supplementation of
the diet with MOS preparation reduced oocyst excretion on days five and twelve of the
177
experiment and diminished the severity of E. acervulina lesions but not those due to E.
maxima and E. tenella infection.
References
Brackett, S. & Bliznick, A. (1952). The reproductive potential of five species of coccidia of the chicken
as demonstrated by oocyst production. Journal of Parasitology, 38, pp. 133139.
Chapman, H.D., Marsler, P. & LaVorgna, M.W. (2004). The effects of salinomycin and roxarsone on the
performance of broilers when included in the feed for four, five, or six weeks and infected with
Eimeria species during the starter or grower phase of production. Poultry Science, 83, pp. 761764.
Conway, D.P., Sasai, K., Gaafar, S.M. & Smothers, C.D. (1993). Effects of different levels of oocyst
lesion scores, and performance in chickens. Avian Diseases, 37, pp. 118123.
Elmusharaf, M.A., Bautista, V. & Beynen, A.C. (2006). Effect of a mannanoligosaccharide preparation
on Eimeria tenella infection in broiler chickens. International Journal of Poultry Science, 5, pp. 583
588.
Ferket, P.R., Parks, C.W. & Grims, J.L. (2002). Benefits of dietary antibiotic and mannanoligosaccharide
supplementation for poultry. In: Proceedings of the Multi-State Poultry Feeding and Nutrition
Conference, Indianapolis, Ind., May 1416.
Parasitology, 28, pp. 99102.
Jeurissen, S.H.M., Janse, E.M., Vermeulen, A.N. & Vervelde, L. (1996). Eimeria tenella infections in
chickens: aspects of hostparasite interaction. Veterinary Immunology and Immunopathology, 54, pp.
231238.
experiments with chickens. Experimental Parasitology, 28, pp. 3036.
Joyner, L.P. & Norton, C.C. (1973). The immunity arising from continuous low-level infection with
Eimeria tenella. Parasitology, 67, pp. 333340.
Joyner, L.P. & Norton, C.C. (1976). The immunity arising from continuous low-level infection with
Eimeria maxima and Eimeria acervulina. Parasitology, 72, pp. 115125.
Loddi, M.M., Nakaghi, L.S.O., Edens, F., Tucci, F.M., Hannas, M.I., Moraes, V.M.B. & Ariki, J. (2002).
Mannanoligosaccharide and organic acids on intestinal morphology of broilers evaluated by scanning
electrons microscopy. In: Proceedings of the 11th European Poultry Science Conference, Bremen,
Germany, September 610, pp. 121.
McDougald, L.R. (2003). Coccidiosis. In: Saif, Y.M., Barnes, H.J., Fadly, A.M., Glisson, J.R.,
McDouglad, L.R., Swayne, D.E. (Eds.), Poultry Diseases. Iowa State Press, Iowa, pp. 974991.
field isolates originating from 1996, 1999 and 2001. Avian Pathology, 32, pp. 391401.
SPSS (2006) SPSS 15.0 Command Syntax reference 2006. SPSS Inc., Chicago Illinois, USA.
van den Ban, E.C.D., Aarts H.J.M., Bokma-Bakker, M.H., Bouwmeester, H. & Jansman, A.J.M. (2005).
AMGBs en coccidiostatica in pluimveevoeders: Zijn er goede en veilige alternatieve
toevoegingsmiddelen? Rapport 05/I00648 Animal Sciences Group van Wageningen UR, Lelystad.
Williams, R.B. (1995). Epidemiological studies of coccidiosis in the domestic fowl (Gallus gallus): IV.
Reciprocity between the immune status of floor-reared chickens and their excretion of oocysts. Applied
Parasitology, 36, pp. 290298.
Williams, R.B. & Andrews, S.J. (2001). The origin and biological significance of the coccidial lesions
that occurs in chickens vaccinated with live attenuated anticoccidial vaccine. Avian Pathology, 30, pp.
215220.
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Williams, R.B. (2002). Anticoccidial vaccines for broilers chickens: pathway to success. Avian
Pathology, 31, pp. 317353.
179
Chapter 4
Influence of live coccidiosis vaccination on the

occurrence of anticoccidial drug resistance
Higher incidence of Eimeria spp. field isolates sensitive

for diclazuril and monensin associated with the use of
live coccidiosis vaccination with Paracox-5 in broiler
farms
Netherlands
Avian Diseases (September 2006), 50(3), 434-439
Summary
Twenty European Eimeria spp. field isolates were subjected to AST. The anticoccidial
drugs tested were diclazuril (Clinacox) and monensin (Elancoban). The assay was
performed in a battery cage trial. Infected medicated birds were compared with an
unmedicated control group. Coccidial lesion scores and oocyst shedding were used as
parameters. The results of the AST show that resistance is common amongst coccidiosis
field isolates especially E. acervulina (68 and 53% resistance for diclazuril and monensin,
respectively). Resistance is less frequent amongst E. maxima (38 and 50% resistance for
diclazuril and monensin, respectively) and E. tenella isolates (23 and 38% resistance for
diclazuril and monensin, respectively). A highly significant influence of the coccidiosis
prevention program (live coccidiosis vaccination with Paracox-5 versus anticoccidial
drugs in feed) on the sensitivity patterns of Eimeria spp. field isolates for both diclazuril
(P = 0.000) and monensin (P = 0.001) was found. Further, when looking at the single
species and each anticoccidial drug level significantly more sensitivity of E. acervulina for
monensin (P = 0.018), E. maxima for diclazuril (P = 0.009) and E. tenella for diclazuril (P
= 0.007) was found in isolates originating from vaccinated flocks. Moreover, for E.
acervulina and diclazuril, E. maxima and monensin, and E. tenella and monensin a trend
towards higher sensitivity of isolates for these products was found when live coccidiosis
vaccination was applied. The present study shows that sensitivity for the anticoccidial
drugs diclazuril and monensin is more frequent in Eimeria spp. field isolates originating
from broiler farms where a coccidiosis vaccination policy is followed.
Keywords: coccidiosis, broiler chickens, vaccination, anticoccidial drugs, sensitivity test,

Eimeria acervulina, E. maxima, E. tenella
CHAPTER 4
Introduction
Coccidiosis in poultry is caused by a protozoan parasite belonging to the phylum
Apicomplexa (Levine, 1988) and is of great economic importance to the poultry industry.
Despite large financial investments involved in the prevention and treatment of coccidiosis
its absolute control is still out of reach.
At present a number of strategies are used for the prevention and control of
coccidiosis in broilers. Until recently anticoccidial drugs in feed has been by far the most
popular approach, while in some cases an anticoccidial drug administered shortly via
drinking water is applied. Also, management measures focusing on good sanitation,
cleaning out contaminated litter and good litter condition in broiler houses are considered
of strategic value in controlling this parasitic disease. Lately, live coccidiosis vaccination
has added to this list and is becoming increasingly popular to control the coccidiosis
problem.
A major drawback of the use of anticoccidial drugs is the development of resistance,
which has been described for all anticoccidial drugs introduced so far (Chapman, 1986a,
1997; Peek & Landman, 2003, 2004). To minimize the occurrence of anticoccidial drug
resistance rotation of various anticoccidial drugs or shuttle programs (ideally based on
knowledge of the anticoccidial sensitivity profiles of field isolates) are used. The rationale
behind it is the reported loss of resistance following propagation of resistant parasites in
unmedicated birds (Chapman, 1986a) and chickens given unrelated anticoccidial drugs
(Ryley, 1980; Chapman, 1982). However, most studies indicate that resistance is stable
even in the absence of drug selection pressure (Gardiner & McLoughlin, 1963; Ball, 1968;
McLoughlin & Chute, 1968; Williams, 1969; Chapman, 1986b) which explains why these
measures have not completely solved the coccidiosis resistance problem in the field.
In case of resistance the use of live coccidiosis vaccines can be considered as an
alternative prophylactic measure against coccidiosis. Furthermore, a few studies have
reported an improved sensitivity of coccidiosis field isolates for anticoccidial drugs as a
welcome side-effect of coccidiosis vaccination due to seeding of the houses with drug
sensitive vaccine oocysts (Mathis & McDougald, 1989; Chapman, 1994; Chapman, 1996;
Newman & Danforth, 2000). However, large scale field studies reporting on the
occurrence of a higher incidence of anticoccidial drug sensitivity of coccidiosis isolates
associated with the use of live coccidiosis vaccination programs are lacking. Therefore, the
aim of the present work was to perform a study on the association between the coccidiosis
prevention program (vaccination with a live attenuated coccidiosis vaccine or anticoccidial
drugs in feed) and the occurrence of sensitivity for diclazuril and monensin of Eimeria
spp. field isolates.
183
INFLUENCE OF
VACCINATION ON DRUG RESISTANCE

Experimental design
Three experimental groups of nine chicks were used for each Eimeria spp. field isolate.
Group 1 was medicated with diclazuril (Clinacox product code 01-080.0) at a dose of 1
mg/kg feed, group 2 received monensin (Elancoban product code 01-126.0) at a dose of
100 mg/kg feed and the third group acted as an infected unmedicated control (IUC). For
all field isolates (n = 20) one uninfected unmedicated control (UUC) group of nine birds
was used as negative control. Both, the IUC and UUC groups were given feed free of
anticoccidial products.
Animals, housing and medicated feed
One-day-old male commercial broilers (Hybro) were reared coccidiosis free. At the age of
six days, two days prior to the inoculation (D -2) the birds were transferred ad random to
battery stain-less-steel cages (height x width x depth = 30 cm x 40 cm x 50 cm) and given
access to the medicated feed. The birds were subjected to 22 hours light per day. Feed and
water were supplied ad libitum.
The feed, a broiler starter (Apparent Metabolizable Energy (AME) 12.0 MJ/kg), was
mixed with commercial anticoccidial premixes and concentrations of in feed medication
was assessed by chemical analyses (Peek & Landman, 2003).
The birds were observed daily and deceased birds were subjected to postmortem
examination.
Eimeria spp. field isolates, identification, assessment of pathogenicity and preparation of
inocula
Twenty Eimeria spp. field isolates from six European countries were analyzed: Germany
(seven isolates), Italy (seven isolates), Greece (two isolates), Portugal (two isolates),
Romania (one isolate) and Denmark (one isolate). Oocysts were purified and sporulated as
described (Peek & Landman, 2003), followed propagation in SPF broilers. The inoculum
for propagation ranged from approximately 104 to 5 x 104 sporulated oocysts per chicken.
Birds were inoculated at eight days of age followed by postmortem analysis seven days
later at which time identification and assessment of the pathogenicity of the species
present in the field isolate were performed. The Eimeria spp. were identified by the
location and appearance of the gross lesions in the intestine and by microscopic
examination and measurement of the oocysts (Johnson & Reid, 1970; Long et al., 1976).
Faeces samples were gathered from day four (D +4) to 8 (D +8) post-inoculation, oocysts
were purified and sporulated for the AST.
At an age of eight days (D 0) chicks were inoculated in the crop with a defined number of
sporulated oocysts. The inoculation dose for the AST was adjusted to induce a lesion score
of 2 to 3 on the scale of Johnson and Reid (1970) and avoid mortality in the IUC (Conway
184
CHAPTER 4
et al., 1993; Peek & Landman, 2003). The oocyst countings of the inocula were performed
using a Fuchs-Rosenthal haemocytometer counting chamber. The inoculation doses are
outlined in Table 1.
Coccidial lesion scores, OPGs and evaluation of sensitivity
Six days post-infection (fourteen days of age) five birds per experimental group were
sacrificed for postmortem examination to determine the individual coccidial lesion scores
(ILSs). The ILSs were determined according to the method of Johnson and Reid (1970).
The mean lesion scores (MLS) per Eimeria spp. and experimental group (n = 5) was
subsequently calculated. In fresh collected faeces material the OPG per experimental
group were performed (Peek & Landman, 2003).
The anticoccidial sensitivity profile of each isolate was based on the percentage reduction
of the MLS per Eimeria spp. compared to the IUC group {McDougald formula: 100% (MLS of treated group / MLS of the IUC group x 100%)}. A reduction percentage of 030% indicates coccidial resistance, 31-49% indicates reduced sensitivity or partial
resistance and 50% or more indicates full sensitivity to the tested anticoccidial product
(McDougald et al., 1986).
Differences in coccidial lesion scores between medicated and non-medicated infected
groups were assessed statistically using the non-parametric Kruskal-Wallis test (StataCorp,
2004). Agreement analysis (Fleiss et al., 1969; Fleiss, 1981) was performed for all field
isolates per Eimeria spp. and anticoccidial drug between the results obtained with the
Kruskal-Wallis test and the sensitivity profiles obtained with the formula of McDougald et
al. (1986). If the Kruskal-Wallis test detected a significant difference in lesion scores
between medicated and non-medicated infected birds the isolate was rated as sensitive for
that particular drug, if the contrary happened, the isolate was rated as resistant. All profiles
of field isolates obtained with both methods were summed up per Eimeria spp. and
anticoccidial drug and subsequently ordered in 2 x 2 tables for agreement analysis.
Reduced sensitive isolates were considered resistant for the agreement analysis.
Then, the overall effect of treatments (anticoccidial drug or preventive vaccination) on the
occurrence of sensitive, reduced sensitive and resistant Eimeria spp. isolates for either
diclazuril or monensin was assessed with the Chi-Square test. For this purpose the total
number of isolates sensitive, reduced sensitive or resistant per anticoccidial drug were
counted and analyzed. Then, follow-up Chi-Square tests were performed counting the
isolates per profile category, species and anticoccidial drug to assess in which cases the
association of vaccination with sensitivity profiles were significant. Pearson Chi-Square
was used if cell frequencies were 5 in 80% of cells, otherwise Fishers exact test was
used (SPSS, 2001). For all tests differences were considered significant if P < 0.05.
185
INFLUENCE OF
Results
Chemical analysis of the anticoccidial products in the feed
The results of the chemical analysis show that the anticoccidial product concentrations in
feed were close to the desired dose. For diclazuril the concentration found was 1.1 mg/kg
and for monensin 103 mg/kg.
Eimeria spp. field isolates and inocula
During multiplication it became apparent that most field isolates consisted of mixtures of
two or three Eimeria spp. Only three isolates comprised a single species, namely E.
acervulina, which was found in all isolates except isolate 14 (Germany). E. maxima was
detected in sixteen field isolates and always occurred in combination with other species. E.
tenella was present in thirteen isolates in combination with one or two other species. The
mixture of three Eimeria spp. was prevailing and occurred eleven times. Field isolate
number, countries of origin, sampling date, coccidiosis prevention program in use,
previously used anticoccidial programs, Eimeria spp. identified in the field isolates and
inoculation dose (number of sporulated oocysts) per bird are given in Table 1.
Coccidial lesion scores, OPGs and evaluation of sensitivity
In Table 2 the MLS, OPGs and anticoccidial sensitivity profile of each Eimeria spp. field
isolate are outlined. In the IUC groups, the overall average lesion score for E. acervulina
was 1.58, for E. tenella it was 1.55 and for E. maxima 1.40. Mortality due to the
coccidiosis infection was only recorded in the IUC group of the Greek field isolate
(number 6). In this group 4/9 birds died due to the E. tenella infection, which seemed to be
quite pathogenic.
Moreover, a summary of the sensitivity profiles per Eimeria spp. in relation to the
coccidiosis prevention program is given in Table 3. Resistance was common amongst the
tested field isolates especially E. acervulina; 68.4% (13/19) isolates were resistant to
diclazuril, while 52.6% (10/19) showed resistance against monensin. Resistance was less
frequent amongst E. maxima and E. tenella field isolates. The former species showed
resistance in 37.5% (6/16) cases for diclazuril and 50% (8/16) for monensin. E. tenella
isolates showed resistance for diclazuril in 23.0% (3/13) cases and 38.5% (5/13) for
monensin.
The Kruskal-Wallis test showed significant differences between medicated and nonmedicated infected groups in a number of cases (Table 2). The strength of agreement
between the significances obtained with the Kruskal-Wallis test and the sensitivity profiles
ranged from good (Kappa () = > 0.6 0.8) to very good ( = > 0.8 1) for the sensitivity
profiles of E. tenella for monensin ( = 0.649 0.22), of E. acervulina for diclazuril ( =
0.759 0.16), of E. maxima for monensin ( = 0.871 0.12) and of E. tenella for
diclazuril ( = 0.831 0.16). The agreement was moderate ( = > 0.4 0.6) only for the
186
CHAPTER 4
profiles of E. acervulina for monensin (( = 0.565 0.22). Kappa-values were not

calculated for the sensitivity profiles of E. maxima and diclazuril, because here lesion
scores were not used to calculate statistical differences between medicated and nonmedicated infected groups or establishing the anticoccidial sensitivity profiles to
McDougalds formula. For the agreement analysis, ten reduced sensitive isolates were
considered resistant, of these seven did not show a significant difference between
medicated and non-medicated infected birds in the Kruskal-Wallis test.
187
Table 1. Field isolate number, countries, age (days) at collection of samples, coccidiosis prevention program in use, previous anticoccidial
(Ac.) programs, Eimeria spp. identified in the field isolates and inoculation dose (number of sporulated oocysts) per bird used in the
anticoccidial sensitivity test
Isolate
Country*
Age of
collection
31
Coccidiosis prevention
program in use
Monensin
Ac. not specified
Paracox-5
(2nd vaccination)
Previous anticoccidial programs
Eimeria spp.
E. acervulina/E. tenella
E. acervulina
E. acervulina/E. maxima/E.
tenella
1
2
3
DEU
GRC
ITA
DNK
39
salinomycin
ITA
34
monensin
GRC
26
Paracox-5
Five flocks or more with monensin

Ac. not specified
Three flocks maduramicin & monensin
and three flocks narasin/nicarbazin &
monensin
One flock narasin and 4 flocks
salinomycin
Four flocks salinomycin and two flocks
monensin
Three or more flocks Paracox-5
ROM
34
Ac. not specified
Ac. not specified
8
9
ITA
ITA
27
21
Paracox-5
Paracox-5
Six or more flocks Paracox-5

10
PRT
21-28
11
PRT
28-35
12
DEU
29
Six flocks narasin/nicarbazin &

salinomycin
Six flocks narasin/nicarbazin &
salinomycin
Ac. not specified
13
DEU
33
narasin/nicarbazin &
robenidine
maduramicin
Paracox-5**
(1st vaccination)
Ac. not specified
14
DEU
19
Paracox-5
27
Ac. not specified
Inoculation
dose (x 103)
21
53
27
E. acervulina/E. maxima
39
85
tenella
tenella
tenella
tenella
tenella
39
tenella
E. maxima/E. tenella
35
63
27
49
15
51
32
26
15
16
DEU
DEU
26
24
17
DEU
30
18
19
20
ITA
ITA
ITA
31
27
27
Paracox-5
monensin
monensin
Paracox-5
Paracox-5
Paracox-5
* = country abbreviations (ISO 3166).

** = considered as non-vaccinated.

One flock narasin/nicarbazin & monensin
and six flocks monensin
One flock narasin/nicarbazin & monensin
and six flocks monensin
Three or more flocks Paracox-5
E. acervulina/E. maxima/E. tenella

E. acervulina
63
62
E. acervulina
45

35
63
48
INFLUENCE OF
Chi-Square test: association with live vaccination

The results of the overall Chi-Square test showed a highly significant influence of the
treatment (vaccination versus other preventive coccidiosis program) on the sensitivity
profiles of the field isolates for both diclazuril (P = 0.000) and monensin (P = 0.001).
In Table 3 the results of the follow up Chi-Square tests of sensitivity profiles per Eimeria
spp. and anticoccidial drug versus treatment are given. When looking at the species level
we find significant results for the association of vaccination and the sensitivity of E.
acervulina for monensin (P = 0.018), of E. maxima for diclazuril (P = 0.009) and of E.
tenella for diclazuril (P = 0.007). These cases are responsible for the significance found
with the overall Chi-Square. For all remaining isolates a trend towards higher sensitivity
for the anticoccidial drugs tested was found if preventive vaccination had taken place on
the farms of origin.
Table 2. Mean lesion score (D +6), sensitivity profile (according to the criteria of
McDougald et al., 1986 and OPG (D +4 till D +8) per treatment and Eimeria spp. field
isolate
Field isolate
Treatment
1. (DEU)
DIC
MON
IUC
DIC
MON
IUC
DIC
MON
IUC
DIC
MON
IUC
DIC
MON
IUC
DIC
MON
IUC
DIC
MON
IUC
DIC
MON
IUC
DIC
2. (GRC)
3. (ITA)
4. (DNK)
5. (ITA)
6. (GRC)
7. (ROM)
8. (ITA)
9. (ITA)
190
Mean lesion scores (Sensitivity profile)**

(n = 5)
E. acervulina
E. maxima
E. tenella
1.0a (R)
0.6a (R)
1.0a (R)
0.6a (R)
a
0.8a
1.2
1.6a (R)
1.4a (R)
1.8a
1.6a (R) (*S)
0.0b (S)
1.6a (R)
a
b
0.8 (RS)
0.2 (S)
0.6a (S)
a
a
1.4
1.2
1.2a
1.2a (R)
1.2a (RS) (*S)
0.8a (RS)
0.4b (S)
1.4a
1.8a
0.4b (S)
1.0b (RS)
a
1.8 (R)
1.4a (R)
1.6a
2.0a
1.0a (R)
0.0b (S)
0.2b (S)
a
a
0.8 (R)
1.0 (R)
1.2b (S)
1.3a
3.2a
1.0a
1.2a (R)
2.6a (R)
1.2a (R)
1.6a (R)
2.0a (R)
2.6a (R)
a
a
1.4
1.2
3.0a
b
a
1.0 (S)
1.0 (R) (*S)
0.6b (S)
0.0b (S)
a
2.8
1.0a
a
1.0 (R)
1.0a (R) (*S)
0.0b (S)
OPG
(x 103)
2390
1470
3690
4890
939
1890
539*
1170
979
1290*
839
3590
839
1180
1690
679
969
1120
1420
1280
2790
569*
889
2090
2290*
CHAPTER 4
10. (PRT)
11. (PRT)
12. (DEU)
13. (DEU)
14. (DEU)
15. (DEU)
16. (DEU)
17. (DEU)
18. (ITA)
19. (ITA)
20. (ITA)
None
MON
IUC
DIC
MON
IUC
DIC
MON
IUC
DIC
MON
IUC
DIC
MON
IUC
DIC
MON
IUC
DIC
MON
IUC
DIC
MON
IUC
DIC
MON
IUC
DIC
MON
IUC
DIC
MON
IUC
DIC
MON
IUC
UUC
0.6a (S)
1.2a
2.0a (R)
1.4b (RS)
2.2a
0.8a (R)
1.0a (R)
1.0a
1.2a (R)
1.4a (R)
1.0a
1.0b (RS)
1.0b (RS)
1.6a
0.0b (S)
1.2a (RS)
1.8a
1.8a (R)
2.0a (R)
2.4a
1.4b (R)
1.8a (R)
2.0a
0.0b (S)
0.0b (S)
1.0a
1.4a (R)
0.0b (S)
1.4a
1.4a (R)
1.0a (R)
1.2a
0
0.0b (S)
1.2a
1.6a (R)
1.4a (R)
1.6a
1.2a (R)
1.2a (R)
1.6a
1.0a (R)
1.6a (R)
1.4a
1.6a (R)
1.2a (R)
1.4a
1.0a (R) (*S)
1.4a (R)
1.4a
1.2a (RS)(*S)
1.2a (RS)
1.8a
0.0b (S)
0.6a
1.2a (RS)
2.8a (R)
2.0a
2.0a (R)
1.0a (RS)
1.6a
1.4a (R) (*S)

1.0b (RS)
1.6a
1.4a (R) (*S)
0.4b (S)
1.2a
1.4a (R) (*S)
0.0b (S)
1.0a
0
0.4b (S)
1.4a (R)
1.8a
0.2b (S)
1.2a (R)
1.2a
0.0b (S)
0.4a (S)
0.8a
0
0.6a (S)
0.4b (S)
1.2a
0.0b (S)
1.0a (RS)
1.6a
0.0b (S)
0.6a (RS)
1.0a
719
2790
5690
719
4090
2590
1120
1990
3990
5790
5590
2690
1370
2630
0*
365
313
0,390*
1650
2790
4890
6890
3590
2990
4720
2990
69*
1360
1490
699*
1000
1290
1740*
930*
1180
0
* = No E. maxima oocysts were found in faeces.

**
DIC = diclazuril; IUC = infected unmedicated control; MON = monensin; OPG = oocyst per gram faeces;
UUC = uninfected unmedicated control.
a, b
Treatment groups with no common superscript within columns differ significantly from IUC group (P
< 0.05) (Kruskal-Wallis test).
191
INFLUENCE OF
Table 3. Summary of the anticoccidial sensitivity profiles per Eimeria spp. and
anticoccidial drug (diclazuril or monensin) in relation to the coccidiosis prevention
program. The significance of the association between the coccidiosis prevention program
and the sensitivity profiles found was assessed using the Chi-Square test (P-values are
found in the left column; P < 0.05 = significant)
Prevention program
Eimeria species
E. acervulina
(n = 19)
P = 0.111
E. maxima
(n = 16)
P = 0.009
E. tenella
(n = 13)
P = 0.007
Eimeria species
E. acervulina
(n = 19)
P = 0.018
E. maxima
(n = 16)
P = 0.060
E. tenella
(n = 13)
P = 0.767
Classification
Sensitive
Reduced sensitive
Resistant
Sensitive/total
Sensitive
Reduced sensitive
Resistant
Sensitive/total
Sensitive
Reduced sensitive
Resistant
Sensitive/total
Vaccination
Sensitivity profile diclazuril
4
0
4
4/8 (50%)
8
0
1
8/9 (89%)
8
0
0
8/8 (100%)
Classification
Sensitive
Reduced sensitive
Resistant
Sensitive/total
Sensitive
Reduced sensitive
Resistant
Sensitive/total
Sensitive
Reduced sensitive
Resistant
Sensitive/total
Sensitivity profile monensin

4
2
2
4/8 (50%)
5
2
2
5/9 (55%)
4
2
2
4/8 (50%)
Anticoccidial drugs
1
1
9
1/11 (9%)
1
1
5
1/7 (14%)
1
1
3
1/5 (20%)
0
3
8
0/11 (0%)
1
0
6
1/7 (14%)
1
1
3
1/5 (20%)
Discussion
The rate at which E. acervulina was found, was similar to previous studies (Peek &
Landman, 2003, 2004). However, E. maxima and E. tenella were detected more often.
Other authors also have found a higher number of Eimeria spp. in vaccinated flocks
compared to anticoccidial drug treated ones (Jenkins et al., 2005).
Ideally a lesion score ranging between 2 and 3 should be obtained. Adjustment of
the inoculum dose to obtain such lesions proved to be difficult in the present study because
most isolates consisted of two or three Eimeria spp. In these cases the most pathogenic
species determines the inoculation dose, which can result in lower lesion scores for other
192
CHAPTER 4
species lowering the average. Despite the fact that the obtained lesion scores were lower
than aimed, it was still possible to adequately calculate the reduction in lesion scores.
The results of the AST show that resistance is still common amongst European
coccidiosis field isolates especially E. acervulina (68 and 53% resistance for diclazuril and
monensin, respectively), although it was lower than in previous studies (Peek & Landman,
2003, 2004). Resistance was less frequent amongst E. maxima (38 and 50% resistance for
diclazuril and monensin, respectively) and E. tenella isolates (23 and 38% resistance for
diclazuril and monensin, respectively), also when compared to previously reported AST
(Peek & Landman, 2003, 2004). The higher incidence of sensitivity found was attributed
to the use of live coccidiosis vaccination on a number of farms from which the field
isolates originated, although a cause-effect remains to be demonstrated. Regarding the
higher incidence of sensitivity of E. maxima for diclazuril found in this study it was in part
explained by the fact that sensitivity here was assessed based on the absence of oocyst
shedding.
In some field isolates exposed to diclazuril E. maxima oocyst shedding was absent, while
at postmortem lesions containing oocysts were found. If so, these isolates were considered
to be sensitive to diclazuril. According to Verheyen et al. (1989) and Maes et al. (1989),
the absence of oocysts during E. maxima and E. brunetti infections is explained by the fact
that the normal pattern of oocyst wall establishment is completely disturbed after treatment
with diclazuril. It results in the formation of an abnormally thickened, incomplete oocyst
wall and the necrosis of the zygote in all fertilized macrogamonts of both species. This
explains the occurrence of E. maxima coccidial lesions without oocyst shedding, in which
case the field isolate is considered sensitive for diclazuril.
A number of manuscripts suggesting the restoration of anticoccidial drug sensitivity
in Eimeria spp. isolates have been published in the past. The first paper portraying this
phenomenon was by Ball (1966), who found that when a small number of oocysts of a
glycarbylamide-resistant E. tenella strain were mixed with a larger number of sensitive
oocysts and the combination was serially passaged in unmedicated chickens, the
percentage of recovered resistant oocysts diminished progressively at each passage. Later
studies (McLoughlin, 1970; Jeffers, 1976; McLoughlin & Chute, 1979) all pointed out that
an infusion of drug-sensitive oocysts into a drug-resistant population causes a decrease of
resistance within the mixed population, confirming Balls results (1966). This loss of
detectable resistance was attributed to dilution and not to a natural loss of resistance, while
Long et al. (1985) also suggested that drug-sensitive parasites are likely to dominate in the
absence of medication outgrowing their resistant counterparts possibly due to a
reproductive advantage.
Restoration of anticoccidial drug sensitivity (for monensin and salinomycin) under
field circumstances has been described after the use of the non-attenuated coccidiosis
vaccine Coccivac-B (Chapman, 1994; Chapman, 1996; Newman & Danforth, 2000),
drug sensitive parasites supposedly replacing the resistant isolates after one to five
vaccinated flocks. A similar effect has been reported briefly without further details on
anticoccidial sensitivity profiles for the vaccine Paracox-8 (Vertommen, 1996), in spite
of its precocious nature, and the attenuated vaccine Livacox (Oliveira, 2001).
193
INFLUENCE OF
How the resistance of Eimeria spp. parasites is reverted under experimental and
field circumstances is currently unknown. As mentioned previously, outgrowing of
resistant parasites by reproductive more advantageous sensitive strains and thereby
replacing the resident population might be a possibility. However, interbreeding between
vaccine and field parasites leading to improved anticoccidial sensitivity should also be
considered. Additionally, in case precocious vaccines are used not only could the
resistance be ameliorated, but the virulence of the field isolates could be reduced as well
(Williams, 2002). It has been shown that the precocious nature of some Eimeria strains is
a genetically stable trait (McDonald et al., 1986; Shirley, 1988), which will recombine
with that of drug resistance described by Shimura & Isobe (1994), resulting in a less
virulent interbreed.
In the present study a significant association between preventive coccidiosis
vaccination with live attenuated vaccines and the occurrence of sensitivity for diclazuril
and monensin of Eimeria spp. field isolates was found. Although a shift in the sensitivity
profiles was not demonstrated (for that matter anticoccidial sensitivity testing should have
been performed before and after the application of live coccidiosis vaccines), it might well
have occurred as demonstrated by others for other non-attenuated vaccines (Mathis &
McDougald, 1989; Chapman, 1994; Newman & Danforth, 2000).
References
Ball, S.J. (1966). The development of resistance to glycarbylamide and 2-chloro-4- nitrobenzamide in
Eimeria tenella in chicks. Parasitology, 56, pp. 25-37.
Ball, S.J. (1968). The stability of resistance to glycarbylamide and 2-chloro-4-nitrobenzamide in Eimeria
tenella in chicks. Research in Veterinary Medicine, 9, pp. 149-151.
Chapman, H.D. (1982). Anticoccidial drug resistance. In: The biology of the coccidia. P.L. Long, ed.
Baltimore, University Park Press. pp. 429-452.
Chapman, H.D. (1986a). Drug resistance in coccidia: recent research. In: Proceedings of the Georgia
Coccidiosis Conference. L.R. McDougald, L.P. Joyner & P.L. Long, eds. Georgia, USA. pp. 330-341.
Chapman, H.D. (1986b). Eimeria tenella: stability of resistance to halofuginone, decoquinate and
aprinocid in the chicken. Research Veterinary Science, 40, pp. 139-140.
Chapman, H.D. (1994). Sensitivity of field isolates of Eimeria to monensin following the use of a
coccidiosis vaccine in broiler chickens. Poultry Science, 73, pp. 476-478.
Chapman, H.D. (1996). Restoration of drug sensitivity following the use of live coccidiosis vaccines.
World Poultry special Supplement Coccidiosis, 2, pp. 20-21.
Chapman, H.D. (1997). Biochemical, genetic and applied aspects of drugs resistance in Eimeria parasites
Conway, D.P., Sasai, K., Gaafar, S.N. & Smothers, C.D. (1993). Effects of different levels of oocyst
lesion score, and performance in chickens. Avian Diseases, 37, pp. 118-123.
Fleiss, J.L. (1981). Statistical methods for rates and proportions. 2nd Edition, John Wiley & Sons, New
York, pp. 221.
Fleiss, J.L., Cohen, J. & Everitt, B.S. (1969). Large sample standard errors of Kappa and weighted Kappa.
Psychological Bulletin, 72, pp. 323-327.
Gardiner, J.L. & McLoughlin, D.K. (1963). Drug resistance in Eimeria tenella. III. Stability of resistance
to glycarbylamide. Journal of Parasitology, 49, pp. 657-659.
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Jeffers, T.K. (1976). Reduction of anticoccidial drug resistance by massive introduction of drug-sensitive
coccidia. Avian Diseases, 20, pp. 649-653.
Jenkins, M.C., Klopp, S., Wilkins, G. & Miska, K. (2005). Comparison of drug-sensitivity and species
composition of Eimeria isolated from poultry farms utilizing either anti-coccidial drugs or a live
oocyst vaccine to control avian coccidiosis. In Proceedings of the IXth International Coccidiosis
Conference, Foz de Ignuassu, Parana, Brazil. pp. 157.
Levine, N.D. (1988). The protozoan phylum Apicomplexa, 2 vols. CRC Press, Inc., Boca Raton, FL.
Long, P.L., Joyner, L.P., Miljard, B.J. & Norton, C.C.(1976). A guide to laboratory techniques in the
study and diagnosis of avian coccidiosis. Folia Veterinary Lat, 6, pp. 201-217.
Long, P.L., Johnson, J. & Baxter, S. (1985). Eimeria tenella: relative survival of drug resistant and drugsensitive populations in floor pen chickens. Poultry Science, 64, pp. 2403-2405.
Maes, L., Coussement, W., Vanparijs, O. & Verheyen, F. (1989). Species- specificity action of diclazuril
(Clinacox) against different Eimeria species in the chicken. In: Proceedings of the Coccidia and
intestinal Coccidiomorphs, Vth International Coccidiosis Conference, Tours, France. pp. 259-264.
Mathis, G.F. & McDougald, L.R. (1989). Restoration of drug sensitivity on turkey farms after
introduction of sensitive coccidia during controlled-exposure immunization. In: Proceedings of
Coccidia and intestinal Coccidiomorphs, Vth International Coccidiosis Conference, Tours, France. pp.
17-20.
McDonald, V., Shirley, M.W. & Bellatti, M.A. (1986). Eimeria maxima: characteristics of attenuated
lines obtained by selection for precocious development in the chicken. Experimental Parasitology, 61,
pp. 192-200.
McLoughlin, D.K. & Chute. M.B. (1968). Drug resistance in Eimeria tenella. VII. Acriflavine-medicated
loss of resistance to amprolium. Journal of Parasitology, 54, pp. 696-698.
McLoughlin, D.K. (1970). Coccidiosis: experimental analysis of drug resistance. Experimental
Parasitology, 28, 129-136.
McLoughlin, D.K. & Chute, M.B. (1979). Loss of amprolium resistance in Eimeria tenella by admixture
of sensitive and resistant strains. Proceedings of the helminthological society of Washington, 14, pp.
138-141.
Newman, L.J. & Danforth, H.D. (2000). Improved sensitivity of field oocyst populations to coccidiostat
following vaccination with live oocyst vaccine. Poultry Science, 79 (supplement 1), pp. 4.
Oliveira, C. de. (2001). Latest thinking on avian coccidiosis. In: Vaccination at Work in Commercial
Broilers. Driffield UK: Positive Action Publications Ltd. pp. 9.
Eimeria spp. veldisolaten voor anticoccidiose middelen. Tijdschrift voor Diergeneeskunde, 129, pp.
210-214.
Ryley, J.F. (1980). Drug resistance in coccidia. Advances in veterinary Science and Comparative
Medicine, 24, pp. 99-120.
Shimura, K. & Isobe, T. (1994). Pathogenicity and drug resistance of a recombinant line between a
precocious line and a drug resistant field isolate of Eimeria tenella. Proceedings Second Asian
Conference Coccidiosis, Guangzhou: China. pp. 119-123.
Shirley, M.W. (1988). Control of coccidiosis with vaccines, In: Proceedings Second Asian/Pacific
Poultry Health Conference, Surfers Paradise, Australia. pp. 129-157.
SPSS (2001). SPSS 11.0 for Windows. SPSS Inc., Chicago, Illinois, USA.
StataCorp. (2004). Stata/SE 8.2 for Windows. StataCorp, College Station, Texas, USA.
195
INFLUENCE OF
Verheyen, A., Maes, L., Coussement, W., Vanparijs, O., Lauwers, F. & Marsboom, R. (1989).
Ultrastructural evaluation of the effects of diclazuril on the endogenous stages of Eimeria maxima and
E. brunetti in experimentally inoculated chickens. Parasitology Research, 75, pp. 604-610.
Vertommen, M.H. (1996). Coccidiosis control methods. In: Proceedings of the Paracox European
Symposium, Annecy. France. pp. 11-20.
Williams, R.B. (1969). The persistence of drug resistance in strains of Eimeria species in broiler chickens
following a change in coccidiostat. Research Veterinary Science, 10, pp. 490-492.
196
Chapter 5
Improvement of live anticoccidial vaccines
Cross protection studies between Eimeria acervulina

and E. maxima, and E. tenella
H.W. Peeka, M.H. Vertommenb and W.J.M. Landmana,c
a
Animal Health Service (GD), Arnsbergstraat 7, 7418 EZ Deventer, the Netherlands

Zeelandia 4, 3901 GZ Veenendaal, the Netherlands
c
Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University,
Yalelaan 7, 3584 CL Utrecht, the Netherlands
b
Submitted
Summary
Three successive animal passages with extraintestinal blood stages of an Eimeria
acervulina strain were performed in commercial broiler chickens to select an E. acervulina
vaccine line. Subsequently, commercial broilers were vaccinated at one day of age by
coarse spray with graded doses of the obtained E. acervulina vaccine line to determine the
vaccination dose and to establish the protection against challenge with virulent strains of
E. acervulina, E. tenella and E. maxima with time. Vaccinated birds showed complete
protection against challenge with E. acervulina, partial cross protection against E. tenella
and no cross protection against E. maxima.
Keywords: Eimeria, chickens, extraintestinal stages, vaccine, cross protection
CHAPTER 5
1. Introduction
Coccidiosis, an intestinal disease of chickens, is caused by a protozoan parasite belonging
to the genus Eimeria within the family Eimeriidae and the Phylum Apicomplexa. In
poultry, this disease has a great economic impact and the worldwide costs for production
losses and prophylaxis against coccidiosis have been estimated at US$ 2400 million per
annum (Shirley et al., 2005).
During the past 50 years, control of avian coccidiosis has mainly relied on
prophylactic chemotherapy. In the broiler industry the largest sector of the poultry
industry chickens are given anticoccidial drugs from hatch until a few days before
slaughter. The intensive prophylactic use of anticoccidial products has resulted in the
development of resistance to all of them despite the strategic use of rotation and shuttle
programs (Chapman, 1997; Peek & Landman, 2003; 2004).
The increasing occurrence of resistance and the possible ban of anticoccidial drugs
as feed additives at least in Europe has further stimulated the use of vaccination, which
was first described in 1925 (Beach & Corl), as an efficient alternative control measure
against coccidiosis. Different types of anticoccidial vaccines have been developed during
the past decades and live vaccines are most frequently used. Although research exploring
the feasibility of subunit vaccines against coccidiosis has been substantial, no commercial
products except CoxAbic have been marketed yet. Identification of vital antigens in
coccidial immunology seems to be the major limiting factor (Shirley et al., 2007).
Live anticoccidial vaccines consist of sporulated oocysts of either non-attenuated
(wild type) or attenuated Eimeria spp. lines. To date two approaches have been used for
the attenuation of Eimeria spp. from chickens. The first approach is the use of a vaccine
composed of parasites obtained after serial passages in embryonated eggs and the second
approach is based on selection for precocity, which is characterized by a shortened
endogenous life cycle due to a decreased number of schizogony cycles. During the last
two decades numerous reports describing anticoccidial vaccines have been published and a
recent overview of available vaccines has been given by Williams (2002a) and Shirley et
al. (2005).
Major disadvantages of live anticoccidial vaccines are: 1/ propagation has to be
performed in the natural host, 2/ yield of oocysts is lowered for attenuated precocious
vaccines due to precocity characteristics and 3/ all relevant Eimeria spp. must be included
in the vaccine further increasing production costs. Coccidiosis vaccines could be produced
more efficiently if cross protection between Eimeria spp. would be relevant enough to
reduce the number of species included in the vaccine and/or reduce the vaccination doses.
Therefore, cross protection studies between an E. acervulina vaccine line obtained through
three successive animal passages with early extraintestinal blood stages of the parasite and
E. tenella, and E. maxima were performed.
199
IMPROVEMENT OF LIVE ANTICOCCIDIAL VACCINES
2. Material and methods

2.1. Eimeria spp. laboratory strains/lines
2.1.1. Challenge strains
The E. acervulina (Weybridge), E. maxima (Weybridge) and E. tenella (Houghton) strains
were obtained from the Veterinary Laboratory Agency (VLA), Weybridge, UK. The E.
acervulina (Weybridge) strain was provided in 1990 while the E. maxima (Weybridge)
and the E tenella (Houghton) strains were obtained in 2000. All Eimeria spp. strains were
rejuvenated approximately every half year.
The terminology for populations of Eimeria spp. used in this manuscript i.e. isolate, strain
and vaccine line are in agreement with the definitions given previously by Chapman et al.
(2005).
2.1.2. Vaccines lines
The E. acervulina (Weybridge) strain was used to obtain an E. acervulina vaccine line by
means of animal passages with extraintestinal blood stages.
All strains/lines were stored as oocyst suspensions at 2-8 C in a 2.5% potassium
dichromate (w/v) solution. During preparation of the inocula the potassium dichromate
was removed by means of centrifugation and assessment of the oocyst concentrations was
performed by a Fuchs-Rosenthal haemocytometer counting chamber. Inoculations were
given individually in the crop with a calibrated syringe without needle using a volume of
1-ml tap water.
2.2. Selection of the E. acervulina vaccine line through animal passages with early
extraintestinal blood stages of the parasite
Three passages of early extraintestinal blood stages of E. acervulina were performed by
subsequently infecting donor birds orally with approximately 107 sporulated oocysts per
bird and debleeding them within 3 hours post infection in order to inoculate recipient birds
with 3 ml heparinized blood as described by Fernando et al. (1987). E. acervulina oocysts
obtained from faeces of recipient birds after the animal passages with the early
extraintestinal blood stages were multiplied in SPF broilers in order to obtain sufficient
oocysts to proceed with further research.
2.3. Purity of Eimeria spp. strains/lines
The E. acervulina vaccine line used in vaccination Experiment 1 was multiplied once after
this trial to rule out contaminations with other coccidiosis species. During this
multiplication a high dose inoculum of 106 sporulated oocysts/bird was used and at 6 days
post inoculation (PI) of ten birds the intestines were thoroughly examined for coccidiosis
lesions of E. acervulina and other species according to the method of Johnson & Reid
(1970).
200
CHAPTER 5
In Experiment 2, at 13 and 20 days post vaccination (PV), five birds from the vaccinated
group were autopsied to determine the coccidial lesion scores and also rule out
contaminations with other species. At 25 days of age, just prior to the challenge infection,
three birds of the vaccinated group and three of the negative control group (non-vaccinated
group in the isolator) were autopsied and investigated for coccidial lesions according to
the method of Johnson & Reid (1970).
Moreover, the E. acervulina vaccine line was subjected to Polymerase Chain Reaction
(PCR) analysis performed as described by Haug et al., 2007 in order to assess its purity
also. Briefly, optimized single PCR arrays targeting the ITS-1 of seven valid Eimeria
species (E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox and E.
tenella) were performed. Additionally, the detection level of contamination of E.
acervulina vaccine line with E. tenella was studied by spiking the E. acervulina line with
decreasing concentrations of E. tenella oocysts (1%, 0.1%, 0.01% and 0.001%).
PCR procedure. The Eimeria spp. stock suspensions were washed to remove the potassium
dichromate by repeated centrifugation and resuspension in water. Subsequently, 1 ml of
the stock suspension of each Eimeria spp. containing 106 sporulated oocysts per ml was
centrifuged at 14,000 x g for 3 min. The pellet was resuspended in 500 l molecular
biological quality water and mechanically disrupted using a Magalyser (Roche) and green
beads (Roche, ref. 03358941) for 1 min at 6,500 x g. Thereafter, 20 l of a lysing reagent
(Lyse-N-Go. Pierce, ref 7882) was added to 20 l supernatant, mixed and incubated in a
heating block at 99 C for 2 min. Then the suspension was centrifuged for 3 min at 14,000
x g after which the supernatant was collected for PCR.
Amplification of the species specific ITS-1 sequences of the rDNA coding region was
based on a protocol described by Haug et al., 2007. Briefly, the PCR was carried out in a
reaction volume of 20 l, containing 200 M of each deoxynucleotide triphosphate, 8
pmol of species-specific forward and reverse primers, 2.75 mM MgCl2, 1x PCR buffer
(Applied Biosystems), 0.4 U DNA polymerase (AmpliTaq Gold, Applied Biosystems),
and 2 l of DNA template (sample). The amplification was performed in a thermocycler
(Variti, Applied Biosystems). The initial activation step was 5 min at 95 C and was
followed by 35 cycles of denaturation during 30 s at 95 C, annealing during 30 s at 65 C
and elongation during 1 min at 72 C. Final extension was done during 5 min at 72 C. Of
each PCR product 10 l was added to 10 l loading buffer and size fractioned by
electrophoresis at 80V during 1 h in 2% agarose gels containing 0.5 g/ml ethidium
bromide. The PCR products were visualized with UV light and identified by size using a
50 bp DNA ladder (Boehringer).
2.4. Experimental animals and housing
Multiplication and rejuvenation of the obtained E. acervulina vaccine lines was performed
in SPF (Specified Pathogen Free) broiler chickens (Animal Health Service (GD),
Deventer, the Netherlands). In the cross protection studies male broiler chickens (Arbor
Acres) purchased from a commercial hatchery (Putten, the Netherlands) were used.
201
Chicks were housed in stainless steel battery cages (height x width x depth = 30 x 40 x 50
cm) for the selection of E. acervulina lines through animal passages with extraintestinal
blood stages, the determination of the vaccination doses and the challenge experiments.
Birds were housed in isolated floor pens with a density of 20 chicks/m2 with wood
shavings as bedding during further vaccination studies.
A complete formulated broiler starter mash (Arkervaart-Twente, Nijkerk, the Netherlands)
(2868 kcal/kg, 203.9 CP/kg) free of anticoccidial products was used in all experiments.
Feed and water were provided ad libitum and during all trials a lighting scheme of 23 h of
light per day was used. The birds were observed daily and deceased birds were collected
for postmortem examination.
2.5. Oocyst counts (oocyst per gram faeces (OPG)), purification and multiplication
In the experiments regarding the selection of E. acervulina vaccine lines through animal
passages with extraintestinal blood stages, faeces from recipient birds were collected per
battery cage from day 3 or 4 until day 6 PI and assessed for the presence of oocysts.
Briefly: about 25 fresh droppings per cage were weighed and suspended in a 10% (w/v)
solution of sodium chloride (density = 1.1) and mixed until a homogeneous suspension
was obtained. In triplicate, 1 ml of this faeces suspension was mixed with 9 ml saturated
salt solution (density = 1.2) and this dilution was used to fill a McMaster counting
chamber for the examination of the presence of oocysts. If oocysts were present in the
counting chamber purification of oocysts from faeces was performed using a modification
of the salt flotation technique as described earlier by Peek & Landman (2003). After
purification, the oocysts were suspended in a 2.5% (w/v) potassium dichromate solution
and sporulated under aeration during 48-72 hours at a temperature of 29 1 C (Ryley et
al., 1976). Subsequently, the oocyst suspensions of the Eimeria spp. lines were multiplied
in SPF broilers for rejuvenation and identification as described by Peek & Landman
(2003) and refrigerated (2-8 C) in a solution of 2.5% potassium dichromate (w/v) until
further use.
2.6. Vaccination and challenge studies
2.6.1. Assessment of vaccination dose
Commercial male broilers were vaccinated with graded doses sporulated oocysts of the E.
acervulina vaccine line. Hereto, six groups of ten broiler chicks were vaccinated with
doses of 9 x 101, 4 x 102 or 6 x 102 sporulated oocysts per chick performed in duplicate.
The vaccination was applied by a coarse spray to day old chicks at a rate of 0.5 ml/chick.
Examination of individual cloacal faeces sampled on day 5 and 6 PV was done to
determine the percentage of coccidiosis positive chickens per vaccination dose. Therefore,
individual cloacal faeces samples ( 0.5 g) were homogenized with 4 ml of a saturated salt
solution using a Vortex mixer. This faeces suspension was then used to fill one
compartment of a McMaster counting chamber. Subsequently, the counting chamber was
examined for the presence of oocysts by light microscopy.
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CHAPTER 5
2.6.2. Experiment 1: vaccination of birds with the E. acervulina vaccine line and challenge
with a mixture of E. acervulina, E. maxima and E. tenella at day 7, 14, 21, 28 or 35 of age
Two groups of birds were used. Group 1, consisting of 50 birds, was housed in an isolator
(Beyer & Eggelaar, Utrecht, the Netherlands) and served as a non-vaccinated group.
Group 2, consisting of 149 birds, was housed in a floor pen and served as a vaccinated (V)
group. Birds of Group 2 were vaccinated at one day of age using a coarse spray. A dose of
6 x 102 sporulated oocysts of the E. acervulina vaccine line was applied using a volume of
0.5 ml per chick. The vaccine uptake was monitored by examining individual cloacal
faeces taken from ninety-six birds of Group 2 at six days PV (as described on the
assessment of the vaccination dose).
After vaccination, oocyst shedding per gram faeces (OPG) of the floor pen group was
monitored. The oocyst shedding was assessed in fresh faeces samples collected at 4, 5, 6,
8, 10, 13, 15, 17, 20, 22, 27, 31, 34, 36, 38 and 41 days of age by means of the oocyst
counting as described earlier.
Birds were challenged at day 7, 14, 21, 28 or 35 of age. At each challenge moment ten
randomly selected birds per group were transferred to the stainless cages. The challenge
inoculum consisted of a mixture of 105 E. acervulina (Weybridge), 3 x 104 E. maxima
(Weybridge) and 5 x 103 E. tenella (Houghton) sporulated oocysts. The inoculation dose
used, was adjusted to obtain a coccidial lesion score of 2 to 3 in the non-vaccinated birds
on the scale of Johnson & Reid (1970) and avoid mortality. Six days after challenge, five
birds (50%) per group were euthanized in order to determine the coccidiosis lesion scores.
Faeces samples were collected of the remaining five birds per group from day 5 until day 9
post challenge for assessment of the OPG.
2.6.3. Experiment 2: vaccination of birds with the E. acervulina vaccine line and challenge
with E. acervulina, E. tenella or with a mixture of both species at 25 days of age
Eighty commercial day-old broiler chicks were divided in two groups. Group 1 (n = 20)
was housed in an isolator while Group 2 (n = 60) was housed in an isolated floor pen.
Chicks in the floor pen group were vaccinated by coarse spray (0.5 ml/bird) with the E.
acervulina vaccine line using a dose of 5 x 102 sporulated oocysts/bird. Birds in the
isolator were used as negative control and were not vaccinated.
The vaccine uptake was monitored by examination of individual cloacal faeces samples
taken from forty birds of Group 2 at six days PV. During the growing phase the oocyst
excretion pattern of birds in the floor pen group was monitored by determining the number
of oocysts per gram of fresh collected faeces at 4, 5, 6, 9, 13, 15, 18, 20, 22 and 25 days of
age. In the non-vaccinated group, three mixed faeces sample were examined for the
presence of oocysts just prior to the challenge infection.
At 13 and 20 days PV, five birds from the vaccinated group were autopsied to determine
the coccidial lesion scores. At 25 days of age, just prior to the challenge infection, three
birds of the vaccinated group and the negative control group (non-vaccinated group in the
isolator) were autopsied and investigated for coccidial lesions.
203
At the age of 25 days, birds from both groups were transferred to the stainless battery
cages and infected with the challenge inocula. The inocula consisted of 9 x 104 sporulated
E. acervulina (Weybridge) oocysts, 8 x 103 sporulated E. tenella (Houghton) oocysts or of
a mixture of both species. For each challenge inoculum, five birds of Group 1 and ten
birds of Group 2 (floor pen) were used. Six days after the challenge, all birds per group
were euthanized in order to determine the coccidial lesion scores and oocyst counts were
performed in faeces collected from day 4 until day 6 PI.
2.7. Statistics
Differences in coccidial lesion score per Eimeria spp. between experimental groups in
both experiments were analyzed using the non-parametric Kruskal-Wallis test (Statistix,
2003). Differences were considered significant if P 0.05.
3. Results
3.1. Selection of the E. acervulina vaccine line through animal passages with early
extraintestinal blood stages of the parasite
During the animal passage with extraintestinal blood stages, recipient birds showed oocyst
positive faeces samples when inoculated with donor blood obtained 3 h PI with the E.
acervulina parent strain. E. acervulina vaccine lines obtained from faeces samples from
recipient birds at day 5 to 6 PI were randomly chosen for subsequent animal passages and
further investigations.
3.2. Vaccination studies
3.2.1. Assessment of vaccination dose
Clinical signs of coccidiosis were not observed in any of the vaccinated birds. At least
50% of the cloacal faeces samples contained oocysts after vaccination with the low dose
(90 sporulated oocysts) on both sampling days (five and six days post spraying). An
increasing number of oocyst positive cloacal faeces samples was found parallel with an
increased vaccination dose (Table 1). Therefore the highest vaccination dose was chosen
for further vaccination studies.
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CHAPTER 5
Table 1. Proportion of broilers (%) with oocyst positive cloacal faeces sample following
spray vaccination with an E. acervulina line at one day of age, related to vaccination dose.
Vaccination doses were studied in duplicate (A, B)
Experimental group
(vaccination dose; # sporulated oocysts)
1 (9 x 101)
2 (4 x 102)
3 (6 x 102)
Duplicate
(n = 10)
A
B
A
B
A
B
5 DPV*
6 DPV
5/10** (50)
4/5 (80)
5/8 (62.5)
8/8 (100)
8/8 (100)
5/7 (71)
3/6 (50)
5/8 (62)
4/8 (50)
6/8 (75)
6/6 (100)
7/8 (87.5)
DPV = days post vaccination.

No. of positive broilers/no. examined.
**
3.2.2. Experiment 1: vaccination with the E. acervulina vaccine line and challenge with a
mixture of E. acervulina, E. maxima and E. tenella with time
No clinical signs of coccidiosis were observed in the vaccinated and control group. The
proportion of individual positive cloacal faeces samples from birds in the vaccinated group
assessed at six days PV was 75/96 (87.1%) which indicates that the vaccination was
performed successfully.
The OPG of pooled faeces samples collected from the vaccinated birds in Group 2 showed
an oocyst shedding pattern that started at 5 days PV and ceased after 8 days PV.
Thereafter, oocyst excretion was evoked again at 10, 13 and 17 days of age.
The results of the challenge experiment showed that vaccination with the E. acervulina
line protected the birds almost completely against the homologous challenge from day 7 of
age onwards. There was no cross protection between E. acervulina and E. maxima. E.
maxima lesion scores did not differ significantly between the control and vaccinated group
at any point in time. In contrast partial cross protection was observed between E.
acervulina and E. tenella based on a reduction in the severity of the coccidial lesions. The
partial cross protection started at 14 days PV with an optimum on day 21 and 28 PV and
declining again at day 35. E. tenella coccidiosis lesions of vaccinated birds were
significantly lower than those of controls at 21 days PV (Table 2).
205
Table 2. Mean lesion score (MLS SEM) per Eimeria spp. and number of oocysts per
gram of faeces (OPG) per experimental group (i.e. vaccinated with an E. acervulina line
at one day of age and non-vaccinated) following a mixed challenge infection at different
ages with E. acervulina, E. maxima and E. tenella. MLS and OPG were assessed at six
days after challenge
Age challenge (days)
7
Vaccinated
Non-vaccinated
14
Vaccinated
Non-vaccinated
21
Vaccinated
Non-vaccinated
28
Vaccinated
Non-vaccinated
35
Vaccinated
Non-vaccinated
n
5
5
E. acervulina
0.4b 0.3
1.2a 0.2
MLS SEM
E. maxima
0.8a 0.2
0.8a 0.2
E. tenella
3.0a 0.0
3.0a 0.0
OPG (x103)
180
260
5
5
0.0b 0.0
2.2a 0.2
1.2a 0.2
1.0a 0.0
1.6a 0.7
2.4a 0.3
580
370
5
5
0.0b 0.0
2.4a 0.3
1.2a 0.2
1.0a 0.0
0.4b 0.3
1.8a 0.4
110
1100
5
5
0.0b 0.0
2.8a 0.2
1.8a 0.2
1.8a 0.2
0.6a 0.4
1.4a 0.3
35
93
5
5
0.0b 0.0
2.0a 0.3
1.8a 0.2
1.8a 0.2
1.4a 0.4
2.2a 0.2
150
610
a,b
Groups with different superscript per time point within column differ significantly.
Lesion scores differ significantly between groups if P 0.05.
3.2.3. Experiment 2: vaccination with the E. acervulina vaccine line and challenge with E.
acervulina, E. tenella or with a mixture of both species at 25 days of age
No clinical signs of coccidiosis were seen in the vaccinated and control group. The
proportion of individual positive cloacal faeces samples from birds in the vaccinated group
assessed at six days PV was 29/40 (72.5%).
The OPG of pooled fresh faeces samples collected from the vaccinated birds showed a
similar oocyst shedding pattern as Experiment 1. Oocyst excretion started at day 5 through
day 18 PV and oocysts were also found at day 22 and 25 PV but at lower numbers. No
oocysts were found in pooled faeces samples from control birds housed in the isolator.
The results of the challenge experiment showed that birds in the control group were fully
susceptible to both E. acervulina and E. tenella. No coccidial lesions of E. acervulina were
observed in vaccinated birds after homologous challenge. After heterologous challenge of
vaccinated birds a significant reduction of lesion scores was seen in comparison to control
birds. The same results were obtained after a mixed challenge with E. acervulina and E.
tenella (Table 3).
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CHAPTER 5
3.3. Purity of Eimeria spp. strains/lines

During the multiplication of the E. acervulina vaccine line, used in Experiment 1, no
intestinal lesions of any other Eimeria spp. than E. acervulina (with a mean score of 2.9)
were found.
Similarly in Experiment 2, also only E. acervulina coccidiosis lesions (with a mean score
of 1.6) were observed. Coccidiosis lesions were not found at other time points in any of
the examined birds.
The PCR generated strong signals for the positive control sample tested included
(Paracox-8). PCR products of the expected size were found for all known Eimeria spp.
(Figure 1). The results of the PCR of the E. acervulina vaccine line and the E. acervulina
(Weybridge) strain only generated products corresponding to these species (Figure 2). The
E. tenella strain used at decreasing concentrations to spike the E. acervulina vaccine line
could be detected up to a concentration of 0.01% (i.e. 102 oocysts E. tenella per 106
oocysts E. acervulina) (Figure 3).
Figure 1. Agarose gel electrophoresis of PCR products of the ITS-1 gene from different Eimeria spp.:
lanes 1-7 correspond to E. brunetti, E. mitis, E. necatrix, E. praecox, E. acervulina, E. tenella and E.
maxima respectively. Lane M is molecular weight marker (50 bp ladder).
207
Figure 2. Agarose gel electrophoresis of PCR products of the ITS-1 gene from different E. acervulina
strain/line: lanes 1-3 correspond to the negative control, the E. acervulina vaccine line and the E.
acervulina (Weybridge) strain, respectively. M lane is molecular weight marker (50 bp ladder).
Figure 3. Agarose gel electrophoresis of PCR products of the ITS-1 gene from different Eimeria species:
lanes 1-7 correspond to E. tenella (Houghton) strain, E. acervulina vaccine contaminated with 1% E.
tenella, 0.1% ibid, 0.01% ibid, 0.001% ibid, the E. acervulina vaccine line and the negative control
respectively. M lane is molecular weight marker (50 bp ladder). The lowest contamination level visible as
a faint line is 0.01% E. tenella (lane 4).
208
Table 3. Mean lesion score (MLS SEM) per Eimeria spp. and number of oocysts per gram of faeces (OPG) per experimental group (i.e.
vaccinated with an E. acervulina line at one day of age and non-vaccinated) following challenge at 25 days of age with E. acervulina, E.
tenella or a mixed inoculum of both species. MLS and OPG were assessed at six days after challenge
Challenge
E. acervulina
E. tenella
MLS SEM
n
Vaccinated
Non-vaccinated
10
5
E. ac
a
0.0 0.0
b
2.8 0.2
E. ten
-
E. acervulina/E. tenella
MLS SEM
OPG
0
10
n
10
6.43
E. ac
-
E. ac = E. acervulina, E. ten = E. tenella.

a,b
Groups with different superscript within columns differ significantly (P 0.05).
MLS SEM
E. ten
a
1.4 0.2
b
3.0 0.0
OPG
10
4,00
10
3.70
n
10
5
E. ac
a
0.0 0.0
b
3.0 0.0
E. ten
OPG
a
102.78
106.40
1.4 0.3
3.0 0.3
4. Discussion
The occurrence of extraintestinal stages of Eimeria spp. in poultry blood was first
described by Kogut & Long (1984), who were able to transfer coccidiosis to chickens by
the administration of blood and other tissues from turkeys and chickens previously
inoculated with Eimeria spp. The transfer of coccidiosis to recipient birds was possible up
to 4 days after inoculation of donor birds. Infective stages of E. acervulina, E. brunetti, E
maxima and E. praecox in blood, liver and spleen were later described by Fernando et al.
(1987). They found that the transfer of blood and tissue stages of the parasite was more
successful if done within a short time after inoculation of donors as confirmed by oocyst
shedding of recipient birds. Although no specific time was mentioned as optimal for the
transfer of stages of the parasite in blood, our results indicate that the highest rate of
successful transfer is achieved at three hours post inoculation of donor birds.
Vaccination of the birds by means of coarse spray using the medium and higher
vaccination dose (4 x 102 and 6 x 102 sporulated oocyst/bird) induced an average
percentage of coccidiosis positive chickens (oocyst positive cloacal faeces samples) of
81.3 and 62.5% for the medium dose at day 5 and 6 PV, respectively. The percentage
positive birds for the higher dose was 85.7 and 93.8% at day 5 and day 6 PV, respectively.
The higher dose was therefore used in the floor pen studies and induced percentages of
coccidiosis positive birds of 87.1% and 72.5% in Experiment 1 and 2 at day 6 PV,
respectively. These percentages are in agreement with results obtained after hatchery spray
vaccinations performed by Cherry T.E. in Chapman et al. (2002) and by Schetters et al.
(1999). The method used to determine oocyst shedding was relatively insensitive as the
yield of cloacal faeces was often below expectation and only one compartment of the
McMaster counting chamber was examined. Therefore, the actual number of chicks
shedding oocysts after vaccination may have been higher.
The OPG counts performed after vaccination of the birds in Experiment 1 and 2
showed oocyst shedding patterns, which have been described for trickle infections, as
recycling of coccidiosis infections is a well-known pattern occurring after spray
vaccination with live coccidiosis vaccine consisting of several Eimeria spp. (Williams,
2002b). In the present study, the trickle infection pattern was dominated by a prominent
peak in the oocyst shedding pattern at day 13, probably because the vaccination was
performed with a single Eimeria spp. line (E. acervulina) with a relative short prepatent
period of 97 h.
The results of Experiment 1 and 2 showed that vaccination of birds with the E.
acervulina vaccine line obtained through animal passages of extraintestinal blood stages
conferred excellent protection against homologous challenge provided as a mixed
infection including also E. maxima and E. tenella. The protection was already noticeable at
7 days PV, showing maximal effect from 14 days PV onwards. The homologous
protection found is in agreement with described literature for other E. acervulina lines
(Shirley & Millard, 1986; Rose, 1987; Williams, 1992).
The E. tenella lesion scores found in the vaccinated chickens of Experiment 1
decreased with time starting to be noticeable at 14 days PV reaching a maximum at 21
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CHAPTER 5
days PV (being significantly different, P = 0.0214) indicating the occurrence of partial

cross protection between the E. acervulina vaccine line tested and E. tenella. The decrease
in E. tenella lesion scores at day 28 PV was lower but still in the same range as that of day
21 PV. However, the partial cross protective effect became less at day 35 PV suggesting
that it probably only lasts a few weeks. Nevertheless in order to get a definitive answer on
this matter longer follow up studies should be performed. The significant differences in
coccidiosis lesion scores between E. acervulina vaccinated and non-vaccinated coccidiose
challenged birds at different time points as shown in Table 2 were calculated using the
non-parametric Kruskal-Wallis test. If corrections for multiple comparisons were made
then 2 out of 6 significant differences (E. acervulina challenged at day 7 and E. tenella
challenged at day 21) became non significant. However, this would not affect the
significance of the reduction in lesion scores found (E. acervulina challenged at day 7 with
a mean lesion score of 0.4 versus 1.2 and of E. tenella challenged at day 21 with a mean
lesion score of 0.4 versus 1.8). This is demonstrated by the fact that a reduction in mean
lesion score of 50% or more would change the classification of an Eimeria spp. field
isolate subjected to an anticoccidial sensitivity test from resistant to sensitive (McDougald
et al., 1986). Therefore, results were presented without the latter corrections. Moreover,
the clinical and statistical significance of our findings was confirmed in Experiment 2
where similar results were obtained.
No signs of reduction in lesion scores were found after challenging birds with E. maxima.
As interaction between Eimeria spp. in a mixed challenge model can not be ruled
out, the challenge in Experiment 2 was performed using both, single species inocula and
inocula consisting of a mixture of E. acervulina and E. tenella. A number of interactions
between species in a mixed infection model have been reported. E. acervulina decreased
the oocyst output of other Eimeria spp. in simultaneous mixed infection (Williams, 1973;
Hein, 1975, while Tyzzer (1929) observed a late onset of immunity of Eimeria spp. during
mixed infections.
In Experiment 2, no coccidiosis lesions (for single and mixed species infection) and
oocyst shedding (for single species infection) were found after homologous challenge at
25 days PV indicating an excellent protection. Partial cross protection as described in the
first experiment was confirmed in Experiment 2, although the reduction in E. tenella
lesions, which were significantly different from those of challenged control birds, was less
pronounced.
Cross protection could have been induced by contamination of the E. acervulina
vaccine line with E. tenella, however, the purity of isolates was assessed by both, the
occurrence and distribution of coccidiosis lesions scores, and by means of PCR analysis.
In neither case, indications for a contamination of the E. acervulina vaccine line with E.
tenella were found. The PCR analysis was able to detect contamination levels of 100
oocysts of E. tenella per million oocysts of the E. acervulina vaccine line (Figure 3)
Protection against Eimeria spp. was for long believed to be species specific (Tyzzer
et al., 1932; Johnson, 1938). However, Rose was the first to report cross protection
between E. tenella and E. necatrix (Rose, 1967a). She also reported on the occurrence of
cross immunisation between E. brunetti and E. maxima (Rose, 1967b), which could not be
211
confirmed by Hein (1971). More recently, Augustine (1999) suggested that E. acervulina
elicits a small degree of protection against infection with E. tenella that is expressed
primarily in increased weight gain. In contrast, reverse immunisation, (i.e. with E. tenella)
had little effect on the response of chickens to E. acervulina infection.
The disadvantages of the conventional coccidiosis vaccines (Lillehoj and Trout,
1993; Jeston et al., 2002) have propelled the quest for the alternative products using a.o.
epitope mapping, hybridoma and recombinant DNA technology. Species cross immunity
to Eimeria is very important for the development of such vaccines and has been focus of
research during recent years. Partial resistance to E. tenella and also to E. acervulina, E.
maxima and E. necatrix was obtained using the recombinant antigen CheY-So7 of E.
tenella (Crane et al., 1991). Thereafter, cross protection against challenge with E.
acervulina but not E. maxima was found after in ovo vaccination with the E. tenella
EtMIC2 gene (Ding et al., 2005). Recently, partial cross protection of an E. tenella DNA
vaccine against challenge with E. necatrix and E. acervulina, but not E. maxima was
shown (Song et al., 2009). However, the new approach has failed to yield a successful
commercial anticoccidial vaccine so far, live attenuated anticoccidial vaccine being most
commonly used to date. Therefore, we pursued to further investigate cross protection
between E. acervulina and E. tenella using live parasites.
In the present study, further research into the cross protection between E. acervulina
and E. tenella was performed. In contrast with Augustines work (1999), in which twoweek-old birds were immunized with five inoculations of 3 x 105 sporulated oocysts of E.
acervulina during a two week period, our chickens were vaccinated once by coarse spray
at day-old and allowed to be auto exposed to a trickle infection by housing them in floor
pens mimicking more closely the situation in the field. Another difference was that
challenge was performed at different time intervals instead of 11 days after the last
immunisation, i.e. at thirty eight days of age, which is too late for the immunisation of
broiler chickens in the field. A third difference was that the birds were housed in cages,
while in this study they were placed in floor pens making repeated immunizations
redundant. A fourth difference was that challenge experiments were performed using a
mixture of E. acervulina, E. maxima and E. tenella (Experiment 1) or single species
consisting of E. acervulina or E. tenella, or a mixture of those (Experiment 2) instead of E.
tenella only. A fifth difference was that the degree of cross protection in our experiments
based on lesion scores was much higher and a last difference is that we used broilers
instead of white leghorn chickens.
Although the lower degree of cross protection found by Augustine (1999) could by
explained by differences in immunisation protocol, housing and breed of chickens, the
selection method to obtain an E. acervulina line trough animal passages of extraintestinal
blood stages may have resulted in a line with enhanced cross protective capabilities.
Further research comparing E. acervulina lines from different sources could unravel
whether differences in cross protective properties exist. E. acervulina lines showing
enhanced cross protection against E. tenella could then be used to improve current live
anticoccidial vaccines.
212
CHAPTER 5
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Beach, J.R. & Corl, J.C. (1925). Studies in the control of avian coccidiosis. Poultry Science, IV, pp. 8393.
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efficacy and savety of live anticoccidial vaccines, and obtaining approval for their use in chickens and
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214
Chapter 6
SUMMARIZING DISCUSSION
The aim of the thesis was to investigate the occurrence of Dutch and other European
Eimeria spp. field isolates resistant against anticoccidial drugs (Chapter 2). This was
preceded by an extensive literature review on anticoccidial products and alternative
prevention strategies (Chapter 1). Other aims of the thesis were to assess the anticoccidial
potency of ibuprofen, a mucolytic enzyme and mannanoligosaccharide as alternative
coccidiosis control strategies (Chapter 3) and to investigate the influence of vaccination on
the anticoccidial drug sensitivity profiles of Eimeria spp. field isolates (Chapter 4).
Finally, cross protection between an E. acervulina vaccine line and E. tenella, and E.
maxima, which may in future help to improve coccidiosis vaccines, was studied (Chapter
5).
In Chapter 1, coccidial infections with Eimeria spp. in poultry are described. After
an introduction, the history and classification of the genus Eimeria is given, followed by a
description of the occurrence of Eimeria spp. in farm and game birds and their clinical
significance. Further attention is paid to the life cycle of avian Eimeria spp., species
determination and diagnosis. In chickens, seven Eimeria spp. are considered of which E.
acervulina, E. maxima and E. tenella are the most relevant as they most frequently affect
broilers and these birds form the major part of the poultry industry. The diagnosis of
coccidiosis is generally performed at postmortem by identifying the localization and
morphology of the lesions in the intestines and is complemented by analyzing native
intestinal scrapings with light microscopy in order to morphologically identify schizonts
and oocysts. Due to the shortcomings in the traditional diagnosis system (visual
identification of species is not accurate), new coccidiosis identification technologies are
being developed. Recently, identification of Eimeria oocysts was greatly improved by
classification through computational analysis of microscopic analysis. Also, molecular
biology techniques have been the focus of research and development by many groups
involved in coccidiology. Various PCR-coupled and other molecular techniques for the
identification of coccidia have been studied during the past decade. Particular attention has
been given to the usefulness of ITS-1 and -2 markers for the diagnosis and the analysis of
genetic variation of Eimeria. It is a dynamic, constantly evolving area of research.
In the second heading of the review, the host and site specifity of Eimeria spp. is
presented. Host specificity of coccidia was thought to be very strict; however, research
showed that this is only true as far as completion of their life cycle is concerned. Eimeria
spp. are able to infect other species of hosts, restrictions being less pronounced the closer
host species are genetically related. Mechanisms responsible for host specificity are
parasite biochemistry and nutrition, the host genome and host immune system. Regarding
site specificity, coccidia invade narrowly defined cell types within a tissue or organ. In
chickens, Eimeria spp. parasitize and develop only in specific defined regions of the
intestine, which not only is the case in the natural host but also in foreign hosts.
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CHAPTER 6
In the third part of the review a historical overview is given of the mode of action,
the occurrence of resistance and efficacious use of anticoccidial products. Most
anticoccidial drugs have more than one biochemical effect on a specific developmental
stage of coccidia. Despite the lack of detailed knowledge on the selective action of
anticoccidials, a broad categorization of their mode of action has been performed. Products
affecting cofactor synthesis are ethopabate, sulphonamides, pyrimethamines and thiamine
analogues like amprolium; products affecting mitochondrial functions are quinolones
(buquinolate, decoquinate and nequinate), pyridinols (meticlorpindol), nicarbizin,
robenidine and toltrazuril; products that affect cell membrane function are polyether
antibiotics or ionophores (monensin, semduramycin, narasin, salinomycin, lasalocid and
maduramicin) and products with unknown mode of action are diclazuril and halofuginone.
The worldwide use of anticoccidial drugs has inevitably led to the development of
resistance to all anticoccidial products used to date as long term exposure to any drug will
result in loss of sensitivity. Resistance against anticoccidial drugs has been reported in
scientific literature in the United States, South America, Europe and China. In order to
minimize the occurrence of resistance, rotation (an anticoccidial product is used during a
maximum of two months or two fattening periods) of various anticoccidial drugs or shuttle
programmes (two or more anticoccidial drugs are used within a growing period) are used.
The rationale behind this is the fact that loss of sensitivity is related to the duration of drug
exposure, which should be kept as short as possible. Due to the occurrence of cross
resistance between anticoccidial drugs, anticoccidial drugs with distinct mode of action
should be used within rotation and shuttle programmes. Ideally, rotation and shuttle
programmes should be designed based on knowledge of anticoccidial sensitivity profiles
of relevant Eimeria spp. field isolates. A relative new approach in the management of
anticoccidial drug resistance is the incorporation of live coccidiosis vaccines into rotation
programmes. Seeding of poultry houses with drug sensitive lines of vaccine coccidia has
shown to increase the incidence of anticoccidial drug sensitive Eimeria spp. field isolates.
In the fourth heading, the influence of feed structure, composition (macro and micro
ingredients, special additives), toxins and feed management on the course of coccidiosis is
reported. Various feed components seem to affect Eimeria parasites, either directly by
affecting the development and multiplication of coccidia or indirectly by stimulating the
immune system, influencing the intestinal flora and mucosa, changing the physiology of
the digestive tract, etc. Some feed components show activity during the acute phase of the
disease, while others favour recovery of infected birds. Feed components that have been
shown to favour recovery from coccidiosis infections are: proteins, vitamin A, C and K.
Feed components which negatively affect Eimeria parasites directly are -3 fatty acids,
while variable effects have been described for protein concentration and feed restriction.
Development of coccidia is negatively influenced in an indirect way by -glucans, vitamin
E, selenium, feed restriction, -3 fatty acids, vitamin A and C and NaHCO3, which boost
the immune system. The development of coccidia is also negatively influenced indirectly
by vitamin K, medium chain fatty acids and, vitamins A and C. These components
promote mucosal health and therefore are of help against coccidiosis. Variable indirect
effects on the parasite have been described for wheat affecting viscosity and intestinal
217
flora, the latter is also influenced by fibres and milk products. Whole cereals indirectly
affect Eimeria by their influence on the physiology of the digestive tract. Finally, feed
components known to stimulate directly Eimeria development are: calcium, aflatoxins,
vitamin B, essential fatty acids and chemotrypsine. These products should be avoided or if
necessary not given at Eimeria promoting concentrations.
In heading five, other preventive strategies against coccidiosis including
management and biosecurity, alternative methods of control like homeopathy,
phytotherapy and vaccines, have been discussed. Management and biosecurity measures
should aim at avoiding the introduction of the parasite to the poultry premises through
isolation, traffic control and sanitation; although in practice it is nearly impossible to raise
chickens free of coccidiosis, unless SPF-like housing conditions are used. Under field
circumstances infected poultry houses are best sanitated by using a lot of cleansing water
and disinfection with a 5-10% solution of ammonia.
Phyto- and aromatherapy have become increasingly popular during the last decade due to
a renewed interest in natural medicine. A number of plant extracts have been studied for
their potential use as dietary supplement against coccidiosis. Products showing a direct
inhibitory effect on the development of Eimeria parasites are artemisinin and citric
extracts. Oregano also showed a direct effect on coccidia, however, it had also some
conflicting results about the inhibitory effect on the development of the Eimeria parasite.
Herbal products showing an indirect effect on the development of coccidia have also been
described. Echinacea, mushrooms (lectin) and turmeric seem to boost the immune
response and therefore soothen the severity of coccidiosis infection, while betain acts
indirectly by stimulating the immune response but also due to its osmoprotective
properties in the intestinal environment. The prebiotic (MOS) has been reported to act
directly on coccidia and either inhibit the development of the parasite or show no effect.
Probiotica have an indirect hindering effect on Eimeria parasites by stimulating the
humoral immune response.
Live vaccines, both attenuated and non-attenuated are becoming increasingly popular as an
alternative control strategy against coccidiosis. Nevertheless, a number of drawbacks such
as high production costs, loss of infectivity with time affecting their expiry and
management shortcomings during application (dosage errors, presence of anticoccidial
drugs in feed frustrating vaccination with drug sensitive vaccine strains, etc.) have limited
their use in practice. Additionally, reversal of virulence of live vaccines may also be a
point of concern. An important beneficial side effect of live coccidiosis vaccines is their
association with increased sensitivity of Eimeria spp. to anticoccidial drugs by seeding of
poultry houses with drug sensitive vaccine parasites. This has promoted their use in
alternation with anticoccidial drugs in rotation programs. Subunit vaccines may
circumvent most shortcomings of live vaccines; however, at present these products
underperform due to lack of immunogenicity. Identification of antigens eliciting protective
immune responses through the E. tenella genome project may hold promise for a more
prominent role of subunit vaccines in the control of coccidiosis.
It can be concluded that the occurrence of anticoccidial drug resistant Eimeria spp.
field isolates will at best remain unchanged, but most likely will further increase if
218
CHAPTER 6
commercial poultry farming keep using anticoccidial products in a non-rational fashion

(i.e. not guided by anticoccidial drug sensitivity tests and timely shift in anticoccidial
drugs). Although some dietary compounds, phytochemicals, pre- and probiotica and even
non-steroidal anti-inflammatory drugs seem capable to reduce the effects of an Eimeria
infection either by a direct effect on the parasite or indirectly by stimulating immune
responses and recovery of the birds, this seems to be insufficient to control the disease.
Therefore, named strategies do not represent an effective alternative to solve the problem
of anticoccidial drug resistance. At present, vaccination seems to be the most solid
anticoccidiosis strategy, despite some drawbacks. In case immunogenicity of subunit
vaccines can be improved substantially, these vaccines might form the core of future
anticoccidial strategy.
In Chapter 2 the anticoccidial sensitivity profiles of a variety of Eimeria spp. field
isolates gathered from broiler farms are outlined.
In two studies fifteen Eimeria spp. field isolates sampled on Dutch broiler farms,
four from 1996, four from 1999 and seven from 2001 and twenty-four Eimeria spp. field
isolates originating from Dutch, German and Spanish broiler farms collected in the period
2000-2002 were subjected to an Anticoccidial Sensitivity Test (AST) in a battery cage
study. The selected anticoccidial drugs were diclazuril, halofuginone, lasalocid,
maduramicin, meticlorpindol/methylbenzoquate, monensin, narasin, narasin/nicarbazin,
nicarbazin, robenidine and salinomycin. The results showed a high degree of resistance
against most tested anticoccidial products. However, despite the high prevalence of
coccidiosis infections in the field in the Netherlands, an increase of severe clinical
coccidiosis was not observed. Possible spontaneous vaccination occurs in the field as
despite anticoccidial drug resistance a residual effect of anticoccidial drugs (especially
ionophores) would mitigate the severeness of coccidiosis without inhibiting immunisation.
Taking together, during the period 1996-2002 a total of thirty nine Eimeria spp.
field isolates, of which the majority contained more than one species, were investigated for
their sensitivity against most commonly used anticoccidial products. The tested field
isolates consisted of thirty six E. acervulina isolates, fourteen E. maxima isolates and
twelve E. tenella isolates (Table 1). The anticoccidial sensitivity-profiles were based on
the criteria of McDougald et al. (1986).
Resistance of E. acervulina isolates to anticoccidial drugs is very common as 145 of
the 221 (66%) assays showed resistance against at least one anticoccidial products. The
number of E. maxima assays showing resistance to one or more products was 39 out of 79
(49%). E. tenella isolates showed resistance to at least one anticoccidial drug in 45 assays
out of 76 (59%).
The coccidial lesion score system according to Johnson & Reid (1970) gives a good
impression of the severity of the pathology induced by a coccidiosis infection and is likely
the most commonly used method to investigate the efficacy of anticoccidial products
(Chapman, 1998). In both anticoccidial sensitivity studies, mentioned coccidiosis lesion
scoring system was used to determine the severity of the coccidiosis infection of
sensitivity against the anticoccidial products. The criteria of McDougald et al. (1986) were
219
used to calculate the extent of resistance/sensitivity to the anticoccidial products tested

using the formula: 100% - (mean lesion score of treated group/mean lesion score of the
infected unmedicated control group x 100%). A reduction in the medicated birds
compared with the infected unmedicated control birds of 0 to 30% indicates coccidial
resistance; 31 to 49% reduction indicates reduced sensitivity or partial resistance and 50%
or more indicates full sensitivity to the tested anticoccidial drug.
Table 1. Anticoccidial sensitivity-profiles expressed in number of resistant (R), reduced
sensitive (RS) and sensitive (S) Eimeria spp. isolates per anticoccidial product. Eimeria
spp. field isolates were obtained from broiler flocks in the Netherlands, Germany and
Spain during 1996-2002
Anticoccidial drug
Diclazuril (Clinacox)
Halofuginone (Stenorol)
Lasalocid (Avatec)
Maduramicin (Cygro)
Meticlorpindol/methylbenzoquate
(Lerbek)
Monensin (Elancoban)
Narasin (Monteban)
Narasin/nicarbazin (Maxiban)
Nicarbazin (Nicarb)
Robenidine (Robenz)
Salinomycine (Sacox)
Total
n
30
16
19
4
19
E. acervulina
R
RS S
27
1
2
10
6
0
17
2
0
3
1
0
1
1
17
n
11
5
3
1
9
E. maxima
R RS S
9
1
1
0
1
4
1
2
0
1
0
0
3
0
6
n
11
3
8
1
7
R
6
0
4
0
0
20
25
16
30
6
36
221
13
16
11
26
3
18
145
6
8
8
13
1
14
79
5
5
2
7
0
6
39
8
10
4
11
1
12
76
7
7
4
7
0
10
45
4
4
4
1
0
7
31
3
5
1
3
3
11
45
1
1
1
2
0
5
14
0
2
5
4
1
3
26
E. tenella
RS
S
1
4
0
3
2
2
0
1
0
7
0
1
0
2
0
1
7
1
2
0
2
1
1
24
However, unanimity on the parameters required for anticoccidial sensitivity testing is

lacking. Moreover, there are concerns about the infection model used in the sensitivity test
(i.e. infection dose versus spontaneous (trickle) infections), rising doubts regarding the
extrapolation of laboratory tests results to the field (Bafundo et al., 2008). Differences
between artificial and natural infection doses may explain conflicting results between
experimental settings and practice (Watkins, 1997), Despite the drawbacks of the
anticoccidial sensitivity test, at present, it is the only tool available to study anticoccidial
drug sensitivity profiles of Eimeria spp. field isolates. Although, as said previously, the
results of sensitivity testing should not be translated directly to the field one on one, they
can be a valuable tool as a predictor that will help to implement timely strategic changes of
the anticoccidial drugs (Ruff et al., 1997).
In Chapter 3 the anticoccidial potential of three alternative compounds is outlined.
In the first section the anticoccidial effect of ibuprofen (IBU), a non-steroidal antiinflammatory drug that inhibits the biosynthesis of prostaglandins with pro-inflammatory
220
CHAPTER 6
and immunosuppressive properties is presented. IBU was administered via the drinking
water in all experiments. IBU at a dose of 15 mg/kg body weight (BW) had no effect on
infections with oocysts of Eimeria acervulina (104.7), E. maxima (104.5) and E. tenella
(103.9). IBU given at a dose of 100 mg/kg BW significantly (P 0.05) decreased oocyst
shedding after infection with doses of sporulated E. acervulina oocysts between 104 and
105 per bird. Coccidial lesion scores were reduced for all infection doses; but significant
reduction only occurred in birds infected with a dose of 104 sporulated E. acervulina
oocysts. A significant effect on body weight gain was not observed and treatment with
IBU did not affect the sporulation and infectivity of excreted oocysts.
In the second section of Chapter 3 two experiments are presented. In the first
experiment the effect of dietary protease on a coccidiosis infection in broilers and their
performance after challenge with E. acervulina (104.0), E. maxima (103.8) or E. tenella
(103.3) was investigated. In the second experiment the influence of dietary protease on the
intestinal mucus layer thickness and brush border enzyme activity was established. In both
studies the protease was supplied at a concentration of 2.5 x 104 DU per kg feed. No
significant interaction was observed on body weight gain between dietary enzyme
supplementation and single Eimeria spp. infection. However, protease addition to the diet
resulted in a significant (P = 0.046) higher weight gain after comparing all supplemented
with non-supplemented groups. Coccidial lesions were not significantly affected by the
dietary protease supplementation, despite the slight tendency to a higher lesion score for E.
acervulina on the enzyme supplemented diets. Dietary protease significantly thickened the
adherent mucus layer of the duodenum, jejunum and caeca. The sucrase-isomaltase
activity in mucosal scrapings of the jejunum was significantly lower in birds fed the
enzyme supplemented diet, indicating a higher villus turnover rate. In conclusion, our data
suggest that dietary supplementation with the protease reduced the negative impact of a
coccidiosis infection on body weight gain of broilers, although the coccidial lesions and
oocyst excretion remained unaffected.
In the third section of Chapter 3 a study to examine the anticoccidial activity of a
mannanoligosaccharide (MOS) preparation is shown. One-day-old broilers were given a
single, mild coccidiosis infection with a mixture of three Eimeria species: E. acervulina
(102.95), E. maxima (102.75) and E. tenella (102.23). Infected and non-infected birds were fed
a diet with or without 10 g MOS/kg. Growth performance, feed intake and feed conversion
were not significantly different (P > 0.05) among the treatment groups. Feeding of the
MOS preparation reduced oocyst excretion and diminished the severity of coccidiosis
lesions induced by E. acervulina (P 0.05), but did not affect the lesions mediated by E.
maxima and E. tenella.
All three tested alternative compounds (IBU, protease and MOS) only showed a slight
anticoccidial effect but could not be considered as anticoccidial drugs. The growth
promoting effect of the protease deserves further research.
221
In Chapter 4 the influence of anticoccidial programs (anticoccidial product in feed

or vaccination with an attenuated anticoccidial sensitive vaccine) on the sensitivity profiles
of Eimeria spp. field isolates is described.
Twenty European Eimeria spp. field isolates were subjected to an AST. The
anticoccidial drugs tested were diclazuril and monensin. The results showed again that
resistance is common amongst coccidiosis field isolates, 68% and 53% of the E.
acervulina isolates were resistant against diclazuril and monensin, respectively, while
resistance was less frequent amongst E. maxima isolates, 38% and 50% were resistant to
diclazuril and monensin, respectively. Resistance of E. tenella isolates occurred in 23%
and 38% of cases against diclazuril and monensin, respectively. A comparison of the total
results obtained in 2004 with anticoccidial sensitivity studies performed in the period
1996-2002 (Chapter 2) showed a decrease in the number of Eimeria spp. isolates resistant
to diclazuril and monensin. Further analysis of the sensitivity study of 2004 with the
overall Chi-square test showed a highly significant influence of the coccidiosis prevention
program (live coccidiosis vaccination with Paracox-5 versus anticoccidial drugs in feed)
on the sensitivity pattern of Eimeria spp. field isolates for both diclazuril (P = 0.000) and
monensin (P = 0.001) (Table 2).
Table 2. Number of diclazuril and monensin resistant Eimeria spp. field isolates obtained
from European broiler flocks in 2004 (related to anticoccidial program) and in 1996-2002
Eimeria spp.
E. acervulina
Year/period
2004
E. maxima
1996-2002
2004
E. tenella
1996-2002
2004
1996-2002
Anticoccidial program
Vaccination (Paracox-5)
Anticoccidial drug
Anticoccidial drug
Anticoccidial drug
Diclazuril (%)
13/19 (68)
4/8 (50)
9/11 (82)
27/30 (90)
6/16 (38)
1/9 (11)
5/7 (71)
9/11 (81)
3/13 (23)
0/8 (0)
3/5 (60)
6/11 (54)
Monensin (%)
10/19 (53)
2/8 (25)
8/11 (73)
13/20 (65)
8/16 (50)
2/9 (22)
6/7 (86)
5/6 (83)
5/13 (38)
2/8 (25)
3/5 (60)
7/8 (88)
Additional analysis (Chi-square test) at the single species level and each anticoccidial drug
showed significantly more sensitivity of E. acervulina for monensin (P = 0.018), of E.
maxima for diclazuril (P = 0.009) and of E. tenella for diclazuril (P = 0.007) in field
isolates originating from vaccinated flocks (Table 3).
222
CHAPTER 6
Table 3. Number of diclazuril and monensin sensitive Eimeria spp. isolates obtained from
European broiler flocks in 2004 in relation to the anticoccidial program
Eimeria spp.
Paracox-5
Anticoccidial drug
Chi-square (P)*
1/11
1/7
1/5
0.111
0.009
0.007
0/11
1/7
1/5
0.018
0.060
0.767
Diclazuril
E. acervulina
E. maxima
E. tenella
4/8
8/9
8/8
E. acervulina
E. maxima
E. tenella
4/8
5/9
4/8
Monensin
*The significance of the association between the coccidiosis prevention program and the anticoccidial
sensitivity profiles was assessed using the Chi-square test (P 0.05 is considered significant).
In Chapter 5 studies on cross protection between an E. acervulina vaccine line

obtained by animal passages of extraintestinal blood stages of the parasite and E. maxima,
and E. tenella are presented. Three successive bird passages with early extraintestinal
blood stages of an E. acervulina strain were performed in commercial broiler chickens in
order to obtain an E. acervulina vaccine line. Subsequently, commercial broilers were
vaccinated at one day of age with graded doses of the obtained E. acervulina vaccine line
by coarse spray to determine a vaccination dose and to establish the protection against a
challenge infection with virulent strains of E. acervulina, E. maxima and E. tenella with
time. Vaccinated birds showed complete protection against challenge with E. acervulina,
partial cross protection against E. tenella but no cross protection against E. maxima.
Further research comparing E. acervulina lines from different sources could unravel
whether differences in cross protective properties exist. E. acervulina lines showing
enhanced cross protection against E. tenella could then be used to improve current live
anticoccidial vaccines.
Conclusions and perspectives

To date coccidiosis control in broilers has relied mainly on chemoprophylaxis, however,
the high degree of resistance against anticoccidial drugs, as shown in Chapter 2,
strengthening regulations, and possible upcoming bans on the use of anticoccidial drugs as
feed additives, have been the principal motivators in the quest for alternative control
strategies.
A wealth of papers concerning the effect of diet composition (ingredient and
nutrient composition), feed structure and the use of alternative feed additives (phyto- and
aromatherapy) on Eimeria parasites have been published to date. However, the results of
these studies are frequently ambivalent, while the number of manuscripts describing a
clear anticoccidial activity against various Eimeria spp. is relatively scarce. It is therefore
223
concluded that future coccidiosis control is unlikely to be achieved solely through feed
composition, structure and the use of alternative feed additives. In agreement herewith are
the results obtained in Chapter 3 of this thesis, in which the anticoccidial potency of
alternative preventive treatments for coccidiosis (a non-steroidal anti-inflammatory drug
(ibuprofen), a mucolytic enzyme (protease) and a prebiotic (MOS)) was investigated. All
three tested alternative compounds only showed a slight anticoccidial effect and could not
be considered as promising anticoccidial drugs.
Despite the high costs and a number of drawbacks associated with the production
and use of live anticoccidial vaccines, their use has increased considerably during the last
decade due to their efficacy and their association with increased sensitivity of Eimeria spp.
field isolates to anticoccidial drugs. To date, they represent the best alternative to
anticoccidial products considering resistance and residues. However, if the
immunogenicity of subunit vaccines can be improved substantially they might form the
core of future anticoccidial strategy.
Based on this thesis the following recommendations relevant to the broiler industry
can be made:
1.
In view of the anticoccidial drug resistance problem as shown in Chapter 2
monitoring of the sensitivity of Eimeria spp. isolates to anticoccidial
products is needed to optimize the use of these drugs in rotation and shuttle
programs.
2.
Data obtained from the literature review (Chapter 1) and studies performed
with mannanoligosaccharide, ibuprofen and a mucolytic enzyme (Chapter
3) indicate that alternative coccidiosis control strategies excluding
vaccination seem insufficient to prevent clinical coccidiosis.
3.
Vaccination with live attenuated anticoccidial drug sensitive Eimeria lines
has shown to be associated with a higher incidence of anticoccidial drug
sensitive coccidiosis field isolates (Chapter 4). Vaccination could therefore
be used in rotation programmes in order to efficiently control clinical
disease and at the same time reduce the anticoccidial drug resistance
problem. Anticoccidial programs should be guided by ASTs in order to use
anticoccidial drugs as well as vaccines, in the (economically) most
efficient manner.
4.
In view of the results obtained in Chapter 5, vaccines could be produced
more efficiently if cross protection can be further exploited. In this context,
research into the mechanisms ruling cross protection and screening of
Eimeria strains for enhanced expression of this property are of crucial
relevance.
224
CHAPTER 6
References
Bafundo, K.W., Cervantes, H.M. & Mathis, G.F. (2008). Sensitivity of Eimeria field isolates in the
United States: responses of nicarbazin-containing anticoccidials. Poultry Sciences, 87, pp. 1760-1767.
Ruff, M.D., Danforth, H.D. & Wilkens, G.C. (1997). Interpreting results of anticoccidial testing. In:
Proceedings of the VIIth International Coccidiosis Conference, Control of coccidiosis into the next
Millennium. Oxford, UK, pp. 52.
Watkins, K.l. (1997). Are sensitivity test results good predictors of anticoccidial field efficacy? In:
225
Samenvatting
Doel van het proefschrift was om de resistentie van Nederlandse en Europese Eimeria spp.
veldisolaten tegen de in gebruik zijnde anticoccidiosemiddelen te onderzoeken (Hoofdstuk
2). Dit onderzoek werd voorafgegaan door een uitgebreide literatuurstudie betreffende
coccidiose, anticoccidiosemiddelen en alternatieve preventieve behandelingen en
strategien (Hoofdstuk 1). Tevens beoogde het proefschrift de anticoccidiose
werkzaamheid van ibuprofen, een mucolytisch enzym en mannanoligosaccharide als
alternatieve coccidiose controle strategie vast te stellen (Hoofdstuk 3). Bovendien werd de
invloed van vaccinatie op de gevoeligheidprofielen van Eimeria spp. veldisolaten ten
opzichte van anticoccidiosemiddelen onderzocht (Hoofdstuk 4). Tenslotte, werd de
kruisbescherming van een E. acervulina vaccinlijn tegen E. tenella en E. maxima
bestudeerd, aangezien kruisbescherming verbetering van de effectiviteit van
coccidiosevaccins zou betekenen (Hoofdstuk 5).
In Hoofdstuk 1 wordt aan de hand van een uitgebreid literatuuroverzicht de ziekte
coccidiose beschreven. De geschiedenis van de ziekte en de classificatie en de
levenscyclus van de parasiet worden gedetailleerd toegelicht alsmede de mogelijkheden tot
identificatie van de species en het stellen van de diagnose. In enkele overzichtstabellen
worden de verschillende Eimeria spp. van het commercieel gehouden pluimvee
beschreven en getoond. Bij de kip zijn zeven Eimeria spp. beschreven waarvan E.
acervulina, E. maxima en E. tenella de belangrijkste zijn aangezien deze species bij
vleeskuikens frequent voorkomen en deze dieren het overgrote deel van de
pluimveehouderij vormen. De diagnose coccidiose wordt doorgaans bij postmortem
onderzoek gesteld door de locatie en morfologie van de letsels in de darm. Dit onderzoek
wordt aangevuld door lichtmicroscopisch onderzoek van natieve darmschraapsels voor
morfologische identificatie van schizonten en ocysten. Aangezien deze traditionele
manier van diagnostiek zijn beperkingen kent, zijn nieuwe identificatiemethoden
ontwikkeld. Zo is recentelijk de identificatie van Eimeria-ocysten sterk verbeterd door
analyse en classificatie van microscopische opnamen met behulp van een
computerprogramma. Daarnaast zijn moleculairbiologische technieken het speerpunt van
veel lopend onderzoek. Op dit moment is het mogelijk om middels PCR techniek, gebruik
makend van de ITS-1 en ITS-2 markers, de bij pluimvee voorkomende Eimeria spp. te
identificeren. Dit onderzoeksgebied is volop in beweging.
In het tweede gedeelte van het overzichtsartikel worden de gastheer- en
plaatsspecificiteit van de parasiet beschreven. Eimeria spp. kunnen genetisch nauw
verwante gastheren infecteren maar voor de complete ontwikkeling van de levenscyclus
blijkt de gastheerspecificiteit zeer strikt te zijn. Het mechanisme dat hiervoor
verantwoordelijk is, wordt toegeschreven aan de biochemie en voeding van de parasiet
enerzijds en het genoom en het immuunsysteem van de gastheer anderzijds. Ten aanzien
van de plaatsspecificiteit is vastgesteld dat sporozoieten van Eimeria spp. zeer specifieke
weefsels of orgaancellen infecteren van niet alleen de natuurlijke gastheer maar ook die
SAMENVATTING
van andere gastheren. De karakteristieken van deze weefsels of organen die door een groot
aantal gastheren gedeeld worden, blijken hiervoor verantwoordelijk te zijn.
In het derde deel wordt een historisch overzicht gegeven van alle
anticoccidiosemiddelen en worden deze middelen ingedeeld op basis van hun chemische
samenstelling en werkingsmechanisme. Ondanks het feit dat de meeste
anticoccidiosemiddelen meer dan n biochemisch effect op de ontwikkeling van een
specifiek stadium van de parasiet hebben en ondanks een gebrek aan gedetailleerde kennis
over een selectieve werking van deze producten is toch een grove indeling gemaakt.
Middelen die de cofactor-synthese benvloeden zijn ethopabaat, sulfonamides,
pyrimethamines en thiamine-analogen zoals amprolium. Middelen met invloed op
mitochondriale functies zijn de quinolonen (buquinolaat, decoquinaat en nequinaat), de
pyridinolen (meticlorpindol), nicarbazine, robenidine en toltrazuril. Middelen die de
celmembraamfuncties benvloeden zijn de polyether-antibiotica (ook wel ionoforen
genoemd) (monensin, semduramicin, narasin, salinomycin, lasalocid en maduramicin).
Daarnaast is van enkele middelen het werkingsmechanisme onbekend (diclazuril en
halofuginone).
Het wereldwijde intensieve gebruik van anticoccidiosemiddelen heeft uiteindelijk geleid
tot de ontwikkeling van resistentie tegen al deze middelen. Resistentie tegen
anticoccidiosemiddelen is in de wetenschappelijke literatuur in de Verenigde Staten, Zuid
Amerika, Europa en China gerapporteerd. Om resistentie zoveel mogelijk te beperken
worden de middelen geroteerd (een bepaald middel gedurende maximaal twee maanden of
twee groeirondes gebruiken) of worden shuttleprogrammas (twee of meer middelen in
n groeiperiode gebruiken) toegepast. De gedachte hierachter is dat verlies van
gevoeligheid gerelateerd is aan de gebruiksduur van het middel. Door het optreden van
kruisresistentie tussen anticoccidiosemiddelen met hetzelfde werkingsmechanisme zouden
producten met verschillende werkingsmechanismen afwisselend gebruikt moeten worden
in de rotatie en shuttleprogrammas. In het ideale geval zouden rotatie en
shuttleprogrammas gebaseerd moeten zijn op kennis van de gevoeligheidsprofielen van
de Eimeria spp. veldisolaten. Een relatief nieuwe benadering in het terugdringen en
beperken van resistentie tegen anticoccidiosemiddelen is het gebruik van levende
coccidiosevaccins in rotatieprogrammas. Verspreiding van gevoelige Eimeria spp.
vaccinlijnen in de pluimveestallen heeft een toename van de incidentie van gevoelige
Eimeria spp. veldisolaten te weeg gebracht.
In het vierde deel van het literatuuroverzicht wordt stilgestaan bij de mogelijkheid
om door middel van voerstructuur, voersamenstelling (micro- en macro-ingredinten,
speciale additieven), toxinen in het voer en voermanagement, coccidiose-infecties te
benvloeden. Verschillende voercomponenten blijken de parasiet direct (invloed op de
vermeerdering van de parasiet) of indirect (stimulatie immuunsysteem, invloed op
darmflora en slijmlaag in de darm, verandering van de fysiologie van de darm, enz.) te
benvloeden. Sommige voercomponenten zijn actief tijdens de acute fase van de ziekte
terwijl andere het herstel bevorderen. Voercomponenten die het herstel bevorderen zijn
eiwitten en de vitaminen A, C en K. Voercomponenten die de ontwikkeling van de
parasiet direct negatief benvloeden zijn -3 vetzuren. Voor eiwitconcentratie en
228
SAMENVATTING
voerrestrictie zijn wisselende effecten beschreven. De ontwikkeling van de parasiet wordt

op een indirecte manier negatief benvloed via stimulatie van het immuunsysteem door glucanen, vitamine E, selenium, voederbeperking, -3 vetzuren, vitaminen A en C en
natriumbicarbonaat (NaHCO3). De ontwikkeling van de parasiet wordt ook indirect
negatief benvloed door middellange keten vetzuren en de vitaminen A, C en K. Deze
componenten bevorderen de gezondheid van de slijmlaag in de darm en beschermen zo
tegen coccidiose-infecties. Wisselende indirecte effecten op de ontwikkeling van de
parasiet zijn beschreven voor tarwe dat de viscositeit en darmflora benvloedt. De
darmflora wordt ook benvloed door vezels en melkproducten. Hele granen hebben een
indirect effect op de Eimeria-parasiet via hun invloed op de fysiologie van de darm. Tot
slot, voercomponenten die de ontwikkeling van de parasiet direct bevorderen zijn calcium,
aflatoxinen, vitamine B, essentile vetzuren en trypsine. Deze componenten moeten dan
ook indien maar enigszins mogelijk niet gebruikt te worden in concentraties die de
ontwikkeling van de parasiet bevorderen.
In deel vijf worden andere alternatieve preventieve methoden zoals management en
bioveiligheid, homeopathie, phytotherapie en vaccinatie beschreven. Management- en
bioveiligheidsmaatregelen zijn ervoor bedoeld om zoveel mogelijk insleep van de parasiet
in pluimveestallen te voorkomen door isolatie, controle van voertuigen (verkeer) en
hyginemaatregelen. In de praktijk is het echter vrijwel onmogelijk om kuikens coccidiose
vrij te houden tenzij gebruik gemaakt wordt van SPF-achtige huisvestingscondities.
Besmette pluimveestallen kunnen het beste met veel water gereinigd worden waarna
ontsmetting met een ammoniak oplossing (5-10%) dient plaats te vinden. Phytotherapie en
aromatherapie kennen gedurende het laatste decennium een groeiende belangstelling door
vernieuwde interesse in natuurgeneeskunde. Een groot aantal plantenextracten (kruiden) is
onderzocht op werkzaamheid tegen coccidiose. Middelen die een negatief effect op de
ontwikkeling van de parasiet vertonen zijn artemisinin, extracten van citrusvruchten en
mengsels van kruidenextracten. Oregano vertoonde een wisselend effect op de
ontwikkeling van de Eimeriaparasiet. Betaine, Echinacea, paddestoelen (lectines) en
turmeric (curcumine) stimuleren het immuunsysteem waardoor de ernst van de coccidioseinfectie afneemt. Betaine heeft bovendien osmoprotectieve eigenschappen in de
darmtractus. Het effect van het prebioticum MOS op coccidiose is niet eenduidig. Soms
werd een directe werking aangetoond door de ontwikkeling van de parasiet te verhinderen
en soms vertoonde het geen effect. Probiotica hebben een indirect belemmerend effect op
de parasiet door stimulatie van de humorale immuunrespons.
Vaccinatie met levende afgezwakte of niet afgezwakte Eimeria spp. lijnen wordt steeds
meer beschouwd als een betrouwbare alternatieve maatregel tegen coccidiose. Levende
coccidiosevaccins hebben echter ook enkele nadelen zoals hoge productiekosten, beperkte
houdbaarheid en fouten bij toediening (doseringsfouten, aanwezigheid van
anticoccidioseproducten in het voer die de vaccinatie met voor deze middelen gevoelige
vaccinlijnen frustreren). Bovendien kan ook herstel/toename van de virulentie van de
levende afgezwakte vaccinlijnen een punt van zorg zijn. Een gunstige nevenwerking van
vaccinatie met levende voor anticoccidiosemiddelen gevoelige vaccinlijnen is dat er
verschuiving plaatsvindt van resistente naar gevoelige veldisolaten. Dit fenomeen heeft het
229
SAMENVATTING
gebruik van deze vaccins in rotatieprogrammas sterk bevorderd. Subunit-vaccins missen

veel van de onvolkomenheden van de levende vaccins, maar thans presteren deze
producten teleurstellend door gebrek aan immunogeniteit. Het E. tenella genoom project
kan ontdekking van essentile immunogene antigenen die wel voldoende bescherming
induceren dichterbij brengen. En dan kunnen de subunit-vaccins de hooggespannen
verwachtingen mogelijk waarmaken.
Geconcludeerd kan worden dat het vrkomen van tegen anticoccidiosemiddelen
resistente Eimeria spp. veldisolaten op zijn minst onveranderd zal blijven. Aannemelijker
is dat resistentie verder zal toenemen vooral als de commercile pluimveehouderij
vasthoudt aan het gebruik van anticoccidiosemiddelen op een niet-rationele manier dat wil
zeggen niet begeleid door gevoeligheidstesten en zonder tijdige afwisseling van
anticoccidiosemiddelen. Hoewel sommige voercomponenten, fytochemicalin,
prebiotica/probiotica en een niet-sterod anti-inflammatoir geneesmiddel in staat bleken te
zijn om de nadelen van een coccidiose-infectie te verminderen, hetzij door een direct
effect op de parasiet hetzij indirect door immuunstimulatie en door bevordering van het
herstel, blijken deze effecten onvoldoende om de ziekte te kunnen controleren. Genoemde
middelen bleken dan ook geen oplossing voor het probleem van de
anticoccidiosemiddelen resistentie. Op dit moment is vaccinatie dan ook de meest voor de
hand liggende en betrouwbare anticoccidiose strategie ondanks zijn onvolkomenheden.
Indien het immuniserend vermogen van de subunit vaccins sterk verbeterd zou kunnen
worden, zouden deze vaccins het belangrijkste onderdeel van de toekomstige
anticoccidiose strategie kunnen zijn.
In Hoofdstuk 2 worden de anticoccidiosemiddelen gevoeligheidprofielen van een
aantal Eimeria spp. veldisolaten afkomstig van vleeskuikenkoppels, gepresenteerd en
bediscussieerd.
In twee onderzoeken werden vijftien Eimeria spp. veldisolaten van Nederlandse
bedrijven, vier uit 1996, vier uit 1999 en zeven uit 2001 en vierentwintig veldisolaten
afkomstig van Nederlandse, Duitse en Spaanse vleeskuikenbedrijven in de periode 20002002 onderzocht op gevoeligheid voor anticoccidiosemiddelen. De onderzochte
anticoccidiosemiddelen waren: diclazuril, halofuginone, lasalocid, maduramicin,
meticlorpindol/methylbenzoquate, monensin, narasin, narasin/nicarbazine, nicarbazine,
robenidine en salinomycin. De resultaten vertoonden een hoge mate van resistentie tegen
de meeste anticoccidiosemiddelen. Desondanks bleven ernstige coccidiose problemen in
het veld uit, terwijl de coccidiose prevalentie in Nederland hoog was. Een mogelijke
verklaring hiervoor is dat de resistentie tegen de anticoccidiosemiddelen niet volledig is en
in het veld natuurlijke vaccinatie en immuniteitsopbouw plaatsvindt onder de partile
protectie van het anticoccidiosemiddel.
In de periode van 1996 tot 2002 werden in totaal negenendertig verschillende Eimeria spp.
veldisolaten, waarvan het merendeel uit meer dan n species bestond, onderzocht op
gevoeligheid voor de meest gebruikte anticoccidiosemiddelen. De onderzochte
veldisolaten bestonden uit zesendertig E. acervulina isolaten, veertien E. maxima isolaten
230
SAMENVATTING
en twaalf E. tenella isolaten (Tabel 1). Voor het vaststellen van de anticoccidiosemiddelen
gevoeligheidprofielen werden de criteria (zie later) van McDougald et al. (1986)
gehanteerd.
Tabel 1. Anticoccidiosemiddelen gevoeligheidprofielen uitgedrukt in aantal resistente (R),
verminderd gevoelige (VG) en gevoelige (G) Eimeria spp. isolaten per
anticoccidiosemiddel. Eimeria spp. isolaten waren afkomstig van vleeskuikenbedrijven in
Nederland, Duitsland en Spanje in de periode 1996-2002
Anticoccidiosemiddel
n
30
16
19
4
19
E. acervulina
R
VG G
27
1
2
10
6
0
17
2
0
3
1
0
1
1
17
n
11
5
3
1
9
R
9
0
1
1
3
4
4
4
1
0
7
31
6
8
8
13
1
14
79
5
5
2
7
0
6
39
Diclazuril (Clinacox)
Halofuginone (Stenorol)
Lasalocid (Avatec)
Maduramicin (Cygro)
Meticlorpindol/methylbenzoquate
(Lerbek)
20 13
Monensin (Elancoban)
25 16
Narasin (Monteban)
Narasin/nicarbazin (Maxiban)
16 11
30 26
Nicarbazin (Nicarb)
Robenidine (Robenz)
6
3
36 18
Salinomycin (Sacox)
Totalen
221 145
3
5
1
3
3
11
45
E. maxima
VG G
1
1
1
4
2
0
0
0
0
6
1
1
1
2
0
5
14
0
2
5
4
1
3
26
n
11
3
8
1
7
R
6
0
4
0
0
8
10
4
11
1
12
76
7
7
4
7
0
10
45
E. tenella
VG G
1
4
0
3
2
2
0
1
0
7
0
1
0
2
0
1
7
1
2
0
2
1
1
24
Resistentie van E. acervulina isolaten tegen de anticoccidiosemiddelen komt veel voor: in

145 van de 221 (66%) bepalingen werd resistentie tegen tenminste n
anticoccidiosemiddel vastgesteld. Het aantal E. maxima isolaten dat resistentie vertoonde
tegen n of meer anticoccidiosemiddelen bedroeg 39 uit 79 (49%). De E. tenella isolaten
vertoonden in 45 van de 76 (59%) bepalingen tegen n of meerdere
anticoccidiosemiddelen resistentie.
Het coccidiose laesie score systeem volgens Johnson and Reid (1970) geeft een
goede indruk van de pathologie van een coccidiose-infectie en is n van de meest
gebruikte methoden voor vaststelling van het effect van anticoccidioseproducten op
coccidiose-infecties (Chapman, 1998). In beide gevoeligheidsonderzoeken is van dit
systeem gebruik gemaakt. Vervolgens werden de criteria van McDougald et al. (1986)
toegepast om het gevoeligheidsprofiel vast te stellen van de afzonderlijke Eimeria spp.
aanwezig in de veldisolaten met betrekking tot de anticoccidiosemiddelen. Hierbij werd
gebruik gemaakt van de formule: laesie reductie = 100% - (gemiddelde laesie score van de
behandelde groep/gemiddelde laesie score van de niet behandelde genfecteerde controle
groep x 100%). Een reductie van 0-30% duidt op resistentie, 31-49% reductie staat voor
verminderde gevoeligheid of gedeeltelijke resistentie en een reductie van 50% of meer
betekent volledige gevoeligheid voor het geteste anticoccidiosemiddel. De vertaling van
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SAMENVATTING
de laboratoriumresultaten naar praktijkomstandigheden is discutabel (Bafundo et al.,

2008). Extrapolatie van de resultaten van de laboratorium gevoeligheidstest naar
praktijkomstandigheden is ingewikkeld aangezien in de laboratoriumtest een eenmalige
hoge infectiedosis gebruikt wordt, die in de praktijk waarschijnlijk nauwelijks voorkomt.
Het is mogelijk dat het anticoccidiosemiddel onder proefomstandigheden minder effectief
is dan in de praktijk. Dit kan aanleiding geven tot fouten bij de interpretatie van de
testresultaten (Watkins, 1997). Desondanks is de test tot op dit moment de enig
beschikbare methode om de anticoccidiosemiddelen gevoeligheidsprofielen van Eimeria
spp. veldisolaten vast te stellen. Overigens kan de test wel degelijk belangrijke informatie
geven over verslechtering van het anticoccidiosemiddelen gevoeligheidspatroon van een
Eimeria spp. veldpopulatie, nog voordat er problemen optreden en dat kan dan reden zijn
om een effectief anticoccidioseprogramma toch te veranderen (Ruff et al., 1997).
In Hoofdstuk 3 wordt de anticoccidiose activiteit van drie alternatieve middelen
gepresenteerd.
In het eerste deel wordt het onderzoek met ibuprofen - een niet-sterod antiinflammatoir geneesmiddel beschreven. Ibuprofen werd via het drinkwater verstrekt.
Toediening van 15 mg/kg lichaamsgewicht vanaf een leeftijd van dag n tot zeven dagen
na de infectie bleek geen effect te hebben op een coccidiose-infectie met een samengesteld
inoculum van 5 x 104 E. acervulina, 3 x 104 E. maxima en 7,5 x 103 E. tenella
gesporuleerde ocysten per kuiken. Ibuprofen, verstrekt in een dosis van 100 mg/kg
lichaamsgewicht vanaf n dag voor de infectie tot vijf dagen na de infectie, verminderde
de ocystenuitscheiding significant (P 0,05) bij infectiedoses tussen de 104 tot 105
gesporuleerde E. acervulina ocysten per kuiken. Hoewel de coccidiose-laesiescores voor
alle genoemde infectiedoses lager waren dan die van de controle groepen, werd alleen bij
de infectie met 104 E. acervulina ocysten per kuiken een significant lagere score
vastgesteld. Een effect op de groei van de kuikens werd niet waargenomen en de
behandeling met ibuprofen had geen invloed op de sporulatie en infectiviteit van de
uitgescheiden E. acervulina ocysten.
In het tweede gedeelte van dit hoofdstuk worden de resultaten van twee
experimenten gepresenteerd. In het eerste experiment werd het effect onderzocht van de
toediening van een mucolytisch enzym (protease) via het voeder op een coccidioseinfectie met 104 E. acervulina, 6,3 x 103 E. maxima of 1,9 x 103 E. tenella gesporuleerde
ocysten per dier. In een tweede experiment werd de invloed van het enzym op de dikte
van de darmslijmlaag en de borstelzoom enzym activiteit bestudeerd. In beide
experimenten werd de protease in een dosering van 2,5 x 104 DU per kg voer verstrekt. In
het eerste experiment werd vastgesteld dat het enzym geen invloed had op de groei noch
op laesie en ocystenuitscheiding bij enkelvoudige Eimeria spp. infecties. Wel werd een
significant verschil (P = 0,046) gevonden tussen de gemiddelde groei van alle dieren ten
gunste van de dieren die voeder met protease kregen. In het tweede experiment werd
vastgesteld dat de slijmlaag van de dunne darm, jejunum en blinde darm significant dikker
was en de sucrase-isomaltase enzym activiteit in de borstelzoom van het jejunum
significant lager was bij de dieren die voeder met protease kregen. Deze resultaten duiden
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SAMENVATTING
op een hogere villus turnover rate en suggereren dat het mucolytisch enzym de negatieve
invloed van een coccidiose-infectie op de groei vermindert hoewel de coccidiose laesies en
ocystenuitscheiding onveranderd bleven.
In het derde gedeelte van dit hoofdstuk wordt het effect van een
mannanoligosaccharide (MOS) preparaat op een milde coccidiose-infectie, beschreven.
En dag oude vleeskuikens werden genoculeerd met een milde gemengde coccidioseinfectie van 900 E. acervulina, 570 E. maxima en 170 E. tenella gesporuleerde ocysten
per kuiken. Genfecteerde en niet-genfecteerde dieren kregen voer met 10 g MOS/kg of
zonder MOS. Geen significante invloed van MOS werd vastgesteld op groei, voeropname
en voederconversie. Wel bleek MOS de ernst van de E. acervulina laesies evenals de
ocystenuitscheiding significant (P 0,05) te verminderen. Het preparaat had echter geen
invloed op de laesies van E. maxima en E. tenella.
De drie onderzochte alternatieve producten (ibuprofen, protease en MOS) vertoonden
slechts een geringe mate van anticoccidiose-activiteit en kunnen derhalve niet als een
anticoccidiosemiddel beschouwd worden. Het groeibevorderende effect van het
mucolytisch enzym verdient nader onderzoek.
In Hoofdstuk 4 wordt de invloed van anticoccidioseprogrammas
(anticoccidioseproduct in het voer of vaccinatie met een afgezwakt coccidiose vaccin dat
gevoelig is voor anticoccidiosemiddelen) op de resistentie van Eimeria spp. veldisolaten
voor anticoccidiosemiddelen beschreven.
Twintig Europese Eimeria spp. veldisolaten werden onderzocht op hun
gevoeligheid voor diclazuril en monensin. Opnieuw bleek dat resistentie veel voorkomt bij
coccidiose veldisolaten. Van de E. acervulina isolaten bleek 68 respectievelijk 53 procent
resistent te zijn tegen diclazuril en monensin. Bij E. maxima isolaten bedroegen deze
percentages 38 en 50 en bij E. tenella isolaten 23 en 38. Uit vergelijking van deze
resultaten met die van eerder uitgevoerde studies (Hoofdstuk 2, periode 1996-2002) blijkt
de resistentie tegen diclazuril en monensin minder te zijn. Statistische analyse met de Chisquare test toonde een significante relatie aan tussen de toegepaste
anticoccidioseprogrammas (levende anticoccidiose vaccinatie met Paracox-5 versus
anticoccidiosemiddelen in het voer) en het gevoeligheidsprofiel van de Eimeria spp.
veldisolaten voor diclazuril (P = 0,009) en monensin (P = 0,001) (Tabel 2).
233
SAMENVATTING
Tabel 2. Aantal diclazuril en monensin resistente Eimeria spp. veldisolaten afkomstig van
Europese vleeskuiken koppels in 2004 (in relatie met anticoccidioseprogramma) en in
1996-2002
Eimeria spp.
E. acervulina
E. maxima
E. tenella
Jaar/periode Anticoccidioseprogramma
2004
Vaccinatie (Paracox-5)
1996-2002
2004
1996-2002
2004
1996-2002
Diclazuril (%)
13/19 (68)
4/8 (50)
9/11 (82)
27/30 (90)
6/16 (38)
1/9 (11)
5/7 (71)
9/11 (81)
3/13 (23)
0/8 (0)
3/5 (60)
6/11 (54)
Monensin (%)
10/19 (53)
2/8 (25)
8/11 (73)
13/20 (65)
8/16 (50)
2/9 (22)
6/7 (86)
5/6 (83)
5/13 (38)
2/8 (25)
3/5 (60)
7/8 (88)
Nadere statische analyse (Chi-square test) op enkelvoudig species niveau en per

anticoccidiosemiddel toonde significant meer gevoeligheid aan bij E. acervulina voor
monensin (P = 0,018), bij E. maxima (P = 0,009) voor diclazuril en bij E. tenella (P =
0,007) voor diclazuril in Eimeria spp. veldisolaten afkomstig van gevaccineerde koppels
(Tabel 3).
Tabel 3. Aantal diclazuril en monensin gevoelige Eimeria spp. veldisolaten afkomstig van
Europese vleeskuiken koppels in 2004 in relatie tot het anticoccidioseprogramma
Eimeria spp.
Paracox-5
E. acervulina
E. maxima
E. tenella
4/8
8/9
8/8
E. acervulina
E. maxima
E. tenella
4/8
5/9
4/8
Diclazuril
1/11
1/7
1/5
Monensin
0/11
1/7
1/5
Chi-square (P)*
0,111
0,009
0,007
0,018
0,060
0,767
*De significantie van de associatie tussen het coccidiose preventieprogramma en de anticoccidiose

gevoeligheidsprofielen is vastgesteld door statistische analyse met de Chi-square test, P 0,05 is
significant.
234
SAMENVATTING
In Hoofdstuk 5 wordt onderzoek naar kruisbescherming tussen een E. acervulina

vaccinlijn, verkregen door dierpassages met vroege extra-intestinale parasitaire
bloedstadia, en E. maxima, en E. tenella beschreven.
Drie achtereenvolgende dierpassages met extra-intestinale bloedstadia van een E.
acervulina stam werden uitgevoerd in commercile vleeskuikens ter verkrijging van een E.
acervulina vaccinlijn. Vervolgens zijn n dag oude commercile vleeskuikens met deze
E. acervulina vaccinlijn per spray gent en is de mate van bescherming tegen een
gecombineerde herinfectie van E. acervulina, E. tenella en E. maxima in de tijd
vastgesteld. Gevaccineerde dieren vertoonden complete bescherming tegen de herinfectie
met E. acervulina, een gedeeltelijke bescherming tegen E. tenella maar geen bescherming
tegen E. maxima. Vervolgonderzoek met andere E. acervulina lijnen zal moeten uitwijzen
of verschillen in kruisbeschermende eigenschappen voorkomen. E. acervulina lijnen die
een kruisbescherming tegen E. tenella vertonen zouden dan gebuikt kunnen worden om de
huidige levende coccidiosevaccins te verbeteren.
Conclusies en perspectief
Tot op heden berust de controle van coccidiose bij vleeskuikens voornamelijk op
chemoprofylaxis. De in hoofdstuk 2 aangetoonde hoge mate van resistentie tegen
anticoccidiosemiddelen, de toenemende wetgeving en het mogelijke verbod op het gebruik
van anticoccidiosemiddelen als voederadditief, vormen echter belangrijke redenen om naar
alternatieve controlemaatregelen te zoeken.
Talrijk zijn de studies betreffende de invloed van voersamenstelling (ingredinten
en samenstelling) voerstructuur en het gebruik van alternatieve voeder additieven (phytoen aromatherapie) op Eimeria parasieten. De resultaten van deze studies zijn echter veelal
ambivalent terwijl het aantal dat een duidelijke directe anticoccidiose werking tegen
verschillende Eimeria spp. beschrijft, relatief gering is. Geconcludeerd kan worden dat het
onwaarschijnlijk is dat toekomstige controle van coccidiose op basis van alleen
voersamenstelling, voerstructuur en het gebruik van alternatieven voeradditieven
gerealiseerd zal worden. In analogie hiermee zijn de in hoofdstuk 3 van dit proefschrift
behaalde resultaten met alternatieve preventieve behandelingen tegen coccidiose. De drie
onderzochte alternatieve producten ((een niet-sterod anti-inflammatoir geneesmiddel
(ibuprofen), een mucolytisch enzym (protease) en een prebioticum (MOS)) vertoonden
slechts een geringe mate van anticoccidiose werking en kunnen derhalve niet als
alternatief voor de huidige chemoprofylaxe worden beschouwd.
Ondanks hoge kosten en een aantal nadelen met betrekking tot de productie en
toepassing van levende coccidiosevaccins, is het gebruik van deze vaccins gedurende het
laatste decennium aanzienlijk toegenomen vanwege hun effectiviteit en het feit dat zij
geassocieerd worden met een terugkerende gevoeligheid van Eimeria spp. veldisolaten
voor anticoccidiosemiddelen. Tot nu toe zijn vaccins op basis van gesporuleerde ocysten
het beste alternatief voor anticoccidiosemiddelen. Wanneer, echter de immunogeniteit van
235
SAMENVATTING
subunit-vaccins verbeterd zou kunnen worden dan zouden deze vaccins een belangrijk
onderdeel kunnen vormen van de toekomstige anticoccidiose strategie.
Gebaseerd op dit proefschrift kunnen de volgende aanbevelingen, relevant voor de

vleeskuikenhouderij gedaan worden:
1. Gegeven het resistentie probleem, zoals aangetoond in hoofdstuk 2, is monitoren
van de gevoeligheid van de Eimeria spp. veldisolaten voor
anticoccidiosemiddelen noodzakelijk om het gebruik van deze middelen in rotatie
en shuttleprogrammas te optimaliseren.
2. Uit de literatuurstudie (Hoofdstuk 1) en het onderzoek met ibuprofen, een
mucolytisch enzym en mannanoligosaccharide (Hoofdstuk 3) blijkt dat
alternatieve coccidiose-controle strategien, met uitzondering van vaccinatie, niet
in staat blijken klinische coccidiose te voorkomen.
3. Vaccinatie met levende, afgezwakte en voor anticoccidiosemiddelen gevoelige
Eimeria spp. lijnen leidt tot een hogere incidentie van anticoccidiosemiddelen
gevoelige veldisolaten (Hoofdstuk 4). Vaccinatie kan derhalve in
rotatieprogrammas gebruikt worden om enerzijds de ziekte zo efficint mogelijk
te controleren en anderzijds gelijktijdig de resistentieproblematiek te verminderen.
Anticoccidioseprogrammas zouden door gevoeligheidstesten begeleid moeten
worden teneinde anticoccidiosemiddelen en vaccins zo efficint mogelijk te
gebruiken.
4. Vaccins die kruisbescherming bieden zijn in principe mogelijk (Hoofdstuk 5).
Screening van Eimeria lijnen op kruisbescherming en onderzoek naar het
mechanisme van deze eigenschap zijn van belang.
236
SAMENVATTING
Literatuur
Bafundo, K.W., Cervantes, H.M. & Mathis, G.F. (2008). Sensitivity of Eimeria field isolates in the
United States: responses of nicarbazin-containing anticoccidials. Poultry Sciences, 87, pp. 1760-1767.
Ruff, M.D., Danforth, H.D. & Wilkens, G.C. (1997). Interpreting results of anticoccidial testing. In:
Watkins, K.l. (1997). Are sensitivity test results good predictors of anticoccidial field efficacy? In:
237
Dankwoord
Het doet me een groot genoegen om allen te bedanken die een belangrijke bijdrage
geleverd hebben bij de onderzoeken die beschreven zijn in mijn proefschrift.
Allereerst gaat mijn dank gaat uit naar mijn promotor Arjan Stegeman, die de
mogelijkheid van de promotie faciliteerde en de voortgang van mijn proefschrift op de
voet volgde. Arjan, onze regelmatige bijeenkomsten lieten mij door jouw ogen zien hoe
mijn proefschrift langzaam maar zeker in de tijd groeide.
Speciale dank voor de totstandkoming van mijn proefschrift gaat uit naar mijn copromotor Wil Landman. Wil, bedankt voor de zeer vele tekstuele correcties in mijn
manuscripten, het vertrouwen in mijn ervaring op het gebied van coccidiose, je nietaflatende stimulans bij het schrijven en je oprechte belangstelling in het onderzoek.
In mijn persoonlijke en wetenschappelijke ontwikkeling heeft Mattie Vertommen een zeer
belangrijke rol gespeeld. Mattie, door jouw toedoen ben ik indertijd bij de
Gezondheidsdienst voor Pluimvee (GVP) in dienst getreden. Bedankt voor het vertrouwen
dat je in mij had en voor de fijne samenwerking gedurende de tijd bij de GVP. Het was
voor mij een zeer actieve en creatieve periode waarbij wij ontzettend veel werk verzet
hebben. Eigenlijk is destijds al, ondermeer door de uitvoering van de vele anticoccidiose
gevoeligheidstesten, de basis van mijn proefschrift gelegd.
Ik wil de GD hartelijk danken voor de mogelijkheid dat ik gedurende een langere
aaneengesloten periode aan n en hetzelfde onderwerp heb kunnen werken. Teun,
bedankt voor het vertrouwen in de goede afloop en de tijd die ik mocht gebruiken voor de
afronding van mijn proefschrift. Verder dank ik alle collegas bij de GD die een bijdrage
hebben geleverd aan de, in mijn proefschrift beschreven, onderzoeken. Een bijzonder
woord voor dank heb ik voor de jongens (Machiel, Johan en Ad) van Lab 2 die altijd
bereid waren om een helpende hand te bieden bij de uitvoering van de dierproeven.
Jongens bedankt voor de inspanningen en vele gedachtenwisselingen die we over
allerhande aangelegen (ook aangaande de GD) gevoerd hebben.
Graag wil ik Brenda Vermeulen bedanken voor haar initiatief in het onderzoek naar de
invloed van ibuprofen op coccidiose. Brenda, bedankt voor je bijdrage bij de aanloop en
afronding van mijn proefschrift en voor de lange en indringende gesprekken over het werk
en de zeer fijne samenwerking. Ik heb altijd veel waardering gehad voor je zichtbaar
onuitputtelijke energie en je grote vertrouwen in mijn kennis van coccidiose. Dank aan
Mohammed Ali Elmusharaf en Anton Beynen bij het onderzoek naar de werkzaamheid
van een prebioticum tegen coccidiose. Ook dank aan Jan Dirk van der Klis die het
onderzoek naar de invloed van een mucolytische enzym op een coccidiose-infectie
mogelijk maakte. Jan Dirk, je snelheid van denken en het (in)zien van oplossingen heeft
me aangenaam verrast. Ik ervoer dat vaak als stimulerend en verfrissend. Op de valreep
wil ik Jo van Eck bedanken voor de allerlaatste correcties in mijn proefschrift en de door
de jaren heen lopende licht filosofische relativerende discussies en gesprekken. Die
DANKWOORD
levenshouding van jou heb ik altijd in je bewonderd en enorm op prijs gesteld en dikwijls
als een verademing (verlichting) van de alledaagse zorgen ervaren.
Tenslotte gaat mijn dank uit naar mijn ouders. Beste Ma, het is jammer dat Pa niet meer
bij ons is, hij zou ongetwijfeld zeer trots geweest zijn en heeft onmiskenbaar zijn aandeel
hierin bijgedragen. Daarnaast wil ik mijn broers en zussen bedanken die ieder op zijn/haar
manier er toe hebben bijgedragen dat ik me ontwikkeld heb tot wie ik nu ben. Gerard, Ria,
Wim, Gerda, Ruud en Corien, Thankx!!!
240
Curriculum vitae
Herman W. Peek werd op 2 december 1952 te Harmelen geboren. Nadat hij in 1972 zijn
HAVO-diploma behaald heeft en daarna in militaire dienst is geweest, heeft hij de
zologische HBO-A opleiding richting histologie van 1975-1978 bij het dr. ir. W.L.
Ghijsen instituut te Utrecht gevolgd. Daarna is hij bij het RIVM (Rijksinstituut voor
Volksgezondheid en Milieu) in dienst getreden en heeft tot 1980 op de afdeling
toxicologie aan onderzoek op het gebied van embryotoxiciteit bij vissen gewerkt.
Vervolgens is hij tot 1983 als analist bij de Faculteit voor Diergeneeskunde Vakgroep
Tropische Diergeneeskunde bij het onderzoek van Toxoplasmose werkzaam geweest. In
augustus 1984 is hij bij de GVP (Gezondheidsdienst voor Pluimvee) in Doorn in dienst
getreden en heeft tot op heden als onderzoeksanalist aan diverse coccidiose projecten bij
pluimvee gewerkt. Uiteindelijk hebben de werkzaamheden op het gebied van coccidiose
bij de GD (Gezondheidsdienst voor Dieren) tot dit proefschrift geleid.
List of publications
Manuscripts
Peek, H.W., Vertommen, M.H., & Landman, W.J.M. (2009). Cross protection studies between Eimeria
acervulina and E. maxima, and E. tenella. Submitted.
Peek, H.W., van der Klis, J.D., Vermeulen, B. & Landman, W.J.M. (2009). Dietary protease can alleviate
negative effects of a coccidiosis infection on production performance in broiler chickens. Animal
Feed Science and Technology, 150, pp. 151-159.
Elmusharaf, M.A., Peek, H.W., Nollet, L. & Beynen, A.C. (2007). Effect of an in-feed
mannanoligosaccharide preparation (MOS) on a coccidiosis infection in broilers. Animal Feed
Science and Technology, 134, pp. 347-354.
Peek, H.W. & Landman, W.J.M. (2006). Higher incidence of Eimeria spp. field isolates sensitive for
diclazuril and monensin associated with the use of live coccidiosis vaccination with Paracox-5 in
broiler farms. Avian Diseases, 50, pp. 434-439.
Eimeria spp. veldisolaten voor anticoccidiosemiddelen. Tijdschrift voor Diergeneeskunde, 129, pp.
210-214.
Vermeulen, B., Peek, H.W., Remon, J.P. & Landman, W.J.M. (2004). Effect of ibuprofen on coccidiosis
in boiler chickens. Avian Diseases, 48, pp. 68-76.
Hornok, S., Heijmans, J.F., Bekesi, L., Peek, H.W., Dobos-Kovacs, M., Dren, Cs.N. & Varga, I. (1998).
Interaction of chicken anaemia virus and Cryptosporidium bailey in experimentally infected chickens.
Veterinary Parasitology, 76, pp. 43-55.
Vertommen, M.H., Peek, H.W. & van der Laan, A. (1990). Efficacy of toltrazuril in broilers and
development of a laboratory model for sensitivity testing of Eimeria field isolates. The Veterinary
Quarterly, 12, pp. 183-92.
Cornelissen, A.W.C.A., Overdulve, J.P. & Peek, H.W. (1982). The role of the stomach and the small
intestine on the development of Isospora (Toxoplasma) gondii in cats infected directly into the small
intestine by laparotomy. In thesis of A.W.C.A. Cornelissen. (Ploidy, meiosis and Sex Differentiation
in Isospora (Toxoplasma) gondii with some considerations on other coccidian parasites. A study
combining the results of monoclonal infection and cytophotometry).
Cornelissen, A.W.C.A., Overedulve, J.P. & Peek, H.W. (1982). Sex determination and sex differentiation
in coccidia: gametogony and oocyst production after monoclonal infection of cats with intermediate
host stagess of Isospora (Toxoplasma) gondii. In thesis of A.W.C.A. Cornelissen. (Ploidy, meiosis
and Sex Differentiation in Isospora (Toxoplasma) gondii with some considerations on other coccidian
parasites. A study combining the results of monoclonal infection and cytophotometry).
LIST OF PUBLICATIONS
Conference proceedings/abstracts/posters
Landman, W.J.M. & Peek, H.W. (2005). Anticoccidial sensitivity profiles of recently obtained Dutch,
German and Spanish Eimeria spp. isolates. In the Proceedings of the IV International Poultry
Symposium, pp. 133-152. Istanbul, Turkey.
Peek, H.W. & Landman, W.J.M. (2005). Anticoccidial sensitivity profiles of recently obtained Dutch,
German and Spanish Eimeria spp. isolates. In the Proceedings of the IXth International Coccidiosis
Conference, pp. 170, Iguau Falls, Brazil.
Landman, W.J.M. & Peek, H.W. (2005). Higher incidence of Eimeria spp. field isolates sensitive for
diclazuril and monensin after live coccidiosis vaccination with Paracox-5. In the Proceedings of the
IXth International Coccidiosis Conference, pp. 157, Iguau Falls, Brazil.
Mohammed Ali Elmusharaf, Herman Peek, Anton Beynen, Lode Nollet and Lucy Tucker. (2005).
Development of a coccidiostat challenge model for screening of gut health promoting additives in
broilers. Alltech symposium.
Landman, W.J.M. & Peek, H.W. (2003). Anticoccidial sensitivity profiles of recent Dutch, German and
Spanish Eimeria spp. isolates. The Poultry Business Unit of Shering-Plough Animal Health, Irene
Meeting Room, Jaarbeurs Exhibition Centre, Utrecht, the Netherlands.
Vermeulen, B., Peek, H.W. & Remon, J.P. (2002). The effect of ibuprofen on sporulation and liveability
of Eimeria acervulina oocysts. In the Proceedings of the 11th European Poultry Conference. pp. 180181, Bremen, Germany.
Vermeulen, B., Peek, H.W. & Remon, J.P. (2002). The effect of ibuprofen on a coccidiosis infection in
boiler chickens. In the Proceedings of the 11th European Poultry Conference. pp. 180, Bremen,
Germany.
Landman, W.J.M. & Peek, H.W. (2002). Anticoccidialsensitivity profiles of West European Eimeria
spp. field isolates. In the Proceedings of the 11th European Poultry Conference. pp. 61, Bremen,
Germany.
Vermeulen, A., Schetters, T.,H. Janssen, H. Peek and W. Braunius. (1998). Oral application of live
oocyst-based coccidiosis vaccine: safety and efficacy. Parasitology International Vol. 47 (Suppl.) pp.
271-271.
Michels, H.J.M., Schoenmaker, G.J.M., Peek, H.W. & Vertommen, M.H. (1993). Field trail results in the
Netherlands using Clinacox in the second phase of the shuttle program. In J.R. Barta & M.A.
Fernando (Eds.), Proceedings of the VIth International Coccidiosis Conference. pp. 148, Guelph,
Canada.
sensitivity tests; resistance development; cross resistance to Baycox. In J.R. Barta & M.A. Fernando
(Eds.), Proceedings of the VIth International Coccidiosis Conference. pp. 154-155, Guelph, Canada.
Hooimeijer, J., Peek, H.W. & Vertommen, M.H. (1993). Coccidiosis in lorikeets infectious for
budgeriger. In the Proceedings of Annual Conference of the Association of Avian Veterinarians, pp.
59-61.
Vertommen, M.H. & Peek, H.W. (1989). Laboratory experiments on the sensitivity of Dutch Coccidia
field isolates to diclazuril. In P. Yvore (Ed.), Proceedings of the Vth International Coccidiosis
Conference, pp. 329-332, Tours, France.
244

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Resistance to anticoccidial drugs: alternative

strategies to control coccidiosis in broilers

Resistance to anticoccidial drugs: alternative strategies to control

Resistance to anticoccidial drugs: alternative

Resistentie tegen anticoccidiosemiddelen: alternatieve strategien

Hermanus Wilhelmus Peek

Prof. dr. J.A. Stegeman

Dr. W.J.M. Landman

Improvement of live anticoccidial vaccines

Aim of the thesis

Coccidiosis in poultry: anticoccidial products and

2. Host and site specificity of the coccidia

5. Other preventive strategies against coccidiosis

6. Conclusions and perspectives

1. Coccidiosis in poultry: history, classification, Eimeria spp. life

1.2. History and classification of the genus Eimeria

1.3. Eimeria spp. in farm and game birds

1.3.1.1. Domestic fowl

1.4. Life cycle of avian Eimeria spp.

Table 1. Diagnostic characteristics of Eimeria spp. in the chicken

Table 2. Diagnostic characteristics of Eimeria spp. in the turkey

Oocyst size (m)

Moore & Brown,

Moore & Brown,

Mitra & Das Gupta,

Figure 1. Life cycle of Eimeria spp. in chickens

1.5. Eimeria spp. determination and diagnosis

1.5.1. Advances in diagnosis

2. Host and site specificity of the coccidia

2.2. Host specificity of Eimeria spp.

Moore & Brown, 1951

The mechanisms responsible for inhibiting the reproduction/multiplication of Eimeria spp.

2.2.1. Parasite biochemistry and nutrition

2.3. Site specificity of Eimeria spp.

McCully et al., 1970

Novilla et al., 1981

Lotze et al., 1964

3. Anticoccidial products: mode of action, the occurrence of

3.2. Anticoccidial products

Aviochina, Quinatrol, SQ,

Waletsky et al. (USA)

Cuckler & Malanga

Nicarb, Nicoxin, Nicrazin

NF-180, Furovag, Furoxane,

Zoamix, DOT, DNOT

Amprol Plus, Amprol Hi-E

Clopidol or meticlorpindol, 0.0125-0.025%

Agribon, Albon, Rofenaid

Vertommen & Peek

Kawazoe & Fabio

Totrazuril, 7 mg/kg bodyweight

Table 7. Contemporary anticoccidial products and recommended doses for prophylactic

Concentration in feed (ppm)

3.2.2. Mode of action

Resistance has been described for all anticoccidial drugs introduced.

3.2.3. Antimicrobial & growth promoting properties of anticoccidial products

As necrotizing enteritis is a multifactorial disease in which coccidiosis may be involved,

3.2.5.1. Anticoccidial products currently available in the Netherlands

3.3. Anticoccidial product resistance

Shuttle programs are characterized by the alternation of anticoccidial compounds within a

4. The influence of feed structure, composition, toxins and

4.2. Feed structure

4.3. Feed composition

Different types of saccharides