UNIVERSITY OF HARGEISA

COLLEGE OF VETERINARY MEDICINE

Seroprevalence of Brucella melitensis in Hargeisa district in Somaliland

A Thesis Submitted to University of Hargeisa College of Agriculture

andVeterinaryMedicine in Partial Fulfillment of the Requirements for the Degree of
Doctor of Veterinary Medicine

By

Rooble Mohamoud Ahmed

supervisor : Dr Abdiqadir Mohamed Aden

August, 2016

HARGEISA ,SOMALILAND

1|Page
DECLRATION

I, Roble Mohamoud Ahmed declares that the work presented here is my original work and has
not been appear anywhere else in any form and has not been presented in any institution of
higher education

2|Page
ACKNOWLEGMENT

All thanks belong to almighty ALLAAH who created us from nothing to human being I would
also like to thank everyone who take part and play even minor role to my studies and writing this
thesis. My especial thanks go to all my dear tutors for their effort and honest education that they
gave me during my studies.

I am so grateful to my supervisor Dr Abdiqadir Mohamed Aden for his encouragement and

friendly support also I am thankful to my uncle who helped me during my field work.

My deepest thanks belong to my beloved parents and all my brothers and sisters for their effort
and wishing to my success

3|Page
DEDICATION

This thesis is dedicated to my dearly loved parents my mother ASHA YOUSUF ALI and my
father MOHAMOUD AHMED H.YOUSUF for their higher contribution to my enjoyable
education and respected life and I say ALLAH my bless you.

4|Page
Contents
DECLRATION ............................................................................................................................... 2
ACKNOWLEGMENT ................................................................................................................... 3
DEDICATION ................................................................................................................................ 4
ABSTRACT .................................................................................................................................... 6
1. INTRODUCTION .................................................................................................................... 10
Background ................................................................................................................................... 10
Prevelance in goat brucellosis....................................................................................................... 12
PROBLEM STATEMENT ........................................................................................................... 12
CHAPTER TWO: LITERATURE REVIEW ............................................................................... 14
2.1 The definition of the disease: -................................................................................................ 14
2.2 Aetiology: ............................................................................................................................... 14
2.3Morphology and characteristics: - ........................................................................................... 14
2.4 Pathogenesis of brucellosis ..................................................................................................... 15
2.5 Public Health Significance of Brucellosis .............................................................................. 17
2.6 Economic Significance of Brucellosis .................................................................................... 18
2.7 Epidemiology: ......................................................................................................................... 19
2.8 Geographic Distribution.......................................................................................................... 19
2.10 Disease transmission ............................................................................................................. 21
2.11Clinical signs .......................................................................................................................... 22
2.12 Pathology of brucellosis........................................................................................................ 23
2.13 Risk Factors .......................................................................................................................... 24
2.14 Environmental and climatic factors ...................................................................................... 25
2.15 Diagnostic Tests .................................................................................................................... 26
2.5.1 Serology ............................................................................................................................... 27
2.5.2 Culture.................................................................................................................................. 28
2.16 Prevention ............................................................................................................................ 29
2.17 Treatment of brucellosis ....................................................................................................... 30
2.18Vaccination of brucellosis...................................................................................................... 31
2.19 Management practices and movement control herd ............................................................ 31
2.20 Communicability.................................................................................................................. 32

5|Page
CHAPTER THREE ...................................................................................................................... 33
3. MATERIALS AND METHODS .............................................................................................. 33
3.1. Description of Study Areas and Population ........................................................................... 33
3.2. Study Area ............................................................................................................................. 33
3.3.Study design ............................................................................................................................ 34
3.4. Animals and data collection ................................................................................................... 34
3.5. Sample Collection and Transportation................................................................................... 34
3.6 Questionnaires......................................................................................................................... 35
3.7 Serological tests ..................................................................................................................... 35
3.8 Modified Rose Bengal Plate Test (mRBPT) ........................................................................... 35
3.9 Complement Fixation Test (CFT) ........................................................................................... 36
3.10 Data analysis ......................................................................................................................... 36
CHAPTER FOUR RESULTS ...................................................................................................... 37
4.1 Results ..................................................................................................................................... 37
4.2 Seroprevalence of brucellosis in goats around hargeisa town of Somaliland ........................ 37
4.3 prevalence distribution of Brucella according to sex .............................................................. 38
4.3 Knowledge of pastoralistson the disease (Brucellosis) and associated risk transmission ...... 39
CHAPTER FIVE: CONCLUSIONS AND RECOMMENDATIONS ......................................... 43
5.1 Conclusion .............................................................................................................................. 43
5.2 Recommendations ................................................................................................................... 44
APPENDEX ( A)REFERENCES ................................................................................................. 44
APPENDEX {B}QUESTIONAIRE ............................................................................................. 47

6|Page
List of Tables

Table 1: this pie chart shows the overall prevalence of brucellosis in goats in the study area.

Table 1.2 shows that 32% of the respondent are secondary and 40% are Primary level and 30%
are university level 30%

Table1.3 shows that 34% of the goats are male and 64% of the goats are female

Table1.4 shows that 34% of the goats are male and 64% of the goats are female

Table 1.5 shows that 17% of the animals aborted while 83% are not aborted

Table 1.6 shows that 17% of the animals aborted in the year while 83% of the animal not aboted
per year

Table 1.7 show that 16% of the animals aborted early gestation and 28% of the animals aborted
mid gestation and 56% of the animals aborted during late

Table1.8 shows that 54% of animal have fair sanitary system while 46% are good sanitary
system

Table 1.9 shows that 15% of the animals have confinement housing system and 35% have partial
confinement system and 50% night stable

Table 10: shows determination of clinical signs related to brucellosis in goats

Figure 1: Study areas (districts) in Hargeisa state of somaliland

7|Page
LIST OF ABBREVIATIONS

asl - above sea level

CFT -Complement Fixation Test

GDP - Gross Domestic Product

RBPT - Rose Bengal Plate Test

MRBPT -Modified Rose Bengal Plate Test

mm - millimeter

SAT - Serum Agglutination Test

WHO - World Health Organization

ILRI - International Livestock Research Institute

NVI -National Veterinary Institute

ELISA -Enzyme Linked Immuo Sorbent Assay

DVM -doctor of veterinary medicine

MVSc -master of veterinary science

8|Page
ABSTRACT

A retrospective study design was employed on previously collected sera samples to investigate
brucellosis in goats from june and july 2016 with the objectives of estimating the seroprevalence
and the occurrence of brucellosis in goats in selected district; ministry of livestock,m.mooge and
kilarka ,hodan village A total of 150 sera samples (male90 and female60 goats ) were tested
using serological tests, screening by RBPT and confirmatory test CFT, The seroprevalence of
brucellosis was calculated as the number in study population testing positive to the serological
test divided by the total study units tested. The Overall seroprevalence of 2%, 1%, and 2.05%
respectively. Data including serological test result and earlier collected questionnaire survey
were recorded and coded in Microsoft Excel spread sheets and then statistically analyzed using
(stata tm 11.0) to determine the strength of potential risk factors associated with the occurrence of
brucellosis by using univariable logistic regression. Mixed flock OR=2.11(1.33-3.36 CI;
p=0.002), agro-pastoral OR=4.01 (2.35-6.84CI; p=0.000) and pastoral OR=2.59(1.37-4.90 CI;
p=0004) production system, larger flock size OR=1.68(1.08-2.60CI; p=0.021) were factors
significantly affecting the prevalence of small ruminant brucellosis. By considering collinearity
of variables, p<0.25 in univariable analysis and independent predictors of goats in brucellosis
were further analyzed using multilogistic regression.

Key words: brucellosis, CFT, RBPT, risk factors, brucella mellitensis, sera, unilogstic
regression, multilogstic regression.

9|Page
 1. INTRODUCTION

1.1Background

Livestock is currently the leadingeconomic sector in all of Somaliland. Livestock production

accounts for 60-65%of gross domestic product(GDP).Based on 1998 estimates of livestock
numbers and past growth rates, Somalilandhas about 1.69million camels, 0.40 million head of
cattle, 8.4 million goatsand 8.75 million sheep in 2011. The Sool, Sanaag and Togdheerregions
account forabout 75% of all livestock. Somalilands major livestock exports aresheep and goats,
accounting for 91% ofall animal exports. In 2010 a total of2.352 million shoats were exported
through the Berbera port (including fromEthiopian sources). Of this total, 1.612 million (69%)
were exported betweenSeptember and November for the Hajj festivities. Assuming an average
exportprice of US$70, the estimated total value would be over US$160 million. Withgovernment
taxes at around US$3.60 per head, this means tax revenue of aroundUS$8.5 million, or 10% of
the Somaliland governments total revenue.Somaliland, and to a certain extent the other
exporters from the Horn of Africa,depends on only a few countries for exports. For -example out
of the 2.585million head of sheep and goat exported through the Port of Berbera in 2010,
about78% went to Saudi Arabia, 20% to Yemen and the rest to Egypt and Oman.
goats and their products are important export commodity significantly contributing national
economy; moreover they support the livelihood of millions of pastoral peoples as a source of
milk and meat. Their adaptability to broad range of environments, short generation cycle and
high reproductive rates that lead to the high production efficiency made goats production an
attractive enterprise in pastoral production system (PFE, 2004). There was a growing export
market for sheep and goats meat in the Middle Eastern Gulf States and African countries (Alemu
and Markel, 2008). The main constraints to live stock development in Ethiopia are nutritional
shortage, traditional husbandry, water shortage, poor marketing and different disease that limit
productivity .
Brucellosis is a worldwide bacterial zoonotic disease affecting both animals and humans. It
causes heavy economiclosses to the livestock industry and also poses serious human health
hazards. The disease is caused bymembers of genus Brucella that are gram-negative, facultative
intracellular, coccobacilli, non-motile and non-spore forming bacteria. The genus Brucella

10 | P a g e
currently consists of ten classified species of Brucella in domestic animals coused by Brucella
abortus(cattle), Brucella melitensis (goats), Brucella suis(pigs), and Brucellaovis(sheep). How
ever,several of other species are also pathogenic for humans.

Brucella infection is transmitted by direct or indirect contact with infected animals. The
organism is mostfrequently acquired by ingestion. Respiratory route, conjunctival and genital
inoculation, skin contamination, intrauterineand venereal transmissions are other possibilities of
the organism . Humans can become infectedindirectly through contact with infected animals or
by consumption of animal products.Person-to-persontransmission is extremely rare. Vertical
transmission, breastfeeding, blood transfusion or tissue transplantation.Brucellosis in animal is
characterized primarily by abortion in late pregnan .frequently followed by retention of foetal
membrane and endometritis which may be a cause of infertility. In bulls the disease usually
causes orchitis, epididymitis, seminal vesiculitis and sterility.

In Africa, brucellosis is considered to be one of th most serious disease problems facing the
veterinary profession. The high prevalence is due to close human-animal contacts, food
consumption customs and the fact that many countries have not yet started control or eradication
schemes. According to STAAK. (1990),Brucellos is is perhaps the most widespread and
economically important disease in tropical and sub-tropical regions. The direct loss of meat (as a
result of abortion, infertility and weight loss) in infected herds of cattle was estimated to be 15%
while that of milk (reduced milk production) was 20%.

Brucella melitensis has a worldwide distribution and it is an important disease among livestock
and people in Sub-Sahara Africa.

In Somaliland brucellosis one most serious of the disease in the livestock which constitutes
amajor impediment for livestock trade though brucellosis is the only disease which is diagnosed
in the port town of Berbera prior to shipment of animals to gulf states such as Saudi Arabia.
It remains an uncontrolled problem and its prevalenceinSomaliland regions is increased in that
almost all domestic species can be affected with brucellosis and cross-transmission can occur
between animal such as camal,cattle,sheep and goat and other species.

11 | P a g e
 1.2 Prevalance of goat Brucella in Somaliland

Brucella melitensis Disease of goats, also Transmission congenital or by ingestion or contact

with infected placenta, vaginal discharge, or milk. Clinical findings, Abortion storms, abortions
often in last 2 months of pregnancy. Weak-born lambs. Important zoonotic disease in humans.
Clinical pathology,Culture of organism.Serological tests and skin hypersensitivity testing for
herd diagnosis. Necropsy findings:Placentitis also Control: Slaughter eradication. Vaccination
with Rev. 1 vaccine. Rev. 1 vaccine will produce abortion in pregnant animals

Prevelance in goat brucellosis

Ghanemet al. (2009) documented prevalence of 4.9% and 3.9% in goats in three main regions of
soamaliland (Awdal, WaqoyiGalbed and Togdheer) byusing RBPT and i-ELISA respectively

PROBLEM STATEMENT
Lack of sufficient knowledge of the disease of brucellosis, and the absent of effective prevention
and management strategies and continuous movement (mixing of the animals).Also lack of
awareness of livestock farmers and butchers about the brucellosis disease contribute to the
continuous and wide spread of the disease. Worst of all, there are no documented information
about goat in hargeisa somalalinad. Therefore, this preliminary study will determine the
prevalence of goat of Brucella is and identify the associated impacts of the disease to livestock
herders under the extensive husbandry systems in hargeisa district. It is a major veterinary public
health challenge, as animals are almost exclusively the source of infection to people. It is often
undiagnosed in both human patients and animal sources and it is widely acknowledged that the
epidemiology of Brucellos is in goat is poorly understood, particularly in Somaliland.
Surveillance and control of brucellosis is not implemented and even the disease is ignored in
humans and in most cases go undiagnosed and untreated.

1.5 General Objectives

12 | P a g e
 To investigate the seroprevelance of Brucella melitensis in Hargeisa district in general.
To examine the disease distribution between goat male and female in Hargeisa
Somaliland

1.6 Specific Objectives

To determine the prevalence of goat brucellosis in Hargeisa district

To find out the socio-economic impacts of the disease for the domestic animals in
Hargeisa
To produce an Epidemiological Map of Brucella which as base to develop control
strategy.

13 | P a g e
CHAPTER TWO: LITERATURE REVIEW

2.1 The definition of the disease: -

Brucellosis is an infectious disease caused by the bacteria of the genus Brucella. These
bacteria are primarily passed among animals, and they cause disease in many different
vertebrates. Various Brucella species affect sheep, goats, cattle, deer, elk, pigs dogs, and
several other animals. Humans become infected by coming in contact with animals or
animal products that are contaminated with these bacteria. In humans brucellosis can
cause a range of symptoms from mild flu-like illness to severe infection of the central
nervous system or lining of the heart. It can also cause long-lasting or chronic symptoms
that include recurrent fevers, joint pain, and depression(Gameel et al., 2002).

2.2 Aetiology:

2.3Morphology and characteristics: -

Brucellosis results from infection by various species of Brucella, a Gram negative,
facultative intracellular coccobacillus or short rod in the family Brucellaceae. Six named
species occur in animals: B. abortus, B. melitensis, B. suis, B. ovis, B. canis and B.
neotomae. One or more unnamed species of Brucella have been found in marine
mammals. Formal names proposed for marine mammal isolates are B. Maris for all
strains, or B. pinnipediae for strains from pinnipeds (seals, sea lions and walruses) and B.
cetaceae for isolates from cetaceans (whales, porpoises and dolphins).

Some species of Brucella contain biovars. Five biovars have been reported for B. suis,
three for B. melitensis, and up to nine for B. abortus. Each Brucella species is associated
most often with certain hosts. B. abortus usually causes brucellosis in cattle, bison and
buffalo. B. melitensis is the most important species in sheep and goats, but B. ovis can
also cause infertility in rams. B. canis causes disease almost exclusively in dogs. B.
neotomae is found in rodents, but has not been linked to disease. B. suis contains more
diverse isolates than other Brucella species, and these isolates have broader host

14 | P a g e
specificity. B. suisbiovars 1, 2 and 3 are maintained in pigs; European hares are also a
reservoir for biovar. Biovar 4 mainly affects reindeer and caribou, and is not normally
found in pigs(OIE2000. El-Sherbini et al., 2007).

This biovar was formerly known as B. rangiferi. Biovar 5 occurs in rodents. In humans,
brucellosis can be caused by B. abortus, B. melitensis, B. suisbiovars 1-4 and, rarely, B.
canis or marine mammal Brucella. Live vaccines for B. abortus and B. melitensis, as well
as the B. canis M- strain (a less virulent strain used as an antigen for serological testing),
are also pathogenic for humans. B. ovis, B. neotomae and B. suisbiovar 5 have not been
linked to human disease. Genetic and immunological evidence suggests that all members
of the genus Brucella are closely related, and some microbiologists have proposed that
this genus be reclassified into a single species (B. melitensis), which contains many
biovars. This proposal is controversial, and both taxonomic systems are currently in use.
The multiple species nomenclature is used in this factsheet(Musa et al., 2008).
2.4 Pathogenesis of brucellosis: -
The ability of Brucella spp. to cause disease requires a few critical steps during infection.
Brucella spp. can invade epithelial cells of the host, allowing infection through mucosal
surfaces: M cells in the intestine have been identied as a portal of entry for Brucella spp.
Once Brucella spp. has invaded, usually through the digestive or respiratory tract, they
are capable of surviving intracellularly within phagocytic or non-phagocytic host cells.
Brucella has the ability to interfere with intracellular trafficking, preventing fusion of the
Brucella-containing vacuole (BCV).
With lysosome markers and directing the vacuole towards a compartment that has rough
endoplasmic reticulum (RER), which is highly permissive to intracellular replication of
Brucella.
The outcome of infection is dependent on the species of Brucella and host. The Brucella
spp. that infects livestock is host restricted. For instance, Brucellamelitensis, B. abortus,
B. suis and B. ovis infect preferentially small ruminants, cattle, pigs and sheep,
respectively. With the exception of B. ovis, this Brucella spp. has zoonotic potential, with
B. melitensis being the most pathogenic for humans (ILCA, 1980).

15 | P a g e
The mechanisms that allow host cell invasion by Brucella spp. are not completely clear,
but although specic host receptors that interact with Brucella have not yet been
identied, internalization of Brucella into host cells requires cytoskeleton changes.
Interestingly, invasion through the digestive tract does not elicit any inammatory
response from the host.
Therefore, Brucella spp. invades silently or unnoticed by the innate immune system of
the host. In fact, Brucella spp. has mechanisms that prevent activation of the host innate
immune system. Indeed, Brucella Toll/interleukin-1 receptor (TIR) domain-containing
protein prevents Toll-like receptor (TLR) 2 signaling by interfering with MyD88, and
also inhibits DC maturation, cytokine secretion and antigen presentation. Brucellaabortus
also induces suppression of the transcription of pro-inammatory mediators in
trophoblastic cells at very early stages of infection. Trophoblasts are placental cells that
are targeted during infection of pregnant cows. After an initial suppression of pro-
inammatory transcripts, B. abortus induces expression of pro-inammatory chemokines
by cultured trophoblastic cells, which correlates with the prole of expression observed
in vivo in the placenta of infected cows (Seifert, 1996; Radostits et al., 2007).

Brucella spp. lack classical bacterial virulence factors such as exotoxins, cytolysins, a
capsule, mbriae, agella, plasmids, lysogenic phages, endotoxic lipopolysaccharide
(LPS), and inducers of host cell apoptosis. However, LPS plays an important role in
Brucella virulence because it prevents complement-mediated bacterial killing and
provides resistance against antimicrobial peptides such as defensins and lactoferrin.
Another important virulence mechanism of Brucella is the BvrR/BvrS two-component
regulatory system, which is required for modulation of the host cell cytoskeleton upon
Brucella invasion, and for regulation of the expression of outer membrane proteins, some
of which are required for full virulence. Cyclic -1, 2-glucans, which are also part of the
outer membrane, is also required for intracellular survival of Brucella. Brucella spp.
express a type IV secretion system (T4SS), encoded by the components of the virB
operon, that is crucial for intracellular survival in host cells and virulence in vivo
(Radostits, 2007).

16 | P a g e
Unlike other type IV systems, which are expressed extracellularly, transcription of the
virB operon is induced specically within macrophages, and phagosome acidication is a
key intracellular signal inducing virB expression.
The Brucella T4SS is required for persistence in mice and induction of the host immune
response. It is also essential for elicitation of inammatory and immune responses during
Brucella infection in mice, and it is required for microgranuloma formation during
Brucella infection. The T4SS is required for Brucella to reach its intracellular replication
niche, and its expression is regulated by the BvrR/BvrS two-component regulatory
system. The T4SS delivers Brucella effector proteins into the host cell cytosol, but the
identities and roles of these effectors have only recently begun to emerge.
A novel strategy used for screening proteins of unknown function that are involved in
proteinprotein interactions was used to identify Brucella effectors. Using this approach,
four putative proteins (BPE043, BPE005, BPE275 and BPE123), which were
translocated into mouse macrophages by B. abortus, are hypothesised to be candidates for
modulation of host cell functions.
2.5 Public Health Significance of Brucellosis: -
Five out of nine known Brucella species can infect humans. The most pathogenic and
invasivespecies for human are, B. melitensis, B. abortus, and B. canis. The zoonotic
nature of marine Brucella has been documented. Human brucellosis caused by B.
melitensis is the most sever one followed by B. suis, B. abortus and B. canis in
decreasing order. They are listed as potential bio-weapons by the contents for disease
control and prevention program in USA. This is due to the highly infectious nature of
three species, as they can be aerosolized(FAO, 2003).
Moreover an outbreak of brucellosis would be difficult to detect because the initial
symptoms are easily confused with those of influenza 24. Occurrence in Humans: Each
year half a million case of brucellosis occurs in humans around the world. The prevalence
of infection in animal reservoir provides a key of its occurrence in humans (Scholz et al.,
2008). B. abortus and B. suis infection usually affect occupational groups. B. melitensis
infection occurs more frequently than others types in the general population. In the Latin
American countries, the greatest number of record are Argentina, Mexico, and Peru. The

17 | P a g e
same pattern holds true to the Mediterranean countries, Iran, the former Soviet Union and
Mongolia.

2.6 Economic Significance of Brucellosis: -

Brucellosis occurring worldwide in domestic and game animals as well as humans creates
a serious economic problem for the intensive and extensive livestock production systems.
Losses in animal production due to this disease can be of major importance primarily
because of 20% decreased milk production in aborting cows. The common sequel of
infertility increases the period between lactations(Cooper, 1992). The average inter
calving period of an infected herd prolonged by several months.

In addition, it results in loss of calves and interference with the breeding program. This is
of the greatest importance in beef herds, where the calves represent the sole source of
income. A high incidence of permanent infertility results in heavy culling of valuable
cows and some deaths occur as a result of acute metritis following retention of the
placenta. The economic losses due to bovine brucellosis include: losses of calves due to
abortion, reduced milk yield, culling and condemnation of valuable cows because of
breeding failure, endangering animal export trading of a nation, loss of man power,
medical costs and government cost for research and eradication programs. Available
information indicates that brucellosis is one of the most serious diseases of cattle in Latin
America and other developing areas(Abbas et al., 1987, Gameel et al., 1993). Official
estimates put annual losses from bovine brucellosis in Latin America at approximately
US$ 600 million 8. Brucellosis in sheep caused by B. ovis has been reported in Australia,
Newzealand the United state, South Africa and Europe.
The incidence has been very high in some areas, and there was much economic loss at
one time. In California, 30-40% of rams were thought to be affected and annual loss of
US $ 2million was estimated. B. suis is a chronic disease of swine manifested by sterility
and abortion in sows, heavy piglet mortality and orchitis in boars. The disease owes its
economic importance to the fertility and reduction in numbers of pigs weaned per litter
that occur in infected herds (FAO/OIE/WHO., 1997).

18 | P a g e
 2.7 Epidemiology: -
The epidemiology of brucellosis is complex. The important factors that could contribute
to the occurrence and spread in livestock include, farming system and practice, farm
sanitation, live stock movement, sharing of grazing lands and moderate changes towards
identification 18. Global Perspective: Brucellosis occurs worldwide in domestic and
game animals 5. Bovine brucellosis has been eradicated from most industrialized
countries such as in Finland, Norway, Sweden, Denmark, Germany, Australia, and
The world the rates of brucellosis caused by B. abortus vary greatly from one country to
another and between regions with in a country(Manture et al., 2007).

The highest prevalence is noticed in dairy cattle. Even in highly developed countries like
USA and France have so far not been able to eradicate brucellosis completelyBrucellosis
caused by B. melitensis occurs in sheep and goat raising regions of the world with
exception of North America, Australia and Newzealand. B. suis infection also occurs
worldwide 12, 14. Brucellosis is an important livestock disease in many African
countries 14. The incidence of infection up to 80% can be found in intensive dairy
production systems of the tropics. The extensive animal production systems of the Sahel,
an average diseases incidence of 25 -30% has been calculated. In eastern Sudan an
infection rate in cattle of almost 22% and in sheep about 13.6% was found.

2.8 Geographic Distribution: -

Brucellosis is found worldwide but it is well controlled in most developed countries.
Clinical disease is still common in the Middle East, Asia, Africa, South and Central
America, the Mediterranean Basin and the Caribbean.
Brucella species vary in their geographic distribution.
B. abortus is found worldwide in cattle-raising regions except in Japan, Canada, and
some European countries, Australia, New Zealand and Israel, where it has been
eradicated. Eradication from domesticated herds is nearly complete in the U.S. B. abortus
persists in wildlife hosts in some regions, including the Greater Yellowstone Area of
North America. B. melitensis is particularly common in the Mediterranean. It also occurs

19 | P a g e
in the Middle East and Central Asia, around the Persian Gulf (also known as the Arabian
Gulf) and in some countries of Central America.
This organism has been reported from Africa and India, but it does not seem to be
endemic in northern Europe, North America (except Mexico), Southeast Asia, Australia
or New Zealand. B. ovis probably occurs in most sheep-raising regions of the world. It
has been reported from Australia, New Zealand, North and South America, South Africa
and many countries in Europe. In the past, B. suis was found worldwide in swine- raising
regions. This organism has been eradicated from domesticated pigs in the U.S., Canada,
many European countries and other nations (Sahin et al., 2008). However, it persists in
wild and/ or feral swine populations in some areas, including the U.S., Europe and
Queensland, Australia. Sporadic outbreaks are reported in domesticated herds or humans
due to transmission from this source. B. suis continues to occur in domesticated herds in
some countries of South and Central America (including Mexico) and Asia.

B. suisbiovars 1 and 3 are found worldwide, but other biovars have a limited geographic
distribution. Biovar 2 occurs in wild boar in much of Europe. Biovar 4 (rangiferine
brucellosis) is limited to the Arctic regions of North America and Russia including
Siberia, Canada and Alaska. Biovar 5 (murine brucellosis) is found in the former USSR.
B. canis probably occurs throughout most of the world; however, New Zealand and
Australia appear to be free of this organism. Brucella species also seem to be widespread
in marine mammal populations. Culture-positive or seropositive animals have been found
in the North Atlantic Ocean, the Mediterranean Sea, and the Arctic including the Barents
Sea. Infected or exposed animals have also been found along the Atlantic and Pacific
coasts of North America; the coasts of Peru, Australia, New Zealand and Hawaii; and in
the Solomon Islands and the Antarctic (AOAD, 1995).
2.9 Prevalence of brucellosis in East Africa countries: -
Brucellosis, particularly due to B. abortus, is considered to be one of the most important
zoonotic diseases of camels and other domestic animals in some countries of East Africa.
Camel brucellosis was recorded to be caused by B. abortus and B. melitensis with a
prevalence of 1.8-20% (Abbas and Agab 2002). Several published literature regarding the
prevalence of domestic animal brucellosis from different countries were.

20 | P a g e
2.10 Disease transmission: -
Transmission B. abortus, B. melitensis, B. suis and B. canis are usually transmitted
between animals by contact with the placenta, fetus, fetal fluids and vaginal discharges
from an infected animal. Animals are infectious after either an abortion or full term
parturition. Although ruminants are usually asymptomatic after their first abortion, they
can become chronic carriers, and continue to shed Brucella in milk and uterine discharges
during subsequent pregnancies (charters, 1980; DFRA, 2002).

Most or all Brucella species are also found in semen. Males can shed these organisms for
long periods or lifelong. The importance of venereal transmission varies with the species.
It is the primary route of transmission for B. ovis. B. suis and B. canis are also spread
frequently by this route. B. abortus and B. melitensis can be found in semen, but venereal
transmission of these organisms is uncommon. Some Brucella species have also been
detected in other secretions and excretions including urine, feces, hygromafluids, saliva,
and nasal and ocular secretions. In most cases, these sources seem to be relatively
unimportant in transmission; however, some could help account for direct non-venereal
transmission of B. ovis between rams (Cooper, 1992). Brucella can be spread on fomites
including feed and water. In conditions of high humidity, low temperatures, and no
sunlight, these organisms can remain viable for several months in water, aborted fetuses,
manure, wool, hay, equipment and clothes.

Brucella can withstand drying, particularly when organic material is present, and can
survive in dust and soil. Survival is longer when the temperature is low, particularly when
it is below freezing. Accidental hosts usually become infected after contact with
maintenance hosts. Although the ruminant udder is usually colonized during the course of
an infection, it can also be infected by direct contact (for example, by bacteria on the
hands of farm workers). This can result in the long- term shedding of species not
normally found in ruminant milk, such as B. suis. Humans usually become infected by
ingesting organisms or by the contamination of mucous membranes and abraded skin.

21 | P a g e
 In the laboratory and probably in abattoirs, Brucella can be transmitted in aerosols.
Common sources of infection for people include contact with animal abortion products;
ingestion of unpasteurized dairy products from cows, small ruminants or camels;
ingestion of undercooked meat, bone marrow or other uncooked meat products; contact
with laboratory cultures and tissue samples; and accidental injection of live brucellosis
vaccines (Perry et al., 2001). Human to human transmission is rare, but has been reported
after blood transfusion, bone marrow transplantation or sexual intercourse. Rare
congenital infections seem to result from transplacental transmission or the ingestion of
breast milk. Congenital infections might also occur if the infant is exposed to organisms
in the mothers blood, urine or feces during delivery.
2.11Clinical signs: -
Brucellosis is a multisystemic disease with a broad spectrum of symptoms.
Asymptomatic infections are common. In symptomatic cases, the disease is extremely
variable and the clinical signs may appear insidiously or abruptly. Typically, brucellosis
begins as an acute febrile illness with nonspecific flu-like signs such as fever, headache,
malaise, back pain, myalgia and generalized aches. Drenching sweats can occur,
particularly at night. Splenomegaly, hepatomegaly, coughing and pleuritic chest pain are
sometimes seen.
Gastrointestinal signs including anorexia, nausea, vomiting, diarrhea and constipation
occur frequently in adults but less often in children. In many patients, the symptoms last
for two to four weeks and are followed by spontaneous recovery. Others develop an
intermittent fever and other persistent symptoms that typically wax and wane at 2 to 14
day intervals (Alton, 1990, Duran-Ferrerr, 1998). Most people with this undulant form
recover completely in three to 12 months. A few patients become chronically ill.
Relapses can occur months after the initial symptoms, even in successfully treated cases.
Hypersensitivity reactions can mimic the symptoms of brucellosis. Complications are
seen occasionally, particularly in the undulant and chronic forms. The most common
complications are arthritis, spondylitis, epididymo-orchitis and chronic fatigue.

Neurological signs occur in up to 5% of cases. They may include personality changes,

meningitis, encephalitis and peripheral neuropathy. Uveitis, optic neuritis and

22 | P a g e
papilledema have been reported. Endocarditis is one of the most serious complications,
and is often the cause of death in fatal cases. Many other organs and tissues can also be
affected, resulting in a wide variety of syndromes including nephritis, dermatitis,
vasculitis, lymphadenopathy, deep vein thrombosis, granulomatous hepatitis,
cholecystitis, osteomyelitis, anemia, leukopenia and thrombocytopenia. Abscesses can
occur in internal organs. The symptoms of congenital brucellosis are variable. Some
congenitally infected infants are delivered prematurely, while others are born at full term.
Common symptoms include low birth weight, fever, failure to thrive, jaundice,
hepatomegaly and Splenomegaly. Some newborns with congenital brucellosis have
respiratory difficulty or severe respiratory distress, hypotension, vomiting and other signs
of sepsis. Other infants may be asymptomatic or have only mild symptoms at birth.
Whether brucellosis can lead to spontaneous abortion in humans is controversial (Young
and Corbel, 1989).
2.12 Pathology of brucellosis: -
Examination of tissue sections from Brucella spp.-positive aborted fetuses and placentas
revealed a severe necropurulentplacentitis and mild bronchointerstitial pneumonia.
Lesions in the placentas were characterized by edema of the chorionic stroma and
multifocal necrosis of the al- lantochorion and placental Trophoblasts accompanied by
large accumulations of neutrophils and degenerate leukocytes. Severe well-demarcated
necrosis of coty- ledonaryvilli was characteristic (Chukwu, 1987).
Large numbers of tiny Gram-positive coccobacilli were present in some Trophoblasts.
Immunohistochemical (IHC) staining of placental sections demonstrated numerous la-
belled bacteria in Trophoblasts and adherent exudates. Lung sections from the three B.
abortus-positive aborted fetuses had mild hypercellularity of interalveolarseptae and
occasional large macro- phages, amnionic debris, few neutrophils, and occasional or rare
binucleated or multinucleated cells in airways. There was par- ticulate IHC labeling of
scattered macro- phages, amnionic debris, and rare neutrophils in airways.
In one fetus, a small irregular area of caseous necrosis in the splenicred pulp contained
numerous IHC-positive coccobacilli. Lesions in Brucella spp.-negative fetuses and
neonates consisted of mild purulent bronchointerstitial pneumonia in one neonate and
mildpneumonia with purulent placentitis of unknown etiology in one aborted fetus.

23 | P a g e
Histopathologic lesions in the 2-wk-old calf consisted of bronchointerstitial pneumonia
with moderate numbers of macro- phages and neutrophils in airways, marked purulent
nephritis, focal splenic infarction and moderate lymphocyte depletion of the splenic white
pulp and lymph node cortices. Immunohistochemical staining for B. abortus resulted in
marked particulate la- belling in pulmonary macrophages and in scattered cells in lymph
node follicles (Gameel et al., 1993). In the adults, histologic lesions of the uterus were
limited to two animals. Examination of uterus and placenta from revealed a marked
endometritis and placentitis characterized by moderate endometrial inltrates of
lymphocytes, plasma cells, and neutrophils; focal uterine epithelial necrosis; adherence of
neutrophils, macrophages, de- generate leukocytes, and brin to the endometrium; and
marked necrosis of placental Trophoblasts with diffuse inltrates of neutrophils in the
chorionic stroma.

Special stains and IHC staining of selected specimens for B. abortus showed labeling of
large numbers of coccobacilli in Trophoblasts, in exudates adherent to the placenta and
endometrium, and within the placental chorion. A seronegative animal had a mild,
diffuse, lymphoplasmacyticinltrate of the endometrium with several, large, focal,
endometrial accumulations of lymphocytes. Special stains for bacteria and IHC staining
for B. abortus were negative.

The cause of the endometritis was undetermined. Lesions were not observed in fetal
tissues and other placentas collected from the bison cows. Micro- scopically, the liver
abscess consisted of a necropurulent center containing large numbers of Gram-positive
lamentous bacteria surrounded by marked brosis and lymphoplasmacytic inltrates.
The lesion was considered unrelated to B. abortus infection (Abou-Eisha, 2000; El-
Ansary et al., 2001).
2.13 Risk Factors: -
Animal risk factors: Susceptibility of cattle to B. abortus infection is influenced by the
age, sex and reproductive status of the individual animal. Sexually mature pregnant cattle
are more susceptible to infection with the organism than sexually immature cattle of
either sex. Susceptibility increases as stage of gestation increases. Pathogen risk factor:

24 | P a g e
B. abortus is a facultative intracellular organism capable of multiplication and survival
within the host phagosome.

The organisms are phagocytised by polymorphonuclar leucocytes in which some survive

and multiply. The organism is able to survive within macrophages because; it has the
ability to survive phagolysosome. The bacterium possesses an unconvenential non-
endotoxin lipopolysaccaride, which confers resistance to antimicrobial attacks 0and
modulates the host immune response. These properties make lipopolysaccharide an
important virulence factor for Brucella survival and replication in the host.
Occupational risk factors: Laboratory workers handling Brucella cultures are at high risk
of acquiring brucellosis trough accidents, aresolization and/or inadequate laboratory
procedures (Fensterbank, 1987b).

In addition to this, abattoir workers, farmers and veterinarians are at high risk of
acquiring the infection. Managemental risk factors: The spread of the disease from one
herd to the other and from one area to another is almost always due to the movement of
an infected animal from infected herd in to a non infected susceptible herd. A case-
control study of brucellosis in Canada indicates that, herds located close to other infected
herds and those herds whose owners made frequent purchase of cattle had an increase
risk of acquiring brucellosis. Once infected, the time required to become free of
brucellosis was increased by large herd size, active abortion and by loss housing (Ray
and Steele, 1979a).

2.14 Environmental and climatic factors: -

Atmospheric conditions and seasons of the year may have influence on the management
and contact of infected and susceptible host. In dry areas water resource meagerly
distributed (Helland1982). As a result, the congregation of large number of variety
ruminants at wells takes part disease spread. The coincidence of parturition in wet season,
(Schwart and Dioll, 1992) increase chance of infecting susceptible animals.

25 | P a g e
2.15 Diagnostic Tests: -
Microscopic examination of stained smears can be useful for a presumptive diagnosis,
particularly if the direct examination is supported by other tests. Brucellae are
coccobacilli or short rods, usually arranged singly but sometimes in pairs or small groups.
They are not truly acid- fast; however, they are resistant to decolorization by weak acids,
and stain red against a blue background with the Stamp's modification of the Ziehl-
Neelsen method. Other organisms such as Coxiellaburnetii can resemble Brucella. In
humans, the definitive diagnosis is by culture or serology (Corbel, 1997).

Brucella species can sometimes be isolated from the blood early in the infection; bone
marrow is often positive at this stage. Occasionally, bacteria can be recovered from the
cerebrospinal fluid, urine or tissues. Brucella spp. can be isolated on a variety of plain
media, or selective media such as Farrell's medium or Thayer- Martins modified
medium. Enrichment techniques can also be used. Colony morphology varies with the
species. Colonies of smooth forms (B. abortus, B. suis, B. melitensis and marine mammal
Brucella) are round with smooth margins.

When the plates are viewed in daylight through a transparent medium, these colonies are
translucent and a pale honey color. From above, they are convex and pearly white. B.
ovis and B. canis are rough (R) forms (Manture et al., 2007). The colonies are round,
shiny and convex, but their rough nature can be seen by examining the colony with
oblique illumination. Most Brucella species form colonies within a few days, but isolates
from seals grow slowly and may take 7 to 10 days to become visible on selective media.
Brucella isolates can be identified to the species and biovar level by phage typing and
cultural, biochemical and serological characteristics. Care should be taken during
identification, as marine mammal isolates are sometimes misidentified initially as
terrestrial strains.

Genetic techniques can also be used for biotyping. Most human infections are diagnosed
by serology. Tests used include serum agglutination, a modified Coombs (antiglobulin)
technique, ELISAs and immunoblotting (Western blotting). Serologic diagnosis is

26 | P a g e
complicated by previous exposures and other factors; a definitive diagnosis usually
requires a fourfold rise in titer. Immunostaining can sometimes demonstrate the presence
of Brucella spp. in a clinical specimen. PCR techniques can also be used for diagnosis.
Chronic brucellosis can be extremely difficult to diagnose, if the serologic results are
equivocal and the organism cannot be cultured (Ahmed, 1993).
2.5.1 Serology:
Brucellosis is often diagnosed by serology. Serological tests are not completely specific
and cannot always distinguish reactions due to B. melitensis from cross- reactions to
other bacteria, particularly Yersinia enterocolitica. In cattle, sheep and goats, serology
can be used for a presumptive diagnosis of brucellosis, or to screen herds. Serological
tests commonly used to test individual cattle or herds include the buffered Brucella
antigen tests (rose bengal test and buffered plate agglutination test), complement
fixation, indirect or competitive enzyme-linked immunosorbent assays (ELISAs) and the
fluorescence polarization assay. Rivanol precipitation, acidified antigen procedures and
the serum agglutination test (tube or microtiter test) are also available. Supplemental tests
such as complement fixation or rivanol precipitation are often used to clarify the results
from plate or card agglutination tests. ELISAs or the Brucella milk ring test (BRT) can be
used to screen herds by detecting antibodies in milk. In vaccinated cattle, the native
hapten -based gel precipitation tests (gel diffusion or radial immunodiffusion tests) are
sometimes used to distinguish vaccination from infection. In sheep and goats, B.
melitensis can be diagnosed with the buffered Brucella antigen tests, complement fixation
or
ELISAs. Native hapten -based gel precipitation tests are also used in vaccinated sheep
and goats. The bulk milk ring test is not used in small ruminants. Serological tests used to
detect B. ovis include ELISAs, agar gel immunodiffusion (AGID) and complement
fixation. Other tests including hemagglutination inhibition and indirect agglutination have
also been described. Serological tests used to detect B. canis in dogs include rapid slide
agglutination (card or RSAT) tests, tube agglutination, an indirect fluorescent antibody
(IFA) test, AGID and ELISA. In swine, serology is generally considered to be more
reliable for identifying infected herds than individual pigs. Serological tests used in swine
include ELISAs, the buffered Brucella antigen tests and complement fixation. A

27 | P a g e
fluorescence polarization assay has been developed. Supplemental serological tests used
in cattle may also be used in swine. The serological tests used in marine mammals have
been adapted from livestock Brucella tests. They include the buffered Brucella antigen
tests, serum agglutination tests, complement fixation, AGID, ELISAs and rivanol test. In
general, these tests have not yet been validated for marine mammals; threshold values
have not been established and can vary between laboratories.
2.5.2 Culture:
Brucella species can be recovered from numerous tissues and secretions, particularly fetal
membranes, vaginal secretions, milk (or udder secretions in nonlactating cows), semen,
arthritis or hygroma fluids, and the stomach contents, spleen and lung from aborted
fetuses. Blood cultures are often used to detect B. canis in dogs. In this species,
bacteremia (which may be intermittent) can persist for up to five years and possibly
longer. Oral, nasal, tracheal, vaginal and anal swabs, as well as feces, can be submitted
for culture from marine mammals. At necropsy, bacteria can be isolated from a variety
of organs including lymph nodes, spleen, uterus, udder, testis, epididymis, joint exudate,
abscesses and other affected tissues. In ruminants with suspected B. abortus or B.
melitensis infections, the spleen, mammary and genital lymph nodes, udder and late
pregnant or early post- parturient uterus are the most reliable samples to collect. The
preferred tissues to collect in rams suspected of B. ovis infection are the epididymis,
seminal vesicles, ampullae and inguinal lymph nodes. In dogs, recommended biopsy or
necropsy samples include lymph nodes, prostate, epididymis, testis, uterus, spleen, liver
and bone marrow.
The lymph nodes and spleen are most likely to be positive in non-bacteremic dogs.
Brucella spp. can be isolated on a variety of plain media, or selective media such as
Farrell's medium or Thayer-Martins modified medium. Enrichment techniques can also
be used. Colony morphology varies with the species. Colonies of smooth forms (B.
abortus, B. suis, B. melitensis and marine mammal Brucella) are round with smooth
margins. When the plates are viewed in daylight through a transparent medium, these
colonies are translucent and a pale honey color. From above, they are convex and pearly
white. B. ovis and B. canis are rough (R) forms. The colonies are round, shiny and
convex, but their rough nature can be seen by examining the colony with oblique

28 | P a g e
illumination. Most Brucella species form colonies within a few days, but isolates from
seals grow slowly and may take 7-10 days to become visible on selective media. Brucella
isolates can be identified to the species and biovar level by phage typing and cultural,
biochemical and serological characteristics. Care should be taken during identification, as
marine mammal isolates are sometimes misidentified initially as terrestrial strains.
Genetic techniques can also be used for biotyping. The vaccine strains (B. abortus strains
S19 and RB51, and B. melitensis Rev-1) can be distinguished from field strains by their
growth characteristics and sensitivity to antibiotics and other additives. Animal
inoculation is rarely used to isolate Brucella, but may be necessary if other techniques
fail. Guinea pigs or mice can be used.

2.16 Prevention: -
Human brucellosis is usually prevented by controlling the infection in animals.
Pasteurization of dairy products is an important safety measure where this disease is
endemic. Unpasteurized dairy products and raw or undercooked animal products
(including bone marrow) should not be consumed. Good hygiene and protective
clothing/equipment are very important in preventing occupational exposure.

Precautions should be taken to avoid contamination of the skin, as well as inhalation or

accidental ingestion of organisms when assisting at a birth, performing a necropsy, or
butchering an animal for consumption. Particular care should be taken when handling an
aborted fetus or its membranes and fluids. Risky agricultural practices such as crushing
the umbilical cord of newborn livestock with the teeth or skinning aborted fetuses should
be avoided. The Strain 19 B. abortus vaccine and B. melitensis Rev-1 vaccine must be
handled with caution to avoid accidental injection or exposure. Adverse events have also
been reported with the B. abortus RB51 vaccine, although it is safer than Strain 19.
Persistent infections with vaccine strains have occasionally been reported in vaccinated
animals.

These strains can be shed in the milk or aborted fetuses and can infect humans.
Obstetricians should also take precautions when assisting at human births, particularly in

29 | P a g e
regions where brucellosis is common. Recently, an obstetrician became infected by
ingesting amniotic fluid and secretions from a congenitally infected infant. In the
laboratory, Brucella spp. should be handled under biosafety level conditions or higher.
Human vaccines are not available.
2.17 Treatment of brucellosis: -
Antibiotics such as tetracyclines, rifampicin, and the amino glycosidesstreptomycin and
gentamicin are effective against Brucella bacteria. However, the use of more than one
antibiotic is needed for several weeks, because the bacteria incubate within cells.
Surveillance using serological tests, as well as tests on milk like the milk ring test, can be
used for screening and play an important role in campaigns to eliminate the disease. As
well individual animal testing both for trade and for disease control purposes is practiced.

In endemic areas, vaccination is often used to reduce the incidence of infection. The gold
standard treatment for adults is daily intramuscular injections of streptomycin 1 g for 14
days and oral doxycycline 100 mg twice daily for 45 days (concurrently). Gentamicin
5 mg/kg by intramuscular injection once daily for seven days is an acceptable substitute
when streptomycin is not available or contraindicated. Another widely used regimen is
doxycycline plus rifampin twice daily for at least six weeks. This regimen has the
advantage of oral administration. A triple therapy of doxycycline, with rifampin and co-
trimoxazole, has been used successfully to treat neurobrucellosis.

Doxycycline is able to cross the bloodbrain barrier, but requires the addition of two
other drugs to prevent relapse. Ciprofloxacin and co-trimoxazole therapy is associated
with an unacceptably high rate of relapse. In brucellic Endocarditis, surgery is required
for an optimal outcome. Even with optimal antibrucellic therapy, relapses still occur in 5
to 10% of patients with Malta fever.
The main way of preventing brucellosis is by using fastidious hygiene in producing raw
milk products, or by pasteurizing all milk that is to be ingested by human beings, either in
its unaltered form or as a derivate, such as cheese. Co-trimoxazole and rifampin are both
safe drugs to use in treatment of pregnant women who have brucellosis.

30 | P a g e
 Central Nervous System cases treat 6-9 months, same for Endocarditis cases plus surgical
replacement of valves. The reason for the length of therapy is that it is not known where
the bacteria may hide in the body, and they could cause infection in places like bone
marrow.

2.18Vaccination of brucellosis:
Yes. The brucellosis vaccine is called RB51. RB51 works by producing an immune
response that increases the animal's resistance to the disease. The vaccine is a live
product and must be administered only by an accredited veterinarian or State or Federal
animal health official. Vaccination is not 100 percent effective in preventing brucellosis;
it typically protects about 70- 80 percent of the vaccinated cattle from becoming infected
by an average exposure. For best results, female calves should be vaccinated when they
are between 4 months and 1 year old. At the time of vaccination, a tattoo is applied in the
ear which identifies the animal as an "official vaccinate." The tattoo identifies the RB51
vaccine and the year in which vaccination took place. Vaccination is an important tool in
the control, management and elimination of brucellosis. Every cattle or domestic bison
owner, regardless of location, should discuss the advantages and disadvantages of
vaccination with his or her veterinarian. Some States do not allow cattle and domestic
bison to be imported for breeding if they are not official brucellosis vaccinates and they
Are beyond the age at which they should have been vaccinated. In brucellosis affected
areas such as the Greater Yellowstone Area, revaccination of female cattle or domestic
bison at 3 to 5 year intervals is recommended. This gives much better protection to the
herd.
2.19 Management practices and movement control herd: -
Improving management practice is one way attempting to control brucellosis, this is
aimed to improve hygiene and reduce contact between infected and non infected animals.
Following points attempted in reducing infection rates (Hanter, 1994; Radostits et al./
1994).
Public awareness, isolation of infected animals for successful control and
prevention brucellosis.

31 | P a g e
 Proper disposal of aborted feotus, placental uterine discharge and disinfecting
contaminated areas and also premises.

2.20 Communicability: -
Brucellosis is not usually transmitted from person to person. Rarely, bacteria have
been transmitted by bone marrow transplantation, blood transfusion or sexual
intercourse. Rare congenital infections have also been documented. In some
cases, the infant appeared to be infected through the placenta and in others by the
ingestion of breast milk. Brucellosis was reported in an obstetrician who
swallowed secretions while trying to clear a congenitally infected infants
respiratory tract at birth.

32 | P a g e
 CHAPTER THREE

3. MATERIALS AND METHODS

3.1. Description of Study Areas and Population

3.2. Study Area

Hargeisa is capital city of Somaliland and located in a valley in the western section of the
country. It is in a mountainous area because Hargeisa area is located in an enclosed valley of the
(Ogo) highlands, at an elevation of 1334 meters (4,377 ft) above sea level. This altitude gives
Hargeisa and the surrounding area a milder climate than the Gulf of Aden coastal area (one of
the hottest areas on earth) and the Hargeisa region has a fairly equable climate. The temperature
ranges between 13 and 32 degrees Celsius (55 and 89 degrees Fahrenheit).

Hargeisa also receives larger amounts of rain, and used to be surrounded by forest when the city
was smaller but the countryside around the city has small juniper forests. Moreover, near
Hargeisa are the fertile Sheikh and Daallo mountains, which receive large amount of rain. Also,
south of Hargeisa is the Sahaley Savannah which attracts many different animals to graze in the
area Hargeisa is also close to another Somaliland town called Arabsiyo. It is a major farming and
agricultural area and it falls into the main boundaries of Hargeisa.

33 | P a g e
Figure 1: Study areas (districts) in Hargeisa state of somaliland

3.3.Study design
A cross-sectional epidemiological study was carried out in goat with serological test using RBPT
(Rose Bengal Plate Test) and a questionnaire from June 2016 to July 2016 to determine the
prevalence of goat brucellosis in Hargeisa district.

During the study two different sampling methods were used, simple random sampling in five
randomly selected villages (saylada , xeroolaha ,ministry of livestoch , m.mooge and hodan
village ), and Purposive sampling was used in Hargeia slaughter house and since the goat that
were slaughtered there were less than 20goat only two night, blood was collected from all
slaughtered animals at the time of slaughter (jugular vein).The goat selected for this study were
all aged above 1-2yrs of age

3.4. Animals and data collection:

The 3 weeks of the study involved 22 herds of different sizes and a total of 150 goat,s heads.
They were of different health status, sex, age and location in Hargeisa District. The number of
goat heads per herd ranged between 10-20 heads. Goat herds were regularly examined and
random blood samples were collected during the period from June to June 2016. The visited
areas included: saylada 9herds (60goat), xeroole three herd (20camels), minestry of livestock
four herd (30camels), m.mooge four herds (30 camels) and hodan vallege two herds (10). 31goat
herders were interviewed asking whether disease was present or absent. Information on location,
herd size, sex, age, history of abortion, camel health and major clinical signs were obtained. The
genital organs were subjected to careful clinical examination by visual observation and
palpation.

3.5. Sample Collection and Transportation

Blood was collected from the jugular vein of goats. goats were aseptically bled (approximately 5
ml) from the jugular vein by using vein-puncture into 10 ml vacutainer tubes which contained no
anti-coagulants or preservatives, (BD Vacutainer Systems, Plymouth, UK). The blood samples
were left for few hours at room temperature to allow clotting, and then centrifuged at 3000 rpm

34 | P a g e
for 10 min. The serum was collected into 1.5 ml Eppendorf tubes (Eppendorf-AG, Hamburg,
Germany), and transported to ministry of livestoch hargeisa, using an ice box and stored at -20C
until serologically tested for the presence of anti brucella antibodies.

3.6 Questionnaires
A questionnaire was prepared and administered with 30 owner between 20 Jun 2016 and 10 July
2016. Data of breed, sex, age, herd size and abortions was recorded. The questionnaire focused
on the awareness of the respondents on the risk of transmission of brucellosis from goat to
humans, contribution of herd management practices and other risk factors as far as the
occurrence and dissemination of brucellosis were concerned.

Further more, knowledge of brucellosis, the impact of the disease and the control and control
methods, if any, and whether they use protection gears while handling aborted material, and
consumption of un-pasteurized milk, history of abortion among animals and among household
members, were included in the questionnaire format.

3.7 Serological tests

3.8 Modified Rose Bengal Plate Test (mRBPT)

All the serum samples were tested for the presence of antibodies against caprine brucellosis
following the protocol of the OIE (2004). In order to improve the sensitivity of the RBPT and
minimize the discrepancies between RBPT and CFT results we used three volumes of serum and
one volume of antigen (e.g. 75 l and 25 l, respectively) in place of an equal volume of each as
recommended by OIE (2004). After mixing of test and control sera with the antigen the
plates/slides were rocked by hand for about 4 minutes. The results were interpreted according to
(Nielson and Punkan, 1990), 0 as negative (No agglutination), + (Barely perceptible
agglutination), ++ (Fine agglutination and some clearing), +++ (Course clumping, definite
with clearing)

35 | P a g e
3.9 Complement Fixation Test (CFT)
Rose Bengal Plate Test positive sera were stored at -20C until tested by CFT for confirmation.
The protocol described by (Mac Millan,1990) which uses standard B. abortus antigen
(Veterinary Laboratories Agency, Addlestone, United Kingdom), Amboceptor (Biomerieux,
France), 1% goat RBC, positive and negative control antisera was used. The complement was
obtained from the Federal Institute for Health Protection of Consumers and Veterinary Medicine,
Berlin, Germany. Sera with strong reaction at dilution of 1:5 with a strong reaction of
approximately 100% fixation of the complement (4+), more than 75% fixation of complement
(3+) at a dilution of 1: 5 and at least 50% fixation of complement (2+) at a dilution of 1:10 and
1:20 were classified as positive ( OIE, 2004). The details of CFT protocol is indicated in Annex

3.10 Data analysis

Data was stored in Microsoft excel spread sheet program and analyzed. The seroprevalence was
calculated by dividing the number of RBPT positive animals by the total number of animals
tested

36 | P a g e
CHAPTER FOUR RESULTS

4.1 Results
The results of the study are summarized in the table 1. Of a total of 150 serum
samples tested, two (2) 1% tested positive for brucellosis infection by RBPT, all
the positive reactors were females.
Table 1: this table shows the number of sampled animals and the number of
positive animals.

Date Location No of Male Female Results

sampled
Goats
24/5/2016 xeroole 50 15 35 1 positive

26/5/2016 Ministery of 30 10 20 0 positive

livestock
27/5/2016 m.mooge and 40 12 28 1 positive
kilarka
28/5/2016 Hodan village 30 5 25 0 positive

150 42 108 2

4.2 Seroprevalence of brucellosis in goats around hargeisa town of

Somaliland
Out of 150 sera tested (42 males and 108 females), 2 animals (1%) were tested
positively by using RBPT. All positive animals were females, above the age of 3
years. The overall prevalence of tested animals is 1%.

37 | P a g e
 prevalence

1%

negative
99%
positive

Figure 1: this pie chart shows the overall prevalence of brucellosis in goats in the study
area.

4.3 prevalence distribution of Brucella according to sex

The current study finds two positive animals which represents 1% prevalence rate.All the
positive (2) animals were females while no positives were found in males.

prevalence distribution according to sex

0.3

0.25

0.2

0.15 prevalence distribution

0.24 according to sex
0.1 0.2

0.05

0
female 1% male 0%

38 | P a g e
4.3 Knowledge of pastoralistson the disease (Brucellosis) and associated
risk transmission
Most participants in the study stated that abattoir workers slaughter animals and make skinning
without wearing gloves and any protective materials and that is endangering their health. 7
(35%) persons out of the 20 people those were interviewed stated that they aware about the risks
of the disease while the 13 (65%) remaining respondents stated that they do not aware about it.
Those respondents that aware about the risk of the disease told that they have attended some
trainings carried out by FAO and ministry of livestock.

40%
35%
30%
25%
40%
20%
30% 30%
15%
10%
5%
0%
Primary school secondry school university

Table 1.2 shows that 32% of the respondent are secondary and 40% are

Primary level and 30% are university level 30%

39 | P a g e
 flock sizes of goat

70%
60%
50%
40% 64%
30%
20% 34%

10%
0%
male female

Table1.3 shows that 34% of the goats are male and 64% of the goats are female

Has there been history of abortion in your flock

100%

80%

60%
83%
40%

20% 17%
0%
aborted Not aborted

Table 1.4 shows that 17% of the animals aborted while 83% are not aborted

40 | P a g e
 how many goats aborted over the last year

100%

80%

60%
83%
40%

20% 17%
0%
aborted Not aborted

Table 1.5 shows that 17% of the animals aborted in the year while 83% of the animal not aboted per
year

what is the approximate age of fetus at time of

abortion of goats

60%
50%
40%
30% 56%
20%
28%
10% 16%
0%
early gestation mid gestation late gestation

Table 1.6 show that 16% of the animals aborted early gestation and 28% of the animals aborted mid
gestation and 56% of the animals aborted during late gestatio

41 | P a g e
 sanitary system of the farm

46%

54% fair
good

Table1.7 shows that 54% of animal have fair sanitary system while 46% are good sanitary system

How do you keep your goat housing?

0.5

0.4

0.3
50%
0.2 35%

0.1 15%

0
Total confinement partial confinement night stable

Table 1.8 shows that 15% of the animals have confinement housing system and 35% have partial
confinement system and 50% night stable

42 | P a g e
 knowledge of the butchers to brucellosis
respondents that do not aware respondents that aware brucellosis

0% 0%

35%

65%

Table 3: shows determination of clinical signs related to brucellosis in goats

Number of Clinical sings

respondents

Abortion Infertility Fever

20 owner 1(5%) 1(5%) 2(10%)

CHAPTER FIVE: CONCLUSIONS AND RECOMMENDATIONS

5.1 Conclusion
The result of the present study showed that the serprevalence of goats brucellosis in the study
area was very low. However, the existence of the disease in the study area has possible risk of
spread in the future. Accordingly, elimination of positive reactors will provide better
considerable success in the control of brucellosis in goats brucella populations.

43 | P a g e
5.2 Recommendations
Since goats is reared and consumed much more in Somaliland, due to the little knowledge
abattoir butchers and animals owners have about the impact of the disease, since the animal
owners have no relevant knowledge about the disease and since butchers do not use protective
gear when handling the animal meat, it is imperative that the risk of the disease spreading further
is great. Thus the following is recommended for its reduction and elimination.

Further study of the disease at a large scope in rural area is recommended.

Provision awareness of zoonotic diseases and their mode of transmission especially
Brucellosis to pastoralists or animal owners
Provision trainings and preventive materials, such as gloves to butchers
Testing the exotic animals before entering the country
Monitoring and screening tests of exotic animals.
It is imperative to investigate the disease in human, especially among the animal handlers
and particularly pastoralists who indulge in the practice of manipulating during
parturition of small ruminants without protective clothing.

APPENDEX ( A)REFERENCES: -

1. M.J. Corbel. Brucellosis in Humans and animals, who and oie

2. http://www.aam.org.ar/brucellosis2011/descargas/brucellosis2011-argentina.pdf , Renato
L. Santos et al
3. Young EJ. An overview of human brucellosis. Clin Infect Dis 1995; 2: 283-90.
4. Pappas G, Papadimitriou P, Akritidis N, Christou L, Tsianos EV: The new global map of
human brucellosis. Lancet Infect Dis 2006, 6(2):91-99.

44 | P a g e
5. Jacques Graffold(http://siteresources.worldbank.org/INTARD/Resources/ PPPWeb.pdf).
people, pathogen and our planet, JoergenVoegele
6. Jacques Godfroidab*, Sascha Al Dahoukcd, Georgios Pappase, Felix Rothf, Gift
Matopeg, John Mumah, Tanguy Marcottyib, Dirk Pfeifferj, EysteinSkjerve,(2013) One
Health surveillance and control of brucellosis in developing countries. Moving away
from improvisation, 2013
7. Daz R, Casanova A, Ariza J, Moriyon I (2011) The Rose Bengal Test in Human
Brucellosis: A Neglected Test for the Diagnosis of a Neglected Disease. PLoSNegl Trop
Dis 5(4): e950. doi:10.1371/journal.pntd.0000950
8. MayadaGwida, Sascha, Al Dahouk, Falk Melzer,UweRsler, Heinrich Neubauer, Herbert
Tomaso, (2010), Brucellosis: regionally emerging zoonotic disease
9. IGAD, 2006. Intergovernmental Authority on Development: Pro-poor livestock policy
initiative. Project summary. European commission.
10. Terra Nuova (International non-governmental Organization), 2003. Qualitative
epidemiological assessment of Somali livestock diseases
11. Bovine brucellosis of southern regions of the Somali Democratic Republic, U. Werney,
A.A. Kerani, P. Viertel, 1979. Serum and vaccine institute, CBPP section. Trop. Anim.
Prod. (1979) 11, 31-35

45 | P a g e
Rose Bengal Plate test during sample processing in ministry of livestoch Laboratory

Positive serum found during serological test of Rose Bengal testing in ministry of
livestoch Laboratory

46 | P a g e
APPENDEX {B}QUESTIONAIRE
ANNEXES
Annex1. Questionnaire survey to investigate potential for brucellosis infection in goats

1. General Information:

a. District ________ Village_________tuulo___________

b. Id No. of male goat____________, Id No. of female goats_________

c. Breed of male goat _______________, Breed of female

goats____________

d. Altitude____, Min. average annual Temp. _____, Max average annual

Temp.___, Average annual Temp. _____

e. Residential place: Urban peri-urban Rural

2. Level of education of herder/owner

Herder Owner

Illiterate

Primary school

Secondary school

Tertiary /university

47 | P a g e
3. Flock sizes of goats

a. Number of male goats in the flock __________

b. Number of female goats in the flock ___________
c. Number of female and male goats in the flock ___________
4. Has there been history of abortion(s) in your flock? Yes No

6. How many goats aborted over the last one year? __________

7. What was the average flock size i.e., females above puberty for goats______________

10. What is the approximate age of fetus at time of abortion (for goats)? _______

- Below 60 days (early gestation)

- 60 120 days (mid gestation)

- >120 days (late gestation)

12. Has there been stillbirth among your goat flock? Yes No

14. Has there been neonatal mortality among your goat flock? Yes No

16. Have there been cases of mummification of fetus among your goats flock?

Yes No

17. Sanitary system of the farm: Poor Fair Good

18. What do you feed your goats?

Forage alone

Forage +mineral (salt)

Forage +salt +atella

Forage +concentrate

Forage +concentrate + mineral supplement

48 | P a g e
19. How do you keep your goats (housing)?

Total confinement

Partial confinement

Night stable with access to pasture day time

20. What is the management system of the flocks?

Extensive

Semi-intensive

Intensive

21. Indicate the water supply for your goats.

Tap (municipality)

Wells

Pond

River

22. Grazing land:

Mountainous
Sloppy
Plain
Water logged

49 | P a g e
23. Housing of goats:

Perforated or slatted floor

Concrete

Soil

Other, specify________________

24. Supplement feeding troughs:

No supplement

Concrete feeding trough

Wooden boxes

Other, specify_________________

50 | P a g e