Transboundary and Emerging Diseases

ORIGINAL ARTICLE

Can Anaplasma ovis in Small Ruminants be Neglected any

Longer?
S. Renneker1,*, J. Abdo2,*, D. E. A. Salih3,*, T. Karagencß4,*, H. Bilgicß4,*, A. Torina5, A. G. Oliva6,
J. Campos6, B. Kullmann1, J. Ahmed1 and U. Seitzer1
1
Division of Veterinary Infection Biology and Immunology, Research Center Borstel, Borstel, Germany
2
Duhok Research Center (DRC), Duhok, Iraq
3
Kassala Veterinary Research Laboratory (KVRL), Kassala, Sudan
4
Faculty of Veterinary Medicine, Department of Parasitology, Adnan Menderes University, Aydin, Turkey
5
Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy
6

gica Experimental e Tecnologia (IBET), Oeiras, Portugal
Instituto de Biolo

Keywords:
Anaplasma ovis; small ruminants; PCR-based
diagnostics
Correspondence:
J. Ahmed. Division of Veterinary Infection
Biology and Immunology, Research Center
Borstel, Parkallee 22, Borstel 23845,
Germany. Tel.: +49 4537 1884280; Fax: +49
4537 1886270;
E-mail: jahmed@fz-borstel.de
*Authors contributed equally.
Received for publication November 15, 2012
Summary
Anaplasma species are obligate intracellular rickettsial pathogens transmitted by
ticks with an impact on human and animal health. Anaplasma ovis infects sheep
and goats in many regions of the world, and it can be diagnosed by different
methods like Giemsa staining, PCR or competitive ELISA. In this study, a PCR
based on the gene coding for major surface protein 4 (MSP-4) was used to exam-
ine field samples collected from sheep in different countries. Altogether, 1161
blood samples from Turkey (n = 830), Iraq (n = 195), Sudan (n = 96) and Por-
tugal (n = 40) were examined, of which 31.4%, 66.6% 41.6% and 82.5%, respec-
tively, were positive. This indicates high prevalence of A. ovis in the countries
under investigation, and it can be assumed that the situation in other areas of the
world might be similar. Thus, A. ovis should be considered as an important con-
straint of livestock production, and further efforts are needed to better under-
stand the epidemiology and to implement suitable control measures.

doi:10.1111/tbed.12149

identified in the vector ticks (Aktas et al., 2009). Although

Introduction
Anaplasmosis is a tickborne disease (TBD) affecting human
and animals (Torina et al., 2007). In small ruminants,
the intra-erythrocytic Gram-negative rickettsial bacteria
Anaplasma ovis is transmitted by Rhipicephalus bursa and
other ticks in the Old World and by Dermacentor andersoni
in the New World (Friedhoff, 1997). A. ovis was first
described in 1912 by Bevan (Bevan, 1912). It has been
found in different regions of the world (Ndung′u et al.,
1995) including Italy (Barboni, 1931; Cerruti, 1934),
Turkey (Lestoquard and Ekrem, 1931; G€ oksu, 1965), Iraq
(Khayyat and Gilder, 1947; Naqid and Zangana, 2011),
India (Sinha and Pathak, 1966), France (Cuille and Chelle,
1936) and USA (Splitter et al., 1955) among others (Neitz,
1968; Naqid and Zangana, 2011) (Table 1). It has also been
the disease appears to be widespread, the extent of the
infection and the loss of livestock productivity remain
poorly understood. One reason for this situation could be
that infection with A. ovis was neglected, as it is considered
to induce only mild clinical symptoms and thus being of
minor economic importance. On the other hand, infection
has been reported to cause severe disease in bighorn sheep
(Tibbitts et al., 1992) and goats (Sinha and Pathak, 1966;
Ndung′u et al., 1995), and acute diseases are described to
be associated with stress factors like co-infection, hot
weather, vaccination, deworming, heavy tick infestation,
long-distance transportation and animal movement (Kha-
yyat and Gilder, 1947; Manickam, 1987; Friedhoff, 1997).
An aspect that has been neglected in this and many other
tickborne pathogens is the occurrence of possible co-infec-

© 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 60 (Suppl. 2) (2013) 105–112 105
The Implication of A. ovis Infection S. Renneker et al.

Table 1. Overview of previous records and follow-up studies regarding the detection of A. ovis in small ruminants in different countries

Country First description Method (% pos. samples) Follow-up studies Method (% pos. samples)

Algeria Lestoquard (1924) Giemsa

Belgian Congo Jussiant (1948) Giemsa
China Lu et al. (1997) CFT (18–55.3 in 2813 Liu et al. (2011) PCR (15.3 in goats)
sheep and goats) Ma et al. (2011) LAMP (69.2 in 227 sheep)
Former German-East Schellhase (1912) Giemsa Schellhase (1913) Giemsa
Africa (today Tanzania, Trautmann (1913)
Rwanda, Burundi
and parts
of Mozambique)
France Cuille et al. (1935) Giemsa Cuille and Chelle (1936) Giemsa
French Congo Malbrant (1938) Giemsa
Greece, Cyprus Chochlakis et al. (2009) (A. sp.) PCR (16S) (51, having
tested 71 DNA pools)
Hungary Hornok et al. (2007) cELISA (99.4)
India Sinha and Pathak (1966)
Indochina Jacotot and Evanno (1931) Giemsa
Iran Razmi et al. (2006) Giemsa (80.3)
Iraq Khayyat and Gilder (1947) Giemsa Yousif et al. (1983) Giemsa
Al-Amerey and Hasso (2002) Giemsa (32.2)
(Baghdad) Giemsa (24.7)
Alsaad et al. (2009) (Mosul)
Italy Torina et al., 2008; MSP-5 cELISA (70) & Torina et al. (2010) MSP-4 PCR (37) in one
MSP-4 PCR (7) flock with poor health
Italy, Sardinia Cerruti (1934) Giemsa
Italy, Umbria Barboni (1931) Giemsa
Jordan Sherkov et al. (1976) Giemsa (8.6)
Kenya Shompole et al. (1989) DNA Probes (22–87)
Korea Hur et al. (1995) Giemsa (goats) (71.7) Park et al. (1997) Giemsa (22.9) and
CFT (76.7)
Pakistan Talat et al. (2005) Giemsa (13.2)
Palestine Gilbert (1926) Giemsa
Russia Yakimoff et al. (1929) (sheep) Giemsa Yakimoff and Bassilia (1930) Giemsa
(goats)
South Africa de Kock and Quinlan, 1924; Giemsa
Turkey Lestoquard and Ekrem (1931) Giemsa Noyan (1955) Giemsa
€ksu (1965)
Go Giemsa (1.3)
Hoffmann et al. (1971) Giemsa (2–30)
USA Splitter et al. (1955) Giemsa Splitter et al. (1956) Giemsa
de la Fuente et al. (2006) cELISA (39)
PCR (14)

tions. Khayyat and Gilder (1947) described a co-infection
of A. ovis with B. motasi in six clinical cases, of which five
did not survive, indicating that the outcome of a co-infec-
tion can be more severe than a single infection with this
pathogen. Other authors also described co-infection of
sheep with A. ovis and B. ovis (Myalo, 1957; G€ oksu, 1965).
The phenomenon of co-infection has been neglected in
many other tickborne pathogens. Recently, research on the
epidemiology of A. ovis has gained more importance in
light of findings indicating its zoonotic potential, as a vari-
ant of A. ovis could be detected in a human patient from
Cyprus (Chochlakis et al., 2010). This zoonotic aspect
should also be considered regarding potential new vectors
of the pathogen and the possible effects of climatic changes
on vector biology. In this respect, Hornok et al. (2011)
recently detected A. ovis in sheep keds (Melophagus ovinus),
indicating that these hippoboscid flies may have a reservoir
role. This finding is supported by de Silva and Fikrig
(1997)stating that the diversity of pathogenic bacteria
spread by arthropods is probably underestimated. Has-
hemi-Fesharki (1997) reported that biting flies are involved
in the transmission of A. ovis, and Uilenberg (1997) sug-
gested that several genera and many species can be involved
in the transmission of A. ovis.
It is important to assess in more detail the presence of
A. ovis in different geographical areas, especially in those

106 © 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 60 (Suppl. 2) (2013) 105–112
S. Renneker et al.
where A. ovis has been sporadically detected or reported. In
Iraq, for example, a first report on A. ovis was in 1947, a
second report was published in 1983. Therefore, a system-
atic study is required to assess the prevalence of A. ovis by
examining a greater number of samples. The following
study has been conducted by testing more than 1000 sam-
ples from different regions including Europe, the Middle
East and Africa using a PCR targeting the MSP-4 gene of
A. ovis (de la Fuente et al., 2002, 2007; Torina et al., 2010).
Materials and Methods
Collection of samples and DNA extraction
Samples collected in Italy (69 DNA samples derived from
19 sheep and from 50 goats) that have previously been
tested positive by MSP-4 PCR were used for inter-labora-
tory comparison. Forty blood samples were collected from
sheep in five different counties in Portugal; 24 were from
the northern half of the country (Vila Real, Torre de Mon-
corvo and Gouveia) and 16 were from the southern part
(Beja and Montemor-o-Novo). A number of 96 blood sam-
ples were collected from sheep in Sudan; 48 were in Atbara
and 48 were in Khartoum. In addition, 195 blood samples
were collected from sheep in the Kurdistan Region of Iraq,
of which 78 were collected in the field and 117 were derived
from animals brought to abattoirs within the governorates.
Finally, 830 blood samples were collected from sheep in
Turkey. Of these, 624 samples were derived from the south-
western part of the country (Aydin, Mugla, Denizli, Burdur
and Usak), 74 samples from the centre (Aksaray and Ko-
nya) and 132 samples from the south-eastern part of Tur-
key (Van and Urfa). In all occasions, blood was either
spotted on Whatman FTA cards (Roth, Karlsruhe, Ger-
many) or collected into K-EDTA tubes. DNA was extracted
using, for example, the DNeasyâ Tissue kit (Qiagen, Hil-
den, Germany) following the protocol of the manufacturer.
Detection of A. ovis DNA using PCR targeting the MSP-4
gene
Conventional PCR for detection of A. ovis was performed
as described before (de la Fuente et al., 2002, 2007; Torina
et al., 2010). The MSP-4 gene was amplified with the
oligonucleotides MSP45 5′-GGGAGCTCCTATGAATTA-
CAGAGAATTGTTTAC-3′ and MSP43 5′-CCGGATCCT-
TAGCTGAACAGGAATCTTGC-3′ in a final volume of
35 ll, which included 3.5 ll 109 reaction buffer, 7 ll 59
enhancer solution, 0.7 ll of a 10 mM dNTP mix (200 lM
final concentration of each dNTP), 1.6 ll of each primer
having a concentration of 10 lM (0.46 lM final concen-
tration of each primer) and 0.175 ll of 5 U/ll Taq
polymerase (0.025 U/ll final concentration) (PEQLAB
Biotechnologie GmbH, Erlangen, Germany). The PCR
© 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 60 (Suppl. 2) (2013) 105–112
The Implication of A. ovis Infection
profile consisted of an initial denaturing step of 1 min at
94°C followed by 35 cycles of denaturing for 30 s at 94°C,
annealing for 30 s at 60°C and an extension step for 1 min
at 68°C. Final extension was performed for 5 min at 72°C.
The 870-bp-long amplification product was identified by
electrophoresis on a 1.5% agarose gel containing ethidium
bromide for visualization under UV light.
Results
Inter-laboratory comparison
Regarding the MSP-4 PCR, the results obtained for the
Italian samples in the Centro Nazionale di Referenza per
Anaplasma, Babesia, Rickettsia e Theileria (C.R.A.Ba.R.T.)
at the Istituto Zooprofilattico Sperimentale della Sicilia
could be confirmed. All 69 DNA samples were positive
using the described PCR protocol.
PCR results
Of the 24 samples collected from the northern part of Por-
tugal (Vila Real, Torre de Moncorvo and Gouveia), 22 were
found positive, while 11 of the 16 samples collected from
animals originating from the southern part of the country
(Beja and Montemor-o-Novo) tested positive. Taken
together, in 33 of 40 samples from Portugal, DNA of
A. ovis could be detected (82.5%).
Twenty-four of the 48 samples collected from sheep in
Atbara, Sudan, gave positive PCR results (50%), while 16
of the 48 samples from Khartoum tested PCR positive
(33.3%). Thus, altogether 40 of 96 DNA samples from
Sudan were found to be positive (41.6%).
Of the 195 DNA samples collected in the northern part
of Iraq in three different governorates (Dohuk, Erbil and
Sulaimaniya), 59 of the 78 field samples were positive in
PCR (75.6%). The analysis of the 117 samples from abattoir
animals demonstrated 71 to be positive (60.7%). Taken
together, 130 of the 195 DNA samples collected in Iraq
were positive in PCR (66.6%).
The results for the 830 samples collected in Turkey were
as follows: of the 624 DNA samples collected in the south-
western part of the country (Aydin, Mugla, Denizli, Burdur
and Usak), 236 were positive (37.8%). The 74 samples from
the centre (Aksaray and Konya) revealed 11 positive sam-
ples (14.9%), and the 132 samples from the south-eastern
part (Van and Urfa) gave 14 positive results (31.4%). Alto-
gether, 31.4% of the 830 studied samples tested PCR posi-
tive. The results are summarized in Table 2.
Discussion
Ovine anaplasmosis is considered to be widely distributed.
However, the data supporting this notion have been
107
The Implication of A. ovis Infection S. Renneker et al.

Table 2. Summary of PCR results detecting Anaplasma ovis DNA in respectively. However, A. ovis was only sporadically stud-
samples collected in the different indicated countries ied, and no systematic surveillance exists in these countries.
A. ovis PCR pos (%) In Iraq, A. ovis was recorded for the first time in 1947
(Khayyat and Gilder, 1947) and has also been described
Portugal in a flock of local breed goats in 1983 (Yousif et al., 1983).
North 22/24 (91.7)
Al-Amerey and Hasso (2002) reported a prevalence of
South 11/16 (68.8)
Total 33/40 (82.5)
A. ovis in goats from Baghdad of 32.2% as determined by
Giemsa staining. Alsaad et al. (2009) found A. ovis to be
Sudan present in goats in the Mosul province with 24.7% positive
Atbara 24/48 (50) samples as determined by Giemsa staining. A study from
Khartoum 16/48 (33.3) 2010 performed in Duhok revealed that 55.9% and 75.2%
Total 40/96 (41.7) of goats examined were infected with A. ovis using Giemsa
staining and cELISA, respectively (Naqid and Zangana,
Iraq
Field 59/78 (75.6)
2011). The present study is the first one using a PCR-based
Abbatoir 71/117 (60.7) detection approach in Iraq and demonstrates a high rate of
Total 130/195 (66.7) infection with A. ovis in sheep in the Kurdistan Region of
Iraq (66.6%). The present study and that of Naqid and
Turkey Zangana (2011) who documented an infection rate of
South-west 236/624 (37.8) 55.9% (Giemsa staining) and 75.2% (cELISA), respectively,
Centre 11/74 (14.9)
record a conspicuously higher infection rate with A. ovis in
South-east 14/132 (10.6)
Total 261/830 (31.4)
this region compared to the study of Alsaad et al. in 2009
(29.3% with Giemsa staining). It is worth noting that in the
time of 2008–2009, a huge movement of small ruminants
from the south to the north of the country occurred (Mu-
raised sporadically and using different methodological rad (Elam) T, 2011). These movements imply general stress
approaches, complicating the evaluation of currently avail- for the animals in a way that they had to walk long dis-
able data. For instance, detection using Giemsa-stained tances, get accustomed to different food and climate and
blood smears does not reflect the real situation of A. ovis had to cope with various pathogens as well as ticks and tick-
infections as it is insensitive method and requires great borne diseases (TTBDs). This fact in addition to the higher
expertise. Likewise, serological approaches still bear the risk sensitivity of PCR could be a reason for the high percentage
of cross-reactivity with closely related pathogens. In case of of animals positive for A. ovis in Northern Iraq. Regarding
the competitive inhibition ELISA for the detection of the overall economic importance of the pathogen, it can be
A. marginale on the basis of MSP-5, it is evident that this assumed that stressful factors such as drought and heavy
assay detects serum antibodies against A. marginale as well tick infestation promote clinical cases of A. ovis, and it is
as A. centrale, A. ovis and A. phagocytophilum (Palmer likely that the pathogen contributes to economic losses to
et al., 1994; Dreher et al., 2005). Also a complement fixa- the livestock industry in Iraq. In summer, the temperatures
tion test specific for the detection antibodies against climb above 40°C, and water and pasture are often scarce –
A. marginale antigen is known to cross-react with A. ovis conditions that are supportive for outbreaks of anaplasmo-
antigen (Kuttler, 1984). Progress in molecular biology sis. Furthermore, the bacterium could also favour infections
including PCR and sequencing has greatly advanced the with other pathogens as the immune system of A. ovis-
sensitive and specific detection of pathogens. Since the infected sheep is weakened. A previous study conducted in
establishment of a specific PCR for the detection of A. ovis Sicily comes to the conclusion that animals under poor
DNA (de la Fuente et al., 2002, 2007), new data have been health conditions may show a higher infection rate. More-
generated on this pathogen, as for example in Italy, where over, in these animals, concurrent infection with A. phago-
samples collected in a naturally infected sheep flock with cytophilum and A. ovis was found, suggesting that poor
poor health condition showed that 37% of these were posi- health conditions also contribute to multiple Anaplasma
tive for A. ovis (Torina et al., 2010). These and other PCR infections (Torina et al., 2010), and own data indicate that
data (de la Fuente et al., 2005; Hornok et al., 2007; Liu indeed co-infection with A. ovis and other pathogens in
et al., 2011) show that A. ovis is endemic in a number of sheep (Theileria uilenbergi, T. ovis, T. lestoquardi and Babesia
geographical regions. The present study confirms this ovis) occurs in Iraq (Renneker et al., submitted).
notion in the areas where the samples originated. Thus, in In Sudan, studies on TTBDs are mainly focused on
Iraq, Sudan, Portugal and Turkey, the percentage of PCR- cattle, where the occurrence of Theileria, Babesia and
positive samples was 66.6%, 41.6%, 84.2% and 31.4%, Anaplasma species has previously been reported using

108 © 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 60 (Suppl. 2) (2013) 105–112
S. Renneker et al.
molecular and serological approaches (Birdane et al., 2006;
Salih et al., 2007, 2009; Awad et al., 2011). Although
TTBDs have been described to cause considerable losses to
livestock industry in Sudan, reported studies lacked exten-
sive surveillance to determine their magnitude and contri-
bution to retardation of livestock development in the
country (Salih et al., 2009), and this is more so the case
regarding TTBDs of small ruminants. The present study is
thus the first to estimate the presence of A. ovis in sheep in
Sudan (41.7%). Climatic pressure on the animals especially
during the very hot and dry summer months increases the
incidence of clinical cases of anaplasmosis and likely results
in economic losses due to A. ovis and/or other pathogens.
The range of the geographical locations where sampling
was carried out and the high percentage of positive samples
(84.2%) suggest that ovine anaplasmosis caused by A. ovis
may be frequent in Portugal. This reflects the assessment of
local veterinarians stating that anaplasmosis in small rumi-
nants is suspected to occur (Joana Campos, personal com-
munication; Santos et al., 2004), however, so far no studies
are available regarding this disease. On one hand, which is
supported by the data of the present study, A. ovis may
likely be the reason for anaplasmosis in small ruminants in
Portugal. On the other hand, A. phagocytophilum as causa-
tive agent should also be taken into consideration.
A. phagocytophilum was identified in a small number of
I. ricinus ticks on Madeira Island (Santos et al., 2004,
2009a,b; de Carvalho et al., 2008) and in I. ventalloi col-
lected on the mainland of Portugal (Santos et al., 2004,
2009b). Furthermore, serological data indicate that besides
rodents, horses,dogs and humans have been exposed to
A. phagocytophilum, whereby cross-reactivity reactions with
related pathogens cannot be ruled out (Santos et al.,
2009a). In light of the findings of Chochlakis et al. (2010),
it may thus be worth considering that variants of A. ovis
may also play a role in human patients in Portugal.
The first description of A. ovis in Turkey was done as
early as 1931 by Lestoquard (Lestoquard and Ekrem, 1931).
After that, G€oksu reported a prevalence of 1.3% of A. ovis
in sheep having investigated 520 clinically suspected ani-
mals by Giemsa staining. In clinically healthy sheep, A. ovis
was found in four of 236 animals (1.7%), and he also
reported the observation of co-infection of A. ovis and
B. ovis in 0.4% of the investigated sheep (G€ oksu, 1965).
Also in a previous study in 1955, co-infection of A. ovis
and B. ovis was found in sheep in Turkey (Myalo, 1957).
Regarding the situation concerning A. ovis infection in
small ruminants, the number of animals that tested positive
in this study was 31.4%. This is much higher than reported
previously and indicates that the infestation rate seems to
have been underestimated until now due to relatively
unspecific detection methods. A higher incidence of ana-
plasmosis among sheep and goats in Turkey was also
© 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 60 (Suppl. 2) (2013) 105–112
The Implication of A. ovis Infection
suggested by G€ oksu (1965) as after splenectomy of clinically
healthy animals, Anaplasma species were detectable in the
peripheral blood. Sayin et al. (1997) reported a number of
42 million sheep in Turkey and the relatively low produc-
tivity of local breeds which might not only be related to the
genetic configuration of the sheep but also to the infection
with various tickborne pathogens, for example A. ovis, hav-
ing an impact on the sheep′s health and thus on their milk
and meat production (Ndung′u et al., 1995).
In conclusion, it can be stated that A. ovis seems to be
widely distributed in the investigated geographical regions.
Although it is assumed that this bacterium causes only mild
clinical symptoms (Friedhoff, 1997), however, its adverse
effect is aggravated in infected sheep and goats, especially if
the animals are stressed by other factors such as co-
infection, poor health conditions, hot weather, vaccination,
deworming or heavy tick infestation (Khayyat and Gilder,
1947; Manickam, 1987). As small ruminants are a major
source of meat, milk, hide and wool in many areas of the
world, especially where the climate is rather dry and hot
and where pasture is scarce (Sherman, 2011), there is an
increasing demand for a better understanding of the dis-
eases affecting these animals as they seem to contribute to a
decrease in productivity. Therefore, further investigations
should allow establishing epidemiologically sound data on
A. ovis to analsze the bacterium′s impact on the health of
small ruminants with special regard to its influence on co-
infection with other pathogens as described previously
(Khayyat and Gilder, 1947; Myalo, 1957; G€ oksu, 1965) and
to assess the socio-economic impact of ovine anaplasmosis.
Furthermore, host specificity and transmission pathways
including arthropod vectors are areas that are obviously
underestimated and where subsequent investigations are
necessary (de Silva and Fikrig, 1997). These aspects are a
precondition for developing integrated control strategies,
which could benefit the livestock industry. Moreover, the
zoonotic potential of A. ovis (Chochlakis et al., 2010)
should be considered, whereby further studies should be
aimed at the isolation and sequencing of selected genes of
A. ovis strains from different geographical regions followed
by phylogenetic analyses. To summarize, the economic and
public health implications of A. ovis infection necessitate
more research and should no longer be neglected.
Conflicts of interest
The authors declare no conflicts of interest in relation to
this work.
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