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 2 3 4 5 6
Division of Veterinary Infection Biology and Immunology, Research Center Borstel, Borstel, Germany Duhok Research Center (DRC), Duhok, Iraq Kassala Veterinary Research Laboratory (KVRL), Kassala, Sudan Faculty of Veterinary Medicine, Department of Parasitology, Adnan Menderes University, Aydin, Turkey Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy 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: [email protected] *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 examine field samples collected from sheep in different countries. Altogether, 1161 blood samples from Turkey (n = 830), Iraq (n = 195), Sudan (n = 96) and Portugal (n = 40) were examined, of which 31.4%, 66.6% 41.6% and 82.5%, respectively, 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 constraint of livestock production, and further efforts are needed to better understand the epidemiology and to implement suitable control measures.
doi:10.1111/tbed.12149
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
identified in the vector ticks (Aktas et al., 2009). Although 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 (Khayyat 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-
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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)
Algeria Belgian Congo China
Lestoquard (1924) Jussiant (1948) Lu et al. (1997)
Former German-East Africa (today Tanzania, Rwanda, Burundi and parts of Mozambique) France French Congo Greece, Cyprus
Schellhase (1912)
Giemsa Giemsa CFT (18–55.3 in 2813 sheep and goats) Giemsa
Cuille et al. (1935) Malbrant (1938) Chochlakis et al. (2009) (A. sp.)
Hungary India Indochina Iran Iraq
Hornok et al. (2007) Sinha and Pathak (1966) Jacotot and Evanno (1931) Razmi et al. (2006) Khayyat and Gilder (1947)
Italy
Torina et al., 2008;
Italy, Sardinia Italy, Umbria Jordan Kenya Korea
Cerruti (1934) Barboni (1931) Sherkov et al. (1976) Shompole et al. (1989) Hur et al. (1995)
MSP-5 cELISA (70) & MSP-4 PCR (7) Giemsa Giemsa Giemsa (8.6) DNA Probes (22–87) Giemsa (goats) (71.7)
Pakistan Palestine Russia
Talat et al. (2005) Gilbert (1926) Yakimoff et al. (1929) (sheep)
Giemsa (13.2) Giemsa Giemsa
South Africa Turkey
de Kock and Quinlan, 1924; Lestoquard and Ekrem (1931)
Giemsa Giemsa
USA
Splitter et al. (1955)
Giemsa
Giemsa Giemsa PCR (16S) (51, having tested 71 DNA pools) cELISA (99.4) Giemsa Giemsa (80.3) Giemsa
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-infection 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 variant 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 106
Follow-up studies
Method (% pos. samples)
Liu et al. (2011) Ma et al. (2011) Schellhase (1913) Trautmann (1913)
PCR (15.3 in goats) LAMP (69.2 in 227 sheep) Giemsa
Cuille and Chelle (1936)
Giemsa
Yousif et al. (1983) Al-Amerey and Hasso (2002) (Baghdad) Alsaad et al. (2009) (Mosul) Torina et al. (2010)
Giemsa Giemsa (32.2) Giemsa (24.7)
Park et al. (1997)
Giemsa (22.9) and CFT (76.7)
Yakimoff and Bassilia (1930) (goats)
Giemsa
Noyan (1955) €ksu (1965) Go Hoffmann et al. (1971) Splitter et al. (1956) de la Fuente et al. (2006)
Giemsa Giemsa (1.3) Giemsa (2–30) Giemsa cELISA (39) PCR (14)
MSP-4 PCR (37) in one flock with poor health
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. Hashemi-Fesharki (1997) reported that biting flies are involved in the transmission of A. ovis, and Uilenberg (1997) suggested 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
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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 systematic 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 samples 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-laboratory 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 Moncorvo and Gouveia) and 16 were from the southern part (Beja and Montemor-o-Novo). A number of 96 blood samples 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 southwestern part of the country (Aydin, Mugla, Denizli, Burdur and Usak), 74 samples from the centre (Aksaray and Konya) and 132 samples from the south-eastern part of Turkey (Van and Urfa). In all occasions, blood was either spotted on Whatman FTA cards (Roth, Karlsruhe, Germany) or collected into K-EDTA tubes. DNA was extracted using, for example, the DNeasyâ Tissue kit (Qiagen, Hilden, 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′-GGGAGCTCCTATGAATTACAGAGAATTGTTTAC-3′ and MSP43 5′-CCGGATCCTTAGCTGAACAGGAATCTTGC-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 concentration 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
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 Portugal (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 southwestern 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 samples (14.9%), and the 132 samples from the south-eastern part (Van and Urfa) gave 14 positive results (31.4%). Altogether, 31.4% of the 830 studied samples tested PCR positive. The results are summarized in Table 2. Discussion Ovine anaplasmosis is considered to be widely distributed. However, the data supporting this notion have been
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Table 2. Summary of PCR results detecting Anaplasma ovis DNA in samples collected in the different indicated countries A. ovis PCR pos (%) Portugal North South Total
22/24 (91.7) 11/16 (68.8) 33/40 (82.5)
Sudan Atbara Khartoum Total
24/48 (50) 16/48 (33.3) 40/96 (41.7)
Iraq Field Abbatoir Total
59/78 (75.6) 71/117 (60.7) 130/195 (66.7)
Turkey South-west Centre South-east Total
236/624 (37.8) 11/74 (14.9) 14/132 (10.6) 261/830 (31.4)
raised sporadically and using different methodological approaches, complicating the evaluation of currently available data. For instance, detection using Giemsa-stained blood smears does not reflect the real situation of A. ovis infections as it is insensitive method and requires great expertise. Likewise, serological approaches still bear the risk of cross-reactivity with closely related pathogens. In case of the competitive inhibition ELISA for the detection of A. marginale on the basis of MSP-5, it is evident that this assay detects serum antibodies against A. marginale as well as A. centrale, A. ovis and A. phagocytophilum (Palmer et al., 1994; Dreher et al., 2005). Also a complement fixation test specific for the detection antibodies against A. marginale antigen is known to cross-react with A. ovis antigen (Kuttler, 1984). Progress in molecular biology including PCR and sequencing has greatly advanced the sensitive and specific detection of pathogens. Since the establishment of a specific PCR for the detection of A. ovis DNA (de la Fuente et al., 2002, 2007), new data have been generated on this pathogen, as for example in Italy, where samples collected in a naturally infected sheep flock with poor health condition showed that 37% of these were positive for A. ovis (Torina et al., 2010). These and other PCR data (de la Fuente et al., 2005; Hornok et al., 2007; Liu et al., 2011) show that A. ovis is endemic in a number of geographical regions. The present study confirms this notion in the areas where the samples originated. Thus, in Iraq, Sudan, Portugal and Turkey, the percentage of PCRpositive samples was 66.6%, 41.6%, 84.2% and 31.4%, 108
respectively. However, A. ovis was only sporadically studied, and no systematic surveillance exists in these countries. In Iraq, A. ovis was recorded for the first time in 1947 (Khayyat and Gilder, 1947) and has also been described in a flock of local breed goats in 1983 (Yousif et al., 1983). Al-Amerey and Hasso (2002) reported a prevalence of A. ovis in goats from Baghdad of 32.2% as determined by Giemsa staining. Alsaad et al. (2009) found A. ovis to be present in goats in the Mosul province with 24.7% positive samples as determined by Giemsa staining. A study from 2010 performed in Duhok revealed that 55.9% and 75.2% of goats examined were infected with A. ovis using Giemsa staining and cELISA, respectively (Naqid and Zangana, 2011). The present study is the first one using a PCR-based detection approach in Iraq and demonstrates a high rate of infection with A. ovis in sheep in the Kurdistan Region of Iraq (66.6%). The present study and that of Naqid and Zangana (2011) who documented an infection rate of 55.9% (Giemsa staining) and 75.2% (cELISA), respectively, record a conspicuously higher infection rate with A. ovis in 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 (Murad (Elam) T, 2011). These movements imply general stress for the animals in a way that they had to walk long distances, get accustomed to different food and climate and had to cope with various pathogens as well as ticks and tickborne diseases (TTBDs). This fact in addition to the higher sensitivity of PCR could be a reason for the high percentage of animals positive for A. ovis in Northern Iraq. Regarding the overall economic importance of the pathogen, it can be assumed that stressful factors such as drought and heavy tick infestation promote clinical cases of A. ovis, and it is likely that the pathogen contributes to economic losses to the livestock industry in Iraq. In summer, the temperatures climb above 40°C, and water and pasture are often scarce – conditions that are supportive for outbreaks of anaplasmosis. Furthermore, the bacterium could also favour infections with other pathogens as the immune system of A. ovisinfected sheep is weakened. A previous study conducted in Sicily comes to the conclusion that animals under poor health conditions may show a higher infection rate. Moreover, in these animals, concurrent infection with A. phagocytophilum and A. ovis was found, suggesting that poor health conditions also contribute to multiple Anaplasma infections (Torina et al., 2010), and own data indicate that indeed co-infection with A. ovis and other pathogens in sheep (Theileria uilenbergi, T. ovis, T. lestoquardi and Babesia ovis) occurs in Iraq (Renneker et al., submitted). In Sudan, studies on TTBDs are mainly focused on cattle, where the occurrence of Theileria, Babesia and Anaplasma species has previously been reported using
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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 extensive surveillance to determine their magnitude and contribution 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 ruminants is suspected to occur (Joana Campos, personal communication; 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 causative 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 collected 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 animals 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 anaplasmosis among sheep and goats in Turkey was also
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 productivity 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, having 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 coinfection, 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 diseases 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 coinfection 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. References Aktas, M., K. Altay, N. Dumanli, and A. Kalkan, 2009: Molecular detection and identification of Ehrlichia and Anaplasma spe-
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Tibbitts, T., W. Goff, W. Foreyt, and D. Stiller, 1992: Stiller Susceptibility of two Rocky Mountain bighorn sheep to experimental infection with Anaplasma ovis. J. Wildl. Dis. 28, 125–129. Torina, A., J. Vicente, A. Alongi, S. Scimeca, R. Turla, S. Nicosia, V. Di Marco, S. Caracappa, and J. de la Fuente, 2007: Observed prevalence of tick-borne pathogens in domestic animals in Sicily, Italy during 2003–2005. Zoonoses Public Health 54, 8–15. Torina, A., A. Alongi, V. Naranjo, S. Scimeca, S. Nicosia, V. Di Marco, S. Caracappa, K. M. Kocan, and J. Fuente, 2008: Characterization of Anaplasma infections in Sicily, Italy. Ann. N. Y. Acad. Sci. 1149, 90–93. Torina, A., R. C. Galindo, J. Vicente, V. Di Marco, M. Russo, V. Aronica, M. Fiasconaro, S. Scimeca, A. Alongi, S. Caracappa, K. M. Kocan, C. Gortazar, and J. de la Fuente, 2010: Characterization of Anaplasma phagocytophilum and A. ovis infection
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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 2 3 4 5 6
Division of Veterinary Infection Biology and Immunology, Research Center Borstel, Borstel, Germany Duhok Research Center (DRC), Duhok, Iraq Kassala Veterinary Research Laboratory (KVRL), Kassala, Sudan Faculty of Veterinary Medicine, Department of Parasitology, Adnan Menderes University, Aydin, Turkey Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy 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: [email protected] *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 examine field samples collected from sheep in different countries. Altogether, 1161 blood samples from Turkey (n = 830), Iraq (n = 195), Sudan (n = 96) and Portugal (n = 40) were examined, of which 31.4%, 66.6% 41.6% and 82.5%, respectively, 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 constraint of livestock production, and further efforts are needed to better understand the epidemiology and to implement suitable control measures.
doi:10.1111/tbed.12149
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
identified in the vector ticks (Aktas et al., 2009). Although 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 (Khayyat 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-
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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)
Algeria Belgian Congo China
Lestoquard (1924) Jussiant (1948) Lu et al. (1997)
Former German-East Africa (today Tanzania, Rwanda, Burundi and parts of Mozambique) France French Congo Greece, Cyprus
Schellhase (1912)
Giemsa Giemsa CFT (18–55.3 in 2813 sheep and goats) Giemsa
Cuille et al. (1935) Malbrant (1938) Chochlakis et al. (2009) (A. sp.)
Hungary India Indochina Iran Iraq
Hornok et al. (2007) Sinha and Pathak (1966) Jacotot and Evanno (1931) Razmi et al. (2006) Khayyat and Gilder (1947)
Italy
Torina et al., 2008;
Italy, Sardinia Italy, Umbria Jordan Kenya Korea
Cerruti (1934) Barboni (1931) Sherkov et al. (1976) Shompole et al. (1989) Hur et al. (1995)
MSP-5 cELISA (70) & MSP-4 PCR (7) Giemsa Giemsa Giemsa (8.6) DNA Probes (22–87) Giemsa (goats) (71.7)
Pakistan Palestine Russia
Talat et al. (2005) Gilbert (1926) Yakimoff et al. (1929) (sheep)
Giemsa (13.2) Giemsa Giemsa
South Africa Turkey
de Kock and Quinlan, 1924; Lestoquard and Ekrem (1931)
Giemsa Giemsa
USA
Splitter et al. (1955)
Giemsa
Giemsa Giemsa PCR (16S) (51, having tested 71 DNA pools) cELISA (99.4) Giemsa Giemsa (80.3) Giemsa
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-infection 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 variant 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 106
Follow-up studies
Method (% pos. samples)
Liu et al. (2011) Ma et al. (2011) Schellhase (1913) Trautmann (1913)
PCR (15.3 in goats) LAMP (69.2 in 227 sheep) Giemsa
Cuille and Chelle (1936)
Giemsa
Yousif et al. (1983) Al-Amerey and Hasso (2002) (Baghdad) Alsaad et al. (2009) (Mosul) Torina et al. (2010)
Giemsa Giemsa (32.2) Giemsa (24.7)
Park et al. (1997)
Giemsa (22.9) and CFT (76.7)
Yakimoff and Bassilia (1930) (goats)
Giemsa
Noyan (1955) €ksu (1965) Go Hoffmann et al. (1971) Splitter et al. (1956) de la Fuente et al. (2006)
Giemsa Giemsa (1.3) Giemsa (2–30) Giemsa cELISA (39) PCR (14)
MSP-4 PCR (37) in one flock with poor health
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. Hashemi-Fesharki (1997) reported that biting flies are involved in the transmission of A. ovis, and Uilenberg (1997) suggested 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
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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 systematic 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 samples 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-laboratory 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 Moncorvo and Gouveia) and 16 were from the southern part (Beja and Montemor-o-Novo). A number of 96 blood samples 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 southwestern part of the country (Aydin, Mugla, Denizli, Burdur and Usak), 74 samples from the centre (Aksaray and Konya) and 132 samples from the south-eastern part of Turkey (Van and Urfa). In all occasions, blood was either spotted on Whatman FTA cards (Roth, Karlsruhe, Germany) or collected into K-EDTA tubes. DNA was extracted using, for example, the DNeasyâ Tissue kit (Qiagen, Hilden, 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′-GGGAGCTCCTATGAATTACAGAGAATTGTTTAC-3′ and MSP43 5′-CCGGATCCTTAGCTGAACAGGAATCTTGC-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 concentration 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
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 Portugal (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 southwestern 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 samples (14.9%), and the 132 samples from the south-eastern part (Van and Urfa) gave 14 positive results (31.4%). Altogether, 31.4% of the 830 studied samples tested PCR positive. The results are summarized in Table 2. Discussion Ovine anaplasmosis is considered to be widely distributed. However, the data supporting this notion have been
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Table 2. Summary of PCR results detecting Anaplasma ovis DNA in samples collected in the different indicated countries A. ovis PCR pos (%) Portugal North South Total
22/24 (91.7) 11/16 (68.8) 33/40 (82.5)
Sudan Atbara Khartoum Total
24/48 (50) 16/48 (33.3) 40/96 (41.7)
Iraq Field Abbatoir Total
59/78 (75.6) 71/117 (60.7) 130/195 (66.7)
Turkey South-west Centre South-east Total
236/624 (37.8) 11/74 (14.9) 14/132 (10.6) 261/830 (31.4)
raised sporadically and using different methodological approaches, complicating the evaluation of currently available data. For instance, detection using Giemsa-stained blood smears does not reflect the real situation of A. ovis infections as it is insensitive method and requires great expertise. Likewise, serological approaches still bear the risk of cross-reactivity with closely related pathogens. In case of the competitive inhibition ELISA for the detection of A. marginale on the basis of MSP-5, it is evident that this assay detects serum antibodies against A. marginale as well as A. centrale, A. ovis and A. phagocytophilum (Palmer et al., 1994; Dreher et al., 2005). Also a complement fixation test specific for the detection antibodies against A. marginale antigen is known to cross-react with A. ovis antigen (Kuttler, 1984). Progress in molecular biology including PCR and sequencing has greatly advanced the sensitive and specific detection of pathogens. Since the establishment of a specific PCR for the detection of A. ovis DNA (de la Fuente et al., 2002, 2007), new data have been generated on this pathogen, as for example in Italy, where samples collected in a naturally infected sheep flock with poor health condition showed that 37% of these were positive for A. ovis (Torina et al., 2010). These and other PCR data (de la Fuente et al., 2005; Hornok et al., 2007; Liu et al., 2011) show that A. ovis is endemic in a number of geographical regions. The present study confirms this notion in the areas where the samples originated. Thus, in Iraq, Sudan, Portugal and Turkey, the percentage of PCRpositive samples was 66.6%, 41.6%, 84.2% and 31.4%, 108
respectively. However, A. ovis was only sporadically studied, and no systematic surveillance exists in these countries. In Iraq, A. ovis was recorded for the first time in 1947 (Khayyat and Gilder, 1947) and has also been described in a flock of local breed goats in 1983 (Yousif et al., 1983). Al-Amerey and Hasso (2002) reported a prevalence of A. ovis in goats from Baghdad of 32.2% as determined by Giemsa staining. Alsaad et al. (2009) found A. ovis to be present in goats in the Mosul province with 24.7% positive samples as determined by Giemsa staining. A study from 2010 performed in Duhok revealed that 55.9% and 75.2% of goats examined were infected with A. ovis using Giemsa staining and cELISA, respectively (Naqid and Zangana, 2011). The present study is the first one using a PCR-based detection approach in Iraq and demonstrates a high rate of infection with A. ovis in sheep in the Kurdistan Region of Iraq (66.6%). The present study and that of Naqid and Zangana (2011) who documented an infection rate of 55.9% (Giemsa staining) and 75.2% (cELISA), respectively, record a conspicuously higher infection rate with A. ovis in 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 (Murad (Elam) T, 2011). These movements imply general stress for the animals in a way that they had to walk long distances, get accustomed to different food and climate and had to cope with various pathogens as well as ticks and tickborne diseases (TTBDs). This fact in addition to the higher sensitivity of PCR could be a reason for the high percentage of animals positive for A. ovis in Northern Iraq. Regarding the overall economic importance of the pathogen, it can be assumed that stressful factors such as drought and heavy tick infestation promote clinical cases of A. ovis, and it is likely that the pathogen contributes to economic losses to the livestock industry in Iraq. In summer, the temperatures climb above 40°C, and water and pasture are often scarce – conditions that are supportive for outbreaks of anaplasmosis. Furthermore, the bacterium could also favour infections with other pathogens as the immune system of A. ovisinfected sheep is weakened. A previous study conducted in Sicily comes to the conclusion that animals under poor health conditions may show a higher infection rate. Moreover, in these animals, concurrent infection with A. phagocytophilum and A. ovis was found, suggesting that poor health conditions also contribute to multiple Anaplasma infections (Torina et al., 2010), and own data indicate that indeed co-infection with A. ovis and other pathogens in sheep (Theileria uilenbergi, T. ovis, T. lestoquardi and Babesia ovis) occurs in Iraq (Renneker et al., submitted). In Sudan, studies on TTBDs are mainly focused on cattle, where the occurrence of Theileria, Babesia and Anaplasma species has previously been reported using
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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 extensive surveillance to determine their magnitude and contribution 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 ruminants is suspected to occur (Joana Campos, personal communication; 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 causative 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 collected 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 animals 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 anaplasmosis among sheep and goats in Turkey was also
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 productivity 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, having 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 coinfection, 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 diseases 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 coinfection 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. References Aktas, M., K. Altay, N. Dumanli, and A. Kalkan, 2009: Molecular detection and identification of Ehrlichia and Anaplasma spe-
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