Survey of Theileria lestoquardi antibodies among Sudanese sheep

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1 Veterinary Parasitology 111 (2003) Short communication Survey of Theileria lestoquardi antibodies among Sudanese sheep D.A. Salih a, A.M. ElHussein a,, M. Hayat a, K.M. Taha b a Central Veterinary Research Laboratories, P.O. Box 8067, Alamarat, Khartoum, Sudan b Atbara Veterinary Research Laboratory, P.O. Box 121, Atbara, Sudan Received 25 September 2002; accepted 18 October 2002 Abstract The prevalence of Theileria lestoquardi antibodies in Sudanese sheep from nine geographical areas in Sudan was determined using indirect fluorescent antibody IFA test. Out of 315 samples examined, 51 (16.2%) were found positive and ranged between 23.4% in River Nile State and 10% in Kasala and Darfour Provinces with an overall prevalence of 16.2% indicating widespread distribution of the infection. We also report on presence of antibodies reactive to Theileria annulata in sheep sera Elsevier Science B.V. All rights reserved. Keywords: Sheep-protozoa; Theileria lestoquardi; IFA test; Schizont antigen; Antibodies 1. Introduction Theileria lestoquardi (Morel and Uilenberg, 1981), previously known as Theileria hirci is a tick-borne protozoan parasite of sheep, which occurs in south-eastern Europe, northern Africa, western and central Asia (Uilenberg, 1981b) and in India (Sisodia, 1981). Of the three Theileria species occurring in sheep, T. lestoquardi is considered the only one that is highly pathogenic (Hooshmand-Rad and Hawa, 1973; Uilenberg, 1981b). The parasite causes malignant ovine theileriosis, which assumes acute, subacute or chronic forms causing high mortality rates among sheep. Theileria ovis and Theileria seperata, on the other hand, are less pathogenic and have lower importance than T. lestoquardi (Soulsby, 1982; Arnold and Dias, 1983; Uilenberg, 1983). Malignant ovine theileriosis was first described in Sudan by Mason (1915), then reported in Khartoum State (Nagwa, 1986; Tageldin et al., 1992; Latif et al., 1994). In northern Sudan Corresponding author. Tel.: ; fax: address: abdelhussein@hotmail.com (A.M. ElHussein) /02/$ see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S (02)

2 362 D.A. Salih et al. / Veterinary Parasitology 111 (2003) it was reported by ElHussein et al. (1993), ElGhali and ElHussein (1995) and Ahmed (1999) who showed that the disease caused substantial losses among sheep. Many serological tests have been used for detection of antibodies produced by Theileria species. These include complement fixation, capillary agglutination and indirect haemagglutination tests, but their reliability is not always proven (Uilenberg, 1981a). Indirect fluorescent antibody (IFA) test has for long time been used and currently represents the reference test used in investigation of tropical theileriosis (Darghouth et al., 1996). The test has also been used for detection of antibodies produced by T. lestoquardi (Hawa et al., 1976). More recently, Leemans et al. (1997) used schizont-based IFA test to detect antibodies against T. lestoquardi in sheep sera collected from the field. In the present study, we report the prevalence of T. lestoquardi antibodies in sheep in Sudan. 2. Materials and methods 2.1. Samples collection Serum samples were collected from apparently healthy adult sheep, in nine geographical areas in Sudan, viz. River Nile, Khartoum, White Nile, Blue Nile, Western Kordofan, South Kordofan, Kasala, Red Sea and Darfour States (Fig. 1). The samples were labeled and stored at 20 C until used Antigen preparation The schizont antigen was prepared from a local T. lestoquardi and Theileria annulata cell line at low passage (<20 passage) according to the method described by FAO (1984) in 12-well Teflon-coated multispot slides (Highveld Biological, USA). Antigen-coated slides were individually wrapped in tissue paper and packed in aluminum foil with five slides in each packet. The slide packets were labeled and stored in airtight, waterproof plastic containers at 20 C until used Conjugate Rabbit anti-sheep immune gammaglobulin (IgG) conjugated to fluoresein isothiocyanate (FITC) was obtained from Nordic Immunological Laboratory, The Netherlands. The conjugate was used at dilution in phosphate buffer saline (PBS) that gives no loss of titre of positive control serum in the IFA test, but which at the same time gives a reaction not greater than 1/10 with the negative control. This was achieved by dilution 1/80. Evans blue at a concentration of 0.01% was added to the conjugate as a counterstain Control sera Positive control (C +ve) sera was obtained from Razzi institute, Iran. Negative control (C ve) sera was obtained from BDSL, UK. Control sera (C ve and C +ve) were diluted directly to 1/80.

3 D.A. Salih et al. / Veterinary Parasitology 111 (2003) Fig. 1. Map of Sudan showing the nine geographical areas where serum samples were collected Test sera Test sera were diluted in PBS 1/80 for screening the antibodies to T. lestoquardi and T. annulata according to FAO (1984).

4 364 D.A. Salih et al. / Veterinary Parasitology 111 (2003) Indirect fluorescent antibody IFA test The procedures followed in materials preparation and running the test were essentially as described by Burridge et al. (1974) and FAO (1984). Examination was carried out using Olympus Vanox incident-light excitation fluorescent microscope (Japan). The slides were examined under 40 objective using a mountant composed of nine parts glycerol and one part PBS. 3. Results The results of examining 315 serum samples obtained from nine geographical areas are shown in Table 1. The prevalence rate of T. lestoquardi antibodies ranged between 23.4% in River Nile State and 10% in Kasala and Darfour Provinces with an overall prevalence of 16.2%. On the other hand, the results of the reaction with T. annulata antigen are shown in Table 2. The prevalence rate ranged between 10.6% in River Nile State and 5% in Kordofan State with an overall prevalence of 9.3%. Table 1 The prevalence rate of T. lestoquardi antibodies using the IFA test based on schizont antigen among sheep in Sudan Location Number examined Number positive % River Nile White Nile Blue Nile Western Kordofan South Kordofan Khartoum Kasala Darfour Red Sea Total Table 2 The prevalence rate of T. annulata antibodies using the IFA test based on schizont antigen among sheep in Sudan Location Number examined T. lestoquardi T. annulata River Nile (23.4) 5 (10.6) Kasala 20 2 (10) 1 (5) Western Kordofan 30 4 (13.3) 3 (10) Total (17.5) 9 (9.3) The values in parentheses are percentages.

5 D.A. Salih et al. / Veterinary Parasitology 111 (2003) Discussion This preliminary survey was carried out in nine geographical areas in Sudan (Fig. 1), known to be natural grazing areas for sheep breeding. Results presented here show that T. lestoquardi is widely distributed in Sudan. Findings reported here in regard to antibodies prevalence rate, extend results obtained by different authors (Nagwa, 1986; ElHussein et al., 1993; Latif et al., 1994; ElGhali and ElHussein, 1995; Ahmed, 1999; Taha, 2000). Among the nine investigated areas in Sudan, River Nile State showed the highest prevalence rate (23.4%). This finding could be attributed to the fact that, sheep in River Nile State are mainly raised along the River Nile and Atbara River banks, where favorable microhabitat for survival and reproduction of the dominant tick vector Hyalomma annatolicum annatolicum exists (Hoogstraal, 1956; Ahmed, 1999). These ticks are usually found in animal sheds which are largely made of mud bricks and tree branches. This finding corresponds largely with the results obtained by Ahmed (1999) who reported that 22% out of 800 apparently healthy sheep showed patent theilerial piroplasmosis in River Nile State, northern Sudan. The prevalence rate 16.7% reported here from Khartoum State was much lower than that reported by Nagwa (1986). This may reflect sampling factors as animals sampled in the latter study were obtained from resident animals, while our samples were obtained from markets, where mostly non-resident animals are brought for sale. Although Blue Nile area is reported to be free of H. annatolicum annatolicum ticks (Jongejan et al., 1987), the present study showed 14% prevalence rate among sheep in the area. This may indicate either recent introduction of the known vector, H. annatolicum annatolicum, into the area or alternative vectors (e.g. Rhipicephalus sanguineus group, Rhipicephalus evertsi evertsi, Hyalomma imepltatum and Hyalomma rufipes, Jongejan et al., 1987) may be involved in the transmission cycle. Further investigation regarding this subject in the Blue Nile State is needed. Inspite of the fact that sheep is not considered to be a natural host of T. annulata (Uilenberg, 1981a,b; Robinson, 1982), it can become infected with this parasite. However, the developmental cycle of T. annulata in the sheep seemed to be incomplete since no piroplasms were detected (Leemans et al., 1999). Hence, the presence of antibodies to T. annulata may not be surprising since the known vector for T. annulata and T. lestoquardi in Sudan is H. annatolicum annatolicum and in the areas investigated, both cattle and sheep are raised together. These findings imply that, under field condition in Sudan, sheep are at risk of theileriosis where tick management is not strictly practiced. As mortality due to malignant ovine theileriosis is high and may reach 100% (Tageldin et al., 1992), the economic impact and control measures of the disease should be further investigated. In conclusion, the present study showed that infection with T. lestoquardi is widely distributed in Sudan. Acknowledgements The authors are indebted to the Director of Central Veterinary Research Laboratories, Prof. Ali M.A. Majid for his unlimited help and support. We are grateful to Dr. Shawgi M.

6 366 D.A. Salih et al. / Veterinary Parasitology 111 (2003) Hassan for reading the manuscript. We also thank the Director General of Animal Resources Research Corporation for permission to publish this work. References Ahmed, B.M., Studies on epizootiology of Theileria lestoquardi (nomen novem) in River Nile State, Sudan. M.Sc. Thesis, Nile Valley University, Sudan. Arnold, R.M., Dias, T.S., Ticks and tick-borne haemoparasites of sheep and goats. Seasonal distribution in Mozambique. FAO World Anim. Rev. 45, Burridge, M.J., Brown, C.G.D., Kimber, C.D., Theileria annulata: cross-reaction between a cell culture schizont antigen and antigen of East-Africa species in the indirect fluorescent antibody test. Exp. Parasiol. 35, Darghouth, M.E.A., Bouattour, A., Ben Miled, L., Sassi, L., Diagnosis of Theileria annulata infection of cattle in Tunisia: comparison of serology and blood smears. Vet. Res. 27, ElGhali, A.A., ElHussein, A.M., Diseases of livestock in EdDamer Province, River Nile State, Sudan: a two-years retrospective study. Sud. J. Vet. Sci. Anim. Husb. 34, ElHussein, A.M., ElGhali, A.A., Mohammed, S.A., Efficacy of buparvaquone in the treatment of malignant theileriosis of sheep in Ed-Damer Province, N. State, Sudan: a field trial. Sud. J. Vet. Res. 12, FAO, Tick and tick-borne disease control. A Practical Field Manual, vols. I/II. Food and Agriculture Organisation of the United Nations, Rome, 621 pp. Hawa, N., Latif, B.M.A., Bakir, F.A., Application of the indirect fluorescent antibody test for diagnosis of Theileria hirci infection of sheep: using cell culture schizont antigen. Trop. Anim. Health Prod. 8, Hoogstraal, H., African Ixodoidea. I. Ticks of the Sudan. US Navy, Washington, DC. Hooshmand-Rad, P., Hawa, N.J., Malignant theileriosis of sheep and goats. Trop. Anim. Health Prod. 5, Jongejan, F., Zivkovic, D., Pegram, R.G., Tatchell, R.J., Fison, T., Latif, A.A., Paine, G., Ticks (Acari, Ixodidae) of the Blue and White Nile ecosystem in the Sudan with particular reference to the Rhipicephalus sanguineus group. Exp. App. Acarol. 3, Latif, A.A., Abdulla, H.M., Hassan, S.M., Zubeir, N., Morazaria, S.P., Osman, A.M., Mustafa, U.E., Theileriosis of sheep in the Sudan. In: Proceedings of the Workshop Held at Sudan Veterinary Association Residence, Khartoum, Sudan, 4 5 May pp Leemans, I., Hooshmand-Rad, P., Uggla, A., The indirect fluorescent antibody test based on schizont antigen for study of the sheep parasite Theileria lestoquardi. Vet. Parasitol. 69, Leemans, I., Brown, D., Hooshmand-Rad, P., Infectivity and cross-immunity studies of Theileria lestoquardi and Theileria annulata in sheep and cattle. I. In vivo responses. Vet. Parasitol. 82, Mason, F.E., Annual report (1914) of Vet, Service, Ministry of Agriculture. Veterinary and Pathological Report, vol. 44. Govt. Press, Cairo, Egypt. Morel, P.C., Uilenberg, G., The nomenclature of some Theileria species (Sporozoa, Babesia) of some domestic ruminants. Rev. Elev. Med. Vet. Pays. Trop. 34, Nagwa, Z.G., A survey of sheep piroplasmosis in Khartoum Province (Sudan). M.Sc. Thesis, University of Khartoum, Sudan. Robinson, P.M., Theileria annulata and its transmission. A review. Trop. Anim. Health Prod. 14, Sisodia, R.S., Present Status of sheep theileriasis in India a review. Livestock Advisor 6, Soulsby, E.J.L., Helminths, Arthropods and Protozoa of Domesticated Animals. Baillière Tindall, London, pp Tageldin, M.H., Zakia, A.M., Nagwa, E.G., ElSawi, S.A.S., An outbreak of theileriosis in sheep in Sudan. Trop. Anim. Health Prod. 24, Taha, K.M., Some studies on the role of goats in the transmission of malignant ovine theileriosis. M.V.Sc. Thesis, University of Khartoum, Sudan.

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