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Haemaphysalis qinghaiensis ticks transmit at least two different Theileria species: one is infective to yaks, one is infective to sheep
Veterinary Parasitology 107 (2002) 29–35
Haemaphysalis qinghaiensis ticks transmit at least two different Theileria species: one is infective to yaks, one is infective to sheep Hong Yin a,b,∗ , Guiquan Guan a , Miling Ma a , Jianxun Luo a , Bingyi Lu a , Gailing Yuan a , Qi Bai a , Chenping Lu b , Zhengpu Yuan c , Patricia Preston d a
d
Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 11, Lanzhou, Gansu 730046, PR China b Department of Veterinary Medicine, Nanjing Agricultural University, Weigang, Nanjing, Jiangsu 210095, PR China c Gannan Institute of Animal Health and Husbandry, Hezuo, Gannan 747000, PR China Division of Biological Sciences, Institute of Cell, Animal and Population Biology, King’s Buildings, West Mains Road, University of Edinburgh, Edinburgh EH9 3JT, UK Received 14 November 2001; received in revised form 26 March 2002; accepted 5 April 2002
Abstract Adult Haemaphysalis qinghaiensis ticks from Gannan area, where ovine theileriosis is prevalent but bovine theileriosis has not been reported, transmitted Theileria parasites to yaks and sheep. Blood from the infected yak contained Theileria parasites that were infective to yaks, but not sheep. In contrast, blood from the infected sheep did not contain Theileria parasites infective to yaks. These results suggested that the ticks from the Gannan area were infected with two species of Theileria one which specifically parasitises yaks, one which parasitises sheep. Both infections were more aggressive in splenectomised hosts as compared to intact host. Adult H. qinghaiensis ticks from Zhanjiachuan area, where ovine theileriosis is prevalent but where no yaks are raised, transmitted Theileria parasites to ovine, but not yaks. Taken together these results indicate that H. qinghaiensis ticks may be infected with two different Theileria species. The species infective for sheep could be related to the newly recognised, but not yet named, pathogen recently reported in small ruminants in China. The species infective for yaks appears to be benign and is morphologically similar to parasites of the Theileria sergenti/orientalis/buffeli group. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Theileria spp.; Yak; Haemaphysalis qinghaiensis; Transmission
∗ Corresponding author. Tel.: +86-931-8342515; fax: +86-931-8340977. E-mail address: [email protected] (H. Yin).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 2 ) 0 0 0 9 6 - 1
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H. Yin et al. / Veterinary Parasitology 107 (2002) 29–35
1. Introduction Theileriosis, caused by various intraerythrocytic protozoan parasites of the genus Theileria, is a tick-borne disease of domestic and wild animals (Barnett, 1977; Uilenberg, 1981; Mehlhorn et al., 1994). At least five species of Theileria (including T. parva, T. annulata, T. mutans, T. taurotragi, T. velifera—and members of the T. sergenti/orientalis/buffeli group) have been found to infect cattle. T. parva and T. annulata are known therefore as ‘malignant’ Theileria because both cause lethal disease in susceptible cattle. The other species are less important because of low pathogenicity and are known as ‘benign’ Theileria. T. annulata, T. sergenti and an unnamed species of Theileria infective for small ruminants have been identified in China (Yang et al., 1958; Li et al., 1983; Lu et al., 1995). T. annulata and T. sergenti cause substantial losses in the cattle industry and the unnamed Theileria sp. is pathogenic for sheep and goats. However, very little is known about tick-borne diseases of the yak, Bos mutus (syn grunniens), which is the major source of income for most farmers in the highlands of west China, especially the Tibetan people living on the Tibetan plateau. In present studies, the transmission of Theileria by Haemaphysalis qinghaiensis ticks to yaks and sheep was investigated.
2. Materials and methods 2.1. Animals Six- to 12-month-old yaks were purchased from a Theileria-free area in Tianzhu county in Gansu province. The sheep described here were born in winter, purchased when they were 1- or 2-month-old and transferred to the stable at the Lanzhou Veterinary Research Institute. Four na¨ıve yaks (nos. 0103, 0105, 0108, 0118) and one sheep (no. 2107) were splenectomised for this study. Ten days prior to the experiments, blood films taken from the ears of both intact and splenectomised yaks were fixed with methanol, stained with Giemsa stain and examined microscopically for the presence of haemoprotozoan parasites. Only those animals negative for haemoparasites were used in the studies. 2.2. Ticks All the H. qinghaiensis ticks used in this study were collected from grass. Two batches of ticks were used. Batch 1 was collected from Gannan region in Gansu province, PR China, where yaks are raised by Tibetan people. Theileriosis has not yet been recorded in yaks but where ovine theileriosis is prevalent in this region. Batch 2 was collected from Zhanjiachuan in the Tianshui region in Gansu province, where ovine theileriosis has been described but yaks are not raised. 2.3. Infestation of yaks and sheep with adult H. qinghaiensis ticks One or two bags containing ticks were pasted onto the backs of animals, at sites from which hair had been removed. An intact yak (no. 0109) and a splenectomised yak (no. 0103)
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were each infested with 1000 adult ticks from Gannan and an intact sheep (no. 2007) was infested with 200 adult ticks. An intact yak (no. 0107) and a splenectomised yak (no. 0108) were each infested with 250 adult ticks from Zhangjiachuan and three intact sheep were infested with 300 adult ticks. 2.4. Inoculation of blood from Theileria-infected animals to na¨ıve yaks or sheep When the parasitaemia of experimentally infected intact yaks or sheep reached more than 10 per 1000 red blood cells (RBCs), the animals were bled, using heparin as an anticoagulant, and the piroplasm-infected blood immediately inoculated intravenously into recipient animals. Seventy days post-infestation (p.i.) with ticks, yak (no. 0109) was splenectomised and 13 days later, when the parasitaemia had reached 25 per 1000 RBCs, 20 ml of blood was inoculated intravenously into splenectomised yak (no. 0118) and 50 ml into splenectomised sheep (no. 2107) (50 ml of blood). Fifty milliliters of infected blood was taken from a splenectomised sheep (no. 2007), when the parasitaemia of 400 per 1000 RBCs, and inoculated into splenectomised yak (no. 0105). 2.5. Assessment of the transmission of Theileria sp. by ticks or inoculation of blood The daily rectal temperatures of the experimental animals were taken daily p.i. with ticks or inoculation of infected blood. Blood smears were made daily using blood from the ear vein. Smears were fixed with methanol, stained with Giemsa stain and examined microscopically for the presence of haemoprotozoans. RBCs and haemoglobin level were also measured daily using the blood collected from the ear vein. If blood smears made from the blood of the yak or sheep remained negative for Theileria for 60 days and no clinical symptoms were observed, the animals were considered not to have become infected with Theileria.
3. Results 3.1. Transmission of Theileria sp. to yaks by adult H. qinghaiensis collected from grass in Gannan 3.1.1. Successful transmission of Theileria sp. to yaks by H. qinghaiensis Of the two yaks used in this experiment, one was splenectomised; the other left intact. One thousand adults of H. qinghaiensis collected from grass in Gannan were applied in a bag to the splenectomised yak (no. 0103). Engorged females began to drop 6 days p.i. and 584 engorged adult ticks were collected between 6 and 10 days p.i. Haemoprotozoans were discovered in the blood films on day 9 p.i. and identified as a species of Theileria. Parasitaemia reached 300 per 1000 RBCs on day 8 after the appearance of parasites. Haemoglobin level fell from 80 to 65 g/l; RBC count fell from 8.89 × 1012 to 5.3 × 1012 /l. The yak died on day 19 p.i. Fig. 1. One thousand adults of H. qinghaiensis were put into a bag on the intact yak (no. 0109). Four hundred eighty-six engorged females were collected from the bag between 5 and 11 days p.i. On day 13 p.i., piroplasms of Theileria were found in the blood film and identified
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H. Yin et al. / Veterinary Parasitology 107 (2002) 29–35
Fig. 1. A schematic diagram of the sequence of experiments. Adult H. qinghaiensis ticks were from Gannan area, where ovine theileriosis is prevalent but bovine theileriosis has not been reported, and Zhanjiachuan area, where ovine theileriosis is prevalent but where no yaks are raised. The (+) represents that the animal is infected with Theileria parasites and (−) indicates that the animal is not infected with Theileria parasites.
morphologically as the same parasite as that found in the splenectomised yak (no. 0103). Parasitaemia reached 50 per 1000 RBCs on day 2 after the appearance of parasites and decreased gradually; no piroplasms could be seen on day 45 p.i. No obvious changes in haemoglobin level or RBC count were observed. The yak was splenectomised on day 70 p.i. with ticks. By day 13 after splenectomy, when the parasitaemia had reached 25 per 1000 RBCs, blood was transfused from this animal into a second splenectomised yak (no. 0118) and a splenectomised sheep (no. 2107). Yak (no. 0109) died on day 23 after splenectomy. 3.1.2. Successful transmission of Theileria sp. by inoculating blood from an infected splenectomised yak to a na¨ıve, splenectomised yak Piroplasms of Theileria sp. were seen in a film of blood taken from a splenectomised yak (no. 0118) only 2 days after the yak had been inoculated with blood from an infected, splenectomised yak (no. 0109). Parasitaemia reached a maximum of 5 per 1000 RBCs on the day 9 after blood transfusion; then declined gradually. The yak survived the infection. 3.1.3. Failure to transmit Theileria sp. with blood from an infected yak into a na¨ıve, splenectomised sheep No piroplasms were seen in the blood films prepared for 60 days after inoculation of a splenectomised sheep (no. 2107) with Theileria-infected blood from a splenectomised yak (no. 0109). No febrile responses were observed in the sheep during this period. 3.2. Transmission of Theileria sp. to sheep by adult H. qinghaiensis collected from grass in Gannan 3.2.1. Successful transmission of Theileria sp. to sheep by H. qinghaiensis An intact sheep (no. 2007) was infested with 45 adult females and 5 adult males of H. qinghaiensis that had been collected from grass in Gannan region. Ten engorged females were collected from the experimental sheep from 5 to 10 days p.i. On day 8 p.i., a slight swelling of lymph nodes and a mild febrile response were recorded. From day 13 p.i., the sheep showed high rectal temperatures (above 40.0 ◦ C). On day 18 p.i., piroplasms of
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Theileria were seen in the blood. The maximum parasitaemia, recorded on day 22 p.i., was 150 per 1000 RBCs. The animal was treated with primaquine phosphate and the parasitaemia decreased gradually. Thirteen months p.i., the sheep was splenectomised. Parasitaemia was 400 per 1000 RBCs on day 26 after splenectomy, when blood from the sheep was inoculated into a splenectomised yak (no. 0105). 3.2.2. Failure to transmit Theileria sp. by inoculating blood from an infected sheep into a na¨ıve, splenectomised yak No piroplasms were seen in the blood films prepared for 60 days after inoculation of a splenectomised yak (no. 0105) with Theileria-infected blood from a splenectomised sheep (no. 2007). No febrile responses were observed in the sheep during this period. 3.3. Transmission of Theileria sp. by H. qinghaiensis collected from grass in Zhangjiachuan Three sheep (nos. 3218, 3245, 0005) were each infested with 300 adult H. qinghaiensis ticks collected from grass in Zhangjiachuan. From 11 to 15 days p.i. 43, 50 and 63 engorged females were recovered from the three sheep, respectively. Piroplasms were observed in the blood films made from all three sheep. The maximum parasitaemias observed were: 0.1, 0.2, 20 per 1000 RBCs, respectively. Two yaks—an intact yak (no. 0107) and a splenectomised yak (no. 0108)—were each infested with 250 adults of the same batch of H. qinghaiensis collected from grass in Zhangjiachuan. From 5 to 20 days p.i. 134 and 97 engorged females were recovered from the intact yak and splenectomised yak, respectively. The animals were examined for 60 days but no piroplasms were seen in blood films and no febrile responses were observed.
4. Discussion This study provided the first evidence that H. qinghaiensis can transmit Theileria species to yaks as well as sheep. Results also and indicated that two different species of Theileria occurred in H. qinghaiensis ticks collected in the Gannan region of Gansu province. When blood containing Theileria piroplasms was passaged from a tick infected yak, into a na¨ıve, splenectomised yak, piroplasms were found in the blood just 2 days after inoculation. When the Theileria-infected blood from the same yak was inoculated into sheep, no piroplasms were seen in the blood of the recipient sheep. The latter study suggests that the species of Theileria infective for yaks was not infective for sheep. Conversely, when Theileria-infected blood from a sheep was inoculated into a splenectomised yak, no piroplasms were detected in the blood of the recipient yak. However, in our previous experiment, when blood from infected sheep, which were infested with different batch of H. qinghaiensis ticks, was inoculated into splenectomised sheep, piroplasms were easily seen in blood films, reaching a parasitaemia of 400 piroplasms per 1000 RBCs. These observations indicated that the Theileria piroplasms which had established in the sheep were not infective to yaks. The comparison of these observations suggests that the batch of H. qinghaiensis from Gannan
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was infected by two species of Theileria, one infective for yaks, the other infective for small ruminants. The idea that a species that infects sheep may not be infective for yaks was supported by finding that the Theileria species carried by the ticks from Zhangjiachuan became established in sheep but not in yaks. The Theileria sp. infective for sheep could be the unnamed Theileria sp. previously described in small ruminants (Yang et al., 1958; Schnittger et al., 2000; Yin et al., 2002). The Theileria sp. infective for yaks may be a previously unrecognised species. Only the batch of adult H. qinghaiensis ticks from Gannan successfully transmitted Theileria sp. to yaks and sheep. In the area where the ticks were collected, H. qinghaiensis was the dominant tick and ovine theileriosis the most important disease of small ruminants. While maintaining H. qinghaiensis in the laboratory, using routine techniques, the yak was found to be a more suitable host than sheep or goats for all stages of the tick, especially the larval stage. When investigating ovine theileriosis in Gannan, yaks were seen to be grazed on the same grassland as sheep and goats suggesting that yaks in Gannan might be infested by H. qinghaiensis. This was confirmed by local veterinarians. Since all the ticks used in this study were collected from grass, it appears that H. qinghaiensis is the natural vector for both species of Theileria sp. described here. The Theileria sp. found infective for yaks was not very pathogenic, which may be the reason why theileriosis has not been described in yaks in Gannan. The development of a lethal infection in a splenectomised yak raises the question as to whether this Theileria species might possibly cause clinical disease in hosts undergoing periods of immunosuppression, induced for example, by pregnancy or parturition, as reported for other ‘benign’ Theileria species in cattle (Shimizu et al., 2000). The morphology of the parasite that became established in the yaks was very similar to T. sergenti described in China (Li et al., 1983). The polymorphic piroplasms were exhibited rings, ovals, rods, comma-shapes and trilobed form. In the early stage of infection, the rod form was dominant; during the carrier state rings and ovals were dominant. The relationship of the Theileria sp., which established in the yak, to the other Theileria species infecting bovids in China and east Asia, i.e. members of the T. buffeli/sergenti/orientalis group and T. annulata, remains to be clarified. The only other known record, as far as is known, of thelerial infections in yaks—in two yaks in a zoo in the Ukraine—is considered to have been caused by a local (Ukrainian) Theileria species and not by a natural parasite of the yak, i.e. from the contemporary range of wild or domesticated yaks (Uilenberg, 1981). Considerably more work is needed to elucidate the mode of transmission of Theileria sp. to yaks and the outcome of infection.
Acknowledgements This study was partially supported by the International Foundation for Sciences (B/2578-1), Stockholm, Sweden; the Natural Science Foundation of Gansu Province (A21-039-N), Lanzhou, Gansu, China; the ADDAV project (ICA4-CT-2000-30028) coordinated by Prof. J.S. Ahmed; the ICTTD-2 Concerted Action Project (ICA4-CT-2000-30006) coordinated by Prof. F. Jongejan; the INCO-DEV programme of the European Commission, Brussels, Belgium.
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References Barnett, S.F., 1977. Theileria. In: Kreier, J.P. (Ed.), Parasitic Protozoa, Vol. 4. Academic Press, New York, pp. 77–113. Li, D.C., Liu, W.D., Hao, L.C., Yan, Z.G., Li, W.S., Wang, Z.G., Dun, Y., 1983. Confirmation of the existence of Theileria sergenti with transmission and morphological observation of the pathogen. Acta Changchun Vet. Univ. 1, 1–9 (in Chinese). Lu, W.X., Lu, W.S., Zhang, Q.C., Luo, J.X., Yin, H., 1995. Investigation on the distribution of tick-borne disease in China. Chin J. Vet. Sci. Technol. 25, 13–16 (in Chinese). Mehlhorn, H., Schein, E., Ahmed, J.S., 1994. Theileria. In: Kreier, J.P. (Ed.), Parasitic Protozoa, Vol. 7. Academic Press, London, pp. 217–304. Schnittger, L., Yin, H., Jianxun, L., Ludwig, W., Shayan, P., Rahbari, S., Voss-Holtmann, A., Ahmed, J.S., 2000. Ribosomal small-subunit RNA gene-sequence analysis of Theileria lestoquardi and a Theileria species highly pathogenic for small ruminants in China. Parasitol. Res. 86, 352–358. Shimizu, S., Nojiri, K., Matsunaga, N., Yamane, I., Minami, T., 2000. Reduction in tick numbers (Haemaphysalis longicornis), mortality and incidence of Theileria sergenti infected in field-grazed calves treated with flumethrin pour-on. Vet. Parasitol. 92, 129–138. Uilenberg, G., 1981. Theileria species of domestic livestock. In: Irvin, A.D., Cunningham, M.P., Young, A.S., (Eds.), Advances in the Control of Theileriosis. Martinus Nijhoff Publishers, The Hague, pp. 4–37. Yang, F.G., Feng, Z.G., Yu, G.H., Liu, J.X., Wei, Z.Q., X, Z., 1958. A report on ovine theileriosis in ganning animal farm station in Ganzi district. Chin. J. Vet. Med. 2, 33–37 (in Chinese). Yin, H., Luo, J.X., Guan, G.Q., Gao, Y.L., Lu, B.Y., Zhang, Q.C., Ma, M.L., Lu, W.S., Lu, W.X., Yuan, Z.P., Guo, S.Z., Wang, B., Du, H.D., Schnittger, L., Ahmed, J.S., Jongejan, F., 2002. Transmission of an unidentified Theileria species to small ruminants with Haemaphysalis qinghaiensis ticks collected from field. Parasitol. Res. 88, 25–27.
Haemaphysalis qinghaiensis ticks transmit at least two different Theileria species: one is infective to yaks, one is infective to sheep Hong Yin a,b,∗ , Guiquan Guan a , Miling Ma a , Jianxun Luo a , Bingyi Lu a , Gailing Yuan a , Qi Bai a , Chenping Lu b , Zhengpu Yuan c , Patricia Preston d a
d
Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 11, Lanzhou, Gansu 730046, PR China b Department of Veterinary Medicine, Nanjing Agricultural University, Weigang, Nanjing, Jiangsu 210095, PR China c Gannan Institute of Animal Health and Husbandry, Hezuo, Gannan 747000, PR China Division of Biological Sciences, Institute of Cell, Animal and Population Biology, King’s Buildings, West Mains Road, University of Edinburgh, Edinburgh EH9 3JT, UK Received 14 November 2001; received in revised form 26 March 2002; accepted 5 April 2002
Abstract Adult Haemaphysalis qinghaiensis ticks from Gannan area, where ovine theileriosis is prevalent but bovine theileriosis has not been reported, transmitted Theileria parasites to yaks and sheep. Blood from the infected yak contained Theileria parasites that were infective to yaks, but not sheep. In contrast, blood from the infected sheep did not contain Theileria parasites infective to yaks. These results suggested that the ticks from the Gannan area were infected with two species of Theileria one which specifically parasitises yaks, one which parasitises sheep. Both infections were more aggressive in splenectomised hosts as compared to intact host. Adult H. qinghaiensis ticks from Zhanjiachuan area, where ovine theileriosis is prevalent but where no yaks are raised, transmitted Theileria parasites to ovine, but not yaks. Taken together these results indicate that H. qinghaiensis ticks may be infected with two different Theileria species. The species infective for sheep could be related to the newly recognised, but not yet named, pathogen recently reported in small ruminants in China. The species infective for yaks appears to be benign and is morphologically similar to parasites of the Theileria sergenti/orientalis/buffeli group. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Theileria spp.; Yak; Haemaphysalis qinghaiensis; Transmission
∗ Corresponding author. Tel.: +86-931-8342515; fax: +86-931-8340977. E-mail address: [email protected] (H. Yin).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 2 ) 0 0 0 9 6 - 1
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H. Yin et al. / Veterinary Parasitology 107 (2002) 29–35
1. Introduction Theileriosis, caused by various intraerythrocytic protozoan parasites of the genus Theileria, is a tick-borne disease of domestic and wild animals (Barnett, 1977; Uilenberg, 1981; Mehlhorn et al., 1994). At least five species of Theileria (including T. parva, T. annulata, T. mutans, T. taurotragi, T. velifera—and members of the T. sergenti/orientalis/buffeli group) have been found to infect cattle. T. parva and T. annulata are known therefore as ‘malignant’ Theileria because both cause lethal disease in susceptible cattle. The other species are less important because of low pathogenicity and are known as ‘benign’ Theileria. T. annulata, T. sergenti and an unnamed species of Theileria infective for small ruminants have been identified in China (Yang et al., 1958; Li et al., 1983; Lu et al., 1995). T. annulata and T. sergenti cause substantial losses in the cattle industry and the unnamed Theileria sp. is pathogenic for sheep and goats. However, very little is known about tick-borne diseases of the yak, Bos mutus (syn grunniens), which is the major source of income for most farmers in the highlands of west China, especially the Tibetan people living on the Tibetan plateau. In present studies, the transmission of Theileria by Haemaphysalis qinghaiensis ticks to yaks and sheep was investigated.
2. Materials and methods 2.1. Animals Six- to 12-month-old yaks were purchased from a Theileria-free area in Tianzhu county in Gansu province. The sheep described here were born in winter, purchased when they were 1- or 2-month-old and transferred to the stable at the Lanzhou Veterinary Research Institute. Four na¨ıve yaks (nos. 0103, 0105, 0108, 0118) and one sheep (no. 2107) were splenectomised for this study. Ten days prior to the experiments, blood films taken from the ears of both intact and splenectomised yaks were fixed with methanol, stained with Giemsa stain and examined microscopically for the presence of haemoprotozoan parasites. Only those animals negative for haemoparasites were used in the studies. 2.2. Ticks All the H. qinghaiensis ticks used in this study were collected from grass. Two batches of ticks were used. Batch 1 was collected from Gannan region in Gansu province, PR China, where yaks are raised by Tibetan people. Theileriosis has not yet been recorded in yaks but where ovine theileriosis is prevalent in this region. Batch 2 was collected from Zhanjiachuan in the Tianshui region in Gansu province, where ovine theileriosis has been described but yaks are not raised. 2.3. Infestation of yaks and sheep with adult H. qinghaiensis ticks One or two bags containing ticks were pasted onto the backs of animals, at sites from which hair had been removed. An intact yak (no. 0109) and a splenectomised yak (no. 0103)
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were each infested with 1000 adult ticks from Gannan and an intact sheep (no. 2007) was infested with 200 adult ticks. An intact yak (no. 0107) and a splenectomised yak (no. 0108) were each infested with 250 adult ticks from Zhangjiachuan and three intact sheep were infested with 300 adult ticks. 2.4. Inoculation of blood from Theileria-infected animals to na¨ıve yaks or sheep When the parasitaemia of experimentally infected intact yaks or sheep reached more than 10 per 1000 red blood cells (RBCs), the animals were bled, using heparin as an anticoagulant, and the piroplasm-infected blood immediately inoculated intravenously into recipient animals. Seventy days post-infestation (p.i.) with ticks, yak (no. 0109) was splenectomised and 13 days later, when the parasitaemia had reached 25 per 1000 RBCs, 20 ml of blood was inoculated intravenously into splenectomised yak (no. 0118) and 50 ml into splenectomised sheep (no. 2107) (50 ml of blood). Fifty milliliters of infected blood was taken from a splenectomised sheep (no. 2007), when the parasitaemia of 400 per 1000 RBCs, and inoculated into splenectomised yak (no. 0105). 2.5. Assessment of the transmission of Theileria sp. by ticks or inoculation of blood The daily rectal temperatures of the experimental animals were taken daily p.i. with ticks or inoculation of infected blood. Blood smears were made daily using blood from the ear vein. Smears were fixed with methanol, stained with Giemsa stain and examined microscopically for the presence of haemoprotozoans. RBCs and haemoglobin level were also measured daily using the blood collected from the ear vein. If blood smears made from the blood of the yak or sheep remained negative for Theileria for 60 days and no clinical symptoms were observed, the animals were considered not to have become infected with Theileria.
3. Results 3.1. Transmission of Theileria sp. to yaks by adult H. qinghaiensis collected from grass in Gannan 3.1.1. Successful transmission of Theileria sp. to yaks by H. qinghaiensis Of the two yaks used in this experiment, one was splenectomised; the other left intact. One thousand adults of H. qinghaiensis collected from grass in Gannan were applied in a bag to the splenectomised yak (no. 0103). Engorged females began to drop 6 days p.i. and 584 engorged adult ticks were collected between 6 and 10 days p.i. Haemoprotozoans were discovered in the blood films on day 9 p.i. and identified as a species of Theileria. Parasitaemia reached 300 per 1000 RBCs on day 8 after the appearance of parasites. Haemoglobin level fell from 80 to 65 g/l; RBC count fell from 8.89 × 1012 to 5.3 × 1012 /l. The yak died on day 19 p.i. Fig. 1. One thousand adults of H. qinghaiensis were put into a bag on the intact yak (no. 0109). Four hundred eighty-six engorged females were collected from the bag between 5 and 11 days p.i. On day 13 p.i., piroplasms of Theileria were found in the blood film and identified
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Fig. 1. A schematic diagram of the sequence of experiments. Adult H. qinghaiensis ticks were from Gannan area, where ovine theileriosis is prevalent but bovine theileriosis has not been reported, and Zhanjiachuan area, where ovine theileriosis is prevalent but where no yaks are raised. The (+) represents that the animal is infected with Theileria parasites and (−) indicates that the animal is not infected with Theileria parasites.
morphologically as the same parasite as that found in the splenectomised yak (no. 0103). Parasitaemia reached 50 per 1000 RBCs on day 2 after the appearance of parasites and decreased gradually; no piroplasms could be seen on day 45 p.i. No obvious changes in haemoglobin level or RBC count were observed. The yak was splenectomised on day 70 p.i. with ticks. By day 13 after splenectomy, when the parasitaemia had reached 25 per 1000 RBCs, blood was transfused from this animal into a second splenectomised yak (no. 0118) and a splenectomised sheep (no. 2107). Yak (no. 0109) died on day 23 after splenectomy. 3.1.2. Successful transmission of Theileria sp. by inoculating blood from an infected splenectomised yak to a na¨ıve, splenectomised yak Piroplasms of Theileria sp. were seen in a film of blood taken from a splenectomised yak (no. 0118) only 2 days after the yak had been inoculated with blood from an infected, splenectomised yak (no. 0109). Parasitaemia reached a maximum of 5 per 1000 RBCs on the day 9 after blood transfusion; then declined gradually. The yak survived the infection. 3.1.3. Failure to transmit Theileria sp. with blood from an infected yak into a na¨ıve, splenectomised sheep No piroplasms were seen in the blood films prepared for 60 days after inoculation of a splenectomised sheep (no. 2107) with Theileria-infected blood from a splenectomised yak (no. 0109). No febrile responses were observed in the sheep during this period. 3.2. Transmission of Theileria sp. to sheep by adult H. qinghaiensis collected from grass in Gannan 3.2.1. Successful transmission of Theileria sp. to sheep by H. qinghaiensis An intact sheep (no. 2007) was infested with 45 adult females and 5 adult males of H. qinghaiensis that had been collected from grass in Gannan region. Ten engorged females were collected from the experimental sheep from 5 to 10 days p.i. On day 8 p.i., a slight swelling of lymph nodes and a mild febrile response were recorded. From day 13 p.i., the sheep showed high rectal temperatures (above 40.0 ◦ C). On day 18 p.i., piroplasms of
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Theileria were seen in the blood. The maximum parasitaemia, recorded on day 22 p.i., was 150 per 1000 RBCs. The animal was treated with primaquine phosphate and the parasitaemia decreased gradually. Thirteen months p.i., the sheep was splenectomised. Parasitaemia was 400 per 1000 RBCs on day 26 after splenectomy, when blood from the sheep was inoculated into a splenectomised yak (no. 0105). 3.2.2. Failure to transmit Theileria sp. by inoculating blood from an infected sheep into a na¨ıve, splenectomised yak No piroplasms were seen in the blood films prepared for 60 days after inoculation of a splenectomised yak (no. 0105) with Theileria-infected blood from a splenectomised sheep (no. 2007). No febrile responses were observed in the sheep during this period. 3.3. Transmission of Theileria sp. by H. qinghaiensis collected from grass in Zhangjiachuan Three sheep (nos. 3218, 3245, 0005) were each infested with 300 adult H. qinghaiensis ticks collected from grass in Zhangjiachuan. From 11 to 15 days p.i. 43, 50 and 63 engorged females were recovered from the three sheep, respectively. Piroplasms were observed in the blood films made from all three sheep. The maximum parasitaemias observed were: 0.1, 0.2, 20 per 1000 RBCs, respectively. Two yaks—an intact yak (no. 0107) and a splenectomised yak (no. 0108)—were each infested with 250 adults of the same batch of H. qinghaiensis collected from grass in Zhangjiachuan. From 5 to 20 days p.i. 134 and 97 engorged females were recovered from the intact yak and splenectomised yak, respectively. The animals were examined for 60 days but no piroplasms were seen in blood films and no febrile responses were observed.
4. Discussion This study provided the first evidence that H. qinghaiensis can transmit Theileria species to yaks as well as sheep. Results also and indicated that two different species of Theileria occurred in H. qinghaiensis ticks collected in the Gannan region of Gansu province. When blood containing Theileria piroplasms was passaged from a tick infected yak, into a na¨ıve, splenectomised yak, piroplasms were found in the blood just 2 days after inoculation. When the Theileria-infected blood from the same yak was inoculated into sheep, no piroplasms were seen in the blood of the recipient sheep. The latter study suggests that the species of Theileria infective for yaks was not infective for sheep. Conversely, when Theileria-infected blood from a sheep was inoculated into a splenectomised yak, no piroplasms were detected in the blood of the recipient yak. However, in our previous experiment, when blood from infected sheep, which were infested with different batch of H. qinghaiensis ticks, was inoculated into splenectomised sheep, piroplasms were easily seen in blood films, reaching a parasitaemia of 400 piroplasms per 1000 RBCs. These observations indicated that the Theileria piroplasms which had established in the sheep were not infective to yaks. The comparison of these observations suggests that the batch of H. qinghaiensis from Gannan
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was infected by two species of Theileria, one infective for yaks, the other infective for small ruminants. The idea that a species that infects sheep may not be infective for yaks was supported by finding that the Theileria species carried by the ticks from Zhangjiachuan became established in sheep but not in yaks. The Theileria sp. infective for sheep could be the unnamed Theileria sp. previously described in small ruminants (Yang et al., 1958; Schnittger et al., 2000; Yin et al., 2002). The Theileria sp. infective for yaks may be a previously unrecognised species. Only the batch of adult H. qinghaiensis ticks from Gannan successfully transmitted Theileria sp. to yaks and sheep. In the area where the ticks were collected, H. qinghaiensis was the dominant tick and ovine theileriosis the most important disease of small ruminants. While maintaining H. qinghaiensis in the laboratory, using routine techniques, the yak was found to be a more suitable host than sheep or goats for all stages of the tick, especially the larval stage. When investigating ovine theileriosis in Gannan, yaks were seen to be grazed on the same grassland as sheep and goats suggesting that yaks in Gannan might be infested by H. qinghaiensis. This was confirmed by local veterinarians. Since all the ticks used in this study were collected from grass, it appears that H. qinghaiensis is the natural vector for both species of Theileria sp. described here. The Theileria sp. found infective for yaks was not very pathogenic, which may be the reason why theileriosis has not been described in yaks in Gannan. The development of a lethal infection in a splenectomised yak raises the question as to whether this Theileria species might possibly cause clinical disease in hosts undergoing periods of immunosuppression, induced for example, by pregnancy or parturition, as reported for other ‘benign’ Theileria species in cattle (Shimizu et al., 2000). The morphology of the parasite that became established in the yaks was very similar to T. sergenti described in China (Li et al., 1983). The polymorphic piroplasms were exhibited rings, ovals, rods, comma-shapes and trilobed form. In the early stage of infection, the rod form was dominant; during the carrier state rings and ovals were dominant. The relationship of the Theileria sp., which established in the yak, to the other Theileria species infecting bovids in China and east Asia, i.e. members of the T. buffeli/sergenti/orientalis group and T. annulata, remains to be clarified. The only other known record, as far as is known, of thelerial infections in yaks—in two yaks in a zoo in the Ukraine—is considered to have been caused by a local (Ukrainian) Theileria species and not by a natural parasite of the yak, i.e. from the contemporary range of wild or domesticated yaks (Uilenberg, 1981). Considerably more work is needed to elucidate the mode of transmission of Theileria sp. to yaks and the outcome of infection.
Acknowledgements This study was partially supported by the International Foundation for Sciences (B/2578-1), Stockholm, Sweden; the Natural Science Foundation of Gansu Province (A21-039-N), Lanzhou, Gansu, China; the ADDAV project (ICA4-CT-2000-30028) coordinated by Prof. J.S. Ahmed; the ICTTD-2 Concerted Action Project (ICA4-CT-2000-30006) coordinated by Prof. F. Jongejan; the INCO-DEV programme of the European Commission, Brussels, Belgium.
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