Ticks and Tick-borne Diseases 5 (2014) 105–106
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Short communication
PCR-based detection of Theileria annulata in Hyalomma asiaticum ticks in northwestern China K. Meng a,1 , Z. Li a,1 , Y. Wang b,1 , Z. Jing c,1 , X. Zhao a , J. Liu a,∗ , D. Cai a , L. Zhang a , D. Yang a , S. Wang a a College of Veterinary Medicine, Research Center for Animal Disease Control Engineering Shandong Province, Shandong Agricultural University, Tai’an 271018, China b Key Laboratory of Tarim Animal Husbandry Science & Technology of Xinjiang Production and Construction Groups/College of Animal Science, Tarim University, Alar, Xinjiang Uygur Autonomous Region 843300, China c College of Foreign Languages, Shandong Agricultural University, Tai’an 271018, China
a r t i c l e
i n f o
Article history: Received 24 May 2013 Received in revised form 11 September 2013 Accepted 11 September 2013 Available online 24 October 2013
a b s t r a c t This study aimed to detect Theileria annulata infection using polymerase chain reaction (PCR) amplification in Hyalomma asiaticum ticks. Sequence analysis showed that the 28 analyzed sequences obtained from 81 H. asiaticum ticks had an identical length and sequence which were closely related to that of T. annulata. This study is the first to report on the presence of T. annulata in H. asiaticum ticks in China. Crown Copyright © 2013 Published by Elsevier GmbH. All rights reserved.
Keywords: Tropical theileriosis Cattle Hyalomma asiaticum Tick vector PCR Theileria annulata
Introduction Theileria annulata, transmitted by hard ticks of the genus Hyalomma, can cause tropical theileriosis, making an important economic loss on domestic animal production from the global perspective (Mehlhorn and Schein, 1984; Schnittger et al., 2012). Theileria annulata has been found in Turkey, Tunisia, China, India, Iran, the UK, and the USA (Collins et al., 1996; Dumanli et al., 2005; Gajadhar et al., 1991; Gharbi et al., 2006; Hashemi-Fesharki, 1988; He et al., 2012; Pain et al., 2005). In addition, Hyalomma detritum, H. anatolicum anatolicum, H. anatolicum excavatum, H. dromedarii, and H. marginatum marginatum have been reported to be associated with the transmission of T. annulata (Sayin et al., 2003). Hyalomma asiaticum, a 3-host tick, is widely distributed in northern China (arid or semi-arid regions), Mongolia, and Russia. The hosts of adult H. asiaticum are cattle, goats, camels, sheep, pigs, horses, hedgehogs, and wild boar. In recent years, the ticks were also found on humans (Chen et al., 2009). However, H. asiaticum ticks have not yet been
reported as a potential vector of theileriosis in China. Thus, this study aimed to use polymerase chain reaction (PCR) and sequence analysis to detect T. annulata in H. asiaticum ticks in northwestern China. Materials and methods Collection of ticks and DNA isolation Sixty-one cattle were examined for the presence of H. asiaticum ticks in a randomly selected dairy farm in northwestern China. The H. asiaticum ticks were manually collected from the body of the cattle. A total of 81 adult ticks were obtained from 61 cattle. All 81 ticks were placed in 75% ethanol and transported to the laboratory for molecular analysis. DNA was extracted from the H. asiaticum ticks using an E.Z.N.A.TM Insect DNA Kit (D0926-01, Omega) following the manufacturer’s protocol. PCR amplification and sequencing analysis
∗ Corresponding author. Tel.: +86 538 8246287; fax: +86 538 8241419. E-mail address: [email protected] (J. Liu). 1 These authors contributed equally to the work.
To amplify the Tams1 gene of T. annulata described by Katzer et al. (1998), the primers (Forward 5 -GTAACCTTTAAAAACGT-3 , Reverse 5 -CAGTTACGAACATGGGTTT-3 ) and the PCR procedure
1877-959X/$ – see front matter. Crown Copyright © 2013 Published by Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2013.09.006
106
K. Meng et al. / Ticks and Tick-borne Diseases 5 (2014) 105–106
Table 1 The vectors of Theileria species. Theileria species
The vectors of Theileria species
Theileria annulata
Hyalomma detritum ticks Hyalomma anatolicum anatolicum ticks Hyalomma dromedarii ticks Hyalomma anatolicum excavatum ticks Hyalomma marginatum marginatum ticks Hyalomma asiaticum asiaticum ticks Hyalomma anatolicum anatolicum ticks Hyalomma anatolicum anatolicum ticks Hyalomma anatolicum anatolicum ticks Hyalomma dendritum ticks
Theileria lestoquardi Theileria ovis Theileria orientalis
described by Santos et al. (2012) was used in this study. All of the 81 PCR products were analyzed by 1% agarose gel electrophoresis. Twenty-eight positive products were retrieved and purified using a DNA Fragment Quick Purification/Retrieval Kit (Axygen) following the manufacturer’s instructions. The purified DNA fragments were cloned into the plasmid Pmd18-T vector (Takara) and transformed into Escherichia coli TOP10 competent cells. The recombinant plasmids were extracted and purified from an overnight culture and then sent to a private company [Sangon Biotech (Shanghai) Co., Ltd.] for sequencing. Sequence results were identified by checking the specified sequence against the registered sequences in GenBank through the BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Results and discussion Twenty-eight of 81 ticks (34.5%) collected from 61 cattle were found to be infected with T. annulata in the PCR assay. Sequence analysis showed that the analyzed Tams1 gene had an identical sequence (GenBank accession no. JX475044). Theileriosis is a tick-borne disease of wild and domestic animals caused by infection with T. annulata (Weir et al., 2011). Theileria annulata has numerous hosts, such as cattle (d’Oliveira et al., 1995), sheep (Altay et al., 2005), goat (Schnittger et al., 2003), and buffalo (Sibeko et al., 2008). Theileria annulata is widely distributed, for example, in Africa, Europe, and Asia (Saravanan et al., 2003). In China, T. annulata is often found in semi-arid or arid regions. The vectors of Theileria species are shown in Table 1. However, T. annulata infection in H. asiaticum ticks has not yet been investigated. This study found that 28 out of 81 H. asiaticum tick samples were infected with T. annulata. However, the infection rate was difficult to evaluate epidemiologically because the samples were collected from only 61 cattle in a single dairy farm. This study is the first to report on the presence of T. annulata in H. asiaticum ticks in China. Acknowledgements This work was supported by a Project of Shandong Province Higher Educational Science and Technology Program
(No. J10LC06), the Natural Science Foundation of Shandong Province (ZR2012CQ001), the Research Fund for the Doctoral Program of Higher Education (20123702120008). References Altay, K., Dumanli, N., Holman, P.J., Aktas, M., 2005. Detection of Theileria ovis in naturally infected sheep by nested PCR. Vet. Parasitol. 127, 99–104. Chen, Z., Yu, Z., Yang, X., Zheng, H., Liu, J., 2009. The life cycle of Hyalomma asiaticum kozlovi Olenev, 1931 (Acari: Ixodidae) under laboratory conditions. Vet. Parasitol. 160, 134–137. Collins, R., Sopp, P., Gelder, K., Morrison, W., Howard, C., 1996. Bovine ␥/␦ TcR+ T lymphocytes are stimulated to proliferate by autologous Theileria annulatainfected cells in the presence of interleukin-2. Scand. J. Immunol. 44, 444–452. D’Oliveira, C., van der Weide, M., Habela, M.A., Jacquiet, P., Jongejan, F., 1995. Detection of Theileria annulata in blood samples of carrier cattle by PCR. J. Clin. Microbiol. 33, 2665–2669. Dumanli, N., Aktas, M., Cetinkaya, B., Cakmak, A., Koroglu, E., Saki, C., Erdogmus, Z., Nalbantoglu, S., Ongor, H., Sims¸ek, S., 2005. Prevalence and distribution of tropical theileriosis in eastern Turkey. Vet. Parasitol. 127, 9–15. Gajadhar, A.A., Marquardt, W.C., Hall, R., Gunderson, J., Ariztia-Carmona, E.V., Sogin, M.L., 1991. Ribosomal RNA sequences of Sarcocystis muris, Theileria annulata and Crypthecodinium cohnii reveal evolutionary relationships among apicomplexans, dinoflagellates, and ciliates. Mol. Biochem. Parasitol. 45, 147–154. Gharbi, M., Sassi, L., Dorchies, P., Darghouth, M.A., 2006. Infection of calves with Theileria annulata in Tunisia: economic analysis and evaluation of the potential benefit of vaccination. Vet. Parasitol. 137, 231–241. Hashemi-Fesharki, R., 1988. Control of Theileria annulata in Iran. Parasitol. Today 4, 36–40. He, L., Feng, H.-H., Zhang, W.-J., Zhang, Q.-L., Fang, R., Wang, L.-X., Tu, P., Zhou, Y.-Q., Zhao, J.-L., Oosthuizen, M.C., 2012. Occurrence of Theileria and Babesia species in water buffalo (Bubalus babalis, Linnaeus, 1758) in the Hubei province, South China. Vet. Parasitol. 186, 490–496. Katzer, F., McKellar, S., Kirvar, E., Shiels, B., 1998. Phylogenetic analysis of Theileria and Babesia equi in relation to the establishment of parasite populations within novel host species and the development of diagnostic tests. Mol. Biochem. Parasitol. 95, 33–44. Mehlhorn, H., Schein, E., 1984. The piroplasms: life cycle and sexual stages. Adv. Parasitol. 23, 37–103. Pain, A., Renauld, H., Berriman, M., Murphy, L., Yeats, C.A., Weir, W., Kerhornou, A., Aslett, M., Bishop, R., Bouchier, C., 2005. Genome of the host-cell transforming parasite Theileria annulata compared with T. parva. Science 309, 131–133. Santos, M., Soares, R., Costa, P., Amaro, A., Inácio, J., Gomes, J., 2012. Revisiting the Tams1-encoding gene as a species-specific target for the molecular detection of Theileria annulata in bovine blood samples. Ticks Tick Borne Dis. 4, 72–77. Saravanan, B.C., Sreekumar, C., Bansal, G.C., Ray, D., Rao, J.R., Mishra, A.K., 2003. A rapid MTT colorimetric assay to assess the proliferative index of two Indian strains of Theileria annulata. Vet. Parasitol. 113, 211–216. Sayin, F., Karaer, Z., Dincer, S., Cakmak, A., Inci, A., Yukari, B.A., Eren, H., Vatansever, Z., Nalbantoglu, S., Melrose, T.R., 2003. A comparison of susceptibilities to infection of four species of Hyalomma ticks with Theileria annulata. Vet. Parasitol. 113, 115–121. Schnittger, L., Rodriguez, A., Florin-Christensen, M., Morrison, D., 2012. Babesia: a world emerging. Infect. Genet. Evol. 12, 1788–1809. Schnittger, L., Yin, H., Gubbels, M.J., Beyer, D., Niemann, S., Jongejan, F., Ahmed, J.S., 2003. Phylogeny of sheep and goat Theileria and Babesia parasites. Parasitol. Res. 91, 398–406. Sibeko, K.P., Oosthuizen, M.C., Collins, N.E., Geysen, D., Rambritch, N.E., Latif, A.A., Groeneveld, H.T., Potgieter, F.T., Coetzer, J.A., 2008. Development and evaluation of a real-time polymerase chain reaction test for the detection of Theileria parva infections in Cape buffalo (Syncerus caffer) and cattle. Vet. Parasitol. 155, 37–48. Weir, W., Karagenc¸, T., Gharbi, M., Simuunza, M., Aypak, S., Aysul, N., Darghouth, M.A., Shiels, B., Tait, A., 2011. Population diversity and multiplicity of infection in Theileria annulata. Int. J. Parasitol. 41, 193–203.
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Short communication
PCR-based detection of Theileria annulata in Hyalomma asiaticum ticks in northwestern China K. Meng a,1 , Z. Li a,1 , Y. Wang b,1 , Z. Jing c,1 , X. Zhao a , J. Liu a,∗ , D. Cai a , L. Zhang a , D. Yang a , S. Wang a a College of Veterinary Medicine, Research Center for Animal Disease Control Engineering Shandong Province, Shandong Agricultural University, Tai’an 271018, China b Key Laboratory of Tarim Animal Husbandry Science & Technology of Xinjiang Production and Construction Groups/College of Animal Science, Tarim University, Alar, Xinjiang Uygur Autonomous Region 843300, China c College of Foreign Languages, Shandong Agricultural University, Tai’an 271018, China
a r t i c l e
i n f o
Article history: Received 24 May 2013 Received in revised form 11 September 2013 Accepted 11 September 2013 Available online 24 October 2013
a b s t r a c t This study aimed to detect Theileria annulata infection using polymerase chain reaction (PCR) amplification in Hyalomma asiaticum ticks. Sequence analysis showed that the 28 analyzed sequences obtained from 81 H. asiaticum ticks had an identical length and sequence which were closely related to that of T. annulata. This study is the first to report on the presence of T. annulata in H. asiaticum ticks in China. Crown Copyright © 2013 Published by Elsevier GmbH. All rights reserved.
Keywords: Tropical theileriosis Cattle Hyalomma asiaticum Tick vector PCR Theileria annulata
Introduction Theileria annulata, transmitted by hard ticks of the genus Hyalomma, can cause tropical theileriosis, making an important economic loss on domestic animal production from the global perspective (Mehlhorn and Schein, 1984; Schnittger et al., 2012). Theileria annulata has been found in Turkey, Tunisia, China, India, Iran, the UK, and the USA (Collins et al., 1996; Dumanli et al., 2005; Gajadhar et al., 1991; Gharbi et al., 2006; Hashemi-Fesharki, 1988; He et al., 2012; Pain et al., 2005). In addition, Hyalomma detritum, H. anatolicum anatolicum, H. anatolicum excavatum, H. dromedarii, and H. marginatum marginatum have been reported to be associated with the transmission of T. annulata (Sayin et al., 2003). Hyalomma asiaticum, a 3-host tick, is widely distributed in northern China (arid or semi-arid regions), Mongolia, and Russia. The hosts of adult H. asiaticum are cattle, goats, camels, sheep, pigs, horses, hedgehogs, and wild boar. In recent years, the ticks were also found on humans (Chen et al., 2009). However, H. asiaticum ticks have not yet been
reported as a potential vector of theileriosis in China. Thus, this study aimed to use polymerase chain reaction (PCR) and sequence analysis to detect T. annulata in H. asiaticum ticks in northwestern China. Materials and methods Collection of ticks and DNA isolation Sixty-one cattle were examined for the presence of H. asiaticum ticks in a randomly selected dairy farm in northwestern China. The H. asiaticum ticks were manually collected from the body of the cattle. A total of 81 adult ticks were obtained from 61 cattle. All 81 ticks were placed in 75% ethanol and transported to the laboratory for molecular analysis. DNA was extracted from the H. asiaticum ticks using an E.Z.N.A.TM Insect DNA Kit (D0926-01, Omega) following the manufacturer’s protocol. PCR amplification and sequencing analysis
∗ Corresponding author. Tel.: +86 538 8246287; fax: +86 538 8241419. E-mail address: [email protected] (J. Liu). 1 These authors contributed equally to the work.
To amplify the Tams1 gene of T. annulata described by Katzer et al. (1998), the primers (Forward 5 -GTAACCTTTAAAAACGT-3 , Reverse 5 -CAGTTACGAACATGGGTTT-3 ) and the PCR procedure
1877-959X/$ – see front matter. Crown Copyright © 2013 Published by Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2013.09.006
106
K. Meng et al. / Ticks and Tick-borne Diseases 5 (2014) 105–106
Table 1 The vectors of Theileria species. Theileria species
The vectors of Theileria species
Theileria annulata
Hyalomma detritum ticks Hyalomma anatolicum anatolicum ticks Hyalomma dromedarii ticks Hyalomma anatolicum excavatum ticks Hyalomma marginatum marginatum ticks Hyalomma asiaticum asiaticum ticks Hyalomma anatolicum anatolicum ticks Hyalomma anatolicum anatolicum ticks Hyalomma anatolicum anatolicum ticks Hyalomma dendritum ticks
Theileria lestoquardi Theileria ovis Theileria orientalis
described by Santos et al. (2012) was used in this study. All of the 81 PCR products were analyzed by 1% agarose gel electrophoresis. Twenty-eight positive products were retrieved and purified using a DNA Fragment Quick Purification/Retrieval Kit (Axygen) following the manufacturer’s instructions. The purified DNA fragments were cloned into the plasmid Pmd18-T vector (Takara) and transformed into Escherichia coli TOP10 competent cells. The recombinant plasmids were extracted and purified from an overnight culture and then sent to a private company [Sangon Biotech (Shanghai) Co., Ltd.] for sequencing. Sequence results were identified by checking the specified sequence against the registered sequences in GenBank through the BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Results and discussion Twenty-eight of 81 ticks (34.5%) collected from 61 cattle were found to be infected with T. annulata in the PCR assay. Sequence analysis showed that the analyzed Tams1 gene had an identical sequence (GenBank accession no. JX475044). Theileriosis is a tick-borne disease of wild and domestic animals caused by infection with T. annulata (Weir et al., 2011). Theileria annulata has numerous hosts, such as cattle (d’Oliveira et al., 1995), sheep (Altay et al., 2005), goat (Schnittger et al., 2003), and buffalo (Sibeko et al., 2008). Theileria annulata is widely distributed, for example, in Africa, Europe, and Asia (Saravanan et al., 2003). In China, T. annulata is often found in semi-arid or arid regions. The vectors of Theileria species are shown in Table 1. However, T. annulata infection in H. asiaticum ticks has not yet been investigated. This study found that 28 out of 81 H. asiaticum tick samples were infected with T. annulata. However, the infection rate was difficult to evaluate epidemiologically because the samples were collected from only 61 cattle in a single dairy farm. This study is the first to report on the presence of T. annulata in H. asiaticum ticks in China. Acknowledgements This work was supported by a Project of Shandong Province Higher Educational Science and Technology Program
(No. J10LC06), the Natural Science Foundation of Shandong Province (ZR2012CQ001), the Research Fund for the Doctoral Program of Higher Education (20123702120008). References Altay, K., Dumanli, N., Holman, P.J., Aktas, M., 2005. Detection of Theileria ovis in naturally infected sheep by nested PCR. Vet. Parasitol. 127, 99–104. Chen, Z., Yu, Z., Yang, X., Zheng, H., Liu, J., 2009. The life cycle of Hyalomma asiaticum kozlovi Olenev, 1931 (Acari: Ixodidae) under laboratory conditions. Vet. Parasitol. 160, 134–137. Collins, R., Sopp, P., Gelder, K., Morrison, W., Howard, C., 1996. Bovine ␥/␦ TcR+ T lymphocytes are stimulated to proliferate by autologous Theileria annulatainfected cells in the presence of interleukin-2. Scand. J. Immunol. 44, 444–452. D’Oliveira, C., van der Weide, M., Habela, M.A., Jacquiet, P., Jongejan, F., 1995. Detection of Theileria annulata in blood samples of carrier cattle by PCR. J. Clin. Microbiol. 33, 2665–2669. Dumanli, N., Aktas, M., Cetinkaya, B., Cakmak, A., Koroglu, E., Saki, C., Erdogmus, Z., Nalbantoglu, S., Ongor, H., Sims¸ek, S., 2005. Prevalence and distribution of tropical theileriosis in eastern Turkey. Vet. Parasitol. 127, 9–15. Gajadhar, A.A., Marquardt, W.C., Hall, R., Gunderson, J., Ariztia-Carmona, E.V., Sogin, M.L., 1991. Ribosomal RNA sequences of Sarcocystis muris, Theileria annulata and Crypthecodinium cohnii reveal evolutionary relationships among apicomplexans, dinoflagellates, and ciliates. Mol. Biochem. Parasitol. 45, 147–154. Gharbi, M., Sassi, L., Dorchies, P., Darghouth, M.A., 2006. Infection of calves with Theileria annulata in Tunisia: economic analysis and evaluation of the potential benefit of vaccination. Vet. Parasitol. 137, 231–241. Hashemi-Fesharki, R., 1988. Control of Theileria annulata in Iran. Parasitol. Today 4, 36–40. He, L., Feng, H.-H., Zhang, W.-J., Zhang, Q.-L., Fang, R., Wang, L.-X., Tu, P., Zhou, Y.-Q., Zhao, J.-L., Oosthuizen, M.C., 2012. Occurrence of Theileria and Babesia species in water buffalo (Bubalus babalis, Linnaeus, 1758) in the Hubei province, South China. Vet. Parasitol. 186, 490–496. Katzer, F., McKellar, S., Kirvar, E., Shiels, B., 1998. Phylogenetic analysis of Theileria and Babesia equi in relation to the establishment of parasite populations within novel host species and the development of diagnostic tests. Mol. Biochem. Parasitol. 95, 33–44. Mehlhorn, H., Schein, E., 1984. The piroplasms: life cycle and sexual stages. Adv. Parasitol. 23, 37–103. Pain, A., Renauld, H., Berriman, M., Murphy, L., Yeats, C.A., Weir, W., Kerhornou, A., Aslett, M., Bishop, R., Bouchier, C., 2005. Genome of the host-cell transforming parasite Theileria annulata compared with T. parva. Science 309, 131–133. Santos, M., Soares, R., Costa, P., Amaro, A., Inácio, J., Gomes, J., 2012. Revisiting the Tams1-encoding gene as a species-specific target for the molecular detection of Theileria annulata in bovine blood samples. Ticks Tick Borne Dis. 4, 72–77. Saravanan, B.C., Sreekumar, C., Bansal, G.C., Ray, D., Rao, J.R., Mishra, A.K., 2003. A rapid MTT colorimetric assay to assess the proliferative index of two Indian strains of Theileria annulata. Vet. Parasitol. 113, 211–216. Sayin, F., Karaer, Z., Dincer, S., Cakmak, A., Inci, A., Yukari, B.A., Eren, H., Vatansever, Z., Nalbantoglu, S., Melrose, T.R., 2003. A comparison of susceptibilities to infection of four species of Hyalomma ticks with Theileria annulata. Vet. Parasitol. 113, 115–121. Schnittger, L., Rodriguez, A., Florin-Christensen, M., Morrison, D., 2012. Babesia: a world emerging. Infect. Genet. Evol. 12, 1788–1809. Schnittger, L., Yin, H., Gubbels, M.J., Beyer, D., Niemann, S., Jongejan, F., Ahmed, J.S., 2003. Phylogeny of sheep and goat Theileria and Babesia parasites. Parasitol. Res. 91, 398–406. Sibeko, K.P., Oosthuizen, M.C., Collins, N.E., Geysen, D., Rambritch, N.E., Latif, A.A., Groeneveld, H.T., Potgieter, F.T., Coetzer, J.A., 2008. Development and evaluation of a real-time polymerase chain reaction test for the detection of Theileria parva infections in Cape buffalo (Syncerus caffer) and cattle. Vet. Parasitol. 155, 37–48. Weir, W., Karagenc¸, T., Gharbi, M., Simuunza, M., Aypak, S., Aysul, N., Darghouth, M.A., Shiels, B., Tait, A., 2011. Population diversity and multiplicity of infection in Theileria annulata. Int. J. Parasitol. 41, 193–203.
