Abstract
Coinfections with the tick-borne pathogens Theileria luwenshuni and Anaplasma phagocytophilum can cause significant economic losses in sheep and goat farming. The difficulty in detecting these two pathogens by microscopic examination warrants the development of a rapid detection test to discriminate them. In this study, a duplex polymerase chain reaction (PCR) assay was developed to simultaneously detect T. luwenshuni and A. phagocytophilum. Alignment of the sequences from related pathogens allowed us to design a primer pair targeting the 18S ribosomal RNA gene in T. luwenshuni and generate a target product of 962 bp, whereas a previously reported species-specific primer (SSAP2f/SSAP2r) for A. phagocytophilum was used in the same reaction to generate a product of 641 bp. Genomic DNA from T. luwenshuni and A. phagocytophilum was 10-fold serially diluted for testing PCR sensitivity. Under the optimal PCR conditions we established, the lower limit of detection of the assay was 29.13 fg/μL for T. luwenshuni and 1.53 fg/μL for A. phagocytophilum, and PCR primers used in this study were confirmed to be 100% species-specific using other hemoparasites previously identified by other methods. No significant difference was found between conventional and duplex PCR protocols used to detect the two species. Our study provides an effective, sensitive, specific, and accurate tool for the diagnosis and epidemiological surveillance of mixed infections of the two pathogens in sheep and goats.
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References
Ahmed J, Yin H, Schnittger L, Jongejan F (2002) Ticks and tick-borne diseases in Asia with special emphasis on China. Parasitol Res 88:S51–S55. https://doi.org/10.1007/s00436-001-0574-3
Ahmed JS, Luo J, Schnittger L, Seitzer U, Jongejan F, Yin H (2006) Phylogenetic position of small-ruminant infecting piroplasms. Ann N Y Acad Sci 1081:498–504. https://doi.org/10.1196/annals.1373.074
Barlough JE, Madigan JE, DeRock E, Bigornia L (1996) Nested polymerase chain reaction for detection of Ehrlichia equi genomic DNA in horses and ticks (Ixodes pacificus). Vet Parasitol 63:319–329. https://doi.org/10.1016/0304-4017(95)00904-3
Bilgiç HB, Karagenç T, Simuunza M, Shiels B, Tait A, Eren H, Weir W (2013) Development of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle. Exp Parasitol 133:222–229. https://doi.org/10.1016/j.exppara.2012.11.005
Chi Q, Liu Z, Li Y, Yang J, Chen Z, Yue C, Luo J, Yin H (2013) Development of a real-time PCR assay for detection and quantification of Anaplasma ovis infection. Transbound Emerg Dis 60(Suppl. 2):119–124. https://doi.org/10.1111/tbed.12139
Cui Y, Zhang Y, Jian F, Zhang L, Wang R, Cao S et al (2017) Development of duplex PCR for simultaneous detection of Theileria spp. and Anaplasma spp. in sheep and goats. Exp Parasitol 176:1–7. https://doi.org/10.1016/j.exppara.2017.01.011
de la Fuente J, Massung RF, Wong SJ, Chu FK, Lutz H, Meli M (2005) Sequence analysis of the msp4 gene of Anaplasma phagocytophilum strains. J Clin Microbiol 43:1309–1317. https://doi.org/10.1128/JCM.43.3.1309-1317.2005
Decaro N, Carelli G, Lorusso E, Lucente MS, Greco G, Lorusso A et al (2008) Duplex real-time polymerase chain reaction for simultaneous detection and quantification of Anaplasma marginale and Anaplasma centrale. J Vet Diagn Invest 20:606–611. https://doi.org/10.1177/104063870802000511
Djiba ML, Mediannikov O, Mbengue M, Thiongane Y, Molez JF, Seck MT et al (2013) Survey of anaplasmataceae bacteria in sheep from senegal. Trop Anim Health Prod 45:1557–1561. https://doi.org/10.1007/s11250-013-0399-y
Ge Y, Pan W, Yin H (2012) Prevalence of Theileria infections in goats and sheep in southeastern China. Vet Parasitol 186:466–469. https://doi.org/10.1016/j.vetpar.2011.11.066
Gebrekidan H, Hailu A, Kassahun A, Rohousova I, Maia C, Talmi-Frank D et al (2014) Theileria infection in domestic ruminants in northern ethiopia. Vet Parasitol 200:31–38. https://doi.org/10.1016/j.vetpar.2013.11.017
Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH (1997) Multiplex PCR: critical parameters and step-by-step protocol. Biotechniques 23:504–511. https://doi.org/10.2144/97233rr01
Kawahara M, Rikihisa Y, Lin Q, Isogai E, Tahara K, Itagaki A (2006) Novel Genetic Variants of Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma centrale, and a Novel Ehrlichia sp. in Wild Deer and Ticks on Two Major Islands in Japan. Appl Environ Microb 72:1102–1109. https://doi.org/10.1128/AEM.72.2.1102-1109.2006
Lee SH, Park S, Lee YS, Lee HK, Hwang SD (2018) Diagnosis and molecular characteristics of human infections caused by Anaplasma phagocytophilum in South Korea. J Microbiol 56:847–853. https://doi.org/10.1007/s12275-018-8385-8
Lew AE, Gale KR, Minchin CM, Shkap V, de Waal DT (2003) Phylogenetic analysis of the erythrocytic Anaplasma species based on 16S rDNA and GroEL (HSP60) sequences of A. marginale, A. centrale, and A. ovis and the specific detection of A. centrale vaccine strain. Vet Microbiol 92:145–160. https://doi.org/10.1016/s0378-1135(02)00352-8
Li H, Jiang JF, Liu W, Zheng YC, Huo QB, Tang K (2012) Human infection with Candidatus Neoehrlichia mikurensis, China. Emerg Infect Dis 18:1636–1639. https://doi.org/10.3201/eid1810.120594
Li Y, Guan G, Liu A, Peng Y, Luo J, Yin H (2010) Experimental transmission of Theileria ovis by Hyalomma anatolicum anatolicum. Parasitol Res 106:991–994. https://doi.org/10.1007/s00436-010-1763-8
Li Y, Luo J, Guan G, Ma M, Liu A, Liu J et al (2009) Experimental transmission of Theileria uilenbergi infective for small ruminants by haemaphysalis longicornis and haemaphysalis qinghaiensis. Parasitol Res 104:1227–1231. https://doi.org/10.1007/s00436-009-1347-7
Li Y, Zhang X, Liu Z, Chen Z, Yang J, He H (2014) An epidemiological survey of Theileria infections in small ruminants in central China. Vet Parasitol 200:198–202. https://doi.org/10.1016/j.vetpar.2013.07.023
Liu Z, Ma M, Wang Z, Wang J, Peng Y, Li Y et al (2012) Molecular survey and genetic identification of Anaplasma species in goats from central and southern China. Appl Environ Microbiol 78:464–470. https://doi.org/10.1128/AEM.06848-11
M’Ghirbi Y, Beji M, Oporto B, Khrouf F, Hurtado A, Bouattour A (2016) Anaplasma marginale and A. phagocytophilum in cattle in Tunisia. Parasites Vectors 9(1):556. https://doi.org/10.1186/s13071-016-1840-7
Mans BJ, Pienaar R, Latif AA (2015) A review of Theileria diagnostics and epidemiology. Int J Parasitol Parasites Wildl 4(1):104–118. https://doi.org/10.1016/j.ijppaw.2014.12.006
Markoulatos P, Siafakas N, Moncany M (2002) Multiplex polymerase chain reaction: a practical approach. J Clin Lab Anal 16(1):47–51. https://doi.org/10.1002/jcla.2058
Nasreen Khan A, Niaz S, Hassan Shah M, Khan A, Ahmed H, Khattak I (2020) Molecular detection of small ruminant piroplasmosis and first report of Theileria luwenshuni (apicomplexa: Theileridae) in small ruminants of pakistan. Exp Parasitol 212:107872. https://doi.org/10.1016/j.exppara.2020.107872
Rodriguez I, Burri C, Noda AA, Douet V, Gern L (2015) Multiplex PCR for molecular screening of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp. Ann Agric Environ Med Aaem 22:642–646. https://doi.org/10.5604/12321966.1185767
Schnittger L, Yin H, Gubbels MJ, Beyer D, Niemann S, Jongejan F et al (2003) Phylogeny of sheep and goat Theileria and Babesia parasites. Parasitol Res 91:398–406. https://doi.org/10.1007/s00436-003-0979-2
Sint D, Raso L, Traugott M (2012) Advances in multiplex PCR: balancing primer efficiencies and improving detection success. Methods Ecol Evol 3:898–905. https://doi.org/10.1111/j.2041-210X.2012.00215.x
Spitalska E, Torina A, Cannella V, Caracappa S, Sparagano OA (2004) Discrimination between Theileria lestoquardi and Theileria annulata in their vectors and hosts by RFLP based on the 18S rRNA gene. Parasitol Res 94:318–320. https://doi.org/10.1007/s00436-004-1217-2
Stuen S, Granquist EG, Silaghi C (2013) Anaplasma phagocytophilum–a widespread multi-host pathogen with highly adaptive strategies. Front Cell Infect Microbiol 3:31. https://doi.org/10.3389/fcimb.2013.00031
Wang HN, Yang JF, Mukhtar MU, Liu ZJ, Zhang MH, Wang XL (2019) Molecular detection and identification of tick-borne bacteria and protozoans in goats and wild Siberian roe deer (Capreolus pygargus) from Heilongjiang Province, northeastern China. Parasites Vectors 12(1):296. https://doi.org/10.1186/s13071-019-3553-1
Woldehiwet Z (2010) The natural history of Anaplasma phagocytophilum. Vet Parasitol 167:108–122. https://doi.org/10.1016/j.vetpar.2009.09.013
Yin H, Liu Z, Guan G, Liu A, Ma M, Ren Q et al (2008) Detection and differentiation of Theileria luwenshuni and T. uilenbergi infection in small ruminants by PCR. Transbound Emerg Dis 55:233–237. https://doi.org/10.1111/j.1865-1682.2008.01031.x
Yin H, Schnittger L, Luo J, Seitzer U, Ahmed JS (2007) Ovine theileriosis in china: a new look at an old story. Parasitol Res 101(Suppl 2):S191-195. https://doi.org/10.1007/s00436-003-0979-2
Zhang L, Liu H, Xu B, Lu Q, Li L, Chang L (2012) Anaplasma phagocytophilum infection in domestic animals in Ten Provinces/Cities of China. Am J Trop Med Hyg 87:185–189. https://doi.org/10.4269/ajtmh.2012.12-0005
Zhang X, Liu Z, Yang J, Chen Z, Guan G, Ren Q (2014) Multiplex PCR for diagnosis of Theileria uilenbergi, Theileria luwenshuni, and Theileria ovis in small ruminants. Parasitol Res 113:527–531. https://doi.org/10.1007/s00436-013-3684-9
Zhang Y, Lv Y, Zhang F, Zhang W, Wang J, Cui Y et al (2016) Molecular and phylogenetic analysis of Anaplasma spp. in sheep and goats from six provinces of China. J Vet Sci 17:523–529. https://doi.org/10.4142/jvs.2016.17.4.523
Zhou M, Cao S, Sevinc F, Sevinc M, Ceylan O, Ekici S et al (2017) Molecular detection and genetic characterization of Babesia, Theileria and Anaplasma amongst apparently healthy sheep and goats in the central region of turkey. Ticks Tick-Borne Dis 8:246–252. https://doi.org/10.1016/j.ttbdis.2016.11.006
Acknowledgements
This work was supported by the Earmarked Fund for China Modern Agro-industry Technology Research System (nycytx-38) and The Key Scientific and Technological Project of Henan Province (202102110104). We gratefully acknowledge Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences and Tarim University for providing the negative and positive control samples. We thank Sandra Cheesman, PhD, from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
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This research was conducted according to the Chinese Laboratory Animal Administration Act (1988) after review and approval of its protocol by the Research Ethics Committee of Henan Agricultural University. Appropriate permission was obtained from the farm owners before any blood samples were collected from their sheep and goats.
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Yan, Y., Cui, Y., Zhao, S. et al. Development of a duplex PCR assay for detecting Theileria luwenshuni and Anaplasma phagocytophilum in sheep and goats. Exp Appl Acarol 85, 319–330 (2021). https://doi.org/10.1007/s10493-021-00662-y
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DOI: https://doi.org/10.1007/s10493-021-00662-y