Abstract
The sensitivity of LAMP, PCR and in vitro culture methods for the detection of Theileria equi and Babesia caballi was evaluated using tenfold serially diluted culture parasites. On day 1 post-culture, both T. equi and B. caballi parasites could only be observed at 1% parasite dilution from the in vitro culture method, whereas LAMP could detect up to 1 × 10−3% of both T. equi and B. caballi parasite dilutions, whilst PCR could detect 1 × 10−3% T. equi and 1 × 10−1% B. caballi parasite dilutions. On day 7 post-culture, the detection limit for T. equi and B. caballi in the in vitro culture increased up to 1 × 10−6%, whereas LAMP detection limit increased to 1 × 10−10% for both parasites, whilst the PCR detection limit increased to 1 × 10−10% and 1 × 10−6% for T. equi and B. caballi, respectively. Furthermore, LAMP and PCR amplified the T. equi DNA extracted from the organs of an experimentally infected horse. This study further validates LAMP as an alternative molecular diagnostic tool, which can be used in the diagnosis of early infections of equine piroplasmosis and together with PCR can also be used as supplementary methods during post-mortems.
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References
Alhassan A, Pumidonming W, Okamura M, Hirata H, Battsetseg B, Fujisaki K, Yokoyama N, Igarashi I (2005) Development of a single-round and multiplex PCR method for the simultaneous detection of Babesia caballi and Babesia equi in horse blood. Vet Parasitol 129:43–49
Alhassan A, Thekisoe OMM, Yokoyama N, Inue N, Motloang M, Mbati P, Igarashi I (2006) Development of loop-mediated isothermal amplification (LAMP) method for the detection of equine piroplasmosis. Vet Parasitol (in press)
Avarzed A, Igarashi I, Kanemaru T, Hirumi K, Omata Y, Saito A, Oyamada T, Nagasawa H, Toyoda Y, Suzuki N (1997) Improved in vitro cultivation of Babesia caballi. J Vet Med Sci 59:479–481
Bashiruddin JB, Camma C, Rebelo E (1999) Molecular detection of Babesia equi and Babesia caballi in horse blood by PCR amplification of part of the 16S rRNA gene. Vet Parasitol 84:75–83
Battsetseg B, Lucero S, Xuan X, Claveria F, Byambaa B, Battur B, Boldbaatar D, Batsukh Z, Khaliunaa T, Battsetseg G, Igarashi I, Nagasawa H, Fujisaki K (2002) Detection of equine Babesia spp. gene fragments in Dermacentor nuttalli Olenev 1929 infesting Mongolian horses, and their amplification in egg and larval progenies. J Vet Med Sci 64:727–730
De Waal DT (2000) Global importance of piroplasmosis. J Protozool Res 10:106–127
Friedhoff KT (1982) The piroplasms of equidae. Significance for the international equine trade. Berl Muench Tierarztl Wochenschr 95:368–374
Holman PJ, Frerichs WM, Chieves L, Wagner GG (1993) Culture confirmation of the carrier status of Babesia caballi-infected horses. J Clin Microbiol 31:698–701
Holman PJ, Hietala SK, Kayashima LR, Olson D, Waghela SD, Wagner GG (1997) Case report: field-acquired subclinical Babesia equi infection confirmed by in vitro culture. J Clin Microbiol 35:474–476
Ikadai H, Martin MD, Nagasawa H, Fujisaki K, Suzuki N, Mikami T, Kudo N, Oyamada T, Igarashi I (2001) Analysis of a growth-promoting factor for Babesia caballi cultivation. J Parasitol 87:1484–1486
Mehlhorn H, Schein E (1998) Redescription of Babesia equi Laveran, 1901 as Theileria equi Mehlhorn Schein 1998. Parasitol Res 84:467–475
Nicolaiewsky TB, Richte MF, Lunge VR, Cunha CW, Delagostin O, Ikuta N, Fonseca AS, da Silva SS, Ozaki LS (2001) Detection of Babesia equi (Laveran, 1901) by nested polymerase chain reaction. Vet Parasitol 101:9–21
Notomi T, Hiroto O, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:E63
Rampersad J, Cesar E, Campbell MD, Samlal M, Ammons DA (2003) A field evaluation of PCR for the routine detection of Babesia equi in horses. Vet Parasitol 114:81–87
Roby TO, Anthony DW (1963) The transmission of equine piroplasmosis by the tropical horse tick (Dermacentor nitens Neumann). Am J Vet Res 142:768–769
Schein E (1988) Equine babesiosis. In: Ristic M (eds) Babesiosis of domestic animals and man. CRC, Boca Raton, FL, pp 197–208
Stiller D, Frerichs WM, Leatch G, Kuttler KL (1980) Transmission of equine babesiosis and bovine anaplasmosis by Dermacentor albipictus (Packard) (Acari: Ixodidae). J N Y Entomol Soc 88:75–76
Tenter AM, Friedhoff KT (1986) Serodiagnosis of experimental and natural B. equi and B. caballi infection. Vet Parasitol 21:139
Xu Y, Zhang S, Huang X, Bayin C, Xuan X, Igarashi I, Fujisaki K, Kabeya H, Maruyama S, Mikami T (2003) Seroepidemiologic studies on Babesia equi and Babesia caballi infections in horses in Jilin province of China. J Vet Med Sci 65:1015–1017
Xuan X, Larsen A, Ikadai H, Tanaka T, Igarashi I, Nagasawa H, Fujisaki K, Toyoda Y, Suzuki N, Mikami T (2001) Expression of Babesia equi merozoite antigen 1 in insect cells by recombinant baculovirus and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay. J Clin Microbiol 39:705–709
Zweygarth E, Lopez-Rebollar LM, Nurton J, Guthrie AJ (2002) Culture isolation and propagation of Babesia caballi from naturally infected horses. Parasitol Res 88:460–462
Acknowledgments
We thank the Japanese Racing Association (JRA), Tochigi, Japan for the provision of T. equi experimental materials. This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS), the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), the 21st Century COE Program from the Japanese Ministry of Education, Culture, Sports, Science and Technology and the Japan International Cooperation Agency (JICA).
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Alhassan, A., Govind, Y., Tam, N.T. et al. Comparative evaluation of the sensitivity of LAMP, PCR and in vitro culture methods for the diagnosis of equine piroplasmosis. Parasitol Res 100, 1165–1168 (2007). https://doi.org/10.1007/s00436-006-0430-6
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DOI: https://doi.org/10.1007/s00436-006-0430-6