Abstract
Anaplasma genus has been classified under the family Anaplasmataceae of order Rickettsiales, which contains the spp. Anaplasma marginale, Anaplasma bovis, Anaplasma centrale, Anaplasma platys and Anaplasma phagocytophilum is accountable for a broad range of diseases in both man and animal medicine around the globe. A multiplex PCR procedure was designed to identify A. marginale, A. bovis, and A. platys simultaneously by employing species-specific primers targeting the msp4 (Anaplasma marginale), 16S rRNA (Anaplasma bovis), and groEL (Anaplasma platys) genes. The sensitivity of the assay was ascertained by tenfold dilutions of DNA obtained from cattle blood infected with A. marginale, A. bovis, and A. platys was used to evaluate the test sensitivity. A total of 31 Anaplasma genus positive samples were subjected to mPCR by using species specific primers for simultaneous detection of all the three species. All the 31 samples showed amplification of 753 bp fragment specific for A. marginale and 61.29% of the samples showed amplification of 547 bp fragment specific for A. bovis. The 470 bp DNA fragment specific for A. platys was found in 96.7% of samples. The lower limit of detection of the method for A. platys, A. marginale and A. bovis was found to be 4.4, 0.44 and 0.044 ng/µl, respectively.
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References
Bisen S, Aftab A, Jeeva K, Silamparasan M, Yadav S, Chandra D, Sankar M, Garg R, Raina OK (2021) Molecular and serological detection of Anaplasma infection in carrier cattle in north India. Vet Parasitol 24(1):100550
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(1):222–229
Bowman DD (2014) Georgi’s Parasitology for Veterinarians, 10th ed. Saunders, 3251 Riverport lane, St. Louis, Missouri 63043, pp 246–249
Cui Y, Zhang Y, Jian F, Zhang L, Wang R, Cao S, Wang X, Yan Y, Ning C (2017) Development of duplex PCR for simultaneous detection of Theileria spp. and Anaplasma spp. in sheep and goats. Exp Parasitol 176(1):1–7
Edwards MC, Gibbs RA (1994) Multiplex PCR: advantages, development, and applications. Genome Res 3(4):S65–S75
Gale KR, Dimmock CM, Gartside M, Leatch G (1996) Anaplasma marginale: detection of carrier cattle by PCR-ELISA. Int J Parasitol 26(10):1103–1109
George N, Bhandari V, Sharma P (2017) Phylogenetic relationship and genotypic variability in Anaplasma marginale strains causing anaplasmosis in India. Infect Genet Evol 48(1):71–75
Joazeiro AC, Martins J, Masuda A, Seixas A, Junior IDSV (2015) A PCR for differentiate between Anaplasma marginale and A. centrale. Acta Sci Vet 43(1):1–7
Kundave VR, Ram H, Banerjee PS, Garg R, Mahendran K, Ravikumar GVPPS, Tiwari AK (2018) Development of multiplex PCR assay for concurrent detection of tick borne haemoparasitic infections in bovines. Acta Parasitol 63(4):759–765
Nguyen AH, Tiawsiriup S, Kaewthamasorn M (2020) Molecular detection and genetic characterisation of Anaplasma maginale and Anaplasma platys-like (Rickettsiales: Anaplasmataceae) in water buffalo from eight provinces of Thailand. BMC Vet Res 16(1):1–12
Peng Y, Zhao S, Wang K, Song J, Yan Y, Zhou Y, Shi K, Jian F, Wang R, Zhang L, Ning C (2020) A multiplex PCR detection assay for the identification of clinically relevant Anaplasma species in field blood samples. Front Microbiol 11(1):606
Pradeep RK, Nimisha M, Sruthi MK, Vidya P, Amrutha BM, Kurbet PS, Kumar KGA, Varghese A, Deepa CK, Dinesh CN, Chandrasekhar L (2019) Molecular characterisation of South Indian field isolates of bovine Babesia spp. and Anaplasma spp. Parasitol Res 118(2):617–630
Rodriquez 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 22(4):642–646
Sacchi ABV, Duarte JMB, Andre MR, Machado RZ (2012) Prevalence and molecular characterization of Anaplasmataceae agents in free-ranging Brazilian marsh deer (Blastocerus dichotomus). Comp Immunol Microbiol Infect Dis 35(4):325–334
Zobba R, Anfossi AG, Pinna Parpaglia ML, Dore GM, Chessa B, Spezzigu A, Rocca S, Visco S, Pittau M, Alberti A (2014) Molecular investigation and phylogeny of Anaplasma spp. in Mediterranean ruminants reveal the presence of neutrophil-tropic strains closely related to A. platys. Appl Environ Microbiol 80(1):271–280
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No funding was received to conduct research but facilities available at Veterinary College, Bengaluru, IAH & VB were used.
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Teja, M.M.S., Mamatha, G.S., Lakkundi, J.N. et al. Multiplex PCR for detection of Anaplasma marginale, A. bovis and A. platys in cattle. J Parasit Dis 47, 659–663 (2023). https://doi.org/10.1007/s12639-023-01606-6
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DOI: https://doi.org/10.1007/s12639-023-01606-6