Diagnostic evaluation of nested PCR and microscopy for Cryptosporidiosis in goats: can COWP gene based qRT-PCR be useful in assessment of active Cryptosporidial infections?
DOI:
https://doi.org/10.12834/VetIt.3056.22638.1Keywords:
Cryptosporidium, Goat-kids, PCR-RFLP, zoonoses, COWP transcriptsAbstract
The present research delved into the transmission patterns, diagnostic methods, molecular traits, and phylogenetic analysis of Cryptosporidium species. The research was undertaken to enhance comprehension of the epidemiology and the potential for zoonotic transmission. A total of 80 goat-kid samples were tested, 7 were confirmed positive by mZN microscopy and 12 by nested-PCR. By PCR, 18SSUrRNA, HSP70, and GP60 amplicons were tested for Cryptosporidium. The restriction enzymes viz., SspI, VspI and MboII were used to genotype 12 Cryptosporidium positive samples by which C. parvum and C. bovis mixed infections were detected. Quantitative reverse transcription real-time PCR was used to transcriptionally screen the COWP-subunit genes to assess the severity of the infection in goat-kids, which showed upregulation of COWP6 and COWP4, while COWP9 and COWP3 genes were downregulated. A silent mutation was found at the codon CCA→CCC, which is being reported for the first time in goat field isolates. Phylogenetic and sequencing analyses confirmed the presence of the anthropozoonotic IIe subtype.
References
Ahamed I. 2012. Epidemiological studies on cryptosporidiosis in sheep and goats. M.V.Sc. & A. H. Thesis (Veterinary Parasitology), Sher-e-Kashmir University of Agricultural Sciences and Technology, Jammu.
Bejan A., MirceanV., Radu C., Smaro S. & Cozma V. 2009. Epidemiology of Cryptosporidium spp. infection in goat kids in the central and the northwest part of Romania. Revista Scientia Parasitologica, 10, 32-36.
Castro–Hermida J.A., Warleta M.G.A. & Mezo M. 2007. Natural infection by Cryptosporidium parvum and Giardia duodenalis in sheep and goats in Galicia (NW Spain). Small Ruminant Res, 72, 96–100.
Castro-Hermida J.A., Yolanda A., Gonzalez-Losada A. & Ares-Mazas E. 2002a. Prevalence of and risk factors involved in the spread of neonatal bovine cryptosporidiosis in Galicia (NW Spain). Vet Parasitol, 106, 1-10.
Castro-Hermida J.A., Yolanda A., Gonzalez-Losada A., Mezo-Menendez M. & Ares-Mazas E. 2002b. A study of cryptosporidiosis in a cohort of neonatal calves. Vet Parasitol, 106, 11-17.
Causape A.C., Quıìlez J., Sánchez-Acedo C., delCacho E. & López-Bernad F. 2002. Prevalence and analysis of potential risk factors for Cryptosporidium parvum infection in lambs in Zaragoza (North eastern Spain). Vet Parasitol, 104(4), 287- 298.
Chatterjee A., Banerjee S., Steffen M., O’Connor, R.M., Ward H.D., Robbins P.W. & Samuelson J. 2010. Evidence for mucin-like glycoproteins that tether sporozoites of Cryptosporidium parvum to the inner surface of the oocyst wall. Eukaryot Cell, 9, 84–96.
Chen X.M., Keithly J.S., Paya C.V. & LaRusso N.F. 2002. Cryptosporidiosis. N Engl J Med, 346, 1723-1731.
Chomczynski P. 1993. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques, 15(3), 532-4.
Del Coco V.F., Córdoba M.A., Bilbao G., de Almeida Castro A.P., Basualdo J.A., Fayer R. & Santín M. 2014. Cryptosporidium parvum GP60 subtypes in dairy cattle from Buenos Aires, Argentina. Res Vet Sci, 96(2), 311-314.
Dixit P., Rao M.L.V., Dixit A.K., Gupta R. & Shukla P.C. 2018. Prevalence and molecular characterization of Cryptosporidium spp. in goat kids. Indian J Anim Res, 53, 1234-1238.
Fall A., Thompson R.C., Hobbs R.P. & Morgan-Ryan U. 2003. Morphology is not a reliable tool for delineating species within Cryptosporidium. J Parasitol, 89, 399–402.
Fayer R., Trout J.M., Graczyk T.D. & Lewis E.J. 2000. Prevalence of Cryptosporidium, Giardia and Eimeria infection in post-weaned and adult cattle in three Maryland farms. Vet Parasitol, 93, 103-112.
Feltus C.D., Giddings C.W., Khaitsa M.L. & McEvoy J.M. 2008. High prevalence of Cryptosporidium bovis and the deer-like genotype in calves compared to mature cows in beef cow-calf operations. Vet Parasitol, 151, 191-195.
Feng Y., Ortega Y., He G., Das P., Xu M., Zhang X., Fayer R., Gatei W., Cama V. & Xiao L. 2007. Wide geographic distribution of Cryptosporidium parvum and deer like genotype in bovines. Vet Parasitol, 144, 1-9.
Geurden T., Thomas P., Casaert S., Vercruysse J. & Claerebout E. 2008. Prevalence and molecular characterization of Cryptosporidium and Giardia in lambs and goat kids in Belgium. Vet Parasitol, 155, 142-145.
Hall M.C. 1919. The adult taenioid cestodes of dogs and cats and related carnivores in North America.In: Proceedings of the United States National Museum. 55, 1–94.
Henricksen S.A. & Pohlenz J.F.L. 1981. Staining of cryptosporidia by a modified Ziehl-Neelsen technique. Acta Vet Scand, 22, 594-596.
Jafari R., Maghsood A.H. & Fallah M. 2012. Prevalence of Cryptosporidium infection among livestock and humans in contact with livestock in Hamadan district, Iran. J Res Health Sci, 13(1), 86-89.
Kaupke A., Michalski M.M. & Rzeżutka A. 2017. Diversity of Cryptosporidium species occurring in sheep and goat breeds reared in Poland. Parasitol Res, 116, 871–879.
Khalil S., Mirdha B.R., Panda A., Singh Y., Makharia G. & Paul J. 2017. Cryptosporidium species subtypes and associated clinical manifestations in Indian patients. Gastroenterol Hepatol Bed to Bench, 10(4), 311-318.
Khursheed A., Yadav A., Rafiqi S.I., Katoch R., Godara R., Sood S. & Saleem T. 2018. Periparturient rise in the Cryptosporidium oocyst count in Beetal goats and evaluation of infection in new born kids. Indian J Anim Sci, 88 (9), 994–997.
Koinari M., Lymbery A.J. & Ryan U.M. 2014. Cryptosporidium species in sheep and goats from Papua New Guinea. Exp Parasitol, 141, 134-137.
Landis J.R. & Koch G.G. 1977. An application of hierarchical kappa-type statistics in the assessment of majority agreement among multiple observers. Biometrics, 363-374.
Lassen B., Viltrop A., Raaperi K. & Jarvis T. 2009. Eimeria and Cryptosporidium in Estonian dairy farms in regard to age, species and diarrhoea. Vet Parasitol, 166, 212-219.
Lippuner C., Ramakrishnan C., Basso W.U., Schmid M.W., Okoniewski M., Smith N.C. & Hehl A.B. 2018. RNA-Seq analysis during the life cycle of Cryptosporidium parvum reveals significant differential gene expression between proliferating stages in the intestine and infectious sporozoites. Int J Parasitol, 48(6), 413-422.
Lorenz I., Fagan J. & More S.J. 2011. Calf health from birth to weaning. II. Management of diarrhoea in pre-weaned calves. Ir Vet J, 64(1), 1.
Mahmoud A.E., Abdel-Rahman H.G., Abouelhassan E.M. & Sallam N.H. 2018. Cryptosporidium parvum in clinically examined dairy calves; molecular and biochemical studies. J Egypt Soc Parasitol, 48(3), 535 – 542.
Mammeri M., Obregón D.A., Chevillot A., Polack B., Julien C., Pollet T, Cabezas-Cruz A. & Adjou K.T. 2020. Cryptosporidium parvum infection depletes butyrate producer bacteria in goat kid microbiome. Front Microbiolo, 11, 548737.
Marreros N., Frey C.F., Willisch C.S., Signer C. & Ryser-Degiorgis M.P. 2012. Coprological analyses on apparently healthy Alpine ibex (Capra ibex ibex) from two Swiss colonies. Vet Parasitol, 186(3), 382-389.
Mason R.W., Hartley W.J. & Tilt L. 1981. Intestinal cryptosporidiosis in a kid goat. Aust Vet J, 57, 386–388.
Maurya P.S., Rakesh R.L., Pradeep B., Kumar S., Kundu K., Garg R., Ram H., Kumar A. & Banerjee P.S. 2013. Prevalence and risk factors associated with Cryptosporidium spp. infection in young domestic livestock in India. Trop Anim Health Pro, 45, 941–946.
Mendonca C., Almeida A., Castro A., de L Delgado M, Soares S., da Costa J.M.C. & Canada N. 2007. Molecular characterization of Cryptosporidium and Giardia isolates from cattle from Portugal. Vet Parasitol, 147, 47-50.
Misic Z.B., Katic-Radivojevic S.P. & Kulisic Z. 2006.Cryptosporidium infection in lambs and goat kids in Serbia. Acta Vet Scand, 56(1), 49-54.
Morgan U.M., Monis P.T., Xiao L., Limor J., Sulaiman I., Raidal S., O’Donoghue P., Gasser R., Murray A., Blagburn B.L., Lal A.A. & Thompson R.C. 2001 .Molecular and phylogenetic characterization of Cryptosporidium from birds. Int J Parasitol, 31, 203-215.
Navin T.R. & Juranek D.D. 1984. Cryptosporidiosis; clinical, epidemiological and parasitological review. Rev Infect Dis, 6, 313–327.
Noordeen F., Rajapakse R.P.V.J., Faizal A.C.M., Horadagoda N.U. & Arulkanthan A. 2000. Prevalence of Cryptosporidium infection in goats in selected locations in three agroclimatic zones of Sri Lanka. Vet Parasitol, 9, 95–101.
Olson M.E., Thorlakson C.L., Deselliers L., Morck D.W. & McAllister T.A. 1997. Giardia and Cryptosporidium in Canadian farm animals. Vet Parasitol, 68, 375–381.
Paraud C. & Chartier C. 2012. Cryptosporidiosis in small ruminants. Small Ruminant Res, 103, 93–97.
Paul S., Chandra D., Tewari A.K., Banerjee P.S., Ray D.D, Boral R. & Rao J.R. 2009. Comparative evaluation and economic assessment of coprological diagnostic methods and PCR for detection of Cryptosporidium spp. in bovines. Vet Parasitol, 164, 291–295.
Paul S., Reddy M.G.B. & Sharma D.K. 2013. Cryptosporidiosis in neonatal goat kids in north–western India. Indian Vet J, 90, 142–143.
Paul S., Sharma D.K., Boral R., Mishra A.K., Shivsharanappa N., Banerjee P.S. & Pawaiya R.V.S. 2014. Cryptosporidiosis in goats; a review. Adv Anim Vet Sci, 2 (3S), 49 – 54.
Quílez J., Sanchez-Acedo C., Clavel A., delCacho E. & Lopez-Bernad F. 1996. Comparison of an acid-fast stain and a monoclonal antibody-based immunofluorscence reagent for the detection of Cryptosporidium oocysts in faecal specimens from cattle and pigs. Vet Parasitol, 67, 75-81.
Quílez J., Torres E., Chalmers R.M., Hadfield S.J., Del Cacho E. & Sánchez-Acedo C. 2008. Cryptosporidium genotypes and subtypes in lambs and goat kids in Spain. Appl Environ Microbiol, 74, 6026–6031.
Rieux A., ParaudC., Pors I. & Chartier C. 2013. Molecular characterization of Cryptosporidium spp. in pre-weaned kids in a dairy goat farm in western France. Vet Parasitol, 192, 268–72.
Rzhetsky A. & Nei M. 1992. Statistical properties of the ordinary least-squares, generalized least-squares, and minimum-evolution methods of phylogenetic inference. J Mol Evol, 35(4), 367-375.
Saitou N. & Nei M. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees, Mol Biol Evol, 4, 406-425.
Sambrook J. & Russel D. 2001. Molecular cloning- A laboratory manual 3rd ed., Cold spring harbor laboratory press, Cold spring harbor, New York, USA, 5.1-5.14.
Santı´n M. & Trout J.M. 2008. Livestock, in: Fayer R, Xiao L (Eds.), Cryptosporidium and Cryptosporidiosis, CRC Press, Boca Raton, FL, USA, 451–483.
Shanmathi S., Yashica K.A. & Gunasekaran P. 2020. Epidemiology and diagnosis of cryptosporidiosis: A review. J Entomol Zool Stud, 8(4), 471-474.
Sharma P., Sharma A., Sehgal R., Malla N. & Khurana S. 2013. Genetic diversity of Cryptosporidium isolates from patients in North India. Int J Infect Dis, 17, 601-05.
Sharma A.K., Gururaj K., Sharma R., Goel A., Paul S. & Sharma D.K., 2022. Development of duplex real‐time PCR for quick detection of cryptosporidiosis in goats. Cell Biochem Funct, https://doi.org/10.1002/cbf.3759.
Singla L.D., Gupta M.P., Singh H., Singh S.T., Kaur P. & Juyal P.D. 2013. Antigen based diagnosis of Cryptosporidium parvum infection in faeces of cattle and buffalo calves. Indian J Anim Sci, 83(1), 37-39.
Snedecor G.W. & Cochran W.G. 1994. Statistical Methods, 7th Edn., Oxford and IBH Publishing Co., New Delhi, 1-593.
Tamura K., Nei M. & Kumar S. 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. PNAS (USA), 101, 11030-11035.
Tamura K., Stecher G., Peterson D., Filipski A. & Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol, 30, 2725-2729.
Templeton T.J., Lancto C.A., Vigdorovich V., Liu C., London N.R., Hadsall K.Z. & Abrahamsen M.S. 2004. The Cryptosporidium oocyst wall protein is a member of a multigene family and has a homolog in Toxoplasma. Infect Immun, 72(2), 980-987.
Utaaker K.S., Myhr N., Bajwa R.S., Joshi H., Kumar A. & Robertson L.J. 2017. Goats in the city: prevalence of Giardia duodenalis and Cryptosporidium spp. in extensively reared goats in Northern India. Acta Vet Scand, 59(1), 86.
Venu R., Latha B.R., Abdul Basith S., Sreekumar C., Dhinakar Raj G. & Raman M. 2013. Comparative evaluation of conventional diagnostic methods for detection of bovine cryptosporidiosis. Indian J Anim Sci, 83(2), 124-128.
Viel H., Rocques H., Martin J. & Chartier C. 2007. Efficacy of nitazoxanide against experimental cryptosporidiosis in goat neonates. Parasitol Res, 102, 163–166.
Wang R., Li G., Cui B., Huang J., Cui Z, Zhang S., Dong H., Yue D., Zhang L., Ning C. & Wang M. 2014. Prevalence, molecular characterization and zoonotic potential of Cryptosporidium spp. in goats in Henan and Chongqing, China. Exp Parasitol, 142, 11-16.
Wielinga P.R., de Vries A., van der Goot T.H., Mank T., Mars M.H., Kortbeek L.M. & van der Giessen J.W.B. 2008. Molecular epidemiology of Cryptosporidium in humans and cattle in The Netherlands. Int J Parasitol, 38, 809-817.
Xiao L. 2010. Molecular epidemiology of cryptosporidiosis: an update. Exp Parasitol, 124, 80–89.
Xiao L. & Fayer R. 2008. Molecular characterization of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission. Int J Parasitol, 38, 1239–1255.
Yadav A., Katoch R., Godara R., Ganai A., Khan A., Bhat M.A., Nazim A., Khursheed A. and Kaur D. 2016. Cryptosporidiosis in lambs and kids in Jammu with special reference to Cryptosporidium ubiquitum in goat kids. Proceedings of 25thNCVP, Chennai, 17–19th February, 151.
Yang R., Jacobson C., Gardner G., Carmichael I., Campbell A.J.D., Ng-Hublin J. & Ryan U. 2014. Longitudinal prevalence, oocyst shedding and molecular characterization of Cryptosporidium species in sheep across four states in Australia. Vet Parasitol, 200, 50-58.
Zorana M., Sofija K. & Kulisicz. 2006. Cryptosporidium infection in lambs and goat kids in Serbia. Acta Vet (Beograd), 56 (1), 49-54.