Lucy SabuASHA RAJAGOPAL2023-01-192023-01-192017-06-23https://krishikosh.egranth.ac.in/handle/1/5810192562Thesis Submitted in partial fulfilment of the requirement for the degree of Doctor of Philosophy in Veterinary Parasitology.The study was conducted with the objectives of assessing the status of benzimidazole resistance in gastrointestinal (GI) nematodes of goats in Kerala, detection of single nucleotide polymorphisms (SNPs) associated with benzimidazole resistance in the β-tubulin gene of predominant GI nematode species and to evaluate the efficacy of the egg hatch assay, larval development assay and PCR-RFLP in detection of benzimidazole resistance. Microscopical examination of 520 faecal samples collected from goats from 10 organized farms and 16 small holder farmers’ flocks in eight agro-ecological zones of Kerala revealed an overall prevalence of 81.5 + 5.54 per cent strongyles in goats. There was significant difference between the prevalence of strongylosis in organized farms (91.19 + 4.33 %) and small holder farmers’ flocks (65.34 + 10.27 %). The mean faecal egg counts (FECs) also differed significantly between organized farms and small holder farmers’ flocks. The prevalent genera of strongyles identified on coproculture were Haemonchus spp., Trichostrongylus spp. and Oesophagostomum spp. Faecal egg count reduction test (FECRT) done for screening the benzimidazole resistance status in 10 organized farms and 16 small holder farmers’ flocks identified benzimidazole resistance in all the organized farms. Among the small holder farmers’ flocks, 43.75 per cent were found to be resistant to benzimidazoles. Susceptibility was identified in 37.5 per cent of the small holder farmers’ flocks while resistance was suspected in 18.75 per cent of the flocks. Statistical analysis revealed significant association between the resistance status and farm type. Haemonchus spp. was found to be the most predominant GI nematode in post-treatment faecal cultures indicating that it is the major species responsible for benzimidazole resistance. Resistance status was found to be significantly correlated with the frequency of deworming in flocks. Molecular genotyping by PCR-RFLP revealed E198A polymorphism in isotype 1 β-tubulin gene in Haemonchus spp. with an overall frequency of 0.516 for the resistant allele (r). The overall prevalence of homozygous resistant genotype (rr) at codon 198 in Haemonchus spp. was 25.6 per cent. No polymorphism was identified at codons 167 and 200 in Haemonchus spp. in this study. In Trichostrongylus spp., F200Y polymorphism was identified in isotype 1 β-tubulin gene with an overall gene frequency of 0.337 for the resistant allele and with 28 per cent of the larvae genotyped being homozygous resistant (rr). Susceptible genotype was identified at codons 167 and 198. All the Oesophagostomum spp. larvae genotyped were found to be of the susceptible genotype at codons 198 and 200. In vitro detection of benzimidazole resistance was done by egg hatch assay (EHA) and larval development assay (LDA). Correlation of the results of FECRT, EHA, LDA and PCR-RFLP revealed significant correlation (p < 0.05) between FECR per cent, ED50 in EHA, Pdd (proportion of larvae hatching at the discriminating dose of 0.02 µg/ml) in LDA and the percentage of homozygous resistant (rr) genotype in PCR-RFLP. There was significant correlation between ED50 and Hdd (hatching ratio of strongyle eggs at the discriminating dose of 0.1 µg/ml) values in egg hatch assay. Pdd values were found to be significantly correlated with other resistance parameters indicating that it is a better criterion for resistance detection than LD50 in LDA. To predict the genotypic resistance using phenotypic resistance indicators, regression equations were derived with rr genotype per cent as dependent variable and FECR per cent, ED50 and Pdd as the independent variables.EnglishThesis