SREEDEVI, C(Major)MALAKONDAIAH, PSRINIVASA RAO, GSUDHAKAR, KSATHISH, K2017-08-192017-08-192016-12http://krishikosh.egranth.ac.in/handle/1/5810028999THESESABSTRACT: The present study was carried out for simultaneous detection of point mutations (SNPs) at codon 167 and 200 of β-tubulin isotype 1 gene of H. contortus that are linked to benzimidazole (BZ) resistance and for identifying the PCR-RFLP polymorphism of β-tubulin isotype 1 gene of H. contortus in sheep in Ibrahimpatnam, Vijayawada, Nuziveedu, Hanuman Junction, Gannavaram, Gudiwada and Machilipatnam regions of Krishna district, Andhra Pradesh. A total of 226 adult male H. contortus were genotyped by PCR-RFLP at codons 167 (P167Y) and 200 (P200Y) for detection of mutation in β-tubulin isotype 1 gene. The isolated genomic DNA of each single worm was separately used to amplify the β-tubulin isotype 1 gene that yielded PCR product of 629 bp. Further each PCR product was amplified with modified forward primer that yielded an expected fragment of 488 bp product. The PCR product (488 bp) was subsequently digested with restriction endonuclease SnaBI and TaaI to detect mutation at codon 167 and 200 respectively. On digestion with SnaBI RFLP pattern revealed two alleles encoding for phenylalanine (BZ ‘SS’) at codon 167 in all 226 worms. No resistant allele was demonstrable at this codon. On digestion with restriction endonuclease TaaI three different patterns were observed, one with 215 bp, 206 bp and 67 bp, other with 282 bp, 215 bp, 206 bp and 67 bp, and another with 282 bp and 206 bp fragment which were referred to ‘rr’ (homozygous resistant), ‘rS’ (heterozygous) and ‘SS’ (homozygous susceptible) genotypes respectively. Thirty two individual worms genotyped from Ibrahimpatnam region revealed two (6.25 %) homozygous resistant (‘rr’), 24 (75.0 %) heterozygous (‘rS’) and six (18.75 %) homozygous susceptible (‘SS’) type. Thirty one individuals from Vijayawada region were genotyped, of which four (12.9 %) were homozygous resistant (‘rr’) and 24 (77.42 %) were heterozygous (‘rS’) and three (9.68 %) were homozygous susceptible (‘SS’). Out of 23 individuals genotyped from Nuziveedu region, the observed genotypic frequencies were 30.43 per cent ‘rr’, 34.78 per cent ‘rS’ and 34.78 ‘SS’ type. Out of 31 individuals genotyped from Hanuman Junction region, nine (29.03 %) individuals were homozygous resistant (‘rr’), 21 (67.74 %) were heterozygous resistant (‘rS’) and one (3.22 %) was homozygous susceptible type (‘SS’). Among the worms collected from Gannavaram region 14 (23.33 %) individuals were homozygous resistant (‘rr’), 40 (66.67 %) were heterozygous (‘rS’) and six (10.0 %) were homozygous susceptible (‘SS’). Twenty two individuals from Gudiwada region were genotyped, of which seven (31.82 %) were homozygous resistant (‘rr’) and 15 (68.18 %) were heterozygous (‘rS’) type. No homozygous susceptible (‘SS’) genotypes could be observed. Out of 27 individuals genotyped in Machilipatnam region five (18.52 %) individuals were homozygous resistant (‘rr’), 14 (51.85 %) were heterozygous (‘rS’) and eight (29.63 %) were homozygous susceptible type (‘SS’). The overall genotypic frequencies varied significantly (P<0.01) with respect to β-tubulin gene/TaaI locus in the study area. The frequency of ‘rS’ (64.60 %) genotypes was high compared to that of ‘rr’ (21.24 %) and ‘SS’ (14.16 %) genoytpes in all the regions under study. Observation of genotypic frequencies in different regions of Krishna district revealed that there was no significant difference (P>0.01) between the genotypes only in Nuziveedu and Machilipatnam. The frequency of ‘rr’ genotypes were more (31.82 %) in Gudiwada whereas the frequency of ‘rS’ was more in Vijayawada (77.42 %). As a whole the prevalence of benzimidazole resistance allele (‘r’) was found significantly (P<0.01) higher (54.0 %) compared to 46.0 per cent benzimidazole susceptible allele (‘S’). Results revealed absence of P167Y and the presence of P200Y β-tubulin isotype 1 BZ resistance associated polymorphism in H. contortus at low frequency indicating emergence of resistance in this parasite in sheep in the study area.ennullThesis