SIMULTANEOUS DETECTION OF POINT MUTATIONS (SNPs) AT CODON167 AND 200 OF β-TUBULIN ISOTYPE 1GENE LINKED TO BENZIMIDAZOLE RESISTANCE IN HaemonchuscontortusIN SHEEP IN KRISHNA DISTRICT, ANDHRA PRADESH.
Loading...
Date
2016-12
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI – 517 502. (A.P) INDIA
Abstract
ABSTRACT:
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.
Description
THESES
Keywords
null