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Authors: Girish, Haris
Advisor: Rahumathulla, P.S.
Sivaselvam, S.N.
Kumarasamy, P
Raj, G. Dhinakar
Title: Toll-Like Receptor (TLR) Gene Polymorphism And Its Relationship With Somatic Cell Count In Cattle
Publisher: Tamil Nadu Veterinary and Animal Sciences University
Language: en
Type: Thesis
Abstract: Mastitis, an inflammatory disease of the mammary gland generally caused by intramammary infections, is the most frequently occurring disease in dairy industry worldwide. Mastitis is a major source of economic loss in dairy farms. During intramammary infections, cells of the innate immune system become activated through pattern recognition receptors that recognize the conserved molecular signatures associated with the invading pathogens. In this study, the bovine TLR2 and TLR4 genes were taken as candidate genes for mastitis resistance. The coding sequence of TLR2 gene is divided into two regions, exon 1 and exon 2. We amplified exon 1 with one primer pair and the exon 2 with six sets of primer pairs by PCR. Whereas TLR4 had three exons and was amplified by nine sets of primer pairs one for exon 1, one for exon 2 and the rest for exon 3. All these amplicons were screened for SNPs through DNA sequencing, which detected 24 mutations in TLR2 gene, averaging to one SNP per 109 bp of coding sequence and 11 mutations in TLR4 gene. Genotyping was carried out for 18 SNPs out of 22, of the TLR2 gene. PCR- RFLP and Tetra-primer ARMS-PCR were used for genotyping. All the SNPs in TLR2 excepting for one SNP (199C>T) were in coding regions, while the locations of SNPs in TLR4 gene were one in 5’UTR region, two in intron 1, one in exon 2 and the rest in exon3. Out of these 35 mutations, eight were non-synonymous and 25 were synonymous mutations. These mutations substituted the aminoacids at positions 63, 68, 211, 326, 337, 605, 665 in the mature protein of TLR2 and at 674th position in TLR4 mature protein. All these aminoacids are positioned in functionally critical regions of the protein and may potentially alter specificity of pathogen recognition or efficiency of signaling. Altogether 221 dairy cattle (Jersey crossbred, Holstein Friesian crossbred and Indian native breeds) were genotyped and allele frequencies determined. Somatic cell count (SCC) was estimated and log transformed to SCS. Selection for lower SCS is consistent with the goal of maximizing genetic improvement for total economic merit and is being included in breeding programs. The SCC values (x10 5 /ml) for normal samples ranged from 1.53 to 2.85 with the mean of 2.31±0.10 and for mastitic samples it ranged from 26.35 to 179, with the mean value being 68.85 ± 3.9. The milk samples were subjected to California Mastitis Test (CMT) and bacteriological analysis. The CMT being a rapid cowside test whose values are in correlation with SCC and the normal samples showed negative to trace and mastitis samples showed 2+ or 3+ depending upon the severity. The most common pathogens found in the samples were, Staphylococcus. sp., E.coli, Streptococcus sp., Klebsiella sp., and Pseudomonas sp. While some milk samples showed mixed infection. The most prevalent organism was Staphylococcus sp. followed by E.coli. The organism which caused highest (114.57±4.2 x10 5 /ml) SCS was E.coli. The effects of TLR2 and TLR4 polymorphisms were analysed using least- squares analysis and a significant association was (P<0.01) found between the SNP 10095G>T, and SCS. However, the genetic group (breed) did not show any variation in the SCS. Among the three genotypes in 10095G>T, ‘GG’ and ‘GT’ genotypes were found to have lower mean SCS than the ‘TT’ genotypes. The results in this research will be useful in further studies to determine the role of TLR2 and TLR4 genes in mastitis resistance and further work is necessary to investigate whether the TLR2 and TLR4 genes play a role in defending the host from intramammary infections.
Issue Date: 2013
Appears in Collections:Theses & Dissertations

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