Toll-Like Receptor (TLR) Gene Polymorphism And Its Relationship With Somatic Cell Count In Cattle
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Date
2013
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Tamil Nadu Veterinary and Animal Sciences University
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.