COMPARISON OF INNATE IMMUNITY RELATED GENES IN VECHUR AND CROSSBRED CATTLE AND THEIR EXPRESSION PROFILE IN CLINICAL AND SUBCLINICAL MASTITIS
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Date
2016-12-30
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR
Abstract
Bovine mastitis is considered as the most economically imposing diseases of
dairy cattle. Vechur cattle an indigenous breed of Kerala are generally not
susceptible to mastitis. Investigation of innate immune mechanism of this breed
might provide an insight into the mechanisms involved in the disease resistance. In
light of this premise, the present study was carried out to investigate the expression
of TLR2, TLR4, and TLR9 in crossbred and Vechur cattle using RT-qPCR, to better
understand the immune resistance mechanisms against mastitis and also
characterized the promoter and mRNA sequence of TLR genes in Vechur cattle.
RT-qPCR analysis showed a significant up fold increase in the TLR2 gene
expression in mastitis caused by S. aureus, whereas expression level of TLR4 mRNA
was relatively higher in E. coli caused samples. In both S. aureus and E. coli caused
mastitis milk samples, relative expression of all three TLRs was found to be
significantly high (P ≤ 0.01) in sub-clinical mastitis than the clinical mastitis. So,
during early stage of mammary infection these TLRs are expressed at high level to
subside the sub-clinical mastitis without precipitating into clinical mastitis.
After the challenging the PBMCs with TLR agonist in vitro, relative
expression of mRNA of all three TLR genes was higher in Vechur cattle than the
crossbred cattle. Furthermore, the expression of TLR2 mRNA was relatively higher
in Vechur breed as compared with other TLRs. These findings suggest that one of the
reasons for the development of resistance to mastitis in Vechur cattle is associated
with the level of expression of TLRs in response to infection.
The sequence of promoter region of TLR2 of Vechur cattle with the Bos
taurus sequence showed 98 per cent similarity whereas TLR4 and TLR9 revealed 99
per cent similarity. TLR2 and TLR9 revealed variations for three sequence motifs.
Significant variants observed for TATA and CAT boxes and multiple putative
binding sites in the promoter region of TLR2 and TLR9 genes in Vechur cattle breed,
may potentially link the influence the innate immunity response against mastitis
diseases.
All three TLR mRNA sequences showed 99 per cent homology with Bos
taurus sequence and exposed variations for 17 nucleotide in TLR2, 7 nucleotide in
TLR4 and 5 nucleotide in TLR9 mRNA. The ectodomain of Vechur cattle displayed
10 LRRs for TLR2, 13 LRRs for TLR4 and 18 LRRs for TLR9. The variation in the
extracellular domain of LRRs, which may promote the recognition of pathogen
ligand specificity. The primary structure of protein showed highest per cent of
leucine amino acid for all three TLRs and alpha helix is the prominent secondary
structure seen in all TLRs followed by beta turn and random coil. Phylogenetic tree
for TLR genes showed all Bovidae family falling under the same group, indicated
conserved nature of TLR genes. The presence of unique structural features and
substantial variation for TLR genes in Vechur cattle, may change the confirmation of
TLR proteins, which may influence the binding affinity and interaction with pathogen
to boost the innate host disease resistance in Vechur cattle.
Description
Submitted in partial fulfillment of the requirement for the degree of
Doctor of Philosophy in Veterinary Biochemistry