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Mastitis is one of the most important disease occurring among cross-bred cows, which is one of the important cause of
declining milk production. The candidate gene approach may provide a more direct understanding of the genetic basis
for the expression of quantitative differences between individuals and revealing genomic regions and specific markers that are
associated with mastitis traits. Although various reports are available that cytokine genes like TLR4, TLR2 etc. are strongly
associated with mastitis occurring among cattle but there is scanty of information related to non cytokine genes and their
association with bovine mastitis. In the present study, A calcium channel, voltage-dependent, alpha-2/delta subunit 1 (CANA2D1)
gene was considered to be an important non cytokine candidate gene influencing mastitis, because the cattle CACNA2D1 gene
has been mapped to BTA 4q 18 and located within the genomic region of QTL for SCS and near by SCC. Primers were designed
from the flanking regions of the SNP (G519663A) of bovine CACNA2D1 gene. The amplified products were digested with HpaII
restriction enzyme. PCR RFLP products were analyzed in 2.5% agarose gel. Further SSCP was also conducted for analysis of each
genotype. We have also sequenced the PCR products of each genotype and the accession numbers were obtained (JX524782 for
GG genotype and JX524783 for AA genotype). Genotype frequency of AA (0.51) was significantly higher than AG (0.34) and
GG (0.15). The effects of CACNA2D1 polymorphism on somatic cell score (SCS) were analyzed. Our result shows that AG (3.76
?0.56) and AA (3.21?0.47) genotypes were associated with higher SCC compared to GG (2.64?0.33). We have also screened
some clinical mastitis case and in all the cases were associated with AA and AG genotypes, no GG genotypes were identified.
On the basis of SCC association with genotypes come to the conclusion that animals with GG genotypes are resistant to mastitis
whereas AA and AG are susceptible to the disease. Further on the basis of genotypes we have classified the animals and the milk
samples were collected and mRNA expression was analyzed by Real Time PCR in each group. Our results revealed that GG
genotypes were significantly showing higher expression than AG and AA genotypes. Further we have isolated blood sample from
the all groups and (Peripheral blood mononuclear cells) PBMCs cells were cultured from each blood sample as per the standard
protocol. They were treated with Calcium channel blocker and the expression level of the CACNA2D1 gene was evaluated by
Real Time PCR. Results show that expression level decline in each genotypic group after treatment and expression level of GG are
again significantly higher than AA and AG.
Rajib Deb has completed his post graduation and PhD from Indian Veterinary Research Institute, India. Presently he is a Scientist at Project
Directorate on Cattle, Indian Council of Agricultural Research, India. He has published few research papers, review papers in reputed international
and national journals. He is also published few books and monographs. Presently he is handling two research projects funded by ICAR. He is also
serving as an editorial board member of Journal of Veterinary Science & Technology under OMICS Publishing Group.
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