HAPTOGLOBIN GENE EXPRESSION IN SPONTANEOUS BOVINE SUBCLINICAL MASTITIS CAUSED BY STAPHYLOCOCCUS AND COLIFORMS MICROBES

Pro-inflammatory cytokines arbitrate the synthesis of haptoglobin (Hp), which contributes towards reinstatement of homeostasis and restriction of microbial growth in case of mastitis in the host. It has also immense diagnostic significance. Host-pathogen interaction with respect to Hp gene expression has been amply studied in case of induced mastitis in bovines. There is scanty knowledge about their expression and kinetics in natural infection. The present study depicts the cytokine (TNF-a, IL-6) and Hp gene expression and their interaction with pathogens, in naturally infected cases of bovine subclinical mastitis (SCM). The milk microbiome revealed that 22 samples (68.75%) out of 32, were positive for Staphylococcus spp. (gram positive microorganisms), out of which 18 (81.8%) were positive for S. aureus, which was confirmed by PCR amplification using primers, specific for nuc gene (270 bp) of S. aureus. The rest 10 (31.25%) samples tested positive for coliforms (gram-negative microorganisms), of which 4 (40%) were positive for Escherichia coli, and was confirmed by PCR amplification using primers, specific for Alr gene (336 bp) of E. coli. Total RNA isolated from the somatic cells (Leukocytes) of culture-positive milk samples, revealed two distinct bands corresponding to 28S and 18S ribosomal RNA. The expressions of Actin (Positive control), TNF-a, IL-6, and Hp genes were studied using RT-PCR. The expression of Actin gene corresponded to the expected size (228bp) in S. aureus and E. coli. There was no significant expression of TNF-a gene in both the microorganisms. The expression of IL-6 gene was evident, and the expected product size corresponded to 304 bp in both the microorganisms. The product size of Hp gene corresponded to the expected size of 174 bp in case of S. aureus as well as E. coli. The semi-quantified level of expression of IL-6 gene and Hp gene in terms of arbitrary units revealed that the expression of both the genes was significantly (P0.001) higher in case of S. aureus than in E. coli, which could serve as a reference point in the diagnosis of bovine sub clinical mastitis. It is inferred that IL-6 arbitrated in the induction and expression of Hp gene in bovines.