Short communication: Locus-specific interrelations between gene expression and DNA methylation patterns in bovine mammary gland infected by coagulase-positive and coagulase-negative staphylococci.

Pathogens are able to alter the cell cycle program and immune response of the host by changing the transcription and epigenetics of genes responsible for cell cycle control and inflammation. In this regard, we evaluated interrelations between DNA methylation and expression of autophagy, apoptosis, and lipid metabolism-related genes in a sample set of mammary gland secretory tissue sections derived from bovine mammary glands infected with coagulase-negative and coagulase-positive staphylococci. We assessed relative transcript abundance and DNA bisulfite sequencing in loci of the ATG5, IGF1R, TERT, and DGAT1 genes. Lack of DNA methylation in ATG5 and DGAT1 loci might be associated with maintenance of ATG5 and DGAT1 expression regardless of the health status of bovine mammary gland. Complete methylation of intragenic CpG regions in the IGF1R locus was apparently not related to the presence of its transcript in the investigated udder parenchyma samples. Detected hypermethylation of the TERT upstream element was associated with a small amount of TERT mRNA in bovine mammary gland, regardless of the presence, or absence, of the pathogen. A significant decrease in TERT gene expression in tissue sections of mammary gland free of bacteria and in those infected with coagulase-positive staphylococci was observed in parenchyma samples infected with coagulase-negative staphylococci. Two possible explanations are the direct involvement of the TERT gene in the etiology of bovine mastitis or the increase of TERT mRNA due to activation of the MAPK signaling pathway in response to release of exotoxins by coagulase-negative bacteria in the bovine mammary gland.

Acute phase protein expressions in secretory and cistern lining epithelium tissues of the dairy cattle mammary gland during chronic mastitis caused by staphylococci.

BACKGROUND: Mastitis is the most common disease in dairy cattle and the costliest for the dairy farming industry, as it lowers milk yield and quality. Mastitis occurs as a result of interactions between microorganisms and the individual genetic predispositions of each animal. Thus, it is important to fully understand the mechanisms underlying these interactions. Elucidating the immune response mechanisms can determine which genetic background makes an animal highly resistant to mastitis. We analyzed the innate immune responses of dairy cows naturally infected with coagulase-positive staphylococci (CoPS; N = 8) or coagulase-negative staphylococci (CoNS; N = 7), causing persistent mastitis (after several failed treatments) vs. infection-free (i.e., healthy [H]; N = 8) dairy cows. The expressions of the acute phase protein genes serum amyloid A3 (SAA3), haptoglobin (HP), ceruloplasmin (CP) genes in the tissues most exposed to pathogens- mammary gland cistern lining epithelial cells (CLECs) and mammary epithelial cells (MECs)-were analyzed. RESULTS: We found constitutive and extrahepatic expressions of the studied genes in both tissue types. HP expression in the MECs of the CoPS-infected group was higher than in the H group (p ≤ 0.05). Moreover, higher SAA3 expression in the CoPS and CoNS groups than in the H group (p = 0.06 and 0.08, respectively) was found. No differences between SAA3 and HP in CLECs were revealed, regardless of the pathogen type. However, higher expression of CP (p ≤ 0.05) in the CoPS group than in the H group was noted. CONCLUSIONS: The expressions of selected acute phase proteins were similar between CLECs and MECs, which means that CLECs are not only a mechanical barrier but are also responsible for the biological immune response. Our findings agree with the results of other authors describing the immunological response of MECs during chronic mastitis, but the results for CLECs are novel.

Structural and functional analysis of the signaling lymphocytic activation molecule family 7 (SLAMF7) gene in response to infection with coagulase-negative and coagulase-positive staphylococci.

Splice variants of the signaling lymphocytic activation molecule family 7 (SLAMF7) gene have been identified, and differences in the expression of this gene have been demonstrated at the mRNA level in the mammary glands of healthy and mastitis-infected dairy cows. At the same time, significant associations have been found between a deletion in the SLAM7 gene exon, the occurrence of different splice variants, and the occurrence of mastitis in one group of dairy cows. An expression study was conducted on 40 Polish Holstein-Friesian dairy cows of the Black and White variety (group I). Milk samples were taken for microbiological analysis 2 d before slaughter and examined for the presence of bacteria. Immediately after slaughter, mammary tissue samples were taken and divided into 3 groups according to the health status of the mammary gland: healthy (without pathogenic bacteria in milk), coagulase-negative staphylococci (CNS), and coagulase-positive staphylococci (CPS). Based on different SLAMF7 gene DNA fragments, 2 alternative variants of this gene (V1 and V2) and complete gene expression were identified. Separate analyses performed for each isoform showed that the health status of the cow was strongly associated with the expression level of individual variants. The highest expression was detected for the SLAMF7 complete amplicon in healthy cows, and in the CNS and CPS cows the expression of this variant was also higher than V1 and V2. Sanger sequencing was applied to detect the polymorphism/indel variant in the second exon of the SLAMF7 gene probably having the greatest effect on the protein structure and function of SLAMF7. Two genotypes were detected: AA (wild-type) and AB (insertion A). In healthy cows, the frequency of homozygotes AA was higher than the heterozygotes, whereas in the infected animals, the genotypic distribution was the opposite. An association analysis between the identified polymorphism and production traits-including somatic cell count, as well as lactose, protein, and casein content and yield as indicators of subclinical mastitis occurrence-was performed on the group II cows (166 Polish Holstein-Friesian dairy cows). Unfortunately, due to the low number of AB animals, no relationship was demonstrated between genotype in the second exon and the health status of cows. Additionally, the difference in the percentage of SLAMF7-targeted DNA methylation between the groups of animals was not significant, with an average of ∼66 to 68%.