Peptidoglycan and Lipoteichoic Acid Induces Differential mRNA Response of Immune-Related Genes in PBMC of Crossbred, Tharparkar Cattle and Murrah Buffalo.

Subclinical mastitis, generally caused by Staphylococcus aureus, has a global economic impact all over the world. Hence, it needs to be resolved on higher priority which may be attained via. selection of mastitis resistant animals or inclusion of mastitis resistant trait into herd apart from management care. Diverse hosts with various genetic make-ups encounter pathogens in a diverse manner which in turn leads to contradicting outcome of the disease. Identification of species-wise or breed-wise differential expressed genes in response to S. aureus through relative evaluation of transcripts may be useful for judging the immuno-competency of a species or breed toward mastitis. The present study was undertaken to examine the stimulant effect of S. aureus peptidoglycan (PGN) and lipoteichoic acid (LTA) on Peripheral blood mononuclear cells (PBMC) harvested from blood samples of crossbred cattle, Tharparkar cattle, and Murrah buffaloes. After 6 h of in vitro stimulation qRT-PCR was used to measure the relative mRNA expression of TLR-2, TNF-α, IL-8, IFN-γ and IL-10 genes in stimulated and un-stimulated PBMC. The selected genes revealed significant differences in the pattern of immune response among crossbred cattle, Tharparkar cattle and Murrah buffalo in spite of the same stimulant dose.

Differential expression of ten candidate genes regulating prostaglandin action in reproductive tissues of buffalo during estrous cycle and pregnancy.

Prostaglandins (PGs) are the key mediators of several female reproductive functions, including luteolysis, ovulation, fertilization, implantation, pregnancy, and parturition. The present study was conducted in buffalo endometrial and luteal tissues between nonpregnant and two stages of pregnancy (29-38 days of pregnancy, 48-56 days of pregnancy) tissue samples. The genes involved from synthesis upto receptor level effect of PGs (PGF2α and PGE2) were studied for their relative mRNA expression. We have collected the endometrial and luteal tissues from slaughtered animals and confirmed the stages by external examination and crown vertebral rump length measurement of the foetus. The mRNA expression of COX-2 and PGFS genes revealed high significant rise in the transcript at pregnancy stage I as compared to the late luteal phase of nonpregnant. However, EP2 and EP3 genes were highly upregulated in pregnancy stage II. The expression of PLA2G4A and PGT genes showed difference in their transcripts in pregnancy, however, the difference was nonsignificant as compared to the nonpregnant stage. The findings emerged from this study also suggested the strict regulation at COX-2 mRNA level than at synthase enzyme's level. Among the four subtypes of EP gene, we have observed highly significant expression difference in EP2 followed by EP3 after implantation.

Study of lysophosphatidic acid receptors (LPARs) in buffalo uterus demonstrated upregulation of LPAR1 and LPAR6 in early pregnancy.

Lysophosphatidic acid (LPA) is an important factor involved in embryo implantation and pregnancy establishment in humans and domestic livestock. LPA exerts its action through six G-protein-coupled receptors (LPA1-LPA6). We investigated the types of LPA receptors expressed in buffalo uterus and also their differential expression in the nonpregnant, and early-pregnant endometrium. The nonpregnant, and early-pregnant (<42 days) uteri were collected from the local slaughterhouse. RT-PCR experiments detected mRNAs of all the six LPA receptors (LPAR1-LPAR6) in both nonpregnant, and early-pregnant endometrial tissues. Their comparative profiling by real-time PCR revealed that the early pregnant endometrium expressed more mRNAs of LPAR1 and LPAR6. All the mRNA fragments were sequenced and submitted to Genbank, NCBI. Western blot studies also showed a similar expression pattern of these two receptor proteins, including higher expression of both LPA1 and LPA6 proteins during early pregnancy. And between these two receptors, LPA6 upregulation was more pronounced than LPA1. In immunohistochemistry, these receptors were found to be localized in the endometrial glandular epithelial cells of both types of uterus. Level of LPA was also higher in early pregnant endometrial tissues. In summary, our study demonstrated expression of all the six LPAR mRNAs in buffalo uterus, wherein the early-pregnant uterus did express comparatively higher mRNA as well as protein of LPA1 and LPA6, indicating their role in pregnancy. The more pronounced expression of LPA6 possibly indicates its greater contribution to mediating LPA signaling in early pregnancy (29-42 days) of buffalo.

Differential response of immune-related genes to peptidoglycan and lipoteichoic acid challenge in vitro.

AIM: To study the effect of Staphylococcus aureus cell wall antigens, peptidoglycan (PGN) and lipoteichoic acid (LTA) challenge on immune cells present in bovine peripheral blood mononuclear cells (PBMCs). MATERIALS AND METHODS: In this study, efforts have been made to investigate the effects of three combinations (10+10, 20+20 and 30+30 µg/ml) of PGN and LTA obtained from S. aureus. These antigens were used to challenge the bovine PBMCs. After 6 h of incubation quantitative, real time-polymerase chain reaction was used to study toll-like receptor 2 (TLR-2) and major cytokine mRNA expression in bovine PBMC challenged with three different antigen blends. RESULTS: The results indicated that mRNA level of interferon gamma is influenced by the expression of TLR-2 gene. Tumor necrosis factor-alpha (TNF-α), interleukin 10 (IL-10), and IL-8 genes showed a maximum response at a dose of 10 µg of PGN and 10 µg of LTA challenge per ml of culture medium. The outcome also suggests that both IL-10 and IL-8 followed the expression pattern of TNF-α. CONCLUSION: A dose of 10 µg of PGN and 10 µg of LTA per ml of culture medium was found to be most suitable for challenging PBMC.

Hepatotoxicity Induced by Subchronic Exposure of Fluoride and Chlorpyrifos in Wistar Rats: Mitigating Effect of Ascorbic Acid.

The aim of the study was to investigate the ameliorative properties of ascorbic acid against the subchronic effect of co-exposure of fluoride (F) and chlorpyrifos (CPF) on oxidative damage markers such as lipid peroxidation (MDA) and antioxidant defense system in the liver of adult Wistar rats. The animal groups were provided with either vehicle or ascorbic acid (60 mg/kg, b.w.) or NOAEL dose of fluoride (1 ppm) or CPF (1 mg/kg, b.w.) or ten times of such doses orally alone and in combination or pre-treated with ascorbic acid along with co-exposure of F and CPF every consecutive day for 28 days. Hepatic damage marker analysis in blood revealed that aspartate and alanine aminotransferases, alkaline phosphatase, and lactate dehydrogenase were significantly (P < 0.05) increased with single or combined exposure of F or CPF at either dose levels. Significant increased oxidative damage of hepatocytes as indicated by increased MDA levels with decrease in tissue ascorbate and free radical scavenging enzymes like catalase, superoxide dismutase, and glutathione peroxidase was observed in groups treated with either F or CPF as well as in combinedly treated animals as compared to control animals. Supplementation of ascorbic acid restored the hepatic specific marker enzymes in blood following co-exposure of F and CPF at lower doses which were otherwise increased in the F and CPF co-exposed rats. The results show that ascorbic acid supplementation with F and CPF prevents or diminishes the hepatic damage in rats co-exposed to toxicants and may act as a putative protective agent against toxicant-induced liver tissue injury.

Bacteriological and Molecular Detection of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus in Equines of Northern India.

Present study was undertaken to study the prevalence of ß-haemolytic streptococci in equine of northern temperate region of Jammu and Kashmir, India. One hundred and forty one samples were collected in duplicate from nasopharyngeal tract of diseased (53) and apparently healthy equine (88) for isolation and direct PCR. A total of 77 isolates of streptococci were recovered from 141 samples with an overall prevalence rate of 54.60%. Out of these 77 isolates, 52 were from diseased and 25 from apparently healthy animals. Of the 77 isolates, 4 were identified as Streptococcus equi subsp. equi, 56 as S. equi subsp. zooepidemicus and 17 as S. dysgalactiae subsp. equisimilis. Thus the overall prevalence of S. equi subsp. equi, S. equi subsp. zooepidemicus and S. dysgalactiae subsp. equisimilis was 2.83, 39.71 and 12.05% respectively. The sensitivity of the PCR for the detection of S. equi species was found higher when attempted from direct swab samples.