Differential expression of immune response genes associated with subclinical mastitis in dairy buffaloes

Buffalo milk production has become of significant importance on the world scale, however, there are few studies involving biotechnological tools specifically for buffalo. To verify the effects caused by subclinical mastitis on the components of milk and to study the innate immune system in the udder of dairy buffaloes with subclinical mastitis, we evaluated the levels of expression of the lactoferrin (LTF), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-8 (IL-8), and toll-like receptors 2 (TLR-2) and 4 (TLR-4) genes in buffaloes with and without subclinical mastitis. Milk samples were collected for the determination of milk components: somatic cell score (SCS), fat, protein, lactose, total solids and solids-not-fat (SNF), as well as for RNA extraction of milk cells, complementary DNA synthesis, and expression profile quantification by quantitative real-time PCR. For gene expression, the ΔΔCt was estimated using contrasts of the target genes expression adjusted for the expression of the housekeeping genes between both groups. Linear regression analysis was performed to determine the relationship between the genes studied and the milk components. Subclinical mastitis induced changes in the fat, lactose and SNF in milk of buffaloes, and the messenger RNA abundance was upregulated for TLR-2, TLR-4, TNF-α, IL-1β and IL-8 genes in milk cells of buffaloes with subclinical mastitis, whereas the LTF gene was not differentially expressed. Results of linear regression analysis showed that TLR-2 gene expression most explains the variation in SCS, and the change in a unit of ΔCt of the TNF-α gene would result in a higher increase in SCS. The study of these immune function genes that are active in the mammary gland is important to characterize the action mechanism of the innate immunity that occurs in subclinical mastitis in dairy buffaloes and may aid the development of strategies to preserve the health of the udder.     

Polymorphisms of Mitochondrial ATPASE 8/6 Genes and Association with Milk Production Traits in Holstein Cows

The maternal effect has been widely proposed to affect the production traits in domestic animals. However, the sequence polymorphisms of mitochondrial DNA (mtDNA) and association with milk production traits in Holstein cows have remained unclear. In this study, we investigated the single nucleotide polymorphisms (SNPs) of mtDNA ATPase 8/6 genes and association with four milk production traits of interest in 303 Holstein cows. A total of 18 SNPs were detected among the 842 bp fragment of ATPase 8/6 genes, which determined six haplotypes of B. taurus (H1-H4) and B. indicus (H5-H6). The mixed model analysis revealed that there was significant association between haplotype and 305-day milk yield (MY). The highest MY was observed in haplotype H4. However, we did not detect statistically significant differences among haplotypes for the traits of milk fat (MF), milk protein (MP), and somatic cell count (SC). The overall haplotype diversity and nucleotide diversity of ATPase 8/6 genes were 0.563 ± 0.030 and 0.00609 ± 0.00043, respectively. The results suggested that mitochondrial ATPase 8/6 genes could be potentially used as molecular marker to genetically improve milk production in Holstein cows.     

Seasonal variation in in vitro immune activity of milk leukocytes in elite and non-elite crossbred cows of Indian sub-tropical semi-arid climate

Immunity of mammary gland in terms of in vitro activity of milk leukocytes has been evaluated during hot-humid, summer, and winter season in elite (n = 10) and non-elite (n = 10) crossbred cows. Milk samples were collected from all the cows throughout the year at 15-day interval. Milk somatic cell counts (SCC) and differential leukocyte counts (DLC) were evaluated microscopically. Milk neutrophils, macrophages, and lymphocytes were isolated and cultured in vitro. In vitro PI of milk neutrophils and macrophages was evaluated by colorimetric NBT (nitro-blue tetrazolium) reductive assay. Mitogen-induced milk lymphocyte blastogenic response was measured by colorimetric MTT (thiazolyl blue tetrazolium bromide) assay. Milk SCC was found to be significantly (p < 0.01) higher in elite cows compared to non-elite cows irrespective of season. There was significant (p < 0.05) increase in milk SCC during hot-humid season compared to winter season in both the group of the cows. There was no significant difference between group and season in terms of DLC. In vitro phagocytic index of elite cows was significantly (p < 0.01) higher than non-elite cows. The phagocytic index was significantly (p < 0.01) decreased in summer and hot-humid season compared to winter season in both the group of animals. Macrophages isolated from elite cows having significantly (p < 0.01) lower phagocytic index than non-elite cows which significantly (p < 0.01) decreased during summer and hot-humid season compared to winter. In vitro milk lymphocyte proliferative response was significantly (p < 0.01) lower in elite cows. Activity of B-lymphocytes decreased significantly (p < 0.01) during summer and hot-humid season than winter, but activity of T-lymphocytes remains unaltered during different seasons. In conclusion, the mammary immunity in terms of in vitro activity of milk leukocytes is compromised during summer and hot-humid season in elite crossbred cows; therefore, better care and management should be taken in high-yielding cows during summer and hot-humid season to minimize intramammary infections.     

Incidence of Subclinical Mastitis and Prevalence of Major Mastitis Pathogens in Organized Farms and Unorganized Sectors

Subclinical mastitis (SCM) represents a major proportion of the burden of mastitis. Determining somatic cell count (SCC) and electrical conductivity (EC) of milk are useful approaches to detect SCM. In order to correlate grades of SCM with the load of five major mastitis pathogens, 246 milk samples from a handful of organized and unorganized sectors were screened. SCC (>5 × 10⁵/mL) and EC (>6.5 mS/cm) identified 110 (45 %) and 153 (62 %) samples, respectively, to be from SCM cases. Randomly selected SCM-negative samples as well as 186 samples positive by either SCC or EC were then evaluated for isolation of five major mastitis-associated bacteria. Of the 323 isolates obtained, 95 each were S. aureus and coagulase-negative staphylococci (CoNS), 48 were E. coli and 85 were streptococci. There was no association between the distribution of organisms and (a) the different groups of SCC, or (b) organised farms and unorganised sectors. By contrast, there was a significant difference in the distribution of CoNS, and not other species, between organized farms and unorganized sectors. In summary, bacteria were isolated irrespective of the density of somatic cells or the type of farm setting, and the frequency of isolation of CoNS was higher with organized farms. These results suggest the requirement for fine tuning SCC and EC limits and the higher probability for CoNS to be associated with SCM in organized diary sectors, and have implications for the identification, management and control of mastitis in India.     

Infradian rhythmicity in milk leukocyte activity together with plasma cortisol and prolactin levels throughout the lactation period in high-yielding crossbred cows

Infradian rhythmicity in milk leukocyte activity together with plasma cortisol and prolactin levels throughout the lactation period in high-yielding crossbred cows has been studied in 10 high-yielding (milk production: 5000 l per lactation) Karan Fries crossbred (Holstein Friesian × Tharparkar) cows. Milk and blood samples were collected from all the experimental animals. Isolation of milk phagocytes (neutrophils and macrophages) and lymphocytes were done by density gradient centrifugation. In vitro phagocytic index of milk neutrophils and macrophages was performed by colorimetric NBT reductive assay. Mitogen-induced milk lymphocyte blastogenic response was estimated by colorimetric MTT (tetrazolium) assay. Total plasma cortisol and prolactin were estimated by enzyme immune assay. Highest value of plasma cortisol and prolactin was observed at calving which decreased significantly (p < 0.01) on 15th day postpartum for both prolactin and cortisol. Immune activity of milk leukocytes was highest on day 0 colostrum and decreased significantly (p < 0.01) on 7th day postpartum. A significant (p < 0.01) rise of plasma prolactin was observed around 135th and 225th days postpartum, whereas a peak level of plasma cortisol was observed at 105th, 180^th, and 270th days postpartum. Phagocytic index of milk neutrophils and macrophages remains almost in a steady state during mid-lactation period (between 100 and 200 days postpartum). A decline in increasing trend of milk phagocytic activity was observed during late lactation. Mitogen-induced milk lymphocyte blastogenic response was highest on day 0 colostrum which decreased significantly (p < 0.01) on 15th day postpartum. Con A-induced milk lymphocyte blastogenic response showed an increasing trend from 120th to 210th days postpartum. Upon correlation study, it showed that the plasma cortisol has a negative effect on milk leukocyte activity, while prolactin has a positive effect, though the effect is lactation stage specific.     

Alteration in the in vitro activity of milk leukocytes during different parity in high yielding cross-bred cows

In vitro activity of milk leukocytes (viz. neutrophils, lymphocytes and macrophages) was evaluated in forty-eight (48) clinically healthy high-yielding cross-bred cows of mid-lactation stage (100–200 days of lactation), divided into four groups namely 1st parity (n = 12), 2nd parity (n = 12), 3rd parity (n = 12) and 4th and above parity (n = 12). Milk samples were taken (250 ml/cow) were taken. Milk somatic cell counts (SCC) and differential leukocyte counts (DLC) were performed microscopically. In vitro phagocytic index (PI) of milk neutrophils and macrophages was evaluated by colorimetric nitro blue tetrazolium reductive assay. Mitogen-induced milk lymphocyte blastogenic response was measured by colorimetric MTT (tetrazolium) assay after isolation of the milk leukocytes by density gradient centrifugation. Milk SCC differed significantly (p < 0.01) between different parity. Cows of 4 and above parity showed significantly (p < 0.01) higher milk SCC compared to primiparous cows. There was no significant difference in milk DLC during different parities in high-yielding cross-bred cows. There was a significant (p < 0.01) variation in lymphocyte blastogenesis amongst parity. The highest value of lymphocyte blastogenesis was seen at 3rd parity, whereas lowest value was obtained in the cows of both 1st and 4th or above parity. PI of milk neutrophils did not differ significantly between parity. PI of milk macrophages was significantly (p < 0.01) higher in 3rd parity and lower (p < 0.01) in 1st and 4th parities. The study indicated that depressed activity of milk lymphocytes and macropages was lower and SCC was higher in the cows of 4th and above parity indicating more mammary stress and hence susceptible to udder infection and mastitis. Therefore, better care and managemental interventions should be taken around these periods.     

Colostrum and milk selenium,antioxidative capacity and immune status of dairy cows fed sodium selenite or selenium yeast

Dietary selenium (Se) can be supplemented from organic or inorganic sources and this may affect Se metabolism and functional outcome such as antioxidative status and immune functions in dairy cows. A feeding trial was performed with 16 Holstein-Friesian dairy cows fed with a total mixed ration (0.18 mg Se/kg dry matter (DM)) either without Se supplement (Control, n = 5), or with Se from sodium selenite (Group SeS, n = 5) or Se yeast (Group SeY, n = 6). In Groups SeS and SeY, the Se supplementation amounted to an additional intake of 4 mg Se and 6 mg Se/d during gestation and lactation, respectively. The effect of both Se sources was characterised by milk Se and antioxidant levels, and the phenotyping and functional assessment of phagocytic activity of milk immune cells. Se yeast has been found to increase (p ≤ 0.001) the milk Se and antioxidant levels markedly compared to the control group. The experimental treatment did not affect the immune parameters of the cows. Lymphocyte subpopulations and phagocytosis activity of neutrophilic granulocytes were affected neither by the Se intake nor by the two different dietary supplements. It can be concluded that sodium selenite and Se yeast differ considerably in their effects on antioxidant status in dairy cows. However, the basal dietary Se concentration of 0.18 mg/kg DM seemed to be high enough for the measured immune variables.     

Differential expression of genes during mastitis in Holstein-Zebu crossbreed dairy cows

Among the potential public health problems of animal production, infectious-contagious diseases stand out. Mastitis is among the main diseases affecting dairy cattle. One of the most promising options to reduce the problems caused by this disease, besides proper sanitary and management practices, is selective breeding of resistant animals. To shed light on the immune response mechanisms involved in the resistance/susceptibility phenotype to this disease, we quantified the relative expression of the genes IL-2, IL-6, IL-8, IL-12, IFN-γ, TNF-α, TLR-2, SEMA5A, and FEZL in cells of crossbreed dairy cows, divided into two groups, one healthy and the other suffering from clinical mastitis. Total RNA was extracted from the cells in the milk from the animals in each group (with and without clinical mastitis). Gene expression was determined using the real-time PCR method. The levels of gene expression were compared, and the cows with mastitis were found to express 2.5 times more TLR-2 than those free of mastitis (P < 0.05). There were no significant differences in the expression of the other genes.     

Antimicrobial resistance and toxin gene profiles of Staphylococcus aureus strains from Holstein milk

Isolation of Staphylococcus aureus (Staph. aureus) from Holstein milk samples with mastitis and nonmastitis was conducted to estimate its prevalence, antimicrobial resistance and toxin genes. A total of 353 milk samples were collected from three Chinese Holstein herds. Fifty‐three Staph. aureus isolates collected from 29 Staph. aureus‐positive samples were characterized via antimicrobial susceptibility, toxin genes and Pulsed‐field Gel Electrophoresis (PFGE) profiles. The prevalence of Staph. aureus was 4·0–9·5% in mastitic and 7·3–11·5% in nonmastitic samples in the analysed herds. Approximately 61·0% of Staph. aureus strains isolated from mastitis cows were resistant to ≥10 antimicrobials compared with 0% of isolates with nonmastitis. The most frequently observed super antigenic toxin gene was pvl (41·5%) followed by seh + pvl (13·2%). We did not find mecA‐positive methicillin‐resistant Staph. aureus (MRSA) strains, while mecA‐negative MRSA strains were identified in the three herds. PFGE results suggested potential transmission of Staph. aureus strains in different farms. These results open new insights into Staph. aureus transmission and antimicrobial resistance of Holstein dairy cows and into developing strategies for udder health improvement of dairy cattle.     

Alterations in the relative abundance of Haptoglobin (Hp) transcripts in total milk somatic cells during different stages of lactation cycle in high yielding cross-bred cows

The expression profile of Haptoglobin (Hp) gene in total milk somatic cells (SCC) of high-yielding cross-bred Karan Fries (KF) was studied during early, mid, and late lactation cycle. Milk samples (200 ml/animals) were collected from 10 high-yielding and 10 low-yielding cows throughout the lactation cycle (from day 7 to day 300) with an interval of one month. Relative mRNA expression profiles of Hp by RT polymerase chain reaction was studied in high-yielding cows, whereas low-yielding cows were taken as control. The folds of induction of Hp was significantly (p < 0.001) downregulated by a mean factor of 0.207 in milk SCC during early lactating cows. Whereas, it was significantly (p < 0.01) upregulated by a mean factor of 20.888 during mid lactation. The expression was unaltered during the late lactation. The study demonstrates that Hp is synthesized within the mammary gland and significantly upregulated during mid-lactation period compared to other stages of lactation cycle.     

BmMD-2A responds to 20-hydroxyecdysone and regulates Bombyx mori silkworm innate immunity in larva-to-pupa metamorphosis

20E-hydroxyecdysone (20E) plays important roles in larval molting and metamorphosis in insects and is also involved in the insect innate immune response. Insect metamorphosis is a highly successful strategy for environmental adaptation and is the most vulnerable stage during which the insect is susceptible to various pathogens. 20E regulates a series of antimicrobial peptides (AMPs) through the immunodeficiency (IMD) pathway activation in Drosophila; nevertheless, whether other immune pathways are involved in 20E-regulated insect immunity is unknown. Our previous studies showed that BmMD-2A is a member of the MD-2-related lipid recognition (ML) family of proteins that are involved in the Bombyx mori innate immunity Toll signaling pathway. In this study, we further demonstrate that BmMD-2A is also positively regulated by 20E, and the BmMD-2A neutralization experiment suggested that 20E activates some downstream immune effect factors, the AMP genes against Escherichia coli and Staphylococcus aureus, through the regulation of BmMD-2A in larval metamorphosis, implying that B. mori may use the Toll-ML signaling pathway to maintain innate immune balance in the larval-pupal metamorphosis stage, which is a different innate immunity pathway regulated by 20E compared to the IMD pathway in Drosophila.     

Pathogenic bacteria prime the induction of Toll-like receptor signalling in human colonic cells by the Gal/GalNAc lectin Carbohydrate Recognition Domain of Entamoeba histolytica

In mixed intestinal infections with Entamoeba histolytica trophozoites and enteropathogenic bacteria, which are wide-spread in areas of endemic amoebiasis, interaction between the pathogens could be an important factor in the occurrence of invasive disease. It has been reported that exposure of human colonic cells to enteropathogenic bacteria increased trophozoite adherence to the cells and their subsequent damage. We report here that the Carbohydrate Recognition Domain (CRD) of the amoebic Gal/GalNAc lectin binds to Toll-like receptors TLR-2 and TLR-4 in human colonic cells, activating the “classic” signalling pathway of these receptors. Activation induced expression of TLR-2 and TLR-4 mRNAs and the mRNAs of pro-inflammatory cytokines, as well as an increase in the corresponding proteins. Direct correlation was observed between the increased expression of TLRs and pro-inflammatory cytokines, the enhanced adhesion of trophozoites to the cells and the inflicted cell damage. When cells were exposed to pathogenic bacteria Staphylococcus aureus (Gram+) or Shigella dysenteriae (Gram−), elements of an innate immune response were induced. CRD by itself elicited a similar cell response, while exposure to a commensal Escherichia coli had a null effect. Pre-exposure of the cells to pathogenic bacteria and then to CRD rendered an inflammatory-like microenvironment that after addition of trophozoites facilitated greater cell destruction. Our results suggest that CRD is recognised by human colonic cells as a pathogen-associated-molecular-pattern-like molecule and as such can induce the expression of elements of an innate immune response. In the human host, an exacerbated inflammatory environment, derived from pathogen interplay, may be an important factor for development of invasive disease.     

Effect of palmitic acid on the mitigation of milk fat depression syndrome caused by trans-10, cis-12-conjugated linoleic acid in grazing dairy cows

The objective of the study was to evaluate the effect of adding protected palmitic acid (PA) to the ration of grazing dairy cows supplemented with protected conjugated linoleic acid (CLA) on milk production, chemical composition and fat profile. Six cows were used, 3/4 American Swiss × Zebu, under a rotational grazing system in a mixed sward with Cynodon plectostachyus, Brachiaria decumbens and Brachiaria brizantha. Furthermore, each cow received daily 4 kg concentrates and 8 kg sorghum silage, which made up the basal diet. The cows were distributed into three two-cow groups. Three treatments were randomly assigned to the groups, using a cross design: (1) control (basal diet), (2) basal diet + CLA (50 g/d) and (3) basal diet + CLA (50 g/d) + PA (412 g/d). The following variables were evaluated: forage intake, milk production, protein, fat and lactose concentration in milk, and milk fatty acid (FA) profile. There were no differences in forage intake between treatments; however, there were differences in milk production, protein, fat and lactose yield and fat concentration, which increased significantly in group CLA + PA when compared with group CLA. The concentration of FA synthesised de novo was lower when PA was included in the diet. Adding PA to the diet of grazing cows mitigates the milk fat decline caused by including trans-10, cis-12 CLA in the diet.     

Expression profile of genes associated with mastitis in dairy cattle

In order to characterize the expression of genes associated with immune response mechanisms to mastitis, we quantified the relative expression of the IL-2, IL-4, IL-6, IL-8, IL-10, IFN-γ and TNF- α genes in milk cells of healthy cows and cows with clinical mastitis. Total RNA was extracted from milk cells of six Black and White Holstein (BW) cows and six Gyr cows, including three animals with and three without mastitis per breed. Gene expression was analyzed by real-time PCR. IL-10 gene expression was higher in the group of BW and Gyr cows with mastitis compared to animals free of infection from both breeds (p < 0.05). It was also higher in BW Holstein animals with clinical mastitis (p < 0.001), but it was not significant when Gyr cows with and without mastitis were compared (0.05 < p < 0.10). Among healthy cows, BW Holstein animals tended to present a higher expression of all genes studied, with a significant difference for the IL-2 and IFN- γ genes (p < 0.001). For animals with mastitis no significant difference in gene expression was observed between the two breeds. These findings suggest that animals with mastitis develop a preferentially cell-mediated immune response. Further studies including larger samples are necessary to better characterize the gene expression profile in cows with mastitis.     

Differential Effect of Lactobacillus johnsonii BFE 6128 on Expression of Genes Related to TLR Pathways and Innate Immunity in Intestinal Epithelial Cells

Probiotics have been shown to enhance immune defenses, but their mechanisms of action are only partially understood. We investigated the modulation of signal pathways involved in innate immunity in enterocytes by Lactobacillus johnsonii BFE 6128 isolated from ‘Kule naoto’, a Maasai traditional fermented milk product. This lactobacillus sensitized HT29 intestinal epithelial cells toward recognition of Salmonella enterica serovar Typhimurium by increasing the IL-8 levels released after challenge with this pathogen and by differentially modulating genes related to toll-like receptor (TLR) pathways and innate immunity. Thus, the modulation of pro-inflammatory mediators and TLR-pathway-related molecules may be an important mechanism contributing to the potential stimulation of innate immunity by lactobacilli at the intestinal epithelial level.     

Milk microbiome signatures of subclinical mastitis-affected cattle analysed by shotgun sequencing

Aims: Metagenomic analysis of milk samples collected from Kankrej, Gir (Bos indicus) and crossbred (Bos taurus × B. indicus) cattle harbouring subclinical mastitis was carried out by next‐generation sequencing 454 GS‐FLX technology to elucidate the microbial community structure of cattle milk. Methods and Results: Milk samples from Kankrej, Gir and crossbred cattle were subjected to metagenomic profiling by pyrosequencing. The Metagenomic analysis produced 63·07, 11·09 and 7·87 million base pairs (Mb) of sequence data, assembled in 264 798, 56 114 and 36 762 sequences with an average read length of 238, 197 and 214 nucleotides in Kankrej, Gir and crossbred cattle, respectively. Phylogenetic and metabolic profiles by the web‐based tool MG‐RAST revealed that the members of Enterobacteriales were predominant in mastitic milk followed by Pseudomonadales, Bacillales and Lactobacillales. Around 56 different species with varying abundance were detected in the subclinically infected milk. Escherichia coli was found to be the most predominant species in Kankrej and Gir cattle followed by Pseudomonas aeruginosa, Pseudomonas mendocina, Shigella flexneri and Bacillus cereus. In crossbred cattle, Staphylococcus aureus followed by Klebsiella pneumoniae, Staphylococcus epidermidis and E. coli were detected in descending order. Metabolic profiling indicated fluoroquinolones, methicillin, copper, cobalt–zinc–cadmium as the groups of antibiotics and toxic compounds to which the organisms showed resistance. Sequences indicating potential of organisms exhibiting multidrug resistance against antibiotics and resistance to toxic compounds were also present. Interestingly, presence of bacteriophages against Staph. aureus, E. coli, Enterobacter and Yersinia species was also observed. Conclusions: The analysis identified potential infectious organisms in mastitis, resistance of organisms to antibiotics and chemical compounds and the natural resistance potential of dairy cows. Significance and Impact of the Study: The findings of this study may help in formulating strategies for the prevention and treatment of mastitis in dairy animals and consequently in reducing economic losses incurred because of it.     

Effects of different doses of free 𝛂-linolenic acid infused to the duodenum on the immune function of lactating dairy cows

This study investigated the effect of a duodenal infusion of a C18:3 free fatty acid on the immune function of lactating dairy cows. Four primiparous Chinese Holstein cows fitted with duodenal cannulas received 0, 100, 200, 300, and 400 g/d of α-linolenic acid (LNA) in a two-treatment crossover design. Blood was collected and serum IgA, IgG, IgM, prostaglandin E₂ (PGE₂) and Th1/Th2 cytokines were determined. Results showed that increasing the supply of LNA to the small intestine of dairy cows linearly increased serum IgG and quadratically enhanced interferon-γ (p < 0.05), whereas the concentrations of PGE₂ declined linearly (p < 0.05) and those of interleukin (IL)-4 tended to decrease (p = 0.08). No difference was observed in serum IgA, IgM or other cytokines, such as IL-2, IL-6 and IL-10. This study demonstrated that in dairy cows, a post-ruminal infusion of high doses of LNA has immunomodulatory effects, possibly associated with a predisposition to a Th1-type response.