Physical and chemical characteristics of yoghurt produced from whole milk with different levels of somatic cell counts.

For yoghurts were made from milk with different levels of somatic cell count (SCC) (low 95,000 cells ml(- 1), intermediate 398,000 cells ml(- 1) and high 1,150,000 cells ml(- 1)). Yoghurt samples were analysed for the degree of proteolysis, lipolysis (free fatty acid (FFA) content), acidity, pH and apparent viscosity on days 1, 14 and 28. The SCC had no significant effect (p>0.05) on either the acidity or the pH of the yoghurt after 1 day of cold storage. However, significant effects (p < 0.05) of SCC were observed after 14 and 28 days of storage. Yoghurt samples made from intermediate and high SCC milk showed higher viscosity (p < 0.05) and lower (p < 0.05) casein content on days 14 and 28 of cold storage than yoghurt made from low SCC milk. High FFA concentrations (p < 0.05) were observed only in yoghurt made from high SCC milk. High SCC in milk increased both proteolysis and lipolysis in yoghurt during storage.

A sentinel function for teat tissues in dairy cows: dominant innate immune response elements define early response to E. coli mastitis.

Escherichia coli intramammary infection elicits localized and systemic responses, some of which have been characterized in mammary secretory tissue. Our objective was to characterize gene expression patterns that become activated in different regions of the mammary gland during the acute phase of experimentally induced E. coli mastitis. Tissues evaluated were from Fürstenburg's rosette, teat cistern (TC), gland cistern (GC), and lobulo-alveolar (LA) regions of control and infected mammary glands, 12 and 24 h after bacterial (or control) infusions. The main networks activated by E. coli infection pertained to immune and inflammatory response, with marked induction of genes encoding proteins that function in chemotaxis and leukocyte activation and signaling. Genomic response at 12 h post-infection was greatest in tissues of the TC and GC. Only at 24 h post-infection did tissue from the LA region respond, at which time the response was the greatest of all regions. Similar genetic networks were impacted in all regions during early phases of intramammary infection, although regional differences throughout the gland were noted. Data support an important sentinel function for the teat, as these tissues responded rapidly and intensely, with production of cytokines and antimicrobial peptides.

Evaluation of breed-dependent differences in the innate immune responses of Holstein and Jersey cows to Staphylococcus aureus intramammary infection.

Mastitis is one of the most prevalent diseases of cattle. Various studies have reported breed-dependent differences in the risk for developing this disease. Among two major breeds, Jersey cows have been identified as having a lower prevalence of mastitis than Holstein cows. It is well established that the nature of the initial innate immune response to infection influences the ability of the host to clear harmful bacterial pathogens. Whether differences in the innate immune response to intramammary infections explain, in part, the differential prevalence of mastitis in Holstein and Jersey cows remains unknown. The objective of the current study was to evaluate several parameters of the innate immune response of Holstein and Jersey cows to intramammary infection with Staphylococcus aureus, a common mastitis-inducing pathogen. To control for non-breed related factors that could influence these parameters, all cows were of the same parity, in similar stages of milk production, housed and managed under identical conditions, and experimentally infected and sampled in parallel. The following parameters of the innate immune response were evaluated: acute phase protein synthesis of serum amyloid A and lipopolysaccharide-binding protein; total and differential circulating white blood cell counts; milk somatic cell counts; mammary vascular permeability; milk N-acetyl-beta-d-glucosaminidase (NAGase) activity; and production of the cytokines, interferon (IFN)-gamma, interleukin (IL)-12, tumour growth factor(TGF)-alpha, and TGF-beta1. The temporal response of all of these parameters following infection was similar between Holstein and Jersey cows. Further, with the exception of changes in circulating neutrophils and NAGase activity, the overall magnitude of these parameters were also comparable. Together, these data demonstrate that the innate immune response of Holstein and Jersey cows to Staph. aureus intramammary infection remains highly conserved despite previously reported differences in mastitis prevalence, as well as genotypic and phenotypic traits, that exist between the two breeds.

Differential alterations in the ability of bovine neutrophils to generate extracellular and intracellular reactive oxygen species during the periparturient period.

The periparturient period of a dairy cow is associated with increased incidence and/or severity of certain infectious diseases, including mastitis. It is believed that the heightened physiological demands of calving and initiation of milk production contribute to a state of immunosuppression during this period. Previous studies have indicated that neutrophil production of reactive oxygen species (ROS), which is a critical element of the host innate immune response to bacterial infection, is impaired in the 1-2week period following calving. However, whether there is comprehensive inhibition of ROS production or selective inhibition of particular ROS remains unknown. The present study provides evidence that neutrophils isolated from cows (n=20) after calving have an increased capacity to generate intracellular ROS and an impaired ability to release extracellular superoxide anion and hydrogen peroxide.

Bacterial lipopolysaccharide induces increased expression of toll-like receptor (TLR) 4 and downstream TLR signaling molecules in bovine mammary epithelial cells.

Bovine mammary epithelial cells contribute to the innate immune response to intramammary infections by recognizing pathogens through specialized pattern recognition receptors. Toll-like receptor 4 (TLR4) is one such receptor that binds and is activated by lipopolysaccharide (LPS), a component of the outer envelope of Gram-negative bacteria. In this study, MAC-T cells (a bovine mammary epithelial cell line) were incubated in the presence or absence of increasing concentrations of LPS for 24 h. Expression of TLR2 and TLR4 were analyzed at both mRNA and protein levels by quantitative real-time PCR (qPCR) and flow cytometry, respectively. The mRNA of both receptors were up-regulated by all concentrations of LPS used (P<0.01). Similarly, flow cytometry with specific antibodies against TLR2 and TLR4 detected increased surface expression of these proteins. Furthermore, expression of downstream TLR4 signaling molecules was examined by qPCR following varying exposure times to 1 mug/mL of LPS. Results demonstrate that the required adaptor molecules and transcription factors were up-regulated in a time-dependent manner. Both the MyD88 dependent and independent pathways in TLR4 signaling were activated in MAC-T cells. Expression of TOLLIP increased in response to LPS as did the pro-apoptotic protease, CASP8. These results suggest that the bovine mammary epithelium possesses the necessary immune repertoires required to achieve a robust defense against E. coli. The current findings, coupled with previous findings that S. aureus ligands induce up-regulation of TLR4, may indicate a positive adaptation by mammary epithelial cells to effectively respond to different types of mastitis pathogens.

The toll-like receptor-4 (TLR-4) pathway and its possible role in the pathogenesis of Escherichia coli mastitis in dairy cattle.

Mastitis is one of the most costly production diseases in the dairy industry that is caused by a wide array of microorganisms. In this review, we focus on the Gram-negative Escherichia coli infections that often occur at periods when the innate immune defence mechanisms are impaired (i.e., parturition through the first 60 days of lactation). There is substantial evidence demonstrating that at these periods, the expected influx of polymorphonuclear neutrophil leukocytes (PMN) into the mammary gland is delayed during inflammation after intramammary infection with E. coli. Here, we provide some hypotheses on the potential mechanisms of action on how the disease may develop under circumstances of immunosuppression, and describe the potential involvement of the toll-like receptor-4 signal transduction pathway in the pathogenesis of E. coli mastitis. In addition, some ideas are proposed to help prevent E. coli mastitis and potentially other diseases caused by Gram-negative infections in general.

Veterinary field test as screening tool for mastitis and HIV-1 viral load in breastmilk from HIV-infected Zambian women.

Clinical and subclinical mastitis increase the risk of mother-to-child transmission (MTCT) of HIV-1 through breastfeeding. We hypothesized that a field test for mastitis used for bovine milk, the California Mastitis Test, would detect high cell counts in milk of HIV-infected women. We also investigated whether total milk cell count would positively correlate with viral HIV-1 RNA in the milk of 128 HIV-positive Zambian women. Mean cell counts in each California Mastitis Test scoring category were significantly different (p < 0.01, n = 232). In a subset of 4-month postpartum milk samples tested for HIV-1 RNA, viral RNA levels did not significantly correlate with total cell count (r = 0.166, p = .244). The CMT may serve as a screening tool for mastitis in breastmilk, but total cell count does not correlate with HIV-1 RNA levels. Since both cell-free and cell-associated virus are associated with increased risk of MTCT, investigation of the relationship between total milk cell count and HIV-1 proviral DNA is warranted before a conclusive determination is made regarding use of the CMT as a clinical screening tool to detect cases at high risk for breastmilk transmission.

Predicting perchlorate exposure in milk from concentrations in dairy feed.

Perchlorate has been detected in U.S. milk samples from many different states. Applying data from a recently reported 9-week experiment in which 16 Holstein dairy cows were administered perchlorate allowed us to derive an equation for the dose-response relationship between perchlorate concentrations in feed/drinking water and its appearance in milk. Examination of background concentrations of perchlorate in the total mixed ration (TMR) fed in addition to the variable dose supplied to treated cows as a ruminal infusate revealed that cows receive significant and variable exposure to perchlorate from the TMR. Weekly examination of the TMR disclosed that a change in ingredients midway through the experiment caused a significant (78%) change in TMR perchlorate concentration. Analyses of the ingredients comprising the TMR revealed that 41.9% of the perchlorate came from corn silage, 22.9% came from alfalfa hay and 11.7% was supplied by sudan grass. Finally, USDA Food and Nutrition Survey data on fluid milk consumption were used to predict potential human exposure from milk that contained concentrations of perchlorate observed in our previous dosing study. The study suggests that reducing perchlorate concentration in dairy feed may reduce perchlorate concentrations in milk as well as the potential to reduce human exposure to perchlorate in milk.

Bacterial lipopolysaccharide stimulates bovine neutrophil production of TNF-alpha, IL-1beta, IL-12 and IFN-gamma.

After intramammary infection, polymorphonuclear neutrophil leukocytes (PMN) are the first cells recruited into the mammary gland. Rapid recruitment of and bacterial phagocytosis and killing by PMN are the most effective defenses against establishment of bacterial infection. In addition to their phagocytic and bactericidal properties, PMN may play a key supportive role through secretion of cytokines during the innate immune response. We sought to determine whether bovine PMN produce cytokines in response to stimulation by lipopolysaccharide (LPS). To investigate the effects of LPS on the expression of cytokines secreted by bovine PMN, we measured the expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-12, and interferon (IFN)-gamma by ELISA after stimulation with different concentrations of LPS, and secretion of IL-8 after co-stimulation with LPS and either TNF-alpha or IL-1beta. Bovine PMN were shown to secrete TNF-alpha , IL-1beta, IL-12, IL-8 and IFN-gamma in response to LPS. Co-incubation of PMN with LPS and TNF-alpha increased secretion of IL-8 when compared to LPS alone. It was concluded that LPS stimulation up-regulates the secretion of cytokines by bovine PMN, and that co-incubation of LPS with TNF-alpha had an additive effect on the secretion of IL-8. These data show that bovine PMN, in addition to their phagocytic and bactericidal properties, may play a supportive role in the innate immune response to infection by Gram-negative bacteria through their ability to produce immuno-regulating cytokines.

Evaluation of assays for the measurement of bovine neutrophil reactive oxygen species.

During mastitis and other bacterial-mediated diseases of cattle, neutrophils play a critical role in the host innate immune response to infection. Neutrophils are among the earliest leukocytes recruited to the site of infection and contribute to host innate immune defenses through their ability to phagocytose and kill bacteria. The bactericidal activity of neutrophils is mediated, in part, through the generation of reactive oxygen species (ROS). Extracellular release of ROS can induce injury to host tissue as well, and aberrant release of ROS has been implicated in the pathogenesis of certain inflammatory-mediated diseases. Due to their essential role in bacterial clearance and implicated involvement in the pathogenesis of other diseases, there is much interest in the study of neutrophil-generated ROS. Several assays have been developed to measure ROS production, however, many of these have not been evaluated with bovine neutrophils. The objectives of the current study were to evaluate different assays capable of measuring bovine neutrophil ROS, and to compare the results of assays never previously tested with bovine neutrophils to those obtained from more well-established assays frequently used with these cells. Eight different assays were evaluated, including: luminol, isoluminol, and methyl cypridina luciferin analog (MCLA) chemiluminescence assays; Amplex Red, dihydroethidium (DHE), dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), and dihydrorhodamine 123 fluorescence assays; and the cytochrome c absorbance assay. The assays were evaluated in the context of their abilities to detect ROS produced in response to two agonists commonly used to induce neutrophil activation, phorbol 12-myristate, 13-acetate (PMA) and opsonized zymosan. Diphenyleneiodonium chloride, a NADPH oxidase inhibitor, was used to assess the specificity of the assays to detect ROS. The ability of these assays to discriminate between intra- and extracellular ROS and to specifically detect distinct ROS was evaluated using superoxide dismutase and catalase, which scavenge extracellular superoxide and hydrogen peroxide, respectively. With the exception of the DHE assay, all assays detected bovine neutrophil ROS generation elicited by PMA and zymosan. PMA, but not zymosan, was able to stimulate neutrophil generation of ROS at levels that were detectable with DHE. The MCLA chemiluminescence assay was the only assay that detected ROS produced in response to each of the lowest concentrations of PMA and zymosan tested. To our knowledge, this is the first study to evaluate DHE-, MCLA-, Amplex Red-, and isoluminol-based assays for the measurement of bovine neutrophil ROS, and the most comprehensive comparative study of ROS assays under similar experimental conditions.