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.

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.

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.

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.

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.

Shedding of sCD14 by bovine neutrophils following activation with bacterial lipopolysaccharide results in down-regulation of IL-8.

CD14, the leukocyte co-receptor for lipopolysaccharide (LPS), is important in the response of bovine polymorphonuclear neutrophil leukocytes (PMN) to Gram-negative bacteria. In other species, the expression of CD14 on the surface of PMN was shown to increase after exposure to inflammatory stimuli. These newly expressed molecules may originate from either an intracellular pool or through new gene expression. We sought to characterize bovine PMN cell surface expression and shedding of CD14 molecules, and CD14's effect on secretion of the chemoattractants IL-8 and IL-1beta by PMN. Bovine PMN were incubated in RPMI for 20 h at 37 degrees C with LPS (1, 10, 100 microg/mL). IL-8 release increased with treatment of 1 microg/mL LPS, but decreased 41.5 and 95% at the 10 and 100 microg/mL concentrations of LPS, respectively. In contrast, shedding of CD14 from the surface of PMN only increased at the highest concentration of LPS (100 microg/mL). Secretion of IL-1beta was similar regardless of the LPS concentration used to stimulate PMN. The effect of PMN concentration (1 x 10(7), 2.5 x 10(7), 5 x 10(7), and 10 x 10(7)/mL) on CD14 cell surface expression and shedding of IL-8 and IL-1beta were also determined. Shedding of CD14 by PMN increased with increasing concentration of PMN after exposure to 0.1 and 10 microg/mL of LPS, while secretion of IL-8 decreased. IL-1beta increased at the highest concentration of PMN. The use of real time polymerase chain reaction showed that CD14 mRNA expression was not different between control and LPS-stimulated cells, indicating that the sCD14 came from either membrane bound CD14 or a preformed pool. Our results demonstrate that release of CD14 from PMN suppresses secretion of IL-8, and may be an important regulatory mechanism for controlling excessive migration of PMN into the bovine mammary gland.

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.

Bovine CD14 receptor produced in plants reduces severity of intramammary bacterial infection.

CD14 is a high-affinity receptor protein for the complex of bacterial LPS (LPS) and LPS binding protein in animals. Binding of the soluble form of CD14 (sCD14) to LPS, found in the outer membrane of Escherichia coli and other Gram-negative bacteria, enhances host innate immune responses, reduces the severity of mastitis, and facilitates clearance and neutralization of LPS, thus protecting against an excessive immune response to LPS and development of endotoxic shock. A truncated form of sCD14, carrying a histidine residue affinity tag for purification, was incorporated into Potato virus X for transient expression in Nicotiana benthamiana plants. Western blots probed with CD14-specific antibodies demonstrated that crude plant extracts and affinity-purified samples contained immunoreactive sCD14. Biological activity of plant-derived recombinant bovine sCD14 (PrbosCD14) was demonstrated in vitro by LPS-induced apoptosis and interleukin (IL) -8 production in bovine endothelial cells, and in vivo by enhancement of LPS-induced neutrophil recruitment. Finally, in PrbosCD14-infused glands subsequently infected with E. coli, lower numbers of viable bacteria were recovered and there was an absence of clinical symptoms, demonstrating prophylactic efficacy of PrbosCD14. This is the first report of a functionally active animal receptor protein made in plants and the prophylactic use of a plant-derived protein to reduce the severity of bacterial infections in animals.

Staphylococcus aureus intramammary infection elicits increased production of transforming growth factor-alpha, beta1, and beta2.

In contrast to other mastitis pathogens, the host response evoked during Staphylococcus aureus intramammary infection is marked by the absence of the induction of critical cytokines, including IL-8 and TNF-alpha, which have established roles in mediating host innate immunity. The elucidation of changes in the expression of other mediators with the potential to regulate mammary inflammatory responses to S. aureus remains lacking. Transforming growth factor (TGF)-alpha, TGF-beta1, and TGF-beta2 are cytokines that regulate mammary gland development. Because these cytokines also have a demonstrated role in mediating inflammation, the objective of the current study was to determine whether S. aureus intramammary infection influences their expression. Ten cows were challenged with S. aureus and milk samples collected. Increases in milk levels of TGF-alpha were evident within 32h of infection and persisted for 16h. Increases in TGF-beta1 and TGF-beta2 levels were detected within 40h of S. aureus infection and persisted through the end of the study. Thus, in contrast to IL-8 and TNF-alpha, S. aureus elicits host production of TGF-alpha, TGF-beta1, and TGF-beta2. This finding may suggest a role for these cytokines in mediating mammary gland host innate immune responses to S. aureus.

Characterization of cytokine expression in milk somatic cells during intramammary infections with Escherichia coli or Staphylococcus aureus by real-time PCR.

The expression of inflammatory cytokines, including interleukin (IL)-6, IL-8, IL-12, granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma, by milk somatic cells was characterized by real-time polymerase chain reaction (PCR) in dairy cows experimentally challenged with either E. coli (n = 8) or S. aureus (n = 8). The mRNA abundance of a target gene was calibrated with that of a reference gene (beta-actin) and expressed as fold of induction over the control quarter at each time point. At no single time point did all eight quarters challenged with the same type of bacteria demonstrated increased expression of a target gene and there was large variation among animals at each given time. As a consequence, most tested comparisons were not statistically significant except the peak time points of IL-8 expression (75- and 29- fold in glands challenged with E. coli and S. aureus, respectively). However, the average fold induction of all targeted cytokines was increased in response to both bacterial challenges with the exception of IFN-gamma. The expression of IFN-gamma was only increased in milk somatic cells isolated from E. coli, but not S. aureus, challenged mammary glands. Moreover, upregulated expression of cytokine genes had higher magnitudes and/or faster responses in glands challenged with E. coli in comparison with those challenged with S. aureus. We propose that the compromised upregulation of inflammatory cytokines in S. aureus infected glands may, at least partially, contribute to the chronic course of infection caused by this pathogen. Further research on identifying factors responsible for the differentially expressed cytokine profiles may be fundamental to developing strategies that mitigate the outcome of bovine mastitis.

Expression of lymphocyte homing and adhesion molecules during intramammary infection of cows with Serratiamarcescens or Streptococcusuberis: correlation with bacterial colonization and clinical signs.

We wished to determine the expression of trafficking/adhesion molecules on the surface of lymphocytes isolated from infected mammary glands of cows challenged with either Serratia marcescens or Staphylococcus uberis. Healthy Holstein cows in mid lactation were infected by intramammary infusion with S. marcescens or S. uberis. Following infection, milk samples were collected at various time points. Body temperatures of the cows were taken, and milk was analyzed for colony forming units (CFU) of bacteria and somatic cell counts (SCC). Leukocytes were isolated from the milk and analyzed by flow cytometry. Percentages and types of lymphocytes were determined as well as expression of CD62L, CD11a, LPAM-1 and CD44 on these cells. We found that the percentage of lymphocytes expressing either CD62L or CD11a showed a marked increase 12 h post infection (PI) with S. marcescens that was not seen in cows infected with S. uberis. Conversely, the percentage of lymphocytes expressing CD44 increased in cows infected with S. uberis at 12 h PI, but the increase was not seen in cows infected with S. marcescens. Expression of LPAM-1 was low at all time points in both groups of cows. Body temperatures became elevated in both groups of cows, peaking at 24 h PI in S. marcescens-infected cows and dropping thereafter. In contrast, temperatures of S. uberis-infected cows continued to rise and were still elevated 96 h PI. CFU of bacteria isolated from mammary glands of S. marcescens-infected cows dropped precipitously 24 h PI but continued at high levels in S. uberis-infected cows. SCC began falling in S. marcescens-infected cows 48 h PI but continued to increase in S. uberis-infected cows. Thus, a greater percentage of lymphocytes in milk had a phenotype consistent with recruitment from the peripheral pool following infection with S. marcescens than was seen following infection with S. uberis. Concurrent with the increases seen in percentages of this lymphocyte phenotype, clinical signs lessened in the S. marcescens-infected cows.

The bovine innate immune response during experimentally-induced Pseudomonas aeruginosa mastitis.

Almost half of all clinical cases of mastitis are caused by Gram-negative bacteria. Among these bacteria, intramammary infection by Pseudomonas aeruginosa remains one of the most refractory to antibiotic therapy. The ability to recognize potentially harmful pathogens whether previously encountered or not, as well as the induction of an initial pro-inflammatory response to these pathogens, are critical components of host innate immunity. Although the innate immune response to another Gram-negative mastitis-causing pathogen, Escherichia coli, has been well-characterized, little is known about the response to other Gram-negative bacteria, including P. aeruginosa. The objective of the current study was to characterize the systemic and localized bovine innate immune response to intramammary infection with P. aeruginosa. The contralateral quarters of ten mid-lactating Holstein cows were challenged with either saline or P. aeruginosa. Following the establishment of infection, milk samples were collected and assayed for changes in cytokine and growth factor concentrations, complement activation, and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), two accessory molecules involved in host recognition of Gram-negative bacteria. Initial increases in milk somatic cell counts were evident within 12h of experimental challenge and remained elevated for >or=3 weeks. Increased permeability of the mammary gland vasculature, as evidenced by elevated milk levels of BSA, was initially observed 20 h post-infection and persisted for 2 weeks. Within 32 h of challenge, increased levels of IL-8, TNF-alpha, IL-10, and IL-12 were detected, however, the elevated levels of these cytokines were not sustained for longer than a 24h period. In contrast, elevations in IL-1beta, IFN-gamma, TGF-alpha, TGF-beta1, TGF-beta2, sCD14, LBP, and activated complement factor 5 (C5a) were sustained for periods of >48 h. Systemic changes were characterized by elevated body temperature, induction of the acute phase protein synthesis of serum amyloid A and LBP, and a transient decrease in circulating neutrophils and lymphocytes. Together, these data demonstrate the capability of the mammary gland to mount a robust innate immune response to P. aeruginosa that is characterized by the induction of pro-inflammatory cytokines, complement activation, and increased levels of accessory molecules involved in Gram-negative bacterial recognition.

Genetically enhanced cows resist intramammary Staphylococcus aureus infection.

Mastitis, the most consequential disease in dairy cattle, costs the US dairy industry billions of dollars annually. To test the feasibility of protecting animals through genetic engineering, transgenic cows secreting lysostaphin at concentrations ranging from 0.9 to 14 micrograms/ml [corrected] in their milk were produced. In vitro assays demonstrated the milk's ability to kill Staphylococcus aureus. Intramammary infusions of S. aureus were administered to three transgenic and ten nontransgenic cows. Increases in milk somatic cells, elevated body temperatures and induced acute phase proteins, each indicative of infection, were observed in all of the nontransgenic cows but in none of the transgenic animals. Protection against S. aureus mastitis appears to be achievable with as little as 3 micrograms/ml [corrected] of lysostaphin in milk. Our results indicate that genetic engineering can provide a viable tool for enhancing resistance to disease and improve the well-being of livestock.

Effect of a trivalent vaccine against Staphylococcus aureus mastitis lymphocyte subpopulations, antibody production, and neutrophil phagocytosis.

The effect of a novel bovine mastitis trivalent vaccine, containing Staphylococcus aureus capsular polysaccharide type 5 (T5), 8 (T8), and 336 (T336), on lymphocyte subpopulations, antibody production, and neutrophil phagocytosis was evaluated. Twenty pregnant heifers were immunized with either the trivalent alone, trivalent emulsified in Freund's incomplete adjuvant (FICA), trivalent in aluminum hydroxide, or adjuvant only (FICA). Immunization was done 30 d before the expected calving date followed by 2 boosts in a 2-week interval. Compared to FICA, serum antigen-specific immunoglobulin (Ig)G1 and IgG2 were significantly increased in all the vaccinated groups before parturition and sustained until 3 wk postpartum. In comparison with the trivalent alone, formulation with either adjuvant enhanced production of IgG2, but not IgG1. Immune sera, which contained the highest amount of antibodies, slightly increased neutrophil phagocytosis to the 3 serotypes of killed S. aureus, but most of the differences were not significant due to large variation between the cows. The percentage of CD4+ lymphocyte was significantly higher in vaccinated groups than that of FICA 4 wk after the primary immunization. In comparison with FICA, cows inoculated with trivalent vaccine and adjuvants had an increased percentage of CD8+ lymphocytes at 2 time points, 2 wk before and after calving. Our results indicated that the whole cell trivalent vaccine, with or without adjuvants, is able to elicit antibody responses specific to the 3 capsular polysaccharide antigens. The increase of T8-specific IgG2 was more noticeable when the vaccine was emulsified with adjuvants.

Innate immune response to intramammary infection with Serratia marcescens and Streptococcus uberis.

Streptococcus uberis and Serratia marcescens are Gram-positive and Gram-negative bacteria, respectively, that induce clinical mastitis. Once initial host barrier systems have been breached by these pathogens, the innate immune system provides the next level of defense against these infectious agents. The innate immune response is characterized by the induction of pro-inflammatory cytokines, as well as increases in other accessory proteins that facilitate host recognition and elimination of the pathogens. The objective of the current study was to characterize the innate immune response during clinical mastitis elicited by these two important, yet less well-studied, Gram-positive and Gram-negative organisms. The pro-inflammatory cytokine response and changes in the levels of the innate immune accessory recognition proteins, soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), were studied. Decreased milk output, induction of a febrile response, and increased acute phase synthesis of LBP were all characteristic of the systemic response to intramammary infection with either organism. Infection with either bacteria similarly resulted in increased milk levels of IL-1 beta, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha, sCD14, LBP, and the complement component, C5a. However, the duration of and/or maximal changes in the increased levels of these inflammatory markers were significantly different for several of the inflammatory parameters assayed. In particular, S. uberis infection was characterized by the sustained elevation of higher milk levels of IL-1 beta, IL-10, IL-12, IFN-gamma, and C5a, relative to S. marcescens infection. Together, these data demonstrate the variability of the innate immune response to two distinct mastitis pathogens.

The production and characterization of anti-bovine CD14 monoclonal antibodies.

To characterize further the chemical and biological properties of bovine soluble (bos) CD14, a panel of ten murine monoclonal antibodies (mAb) reactive with recombinant (r) bosCD14 were produced. A sandwich ELISA, using murine mAb and rabbit polyclonal antibodies reactive with rbosCD14 was developed. All the mAb were reactive by ELISA with baculovirus-derived rbosCD14 and they recognized rbosCD14 (40 kDa) by western blot analysis. The mAb also identified by western blot sCD14 (53 and 58 kDa) in milk and blood and sCD14 (47 kDa) in a lysate of macrophages obtained from involuted bovine mammary gland secretions. Analysis by ELISA of whey samples after intramammary injection of lipopolysaccharide (LPS) (10 micro g) revealed increased sCD14 levels between 8 to 48 h after injection. Flow cytometric analysis showed that the mAb bound to macrophages isolated from involuted mammary gland secretions and mouse macrophages but not to swine or horse monocytes. Addition of anti-rbosCD14 mAb to monocytes stimulated with LPS reduced in vitro production of TNF-alpha. The anti-rbosCD14 antibodies generated in this study will be useful in studying CD14, an accessory molecule that contributes to host innate recognition of bacterial cell wall components in mammary secretions produced during mastitis.