T cell populations and cytokine expression in milk derived from normal and bacteria-infected bovine mammary glands.

This study investigated the effect of naturally acquired bacterial infection of the bovine mammary gland on subpopulations of T lymphocytes and cytokine expression in milk. Twenty-nine lactating cows with mastitis were compared to 12 normal animals. CD4+ lymphocytes represented a significantly greater percentage of the milk-derived lymphocytes in infected mammary glands compared to normal controls. Cytokine mRNA expression by cells derived from milk was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR). No IL-2 or IL-4 mRNA was detected in any samples, while IFN-gamma mRNA was detected in all milk samples. IL-10 mRNA was detected in cells from the milk of 2 mastitic cows and 1 normal cow, and IL-12 mRNA was detected in 2 cows with mastitis. While TNF-alpha mRNA was not detected in this study, IL-6 mRNA was identified in cells from the milk of all animals, with levels being greater in mastitic animals.

Associations among prostaglandin F2alpha, plasma zinc, copper and iron concentrations and fetal loss in cows and mares.

The objective of this study was to test the hypothesis that PGF2alpha is associated with abortion and changes in plasma Zn, Cu, and Fe concentrations in cows and mares in their first trimester of pregnancy. Eleven pregnant cows were infused with endotoxin (n = 5) or endotoxin plus an inhibitor of cycloxygenase, flunixin meglumine (n = 6). Blood was collected over a 5-d period. Additionally, 4 mares were treated every 24 h with cloprostenol sodium and blood was collected hourly until abortion. Plasma Zn, Cu, and Fe were determined. Three of five cows treated with endotoxin aborted, but none of the six cows treated with endotoxin and flunixin meglumine aborted. Aborting cows had lower plasma Zn (P = 0.048) over the 5-d study period compared with the nonaborting cows. The changes in Zn corresponded to release of PGF2alpha. All 4 mares aborted and plasma Zn concentrations were lower (P = 0.008) and Cu/Zn was higher (P = 0.02) 12 h after cloprostenol treatment. Plasma Zn may be a useful biomarker for risk of spontaneous abortion, and the decline in plasma Zn may be caused by PGF2alpha.

Bovine milk lymphocytes display the phenotype of memory T cells and are predominantly CD8+.

The phenotype of bovine milk lymphocytes was investigated and compared to peripheral blood lymphocytes using monoclonal antibodies specific for bovine leukocyte differentiation antigens and flow cytometry. T lymphocytes traffic selectively into bovine milk while B lymphocytes represent a minor population in milk by comparison to peripheral blood. The vast majority of T cells in milk express alpha beta T cell receptors and are predominantly CD8+. T cells in milk express twofold higher levels of CD2 and fivefold lower levels of CD45R, characteristics associated with memory T cells. Grouping of cows by lactational stage and analysis of lymphocyte subpopulation percentages indicated that CD4+ T cells are present in relatively low numbers in milk of cows in the first 50 days of lactation and have a significant tendency to increase in number as lactation progresses.

Hematologic and bone marrow changes after short- and long-term administration of two recombinant bovine granulocyte colony-stimulating factors.

Colony-stimulating factors are a category of glycoproteins that are instrumental in the regulation of hematopoiesis and inflammation. This investigation documented the clinical bone marrow and peripheral blood responses to short-term and long-term administration of a recombinant bovine granulocyte colony-stimulating factor (rb-GCSF) and an analog, where the cysteine at position 17 was substituted with a serine (rb-GCSF ser17). The colony-stimulating factors produced the expected changes in the hematologic findings of the bovine subjects in the study, and there was a cell-specific response to the compounds. The sustained neutrophilia in the long-term study indicates that the bovine species can tolerate the administration of recombinant forms of bovine GCSF for extended periods of time without detectable adverse side effects. The neutrophils from the short-term study revealed no apparent fluctuation, either as enhanced or reduced capability to reduce nitro blue tetrazolium as compared to pretreatment neutrophils. The administration of both recombinant forms of GCSF produced large increases in the bone marrow myeloid:erythroid (M:E) ratio concomitantly with the neutrophilias. This is the first preliminary report documenting the bone marrow response of cattle to the native and recombinant (rb-GCSF ser17) forms of bovine GCSF.

Hemogram changes in lactating dairy cows given human recombinant granulocyte colony stimulating factor (r-MethuG-CSF).

As a prelude to mammary gland challenge experiments, this investigation was implemented to assess the hematologic changes in lactating dairy cattle induced by two dosage regimes of human recombinant colony stimulating factor (Hr-GCSF). This study documents the capability of the human recombinant colony stimulating factor to produce hematologic changes in both a time and dose dependent manner when administered to the adult lactating bovine. A screening dose of 1 microgram/kg of Hr-GCSF administered to three study subjects produced a three- to four-fold increase in peripheral blood mature neutrophil counts (P less than 0.043) by day 12 of the trial. The priming dose treatment group of four lactating cows (3 micrograms/kg of Hr-GCSF) exhibited a three- to five-fold increase in peripheral blood mature neutrophil counts (P less than 0.05) and two- to three-fold increases in white blood cell counts by day 5 of the trial. Hematologic examinations of the control group (n = 4; no Hr-GCSF administration) did not detect significant changes in their neutrophil counts over baseline values. The milk somatic cell counts did not statistically shift over baseline values in any of the control or Hr-GCSF treatment groups. When attempting to alter the course of infectious disease processes, potential applications of colony stimulating factors provide interesting speculations about new therapeutic modalities.

Cross-reactive affinity purification of immunoglobulin recognizing common gram-negative bacterial core antigens.

A procedure isolating immunoglobulins specific for common gram-negative bacterial core antigens is described. A polyclonal reagent was purified by ammonium sulfate precipitation, dialysis, and column affinity chromatography. The initial vaccinal antigen was an Ra mutant Escherichia coli O111:B4 (strain J5). The capture antigen was lipopolysaccharide derived from an Ra mutant, Salmonella typhimurium TV119 covalently-linked to an agarose matrix. Column eluants were characterized in terms of total protein concentration, IgG concentration, and EIA titer recognizing E. coli (J5). Low protein, low IgG, high EIA reading fractions were isolated, demonstrating the utility of the described technique to purify broad spectrum cross-reactive immunoglobulin reagents.

Immunobinding assay for detection of Mycoplasma bovis in milk.

An immunobinding dot-blot assay (IBA) was developed for the detection of mycoplasma in milk. The test was highly species specific when monoclonal antibody preparations were employed in the assay system. Reactions were obtained with all mycoplasma species tested when polyclonal antisera preparations were used. Preincubation for 48-72 hours was necessary with milk samples containing only a few mycoplasma. Time from sample receipt to diagnosis in most positive samples could be reduced from several days by culture to a few hours by the IBA, thus enabling control procedures to be quickly initiated.

Clinical mastitis in two California dairy herds participating in contagious mastitis control programs.

The occurrence of clinical mastitis in two large California dairy herds over a 3-yr period is described. Herds had been participating for 15 or 22 yr in mastitis control programs against Streptococcus agalactiae and Staphylococcus aureus, had low bulk tank SCC, and had maintained good standards of hygiene and husbandry, but clinical mastitis remained a serious problem. A total of 1654 clinical mastitis cases were detected; the annual incidence in each herd was 49%. Coliform bacteria and environmental streptococci were etiological agents in 60% of the total clinical mastitis cases; coliforms produced 1.6 times more clinical mastitis than environmental streptococci. A higher susceptibility to clinical mastitis, primarily from coliform bacteria and environmental streptococci, was found in the first months of lactation. Clinical mastitis incidence peaked for cows in lactations 4 and 5 and was lowest during the first lactation. Highest incidence of clinical mastitis due to coliform bacteria and environmental streptococci at each dairy occurred during the rainy season (late fall and winter).

Effects of human recombinant granulocyte colony stimulating factor (HR-GCSF) on the hemogram of lactating dairy cattle.

Mastitis commonly occurs in the dairy cow and results in an influx of granulocytes into the mammary gland. Presently, colony stimulating factors have been isolated. One factor, granulocyte colony stimulating factor (GCSF), has been identified and reproduced using recombinant DNA technologies. In an experiment, lactating bovine were given a bacterially-synthesized human recombinant granulocyctic colony stimulating factor (Hr-GCSF) at a specified dose rate to monitor and characterize their hematological responses. Doses of HR-GCSF were administered subcutaneously and blood samples collected from the tail vein into vacutainer tubes. Results of the study indicated a toleration by the bovine for the HR-GCSF for the tested period, and that the HR-GCSF can stimulate a sustained elevation of circulating neutrophils.

Humoral response in neonatal calves following immunization with Escherichia coli (strain J5): the effects of adjuvant, age and colostral passive interference.

Serologic responses in 61 calves 3 to 34 days of age following immunization with bacterins containing a heat-killed rough mutant, Escherichia coli 0111:B4 (strain J5) were determined by an enzyme-linked immunosorbent assay specific for the IgG isotype. Administration of either heat-killed bacteria or oil-based adjuvants alone failed to enhance serologic recognition of common core antigens when comparing to nonvaccinate controls. Increased titers were uniquely and specifically limited to calves receiving the antigen in an oil emulsion. In a second experiment, age and initial, passively acquired titer recognizing the vaccinal antigen were not found to have any effect on the magnitude of the humoral response of 57 calves following immunization.

Detection of bovine serum antibody specific to Mycoplasma bovis and Mycoplasma californicum by enzyme-linked immunosorbent assay (ELISA).

ELISA for use in epidemiologic field studies of bovine mastitis, were developed to measure serum antibody to Mycoplasma bovis and M. californicum. Varying levels of serological cross-reactivity to seven heterologous bovine mycoplasmal species were demonstrated in each assay. Cross-reactivity was minimized by preincubation of cattle sera with suspensions of heterologous mycoplasma antigens, prior to measuring serum antibody to solid-phase antigen. Heterologous absorption improved the immunological specificity of the assays while avoiding the need to prepare species-unique antigens. Serum antibody was measured at one serum dilution. Test results were expressed as a ratio of the reactivity of a positive and a negative reference serum. A negative reference population (n = 127) was assembled. The percentile distribution of ELISA reactivity of these 127 sera were used to establish the classification criteria for each assay. The statistical methods used, while easily applied, were found to be sensitive to outlying values in the reference population. The resulting classification criteria provided controlled or known probabilities of false-positive misclassification in the two ELISA test systems. Sera from cattle with defined exposure histories were tested and classified according to these criteria.

Enzyme-linked immunosorbent assay for detection of Mycoplasma californicum-specific antibody in bovine serum: optimization of assay determinants and control of serologic cross-reactions.

An enzyme-linked immunosorbent assay (ELISA) was adapted to detect Mycoplasma californicum-specific antibodies in bovine serum. Cross-reactive antibody was found in the M californicum-positive reference serum when assayed against each of 7 solid-phase antigens of heterologous mycoplasma species. Cross-reactivity was further demonstrated by inhibition of ELISA reactivity to M californicum solid-phase antigen by incubation of sera with antigen suspensions of each heterologous species. Incubation of test sera with a cross-reacting antigen mixture containing equal proportions of the 7 cross-reactive mycoplasmas was used to minimize cross-reactivity in the M californicum-specific ELISA. Specificity of antibody reactivity to M californicum, as measured by ELISA, was determined by enzyme-linked immunosorbance inhibition, in which sera were incubated with M californicum antigen suspensions before determining ELISA reactivity to M californicum solid-phase antigen. Seropositive and suspect sera (n = 55) were obtained from 3 dairies that had bacteriologically verified epizootics of M californicum mastitis. The percentage of inhibition demonstrated in enzyme-linked immunosorbance inhibition was determined for each serum. Inhibition percentages below the 15th percentile (61% inhibition) of this distribution were classified as nonspecific.

Efficacy of the API Staph-Ident system for identification of Staphylococcus species from milk.

A total of 524 staphylococcal isolates from bovine milk were identified, using the API Staph-Ident system and conventional biochemical methods. The API Staph-Ident system correctly identified 192 of 201 (95.5%) Staphylococcus aureus isolates, but was correct on only 23 of 323 (7.1%) non-S aureus isolates.

Relationships among the results of coagulase, staphylococcal toxin, and thermonuclease tests on staphylococci from cow milk.

Production of staphylococcal alpha- or alpha-beta-toxins correlated well with production of coagulase or thermonuclease (or both) in 203 Staphylococcus aureus isolates from milk and should be reliable indicators of S. aureus in the absence of Staphylococcus intermedius. Failures to produce toxin, tube coagulase, or thermonuclease occurred in only 1 to 2% of S. aureus. Evidence of beta- or alpha-beta-toxins was not found among 321 other staphylococci isolated from milk. A few coagulase- or thermonuclease-positive isolates not producing beta- or alpha-beta-toxins were found among the Staphylococcus hyicus isolates.

Evaluation of latex agglutination tests for establishing coagulase status of staphylococci from milk.

A total number of 640 staphylococci isolated from cows' milk were tested by latex agglutination and coagulase tests. About 50% of coagulase positive and 5% of coagulase negative staphylococci were positive to the latex agglutination tests. Latex agglutination tests were found to be not satisfactory for determining the coagulase status of staphylococci isolated from cows' milk.

Prevalence of mycoplasmas and immune responses to Mycoplasma bovis in feedlot calves.

Microbiological cultural, cytologic, and immunologic observations were made on 30 calves. The eyes, nares, and bronchioalveolar region were subjected to microbiological cultural examination for mycoplasmas. Four of the examinations of 30 eyes, 15 of those of 30 nasal tissues, and 25 of those of the 30 bronchioalveolar regions from the 30 calves were positive for mycoplasmas. Mycoplasma bovis and M bovirhinis were the most prevalent species. Cytologic examinations of peripheral blood and bronchioalveolar washes did not show pathologic changes. Results of indirect hemagglutination, enzyme-linked immunosorbent assay, lymphocyte-stimulation tests on peripheral blood cells, and skin testing demonstrated only a low prevalence of immune recognition of M bovis. Infection and immune response were studied in 3 calves for 10 weeks before, and for 4 weeks after, intratracheal administration of live M bovis.

Prevalence of mycoplasmal bovine mastitis in California.

Seven species of mycoplasma plus one or more unknown species were found to cause bovine mastitis in California. Both the frequency of cases and number of species of mycoplasma in samples received at the laboratory have increased from 1976 to 1978. By survey, nearly 4% of samples of bulk tank milk from dairy farms were found to contain mycoplasma of potential pathogenic significance. Acholeplasma laidlawii was frequently isolated from samples both from cows and from farm bulk tanks during wet, rainy weather in the spring of 1978, apparently as contaminants only. The prevalence of positive bulk tank milk samples in an area appeared to parallel the prevalence of clinical mycoplasmal mastitis problem herds.

Characterization studies on mycoplasmas isolated from bovine mastitis and the bovine respiratory tract.

Mycoplasmas isolated from bovine mastitis in California were classified into five distinct species. These included Mycoplasma bovis, M. bovigenitalium, M. alkalescens, M. canadenfe, and an unidentified strain, ST-6. Strains frequently recovered from the nose of young calves proved to be M. arginini, M. bovirhinis was recovered from the respiratory tract but was not a common finding.

Effectiveness of certain teat dips and sanitizers in vitro and on teat skin against Mycoplasma agalactiae subsp. bovis.

Seven teat dip and sanitizer products were tested in vitro and in vivo for mycoplasmacidal activity against Mycoplasma agalactiae subsp. bovis (M. bovimastitidis). Most, but not all products tested appeared to kill the mycoplasma at satisfactory dilutions. These mycoplasma survived longer on teat skin during humid, rainy weather than during warm, dry weather. Acholeplasma laidlawii was frequently found on normal teat skin.

Teat apex coliform populations and coliform mastitis--a herd study.

A dairy herd having a high incidence of coliform mastitis was observed by means of pre- and post-milking samples and teat apex culture. Infected quarters were the major source of teat apex contamination with coliform bacteria and therefore constituted a risk to unifected quarters and to other cows. Coliform populations on the apex from environmental sources appeared to be transitory.