Evaluation of milk haptoglobin and amyloid A in high producing dairy cattle with clinical and subclinical mastitis in Shiraz

Mastitis is one of the most common diseases in dairy cattle and results in considerable loss of animals. This study was designed to evaluate milk haptoglobin (Hp) and milk amyloid A (MAA) as an inflammatory indicator for clinical and subclinical mastitis of cattle in dairy farms in Shiraz, Iran. Forty-three subclinical mastitic cows with a positive California Mastitis Test (CMT) and no clinical signs of mastitis, 28 clinical mastitic cows, and 10 healthy cows with negative CMT were selected. After confirmation of clinical and subclinical mastitis by bacterial identification, milk samples were taken from four quarters of each cow and mixed, and one sample was taken from the pooled milk. The most dominant isolated bacterium from clinical and subclinical samples was Staphylococcus aureus (n = 25; 35.2%). The most dominant isolated bacterium from clinical (19/28) and subclinical (11/43) samples was Staphylococcus spp. Of isolated bacteria of milk in cattle with clinical mastitis, 67.8% (n = 19) was S. aureus. There was no bacterial growth in 37.1% (n = 16) of cattle with subclinical mastitis. Of isolated bacteria of milk in cattle with subclinical mastitis, 13.9% (n = 6) and 11.6% (n = 5) was S. aureus and Staphylococcus epidermidis, respectively. There were significant differences (P < 0.05) in concentrations of milk Hp, MAA, and somatic cell count between clinically healthy cattle and cows with clinical and subclinical mastitis. The concentrations of milk Hp, MAA, and somatic cell count in clinical mastitic cows were significantly higher than those in subclinical mastitic cows and control group. The optimal cutoff point was set, using the receiver operating characteristic curve analysis method, to >13.43 μg/ml for MAA, >9.71 ng/ml for milk Hp, and >14 × 10⁴ cell per millilitre for somatic cell count with corresponding 100% sensitivity and 100% specificity for MAA, 83.72% sensitivity and 100% specificity for milk Hp, and 88.37% sensitivity and 100% specificity for somatic cell count. The results of this study reveal that MAA is a sensitive factor for diagnosis of subclinical mastitis in cattle.     

Role of bovine herpesvirus 4 in bacterial bovine mastitis

In order to study the role played by bovine herpesvirus 4 (BoHV-4) in bovine mastitis, PCR experiments were performed on a Hungarian dairy herd of 2000 cows. Milk cells were tested with a nested PCR adjusted to detect the virus in the milk. Thirty to forty-one percentage of the udders of 101 cows with bacterial mastitis (Escherichia coli, Streptococcus uberis or Staphylococcus aureus) gave positive results, whereas less than 6% of the milk samples were positive for BoHV-4 from 118 animals with healthy udders. The mastitis status of these 118 healthy cows was followed throughout the milking period; 4.2% had clinical, and 25.42% had subclinical mastitis. By the end of the milking period, more than 90% of the cows shed the virus in their milk, regardless of the bacterial status of the udder. No correlation was found between the virus shed, the somatic cell count, and the bacterial status of the udder. Viral DNA was detected in the wall of the milk duct. These results demonstrate that BoHV-4 neither causes mastitis directly nor plays a role in the initiation of the process, but later, when bacterial infection of the udder occurs, the reactivated virus replicates in the immune cells of the udder and/or in the epithelial cells of the milk ducts and may be responsible for more severe, prolonged mastitis. As mastitis is a crucial problem of milk production, this virus may be considered a possible predisposing factor and also an agent of secondary udder infections in prolonged mastitis cases.     

Effect of enrofloxacin treatment on plasma endotoxin during bovine Escherichia coli mastitis

OBJECTIVE AND DESIGN: To investigate the effect of enrofloxacin on endotoxin resorption during bovine Escherichia coli mastitis. ANIMALS: 12 healthy early post partum Holstein cows. TREATMENT: Mastitis was induced by intramammary infusion of 10(4) cfu E. coli P4:032. Six cows were treated twice according to the usual enrofloxacin therapy: 5 mg/kg enrofloxacin 1) intravenously at 10 h and 2) subcutaneously at 30 h after challenge. The other 6 cows served as non-treated controls. METHODS: Blood and milk samples were collected at several time points after challenge. LPS in plasma was quantified using the limulus amoebocyte lysate (LAL) assay. The somatic cell count (SCC) and cfu of milk samples were also analysed. RESULTS: Occasional LPS peaks were detected in the plasma of 2 control cows at 6 h post-challenge and of 1 enrofloxacin-treated cow at 10 h post-challenge (P < 0.01 and P < 0.05, respectively, in comparison with time 0), just before enrofloxacin treatment. After enrofloxacin treatment, no significant LPS amounts were detected in the plasma of treated cows, but neither in the control cows. CONCLUSION: During induced coliform mastitis, LPS resorption in plasma occured only sporadically and within 10 h post-challenge. Whereas enrofloxacin treatment clearly limited bacterial growth in milk, significant effects on LPS resorption could not be detected. This suggests that enrofloxacin treatment of E. coli mastitis is predominantly beneficial by its bactericidal activity and is not associated with enhanced resorption of endotoxins.     

Acridine orange staining for diagnosis of Mycoplasma bovis infection in cow milk.

Mycoplasma organisms were readily recognized in samples of milk or udder secretions from cows with clinical Mycoplasma bovis mastitis when these samples were stained with 0.01% acridine orange at pH 3.0. Samples could be stored at -4 degrees C for several days or subjected to repeated freezing and thawing without loss of staining or fluorescence properties. Use of this procedure in diagnostic laboratories on suspect samples from cows with clinical mastitis could hasten inauguration of control measures against this highly contagious disease by several days; however, definitive diagnosis still requires standard culture methods.     

Selective recruitment of T-cell subsets to the udder during staphylococcal and streptococcal mastitis: analysis of lymphocyte subsets and adhesion molecule expression

During bacterial infection of the bovine mammary gland, large numbers of leukocytes migrate into the udder, resulting in the establishment of a host response against the pathogen. Currently, the specific leukocyte populations mediating this immune response are not well defined. In the studies described here, we analyzed blood and milk from healthy cows and cows with naturally occurring mastitis to determine if distinct alphabeta and gammadelta T-lymphocyte subsets were involved in the response of the udder to a mastitis pathogen and if the type of mastitis pathogen influenced the subset composition of these responding leukocytes. Although blood samples from cows with confirmed staphylococcal and streptococcal mastitis were characterized by increased numbers of gammadelta T cells, the most dramatic changes in leukocyte distributions occurred in milk samples from these cows, with a 75% increase in alphabeta T-cell levels and a 100% increase in gammadelta T-cell levels relative to the levels in milk samples from healthy animals. Interestingly, the increase in alphabeta T-cell numbers observed in milk from cows with staphylococcal mastitis was primarily due to increased numbers of CD4(+) T cells, while the increase in alphabeta T-cell numbers observed in cows with streptococcal mastitis was due to a parallel increase in both CD4(+) and CD8(+) T-cell numbers. The increased numbers of gammadelta T cells in milk from cows with staphylococcal and streptococcal mastitis were due to a selective recruitment of a distinct gammadelta T-cell subset (GD3.1(+)), while no change in the numbers of GD197(+) gammadelta T cells was observed. We also analyzed adhesion protein expression on blood and milk leukocytes and found that, in comparison to the situation for healthy cows, L-selectin was down-regulated and CD18 was up-regulated on leukocytes from cows with mastitis. Thus, shedding of L-selectin and up-regulation of CD18 by neutrophils may provide a sensitive indicator of early inflammatory responses during bovine mastitis. Overall, these studies suggest that distinct alphabeta and gammadelta T-cell subsets are involved in the host defense of the udder against mastitis infection and that selective recruitment of these T-cell subsets depends on the infectious agent involved.     

Transgenic cows that produce recombinant human lactoferrin in milk are not protected from experimental Escherichia coli intramammary infection

This is the first study describing an experimental mastitis model using transgenic cows expressing recombinant human lactoferrin (rhLf) in their milk. The aim of the study was to investigate the concentrations in milk and protective effects of bovine and recombinant human lactoferrin in experimental Escherichia coli mastitis. Experimental intramammary infection was induced in one udder quarter of seven first-lactating rhLf-transgenic cows and six normal cows, using an E. coli strain isolated from cows with clinical mastitis and known to be susceptible to Lf in vitro. Clinical signs were recorded during the experimental period, concentrations of human and bovine Lf and indicators of inflammation and bacterial counts were determined for milk, and concentrations of acute-phase proteins and tumor necrosis factor alpha were determined for sera and milk. Serum cortisol and blood hematological and biochemical parameters were also determined. Expression levels of rhLf in the milk of transgenic cows remained constant throughout the experiment (mean, 2.9 mg/ml). The high Lf concentrations in the milk of transgenic cows did not protect them from intramammary infection. All cows became infected and developed clinical mastitis. The rhLf-transgenic cows showed milder systemic signs and lower serum cortisol and haptoglobin concentrations than did controls. This may be explained by lipopolysaccharide-neutralizing and immunomodulatory effects of the high Lf concentrations in their milk. However, Lf does not seem to be a very efficient protein for genetic engineering to enhance the mastitis resistance of dairy cows.     

Therapeutic Chlamydophila abortus and C. pecorum vaccination transiently reduces bovine mastitis associated with Chlamydophila infection

Infections with Chlamydophila abortus and C. pecorum are highly prevalent in cattle and have been associated with bovine mastitis. A prospective cohort study was conducted with a herd of 140 Holstein dairy cows to investigate the influence of Chlamydophila infection on subclinical inflammation of the bovine mammary gland as characterized by somatic cell numbers in milk. PCR detection of C. abortus and low serum antibody levels against Chlamydophila spp. were significantly associated with subclinical mastitis. To examine the effect of the infection by response modification, immune perturbation was done by two subcutaneous administrations of an experimental vaccine preparation of inactivated C. abortus and C. pecorum elementary bodies. Vaccination against Chlamydophila highly significantly decreased milk somatic cell numbers, thus reducing bovine mastitis, and increased antibody levels against Chlamydophila but did not eliminate shedding of C. abortus in milk as detected by PCR. The protective effect peaked at 11 weeks after vaccination and lasted for a total of 14 weeks. Vaccination with the Chlamydophila vaccine, a mock vaccine, or a combination vaccine against bovine viral diseases highly significantly increased C. abortus shedding in milk for 1 week, presumably mediated by the vaccine adjuvant. In summary, this study shows an etiological involvement of the widespread Chlamydophila infections in bovine mastitis, a herd disease of critical importance for the dairy industry. Furthermore, this investigation shows the potential for temporary improvement of chlamydial disease by therapeutic vaccination. Chlamydophila vaccination of cattle might serve as a testing ground for vaccines against human chlamydial infections.     

Differential induction of complement fragment C5a and inflammatory cytokines during intramammary infections with Escherichia coli and Staphylococcus aureus

The prompt recruitment of neutrophils to the site of infection is essential for the defense of the bovine mammary gland against invading pathogens and is determinant for the outcome of the infection. Escherichia coli is known to induce clinical mastitis, characterized by an intense neutrophil recruitment leading to the eradication of the bacteria, whereas Staphylococcus aureus induces subclinical mastitis accompanied by a moderate neutrophil recruitment and the establishment of chronic mastitis. To elicit the neutrophil recruitment into the udder, inflammatory mediators must be produced after recognition of the invading pathogen. To our knowledge, those mediators have never been studied during S. aureus mastitis, although understanding of the neutrophil recruitment mechanisms could allow a better understanding of the differences in the pathogeneses elicited by E. coli and S. aureus. Therefore, we studied, at several time points, the accumulation of neutrophils and the presence of the chemoattractant complement fragment C5a and of the cytokines interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and IL-8 in milk after inoculation of E. coli or S. aureus in lactating bovine udders. The low levels of C5a and the absence of cytokines in milk from S. aureus-infected cows, compared to the high levels found in milk from E. coli-infected animals, mirror the differences in the severities of the two inflammatory reactions. The cytokine deficit in milk after S. aureus inoculation in the lactating bovine mammary gland could contribute to the establishment of chronic mastitis. This result could help in the design of preventive or curative strategies against chronic mastitis.     

Longitudinal analysis of Prototheca zopfii-specific immune responses: correlation with disease progression and carriage in dairy cows

In order to characterize the humoral and cellular immune responses to bovine mammary protothecosis, serum and whey samples obtained from 72 dairy cows assigned to four different clinical stages of infection were examined for specific antibodies by indirect enzyme-linked immunosorbent assay techniques. Milk samples were analyzed for the total numbers of excreted algal cells and somatic cells. After characterization of the course of immune induction in bovine protothecal mastitis, a long-term sentinel study was performed in an affected herd in order to investigate disease progression. A total of 61 dairy cows with protothecal mastitis were examined for shedding of algae cells and for local immune responses three times in 6-month intervals. During acute and chronic stages of protothecosis, significantly elevated specific antibody activities in sera were detected. A strong correlation of whey immunoglobulin A (IgA) and whey IgG1 antibody activity with the total counts of somatic cells in milk was observed, whereas only a weak correlation of whey IgA and whey IgG1 concentrations to the number of algal cells excreted with the milk was seen. Our results from the sentinel long-term study of infected cows revealed that 70.5% of the persistently infected animals were continuously shedding the pathogen. About 4.9% of the animals showed an intermittent shedding, whereas 18% of the cows were tested culturally negative throughout the study. It can be assumed that Prototheca zopfii mastitis in dairy cows is maintained on the herd level by subclinically infected alga-shedding cows.     

Prenatal allergen contact with milk proteins

Background Cellular proliferation to various allergens (Dermatophagoides pleronyssinus, β-lactoglobulin, bovine serum albumin, ovalbumin) has been found in cord blood cells. Whether this reflects a sensitization during foetal life is uncertain. Objective We studied the cellular reactivity and cytokine production of cord blood cells in response to cow's milk proteins in a randomely selected group of newborns. The delineation of possible in utero allergen contact was attempted. Objective Cord blood mononuclear cells from 39 neonates were incubated with cow's milk proteins (α-lactalbumin, β-lactoglobulin, casein, α-casein, β-casein, k-casein, bovine serum albumin) for 7 days, and proliferation was assessed by incorporation of [³H]thymidine. Cord blood cell-derived interferon-γ (IFNγ) and interleukin-4 (IL-4) secretion was evaluated in response to allergen or phytohaemagglutinin (PHA) stimulation. Results A pronounced proliferation of cells stimulated with α-lactalbumin (ALA; mean stimulation index 8.0, 95% confidence interval 5.2–10.8), β-lactoglobulin (BLG; mean stimulation index 5.9, 95% confidence interval 3.2–8.6) and α-casein (2.6, 95% confidence interval 2.9–9.1), as opposed to unstimulated cells in medium, was found. No correlation was found between cellular proliferation to milk proteins and parental atopy, maternal total IgE or cord blood IgE. IFNγ production (but not IL-4) was inducible by PHA (range 429–1810 pg/ml), but only in one individual upon stimulation with BLG. Preferentially, reduced IFNγ levels were found in individuals with positive parental allergic history. Conclusion The recognition of allergen by cord blood cells indicates that allergen priming must occur prenatally. The relevance for subsequent sensitization is unclear.     

Differential Induction of Complement Fragment C5a and Inflammatory Cytokines during Intramammary Infections with Escherichia coli and Staphylococcus aureus

The prompt recruitment of neutrophils to the site of infection is essential for the defense of the bovine mammary gland against invading pathogens and is determinant for the outcome of the infection. Escherichia coli is known to induce clinical mastitis, characterized by an intense neutrophil recruitment leading to the eradication of the bacteria, whereas Staphylococcus aureus induces subclinical mastitis accompanied by a moderate neutrophil recruitment and the establishment of chronic mastitis. To elicit the neutrophil recruitment into the udder, inflammatory mediators must be produced after recognition of the invading pathogen. To our knowledge, those mediators have never been studied during S. aureus mastitis, although understanding of the neutrophil recruitment mechanisms could allow a better understanding of the differences in the pathogeneses elicited by E. coli and S. aureus. Therefore, we studied, at several time points, the accumulation of neutrophils and the presence of the chemoattractant complement fragment C5a and of the cytokines interleukin-1β (IL-1β), tumor necrosis factor alpha, and IL-8 in milk after inoculation of E. coli or S. aureus in lactating bovine udders. The low levels of C5a and the absence of cytokines in milk from S. aureus-infected cows, compared to the high levels found in milk from E. coli-infected animals, mirror the differences in the severities of the two inflammatory reactions. The cytokine deficit in milk after S. aureus inoculation in the lactating bovine mammary gland could contribute to the establishment of chronic mastitis. This result could help in the design of preventive or curative strategies against chronic mastitis.     

An enzymatic cow immunity-targeted approach to reducing milk somatic cell count: 3. A comparative field trial

The effectiveness of lysosubtilin and lysozyme, a combination thereof and a combination of these enzyme preparations (each alone and in combination) with vitamins as possible coimmunostimulants, which reduced the milk somatic cell count (SCC), were compared in a field trial. Seventy second to third lactation Lithuanian Black and White cows with a similar milk SCC ([750±200]×10³?cells?ml^?1) and of a similar weight (550±50?kg) were involved in the trial and were randomly allocated into seven groups (n=10). Lysosubtilin and/or lysozyme at doses of 0.02?g?kg?wt^?1 and 0.2?g?kg?wt^?1, respectively, and vitamins A, C and E (if any) at doses twice as high as required for nutritional adequacy were given, except for control group cows, once daily with feed for ten successive days. After four-, seven-, and ten-day periods of giving enzymes (with or without vitamins) a significant reduction of SCC (p<0.001) was observed in the milk of cows that received a combination of lysozyme with vitamins. On the tenth day a significant reduction of SCC (p<0.001) was also observed in the milk of cows that received lysozyme and lysosubtilin (each alone; without vitamins) or lysosubtilin in combination with vitamins. At the end of the trial (on the 15th day) SCC in milk of cows of all of the study groups was significantly lower (p<0.001) when compared with that of the control group.     

Detection of bovine herpesvirus 4 glycoprotein B and thymidine kinase DNA by PCR assays in bovine milk

A polymerase chain reaction (PCR) assay was developed to detect bovine herpesvirus 4 (BHV4) glycoprotein B (gB) DNA, and a nested-PCR assay was modified for the detection of BHV4 thymidine kinase (TK) DNA in bovine milk samples. To identify false-negative PCR results, internal control templates were constructed, added to milk samples, and co-amplified with viral DNA using the same primers for both templates. Specificity, sensitivity, and reproducibility of the two PCR assays were examined. In both PCR assays, all 31 BHV4 strains examined were scored positive, whereas 14 unrelated viruses scored negative. Sensitivity studies showed that two-ten copies of BHV4 DNA were detectable by the gB-PCR, while one-three copies could be detected by the TK-PCR. For the detection of BHV4 in milk samples, the gB-PCR amplification was found to be ten-times, and the TK-PCR was found to be 55-times more sensitive than virus isolation. BHV4 DNA was detected by gB-PCR and TK-PCR in 93 and 95%, respectively, of 61 milk samples collected from cows infected intramammarily with BHV4, while only 61% were positive by virus isolation. Four out of 48 cows with clinical mastitis were positive for BHV4-gB and BHV4-TK DNA, whereas no BHV4 DNA was detected in milk from control cows. Considerable agreement was seen between the results of the two PCR assays, and both methods were considered as rapid and reliable tests for the screening of BHV4 DNA in bovine milk. The less laborious gB-PCR might be the recommended test of choice for screening large amounts of milk samples for the presence of BHV4.     

Generation of an anti-NAGase single chain antibody and its application in a biosensor-based assay for the detection of NAGase in milk

Bovine mastitis, an inflammation of the mammary gland in cows, is a major challenge for the dairy industry worldwide as it lowers milk yield, reduces milk quality and increases overall production costs. Early diagnosis is of the utmost importance. N-acetyl-β-D-glucosaminidase (NAGase) is an enzyme released into milk during inflammation and acts as an early indicator of mastitis. This paper describes the selection of anti-NAGase single chain fragment variable antibodies (scFv) from na?ve human antibody libraries and their incorporation into an automated optical biosensor-based immunoassay to detect NAGase in milk. The scFv with the highest affinity for NAGase was first characterized by inhibition ELISA, followed by further evaluation using a surface plasmon resonance platform. Purified NAGase was immobilized on the surface of a CM5 chip and spiked NAGase milk samples were analyzed. The limit of detection for the assay for the assay was determined as 1μg/ml.     

Enzyme-linked immunosorbent assay for detection of leukocidin toxin from Staphylococcus aureus in bovine milk samples.

An enzyme-linked immunosorbent assay was developed for the detection of leukocidin toxin from Staphylococcus aureus. The minimum concentration of leukocidin detectable with the assay was 30 ng/ml. The enzyme-linked immunosorbent assay was found to be a more sensitive method, by a mean of 45-fold, for leukocidin detection than was observation of cytolytic effects of the toxin on bovine neutrophils. A mean toxin concentration of 974 ng/ml was required to produce observable cytolytic effects on neutrophils. Although the enzyme-linked immunosorbent assay was able to detect leukocidin in milk samples from toxin-infused mammary glands, the toxin was detectable in only 2 of 27 S. aureus-infected milk samples (7%) from cows with chronic staphylococcal mastitis. To determine whether leukocidin antibodies in the mastitic milk samples were preventing toxin detection, leukocidin was mixed with milk with a high antileukocidin antibody titer (from a vaccinated cow) and evaluated with the immunoassay. Leukocidin was readily detected in this sample, indicating that milk antileukocidin antibodies were not sufficient to prevent detection of any leukocidin present in the mastitic milk samples. Failure to detect leukocidin in most mastitic milk samples with this assay indicated that, if leukocidin is produced in the bovine mammary gland during chronic staphylococcal mastitis, the concentration of the toxin may be too low to produce cytolytic effects on neutrophils.     

Application of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of an inflammatory response antigen in subclinical mastitic milk samples.

A monoclonal antibody to a 23.5-kDa bovine inflammatory antigen present in high levels in mastitic milk has been used in an antigen-capture enzyme-linked immunosorbent assay (ELISA) to screen milk samples from herds of cattle for subclinical mastitis. The results from 20 herds with a total of 2,612 quarter samples are presented. Good correlation was observed between the ELISA level and the milk cell count (MCC). The results demonstrated an average of 5% false negatives (1.8% associated with isolates of Staphylococcus aureus and/or Streptococcus spp.) and 7.7% false positives for each herd in relation to mastitic (greater than 400,000 cells per ml) or nonmastitic (less than 400,000 cells per ml) MCCs.     

Characterisation of isolates of Staphylococcus aureus from acute, chronic and subclinical mastitis in cows in Norway

Eighty-six Staphylococcus aureus isolates from cases of bovine mastitis were characterised biochemically and with respect to serotype, multilocus enzyme electrophoresis genotypes, antibiotic sensitivity, and production of enterotoxins A through D (SEA-D) and toxic shock syndrome toxin-1 (TSST-1). The samples were obtained from 81 different cows from 79 Norwegian dairy herds in 10 different counties in southern Norway. There was an equal representation of isolates from cases of acute, chronic and subclinical mastitis. Multilocus enzyme electrophoresis using 13 genetic loci showed that 69 of 86 isolates had the same electrophoretic type. This common electrophoretic type comprised isolates that differed in the expression of other phenotypical characteristics studied. Fifty-eight percent of the isolates produced one or more enterotoxins, predominantly a combination of SEC and TSST-1. Capsular serotyping revealed that 95% of the isolates belonged to serotype 8. No correlation was found between the factors studied and the clinical classification of mastitis. It appears that the majority of S. aureus isolates recovered from cases of bovine mastitis in Norway are genetically closely related and express common phenotypical characteristics.     

MtuA,a lipoprotein receptor antigen from Streptococcus uberis,is responsible for acquisition of manganese during growth in milk and is essential for infection of the lactating bovine mammary gland

A mutant strain of Streptococcus uberis (AJS001) that was unable to grow in bovine milk was isolated following random insertional mutagenesis. The level of growth in milk was restored to that of the parental strain (strain 0140J) following addition of MnSO(4) but not following addition of other metal ions. The mutant contained a single insertion within mtuA, a homologue of mtsA and psaA, which encode metal-binding proteins in Streptococcus pyogenes and Streptococcus pneumoniae, respectively. Strain AJS001 was unable to infect any of eight quarters on four dairy cows following intramammary challenge with 10(5) CFU. Bacteria were never recovered directly from milk of these animals but were detected following enrichment in Todd-Hewitt broth in three of eight milk samples obtained within 24 h of challenge. The animals showed no inflammatory response and no signs of mastitis. Three mammary quarters on two different animals simultaneously challenged with 600 CFU of the parental strain, strain 0140J, became colonized, shed high numbers of S. uberis organisms in milk, displayed a marked inflammatory response to infection, and showed overt signs of mastitis. These data indicate that mtuA was required for efficient uptake of Mn(2+) during growth in bovine milk and infection of the lactating bovine mammary gland.     

Ingestion and killing of Listeria monocytogenes by blood and milk phagocytes from mastitic and normal cattle.

Human listeriosis resulting from consumption of listeria-contaminated dairy products is emerging as a significant public health concern. There is a need to understand better the processes involved in the pathogenesis of Listeria monocytogenes-induced bovine mastitis. In the present report, we describe the results of the in vitro interaction of L. monocytogenes with bovine blood and milk leukocytes. Induction of an experimental L. monocytogenes mastitis resulted in a rapid and dramatic increase in neutrophils in the milk of infected cows. Blood neutrophils and mononuclear cells and milk leukocytes from listeria-infected and uninfected cows readily ingested L. monocytogenes in the presence of serum opsonins. These leukocytes also killed a portion of the ingested listeriae. Ingestion of listeriae evoked a vigorous chemiluminescence response by blood neutrophils and a relatively weak response by blood mononuclear cells. Ingestion, killing, and chemiluminescence by milk leukocytes were directly related to the percentage of neutrophils that were present. Blood neutrophils from healthy donor cattle ingested and killed L. monocytogenes when leukocyte-depleted milk and whey from mastitic cows were the sole sources of opsonins, although fewer listeriae were ingested than when normal bovine serum was present. These results indicate that bovine blood and milk phagocytes, like blood and inflammatory phagocytes from other mammalian species, can ingest and kill L. monocytogenes in vitro.     

Immune reactivity of cow-asthmatic dairy farmers to the major allergen of cow (BDA20) and to other cow-derived proteins. The use of purified BDA20 increases the performance of diagnostic tests in respiratory cow allergy

Background Cow dust is one of the most important inducers of occupational allergic diseases in Finland. For example, in 1991 it accounted for almost 40% of the new occupational asthma cases. Objective This study compares the performance of the purified major cow allergen (BDA20) and crude bovine epithelial extract (BEA) in diagnostic tests and examines the role of milk allergy-associated bovine proteins (bovine serum albumin, α-lactalbumin, β-lactoglobulin, casein) in respiratory cow allergy. Methods The humoral responses of cow-asthmatic and healthy farmers to the various components of BEA were analysed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The levels of specific Ige and IgG antibodies were quantifieated with enzyme-linked inimunosorbent assays (FLISAs). The cellular responses were analysed with antigen-specific lymphoeyte proliferation tests. Results The specific anti-BDA20 IgE measurement was found to be best in distinguishing between the asthmatic farmers and their healthy colleagues. It proved possible to determine a cut-off value that gave the analysis a specificity and sensitivity of 100%; the distinction between the two groups was highly significant (P 0.0001). In the lymphocyte proliferation analysis, cow asthma was more closely associated with reactivity to BDA20 than to BEA. In the measurement of anti-BDA20 and anti-BEA IgG antibody levels, considerable overlap between the groups was observed, suggesting that these antibodies are not directly involved in cow allergy. When proteins associated with milk allergy were used as test reagents, no statistically significant differences could be observed between the groups, except for anti-casein IgE antibodies the level of which, however, overlapped considerably between the farmer groups. Conclusion These findings suggest that purified BDA20 is better than BEA for diagnosing cow asthma and that proteins associated with milk allergy are of only marginal significance in this disease.