The survival of serum resistant Escherichia coli in the bovine mammary gland following experimental infection.

Serum resistant strains of Escherichia coli were injected into one or two quarters of the udders of eight healthy dairy cows. Animals receiving infection into two quarters showed variation in their ability to eliminate the bacteria. This variation extended from elimination from both glands to complete failure to remove the organisms from either gland. In most cases, the organisms were removed from one gland before the clinical signs of infection were observed, but persisted in the other gland for three to four days. Following a single infection most animals eliminated the organisms before the appearance of clinical signs, but one retained the bacteria for four days. The retention of bacteria within the gland for a period longer than the initial inflammatory response resulted in their survival within neutrophils; and in some of these glands spasmodic clinical signs of mastitis reappeared for up to at least 40 days after the initial infection. These signs were associated with the reappearance of the same serological strain of E coli. Bovine serum albumin levels in the milk were found not to constitute the most effective marker of the serum components involved in the bactericidal activity.     

The innate immune response of the bovine mammary gland to bacterial infection

Intra-mammary (IM) bacterial infection in cattle can result in clinical outcomes that range from being acute and life-threatening to those that are chronic and sub-clinical. The typical bacteria involved in IM bacterial infections activate the mammary immune system in different ways which can influence the severity of the outcome. A clear understanding of the mechanisms that activate and regulate this response is central to the development of effective preventative and treatment regimes. This review focuses on the different immune responses of the bovine mammary gland to common mastitis-causing pathogens. There is special emphasis on comparing the responses to Escherichia coli and Staphylococcus aureus infections, as these are typically associated, respectively, with acute/severe and chronic/sub-clinical forms of the disease.     

Persistence of Mycoplasma bovis infection in the mammary glands of lactating cows inoculated experimentally

To study the course of clinical mycoplasma mastitis and investigate its potential for persistence, 10(8) colony-forming units (cfu) of an Irish isolate of Mycoplasma bovis was inoculated aseptically into the right fore teat canal of three lactating cows. M bovis rapidly colonised the infected quarters and grew exponentially to more than 10(10) cfu/ml within the first three days, and spread to other quarters of each of the three cows within five to 10 days. After periods of between 24 and 72 hours the infected quarters became distended and sensitive to touch, and their secretions changed from containing visible particles, to a seropurulent exudate, to an aqueous suspension of fine particles which formed a sediment after a sample was collected. M bovis-specific antibody levels increased to varying degrees in all three cows. Subsequently, the concentrations of mycoplasma decreased to less than 10(7) cfu/ml in two of the cows, but remained at more than 10(8) cfu/ml to the end of the lactation of the other cow. Apparently normal milk was secreted by one of the cows within a month of the challenge, and by the other two cows at the start of their next lactation. However, in two of the cows subclinical M bovis infection persisted through the dry periods and into their next lactations.     

Biopsy of the bovine mammary gland.

A technique is described for biopsy of the bovine udder, employing sedation and local anaesthesia. Tissue samples of approximately 5 g were obtained by electrocautery from two quarters of the udder of a cow laterally recumbent. Care was taken to ensure complete haemostasis which was achieved by electrocoagulation and ligation. Postoperative recovery was rapid, and loss of yield was no greater in biopsied glands than in control glands of the same cow. Yield from all quarters returned to preoperative levels within 48 h.     

Systemic and local immune responses of gnotobiotic calves to respiratory infection with Mycoplasma bovis

Gnotobiotic calves were inoculated by the intratracheal route with Mycoplasma bovis and the specific antibody response in sera and tracheo-bronchial washings examined by radioimmunoassay. In sera an IgM response which reached a peak two weeks post infection was followed by IgG1 and IgG2 antibody responses. Low levels of IgA antibody were detected in sera three and four weeks after infection. The predominant antibody in tracheo-bronchial washings 2 weeks after infection was IgA. Four weeks after infection IgG1 antibody predominated, but IgG2 and IgA antibodies were also present. Cells containing Ig were present in the cellular accumulations around the necrotic zones produced by M. bovis in the lung parenchyma two and four weeks after infection. IgG1 containing cells predominated in these cellular infiltrates. IgG2 producing cells were the next in frequency. It is concluded that the lung lesion caused by M. bovis is partly due to the host's immune response, presumably contributing to the control of the infection, and that the cells infiltrating the lung are a major source of the local and systemic IgG antibody that is detected after infection. IgA staining cells were observed in the submucosa of tissues from nasal cavity and trachea. These cells are probably the source of IgA in tracheo-bronchial washings and sera since IgA-producing cells were not a predominant component of the lesion in the lung parenchyma.     

Mycoplasma agalactiae subsp. bovis in pneumonia and arthritis of the bovine.

The pneumonic lungs of 42 cattle from 26 feedlots were examined for the presence of mycoplasma, pathogenic bacteria and viruses. Four animals representative of two lots failed to yield mycoplasma. One of these yielded the virus of infectious bovine rhinotracheitis and Pasteurella hemolytica, the other yielded only P. P. multocida. Nine animals in eight lots yielded Mycoplasma sp.: five of these were M. bovirhinis, two were M. arginini and two were untypable. All of these animals yielded one or more of P. hemolytica, P. multiocida, infectious bovine rhinotracheitis virus or bovine virus diarrhea virus. Twenty-five of 29 animals in 16 lots yieled M. agalactiae subsp. bovis from lung tissues. The same organism was recovered from the arthritic joints of 12 of these animals. Eight of the 25 animals yielded no other pathogen and all of these had not received any treatment. Nine of the 25 M. agalactiae subsp. bovis positive animals also yielded one or more of P. hemolytica, P. multocida, Corynebacterium pyogenes or infectious bovine rhinotracheitis virus. Bacteriological and virological studies were not completed for the remaining eight of the 25 positive animals. In five lots of cattle which had not received medication for pneumonia and for arthritis only M. agalactiae subsp. bovis was recovered. Twenty-five grossly normal lungs obtained from normal cattle at the time of slaughter were cultured and all were negative. The possible role of M. agalactiae subsp. bovis in pneumonia and arthritis was discussed.     

Immune responses of cattle following vaccination with living and non-living Babesia bovis antigens

Twenty-four yearling Hereford (Bos taurus) cattle were vaccinated against Babesia bovis using either live parasites or non-living antigens obtained from the supernatant of in vitro cultures. A single dose of live parasites was given subcutaneously, while the non-living supernatant antigen (NLSA) was combined with saponin and 2 doses given, 2 weeks apart. Following vaccination with live parasites, serum antibodies remained at high levels for 6 months, but the lymphocyte transformation response was low and lasted only 10-18 days. In contrast, NLSA vaccination was followed, after 21-28 days, by a peak of serum antibodies which then slowly declined. The lymphocyte transformation response in these animals was much higher and persisted for 6 months. Following heterologous challenge all unvaccinated cattle had severe reactions and required treatment to prevent death. Cattle vaccinated with live parasites had mild reactions with only 1 of the 12 requiring treatment. Cattle vaccinated with NLSA were only partially protected and 6 of the 12 required treatment.     

Mycoplasma bovis suppression of bovine lymphocyte response to phytohemagglutinin

The effect of viable Mycoplasma bovis on the in vitro bovine peripheral blood lymphocyte response to phytohemagglutinin (PHA) was studied. Results showed that M. bovis did not act as a mitogen for bovine lymphocytes. Viable M. bovis produced a dose and time dependent suppression of the PHA stimulated lymphocyte response. Suppression was not a result of differences in the viability of infected or control lymphocyte cultures. The suppressive effect of M. bovis was found to be independent of the concentration of PHA used in the test and the lymphocyte response could not be restored by supplementation of the culture medium with arginine. Delay for 48 h after PHA stimulation before adding M. bovis to the lymphocyte cultures diminished, but did not prevent, the suppression of the lymphocyte response. These results show that suppression of the lymphocyte response does not require the presence of M. bovis during the period of PHA stimulation, and that M. bovis was capable of interrupting [3H]-thymidine incorporation in lymphocytes which were actively synthesizing DNA.     

Punch-excised explants of bovine mammary gland to model early immune response to infection

Background Mammary gland(MG) infections(mastitis) are frequent diseases of dairy cows that affect milk quality, animal welfare and farming profitability. These infections are commonly associated with the bacteria Escherichia coli and Staphylococcus aureus. Different in vitro models have been used to investigate the early response of the MG to bacteria, but the role of the teat in mastitis pathogenesis has received less attention. In this study, we used punchexcised teat tissue as an ex vivo mode...     

The course of mastitides in cows infected into the mammary gland with strains of Mycoplasma bovigenitalium and M. bovis]

Six cows were inoculated into the mammary gland with eight mycoplasma strains isolated from the genital tract of bulls and two type strains. The milk of cows infected with Mycoplasma bovigenitalium strains isolated from the genital tracts of bulls showed a change in the appearance and contained large quantities of mycoplasmas and specific antibodies. The mastitis was most intense in about 9 days and began to subside in 17 days infection. The type strain of M. bovigenitalium PG11 failed to produce mastitis. On the other hand, the type strain of M. bovis PG45 produced severe mastitis after a 14-day latency period, with the infection spreading to the uninoculated quarters, causing atrophy of the mammary gland, and persisting till slaughter. The sera of all cows that developed mastitis after experimental infection contained high titres of specific antibodies. The two infecting mycoplasma species were recovered from the inner organs and mammary glands of these cows after slaughter.     

Tuberculin elicited cellular immune response in the lactating bovine mammary gland vaccinated intramammarily with Mycobacterium bovis

The development of a local antigen-specific sensitivity was monitored histologically and in secretions of the bovine mammary gland. Three cows in mid-lactation were immunized by injecting 50 microliter of a killed Mycobacterium bovis vaccine into the dorsal secretory tissue of the rear mammary glands; two cows served as unvaccinated controls. Ten weeks after vaccination, all cows were challenged by intramammary infusion of 1.0 microgram tuberculin in 5 ml phosphate-buffered saline (PBS). Three quarters of each cow received tuberculin at 72, 48, and 24 hours before slaughter; a control quarter received PBS at 72 hours. Vaccinated cows exhibited an intense, local cellular reaction to tuberculin in teat-end tissues at all times post infusion; PBS-infused glands were normal. A moderate leukocyte response in parenchymal tissues adjacent to the gland cistern of tuberculin-infused quarters was observed, but deep parenchymal tissues were normal; no effect on milk yield was found. Tuberculin-infused quarters exhibited histological responses in teat cisternal tissues similar to those in delayed-type hypersensitivity reactions. Leukocytic accumulation was primarily macrophages and lymphocytes with few neutrophils. Erythema was observed in the distal half of the cistern, and fibrin deposits were found in subepithelial connective tissues. The epithelium, although distended with leukocytes, was intact and numerous mitotic figures were present. Unvaccinated cows showed no response to challenge. Results demonstrated a marked and specific cellular response in sensitized cows to challenge with tuberculin.     

Characterization of the immune response to Mycoplasma bovis lung infection

To better understand the interaction between Mycoplasma bovis and its bovine host, we have characterized the immune response generated during an experimental lung infection with M. bovis. Proliferation ([3H]-thymidine blastogenesis) and Th1/Th2 cytokine production were used to monitor peripheral cellular immune responses. Flow cytometry analysis was used to determine T-cell subset activity by CD25 expression. Humoral immune response was monitored by the identification of antigen-specific IgG1 and IgG2 isotypes over time. Herein, we show that M. bovis antigen stimulates activation of CD4+ and CD8+ cells in vitro in a manner consistent with memory, and that gammadelta-T cells are activated by antigen in a manner consistent with innate immunity. In addition, the percentage of cells producing IFN-gamma during recall response is equal to that of IL-4 producing cells. Serological analysis shows M. bovis stimulates increased production of antigen-specific IgG1 while very little IgG2 is produced. We therefore submit that experimental lung infection of cattle with M. bovis results in a Th2-skewed immune response.     

Defences of the bovine mammary gland against infection and prospects for their enhancement

The equine alternative complement pathway has been partially characterized and compared to the equine classical activation pathway. A dose-dependent lysis of RbRBC was observed with peak lytic values noted within 10 minutes at 37°C when rabbit red blood cells (RbRBC) were used as an alternative pathway activator. Sheep red blood cells (SRBC) sensitized with rabbit hemolysin or partially purified equine IgM antibodies were equally sensitive to lysis. Dilution of the commercial hemolysin by $\text{1}\text{5}$ reduced lysis from 90% to 38% in the presence of constant cell numbers. Hemolysis of SRBC peaked at 10 minutes and the majority of lysis occurred within 10 minutes. Dilution of equine sera by as little as $\text{1}\text{5}$ decreased hemolytic activity for SRBC to 21.5% from greater than 90% with undiluted sera. The alternative pathway protein, equine factor B, was tested using RbRBC and monitored by its differential susceptibility to heat treatment at 50°C. This treatment led to almost complete inactivation after a 15-minute incubation. An apparent heat-dependent decay of certain classical pathway components was also observed after 50°C treatment. This sensitivity was indicated by a reduction in the lytic activity for sensitized SRBC. Treatment for 15 minutes at 56°C with either RbRBC or SRBC was sufficient to abolish hemolytic activity in all equine sera tested. Chelation of cations with 0.04 M EDTA blocked expression of alternative and classical pathway activation; however, chelation of Ca⁺⁺ ions with 10 mM EGTA containing 1 mM Mg⁺⁺ ions permitted lysis of the RbRBC but not the SRBC. A dose-related Mg⁺⁺-ion dependence for RbRBC hemolytic activity was observed as the concentration of Mg⁺⁺ was increased to 1.0 mM. In addition, our results obtained with pre-colostral foal serum strongly suggest that natural antibody to RbRBC was of little importance in the lysis observed with these cells. These results also show that the equine alternative pathway activation may require Ca⁺⁺ ions. If Ca⁺⁺ ions are required, the equine alternative pathway is quite different from any other mammalian complement system so far described. Our results suggest that the alternative pathway of activation is of major importance in the equine complement system. Confirmation of this hypothesis requires both purification of the components involved as well as further characterization.