M.J. Lewis

Endocrine profiles of dairy cows following experimentally induced clinical mastitis during early lactation

Concentrations of LH, cortisol, estradiol-17beta (E(2)), prolactin and 13,14-dihydro-15-keto-prostaglandin F(2alpha) (PGFM) were determined in cows with experimentally induced clinical mastitis during early lactation. Cows free of intramammary infection (IMI) and in the luteal phase of the estrous cycle were balanced by lactation number and days in milk and assigned to either control (n=5) or treatment (n=5) groups. Treated cows were infected experimentally (day 0), in two mammary quarters, with Streptococcus uberis and developed clinical mastitis within 60 h after inoculation as evidenced by increased mastitis scores, elevated rectal temperatures, mammary swelling and isolation of S. uberis pathogen. Four days following bacterial challenge, blood samples were collected every 20 min for 8 h for determination of PGFM and LH following administration of oxytocin and GnRH, respectively. Blood samples were also collected on days 0, 4 and 7 of the experiment to determine concentrations of E(2), prolactin and cortisol. Four days after bacterial challenge, concentrations of cortisol were higher (P=0.04) in experimentally infected cows than controls. Experimentally challenged cows had increased (P=0.02) concentrations of cortisol on days 4 and 7 compared with day 0. Control cows had no significant increase in blood cortisol during the experimental period. Baseline concentrations of PGFM did not differ between groups; however, peak concentrations of PGFM following oxytocin challenge were elevated (P=0.006) in cows with clinical mastitis compared with control animals. Prolactin, E(2) and LH did not differ between cows with clinical mastitis or controls. Experimentally induced mastitis during early lactation elevated concentrations of cortisol during the luteal phase of the estrous cycle. Furthermore, mastitic cows demonstrated an increased PGFM response following oxytocin administration. Altered reproductive efficiency in cows with clinical mastitis caused by Gram-positive pathogens may be the result of increased uterine sensitivity to prostaglandin F(2alpha) (PGF(2alpha)).

Vaccination of dairy cows with recombinant Streptococcus uberis adhesion molecule induces antibodies that reduce adherence to and internalization of S. uberis into bovine mammary epithelial cells.

Streptococcus uberis is an important environmental mastitis pathogen that causes subclinical and clinical mastitis in lactating and nonlactating cows and heifers throughout the world. Previous work from our laboratory suggests that S. uberis adhesion molecule (SUAM) is involved in S. uberis pathogenesis and may be an excellent target for vaccine development. The objective of this study was to evaluate the antibody response of cattle vaccinated with recombinant SUAM (rSUAM). Uninfected primiparous dairy cows (n=30) in late lactation were divided randomly into three groups of 10 cows each: control, 200 μg rSUAM, and 400 μg rSUAM. Cows in groups vaccinated with 200 μg and 400 μg rSUAM received an emulsion containing adjuvant, phosphate-buffered saline (PBS) and affinity purified rSUAM. Cows in the control group received an emulsion containing adjuvant and PBS. Cows were vaccinated subcutaneously in the neck region at drying off (D-0), 28 d after drying off (D+28) and within 7 d after calving. Serum was collected at D-0, D+28, at calving (C-0), calving vaccination (CV), and during early lactation (CV+14). Serum antibody responses were measured by an ELISA against rSUAM. Following the first vaccination a significant increase in anti-rSUAM antibodies was detected at D+28 in cows from groups vaccinated with 200 μg and 400 μg rSUAM when compared to the control group. This increase in anti-rSUAM antibodies continued following the second immunization at D+28; reaching the highest levels in the post-parturient sampling period (C0), after which antibodies appeared to plateau. S. uberis UT888 pretreated with several dilutions of heat-inactivated serum from cows vaccinated with rSUAM, affinity purified antibodies against rSUAM, and to a 17 amino acid long peptide from the N terminus of SUAM (pep-SUAM) were co-cultured with bovine mammary epithelial cells and adherence to and internalization of S. uberis into epithelial cells was measured. Compared to untreated controls, opsonization of two strains of S. uberis with sera from cows vaccinated with rSUAM, with affinity purified rSUAM antibodies, or with affinity purified pep-SUAM antibodies significantly reduced adherence to and internalization of this pathogen into bovine mammary epithelial cells. In conclusion, subcutaneous vaccination of dairy cows with rSUAM during physiological transitions of the mammary gland either from or to a state of active milk synthesis induced antibodies in serum and milk and these antibodies reduced adherence to and internalization of S. uberis into mammary epithelial cells under in vitro conditions. SUAM appears to be an excellent candidate for vaccine development.

Short communication: Evaluation of bulk tank milk microbiological quality of nine dairy farms in Tennessee

The purpose of this study was to evaluate the bulk tank milk (BTM) quality of 9 East Tennessee dairy farms and to determine its relationship with selected quality milk parameters. Bulk tank milk samples (n=1,141) were collected over a 42-mo period (June 2006 through November 2009) from farms, based on their preliminary incubation count (PIC) history. Parameters of BTM quality evaluated in this study included somatic cell count (SCC), standard plate count (SPC), PIC, laboratory pasteurization count (LPC), Staphylococcus spp. count, Streptococcus spp. count, and coliform count. Strong correlations between SPC and Streptococcus spp. counts (0.72) and between SPC and PIC (0.70) were found. However, moderate correlations were seen among other milk quality parameters. In addition, seasonal variations for some milk quality parameters were noted. For example, milk quality parameters such as SCC, SPC, LPC, and coliform count were significantly higher in summer, whereas Streptococcus spp. counts were significantly higher in winter. No seasonal variation in PIC or Staphylococcus spp. counts was observed. Summarizing, results from this investigation showed the importance of using several bacterial counts (SCC, SPC, PIC, LPC, Streptococcus spp. count, Staphylococcus spp. count, and coliform counts) as simultaneous indicators of milk quality.

Persistence of antibiotics in bovine mammary secretions following intramammary infusion at cessation of milking

Abstract A study was conducted to determine the persistence of antibiotic preparations for use in nonlactating cows in bovine mammary secretions following intramammary infusion at cessation of milking. Five commercially available antibiotic formulations were evaluated using 311 cows. All quarters of each cow were sampled once only during the nonlactating period and most cows were sampled at or near parturition. Antibiotic residues were detected qualitatively by the Bacillus stearothermophilus disc assay. Great variation between different antibiotics in persistence in mammary secretion was observed. In general, mammary secretions from most mammary glands infused with cloxacillin or penicillin-dihydrostreptomycin were positive at 28–35 days after infusion and some were positive at 42–49 days after infusion. On the other hand, B. stearothermophilus and influence the interpretation of results. Colostrum samples from all quarters except one were negative for antibiotics. These data suggest that nonlactating-cow antibiotic formulations persist primarily during the early to mid-nonlactating period. Based upon present methods of formulation, it would appear that antibiotic preparations for use in nonlactating cows most likely provide little protection during the periparturient period, at a time when mammary glands are highly susceptible to new intramammary infections.

Intramammary infections in heifers during early lactation following intramammary infusion of pirlimycin hydrochloride or penicillin-novobiocin at the first milking after parturition

A study was conducted to determine whether intramammary antibiotic treatment of heifer mammary glands following the first milking after calving was effective for reducing the percentage of mammary quarters infected during early lactation. Jersey and Holstein heifers from two research herds were assigned to one of three treatment groups: (1) no intramammary infusion following the first milking after parturition, (2) intramammary infusion of all quarters with pirlimycin hydrochloride following the first milking after parturition and (3) intramammary infusion of all quarters with novobiocin sodium plus penicillin G procaine following the first milking after parturition. Almost 93% of Jersey heifers (40/43) and 73.1% of quarters (125/171) were infected at the first milking. Almost 77% of quarters (33/43) were cured following treatment with pirlimycin, 61.8% (21/34) were cured following treatment with penicillin-novobiocin and 39.6% (19/48) of infections were eliminated spontaneously in the untreated control group. Significantly fewer infections were observed in pirlimycin or penicillin-novobiocin treated mammary glands of Jersey heifers during early lactation than in untreated control mammary glands. Almost 89% of Holstein heifers (32/36) and 52.8% of quarters (76/144) were infected at the first milking. About 57% (12/21) of quarters were cured following treatment with pirlimycin, 41.4% (12/29) were cured following treatment with penicillin-novobiocin and 23.1% (6/26) of infections were eliminated spontaneously in the untreated negative control group. Significantly fewer infections were observed in pirlimycin treated mammary glands of Holstein heifers during early lactation than in untreated control mammary glands. However, no significant differences were observed following penicillin-novobiocin treatment of Holstein heifers after the first milking of lactation compared with untreated control quarters. Coagulase-negative staphylococci, Streptococcus uberis and Streptococcus dysgalactiae subsp dysgalactiae were isolated most frequently in heifers from both herds.

Protective effect of anti-SUAM antibodies on Streptococcus uberis mastitis

In the present study, the effect of anti-recombinant Streptococcus uberis adhesion molecule (SUAM) antibodies against S. uberis intramammary infections (IMI) was evaluated using a passive protection model. Mammary quarters of healthy cows were infused with S. uberis UT888 opsonized with affinity purified anti-rSUAM antibodies or hyperimmune sera. Non-opsonized S. uberis UT888 were used as a control. Mammary quarters infused with opsonized S. uberis showed mild-to undetectable clinical symptoms of mastitis, lower milk bacterial counts, and less infected mammary quarters as compared to mammary quarters infused with non-opsonized S. uberis. These findings suggest that anti-rSUAM antibodies interfered with infection of mammary gland by S. uberis which might be through preventing adherence to and internalization into mammary gland cells, thus facilitating clearance of S. uberis, reducing colonization, and causing less IMI.

Role of Streptococcus uberis adhesion molecule in the pathogenesis of Streptococcus uberis mastitis.

Adherence to and internalization into mammary epithelial cells are central mechanisms in the pathogenesis of S. uberis mastitis. Through these pathogenic strategies, S. uberis reaches an intracellular environment where humoral host defenses and antimicrobials in milk are essentially ineffective, thus allowing persistence of this pathogen in the mammary gland. We reported that S. uberis expresses a surface adhesion molecule (SUAM) that has affinity for lactoferrin (LF) and a central role adherence to and internalization of S. uberis into bovine mammary epithelial cells. To define the role of SUAM in the pathogenesis of S. uberis mastitis, we created a sua gene deletion mutant clone of S. uberis UT888 (Δsua S. uberis UT888) unable to express SUAM. When tested in vitro, Δsua S. uberis UT888 was defective in adherence to and internalization into bovine mammary epithelial cells. To prove that the absence of SUAM reduces bacterial attachment, subsequent colonization and infection of bovine mammary glands, the wild type S. uberis UT888 and its isogenic Δsua S. uberis UT888 were infused into mammary quarters of dairy cows. Results showed that fewer mammary glands infused with Δsua S. uberis UT888 become infected than those infused with the isogenic parental strain. Furthermore, mammary glands infused with Δsua S. uberis UT888 had less severe clinical symptoms as compared to those infused with the isogenic parental strain. These results suggest that the SUAM mutant clone was less virulent than the isogenic parental strain which further substantiates the role of SUAM in the pathogenesis of S. uberis mastitis.