In vitro growth of enterococci of bovine origin in bovine mammary secretions from various stages of lactation

In vitro growth responses of Enterococcus faecium and Enterococcus faecalis were tested in cell-free, fat-free bovine mammary secretions. Mammary secretions were collected during the dry period, and during early, late, and extended lactation. Sixty-three enterococcal isolates from aseptically collected bovine quarter milk samples and bedding samples from a commercial dairy herd were tested. Isolates from bovine quarter milk samples originated from mammary glands with clinical mastitis, cows with composite somatic cell score >4, postpartum milk samples, or from routine milk samples submitted to a mastitis diagnostic laboratory. Source of enterococcal isolates and the species significantly contribute to the ability of organisms to multiply in mammary secretions from various stages of lactation. Isolates collected from milk samples of the commercial herd and isolates from milk submitted to a mastitis diagnostic lab did not display enhanced growth in mammary secretions compared with isolates from bedding. Growth responses of E. faecalis were greater than those for E. faecium in secretions collected during the dry period, late lactation, and extended lactation. Bacterial growth did not differ between enterococcal species in mammary secretion collected from cows in early lactation. Differences in bacterial growth between E. faecalis and E. faecium in mammary secretions may indicate differences between species in susceptibility of mammary glands during the lactation cycle.     

Effects of Parity,Bacteriological Status,Stage of Lactation,and Dry Period on N-Acetyl-β-D-Glucosaminidase Activity of Milk and Dry Secretion

Quarter foremilk samples from 61 cows were obtained at 1.5, 3, 21, and 35 wk of lactation and at 7 d after drying off. Measurements for each sample were milk SCC, NAGase activity in the milk and ability of milk to promote phagocytosis of Staphylococcus aureus by polymorphonuclear leukocytes. Milk SCC and NAGase were correlated (r = .62). The NAGase in dry cow secretion was 10-fold higher than in milk. Parity differences in NAGase activity were not significant. There were large stage of lactation trends in NAGase: NAGase activity was high in early lactation, decreased in midlactation, and increased in late lactation and in dry secretion. The increase in activity of the enzyme in milk of first parity cows in late lactation was not as great as in milk of second and third lactation cows. N-Acetyl-β-D-glucosaminidase activity of milk from quarters with intramammary infections was higher than that of milk from quarters free of pathogens.     

Association of coagulase-negative staphylococcal species,mammary quarter milk somatic cell count,and persistence of intramammary infection in dairy cattle

This study was conducted to evaluate the association between subclinical intramammary infection (IMI) with coagulase-negative staphylococci (CNS), mammary quarter milk somatic cell count (SCC), and persistence of IMI in dairy cattle. Convenience samples of CNS isolates harvested from milk samples of subclinically infected mammary quarters collected between 4 and 2 wk before drying-off, between 2 wk before drying-off and the day of drying-off, within 24 h after calving, between 1 and 2 wk after calving, and during lactation were evaluated. Isolates were obtained from the Canadian Bovine Mastitis Research Network culture bank and were identified to the species level using rpoB gene sequencing. Cow and quarter-level data were obtained from the Canadian Bovine Mastitis Research Network database and used for statistical analyses. In addition, for mammary quarters that had more than one isolation of the same CNS species at different time points, the isolates were evaluated using pulsed-field gel electrophoresis to identify persistent IMI. Milk SCC was compared between mammary quarters infected with different CNS species and to a cohort of uninfected mammary quarters. A total of 877 isolates from 643 mammary quarters of 555 cows on 89 Canadian dairy farms were identified to the species level. Twenty different species were identified, with Staphylococcus chromogenes being the most common species identified (48% of isolates), followed by Staphylococcus simulans (19%) and Staphylococcus xylosus (10%). Of the 20 species identified, only 9 species were found in persistently infected quarters. Milk SCC was significantly higher in the CNS-infected mammary quarters than in the uninfected control quarters for 8 of the 20 species studied. Also, mean SCC differed significantly between mammary quarters infected with different CNS species. Within a given species, a high degree of variability was noted in milk SCC. These data corroborate recent data from Europe with regard to the predominance of certain species of CNS (e.g., Staph. chromogenes). In addition, some species of CNS appear to have a greater effect on milk SCC. Finally, some CNS species are associated with persistent IMI suggesting that some species (e.g., Staph. chromogenes and Staph. simulans) are better host-adapted, whereas others may have an environmental reservoir.     

Elevated milk soluble CD14 in bovine mammary glands challenged with Escherichia coli lipopolysaccharide

The purpose of this study was to determine whether soluble CD14 (sCD14) in milk was affected by stage of lactation, milk somatic cell count (SCC), presence of bacteria, or lipopolysaccharide (LPS)-induced inflammation. Milk samples from 100 lactating cows (396 functional quarters) were assayed for sCD14 in milk to determine effects of stage of lactation, SCC, and intramammary infection. The concentration of sCD14 was highest in transitional milk (0 to 4 d postpartum) and in milk with high SCC (> 750,000 cells/ml). Most of the infected quarters (> 80%) were infected by coagulase-negative staphylococci and yeast. No difference was found between noninfected and infected quarters. One quarter of six healthy lactating cows was challenged with 100 microg LPS in order to study the kinetics of sCD14 during an LPS-induced inflammation. Milk samples were collected at various intervals until 72 h after injection. Rectal temperature, milk tumor necrosis factor-alpha, and interleukin-8 increased immediately after challenge. The increase in sCD14 paralleled the increase in SCC, peaked at 12 h, and started to decline after 24 h. Serum leakage, as characterized by the level of bovine serum albumin in milk, peaked at 4 h and then gradually decreased. All parameters remained at basal levels in control quarters throughout the study. In vitro experiments indicated that neutrophils released sCD14 in response to LPS in a dose-dependent manner. The results indicate that the concentration of sCD14 was significantly increased in milk after LPS challenge. The increase was not likely due to serum leakage. Instead, infiltrated neutrophils might be the main source of increased sCD14 in milk during inflammation.     

Effects of intramammary infusions of casein hydrolysate,ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid,and lactose at drying-off on mammary gland involution

The transition from the lactation to the dry period in dairy cows is a period of high risk for acquiring new intramammary infections. This risk is reduced when involution of mammary glands is completed. Consequently, strategies that accelerate the involution process after drying-off could reduce the incidence of mastitis. The objective of this study was to assess the effect of 3 different treatments on mammary gland involution. Each quarter of 8 Holstein cows in late lactation was randomly assigned at drying-off to an intramammary infusion of casein hydrolysate (CNH; 70 mg), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA; 5.7 g), lactose (5.1 g), or saline 0.9% (control) solutions. Milk samples were collected on the last 2 d before and 1, 3, 5, 7, 10, and 14 d after the last milking for determining concentrations of mammary gland involution markers. Lactoferrin, somatic cell counts (SCC), BSA, and Na⁺ concentrations, as well as matrix metalloproteinase-2 and matrix metalloproteinase-9 activities gradually increased in mammary secretions during the first 2 wk following the last milking, whereas milk citrate and K⁺ concentrations decreased. As involution advanced, the Na⁺:K⁺ ratio increased, whereas the citrate:lactoferrin ratio decreased. Compared with mammary secretions from control quarters, mammary secretions of quarters infused with CNH had higher SCC on d 1, 3, 5, and 7, and greater BSA concentrations on d 1, 3, and 5. Similarly, the CNH treatment induced a faster increase in lactoferrin concentrations, which were greater than in milk from control quarters on d 3, 5, and 7 after drying-off. Milk citrate concentrations were unaffected by CNH but the citrate:lactoferrin ratio was lower in CNH-treated quarters on d 3 and 5 than in control quarters. Moreover, CNH treatment hastened the increase in Na⁺ concentration and in the Na⁺:K⁺ ratio on d 1. Infusion of CNH also led to an increase in proteolytic activities, with greater matrix metalloproteinase 9 activities on d 1 and 3. The EGTA infusion increased SCC above that of control quarters on d 1 and 3 but it had no effect on the other parameters. Lactose infusion had no effect on any of the involution markers. In this study, intramammary infusions of CNH were the most efficient treatment to accelerate mammary gland involution, suggesting a potential role of CNH as a local milk secretion inhibitor during milk stasis.     

Effect of stage of lactation and parity on mammary gland cell renewal

Milk production is a function of the number and activity of mammary epithelial cells, regardless of stage of lactation. Milk yield is generally higher in multiparous cows than in primiparous cows, but persistency is usually greater in the latter group. We compared several measures related to metabolic activity, apoptosis, and endocrine control of mammary cell growth in 8 primiparous and 9 multiparous cows throughout lactation. Mammary gland biopsies were taken in early [10 d in milk (DIM)], peak (50 DIM), and late (250 DIM) lactation to evaluate gene expression and determine DNA and fatty acid synthase (FAS) content. Milk samples taken the day before the biopsies were used to detect protease activities and to determine stanniocalcin-1 (STC) concentrations. Blood samples served to measure insulin-like growth factor-1, prolactin, and STC concentrations. Milk yield was higher in multiparous cows than in primiparous cows at the 10 DIM (32.8 ± 1.3 and 25.2 ± 0.8 kg/d) and 50 DIM (38.0 ± 1.2 and 29.8 ± 1.1 kg/d), but it was the same for both groups at 250 DIM (23.9 ± 1.5 and 23.8 ± 1.1 kg/d). Except for stearoyl-coenzyme A desaturase, expression of genes related to milk synthesis was not affected by stage of lactation. However, gene expression of acetyl-coenzyme A carboxylase, β-casein, and FAS was lower in early lactation in primiparous cows. Expression of both proapoptotic bax and antiapoptotic bcl-2 genes was higher in primiparous cows, whereas the bax-to-bcl-2 ratio was not changed. Mammary DNA concentration was higher in multiparous cows, as was the amount of FAS protein in early lactation. Two bands of protease activity were found in milk samples, and one of the bands had an apparent molecular weight similar to gelatinase A and was dependent on the stage of lactation. Serum insulin-like growth factor-1 increased with day of lactation and was higher in primiparous cows. Serum prolactin decreased in late lactation, but peak values were observed in early lactation for primiparous cows and peak lactation for multiparous cows. Milk STC content increased with advancing lactation. The results are consistent with a lower degree of differentiation and a greater capacity for cell renewal in the mammary gland of primiparous cows.     

Effect of a biological response modifier on expression of growth factors and cellular proliferation at drying off

Agents that increase natural protective mechanisms have been proposed for the prevention and treatment of intramammary infections. Staphylococcus aureus is a major pathogen causing primarily subclinical chronic mastitis that responds poorly to antibiotic therapy. The objectives of this study were to describe the effects of a single intramammary infusion of a lipopolysaccharide-based biological response modifier (BRM) on mammary epithelial cellular proliferation and expression of insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor (VEGF) in uninfected and Staph. aureus-infected bovine mammary glands during involution. Three groups of 12 cows, 6 Staph. aureus-infected and 6 uninfected, were infused with BRM or placebo in 2 mammary quarters and killed at 7, 14, and 21 d of involution. The proportion of infected quarters, mammary cell proliferation, and IGF-I and VEGF expression were evaluated. Biological response modifier treatment decreased the proportion of Staph. aureus-infected mammary quarters at 7 d of involution, but a similar number of isolations were observed at 14 and 21 d of involution in either treated or control quarters. The percentage of proliferating mammary epithelial cells was higher in infected than uninfected quarters at every observation period, irrespective of the treatment administered, whereas uninfected BRM-treated quarters showed increased cell proliferation at 7 d of involution. Insulin-like growth factor-I expression in uninfected quarters was not affected by treatment and showed a decrease at 21 d of involution. Expression of IGF-I was greater in infected than uninfected quarters at every observation period, irrespective of the treatment received. Expression of VEGF was greater in BRM-treated uninfected quarters at 7 d of involution compared with controls. In infected quarters, VEGF expression was lowest in BRM-treated quarters at 7 d of involution and increased throughout the observation period. Conversely, untreated infected quarters showed the highest VEGF expression at 7 d and decreased at 21 d of involution. Mammary cell proliferation and expression of IGF-I and VEGF were increased in Staph. aureus-infected quarters. Increased mammary cell proliferation and VEGF expression were observed in BRM-treated quarters during the first week of involution.     

Pathological changes in bovine mammary glands following intramammary infusion of recombinant interleukin-2.

Eighteen lactating dairy cows were used to evaluate the physiological response of mammary glands to increasing doses of recombinant bovine interleukin-2. Right front and rear quarters were intramammarily infused with five different doses (.1 to 100 micrograms per quarter) of interleukin-2 as either a single or multiple treatment. Left front and rear quarters were intramammarily infused with a saline placebo and served as within-animal controls. Milk secretion samples for compositional analysis were collected from each quarter prior to infusion and at 12, 24, 36, and 48 h following infusion. Animals were slaughtered by exsanguination immediately following the 48-h sampling period, and mammary gland tissue was obtained for morphometric analysis. No changes in milk composition were observed between control quarters and those infused with up to 10 micrograms of interleukin-2 per quarter, administered as either a single or multiple treatment. Quarters infused with a single 100-micrograms dose of interleukin-2 or three consecutive doses of 25 and 100 micrograms of interleukin-2 had significantly lower lactose concentrations; there was a concomitant increase in bovine serum albumin, pH, and SCC compared with preinfusion concentrations or with control quarters. Morphometric analysis of tissue demonstrated an increase in stroma, a decrease in lumenal area, and a marked increase in the number of infiltrating leukocytes in those quarters infused with the higher doses of interleukin-2. Results suggest that interleukin-2 can be intrammammarily infused at doses as high as 10 micrograms per quarter without adversely affecting milk quality or normal mammary gland function.     

Milk lactoferrin in heifers: Influence of health status and stage of lactation

The objective of the current study was to analyze the variations in lactoferrin (LF) concentrations in primiparous cows with intramammary infection and to study how the lactation stage affects these variations. In addition, we aimed to study the potential of the LF concentration in early lactation as a predictive factor for future infections. To accomplish this goal, a longitudinal analysis was performed for 96 primiparous cows. Milk samples were collected each month from individual quarters, and the LF concentration was determined for each sample. Criteria that included both somatic cell count (SCC) and a microbiological analysis were used to assess the health status of the quarters. Of the diseased quarters (SCC >200,000 or positive for pathogen isolation, or both), 62% corresponded to nonspecific mastitis (SCC >200,000 but microbiologically negative) and 25% corresponded to the category “presence of bacterial growth” (SCC <200,000 but microbiologically positive). Diseased quarters showed increased concentrations of LF compared with healthy quarters. However, this increase was greater during the first days of lactation compared with later periods. Kaplan-Meier analysis of time free of infection demonstrated that quarters with LF concentrations at early lactation (3–10 d in milk) greater than 0.1 mg/mL are more likely to become infected during the following lactation compared with quarters with lower LF concentrations in early lactation. The results support that LF plays a relevant role in combating intramammary infection, particularly during the first days of lactation. In addition, we present evidence of the potential use of LF as a predictive marker of future infections in the individual quarters of dairy heifers.     

Streptococcus dysgalactiae isolates at calving and lactation performance within the same lactation

The objective of the study was to investigate the association between early lactation Streptococcus dysgalactiae isolates and milk yield, somatic cell count (SCC), clinical mastitis, and culling in the same lactation. The 178 commercial dairy herds were randomly placed into 3 penicillin- or penicillin-dihydrostreptomycin-based dry-cow treatments and 3 different postmilking teat disinfection groups—negative control, iodine, or external teat sealant. All cows were sampled in early lactation, and Strep. dysgalactiae-positive and culture-negative cows were followed throughout the remainder of the lactation. Mixed models, including repeated measurements, with test-day observation as dependent variable, were used to compare milk yield, SCC, and available milk quality variables throughout the remaining lactation. Survival analyses, using a positive frailty model to account for any herd random effects, were used to estimate the hazard ratio for clinical mastitis and culling. Streptococcus dysgalactiae-positive cows had a significantly higher SCC throughout the lactation compared to culture-negative cows. For primiparous or multiparous cows, respectively, the differences in the geometric mean SCC between Strep. dysgalactiae-positive and culture-negative cows was 197,000 or 280,000 cells/mL at the beginning of the lactation, 24,000 or 46,000 cells/mL in mid lactation, and 39,000 or 111,000 cells/mL at the end of the lactation. Streptococcus dysgalactiae-positive primiparous or multiparous cows produced 334 or 246 kg less milk, respectively, during a 305-d lactation compared with culture-negative cows. Compared with culture-negative cows, the hazard ratios for clinical mastitis in Strep. dysgalactiae-positive cows were 2.3 (1.9 to 2.9) and 1.6 (1.3 to 2.0) for culling. For cows with both Strep. dysgalactiae and Staphylococcus aureus isolates, the hazard ratio for culling significantly increased to 2.5 (1.9 to 3.2).     

Evaluation of somatic cell count thresholds to detect subclinical mastitis in Gyr cows

The objectives of this study were (1) to determine the sensitivity (Se) and specificity (Sp) of somatic cell count (SCC) thresholds to identify subclinical mastitis in Gyr cows caused by major and minor pathogens; (2) to study the effects of month of sampling, rear or front mammary quarters, herd, intramammary infection (IMI), and bacterial species on SCC at quarter level; and (3) to describe the prevalence of IMI in Gyr cows in commercial dairy herds. In total, 221 lactating Gyr cows from 3 commercial dairy farms were selected. Milk samples were collected from individual quarters once a month for 1 yr from all lactating cows for SCC and bacteriological analysis. Mammary quarters were considered the experimental units and the SCC results were log₁₀-transformed. Four SCC thresholds (100, 200, 300 and 400 × 10³ cells/mL) were used to determine Se and Sp to identify infected mammary quarters. The overall prevalence of IMI in quarter milk samples of Gyr cows was 49.8%, and the prevalence of minor pathogens was higher (31.9%) than that of major pathogens (17.8%). Quarter samples with microbial isolation presented higher SCC compared with negative samples. Sensitivity and Sp of selected SCC thresholds varied according to the group of pathogen (major and minor) involved in the IMI definition. Sensitivity increased and Sp decreased when mammary quarters with only major pathogens isolation were considered positive. The use of a single SCC analysis to classify quarters as uninfected or infected in Gyr cows may not be a useful test for this breed because Se and Sp of SCC at the studied thresholds were low. The occurrence of IMI and the bacterial species are the main factors responsible for SCC variation in mammary quarters of Gyr cows. Milk samples with major pathogens isolation elicited higher SCC than those with minor pathogens.     

Bovine intramammary infections caused by coagulase-negative staphylococci may persist throughout lactation according to amplified fragment length polymorphism-based analysis

Persistence of coagulase-negative staphylococci (CNS) in intramammary infections during lactation was studied in a research dairy herd of University of Helsinki. Milk samples from 328 udder quarters of 82 dairy cows (30 primiparous, 52 multiparous) were collected 2 wk before calving, at calving, and every 4 wk thereafter until the end of lactation or until the cow left the herd. The CNS isolated from the milk samples were analyzed with the API Staph ID 32 (bioMérieux, Marcy l’Etoile, France) test (API) and genotyped using amplified fragment length polymorphism (AFLP) analysis. The AFLP patterns were used for similarity analysis between CNS isolates and for species identification. For the latter, AFLP patterns of CNS isolates and staphylococcal type strains were used as operational taxonomic units in numerical analysis. In addition, the somatic cell count (SCC) of the milk samples was measured during lactation. A CNS infection was considered persistent when isolates originating from the same quarter had identical AFLP patterns on at least 3 consecutive samplings. In total, 63 CNS infections were detected during lactation in 30 and 33 quarters in the first and later lactations, respectively. Twenty-nine of these infections persisted and 34 were transient. Most of the persistent infections lasted until the end of lactation. In 57 quarters, CNS infection was detected before calving, at calving, or both, but only half of these quarters were infected by CNS during subsequent lactation. The geometric mean of SCC in quarters during persistent CNS infection was 657,600 cells/mL, and the mean of SCC in quarters with transient CNS infection was 619,100 cells/mL. The median of SCC in quarters during persistent CNS infection was 355,400 cells/mL, and the median of SCC in quarters with transient CNS infection was 133,500 cells/mL. According to both the API test and AFLP results, Staphylococcus chromogenes and Staphylococcus simulans were the CNS species isolated most often. Identification results for API and AFLP corresponded in 71.9% of the isolates.     

Bovine intramammary Escherichia coli challenge infections in late gestation demonstrate a dominant antiinflammatory immunological response

Coliform mastitis that presents itself at parturition or in the early weeks of bovine lactation is often characterized by severe inflammation and impaired milk production and can lead to death of the animal. Chronic intramammary infections caused by persistent strains of Escherichia coli may result in high production losses. The aim of this study was to determine the inflammatory response to a teat-canal challenge of bovine mammary glands with a persistent strain of E. coli during late gestation (dry period) and into early lactation. Two weeks before parturition, animals were challenged in 2 quarters with 30 cfu of a persistent strain of E. coli; control quarters were vehicle-infused and not infused, respectively. Samples of dry cow secretions were taken from all quarters before challenge and at 6, 12, 18, 24, 48, 72, 96, and 120 h following challenge. Colostrum samples and milk samples were taken from all quarters at parturition and 6, 12, 18, 24, 48, 72, 96 and 120 h postpartum. Bacterial culture, combined with random amplified polymorphic DNA genetic strain-typing analysis, indicated recovery of the bacterial challenge strain until 48 to 96 h postchallenge, and again at parturition and up to 6 and 12h postpartum. One animal exhibited clinical mastitis and the bacterial challenge strain was evident to at least 12 d postpartum. During twice-daily milkings, production levels were lower in bacteria-challenged quarters compared with controls. Somatic cell counts decreased to normal levels at a slower rate in challenged quarters compared with control quarters. Cytokine analysis indicated a minimal proinflammatory cytokine response, including interleukin-1β and tumor necrosis factor-α in challenged-quarter dry cow samples up to 120 h postchallenge. Interleukin-10 levels were significantly increased by 12h postchallenge in secretions from challenged and control quarters. These preliminary results in 2 cows indicate that proinflammatory signaling after intramammary bacterial infection may be actively suppressed during late gestation. We hypothesize that this immune-inhibitory response allows intramammary infections to become persistent in the dry period and cause clinical signs immediately after parturition.     

Apoptosis and proliferation in Staphylococcus aureus-challenged,nonlactating mammary glands stimulated to grow rapidly and develop with estradiol and progesterone

Bovine mastitis is a common and costly disease in the dairy industry and is known to negatively affect the amount of epithelium in nonlactating mammary glands. Despite this recognition, an understanding of the mechanisms contributing to reductions in epithelium is lacking. The objective of this study was to evaluate cellular apoptosis and proliferation in uninfected and Staphylococcus aureus-infected mammary glands that were stimulated to rapidly grow and develop. Estradiol and progesterone injections were administered to 18 nonlactating dairy cows to induce mammary growth, and 2 quarters from each animal were infused with saline or Staph. aureus. Mammary tissues were collected at 5 (n = 9) and 10 d (n = 9) postinfusion and examined using quantitative bright field and florescent immunohistochemistry. Staphylococcus aureus mammary glands tended to have a greater number of mammary epithelial cells undergoing apoptosis than saline quarters. In the stromal compartment, challenged quarters contained a lower proportion of cells undergoing apoptosis than saline quarters overall; however, cell types undergoing apoptosis were differentially affected. Staphylococcus aureus quarters contained a lesser percentage of apoptotic fibroblasts while also containing more nonapoptotic immune cells than saline quarters in the intralobular stroma compartment. A similar number of proliferating epithelial cells were present in Staph. aureus and saline mammary tissues, but more proliferating cells were present in the intralobular stroma compartment of Staph. aureus-infused quarters than those infused with saline. When these cellular responses are considered together, it indicates that changes in cellular apoptosis and proliferation contribute to changes in the gland structure by potentiating the expansion of the intralobular stromal compartment, via cellular accumulation, and limiting the amount of epithelium due to increases in cellular apoptosis in affected glands. Reductions in mammary epithelium are expected to reduce future milk yields and productive herd life.     

Effects of Staphylococcus aureus intramammary infection on the expression of estrogen receptor α and progesterone receptor in mammary glands of nonlactating cows administered estradiol and progesterone to stimulate mammary growth

Intramammary infections (IMI) are prevalent in nonlactating dairy cattle and are known to alter mammary structure and negatively affect the amount of mammary epithelium in the gland. Mechanisms responsible for the observed changes in mammary growth during an IMI are poorly understood, yet the importance of the key mammogenic hormones driving mammary growth is well recognized. This study's objective was to characterize the expression of estrogen receptor α (ESR1) and progesterone receptor (PGR) in mammary glands stimulated to grow and develop in the presence or absence of an IMI as well as preliminarily characterize myoepithelial cell response to IMI. Mammary growth was stimulated in 18 nonpregnant, nonlactating dairy cows using subcutaneous estradiol and progesterone injections, and 2 culture-negative quarters of each cow were subsequently infused with either saline (n = 18) or Staphylococcus aureus (n = 18). Mammary parenchyma tissues were collected 5 d (n = 9) or 10 d (n = 9) postchallenge and examined using immunofluorescence microscopy to quantify positive nuclei and characterize staining features. There tended to be a greater number of ESR1-positive nuclei observed across 8 random mammary parenchyma fields of view in saline quarters than in Staph. aureus quarters (201 vs. 163 ± 44 nuclei). Saline quarters also contained a greater number of PGR-positive nuclei (520 vs. 440 ± 45 nuclei) and myoepithelial cells (971 vs. 863 ± 48 nuclei) than Staph. aureus-challenged quarters. However, when ESR1, PGR, and myoepithelial nuclei counts were adjusted for Staph. aureus quarters containing less epithelium, differences between quarter treatments abated. The examined ESR1 and PGR staining characteristics were similar between saline and Staph. aureus quarters but were differentially affected by day of tissue collection. Additionally, nuclear staining area of myoepithelial cells was greater in Staph. aureus quarters than in saline quarters. These results indicate that IMI had little effect on the number or staining characteristics of ESR1- or PGR-positive nuclei relative to epithelial area, but myoepithelial cells appear to be affected by IMI and the associated inflammation in nonlactating mammary glands that were stimulated to grow rapidly using mammogenic hormones. Accordingly, reductions in mammary epithelium in affected glands are not suspected to be resultant of alterations in the number or staining characteristics of ESR1- or PGR-positive mammary epithelial cells.     

Effects of local or systemic administration of meloxicam on mammary gland inflammatory responses to lipopolysaccharide-induced mastitis in dairy cows

Nonsteroidal anti-inflammatory drugs (NSAID) are commonly used in combination with antimicrobial mastitis treatments to reduce pain. Little is known about whether meloxicam, an NSAID designed for the preferential inhibition of cyclooxygenase-2 over cyclooxygenase-1, affects the mammary immune response. The objective of this study was to analyze the mammary immune response to intramammary (local) or intravenous (systemic) administration of meloxicam with or without immune activation by lipopolysaccharide (LPS). We challenged 108 quarters of 30 cows with or without a low or high dose of LPS from Escherichia coli (0.1 or 0.2 µg/quarter), with or without meloxicam via intramammary administration (50 mg/quarter) or intravenous injection (0.5 mg/kg of body weight; ~300 mg/cow). Intramammary administration of meloxicam alone did not trigger an acute inflammatory response, verified by unchanged somatic cell count (SCC) and lactate dehydrogenase (LDH), BSA, and IgG concentrations in milk, which are normally augmented during mastitis due to an opening of the blood–milk barrier. Similarly, intramammary meloxicam did not change the mRNA abundance of inflammatory factors in mammary gland tissue. As expected, quarters challenged with either dose of LPS showed increased leukocyte infiltration (SCC); increased LDH, BSA, IgG, Na, and Cl concentrations; and diminished K concentrations in milk. In contrast to our hypothesis, the addition of intramammary or intravenous meloxicam did not reduce these markers of mastitis in milk. Instead, intramammary meloxicam appeared to accelerate the SCC response to LPS, but only at the lower LPS dose. Moreover, the mRNA expression of inflammatory factors in mammary tissue was not modified by the intramammary application of meloxicam compared with the contralateral quarters that were challenged with LPS only. We demonstrated for the first time that intramammary meloxicam at a dose of 50 mg/quarter did not trigger an immune response in the mammary glands of dairy cows. At the doses we used, meloxicam (intramammary or systemic) did not lower inflammatory responses. The intramammary administration of meloxicam seemed to stimulate leukocyte recruitment into the milk in quarters challenged with a low dose of LPS. The integrity of the blood–milk barrier was not protected by meloxicam in LPS-stimulated quarters. This study provides the first indications that meloxicam does not limit the inflammatory response in the mammary gland, although it does not impair the mammary immune system.     

Technical note: Therapeutic cessation of lactation of Staphylococcus aureus-infected mammary quarters

The objective of the present study was to compare the ability of chlorhexidine and povidone-iodine to cause cessation of lactation in Staphylococcus aureus-infected mammary quarters, assess milk production in the treated quarter in the subsequent lactation, and evaluate whether microbiological cure was obtained. Fourteen mid- to late-lactation Holstein-Friesian dairy cattle from the Washington State University dairy herd with single mammary quarter S. aureus intramammary infections were studied. Cows were randomly assigned to one of two treatment groups, povidone-iodine or chlorhexidine. Cows in the povidone-iodine group were infused with 120 ml of 5% povidone-iodine solution (0.5% iodine) after complete milk-out. Chlorhexidine-treated cows were infused with a proprietary chlorhexidine suspension after two milkings 24 h apart. Treated mammary quarters were not milked for the rest of the lactation. Milk production from each mammary quarter (kg of milk/quarter) was measured using in-line volume flow meters for 5 consecutive days before treatment and again at the start of the subsequent lactation. Povidoneiodine caused permanent cessation of lactation in the treated quarter, whereas 71% of the chlorhexidine-treated mammary quarters returned to function in the subsequent lactation. Hence, if the primary objective is to eliminate the mammary quarter from lactation, and thereby presumably lower the risk of herdmates acquiring new S. aureus intramammary infection, then povidone-iodine appears to be the best of the two methods. No difference in total milk production between lactation one and two in either group was found, suggesting that permanent loss of a quarter was not detrimental to overall milk production.     

Between-cow variation in dermal fibroblast response to lipopolysaccharide reflected in resolution of inflammation during Escherichia coli mastitis

Effective response to mammary gland infection depends on efficient early innate immune response. The desired response would be one that is sufficient to clear the infection with a rapid return to the production of high-quality milk and limited tissue damage. In this study, 43 early lactation cows were ranked based on the ability of their fibroblasts to produce IL-8 in response to Escherichia coli lipopolysaccharide. Subsequently, the effect of a low or high response phenotype on the response to E. coli mastitis was determined. Untreated fibroblasts produced no detectable IL-8, whereas the range of IL-8 production in response to LPS (100 ng/mL) was approximately 7-fold between the lowest and highest responding cultures. Similar patterns of between-cow variation were observed in fibroblast production of IL-8 and IL-6 in response to IL-1β and Pam2CSK4 (a synthetic diacylated lipopeptide ligand). Four low and 4 high responder cows were challenged in late lactation with intramammary infusion of E. coli. All cows developed clinical mastitis in the challenged quarters and all cows cleared the infection within 8 d. However, somatic cell count began to decline earlier in the low responder group, and milk BSA concentration (an indicator of tissue damage) was also lower in low responders compared with high responders. Milk production from the challenged quarter was markedly depressed in both groups, but returned toward prechallenge values earlier in low responder cows. Dermal fibroblast cells appear predictive of a cow's response to mastitis. In this study, the low responder phenotype was sufficient to contain an E. coli infection with a more rapid return to the production of high quality milk.     

Mammary infection with Staphylococcus aureus in cows: progress from inoculation to chronic infection and its detection

The progress of Staphylococcus aureus infection from inoculation to the early chronic stage was examined in 12 Israeli-Holstein cows (four primiparous and eight multiparous) for up to 48 d after inoculation. Before inoculation, the primiparous cows were free from any infection and the multiparous cows were infected by coagulase-negative staphylococci. Two quarters in each cow were inoculated intracisternally following milking with 2000 cfu of a local prevailing Staph. aureus strain, VL-8407. Infection was established in 21 out of 24 quarters. The control quarters remained free from infection during the study, with no significant change in function. No statistically significant differences were found between primiparous and multiparous cows in the responses examined. Somatic cell count (SCC) increased within 24 h of inoculation and remained high for the duration of the study. In the infected quarters mean ln (SCC) increased within 24 h from 9.9 +/- 0.5 before inoculation to 13.0 +/- 0.2 after inoculation; most of the cells were neutrophils. N-acetyl-beta-glucosaminidase activity, expressed as ln (nnmol/min per l), was increased from 0.9 +/- 0.6 to 2.4 +/- 0.2 by inoculation, and was highly correlated with SCC. The Staph. aureus count fluctuated with no particular relationship with SCC. The phagocytic activity of neutrophils was significantly lower in the inoculated than in the control quarters and this difference increased with time after inoculation. CD8+ T lymphocytes were the main subpopulation of lymphocytes found in inoculated quarters. After inoculation, maximum but not minimum electrical conductivity (EC) recorded during milking increased significantly. The rises in maximum EC varied significantly among cows. The rises in SCC were associated with a persistent increase in EC in only one of the eight cows examined. No clinical signs were observed, and milk yield and composition were not affected during the study period. The results suggest that some strains of Staph. aureus may induce a relatively mild response in mammary glands of cows in mid lactation, and that the concomitant development of such chronic Staph. aureus infections in two quarters may not be detected by changes in the EC of composite milk and in the yield of the cow.