Bactericidal activity of macrophages against Streptococcus uberis is different in mammary gland secretions of lactating and drying off cows

The aim of this study was to compare the ability of milk macrophages and macrophages from the mammary gland secretions during the mid-dry period for their interaction with the mastitis-causing Streptococcus uberis. We also aimed to determine if S. uberis induced the release of the cytokine tumour necrosis alpha (TNF-alpha) and the bactericidal moiety nitric oxide (NO) from milk macrophages of lactating cows and macrophages from the mammary gland secretions at the mid-dry period. Macrophages were isolated from the mammary gland secretions of cows during the mid-lactation or mid-dry period, and compared with blood monocytes for their interaction with the important mastitis-causing pathogen S. uberis. When infected in vitro with S. uberis, milk macrophages from lactating cows with S. uberis released modest amounts of the cytokine tumour necrosis factor alpha (TNF-alpha) (139 pg/ml) and the bactericidal moiety nitric oxide (NO) (3-4 microM of nitrite). Blood monocytes from lactating cows released significantly higher amounts of TNF-alpha (345 +/- 143 pg/ml) and NO (7 +/- 2 microM of nitrite) after interaction with S. uberis, compared to milk macrophages (P < 0.01 for both TNF-alpha and NO). Stimulation of blood monocytes with the cytokine interferon-gamma (IFN-gamma) enhanced significantly the release of NO and TNF-alpha, but IFN-gamma did not significantly enhance the production of NO and TNF-alpha by milk macrophages from lactating cows. Milk macrophages from all lactating cows failed to kill S. uberis efficiently, and this lack of killing was unaffected by prior treatment with gamma interferon (IFN-gamma) (P > 0.05). Rather, S. uberis multiplied significantly inside infected milk macrophages from lactating cows, with a two-fold increase in bacterial numbers at 2 h post-infection. Milk macrophages from lactating cows were able however, to kill a significant proportion (50-60%, P < 0.01) of phagocytosed Staphylococcus aureus. Blood monocytes from all cows were found to exert significant bactericidal activity against S. uberis. There were no significant differences in the bactericidal activity of milk macrophages obtained from lactating cows with low somatic cell counts (SCC; < 10(5) ml(-1)) compared with those with a mildly elevated SCC (> 10(5) ml(-1)) (P > 0.05). In contrast, mammary gland secretion macrophages isolated from the same cows in the mid-dry period killed a significant proportion of phagocytosed S. uberis (50-65% of ingested S. uberis killed, P < 0.01) although cytokine production in response to in vitro bacterial infection was low. We conclude that the bactericidal activity of mammary gland secretion macrophages against a virulent strain of S. uberis is low during the lactation period. In addition, our data indicate that S. uberis is not a strong inducer of NO and TNF-alpha in macrophages from the milk or mammary gland secretions of cows during the drying off period. Finally, IFN-gamma does not activate milk macrophages or macrophages from cows during the lactating period or mammary gland secretions during the drying off period.     

Prevalence and associations between bacterial isolates from dry mammary glands of dairy cows

To assess the prevalence and patterns of bacterial isolates, cultures were made from the dry mammary glands of dairy cows in six commercial dairy herds in the UK. Milk samples were taken from all four quarters of 480 cows at drying off and at weekly intervals from 14 days before to seven days after calving. A major mastitis pathogen was isolated from at least one quarter of 220 (45.8 per cent) of the cows and from more than one quarter of 90 (18.8 per cent) of them. During the late dry to calving period, of the 957 quarters with three culture results, a major mastitis pathogen was cultured from 236 (24.7 per cent) quarters of 186 (38.8 [corrected] per cent) cows. The most commonly isolated major pathogen was Escherichia coli, followed by Streptococcus uberis and coagulase-positive staphylococci. There were significant differences between the patterns of isolates from different farms and in different calving months, suggesting that the rate of infection was partially dependent on external conditions. The isolation of E. coli, S. uberis or coagulase-positive staphylococci from a cow during the late dry/periparturient period was associated with an increased risk of that cow being culled in the next lactation. Bayesian general linear mixed models were used to assess the associations between the different bacterial species. The probability of isolating either E. coli or S. uberis was significantly greater when the other organism was cultured in a milk sample; this was also true of coagulase-positive staphylococci and S. uberis. When Corynebacterium species were isolated from a milk sample, the probability of isolating coagulase-positive staphylococci or S. uberis decreased significantly, and when coagulase-negative staphylococci were isolated the probability of isolating coagulase-positive staphylococci was reduced.     

Macrophage function in mammary glands of Brucella abortus-infected cows and cows that resisted infection after inoculation of Brucella abortus

Nonvaccinated pregnant cows were segregated retrospectively into 2 groups following inoculation with Brucella abortus strain 2308. One group resisted infection (resistant cows) and the other group developed active infections (susceptible cows) and subsequently aborted. Mammary gland macrophages collected from the 2 groups of cows were compared, using in vitro functional assays. In a chemiluminescence assay, mammary gland macrophages from resistant cows produced significantly (P = 0.014) higher oxidative burst activity than did macrophages from susceptible cows. Macrophages from resistant cows had significantly (P = 0.038) greater bacteriostatic activity against B abortus than did macrophages from susceptible cows. Differences in lysosomal enzymatic activity or Fc receptor expression were not observed for macrophages from the 2 groups of cows. Differences in macrophage function may be one factor responsible for natural resistance to Brucella infection in cattle.     

Modifications of the defense and remodeling functionalities of bovine neutrophils inside the mammary gland of milk stasis cows received a commercial dry-cow treatment

An appropriate length of milk stasis between two consecutive lactations of dairy cows is crucial for sustainable milk production. This dry period of cows allows extensive remodeling and sufficient cell renewal in mammary gland. Nevertheless, early dry period is one of the most risky stages in cow lactation cycle to intramammary infection. Dry-cow treatment through teats is, therefore, widely practiced at the commencement of milk stasis. Neutrophils are the most abundant cellular components in cow mammary secretion during early dry period, which in turn attribute to the meanwhile elevation of somatic cell counts and matrix metalloproteinase-9 (MMP-9) level. This study used bovine peripheral neutrophils as a cell model to examine the mode of modifications in their defense and remodeling functionalities after infiltration into mammary gland during early dry period. Results indicate a dose-dependent suppression of phorbol 12-myristate 13-acetate-stimulated free radical production and induction of MMP 9 degranulation in bovine peripheral neutrophils exposure to the d 7-dry secretion of cows received dry cow treatment at d 0 in milk stasis. Meanwhile, an enhancement of plasminogen activation and TNF-α shedding on bovine peripheral neutrophils were also observed. These two cellular events might be involved in the functional modifications on infiltrated neutrophils during early dry period. In conclusion, the opposite trend of modifications in the defense and matrix remodeling functionalities of neutrophils inside the mammary gland of cows at early dry period reflect the collaboration of infiltrated neutrophils for promoting extensive glandular remodeling at minimum compromise of local defense during the acute involution period without apparent disturbance by dry cow treatment.     

Involvement of TNF-α and MAPK pathway in the intramammary MMP-9 release via degranulation of cow neutrophils during acute mammary gland involution

Neutrophil-derived MMP-9 activity is regulated more promptly and efficiently at the level of degranulation than at other levels of regulation. In human neutrophils, degranulation is one of the earliest responses to TNF-α stimulation, which involves protein kinase C and mitogen-activating protein kinase (MAPK) pathways. The level of MMP-9 in mammary secretion of cows increases drastically following milk-stasis, which is partially explained by increases of both neutrophil infiltration and neutrophil degranulation per se. Since MMP-9 represents one of the major remodeling capacities in the mammary gland of cows during early dry period, the current study attempted to explore the involved intracellular mechanisms in the up-regulated MMP-9 secretion. We repeatedly measured on the somatic cells of mammary secretion along the early dry period of cows the expression of TNF-α protein and the phosphorylation of p38 MAPK, ERK, and JNK. Also, cultures of bovine peripheral neutrophils were conducted to examine the mode of short-term MMP-9 secretion in response to TNF-α stimulation and the blocking effects of TNF-α antibody and inhibitors of MAPK pathways. Ex vivo measurements show that conventional cow milk has fully transformed into a neutrophil-abundant, lactoferrin-rich, and high-MMP-9 mammary secretion by d 7 in milk-stasis. No significant (P>0.05) change, however, was found in the expression of TNF-α or the phosphorylation extent of MAPK pathway intermediates on the somatic cells of mammary secretion during the first 3 weeks in milk-stasis. In vitro studies indicate linear increase of short-term MMP-9 release in response to TNF-α stimulation in dosages between 0.1 and 10 ng/ml. In the presence of preparations of d 7-dry secretion of cows, the short-term release of MMP-9 from bovine peripheral neutrophils was significantly (P<0.05) blocked by inhibitor of p38 MAPK but was significantly (P<0.05) promoted by ERK inhibitor while TNF-α antibody or JNK inhibitor exerted no effect. In conclusion, the current ex vivo measurements suggest no apparent association of TNF-α and MAPK pathway with long term intramammary accumulation of MMP-9 during the early dry period of cows, whereas cultures of bovine peripheral neutrophils under a simulated acute involution intramammary environment of cows suggest a role played by TNF-α and MAPK pathways in the short-term MMP-9 release via degranulation.     

Effects of intracisternal bead devices on lacteal secretion components, plaque formation, and bacterial infection during the nonlactating period

Effects of placing intracisternal bead devices (ICB) into teat cisterns of 6 dairy cows, from the end of lactation through parturition, were studied. Lacteal secretion samples were collected weekly from each mammary quarter during the nonlactating period to monitor composition changes in ICB-fitted and nonfitted quarters. In quarters remaining uninfected (n = 15), there were significantly higher mean somatic cell counts (P less than 0.05), percentage of neutrophils (P less than 0.019), and cell viability (P less than 0.038), but significantly lower percentage of macrophages (P less than 0.013) in ICB-fitted quarters compared with those in nonfitted quarters. The ICB had no significant effect on mean weekly values for percentage of lymphocytes, pH, lactoferrin, citrate, citrate/lactoferrin molar ratio, serum albumin, alpha-lactalbumin, and N-acetyl-beta-D-glucosaminidase. In infected quarters (n = 9), pH of mammary secretions was significantly (P less than 0.004) higher in ICB-fitted quarters, but concentrations of lactoferrin (P less than 0.004), alpha-lactalbumin (P less than 0.013), and N-acetyl-beta-D-glucosaminidase (P less than 0.028) were significantly lower, compared with those in nonfitted quarters. Coagulase-negative staphylococci comprised approximately 90% of all infections. Over the nonlactating period, 16.4 and 41.5% of samples from nonfitted and ICB-fitted quarters, respectively, contained coagulase-negative staphylococci. Microscopic examination of ICB from uninfected quarters revealed a thin coating of plaque with adhering neutrophils, macrophages, and multinucleated giant cells. Microscopic examination of plaque on devices from ICB-fitted quarters harboring coagulase-negative staphylococci revealed numerous adherent cocci and neutrophils.     

Effects of bovine lactoferrin by the intramammary infusion in cows with staphylococcal mastitis during the early non-lactating period

To evaluate the clinical effects of bovine lactoferrin on staphylococcal mastitis in Holstein cows during the early non-lactating period, 41 mammary quarters were selected randomly from 36 cows on 3 dairy farms. Twelve quarters were infused intramammarily with bovine lactoferrin. Twenty-nine quarters were infused with antibiotic as a control. In the bovine lactoferrin-infused group, 91.7% of mastitic quarters were cured at 7 days after calving, compared with 48.3% in the control group. Furthermore, the changes in mammary secretion induced by the infusion of bovine lactoferrin were investigated. Mean numbers of staphylococci in mammary gland secretions were significantly decreased in both 5 bovine lactoferrin-infused quarters and 5 antibiotic-infused control quarters (p<0.05). Unlike in the control quarters, the mean total cell concentration in the mammary gland secretions increased in bovine lactoferrin-infused quarters. Similar results were obtained in 6 healthy quarters which were infused with bovine lactoferrin. In these quarters, the cell population contained mainly phagocytes such as polymorphonuclear leukocytes and cells positive for CD11b which is known as a complement receptor. The mean concentration of C3 in mammary gland secretions was significantly increased in 5 mastitic quarters infused with bovine lactoferrin (p<0.05), but showed no significant change in 5 mastitic control quarters. These results suggested that bovine lactoferrin treatment for staphylococcal mastitis in the early non-lactating period might increase the rate of cure through the induction of innate immunity in the host.     

Drying off cows: a comparative study with two dry cow products

From September 1998 to September 1999, at drying off 397 cows on 73 Dutch farms were treated, with two antibiotic containing dry cow products (cloxacillin; penicillin-neomycin). Farms and cows were selected on an increased bulk milk somatic cell count (BMSCC) and individual cow somatic cell count. The objective of the trial was to compare the efficacy of both products under field conditions. Both products proved to be highly effective against streptococci and penicillin sensitive staphylococci. Because of the low incidence of infections caused by Escherichia coli and penicillin resistant staphylococci no conclusions could be drawn on differences in activity for these two pathogens. No significant differences were found between the two products in cure rate of existing infections at drying off, incidence of new infections during the dry period, incidence of clinical mastitis during the dry period and the course of the individual cow cell counts during the trial.     

奶牛乳腺免疫细胞防御机理研究进展

奶牛乳腺中的初级吞噬细胞、嗜中性粒细胞(PMN)和巨噬细胞构成了抵御病原体入侵的第1道防线。在健康奶牛的乳腺中,巨噬细胞占主导地位且充当哨兵的角色。当病原体侵入乳腺时,巨噬细胞及乳腺上皮细胞就会释放出直接将PMN迁移到该区域的趋化剂,使PMN从循环中迅速流入并吞噬和杀灭细菌,起到保护的作用。抵御病原体入侵的第2道防线是由记忆细胞和免疫球蛋白组成的网络,它们与第1道防线相互作用。随着分子生物学技术的发展,更好地了解炎症反应的调节机制可为研究和调节宿主与病原体的相互作用提供理论基础,因此本文就奶牛乳腺免疫细胞防御机理的研究进展进行了综述。     

The effect of interferon-gamma intramammary administration on mammary phagocyte function

The effect of recombinant bovine interferon-gamma on intramammary phagocyte function of mammary gland was studied in 4 Holstein cows (Study 1) and 7 Holstein cows (Study 2). Recombinant bovine interferon-gamma was intramammarily infused on day 6 of the dry period and phagocytes were collected from lacteal secretions and tested in vitro 24 hours later. Results from Study 1 indicate that phagocytosis of Escherichia coli and Staphylococcus aureus was significantly increased after than before interferon treatment. Similarly, the number of bacteria killed/active phagocyte was enhanced by treatment. Results from Study 2 suggested a trend towards increased production of oxygen dependent bactericidal components and increased killing ability by phagocytes exposed to interferon as compared to control phagocytes. These results from both studies suggest that intramammary infusions of bovine interferon-gamma can stimulate phagocyte function during the early phase of the dry period.     

Coliform mastitis

Gram-negative bacteria that commonly cause bovine mastitis are classified as environmental pathogens. The point sources of coliform bacteria that cause infections include bedding materials, soil, manure and other organic matter in the environment of cows. Rates of coliform mastitis increase during climatic periods that maximize populations in the environment. The portal of entry into the mammary gland for Gram-negative bacteria is the teat canal. Once in the gland, bacteria must utilize available substrates in the mammary secretion to replicate and evade host defenses. Rates of coliform mastitis are greater during the transitional phases of the non- lactating period than during lactation. The ability to infect the non-lactating gland is directly related to the ability of bacteria to acquire iron from the mammary secretion. The primary host defense against coliform mastitis during lactation is the elimination of bacteria by neutrophils migrating into the gland in response to inflammation. Damage to the host is mediated by the release of endotoxin. The severity and duration of clinical signs associated with coliform mastitis are reduced by the use of core-antigen bacterins.     

The Effect of Added Serum and Glucose, and Some Inherent Factors, on Phagocytosis in vitro by Milk Leukocytes from Several Cows

On four occasions leukocytes were obtained from mammary secretions of four cows following irritation by sterile distilled water.

The effects of three levels of bovine immune serum, three levels of glucose, and combinations of these on the ability of the leukocytes to ingest staphylococci in vitro were studied. Phagocytosis was estimated by examination of stained slides prepared after four hours contact at 37°C.

While 1% immune serum increased the percentage of milk leukocytes ingesting staphylococci, no improvement resulted from the addition of 3% or 5%. Addition of 20mg% glucose produced a larger increase than did 1% serum, and 40 and 60mg% gave further increases. Little advantage was obtained by combining serum and glucose at any of the levels studied.

Overall significant differences were found in the ability to ingest staphylococci by milk leukocytes from the four cows, and the previous observation that cells from some cows in vitro do not respond fully to added serum and glucose was confirmed. Neither of these phenomena was related to glycogen levels in blood or milk leukocytes as determined by the anthrone method, blood glucose levels, leukocyte viability, relative proportions of PMN's in circulating blood white cells, nor the amount of homologous blood serum in the mammary secretions. In two cows whose milk cells did not respond fully, very few blood PMN's were active.

     

Changes in gene expression of glucose transporters in lactating and nonlactating cows

Glucose delivery and uptake by the mammary gland are a rate-limiting step in milk synthesis. It is thought that insulin-independent glucose uptake decreases in tissues, except for the mammary gland, and insulin resistance in the whole body increases following the onset of lactation. To study glucose metabolism in peak-, late-, and nonlactating cows, the expression of erythrocyte-type glucose transporter (GLUT1) and the insulin-responsive glucose transporter (GLUT4) in the mammary gland, adipose tissue, and muscle were assessed by Western blotting and real-time PCR. Our results demonstrated that the mammary gland of lactating cows expressed a large amount of GLUT1, whereas the mammary gland of nonlactating cows did not (P < 0.05). On the other hand, adipose tissue of late and nonlactating cows expressed a large amount of GLUT1, whereas the adipose tissue of peak-lactating cows did not (P < 0.05). There were no significant differences in the abundance of GLUT4 mRNA in adipose tissue and muscle, whereas GLUT4 mRNA was not detected in the mammary gland. The plasma insulin concentration was greater (P < 0.05) in nonlactating cows than in peak- and late-lactating cows. The results of the present study indicate that in lactation, GLUT1 expression in the mammary gland and adipose tissue is a major factor for insulin-independent glucose metabolism, and the expression of GLUT4 in muscle and adipose tissue is not an important factor in insulin resistance in lactation; however, the plasma insulin concentration may play a role in insulin-dependent glucose metabolism. Factors other than GLUT4 may be involved in insulin resistance.     

Mastitis and other diseases of the goat's udder.

Udder problems of modern dairy goats are similar to those seen in dairy cows. Anomalies of the goat's udder and teats are common, and many may be hereditary. Skin diseases of the udder include viral infections, mange, sunburn, wounds, and staphylococcal dermatitis. There are numerous known causes of caprine mastitis. These include streptococci, hemolytic and nonhemolytic staphylococci, corynebacteria, and mycoplasmas. Diagnosis of mastitis in goats is often difficult, as the udder secretion may remain grossly normal and somatic cell counts in nonmastitic goats are higher than the recognized normal range for cows. The importance of nonhemolytic staphylococcal cultures remains uncertain. Nonhemolytic staphylococci were isolated at the New York State Mastitis Laboratory from 30% of normal halves and from 22% of halves of udders from goats with assorted clinical problems. Treatment and prophylaxis of caprine mastitis closely parallel the standard technique recommended for bovine mastitis.     

Effect of metoclopramide on luteinizing hormone secretion in postpartum anestrous cows.

The effect of metoclopramide (MC), a dopamine antagonist on luteinizing hormone (LH), was examined in anestrous primaparous cows. Metoclopramide has been found to be beneficial in overcoming fescue toxicosis; increasing LH secretion stimulates return to ovulatory function after parturition. Consequently, if MC had negative effect on LH secretion, it would indicate that administration of MC to reproducing animals might be limited. Of 14 postpartum (47 to 66 days) cows, 7 were given MC (4 mg/kg of body weight, IV), and 7 served as controls. Blood was obtained via jugular cannulas at 15-minute intervals for 8 hours; MC was given at the end of the first hour, and gonadotropin-releasing hormone (GnRH, 7 mg/kg), was given IV at the end of hour 7 as a challenge stimulus for LH secretion. Prior to GnRH administration, MC did not have significant effect on LH secretion, as judged by mean serum LH concentration, LH pulse frequency, and LH pulse amplitude. Administration of MC resulted in greater (P less than 0.05) LH response to GnRH, indicating enhanced secretory ability when the pituitary gland was challenged. Serum prolactin concentration was increased (P less than 0.01) by MC administration. Therefore, MC did not have adverse effect on LH secretion in postpartum cows.     

Infection following challenge of the lactating and dry udder of dairy cows with Actinomyces pyogenes and Peptostreptococcus indolicus

Challenge of 12 mammary glands of cows in mid-lactation with 10(7) colony forming units (cfu) of Peptostreptococcus indolicus on two occasions led to clinical mastitis in only four quarters. The bacteria were rarely recovered and disappeared from the secretion within 14 days. In challenges 7 days prior to drying off eight of 12 quarters became infected and at drying off all quarters challenged became infected. The infections established at drying off persisted well into the dry period. P. indolicus infection was also established in all of 12 dry glands challenged, but usually eliminated at calving or early in the next lactation. Isolation of P. indolicus was accompanied in about one-third of cases by changes in the appearance of the secretion. Intramammary challenge with Actinomyces (formally Corynebacterium) pyogenes led to clinical and subclinical infections in nine of 12 lactating glands and in all of six dry glands. Dry period infections with A. pyogenes were more severe and rarely eliminated even by antibiotic therapy. Infections during lactation were often eliminated either naturally or by antibiotic therapy. Intermittent recovery of A. pyogenes from the lactating mammary gland, without clinical signs of infection, was possible for up to 90 days after challenge. Combined infections with A. pyogenes and P. indolicus were clinically more severe with a higher frequency of systemic involvement. It was shown that in the non-lactating gland an acute mastitis, similar to 'summer mastitis' could be established either by simultaneous inoculation with A. pyogenes and P. indolicus or by subsequent inoculation of quarters excreting P. indolicus with A. pyogenes.     

Utilization of intestinal triglyceride-rich lipoproteins in mammary gland of cows

Elution profiles of total lipoproteins, apolipoprotein B (apoB) concentrations in lipoproteins, and plasma triglyceride (TG) levels were examined in early-, late-, and non-lactating cows. Additionally, arteriovenous (A-V) differences were also measured to elucidate the uptake of TG and apoB-containing lipoproteins in mammary gland. Non-lactating cows showed three major peaks corresponding to triglyceride-rich lipoprotein (TRL), low density lipoprotein (LDL), and high density lipoprotein (HDL) fraction, whereas both early- and late-lactating cows revealed two peaks corresponding to TRL and HDL. The peak area of TRL in early- and late-lactating cows were significantly (p < 0.05) smaller than that in non-lactating cows. The plasma TG levels and apoB-48 concentrations of TRL in early- and late-lactating cows were also significantly (p < 0.01) lower. Furthermore, early lactating cows showed significantly (p < 0.05) larger A-V differences in both plasma TG and apoB-48 concentration of TRL than those in late- and non-lactating cows. These results suggested that TG in exogenous (intestinal) TRL was utilized for milk fat synthesis in lactating mammary gland of cows by the receptor-mediated uptake.     

Antimicrobial resistance and genotypic characterization of coagulase-negative staphylococci over the dry period

Coagulase-negative staphylococci (CNS) have become the most frequently isolated organisms from bovine intramammary infections in recent years. While antimicrobial resistance (AR) is not considered a major problem among mastitis pathogens, concerns over emerging AR in general are increasing worldwide. Little information exists about the association between AR and one of the most common mastitis control measures, antibiotic dry cow therapy. The primary objective of the current study was to determine the prevalence of AR in CNS isolated before and after antimicrobial dry cow therapy. An additional objective was to genotypically characterize selected CNS isolates using pulsed-field gel electrophoresis (PFGE), to assess diversity and persistence of organisms over the dry period. Resistance against 10 antimicrobials was determined using a broth microdilution method and compared between CNS isolates collected at dry-off and at calving and from cows treated or not treated with intramammary antimicrobial products at dry-off (752 cows in total). Results suggested that increasing age of a cow and dry cow treatment when combined with high milk somatic cell count at dry-off and positive clinical mastitis history, were associated with increased AR to most beta-lactam antimicrobials and sulfadimethoxine. PFGE results suggested considerable diversity among the tested isolates as well as some clusters within cows and herds. PFGE would be useful in distinguishing between potentially persisting infections and cures and reinfections with different CNS strains.     

Teat skin normal flora and colonization with mastitis pathogen inhibitors

Isolates of bacteria from normal teats were used to attempt colonization of teats of dry cows or neonatal calves. Isolates for inoculation were chosen on the basis of ability to inhibit mastitis pathogens in vitro, with the ultimate goal of in vivo inhibition of mastitis pathogens at the teat surface. Three bacterial normal flora isolates (Corynebacterium xerosis, Bacillus sp. and Aerococcus viridans) persisted less than 10 days on the teats of dry cows. The fourth isolate, Staphylococcus hominis 1, was studied in greatest detail because studies characterizing the normal teat flora showed staphylococci to be the predominant flora. The S. hominis 1 isolated used for inoculation was an inhibitor of Gram-positive mastitis pathogens. It was a biotype not found on these teats prior to inoculation, thus facilitating identification of the inoculated isolate on sequential sampling. Colonization of newborn calves, before other bacterial floral became established, resulted in recovery of inoculated S. hominis 1 for an average of 51 days or longer. On dry cow teats it was detected for up to 28 days. On several occasions the inoculated S. hominis 1 was found in pure culture. Since many new infections occur during the dry period, the colonization of dry cow tests with S. hominis 1 organisms inhibitory for Gram-positive pathogens should be tested as an adjunct to other methods of mastitis prevention.