Biofilm production by Staphylococcus aureus associated with intramammary infection

Biofilm production by 221 Staphylococcus aureus isolates from 45 dairy herds was evaluated. Isolates were from composite milk of 117 cows, from teat skin of 70 cows, and from 34 milking machine unit liners. Of S. aureus from milk samples, 41.4% were biofilm producers, as compared to 24.7 and 14.7% of the isolates collected from skin and liners. Pulsed field gel electrophoresis (PFGE) best categorized S. aureus biofilm producers as compared to phage typing and binary typing. PFGE types that were significantly associated with isolation from milk as opposed to teat skin or liners, had isolates that were more likely to produce biofilm than PFGE types that were isolated from milk, skin and liners at similar frequencies. By contrast, PFGE type A was significantly associated with isolation from teat skin and had few biofilm producers. PFGE type Q, which is exclusively a milk, isolate produced more biofilm as evidenced by absorbance values. Given S. aureus that are associated with milk are more likely to produce biofilm as compared to extramammary sources (teat skin and milking unit liners), suggests that biofilm production is a risk factor for infection.     

Effect of intramammary infusion of bacterial lipopolysaccharide on experimentally induced Staphylococcus aureus intramammary infection

Mastitis due to Staphylococcus aureus is a significant problem in the dairy industry and is refractory to antibiotic treatment and/or vaccine prevention. Relative to other mastitis-causing pathogens, S. aureus elicits a diminutive host inflammatory response during intramammary infection. To determine whether induction of a heightened inflammatory response could influence outcome of infection, the highly pro-inflammatory molecule bacterial lipopolysaccharide (LPS) was infused into udder quarters experimentally infected with S. aureus. Relative to S. aureus-infected udder quarters receiving saline, quarters infused with LPS demonstrated a heightened inflammatory response as demonstrated by the induction of TNF-alpha and higher milk somatic cell counts and albumin levels. Although there was no overall effect on bacterial clearance, a trend toward reduced bacterial numbers during the immediate pro-inflammatory response following LPS infusion was observed suggesting that this novel approach to treating S. aureus intramammary infection may warrant further investigation.     

The dynamics of Staphylococcus aureus intramammary infection in nine Danish dairy herds

The aim of the present study was to examine the diversity of Staphylococcus aureus isolates from bovine intramammary infections (IMI) in nine dairy herds, and compare these with isolates from other sites on the cows by phage- and ribotyping. Whether colonisation of milkers with S. aureus could be a source of infection for bovine IMI was investigated. In addition, 100 epidemiologically unrelated S. aureus isolates from asymptomatic human carriers were also phage- and ribotyped to compare the human and bovine reservoir of S. aureus in Denmark. A total of 625 S. aureus isolates from bovine IMI, bovine skin lesions, milking personnel, and non-farm-related human carriers were included in the study. Certain types predominated in one or several herds during the study period of one-and-a-half to two years, whereas the presence of other types was of a more sporadic nature. Within the individual herds, there was a close correspondence between ribo- and phage types of S. aureus isolated from bovine intramammary infections and skin lesions. Isolates from milking personnel, however, were not identical to any of the predominant intramammary strains. Furthermore, several of the isolates from milking personnel showed ribo- and phage patterns identical to S. aureus isolates from human carriers. The findings of the present study underline the importance of strict milking hygiene and improvement of current mastitis therapy. The results support the hypothesis that some S. aureus mastitis strains are more contagious, virulent or persistent than others. The human reservoir of S. aureus does not play a major role as a source of bovine intramammary infections.     

Systemic and local immune response of cows to intramammary infection with Staphylococcus aureus

The association between Staphylococcus aureus chronic mammary gland infection and the resulting immune response expressed by the production of specific IgG and IgA antibodies in blood and milk was studied in Israeli Holstein cows. Specific antibodies of the IgG class were detected in sera of 82.6 per cent of the cows chronically infected by S aureus, while in 17.4 per cent no such antibodies could be detected. Specific IgG antibodies to S aureus were neither detected in sera of cows free of mammary infection nor in those infected with different coagulase-negative staphylococci (CNS) such as S intermedius, S chromogenes or S haemolyticus. In milk, specific IgG antibodies to S aureus were detected only in cows with positive serology. The end point dilutions in the milk were 5 to 30 per cent of that of blood from the same cow. No significant difference in IgG titres was found in the same cow if the quarter was infected with S aureus or not. Specific antibodies to S aureus of the IgA class could not be detected in the sera of any of the cows included in this study. In milk, a specific IgA antibody was detected only in the samples from the S aureus infected quarters in which S aureus was isolated at the time of the experiment. In the same cow, quarters infected by S aureus were found to have a significantly higher IgA titre (P < 0.0001) than that of the non-infected ones.     

The effect of intramuscular and intramammary vaccination of cows on antibody levels and resistance to intramammary infection by Staphylococcus aureus.

Cows were vaccinated simultaneously by the intramuscular and intramammary route with formolised Staphylococcus aureus cells of strains BB, Mexi and 3528. Vaccination resulted in slight increases in serum agglutinin titres, but the levels of the agglutinins in milk and colostrum were not higher in vaccinated cows than in the unvaccinated controls. Vaccination did not result in higher levels of IgM, IgG1, IgG2 or IgA immunoglobulins in serum, colostrum or milk as compared with controls. Vaccinated cows, particularly those in which strain 3528 was used, showed some resistance to infection following challenge with low numbers of viable S aureus Mexi, but there was no resistance to infection when similar numbers of the virulent BB strain were used for challenge. It is concluded that vaccination is unlikely to prevent infection of the bovine udder by S aureus.     

Effect of a milk-derived factor on the inflammatory response to Staphylococcus aureus intramammary infection.

Daily injections of an anti-inflammatory milk-derived factor (MDF) into mice increased resistance to Staphylococcus aureus challenge, and reduced leukocyte infiltration. Intraperitoneal injection of MDF into lactating mice prior to S. aureus intramammary challenge resulted in greater milk secretory activity and less inflammation compared with untreated controls, but had little effect on the number of S. aureus recovered from mammary tissue. Infusion of MDF directly into mouse mammary glands prior to challenge reduced S. aureus recovered after challenge. Incubation of bovine mammary macrophages in medium supplemented with MDF enhanced phagocytosis of opsonized S. aureus. In addition, infusion of 5 mg MDF into uninfected bovine mammary glands 24 h prior to S. aureus challenge resulted in fewer infections (five of ten) than in control quarters (seven of nine). Repeated daily injections of 5 mg MDF into S. aureus-infected quarters increased the percent of mammary neutrophils and decreased the recovery of S. aureus, but did not eliminate infections. Intravenous injection of 8 g MDF into cows resulted in pronounced leukopenia while the accompanying effect on mammary leukocytes was less marked but followed a similar course. Results suggest that the use of MDF in mice enhanced resistance to experimental infection and was beneficial in maintaining mammary secretory activity and reducing inflammation after bacterial challenge. In the cow, MDF promoted phagocytosis in vitro and was effective against challenge when infused intramammarily.     

Detection of capsular polysaccharide in milk of cows with natural intramammary infection caused by Staphylococcus aureus

Detection of capsular polysaccharide (CP) in milk of cows with natural intramammary infection caused by Staphylococcus aureus was attempted. Five quarters of 5 cows harboring S aureus strains that produce type-8 CP were selected. Using an ELISA with a monoclonal antibody, type-8 CP was not detected in extracts prepared from fresh milk collected aseptically. By contrast, CP was easily detectable after incubation of infected milk at 38 C for 20 hours. Quantitation of CP in extracts from incubated milk samples by use of ELISA indicated a great variation of CP expression by strains. Although an incubation step was necessary to detect CP, results of the study indicate that CP may be expressed in vivo during intramammary infection caused by S aureus.     

Herd-level risk factors for Staphylococcus aureus and Streptococcus agalactiae intramammary infections

Herd-level risk factors for high herd prevalence of Staphylococcus aureus and Streptococcus agalactiae intramammary infection were studied using data from Holstein herds participating in the New York State Quality Milk Promotion Services Program during the period 1978–1983. Using information from initial surveys and bacteriologic sampling of all herds, a set of possible risk factors for each organism was examined. Comparisons between herds with high and zero prevalence of each infection were made. Failure to teat dip and use of common cloths or sponges for udder preparation increased the odds of high prevalence for both organisms. Selective or no dry cow therapy, having tie stall or stanchion housing and using dry massage or no udder preparation increased the risk for Str. agalactiae, but not for S. aureus infection. Milking machine function played a role in the risk of both infections.     

Comparison of phenotypic and genotypic detection of penicillin G resistance of Staphylococcus aureus isolated from bovine intramammary infection

Resistance of Staphylococcus aureus to penicillin G is common among isolates from bovine mastitis. We determined phenotypic resistance to penicillin G for 151 S. aureus isolates derived from dairy cows with intramammary infection by two methods. The methods were determination of minimum inhibitory concentration (MIC) by a standard agar dilution technique and direct testing of beta-lactamase production using a chromogenic cephalosporin, nitrocefin. The results from these tests were compared with the presence of the beta-lactamase (blaZ) gene in the isolates, which was detected by polymerase chain reaction (PCR). Testing beta-lactamase production with nitrocefin was more predictive for the presence of the blaZ gene than the agar dilution method and the results of the former agreed highly with the presence of the blaZ gene in the isolates. In contrast, the resistance breakpoint generally used in the agar dilution method may be too high for prediction of penicillin resistance in S. aureus isolates with borderline MICs. Using this method, 40% of the isolates possessing the blaZ gene were classified as susceptible; however, majority of these isolates produced beta-lactamase when tested with nitrocefin.     

Multifactorial relationships between intramammary invasion by Staphylococcus aureus and bovine leukocyte markers

We explored the hypothesis that the outcome of bacterial invasion (infection or no infection) may depend on immunologic factors when bacterial and environmental factors are kept constant. Leukocyte surface molecules (CD3, CD2, CD4, CD8, CD11b, and CD45r) were assessed before and 3 times after intramammary infusion of Staphylococcus aureus in 5 dairy cows. The somatic cell count (SCC/mL), bacterial count (colony-forming units [CFUs]/mL), ratio of milk phagocytes (mononuclear [Mphi] plus polymorphonuclear [PMN] cells) to lymphocytes (P/L index), and ratio of PMN to Mphi cells (PMN/Mphi index) were determined. Although all cows showed evidence of inflammation resulting from the infusion (the median P/L ratio was 11 times greater 1 d after infusion than before infusion), bacteria were not obtained from the milk of 2 cows. Threshold-like responses, resulting in bacterial counts that approached zero (indicating no infection) and SCCs of less than 500000/mL, were observed when the milk CD2+ lymphocyte proportion exceeded 73% (P < or = 0.007). At 1 d after infusion, 7 immune factors distinguished infected cows from those without infection with more than 95% confidence: compared with infected cows, uninfected cows had higher proportions of CD3+, CD2+, CD4+, and CD8+ T cells, higher densities of CD3 and CD2 molecules per cell, and a higher density of CD11b molecules on milk Mphi cells. At 7 d after infusion, the PMN/Mphi index was lower (94% confidence) in uninfected than in infected cows. At 14 d, the CD2, CD8, and CD45r marker densities were lower than those at 1 d (P < 0.02), findings compatible with memory function. Synergism was suggested by the combined effects of the proportions of CD3+, CD2+, and CD11b+ cells, which explained 75.5% of the bacterial-count variability (P < 0.001); alone, none of these markers predicted CFU variability. These results support further studies aimed at identifying cows capable (or incapable) of early bacterial clearance.     

Staphylococcus aureus leucotoxin LukM/F' is secreted and stimulates neutralising antibody response in the course of intramammary infection

Leucotoxins are regarded as virulence factors of Staphylococcus aureus, but data supporting their importance in infection are scarce. Experimental infections of the mammary glands of six goats with a leucotoxin-producing strain were used to investigate in vivo production of leucotoxin. Leucotoxin M/F' was monitored in milk as well as antibodies to LukM/F' in serum and milk. Leucotoxin antigen was detected by ELISA in milk samples of two goats. The appearance of neutrophils in these samples showed similarity with the altered appearance of neutrophils incubated with purified LukM/F' leucotoxin. Leucotoxic activity was found in the milk samples of these two goats, in particular those of the only goat which developed gangrenous (necrotic) mastitis, and this activity was inhibited by antibodies to LukM/F'. Increases in specific antibody titers occurred in the serum and milk of infected goats, and neutralizing titers increased in serum. The results indicate that the lukM and lukF' genes were functional in vivo, and actively transcribed and translated in the course of mammary infection. These results suggest that LukM/F' can interfere with neutrophil defense activities during infection, and they prompt further studies to investigate the contribution of leucotoxins to staphylococcal mastitis.     

Genetic diversity of Staphylococcus aureus isolated from ovine intramammary infections in Norway

Three hundred and eighty-four Staphylococcus aureus isolates obtained from mammary secretions from 332 ewes kept for meat production were typed by pulsed-field gel electrophoresis (PFGE). The ewes were from 242 flocks located in 13 counties distributed in four regions of Norway. In total, 64 different pulsotypes were identified, 31 of these were represented by a single isolate. Fifty-nine percent of the isolates belonged to one of five closely related pulsotypes. This group of pulsotypes occurred in all the counties. Although widely disseminated, the proportions of the prevalent and closely related pulsotypes differed between the regions. Nine pulsotypes were unique to single regions but the number of isolates belonging to each of these pulsotypes was low. Resistance to penicillin was found in only 3 of the 384 S. aureus isolates. These represented three different single banding patterns, not related to any of the prevalent pulsotypes found.     

Effect on outcome of intramammary challenge exposure with Staphylococcus aureus of somatic cell concentration and presence of an intramammary device

Results of experimental Staphylococcus aureus intramammary challenge of all quarters of 6 cows, each fitted with an intramammary device (IMD) in 2 quarters, and of 10 quarters of 3 cows not fitted with an IMD were reported. Infection was established in all 34 quarters, regardless of presence or absence of an IMD. Neither the course nor severity of early S aureus intramammary infection were influenced by the presence of an IMD or by differences in milk somatic cell (MSC) concentration in the gland at the time of bacterial challenge infusion, up to a MSC concentration of nearly 1 million/ml. Cumulative success of experimental infection in this and a previous study from our laboratory was nearly 100% in glands in which the MSC concentration was less than 1 million/ml and about 17% when the MSC concentration exceeded 1 million/ml.     

The ability of the enzyme-linked immunosorbent assay to detect antibody against Staphylococcus aureus in milk following experimental intramammary infection

Changes in the milk antibody levels against Staphylococcus aureus were measured at the start of an experimental intramammary instillation of either S. aureus (Study I) or Staphylococcus hyicus (Study II). A commercial enzyme-linked immunosorbent assay system was used. Twenty-one Holstein cows were enrolled in Study I and 15 Holstein cows were used in Study II. Pathogen instillation began 21 days before the start of the non-lactating period. Cows received intramammary antibiotic treatment in all quarters immediately after the last milking, the start of the non-lactating period. Lacteal secretions were collected before the start of the non-lactating period, and during the immediate postpartum period in both studies, and during the non-lactating period in Study I. Milk was cultured for mastitis pathogens and S. aureus antibody levels and somatic cell counts were determined from all samples. There was an approximate 2-week delay in the elevation in antibody levels in response to the instillation of S. aureus. Antibody levels remained elevated in cows with S. aureus intramammary infections postpartum, but were below threshold in cows where intramammary infections were cured during the non-lactating period. Antibody levels were elevated by S. hyicus intramammary infections, remained elevated for the first 12 days postpartum, but were below threshold by day 21 postpartum. Cows with incipient intramammary S. aureus infections might be misclassified as false negatives by the antibody test. However, results suggest that cows with S. hyicus intramammary infections that were not cured would not be misclassified if milk is withheld from test for the first 30 days postpartum, as recommended by the manufacturer of the test.     

Comparison of four test procedures to identify Staphylococcus aureus isolated from bovine intramammary infections

A comparison was made between conventional tube coagulase, macrocupsular coagulase, latex agglutination, and miniaturized biochemical test systems for identification of Staphylococcus aureus of bovine origin. A total of 303 gram-positive, catalase-positive cocci of bovine origin were tested. Agreement between each pair of 4-hour tube coagulase, macrocupsular coagulase, latex agglutination, and miniaturized biochemical test results within isolates was greater than 95.0. Seventeen (5.6%) isolates were test negative for 4-hour tube coagulase, but test positive for 24-hour tube coagulase. Thirteen (76.5%) of these isolates were identified as S hyicus, 3 were S aureus, and 1 was not identified.     

Use of partial budgeting to determine the economic outcome of Staphylococcus aureus intramammary infection reduction strategies in three Ohio dairy herds

Efforts to reduce the prevalence of Staphylococcus aureus intramammary infection were monitored in 3 Ohio dairy herds. Bacteriologic culturing of milk from all lactating cows in each herd was completed multiple times to identify infected cows and monitor reduction. Partial budgeting techniques were used to determine the economic outcome of the reduction program. Of particular emphasis was the economic impact of culling to maintain or achieve milk quality premium payments on the basis of bulk tank somatic cell counts. The prevalence of S aureusinfected cows was reduced in each herd. Culturing of milk from all lactating cows appeared to be an effective method to identify infected cows. Although numbers were limited, it also appeared that culturing of composite quarter samples was effective as a herd screening test to identify S aureus-infected cows. Bacteriologic culturing had a negative financial impact in all 3 herds. Using partial budgeting to assess the economic impact of the programs, it was determined that 2 herds experienced negative financial impacts as a result of an excess culling rate when compared with a 12-month baseline period prior to the initiation of the project. All herds had increased milk production per cow during the study as measured by the mature-equivalent method. However, when actual production was considered, increased milk production in each herd was not as great as that of other Ohio herds enrolled on Dairy Herd Improvement Association testing programs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical expression of p53, fibroblast growth factor-b, and transforming growth factor-alpha in feline vaccine-associated sarcomas

Fifty feline sarcomas associated with vaccine-site injection were evaluated to determine the immunohistochemical expression of p53 protein, basic fibroblast growth factor (FGF-b), and transforming growth factor-alpha (TGF-alpha). Forty-one tumors (82%) were fibrosarcomas (FS), eight (16%) were malignant fibrous histiocytomas (MFH), and one (2%) was a chondrosarcoma (CS). Overexpression of p53 protein was observed in the nuclei of tumor cells in 28 (56%) sarcomas; FGF-b expression was found in the cytoplasm of tumor cells in 40 (80%) feline sarcomas, but the staining was more intense in the spindle-shaped cells of FS than in polygonal or round cells of MFH. The single CS faintly expressed FGF-b. The majority of feline vaccine-associated sarcomas (43 of 50, 86%) expressed moderate or intense staining for TGF-alpha in the cytoplasm of tumor cells. Heterogeneous immunolabeling for p53, FGF-b, and TGF-alpha was present in neoplastic, multinucleated giant cells. Intense expression of FGF-b was statistically associated with younger cats (P < 0.01) and with tumors with nodular growth patterns (P = 0.02). In addition, sarcomas negative for p53 protein expressed FGF-b more frequently than did p53-positive tumors (P = 0.04). The frequency of FGF-b immunostaining was significantly higher in sarcomas with intense expression of TGF-alpha (P = 0.05). Immunohistochemical detection of p53 protein, FGF-b, and TGF-alpha suggests that these growth-regulating proteins may play different roles in the development of sarcomas associated with vaccine sites.     

金黄色葡萄球菌荚膜多糖血清1型和2型的研究进展

金黄色葡萄球菌荚膜多糖在其感染中起着重要的作用。文章主要介绍了荚膜多糖血清1型和2型的发现、生化性质及其在免疫中的作用。天然荚膜多糖只对同株型起到保护作用,而纯化的荚膜多糖只有少量的免疫原性。从而了解到完全抗原只能用灭活金黄色葡萄球菌或者纯化的荚膜多糖结合蛋白质来实现。     

Efficacy of vaccination and antimicrobial treatment to eliminate chronic intramammary Staphylococcus aureus infections in dairy cattle

OBJECTIVE: To determine whether a combination of vaccination and extended intramammary antimicrobial treatment would eliminate chronic intramammary Staphylococcus aureus infections in lactating dairy cows. DESIGN: Randomized controlled clinical trial. ANIMALS: 50 dairy cows with chronic mastitis caused by S aureus. PROCEDURE: Cows were identified and paired within herd on the basis of days in milk, lactation number, milk production, and numbers of quarters infected. Treated cows (n=20) received 3 doses of a polyvalent S aureus bacterin on days 1, 15, and 21 of the study along with intramammary administration of pirlimycin in all 4 quarters once daily for 5 treatments (days 16 to 20). Control cows (n=23) received no treatment. Follow-up samples for bacteriologic culture were collected for at least 3 months after treatment to determine treatment success rates. RESULTS: Significantly more S aureus infections were eliminated from treated cows (8/20 [40%]), compared with control cows (2/23 [9%]). The proportion of infected quarters that yielded negative results throughout the follow-up period was also significantly higher in treated cows (13/28 [46%]) than in control cows (2/41 [5%]). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that a combination of vaccination and antimicrobial treatment can be successful in eliminating some cases of chronic intramammary S aureus infections in dairy cattle. However, it is important to consider extended treatment protocols carefully because many cows are likely to remain infected with S aureus despite treatment and vaccination.