Species identification and some characteristics of coagulase-negative staphylococci isolated from bovine udders.

The species of 203 strains of coagulase-negative staphylococci (CNS), isolated from bovine udder quarters was determined; all were tested for hydrophobicity and encapsulation, attributes that may relate to virulence. Twelve species were identified, of which Staphylococcus simulans, (34.5%), S. chromogenes (16.7%), S. epidermidis (13.8%) and S. xylosus (8.9%) were the most frequent. The majority of strains possessed a hydrophilic cell surface. However, strains from two species (S. chromogenes and S. epidermidis) were more hydrophobic than the others. Only five strains were encapsulated (S. xylosus, 3; S. saprophyticus, 1; and S. sciuri, 1). Judging from the low frequencies of hydrophobic and encapsulated strains, and comparing with strains isolated from clinical cases, it is suggested that these properties are not major virulence determinants of CNS.     

Surface properties of Staphylococcus aureus isolated from caprine mastitis

A total of 53 Staphylococcus aureus strains isolated from caprine mastitis were tested for surface hydrophobicity, surface Protein A (SpA), and binding capacity of fibronectin, fibrinogen and type II collagen. Strong positive correlation was found between surface hydrophobicity and SpA, and between surface hydrophobicity and 125I-fibronectin-binding. Regardless of hydrophobicity, the binding of fibrinogen was moderate and type II collagen binding was low. The results indicate that SpA and fibronectin-binding protein contribute to the high relative surface hydrophobicity of S. aureus associated with caprine mastitis.     

Enhanced virulence of Staphylococcus aureus from bovine mastitis induced by growth in milk whey

The virulence towards mice of Staphylococcus aureus strains from bovine mastitis was enhanced upon growth in milk whey compared to homologous organisms grown in tryptic soy broth (TSB). In the mouse mastitis model, S. aureus grown in milk whey caused more severe lesions than homologous strains grown in TSB. Staphylococcus aureus strain F1440 grown in milk whey induced 75% mortality and local necrotic reaction in subcutaneously inoculated mice, whereas the homologous strain grown in TSB caused only 5% mortality and slight skin reaction. Extracellular capsule on milk whey-grown, S. aureus could not be demonstrated. However, diffuse type colony morphology could be correlated with an increased virulence of S. aureus towards mice.     

The major bovine mastitis pathogens have different cell tropisms in cultures of bovine mammary gland cells

We previously showed that Staphylococcus aureus cells adhered mainly to an elongated cell type, present in cultures of bovine mammary gland cells. Moreover, we showed that this adhesion was mediated by binding to fibronectin. The same in vitro model was used here, to study adhesion of other important mastitis pathogens. Like the S. aureus strains, the Streptococcus dysgalactiae strains adhered mainly to elongated cells, which seemed to be mediated by fibronectin binding. In contrast, Streptococcus uberis strains adhered mainly to cubic cells. Since the cubic cells did not express fibronectin and S. uberis cells bound fibronectin less efficiently, the adhesion of S. uberis cells was independent of fibronectin binding. Streptococcus agalactiae strains adhered poorly to both cell types. The specificity and efficiency of adhesion of Escherichia coli strains was strongly strain dependent. None of the S. agalactiae and E. coli strains tested was able to bind fibronectin efficiently. The results suggest that the different mastitis pathogens have different target cell specificities and use different mechanisms to adhere to cells of the bovine mammary gland.     

In vitro growth inhibition of major mastitis pathogens by Staphylococcus chromogenes originating from teat apices of dairy heifers

Earlier field observations suggest that teat apex colonization by Staphylococcus chromogenes pre-partum in dairy heifers protects udder quarters against elevated somatic cell counts early post-partum. To explain these findings, the in vitro inhibitory capability of S. chromogenes from teat apices of heifers towards some major mastitis pathogens was tested using a modified cross-streaking method. Two out of 10 S. chromogenes isolates, both originating from two different teats from the same heifer, consistently inhibited growth of all Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis strains, but none of the Escherichia coli strains. The present study, therefore, supports the protective effect of teat apex colonization by S. chromogenes by in vitro production of inhibitory substances.     

Persistence of staphylococcal species and genotypes in the bovine udder

Staphylococci are a major cause of intramammary infections (IMI) in ruminants. The main aim of this study was to investigate staphylococcal IMI in dairy cattle with emphasis on persistence and distribution of staphylococcal species and genotypes. With a sampling interval of 4-8 weeks, over a year, 4030 samples from 206 cows in 4 herds were collected. Coagulase-negative staphylococci (CNS) and Staphylococcus aureus were detected in 13.2% and 4.2% of the samples, respectively. Selected CNS isolates from quarter milk samples were identified to species level using sodA sequencing. Staphylococcus chromogenes (32%) and Staphylococcus simulans (25%) predominated. The proportion of S. chromogenes was greater in primiparous (52%) than in multiparous cows (12%), while the opposite was the case for Staphylococcus epidermidis (6% and 21%, respectively). Isolates from possibly persistent IMI were selected for pulsed-field gel electrophoresis (PFGE). Six staphylococcal species were found to cause persistent IMI; S. aureus, S. chromogenes, S. simulans, S. epidermidis, Staphylococcus haemolyticus and Staphylococcus warneri. It was shown that several pulsotypes (PTs) within each species were associated with persistent infections, but only a few were spread and caused persistent IMI in multiple cows within a herd. Of special interest was the observation that only one, or a few, strains of each species caused persistent IMI in multiple cows within a same herd. This indicates strain differences with respect to transmissibility and pathogenicity.     

Influence of Staphylococcus aureus exosecretions isolated from bovine mastitis on leukocyte activity in vitro

The role of Staphylococcus aureus and coagulase-negative staphylococcal exosecretions was tested for its ability to elicit in vitro proliferation of bovine blood lymphocytes, which we determined by means of the 3H-thymidine proliferation assay and by flow cytometry. Exosecretions of 32 field strains of S. aureus isolated from bovine udder infection and one of each of S. intermedius (M2), S. hyicus (M5), S. xylosus (M6) and S. chromogenes (M10) were used. Of the 32 S. aureus bacterial exosecretions, only 14 stimulated bovine mononuclear cells to proliferate. A high degree of association was found when the proliferation indexes were compared with the virulence as determined by intracisternal inoculation. All the six S. aureus strains that were categorized as highly virulent and that were tested in the proliferation assay exhibited a proliferation index > 20, whereas the five S. aureus strains that were categorized as low did not stimulate at all. Cells treated with media or Columbia broth supplemented with 0.1% D-glucose, yeast extract, and 0.5% NaCl (CBs) did not exceed 15% of the T-cells double positive with CD25+, whereas incubation with Con A activated the T-cells to display CD25+ up to 90%. Cells treated with one of the exosecretions that stimulated bovine mononuclear cells to proliferate, stimulated CD3+ and CD4+ T-cells to exhibit CD25+ receptor significantly higher (P < 0.05) than that found in media and CBs treatments, but lower than those found in Con A treatments. The exosecretions that did not stimulate mononuclear cells to proliferate also did not activate T-cells to exhibit CD25+ receptor. Con A activated 74% out of the total CD8+ to exhibit ACT2 receptor and 50% out of the total CD4+ to exhibit ACT3 receptor. A few but not all of the exosecretions that activated the CD25 receptor on T-cells also activated the ACT3 receptor on CD4+ cells.     

Haemolytic and proteolytic activity of coagulase-negative staphylococci isolated from mastitis cows

The aim of the present study was to assess the haemolytic and proteolytic activity of coagulase-negative staphylococci (CNS) isolated from cows with mastitis. The study was conducted on 100 CNS strains: S. xylosus (n=28), S. chromogenes (n=26), S. haemolyticus (n=25), S. sciuri (n=14), S. warneri (n=4), S. hominis (n=2), S. saprophyticus (n=1); 22 CNS were isolated from cows with clinical mastitis and 78 from those with subclinical mastitis. The CNS studied showed the ability to produce only alpha-haemolysin and belonged to one strain - S. haemolyticus (21.0% of isolated CNS strains). Haemolysin-positive CNS were responsible for both clinical and subclinical mastitis (22.7% and 20.5%, respectively). The ability to produce protease was found in 31.0% of CNS belonging to two strains: S. chromogenes and S. sciuri. Protease-positive CNS were the etiological factor of both clinical and subclinical mastitis (31.8% and 30.8%, respectively). All S. xylosus, S. warneri, S. hominis, and S. saprophyticus strains were found protease-negative and haemolysin-negative, irrespective of whether they caused clinical or subclinical mastitis in cows.     

Characterization of two proteins of Staphylococcus aureus isolated from bovine clinical mastitis with homology to glyceraldehyde-3-phosphate dehydrogenase

Staphylococcus aureus is the most common causative agent of bovine mastitis and vaccines developed to control this disease showed limited protection due in part to the lack of common antigens among the mastitis isolates. We isolated and identified two genes encoding proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from a S. aureus strain isolated from bovine clinical mastitis. The GapB and GapC proteins share considerable homology to the GapB and GapC products of human strains of S. aureus. These two proteins could be distinguished by their different GAPDH activities and binding to bovine transferrin properties. Both gapB and gapC genes were conserved in 11 strains tested, and the GapC protein was present on the surface of all S. aureus strains.     

Coagulase-negative staphylococci-emerging mastitis pathogens

Coagulase-negative staphylococci (CNS) have become the most common bovine mastitis isolate in many countries and could therefore be described as emerging mastitis pathogens. The prevalence of CNS mastitis is higher in primiparous cows than in older cows. CNS are not as pathogenic as the other principal mastitis pathogens and infection mostly remains subclinical. However, CNS can cause persistent infections, which result in increased milk somatic cell count (SCC) and decreased milk quality. CNS infection can damage udder tissue and lead to decreased milk production. Staphylococcus simulans and Staphylococcus chromogenes are currently the predominant CNS species in bovine mastitis. S. chromogenes is the major CNS species affecting nulliparous and primiparous cows whereas S. simulans has been isolated more frequently from older cows. Multiparous cows generally become infected with CNS during later lactation whereas primiparous cows develop infection before or shortly after calving. CNS mastitis is not a therapeutic problem as cure rates after antimicrobial treatment are usually high. Based on current knowledge, it is difficult to determine whether CNS species behave as contagious or environmental pathogens. Control measures against contagious mastitis pathogens, such as post-milking teat disinfection, reduce CNS infections in the herd. Phenotypic methods for identification of CNS are not sufficiently reliable, and molecular methods may soon replace them. Knowledge of the CNS species involved in bovine mastitis is limited. The dairy industry would benefit from more research on the epidemiology of CNS mastitis and more reliable methods for species identification.     

Prevalence and herd-level risk factors for intramammary infection with coagulase-negative staphylococci in Dutch dairy herds

In this study, the prevalence of intramammary infection (IMI) with coagulase-negative staphylococci (CNS) in The Netherlands was estimated on 49 randomly selected herds with at least 40 lactating cows. In total, 4220 quarter milk samples were collected. The prevalence of CNS IMI in The Netherlands was estimated at 10.8% at quarter level and 34.4% at cow level, making it the most frequently isolated group of pathogens. Fourteen species of CNS were identified; the most frequently isolated species was Staphylococcus chromogenes (30.3%) followed by Staphylococcus epidermidis (12.9%) and Staphylococcus capitis (11.0%). Prevalence of CNS IMI was higher in heifers compared to older cows. Geometric mean quarter SCC of CNS-positive quarters was 109,000 cells/ml, which was approximately twice as high as culture-negative quarters. Quarters infected with S. chromogenes, S. capitis and Staphylococcus xylosus had a higher SCC (P<0.05) than culture-negative quarters, while quarters that were culture-positive for S. epidermidis and Staphylococcus hyicus tended to have a higher SCC than culture-negative quarters. An increased prevalence of CNS IMI was associated with the herd-level variables source of drinking water not being tap water, housing of dry cows in one group instead of multiple groups, measurement of cow SCC every month, udder health monitoring by the veterinarian, pasturing during outdoor season, percentage of stalls contaminated with milk, and BMSCC>250,000 cells/ml. Although a causal relation between these factors and prevalence of CNS is not proven and for some factors not even likely, knowledge of the associations found may be helpful when approaching CNS problems on dairy farms.     

Prepartum teat apex colonization with Staphylococcus chromogenes in dairy heifers is associated with low somatic cell count in early lactation

A high number of dairy heifers freshen with udder health problems. The prevalence of teat apex colonization (TAC) with Staphylococcus chromogenes, one of the most widespread coagulase-negative staphylococci (CNS) in milk samples from freshly calved dairy heifers, was measured cross-sectionally in non-lactating heifers on eight commercial dairy farms in Belgium. The influence of age on this prevalence, and the association between teat apex colonization with S. chromogenes prepartum and quarter milk somatic cell count (SCC) in early lactation were studied. In total, 492 teat apices were sampled from 123 heifers. The age of the heifers varied from 8 to 34 months. Overall, 20% of the heifers had at least one teat apex colonized with S. chromogenes. Of all teats sampled, 10% were colonized with S. chromogenes. The chance of having at least one teat apex colonized with S. chromogenes increased with age of the heifer. The presence of prepartum teat apex colonization with S. chromogenes was not associated with intramammary infection (IMI) early postpartum with the same bacterium. On the contrary, teat apex colonization with S. chromogenes prepartum appeared to protect quarters in the first few days of lactation from having somatic cell count >or=200000cells/ml milk, commonly accepted as the threshold for intramammary infection.     

Host response in bovine mastitis experimentally induced with Staphylococcus chromogenes

An experimental infection model was developed to study host response to intramammary infection in cows caused by Staphylococcus chromogenes. CNS intramammary infections have become very common in modern dairy herds, and they can remain persistent in the mammary gland. More information would be needed about the pathophysiology of CNS mastitis, and an experimental mastitis model is a means for this research. Six primiparous Holstein-Friesian cows were challenged with S. chromogenes 4 weeks after calving. One udder quarter of each cow was inoculated with 2.1 x 10(6)cfu of S. chromogenes. All cows became infected and clinical signs were mild. Milk production of the challenged quarter decreased on average by 16.3% during 7 days post-challenge. Cows eliminated bacteria in a few days, except for one cow which developed persistent mastitis. Milk indicators of inflammation, SCC and N-acetyl-beta-D-glucosaminidase (NAGase) returned to normal within a week. Milk NAGase activity increased moderately, which reflects minor tissue damage in the udder. Concentrations of serum amyloid A (SAA) and milk amyloid A (MAA) were both elevated at 12h PC. MAA was affected by the milking times, and was at its highest before the morning milking. In our experimental model, systemic acute phase protein response with SAA occurred as an on-off type reaction. In conclusion, this experimental model could be used to study host response in CNS mastitis caused by the main CNS species and also for comparison of the host response in a mild intramammary infection and in more severe mastitis models.     

Vaccine development for the prevention of staphylococcal mastitis in dairy cows

Staphylococci are the most common and costly mammary disease of dairy cattle worldwide. Target of RNAIII Activating Protein (TRAP), a membrane associated 167AA protein, is highly conserved among staphylococci and was shown in Staphylococcus aureus to be involved in bacterial stress response. The aims of this study were to test the safety and efficacy of recombinant TRAP (rTRAP) vaccine in dairy animals. The vaccine was safe as 2-3 subcutaneous injections of rTRAP (54-100 μg) with adjuvant ISA 206 to cows and goats did not lead to any abnormal symptoms of sensitivity to the vaccine. The rTRAP vaccine was immunogenic and caused the induction of a humoral immune response that remained high for at least 160 d post second immunization. The rTRAP vaccine was efficacious; at parturition only 13.5% (5/37) heifers in the immunized group were infected with Staphylococcus chromogenes as compared to 42.9% (18/42) in the non-immunized group. Additionally, when cows were immunized in mid-lactation, the difference between somatic cell count (SCC) in immunized and control animals was profound (45 ± 7 vs. 470 ± 194, respectively). At the same time, the difference in milk yield was also evident (48.3 ± 1.4 L d(-1)vs. 44.3 ± 0.9 L d(-1), respectively). Put together, these studies indicate the value of the rTRAP vaccine in preventing new udder infections by staphylococci, which significantly lead to lowered SCC and some increase in milk yield. TRAP is conserved among all strains and species and is constitutively expressed in any strain of S. aureus or CNS tested so far, including those isolated from cows. TRAP may thus serve as a universal anti-staphylococcus vaccine.     

Adhesion of Streptococcus gallolyticus strains to extracellular matrix proteins

Fourteen pigeon Streptococcus gallolyticus strains of differing virulence, were tested for their ability to adhere to immobilised fibronectin, collagen types I, III and IV. Eight, 2 and 13 strains were able to bind fibronectin, collagen types III and IV, respectively. None of the strains adhered to collagen type I. Heat treatment, proteolytic digestion or periodate treatment reduced the binding of S. gallolyticus to fibronectin and collagen type IV, suggesting that surface receptors contain proteins and carbohydrates. Although binding to these extracellular matrix proteins can play a role in the pathogenesis of streptococcosis in pigeons, binding properties could not be related to virulence, indicating that other factors determine differences in virulence among pigeon S. gallolyticus strains. Adhesion to collagen type IV may account in part for the distribution pattern of the lesions observed in naturally and experimentally infected pigeons.     

Specificity of the anti-inflammatory effect of a staphylococcal cell wall extract in the bovine udder

The cell wall of Staphylococcus aureus (strains 21 and Glaxo) was treated with deoxycholate and the insoluble residue was solubilised with lysozyme. The effect of the extract in modulating the inflammatory response due to infection of the lactating bovine udder was evaluated. Cows were infected with S. aureus strain 21 or Streptococcus agalactiae, with or without the cell wall extracts. The clinical response to infection was assessed, and milk samples collected up to 30 h were assayed for antitrypsin and NAGase levels, somatic cell count, and for the ability of whey to support bacterial growth. The extracts markedly reduced the inflammatory response elicited by both S. aureus and S. agalactiae, indicating the effect was non-specific. The extract from strain 21 was generally more effective than that from strain Glaxo.     

Production of bacteriocins by coagulase-negative staphylococci involved in bovine mastitis

In the present study, 188 coagulase-negative Staphylococcus (CNS) strains were isolated from bovine mastitis cases from 56 different Brazilian dairy herds, located in the Southeast region of the country, and were tested for antimicrobial substance production. Twelve CNS strains (6.4%) exhibited antagonistic activity against a Corynebacterium fimi indicator strain. Most antimicrobial substances were sensitive to proteolytic enzymes suggesting that they might be bacteriocins (Bac). Amongst the CNS producers, six were identified as S. epidermidis, two as S. simulans, two as S. saprophyticus, one as S. hominis and one as S. arlettae. Plasmid profile analysis of these strains revealed the presence of at least one plasmid. The Bac(+) strains presented either no or few antibiotic resistance phenotypes. Three strains were shown to produce a bacteriocin either identical or similar to aureocin A70, a bacteriocin previously isolated from an S. aureus strain isolated from food. The remaining Bac(+) strains produce antimicrobial peptides that seem to be distinct from the best characterised staphylococcal bacteriocins described so far. Some of them were able to inhibit Listeria monocytogenes, an important food-borne pathogen, and several strains of Streptococcus agalactiae associated with bovine mastitis, suggesting a potential use of these bacteriocins either in the prevention or in the treatment of streptococcal mastitis.     

Staphylococci isolated from healthy goats.

A study was made of the staphylococcal population on the skin and on the nasal mucosa and in the milk of 133 healthy goats. Of a total of 346 strains isolated and characterised as belonging to the genus Staphylococcus, 74 (21.4%) were coagulase-positive (68 S. aureus and 6 S. hyicus), and 272 (78.6%) coagulase-negative. The novobiocin-sensitive species S. haemolyticus (23.5%), S. warneri (16.5%), S. epidermidis (11.8%), S. chromogenes (8.5%), S. caprae (6.6%) and S. hyicus (2.6%), and the novobiocin-resistant species S. xylosus (8.5%), S. sciuri (7.4%), S. saprophyticus (4.8%), S. cohnii (2.2%), S. lentus (1.1%), S. equorum (1.1%) and S. kloosii (1.1%) were identified. Twelve (4.4%) of coagulase-negative strains remained unidentified. Strains isolated in the skin of the udder and teats of the 133 goats were mainly novobiocin-sensitive coagulase-negative staphylococci, the most prevalent species being S. haemolyticus, S. warneri and S. epidermidis. Staphylococci indicative of subclinical infection were determined in the milk of 47 (35.3%) of the 133 goats sampled.     

Intra-species diversity and epidemiology varies among coagulase-negative Staphylococcus species causing bovine intramammary infections

Although many studies report coagulase-negative staphylococci (CNS) as the predominant cause of subclinical bovine mastitis, their epidemiology is poorly understood. In the current study, the genetic diversity within four CNS species frequently associated with bovine intramammary infections, Staphylococcus haemolyticus, S. simulans, S. chromogenes, and S. epidermidis, was determined. For epidemiological purposes, CNS genotypes recovered from bovine milk collected on six Flemish dairy farms were compared with those from the farm environment, and their distribution within the farms was investigated. Genetic diversity was assessed by two molecular typing techniques, amplification fragment length polymorphism (AFLP) and random amplification of polymorphic DNA (RAPD) analysis. Subtyping revealed the highest genetic heterogeneity among S. haemolyticus isolates. A large variety of genotypes was found among environmental isolates, of which several could be linked with intramammary infection, indicating that the environment could act as a potential source for infection. For S. simulans, various genotypes were found in the environment, but a link with IMI was less obvious. For S. epidermidis and S. chromogenes, genetic heterogeneity was limited and the sporadic isolates from environment displayed largely the same genotypes as those from milk. The higher clonality of the S. epidermidis and S. chromogenes isolates from milk suggests that specific genotypes probably disseminate within herds and are more udder-adapted. Environmental sources and cow-to-cow transmission both seem to be involved in the epidemiology of CNS, although their relative importance might substantially vary between species.     

Is the biofilm formation and slime producing ability of coagulase-negative staphylococci associated with the persistence and severity of intramammary infection?

Biofilm and slime formation assists bacteria in avoiding the host immune defence and antimicrobial therapy. It is suspected to affect the severity or persistence of mastitis caused by coagulase-negative staphylococci (CNS), which are a common cause of bovine mastitis. The phenotypic biofilm formation ability of 244 CNS isolates (199 isolates from bovine mastitis and 52 type and reference strains) was investigated with a tissue culture plate (TCP) assay and fluorescent in situ hybridization (FISH). Slime production of the strains was assessed using Congo red agar (CRA) plates. Additionally, genes encoding the adhesion proteins MSCRAMM (microbial surface components recognizing adhesive matrix molecules) and biofilm-associated proteins (bap) were detected. The severity of intramammary infection (IMI) in mastitis from which the isolates originated was measured with milk N-acetyl-β-D-glucosaminidase (NAGase) activity. One-third of isolates from mastitis produced biofilm when analysed with TCP or FISH. The kappa test value, measuring the agreement between two tests, differed between CNS species. Slime production was less frequent for isolates of the common mastitis species Staphylococcus chromogenes (0.2% of isolates produced slime) and Staphylococcus simulans (3.5%) compared to Staphylococcus epidermidis (40%). No association was found between the phenotypic ability to form biofilm and the persistence of IMI or severity of mastitis. Slime production was rare in isolates originating from IMI. Only 12.7% of isolates from persistent IMI and 1.8% of isolates from spontaneously eliminated IMI produced slime. The eno gene encoding laminin-binding protein was most frequently detected among the isolates from mastitis, 75% of them having this gene. Only a few other MSCRAMM genes were detected.