Comparison of Virulence Gene Identification, Ribosomal Spacer PCR, and Pulsed-Field Gel Electrophoresis for Typing of Staphylococcus aureus Strains Isolated from Cases of Subclinical Bovine Mastitis in the United States.

Staphylococcus aureus is one of the most important pathogens causing contagious mastitis in dairy cattle worldwide. The objectives of this study were to determine if recently described S. aureus genotype B was present among previously characterized isolates from cases of bovine intramammary infection in the United States and to compare pulsed-field gel electrophoresis (PFGE) to the combination of ribosomal spacer PCR (RS-PCR) and virulence gene identification for typing of S. aureus strains. The hypothesis was that isolates that were previously characterized as contagious would be identified as genotype B and that the results of the two strain-typing methods would be comparable. Isolates were selected from a collection of S. aureus isolates from eight dairy farms. Mammary quarter milk somatic cell count (SCC) and N-acetyl-ß-d-gluconaminidase (NAGase) activity data were known and used to evaluate strain pathogenicity. RS-PCR was performed with conventional gel electrophoresis, and PCR was used for toxin gene identification. RS-PCR patterns were associated with a specific virulence gene pattern, as previously reported. Five RS-PCR banding patterns were identified. None of the isolates were characterized as genotype B. No association between RS-PCR types and milk SCC was found; however, NAGase activity was significantly higher in milk from mammary glands infected with RS-PCR banding type 1 (RSP type 1) than in milk from those infected with RSP type 2. The discriminatory power values were 1.0 and 0.46 for PFGE and RS-PCR, respectively. These data suggest that genotype B may have a limited geographic distribution and that PFGE is more discriminatory than RS-PCR performed with conventional gel electrophoresis for typing of S. aureus isolates of bovine origin.

A Staphylococcus xylosus isolate with a new mecC allotype.

Recently, a novel variant of mecA known as mecC (mecA(LGA251)) was identified in Staphylococcus aureus isolates from both humans and animals. In this study, we identified a Staphylococcus xylosus isolate that harbors a new allotype of the mecC gene, mecC1. Whole-genome sequencing revealed that mecC1 forms part of a class E mec complex (mecI-mecR1-mecC1-blaZ) located at the orfX locus as part of a likely staphylococcal cassette chromosome mec element (SCCmec) remnant, which also contains a number of other genes present on the type XI SCCmec.

Time to clearance of mycoplasma mastitis: the effect of management factors including milking time hygiene and preferential culling.

Factors associated with time to clearance of mycoplasma mastitis were studied in 18 dairy cattle herds. Most herds cleared mycoplasma mastitis within 1 month; < 50% of the herds culled diseased cows preferentially, yet culling was not associated with hastened clearance. Other known mastitis biosecurity and management practices were not associated with clearance time.

Délai de guérison de la mammite à mycoplasmes : effet des facteurs de gestion incluant l'hygiène lors de la traite et la réforme préférentielle. Les facteurs associés au délai de guérison de la mammite à mycoplasmes ont été étudiés dans 18 troupeaux laitiers. La plupart des troupeaux se débarrassaient de la mammite à mycoplasmes dans un délai d'un mois; < 50 % des troupeaux procédaient à une réforme préférentielle des vaches malades, pourtant la réforme n'a pas été associée à une guérison hâtive. Les autres pratiques de biosécurité et de gestion connues pour la mammite n'ont pas été associées au délai de guérison.(Traduit par Isabelle Vallières).

An Enterotoxin-Bearing Pathogenicity Island in Staphylococcus epidermidis.

Cocolonization of human mucosal surfaces causes frequent encounters between various staphylococcal species, creating opportunities for the horizontal acquisition of mobile genetic elements. The majority of Staphylococcus aureus toxins and virulence factors are encoded on S. aureus pathogenicity islands (SaPIs). Horizontal movement of SaPIs between S. aureus strains plays a role in the evolution of virulent clinical isolates. Although there have been reports of the production of toxic shock syndrome toxin 1 (TSST-1), enterotoxin, and other superantigens by coagulase-negative staphylococci, no associated pathogenicity islands have been found in the genome of Staphylococcus epidermidis, a generally less virulent relative of S. aureus. We show here the first evidence of a composite S. epidermidis pathogenicity island (SePI), the product of multiple insertions in the genome of a clinical isolate. The taxonomic placement of S. epidermidis strain FRI909 was confirmed by a number of biochemical tests and multilocus sequence typing. The genome sequence of this strain was analyzed for other unique gene clusters and their locations. This pathogenicity island encodes and expresses staphylococcal enterotoxin C3 (SEC3) and staphylococcal enterotoxin-like toxin L (SElL), as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and immunoblotting. We present here an initial characterization of this novel pathogenicity island, and we establish that it is stable, expresses enterotoxins, and is not obviously transmissible by phage transduction. We also describe the genome sequence, excision, replication, and packaging of a novel bacteriophage in S. epidermidis FRI909, as well as attempts to mobilize the SePI element by this phage.

Clinical Mycoplasma bovis mastitis in prepubertal heifers on 2 dairy herds.

Findings of herd investigations of heifers with prepubertal mastitis are presented. Mycoplasma bovis was isolated from lacteal secretions and tissue samples of necropsied heifers; the same strain infected dams and herd mates. Vertical transmission is suggested in this first report of intramammary infections of M. bovis in peripubertal heifers.

Interactive effects of dexamethasone and opsonized Mycoplasma bovis on bovine neutrophil function in vitro.

Previously we had reported that exposure to high levels of glucocorticoids, and to unopsonized Mycoplasma bovis, has a negative interactive effect on bovine neutrophil function in vitro, and this interactive effect was a function of M. bovis strain differences. Here we hypothesized that in vitro treatment of bovine neutrophils by glucocorticoid would impair phagocytosis of opsonized M. bovis compared to non-treated neutrophils and such impairment would be a function of M. bovis strain differences. Neutrophils isolated from 20 mid-lactation cows were treated with immunosuppressive dose of 5 × 10-4 M dexamethasone or placebo and incubated with one of four opsonized M. bovis strains that had been isolated from bovine origin. After incubation neutrophil function measured included: percentage reduction in log10 of M. bovis CFU/ml, percentage of phagocytizing neutrophils, phagocytized M. bovis per neutrophil, and killed M. bovis per neutrophil. Least square means of all neutrophil groups were contrasted using linear mixed-effects models. Effects due to strain, treatment, and their interaction on neutrophil function measured by the number of phagocytized M. bovis per neutrophil and number of killed M. bovis per neutrophil were different (P < 0.05). However, no significant strain by treatment interaction effect on percentage reduction in log10 of M. bovis CFU/ml was found. Neither a strain nor a strain by treatment interaction was found to affect the percentage phagocytizing neutrophils. These findings might explain in part the association of stressful events with subsequent outbreaks of Mycoplasma bovis associated bovine diseases.

Unique features of bovine lymphocytes exposed to a staphylococcal enterotoxin.

We previously demonstrated that stimulation of bovine peripheral blood mononuclear cells (PBMCs) with staphylococcal enterotoxin C (SEC), led to an inversion of the CD4(+):CD8(+)T cell ratio and generation of an atypical CD8(+)T cell subpopulation expressing CD26. In the present study, we examined T cell apoptosis and proliferation profiles of PBMC subpopulations in cultures stimulated with SEC. Unlike when stimulated with concanavalin A, nucleic acid synthesis in bovine PBMC cultures stimulated with SEC was low during the first four days but increased greatly on day 5. In contrast, nucleic acid synthesis in human PBMC cultures stimulated with SEC increased continuously. To investigate the mechanism of delayed bovine T cell proliferation, various cell phenotypes were monitored. The inversion of the bovine CD4(+):CD8(+)T cell ratio in PBMC cultures stimulated by SEC was associated with higher proliferation and lower apoptosis of CD8(+)T cells compared to CD4(+)T cells. The mRNA levels for interleukin (IL)-4 and IL-13 were sustained over 4 days but IL-12 mRNA levels dropped to background on day 2. These data suggest that SEC induces a prolonged Th-2- biased microenvironment, and together with the inversion of the bovine CD4(+):CD8(+)T cell ratios in bovine PBMC cultures with SEC, may in part explain the inability of the mammary immune system to establish an effective response to Staphylococcus aureus infections.

Immunosuppression by T regulatory cells in cows infected with Staphylococcal superantigen.

Our recent study has provided that the in vitro SEC-induced proliferation of bovine T cells is preceded by a period of a non-proliferative immunoregulation of T cells that may be associated with cytokine production regulated by type 1 or type 2 T cells. Inversion of CD4(+):CD8(+) T cell ratio and induction of CD8(+) T cells with immunoregulatory activity could increase the probability of intracellular survival of Staphylococcus aureus (S. aureus). The increase of activated CD8(+)(ACT2(+) BoCD8(+)) T cells in cows with mastitis caused by S. aureus may be associated with immune-regulatory function in the bovine mammary gland. The difference and similarity between bovine activated CD8(+) T cells (CD8(+)CD26(+)) and well-established human CD4(+)CD25(+) T regulatory (Tr) cells may help to reveal their unique immune regulatory system in the host infected with S. aureus.

Effects of dexamethasone and Mycoplasma bovis on bovine neutrophil function in vitro.

It is well established that exposure either to elevated levels of glucocorticoids, or to Mycoplasma bovis (M. bovis), has a negative effect on bovine neutrophil function. The objective of this research was to determine whether in vitro treatment of bovine neutrophils by M. bovis strains (n=4) and glucocorticoids would additively impair phagocyte function. Twenty, healthy, dairy cows were enrolled. Whole blood was collected from all cows for neutrophil isolation. Phagocytosis and the generation of superoxide anion (O2(-)) were tested in vitro by incubation of neutrophils with FITC labeled Escherichia coli (E. coli) and cytochrome c after treatment. Treatments included: NM1-4D (neutrophils treated with dexamethasone and exposed to one of the four M. bovis strains); NM1-4 (neutrophils exposed to one of the four M. bovis strains only); ND (neutrophils treated with dexamethasone only); and N (non-treated control neutrophils). The overall percentages of neutrophils phagocytizing E. coli were: 32%, 51%, 37%, and 53% ± 5.25% for treatments NM1-4D, NM1-4, ND, and N, respectively. The overall statistically transformed means of phagocytized E. coli per neutrophil were: 1.37, 1.72, 1.33, and 1.67 ± 0.057 for treatments NM1-4D, NM1-4, ND, and N, respectively. The overall statistically transformed means of neutrophil O2(-) production were: 8.60, 11.91, 9.01, and 12.21 ± 0.21 nmol/10(6) for treatments NM1-4D, NM1-4, ND, and N, respectively. Exposure of neutrophils to M. bovis plus dexamethasone had an additive effect on generation of reactive oxygen species (p=0.0057), but not on the percentage of neutrophils phagocytizing E. coli (p=0.0817) or number of E. coli phagocytized per neutrophil (p=0.2946). Only one of the four M. bovis strains had a negative effect on neutrophil phagocytic function. Dexamethasone treatment consistently decreased neutrophil function as indicated by decreased percentage of neutrophils phagocytizing E. coli, decreased number of E. coli phagocytized per neutrophil, and decreased neutrophil O2(-) production, compared to controls (p<0.0001). Results suggested a synergistic effect of in vitro incubation of glucocorticoids and M. bovis on reduction of bovine neutrophil function as measured by generation of reactive oxygen species. These findings may explain in part the interaction between stressful events and outbreak of Mycoplasma bovis associated bovine disease.

Methodology for quantifying residues of chlorhexidine in raw dairy milk.

A residue method was developed as part of a pharmacokinetics study to determine the elimination of chlorhexidine in raw milk after intramammary infusion into dairy cows affected with bovine mastitis. The developed liquid/liquid and solid-phase extraction procedures effectively reduced sources of milk product interferences in the final extract. By optimizing mobile-phase pH buffer/acetonitrile gradient conditions and employing an end-capped reverse-phase polar embedded-phase chromatographic column, excellent peak resolution was achieved without the additional need of mobile-phase amine modifiers or ion-pairing reagents. The combined cleanup and chromatographic method steps reported herein were sensitive and reliable for determining the pharmacokinetic elimination of chlorhexidine following intramammary infusion. The residue method was found to be rugged with a lower detection limit of 0.1 ppm.

Elimination kinetics of chlorhexidine in milk following intramammary infusion to stop lactation in mastitic mammary gland quarters of cows.

OBJECTIVE: To evaluate the elimination kinetics of chlorhexidine in milk when used as an intramammary infusion to stop lactation in cows. DESIGN: Prospective study. ANIMALS: 6 cows. PROCEDURE: The study was performed in 2 phases. Three cows were studied in each phase. All cows were treated with chlorhexidine suspension by infusion into a mastitic mammary gland quarter after 2 milkings 24 hours apart. Foremilk samples (100 mL) were collected from treated and untreated (controls) mammary gland quarters of each cow. Chlorhexidine was extracted from raw milk, and residue concentrations were quantified by use of high-performance liquid chromatography. Foremilk samples from days 2, 5, and 8 were analyzed in phase I, and samples from time 0 and days 3, 7, 14, 21, 28, 35, and 42 were analyzed in phase II. RESULTS: In phases I and II, there was no quantifiable transference of chlorhexidine to milk in untreated mammary gland quarters. Measurable chlorhexidine residues were found in milk from treated mammary gland quarters of 2 cows throughout the 42-day sample period in phase II. Estimated mean elimination half-life for chlorhexidine in milk was 11.5 days. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of the long elimination half-life of chlorhexidine in milk from treated mammary gland quarters, the lack of human dietary exposure data to suggest a food tolerance for chlorhexidine in food products, and the Food and Drug Administration's published zero tolerance for chlorhexidine in uncooked edible calf tissues, we do not recommend extralabel use of chlorhexidine suspension as a treatment to stop lactation in mastitic mammary gland quarters of cows.

Complete Genome Sequence of the Bovine Mastitis Pathogen Mycoplasma californicum Strain ST-6T (ATCC 33461T).

Mycoplasma californicum is one of several mycoplasmal species associated with bovine mastitis. The complete genome sequence of 793,841 bp has been determined and annotated for the M. californicum ST-6 type strain, providing a resource for the identification of surface antigens and putative pathoadaptive features.

Effects of tulathromycin on incidence of various diseases and growth of young heifers.

OBJECTIVE: To determine the effects of administration of 1 dose of tulathromycin on the incidence of various diseases and growth, identify risk factors for slow growth, and determine the association of Mycoplasma bovis status with the incidence of otitis media in calves. DESIGN: Randomized controlled trial and cross-sectional study. ANIMALS: 788 dairy heifer calves (median age, 3 days). PROCEDURES: Calves received tulathromycin or a saline (0.9% NaCl) solution control treatment once. Calves were observed daily for 8 weeks by farm staff to detect diseases. Nasal swab specimens were collected from some calves for Mycoplasma spp culture. RESULTS: Tulathromycin-treated calves had significantly lower odds of developing otitis media (OR, 0.41; 95% confidence interval, 0.58 to 0.82) versus control calves. Control calves had significantly higher odds of developing diarrhea (OR, 1.8; 95% confidence interval, 1.2 to 2.6) versus tulathromycin-treated calves. Control calves and those with failure of passive transfer, fever, lameness, respiratory tract disease, or diarrhea had significantly lower average daily gain versus other calves. Seventeen of the 66 (26%) calves that underwent repeated testing had positive Mycoplasma spp culture results, but positive results were not associated with otitis media. One of 42 calves with otitis media tested for Mycoplasma spp had positive results, and 1 of 43 age-matched calves without otitis media had positive results. CONCLUSIONS AND CLINICAL RELEVANCE: Tulathromycin-treated calves in this study had a lower incidence of diarrhea and otitis media versus control calves. Various diseases had negative effects on average daily gain. Mycoplasma bovis status was not associated with otitis media in calves.

Dissimilarity of ccrAB gene sequences between methicillin-resistant Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus among bovine isolates in Korea.

The sequences of the ccrAB genes from bovine-, canine- and chicken-originating methicillin-resistant Staphylococcus (S.) epidermidis (MRSE) and bovine methicillin-resistant Staphylococcus (S.) aureus (MRSA) were compared to investigate the frequency of intra-species horizontal transfer of the staphylococcal cassette chromosome mec (SCCmec) complex. Nineteen MRSE strains were isolated from bovine milk, chickens, and dogs, and their genetic characteristics were investigated by multilocus sequence typing and SCCmec typing. Among the animal MRSE strains, the most frequent SCCmec type was type IV, which consisted of the type B mec complex and ccrAB type 2. The ccrA2 and ccrB2 genes were sequenced from the bovine, chicken and canine MRSE strains and compared with those of the bovine MRSA strains. The sequences generally clustered as MRSA and MRSE groups, regardless of the animal source. Additionally, no bovine MRSE sequence was associated with the bovine MRSA groups. Although most of the bovine MRSE and MRSA isolates possessed SCCmec type IV sequences, our results suggest that the intra-species gene transfer of the SCCmec complex between bovine S. aureus and bovine S. epidermidis strains is not a frequent event.

Mycoplasma mastitis: causes, transmission, and control.

Mycoplasma mastitis is an emerging mastitis pathogen. Herd prevalence has increased over the past decade, and this increase parallels the increase in average dairy herd size. It has been documented that the importation of cattle into a herd can result in new cases of Mycoplasma disease in general and Mycoplasma mastitis specifically. Thus, expanding herds are likely to have a greater incidence of this disease. Transmission of the agent can result from either contact with diseased animals or with colonized or asymptomatically infected cattle. Initial transmission might occur via nose-to-nose contact and result in an outbreak of Mycoplasma mastitis, or it might occur during the milking time. This would suggest that new, incoming animals should be quarantined before being comingled with original herd animals. Quarantining does not seem to be a biosecurity strategy often practiced in control of Mycoplasma mastitis and may not be warranted in herds with excellent milking time hygiene practices. The ability to monitor for the incipient stages of an outbreak, often done through bulk tank milk culturing, is recommended.

Prevalence and antibiotic resistance of mastitis pathogens isolated from dairy herds transitioning to organic management.

Changes in udder health and antibiotic resistance of mastitis pathogens isolated from dairies upon conversion from conventional to organic management over a 3-year period was studied. Coagulase-negative staphylococci (CNS) were the most prevalent mastitis pathogens isolated. CNS were significantly less resistant to ß-lactam antibiotics when isolated from milk after the herd transitioned to organic management. Cessation of the use of antimicrobial therapies in dairies in combination with organic management could lead to a reduction in the antimicrobial resistance of mastitis pathogens.

Discrimination between Mycoplasma and Acholeplasma species of bovine origin using digitonin disc diffusion assay, nisin disc diffusion assay, and conventional polymerase chain reaction.

Microbiological culture of milk samples has been used as a standard diagnosis for Mycoplasma mastitis. This technique is effective in isolating mollicutes that are Mycoplasma-like; however, isolates may be misinterpreted as Acholeplasma species, which are indistinguishable from Mycoplasma species by culture. A study to contrast the abilities of 2 culture-based tests, digitonin and nisin disc diffusion assays and a conventional polymerase chain reaction (PCR) technique, to discriminate between Mycoplasma and Acholeplasma was performed using 16S ribosomal RNA gene partial sequencing as the gold standard of comparison. A total of 288 bovine mollicute field isolates (248 from milk and 40 from other organ sites) and 13 reference strains were tested. Results obtained from the digitonin disc diffusion assay when it was performed with all field isolates were 92.7% and 99.0% in agreement with the gold standard using 5 mm and 3 mm of zone of growth inhibition as thresholds, respectively. Considering only milk isolates, agreements between the digitonin disc diffusion assay with the gold standard were 97.2% and 100% using 5 mm and 3 mm of zone of growth inhibition as thresholds, respectively. Culture identification using the nisin disc diffusion assay and the PCR was in a 100% agreement with the gold standard. Comparable results using culture-based nisin and digitonin disc diffusion assays, and PCR, to distinguish Mycoplasma and Acholeplasma species was found, especially for isolates from bovine milk.

Detection of classical and newly described staphylococcal superantigen genes in coagulase-negative staphylococci isolated from bovine intramammary infections.

The coagulase-negative staphylococci (CNS) are the most prevalent mastitis pathogen group yet their virulence characteristics have not been well described. We investigated the presence of 19 classical and newly described staphylococcal superantigen (SAg) genes in CNS isolates from bovine intramammary infections (IMI). A total of 263 CNS representing 11 different Staphylococcus spp. were examined, and 31.2% (n=82) of CNS isolates had one or more SAg genes; there were 21 different SAg gene combinations. The most prevalent combination of SAg genes (seb, seln and selq; n=45) was found in S. chromogenes, S. xylosus, S. haemolyticus, S. sciuri subsp. carnaticus, S. simulans and S. succinus. The genes for SAgs appear to be widely distributed amongst CNS isolated from bovine IMI.

Comparison of phenotypic and genotypic methods for the species identification of coagulase-negative staphylococcal isolates from bovine intramammary infections.

Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens from cows with intramammary infection (IMI). Although API STAPH ID 20, a commercially available identification system, and PCR-restriction fragment length polymorphism (PCR-RFLP) of the gap gene (gap PCR-RFLP) have been successfully applied for the identification of CNS isolates from human specimens, their accuracy in the identification of veterinary isolates has not been fully established. In this study, we identified 263 CNS isolates from bovine IMI at species level by partial 16S rRNA gene sequence analysis as the definitive test. Species identification obtained using partial 16S rRNA gene sequence analysis was compared to results from the API STAPH ID 20 and gap PCR-RFLP analysis. Eleven different CNS species were identified by partial 16S rRNA gene sequence analysis. Only 76.0% (200/263) of the species identification results obtained by API STAPH ID 20 matched those obtained by partial 16S rRNA gene sequence analysis, whereas 97.0% (255/263) of the species identification results obtained by the gap PCR-RFLP analysis matched those obtained by partial 16S rRNA gene sequence analysis. The gap PCR-RFLP analysis could be a useful and reliable alternative method for the species identification of CNS isolates from bovine IMI and appears to be a more accurate method of species identification than the API STAPH ID 20 system.

Characterization of the diversity and temporal stability of bacterial communities in human milk.

Recent investigations have demonstrated that human milk contains a variety of bacterial genera; however, as of yet very little work has been done to characterize the full diversity of these milk bacterial communities and their relative stability over time. To more thoroughly investigate the human milk microbiome, we utilized microbial identification techniques based on pyrosequencing of the 16S ribosomal RNA gene. Specifically, we characterized the bacterial communities present in milk samples collected from 16 women at three time-points over four weeks. Results indicated that milk bacterial communities were generally complex; several genera represented greater than 5% of the relative community abundance, and the community was often, yet not always, stable over time within an individual. These results support the conclusion that human milk, which is recommended as the optimal nutrition source for almost all healthy infants, contains a collection of bacteria more diverse than previously reported. This finding begs the question as to what role this community plays in colonization of the infant gastrointestinal tract and maintaining mammary health.