Staphylococcus (S.) aureus is a common infectious agent of bovine chronic mastitis, a disease that is difficult to eradicate. The abilities of Staphylococci to be internalized and form a biofilm can contribute to host immunological defence evasion that subsequently impairs antimicrobial therapy. The invasive capability of six S. aureus field isolates with different biofilm-forming profiles was compared in vitro using a bovine mammary epithelial cell line. This was further confirmed in primary cell cultures using fluorescent rRNA probes against S. aureus. The results suggest that S. aureus invasion levels are not related to biofilm formation.
Animals ; *Biofilms ; Cattle ; Cell Line ; Colony Count, Microbial/veterinary ; Epithelial Cells/microbiology ; Female ; In Situ Hybridization, Fluorescence ; Mastitis, Bovine/*microbiology ; Portugal ; Staphylococcal Infections/*veterinary ; Staphylococcus aureus/classification/genetics/immunology/*physiology ; Virulence Factors/i

One hundred seven isolates of Staphylococcus aureus from bovine mastitis were investigated for colony morphology in serum-soft agar (SSA), autoagglutination in salt, and capsular serotype. Capsular polysaccharide (CP) was purified and quantified from the extracts of clinical isolates. Overall, 89 isolates (83.2%) were diffuse in the SSA, without any difference in the proportion of diffuse colony between type 5 and type 8 strains. Some strains exhibited compact colonies in the SSA and expressed CP as determined by an enzyme-linked immunosorbent assay, indicating that compact morphology does not exclude encapsulation. The majority of the strains (11/12) showed autoagglutination in the salt aggregation test. The serotype 336 accounted for 46.7% of the isolates followed by serotype 5 (12.1%) and serotype 8 (12.1%). Particularly, twenty-six (24.3%) isolates reacted with two serotypes; 7 for type 8/336 and 19 for type 5/336. Five isolates (4.7%) were nontypeable with monoclonal antibodies specific for CP serotype 5, 8, or 336. The CP concentration in culture supernatants varied with the serotypes, and the total amount of CP produced by cells grown in a liquid medium was much less than that produced by cells grown on a solid medium. The Western blotting indicated that the CP bands of S. aureus serotype 5 and 8 were ranged in the molecular mass of 58-84 kilodalton (kDa), with additional bands in the region of approximately >or= 48 or Agglutination Tests ; Animals ; Bacterial Vaccines/administration & dosage ; Cattle ; Cattle Diseases/immunology/microbiology ; Female ; Mastitis, Bovine/*microbiology ; Polysaccharides, Bacterial/*classification/isolation & purification ; Serotyping/methods/veterinary ; Staphylococcal Infections/immunology/*veterinary ; Staphylococcus aureus/*classification/*immunology/isolation & purification

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.
Animals ; Cattle ; Cell Proliferation ; Female ; Lymphocyte Activation/immunology ; Mastitis, Bovine/*immunology/microbiology ; Staphylococcal Infections/immunology/*veterinary ; Staphylococcus/*immunology ; *Superantigens ; T-Lymphocytes/*immunology

This study was conducted to determine the prevalence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in pigs, farm workers, and the environment in northern Thailand, and to assess LA-MRSA isolate phenotypic characteristics. One hundred and four pig farms were randomly selected from the 21,152 in Chiang Mai and Lamphun provinces in 2012. Nasal and skin swab samples were collected from pigs and farm workers. Environmental swabs (pig stable floor, faucet, and feeder) were also collected. MRSA was identified by conventional bacterial culture technique, with results confirmed by multiplex PCR and multi locus sequence typing (MLST). Herd prevalence of MRSA was 9.61% (10 of 104 farms). Among pigs, workers, and farm environments, prevalence was 0.68% (two of 292 samples), 2.53% (seven of 276 samples), and 1.28% (four of 312 samples), respectively. Thirteen MRSA isolates (seven from workers, four from environmental samples, and two from pigs) were identified as Staphylococcal chromosomal cassette mec IV sequences type 9. Antimicrobial sensitivity tests found 100% of the MRSA isolates resistant to clindamycin, oxytetracycline, and tetracycline, while 100% were susceptible to cloxacillin and vancomycin. All possessed a multidrug-resistant phenotype. This is the first evidence of an LA-MRSA interrelationship among pigs, workers, and the farm environment in Thailand.
*Animal Husbandry ; Animals ; Cross-Sectional Studies ; Genotype ; Humans ; Methicillin-Resistant Staphylococcus aureus/classification/*genetics/*isolation & purification ; Microbial Sensitivity Tests/veterinary ; Molecular Sequence Data ; Multilocus Sequence Typing/veterinary ; Multiplex Polymerase Chain Reaction/veterinary ; Occupational Diseases/*epidemiology/microbiology ; Phylogeny ; Prevalence ; Sequence Analysis, DNA/veterinary ; Staphylococcal Infections/*epidemiology/microbiology ; Swine ; Swine Diseases/*epidemiology/microbiology ; Thailand/epidemiology

The present study determined the genetic relationships between 41 Staphyloccocus (S.) aureus isolates from bovines, humans, and food using a single enzyme amplified fragment length polymorphism (AFLP) technique. We evaluated the prevalence of staphylococcal enterotoxin (SE) genes and other virulence gene determinants by PCR. The identification of S. aureus was based on culturing and biochemical tests, and by amplifying a specific section of the 23S rRNA gene. PCR amplification of the SE genes (sea, seb, sec, see, seg, seh, and sei) singly or in combination was observed. Most isolates of bovine origin harbored hla (84%) and cap5 (74%), while most isolates from humans harbored hla (73%), cap8 (91%), and fnbA (100%). Strains from food sources were positive for hla (100%), cap5 (100%), and cap8 (64%) unlike isolates from humans or bovines. A single enzyme AFLP analysis revealed a correlation between AFLP clusters of some strains and the source of the isolates The genotypic results of the present study might help to better understand the distribution of prevalent S. aureus clones among humans, bovines, and food and will help control S. aureus infections in Indonesia.
Amplified Fragment Length Polymorphism Analysis ; Animals ; Bacterial Proteins/genetics/metabolism ; Cattle ; Female ; *Food Microbiology ; Gene Expression Regulation, Bacterial ; Humans ; Indonesia/epidemiology ; Mastitis, Bovine/epidemiology/microbiology ; Phylogeny ; Staphylococcal Infections/epidemiology/microbiology/*veterinary ; Staphylococcus aureus/*genetics

The prevalence, virulence potential, and antibiotic resistance of ophthalmic Staphylococcus pseudintermedius (SP) isolated from dogs were examined. Sixty-seven Staphylococcus species were isolated from ophthalmic samples and surveyed for species-specific sequences in the Staphylococcus intermedius group (SIG) nuclease gene (SInuc), exfoliative toxin gene for SIG (siet), and antibiotic resistance genes (blaZ and mecA). PCR-restriction fragment length polymorphism analysis of the pta gene was also performed. Fifty isolates were identified as SIG strains, all of which were found to be SP. The blaZ gene was detected in 42 of the 50 SP strains and mecA gene was observed in 18 of the 50 SP strains. The 50 SP strains were most susceptible to amoxicillin/clavulanic acid (94%) and chlorampenicol (70%), and highly resistant to tetracycline (94%) and penicillin (92%). It was also found that 16 (88.9%) mecA-positive SP strains were resistant to oxacillin, tetracycline and penicillin. All mecA-positive SP were resistant to more than four of the eight tested antibiotics and therefore considered SP with multi-drug resistance (MDR). Our results indicate a high prevalence of antibiotic resistance genes in ophthalmic SP along with a close relationship between MDR SP strains and the mecA gene. Based on our findings, judicious administration of antibiotics to companion dogs is necessary.
Animals ; Anti-Bacterial Agents/*therapeutic use ; Dog Diseases/drug therapy/*microbiology ; Dogs ; Drug Resistance, Bacterial ; Drug Resistance, Multiple, Bacterial ; Eye Infections, Bacterial/drug therapy/microbiology/*veterinary ; Microbial Sensitivity Tests/veterinary ; Multiplex Polymerase Chain Reaction ; Staphylococcal Infections/drug therapy/microbiology/*veterinary ; Staphylococcus/*drug effects/isolation & purification

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.
Animals ; Apoptosis/drug effects/immunology ; CD4-CD8 Ratio/veterinary ; CD4-Positive T-Lymphocytes/drug effects/*immunology/microbiology ; CD8-Positive T-Lymphocytes/drug effects/*immunology/microbiology ; Cattle ; Concanavalin A/pharmacology ; Cytokines/genetics/immunology ; Enterotoxins/*pharmacology ; Female ; Lymphocyte Activation/drug effects ; Mastitis, Bovine/immunology/*microbiology ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Staphylococcal Infections/immunology/microbiology/*veterinary ; Staphylococcus aureus/*immunology

In the present study, 35 Staphylococcal strain isolated from milk samples of 16 cows from eight farms of three different geographic locations in Central Java, Indonesia, and from milk samples of 19 cows from 19 farms of different geographic locations in Hesse, Germany, were compared pheno- and genotypically. On the basis of cultural and biochemical properties as well as by amplification of the 23S rRNA specific to Staphylococcus aureus, all isolates could be identified as S. aureus. In addition, all S. aureus isolates harboured the genes clfA and coa encoding staphylococcal clumping factor and coagulase, and the gene segments encoding the immunoglobulin G binding region and the X-region of protein A gene spa. By PCR amplification, the genes seb, seg, seh, and sei was observed for the S. aureus cultures isolated in Central Java, Indonesia and the genes sec, sed, seg, seh, sei, sej and tst for the S. aureus cultures isolated in Hesse, Germany. None of the S. aureus of both origins harboured the genes sea, see, eta and etb. All isolates were additionally positive for the genes nuc, fnbA, hla, and set1. The gene hlb was found for 6 cultures from Central Java, Indonesia and 16 cultures from Hesse, Germany. However, the gene fnbB and the gene segments cnaA and cnaB were not present among the strains isolated in Central Java, Indonesia and rare among the strains isolated in Hesse, Germany. It was of interest that most of the S. aureus isolated in Central Java, Indonesia harboured the gene cap5 and most of the strains isolated in Hesse, Germany the gene cap8. The phenotypic and genotypic results of the present study might help to understand the distribution of prevalent S. aureus clones among bovine mastitis isolates of both countries and might help to control S. aureus infections in dairy herds.
Animals ; Bacterial Typing Techniques ; Cattle ; Female ; Genes, Bacterial ; Genotype ; Germany ; Indonesia ; Mastitis, Bovine/*microbiology ; Phenotype ; Polymerase Chain Reaction ; RNA, Bacterial/genetics ; RNA, Ribosomal, 23S/genetics ; Staphylococcal Infections/microbiology/*veterinary ; Staphylococcus aureus/classification/*genetics/isolation&purification

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 beta-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.
Ampicillin/pharmacology ; Animals ; Anti-Bacterial Agents/pharmacology ; Cattle ; Cephalothin/pharmacology ; Cloxacillin/pharmacology ; Drug Resistance, Microbial ; Female ; Lactation ; Mastitis, Bovine/*microbiology ; Microbial Sensitivity Tests ; Organic Agriculture ; Penicillins/pharmacology ; Prevalence ; Staphylococcal Infections/microbiology/*veterinary ; Staphylococcus/*drug effects/*isolation & purification

Sensitivity to commercial teat dips (nonoxinol-9 iodine complex and chlorhexidine digluconate) of 56 Staphylococcus (S.) aureus strains isolated from quarter milk samples of various German dairy herds treated with different teat dipping schemes was investigated in this study. The minimum inhibitory concentration was determined using a broth macrodilution method according to the German Veterinary Association guidelines. The main objective of the current study was to induce in vitro resistance induction of S. aureus to chemical disinfectants. Ten different strains were repeatedly passed ten times in growth media with sub-lethal concentrations of disinfectants. Nine strains showed a significant reduction in susceptibility to the nonoxinol-9 iodine complex but only one strain developed resistance to chlorhexidine digluconate. Stability of the acquired resistance was observed in all S. aureus strains adapted to the nonoxinol-9 iodine complex and chlorhexidine digluconate. In contrast, simultaneous resistance to different antibiotics was not observed in any of the ten investigated S. aureus strains. However, the isolates exhibited a high degree of resistance to penicillin G. Based on these results, resistance of S. aureus to chemical disinfectants may be more likely to develop if the chemicals are used at concentrations lower than that required for an optimal biocidal effect.
Animals ; Anti-Bacterial Agents/pharmacology ; Cattle ; Chlorhexidine/*pharmacology ; Disinfectants/pharmacology ; Dose-Response Relationship, Drug ; Drug Resistance, Bacterial ; Female ; Germany/epidemiology ; Iodine/chemistry/*pharmacology ; Mastitis, Bovine/epidemiology/*microbiology ; Nonoxynol/*pharmacology ; Staphylococcal Infections/epidemiology/microbiology/*veterinary ; Staphylococcus aureus/classification/*drug effects