Genotyping of long term persistent Staphylococcus aureus in bovine subclinical mastitis.

Bovine mastitis affects dairy cattle worldwide and Staphylococcus aureus is one of the most common microorganisms involved in subclinical and chronic disease. Superantigens, such as enterotoxins contribute to S. aureus persistence and pathogenicity in this disease. Subclinical and chronic mastitis cases were diagnosed and S. aureus isolates from sub-clinical cases were investigated for carriage of virulence and antibiotic resistance genes that may contribute to long-term carriage and infection. Over a 12-month period, 116 S. aureus strains were recovered from 68 cows with subclinical mastitis. Classical enterotoxin genes (sea-see) were detected in 24.1% of isolates, and pvl and tsst-1 were identified in 3.4% and 46.6% the isolates, respectively. 18.1% that were persistent isolates were identified and characterized by pulsed field gel electrophoresis (PFGE), MLST, spa typing. Four isolates were methicillin-resistant S. aureus (MRSA) and belonged to SCCmec type I. Molecular typing showed that the agrI group was the most frequent, and a rare isolate was positive for both agrI and agrIII groups. Molecular characterization revealed the persistence of the spa type t10856 (ST133, clonal complex CC133, agr I), in a single animal for nine months and the persistence t605 (ST126, CC126) colonizing four animals for four months. These strains have been described recently in other herds in the same region, indicating their transmissibility and clonal expansion. We conclude that animals with subclinical mastitis are an important and somewhat overlooked reservoir for transmission within and between herds, and may carry virulence and antibiotic resistance genes contributing to persistent colonization, hinder the control of mastitis and may cause risks to the public health.

Molecular epidemiology of methicillin-susceptible Staphylococcus aureus (MSSA) isolated from milk of cows with subclinical mastitis.

Bovine mastitis has been a concern for dairy herd for decades. The adaptation capacity of one of the main species responsible for this disease, Staphylococcus aureus (S. aureus), plays a pivotal role in this issue. The aim of this study was to establish a molecular and phenotypic profile of 285 S. aureus strains isolated from milk of subclinical mastitis cows from 18 different farms in São Paulo State using spa typing, multilocus sequence typing (MLST), pulsed field gel electrophoresis (PFGE), agr cluster (I, II, III and IV) typing, PCR for genes including enterotoxins (sea, seb, sec, sed, see, seg, seh, sei), toxic shock syndrome toxin (tsst-1), and Panton-Valentine leucocidin (pvl), as well as in vitro resistance assays for 12 antibiotics. The results showed a wide variety of strains with a high toxigenic potential; concomitantly, sec, seg and seh were prevalent. In addition, we observed a predominance of the spa types t605 (ST 126, CC126) and t127 (ST1, CC1) and the unusual presence of t321 causing bovine mastitis, which has been previously reported only in swine. The most frequent ST were ST126 (70.5%) and ST1 (10.5%). Regarding PFGE, we observed four major groups and six profile patterns. The highest resistance was observed for streptomycin (9.5%), followed by tetracycline (3.5%), clindamycin (9.3%), and erythromycin (2.8%). The tsst-1 gene was detected in 36.8% of isolates and pvl was not observed. One hundred and thirty-six (47.7%) isolates possessed agr type II, followed by types III (20%) and I (8.1%), with type IV not being detected. We observed that the same spa type could result in different PFGE profiles, so the exclusive use of spa type sequences can lead to incorrect interpretations regarding the spread of clones in an epidemiological context.

Diagnostic accuracy of Somaticell, California Mastitis Test, and microbiological examination of composite milk to detect Streptococcus agalactiae intramammary infections.

The objectives of this study were to estimate the accuracy of Somaticell (Idexx Laboratories Inc., Westbrook, ME), California Mastitis Test (CMT), and microbiological examination of composite milk (MEC) to diagnose Streptococcus agalactiae intramammary infections (IMI), and to assess the agreement between Somaticell and CMT to detect these infections. A secondary objective was to estimate quarter- and cow-level prevalence of S. agalactiae IMI in the herds included in the study. Seven farms were included in the study. The CMT was performed and aseptic milk samples were collected from all quarters of all lactating cows. Composite milk samples were produced in the laboratory by mixing milk from all quarters of each sampled cow. The Somaticell test was performed on a subset of S. agalactiae-positive (n = 167) and S. agalactiae-negative (n = 152) quarter milk samples. Microbiological examination of quarter milk samples (MEQ) was considered the reference test for diagnosing S. agalactiae IMI. The accuracy of all tests at various thresholds was estimated using Bayesian latent class models. Apparent prevalence of S. agalactiae IMI was 15.8% (n = 184/1,164) at the quarter level (based on MEQ) and 28.5% (n = 83/291) at the cow level (based on MEC). True prevalence, as determined by Bayesian models, was 13.0% [95% credible interval (CR): 6.4-24.4%] at the quarter level, and 25.6% (95% CR: 15.3-39.5%) at the cow level. At the cow level (n = 285), sensitivity and specificity of MEC were 95.6 and 99.5%, respectively. The accuracy of Somaticell (n = 319 quarters) to identify S. agalactiae-infected quarters was 75.4, 86.4, 88.9, 89.4, and 91.0% at thresholds of 98,000, 147,000, 205,000, 244,000, and 282,000 cells/mL, respectively. The accuracy of CMT was 87.6, 90.7, 90.8, and 87.4% at thresholds of trace, 1, 2, and 3, respectively. The areas under the receiver operating characteristic curve for Somaticell and CMT were 94.5% (95% confidence interval: 91.8-97.2%) and 92.0% (88.6-95.4%), respectively. At the tested thresholds, the sensitivity of Somaticell ranged from 94.9 to 99.5% to detect S. agalactiae IMI, and specificity ranged from 48.1 to 87.1%. The sensitivity of Somaticell at the lowest threshold (69,000 cells/mL; sensitivity = 99.9%; 95% CR: 98.2-100%) was higher than that of CMT at any tested threshold. Results of this study could be used at the farm level to reduce the use of antimicrobials and reach specific goals in S. agalactiae eradication programs.