Short communication: Prevalence of methicillin resistance in coagulase-negative staphylococci and Staphylococcus aureus isolated from bulk milk on organic and conventional dairy farms in the United States

The objective of this study was to evaluate the presence of methicillin-resistant Staphylococcus aureus and coagulase-negative Staphylococcus spp. in bulk tank milk samples from 288 organic and conventional dairy farms located in New York, Wisconsin, and Oregon from March 2009 to May 2011. Due to recent publications reporting the presence mecC (a mecA homolog not detected by traditional mecA-based PCR methods), a combination of genotypic and phenotypic approaches was used to enhance the recovery of methicillin-resistant organisms from bulk tank milk. In total, 13 isolates were identified as methicillin resistant: Staph. aureus (n = 1), Staphylococcus sciuri (n = 5), Staphylococcus chromogenes (n = 2), Staphylococcus saprophyticus (n = 3), Staphylococcus agnetis (n = 1), and Macrococcus caseolyticus (n = 1). The single methicillin-resistant Staph. aureus isolate was identified from an organic farm in New York, for an observed 0.3% prevalence at the farm level. The methicillin-resistant coagulase-negative staphylococci prevalence was 2% in the organic population and 5% in the conventional population. We did not identify mecC in any of the isolates from our population. Of interest was the relatively high number of methicillin-resistant Staph. sciuri recovered, as the number of isolates from our study was considerably higher than those recovered from other recent studies that also assessed milk samples. Our research suggests that the presence of a potential methicillin-resistant Staphylococcus reservoir in milk, and likely the dairy farm population in the United States, is independent of the organic or conventional production system.     

Evaluation of the University of Minnesota Tri-plate and 3M Petrifilm for the isolation of Staphylococcus aureus and Streptococcus species from clinically mastitic milk samples

The primary objective was to compare microbiological results of the University of Minnesota Tri-plate and the 3M Petrifilm Staph Express (STX) Count Plate to standard culture techniques for identification of clinical mastitis caused by Staphylococcus aureus. The secondary objective was to evaluate the Tri-plate's ability to differentiate Streptococcus spp. from other gram-positive organisms. The tests were evaluated using clinically positive mastitic milk samples (n = 282) to determine their ability to diagnose the pathogens of interest. A Tri-plate was classified positive for Staph. aureus when at least 1 colony exhibiting β-hemolysis was present on the Factor media portion of the plate. When the plate was used in this manner and read by a trained laboratory technician, the sensitivity of the Tri-plate was 97.9% and the specificity was 81.8%. When the Tri-plate was evaluated by the laboratory technician for its ability to diagnose Streptococcus spp., both sensitivity and specificity of the test were very good (92.6 and 89.5%, respectively). Using the Petrifilm, samples were classified as positive for Staph. aureus if any red-violet colonies were present on the Petrifilm after an initial 24-h incubation. When used in this manner, the Petrifilm had a sensitivity of 97.4% and a specificity of 76.1%. Further evaluation of the Petrifilm was done using the STX disk, which was used to confirm the presence of Staph. aureus. When using the presence of 1 pink colony on the disk, the sensitivity of the Petrifilm was 92.1% and the specificity was 93.1%. Both the Tri-plate and the 3M STX Petrifilm successfully diagnosed Staph. aureus in clinical milk samples when used in a laboratory setting and the Tri-plate successfully differentiated Streptococcus spp. from other gram-positive organisms.     

Relationship between antimicrobial drug usage and antimicrobial susceptibility of gram-positive mastitis pathogens

The objective of this study was to analyze relationships between usage of antimicrobial drugs on dairy farms and results of antimicrobial susceptibility testing of mastitis pathogens. Exposure to selected antimicrobial drugs (n = 10) was standardized by calculation of the number of defined daily doses used per cow. Farms (n = 40) were categorized based on amount of antimicrobial exposure: organic (no usage); conventional-low usage (conventional farms not using or using less than or equal to the first quartile of use of each compound); and conventional-high usage (conventional farms using more than the first quartile of a particular compound). The minimum inhibitory concentration (MIC) of selected antimicrobial drugs was determined using a commercial microbroth dilution system for isolates of Staphylococcus aureus (n = 137), coagulase-negative staphylococci (CNS, n = 294), and Streptococcus spp. (n = 95) obtained from subclinical mastitis infections. Most isolates were inhibited at the lowest dilution tested of most antimicrobial drugs. Survival curves for Staph. aureus and CNS demonstrated heterogeneity in MIC based on the amount of exposure to penicillin and pirlimycin. For CNS, farm type was associated with the MIC of ampicillin and tetracycline. For Streptococcus spp., farm type was associated with MIC of pirlimycin and tetracycline. For all mastitis pathogens studied, the MIC of pirlimycin increased with increasing exposure to defined daily doses of pirlimycin. The level of exposure to most other antimicrobial drugs was not associated with MIC of mastitis pathogens. A dose-response effect between antimicrobial exposure and susceptibility was observed for some pathogen-antimicrobial combinations, but exposure to other antimicrobial drugs commonly used for prevention and treatment of mastitis was not associated with resistance.     

Biofilm-producing ability of Staphylococcus aureus isolates from Brazilian dairy farms

This study aimed to investigate the in silico biofilm production ability of Staphylococcus aureus strains isolated from milking parlor environments on dairy farms from São Paulo, Brazil. The Staph. aureus isolates were obtained from 849 samples collected on dairy farms, as follows: milk from individual cows with subclinical mastitis or history of the disease (n = 220); milk from bulk tank (n = 120); surfaces of milking machines and utensils (n = 389); and milk handlers (n = 120). Thirty-one Staph. aureus isolates were obtained and categorized as pulsotypes by pulsed-field gel electrophoresis and submitted to assays for biofilm formation on polystyrene, stainless steel, rubber, and silicone surfaces. Fourteen (45.2%) pulsotypes were considered producers of biofilm on the polystyrene microplate assay, whereas 13 (41.9%) and 12 (38.7%) pulsotypes were biofilm producers on stainless steel and rubber, respectively. None of the pulsotypes evaluated produced biofilms on silicone. Approximately 45% of Staph. aureus pulsotypes isolated from different sources on dairy farms showed the ability to produce biofilms in at least one assay, indicating possible persistence of this pathogen in the milking environment. The potential involvement of Staph. aureus in subclinical mastitis cases and its occurrence in milk for human consumption emphasize the need to improve hygiene practices to prevent biofilm formation on the farms studied.     

Risk factors associated with short-term post-treatment outcomes of clinical mastitis

The objectives of this study were to characterize 60-d outcomes after treatment of mild (abnormal milk) and moderate (abnormal milk and abnormal udder) cases of clinical mastitis (CM) occurring in a single quarter of cows on Wisconsin farms (n = 4) and to determine risk factors associated with those outcomes. Duplicate milk samples were collected from the affected quarter of each cow for microbiological analysis at the onset of CM (PRE) and 21 d later (POST). Cows were treated only in the affected quarter using an intramammary product containing 125 mg of ceftiofur. Bacteriological cure was defined as absence of pathogens in the POST sample obtained from the enrolled quarter. Recurrence was defined for the cow when CM occurred after the milk-withholding period for the enrolled case of CM. Retention in the herd was defined when a cow was retained within the herd for the 60-d follow-up period. Somatic cell count reduction (SCCR) was defined at the cow level as somatic cell count (SCC) below 200,000 cells/mL at the Dairy Herd Improvement Association test day occurring between 21 to 55 d post-treatment. The effects of farm, days in milk, parity, severity, microbiological diagnosis at PRE, previous milk yield, previous SCC, previous occurrence of CM and treatment duration on selected post-treatment outcomes were assessed using Chi-squared analysis and logistic regression. Microbiological results at PRE were distributed as: Escherichia coli (n = 14), Klebsiella spp. (n = 11), Enterobacter spp. (n = 8), Serratia spp. (n = 7), other gram-negative species (n = 3), Streptococcus spp. (n = 25), coagulase-negative staphylococci (n = 4); Staphylococcus aureus (n = 1); Streptococcus agalactiae (n = 1), other gram-positive species (n = 9), and culture negative (n = 60). Treated quarters were more likely to experience bacteriological cure when the cow experienced CM for the first time in the lactation and when no pathogen was recovered from PRE milk samples obtained from the enrolled quarter. Parity and bacteriological cure were associated with the probability of recurrence. Greater milk yield at previous Dairy Herd Improvement test was the most important predictor for retention within the herd. When SCC before CM was >200,000 cells/mL the probability of having SCCR after treatment was decreased. When the case experienced bacteriological cure, the cow was less likely to experience recurrent cases and was more likely to have SCCR below 200,000 cells/mL. Post-treatment outcomes, such as recurrence and SCCR, are strongly associated with bacteriological cure and, when monitored, can be used to help determine if a treatment has been successful. Information about the etiology of CM, history of clinical and subclinical mastitis, and parity are useful to review when making strategic treatment decisions.     

Identification of virulence factors in 16S-23S rRNA intergenic spacer genotyped Staphylococcus aureus isolated from water buffaloes and small ruminants

Staphylococcus aureus is an important human and animal pathogen, and is regarded as an important cause of intramammary infection (IMI) in ruminants. Staphylococcus aureus genetic variability and virulence factors have been well studied in veterinary medicine, especially in cows as support for control and management of IMI. The aim of the present study was to genotype 71 Staph. aureus isolates from the bulk tank and foremilk of water buffaloes (n = 40) and from udder tissue (n = 7) and foremilk (n = 24) from small ruminants. The method used was previously applied to bovine Staph. aureus and is based on the amplification of the 16S-23S rRNA intergenic spacer region. The technique applied was able to identify different Staph. aureus genotypes isolated from dairy species other than the bovine species, and cluster the genotypes according to species and herds. Virulence gene distribution was consistent with genotype differentiation. The isolates were also characterized through determination of the presence of 19 virulence-associated genes by specific PCR. Enterotoxins A, C, D, G, I, J, and L were associated with Staph. aureus isolates from buffaloes, whereas enterotoxins C and L were linked to small ruminants. Genes coding for methicillin resistance, Panton-Valentine leukocidin, exfoliative toxins A and B, and enterotoxins B, E, and H were undetected. These findings indicate that RNA template-specific PCR is a valid technique for typing Staph. aureus from buffaloes and small ruminants and is a useful tool for understanding udder infection epidemiology.     

Relationships between milk culture results and milk yield in Norwegian dairy cattle

Associations between test-day milk yield and positive milk cultures for Staphylococcus aureus, Streptococcus spp., and other mastitis pathogens or a negative milk culture for mastitis pathogens were assessed in quarter milk samples from randomly sampled cows selected without regard to current or previous udder health status. Staphylococcus aureus was dichotomized according to sparse (≤1,500 cfu/mL of milk) or rich (>1,500 cfu/mL of milk) growth of the bacteria. Quarter milk samples were obtained on 1 to 4 occasions from 2,740 cows in 354 Norwegian dairy herds, resulting in a total of 3,430 samplings. Measures of test-day milk yield were obtained monthly and related to 3,547 microbiological diagnoses at the cow level. Mixed model linear regression models incorporating an autoregressive covariance structure accounting for repeated test-day milk yields within cow and random effects at the herd and sample level were used to quantify the effect of positive milk cultures on test-day milk yields. Identical models were run separately for first-parity, second-parity, and third-parity or older cows. Fixed effects were days in milk, the natural logarithm of days in milk, sparse and rich growth of Staph. aureus (1/0), Streptococcus spp. (1/0), other mastitis pathogens (1/0), calving season, time of test-day milk yields relative to time of microbiological diagnosis (test day relative to time of diagnosis), and the interaction terms between microbiological diagnosis and test day relative to time of diagnosis. The models were run with the logarithmically transformed composite milk somatic cell count excluded and included. Rich growth of Staph. aureus was associated with decreased production levels in first-parity cows. An interaction between rich growth of Staph. aureus and test day relative to time of diagnosis also predicted a decline in milk production in third-parity or older cows. Interaction between sparse growth of Staph. aureus and test day relative to time of diagnosis predicted declining test-day milk yields in first-parity cows. Sparse growth of Staph. aureus was associated with high milk yields in third-parity or older cows after including the logarithmically transformed composite milk somatic cell count in the model, which illustrates that lower production levels are related to elevated somatic cell counts in high-producing cows. The same association with test-day milk yield was found among Streptococcus spp.-positive pluriparous cows.     

Diagnosing intramammary infections: comparison of multiple versus single quarter milk samples for the identification of intramammary infections in lactating dairy cows

The objective was to examine the potential benefits of using different combinations of multiple quarter milk samples compared with a single sample for diagnosing intramammary infections (IMI) in dairy cattle. Data used in the analyses were derived from 7,076 samples from 667 quarters in 176 cows in 8 herds in 4 locations (Minnesota/Wisconsin, n = 4; Prince Edward Island, n = 2; Ontario, n = 1; New York, n = 1). Duplicate quarter milk samples were collected at morning milking for 5 consecutive days. Cows were evenly distributed between early postparturient and mid- to late-lactation cows. All samples were frozen for shipping and storage, thawed once, and cultured in university laboratories using standardized procedures consistent with National Mastitis Council guidelines. The presence of specific pathogens was confirmed and identified using the API identification system (bioMerieux, Marcy l’Etoile, France) in each laboratory. A previously developed gold standard was applied to the first sample from d 1, 3, and 5 to classify infected quarters. The data were analyzed separately for coagulase-negative staphylococci (CNS) and Streptococcus spp. Various combinations of test results from d 2 and 4 were used in the test evaluation. These consisted of single samples (n = 4), 2 sets of duplicate samples (2 samples collected on the same day), 2 sets of consecutive samples (2 samples collected 2 d apart), and 2 sets of triplicate samples (2 samples on the same day and a third sample 2 d apart). Series interpretation of duplicate or consecutive samples (i.e., positive = same pathogen isolated from both samples) resulted in the highest specificity (Sp; CNS Sp = 92.1-98.1%; Streptococcus spp. Sp = 98.7-99.6%), but lowest sensitivity (Se; CNS Se = 41.9-53.3%; Streptococcus spp. Se = 7.7-22.2%). Parallel interpretation of duplicate or consecutive samples (i.e., positive = pathogen isolated from either) resulted in the highest Se (CNS Se = 70.8-80.6%; Streptococcus spp. Se = 31.6-48.1%), but lowest Sp (CNS Sp = 72.0-77.3%; Streptococcus spp. Sp = 89.5-93.3%). The difference in estimates between single and duplicate samples was larger than between single and consecutive samples. Overall, triplicate samples provided the best combination of Se and Sp, but compared with a single sample, provided only a modest gain in Sp and little or no gain in Se.     

Genotype-specific risk factors for Staphylococcus aureus in Swiss dairy herds with an elevated yield-corrected herd somatic cell count

Bovine mastitis is a frequent problem in Swiss dairy herds. One of the main pathogens causing significant economic loss is Staphylococcus aureus. Various Staph. aureus genotypes with different biological properties have been described. Genotype B (GTB) of Staph. aureus was identified as the most contagious and one of the most prevalent strains in Switzerland. The aim of this study was to identify risk factors associated with the herd-level presence of Staph. aureus GTB and Staph. aureus non-GTB in Swiss dairy herds with an elevated yield-corrected herd somatic cell count (YCHSCC). One hundred dairy herds with a mean YCHSCC between 200,000 and 300,000 cells/mL in 2010 were recruited and each farm was visited once during milking. A standardized protocol investigating demography, mastitis management, cow husbandry, milking system, and milking routine was completed during the visit. A bulk tank milk (BTM) sample was analyzed by real-time PCR for the presence of Staph. aureus GTB to classify the herds into 2 groups: Staph. aureus GTB-positive and Staph. aureus GTB-negative. Moreover, quarter milk samples were aseptically collected for bacteriological culture from cows with a somatic cell count ≥150,000 cells/mL on the last test-day before the visit. The culture results allowed us to allocate the Staph. aureus GTB-negative farms to Staph. aureus non-GTB and Staph. aureus-free groups. Multivariable multinomial logistic regression models were built to identify risk factors associated with the herd-level presence of Staph. aureus GTB and Staph. aureus non-GTB. The prevalence of Staph. aureus GTB herds was 16% (n = 16), whereas that of Staph. aureus non-GTB herds was 38% (n = 38). Herds that sent lactating cows to seasonal communal pastures had significantly higher odds of being infected with Staph. aureus GTB (odds ratio: 10.2, 95% CI: 1.9–56.6), compared with herds without communal pasturing. Herds that purchased heifers had significantly higher odds of being infected with Staph. aureus GTB (rather than Staph. aureus non-GTB) compared with herds without purchase of heifers. Furthermore, herds that did not use udder ointment as supportive therapy for acute mastitis had significantly higher odds of being infected with Staph. aureus GTB (odds ratio: 8.5, 95% CI: 1.6–58.4) or Staph. aureus non-GTB (odds ratio: 6.1, 95% CI: 1.3–27.8) than herds that used udder ointment occasionally or regularly. Herds in which the milker performed unrelated activities during milking had significantly higher odds of being infected with Staph. aureus GTB (rather than Staph. aureus non-GTB) compared with herds in which the milker did not perform unrelated activities at milking. Awareness of 4 potential risk factors identified in this study guides implementation of intervention strategies to improve udder health in both Staph. aureus GTB and Staph. aureus non-GTB herds.     

Milk culture results in a large Norwegian survey--effects of season, parity, days in milk, resistance, and clustering

A nationwide random computerized assignment survey that included 3,538 sets of 4 quarter milk samples from 2,834 dairy cows was conducted during 2000. Every fifth cow from every 50th herd was randomly selected for sampling and culture during each quarter of the year. Milk culture results of pathogens known to be related to mastitis were recorded regardless of whether mastitis had been indicated by any inflammatory measure or not. Farmers were blinded to all test results to minimize any potential interventions that might be prompted by the results. The most prevalent isolate was Staphylococcus aureus, which was identified in 8.2% of the quarter milk samples. More than 15 colony-forming units/0.01 mL of Staph. aureus were found in 4.3% of the quarter milk samples, whereas 3.5% had only 1 to 3 colony-forming units/0.01 mL. Streptococcus dysgalactiae, coagulase-negative staphylococci (CNS), and Streptococcus uberis were isolated from 1.2, 3.3, and 0.4% of quarter milk samples, respectively. No isolates were found in 76.6% of the quarter milk samples tested. Among individual cows, 22.2% had an isolate of Staph. aureus in ≥ 1 quarter. Only Strep. dysgalactiae exhibited a higher prevalence with increased parity. Prevalence of Staph. aureus decreased throughout days in milk, but prevalence of Strep. dysgalactiae increased. There was a strong seasonal effect; the highest prevalence of Strep. dysgalactiae and CNS was observed during April and May (late indoor season), and the highest prevalence of Staph. aureus and Strep. uberis was observed during June and July (the outdoor season). A substantial within-cow clustering effect was found for Strep. dysgalactiae, Staph. aureus, and CNS. Additionally, a within-herd effect was found for Strep. uberis, penicillin-resistant Staph. aureus, total Staph. aureus, and CNS. No within-county cluster effect was found. Lastly, both Staph. aureus and CNS exhibited a surprisingly high seasonal effect regarding the prevalence of resistance to penicillin G. Penicillin resistance of Staph. aureus was likely due to higher prevalence of Staph. aureus as a whole, but for CNS, there was also an additional increase caused by a higher proportional rate of penicillin resistance during the late indoor season.     

Detection and enumeration of Staphylococcus aureus from bovine milk samples by real-time polymerase chain reaction

The aim of this study was to develop and evaluate a real-time quantitative PCR (qPCR)-based method to detect and quantify Staphylococcus aureus in bronopol-preserved milk samples from subclinical intramammary infections (IMI). Serial dilutions of milk artificially inoculated with Staph. aureus ATCC 29213 were used to establish a standard curve (cfu/mL) of the qPCR assay targeting the Staph. aureus thermonuclease-encoding gene nuc according to the strain plate count. The analytical sensitivity, specificity, and repeatability of the qPCR assay were determined. A total of 60 milk samples, collected from mammary quarters without abnormal appearance and with positive isolation of Staph. aureus, were submitted to both the qPCR protocol and Staph. aureus plate counting and results from both methods were compared. Staphylococcus aureus from bronopol-preserved, subclinical IMI milk samples were not accurately enumerated by qPCR compared with plate counting of the nonpreserved, raw milk sample. The detection limit of the qPCR protocol of inoculated Staph. aureus ATCC 29213 in bronopol-preserved milk samples was 1.04 × 10¹ cfu/mL. The qPCR protocol can be a high-throughput and rapid diagnostic assay to accurately detect Staph. aureus IMI from bronopol-preserved milk samples compared with a traditional culturing method. However, the proposed qPCR protocol is not accurate for counting of Staph. aureus in bronopol-preserved milk samples from naturally infected mammary glands.     

Short communication: Methicillin-resistant Staphylococcus aureus detection in US bulk tank milk

Staphylococcus aureus is a major cause of mastitis in dairy cattle. This study estimated the herd prevalence of methicillin-resistant Staph. aureus (MRSA) among US dairy herds by testing bulk tank milk (BTM) samples using genotypic and phenotypic methods. A nationally representative sample of 542 operations had BTM cultured for Staph. aureus, and 218 BTM samples were positive upon initial culture. After 4 wk to 4 mo of frozen storage, 87% of 218 samples (n = 190) were still culture positive for Staph. aureus on blood agar, but none were positive for MRSA on the selective indicator medium CHROMagar MRSA. A duplex PCR was used to detect the Staph. aureus-specific nuc gene and the methicillin resistance gene, mecA, in mixed staphylococcal isolates from the 190 BTM samples that were positive for Staph. aureus after storage. Seven samples tested positive for nuc and mecA, and 2 samples tested positive for mecA only. MecA-positive Staphylococcus spp., but not MRSA, were subsequently isolated from 5 samples, whereas neither mecA-positive Staphylococcus spp. nor MRSA was isolated from the remaining 4 samples. Presence of methicillin-resistant, coagulase-negative Staphylococcus spp. may complicate the detection of MRSA by means of PCR on BTM. Bulk tank milk in the United States is not a common source of MRSA.     

Relationships between milk culture results and treatment for clinical mastitis or culling in Norwegian dairy cattle

In quarter milk samples from 2,492 randomly sampled cows that were selected without regard to their current or previous udder health status, the relationships between the following outcome variables were studied: treatment of clinical mastitis; the joint event of either treatment or culling for mastitis; culling for all reasons; culling specifically for mastitis; and the covariates of positive milk culture for Staphylococcus aureus, Streptococcus spp., and coagulase-negative Staphylococcus spp., or other pathogens, or of negative culture for mastitis pathogens. Microbiological diagnoses were assigned at the cow level, and altogether 3,075 diagnoses were related to the outcome variables. The relation between the absence of pathogens and rich (>1,500 cfu/mL of milk) or sparse (≤1,500 cfu/mL of milk) growth of Staph. aureus were also assessed separately for each outcome variable. The hazard of treatment of clinical mastitis was greater for cows diagnosed with Staph. aureus compared with cows with no pathogens in all analyses. Cows with sparse growth of Staph. aureus upon microbiological analysis were more likely to be treated for clinical mastitis, and cows with rich growth of the bacteria experienced a higher overall risk of culling when the models adjusted for cow composite milk somatic cell count. No difference between rich and sparse growth of Staph. aureus was found when mastitis was defined as the joint event of either culling for mastitis or treatment of clinical mastitis, and when the relationship with culling specifically for mastitis was assessed. The combined outcome of treatment and culling for mastitis was related to a positive diagnosis of Strep. spp. after cow composite milk somatic cell count was omitted from the model. Presence of Streptococcus spp. was also related to culling specifically for mastitis, whereas culling for all reasons and treatment of clinical mastitis was not related to a positive culture of Strep. spp. Presence of coagulase-negative Staph. spp. or other pathogens was not associated with either of the outcome variables.     

Systemic prepartum treatment of end-term dairy heifers with penethamate hydriodide: Effect on udder health,milk yield,and culling until 120 days in milk

Prepartum intramammary treatment with antimicrobials of end-term dairy heifers has frequently been proposed as a practice to reduce the prevalence of intramammary infections (IMI) at calving. From a safety standpoint for both animal and administrator, systemic treatment is preferred. A clinical trial was conducted on heifers from 10 well-managed, commercial dairy farms with a low prevalence of heifer mastitis. The aim was to assess both the short- and long-term effects of a systemic prepartum therapy with penethamate hydriodide on udder health and milk production. Because it was hypothesized that some herds would benefit more from this treatment than others, specific herd-level information was collected before the start of the actual trial to screen for and explain potential herd-specific treatment effects. Further, the effect of treatment on antimicrobial susceptibility of staphylococcal isolates was monitored. End-term heifers were either treated systemically (over 3 consecutive days) 2 wk before expected calving date with penethamate hydriodide (n = 76) or remained untreated (n = 73). Systemic prepartum treatment of end-term heifers with penethamate hydriodide resulted in fewer IMI in early lactation. However, all 6 cases of clinical mastitis in early lactation occurred in the treatment group [Streptococcus uberis (n = 1), Corynebacterium bovis (n = 1), Staphylococcus aureus (n = 1); 1 sample was contaminated; 2 samples remained culture negative]. No long-term treatment effects (from 4 to 120 d in milk) on milk production, udder health, or culling hazard during later lactation were detected, although treated heifers belonging to herds classified as having low-yielding heifers out-produced the control heifers. Moreover, penicillin susceptibility of staphylococci isolated from milk samples of treated or control heifers did not differ. Herds with a low prevalence of heifer mastitis are not likely to benefit from prepartum systemic antimicrobial treatment of the end-term heifers.     

Risk factors associated with bulk tank standard plate count,bulk tank coliform count,and the presence of Staphylococcus aureus on organic and conventional dairy farms in the United States

The purpose of this study was to assess the association of bulk tank milk standard plate counts, bulk tank coliform counts (CC), and the presence of Staphylococcus aureus in bulk tank milk with various management and farm characteristics on organic and conventional dairy farms throughout New York, Wisconsin, and Oregon. Data from size-matched organic farms (n = 192), conventional nongrazing farms (n = 64), and conventional grazing farms (n = 36) were collected at a single visit for each farm. Of the 292 farms visited, 290 bulk tank milk samples were collected. Statistical models were created using data from all herds in the study, as well as exclusively for the organic subset of herds. Because of incomplete data, 267 of 290 herds were analyzed for total herd modeling, and 173 of 190 organic herds were analyzed for the organic herd modeling. Overall, more bulk tanks from organic farms had Staph. aureus cultured from them (62% of organic herds, 42% conventional nongrazing herds, and 43% of conventional grazing herds), whereas fewer organic herds had a high CC, defined as ≥50 cfu/mL, than conventional farms in the study. A high standard plate count (×1,000 cfu/mL) was associated with decreased body condition score of adult cows and decreased milk production in both models. Several variables were significant only in the model created using all herds or only in organic herds. The presence of Staph. aureus in the bulk tank milk was associated with fewer people treating mastitis, increased age of housing, and a higher percentage of cows with 3 or fewer teats in both the organic and total herd models. The Staph. aureus total herd model also showed a relationship with fewer first-lactation animals, higher hock scores, and less use of automatic takeoffs at milking. High bulk tank CC was related to feeding a total mixed ration and using natural service in nonlactating heifers in both models. Overall, attentive management and use of outside resources were useful with regard to CC on organic farms. In all models except the organic CC model, we observed an association with the average reported somatic cell count from 3 mo before the herd visit, indicating that many of the regularly tested milk quality parameters are interconnected. In conclusion, we found that conventional and organic farms are similar in regard to overall herd management, but each grazing system faces unique challenges when managing milk quality.     

Evaluation of the Minnesota Easy Culture System II Bi-Plate and Tri-Plate for identification of common mastitis pathogens in milk

The objective of this study was to validate use of the Minnesota Easy Culture System II Bi-Plate and Tri-Plate (University of Minnesota Laboratory for Udder Health, St. Paul) to identify common mastitis pathogens in milk. A total of 283 quarter and composite milk samples submitted to the University of Minnesota Laboratory for Udder Health during the spring of 2010 were cultured simultaneously using 3 methods: standard laboratory culture (reference method) and the Minnesota Easy Culture System II Bi-Plate and Tri-Plate methods. Bi-Plate and Tri-Plate cultures were incubated for 18 to 24 h and interpreted by 2 independent, untrained readers within 5 h of each other. An experienced technician completed the standard laboratory culture. For each sample, all 3 study personnel recorded the culture result (yes/no) for each of the following diagnostic categories: no bacterial growth (NG), mixed (2 organisms), contaminated (3 or more organisms), gram-positive (GP), gram-negative (GN), Staphylococcus spp., Streptococcus spp., Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Enterococcus spp., Staphylococcus aureus, coagulase-negative staphylococci, Escherichia coli, Klebsiella spp., and other. For each category, the prevalence, sensitivity, specificity, accuracy, and predictive values of a positive and negative test were calculated, and the agreement between readers and between each reader and the laboratory was assessed. Specificity, overall accuracy, and negative predictive values were generally high (>80%) for the Bi-Plate and Tri-Plate for each category. Sensitivity and positive predictive values were intermediate (>60%) or high (>80%) for the broad categories of NG, GP, GN, Staphylococcus spp. and Streptococcus spp., and for Staph. aureus, but were generally lower (<60%) for other more specific categories. Similarly, interreader agreement (kappa value) was moderate to substantial (40–80%) for the broad categories of NG, GP, GN, Staphylococcus spp. and Streptococcus spp., and for Staph. aureus and E. coli, but was lower for other categories. The Tri-Plate had a higher sensitivity, accuracy, and negative predictive value for Streptococcus spp., and higher interreader agreement for some of the more specific categories. Our conclusion was that Bi-Plate and Tri-Plate results will be most reliable when used to classify infections in broad diagnostic categories such NG, GP, or GN. The Bi-Plate and Tri-Plate will have intermediate ability to identify infections as being caused by Staphylococcus spp., Streptococcus spp., or Staph. aureus.     

Clinical outcomes and molecular genotyping of Staphylococcus aureus isolated from milk samples of dairy primiparous Mediterranean buffaloes (Bubalus bubalis)

Staphylococcus aureus is one of the most important pathogens causing mastitis in dairy cows and in Mediterranean buffaloes. Genotype B (GTB) is contagious in dairy cows and may occur in up to 87% of cows of a dairy herd. It was the aim of this study to evaluate genotypes present, clinical outcomes, and prevalence of Staph. aureus in milk samples of primiparous Mediterranean dairy buffaloes. Two hundred composite milk samples originating from 40 primiparous buffaloes were collected from May to June 2012, at d 10, 30, 60, 90, and 150 d in milk (DIM) to perform somatic cell counts and bacteriological cultures. Daily milk yields were recorded. Before parturition until 40 to 50 DIM, all primiparous animals were housed separated from the pluriparous animals. Milking was performed in the same milking parlor, but the primiparous animals were milked first. After 50 DIM, the primiparous were mixed with the pluriparous animals, including the milking procedure. Individual quarter samples were collected from each animal, and aliquots of 1 mL were mixed and used for molecular identification and genotyping of Staph. aureus. The identification of Staph. aureus was performed verifying the presence of nuc gene by nuc gene PCR. All the nuc-positive isolates were subjected to genotype analysis by means of PCR amplification of the 16S-23S rRNA intergenic spacer region and analyzed by a miniaturized electrophoresis system. Of all 200 composite samples, 41 (20.5%) were positive for Staph. aureus, and no genotype other than GTB was identified. The prevalence of samples positive for Staph. aureus was 0% at 10 DIM and increased to a maximum of 22/40 (55%) at 90 DIM. During the period of interest, 14 buffaloes tested positive for Staph. aureus once, 6 were positive twice, and 5 were positive 3 times, whereas 15 animals were negative at every sampling. At 90 and 150 DIM, 7 (17.5%) and 3 buffaloes (7.5%), respectively, showed clinical mastitis (CM), and only 1 (2.5%) showed CM at both samplings. At 60, 90, and 150 DIM, 1 buffalo was found with subclinical mastitis at each sampling. At 30, 60, 90, and 150 DIM, 2.5 (1/40), 22.5 (9/40), 35 (14/40), and 10% (4/40) were considered affected by intramammary infection, respectively. Buffaloes with CM caused by Staph. aureus had statistically significantly higher mean somatic cell count values (6.06 ± 0.29, Log₁₀ cells/mL ± standard deviation) and statistically significantly lower mean daily milk yields (7.15 ± 1.49, liters/animal per day) than healthy animals (4.69 ± 0.23 and 13.87 ± 2.64, respectively), buffaloes with IMI (4.82 ± 0.23 and 11.16 ± 1.80, respectively), or with subclinical mastitis (5.47 ± 0.10 and 10.33 ± 0.68, respectively). Based on our knowledge, this is the first time that Staph. aureus GTB has been identified in milk samples of dairy Mediterranean buffaloes.     

Short communication: Comparing real-time PCR and bacteriological cultures for Streptococcus agalactiae and Staphylococcus aureus in bulk-tank milk samples

For more than 30 yr, a control plan for Streptococcus agalactiae and Staphylococcus aureus has been carried out in more than 1,500 dairy herds of the province of Brescia (northern Italy). From 2010 to 2011, the apparent prevalence of Strep. agalactiae has been relatively stable around 10%, but the apparent prevalence of Staph. aureus has been greater than 40% with an increasing trend. The aim of this paper was to estimate and compare the diagnostic accuracy of 3 assays for the detection of Strep. agalactiae and Staph. aureus in bulk-tank milk samples (BTMS) in field conditions. The assays were a qualitative and a quantitative bacteriological culture (BC) for each pathogen and a homemade multiplex real-time PCR (rt-PCR). Because a gold standard was not available, the sensitivities (Se) and specificities (Sp) were evaluated using a Bayesian latent class approach. In 2012 we collected one BTMS from 165 dairy herds that were found positive for Strep. agalactiae in the previous 2-yr campaigns of eradication plan. In most cases, BTMS collected in these herds were positive for Staph. aureus as well, confirming the wide spread of this pathogen. At the same time we also collected composite milk samples from all the 8,624 lactating cows to evaluate the within-herd prevalence of Strep. agalactiae. Streptococcus agalactiae samples were cultured using a selective medium Tallium Kristalviolette Tossin, whereas for Staph. aureus, we used Baird Parker modified medium with added Rabbit Plasma Fibrinogen ISO-Formulation. In parallel, BTMS were tested using the rt-PCR. Regarding Strep. agalactiae, the posterior median of Se and Sp of the 2 BC was similar [qualitative BC: Se = 98%, posterior credible interval (95%PCI): 94–100%, and Sp = 99%, 95%PCI: 96–100%; quantitative BC: Se = 99%, 95%PCI: 96–100%, and Sp = 99%, 95%PCI: 95–100%] and higher than those of the rt-PCR (at 40 cycle threshold, Se = 92%, 95%PCI: 85–97%; Sp = 94%, 95%PCI: 88–98%). Also in case of Staph. aureus, the posterior medians of BC were generally higher than those of rt-PCR. In fact, although the Se of BC was slightly lower (rt-PCR at 40 cycle threshold, median Se = 99%, 95%PCI: 97–100%, and qualitative BC, median Se = 94%, 95%PCI: 87–99%), the Sp was much higher (rt-PCR at 40 cycle threshold, median Sp = 67%, 95%PCI: 38–97%; qualitative BC, median Sp = 95%; 95%PCI: 76–100%). Our study confirms that BC and rt-PCR are reliable diagnostic tools to detect Strep. agalactiae and Staph. aureus, and rt-PCR results should be confirmed by BC carried out on BTMS and possibly on composite milk samples.     

Distribution of coagulase-negative Staphylococcus species from milk and environment of dairy cows differs between herds

In many parts of the world, coagulase-negative staphylococci (CNS) are the predominant pathogens causing intramammary infections (IMI) in dairy cows. The cows’ environment is thought to be a possible source for CNS mastitis and this was investigated in the present paper. A longitudinal field study was carried out in 6 well-managed dairy herds to determine the distribution and epidemiology of various CNS species isolated from milk, causing IMI and living freely in the cows’ environment, respectively. In each herd, quarter milk samples from a cohort of 10 lactating cows and environmental samples from stall air, slatted floor, sawdust from cubicles, and sawdust stock were collected monthly (n = 13). Isolates from quarter milk samples (n = 134) and the environment (n = 637) were identified to species level using amplified fragment length polymorphism (AFLP) genotyping. Staphylococcus chromogenes, S. haemolyticus, S. epidermidis, and S. simulans accounted for 81.3% of all CNS milk isolates. Quarters were considered infected with CNS (positive IMI status) only when 2 out of 3 consecutive milk samples yielded the same CNS AFLP type. The species causing IMI were S. chromogenes (n = 35 samples with positive IMI status), S. haemolyticus (n = 29), S. simulans (n = 14), and S. epidermidis (n = 6). The observed persistent IMI cases (n = 17) had a mean duration of 149.4 d (range 63.0 to 329.8 d). The CNS species predominating in the environment were S. equorum, S. sciuri, S. haemolyticus, and S. fleurettii. Herd-to-herd differences in distribution of CNS species were observed in both milk and the environment, suggesting that herd-level factors are involved in the establishment of particular species in a dairy herd. Primary reservoirs of the species causing IMI varied. Staphylococcus chromogenes and S. epidermidis were rarely found in the environment, indicating that other reservoirs were more important in their epidemiology. For S. haemolyticus and S. simulans, the environment was found as a reservoir, suggesting that IMI with these species were possibly environmental in origin.     

Staphylococcus aureus adlb gene is associated with high prevalence of intramammary infection in dairy herds of northern Italy: A cross-sectional study

Staphylococcus aureus is a major mastitis pathogen in dairy cattle worldwide, responsible for substantial economic losses. Environmental factors, milking routine, and good maintenance of milking equipment have been described as important factors to prevent intramammary infections (IMI). Staphylococcus aureus IMI can be widespread within the farm or the infection can be limited to few animals. Several studies have reported that Staph. aureus genotypes differ in their ability to spread within a herd. In particular, Staph. aureus belonging to ribosomal spacer PCR genotype B (GTB)/clonal complex 8 (CC8) is associated with high within-herd prevalence of IMI, whereas other genotypes are generally associated with individual cow disease. The adlb gene seems to be strictly related to Staph. aureus GTB/CC8, and is a potential marker of contagiousness. We investigated Staph. aureus IMI prevalence in 60 herds in northern Italy. In the same farms, we assessed specific indicators linked to milking management (e.g., teat condition score and udder hygiene score) and additional milking risk factors for IMI spread. Ribosomal spacer-PCR and adlb-targeted PCR were performed on 262 Staph. aureus isolates, of which 77 underwent multilocus sequence typing. In most of the herds (90%), a predominant genotype was identified, especially Staph. aureus CC8 (30%). In 19 of 60 herds, the predominant circulating Staph. aureus was adlb-positive and the observed IMI prevalence was relevant. Moreover, the adlb gene was detected only in genotypes of CC8 and CC97. Statistical analysis showed a strong association between the prevalence of Staph. aureus IMI, the specific CCs, and carriage of adlb, with the predominant circulating CC and presence of the gene alone explaining the total variation. Interestingly, the difference in the odds ratio obtained in the models for CC8 and CC97 suggests that it is carriage of the adlb gene, rather than the circulation of these CCs per se, that leads to higher within-herd prevalence of Staph. aureus. In addition, the model showed that environmental and milking management factors had no or minimal effect on Staph. aureus IMI prevalence. In conclusion, the circulation of adlb-positive Staph. aureus strains within a herd has a strong effect on the prevalence of IMI. Thus, adlb can be proposed as a genetic marker of contagiousness for Staph. aureus IMI in cattle. However, further analyses using whole-genome sequencing are required to understand the role of genes other than adlb that may be involved in the mechanisms of contagiousness of Staph. aureus strains associated with high prevalence of IMI.