Simultaneous detection of mastitis pathogens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by multiplex real-time polymerase chain reaction

The objective of this study was to develop a multiplex real-time polymerase chain reaction (PCR) method for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis directly from milk. A genetic marker specific for Staph. aureus was used for primers and dual-labeled probe design. The target for Strep. agalactiae primers and dual-labeled probe was selected from the cfb gene encoding the Christie-Atkins-Munch-Petersen factor. The plasminogen activator gene was the target for primers and dual-labeled probe design for Strep. uberis. Quarter milk samples (n = 192) were analyzed by the multiplex real-time PCR assay and conventional microbiological methods. An additional 57 quarter milk samples were analyzed in a separate real-time PCR assay for Strep. agalactiae only. Using an overnight enrichment step, the real-time PCR technique correctly identified 96.4% of all quarter milk samples; 91.7% of Staph. aureus, 98.2% of Strep. agalactiae, and 100% of Strep. uberis. Results of conventional microbiological methods were used to determine the sensitivity and specificity of the multiplex real-time PCR procedure. The sensitivity of the procedure to correctly identify Staph. aureus, Strep. agalactiae, and Strep. uberis directly from milk was 95.5%, and the specificity was 99.6%. Results of this study indicate that the multiplex real-time PCR procedure has the potential to be a valuable diagnostic technique for simultaneous identification of Staph. aureus, Strep. agalactiae, and Strep. uberis directly from quarter milk samples.     

Pathogen-specific production losses in bovine mastitis

Reduction in long-term milk yields represents a notable share of the economic losses caused by bovine mastitis. Efficient, economic, and safe measures to prevent these losses require knowledge of the causal agent of the disease. The aim of this study was to investigate pathogen-specific impacts of mastitis on milk production of dairy cows. The materials consisted of milk and health recording data and microbiological diagnoses of mastitic quarter milk samples of 20,234 Finnish dairy cows during 2010, 2011, and 2012. The 6 most common udder pathogens were included in the study: Staphylococcus aureus, non-aureus staphylococci (NAS), Escherichia coli, Corynebacterium bovis, Streptococcus uberis, and Streptococcus dysgalactiae. We used a 2-level multilevel model to estimate curves for lactations with and without mastitis. The data on lactation periods to be compared were collected from the same cow. To enable comparison among lactations representing diverse parities, the estimated lactation curves were adjusted to describe the cow's third lactation. Mastitis caused by each pathogen resulted in milk production loss. The extent of the reduction depended on the pathogen, the timing of mastitis during lactation, and the type of mastitis (clinical vs. subclinical). The 2 most commonly detected pathogens were NAS and Staph. aureus. Escherichia coli clinical mastitis diagnosed before peak lactation caused the largest loss, 10.6% of the 305-d milk yield (3.5 kg/d). The corresponding loss for Staph. aureus mastitis was 7.1% (2.3 kg/d). In Staph. aureus mastitis diagnosed between 54 and 120 d in milk, the loss was 4.3% (1.4 kg/d). The loss was almost equal in both clinical and subclinical mastitis caused by Staph. aureus. Mastitis caused by Strep. uberis and Strep. dysgalactiae resulted in losses ranging from 3.7% (1.2 kg/d) to 6.6% (2.1 kg/d) depending on type and timing of mastitis. Clinical mastitis caused by the minor pathogens C. bovis and NAS also had a negative effect on milk production: 7.4% (2.4 kg/d) in C. bovis and 5.7% (1.8 kg/d) in NAS when both were diagnosed before peak lactation. In conclusion, minor pathogens should not be underestimated as a cause of milk yield reduction. On single dairy farms, control of E. coli mastitis would bring about a significant increase in milk production. Reducing Staph. aureus mastitis is the greatest challenge for the Finnish dairy sector.     

Non-culture-based identification of mastitis-causing bacteria by MALDI-TOF mass spectrometry

The purpose of this study was to evaluate the detection limit of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for direct identification, without previous microbiological culture, of bovine mastitis-causing bacteria from milk samples. Milk samples (n = 15) were experimentally contaminated with Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, and Escherichia coli to have bacterial counts ranging from 10³ to 10⁹ cfu/mL. These contaminated milk samples were subjected to a preparation protocol for bacterial ribosomal protein extraction using the MALDI Sepsityper kit (Bruker Daltonik, Bremen, Germany), which allowed MALDI-TOF MS coupled with Biotyper software (Bruker Daltonik) to identify bacterial fingerprints based on intact ribosomal proteins. The ability of MALDI-TOF MS to correctly identify bacterial strains from experimentally contaminated milk (without previous microbiological culture) depended on the bacterial count of the samples and on the species of the bacteria evaluated. Adequate identification at the bacterial species level (score ≥2.0) directly from milk samples required bacterial counts in the following ranges: ≥10⁶ cfu/mL of Staph. aureus, ≥10⁷ cfu/mL of E. coli, and ≥10⁸ cfu/mL of Strep. agalactiae, Strep. dysgalactiae, and Strep. uberis. We concluded that direct identification of mastitis-causing pathogens is possible for Staph. aureus, E. coli, Strep. agalactiae, Strep. dysgalactiae, and Strep. uberis, but correct identification depended on the bacterial count in the milk samples.     

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.     

Association between teat skin colonization and intramammary infection with Staphylococcus aureus and Streptococcus agalactiae in herds with automatic milking systems

The objective of this study was to investigate the association between teat skin colonization and intramammary infection (IMI) with Staphylococcus aureus or Streptococcus agalactiae at the quarter level in herds with automatic milking systems. Milk and teat skin samples from 1,142 quarters were collected from 300 cows with somatic cell count >200,000 cells/mL from 8 herds positive for Strep. agalactiae. All milk and teat skin samples were cultured on calf blood agar and selective media. A subset of samples from 287 quarters was further analyzed using a PCR assay (Mastit4 PCR; DNA Diagnostic A/S, Risskov, Denmark). Bacterial culture detected Staph. aureus in 93 (8.1%) of the milk samples and 75 (6.6%) of the teat skin samples. Of these, 15 (1.3%) quarters were positive in both the teat skin and milk samples. Streptococcus agalactiae was cultured in 84 (7.4%) of the milk samples and 4 (0.35%) of the teat skin samples. Of these, 3 (0.26%) quarters were positive in both the teat skin and milk samples. The PCR detected Staph. aureus in 29 (10%) of the milk samples and 45 (16%) of the teat skin samples. Of these, 2 (0.7%) quarters were positive in both the teat skin and milk samples. Streptococcus agalactiae was detected in 40 (14%) of the milk samples and 51 (18%) of the teat skin samples. Of these, 16 (5.6%) quarters were positive in both the teat skin and milk samples. Logistic regression was used to investigate the association between teat skin colonization and IMI at the quarter level. Based on bacterial culture results, teat skin colonization with Staph. aureus resulted in 7.8 (95% confidence interval: 2.9; 20.6) times higher odds of Staph. aureus IMI, whereas herd was observed as a major confounder. However, results from the PCR analyses did not support this association. Streptococcus agalactiae was isolated from the teat skin with both PCR and bacterial culture, but the number of positive teat skin samples detected by culture was too low to proceed with further analysis. Based on the PCR results, Strep. agalactiae on teat skin resulted in 3.8 (1.4; 10.1) times higher odds of Strep. agalactiae IMI. Our results suggest that Staph. aureus and Strep. agalactiae on teat skin may be a risk factor for IMI with the same pathogens. Focus on proper teat skin hygiene is therefore recommended also in AMS.     

Assessment of an extraction protocol to detect the major mastitis-causing pathogens in bovine milk

Despite all efforts to control its spread, mastitis remains the most costly disease for dairy farmers worldwide. One key component of better control of this disease is identification of the causative bacterial agent during udder infections in cows. Mastitis is complex, however, given the diversity of pathogens that must be identified. Development of a rapid and efficient bacterial species identification tool is thus necessary. This study was conducted to demonstrate the feasibility of bacterial DNA extraction for the automated molecular detection of major mastitis-causing pathogens directly in milk samples to complement traditional microbiological identification. Extraction and detection procedures were designed and optimized to achieve detection in a respectable time frame, at a reasonable cost, and with a high throughput capacity. The following species were identified: Staphylococcus aureus, Escherichia coli, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, and Klebsiella spp. (including Klebsiella oxytoca and Klebsiella pneumoniae). The detection procedure includes specific genomic DNA amplification by multiplex PCR for each species, separation by capillary electrophoresis, and laser-assisted automated detection. The specificity of the primers was assessed with a panel of bacteria representing mastitis-negative control species. The extraction protocol comprised multiple steps, starting with centrifugation for fat removal, followed by heating in the presence of a cation exchange resin to trap divalent ions. The analytical sensitivity was 100 cfu/mL for milk samples spiked with Staph. aureus, Strep. dysgalactiae, and E. coli, with a tendency for K. pneumoniae. The detection limit was 500 cfu/mL for Strep. uberis and Strep. agalactiae. The overall diagnostic sensitivity (95.4%) and specificity (97.3%) were determined in a double-blind randomized assay by processing 172 clinical milk samples with microbiological characterization as the gold standard. When the physical nature of the milk samples was too altered, DNA purification with a magnetic bead-based system was used. Of the apparent false-positive samples, 5 were identified by specific microbiological analysis as true-positive Staph. aureus co-infections, with further confirmation by ribosomal 16S sequencing. The proposed methodology could, therefore, become an interesting tool for automated PCR detection of major mastitis pathogens in dairy cattle.     

Short communication: Antimicrobial efficacy of intramammary treatment with a novel biphenomycin compound against Staphylococcus aureus, Streptococcus uberis, and Escherichia coli-induced mouse mastitis

Bovine mastitis undermines udder health, jeopardizes milk production, and entails prohibitive costs, estimated at $2 billion per year in the dairy industry of the United States. Despite intensive research, the dairy industry has not managed to eradicate the 3 major bovine mastitis-inducing pathogens: Staphylococcus aureus, Streptococcus uberis, and Escherichia coli. In this study, the antimicrobial efficacy of a newly formulated biphenomycin compound (AIC102827) was assessed against intramammary Staph. aureus, Strep. uberis, and E. coli infections, using an experimental mouse mastitis model. Based on its effective and protective doses, AIC102827 applied into the mammary gland was most efficient to treat Staph. aureus, but also adequately reduced growth of Strep. uberis or E. coli, indicating its potential as a broad-spectrum candidate to treat staphylococcal, streptococcal, and coliform mastitis in dairy cattle.     

Within-herd prevalence of intramammary infection caused by Mycoplasma bovis and associations between cow udder health,milk yield,and composition

Subclinical mastitis is one of the major health problems in dairy herds due to decreased milk production and reduced milk quality. The aim of this study was to examine the within-herd prevalence of subclinical intramammary infection caused by Mycoplasma bovis and to evaluate associations between M. bovis and cow daily milk yield, udder health, and milk composition. Individual cow composite milk samples (n = 522) were collected from all lactating dairy cows in 1 Estonian dairy farm in November 2014. Daily milk yield, days in milk, and parity were recorded. Collected milk samples were analyzed for somatic cell count, milk protein, fat, and urea content. The presence of M. bovis, Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis in the milk samples was confirmed by quantitative PCR analysis. The within-herd prevalence of M. bovis was 17.2% in the study herd. No association was observed between days in milk and parity to the presence of M. bovis in milk. According to linear regression analysis, the daily milk yield from cows positive for M. bovis was on average 3.0 kg lower compared with cows negative for M. bovis. In addition, the presence of M. bovis in milk samples was significantly associated with higher somatic cell count and lower fat and urea content compared with milk samples negative for M. bovis. In conclusion, subclinical M. bovis intramammary infection is associated with decreased milk yield and lower milk quality.     

Evaluating a commercial PCR assay against bacterial culture for diagnosing Streptococcus uberis and Staphylococcus aureus throughout lactation

The performance of a commercial, real-time PCR assay was compared with traditional bacterial culture for the identification of Streptococcus uberis and Staphylococcus aureus in bovine milk collected at different stages of lactation. Initial validation tests using fresh and frozen quarter milk samples identified factors that affected the success of the PCR. Therefore, the standard protocol was adjusted for samples collected at the first milking postpartum (colostrum) and from clinical mastitis cases. The adjustment involved PCR testing both undiluted and diluted (1 in 10 with sterile water) DNA extracts. The performance comparison between culture and the PCR assay used milk samples collected aseptically from individual quarters of mixed-age spring-calving dairy cows, during early, mid, and late lactation. Bacterial culture results were used to select a subset of samples for PCR testing (n = 315) that represented quarters with a current or prior Strep. uberis or Staph. aureus infection. Compared with culture, PCR had a sensitivity of 86.8% and specificity of 87.7% for detecting Strep. uberis (kappa = 0.74) and 96.4% and 99.7%, respectively, for detecting Staph. aureus (kappa = 0.96). The dilution of DNA extracts for colostrum and clinical samples increased the relative sensitivity from 79.2% to 86.8% for Strep. uberis detection and from 92.9% to 96.4% for Staph. aureus, presumably through diluting unidentified PCR inhibitors. The sensitivity for detecting Strep. uberis using PCR, relative to culture, was similar throughout lactation (85–89%), whereas relative specificity was lowest immediately postcalving (64%) but improved in mid and late lactation (98%). Specificity estimates for samples collected in early lactation can be optimized by reducing the cutoff cycle threshold (Ct) value from the recommended value of 37 to 34. Although using this value improved specificity (77%), it reduced test sensitivity (77%). The PCR assay lacked agreement with culture in early lactation, specifically for diagnosing Strep. uberis. Thus, PCR should not be used as the only tool for diagnosing mastitis in early lactation.     

Antimicrobial activity of bovine β-lactoglobulin against mastitis-causing bacteria

Bovine mastitis is one of the most economically deleterious diseases affecting dairy herds and results from an infection of the udder by pathogenic microorganisms such as Staphylococcus aureus, Streptococcus uberis, and Escherichia coli. The mammary gland is capable of preventing and combating bacterial infection by means of a complex network of innate and adaptive immune mechanisms. Lactoferrin is an 86-kDa protein with antibacterial activity that plays a role in the mammary gland's defense against infection. β-Lactoglobulin (β-LG) is an 18-kDa protein that is present in most mammals but is notably absent in humans, rodents, and lagomorphs. Different genetic variants of this protein exist, with β-LG A and β-LG B being the most common. In spite of being well studied, the biological function of β-LG is not thoroughly understood, and most noticeably, no reports exist on the effects of the native protein on bacterial growth. Hence, the objective of this study was to assess the potential antibacterial activity of β-LG against mastitis agents. To do this, we purified β-LG from normal bovine milk using a mild, nondenaturing method and performed in vitro growth inhibition assays with Staph. aureus, E. coli, and Strep. uberis. β-Lactoglobulin inhibited the growth of Staph. aureus and Strep. uberis but had no effect on E. coli. The antimicrobial activity against Staph. aureus and Strep. uberis was concentration dependent and was elicited by the intact protein because Tricine-sodium dodecyl sulfate-PAGE and analytical gel filtration chromatography did not reveal the presence of short degradation peptides. Analysis of the genetic variants of β-LG showed that β-LG A has higher inhibitory activity against Staph. aureus and Strep. uberis than β-LG B. Coincubation of β-LG and lactoferrin resulted in an augmented antibacterial activity against Staph. aureus, suggesting an additive effect of the proteins. This result, along with the proteins’ complementary spectrum of action, suggests that β-LG and lactoferrin may complement each other in the mammary gland's defenses against bacterial infection.     

Relationship between milk lactoferrin and etiological agent in the mastitic bovine mammary gland

Bovine mastitis is one of the most deleterious diseases for dairy herds and is mainly caused by contagious and environmental bacterial pathogens. Among contagious bacteria, Staphylococcus aureus is the most prevalent, whereas the main environmental mastitis pathogens are Streptococcus uberis and Escherichia coli. Bovine lactoferrin (bLF) is an approximately 80-kDa glycoprotein present in milk that participates in the innate response of the mammary gland against bacterial infection. The objectives of the current study were to analyze potential changes in bLF milk concentration, which would constitute a response of the mammary gland toward mastitis induced by different etiologic agents, and to evaluate a possible relation between this response and pathogen susceptibility to bLF. Microbiology analysis and bLF quantification in milk from different bovine mammary gland quarters were performed. Infected quarters presented greater concentrations of bLF compared with those from microbiologically negative quarters. Analysis of individual pathogen contributions showed that most of this increase was attributable to Strep. uberis intra-mammary infection. The ability of mammary gland cells to synthesize bLF in response to Strep. uberis challenge was demonstrated by immunodetection of the protein in in vitro infection experiments. Susceptibility of Strep. uberis, E. coli, and Staph. aureus to the antimicrobial activity of bLF was determined by growth inhibition assays conducted with 4 different isolates of each species. Whereas Staph. aureus and E. coli were shown to be susceptible to this protein, Strep. uberis appeared to be resistant to the antimicrobial activity of bLF. Molecular typing of the 4 Strep. uberis isolates used throughout this study showed that this result was representative of the species and not exclusive of a particular strain. Results presented herein suggest that different bacteria species may elicit different mammary gland responses mediated by bLF secretion and that Strep. uberis has probably adapted to this immune reaction by developing resistance to bLF inhibitory action.     

A survey of mastitis pathogens including antimicrobial susceptibility in southeastern Australian dairy herds

The objectives for this study were to (1) describe the pathogen profile in quarters from cows with clinical mastitis and in cows with subclinical mastitis in southeastern Australia; and (2) describe antimicrobial susceptibility among isolated pathogens. As a secondary objective, we aimed to compare antimicrobial resistance prevalence in pathogens isolated from clinical and subclinical mastitis samples. A convenience sample of dairy herds (n = 65) from 4 regions in southeastern Australia (Gippsland, Northern Victoria, Tasmania, Western Victoria) were invited to submit milk samples from cows with clinical and subclinical mastitis over a 14-mo period (January 2011 to March 2012). Farmers were instructed to collect aseptic quarter milk samples from the first 10 cases of clinical mastitis for each month of the study. In addition, farmers submitted composite milk samples from cows with subclinical mastitis at 1 or 2 sampling occasions during the study period. Aerobic culture and biochemical tests were used to identify isolates. Isolates were classified as susceptible, intermediate, or resistant to a panel of antimicrobial agents based on the zone of growth inhibition around antimicrobial-impregnated disks, with antimicrobial resistance (AMR) classified as nonsusceptibility by combining intermediate and resistant groups into a single category. Generalized linear mixed models were used to compare the prevalence of AMR between clinical and subclinical mastitis isolates. For clinical mastitis samples (n = 3,044), 472 samples (15.5%) were excluded for contamination. Of the remaining samples (n = 2,572), the most common results were Streptococcus uberis (39.2%), no growth (27.5%), Staphylococcus aureus (10.6%), Escherichia coli (8.4%), and Streptococcus dysgalactiae (6.4%). For subclinical mastitis samples (n = 1,072), 425 (39.6%) were excluded due to contamination. Of the remaining samples (n = 647), the most common results were no growth (29.1%), Staph. aureus (29.1%), and Strep. uberis (21.6%). The prevalence of AMR among common isolates was low for the majority of antimicrobial agents. Exploratory analysis found that the probability of Staph. aureus demonstrating resistance to penicillin was 5.16 times higher (95% confidence interval: 1.68, 15.88) in subclinical isolates relative to clinical Staph. aureus isolates. A similar association was observed for amoxicillin with subclinical Staph. aureus isolates being 4.70 times (95% confidence interval: 1.49, 14.75) more likely to be resistant than clinical Staph. aureus isolates. We concluded that the most common bacteria causing clinical mastitis in dairy herds in Australia is likely to be Strep. uberis, whereas Staph. aureus is likely to be the most common cause of subclinical mastitis. Despite decades of antimicrobial use to control these organisms, AMR appears to be uncommon.     

Canadian National Dairy Study: Herd-level milk quality

The objective of this study was to estimate Canadian national milk quality parameters and estimate the bulk tank milk (BTM) prevalence of 4 mastitis pathogens, Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma bovis, and Prototheca spp., on Canadian dairy farms. A questionnaire was sent to all Canadian dairy producers. Of the 1,062 producers who completed the questionnaire, 374 producers from across the country were visited and milking hygiene was assessed. Farm-level milk quality data for all Canadian dairy producers was collected from the provincial marketing boards and combined with the questionnaire and farm visit data. In addition, a BTM sample was collected either during the farm visit or by the marketing board in November of 2015 and was tested for 4 major mastitis pathogens using the PathoProof Mastitis Major 4 PCR Assay (Thermo Fisher Scientific Inc., Waltham, MA). Apparent herd-level prevalence was 46% for S. aureus, 6% for Prototheca spp., 0% for M. bovis, and 0% for Strep. agalactiae. Due to the low prevalence of M. bovis and Strep. agalactiae and a lack of significant factors associated with farms testing positive for Prototheca spp., an association analysis could only be carried out for Staph. aureus-positive farms. Factors associated with Staph. aureus-positive farms were not fore-stripping cows before milking (odds ratio = 1.87), milking with a pipeline system (odds ratio = 2.21), and stall bases made of a rubberized surface (mats and mattresses), whereas protective factors were using blanket dry cow therapy (odds ratio = 0.49) and applying a tag or visible mark on cows known to have chronic mastitis infections (odds ratio = 0.45). The Canadian national production-weighted geometric mean somatic cell count was determined to be 208,000 cells/mL. This is the first national dairy study conducted in Canada. Participating farms had higher milk yield; were more likely to have a loose housing system, parlor, or automated milking system; and had lower weighted mean BTM somatic cell count than the national level. Sampling larger farms with better milk quality means the apparent prevalence of the 4 mastitis pathogens likely underestimates the true levels.     

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.     

Within-herd prevalence thresholds for herd-level detection of mastitis pathogens using multiplex real-time PCR in bulk tank milk samples

The objective of the study was to assess the value of quantitative multiplex real-time PCR examination of bulk tank milk samples for bovine mastitis pathogens as a tool for herd level diagnosis. Using a logistic regression model, this study is aimed at calculating the threshold level of the apparent within-herd prevalence as determined by quarter milk sample cultivation of all lactating cows, thus allowing the detection of a herd positive for a specific pathogen within certain probability levels. A total of 6,335 quarter milk samples were collected and cultured from 1,615 cows on 51 farms in Germany. Bulk tank milk samples were taken from each farm and tested by bacterial culture as well as the commercial PCR assay Mastit 4A (DNA Diagnostic A/S, Risskov, Denmark) identifying Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus agalactiae, and Streptococcus uberis. In addition, PCR was performed on pooled herd milk samples containing milk aliquots from all lactating cows in each of the 51 herds. Only 1 out of the 51 herds was found PCR positive for Streptococcus agalactiae in bulk tank and pooled herd milk samples, and cultured quarter milk samples. Spearman's rank correlations between the cycle threshold value of bulk tank milk PCR and the apparent within-herd prevalence were calculated in regard to Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis. For these pathogens, significant correlations were found. If 1 bulk tank milk sample per herd was tested, the estimated within-herd prevalence thresholds for 90% probability of detection were 27.6% for Staphylococcus aureus, 9.2% for Streptococcus dysgalactiae, and 13.8% for Streptococcus uberis on the cow level. On the quarter level, the within-herd prevalence had to be at least 32.6% for Staphylococcus aureus, 1.7% for Streptococcus dysgalactiae, and 4.3% for Streptococcus uberis to detect a herd as positive using a single bulk milk sample. The results indicate that mastitis pathogens in bulk tank milk can be identified by the applied PCR assay. Bulk tank milk examination is not a reliable tool for the identification of the named pathogens by single testing, but might be a valuable monitoring tool when used frequently with repeated testing. Furthermore, this approach could be a useful monitoring tool for detecting new pathogen occurrence in the herd.     

Antibacterial effect of plant-derived antimicrobials on major bacterial mastitis pathogens in vitro

The objective of this study was to investigate the antimicrobial effect of plant-derived antimicrobials including trans-cinnamaldehyde (TC), eugenol, carvacrol, and thymol on major bacterial mastitis pathogens in milk. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the aforementioned compounds on Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus, and Escherichia coli were determined. In addition, the bactericidal kinetics of TC on the aforementioned pathogens and the persistence of the antimicrobial activity of TC in milk over a period of 2 wk were investigated. All 4 plant-derived molecules exhibited antimicrobial activity against the 5 mastitis pathogens tested, but TC was most effective in killing the bacteria. The MIC and MBC of TC on Staph. aureus, E. coli, and Strep. uberis were 0.1 and 0.45%, respectively, whereas that on Strep. agalactiae and Strep. dysgalactiae were 0.05 and 0.4%, respectively. The MIC and MBC of the other 3 molecules ranged from 0.4 to 0.8% and 0.8 to 1.5%, respectively. In time-kill assays, TC at the MBC reduced the bacterial pathogens in milk by 4.0 to 5.0 log₁₀ cfu/mL and to undetectable levels within 12 and 24 h, respectively. The antimicrobial effect of TC persisted for the duration of the experiment (14 d) without any loss of activity. Results of this study suggest that TC has the potential to be evaluated as an alternative or adjunct to antibiotics as intramammary infusion to treat bovine mastitis.     

Strain diversity and infection durations of Staphylococcus spp. and Streptococcus spp. causing intramammary infections in dairy cows

To effectively prevent and control bovine mastitis, farmers and their advisors need to take infection pathways and durations into account. Still, studies exploring both aspects through molecular epidemiology with sampling of entire dairy cow herds over longer periods are scarce. Therefore, quarter foremilk samples were collected at 14-d intervals from all lactating dairy cows (n = 263) over 18 wk in one commercial dairy herd. Quarters were considered infected with Staphylococcus aureus, Streptococcus uberis, or Streptococcus dysgalactiae when ≥100 cfu/mL of the respective pathogen was detected, or with Staphylococcus epidermidis or Staphylococcus haemolyticus when ≥500 cfu/mL of the respective pathogen was detected. All isolates of the mentioned species underwent randomly amplified polymorphic DNA (RAPD)-PCR to explore strain diversity and to distinguish ongoing from new infections. Survival analysis was used to estimate infection durations. Five different strains of Staph. aureus were isolated, and the most prevalent strain caused more than 80% of all Staph. aureus infections (n = 46). In contrast, 46 Staph. epidermidis and 69 Staph. haemolyticus strains were isolated, and none of these caused infections in more than 2 different quarters. The 3 most dominant strains of Strep. dysgalactiae (7 strains) and Strep. uberis (18 strains) caused 81% of 33 and 49% of 37 infections in total, respectively. The estimated median infection duration for Staph. aureus was 80 d, and that for Staph. epidermidis and Staph. haemolyticus was 28 and 22 d, respectively. The probability of remaining infected with Strep. dysgalactiae or Strep. uberis for more than 84 and 70 d was 58.7 and 53.5%, respectively. Staphylococcus epidermidis and Staph. haemolyticus were not transmitted contagiously and the average infection durations were short, which brings into question whether antimicrobial treatment of intramammary infections with these organisms is justified. In contrast, infections with the other 3 pathogens lasted longer and largely originated from contagious transmission.     

Quarter-level analyses of the associations among subclinical intramammary infection and milk quality,udder health,and cheesemaking traits in Holstein cows

In this study, we investigated associations among subclinical intra-mammary infection (IMI) and quarter-level milk composition, udder health indicators, and cheesemaking traits. The dataset included records from 450 Holstein cows belonging to three dairy herds. After an initial screening (T0) to identify animals infected by Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus, and Prototheca spp., 613 quarter milk samples for 2 different sampling times (T1 and T2, 1 mo after T1) were used for analysis. Milk traits were analyzed using a hierarchical linear mixed model including the effects of days in milk, parity and herd, and bacteriological and inflammatory category [culture negative with somatic cell count (SCC) <200,000 cells/mL; culture negative with SCC ≥200,000 cells/mL; or culture positive]. All udder health indicators were associated with increased SCC and IMI at both sampling times. The largest effects were detected at T2 for milk lactose (?7% and ?5%) and milk conductivity (+9% and +8%). In contrast, the increase in differential SCC (DSCC) in samples with elevated SCC was larger at T1 (+17%). Culture-negative samples with SCC ≥200,000 cells/mL had the highest SCC and greatest numbers of polymorphonuclear-neutrophils-lymphocytes and macrophages at both T1 and T2. Regarding milk cheesemaking ability, samples with elevated SCC showed the worst pattern of curd firmness at T1 and T2. At T2, increased SCC and IMI induced large decreases in recoveries of nutrients into the curd, in particular recovered protein (?14% and ?16%) and recovered fat (?12% and ?14%). Different behaviors were observed between Strep. agalactiae and Prototheca spp., especially at T2. In particular, samples that were positive for Strep. agalactiae had higher proportions of DSCC (+19%) compared with negative samples with low SCC, whereas samples that were positive for Prototheca spp. had lower DSCC (?11%). Intramammary infection with Prototheca spp. increased milk pH compared with culture-negative samples (+3%) and negative samples that had increased SCC (+2%). The greatest impairment in curd firmness at 30 min from rennet addition was observed for samples that were positive for Prototheca spp. (?99% compared with negative samples, and ?98% compared with negative samples with high SCC). These results suggest that IMI caused by Prototheca spp. have detrimental effects on milk technological traits that deserve further investigation of the mechanisms underlying animals' responses to infection.     

Genetic correlations between pathogen-specific mastitis and somatic cell count in Danish Holsteins

The aim of this study was to estimate genetic correlations (r_a) between 2 lactation average somatic cell count (LASCC) traits and 6 different mastitis traits in 226,482 first-parity Danish Holstein cows that calved between 1998 and 2008. The LASCC traits were defined from 5 to either 170 d (LASCC_170) or 300 d (LASCC_300) after calving, and the mastitis traits were unspecific mastitis (all mastitis treatments, both clinical and subclinical, regardless of the causative pathogen) and mastitis caused by either Streptococcus dysgalactiae, Escherichia coli, coagulase-negative staphylococci (CNS), Staphylococcus aureus, or Streptococcus uberis. Variance components were estimated using bivariate threshold-Gaussian models via Gibbs sampling. The posterior means of r_a between LASCC_170 and the mastitis traits were greatest for unspecific mastitis (r_a = 0.71), followed by CNS, Strep. dysgalactiae, Strep. uberis, and E. coli (r_a = 0.54 to 0.69) and were lowest for Staph. aureus mastitis (r_a = 0.44). The genetic correlation between LASCC_300 and the mastitis traits were generally smaller (r_a = 0.47 to 0.69). Caution should be taken when interpreting the results, however, because some posterior density intervals for r_a were large (between 0.14 and 0.47 units). Phenotypically, Staph. aureus is known to be associated with high SCC and especially with subclinical mastitis through chronic infections, so the low r_a between Staph. aureus mastitis and LASCC, compared with r_a for the other pathogens, was not expected. Subclinical cases are usually submitted to dry cow therapy (not included in the present study), not treated at all, or wrongly recorded as clinical cases. Thus, the incidence of Staph. aureus mastitis is likely too low, and the genetic correlation between Staph. aureus mastitis and LASCC may therefore be underestimated in the present study. The results for the remaining pathogens were as expected, smallest for E. coli and larger but similar for Strep. dysgalactiae, Strep. uberis, and CNS. Selection for lower LASCC is expected to decrease the incidence of pathogen-specific mastitis, especially for Strep. uberis, Strep. dysgalactiae, and CNS and, to a lesser extent, for Staph. aureus and E. coli. Data recording should preferably be improved, and economic weights for the pathogen-specific mastitis traits should be estimated before implementing an udder health index that includes pathogen-specific mastitis traits.     

Detection of mastitis pathogens by analysis of volatile bacterial metabolites

The ability to detect mastitis pathogens based on their volatile metabolites was studied. Milk samples from cows with clinical mastitis, caused by Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus uberis, Streptococcus dysgalactiae, and Escherichia coli were collected. In addition, samples from cows without clinical mastitis and with low somatic cell count (SCC) were collected for comparison. All mastitis samples were examined by using classical microbiological methods, followed by headspace analysis for volatile metabolites. Milk from culture-negative samples contained a lower number and amount of volatile components compared with cows with clinical mastitis. Because of variability between samples within a group, comparisons between pathogens were not sufficient for classification of the samples by univariate statistics. Therefore, an artificial neural network was trained to classify the pathogen in the milk samples based on the bacterial metabolites. The trained network differentiated milk from uninfected and infected quarters very well. When comparing pathogens, Staph. aureus produced a very different pattern of volatile metabolites compared with the other samples. Samples with coagulase-negative staphylococci and E. coli had enough dissimilarity with the other pathogens, making it possible to separate these 2 pathogens from each other and from the other samples. The 2 streptococcus species did not show significant differences between each other but could be identified as a different group from the other pathogens. Five groups can thus be identified based on the volatile bacterial metabolites: Staph. aureus, coagulase-negative staphylococci, streptococci (Strep. uberis and Strep. dysgalactiae as one group), E. coli, and uninfected quarters.