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.     

Elimination of selected mastitis pathogens during the dry period

We aimed to evaluate the elimination of 4 different mastitis pathogens, Streptococcus agalactiae, Mycoplasma bovis, Staphylococcus aureus, and Streptococcus uberis, from infected udder quarters during the dry period using quantitative PCR. The second purpose of this study was to evaluate the association between milk haptoglobin (Hp) concentration and the presence of udder pathogens (Strep. agalactiae, Staph. aureus, M. bovis, and Strep. uberis) in udder quarter milk samples before and after dry period. Aseptic udder quarter milk samples (n = 1,001) were collected from 133 dairy cows at dry off and at the first milking after calving from 1 large dairy herd. Bacterial DNA of Strep. agalactiae, Staph. aureus, Strep. uberis, and M. bovis in the udder quarter milk samples was identified with commercial quantitative PCR analysis Mastitis 4B (DNA Diagnostic A/S, Risskov, Denmark). Milk Hp concentration (mg/L) was measured from udder quarter milk samples. The elimination rates during the dry period for M. bovis, Staph. aureus, Strep. agalactiae, and Strep. uberis were 86.7, 93.6, 96.2, and 100.0%, respectively. The new IMI rate was 3.0% for M. bovis, 2.9% for Staph. aureus, 2.4% for Strep. agalactiae, and 3.1% for Strep. uberis. The milk Hp concentration was significantly higher in udder quarter milk samples with blood and in samples positive for Strep. agalactiae at dry off and for Staph. aureus postcalving. Elevated milk Hp concentration was not associated with the presence of M. bovis in the udder quarter milk samples. In conclusion, elimination of Staph. aureus, Strep. agalactiae, and Strep. uberis during the dry period was high; the elimination of M. bovis from infected udder quarters was lower, but probably spontaneous. Additionally, milk Hp concentration may be used as a marker for udder inflammation when combined with the bacteriological results at dry off and postpartum.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Evaluation of chromogenic culture media for rapid identification of microorganisms isolated from cows with clinical and subclinical mastitis

This study aimed to evaluate the diagnostic performance (specificity, Sp; sensitivity, Se; accuracy; positive predictive value; negative predictive value; and Cohen's kappa coefficient, κ, of agreement) of chromogenic culture media for rapid identification of microorganisms isolated from cows with clinical (CM) and subclinical mastitis (SCM). For this, 2 experiments were carried out: evaluation of (1) biplate, and (2) triplate of chromogenic culture media for rapid identification of mastitis-causing microorganisms. For the evaluation of diagnostic performance, identification of microorganisms by MALDI-TOF mass spectrometry was considered the standard methodology. In experiment 1, 476 milk samples collected from cows with CM and 660 from cows with SCM were evaluated by inoculation in 2 selective chromogenic culture media (CHROMagar) for gram-positive bacteria and another for gram-negative bacteria. In experiment 2, 476 milk samples from cows with CM and 500 from cows with SCM were evaluated by inoculation in triplate chromogenic culture media (Smartcolor2, Onfarm), selective for Streptococcus and Strep-like organisms, Staphylococcus, and gram-negative bacteria. In experiment 1 for the CM samples, the use of biplates with gram-positive and gram-negative culture media showed Se that ranged from 0.56 (0.32–0.81; Staphylococcus aureus) to 0.90 (0.80–0.99 Streptococcus uberis), Sp varied from 0.94 (0.92–0.96; Strep. uberis) to 1.00 (Prototheca spp. or yeast), and κ ranged from 0.47 (0.26–0.67; Staph. aureus) to 0.84 (0.78–0.9; Escherichia coli). The Se of biplates for SCM samples ranged from 0.50 (0.15–0.85; E. coli) to 0.94 (0.87–1.00; Staph. aureus), Sp varied from 0.95 (0.93–0.97; Strep. uberis) to 0.99 (0.98–1.00; Staph. aureus and Strep. Agalactiae or dysgalactiae), and κ ranged from 0.18 (0.00–0.40; Escherichia coli) to 0.88 (0.80–0.95; Staph. aureus). In experiment 2, the Se of the triplate chromogenic media in CM samples ranged from 0.09 (0.00–0.26; Serratia spp.) to 0.94 (0.85–1.00; Klebsiella spp. and Enterobacter spp.), Sp varied from 0.94 (0.92–0.96; Strep. agalactiae and Strep. dysgalactiae) to 1.00 (Serratia spp.) and κ ranged from 0.07 (0.00–0.24; Serratia spp.) to 0.85 (0.75–0.94; Klebsiella spp. and Enterobacter spp.). For SCM samples, the use of the triplate with the chromogenic culture media showed Se that varied from 0.25 (0.10–0.40; Lactococcus spp.) to 1.00 (Strep. Agalactiae or dysgalactiae), Sp ranged from 0.92 (0.90–0.94; Strep. Agalactiae and Strep. dysgalactiae) to 0.99 (0.98–1.00; Klebsiella spp. and Enterobacter spp.), and κ varied from 0.28 (0.00–0.72; E. coli) to 0.72 (0.60–0.82; Staph. aureus). Our results suggest that the diagnostic accuracy of the biplate and triplate of chromogenic culture media varies according to pathogen, and the results of chromogenic culture media may be useful for rapid decision-making on mastitis treatment protocols of the main mastitis-causing microorganisms, but their use for implementation of mastitis control measures will depend on each farm specific needs.     

Incidence rate of clinical mastitis on Canadian dairy farms

No nationwide studies of the incidence rate of clinical mastitis (IRCM) have been conducted in Canada. Because the IRCM and distribution of mastitis-causing bacteria may show substantial geographic variation, the primary objective of this study was to determine regional pathogen-specific IRCM on Canadian dairy farms. Additionally, the association of pathogen-specific IRCM with bulk milk somatic cell count (BMSCC) and barn type were determined. In total, 106 dairy farms in 10 provinces of Canada participated in the study for a period of 1 yr. Participating producers recorded 3,149 cases of clinical mastitis. The most frequently isolated mastitis pathogens were Staphylococcus aureus, Escherichia coli, Streptococcus uberis, and coagulase-negative staphylococci. Overall mean and median IRCM were 23.0 and 16.7 cases per 100 cow-years in the selected herds, respectively, with a range from 0.7 to 97.4 per herd. No association between BMSCC and overall IRCM was found, but E. coli and culture-negative IRCM were highest and Staph. aureus IRCM was lowest in low and medium BMSCC herds. Staphylococcus aureus, Strep. uberis, and Streptococcus dysgalactiae IRCM were lowest in the Western provinces. Staphylococcus aureus and Strep. dysgalactiae IRCM were highest in Québec. Cows in tie-stalls had higher incidences of Staph. aureus, Strep. uberis, coagulase-negative staphylococci, and other streptococcal IRCM compared with those in free-stalls, whereas cows in free stalls had higher Klebsiella spp. and E. coli IRCM than those in tie-stall barns. The focus of mastitis prevention and control programs should differ between regions and should be tailored to farms based on housing type and BMSCC.     

Factors associated with intramammary infection in dairy cows caused by coagulase-negative staphylococci,Staphylococcus aureus,Streptococcus uberis,Streptococcus dysgalactiae,Corynebacterium bovis,or Escherichia coli

The aim of this study was to determine risk factors for bovine intramammary infection (IMI) associated with the most common bacterial species in Finland. Large databases of the Finnish milk-recording system and results of microbiological analyses of mastitic milk samples from Valio Ltd. (Helsinki, Finland) were analyzed. The study group comprised 29,969 cows with IMI from 4,173 dairy herds. A cow with a quarter milk sample in which DNA of target species was detected in the PathoProof Mastitis PCR Assay (Thermo Fisher Scientific, Waltham, MA) was determined to have IMI. Only cows with IMI caused by the 6 most common pathogens or groups of pathogens, coagulase-negative staphylococci (CNS), Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, Corynebacterium bovis, and Escherichia coli, were included. The control group comprised 160,176 IMI-free cows from the same herds as the study group. A multilevel logistic regression model was used to study herd- and cow-specific risk factors for incidence of IMI. Pathogen-specific results confirmed those of earlier studies, specifically that increasing parity increases prevalence of IMI regardless of causative pathogen. Holsteins were more susceptible to IMI than Nordic Reds except when the causative pathogen was CNS. Occurrence of IMI caused by C. bovis was not related to milk yield, in contrast to IMI caused by all other pathogens investigated. Organic milk production was associated with IMI only when the causative pathogen of IMI was Staph. aureus; Staph. aureus IMI was more likely to occur in conventional than in organic production. Cows in older freestall barns with parlor milking had an increased probability of contracting an IMI compared with cows in tiestall barns or in new freestall barns with automatic milking. This was the case for all IMI, except those caused by CNS, the prevalence of which was not associated with the milking system, and IMI caused by Staph. aureus, which was most common in cows housed in tiestall barns. A better breeding index for milk somatic cell count was associated with decreased occurrence of IMI, indicating that breeding for improved udder health has been successful in reducing the incidence of IMI caused by the most common pathogens in Finland. In the Finnish dairy sector, the importance of other measures to control IMI will increase as the Holstein breed progressively takes the place of the Nordic Red breed. Attention should be paid to hygiene and cleanliness, especially in old freestall barns. Based on our results, the increasing prevalence of automatic milking is not a reason for special concern.     

Prevalence of mastitis pathogens in Ragusa, Sicily, from 2000 to 2006

The objective of this study was to report the prevalence of intramammary infections (IMI) in Ragusa, Sicily, from milk samples (n = 18,711) collected between October 2000 and June 2006 from 101 dairy herds. Milk samples were collected at 9,747 cow sampling events from 5,285 individual cows. Samples were individual quarter (92.8%) or composite samples (7.2%) from an individual cow. Logistic regression was used to examine the prevalence of IMI at the level of milk sample and at the level of cow, controlling for year and season of collection, type of sample (individual quarter or composite), and type of housing and bedding of the cow at the time of collection. Bedding and housing types were as follows, respectively (number of herd groups): bedding: none (61), organic [51 (straw, 50; sawdust, 1)], and sand (3); housing: bedded pack (37), free stalls (57), tie stalls (4), and paddock (17). Raw prevalence of cow IMI for a sample event was as follows (percentage of cow samples): no growth, 47.4%; coagulase-negative staphylococci, 22.6%; Staphylococcus aureus, 20.6%; other Streptococcus spp., 11.1%; Streptococcus agalactiae, 2.3%; coliform bacteria, 2.9%; and other organisms, 5.8%. Prevalence of IMI differed by bedding type for Staph. aureus (none, 24.5%; organic, 12.7%; sand, 12.3%) and coagulase-negative staphylococci (none, 13.1%; organic, 27.4%; sand, 26.9%) but not for Streptococcus spp. or coliform bacteria. Prevalence of Streptococcus spp. IMI differed by housing type (tie stalls, 22.2%; bedded pack, 12.8%; free stalls, 8.4%; paddock, 7.1%). Housing was not associated with the prevalence of IMI for other bacteria. Herd monthly prevalence of Staph. aureus and Streptococcus spp. IMI was associated with decreased mean milk production (Staph. aureus, −1.42 kg/d per cow, SEM 0.51; Streptococcus spp., −1.31 kg/d per cow, SEM 0.64) and increased mean linear score (Staph. aureus, 1.01 units/d per cow, SEM 0.16; Streptococcus spp., 0.59 units/d per cow, SEM 0.22). Herds (n = 11) with a mean linear score (MLS) less than 3.3 units had the lowest prevalence of Staph. aureus IMI and monthly MLS and the greatest monthly mean milk production compared with other herds grouped by MLS [MLS 3.31 to 3.99 (n = 20), MLS 4.00 to 4.46 (n = 20), MLS >4.46 (n = 17), and MLS not available (n = 33)]. Implementation of a milk quality program to control gram-positive organisms is important for Ragusa.     

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.     

Pathogen-specific effects of quantitative trait loci affecting clinical mastitis and somatic cell count in Danish Holstein cattle

The aim of this study was to investigate whether quantitative trait loci (QTL) affecting the risk of clinical mastitis (CM) and QTL affecting somatic cell score (SCS) exhibit pathogen-specific effects on the incidence of mastitis. Bacteriological data on mastitis pathogens were used to investigate pathogen specificity of QTL affecting treatments of mastitis in first parity (CM1), second parity (CM2), and third parity (CM3), and QTL affecting SCS. The 5 most common mastitis pathogens in the Danish dairy population were analyzed: Streptococcus dysgalactiae, Escherichia coli, coagulase-negative staphylococci, Staphylococcus aureus, and Streptococcus uberis. Data were analyzed using 2 approaches: an independence test and a generalized linear mixed model. Three different data sets were used to investigate the effect of data sampling: all samples, only samples that were followed by antibiotic treatment, and samples from first-crop daughters only. The results showed with high certainty that 2 QTL affecting SCS exhibited pathogen specificity against Staph. aureus and E. coli, respectively. The latter result might be explained by a pleiotropic QTL that also affects CM2 and CM3. Less certain results were found for QTL affecting CM. A QTL affecting CM1 was found to be specific against Strep. dysgalactiae and Staph. aureus, a QTL affecting CM2 was found to be specific against E. coli, and finally a QTL affecting CM3 was found to be specific against Staph. aureus. None of the QTL analyzed was found to be specific against coagulase-negative staphylococci and Strep. uberis. Our results show that particular mastitis QTL are highly likely to exhibit pathogen-specificity. However, the results should be interpreted carefully because the results are sensitive to the sampling method and method of analysis. Field data were used in this study. These kind of data may be heavily biased because there is no standard procedure for collecting milk samples for bacteriological analysis in Denmark. Furthermore, using only the mean SCS from d 10 to 180 after parturition may lead to truncated effects of SCS-QTL when samples collected after d 180 are used. Additionally, repeated samples were used, which could boost the difference in incidence of pathogens between daughters of sires inheriting the positive and negative QTL allele, respectively. However, the magnitude of these effects in this study is unclear.     

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.     

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.