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.     

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.     

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.     

Test characteristics of milk amyloid A ELISA,somatic cell count,and bacteriological culture for detection of intramammary pathogens that cause subclinical mastitis

Bovine mastitis is an important disease in the dairy industry, causing economic losses as a result of withheld milk and treatment costs. Several studies have suggested milk amyloid A (MAA) as a promising biomarker in the diagnosis of mastitis. In the absence of a gold standard for diagnosis of subclinical mastitis, we estimated the diagnostic test accuracy of a commercial MAA-ELISA, somatic cell count (SCC), and bacteriological culture using Bayesian latent class modeling. We divided intramammary infections into 2 classes: those caused by major pathogens (e.g., Escherichia coli, Staphylococcus aureus, streptococci, and lacto-/enterococci) and those caused by all pathogens (major pathogens plus Corynebacterium bovis, coagulase-negative staphylococci, Bacillus spp., Streptomyces spp.). We applied the 3 diagnostic tests to all samples. Of 433 composite milk samples included in this study, 275 (63.5%) contained at least 1 colony of any bacterial species; of those, 56 contained major pathogens and 219 contained minor pathogens. The remaining 158 samples (36.5%) were sterile. We determined 2 different thresholds for the MAA-ELISA using Bayesian latent class modeling: 3.9 µg/mL to detect mastitis caused by major pathogens and 1.6 µg/mL to detect mastitis caused by all pathogens. The optimal SCC threshold for identification of subclinical mastitis was 150,000 cells/mL; this threshold led to higher specificity (Sp) than 100,000 cells/mL. Test accuracy for major-pathogen intramammary infections was as follows: SCC, sensitivity (Se) 92.6% and Sp 72.9%; MAA-ELISA, Se 81.4% and Sp 93.4%; bacteriological culture, Se 23.8% and Sp 95.2%. Test accuracy for all-pathogen intramammary infections was as follows: SCC, sensitivity 90.3% and Sp 71.8%; MAA-ELISA, Se 88.0% and Sp 65.2%; bacteriological culture, Se 83.8% and Sp 54.8%. We suggest the use of SCC and MAA-ELISA as a combined screening procedure for situations such as a Staphylococcus aureus control program. With Bayesian latent class analysis, we were able to identify a more differentiated use of the 3 diagnostic tools. The MAA-ELISA is a valuable addition to existing tools for the diagnosis of subclinical mastitis.     

Acute-phase inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) levels in serum and milk of cows with subclinical mastitis caused by Streptococcus species and coagulase-negative Staphylococcus species

The aim of the study was to investigate the concentrations of acute-phase inter-α-trypsin inhibitor heavy chain 4 (ITIH4) in serum and milk of cows with subclinical mastitis caused by Streptococcus spp. (STR) and coagulase-negative Staphylococcus spp. (CNS) and healthy cows. The blood and milk samples were obtained from 60 mid-lactation, multiparous Holstein-Friesian cows from 7 herds in the Lublin region of Poland. In the milk samples from 40 cows with subclinical mastitis, Streptococcus spp. and CNS were isolated. The ITIH4 was significantly higher in serum of cows with subclinical mastitis caused both by STR and CNS compared with healthy cows. One hundred percent of animals infected with Streptococcus spp. and 89% of animals infected with Staphylococcus spp. showed ITIH4 concentration in sera higher than 0.5 mg/mL. The concentration of ITIH4 in milk also was significantly higher in cows with subclinical mastitis caused by Streptococcus spp. and Staphylococcus spp. compared with the control group. Seventy percent of cows infected by STR and CNS showed ITIH4 concentration in milk higher than 2.5 μg/mL. Milk ITIH4 concentration higher than 5 µg/mL was found in 55% of animals infected with Streptococcus spp. and in 40% of animals infected with Staphylococcus spp. No statistically significant differences were observed in ITIH4 concentrations both in serum and in milk between the studied unhealthy animal groups. These results suggest that ITIH4 may be used in the future as a novel diagnostic marker in serum and in milk of subclinical mastitis in cows.     

Postcalving udder health and productivity in cows approaching dry-off with intramammary infections caused by non-aureus Staphylococcus,Aerococcus, Enterococcus,Lactococcus, and Streptococcus species

The objective of this prospective cohort study was to explore associations between intramammary infection (IMI) in late-lactation cows and postcalving udder health and productivity. Cows (n = 2,763) from 74 US dairy herds were recruited as part of a previously published cross-sectional study of bedding management and IMI in late-lactation cows. Each herd was visited twice for sampling. At each visit, aseptic quarter milk samples were collected from 20 cows approaching dry-off (>180 d pregnant), which were cultured using standard bacteriological methods and MALDI-TOF for identification of isolates. Quarter-level culture results were used to establish cow-level IMI status at enrollment. Cows were followed from enrollment until 120 d in milk (DIM) in the subsequent lactation. Herd records were used to establish whether subjects experienced clinical mastitis or removal from the herd, and DHIA test-day data were used to record subclinical mastitis events (somatic cell count >200,000 cells/mL) and milk yield (kg/d) during the follow-up period. Cox regression and generalized estimating equations were used to evaluate the associations between IMI and the outcome of interest. The presence of late-lactation IMI caused by major pathogens was positively associated with postcalving clinical mastitis [hazard ratio = 1.5, 95% confidence interval (CI): 1.2, 2.0] and subclinical mastitis (risk ratio = 1.5, 95% CI: 1.3, 1.9). Species within the non-aureus Staphylococcus (NAS) group varied in their associations with postcalving udder health, with some species being associated with increases in clinical and subclinical mastitis in the subsequent lactation. Late-lactation IMI caused by Streptococcus and Streptococcus (Strep)-like organisms, other than Aerococcus spp. (i.e., Enterococcus, Lactococcus, and Streptococcus spp.) were associated with increases in postcalving clinical and subclinical mastitis. Test-day milk yield from 1 to 120 DIM was lower (?0.9 kg, 95% CI: ?1.6, ?0.3) in late-lactation cows with any IMI compared with cows without IMI. No associations were detected between IMI in late lactation and risk for postcalving removal from the herd within the first 120 DIM. Effect estimates reported in this study may be less than the underlying quarter-level effect size for IMI at dry-off and postcalving clinical and subclinical mastitis, because of the use of late-lactation IMI as a proxy for IMI at dry-off and the use of cow-level exposure and outcome measurements. Furthermore, the large number of models run in this study (n = 94) increases the chance of identifying chance associations. Therefore, confirmatory studies should be conducted. We conclude that IMI in late lactation may increase risk of clinical and subclinical mastitis in the subsequent lactation. The relationship between IMI and postcalving health and productivity is likely to vary among pathogens, with Staphylococcus aureus, Streptococcus spp., Enterococcus spp., and Lactococcus spp. being the most important pathogens identified in the current study.     

Relationships between milk culture results and composite milk somatic cell counts in Norwegian dairy cattle

Associations between test-day composite milk somatic cell counts (CMSCC) and results from quarter milk cultures for various pathogens associated with mastitis, including Staphylococcus aureus, Streptococcus spp., coagulase-negative staphylococci (CNS), were investigated. S. aureus was dichotomized according to sparse (≤1,500 colony forming units/mL of milk) or rich (>1,500 colony forming units/mL of milk) growth of the bacteria. Quarter milk samples were obtained on between 1 and 4 occasions from 2,714 cows in 354 Norwegian dairy herds, resulting in a total of 3,396 samples. Cows included in the study were randomly selected, without regard to current or previous udder health status. Measures of test-day CMSCC were obtained every second month, and related to 3528 microbiological diagnoses at the cow level. Mixed linear regression models incorporating a compound symmetry covariance structure accounting for repeated test-day CMSCC within cow, and a random effect variable on herd level, was used to quantify the relationship between a positive milk culture and the natural logarithm of test-day CMSCC (LnCMSCC). The material was stratified in time periods before 151 d in milk (DIM) and after 150 DIM. A positive diagnosis for any category of mastitis pathogen was significantly associated with elevated CMSCC. Pathogen positive cows sampled for microbiological diagnosis during the first 150 DIM had higher levels of CMSCC throughout lactation than cows with a positive diagnosis after 150 DIM. Streptococcus spp.-positive milk cultures were associated with steadily elevated values for CMSCC throughout lactation both when sampled before and after 150 DIM. Cows diagnosed with rich growth of S. aureus after 150 DIM experienced a characteristic and sharp increase in CMSCC, but this effect was not observed in cows with a positive diagnosis for rich growth of S. aureus during the first 150 DIM. A considerable increase in CMSCC in cows positive for CNS during the first part of the lactation period was also observed. The practicability of using CMSCC in a diagnostic test to identify cows with a positive milk culture for mastitis pathogens was also assessed. The sensitivity, specificity, and positive predictive values of the tests were regarded as low when sampling for milk culture was conducted, irrespective of cow level characteristics.     

Antimicrobial resistance profiles of 5 common bovine mastitis pathogens in large Chinese dairy herds

The prevalence of antimicrobial resistance (AMR) is increasing in human and animal pathogens, becoming a concern worldwide. However, prevalence and characteristics of AMR of bovine mastitis pathogens in large Chinese dairy herds are still unclear. Therefore, our objective was to determine the AMR profile of bacteria isolated from clinical mastitis in large (>500 cows) Chinese dairy herds. A total of 541 isolates of the 5 most common species, Staphylococcus aureus (n = 103), non-aureus staphylococci (NAS; n = 107), Streptococcus species (n = 101), Klebsiella species (n = 130), and Escherichia coli (n = 100), isolated from bovine clinical mastitis on 45 dairy farms located in 10 provinces of China were included. Presence of AMR was determined by minimum inhibitory concentrations using the microdilution method. Prevalence of multidrug resistance (resistance to >2 antimicrobials) was 27% (148/541). A very wide distribution of minimum inhibitory concentrations was screened in all isolates, including Staph. aureus isolates, which were resistant to penicillin (66%). In addition, NAS (30%) were more resistant than Staph. aureus to oxacillin (84%), penicillin (62%), tetracycline (34%), and clindamycin (33%). Prevalence of resistance to tetracycline was high (59%) in Streptococcus spp. Additionally, prevalence of resistance of both E. coli and Klebsiella spp. was high to amoxicillin/clavulanate potassium (81 and 38%, respectively), followed by tetracycline (only Klebsiella spp. 32%). A high proportion (27%) of isolates were multidrug resistant; the most frequent combinations were clindamycin-cefalexin-tetracycline or enrofloxacin-cefalexin-penicillin patterns for Staph. aureus; enrofloxacin-oxacillin-penicillin-tetracycline patterns for NAS; clindamycin-enrofloxacin-tetracycline patterns for Streptococcus spp.; amoxicillin/clavulanate potassium-ceftiofur-polymyxin B patterns for Klebsiella spp.; and amoxicillin/clavulanate potassium-ceftiofur-polymyxin B patterns for E. coli. Resistance for 4 kinds of antimicrobials highly critical for human medicine, including daptomycin, vancomycin, imipenem, and polymyxin B, ranged from 0 to 24%. In conclusion, prevalence of AMR in mastitis pathogens was high on large Chinese dairy farms, potentially jeopardizing both antimicrobial efficacy and public health. Results of this study highlighted the need for improvements in antimicrobial stewardship and infection control programs in large Chinese dairy farms to reduce emergence of AMR.     

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.     

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.     

Diversity of Staphylococcus species and prevalence of enterotoxin genes isolated from milk of healthy cows and cows with subclinical mastitis

The objectives of this study were to determine the occurrence and diversity of Staphylococcus spp. in milk from healthy cows and cows with subclinical mastitis in Brazil and to examine the profile of enterotoxin genes and some enterotoxins produced by Staphylococcus spp. A total of 280 individual mammary quarter milk samples from 70 healthy cows and 292 samples from 73 cows with subclinical mastitis were collected from 11 farms in the state of São Paulo, Brazil. Staphylococcus spp. were recovered from 63 (22.5%) samples from healthy cows and from 80 samples (27.4%) from cows with mastitis. The presence of Staphylococcus aureus was significantly different between these 2 groups and was more prevalent in the cows with mastitis. The presence of Staphylococcus saprophyticus was also significantly different between these 2 groups, but this organism was more prevalent in healthy cows. No statistically significant differences were observed in the numbers of other staphylococci in milk samples from the 2 groups. The sea gene was the most prevalent enterotoxin gene in both groups. Eight of 15 (53.3%) Staph. aureus carried this gene and all produced the SEA toxin. In the coagulase-negative staphylococci (CNS) group, 61 of 128 (47.5%) had the same gene and just 1 (1.6%) Staphylococcus epidermidis strain produced the enterotoxin in vitro. Because CNS were isolated from both groups of cows and most CNS contained enterotoxin genes but did not produce toxins, the role of CNS in mastitis should be carefully defined.     

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.     

Short communication: Streptococcus species isolated from mastitis milk samples in Germany and their resistance to antimicrobial agents

Mastitis is one of the most frequent infectious diseases in dairy cattle and is a reason for antimicrobial drug usage in dairy cows. The bacteria involved in bovine mastitis are mainly Streptococcus spp., Staphylococcus spp., and coliforms. The aim of this study was to determine antimicrobial resistance among Streptococcus spp. isolated from bovine mastitis milk. Antimicrobial resistance in Strep. uberis (n = 227), Strep. dysgalactiae (n = 49), and Strep. agalactiae (n = 3) was determined for 9 antimicrobial agents using the broth microdilution method in accordance with Clinical and Laboratory Standards Institute recommendations. Of all Streptococcus spp., 13% were multidrug resistant. The rate of multidrug resistance was higher among Strep. uberis (15%) than among Strep. dysgalactiae (6%) and Strep. agalactiae (0%). Resistance to tetracycline was the most common, followed by resistance to erythromycin, pirlimycin, and gentamicin. Resistance rates were higher on farms with more than 80 cows compared with those with fewer than 20 cows. β-Lactams should remain the drugs of choice in the treatment of streptococcal mastitis. The slightly elevated minimum inhibitory concentrations determined for these antibiotics may indicate, however, the emergence of resistant streptococci. To identify such changes in susceptibility as early as possible, antimicrobial resistance in streptococci should be surveyed regularly.     

Real-time polymerase chain reaction-based identification of bacteria in milk samples from bovine clinical mastitis with no growth in conventional culturing

In more than 30% of milk samples from clinical and subclinical bovine mastitis, bacteria fail to grow even after 48 h of conventional culture. The “no-growth” samples are problematic for mastitis laboratories, veterinarians, and dairy producers. This study provides the first investigation of the bacteriological etiology of such samples, using a real-time PCR-based commercial reagent kit. The assay targets the DNA of the 11 most common bacterial species or groups in mastitis and the staphylococcal blaZ gene (responsible for penicillin resistance) and can identify and quantify bacterial cells even if dead or growth-inhibited. A study was made of 79 mastitic milk samples with no-growth bacteria in conventional culture, originating from cows with clinical mastitis. Of the 79 samples, 34 (43%) were positive for 1 (32 samples) or 2 (2 samples) of the target bacteria. The positive findings included 11 Staphylococcus spp. (staphylococci other than Staphylococcus aureus), 10 Streptococcus uberis, 2 Streptococcus dysgalactiae, 6 Corynebacterium bovis, 3 Staph. aureus, 1 Escherichia coli, 1 Enterococcus, and 1 Arcanobacterium pyogenes. The positive samples contained as many as 10³ to 10⁷ bacterial genome copies per milliliter of milk. This study demonstrates that in nearly half of the clinical mastitis cases in which conventional culture failed to detect bacteria, mastitis pathogens were still present, often in substantial quantities. The clearly elevated N-acetyl-β-d-glucosaminidase activity values of the milk samples, together with clinical signs of the infected cows and quarters, confirmed the diagnosis of clinical mastitis and indicated that real-time, PCR-based bacterial findings are able to reveal bacteriological etiology. We conclude that all common mastitis bacteria can occur in large quantities in clinical mastitis samples that exhibit no growth in conventional culture, and that the real-time PCR assay is a useful tool for bacteriological diagnosis of such milk samples. Low bacterial concentration is commonly speculated to explain the no-growth milk samples. This hypothesis is not supported by the results of the current study.     

Optimal insemination and replacement decisions to minimize the cost of pathogen-specific clinical mastitis in dairy cows

Mastitis is a serious production-limiting disease, with effects on milk yield, milk quality, and conception rate, and an increase in the risk of mortality and culling. The objective of this study was 2-fold: (1) to develop an economic optimization model that incorporates all the different types of pathogens that cause clinical mastitis (CM) categorized into 8 classes of culture results, and account for whether the CM was a first, second, or third case in the current lactation and whether the cow had a previous case or cases of CM in the preceding lactation; and (2) to develop this decision model to be versatile enough to add additional pathogens, diseases, or other cow characteristics as more information becomes available without significant alterations to the basic structure of the model. The model provides economically optimal decisions depending on the individual characteristics of the cow and the specific pathogen causing CM. The net returns for the basic herd scenario (with all CM included) were $507/cow per year, where the incidence of CM (cases per 100 cow-years) was 35.6, of which 91.8% of cases were recommended for treatment under an optimal replacement policy. The cost per case of CM was $216.11. The CM cases comprised (incidences, %) Staphylococcus spp. (1.6), Staphylococcus aureus (1.8), Streptococcus spp. (6.9), Escherichia coli (8.1), Klebsiella spp. (2.2), other treated cases (e.g., Pseudomonas; 1.1), other not treated cases (e.g., Trueperella pyogenes; 1.2), and negative culture cases (12.7). The average cost per case, even under optimal decisions, was greatest for Klebsiella spp. ($477), followed by E. coli ($361), other treated cases ($297), and other not treated cases ($280). This was followed by the gram-positive pathogens; among these, the greatest cost per case was due to Staph. aureus ($266), followed by Streptococcus spp. ($174) and Staphylococcus spp. ($135); negative culture had the lowest cost ($115). The model recommended treatment for most CM cases (>85%); the range was 86.2% (Klebsiella spp.) to 98.5% (Staphylococcus spp.). In general, the optimal recommended time for replacement was up to 5 mo earlier for cows with CM compared with cows without CM. Furthermore, although the parameter estimates implemented in this model are applicable to the dairy farms in this study, the parameters may be altered to be specific to other dairy farms. Cow rankings and values based on disease status, pregnancy status, and milk production can be extracted; these provide guidance when determining which cows to keep or cull.     

Diagnosing intramammary infections: evaluation of composite milk samples to detect intramammary infections

Composite milk samples, in which milk from all 4 bovine quarters is collected in a single vial, are widely used in many developed dairy industries for detection of intramammary infections (IMI). These samples are more economical for use in culturing protocols than individual quarter samples, and may be useful when considering management options at the cow and herd level. The dilution effect may be problematic, however, resulting in lower sensitivity (Se) in IMI detection on composite samples. Relative Se and specificity (Sp) in composite samples have previously been described for some major pathogens, but because the causative organism for IMI is initially unknown, it is beneficial to investigate the reliability of composite samples for detection of all types of mastitis-causing bacteria. The Canadian Bovine Mastitis Research Network has a large data collection platform—the National Cohort of Dairy Farms—containing a vast amount of data on mastitis in Canada. These data have been used to further investigate the Se and Sp of composite samples in detecting IMI caused by specific mastitis pathogens. Milk samplings of selected cows before dry-off, after calving, and during lactation (n = 48,835 samples) were employed to this end. Composite samples showed moderately high Se for Staphylococcus aureus (77.1%, 95% CI = 73.3-80.5) and Streptococcus dysgalactiae (73.4%, 95% CI = 60.9-83.7), with moderate Se for Streptococcus uberis (62.1%, 95% CI = 49.3-73.8) and coagulase-negative staphylococci (59.8%, 95% CI = 58.4-61.2). Sensitivities always increased as the number of affected quarters increased. Composite samples also showed high Sp (>97%) for most organisms. Factors such as lactation number and stage of lactation were evaluated for their influence on the Se and Sp of composite sampling, but were only found to be significant for coagulase-negative staphylococci. Predictive values using the herd prevalences found across Canada were calculated and can be useful in field scenarios when composite sampling is employed to assist mastitis management. When used to detect newly occurring IMI in pairs of samples taken before dry-off, post-calving, and also prior to and subsequent to the dry period, composite samples were shown to have lower Se but similar Sp for all pathogens investigated. Composite samples can be used to detect IMI and new IMI in dairy cows, but the Se and Sp of the procedure should be taken into account.     

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.     

Prevalence of mastitis pathogens and their resistance against antimicrobial agents in dairy cows in Brandenburg, Germany

The primary objective of this study was to determine management practices concerning mastitis in Brandenburg, Germany, the prevalence of mastitis pathogens in dairy cows, and their resistance to selected antimicrobial agents. A further objective was to study the potential effect of parity and stage of lactation on the resistance of Staphylococcus aureus isolates against ampicillin. Milk samples for microbiological culture were collected from 4 groups of clinically healthy cows (first lactation, >1 lactation, >50 d in milk, and >250 d in milk; 8 cows/group) in 80 dairy herds. Resistance of gram-positive pathogens against 6 antimicrobial agents was tested using the broth microdilution method. Mastitis pathogens were isolated from 26.4% of the milk samples. Coagulase-negative staphylococci (CNS, 9.1% of quarters) and Corynebacterium bovis (7.3%) were the pathogens most frequently isolated. Among the major pathogens, Staph. aureus (5.7%) and Streptococcus uberis (1.0%) had the highest prevalence. Streptococcus agalactiae was isolated in samples from 29% of the herds. Although the prevalence of most pathogens was higher in older cows, the prevalence of CNS was higher in primiparous cows. Results of the mastitis control questionnaire showed that cows with clinical mastitis were transferred to a sick cow pen in 70% of the herds. Cephalosporins were the drug of first choice for treatment of clinical mastitis cases followed by fixed combinations of antimicrobial agents, β-lactamase-resistant penicillins, and penicillin. Most farmers treated cows 3 to 4 times per case. Cloxacillin, alone or in combination, and penicillin were most often used for dry-cow therapy. Antimicrobial resistance of the pathogens was within the range of other reports. Resistance of Staph. aureus to ampicillin increased significantly during the first lactation. Further research is required to determine the factors that lead to the selection of Staph. aureus strains that are resistant to ampicillin during the first lactation.     

The value of the biomarkers cathelicidin,milk amyloid A,and haptoglobin to diagnose and classify clinical and subclinical mastitis

Timely and objective diagnosis and classification of mastitis is crucial to ensure adequate management and therapeutic decisions. Analyzing specific biomarkers in milk could be advantageous compared with subjective or semiquantitative criteria, such as palpation of the udder in clinical mastitis cases or evaluation of somatic cell count using cow side tests (e.g., California Mastitis Test) in subclinical mastitis quarters. The objective of this study was to investigate the diagnostic value of 3 biomarkers; cathelicidin, milk amyloid A, and haptoglobin for the diagnosis of subclinical and clinical mastitis. Furthermore, the suitability of these biomarkers to differentiate between mild, moderate, and severe clinical mastitis and the influence of different pathogens on biomarker levels was tested. A total of 67 healthy cows, 119 cows with subclinical mastitis, and 212 cows with clinical mastitis were enrolled in the study. Although cathelicidin, haptoglobin, and milk amyloid A were measured in all samples from healthy cows and those with subclinical mastitis, haptoglobin, and cathelicidin results were only available from 121 out of 212 cows with clinical mastitis. Milk amyloid A was measured in all samples. In cows with clinical mastitis, the mastitic quarter and a second healthy quarter serving as a healthy in-cow control quarter were sampled. It was possible to differentiate between healthy quarters, quarters with subclinical mastitis, and quarters with clinical mastitis using all 3 biomarkers. Concerning cathelicidin, thresholds were 0.000 [sensitivity (Se) = 0.83, specificity (Sp) = 0.97] and 0.053 (Se = 0.98, Sp = 0.99) for normalized optical density at 450 nm (NOD450) for differentiating between healthy quarters and quarters with subclinical or clinical mastitis, respectively. Thresholds of 1.28 µg/mL (Se = 0.65, Sp = 0.76) and 1.81 µg/mL (Se = 0.77, Sp = 0.83) for milk amyloid A and 3.65 µg/mL (Se = 0.92, Sp = 0.94) and 5.40 µg/mL mL (Se = 0.96, Sp = 0.99) for haptoglobin were calculated, respectively. Healthy in-cow control quarters from cows with CM showed elevated milk amyloid A and haptoglobin levels compared with healthy quarters from healthy cows. Only the level of milk amyloid A was higher in severe clinical mastitis cases compared with mild ones. In contrast to clinical mastitis, cathelicidin and haptoglobin in subclinical mastitis quarters were significantly influenced by different bacteriological results. The measurement of cathelicidin, milk amyloid A, and haptoglobin in milk proved to be a reliable method to detect quarters with subclinical or clinical mastitis.     

Evaluation of 4 predictive algorithms for intramammary infection status in late-lactation cows

The objective of this observational study was to compare 4 cow-level algorithms to predict cow-level intramammary infection (IMI) status (culture and MALDI-TOF) in late-lactation US dairy cows using standard measures of test performance. Secondary objectives were to estimate the likely effect of each algorithm, if used to guide selective dry cow therapy (SDCT), on dry cow antibiotic use in US dairy herds, and to investigate the importance of including clinical mastitis criteria in algorithm-guided SDCT. Cows (n = 1,594) from 56 US dairy herds were recruited as part of a previously published cross-sectional study of bedding management and IMI in late-lactation cows. Each herd was visited twice for sampling. At each farm visit, aseptic quarter-milk samples were collected from 20 cows approaching dry-off (>180 d pregnant), which were cultured using standard bacteriological methods and MALDI-TOF for identification of isolates. Quarter-level culture results were used to establish cow-level IMI status, which was considered the reference test in this study. Clinical mastitis records and Dairy Herd Improvement Association test-day somatic cell count data were extracted from herd records and used to perform cow-level risk assessments (low vs. high risk) using 4 algorithms that have been proposed for SDCT in New Zealand, the Netherlands, United Kingdom, and the United States. Agreement between aerobic culture (reference test; IMI vs. no-IMI) and algorithm status (high vs. low risk) was described using Cohen's kappa, test sensitivity, specificity, negative predictive value, and positive predictive value. The proportion of cows classified as high risk among the 4 algorithms ranged from 0.31 to 0.63, indicating that these approaches to SDCT could reduce antibiotic use at dry-off by 37 to 69% in the average US herd. All algorithms had poor agreement with IMI status, with kappa values ranging from 0.05 to 0.13. Sensitivity varied by pathogen, with higher values observed when detecting IMI caused by Streptococcus uberis, Streptococcus dysgalactiae, Staphylococcus aureus, and Lactococcus lactis. Negative predictive values were high for major pathogens among all algorithms (≥0.87), which may explain why algorithm-guided SDCT programs have been successfully implemented in field trials, despite poor agreement with overall IMI status. Removal of clinical mastitis criteria for each algorithm had little effect on the algorithm classification of cows, indicating that algorithms based on SCC alone may have similar performance to those based on SCC and clinical mastitis criteria. We recommend that producers implementing algorithm-guided SDCT use algorithm criteria that matches their relative aspirations for reducing antibiotic use (high specificity, positive predictive value) or minimizing untreated IMI at dry-off (high sensitivity, negative predictive value).