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.     

Milk and serum IL-4, IL-6, IL-10, and amyloid A concentrations in cows with subclinical mastitis caused by coagulase-negative staphylococci

The aim of the study was to evaluate the concentrations of cytokines IL-4, IL-6, and IL-10 and acute phase protein amyloid A in milk and in serum from cows with subclinical mastitis caused by coagulase-negative staphylococci and from healthy cows. The blood and milk samples were obtained from 35 midlactation, multiparous (between parities 2 and 4) Holstein-Friesian cows. In the milk samples from 20 cows with subclinical mastitis, the following species of Staphylococcus were detected: Staphylococcus xylosus (8 samples), Staphylococcus chromogenes (6 samples), Staphylococcus haemolyticus (2 samples), Staphylococcus simulans (2 samples), and Staphylococcus sciuri (2 samples). The results of the present study indicate that the level of IL-6 in cows suffering from subclinical mastitis tended to be high in both serum and milk (432.09 and 254.32 pg/mL) compared with the level in healthy cows (164.47 and 13.02 pg/mL, respectively). Amyloid A value also was significantly higher in milk of unhealthy cows compared with cows without subclinical mastitis (790.2 and 360.5 ng/mL). No significant differences were found in levels of amyloid A in serum of both tested groups of cows (2,680.0 and 2,720.0 ng/mL). In contrast, concentration of IL-4 was significantly lower both in serum and in milk of cows with staphylococcal mastitis (86.1 and 123.17 pg/mL) compared with control animals (413.5 and 670.2 pg/mL). The level of IL-10 also was significantly higher in milk of healthy cows than in infected cows (39.78 and 22.5 pg/mL); however, differences in serum levels of this cytokine between tested groups were significantly less important (220.6 and 175.1 pg/mL).     

Some coagulase-negative Staphylococcus species affect udder health more than others

A longitudinal study in 3 dairy herds was conducted to profile the distribution of coagulase-negative Staphylococcus (CNS) species causing bovine intramammary infection (IMI) using molecular identification and to gain more insight in the pathogenic potential of CNS as a group and of the most prevalent species causing IMI. Monthly milk samples from 25 cows in each herd as well as samples from clinical mastitis were collected over a 13-mo period. Coagulase-negative staphylococci were identified to the species level using transfer-RNA intergenic spacer PCR. The distribution of CNS causing IMI was highly herd-dependent, but overall, Staphylococcus chromogenes, Staphylococcus xylosus, Staphylococcus cohnii, and Staphylococcus simulans were the most prevalent. No CNS species were found to cause clinical mastitis. The effect of the most prevalent species on the quarter milk somatic cell count (SCC) was analyzed using a linear mixed model, showing that Staph. chromogenes, Staph. simulans, and Staph. xylosus induced an increase in the SCC that is comparable with that of Staphylococcus aureus. Almost all CNS species were able to cause persistent IMI, with Staph. chromogenes causing the most persistent infections. In conclusion, accurate species identification cannot be ignored when studying the effect of CNS on udder health, as the effect on SCC differs between species and species distribution is herd-specific. Staphylococcus chromogenes, Staph. simulans, and Staph. xylosus seem to be the more important species and deserve special attention in further studies. Reasons for herd dependency and possible cow- and quarter-level risk factors should be examined in detail for the different species, eventually leading to cost-benefit analyses for management changes and, if needed, treatment recommendations.     

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.     

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.     

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.     

Bovine subclinical mastitis caused by different types of coagulase-negative staphylococci

Subclinical mastitis caused by intramammary infections (IMI) with coagulase-negative staphylococci (CNS) is common in dairy cows and may cause herd problems. Control of CNS mastitis is complicated by the fact that CNS contain a large number of different species. The aim of the study was to investigate the epidemiology of different CNS species in dairy herds with problems caused by subclinical CNS mastitis. In 11 herds, udder quarter samples were taken twice 1 mo apart, and CNS isolates were identified to the species level by biochemical methods. The ability of different CNS species to induce a persistent infection, and their associations with milk production, cow milk somatic cell count, lactation number, and month of lactation in cows with subclinical mastitis were studied. Persistent IMI were common in quarters infected with Staphylococcus chromogenes, Staphylococcus epidermidis, and Staphylococcus simulans. The results did not indicate differences between these CNS species in their association with daily milk production, cow milk somatic cell count, and month of lactation in cows with subclinical mastitis. In cows with subclinical mastitis, S. epidermidis IMI were mainly found in multiparous cows, whereas S. chromogenes IMI were mainly found in primiparous cows.     

Molecular characterization and clonal diversity of methicillin-susceptible Staphylococcus aureus in milk of cows with mastitis in Brazil

Mastitis is an important disease for the dairy industry worldwide, causing economic losses and reducing milk quality and production. Staphylococcus aureus is a worldwide agent of this intramammary infection, which also causes foodborne diseases. The objective of this study was to determine the frequency of methicillin-susceptible Staphylococcus aureus (MSSA) isolates in milk of mastitis cows in Brazil and to analyze the genetic lineages and the content of antimicrobial resistance genes and virulence factors among these isolates. Fifty-six MSSA isolates were recovered from 1,484 milk samples (positive for the California mastitis test) of 518 cows from 11 different farms in Brazil (representing 51% of total Staph. aureus obtained), and they were further characterized. Methicillin-susceptible Staphylococcus aureus were isolated from 3.7% of California mastitis test-positive tested milk samples and from 6.2% of tested mastitic cows. Methicillin-susceptible Staphylococcus aureus isolates were characterized by spa typing, agr typing, and multilocus sequence typing, and resistance and virulence traits were investigated by PCR. Seven spa types were identified among MSSA (% of isolates): t127 (44.6), t605 (37.5), t002, t1784, t2066 (1.8), and 2 new ones: t10856 (10.7) and t10852 (1.8). Five distinct sequence types (ST) were detected (% of isolates): ST1 (46.4), ST126 (37.5), ST133 (10.7), ST5 (3.6), and a novel ST registered as ST2493 (1.8). Resistances were detected for streptomycin, chloramphenicol, and tetracycline. One strain contained the chloramphenicol resistance gene (fexA; included within transposon Tn558) and 3 strains contained the tetracycline resistance gene [tet(K)]. Methicillin-susceptible Staphylococcus aureus strains were susceptible to most of the antibiotics studied and lacked the virulence genes of Panton-Valentine leukocidin (lukF/S-PV), toxic shock syndrome toxin 1 (tst), exfoliative toxin A (eta), and exfoliative toxin B (etb), as well as the genes of the immune evasion cluster. Methicillin-susceptible Staphylococcus aureus isolates were detected in a relatively low proportion of cows with mastitis (6.2%) and recovered isolates presented high diversity of genetic lineages, with CC1 and CC126 the predominant clonal complexes, and CC133 also being detected. Larger epidemiological studies with molecular characterization of isolates are required to deepen the knowledge on the circulating genetic lineages among the cow population with mastitis.     

Short communication: Lack of intramammary niche recolonization during a sanitation program for the contagious mastitis pathogen Staphylococcus aureus genotype B

In Switzerland, sanitation programs of dairy herds infected with the contagious mastitis pathogen Staphylococcus aureus genotype B (GTB) have been established for several years. In recent years, Streptococcus uberis and non-aureus staphylococci have emerged as the bacteria most frequently isolated from bovine milk samples. The latter cause subclinical mastitis, and some species are more persistent or pathogenic than others. The present study aimed to investigate the developments in the intramammary colonization spectrum of 5 dairy herds undergoing a sanitation program for Staph. aureus GTB. We collected single-quarter milk samples aseptically from all lactating cows at 3-mo intervals during the sanitation period; after classical bacteriological analysis, MALDI-TOF mass spectrometry was used to identify the isolates to the species level. Non-aureus staphylococci were found to be the bacterial group most frequently occurring on the selected farms, with Staphylococcus chromogenes and Staphylococcus xylosus being predominant. The present study demonstrated that GTB-infected cows treated with antibiotics lacked systematic recolonization with other bacteria during herd sanitation for the contagious Staph. aureus GTB.     

Intramammary infections with different non-aureus staphylococci in dairy cows

Subclinical mastitis causes an increase in milk somatic cell count (SCC) and can lead to reduced milk production and early culling. In many countries, non-aureus staphylococci (NAS) is the most common bacterial finding in subclinical mastitis of dairy cows. New methodology makes it possible to identify NAS species, but knowledge about the epidemiology is limited. The objective of this project was to improve advisory services for mastitis control by investigating associations between NAS and SCC, milk production, and persistence of intramammary infections (IMI). Farmers who had sent milk samples to the Swedish National Veterinary Institute (Uppsala, Sweden) were asked to participate if NAS was identified in the samples. Participating farmers were asked to resample all udder quarters of the cow once within 1 mo. Regression models were used to investigate associations between NAS and cow factors, udder quarter California mastitis test and SCC, and persistence of IMI. Associations with cow composite milk yield and SCC were also investigated. In total, 671 cows from 201 herds were enrolled in the study, and 19 NAS species were identified, of which the 4 most common were Staphylococcus epidermidis, Staphylococcus simulans, Staphylococcus chromogenes, and Staphylococcus haemolyticus. Persistent IMI was more common in udder quarters with Staphylococcus hyicus and S. simulans and less common in those with Staphylococcus saprophyticus IMI. β-Lactamase production by the different NAS species varied from 0 to 100%. There was a significant association between NAS species and California mastitis test and SCC of udder quarters, and this varied depending on parity. The cow composite milk SCC at the test milking before the initial sample was taken differed significantly with NAS species, but not at the subsequent test milking. Milk yield—at the test milking before or after the initial sample—did not differ significantly for NAS species. There were no significant associations between milk yield or SCC and persistent NAS IMI. In conclusion, the NAS species affects SCC and persistent IMI differently but not milk yield.     

Quarter- and cow-level risk factors for intramammary infection with coagulase-negative staphylococci species in Swiss dairy cows

Bacteriological status, evaluation of udder symmetry, udder hygiene, and teat end scores of 92 dairy cows were assessed on 3 Swiss dairy farms in a longitudinal 1-yr study to determine risk factors for intramammary infection (IMI) with coagulase-negative staphylococci (CNS) species. Farm visits were performed monthly including sterile quarter milk sampling and udder evaluation of all lactating cows. Milk samples were evaluated for the presence of staphylococci using selective agar plates. Species identification was performed using MALDI-TOF mass spectrometry. Intramammary infection was defined as milk samples having ≥100 cfu per mL of milk according to culture results. Overall, 3,151 quarter samples were included in the statistical analysis. Staphylococcus chromogenes, Staphylococcus haemolyticus, Staphylococcus xylosus, and a Staphylococcus warneri-like species were the 4 most prevalent CNS species found. Hierarchical multivariable logistic regression models were built to evaluate risk factors for species-specific CNS IMI. Risk factors for Staph. chromogenes IMI were presence in herd B, the period from June 2014 to August 2014 and December 2014 to February 2015, and presence of udder edema. For Staph. haemolyticus, the relevant risk factor included coinfection with Staph. xylosus coinfection with other than the above-mentioned CNS species (“others”) and the period from June 2014 to November 2014. Coinfection with Staph. haemolyticus and “others,” the periods from June 2014 to August 2014 and December 2014 to February 2015, early phase of lactation (1–60 d in milk), and belonging to herd B were significantly associated with Staph. xylosus IMI. Mid and late lactation, coinfection with Staph. xylosus, and the period September 2014 to May 2015 were identified as significant risk factors for Staph. warneri-like IMI. For Staph. chromogenes, 60.6 and 26% of the variance was observed at the quarter and cow level, respectively, whereas for the other investigated species the highest variance was observed at the sample level. The predominant species within herds differed and was most pronounced for the Staph. warneri-like species.     

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

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

Relationships between milk culture results and 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.     

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.     

Characteristics of Enterotoxigenic Coagulase Positive Staphylococci Isolated from Bovine Milk in Cases of Subclinical Mastitis

Coagulase positive staphylococci are the most common cause of bovine subclinical mastitis. The goal of this study was to investigate if isolates from milk in cases of mastitis synthesize enterotoxins and express resistance to methicillin. A total of 50 strains of coagulase positive staphylococci were tested using immunoassay ELFA technique. Ten isolates were enterotoxin positive and RealTime PCR was used to identify genes encoding enterotoxins and methicillin resistance. One isolate carried the enterotoxin B encoding gene, but none were positive for the methicillin resistance gene. All isolates were confirmed as Staphylococcus aureus biochemically and by PCR.     

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

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

Comparisons of milk quality on North Carolina organic and conventional dairies

The organic dairy industry is growing rapidly across the United States and has recently expanded into the southeastern states. To date, no published comparisons of milk quality exist between organic and conventional dairies in the Southeastern United States. Maintaining high milk quality is challenging in this region due to the longer periods of high heat and humidity. The objective of this observational study was to compare milk quality on organic and conventional dairies in North Carolina during the warm summer months of the year. Data were compared from 7 organically and 7 conventionally managed herds in North Carolina. To assess milk quality, milk samples were aseptically collected from each functional quarter of each cow in the milking herds at the time of sampling and linear somatic cell scores (SCS) were obtained for individual cows. A total of 4,793 quarter milk samples (2,526 conventional and 2,267 organic) were collected from 1,247 cows (652 conventional and 595 organic). Milk samples were cultured and bacterial growth was identified using protocols consistent with those of the National Mastitis Council (Verona, WI). Subclinical mastitis was defined as the presence of SCS ≥4 and also a microbiological infection in at least 1 quarter. The proportion of cows with subclinical mastitis did not differ between conventional (20.8%) and organic (23.3%) herds. No significant difference was observed between herd management types in the proportion of cows without microbiological growth in milk samples. Also, no significant differences were observed between organic and conventional herds for cow-level prevalence of Staphylococcus aureus, coagulase-negative Staphylococcus spp., Streptococcus spp., or Corynebacterium spp. Two of the organic herds had a notably higher prevalence of Corynebacterium spp. and higher SCS. Coliforms were found in 5 of 7 conventional herds and in only 1 of 7 organic herds. Mean SCS did not differ between conventional (3.3 ± 0.2) and organic (3.5 ± 0.2) herds. Despite differences in herd management, milk quality was remarkably similar between the organic and conventional dairies compared for this study.     

Pathogen-specific effects on milk yield in repeated clinical mastitis episodes in Holstein dairy cows

The objective of this study was to estimate the effects of clinical mastitis (CM) cases due to different pathogens on milk yield in Holstein cows. The first 3 CM cases in a cow’s lactation were modeled. Eight categories of pathogens were included: Streptococcus spp.; Staphylococcus aureus; coagulase-negative staphylococci (CNS); Escherichia coli; Klebsiella spp.; cases with CM signs but no bacterial growth (above the level detectable by our microbiological procedures) observed in the culture sample, and cases with contamination (≥3 pathogens in the sample); other pathogens that may be treated with antibiotics (included Citrobacter, Corynebacterium bovis, Enterobacter, Enterococcus, Pasteurella, Pseudomonas; “other treatable”); and other pathogens not successfully treated with antibiotics (Trueperella pyogenes, Mycoplasma, Prototheca, yeasts; “other not treatable”). Data from 38,276 lactations in cows from 5 New York State dairy herds, collected from 2003–2004 until 2011, were analyzed. Mixed models with an autoregressive correlation structure (to account for correlation among the repeated measures of milk yield within a lactation) were estimated. Primiparous (lactation 1) and multiparous (lactations 2 and 3) cows were analyzed separately, as the shapes of their lactation curves differed. Primiparas were followed for up to 48 wk of lactation and multiparas for up to 44 wk. Fixed effects included parity, calving season, week of lactation, CM (type, case number, and timing of CM in relation to milk production cycle), and other diseases (milk fever, retained placenta, metritis, ketosis, displaced abomasum). Herd was modeled as a random effect. Clinical mastitis was more common in multiparas than in primiparas. In primiparas, Streptococcus spp. occurred most frequently as the first case. In multiparas, E. coli was most common as the first case. In subsequent cases, CM cases with no specific growth or contamination were most common in both parity groups. The hazard of CM increased with case number. Mastitic cows were generally higher producers before the CM episode than their nonmastitic herdmates. Milk loss varied with pathogen and case number. In primiparas, the greatest losses were associated with E. coli and “other not treatable” organisms. In multiparas, the greatest losses were associated with Klebsiella spp. and “other not treatable” organisms. Milk loss was not associated with occurrence of CNS. The findings may help farmers to make optimal management decisions for their cows.     

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.     

Antimicrobial resistance and genetic characterization of coagulase-negative staphylococci from bovine mastitis milk samples in Korea

Coagulase-negative staphylococci (CNS) are one of the most common bovine mastitis pathogens found worldwide. In this study, we investigated the prevalence and distribution of CNS species in mastitis milk samples and further characterized the methicillin-resistant (MR) CNS. A total of 311 CNS were isolated from 3,692 quarter milk samples from 1,373 dairy cattle at 81 farms between 2013 and 2017. Further evaluation of the CNS isolates revealed 14 CNS species among the samples and 3 predominant species—namely, Staphylococcus chromogenes, Staphylococcus simulans, and Staphylococcus epidermidis. Resistance was higher in S. epidermidis than in other CNS species except for resistance against oxacillin in Staphylococcus sciuri. Resistance to β-lactams was the most common in all CNS species (8.4% in ampicillin, 21.2% in oxacillin, and 13.5% in penicillin). Conversely, only minimal resistance to cephalothin, ceftiofur, and pirlimycin/novobiocin was found. Twenty-one isolates from 4 species were mecA-carrying MRCNS strains, including 18 S. epidermidis and 1 each of S. sciuri, Staphylococcus equorum, and Staphylococcus hominis. The majority of the mecA-carrying MRCNS isolates were produced in the biofilm. Furthermore, multidrug-resistant sequence type 179 isolate produced the strongest biofilm. Seven genotypes were detected in the 18 MR S. epidermidis strains, the most predominant of which persisted on a farm for 2 yr. Our findings for the antimicrobial susceptibility profiles and genotypic characterization of the MRCNS isolates could provide valuable information for controlling the spread of resistance and the selection of appropriate antimicrobial therapies for mastitis in the future. Further, strategic antibiotic use for mastitis treatment and hygienic management practices aimed at the prevention of the growth of resistant bacteria are urgently needed on dairy farms.