Conventional identification of Streptococcus uberis isolated from bovine mastitis in Argentinean dairy herds

The objective of this study was to evaluate a conventional scheme for identifying Streptococcus uberis strains isolated from bovine mastitis. Seventy-five gram-positive, catalase-negative cocci were collected from cows with mastitis from 19 dairy herds located in the east-central region of Argentina. Five American Type Culture Collection strains and bovine isolates were identified by the API 20 Strep system and by restriction fragment length polymorphism analysis of 16S rDNA. A conventional scheme based on 11 biochemical tests was selected for identification of Strep. uberis strains: the Christie-Atkins-Munch-Petersen reaction; hydrolysis of Arg, esculin, and sodium hippurate; growth in inulin, mannitol, raffinose, salicin, and sorbitol; and growth at 45°C and in 6.5% NaCl. Reference strains and 25 bovine isolates were classified accurately to the species level by the conventional scheme in a blind assay. Each reference strain and each bovine isolate were identified as belonging to the same species following these 3 methods. The remaining 50 isolates identified as Strep. uberis by the API 20 Strep system and 16S rDNA RFLP were assayed by the conventional scheme. This scheme correctly identified 47 (94%) of 50 isolates as Strep. uberis by comparing their biochemical profile with that of the reference strain. Three (6%) of the 50 isolates were classified as Strep. uberis by the API 20 Strep system and by 16S rDNA RFLP and were identified as Enterococcus faecalis by the conventional scheme. Thirty percent of the Strep. uberis strains showed biochemical profiles identical to the Strep. uberis American Type Culture Collection 27958 strain. Seventy percent of the Strep. uberis strains demonstrated variability compared with the reference strain, resulting in 19 different biochemical profiles. The conventional scheme proposed in this study resulted in a relatively low number of misidentifications and could biochemically identify not only typical, but also atypical Strep. uberis strains. This conventional scheme can be considered an adequate method for identifying Strep. uberis strains isolated from bovine mastitis because of its affordable cost in developing countries, and it may contribute to determining the frequency of isolation of Strep. uberis strains in Argentinean dairy herds.     

Short communication: Outbreak of Nocardia neocaledoniensis mastitis in an Italian dairy herd

Nocardia spp. are an uncommon cause of mastitis, and outbreaks have typically been reported in dairy farms with poor hygienic and management conditions. The outbreak described herein involved a dairy farm with 43 lactating cows that, after a long period with low bulk milk somatic cell counts (<180,000 cells/mL), experienced an increasing incidence of clinical mastitis with bulk milk somatic cell counts greater than 300,000 cells/mL. Fifteen mastitic quarters milk samples from 9 dairy cows were found to be infected by a member of the genus Nocardia, as identified on the basis of selected phenotypic and chemotaxonomic characteristics. The isolates were confirmed as Nocardia neocaledoniensis by 16S rDNA gene sequencing. Average quarter milk somatic cell count for infected udders was 863,057 cells/mL, significantly greater than the average value in noninfected quarters (189,710 cells/mL).     

Extended ceftiofur therapy for treatment of experimentally-induced Streptococcus uberis mastitis in lactating dairy cattle

Streptococcus uberis is an important cause of mastitis in dairy cows throughout the world, particularly during the dry period, the period around calving, and during early lactation. Strategies for controlling Strep. uberis mastitis are poorly defined and are currently inadequate. Objectives of the present study were to evaluate efficacy of ceftiofur, a new broad-spectrum cephalosporin antibiotic, for treatment of experimentally induced Strep. uberis intramammary infections (IMI) in lactating dairy cows during early lactation and to determine whether extended therapy regimens enhanced efficacy of ceftiofur. Efficacy of extended ceftiofur intramammary therapy regimens was investigated in 37 mammary quarters of 23 dairy cows that developed clinical mastitis following experimental infection with Strep. uberis during early lactation. Cows that developed clinical mastitis during the challenge period were allocated randomly to 3 groups representing 3 different ceftiofur treatment regimens: 2-d (n = 7 mammary quarters), 5-d (n = 16 mammary quarters), and 8-d (n = 14 mammary quarters) treatment regimens. For all groups, 125 mg of ceftiofur hydrochloride was administered via intramammary infusion. A bacteriological cure was defined as an experimentally infected quarter that was treated and was bacteriologically negative for the presence of Strep. uberis at 7, 14, 21, and 28 d posttreatment. Percentage of Strep. uberis IMI eliminated was 43, 88, and 100% for the 2-, 5-, and 8-d ceftiofur treatment regimens, respectively. Both the 5- and 8-d ceftiofur extended therapy treatment regimens had significantly higher bacterial cure rates than the standard 2-d ceftiofur treatment regimen. The bacterial cure rate of the 8-d ceftiofur extended therapy group was marginally better (P = 0.052) than the 5-d ceftiofur extended therapy group. Results of this study indicate that ceftiofur therapy was effective for eliminating Strep. uberis experimental IMI, and 5- and 8-d extended ceftiofur therapy regimens were more effective than the standard 2-d treatment.     

Field observations on the variation of Streptococcus uberis populations in a pasture-based dairy farm

Microbiological and molecular tools were used to monitor Streptococcus uberis populations on farm tracks and paddocks on a dairy farm during different seasons of a year to identify and profile potential environmental niches of Strep. uberis in a pasture-based dairying system. Farm tracks of high or low cow traffic were sampled every 2 wk for an entire year and Strep. uberis numbers were enumerated from a selective medium. During each season of the year, paddocks were sampled for the presence of Strep. uberis before and after grazing by dairy cows. Farm tracks of high cow traffic generally had greater concentrations of Strep. uberis isolated compared with tracks with less cow traffic, but there was also significant variation in the concentrations of Strep. uberis contamination among seasons, being highest in winter and lowest in summer. The bacterium was detected in paddocks only after cow grazing had occurred, but the bacteria could still be detected in soil for up to 2 wk following grazing in winter. Multilocus sequence typing showed great heterogeneity, with some commonality between farm track and milk isolates, which may help explain cow-to-environment or environment-to-cow transmission of the bacterium in the dairy setting.     

Efficacy of extended ceftiofur intramammary therapy for treatment of subclinical mastitis in lactating dairy cows

Little research has focused on treatment of cows with subclinical mastitis during lactation. Ceftiofur is a new broad-spectrum, third-generation cephalosporin antibiotic for veterinary use that inhibits bacterial cell wall synthesis by interfering with enzymes essential for peptidoglycan synthesis. Ceftiofur should be effective against a wide range of contagious and environmental mastitis pathogens. Objectives of the present study were to evaluate the efficacy of ceftiofur for treatment of subclinical mastitis in lactating dairy cows, and to determine if extended therapy regimens enhanced efficacy of ceftiofur. Holstein and Jersey dairy cows (n = 88) from 3 dairy research herds were used. Cows were enrolled in the study based on milk somatic cell counts >400,000/mL and isolation of the same mastitis pathogen in 2 samples obtained 1 wk apart. Cows with one or more intramammary infections (IMI) were blocked by parity and DIM and allocated randomly to 1 of 3 different ceftiofur treatment regimens: 2-d (n = 49 IMI), 5-d (n = 41 IMI), and 8-d (n = 38 IMI) treatment regimens. For all groups, 125 mg of ceftiofur hydrochloride was administered via intramammary infusion. Eighteen cows with 38 IMI were included as an untreated negative control group. A bacteriological cure was defined as a treated infected mammary quarter that was bacteriologically negative for the presence of previously identified bacteria at 14 and 28 d after the last treatment. Efficacy of ceftiofur therapy against all subclinical IMI was 38.8, 53.7, and 65.8% for the 2-, 5-, and 8-d ceftiofur treatment regimens, respectively. Four of 38 (10.5%) IMI in control cows were cured spontaneously without treatment. All 3 ceftiofur treatment regimens were significantly better than the negative control, and the 8-d extended ceftiofur treatment regimen treatment group was significantly better than the standard 2-d treatment group. Pathogen groups had significantly different cure rates from one another. The cure rate for the 8-d extended ceftiofur treatment regimen was 70% for Corynebacterium bovis, 86% for coagulase-negative Staphylococcus species, 36% for Staph. aureus, 80% for Streptococcus dysgalactiae ssp. dysgalactiae, and 67% for Strep. uberis.     

Genetic diversity of mastitis-associated Klebsiella pneumoniae in dairy cows

The objectives of this study were to determine the level of genetic diversity of Klebsiella pneumoniae isolated from clinical mastitis cases and to define genotypes most commonly associated with the disease. Individual quarter milk samples were collected from a single privately owned dairy herd over a 2-yr period and submitted to the Laboratory for Udder Health, Minnesota Veterinary Diagnostic Laboratory, University of Minnesota, for bacteriological culture. Eighty-four K. pneumoniae isolates were obtained and fingerprinted by repetitive DNA sequence PCR, 43 by pulsed-field gel electrophoresis (PFGE), and 29 by multilocus sequence typing (MLST). Significant genetic diversity was observed among the isolates regardless of the fingerprinting method used. Simpson's diversity index was 93.5, 96.1, and 97.0% when analyzed by repetitive DNA sequence PCR (n = 84), pulse field gel electrophoresis (n = 43), and MLST (n = 29), respectively. In some cases more than 1 genotype was obtained from a single milk sample originating from an individual quarter. The majority of infections were observed during the winter and accounted for 69.0% of K. pneumoniae mastitis cases. There was a negative correlation between a matrix of fingerprints similarity and a matrix of temporal distances. The MLST results revealed 5 new and novel allelic types, which have not been previously reported in the MLST database. Three isolates shared MLST types with human clinical isolates, raising the possibility that some K. pneumoniae isolates, of bovine origin, may be capable of causing disease in humans. There were 21 genotypes present within the herd, and there was no evidence for nonrandom distribution of genotypes uniquely associated with mastitis. We have shown, using 3 distinct genotyping methods, that K. pneumoniae isolated from clinical mastitis within a single dairy herd is caused by a genetically diverse population and that multiple genotypes can be isolated from a mastitic quarter. The data suggest that mastitis can be caused by a variety of K. pneumoniae genotypes. Diverse genotypes may have different levels of invasiveness and virulence and may originate from various sources within the dairy.     

Molecular epidemiology,antimicrobial activity,and virulence gene clustering of Streptococcus agalactiae isolated from dairy cattle with mastitis in China

Streptococcus agalactiae is a contagious pathogen that causes bovine mastitis worldwide, resulting in considerable economic losses. In this study, we isolated 42 S. agalactiae strains in 379 milk samples from cows with subclinical mastitis on 15 dairy farms in 12 Chinese provinces. Analysis based on capsular typing and multilocus sequence typing, combined with patterns of virulence gene scanning and antimicrobial resistance, identified the lineages and populations of the isolates. We grouped the 42 isolates into 7 sequence types belonging to 6 clonal complexes, mainly CC103 (31/42 isolates; 73.8%). We identified an ST-23 strain named Sa 129 for the first time on Chinese dairy farms—this strain is usually associated with human isolates. Capsular types Ia and II were predominant in capsular typing. The prevalence of virulence profile 1 (bibA, cfb, cspA, cylE, fbsA, fbsB, hylB, and pavA) was 64.3%, and represented the main trend in China. With respect to antimicrobial resistance, most isolates were susceptible to β-lactams, rifamycin, glycopeptides, and oxazolidone; resistance to several antimicrobial agents, including lincomycin, clindamycin, and doxycycline, varied in 4 different regions. Our research provides a profile for the molecular epidemiology, multilocus sequence typing, antimicrobial resistance, and virulence gene clustering of S. agalactiae, and may be beneficial for the clinical monitoring, prevention, and control of mastitis in dairy cattle.     

Herd-level relationship between antimicrobial use and presence or absence of antimicrobial resistance in gram-negative bovine mastitis pathogens on Canadian dairy farms

Concurrent data on antimicrobial use (AMU) and resistance are needed to contain antimicrobial resistance (AMR) in bacteria. The present study examined a herd-level association between AMU and AMR in Escherichia coli (n = 394) and Klebsiella species (n = 139) isolated from bovine intramammary infections and mastitis cases on 89 dairy farms in 4 regions of Canada [Alberta, Ontario, Québec, and Maritime Provinces (Prince Edward Island, Nova Scotia, and New Brunswick)]. Antimicrobial use data were collected using inventory of empty antimicrobial containers and antimicrobial drug use rate was calculated to quantify herd-level AMU. Minimum inhibitory concentrations (MIC) were determined using Sensititre National Antimicrobial Resistance Monitoring System (NARMS) gram-negative MIC plate (Trek Diagnostic Systems Inc., Cleveland, OH). Isolates were classified as susceptible, intermediate, or resistant. Intermediate and resistant category isolates were combined to form an AMR category, and multivariable logistic regression models were built to determine herd-level odds of AMR to tetracycline, ampicillin, cefoxitin, chloramphenicol, trimethoprim-sulfamethoxazole combination, sulfisoxazole, streptomycin and kanamycin in E. coli isolates. In the case of Klebsiella species isolates, logistic regression models were built for tetracycline and sulfisoxazole; however, no associations between AMU and AMR in Klebsiella species were observed. Ampicillin-intermediate or -resistant E. coli isolates were associated with herds that used intramammarily administered cloxacillin, penicillin-novobiocin combination, and cephapirin used for dry cow therapy [odds ratios (OR) = 26, 32, and 189, respectively], and intramammary ceftiofur administered for lactating cow therapy and systemically administered penicillin (OR = 162 and 2.7, respectively). Use of systemically administered penicillin on a dairy farm was associated with tetracycline and streptomycin-intermediate or -resistant E. coli isolates (OR = 5.6 and 2.8, respectively). Use of cephapirin and cloxacillin administered intramammarily for dry cow therapy was associated with increasing odds of having at least 1 kanamycin-intermediate or -resistant E. coli isolate at a farm (OR = 8.7 and 9.3, respectively). Use of systemically administered tetracycline and ceftiofur was associated with cefoxitin-intermediate or -resistant E. coli (OR = 0.13 and 0.16, respectively); however, the odds of a dairy herd having at least 1 cefoxitin-intermediate or -resistant E. coli isolate due to systemically administered ceftiofur increased with increasing average herd parity (OR = 3.1). Association between herd-level AMU and AMR in bovine mastitis coliforms was observed for certain antimicrobials. Differences in AMR between different barn types and geographical regions were not observed.     

Epidemiological investigation of Streptococcus equi subspecies zooepidemicus involved in clinical mastitis in dairy goats

An outbreak of clinical mastitis was observed in dairy goats due to the zoonotic pathogen Streptococcus equi ssp. zooepidemicus. Affected goats were culled to prevent transmission of infection to other animals or humans. The objective of the study was to determine whether horses on the same farm were the source of the pathogen. Streptococcus equi ssp. zooepidemicus was obtained from milk of 10% of goats in the herd and from feces of 3 of 7 healthy horses that shared pasture and housing with the goats. Isolates of caprine and equine origin had identical biochemical profiles, including the ability to ferment sorbitol and lactose, which distinguishes S. equi ssp. zooepidemicus from S. equi ssp. equi. Sequencing of the 16S-23S intergenic spacer region and results from sodA-seeI multiplex PCR supported identification of isolates as S. equi ssp. zooepidemicus. Based on random amplified polymorphic DNA typing and rpoB and sodA sequencing, caprine isolates were indistinguishable from each other, but distinct from equine isolates. Further analysis of equine fecal samples showed that multiple strains of S. equi ssp. zooepidemicus can be present in a single sample or in sequential samples obtained from a single horse. Failure to detect the mastitis-causing strain in equine feces may indicate that horses were not the source of the mastitis outbreak in goats. Alternatively, the outbreak may be due to presence of multiple S. equi ssp. zooepidemicus strains in equine feces and a failure to detect all strains when analyzing a limited number of isolates per sample.     

Subclinical and clinical mastitis in heifers following the use of a teat sealant precalving

This study investigated the effect in heifers of infusion of a bismuth subnitrate teat-canal sealant and bacterial intramammary infection (IMI) precalving on prevalence of postcalving IMI and incidence of clinical mastitis in the first 2 wk postcalving. Glands (n = 1,020) from heifers (n = 255) in 5 seasonally calving, pasture-fed dairy herds were randomly assigned within heifer to 1 of 4 treatment groups (no treatment; mammary gland secretion collection; infusion of a teat sealant; or sample collection with infusion of teat sealant). Heifers within a herd were enrolled on one calendar day, 31 d on average before the planned start of the seasonal calving period. Duplicate milk samples were collected from each gland within 4 d after calving for bacterial culture. Herd owners collected duplicate milk samples, before treatment, for bacterial culture from glands they defined as having clinical mastitis. The gland prevalence of IMI precalving was 15.5% and did not differ between herds. Bacteria isolated precalving included coagulase-negative staphylococci (76.9% of all bacteriologically positive samples), Streptococcus uberis (14.1%), Staphylococcus aureus (5.1%), Corynebacterium spp. (3.8%), and others (0.1%). The presence of an IMI precalving increased the risk of an IMI postcalving 3.6-fold and the risk of clinical mastitis 4-fold, relative to no IMI precalving. Infusion of the teat sealant reduced the risk of postcalving IMI due to Strep. uberis by 84%, and of clinical mastitis by 68%. Sampling the glands precalving had no effect on postcalving IMI or on clinical mastitis incidence. Use of an internal teat canal sealant in heifers precalving may be a useful tool for reducing the risk of subclinical and clinical mastitis in heifers.     

Technical note: Development of a challenge model for Streptococcus uberis mastitis in dairy heifers

A challenge model for experimentally inducing Streptococcus uberis mastitis in bred dairy heifers was developed. Qualifying heifers (n = 7) exhibited antibody titers of < 1:10,000 against Strep. uberis antigens and were free of intramammary infections (IMI). Two contralateral quarters of each heifer were assigned to receive an infusion of Strep. uberis (1,000 to 2,000 cfu); remaining quarters served as unchallenged controls. For a successful challenge and infection, 3 of 4 consecutive mammary secretion samples had to culture positive for Strep. uberis. Six of the 7 heifers were challenged successfully in both infused quarters with a mean dose of 1,080 cfu; once confirmed, infections were treated with a one-time infusion of nonlactating cow therapy. Before challenge, mammary secretion leukocyte counts averaged 8.4 × 10⁶/mL in all quarters. At 24 h after challenge, leukocyte count increased to 18.4 × 10⁶/mL in challenged quarters, peaking on d 5 at 24.3 × 10⁶/mL; unchallenged quarters remained at ≤ 10.4 × 10⁶/mL, but increased to 15.2 × 10⁶/mL on d 7 and then decreased. Before challenge, macrophages predominated (81%) in mammary secretions followed by lymphocytes (15.3%) and neutrophils (3.7%). By 24 h after challenge, neutrophils increased in challenged quarters and predominated for the duration of the trial (65.3 to 70%), whereas macrophages predominated in unchallenged control quarters (65.2 to 75.2%). The challenge model was successful in establishing Strep. uberis IMI in 85.7% of animals, and IMI were controlled (100% cure) by administering nonlactating cow therapy. All heifers calved free of IMI and antimicrobial residues, with milk production similar to that of herd mates and with somatic cell counts (SCC) < 200,000 cells/mL.     

Fecal shedding of Klebsiella pneumoniae by dairy cows

Klebsiella pneumoniae is a common cause of clinical mastitis in dairy cattle. Wood products are considered to be the main source of Klebsiella on dairy farms. Environmental hygiene and use of inorganic bedding materials such as sand are recommended to control Klebsiella mastitis. However, Klebsiella mastitis still occurs on well-managed dairy farms that use sand as bedding material. In a 5-mo study in a New York State dairy herd performed during the summer of 2005, all of 9 samples of unused sand bedding tested negative for Klebsiella, whereas 14 of 18 samples of used sand bedding contained Klebsiella at a median level of 10^4.6 cfu/g. We hypothesized that fecal shedding of Klebsiella by dairy cows contributes to the presence of Klebsiella in the environment. Using a cheap and simple method based on ampicillin-containing MacConkey agar for screening, and biochemical tests for confirmation of species identity, 595 fecal samples from healthy dairy cattle were screened for presence of Klebsiella. In a longitudinal study of 100 cows followed over 5 mo, more than 80% of fecal samples tested positive for K. pneumoniae. The average prevalence of K. pneumoniae-positive fecal samples was also above 80% in a cross-sectional study of 100 cows from 10 herds across New York and Massachusetts. Fecal shedding of K. pneumoniae by a large proportion of dairy cows may explain why Klebsiella mastitis occurs in herds that use inorganic bedding material or other bedding material that is free from Klebsiella upon introduction into the barn.     

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.     

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.     

Longitudinal study of herd udder hygiene and its association with clinical mastitis in pasture-based dairy cows

The objectives of this exploratory study were to (1) describe the association between herd-level udder hygiene and clinical mastitis and (2) investigate how sample size and milking stage affect the accuracy and precision of herd udder hygiene assessments made at milking time. A prospective longitudinal study was conducted in a dairy herd in Northern Australia as part of a previously published clinical trial of premilking teat disinfection. Video footage from 35 afternoon milkings was used to conduct 12,544 udder hygiene scores from 504 cows during an 89-d period and measure udder hygiene of the herd (proportion of cows with udder hygiene ≥3 out of 4). Linear interpolation was used to estimate herd udder hygiene on the days that were not scored, such that a herd-level udder hygiene measure was available for all cow-days in the study. Clinical mastitis events occurring during the study period were detected and recorded by farm staff according to a standardized definition. The relationship between herd udder hygiene on each of 1, 2, and 3 d before each study day (d ?1, ?2, and ?3, respectively) and clinical mastitis at the cow level on each study day (each in turn being set as d 0) was determined using multivariable generalized estimating equations (family = Poisson, link = log), with the unit of analysis being the cow-day, adjusting for potential confounders and the clustering within the data. In addition, sampling strategies were evaluated by simulating herd udder hygiene assessments using a subset of cows in the herd. Herd udder hygiene from d ?1, ?2, and ?3 was positively associated with clinical mastitis on d 0 (incidence rate ratio = 1.4 per 10-point increase in the percentage of cows with poor udder hygiene). Sampling strategy simulation found that at least 80 cows needed to be scored to achieve sufficiently precise estimations of herd udder hygiene. Furthermore, cows scored later during milking were slightly more likely to have poor udder hygiene than those scored earlier (risk ratio = 1.02 for cows that were 10% later in the milking order). More research is needed to evaluate risk factors for poor udder hygiene and potential interventions in pasture-based dairy 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.     

Duration of infection and strain variation in Streptococcus uberis isolated from cows' milk

The duration of infection and pulsed-field gel electrophoresis (PFGE) types of bovine intramammary Streptococcus uberis isolates were examined. Milk samples were collected in duplicate from all 4 glands of 503 cows from 5 herds within 1 to 3 d of parturition and from 113 cows with clinical mastitis in the same herds throughout lactation. Glands from which S. uberis was isolated were resampled at 28-d intervals.The prevalence of S. uberis was 12% for cows around parturition, and the median duration of infection was 16 d. Cows >2 yr old had a longer duration of infection than 2 yr old cows, and duration varied among herds. A total of 173 different PFGE types were identified from a total of 234 S. uberis isolates. Each farm had a unique set of PFGE types. Only 3 PFGE types were common to each of 3 pairs of cows, and these occurred on the same farm. Where S. uberis was isolated on more than one occasion from a gland, only 55% of the PFGE types were the same across time. For cows with multiple glands infected, only one-half (9 of 18) had the same PFGE type in more than one gland. No predominant PFGE type was identified in any herd.It is concluded that there was wide heterogeneity of PFGE types, that the environment rather than other cows was the likely source of S. uberis infections, and that glands may be infected with multiple S. uberis PFGE types over a lactation.     

Streptococcus dysgalactiae isolates at calving and lactation performance within the same lactation

The objective of the study was to investigate the association between early lactation Streptococcus dysgalactiae isolates and milk yield, somatic cell count (SCC), clinical mastitis, and culling in the same lactation. The 178 commercial dairy herds were randomly placed into 3 penicillin- or penicillin-dihydrostreptomycin-based dry-cow treatments and 3 different postmilking teat disinfection groups—negative control, iodine, or external teat sealant. All cows were sampled in early lactation, and Strep. dysgalactiae-positive and culture-negative cows were followed throughout the remainder of the lactation. Mixed models, including repeated measurements, with test-day observation as dependent variable, were used to compare milk yield, SCC, and available milk quality variables throughout the remaining lactation. Survival analyses, using a positive frailty model to account for any herd random effects, were used to estimate the hazard ratio for clinical mastitis and culling. Streptococcus dysgalactiae-positive cows had a significantly higher SCC throughout the lactation compared to culture-negative cows. For primiparous or multiparous cows, respectively, the differences in the geometric mean SCC between Strep. dysgalactiae-positive and culture-negative cows was 197,000 or 280,000 cells/mL at the beginning of the lactation, 24,000 or 46,000 cells/mL in mid lactation, and 39,000 or 111,000 cells/mL at the end of the lactation. Streptococcus dysgalactiae-positive primiparous or multiparous cows produced 334 or 246 kg less milk, respectively, during a 305-d lactation compared with culture-negative cows. Compared with culture-negative cows, the hazard ratios for clinical mastitis in Strep. dysgalactiae-positive cows were 2.3 (1.9 to 2.9) and 1.6 (1.3 to 2.0) for culling. For cows with both Strep. dysgalactiae and Staphylococcus aureus isolates, the hazard ratio for culling significantly increased to 2.5 (1.9 to 3.2).     

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.