Intramammary infections in lactating Jersey cows: Prevalence of microbial organisms and association with milk somatic cell count and persistence of infection.

There are limited data available regarding pathogens causing intramammary infections (IMI) in Jersey cows. The objectives of this study were to characterize the prevalence of IMI caused by different microorganisms in lactating Jersey cattle and evaluate the associations among microbes and somatic cell count (SCC) and persistence of IMI. This prospective, observational, longitudinal study included lactating Jersey cows (n = 753) from 4 farms within a 415 km radius of Columbia, Missouri. Quarter foremilk samples were aseptically collected monthly for 3 consecutive months. Microorganisms were identified using aerobic milk culture and MALDI-TOF mass spectrometry. A commercial laboratory measured SCC using flow cytometry. Milk culture results were used to classify single microorganism infections as persistent (same microorganism species identified at first sampling and one other sampling) or nonpersistent infection. Mixed models were built to evaluate the associations between IMI status and SCC natural logarithm (lnSCC), as well as persistence and lnSCC. Overall, staphylococci were the most commonly isolated microorganisms among the 7,370 quarter-level milk samples collected. Median prevalence (using all 3 samplings) of specific microbes varied among farms; however, Staphylococcus chromogenes was a common species found at all farms. The most common microbial species that persisted were Staph. chromogenes, Staphylococcus aureus, Staphylococcus simulans, and Streptococcus uberis. Streptococcus dysgalactiae and Staph. aureus were the IMI associated with the most inflammation based on lnSCC. The small number of herds included in this study with the large variation in herd type limits the generalizability of the data. However, results of this study seem to be similar to those of previous studies in other breeds, suggesting management factors are more important than breed-specific differences when evaluating causes of IMI and associated subclinical mastitis.

Distribution of staphylococcal and mammaliicoccal species from compost-bedded pack or sand-bedded freestall dairy farms.

The purpose of this prospective observational study was to determine whether dairy cattle housing types were associated with staphylococcal and mammaliicoccal populations found on teat skin, bedding, and in bulk tank milk. Twenty herds (n = 10 sand-bedded freestall herds; n = 10 compost-bedded pack herds) were enrolled. Each herd was visited twice for sample collection, and at each visit, 5 niches were sampled, including bulk tank milk, composite teat skin swab samples collected before premilking teat preparation, composite teat skin swab samples collected after premilking teat preparation, unused fresh bedding, and used bedding. All samples were plated on Mannitol salt agar and Columbia blood agar and staphylococcal-like colonies were selected for further evaluation. Bacterial colonies were speciated using MALDI-TOF mass spectrometry. All species were grouped into 4 categories included host-adapted, opportunistic, environmental, and unclassified. Absolute numbers and proportions of each genus and species were calculated. Proportional data were compared between groups using Fisher's exact test. Data representing 471 staphylococcal-like organisms were analyzed. Overall, 27 different staphylococcal and mammaliicoccal species were identified. Staphylococcus chromogenes was the only species identified from all 20 farms. A total of 20 different staphylococcal-like species were identified from bulk tank milk samples with the most prevalent species being S. chromogenes, followed by Staphylococcus aureus and Mammaliicoccus sciuri. Overall, more staphylococcal and mammaliicoccal isolates were identified among used bedding than unused bedding. The increased numbers of isolates within used bedding were primarily from used sand bedding samples, with 79% (76/96) of used bedding isolates being identified from sand bedding and only 20.8% (20/96) from used compost-bedded pack samples. When comparing categories found among sample types, more unclassified species were found in used sand bedding than in used compost-bedded pack samples. This finding is possibly related to the composting temperatures resulting in reduced growth or destruction of bacterial species. The prevalence of S. aureus was high in bulk tank milk for all herds, regardless of herd type, which may represent the influence of unmeasured management factors. Overall, staphylococcal and mammaliicoccal species were highly prevalent among samples from both farm types.

Administration of internal teat sealant in primigravid dairy heifers at different times of gestation to prevent intramammary infections at calving.

Intramammary infections (IMI) are common in primigravid dairy heifers and can negatively affect future milk production. Bismuth subnitrate-based internal teat sealants (ITS) have been used to prevent prepartum IMI in dairy heifers by creating a physical barrier within the teat, preventing pathogens from entering the gland, though determination of when to administer ITS in heifers has yet to be investigated. The objectives of this study were to determine if administration of ITS in primigravid heifers reduced the odds of IMI at calving and if administration of ITS at different stages of gestation (75 vs. 35 d prepartum) affected the odds of IMI at calving. A total of 270 heifers were used at a single farm. One quarter of each heifer was randomly chosen to be aseptically sampled and administered ITS 75 d prepartum (ITS75), another quarter of each heifer was sampled and received ITS 35 d prepartum (ITS35), whereas the remaining 2 quarters of each heifer served as control quarters (CON) and were not sampled before calving. Within 12 h of calving, aseptic colostrum samples were collected from all quarters to determine quarter infection status. When an IMI was caused by mastitis pathogens other than non-aureus staphylococci (NAS), CON quarters were 3 times [95% confidence interval (CI): 1.4-6.3] and 2.5 times (95% CI: 1.2-4.9) more likely to be infected at calving than ITS75 and ITS35 quarters, respectively. For IMI with NAS, CON quarters were 5.8 (95% CI: 3.2-10.5) and 6.4 (95% CI: 3.4-12.0) times more likely to be infected than ITS75 and ITS35 quarters, respectively. Odds of IMI at calving was similar between ITS75 and ITS35 quarters for both NAS (odds ratio = 0.9) and other pathogens (odds ratio = 1.2). Results indicate that ITS administration at either 75 and 35 d prepartum reduced IMI prevalence at calving in primigravid dairy heifers. Farm specific factors may influence prevalence and timing of heifer IMI and earlier administration of ITS provides an extended period of protection for the developing gland.

Evaluation of 4 different teat disinfection methods prior to collection of milk samples for bacterial culture in dairy cattle.

The first objective of this study was to determine whether differences would occur among teat end preparation techniques with regard to potential contamination of milk samples collected for bacterial culture. The second objective was to determine whether differences would be detected in genus or species of bacteria isolated from samples collected using the various methods as well as from contaminated or uncontaminated samples. Mammary quarter foremilk samples were collected from lactating dairy cattle at the University of Missouri Foremost Research Dairy Farm (Columbia). Four different teat end preparation methods were used to compare contamination rates in milk samples. Sampling techniques used before milk collection included (1) no preparation, (2) pre-milking disinfection and single-use towel drying of teats only, (3) scrubbing of the teat end with alcohol only, and (4) pre-milking disinfection, single-use towel drying, and scrubbing of the teat end with alcohol. Milk was plated on Columbia blood agar. Cultures were read at 48 h, with the number of morphologically different bacterial colony types quantified and isolated. Isolates were identified using MALDI-TOF mass spectrometry. Median numbers of colony types were compared among groups using Kruskal-Wallis ANOVA with post-hoc pairwise comparisons, and proportional data were compared using the chi-squared test. A total of 168 cows, including 665 quarters, were sampled, and 1,614 isolates resulted. Analysis with MALDI-TOF identified 29 unique genera and 81 unique species among the samples. More contaminated samples occurred in groups 1 and 2 compared with groups 3 and 4. Group 3 had more contaminated samples than group 4. The majority of Pseudomonas spp. isolates were identified within group 2. When applying previously described niches to Staphylococcus spp., environmental species were more likely to be identified among contaminated samples, whereas host-adapted species were more likely to be identified among uncontaminated samples. These data confirm that scrubbing the teat end with alcohol after pre-milking disinfection with an iodine-based teat disinfectant and drying of the teat minimizes contamination of the milk sample.

The effect of intramammary pirlimycin hydrochloride on the fecal microbiome of early-lactation heifers.

The purpose of this study was to determine the effect of intramammary pirlimycin on the fecal microbiome of dairy cattle. Primiparous heifers were enrolled and assigned to a treatment or control group at a ratio of 2:1. In part 1 of the study, treated heifers (T1) were given intramammary pirlimycin into one infected quarter once daily for 2 d at 24-h intervals, according to the label instructions. Control heifers received no treatment. In part 2 of the study, treated heifers (T2) were given intramammary pirlimycin into one infected quarter once daily for 8 d at 24-h intervals, according to the label instructions. All enrolled heifers (T1, T2, and control) had quarter-level milk samples aseptically collected for bacterial culture and fecal samples collected for 16S rRNA gene sequencing on d 0, 2, 7, 14, 21, and 28. Milk samples were plated on Columbia blood agar and incubated at 37°C for 24 h. Bacteria were identified using MALDI-TOF mass spectrometry. The DNA was extracted from feces using PowerFecal kits (Qiagen, Venlo, the Netherlands). The 16S rRNA gene amplicon library construction and sequencing was performed at the University of Missouri DNA Core facility. Testing for differences in fecal community composition was performed via one-way permutational multivariate ANOVA of Bray-Curtis and Jaccard similarities using Past 3.13 (https://folk.uio.no/ohammer/past/). Mean total count of operational taxonomic units and Chao1, Shannon, and Simpson α-diversity indices were determined and compared via t-test or Wilcoxon rank sum test. A treatment-dependent effect was present in the observed and predicted richness of feces from cows in the T1 group at d 2 posttreatment. Additionally, intramammary pirlimycin induced a significant change in the composition of the fecal microbiota by d 2 in the treated groups. Based on calculated intra-subject similarities, intramammary pirlimycin was associated with a significant acute change in the fecal microbiota of dairy heifers and that chance reversed when the antimicrobial exposure was brief, but sustained following longer exposure. Overall, intramammary pirlimycin administration affected the fecal microbiome of lactating dairy heifers. Further work is necessary to determine the effect of these changes on the heifer and the dairy environment as well as if treatment is influencing antimicrobial resistance among enteric and environmental bacteria.

Molecular characterization of non-aureus Staphylococcus spp. from heifer intramammary infections and body sites.

The purpose of this study was to investigate non-aureus Staphylococcus spp. intramammary infections (IMI) in periparturient heifers and determine the relationship of precalving body site isolation with precalving IMI and postcalving IMI using molecular speciation and strain-typing methods. Primiparous heifers were enrolled at approximately 14 d before expected calving date. Precalving mammary quarter secretions and body site swabbing samples (teat skin, inguinal skin, muzzle, and perineum) were collected. Postcalving, mammary quarter milk samples were collected for culture and somatic cell counting. Precalving body site samples were cultured, and up to 10 staphylococcal colonies were saved for characterization. Staphylococcal isolates were speciated using matrix-assisted laser/desorption ionization time-of-flight mass spectrometry or sequencing of rpoB or tuf. Pulsed-field gel electrophoresis was used to strain type a subset of isolates. Overall, Staphylococcus chromogenes, Staphylococcus agnetis, and Staphylococcus simulans were the most common species identified in precalving mammary secretions, whereas S. chromogenes, Staphylococcus xylosus, and S. agnetis were the most common species found in postcalving milk samples. The most common species identified from body site samples were S. chromogenes, S. xylosus, and Staphylococcus haemolyticus. Mammary quarters that had a precalving mammary secretion that was culture positive for S. agnetis, S. chromogenes, or Staphylococcus devriesei had increased odds of having an IMI with the same species postcalving. A S. chromogenes IMI postcalving was associated with higher somatic cell count when compared with postcalving culture-negative quarters. Among heifers identified with a non-aureus Staphylococcus spp. IMI either precalving or postcalving, heifers that had S. agnetis or S. chromogenes isolated from their teat skin had increased odds of having the same species found in their precalving mammary secretions, and heifers with S. chromogenes, S. simulans, and S. xylosus isolated from their teat skin precalving were at increased odds of having an IMI with the same species postcalving. Overall, 44% of all heifers with a S. chromogenes IMI around the time of parturition had the same strain isolated from a body site. Based on pulsed-field gel electrophoresis, a high level of strain diversity was found.

Cross-sectional study to identify staphylococcal species isolated from teat and inguinal skin of different-aged dairy heifers.

The purpose of this study was to describe the prevalence and distribution of staphylococcal species on the teat and inguinal skin of dairy heifers across the various stages of the heifer life cycle. The cross-sectional study included 106 Holstein heifers with an age range of 0 d to 27 mo that were selected from 11 different groups, based on housing type and age, on a single dairy operation. A composite swabbing sample including all 4 teats and a second composite sample including both inguinal regions of each heifer were collected using gas-sterilized electrostatic dusters (Swiffers; Procter and Gamble, Cincinnati, OH). Swabbing samples were mixed with 10 mL of sterile saline, agitated, and cultured on mannitol salt agar plates. At 24 h, plates were read and up to 10 staphylococcal colonies were saved for further analysis. Staphylococcal isolates were speciated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or PCR amplification and partial sequencing of rpoB or tuf. The prevalence of staphylococci was compared between the inguinal and teat regions using the chi-squared or Fisher's exact test, as applicable. Logistic regression models were used to investigate the relationship between a heifer's age (treated as a quantitative continuous variable) and the probability of isolating a given staphylococcal species from a given body site (inguinal region or teats). Overall, the most common species identified were Staphylococcus haemolyticus followed by Staphylococcus chromogenes, Staphylococcus xylosus, Staphylococcus devriesei, and Staphylococcus sciuri. Staphylococcus aureus was more prevalent on the teat than in the inguinal region, whereas Staphylococcus arlettae was more prevalent in the inguinal region than on the teat. All other staphylococcal species were as likely to be found on the teat skin as the inguinal region skin. Isolation from the inguinal and teat skin was associated with age for Staphylococcus agnetis, S. chromogenes, S. devriesei, Staphylococcus equorum, S. haemolyticus, Staphylococcus lentus, S. sciuri, Staphylococcus vitulinus, and S. xylosus. The probability of finding S. chromogenes and S. agnetis on the teat and inguinal region increased with age, whereas the probability of S. devriesei and S. haemolyticus decreased with age. This study provides further insight into the ecology of staphylococcal species involved in heifer mastitis.

Species Identification and Strain Typing of Staphylococcus agnetis and Staphylococcus hyicus Isolates from Bovine Milk by Use of a Novel Multiplex PCR Assay and Pulsed-Field Gel Electrophoresis.

Staphylococcus hyicus and Staphylococcus agnetis are two coagulase-variable staphylococcal species that can be isolated from bovine milk and are difficult to differentiate. The objectives of this study were to characterize isolates of bovine milk origin from a collection that had previously been characterized as coagulase-positive S. hyicus based on phenotypic species identification methods and to develop a PCR-based method for differentiating S. hyicus, S. agnetis, and Staphylococcus aureus Isolates (n = 62) were selected from a previous study in which milk samples were collected from cows on 15 dairy herds. Isolates were coagulase tested and identified to the species level using housekeeping gene sequencing. A multiplex PCR to differentiate S. hyicus, S. agnetis, and S. aureus was developed. Pulsed-field gel electrophoresis was conducted to strain type the isolates. Based on gene sequencing, 44/62 of the isolates were determined to be either S. agnetis (n = 43) or S. hyicus (n = 1). Overall, 88% (37/42) of coagulase-positive S. agnetis isolates were found to be coagulase positive at 4 h. The herd-level prevalence of coagulase-positive S. agnetis ranged from 0 to 2.17%. Strain typing identified 23 different strains. Six strains were identified more than once and from multiple cows within the herd. Three strains were isolated from cows at more than one time point, with 41 to 264 days between samplings. These data suggest that S. agnetis is likely more prevalent on dairy farms than S. hyicus Also, some S. agnetis isolates in this study appeared to be contagious and associated with persistent infections.