The objective of this study was to validate use of the Minnesota Easy Culture System II Bi-Plate and Tri-Plate (University of Minnesota Laboratory for Udder Health, St. Paul) to identify common mastitis pathogens in milk. A total of 283 quarter and composite milk samples submitted to the University of Minnesota Laboratory for Udder Health during the spring of 2010 were cultured simultaneously using 3 methods: standard laboratory culture (reference method) and the Minnesota Easy Culture System II Bi-Plate and Tri-Plate methods. Bi-Plate and Tri-Plate cultures were incubated for 18 to 24h and interpreted by 2 independent, untrained readers within 5h of each other. An experienced technician completed the standard laboratory culture. For each sample, all 3 study personnel recorded the culture result (yes/no) for each of the following diagnostic categories: no bacterial growth (NG), mixed (2 organisms), contaminated (3 or more organisms), gram-positive (GP), gram-negative (GN), Staphylococcus spp., Streptococcus spp., Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Enterococcus spp., Staphylococcus aureus, coagulase-negative staphylococci, Escherichia coli, Klebsiella spp., and other. For each category, the prevalence, sensitivity, specificity, accuracy, and predictive values of a positive and negative test were calculated, and the agreement between readers and between each reader and the laboratory was assessed. Specificity, overall accuracy, and negative predictive values were generally high (>80%) for the Bi-Plate and Tri-Plate for each category. Sensitivity and positive predictive values were intermediate (>60%) or high (>80%) for the broad categories of NG, GP, GN, Staphylococcus spp. and Streptococcus spp., and for Staph. aureus, but were generally lower (<60%) for other more specific categories. Similarly, interreader agreement (kappa value) was moderate to substantial (40-80%) for the broad categories of NG, GP, GN, Staphylococcus spp. and Streptococcus spp., and for Staph. aureus and E. coli, but was lower for other categories. The Tri-Plate had a higher sensitivity, accuracy, and negative predictive value for Streptococcus spp., and higher interreader agreement for some of the more specific categories. Our conclusion was that Bi-Plate and Tri-Plate results will be most reliable when used to classify infections in broad diagnostic categories such NG, GP, or GN. The Bi-Plate and Tri-Plate will have intermediate ability to identify infections as being caused by Staphylococcus spp., Streptococcus spp., or Staph. aureus.

Selective dry-cow therapy (SDCT) could be used to reduce antibiotic use on commercial dairy farms in the United States but is not yet widely adopted, possibly due to concerns about the potential for negative effects on cow health. The objective of this study was to compare culture-and algorithm-guided SDCT programs with blanket dry-cow therapy (BDCT) in a multi-site, randomized, natural exposure, non-inferiority trial for the following quarter-level outcomes: antibiotic use at dry-off, dry period intramammary infection (IMI) cure risk, dry period new IMI risk, and IMI risk at 1 to 13 d in milk (DIM). Two days before planned dry-off, cows in each of 7 herds were randomly allocated to BDCT, culture-guided SDCT (cult-SDCT), or algorithm-guided SDCT (alg-SDCT). At dry-off, BDCT cows received an intramammary antibiotic (500 mg of ceftiofur hydrochloride) in all 4 quarters. Antibiotic treatments were selectively allocated to quarters of cult-SDCT cows by treating only quarters from which aseptically collected milk samples tested positive on the Minnesota Easy 4Cast plate (University of Minnesota, St. Paul, MN) after 30 to 40 h of incubation. For alg-SDCT cows, antibiotic treatments were selectively allocated at the cow level, with all quarters receiving antibiotic treatment if the cow had either a Dairy Herd Improvement Association test somatic cell count >200,000 cells/ mL during the current lactation or 2 or more clinical mastitis cases during the current lactation. All quarters of all cows were treated with an internal teat sealant. Intramammary infection status at enrollment and at 1 to 13 DIM was determined using standard bacteriological methods. The effect of treatment group on dry period IMI cure, dry period new IMI, and IMI risk at 1 to 13 DIM was determined using generalized linear mixed models (logistic), with marginal standardization to derive risk difference (RD) estimates. Quarter-level antibiotic use at dry-off for each group was BDCT (100%), cult-SDCT (45%), and alg-SDCT (45%). The crude dry period IMI cure risk for all quarters was 87.5% (818/935), the crude dry period new IMI risk was 20.1% (764/3,794), and the prevalence of IMI at 1 to 13 DIM was 23% (961/4,173). Non-inferiority analysis indicated that culture-and algorithm-guided SDCT approaches performed at least as well as BDCT for dry period IMI cure risk. In addition, the final models indicated that the risks for each of the 3 IMI measures were similar between all 3 treatment groups (i.e., RD estimates and 95% confidence intervals all close to 0). These findings indicate that under the conditions of this trial, culture-and algorithm-guided SDCT can substantially reduce antibiotic use at dry-off without negatively affecting IMI dynamics.

Bedding is an important source of teat end exposure to environmental mastitis pathogens. To better control environmental mastitis, we need an improved understanding of the relationships among bedding selection and management, bedding bacteria counts (BBC), and udder health (UH). The objectives of this crosssectional observational study were (1) to describe BBC, bedding characteristics, udder hygiene scores, bulk tank milk (BTM) quality, and UH in US dairy herds using 1 of 4 bedding materials; (2) describe the relationship between BBC and herd measures of UH; and (3) identify benchmarks for monitoring bedding hygiene. Local dairy veterinarians and university researchers enrolled and sampled 168 herds from 17 states. Herds were on a Dairy Herd Improvement Association (DHIA) testing program and used 1 of 4 bedding types for lactating cows: new sand, reclaimed sand, manure solids (MNS), or organic non-manure materials. Each herd was sampled twice (winter and summer) in 2016. Samples and data collected included unused and used bedding, BTM samples, udder hygiene scores, DHIA test data, and descriptions of facilities and herd management practices. Bedding was cultured to determine the total bacteria count and counts of Bacillus spp., coliforms, Klebsiella spp., non-coliform gram-negative organisms, streptococci or streptococci-like organisms (SSLO), and Staphylococcus spp. Bedding dry matter, organic matter, and pH were also measured. Bulk tank milk samples were cultured to determine counts of coliforms, NAS, SSLO, Staphylococcus aureus, and Mycoplasma spp. Udder health measures included DHIA test-day average linear score (LS); the proportion of cows with an intramammary infection (IMI), where infection was defined as LS ≥4.0; the proportion of cows with a new IMI, where new IMI was defined as LS changing from <4.0 to ≥4.0 in the last 2 tests; the proportion of cows with a chronic infection, where chronic was defined as LS ≥4.0 on the last 2 tests; and the cumulative incidence of clinical mastitis in the 30-d period preceding sample collection. Although much variation existed within and among bedding types, mixed linear regression showed the use of MNS bedding to be generally associated with higher BBC, dirtier udders, increased coliform and SSLO counts in BTM, and poorer UH measures compared with organic non-manure materials, reclaimed sand, or new sand bedding materials. While controlling for important farm traits and management practices, mixed linear regression showed that increased counts of coliforms, Klebsiella spp., SSLO, and Staphylococcus spp. in both unused and used bedding were associated with poorer values for 1 or more herd-level measures of UH. Achievable benchmarks identified for counts of coliforms (unused: ≤500 cfu/cm 3 ; used: ≤10,000 cfu/ cm 3), Klebsiella spp. (0 cfu/cm 3 for unused and used), Staphylococcus spp. (0 cfu/cm 3 for unused and used), and SSLO (unused: 0 cfu/cm 3 ; used: ≤500,000 cfu/ cm 3) can be used to monitor bedding hygiene in most bedding materials, with minor variations suggested...

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Study objectives were to (1) describe the diagnostic test characteristics of an automated milk leukocyte differential (MLD) test and the California Mastitis Test (CMT) to identify intramammary infection (IMI) in early- (EL) and late-lactation (LL) quarters and cows when using 3 different approaches to define IMI from milk culture, and (2) describe the repeatability of MLD test results at both the quarter and cow level. Eighty-six EL and 90 LL Holstein cows were sampled from 3 Midwest herds. Quarter milk samples were collected for a cow-side CMT test, milk culture, and MLD testing. Quarter IMI status was defined by 3 methods: culture of a single milk sample, culture of duplicate samples with parallel interpretation, and culture of duplicate samples with serial interpretation. The MLD testing was completed in duplicate within 8 h of sample collection; MLD results (positive/negative) were reported at each possible threshold setting (1-18 for EL; 1-12 for LL) and CMT results (positive/negative) were reported at each possible cut-points (trace, ≥1, ≥2, or 3). We created 2 × 2 tables to compare MLD and CMT results to milk culture, at both the quarter and cow level, when using each of 3 different definitions of IMI as the referent test. Paired MLD test results were compared with evaluate repeatability. The MLD test showed excellent repeatability. The choice of definition of IMI from milk culture had minor effects on estimates of MLD and CMT test characteristics. For EL samples, when interpreting MLD and CMT results at the quarter level, and regardless of the referent test used, both tests had low sensitivity (MLD = 11.7-39.1%; CMT = 0-52.2%) but good to very good specificity (MLD = 82.1-95.2%; CMT = 68.1-100%), depending on the cut-point used. Sensitivity improved slightly if diagnosis was interpreted at the cow level (MLD = 25.6-56.4%; CMT = 0-72.2%), though specificity generally declined (MLD = 61.8-100%; CMT = 25.0-100%) depending on the cut-point used. For LL samples, when interpreted at the quarter level, both tests had variable sensitivity (MLD = 46.6-84.8%; CMT = 9.6-72.7%) and variable specificity (MLD = 59.2-79.8%; CMT = 52.5-97.3%), depending on the cut-point used. Test sensitivity improved if interpreted at the cow level (MLD = 59.6-86.4%; CMT = 19.1-86.4%), though specificity declined (MLD = 32.4-56.8%; CMT = 14.3-92.3%). Producers considering adopting either test for LL or EL screening programs will need to carefully consider the goals and priorities of the program (e.g., whether to prioritize test sensitivity or specificity) when deciding on the level of interpretation (quarter or cow) and when selecting the optimal cut-point for interpreting test results. Additional validation studies and large randomized field studies will be needed to evaluate the effect of adopting either test in selective dry cow therapy or fresh cow screening programs on udder health, antibiotic use, and economics.