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Abstract The objective of this study was to evaluate the impact of dietary zinc supplementation in pre-weaned dairy calves on the phenotypic antimicrobial resistance (AMR) of fecal commensal bacteria. A repository of fecal specimens from a random sample of calves block-randomized into placebo (n = 39) and zinc sulfate (n = 28) groups collected over a zinc supplementation clinical trial at the onset of calf diarrhea, calf diarrheal cure, and the last day of 14 cumulative days of zinc or placebo treatment were analyzed. Antimicrobial susceptibility testing was conducted for Enterococcus spp. (n = 167) and E. coli (n = 44), with one representative isolate of each commensal bacteria tested per sample. Parametric survival interval regression models were constructed to evaluate the association between zinc treatment and phenotypic AMR, with exponentiated accelerated failure time (AFT) coefficients adapted for MIC instead of time representing the degree of change in AMR (MIC Ratio, MR). Findings from our study indicated that zinc supplementation did not significantly alter the MIC in Enterococcus spp. for 13 drugs: gentamicin, vancomycin, ciprofloxacin, erythromycin, penicillin, nitrofurantoin, linezolid, quinupristin/dalfopristin, tylosin tartrate, streptomycin, daptomycin, chloramphenicol, and tigecycline (MR = 0.96–2.94, p > 0.05). In E. coli, zinc supplementation was not associated with resistance to azithromycin (MR = 0.80, p > 0.05) and ceftriaxone (MR = 0.95, p > 0.05). However, a significant reduction in E. coli MIC values was observed for ciprofloxacin (MR = 0.17, 95% CI 0.03–0.97) and nalidixic acid (MR = 0.28, 95% CI 0.15–0.53) for zinc-treated compared to placebo-treated calves. Alongside predictions of MIC values generated from these 17 AFT models, findings from this study corroborate the influence of age and antimicrobial exposure on phenotypic AMR.

The aim of this study was to evaluate the effect of dry cow therapy (DCT) on the antimicrobial resistance (AMR) profile of mastitis pathogens post-calving. A repository of isolates based on a DCT trial was utilized for the current study. A stratified random survey sample of cows from the trial were identified within the strata of season, herd, and trial treatment resulting in 382 cows. All isolates from the 382 cows were selected for the current study, which identified 566 isolates from milk samples collected at dry off (S1), post-calving (S2), and at the first clinical mastitis event up to 150 days in milk (S3). The AMR profiles were determined using broth microdilution method. Less than 10% of the coagulase-negative Staphylococcus species (CNS) isolates (n = 421) were resistant to tetracycline, ceftiofur, penicillin/novobiocin or erythromycin, while higher proportions of resistance to sulfadimethoxine (72%) and penicillin (28%) were observed. All Staphylococcus aureus (S. aureus) isolates (n = 4) were susceptible to all tested AMD except sulfadimethoxine, to which all isolates were resistant. Similarly, all Streptococcus spp. (n = 37) were susceptible to penicillin, penicillin/novobiocin, and ampicillin while resistant to tetracycline (17%). All coliforms (n = 21) were susceptible to ceftiofur, but resistance was recorded for sulfadimethoxine (70%), cephalothin (56%), and tetracycline (43%). The increased resistance percent from S1 to S2 was observed in CNS isolates from AMD-treated cows, with the highest increase recorded for penicillin (12.2%). Parametric survival interval regression models were used to explore the association between antimicrobial drug (AMD) therapy at dry off and the AMR phenotype post-calving. The accelerated failure-time metric was adopted to minimum inhibitory concentration measurements to permit interpretation of model exponentiated coefficients. Models for cows with CNS isolated at both S1 and S2 showed increased resistance against cephalothin, oxacillin, and ceftiofur in cows that received DCT from the same drug class, or a class with a shared resistance mechanism. In contrast, resistance of CNS isolates to tetracycline were associated with any AMD therapy at dry off. Resistance of CNS isolates to Penicillin decreased in CNS isolates in cows that received any AMD therapy at dry off compared to those that didn't. The study provided evidence that dry-cow IMM AMD was associated with AMR post-calving.

Weaned dairy heifers are a relatively understudied production group. Bovine respiratory disease (BRD) is the most common cause of antimicrobial drug (AMD) use, morbidity, and mortality in this production group. The study of antimicrobial resistance (AMR) is complicated because many variables that may affect AMR are related. This study generates hypotheses regarding the farm- and animal-level variables (e.g., vaccination, lane cleaning, and AMD use practices) that may be associated with AMR in respiratory isolates from weaned dairy heifers. A cross-sectional study was performed using survey data and respiratory isolates (Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni) collected from 341 weaned dairy heifers on six farms in California. Logistic regression and Bayesian network analyses were used to evaluate the associations between farm- and animal-level variables with minimum inhibitory concentration (MIC) classification of respiratory isolates against 11 AMDs. Farm-level variables associated with MIC classification of respiratory isolates included the number of source farms of a calf-rearing facility, whether the farm practiced onsite milking, the use of lagoon water for flush lane cleaning, and respiratory and pinkeye vaccination practices. Animal-level variables associated with a MIC classification included whether the calf was BRD-score-positive and time since the last phenicol treatment.

Abstract The goals of this study were to evaluate factors affecting recovery and antimicrobial resistance (AMR) in intrauterine E. coli in post-partum dairy cows with and without metritis from commercial California dairy farms. Using a cross-sectional study design, a total of 307 cows were sampled from 25 farms throughout California, from which a total of 162 intrauterine E. coli isolates were recovered. During farm visits, cows within 21 days post-partum were categorized in one of three clinical presentation groups before enrollment: metritis (MET, n = 86), defined as a cow with watery, red or brown colored, and fetid vaginal discharge; cows with purulent discharge (PUS, n = 106), defined as a non-fetid purulent or mucopurulent vaginal discharge; and control cows, (CTL, n = 115) defined as cows with either no vaginal discharge or a clear, non-purulent mucus vaginal discharge. Cows diagnosed as MET had significantly higher odds for recovery of E. coli compared to cows diagnosed as CTL (OR = 2.16, 95% CI: 1.17–3.96), with no significant difference observed between PUS and CTL, and PUS and MET. An increase in days in milk (DIM) at the time of sampling was significantly associated with a decrease in the odds ratio for E. coli recovery from intrauterine swabs (OR = 0.94, 95% CI: 0.89–0.98). All intrauterine E. coli were resistant to ampicillin (AMP), with an AMR prevalence of 30.2% and 33.9% observed for chlortetracycline and oxytetracycline, respectively. Only 8.6% of isolates were resistant to ceftiofur (CEFT), one of the most common drugs used to treat cows on farms sampled. No significant difference in the prevalence of AMR was observed among clinical groups at the individual cow level. At the farm level, a significantly higher odds for isolating intrauterine E. coli resistant to chlortetracycline (OR: 2.6; 95% CI: 3.7–58.0) or oxytetracycline (OR: 1.9; 95% CI: 1.4–33.8) was observed at farms that used an intrauterine infusion of oxytetracycline as a treatment for metritis when compared to those farms that did not use this practice. Findings from this study indicate the need for further research supporting a broader understanding of farm practices driving AMR in cows with metritis, as well as data to increase the accuracy of breakpoints for AMR classification of intrauterine E. coli from cattle.

ABSTRACT: Our objectives were to evaluate the diagnostic accuracy of a rapid and novel immunochromatography-based mastitis kit that includes 3 independent tests to detect coliforms (Escherichia coli or Klebsiella pneumoniae), Streptococcus spp., and Staphylococcus aureus. The kit was developed to facilitate diagnostic-based mastitis treatment. Validation of the kit was based on 154 aseptically collected mastitis samples from 2 clinical herds (clinical population) and 120 milk samples from 3 nonclinical herds (nonclinical population) without clinical cases at the time of enrollment. One herd sampled at different times was common to both populations. A 3-test in 2-population Bayesian latent class model with uniform priors for all test parameters except specificity of culture, which was modeled informatively, was used to estimate sensitivity (Se) and specificity (Sp) of the test kit, culture, and PCR at the cow level. The mastitis test kit's 96.9% Sp for Streptococcus spp. had a low false positive percentage (3.1%), which, together with the kit's rapid turnaround time for results, makes it a suitable initial screening test that producers can use to identify clinical cows to treat based on Streptococcus spp. mastitis in kit-positive results. Due to the 60.4% kit Se, producers should follow up on Streptococcus spp. kit-negative cows using a confirmatory test such as PCR (Sp of 98.4%) or culture (Sp of 99.6%). In contrast, aerobic culture had Se of 76.5% and Sp of 99.6% for Streptococcus spp. Similarly, the Sp of the kit (98.2%) and culture (99.8%) for Staph. aureus were particularly high, and even though the kit's Se (61.0%) was lower than culture (88.4%; posterior probability of difference 98%), the kit could be beneficial before use of a confirmatory test for kit-negative samples due to its ease and rapid turnaround time. Mostly, quantitative real-time (q)PCR outperformed the kit's Se (37.7%) and Sp (92.9%) for coliforms, as well as the kit's Se (60.4%) for Streptococcus spp. However, qPCR may require more technical skills and turnaround time for final results. Use of the on-farm mastitis test kit evaluated in the present study could enhance sustainable antimicrobial drug use by rapidly identifying Streptococcus mastitis for targeted treatment. Furthermore, the kit may be used in a Staph. aureus outbreak where cows can be rapidly screened to identify cases for segregation or culling during an outbreak and kit-negative cows further confirmed by milk culture or qPCR. However, the cost-effectiveness of such an approach has not been investigated.

Abstract Background Microbiota play important roles in the gastrointestinal tract (GIT) of dairy cattle as the communities are responsible for host health, growth, and production performance. However, a systematic characterization and comparison of microbial communities in the GIT of cattle housed in different management units on a modern dairy farm are still lacking. We used 16S rRNA gene sequencing to evaluate the fecal bacterial communities of 90 dairy cattle housed in 12 distinctly defined management units on a modern dairy farm. Results We found that cattle from management units 5, 6, 8, and 9 had similar bacterial communities while the other units showed varying levels of differences. Hutch calves had a dramatically different bacterial community than adult cattle, with at least 10 genera exclusively detected in their samples but not in non-neonatal cattle. Moreover, we compared fecal bacteria of cattle from every pair of the management units and detailed the number and relative abundance of the significantly differential genera. Lastly, we identified 181 pairs of strongly correlated taxa in the community, showing possible synergistic or antagonistic relationships. Conclusions This study assesses the fecal microbiota of cattle from 12 distinctly defined management units along the production line on a California dairy farm. The results highlight the similarities and differences of fecal microbiota between cattle from each pair of the management units. Especially, the data indicate that the newborn calves host very different gut bacterial communities than non-neonatal cattle, while non-neonatal cattle adopt one of the two distinct types of gut bacterial communities with subtle differences among the management units. The gut microbial communities of dairy cattle change dramatically in bacterial abundances at different taxonomic levels along the production line. The findings provide a reference for research and practice in modern dairy farm management.

The objective of this study was to identify alterations in the vaginal discharge (VD) metabolome and potential biomarkers to predict metritis development and a cure in dairy cows. This prospective cohort study was conducted on two dairies located in CA and TX. Vaginal discharge was evaluated and collected using the Metricheck® device. Cows were examined for metritis at 4, 7, and 9 days in milk (DIM). Cows with a fetid, watery, and reddish-brown uterine discharge were classified as having metritis and randomized to receive ceftiofur (n = 10) or remain untreated (n = 7). A cure was defined as the absence of a fetid, watery, reddish-brown uterine discharge at 14 d after enrollment. Vaginal discharge samples were collected from 86 cows within 6 h after parturition, at 4 and 7 DIM, at metritis diagnosis, and at 4 and 7 days after metritis diagnosis. Cows with metritis (MET; n = 17) were paired with counterparts without metritis (HTH) of a similar DIM and parity (n = 34). The uterine metabolome was evaluated using untargeted gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). Metabolomic data were analyzed using the MetaboAnalyst 5.0. Data were log-transformed and auto-scaled for normalization. Univariate analyses, including the fold-change, were performed to identify the metabolites linked to metritis development and its cure and principal component analysis and partial least squares discriminant analysis were performed to explain metabolite variance between animals developing or not developing metritis and being cured or not being cured of metritis. Comparing HTH with MET cows at calving, 12 metabolites were upregulated, and one was downregulated. At four and seven DIM, 51 and 74 metabolites, respectively, were altered between MET and HTH cows. After metritis development, three and five metabolites were upregulated in cows that were cured and in cows that received treatment and were cured, respectively. In all scenarios, the metabolites lignoceric, malic, and maleic acids, ornithine, and hypotaurine, which are associated with arginine/aminoacyl-tRNA biosynthesis and taurine/purine metabolism, were upregulated in HTH cows. Metritis was associated with changes in the uterine metabolome. Cows not being cured of metritis had changes in the uterus metabolome independent of receiving ceftiofur or remaining untreated. Metabolome analysis may be an important tool to understand the vaginal discharge changes during postpartum and the dynamics of metritis development and cures and help to identify biomarkers to predict metritis being cured.

The objectives of this study were to investigate the effects of group housing (three calves per group) on bovine respiratory disease (BRD), diarrhea and antimicrobial resistance (AMR) to fecal commensal Escherichia coli (EC) and enterococci/streptococci (ES). Our study comprised two arms, one experimental and one observational. In the experimental arm, preweaned calves on a California dairy were randomized to either individual (IND; n = 21) or group (GRP; n = 21) housing, using a modified California-style wooden hutch. The study period lasted from birth to 56 days of age, during which calves were health scored daily. Cumulative incidence and hazard ratios were estimated for disease. Antimicrobial resistance outcomes were assessed using a prospective cohort design; feces were collected from each calf three times per week and EC and ES were evaluated for AMR using the broth microdilution method against a panel of 19 antimicrobial drugs (AMD). Analysis of treatment records was used to select calves that had been exposed (EXP) to an AMD-treated calf. In GRP, exposure occurred when a calf was a hutchmate with an AMD-treated calf. In IND, exposure occurred when a calf was a neighbor with an AMD-treated calf (TRT). Age-matched unexposed calves (UNEXP) were then selected for comparison. Proportions of AMR in fecal commensals among EXP, UNEXP, and TRT calves were compared between GRP and IND. Accelerated failure time survival regression models were specified to compare differences in minimum inhibitory concentration (MIC) of fecal commensals between EXP and UNEXP calves within each of GRP and IND calves separately. Group calves had a BRD hazard 1.94 times greater that of IND calves (p = 0.03), using BRD treatment records as the outcome. For AMR in EC isolates, higher resistance to enrofloxacin was detected in enrofloxacin-EXP GRP isolates compared with enrofloxacin-EXP IND isolates, and UNEXP GRP calves had lower resistance to ceftiofur compared with enrofloxacin-EXP and enrofloxacin-TRT calves. A significant housing-by-time interaction was detected for EC ceftiofur MIC in EXP GRP calves at 4–14 days post exposure to enrofloxacin (MIC EXP-UNEXP: µg/mL (95% CI): 10.62 (1.17, 20.07)), compared with UNEXP calves. The findings of this study show an increase in BRD hazard for group-housed calves and an increase in ceftiofur resistance in group-housed calves exposed to an enrofloxacin-treated calf.

Mastitis is the greatest disease challenge for dairy producers, with substantial economic impacts due to lost milk production. Amongst the approaches implemented to control and prevent mastitis on dairies are vaccination, pre- and post-milking teat dips, and treatments at dry-off including intramammary antibiotics and teat sealants. The objectives of our study were to evaluate the effect of different treatments at dry-off on the subsequent lactation’s milk production and somatic cell count (SCC). A single-blinded controlled block randomized clinical trial was conducted between December 2016 and August 2018 on eight herds from four of the top ten milk-producing counties in California: Tulare, Kings, Stanislaus, and San Joaquin. The trial was repeated with cows enrolled during the winter and summer seasons to account for seasonal variability. Eligible cows were treated at dry-off with either intramammary antibiotics (AB), internal teat sealant (TS), AB + TS, or did not receive any treatment (control), and were followed through 150 days in milk (DIM) post-calving. The milk production and SCC data were extracted from monthly test day milk records (Dairy Comp 305, Valley Ag Software, Tulare, CA, USA). Two-piece spline linear mixed models were used to model the milk production (kg) and natural logarithm-transformed SCC. After accounting for parity, breed, season, and dry period duration, the milk model showed a significant increase in milk production (1.84 kg/day) in cows treated with AB + TS at dry-off in comparison to controls. There was no significant difference in the milk produced by cows that received either AB or TS (0.12 kg/day, and 0.67 kg/day, respectively) in comparison to the untreated cows. Different dry cow treatments were associated with a significant reduction in ln SCC during the first 150 DIM. The greatest reduction was associated with using AB + TS, followed by AB, and finally TS in comparison to controls. Dairies with high SCC may benefit from treating cows at dry-off with AB, TS, or both.

Background Antimicrobial drugs (AMD) are critical for the treatment, control, and prevention of diseases in humans and food-animals. Good AMD stewardship practices and judicious use of AMD are beneficial to the preservation of animal and human health from antimicrobial resistance threat. This study reports on changes in AMD use and stewardship practices on California (CA) dairies, following the implementation of CA Senate Bill 27 (SB 27; codified as Food and Agricultural Code, FAC 14400–14408; here onward referred to as SB 27), by modeling the associations between management practices on CA conventional dairies and seven outcome variables relating to AMD use and stewardship practices following SB 27. Methods A survey questionnaire was mailed to 1,282 grade A licensed dairies in CA in spring of 2018. Responses from 132 conventional dairies from 16 counties were included for analyses. Multivariate logistic regression models were specified to explore the associations between survey factors and six outcome variables: producers’ familiarity with the Food and Drug Administration’s (FDA), Silver Spring, WA, USA medically important antimicrobial drugs (MIAD) term; change in over-the-counter (OTC) AMD use; initiation or increased use of alternatives to AMD; changes to prevent disease outbreaks; changes in AMD costs; and better animal health post SB 27. We employed machine learning classification models to determine which of the survey factors were the most important predictors of good-excellent AMD stewardship practices of CA conventional dairy producers. Results Having a valid veterinary-client-patient-relationship, involving a veterinarian in training employees on treatment protocols and decisions on AMDs used to treat sick cows, tracking milk and/or meat withdrawal intervals for treated cows, and participating in dairy quality assurance programs were positively associated with producers’ familiarity with MIADs. Use or increased use of alternatives to AMDs since 2018 was associated with decreased use of AMDs that were previously available OTC prior to SB 27. Important variables associated with good-excellent AMD stewardship knowledge by CA conventional dairy producers included having written or computerized animal health protocols, keeping a drug inventory log, awareness that use of MIADs required a prescription following implementation of SB 27, involving a veterinarian in AMD treatment duration determination, and using selective dry cow treatment. Conclusions Our study identified management factors associated with reported AMD use and antimicrobial stewardship practices on conventional dairies in CA within a year from implementation of SB 27. Producers will benefit from extension outreach efforts that incorporate the findings of this survey by further highlighting the significance of these management practices and encouraging those that are associated with judicious AMD use and stewardship practices on CA conventional dairies.

Background A survey of California (CA) dairies was performed in spring 2018 to characterize antimicrobial stewardship practices, antimicrobial drug (AMD) use, and health management of adult cows on CA dairies since the implementation of the Veterinary Feed Directive (VFD) and the CA Senate Bill 27 (SB 27). Effective January 1, 2017, the U.S. Food and Drug Administration (FDA) implemented regulatory changes requiring veterinary oversight for therapeutic uses of medically-important antimicrobial drugs (MIADs) administered in feed (VFD) and water (veterinary prescription). Similarly, effective January 1, 2018, the CA legislature enacted California Food and Agricultural Code (FAC) 14400–14408, formerly known as Senate Bill 27 (SB 27) requiring veterinary prescriptions for all other dosage forms of MIADs. Methods The questionnaire consisted of 43 questions partitioned into three sections to assess herd information, management practices, and AMD use and perspectives. The questionnaire was mailed to 1,282 grade A licensed dairies in CA and 149 responses (11.6%) were collected from 19 counties across the three defined regions of CA: Northern CA (NCA), Northern San Joaquin Valley (NSJV), and Greater Southern CA (GSCA). Results Most dairies reported treating all dry cows with intramammary AMD and/or teat sealant at the end of a lactation (87.2%). In 92.3% of dairies, producers relied on the veterinarian for information about AMD used to treat cows. Treatment duration for cows treated with AMD was based on the drug manufacturer’s label and veterinarian’s instructions in most dairies (98.6%). Most respondents to the survey confirmed having a valid veterinarian-client-patient-relationship (VCPR) for their dairies (91.7%), participated in animal welfare audit programs (81.8%) and dairy quality assurance programs (52.9%). Approximately 98.6% respondents were aware that all uses of MIADs in livestock required a veterinary feed directive (VFD) or prescription and are no longer sold over-the-counter (OTC) in CA since January 1, 2018. Multiple factor analysis (MFA) was performed and identified seven components composed of 21 variables (questions) that explained 99.7% of the total variance in the data. Hierarchical cluster analysis on the principal coordinates of the MFA based on conventional dairy survey responses identified two clusters characterized as large conventional dairies (median herd size: 1,265 cows) and mid-sized conventional dairies (median herd size: 715 cows) mostly in GSCA and NSJV. The organic dairies grouped into a single cluster of median herd size of 325 cows mostly in NCA. Conclusions The survey results contribute to the knowledge of AMD use and antimicrobial stewardship practices on CA dairies since the implementation of the SB 27 and VFD laws and provide useful information for future evaluation of resistance-related risk in adult cows.

Background Understanding the effects of herd management practices on the prevalence of multidrug-resistant pathogenic Salmonella and commensals Enterococcus spp. and Escherichia coli in dairy cattle is key in reducing antibacterial resistant infections in humans originating from food animals. Our objective was to explore the herd and cow level features associated with the multi-drug resistant, and resistance phenotypes shared between Salmonella, E. coli and Enterococcus spp. using machine learning algorithms. Methods Randomly collected fecal samples from cull dairy cows from six dairy farms in central California were tested for multi-drug resistance phenotypes of Salmonella, E. coli and Enterococcus spp. Using data on herd management practices collected from a questionnaire, we built three machine learning algorithms (decision tree classifier, random forest, and gradient boosting decision trees) to predict the cows shedding multidrug-resistant Salmonella and commensal bacteria. Results The decision tree classifier identified rolling herd average milk production as an important feature for predicting fecal shedding of multi-drug resistance in Salmonella or commensal bacteria. The number of culled animals, monthly culling frequency and percentage, herd size, and proportion of Holstein cows in the herd were found to be influential herd characteristics predicting fecal shedding of multidrug-resistant phenotypes based on random forest models for Salmonella and commensal bacteria. Gradient boosting models showed that higher culling frequency and monthly culling percentages were associated with fecal shedding of multidrug resistant Salmonella or commensal bacteria. In contrast, an overall increase in the number of culled animals on a culling day showed a negative trend with classifying a cow as shedding multidrug-resistant bacteria. Increasing rolling herd average milk production and spring season were positively associated with fecal shedding of multidrug- resistant Salmonella. Only six individual cows were detected sharing tetracycline resistance phenotypes between Salmonella and either of the commensal bacteria. Discussion Percent culled and culling rate reflect the increase in culling over time adjusting for herd size and were associated with shedding multidrug resistant bacteria. In contrast, number culled was negatively associated with shedding multidrug resistant bacteria which may reflect producer decisions to prioritize the culling of otherwise healthy but low-producing cows based on milk or beef prices (with respect to dairy beef), amongst other factors. Using a data-driven suite of machine learning algorithms we identified generalizable and distant associations between antimicrobial resistance in Salmonella and fecal commensal bacteria, that can help develop a producer-friendly and data-informed risk assessment tool to reduce shedding of multidrug-resistant bacteria in cull dairy cows.

Intramammary antibiotic (AB) and internal teat sealants (TS) infusion at dry-off have been used to prevent intramammary infections (IMI) in dairy cows during the dry period and reduce the risk of mastitis during the dry period and subsequent lactation. A randomized clinal trial was completed on eight California dairy herds to estimate the effects of different dry cow therapies (AB, TS, AB + TS or None) on clinical mastitis and culling. A total of 1273 cows were randomized to one of the four treatment groups over summer and winter seasons. For each enrolled cow, microbiological testing was done on quarter milk samples collected from the first detection of clinical mastitis within the first 150 days in milk (DIM) in the subsequent lactation. Statistical analysis was done using generalized linear mixed models. There were no significant differences in the odds of clinical mastitis or culling between cows treated with AB, TS, or AB + TS compared to the controls. Dry cow therapy with AB and/or TS had no statistically significant effect on clinical mastitis and cow culling during the first 150 DIM.

The California (CA) dairy industry was surveyed in July 2017 to evaluate producers' knowledge and perceptions and antimicrobial drug (AMD) use in preweaned dairy calves following the implementation of the nationwide veterinary feed directive final rule (VFD) in January 2017 and prior to statewide implementation of CA Senate Bill (SB) 27 in January 2018. Together, these regulations require veterinary oversight for all uses of medically important antimicrobial drugs (MIADs) administered to livestock in CA. Survey questionnaire was mailed to 1,361 CA Grade A milk producing dairies and calf ranches across CA resulting in a 12% (169) response. Most respondents (83%) were aware of the VFD and SB 27 changes. Use of antibiotics was perceived as important (77%) in raising preweaned dairy calves and judicious use of antibiotics was ranked as the most important antimicrobial stewardship practice, amongst record keeping, observing withdrawal periods, having a valid Veterinarian-Client-Patient-Relationship (VCPR), and use of alternatives to antibiotics. Treating sick calves was the major indication for AMD use (90.5%); however, few producers reported use of antibiotics to control (12.7%) or prevent disease (11%). Neomycin sulfate, chlortetracycline, oxytetracycline and sulfamethazine were the most used AMD. The respondents reported a decreased use of AMD in milk (10%) and in solid feed (5%), and discontinuation of one or more AMDs used in milk (18.6%) or in solid feed (5%) post-VFD rule implementation in 2017. Most respondents reported keeping treatment records and the information recorded included date (82%), dose (44%) and route (15%) of AMD used. A few respondents reported they had initiated use of alternatives to AMDs, such as vitamins (32.6%), minerals (25.6%), herbal remedies (11.6%) and pathogen specific antibodies (7%), post-VFD. The limited changes noted in AMD use could be attributed to the short period between the implementation of the VFD and the time of the survey. Our study outcomes identified opportunities to improve AMD use practices, including record keeping and use of AMD alternatives, and provides a baseline for future evaluation of the impact of these regulatory changes, as well as guidance for the future recommendations on best practices to promote judicious AMD use.

Background This study describes the occurrence of antimicrobial resistance (AMR) in commensal Escherichia coli and Enterococcus/Streptococcus spp. (ES) isolated from fecal samples of dairy cows and assesses the variation of AMR profiles across regions and seasons following the implementation of the Food and Agricultural Code (FAC) Sections 14400–14408 (formerly known as Senate Bill, SB 27) in California (CA). Methods The study was conducted on ten dairies distributed across CA’s three milk sheds: Northern California (NCA), Northern San Joaquin Valley (NSJV), and the Greater Southern California (GSCA). On each study dairy, individual fecal samples were collected from two cohorts of lactating dairy cows during the fall/winter 2018 and spring/summer 2019 seasons. Each cohort comprised of 12 cows per dairy. The fecal samples were collected at enrollment before calving (close-up stage) and then monthly thereafter for four consecutive time points up to 120 days in milk. A total of 2,171 E. coli and 2,158 ES isolates were tested for antimicrobial susceptibility using the broth microdilution method against a select panel of antimicrobials. Results The E. coli isolates showed high resistance to florfenicol (83.31% ± 0.80) and sulphadimethoxine (32.45%), while resistance to ampicillin (1.10% ± 0.21), ceftiofur (1.93% ± 0.29), danofloxacin (4.01% ± 0.42), enrofloxacin (3.31% ± 0.38), gentamicin (0.32% ± 0.12) and neomycin (1.61% ± 0.27) had low resistance proportions. The ES isolates were highly resistant to tildipirosin (50.18% ± 1.10), tilmicosin (48% ± 1.10), tiamulin (42%) and florfenicol (46% ± 1.10), but were minimally resistant to ampicillin (0.23%) and penicillin (0.20%). Multidrug resistance (MDR) (resistance to at least 1 drug in ≥3 antimicrobial classes) was observed in 14.14% of E. coli isolates and 39% of ES isolates. Escherichia coli isolates recovered during winter showed higher MDR prevalence compared to summer isolates (20.33% vs. 8.04%). A higher prevalence of MDR was observed in NSJV (17.29%) and GSCA (15.34%) compared with NCA (10.10%). Conclusions Our findings showed high rates of AMR to several drugs that are not labeled for use in lactating dairy cattle 20 months of age or older. Conversely, very low resistance was observed for drugs labeled for use in adult dairy cows, such as cephalosporins and penicillin. Overall, our findings identified important differences in AMR by antimicrobial class, region and season.

Epidemiological studies of low prevalence disease problems are often hindered by the high cost of diagnostic testing. The objective of this study was to evaluate PCR screening of both individual and pooled fecal samples from culled dairy cows for the invA gene of Salmonella followed by culture to determine if the sensitivity and specificity were comparable to the results from traditional culture methods applied to individual samples. Cows from six different dairies were sampled in all four seasons. A total of 240 individual cow fecal samples, 24 fecal pools and 24 pools of 24-hour tetrathionate enrichment broth were tested. Diagnostic sensitivity of PCR screening followed by culture of PCR positive or indeterminate samples (i.e PCR-CUL method) was lower than that of culture (CUL) when applied to individual fecal samples (94.8%, 99.5%), however the specificity was comparable (99.6% and 97.7% respectively). For pools of five fecal samples and pools of five, 24 h tetrathionate broth samples, the specificity of both tests were comparable (∼98%); however, their sensitivity was only comparable in pooled fecal samples (∼93%) but greater for culture compared to PCR-CUL in pooled broth samples (∼99% versus ∼93%). Compared to culture results from testing of individual fecal samples, testing pooled fecal samples by culture had a relative sensitivity of 74% and relative specificity of 96%, testing pooled fecal samples by PCR-CUL resulted in relative sensitivity of 90% and relative specificity of 96%. Testing of pooled 24-hour enrichment broth by PCR-CUL increased the relative sensitivity and specificity to 100%. PCR testing followed by culture of positive or indeterminate samples is a time saving alternative to traditional methods. In addition, pooling of samples may be a useful method for decreasing cost if study aims can accommodate a moderate loss of relative sensitivity.

Background: The current study is aimed at identifying the factors associated with antimicrobial drug (AMD) use and stewardship practices on conventional California (CA) dairies a year after CA Senate Bill 27. Methods: Responses from 113 out of 1282 dairies mailed a questionnaire in 2019 were analyzed to estimate the associations between management practices and six outcomes including producer familiarity with medically important antimicrobial drugs (MIADs), restricted use of MIADs previously available over the counter (OTC), use of alternatives to AMD, changes in on-farm management practices, changes in AMD costs, and animal health status in dairies. Results: Producers who reported having a veterinarian–client–patient relationship (VCPR) and tracking AMD withdrawal intervals had greater odds of being familiar with the MIADs. Producers who began or increased the use of preventive alternatives to AMD in 2019 had higher odds (OR = 3.23, p = 0.04) of decreased use of MIADs previously available OTC compared to those who did not. Changes in management practices to prevent disease outbreak and the use of diagnostics to guide treatment were associated with producer-reported improved animal health. In addition, our study identified record keeping (associated with familiarity with MIADs), use of alternatives to AMD (associated with management changes to prevent diseases and decreased AMD costs), and use of diagnostics in treatment decisions (associated with reported better animal health) as factors associated with AMD stewardship. Conclusions: Our survey findings can be incorporated in outreach education materials to promote antimicrobial stewardship practices in dairies.

The objective of this study was to investigate the effect of the antimicrobial drugs (AMD) on the shedding of resistant Enterobacteriaceae in feces of pre-weaned dairy calves. The AMD considered were ceftiofur, administered parenterally, and neomycin sulfate added in milk replacer and fed to calves during the first 20 days of life. Fifty-five calves, aged one to three days, were enrolled and followed to 64 days. Fecal samples were collected three times/week and treatments recorded daily. Enterobacteriaceae were quantified for a subset of 33 calves using spiral plating on plain, ceftiofur supplemented, and neomycin supplemented MacConkey agar. Negative binomial models were used to predict the association between treatment with AMD and the gain and loss of Enterobacteriaceae resistance over time. Acquisition of resistance by the Enterobacteriaceae occurred during treatment and peaked between days three to four post-treatment before decreasing to below treatment levels at days seven to eight post-treatment. Acquisition of neomycin resistance was observed on the first sampling day (day four from the start of feeding medicated milk replacer) to day eight, followed by cyclical peaks until day 29, when the Enterobacteriaceae counts decreased below pre-treatment. Enterobacteriaceae resistance against both AMD increased after AMD administration and didn’t return to pre-therapeutic status until seven or more days after therapy had been discontinued. The study findings provide valuable insights into the dynamics of Enterobacteriaceae under routine AMD use in calves.

Antimicrobial resistance (AMR) is a global issue for both human and animal health. Antimicrobial drug (AMD) use in animals can contribute to the emergence of AMR. In January 2018, California (CA) implemented legislation (Senate Bill 27; SB 27) requiring veterinary prescriptions for medically important AMD use in food animals. The objective of our survey was to characterize AMD use, health management, and AMD stewardship practices of adult cows on CA dairies since the implementation of SB 27. In 2019, we mailed a questionnaire to 1282 California dairies. We received a total of 131 (10.2%) survey responses from 19 counties in CA. Our results showed that 45.6% of respondents included a veterinarian in their decision on which injectable AMD to purchase. Additionally, 48.8% of dairy producers included a veterinarian in their decision on which AMDs were used to treat sick cows. The majority (96.8%) of dairy producers were aware that all uses of medically important AMDs require a prescription. Approximately 49% of respondents agreed or strongly agreed that AMD use in livestock does not cause problems in humans. The survey documents antimicrobial use and stewardship practices in CA’s dairy industry and focus areas for future research and education.

Background In this study cull dairy cows from six California dairy herds were sampled seasonally over the course of a year. The objectives were to determine the prevalence of antimicrobial resistant (AMR) Salmonella spp. shed in cull cow feces, and the factors associated with fecal shedding of AMR and multidrug resistant (MDR) Salmonella. Methods Six dairy farms located in the San Joaquin Valley of California were identified and enrolled as a convenience sample. On each dairy, and once during each of the four seasons, 10 cull cows were randomly selected for fecal sampling on the day of their removal from the herd. In addition, study personnel completed a survey based on responses of the herd manager to questions related to the previous 4 month’s herd management and the specific cattle sampled. Fecal samples were submitted to the California Animal Health and Food Safety laboratory for Salmonella isolation. Antimicrobial resistance was evaluated using broth microdilution method and a gram-negative assay plate following Clinical Laboratory Standards Institute (CLSI) guidelines and breakpoint references. All statistical models were survey adjusted for number of animals on sampling day. Results A total of 62 Salmonella were isolated from 60 of the 239 fecal samples collected. For 12% (95% confidence interval (CI) [3–20]) of fecal samples a multidrug resistant Salmonella was isolated. The survey-weighted results for the two most common drug classes for which isolates were resistant were tetracycline (39%; 95% CI [27–51]) and ampicillin (18%; 95% CI [9–27]). An important finding was the identification of cephalosporin as the third most common drug class for which isolates were resistant, with ceftriaxone (10%; 95% CI [2–17]) being the most common drug associated with resistance in that class. At the cow-level, reason for culling, prior treatment with antimicrobial drugs as the reason for culling was associated with higher odds of isolating an AMR Salmonella isolate. At the herd-level, percent of animals monthly culled on the farm as well as number of milking cows in the herd were associated with isolation of antimicrobial resistant Salmonella in cull cows. Discussion Salmonella isolated from fecal samples from cull cows were resistant to important antimicrobials, such as ceftriaxone. The most common drug classes for which isolates were resistant were tetracyclines and beta-lactams, with ampicillin, ceftriaxone and ceftiofur being the three most common drugs within the latter. Cow and herd level factors were associated with isolating antimicrobial resistant Salmonella that should be further investigated for their potential role in promoting occurrence of AMR Salmonella. Our results also highlight the importance of monitoring dairy cattle sent to slaughter for shedding of Salmonella resistant to medically important antimicrobial drugs.

Dairy farm use of antimicrobial drugs (AMD) is a risk for the selection of antimicrobial resistance (AMR); however, these resistance dynamics are not fully understood. A cohort study on two dairy farms enrolled 96 cows with their fecal samples collected three times weekly, for the first 60 days in milk. Enterobacteriaceae were enumerated by spiral plating samples onto MacConkey agar impregnated with 0, 1, 8, 16 and 30 µg/mL ceftiofur. Negative binomial regression analyzed AMR over time. The continuum of ceftiofur concentrations permitted estimation of the minimum inhibitory concentration (MIC) and analysis using interval regression. The most common systemic AMD was ceftiofur, administered in 94% of treatments (15/16 cows). Enterobacteriaceae did not grow in 88% of samples collected from non-AMD treated cows at 8 µg/mL ceftiofur. Samples from AMD treated cows had peak counts of resistant Enterobacteriaceae during AMD treatment and returned to baseline counts by 3–4 days post-treatment at 8 µg/mL. Sensitive Enterobacteriaceae (0–1 µg/mL ceftiofur) were reduced below pre-treated levels for 29–35 days post-AMD treatment. Population MIC peaked during AMD treatment and returned to baseline levels by 7–8 days. We conclude that the effect of systemic ceftiofur on the resistance of Enterobacteriaceae in early lactation dairy cows was limited in duration.

Background The primary objective of this cross-sectional study was to estimate the crude, seasonal and cull-reason stratified prevalence of Salmonella fecal shedding in cull dairy cattle on seven California dairies. A secondary objective was to estimate and compare the relative sensitivity (Se) and specificity (Sp) for pools of 5 and 10 enriched broth cultures of fecal samples for Salmonella sp. detection. Methods Seven dairy farms located in the San Joaquin Valley of California were identified and enrolled in the study as a convenience sample. Cull cows were identified for fecal sampling once during each season between 2014 and 2015, specifically during spring, summer, fall, and winter, and 10 cows were randomly selected for fecal sampling at the day of their sale. In addition, study personnel completed a survey based on responses of the herd manager to questions related to the previous four month’s herd management. Fecal samples were frozen until testing for Salmonella. After overnight enrichment in liquid broth, pools of enrichment broth (EBP) were created for 5 and 10 samples. All individual and pooled broths were cultured on selective media with putative Salmonella colonies confirmed by biochemical testing before being serogrouped and serotyped. Results A total of 249 cull cows were enrolled into the study and their fecal samples tested for Salmonella. The survey-weighted period prevalence of fecal shedding of all Salmonella sp. in the cull cow samples across all study herds and the entire study period was 3.42% (N = 249; SE 1.07). The within herd prevalence of Salmonella shed in feces did not differ over the four study seasons (P = 0.074). The Se of culture of EBP of five samples was 62.5% (SE = 17.12), which was not statistically different from the Se of culture of EBP of 10 (37.5%, SE = 17.12, P = 0.48). The Sp of culture of EBP of five samples was 95.24% (SE = 3.29) and for pools of 10 samples was 100.00% (SE = 0). There was no statistical difference between the culture relative specificities of EBP of 5 and 10 (P > 0.99). Discussion Our study showed a numerically higher prevalence of Salmonella shedding in the summer, although the results were not significant, most likely due to a lack of power from the small sample size. A higher prevalence in summer months may be related to heat stress. To detect Salmonella, investigators may expect a 62.5% sensitivity for culture of EBP of five, relative to individual fecal sample enrichment and culture. In contrast, culture of EBP of 10 samples resulted in a numerically lower Se. Culture of EBP of size 5 or 10 samples, given similar prevalence and limit of detection, can be expected to yield specificities of 95 and 100%, respectively.

Following colostrum management training, a randomized field trial was conducted on a California dairy to determine the effect of supplementing pooled colostrum with either colostrum-derived replacer (CDR) or second-milking colostrum (transition milk) on failure of passive transfer (FPT) and preweaning morbidity risks. A total of 166 calves were randomly assigned to 4L first-milking pooled colostrum (treatment 1), 2L first-milking pooled colostrum and 2L of CDR (treatment 2), or 2L first-milking pooled colostrum and 2L second-milking pooled colostrum (treatment 3). Mean 24-hour serum TP and IgG for treatments 2 (TP 5.2 g/dL, IgG 15.9 g/L) and 3 (TP 5.4 g/dL, IgG 18.3 g/L) did not statistically differ but were significantly lower than for treatment 1 (TP 5.9 g/dL, IgG 24.6 g/L). Risk of FPT did not differ for treatments 1, 2, and 3 (0.0%, 9.3%, and 1.9%, resp.). Similarly, the preweaning risk of diarrhea (81.0%, 92.5%, and 87.0%, resp.) or pneumonia (6.9%, 13.2%, and 18.5%, resp.) did not differ between treatments. Feeding 4L first-milking pooled colostrum resulted in adequate passive transfer. When first-milking pooled colostrum quantity is inadequate, CDR or second-milking pooled colostrum can be used to supplement the required colostrum volume and IgG mass without adversely affecting the risks of FPT or preweaning diarrhea and pneumonia.

Stable flies (Stomoxys calcitrans) are blood-sucking insects commonly associated with cattle production systems worldwide and are known to cause severe irritation to cattle due to painful bites. Cattle react to biting stable flies with an aggregating behavior known as bunching. Bunching behavior reduces grazing or feed consumption and thus reduces cattle productivity and welfare. Cattle's fly-repelling behaviors include foot stomping, head tossing, tail switching and skin twitching. A longitudinal study was conducted in 2017 on 20 California dairies (average lactating herd size = 2,466 (SE±28.392)) during the stable fly season from April to July. The study objectives were to estimate the association between environmental factors and dairy characteristics including facility design, feed and manure management, total mixed ration (TMR) components fed to cattle, and operational pest management procedures and the outcome stable fly activity on California dairies. Stable fly activity was measured by counting stable flies on cow forelimbs (leg count) and on Alsynite traps (trap count) over the 13-week study period. Weekly leg counts were performed for cattle in lactating cow pens (31 pens from 10 study dairies) with counts made during the morning (AM) and again during the afternoon (PM). Trap counts were performed on all 20 study dairies. Data were analyzed using linear mixed models which revealed temporal variation in the average leg and trap counts with stable fly activity increasing from May to June and then decreasing to the lowest activity in July. Leg counts were higher during the afternoon compared to morning. Ambient temperatures ≤30⁰C and relative humidity (RH) measurements

[This corrects the article DOI: 10.1371/journal.pone.0224987.].