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Disbudding damages the germinal horn bud cells and prevents subsequent horn growth in young calves. Hot-iron cautery or caustic paste are the most common disbudding techniques and are unequivocally painful procedures. An important technique in controlling the acute pain experienced during disbudding is the cornual nerve block (CNB) that uses a local anesthetic agent and targets a branch of the trigeminal cranial nerve, the zygomaticotemporal nerve, as it travels along the temporal groove of the skull. Though CNB have been used since 1932, practitioners have reported variability in achieving full desensitization of the horn bud region since its inception. This failure may have led to the establishment of variations in the CNB technique, without consensus on a reliable and repeatable approach. Reasons for CNB failures may include technical errors by the practitioner, such as an injection into subcutaneous or deep muscle bodies; biologically important path variations in the zygomaticotemporal nerve and its cornual branches; and secondary innervation of the horn bud region, particularly the cornual branches of the infratrochlear nerve. Further investigation into documenting and understanding CNB failure and alternatives, such as regional perfusion, is warranted.

Food-producing animals such as dairy cattle are potential reservoirs of antimicrobial resistance (AMR), with multidrug-resistant (MDR) organisms such as Escherichia coli observed in higher frequency in young calves compared to older cattle. In this study, we characterized the genomes of enteric MDR E. coli from pre-weaned dairy calves with and without diarrhea and evaluated the influence of host-level factors on genomic composition. Whole genome sequence comparative analysis of E. coli (n = 43) revealed substantial genomic diversity that primarily clustered by sequence type and was minimally driven by calf diarrheal disease status (healthy, diarrheic, or recovered), antimicrobial exposure, and dietary zinc supplementation. Diverse AMR genes (ARGs)—including extended-spectrum beta-lactamase genes and quinolone resistance determinants—were identified (n = 40), with unique sets of ARGs co-occurring in gene clusters with large AMR plasmids IncA/C2 and IncFIB(AP001918). Zinc supplementation was not significantly associated with the selection of individual ARGs in E. coli, however analysis of ARG and metal resistance gene pairs identified positive associations between certain aminoglycoside, beta-lactam, sulfonamide, and trimethoprim ARGs with acid, tellurium and mercury resistance genes. Although E. coli in this study lacked the typical virulence factors of diarrheagenic strains, virulence genes overlapping with those in major pathotypes were identified. Among the 103 virulence genes detected, the highest abundance and diversity of genes corresponded to iron acquisition (siderophores and heme uptake). Our findings indicate that the host-level factors evaluated in this study were not key drivers of genomic variability, but that certain accessory genes in enteric MDR E. coli may be enriched. Collectively, this work provides insight into the genomic diversity and host-microbe interface of MDR E. coli from pre-weaned dairy calves.

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.

Abstract Background The goal of this study was to assess the microbial ecology and diversity present in the uterus of post-partum dairy cows with and without metritis from 24 commercial California dairy farms using shotgun metagenomics. A set subset of 95 intrauterine swab samples, taken from a larger selection of 307 individual cow samples previously collected, were examined for α and β diversity and differential abundance associated with metritis. Cows within 21 days post-partum were categorized into one of three clinical groups during sample collection: control (CT, n = 32), defined as cows with either no vaginal discharge or a clear, non-purulent mucus vaginal discharge; metritis (MET, n = 33), defined as a cow with watery, red or brown colored, and fetid vaginal discharge; and purulent discharge cows (PUS, n = 31), defined as a non-fetid purulent or mucopurulent vaginal discharge. Results All three clinical groups (CT, MET, and PUS) were highly diverse, with the top 12 most abundant genera accounting for 10.3%, 8.8%, and 10.1% of mean relative abundance, respectively. The α diversity indices revealed a lower diversity from samples collected from MET and PUS when compared to CT cows. PERMANOVA statistical testing revealed a significant difference (P adjusted

Galina Plotnikov,1,2 Saleh Sharif,2,3 Gleb Buturlin,2 Inbal Segal,1 Oleg Gorelik,2 Nadya Kagansky2,4 1Geriatric Assessment Unit, Yitzhak Shamir Medical Center, Zerifin, 7033001, Israel; 2Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel; 3Department of Internal Medicine C, Yitzhak Shamir Medical Center, Zerifin, 7033001, Israel; 4Shmuel Harofe Geriatric Medical Center, Beer Yaakov, IsraelCorrespondence: Galina Plotnikov, Geriatric Assessment Unit, Yitzhak Shamir Medical Center, Zerifin, 7033001, Israel, Tel +972-8-9542271, Fax +972-8-9779597, Email galina.plotnikov@gmail.comPurpose: To evaluate demographic, clinical, and laboratory variables, and their associations with in-hospital mortality, among elderly internal medicine patients with nasogastric tube (NGT) feeding.Patients and Methods: Demographic, clinical, and laboratory data were collected retrospectively for 129 patients aged ≥ 80 years who initiated NGT feeding during their hospitalization in internal medicine wards. The data were compared between survivors and non-survivors. Multivariate logistic regressions were performed to identify the variables most significantly associated with in-hospital mortality.Results: The in-hospital mortality rate was 60.5%. Compared to survivors, non-survivors more often presented with pressure sores (P=0.005) and lymphopenia (P< 0.001), were more often treated with invasive mechanical ventilation (P< 0.001), and less often underwent geriatric assessment (P< 0.001). Non-survivors demonstrated higher mean levels of C-reactive protein, and lower mean values of serum cholesterol, triglycerides, total protein, and albumin (P< 0.001 for all comparisons). On multivariate analysis, the following variables were most significantly associated with in-hospital mortality in the entire cohort: the presence of pressure sores (odds ratio [OR], 4.34; 95% confidence interval [CI], 1.68– 11.48; P=0.003) and lymphopenia (OR, 4.09; 95% CI, 1.51– 11.08; P=0.006), and serum cholesterol (OR, 0.98; 95% CI, 0.96– 0.99; P=0.003).Conclusion: Among elderly acutely ill patients who initiated NGT feeding during hospitalization, in-hospital mortality was extremely high. The factors most strongly associated with in-hospital mortality were the presence of pressure sores and lymphopenia, and lower serum cholesterol levels. These findings may provide useful prognostic information for decision-making regarding initiation of NGT feeding in elderly hospitalized patients.Keywords: aging, enteral feeding, hospitalization, prognosis

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.

Milk production continues to be the main source of income for dairy producers, and mastitis continues to be the major health challenge for dairy cows worldwide. The objective of the current study was to describe the different management practices implemented in Egyptian dairies, which may influence mastitis and improve milk quality. An in-person survey was completed with herd managers and owners of 20 Egyptian dairies selected using a stratified random sample from four of Egypt’s milk sheds. The questionnaire included 80 questions that inquired about herd demographics, mastitis status and control, milking practices, and management practices of the study dairies. Statistical analyses included descriptive statistics, multiple factor analysis (MFA), and hierarchical clustering to identify the important principal components and different dairy clusters. Of the surveyed herds, 69.50% had less than 500 lactating cows, with Holstein as the main cow breed. The reported average milk production on the study herds was 31.1 (SE ± 1.10) Kg/cow/day. Housing of lactating, dry, and close-up cows was in open lots. The majority of the dairies milked cows were three times per day (90.63%), the remaining milked cows four (5.00%) or a mix of four and three times per day. Furthermore, herringbone parlor design was the most common parlor design (66.79%) in the study dairies. The most common disinfectants used for both pre- and post-milking teat dip were iodine-based, 90.0% and 95.0%, respectively, while 52.16% of the dairies reported that their milkers wore gloves during milking. The reported mean annual percentage of mastitis was 52.3%, as a percent of the milking herd. The study dairies reported the inspection of udder health through either visual inspection (95.00%), udder hygiene score (86.88%), teat end score (71.88%), and/or using CMT (54.91%). Contagious mastitis pathogens were reported in 45.2% of the study herds. More than 50% of the study herds relied on importing pregnant cows or pregnant heifers as replacements. Multiple factor analysis identified 20 questions and represented 5 components of variability related to mastitis on dairies. The current survey of Egyptian dairies described the herd demographics and different management practices related to mastitis control and prevention. All the study dairies relied on the blanket intramammary antimicrobial drugs at dry off when current research elsewhere has identified selective dry cow therapy as an integral component of antimicrobial stewardship on dairies. Further research is required to identify the association between different management factors and the occurrence of mastitis.

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.

Cardiomyopathy (CM) is a condition of cardiac dysfunction. It is one of the leading causes of mortality in which both genetic and environmental factors are involved. Cardiotrophin-1 (CT-1) level in plasma is associated with CM. It affects the cardiomyocyte differentiation. To evaluate the expression of CT-1 in cardiomyopathy, this study was done on CM subjects attending the Fatima Memorial Hospital, Lahore, Pakistan, between January and June, 2016. A total of 40 subjects were enrolled who were divided into two groups; CM group (n = 20) and a control group (n = 20). A self-designed questionnaire was filled in by each subject to collect data regarding age, body mass index (BMI) and CM history. RNA was isolated from blood after its quantification, cDNA was prepared and reverse-transcriptase-polymerase chain reaction (RT-PCR) was performed for expression of CT-1. The mean age in CM subjects was 40.1±6.03 years, while it was 35.0±3.7 years in the control group. The mean expression of CT-1 in the CM subjects was 5.2±0.66, while it was 1.00±0.001 in the control group. A highly significant difference was observed in CT-1 expression in the CM group, and expression was significantly correlated with age and BMI in CM subjects.

Background Antibiotic resistance genes (ARGs) are considered to be emerging environmental contaminants of concern potentially posing risks to human and animal health, and this research studied the prevalence of antimicrobial resistance in dairy manure. Methods This study is focused on investigating prevalence of ARGs in California dairy farm manure under current common different manure management. A total of 33 manure samples were collected from multiple manure treatment conditions: (1) flushed manure (FM), (2) fresh pile (FP), (3) compost pile (CP), (4) primary lagoon (PL), and (5) secondary lagoon (SL). After DNA extraction, all fecal samples were screened by PCR for the presence of eight ARGs: four sulfonamide ARGs (sulI, sulII, sulIII, sulA), two tetracycline ARGs (tetW, tetO), two macrolide-lincosamide-streptogramin B (MLSB) ARGs (ermB, ermF). Samples were also screened for two mobile genetic elements (MGEs) (intI1, tnpA), which are responsible for dissemination of ARGs. Quantitative PCR was then used to screen all samples for five ARGs (sulII, tetW, ermF, tnpA and intI1). Results Prevalence of genes varied among sample types, but all genes were detectable in different manure types. Results showed that liquid-solid separation, piling, and lagoon conditions had limited effects on reducing ARGs and MGEs, and the effect was only found significant on tetW (p = 0.01). Besides, network analysis indicated that sulII was associated with tnpA (p < 0.05), and Psychrobacter and Pseudomonas as opportunistic human pathogens, were potential ARG/MGE hosts (p < 0.05). This research indicated current different manure management practices in California dairy farms has limited effects on reducing ARGs and MGEs. Improvement of different manure management in dairy farms is thus important to mitigate dissemination of ARGs into the environment.

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.

Contagious bovine mastitis caused by Mycoplasma bovis and other Mycoplasma species including Mycoplasma californicum, Mycoplasma bovigenitalium, Mycoplasma alkalescens, Mycoplasma arginini, and Mycoplasma canadense is an economical obstacle affecting many dairy herds throughout California and elsewhere. Routine bacteriological culture-based assays for the pathogens are slow and subject to false-positive results due to the presence of the related, non-pathogenic species Acholeplasma laidlawii. To address the need for rapid and accurate detection methods, a new TaqMan multiplex, quantitative real-time PCR (qPCR) assay was developed that targets the 16S rRNA gene of Mycoplasma, rpoB gene of M. bovis, and the 16S to 23S rRNA intergenic transcribed spacer (ITS) region of A. laidlawii. qPCR amplification efficiency and range of detection were similar for individual assays in multiplex as when performed separately. The multiplex assay was able to distinguish between M. bovis and A. laidlawii as well as detect Mycoplasma spp. collectively, including Mycoplasma californicum, Mycoplasma bovigenitalium, Mycoplasma canadense, Mycoplasma arginini and Mycoplasma alkalescens. In milk, the lower limit of detection of M. bovis, M. californicum, and A. laidlawii with the multiplex assay was between 120 to 250 colony forming units (CFU) per mL. The assay was also able to simultaneously detect both M. bovis and A. laidlawii in milk when present in moderate (103 to 104 CFU/mL) to high (106 to 107 CFU/mL) quantities. Compared to laboratory culture-based methods, the multiplex qPCR diagnostic specificity (Sp) was 100% (95% CI [86.8–100]; n = 26) and diagnostic sensitivity (Se) was 92.3% (95% CI [74.9–99.1]; n = 26) for Mycoplasma species in milk samples collected from California dairy farms. Similarly, the Sp was 100% (95% CI [90.5–100]; n = 37) and Se was 93.3% (95% CI [68.1–99.8]; n = 15) for M. bovis. Our assay can detect and distinguish among M. bovis, other prevalent Mycoplasma spp., and non-pathogenic Acholeplasma laidlawii for effective identification and control of mycoplasma mastitis, ultimately supporting dairy cattle health and high-quality dairy products in California.

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.

In recent times, the extinction of fossil fuel resources and emission of harmful pollutants have persuaded the researchers to obtain alternative and renewable fuels. Consequently, in the present experimentation, a ternary fuel blend was acquired and analysed in a modified turbocharged gasoline direct injection (GDI) engine as a feasible alternative to neat fossil fuel. Experiments were carried out using a blend of 8% n-butanol and 8% mass fraction of hydrogen with gasoline fuel at city transport speed mode under stoichiometric and air-fuel ratio of 1.0. Butanol and hydrogen were blended with the gasoline due to their satisfying thermodynamic properties and combustion reactivity. The result shows gradual reduction of particles emitted with n-BH16 by 1.06 × 1011 particles/hr as compared to n-BH00 at 1.89 × 1012 particles/hr. For all fuels, the smallest particles measured by Engine Exhaust Particle Sizer (EEPS) were no longer detectable at phase 2 from 548 s to ~789 s due to low concentrations. However, the gaseous emission of the n-BH16 fueled engine varies with 28 g/kW.h decrement in fuel consumption (FC), while increasing hydrocarbon (HC) emission. As a result, ultra-particles decreased in nucleation mode, which in-turn lead to minimum combustion duration and cylinder gas pressure. Adaptation of n-BH16 fuel blend in light-duty GDI engines might serve as a potential to meet the new particulate emission regulations and improve toxic air contaminant.

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.

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

Waste milk feeding practices have been implicated as a potential source for disseminating antimicrobial resistant bacteria among animals and the environment. Two interventions that have shown potential for degrading antimicrobial drugs in milk are heat and pH treatment. The aim of this study was to evaluate the effect of heat and pH treatments on the degradation of ceftiofur and ceftiofur free acid equivalents in milk at concentrations previously found in waste milk on dairy farms by spiking saleable pasteurized whole milk with ceftiofur sodium. Three heat treatments of ceftiofur sodium spiked milk were evaluated for their ability to degrade ceftiofur: 63°C for 30 min (LTLT), 72°C for 15 s (HTST) and 92°C for 20 min (HTLT). Two pH treatments of ceftiofur sodium spiked milk were evaluated: pH 4.0 (LpH) and pH 10 (HpH). Control samples spiked with ceftiofur sodium were kept at room temperature and samples collected at corresponding times for heat and pH treatments. Four treatment replicates were performed for each treatment group. Ceftiofur was quantified in milk samples using liquid chromatography mass spectrometry (LC-MS/MS) and ceftiofur free acid equivalents (CFAE) were measured using high-performance liquid chromatography (HPLC). HTLT resulted in a degradation of 35.24% of the initial concentration of ceftiofur. Ceftiofur degradation did not differ between control and the remaining two heat treatment groups (LTLT and HTST). HpH resulted in degradation of the 95.72 and 96.28% of the initial concentration of ceftiofur and CFAE, respectively. No significant changes in degradation of ceftiofur or CFAE were observed for control or LpH treatments. In conclusion, our study results were that alkalinizing milk to pH 10 and heating milk to 92°C for 20 min degraded ceftiofur and CFAE in spiked simulated waste milk demonstrated promising potential as treatment options for degrading ceftiofur and CFAE in waste milk, and further research is needed to evaluate the viability for implementation of these treatments in dairy farms.

The present data article is centered on the research work which examines the effect of fuel preheating on DI diesel engine fuelled with Fish Oil Ethyl Ester (FOEE). Kirloskar TV1 model water cooled diesel engine with eddy current dynamometer was used in the experiment. Crude fish oil was converted into FOEE using transesterification process. The physical and chemical properties of FOEE were examined based on American Standards for Testing Materials (ASTM) biodiesel standards and data's were offered. To achieve better engine characteristics, a fuel preheater was designed and fabricated to work at different temperatures (60, 70 and 80 °C).Fuel preheating temperatures were achieved using three different shell and tube heat exchanger at various dimensions. Heat exchanger was designed to work with waste heat obtained from the engine exhaust. Engine tailpipe emission was recorded using AVL make 444 di-gas analyzer and smoke was measured using AVL make 437C free accelerometer smoke meter. Data related to fuel samples like neat diesel, FOEE blends with and without fuel preheating were presented. Keywords: Fish oil ethyl ester, Diesel engine, Fuel preheating, Performance and emission

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

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.

The objectives of this study were to characterize overall genomic antibiotic resistance profiles of fecal Escherichia coli and Enterococcus spp. from dairy cattle at different production stages using whole-genome sequencing and to determine the association between antimicrobial resistance (AMR) phenotypes and their corresponding genotypes. The Comprehensive Antibiotic Resistance Database (CARD) and ResFinder, two publicly available databases of antimicrobial resistance genes, were used to annotate isolates. Based on the ResFinder database, 27.5% and 20.0% of tested E. coli isolates (n = 40) harbored single and ≥3 antimicrobial resistance genes, respectively; for Enterococcus spp., we observed 87.8% and 8.2%, respectively. The highest prevalence of AMR genes in E. coli was for resistance to tetracycline (27.5%), followed by sulphonamide (22.5%) and aminoglycoside (20.0%); the predominant antimicrobial resistance genes in Enterococcus spp. targeted macrolide drugs (77.6%). Based on the CARD database, resistance to ≥3 antimicrobial classes was observed in all E. coli and 77.6% in Enterococcus spp. isolates. A high degree of agreement existed between the resistance phenotype and the presence of resistance genes for various antimicrobial classes for E. coli but much less so for isolates of Enterococcus. Consistent with prior work, fecal E. coli and Enterococcus spp. isolates from calves harbored a wide spectrum of resistance genes, compared to those from cattle at other production stages, based on the cross-sectional samples from the studied farm.

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.

Extended laboratory culture and antimicrobial susceptibility testing timelines hinder rapid species identification and susceptibility profiling of bacterial pathogens associated with bovine respiratory disease, the most prevalent cause of cattle mortality in the United States. Whole-genome sequencing offers a culture-independent alternative to current bacterial identification methods, but requires a library of bacterial reference genomes for comparison. To contribute new bacterial genome assemblies and evaluate genetic diversity and variation in antimicrobial resistance genotypes, whole-genome sequencing was performed on bovine respiratory disease–associated bacterial isolates (Histophilus somni, Mycoplasma bovis, Mannheimia haemolytica, and Pasteurella multocida) from dairy and beef cattle. One hundred genomically distinct assemblies were added to the NCBI database, doubling the available genomic sequences for these four species. Computer-based methods identified 11 predicted antimicrobial resistance genes in three species, with none being detected in M. bovis. While computer-based analysis can identify antibiotic resistance genes within whole-genome sequences (genotype), it may not predict the actual antimicrobial resistance observed in a living organism (phenotype). Antimicrobial susceptibility testing on 64 H. somni, M. haemolytica, and P. multocida isolates had an overall concordance rate between genotype and phenotypic resistance to the associated class of antimicrobials of 72.7% (P < 0.001), showing substantial discordance. Concordance rates varied greatly among different antimicrobial, antibiotic resistance gene, and bacterial species combinations. This suggests that antimicrobial susceptibility phenotypes are needed to complement genomically predicted antibiotic resistance gene genotypes to better understand how the presence of antibiotic resistance genes within a given bacterial species could potentially impact optimal bovine respiratory disease treatment and morbidity/mortality outcomes.

Mycobacterium avium subspecies paratuberculosis (MAP) is a bacterium that can cause substantial economic losses in infected dairy herds due to reduced milk production and increased cow-replacement costs. In order to control MAP in dairies with drylot pens, a standardized environmental sampling protocol to quantify MAP in fecal slurry was developed based on an existing protocol for freestall pens. Specifically, following a 24 h hold of the flush, a grab sample of approximately 10 ml of fecal slurry was collected every 1 m along the flush lane of the drylot pens, avoiding individual cow fecal pats. To determine the reliability and repatability of the new environmental sampling protocol for estimation of MAP bioburden at the pen level, two collectors simultaneously collected fecal slurry samples every day for 3 days from six drylot cow pens on two Central California dairies. During the study period no cow movement between pens was allowed with the exception of sick cows. The study herds had MAP seroprevalence of 5.8% and 3.2%, respectively, based on whole pen serum ELISA results. Variance components models for quantitative real-time PCR (qPCR) results showed samples collected from different pens on different dairies accounted for greater variablitiy in MAP concentration (65%), while samples collected by different collectors had the least variability (0.1%). In contrast, variability in MAP concentration in environmental samples collected on different days had 25% variability. The intraclass correlation coefficient showed high reliability (93%) of environmental sampling simultaneously by different collectors. In contrast, the reliability of environmental sampling at different days was 65%, which was similar to the reliability for sampling by different collectors on different days. Investigators can expect high reliability when employing the new environmental sampling protocol along with qPCR testing of environmental samples from drylot pens.

Background Coagulase negative Staphylococcus (CNS) species are currently the most prevalent intra-mammary pathogens causing subclinical mastitis and occasional clinical mastitis or persistent infection in lactating dairy cattle. More than 10 CNS species have been identified, but they are generally managed as one group on most dairies in the United States. However, improved management decisions and treatment outcomes may be achieved with better understanding of the prevalent species, pathogenicity and strain diversity within and across dairies. Methodology A total of 604 CNS isolates were cultured from milk samples collected during a dry-cow treatment clinical trial conducted on 6 dairy herds in 4 states in the US. All the study cows were randomized to receive 1 of the 3 different intra-mammary antimicrobial infusions (Quatermaster, Spectramast DC or ToMorrow Dry Cow) at dry-off. Milk samples were collected at dry-off, calving (0–6 days in milk, DIM), post-calving (7–13 DIM) and at mastitis events within the first 100 DIM. The CNS isolates were identified to species level by partial sequencing of the rpoβ gene, and genetic relatedness within species was investigated by phylogenetic analysis of the pulse-field gel electrophoresis profiles of the isolates. Results The major CNS species identified were S. chromogenes (48.3%), S. haemolyticus (17.9%), S. simulans and S. epidermidis (each at 6.5%). Other CNS species identified at lower frequencies included S. hominis, S. auricularis, S. sciuri, S. spp KS-SP, S. capitis, S. cohnii, S. warneri, S. pasteuri, S. xylosus, S. hyicus, S. equorum, S. microti, S. rostri, S. gallinarum, S. saprophyticus and S. succinus. Phylogenetic analyses of the major species types demonstrated an association between genetic relatedness and epidemiological distributions of S. chromogenes, S. simulans, S. haemolyticus and S. auricularis. Additionally, identical strains of S. chromogenes and S. simulans were isolated from the same udder quarter of several cows at consecutive sample stages. The rest of the minor species had no deducible genetic-epidemiological link. Discussion The observed association between genetic and epidemiological distributions indicated animal-adapted nature of four CNS species, suggesting possible host-adapted and environmental transmission of these species. Multi-stage isolation of the same udder quarter strain was evidence for chronic intra-mammary infection. Conclusion The different CNS species and strains circulating on US dairy herds were genetically diverse. Four species identified were likely udder-adapted pathogens, 2 of which caused persistent infection. Our findings are important in guiding the design of effective mastitis control strategies.

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.

The objective of this clinical trial was to evaluate the effectiveness of zinc supplementation on diarrhea and average daily weight gain (ADG) in pre-weaned dairy calves. A total of 1,482 healthy Holstein heifer and bull calves from a large California dairy were enrolled at 24 to 48 hours of age until hutch exit at approximately 90 days of age. Calves were block-randomized by time to one of three treatments: 1) placebo, 2) zinc methionine (ZM), or 3) zinc sulfate (ZS) administered in milk once daily for 14 days. Serum total protein at enrollment and body weight at birth, treatment end, and hutch exit were measured. Fecal consistency was assessed daily for 28 days post-enrollment. For a random sample of 127 calves, serum zinc concentrations before and after treatment and a fecal antigen ELISA at diarrhea start and resolution for Escherichia coli K99, rotavirus, coronavirus, and Cryptosporidium parvum were performed. Linear regression showed that ZM-treated bull calves had 22 g increased ADG compared to placebo-treated bulls (P = 0.042). ZM-treated heifers had 9 g decreased ADG compared to placebo-treated heifers (P = 0.037), after adjusting for average birth weight. Sex-stratified models showed that high birth weight heifers treated with ZM gained more than placebo-treated heifers of the same birth weight, which suggests a dose-response effect rather than a true sex-specific effect of ZM on ADG. Cox regression showed that ZM and ZS-treated calves had a 14.7% (P = 0.015) and 13.9% (P = 0.022) reduced hazard of diarrhea, respectively, compared to placebo-treated calves. Calves supplemented for at least the first five days of diarrhea with ZM and ZS had a 21.4% (P = 0.027) and 13.0% (P = 0.040) increased hazard of cure from diarrhea, respectively, compared to placebo-treated calves. Logistic regression showed that the odds of microbiological cure at diarrhea resolution for rotavirus, C. parvum, or any single fecal pathogen was not different between treatment groups. Zinc supplementation delayed diarrhea and expedited diarrhea recovery in pre-weaned calves. Additionally, zinc improved weight gain differentially in bulls compared to heifers, indicating a research need for sex-specific dosing.

Bunching is the term used to describe the protective aggregating behavior of cattle against the painful bites of stable flies (Stomoxys calcitrans), where cattle gather in a group with their heads to the center of the group and their tails to the outside to reduce stable fly attack. Both sexes of the stable fly feed on blood, and their painful bites negatively impact cattle health, productivity and welfare. A longitudinal study was conducted from April to July 2017 to estimate the stable fly activity on 20 California dairies (average herd size = 2466 ± 1050), to determine stable fly activity that induced bunching, and to evaluate the association between management and environmental factors, and cattle bunching. Stable fly activity was recorded weekly using trap counts and leg counts. Data was analyzed using linear mixed models with odds ratio. Cattle bunching at the dairy level was predicted by mean trap counts of ≥150 flies/trap/week, while bunching at the pen level was predicted by mean leg counts >1 fly/leg/cow or mean trap counts >50 flies/trap/week for traps closest to the pen. Additional risk factors predicting cattle bunching at the dairy level were study week (May/June vs July), presence of crops adjacent to dairy >2 sides, and feeding wet distillers grain. Additional risk factors predicting cattle bunching at the pen level were study week (May/June vs July), ambient temperature ≤30°C, pen design (freestall vs open dry lot or bedded pack), production status (lactating/dry vs close-up), presence of crops surrounding cattle pens, feeding rations containing molasses. Cattle bunching was reduced at the pen level by relative humidity >50%, and when the cattle pen was surrounded by other cattle pens or was bordered by a main road. At the dairy level, removal of manure along fence lines of cattle pens was protective against cattle bunching.

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.

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.

Paratuberculosis, also known as Johne's disease (JD), is a chronic contagious disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP). The disease is incurable, fatal and causes economic losses estimated to exceed 200 million dollars to the U.S. dairy industry annually. Several preventive and control measures have been recommended; however, only a few of these measures have been validated empirically. Using a nested compartmental (NC) modeling approach, the main objective of this research was to identify the best combination of control and preventive measures that minimizes the prevalence and incidence of JD and the risk of MAP occurrence in a dairy herd. The NC model employs both MAP transmission estimates and data on pen movement of cattle on a dairy to quantify the effectiveness of control and preventive measures. To obtain reasonable ranges of parameter values for between-pen movements, the NC model was fitted to the movement data of four typical California dairy farms. Using the estimated ranges of the movement parameters and those of JD from previous research, the basic reproduction number was calculated to measure the risk of MAP occurrence in each pen environment as well as the entire dairy. Although the interventions evaluated by the NC model were shown to reduce the infection, no single measure alone was capable of eradicating the infection. The numerical simulations suggest that a combination of test and cull with more frequent manure removal is the most effective method in reducing incidence, prevalence and the risk of MAP occurrence. Other control measures such as limiting calf-adult cow contacts, raising calves in a disease-free herd or colostrum management were less effective.

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.

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.

Recent observations by stakeholders suggested that ecosystem changes may be driving an increased incidence of bovine erythrocytic anaplasmosis, resulting in a reemerging cattle disease in California. The objective of this prospective cohort study was to estimate the incidence of Anaplasma marginale infection using seroconversion in a northern California beef cattle herd. A total of 143 Black Angus cattle (106 prebreeding heifers and 37 cows) were enrolled in the study. Serum samples were collected to determine Anaplasma marginale seroprevalence using a commercially available competitive enzyme-linked immunosorbent assay test kit. Repeat sampling was performed in seronegative animals to determine the incidence density rate from March through September (2013). Seroprevalence of heifers was significantly lower than that of cows at the beginning of the study (P