Two-Dose Ceftiofur Treatment Increases Cephamycinase Gene Quantities and Fecal Microbiome Diversity in Dairy Cows Diagnosed with Metritis.

Antimicrobial resistance is a significant concern worldwide; meanwhile, the impact of 3rd generation cephalosporin (3GC) antibiotics on the microbial communities of cattle and resistance within these communities is largely unknown. The objectives of this study were to determine the effects of two-dose ceftiofur crystalline-free acid (2-CCFA) treatment on the fecal microbiota and on the quantities of second-and third-generation cephalosporin, fluoroquinolone, and macrolide resistance genes in Holstein-Friesian dairy cows in the southwestern United States. Across three dairy farms, 124 matched pairs of cows were enrolled in a longitudinal study. Following the product label regimen, CCFA was administered on days 0 and 3 to cows diagnosed with postpartum metritis. Healthy cows were pair-matched based on lactation number and calving date. Fecal samples were collected on days 0, 6, and 16 and pooled in groups of 4 (n = 192) by farm, day, and treatment group for community DNA extraction. The characterization of community DNA included real-time PCR (qPCR) to quantify the following antibiotic resistance genes: blaCMY-2, blaCTX-M, mphA, qnrB19, and the highly conserved 16S rRNA back-calculated to gene copies per gram of feces. Additionally, 16S rRNA amplicon sequencing and metagenomics analyses were used to determine differences in bacterial community composition by treatment, day, and farm. Overall, blaCMY-2 gene copies per gram of feces increased significantly (p ≤ 0.05) in the treated group compared to the untreated group on day 6 and remained elevated on day 16. However, blaCTX-M, mphA, and qnrB19 gene quantities did not differ significantly (p ≥ 0.05) between treatment groups, days, or farms, suggesting a cephamycinase-specific enhancement in cows on these farms. Perhaps unexpectedly, 16S rRNA amplicon metagenomic analyses showed that the fecal bacterial communities from treated animals on day 6 had significantly greater (p ≤ 0.05) alpha and beta diversity than the untreated group. Two-dose ceftiofur treatment in dairy cows with metritis elevates cephamycinase gene quantities among all fecal bacteria while paradoxically increasing microbial diversity.

Effects of two-dose ceftiofur treatment for metritis on the temporal dynamics of antimicrobial resistance among fecal Escherichia coli in Holstein-Friesian dairy cows.

A pair-matched longitudinal study conducted on three dairy farms in the U.S. High-Plains explored the temporal effects of two-dose ceftiofur crystalline-free acid (CCFA) treatment for metritis on third-generation cephalosporin (3GC) resistance among enteric E. coli in Holstein-Friesian cows. The current 13-day slaughter withholding period does not account for rising populations of third-generation cephalosporin (3GC) resistant bacteria in feces of animals following CCFA treatment. A total of 124 matched-pairs of cows were enrolled in the study. Cows diagnosed with postpartum metritis received the product twice at the labeled dose of 6.6 mg/kg subcutaneously at the base of alternating ears. Untreated cows-absent clinical metritis-were matched on lactation number and calving date. Feces were collected per rectum on days 0 (baseline), 6, 16, 28, and 56. Environmental samples, from watering troughs as well as surface manure from fresh-cow, hospital, maternity, and milking pens, and from the compost pile were collected prior to the animal sample collection period. Historical data on metritis rates and CCFA use were compiled from herd records. On day 0, cows exhibited an overall mean difference of over 4 log10 colony forming units (CFU) comparing 3GC resistant E. coli to the general E. coli population. At the first eligible slaughter date, the difference declined to 3.31 log10 CFU among cows in the CCFA group (P<0.01 compared to control cows). Such differences were no longer observed between the treated and control groups by day 28. Results suggest a 13-day withholding period following the final treatment is insufficient to allow levels of 3GC resistant E. coli to return to baseline. This effect varied by farm and was dependent upon the starting level of resistance. A farm-specific extended slaughter-withholding period could reduce the microbial risk to food products at slaughter.

Development of a method for Bayesian nonparametric ROC analysis with application to an ELISA for Johne's disease in dairy cattle.

Six hundred and sixty-eight dairy cattle were tested for Johne's disease using a direct-fecal real-time polymerase chain reaction (rt-PCR), fecal culture, and serum enzyme-linked immunosorbent assay (ELISA). Likelihood ratios (LRs) were estimated for five categories of sample-to-positive (S/P) values for the ELISA: <0.1, 0.1-0.249, 0.25-0.399, 0.4-0.999, > or =1.0. The statistical method assumed a single multinomial model for cross-classified rt-PCR, fecal culture results, and ELISA category strata. Conditional dependence between tests was investigated by the inclusion of all possible pairwise dependence terms. Sensitivity covariance between ELISA and fecal culture was estimated as 0.017. Estimates for the accuracy of the ELISA at the usual cutoff of 0.25S/P was 67.2% and 95.2% for sensitivity and specificity, respectively for a model that adjusted for the dependence between ELISA and fecal culture. The area under the receiver-operating characteristic (ROC) curve (95% probability interval) for the ELISA was 0.867 (0.796, 0.928). Point estimates for likelihood ratios (95% probability intervals) were 0.24 (0.11, 0.38), 1.52 (0.48, 3.27), 2.49 (0.31, 13.4), 6.33 (2.54, 16.5), and 103 (25.0, 2412) for the categories <0.1, 0.1-0.249, 0.25-0.399, 0.4-0.999, >/=1.0, respectively. Assumptions concerning the underlying distribution of test results for infected and uninfected animals were not necessary and this model can be employed for the general estimation of LRs and ROC curves in absence of knowledge concerning true disease status.

Field testing of an enhanced direct-fecal polymerase chain reaction procedure, bacterial culture of feces, and a serum enzyme-linked immunosorbent assay for detecting Mycobacterium avium subsp paratuberculosis infection in adult dairy cattle.

OBJECTIVE: To estimate the sensitivity (Se) and specificity (Sp) for an enhanced direct-fecal PCR procedure, bacterial culture of feces (BCF), and a serum ELISA for detecting Mycobacterium avium subsp paratuberculosis (MAP) infection in adult dairy cattle. SAMPLE POPULATION: Fecal and serum samples were collected from 669 adult cattle randomly selected from a 4,000-cow dairy herd known to contain animals infected with MAP. PROCEDURES: Serum samples were evaluated for MAP-specific antibodies via ELISA. Fecal samples were evaluated by BCF and enhanced PCR methods (both gel-based [GB]-PCR and quantitative real-time [qRT]-PCR assays). Fecal samples also were pooled (5:1) and then subjected to GB-PCR assay. Bayesian statistical methods were used to estimate Se and Sp for each diagnostic test without knowledge concerning true MAP infection status. RESULTS: Adjusting for Se conditional dependence between serum ELISA and BCR, overall Se and Sp were estimated at 33.7% and 95.9%, 51.3% and 99.0%, and 32.2% and 100% for serum ELISA, qRT-PCR, and BCF, respectively.The GB-PCR assay yielded positive results for 38.3% of the pools known to contain feces from at least 1 cow that had positive GBPCR results. CONCLUSIONS AND CLINICAL RELEVANCE: Estimated Se values for the serum ELISA and BCF were slightly lower than those reported elsewhere. The enhanced qRT-PCR method offered relative improvements in Se of 52% and 59% over serum ELISA and microbial culture, respectively. Pooling of fecal samples and testing with the GB-PCR assay are not recommended. Additional studies with qRT-PCR and fecal pools are required.

Effect of intrauterine administration of ceftiofur on fertility and risk of culling in postparturient cows with retained fetal membranes, twins, or both.

OBJECTIVE: To determine the effect of intrauterine administration of ceftiofur sodium on fertility and the risk of culling in postparturient cows with retained fetal membranes (RFM), twins, or both. DESIGN: Single-blind randomized clinical trial and prospective cohort study. ANIMALS: 2442 cows that calved from January 1, 2000, to May 31, 2001. PROCEDURE: Cows with RFM, twins, or both were randomly allocated to control or treatment (ceftiofur) groups. Ceftiofur-group cows received 1 g of ceftiofur sodium sterile powder reconstituted with 20 mL of sterile water as a single intrauterine infusion once between 14 and 20 days after parturition. Control-group cows received no treatment. Cows that calved but did not have RFM or twins were considered the referent group. Reproductive, culling, and health data were recorded. RESULTS: There was no significant difference in the overall proportion of ceftiofur-group cows confirmed pregnant, compared with cows in the control group. Ceftiofur-group cows were significantly less likely to be culled and were culled at a later time in lactation than control-group cows. In the cohort study, the risk of pregnancy and the risk of being culled in ceftiofur-group cows were not significantly different from cows in the referent group. CONCLUSIONS AND CLINICAL RELEVANCE: Intrauterine treatment of cows with RFM, twins, or both with ceftiofur sodium increased longevity of cows in the herd as measured by the risk of culling and the time to culling. Intrauterine administration of ceftiofur in cattle is considered extralabel drug use, and the attending veterinarian must follow the AMDUCA guidelines for extralabel drug use.