R.V. Pereira

Molecular and epidemiological characterization of bovine intrauterine Escherichia coli

Escherichia coli are believed to be associated with postpartum metritis and endometritis but their role in the pathogenesis of both diseases is still undefined. In this study, uterine swabs for E. coli isolation were collected from 374 lactating Holstein cows housed on 4 commercial farms near Ithaca, New York. A total, 125 of 374 cows (33.4%) were positive for E. coli culture. Standard multiplex PCR protocols were used to screen the isolates for the presence of 32 virulence factor genes. Cows that had twin parturition were 4.4 times more likely to have intrauterine E. coli contamination than those that gave birth to single live female calves. Stillborn parturition and birth of single live male calves also increased the odds of intrauterine contamination by E. coli (3.7- and 1.6-fold, respectively) compared with birth of live female calves. Six virulence factors, common to extraintestinal and enteroaggregative E. coli, were found to be associated with metritis and endometritis: fimH, hlyA, cdt, kpsMII, ibeA, and astA. The virulence factor gene fimH was the most prevalent and the most significant: intrauterine E. coli carrying fimH and at least 1 of the other 5 identified virulence factors were pathogenic, and phylogenetic analysis based on the nucleotide sequence of DNA gyrase from 41 such IUEC revealed 2 clades.

Effect of an injectable trace mineral supplement containing selenium, copper, zinc, and manganese on the health and production of lactating Holstein cows

The objective of this study was to evaluate the effect of a subcutaneous injection of a multimineral preparation containing 300 mg of zinc, 50mg of manganese, 25mg of selenium, and 75 mg of copper at 230 and 260 days of gestation and 35 days postpartum, on the health, milk production and reproductive performance of lactating Holstein cows. A randomized field trial was conducted on three large commercial dairy farms located near Ithaca, New York, USA, with 1416 cows enrolled. All cows were housed and offered a total mixed ration consisting of approximately 55% forage and 45% concentrate on a dry matter basis of the diet, which supplied 2-6 times the NRC requirements for the supplemented elements. Dry cows and pregnant heifers were blocked by parity and randomly allocated to one of two treatments: Trace mineral supplemented (TMS) or control. For multiparous cows, subcutaneous TMS significantly decreased linear somatic cell count scores (normalized data) as compared to control cows. The incidence of subclinical mastitis for TMS and control cows was 10.4% and 8.0%, respectively (P=0.005). The main effect of treatment on clinical mastitis was not significant but the interaction of treatment and parity was significant. For primiparous cows, the incidence of clinical mastitis was 11.8% and 15.6% for control and TMS cows, respectively (P=0.33); for multiparous cows, the incidence of clinical mastitis for control and TMS cows was 25.4% and 19.7%, respectively (P=0.03). Additionally, control cows had increased odds of stillbirth and endometritis (odds ratios 1.69 and 1.30, respectively). The incidence of endometritis was 34.2% and 28.6% for control and TMS cows, respectively (P=0.039) but treatment had no effect on reproductive performance, milk production or other health traits. Further research is required to confirm these findings and to establish whether the response seen in this study was related to the supplementation of a particular mineral.

Efficacy of formalin, copper sulfate, and a commercial footbath product in the control of digital dermatitis

The objective was to evaluate the relative efficacy of a novel, commercially available disinfectant agent (T-Hexx Dragonhyde HBC, Hydromer, Branchburg, NJ) compared with formalin and copper sulfate. The hypothesis was 2 sided; therefore, the hypothesis was that the new agent would be better or worse compared with the industry gold standard footbath agents, formalin and copper sulfate. The study was conducted in a large commercial dairy farm located near Ithaca, New York, from June 18, 2009 to October 26, 2009. Two identical studies were conducted, the first comparing Dragonhyde (5% solution, twice weekly) and formalin (5% solution, twice weekly) and the second comparing Dragonhyde (5% solution, twice weekly) and copper sulfate (10% solution, twice weekly). The study design was identical for both studies with 4 pens (physically identical), 2 treatments (Dragonhyde vs. formalin and Dragonhyde vs. copper sulfate), 2 periods (crossing over the treatment within pen), and 3 repeated measures (3 observations per cow: enrollment, wk 2, and wk 4). For study 1, 406 cows were enrolled (n=201 formalin and 205 Dragonhyde). For study 2, 356 cows were enrolled (n=189 copper sulfate and 167 Dragonhyde). The adjusted odds of digital dermatitis lesion (DDL) throughout the study period were analyzed by mixed logistic regression model. In study 1, the odds of DDL were 1.36 times higher for the formalin group compared with the Dragonhyde group. In study 2, the data were analyzed by a similar statistical model and the variable treatment did not significantly affect the overall prevalence of DDL. In conclusion, the performance of 3 hoof care products was evaluated and it was concluded that Dragonhyde performed better than formalin and that there was no difference between copper sulfate and Dragonhyde.

Microbial diversity in bovine papillomatous digital dermatitis in Holstein dairy cows from upstate New York

Papillomatous digital dermatitis (PDD) is one of the most prevalent diseases of cattle, adversely affecting the dairy industry by its negative effect on milk production and reproductive performance. Our objective was to use culture-independent methods to determine the microbial diversity in different strata of PDD lesions of three Holstein dairy cows, analyzing whether major differences exist compared to foot skin of three non-infected cows. Both group-specific 16S rRNA gene PCR-denaturing gradient gel electrophoresis and clone library sequencing of broad-range 16S rRNA gene showed differences between the microbial composition of healthy dairy cows and the different strata of the lesion. The predominant bacterial community in the lesion, regardless of the stratum, consisted of 166 specific phylotypes belonging to seven bacterial phyla. Spirochetes (particularly, treponemes) was the most prominent group detected in PDD deep biopsies and was only found in samples from the lesion. Additionally, one phylotype phylogenetically affiliated with uncultured Euryarchaeota was detected in two strata of the lesion. Sequences from healthy foot skin samples revealed 86 specific phylotypes that were affiliated with Firmicutes and Proteobacteria. Our study corroborates the theory that treponemes are involved in PDD disease etiology and suggests, for the first time, the presence of archaeal members in this particular bovine infection.

Effect of on-farm use of antimicrobial drugs on resistance in fecal Escherichia coli of preweaned dairy calves.

Respiratory disease and diarrhea are the 2 most common diseases that result in the use of antimicrobial drugs in preweaned calves. Because the use of drugs in food animals, including dairy calves, has the potential for generating cross-resistance to drugs used in human medicine, it is vital to propose farm practices that foster the judicious use of antimicrobials while assuring animal health and productivity. The objective of this study was to use dairy farm calf treatment records to identify antimicrobial drug treatments in calves and to evaluate their effects on the prevalence of antimicrobial-resistant Escherichia coli from rectal swabs of preweaned dairy calves. Eight farms from central New York participated in the study, 3 farms using individual pen housing management and 5 farms using group pen housing management. Eligible study farms could not add antimicrobial drugs to the milk fed to preweaned calves and were required to have farm records documenting antimicrobial drug treatment of calves from birth to weaning. Three fecal E. coli isolates per calf were tested for susceptibility to 12 antimicrobial drugs using a Kirby-Bauer disk diffusion assay. A total of 473 calves were sampled, from which 1,423 commensal E. coli isolates were tested. Of the 9 antimicrobial drugs used on study farms, only enrofloxacin was significantly associated with reduced antimicrobial susceptibility of E. coli isolates, although treatment with ceftiofur was associated with reduced susceptibility to ceftriaxone. The median numbers of days from treatment with ceftiofur and enrofloxacin to rectal swab sampling of calves were 16 d (range: 1-39) and 12 d (range: 6-44), respectively. At the isolate level, treatment with enrofloxacin resulted in odds ratios of 2 [95% confidence interval (CI): 1-4] and 3 (95% CI: 2-6), respectively, for isolation of nonsusceptible E. coli to nalidixic acid and ciprofloxacin compared with calves not treated with enrofloxacin. Treatment with ceftiofur resulted in an odds ratio of 3 (95% CI: 0.9-12) for isolation of nonsusceptible E. coli to ceftriaxone compared with calves not treated with ceftiofur. Treatment with enrofloxacin resulted in selection of isolates that presented phenotypic resistance to both ciprofloxacin and ceftriaxone. Treatment with ceftiofur resulted in a higher prevalence of isolates resistant to ≥3 antimicrobial drugs (97%) compared with no treatment with ceftiofur (73%). These findings reinforce the necessity for continued implementation of practices at the dairy farm that support the sustainable and judicious use of antimicrobial drugs in dairy calves.

The effect of intrauterine administration of mannose or bacteriophage on uterine health and fertility of dairy cows with special focus on Escherichia coli and Arcanobacterium pyogenes

The objective of this study was to evaluate the effects of intrauterine administration of 50 g of ultrapure mannose or a bacteriophage cocktail and the presence of Escherichia coli and Arcanobacterium pyogenes in the uterine lumen on uterine health and reproductive performance of lactating dairy cows. The study was conducted on a commercial dairy farm located near Ithaca, New York, from May 4 to January 20, 2011, and 597 cows were enrolled. The cows were divided randomly into 3 treatment groups, and treatments were administered at 2 ± 1 d in milk (DIM). Treatment 1 consisted of intrauterine administration of 50 g of ultrapure mannose powder divided in 4 pills; treatment 2 consisted of intrauterine administration of a bacteriophage cocktail that included 4 different phages in one pill, with a dose of approximately 10⁷ plaque-forming units; and treatment 3 consisted of intrauterine administration of one empty pill (control). Intrauterine fluid swabs were collected on day of treatment and at 10 ± 3 DIM; uterine lavage samples were collected at 35 ± 3 DIM. Swabs and uterine lavage samples were cultured for E. coli and A. pyogenes. The intrauterine administration of mannose and bacteriophage did not affect uterine health, reproduction performance, or outcome of intrauterine secretion cultures for E. coli and A. pyogenes. Prevalence of intrauterine E. coli at 2 ± 1 DIM and A. pyogenes at 2 ± 1, 10 ± 3, and 35 ± 3 were affected by retained placenta. Additionally, prevalence of intrauterine E. coli at 10 ± 3 and A. pyogenes at 35 ± 3 DIM were associated with metritis, and cows that were diagnosed with clinical endometritis at 35 ± 3 DIM had greater prevalence of intrauterine E. coli at 2 ± 1 DIM and A. pyogenes at 35 ± 3 DIM. Furthermore, cows positive for E. coli at 2 ± 1 and 10 ± 3 DIM and for A. pyogenes at 10 ± 3 DIM had 1.63, 2.34, and 1.54 increased odds of having metritis. Cows positive for A. pyogenes at 35 ± 3 DIM and for E. coli at 2 ± 1 DIM had 19.8 and 2.66 higher odds of being diagnosed with clinical endometritis, respectively. Additionally, cows negative for E. coli at 2 ± 1 DIM were 1.39 times more likely to conceive than cows positive cows for E. coli.

Isolation and Characterization of Faecalibacterium prausnitzii from Calves and Piglets

The goal of our study was to isolate and characterize Faecalibacterium prausnitzii from fecal samples of healthy calves and piglets, in order to develop a novel probiotic for livestock animals. We identified 203 isolates of Faecalibacterium sp., which were clustered in 40 genetically distinct groups. One representative isolate from each cluster was selected for further characterization. The concentrations of the short chain fatty acids (SCFA) acetate, butyrate, propionate and isobutyrate in the culture media were measured by gas chromatography. We observed reduction in the concentration of acetate followed by concomitant increase in the concentration of butyrate, suggesting that the isolates were consuming acetate present in the media and producing butyrate. Butyrate production correlated positively with bacterial growth. Since butyrate has many benefits to the colonic epithelial cells, the selection of strains that produce higher amounts of butyrate is extremely important for the development of this potential probiotic. The effect of pH and concentration of bile salts on bacterial growth was also evaluated in order to mimic the conditions encountered by F. prausnitzii in vivo. The optimal pH for growth ranged between 5.5 and 6.7, while most isolates were inhibited by of the lowest concentration of bile salts tested (0.1%). Antimicrobial resistance profile showed that most isolates of Faecalibacterium sp. were resistant against ciprofloxacin and sulfamethoxazole-trimethoprim. More than 50% of the isolates were resistant to tetracycline, amikacin, cefepime and cefoxitin. A total of 19 different combinations of multidrug resistance were observed among the isolates. Our results provide new insights into the cultural and physiological characteristics of Faecalibacterium prausnitzii illustrating large variability in short chain fatty acid production, in vitro growth, sensitivity to bile salts, and antibiotic resistance and suggesting that future probiotic candidates should be carefully studied before elected for in vivo studies.

In Vivo Selection of Resistant E. coli after Ingestion of Milk with Added Drug Residues

Antimicrobial resistance represents a major global threat to modern medicine. In vitro studies have shown that very low concentrations of drugs, as frequently identified in the environment, and in foods and water for human and animal consumption, can select for resistant bacteria. However, limited information is currently available on the in vivo impact of ingested drug residues. The objective of our study was to evaluate the effect of feeding preweaned calves milk containing antimicrobial drug residues (below the minimum inhibitory concentration), similar to concentrations detected in milk commonly fed to dairy calves, on selection of resistant fecal E. coli in calves from birth to weaning. At birth, thirty calves were randomly assigned to a controlled feeding trial where: 15 calves were fed raw milk with no drug residues (NR), and 15 calves were fed raw milk with drug residues (DR) by adding ceftiofur, penicillin, ampicillin, and oxytetracycline at final concentrations in the milk of 0.1, 0.005, 0.01, and 0.3 µg/ml, respectively. Fecal samples were rectally collected from each calf once a week starting at birth prior to the first feeding in the trial (pre-treatment) until 6 weeks of age. A significantly greater proportion of E. coli resistant to ampicillin, cefoxitin, ceftiofur, streptomycin and tetracycline was observed in DR calves when compared to NR calves. Additionally, isolates from DR calves had a significant decrease in susceptibility to ceftriaxone and ceftiofur when compared to isolates from NR calves. A greater proportion of E. coli isolates from calves in the DR group were resistant to 3 or more antimicrobial drugs when compared to calves in the ND group. These findings highlight the role that low concentrations of antimicrobial drugs have on the evolution and selection of resistance to multiple antimicrobial drugs in vivo.

Antimicrobial resistance and prevalence of virulence factor genes in fecal Escherichia coli of Holstein calves fed milk with and without antimicrobials

Diarrhea in calves has a significant effect on the dairy industry. A common management practice for preventing or decreasing the effects of such disease in preweaned calves is by the use of antimicrobials in milk or milk replacer. In this study, Escherichia coli antimicrobial resistance in fecal samples collected from calves 2 to 8 d of age that had or had not received antimicrobials in the milk and that had or had not signs of diarrhea by inspection of fecal consistency were investigated. Specifically, resistance of E. coli isolates to individual antimicrobials, multiresistance patterns, and presence of virulence factors were analyzed. Escherichia coli isolates were tested for susceptibility to 12 antimicrobials by use of a Kirby-Bauer disk diffusion assay. The study was conducted at 3 farms, 1 administering growth-promoting antimicrobials (GPA) in the milk and 2 not using GPA in the milk (NGPA). All isolates were susceptible to ciprofloxacin and cefepime. From the total isolates tested, 84% (n=251) were resistant to at least 2 antimicrobials and 81% (n=251) were resistant to 3 or more antimicrobials. When antimicrobial resistance was compared between GPA and NGPA, it was observed that the GPA group had higher odds of antimicrobial resistance for most of the individual antimicrobials tested. No significant correlation of virulence factors in GPA or NGPA and diarrheic or non-diarrheic (control) fecal samples was found. Of the 32 virulence factors evaluated, 21 were detected in the study population; the incidence of only 1 virulence factor was statistically significant in each of the diarrheic status (diarrheic or non-diarrheic) and treatment status (NGPA or GPA) groups. Phylogenetic analysis based on the nucleotide sequence of the DNA gyrase gene (gyrB) from 31 fecal E. coli isolates revealed 3 main clades.

Ingestion of Milk Containing Very Low Concentration of Antimicrobials: Longitudinal Effect on Fecal Microbiota Composition in Preweaned Calves

Although antimicrobial drugs are central to combat disease in modern medicine, the use of these drugs can have undesired consequences for human and animal health. One consequence is the post-therapy excretion of pharmacological agents, such as the elimination of drug residues at very low concentrations in the milk of lactating mammals. Limited information is currently available on the impact from the exposure of the gut microbiota to drug residues using in vivo natural models. The objective of our study was to address this knowledge gap and evaluate the effect on the fecal microbiota composition from feeding preweaned dairy calves raw milk with residual concentrations of ampicillin, ceftiofur, penicillin, and oxytetracycline from birth to weaning. At birth, thirty calves were randomly assigned to a controlled feeding trial where: 15 calves were fed raw milk with no drug residues (NR), and 15 calves were fed raw milk with drug residues (DR) by adding ceftiofur, penicillin, ampicillin, and oxytetracycline at final concentrations in the milk of 0.1, 0.005, 0.01, and 0.3 μg/ml, respectively. Fecal samples were rectally collected from each calf once a week starting at birth, prior to the first feeding in the trial (pre-treatment), until 6 weeks of age. Sequencing of the microbial 16S rRNA genes was conducted using the Illumina MiSeq, which provides a high resolution of the microbiota down to the genus level. Discriminant analysis showed that, except for pre-treatment samples, calves fed milk with drug residues and calves fed milk without drug residues easily discriminated at the genus level on their weekly microbial profile. However, analysis comparing the abundance of taxon between NR and DR showed significant differences only at the genus levels, and not at the phylum, class, order or family levels. These results suggest that although drug residues can result in clear discriminate gut microbial communities, they do not result in disruption of taxonomic levels above the genus.