Abstract
OBJECTIVE To determine whether feeding a direct-fed microbial (DFM) to dairy calves would reduce total and antimicrobial-resistant coliform counts in feces and affect average daily gain (ADG).
ANIMALS 21 preweaned Holstein heifer calves.
PROCEDURES The study had a randomized complete block design. Within each block, 3 consecutively born calves were randomly assigned to 1 of 3 treatment groups within 24 hours after birth (day 0). Calves were fed the DFM at 1.0 g (DFM1; n = 7) or 0.5 g (DFM2; 7) twice daily or no DFM (control; 7) from days 0 through 29. A fecal sample was collected from each calf daily on days 0 through 3 and then every other day through day 29. Fecal samples were cultured, and mean numbers of total coliforms and coliforms resistant to ampicillin, ceftiofur, and tetracycline were compared among the 3 treatment groups. Calves were weighed on days 0 and 29 to calculate ADG.
RESULTS Mean total fecal coliform counts did not differ significantly among the 3 treatment groups. Mean ceftiofur-resistant and tetracycline-resistant coliform counts for the control group were significantly lower, compared with those for the DFM1 and DFM2 groups. Mean ADG did not differ significantly between the DFM1 and DFM2 groups; however, the mean ADG for all calves fed the DFM was 0.15 kg less than that for control calves.
CONCLUSION AND CLINICAL RELEVANCE Results suggested that the DFM fed to the preweaned calves of this study did not reduce total or antimicrobial-resistant coliform counts in feces.
Antimicrobial resistance is a worldwide problem in human and animal health because infections caused by bacteria resistant to antimicrobials are often difficult to treat.1 Antimicrobial-resistant bacteria that originate from food-producing livestock are a public health concern because of the potential for transmission of those pathogenic or commensal bacteria and resistance determinants (genes) to humans through the food chain.2 Public health agencies are particularly concerned about the use of antimicrobials that are considered critically important to human health in livestock.3 In response to those concerns, the European Union has banned the use of antimicrobials as growth promotants in livestock.4 In the United States, restrictions have been implemented over the past 15 years on certain uses of antimicrobials in food-producing animals, including restrictions on the use of fluoroquinolones and the extralabel use of third-generation cephalosporins.5,6
Additionally, the FDA is encouraging the pharmaceutical industry to phase out the production of growth promotants and remove over-the-counter labeling for feed antimicrobials used in food-producing animals.7,8 Thus, there is an increasing need for the development of new interventions and strategies to reduce the prevalence and emergence of antimicrobial-resistant bacteria in livestock.
Historically, many of the interventions proposed to reduce antimicrobial-resistant bacteria have focused on decreasing the use of antimicrobials in livestock through voluntary cessation or government regulation or by switching from conventional to organic or antimicrobial-free production.4,9–14 Although the reduction or cessation of the use of antimicrobials has successfully lowered the prevalence of bacteria resistant to some antimicrobial drugs, the removal or prohibition of certain classes of antimicrobials from use in livestock has not resulted in complete cessation of the propagation and accumulation of antimicrobial-resistant pathogens. This may be explained, to some extent, by the coselection for 1 class of antimicrobial-resistance genes caused by the use of other classes of antimicrobials or heavy metals or various management or environmental factors.15 Clonal spread of antimicrobial-resistance genes may also explain the persistence of antimicrobial-resistant bacteria in a herd despite the absence of selective pressure for the development of resistance by cessation of the use of certain antimicrobials.16 In many instances, the cessation of the use of all antimicrobials on organic livestock operations or the exclusion of the use of certain antimicrobials on conventional livestock operations has failed to substantially mitigate antimicrobial resistance.13,14,17–20 Despite the mixed results achieved by banning the use of certain antimicrobials in livestock, research to investigate alternative strategies for reducing antimicrobial-resistant bacteria in livestock is lacking.
One potential intervention to reduce the number of anitmicrobial-resistant bacteria is the feeding of DFMs. Direct-fed microbials are bacteria that, when fed to animals or humans, help positively change the intestinal microbiota to improve or maintain health.21,22 A balanced intestinal microbiota acts as a barrier against pathogen colonization, produces beneficial metabolic substrates, and stimulates the immune system.23–25 Direct-fed microbials have been used as a substitute for antimicrobial growth promotants in livestock and are often fed to preweaned calves to establish and maintain a favorable intestinal microbiota to support growth and performance and reduce disease incidence.26–30 Results of multiple studies26,29,31–35 indicate a reduction in both the incidence and duration of diarrhea in calves fed DFMs that contained Lactobacillus spp, Enterococcus faecium, and Bacillus spp. In other studies,36,37 there was a reduction in the shedding of pathogenic enterohemorrhagic Escherichia coli in the feces of calves that were fed a probiotic that contained E coli. The feeding of a DFM to feedlot steers is associated with a decrease in fecal shedding of E coli O157 and Salmonella spp.38,39 Collectively, the results of those studies26–39 suggest that feeding a DFM to preweaned dairy calves might aid in the development of a favorable intestinal microbiota that could reduce the prevalence of antimicrobial-resistant bacteria in those calves.
Results of a pilot study conducted by our laboratory group in 2012 that was designed to compare and evaluate the effects of 2 DFMs fed to preweaned dairy calves suggested that feeding calves a DFM that contained Bacillus licheniformis, Bacillus subtilis, Lactobacillus acidophilus, Lactobacillus lactis, and Bifidobacterium animalis subsp lactis was associated with a temporary reduction in the absolute numbers of total coliforms and coliforms resistant to ceftiofur, ampicillin, and tetracycline in feces. Coliforms are lactosefermenting, gram-negative bacteria that include E coli and Enterobacter, Klebsiella, and Citrobacter spp.40 The primary objective of the study reported here was to determine the effect of feeding a DFM to preweaned dairy calves on the total and antimicrobial-resistant fecal coliform counts of those calves. Our hypothesis was that preweaned dairy calves fed a DFM twice daily would have lower total and antimicrobial-resistant fecal coliform counts, compared with those of calves that were not fed a DFM. Secondary objectives were to investigate the effect of feeding a DFM on the ADG and fecal scores of preweaned dairy calves.
Materials and Methods
Animals
All study protocols were approved by the Michigan State University Institutional Animal Care and Use Committee. The study was conducted on a commercial dairy farm in Michigan with the owner's consent. Twenty-one Holstein heifer calves were enrolled in the study. Per the herd protocol, all calves were administered an anti–E coli antibody suspensiona (10 mL, PO) and bovine rotavirus and coronavirus vaccineb (3 mL, PO) and fed 3.8 L of colostrum within 2 hours after birth. Each calf was allowed to suckle the colostrum from a bottle, but if it failed to consume the entire volume, the remaining colostrum was administered by an esophageal feeder. A blood sample (10 mL) was obtained by jugular venipuncture from each calf between 24 and 48 hours after birth and submitted to the Michigan State University Diagnostic Center for Population and Animal Health for determination of Hct and serum total protein concentration. Only calves with a serum total protein concentration > 5.5 g/dL were considered to have adequate passive immunity41 and included in the study. Calves were moved to individual hutches at approximately 24 hours old. To reduce the potential for cross-contamination among calves and hutches, the calf hutches were placed in 2 rows with approximately 3 m between the rows and a minimum of 1 m between adjacent hutches within each row. Additionally, investigators changed disposable gloves and boots between hutches when assessing and collecting samples from the calves. Once in the hutches, calves were fed a commercial milk replacerc that was reconstituted in accordance with the label directions every 12 hours. Calves were also provided a starter feedd and water ad libitum. Calves were administered Clostridium perfringens types C and D antitoxine (10 mL, SC) at 3 days old and an injection of a solutionf containing vitamins A, D, and E (5 mL, SC) at 5 days old in accordance with the herd protocol. Calves were monitored for 29 days. Calves that became sick and required antimicrobial treatment were excluded from the study.
Study design
The study had a randomized complete block design with a block size of 3 (ie, 3 treatments). Because we did not have all of the information needed to calculate a sample size that accounted for potential correlation among serially measured outcomes (ie, a study with a repeated measures design), we used a sample size calculation for independent means with information obtained from preliminary studies, which suggested that the SD for the mean total and antimicrobial-resistance fecal coliform counts ranged between 50% and 100%. Thus, we estimated that the mean difference in fecal coliform count among treatment groups would be 101 CFUs/g of feces and that the SD for that mean would be 75%. This resulted in a calculated sample size of 6 calves/treatment group. We enrolled 7 calves/treatment group as insurance against possible uncorrectable attrition.
A block was defined as 3 consecutively born heifer calves. Within 24 hours after birth, each heifer within a block was randomly assigned to 1 of 3 treatment groups by use of a random number generator.g Calves in the DFM1 group were fed 1.0 g of a DFM that was suspended in 15 mL of store-bought, pasteurized whole milk; calves in the DFM2 group were fed 0.5 g of a DFM that was suspended in 15 mL of store-bought, pasteurized whole milk; and calves in the control group were fed 15 mL of store-bought, pasteurized whole milk. The calves were fed the assigned treatment for 29 days. The first treatment was administered within 2 hours after birth prior to oral administration of the anti–E coli antibody suspension, bovine rotavirus and coronavirus vaccine, and colostrum. Subsequently, the assigned treatment was administered to each calf in conjunction with the allotted milk replacer at each feeding. If a calf was administered an antimicrobial within the first week after birth, it was removed from the study and replaced with the next heifer calf that was born; this happened twice. Study personnel were aware of the treatment group assignment for all calves throughout the study (ie, the study personnel were not blinded).
DFM
The DFM fed was a commercially available producth that contained B licheniformis, B subtilis, L acidophilus, L lactis, and B animalis subsp lactis with a guaranteed total bacteria count of 7.2 × 109 CFUs/g. For quality-assurance purposes, the DFM was analyzed by an independent laboratory,i and the results of that analysis were consistent with the guaranteed analysis except that Enterococcus faecium (105 CFUs/g) was also identified.
Fecal sample collection and processing
A fecal sample was collected by digital stimulation of the rectum from each calf daily from day 0 (birth) through day 3 and then every other day through day 29 (ie, 15 fecal samples were obtained from each calf). Each sample was placed in an individual sterile cup and transported in a cooler from the farm to the laboratory. All samples were processed and analyzed within 4 to 6 hours after collection. Outcomes of interest were the total CFUs of coliform bacteria (predominately E coli) per gram of feces and total CFUs of coliforms resistant to each of 3 antimicrobials (ampicillin, ceftiofur, and tetracycline)/g of feces. Those 3 antimicrobials were selected on the basis of the duration or frequency with which they have been used in the dairy industry. Ampicillin and tetracycline have been used by the dairy industry for a long duration (approx 40 to 50 years), whereas ceftiofur has been used for a moderate duration (approx 20 years).
To perform fecal coliform counts, 1 g (wet weight) of each fecal sample was initially diluted in 9 mL of PBSS (pH, 7.3). To ensure CFUs could be counted, subsequent serial dilutions were performed as needed on the basis of the investigators’ experience for determining the CFUs of coliform bacteria in feces obtained from preweaned calves. A spiral autoplaterj was used to inoculate a dilution of each fecal sample onto a MacConkey agar plate without any antimicrobials and 3 additional MacConkey agar plates, each of which contained 1 of the 3 antimicrobials (ampicillin, ceftiofur, and tetracycline) for which resistance was being determined. The MacConkey agar was prepared in accordance with CLSI agar-dilution methodology. Additionally, ampicillink (32 μg/mL), ceftiofurl (8 μg/mL), or tetracyclinem (16 μg/mL) was added to each designated plate at the concentration established by the CSLI as the clinical breakpoint for bacterial resistance to those antimicrobials.40 In accordance with CLSI guidelines, E coli ATCC 25922, Staphylococcus aureus ATCC 29213, Entercoccus faecalis ATCC 29212, E coli ATCC 35218, and Pseudomonas aeroginosa ATCC 27853 were used as quality control strains for all of the prepared agars. Following inoculation, the agar plates were incubated at 37°C for 18 to 24 hours. An automated colony countern (minimum detection limit, 240 CFUs/plate) was used to calculate the number of coliform CFUs on each plate, which was then extrapolated to calculate the CFUs per gram of feces.
Calf health and performance
For each calf, body weight was estimated at birth (day 0) and 29 days old by use of a weight tape.o The health of each calf was monitored approximately every 12 hours by observation of appetite and appearance at feeding. Additionally, rectal temperature, heart rate, respiratory rate, and hydration status were recorded for each calf immediately prior to collection of each fecal sample. The consistency of each fecal sample was subjectively scored on a scale of 0 to 3 as described.p Briefly, a score of 0 was assigned to feces that had a solid consistency, a score of 1 was assigned to feces that were semiformed and pasty, a score of 2 was assigned to feces that had a loose consistency but did not pass through the bedding, and a score of 3 was assigned to feces that had a watery consistency and readily passed through the bedding.
Data analysis
All data were entered into an electronic database.q The primary outcomes of interest were the total fecal coliform count and the respective counts of fecal coliforms resistant to ampicillin, ceftiofur, and tetracycline. Other outcomes of interest included body weight, ADG, and fecal score. The fecal coliform counts were expressed as log10 CFUs per gram of feces. Descriptive and summary data for each outcome were calculated for each treatment group (DFM1, DFM2, and control). Mixed linear regression was used to compare the fecal coliform counts over time among the 3 treatment groups. The model was specified as Yijk = μ + α i + yk + (ay)ik + eijk for the jth subject in the population, where Yijk is the number of fecal coliform CFUs per gram of feces for the jth subject, μ is the overall mean, αi is the fixed effect for treatment group, yk is the fixed effect for sampling day, (ay)ik is the fixed effect for the interaction between treatment group and sampling day, and eijk is the random error with a first-order autoregressive variancecovariance structure to account for repeated sampling within each calf. For each calf, the ADG was calculated as the amount of weight gained between birth and 29 days old divided by 29 days. The mean ADG was compared among the 3 treatment groups by use of a generalized linear model with the Bonferroni method for multiple comparisons. For each calf, the median fecal score for all 15 samples was calculated, and a 2-sided Kruskal-Wallis rank test was used to evaluate differences in median fecal score among calves. For all analyses, model assumptions were evaluated by means of residual diagnostics that included assessment of conditional residuals for normality and outliers with standard diagnostic plots,42 and values of P ≤ 0.05 were considered significant. All analyses were performed with commercially available statistical software.r
Results
Calves
Of the 21 calves that were initially enrolled in the study, 2 were treated with an antimicrobial within 7 days after birth (1 calf assigned to the DFM1 group developed an umbilical infection at 1 day old, and 1 calf assigned to the DFM2 group developed abomasal bloat at 3 days old). Those 2 calves were replaced with 2 other calves. Of the 21 calves that remained in the study, 3 received oral or SC fluid therapy for dehydration, fever, and diarrhea. A calf in the DFM1 group was administered 1.9 L of an oral electrolyte solution twice at 2 days old. A calf in the DFM2 group was administered 3.8 L of an oral electrolyte solution at 25 days old. A calf in the control group was administered lactated Ringer's solution (2 L, SC) twice at 2 days old. Another calf in the DFM2 group was administered antimicrobials and flunixin meglumine for treatment of an infected umbilical hernia at 25 days old and was excluded from the 4 remaining days of the observation period. No adverse events associated with feeding of the DFM were observed in any of the calves. The observed morbidity rate (4/21 [19%]) for the study calves was within expected limits for dairy calves < 1 month old.
Fecal samples
Twenty-one meconium (samples collected on day 0) and 292 fecal samples were collected from the study calves (15 samples from each of 20 calves and 13 samples from the calf that was treated with antimicrobials at 25 days old and subsequently removed from the study). Collectively, coliform bacteria were detected in 312 of the 313 (99.7%) samples collected, and 300 (96.0%) of those samples contained coliform bacteria that were resistant to all 3 antimicrobials (ampicillin, ceftiofur, and tetracycline) evaluated. Coliforms resistant to ampicillin, ceftiofur, or tetracycline were not detected in 6 (1.9%), 12 (3.8%), and 7 (2.2%) samples, respectively. Six (1.9%) samples contained coliforms that were not resistant to any of the 3 antimicrobials evaluated, and all of those samples were collected from calves on day 1. Of the 13 samples that contained coliforms that were not resistant to all 3 antimicrobials, only 4 of those samples were collected after day 1. Growth of all quality control strains was within expected limits for each batch of sample cultures.
Coliform growth was detected in 8 of 21 meconium samples that were collected on day 0. The mean total fecal coliform count for day 0 samples was 101 CFUs/g (median, 0 CFUs/g; range, 0 to 107 CFUs/g), and only 1 calf had a fecal coliform count > 103 CFUs/g, which was suspected to be the result of contamination. The mean total coliform count for fecal samples collected on day 1 was 108 CFUs/g (median, 109 CFUs/g; range, 0 to 1010 CFUs/g) and was significantly increased from that for the meconium samples collected on day 0. The number of coliforms resistant to at least 1 of the 3 antimicrobials evaluated likewise increased significantly between days 0 and 1 (Figure 1). The mean total fecal coliform count and counts for coliforms resistant to ampicillin and tetracycline peaked at day 3, whereas the ceftiofur-resistant coliform count peaked at day 5, after which all counts gradually declined for the duration of the study. Although the interaction between treatment group and sampling day was not significant, it was retained in the linear regression model so that the mean coliform counts could be compared among the 3 treatment groups on each sampling day.
Mean ± 95% confidence interval for the number of total fecal coliforms (A) and fecal coliforms that were resistant to ampicillin (B), ceftiofur (C), and tetracycline (D) over time for Holstein calves that were fed a DFM at 1.0 g (DFM1; n = 7; black diamonds) or 0.5 g (DFM2; 7; light gray squares) twice daily or no DFM (control; 7; dark gray triangles) from birth (day 0) through 29 days old. The DFM fed to calves in the DFM1 and DFM2 groups was a commercially available product that contained Bacillus licheniformis, Bacillus subtilis, Lactobacillus acidophilus, Lactobacillus lactis, and Bifidobacterium animalis subsp lactis with a guaranteed total bacteria count of 7.2 × 109 CFUs/g; independent analysis of the DFM revealed that it also contained Enterococcus faecium (105 CFUs/g). A fecal sample was obtained by digital rectal stimulation from each calf daily from days 0 through 3 and then every other day through day 29. Coliforms were identified as resistant to a given antimicrobial on the basis of growth on MacConkey agar supplemented with that antimicrobial at the concentration (ampicillin, 32 μg/mL; ceftiofur, 8 μg/mL; or tetracycline, 16 μg/mL) established by the CLSI as the clinical breakpoint for bacterial resistance. A calf in the DFM2 group was administered antimicrobials for the treatment of an infected umbilical hernia at 25 days old and was removed from the remaining 4 days of the study. Therefore, in all panels, the values for the DFM2 group on days 27 and 29 represent the mean ± 95% confidence interval for only 6 calves.
Citation: American Journal of Veterinary Research 76, 9; 10.2460/ajvr.76.9.780
The mean total fecal coliform count (P = 0.25) and mean ampicillin-resistant fecal coliform count (P = 0.16) did not differ significantly among the 3 treatment groups at any time during the observation period (Table 1). The mean ceftiofur-resistant and tetracycline-resistant fecal coliform counts did not differ significantly between the DFM1 and DFM2 groups at any time during the study; therefore, the data for those 2 groups were combined for comparison with the control group. For the calves fed the DFM, the mean ceftiofur-resistant and tetracycline-resistant fecal coliform counts were 101.02 (P < 0.01) and 100.57 (P = 0.05) CFUs/g, respectively, greater than those for the calves in the control group across all sampling days.
Least squares mean (SE) values for counts of total fecal coliforms and fecal coliforms resistant to ampicillin, ceftiofur, and tetracycline for Holstein calves that were fed a DFM at 1.0 g (DFM1; n = 7) or 0.5 g (DFM2; 7) twice daily or no DFM (control; 7) for the first 29 days after birth.
| Treatment group | Total fecal coliforms (log10 CFUs/g) | Ampicillin-resistant coliforms (log10 CFUs/g) | Ceftiofur-resistant coliforms (log10 CFUs/g) | Tetracycline-resistant coliforms (log10 CFUs/g) |
|---|---|---|---|---|
| DFM1 | 7.89 (0.13) | 6.90 (0.16) | 5.88 (0.19)* | 6.78 (0.18) |
| DFM2 | 7.77 (0.14) | 7.01 (0.18) | 6.01 (0.15)* | 6.79 (0.18) |
| Control | 7.63 (0.14) | 6.41 (0.20) | 4.92 (0.18) | 6.21 (0.20)† |
The DFM fed to calves in the DFM1 and DFM2 groups was a commercially available product that contained Bacillus licheniformis, Bacillus subtilis, Lactobacillus acidophilus, Lactobacillus lactis, and Bifidobacterium animalis subsp lactis with a guaranteed total bacteria count of 7.2 × 109 CFUs/g; independent analysis of the DFM revealed that it also contained Enterococcus faecium (105 CFUs/g). Fecal samples were obtained by digital rectal stimulation from each calf daily beginning the day of birth (day 0) through day 3 and then every other day through day 29; therefore, 15 fecal samples were obtained from each calf except for 1 calf in the DFM2 group from which only 13 samples were obtained because it was administered antimicrobials at 25 days old and removed from the remainder of the study. Thus, values for the DFM1 and control groups represent the least squares mean values for 105 fecal samples and those for the DFM2 group represent the least squares mean values for 103 fecal samples.
Value differs significantly (P ≤ 0.05) from that for the control group.
Value differs significantly (P = 0.05) from the mean for the DFMI and DFM2 groups combined.
Calf health and performance
The mean ADG did not differ significantly among the 3 treatment groups (Table 2). However, when the data for the calves in the DFM1 and DFM2 groups were combined, the mean ADG for the calves fed the DFM was significantly (P = 0.03) less than that for calves in the control group by approximately 0.15 kg.
Mean ± SD (95% confidence interval) for body weight at birth (day 0) and 29 days old and ADG for the calves of Table 1.
| Treatment group | Birth weight (kg) | Weight at 29 days old (kg) |
|---|---|---|
| DFM1 | 46.85 ± 7.42 (39.99–53.72) | 67.14 ± 5.64 (61.93–72.36) |
| DFM2 | 42.21 ± 6.25 (36.43–47.99) | 64.71 ± 5.85 (59.30–70.13) |
| Control | 40.93 ± 6.34 (35.06–46.79) | 66.50 ± 6.93 (60.09–72.91) |
The ADG for each calf was calculated as the amount of weight gained between birth and 29 days old divided by 29 days.
See Table 1 for remainder of key.
Fecal score data were missing for 4 samples collected from calves in the DFM1 group, 3 samples collected from calves in the DFM2 group, and 6 samples from the calves in the control group. The median fecal score did not differ significantly (P = 0.59) among the 3 treatment groups and was 1 (interquartile range [25th to 75th quartile], 1 to 1; range, 0 to 3) for the DFM1 group, 1 (interquartile range, 1 to 2; range, 0 to 3) for the DFM2 group, and 1 (interquartile range, 1 to 2; range, 0 to 3) for the control group.
Discussion
Results of the present study indicated that the total fecal coliform count and the counts of fecal coliforms resistant to ampicillin, ceftiofur, and tetracycline for dairy calves that were fed 1.0 (DFM1) or 0.5 g (DFM2) of a DFM that contained B licheniformis, B subtilis, L acidophilus, L lactis, and B animalis subsp lactis twice daily during the first 29 days after birth were not significantly decreased, compared with those for calves that were not fed a DFM (control). Moreover, the mean ADG for the calves fed the DFM during the observation period was significantly less than that for the control calves. These findings suggested that the DFM fed to calves of the present study is unlikely to mitigate the prevalence or number of antimicrobial-resistant coliforms in the gastrointestinal tract of preweaned dairy calves. However, these findings may not be applicable to all DFMs, and the effects of other DFMs on the antimicrobial-resistant fecal coliform counts in preweaned dairy calves warrant investigation.
The significantly higher mean counts of ceftiofur-resistant and tetracycline-resistant coliforms in the feces of calves fed the DFM, compared with those for control calves, were unexpected. The published literature contains contradictory findings regarding the role of DFMs on the health and performance of cattle. Results of multiple studies26,27,29,31–33,43,44 suggest that feeding DFMs to cattle improves performance and reduces the incidence and duration of diarrhea, and feeding DFMs to feedlot cattle is associated with a decrease in the prevalence and quantity of E coli O157 shed in the feces.39,45,46 Conversely, results of other studies27,45,47 indicate that feeding DFMs to calves has no beneficial effect on health and performance. Given these conflicting findings, it is likely that the efficacy for decreasing antimicrobial resistance in and improving health and performance of cattle varies among DFMs.
In the present study, calves were randomly assigned to treatment groups, which should have minimized or eliminated selection bias. Unfortunately, we were unable to keep the study personnel responsible for feeding, assessing the health, and collecting samples from calves blinded to the treatment assignments for individual calves, which might have caused potential differential misclassification in the recording of health events. The plating of fecal samples on agar plates and the counting of the number of fecal coliform CFUs on each plate were automated and therefore unlikely to cause any information bias. Also, a larger sample size would have increased the power of the study. Consequently, the results of the present study should be extrapolated with caution to calves raised during different seasons under different housing and management conditions (eg, calves fed medicated milk replacers) because those variables can affect the colonization of antimicrobial-resistant coliforms in the gastrointestinal tract.
In the present study, we administered the DFM at a higher dose than that recommended by the manufacturer at the time the study was conducted in an attempt to ensure a reduction in the colonization of the gastrointestinal tract with antimicrobial-resistant coliforms and improve the ADG of preweaned dairy calves during the first 29 days after birth. Unfortunately, the doses of DFM (0.5 and 1.0 g/calf, q 12 h) administered failed to induce those effects. Although we used a commercially available DFM, it is unknown whether the bacterial strains included in the product were derived from cattle. It is possible that differences in species specificity might affect the ability of bacterial strains in a DFM to colonize the gastrointestinal tract. In human neonates, E coli are among the first bacteria to colonize the gut48; therefore, we speculate that a calf-derived E coli–based DFM might maximize colonization of the gastrointestinal tract of calves with beneficial coliforms that could outcompete the colonization of antimicrobial-resistant bacteria.
We found it interesting that the fecal coliform counts increased exponentially between birth and 3 days old. We expected little, if any, coliform growth in the meconium samples, and it is likely that the meconium sample that had a high coliform count was contaminated in the environment. Few studies16,49 have been conducted to investigate the initial bacterial colonization of the gastrointestinal tract of calves. In the present study, the median number of fecal coliforms increased from 0 CFUs/g on day 0 to 109 CFUs/g on day 1. This finding was similar to that observed for E coli colonization of the gastrointestinal tract of human neonates.48Additionally, the high ampicillin-resistant and tetracycline-resistant fecal coliform counts for the calves of the present study during the first 3 days after birth suggested that most of the coliforms that initially colonize the gastrointestinal tract of the calves on that farm were resistant to those antimicrobials.
Results of other studies27,30,50 suggest that DFMs are most beneficial for preweaned calves when fed during periods of stress. Because the mean counts for total fecal coliforms and fecal coliforms resistant to ampicillin and tetracycline did not differ significantly among the 3 treatment groups of the present study, it is possible that the DFM fed did not affect the antimicrobial resistance of the intestinal microbiome or the calves were not sufficiently stressed for the beneficial effects of the DFM to be realized or detected. The present study was conducted during the spring, and no extreme weather events (large temperature swings or severe inclement weather) occurred during the observation period. Additionally, the specific daily calf management and calf hutch maintenance protocols implemented (eg, a strict 12-hour feeding interval, refilling water buckets with fresh water after each feeding, and the addition of fresh bedding) may have reduced calf stress and affected the study results.
Results of other studies49,51 indicate that fecal coliform counts are high in 1-week-old calves. Findings of the present study supported those results; mean total fecal coliform counts increased from 100 to 108 CFUs/g during the first 24 hours after birth. In the present study, it appeared that the gastrointestinal tract was initially colonized by non–antimicrobial-resistant coliforms; however, by 3 to 5 days of age, most of the coliforms cultured from the fecal samples were resistant to ampicillin, ceftiofur, or tetracycline or some combination of those 3 antimicrobials. This finding was in agreement with the results of other studies52–54 in which antimicrobial-resistant coliforms were prevalent in fecal samples obtained from preweaned calves. Investigators of another study16 reported that the number of total and antimicrobial-resistant E coli in the feces of preweaned calves peaked within 1 week after birth and then gradually decreased over time in a manner similar to that observed for the mean total and antimicrobial-resistant fecal coliform counts in the present study. The results of the present study did not support our hypothesis that preweaned dairy calves fed a DFM twice daily would have lower total and antimicrobial-resistant fecal coliform counts, compared with those of control calves that were not fed a DFM. The reason for this might have been that the DFM was administered during a period when fecal coliforms counts are naturally high or the DFM had a beneficial effect on coliform colonization of the gastrointestinal tract that indirectly resulted in an increase in antimicrobial-resistant fecal coliforms.
The use of antimicrobials in food-producing animals is currently under intense scrutiny by scientists, governmental agencies, and the public. Although much of the debate on the subject focuses on the use of subtherapeutic doses of antimicrobials to promote growth, any use of antimicrobials in food-producing animals for the control or treatment of disease at the group level is considered controversial by some people. One method to potentially decrease the prevalence of antimicrobial-resistant bacteria is to cease the use of all antimicrobials in food-producing animals. However, that method appears to be at a cross-purpose with animal health and well-being, and there are multiple instances in which the cessation of the use of certain antimicrobial classes in food-producing animals has failed to substantially mitigate the prevalence or number of antimicrobial-resistant bacteria.13,14,16–19 Direct-fed microbials are considered a potential alternative for use in food-producing animals to decrease the number of antimicrobial-resistant bacteria. Although the results of the present study suggested that the DFM fed to the study calves had no effect on the total or antimicrobial-resistant fecal coliform counts, further research is warranted to determine whether feeding cattle DFMs with cattle-derived bacterial strains will decrease the colonization of the gastrointestinal tract with antimicrobial-resistant coliforms.
Acknowledgments
Supported by intramural funding from the Michigan State University College of Veterinary Medicine.
The authors declare that they do not have any conflicts of interest regarding the performance of the study reported here.
ABBREVIATIONS
| ADG | Average daily gain |
| ATCC | American Type Culture Collection |
| CLSI | Clinical Laboratory Standards Institute |
| DFM | Direct-fed microbial |
Footnotes
Bovine Ecolizer, Novartis Animal Health US Inc, Larchwood, Iowa.
Calf-Guard, Zoetis Inc, Florham Park, NJ.
Cow's Match milk replacer, Land O'Lakes Inc, Shoreview, Minn.
Dairy Focus Premium Calf Starter 23%, Cargill Inc, Minneapolis, Minn.
Clostridium perfringens types C & D antitoxin, Boehringer Ingleheim, Ridgefield, Conn.
Vitamin E-AD-300, AgriLabs, St Joseph, Mo.
Random number generator, Dr. Mads Haahr, Dublin, Ireland. Available at: www.random.org. Accessed Apr 27, 2015.
Probios Calf Pac, Chr Hansen Inc, Milwaukee, Wis.
Great Lakes Scientific Inc, Stevensville, Mich.
Eddy Jet Spiral Autoplater, Neutec Group Inc, Farmingdale, NY.
Ampicillin (A9393), Sigma-Aldrich Corp, St Louis, Mo.
Ceftiofur (34001), Sigma-Aldrich Corp, St Louis, Mo.
Tetracycline hydrocholoride (T4062), Sigma-Aldrich Corp, St Louis, Mo.
Flash and Go colony counter, Neutec Group Inc, Farmingdale, NY.
Holstein dairy tape, Nasco, Fort Atkinson, Wis.
Calf Heath Scoring Chart, School of Veterinary Medicine, University of Wisconsin, Madison, WIs. Available at: www.vetmed.wisc.edu/dms/fapm/fapmtools/8calf/calf_health_scoring_chart.pdf. Accessed Apr 27, 2015.
Microsoft Access, Microsoft Inc, Redwood, Wash.
SAS, version 9.3, SAS Institute Inc, Cary, NC.
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