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Effect of antibiotics in milk replacer on fecal shedding of Escherichia coli O157:H7 in calves¹

W. Q. Alali^*, J. M. Sargeant^*,2, T. G. Nagaraja and B. M. DeBey

^* Food Animal Health and Management Center and and Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan 66506

	Abstract

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The objective of this study was to compare the concentration and duration of fecal shedding of Escherichia coli O157:H7 between calves fed milk replacer with or without antibiotic (oxytetracycline and neomycin) supplementation. Eighteen 1-wk-old Holstein calves were orally inoculated with a strain of E. coli O157:H7 (3.6 x 10⁸ cfu/calf) made resistant to nalidixic acid (NA). Rectal samples were obtained three times weekly for 8 wk following oral inoculation. Fecal shedding of NA-resistant E. coli O157:H7 was quantified by direct plating or detected by selective enrichment procedure. Eight weeks after inoculation, calves were killed, necropsied, and tissues (tonsils, retropharyngeal and mesenteric lymph nodes, and Peyer’s patches) and gut contents (rumen, omasum, abomasum, ileum, cecum, colon, and rectum) were sampled to quantify or detect NA-resistant E. coli O157:H7. The percentage of calves shedding NA-resistant E. coli O157:H7 in the feces in the antibiotic-fed group was higher (P < 0.001) early in the study period (d 6 and 10) compared with the control group fed no antibiotics. There was no difference between treatment and control groups in the concentration of E. coli O157 in feces that were positive at quantifiable concentrations. A comparison of the duration of fecal shedding between treated and untreated calves showed no significant difference between groups. At necropsy, E. coli O157:H7 was recovered from the rumen and omasum of one calf in the control group and from retropharyngeal lymph node and Peyer’s patch of two calves in the antibiotic group. Supplementation of milk replacer with antibiotics may increase the probability of E. coli O157:H7 shedding in dairy calves, but the effect seems to be of low magnitude and short duration.

Key Words: Antibiotics • Calves • Escherichia coli O157:H7 • Milk Replacer

	Introduction

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Escherichia coli O157:H7 shed in the feces of clinically normal cattle has been linked to hemorrhagic colitis and hemolytic-uremic syndrome in humans (Clarke et al., 1991; Armstrong et al., 1996; Cannon et al., 1996). Reducing concentrations of E. coli O157:H7 at the farm level would require decreasing the number of cattle shedding E. coli O157:H7 in feces or decreasing the magnitude of shedding by animals harboring the organism (Hancock et al., 1994). Therefore, controlling management practices that facilitate transmission of the organism to calves and adult cattle or affect the concentration of shedding is one means of controlling E. coli O157:H7 at the farm level.

A common practice on dairy farms is to feed milk replacer containing antibiotics to newborn calves to prevent disease and improve growth. Escherichia coli O157:H7 has been isolated from calves from farms that used orally administrated antibiotics in water and milk, but not from calves on farms where antibiotics were only occasionally used (Shere et al., 1998). The authors speculated that the use of antibiotics in calves might enhance the growth and persistence of E. coli O157:H7; however, the study was based on four dairy farms. Therefore, further research is necessary before conclusions of the effects of antibiotic supplementation can be made. The objective of this study was to compare the concentration and duration of fecal shedding of E. coli O157:H7 between calves fed milk replacer with or without antibiotic (oxytetracycline and neomycin) supplementation following oral inoculation of E. coli O157:H7.

	Materials and Methods

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Calves and Feeding
Eighteen 1-wk-old Holstein bull calves, weighing approximately 40 kg each, were purchased from a single commercial dairy and housed in individual pens with no calf-to-calf contact in one of two rooms in an Animal Biosafety Level 2 facility. Calves were assigned randomly within room to one of two treatment groups. Group 1 received milk replacer containing no antibiotic and Group 2 received milk replacer containing oxytetracycline (200 mg/kg) and neomycin (400 mg/kg) in the form of neomycin sulfate; this corresponds to approximately 2 mg of oxytetracycline/kg BW and 4 mg of neomycin/kg BW daily. Calves were bucket fed twice daily at 10% of BW/d, to a maximum of 2 L per feeding. Calves in both groups were offered water, hay, and a calf starter (18% CP, corn-based; without antibiotics) throughout the study.

The calves were allowed to acclimate to their new environment for 2 wk before inoculation and were tested four times over the 2-wk period to ensure that they were negative for E. coli O157:H7. Fecal samples were collected from the rectum of each calf. One gram of feces was added to 9 mL of gram-negative (GN) broth (Difco Laboratories, Detroit, MI) containing cefixime (0.05 mg/L; Dynal ASA, Oslo, Norway), cefsoludin (10 mg/L; Sigma Chemical Co., St. Louis, MO), and vancomycin (8 mg/L; Sigma), and then incubated (37°C, 6 h). Following incubation, immunomagnetic separation was performed according to manufacturer’s recommendations (Dynal ASA). The beads in suspension were plated onto sorbitol MacConkey agar (Difco Laboratories) and incubated (37°C, 18 h). If present, up to six sorbitol negative (gray) colonies were picked, cultured on blood agar plates, spot indole tested with Kovac’s reagent (BioMerieux Inc., Hazelwood, MO) for presumptive identification of E. coli, and tested for the O157 antigen by latex agglutination test (Oxoid Ltd., Basingstoke, U.K.). If any isolates were sorbitol negative, indole positive, and latex agglutination positive, a biochemical test strip (API 20E, BioMerieux Inc.) would then be used to confirm the isolate as E. coli.

Animal Inoculation and Sampling Schedule
Calves were inoculated with E. coli O157:H7, strain FRIK 1123, which was made resistant to nalidixic acid (NA) in the laboratory (20 µg/mL). The organism was grown in GN broth (37°C, 7 h; approximately 0.8 to 1.0 absorbance at 600 nm) and colony counts of the culture were done by spread plate technique. The calves were orally inoculated with 1 mL (3.6 x 10⁸ cfu/calf) of NA-resistant E. coli O157:H7 mixed with the milk replacer. Feces were collected from the rectum of each calf three times weekly for 8 wk beginning 2 d after inoculation.

Enumeration and/or Detection of NA-Resistant E. coli O157:H7
The NA-resistant E. coli O157:H7 in each sample was quantified as follows: 1 g of feces was added to 9 mL of GN broth, vortexed, and serially diluted (10^–1 to 10^–3) in peptone water, and then 0.1-mL aliquots from each dilution were spread plated, in triplicate, onto sorbitol MacConkey agar containing 20 µg of NA/mL. After incubation (37°C, 24 h), sorbitol-negative (gray) colonies were counted. One plate was randomly selected out of each dilution, and NA-resistant isolates were confirmed as O157 by latex agglutination.

Selective enrichment technique was used to detect the presence of E. coli O157:H7 when the direct-plating method was negative (detection limit <10² cfu/g). After taking the aliquot for direct plating, the remaining GN broth was incubated at 37°C for 6 h, and then 1 mL was transferred to 9 mL of GN broth and incubated for additional 18 to 24 h. A 0.1-mL aliquot from the incubated broth was spread plated on sorbitol MacConkey agar containing NA, incubated (37°C, 24 h), and the presence or absence of gray colonies was recorded as "yes" or "no." Three gray colonies were picked from each positive plate, streaked on blood agar, and then confirmed as O157 with the latex agglutination test.

At the end of the study (8 wk after inoculation), the calves were killed using sodium pentobarbital (10 mL/45.3 kg). At necropsy, gut contents were sampled from the rumen, omasum, abomasum, ileum, cecum, colon, and rectum. Tissue samples were also collected from the tonsils, retro-pharyngeal and mesenteric lymph nodes, and Peyer’s patches. The gut contents were processed as before to detect or quantify E. coli O157:H7. Tissue samples were cut into approximately 1-g pieces, suspended in GN broth, and homogenized for 1 min with a tissue homogenizer (Brinkman Instruments, Westbury, NY). The suspension was used for enumeration and/or detection of NA-resistant E. coli O157:H7.

Statistical Analysis
The study had three outcomes of interest. First, the proportion of calves shedding E. coli O157:H7 over time was compared between treatment groups (antibiotic or no antibiotic) using a GLM with a binomial distribution and a logit link (GENMOD procedure, SAS Inst., Inc., Cary, NC). The outcome was the presence or absence of E. coli O157:H7 by direct plating or enrichment. The independent variables were treatment, time, and treatment x time interaction. When the interaction term was significant (P 0.05) in the GLM, treatment and control groups were compared at each time point using individual ² tests with the P-values corrected for multiple comparisons using a Bonferroni adjustment.

In the second analysis, the concentration of fecal E. coli O157:H7 shedding was compared between treatment groups. The comparison was done using repeated-measures ANOVA (GLM procedure of SAS). The outcome was the logarithm of the concentration of fecal E. coli O157:H7 (cfu/g) for samples, where the concentration of E. coli O157 was positive at quantifiable levels. Samples positive only on enrichment were not included in this analysis. Antibiotic or no antibiotic was the treatment and time was the repeated measure. The time x treatment interaction was included as a fixed effect.

Finally, the duration of E. coli O157:H7 fecal shedding was compared between the two treatment groups using log-rank survival analysis (LIFETEST procedure of SAS). The outcome was the number of days until each calf stopped detectable shedding by quantification or enrichment. If an animal shed E. coli O157:H7 intermittently, the final date on which the organism was detected using direct plating or enrichment was used. Calves shedding at the end of the study were recorded as censored observations.

	Results

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All calves were negative for E. coli O157:H7 in feces prior to inoculation. Two calves from the group fed milk replacer without antibiotic supplementation were removed from the study because of respiratory disease requiring treatment; data from these calves were not included in the study. A wide variation in the magnitude and duration of fecal shedding of E. coli O157:H7 was observed among individual calves.

One calf in the control group shed E. coli O157:H7 throughout the study. The last calf in the antibiotic-fed group stopped shedding 2 wk before the end of the study. The percentage of calves shedding E. coli O157:H7, as detected by direct plating or enrichment, in each treatment group by sampling day are shown in Figure 1. There was a significant interaction between treatment and time (P < 0.001) over the course of the study. However, when comparing treatment groups at specific sampling days, the proportion of calves shedding fecal E. coli O157:H7 in the antibiotic-fed group was significantly higher (P < 0.001) than the no-antibiotic group only on d 6 and 10.

View larger version (17K): [in this window] [in a new window]   Figure 1. Percentage of calves shedding Escherichia coli O157:H7 in feces from calves fed milk replacer without (, n = 7) or with (•, n = 9) oxytetracycline and neomycin.

View larger version (24K): [in this window] [in a new window]   Figure 2. The mean concentrations of Escherichia coli O157:H7 in feces from positive samples from calves fed milk replacer without () or with (•) oxytetracycline and neomycin. The number of animals contributing to the mean for each data point is shown.

At necropsy, four samples from three calves contained E. coli O157:H7. Positive samples were obtained from the rumen and the omasum of one calf from the no-antibiotic group; this calf was shedding E. coli O157:H7 in the feces throughout the study. The concentrations of E. coli O157:H7 in the rumen and omasum in that calf were 2.6 x 10² and 8.6 x 10² cfu/g, respectively. Two calves from the antibiotic group had positive samples detected by enrichment, one from the retro-pharyngeal lymph node, and another from the Peyer’s patch.

	Discussion

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Oral inoculation of calves with E. coli O157:H7 has been used to measure the magnitude and duration of E. coli O157:H7 shedding by calves, and to investigate factors associated with fecal shedding of these bacteria. For instance, oral inoculation studies in calves have been used to examine the association between dietary stresses, such as fasting, and fecal shedding (Cray and Moon, 1995; Harmon et al., 1999). Oral inoculation of preweaned calves with E. coli O157:H7 in this study did not cause any clinical illness, which agrees with previous challenge studies that E. coli O157:H7 is generally not pathogenic to calves (Cray and Moon, 1995; Sanderson et al., 1999; Woodward et al., 1999). Fecal shedding of E. coli O157:H7 following oral inoculation was highly variable among the individual calves, which is in agreement with previous studies (Cray and Moon, 1995; Harmon et al., 1999; Sanderson et al., 1999). The intermittent fecal-shedding patterns observed in the current study are consistent with a previous challenge study in preweaned calves (Cray and Moon, 1995) and with observations in the field (Besser et al., 1997; Shere et al., 1998).

The percentage of calves shedding E. coli O157:H7 was higher in the antibiotic-fed group compared with the control group for two of the sampling times early in the study. This suggests that the antibiotic supplementation used (oxytetracycline and neomycin) may have enhanced the fecal shedding of E. coli O157:H7 in the preweaned calves. However, there was no significant difference between the concentration of E. coli O157:H7 in the positive calves or the survival time probabilities (duration of shedding) of E. coli O157:H7 for the antibiotic-fed group and the control group. This may mean that the influence of the antibiotics on E. coli O157:H7 shedding did not greatly increase the magnitude of shedding and that the effects were short-term.

Although susceptibility and resistance to E. coli O157:H7 strains with oxytetracycline have not been reported, a high prevalence of resistance to tetracycline has been reported in isolates recovered from cattle (Zhao et al., 1998b; Galland et al., 2001; Schroeder et al., 2002). We tested the NA-resistant E. coli O157:H7 strain used in this study (before passage in the calves) for susceptibility to oxytetracycline and neomycin by broth microdilution method. We observed that the strain was resistant to neomycin (minimum inhibitory concentration >40 µg/mL), oxytetracycline (minimum inhibitory concentration >40 µg/mL), and to the two antibiotics combined in a 1:2 ratio (minimum inhibitory concentration >40 µg/mL). The resistance of the strain could explain why antibiotics in the milk replacer increased the percentage of calves’ E. coli O157:H7 in the early sampling periods. It is possible that the antibiotics inhibited the competitive microorganisms in the gut and, because the E. coli O157:H7 strain was resistant, it was able to proliferate, thus enhancing the shedding of E. coli O157:H7 (Zhao et al., 1998a). However, any effects of the antibiotics were not maintained over the 8-wk study period.

Elder et al. (2002) reported that oral administration of neomycin sulfate at therapeutic doses to cattle that were naturally shedding E. coli O157:H7 reduced their fecal shedding to undetectable concentrations compared with controls. The objective of the Elder et al. (2002) study was to investigate short-term interventions, and the study did not report results after d 7 post-treatment. The apparent difference between this study and our results may be dose related, or may be due to the different strains of E. coli O157:H7 that were used. Our study used a laboratory strain that was resistant to neomycin, which may not be representative of wild-type strains. Results obtained in experimental inoculation studies should be repeated in natural challenge studies to verify the results.

Price et al. (2002) observed that treatment of calves experimentally inoculated with E. coli O157:H7 with tilmicosin resulted in an increase in fecal shedding of E. coli O157:H7 up to 5 d after inoculation, whereas ceftiofur resulted in a decrease by the second day. Longer-term shedding patterns were not included in this report, and additional antimicrobials tested (spectinomycin, tetracycline, and monensin) had no effect on fecal shedding of E. coli O157:H7.

Interestingly, E. coli O157:H7 was identified from two lymphoid tissue samples (Peyer’s patches, and retropharyngeal lymph node) from two different calves in the antibiotic-fed group. Escherichia coli O157:H7 has been isolated from tonsils and mesenteric lymph nodes in low numbers in several calf-challenge studies (Cray and Moon, 1995; Woodward et al., 1999), whereas Brown et al. (1997) did not isolate this organism from calf tonsils or intestinal lymph nodes. None of the studies recovered E. coli O157:H7 from other tissue samples (kidney, spleen, or liver), suggesting that the organism may not be invasive. However, Woodward et al. (1999) recovered E. coli O157:H7 from the lungs of four out of five calves inoculated with E. coli O157:H7. Isolation of E. coli O157:H7 from lymph node and Peyer’s patches may indicate the possibility of invasion to regional lymph nodes and the persistence of this organism in those tissues. In addition, calves may become carriers and shed E. coli O157:H7 intermittently due to the persistence of this organism in the tissues. Identification of E. coli O157:H7 in the retropharyngeal lymph node raises several issues: whether this organism is naturally present in cattle head lymph nodes, which would suggest the need to study the prevalence of E. coli O157:H7 in the lymph nodes, and the necessity for inspection for E. coli O157:H7 in those lymph nodes at slaughter houses.

	Implications

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Escherichia coli O157:H7 is a significant cause of food-borne illness in humans, with cattle commonly implicated in the causal chain. Results of this study indicate that the common use of antibiotic supplementation with oxytetracycline and neomycin in calf milk replacer has limited effects on fecal shedding of E. coli O157:H7. A laboratory strain of the bacteria was used in this study. Further studies using natural challenge with wild-type strains are needed to validate these results.

	Footnotes

 
¹ This research was supported by USDA Grant #99-34359-7474 and is contribution No. 04-066J from the Kansas Agric. Exp. Stn. The authors thank A. Hanson and X. Shi for technical assistance in the laboratory.

² Correspondence and current address: McMaster Univ., HSC 2C15, 1200 Main St. West, Hamilton, ON, Canada L8N 3Z5 (phone: 905-525-9140, ext. 22127; fax: 905-577-0017; e-mail: sargeaj{at}mcmaster.ca).

Received for publication September 15, 2003. Accepted for publication March 22, 2004.

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