Effects of antibiotic use in sows on resistance of E. coli and Salmonella enterica Typhimurium in their offspring.

To determine effects of exposure of parental animals to antibiotics on antibiotic resistance in bacteria of offspring, sows were either treated or not treated with oxytetracycline prior to farrowing and their pigs were challenged with Salmonella enterica Typhimurium and treated or not treated with oxytetracycline and apramycin. Fecal Escherichia coli were obtained from sows, and E. coli and salmonella were recovered from pigs. Antibiotic resistance patterns of isolates were determined using a minimum inhibitory concentration (MIC) analysis. Polymerase chain reaction (PCR) and electroporation were used to characterize the genetic basis for the resistance and to determine the location of resistance genes. Treatments had little effect on resistance of the salmonella challenge organism. The greatest resistance to apramycin occurred in E. coli from pigs treated with apramycin and whose sows had earlier exposure to oxytetracycline. Resistance to oxytetracycline was consistently high throughout the study in isolates from all pigs and sows; however, greater resistance was noted in pigs nursing sows that had previous exposure to that drug. The aac(3)-IV gene, responsible for apramycin resistance, was found in approximately 90% of apramycin-resistant isolates and its location was determined to be on plasmids. Several resistant E. coli bio-types were found to contain the resistance gene. These results indicate that resistance to apramycin and oxytetracycline in E. coli of pigs is affected by previous use of oxytetracycline in sows.

Detection of Salmonella enterica somatic groups C1 and E1 by PCR-enzyme-linked immunosorbent assay.

Objectives of this study were to develop a PCR-based enzyme-linked immunosorbent assay (PCR-ELISA) for identification of Salmonella enterica somatic groups C1 and E1 and to evaluate this procedure along with a PCR-ELISA procedure for S. enterica somatic groups B, C2, and D in a masked study. Primers were selected from the rfb gene cluster, which is responsible for biosynthesis of O antigens of Salmonella lipopolysaccharide. Previously serogrouped Salmonella isolates (n = 169) were used to determine the sensitivity and specificity of the PCR-ELISA procedure. DNA from all isolates was amplified using the PCR procedure for selected somatic groups and amplified products were visualized on agarose gels, as well as subjected to the ELISA procedure. The PCR-ELISA technique correctly identified 97% of somatic group C1 and 87% of somatic group E1. The sensitivity of this procedure to correctly identify S. enterica somatic group C1 was 97% and 88% for somatic group E1. The specificity was 98% for both somatic groups C1 and E1. The PCR-ELISA techniques correctly identified 93% of Salmonella isolates belonging to somatic groups B, C1, C2, D, and E1. The overall sensitivity of this procedure to correctly identify S. enterica somatic groups was 96% and the specificity was 98%. Ninety-one percent of somatic group D, 92% of somatic group B, and 97% of somatic group C2 were identified correctly with this procedure. Results of this study indicate that the PCR-ELISA procedure is a rapid and accurate method for serogrouping Salmonella isolates. Utilization of the PCR-ELISA procedure for Salmonella serogrouping would aide in identification, surveillance, prevention, and control of Salmonella.

Characterization of microbial populations and volatile fatty acid concentrations in the jejunum, ileum, and cecum of pigs weaned at 17 vs 24 days of age.

In a series of five 17-d replicate trials, a total of 54 cannulated and 12 noncannulated pigs were used to determine the effects of weaning age (17 d or 24 d) on pH, dry matter percentage, aerobic and anaerobic microflora, lactate, and volatile fatty acid (VFA) concentrations in the jejunum, ileum, and cecum of weanling pigs. At -14 d of age, cannulated pigs were surgically fitted with T-cannulas in the jejunum (n = 20), ileum (n = 18), or cecum (n = 16). Upon weaning, cannulated pigs were individually caged in an environmentally controlled room with ad libitum access to a phase starter diet and water. Noncannulated pigs were killed at weaning and samples were collected from the jejunum, ileum, and cecum. Digesta and fecal swabs from cannulated pigs were collected twice weekly. The pH of cecal contents was lower (P < 0.05) and dry matter percentage was greater (P < 0.05) than those ofjejunal or ileal contents. Pigs weaned at 24 d of age had increased (P < 0.05) E. coli populations 3 d postweaning compared to preweaning populations, regardless of site of collection, whereas this increase was not observed in pigs weaned at 17 d of age. Unweaned pigs maintained higher (P < 0.05) lactobacilli populations compared to weaned pigs; however, populations declined (P < 0.05) in both groups by 3 d postweaning, with pigs weaned at 24 d of age having lactobacilli populations greater than pigs weaned at 17 d of age. Fecal populations of E. coli and lactobacilli declined (P < 0.05), whereas fecal bifidobacteria populations increased (P < 0.05) postweaning, regardless of weaning age. Concentrations of total fecal anaerobes declined (P < 0.05) in pigs weaned at 17 d of age but were maintained in pigs weaned at 24 d of age. Volatile fatty acid concentrations were greater (P < 0.05) in the cecum than in the jejunum or ileum, and acetic acid concentrations decreased (P < 0.05) postweaning regardless of weaning age. A tendency for L+ lactate concentrations to be greater (P < 0.07) in the ileum and jejunum vs the cecum was observed. Results indicate that weaning and weaning age have significant effects on microbial populations and VFA concentrations.

Three molecular methods to identify Salmonella enterica serotype Typhimurium DT104: PCR fingerprinting, multiplex PCR and rapid PFGE.

Here we report the accuracy with which three molecular techniques (PCR fingerprinting, multiplex PCR and macrorestriction profiling) distinguished Salmonella enterica Typhimurium DT104 (hereafter referred to as DT104) from other related strains of Salmonella. Each technique was tested by screening a set of 20 isolates (10 DT104, eight non-DT104 Typhimurium, one S. enterica Agona, one S. enterica Newport) and each consistently differentiated DT104 from non-DT104 isolates based on visual inspection of band patterns. The accuracy of each technique was confirmed by computer analysis. As such, these data indicate that each technique could be used to presumptively identify DT104 isolates as a precursor to phage typing.

Evaluation of diet as a cause of gastric ulcers in horses.

OBJECTIVE: To measure pH, volatile fatty acid (VFA) concentrations, and lactate concentrations in stomach contents and determine number and severity of gastric lesions in horses fed bromegrass hay and alfalfa hay-grain diets. ANIMALS: Six 7-year-old horses. PROCEDURE: A gastric cannula was inserted in each horse. Horses were fed each diet, using a randomized crossover design. Stomach contents were collected immediately after feeding and 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, and 24 hours after feeding on day 14. The pH and VFA and lactate concentrations were measured in gastric juice Number and severity of gastric lesions were scored during endoscopic examinations. RESULTS: The alfalfa hay-grain diet caused significantly higher pH in gastric juice during the first 5 hours after feeding, compared with that for bromegrass hay. Concentrations of acetic, propionic, and isovaleric acid were significantly higher in gastric juice, and number and severity of nonglandular squamous gastric lesions were significantly lower in horses fed alfalfa hay-grain. Valeric acid, butyric acid, and propionic acid concentrations and pH were useful in predicting severity of nonglandular squamous gastric lesions in horses fed alfalfa hay-grain, whereas valeric acid concentrations and butyric acid were useful in predicting severity of those lesions in horses fed bromegrass hay. CONCLUSIONS AND CLINICAL RELEVANCE: An alfalfa hay-grain diet induced significantly higher pH and VFA concentrations in gastric juice than did bromegrass hay. However, number and severity of nonglandular squamous gastric lesions were significantly lower in horses fed alfalfa hay-grain. An alfalfa hay-grain diet may buffer stomach acid in horses.

Effects of antibiotic regimens on the fecal shedding patterns of pigs infected with salmonella typhimurium.

An experiment was conducted to determine (i) the effects of antibiotic regimens on the shedding patterns of pigs infected with Salmonella Typhimurium and (ii) whether antibiotic resistance increases the incidence of pathogen shedding. The experiment involved 48 50-day-old pigs challenged with Salmonella Typhimurium and receiving one of four antibiotic regimens including (i) intramuscular injection of ceftiofur sodium followed by inclusion of oxytetracycline in the feed; (ii) apramycin in the feed for 14 days followed by oxytetracycline; (iii) carbadox in the feed until pigs reached 35 kg followed by oxytetracycline; (iv) no antibiotics (control). Fecal samples were collected preinoculation, 2 and 4 days postinoculation (DPI) and at weekly and biweekly intervals thereafter to determine shedding patterns. Salmonella Typhimurium isolates from 2, 4, 7, 21, 42, and 70 DPI were analyzed for antibiotic resistance. A time effect (P < 0.05) was observed, indicating that the proportion of isolates resistant to at least one antibiotic varied over time. Overall resistance was determined to be 46% at 2 DPI and increased significantly (P < .05) thereafter. Treatment x time and antibiotic x time interactions were also observed (P < 0.05) as the percentage of isolates resistant to each test antibiotic increased over time. In no case did the development of antibiotic resistance result in an increased incidence of shedding of the original inoculate. The incidence of shedding was reduced in pigs receiving the apramycin-oxytetracycline treatment, when compared to control pigs; however, no differences were observed between antibiotic treatments.

Complications of ileal cannulation in cats.

BACKGROUND AND PURPOSE: The anatomic position of the ileum is such that use of simple, noninvasive sampling techniques is difficult. Sampling of ileal contents by use of a cannula provides information about those contents-digestibility and fermentation patterns; however, complications with use of cannulas in horses and dogs have been reported. Therefore, cats were chosen as a laboratory model in which to study ileal cannulation. METHODS: Simple T-piece cannulas were surgically placed in the ileum of six adult cats. RESULTS: Postsurgical complications included cannula displacement and leakage, with subsequent abscessation and skin inflammation. Owing to these complications, the cannulas were removed from four of the six cats. Similar complications developed after removal of the cannulas. In the only attempt to collect a sample, obtaining a 0.5-ml sample of ileal fluid from one cannula took 3 h. Of the six cats, four died or were euthanized. CONCLUSION: Use of cannulas is not a viable technique of collecting ileal fluid samples in cats.

Multiple antibiotic resistance patterns of Escherichia coli isolates from swine farms.

Antibiotic resistance of Escherichia coli from sows and pigs was determined to compare patterns between pigs of various ages and degrees of antibiotic use. Resistance patterns differed between farm types and pigs of differing ages, indicating that pig age and degree of antibiotic use affect resistance of fecal E. coli.

Effects of a direct-fed yeast culture on enteric microbial populations, fermentation acids, and performance of weanling pigs.

In three replicate trials, a total of 36 pigs that had been cannulated at the terminal ileum were used to determine the effects of a Saccharomyces cerevisiae culture in a phase feeding program (phase I was d 0 to 7 and phase II was d 8 to 21) on performance, ileal microflora, and short-chain fatty acids in weanling pigs. Pigs were cannulated at approximately 12 d of age, weaned at 17 d of age, and randomly assigned to one of three treatments: 1) a pelleted phase feeding program, 2) a similar program with the inclusion of a live S. cerevisiae culture (1 g/ kg), and 3) a nonpelleted feeding program otherwise similar to program 2. Ileal samples were collected at 17, 20, 24, 27, 31, 34, and 38 d of age, and samples were analyzed for total E. coli, streptococci, lactobacilli, yeast, short-chain fatty acids, pH, and dry matter. Performance data were also collected. At 41 d of age, pigs were killed and digesta were collected from various regions of the gastrointestinal tract. Total intake was less for pigs fed the control diet than for pigs fed the yeast diets, and overall gains tended to be greater for pigs fed diets including yeast. Treatment differences were not observed for ileal microflora or short-chain fatty acids in samples obtained from cannulas or from the various sites of the gastrointestinal tract. Inclusion of a live yeast culture in weanling pig diets affected intake and performance but did not alter tested intestinal microflora or net concentrations of fermentation products.

Incidence of antibiotic resistance in fecal Escherichia coli isolated from commercial swine farms.

Sows and pigs from 10 commercial swine farms were sampled to determine patterns of resistance of fecal Escherichia coli to five commonly used antibiotics. Before testing, farms were categorized as high or low antibiotic use based on interviews with the respective producers. On each farm, fecal swabs were obtained from five sows at 7 d postpartum and from five pigs from each sow at 7, 35, and 63 d of age. A total of 6,296 E. coli isolates from 750 pig fecal samples and a total of 462 E. coli isolates from 50 sow fecal samples were tested against apramycin, carbadox, gentamicin, neomycin, and oxytetracycline using a standardized disk diffusion test. Percentage of resistant organisms was compared between pigs at the various stages of growth, between sows and their respective pigs, and between farms of high and low antibiotic use. Incidence of resistance was greater (P < .05) in pigs at 35 or 63 d of age compared with 7 d of age for most antibiotics, and patterns differed between high-use and low-use farms. Resistance differed (P < .05) among E. coli from pigs compared to their respective sows for apramycin and carbadox on low-use farms, whereas incidence of resistance on high-use farms differed (P < .05) between sows and pigs for apramycin and oxytetracycline. These data indicate that patterns of antibiotic resistance are dependent on age of pig and level of antibiotic use.

Influence of galactosyl lactose on energy and protein digestibility, enteric microflora, and performance of weanling pigs.

In three experiments, weanling pigs were used to investigate the effect of feeding .5% galactosyl lactose in a simple corn-soy diet on nutrient digestibility, selected ileal microbial concentrations, fermentation acids, and performance. In Exp. 1, 20 21-d-old weaned pigs in two replicate trials were fed either a 19% CP corn and soybean meal diet or a similar diet containing .5% galactosyl lactose. Feces and urine were collected over a 3-wk period to determine apparent CP and energy digestibilities. Apparent digestibilities of CP and energy were not different between treatments. In Exp. 2, 24 14-d-old nursing pigs were cannulated at the terminal ileum and fed the diets described above after weaning at 21 d of age. Ileal samples were collected at 20, 23, 27, 30, 34, 37, and 41 d of age. Samples were assayed for VFA, lactate, and selected microflora. Ileal fermentation acid concentrations and selected microflora were affected by age of pig but were not different between feed treatments. In Exp. 3, 91 21- to 24-d-old weaned pigs were fed the control and test diets already described; intake, BW gain, and feed efficiency were determined for 9 wk after weaning. Intake was greater in pigs fed galactosyl lactose, but ADG and gain/feed did not differ between treatments. Inclusion of galactosyl lactose in weanling pig diets did not affect protein or energy digestibility, microflora, short-chain fatty acids, or performance of young pigs.

A PCR-based method of detection and differentiation of K88+ adhesive Escherichia coli.

The objective of this study was to develop a polymerase chain reaction (PCR)-based method to detect and differentiate among Escherichia coli strains containing genes for the expression of 3 antigenic variants of the fimbrial adhesin K88 (K88ab, K88ac, and K88ad). Five primers were designed that allowed detection of K88+ E. coli, regardless of antigenic variant, and the separate detection of the ab, ac, and ad variants. Primers AM005 and AM006 are 21 base pair (bp) oligomers that correspond to a region of the K88 operon that is common to all 3 antigenic variants. Primers MF007, MF008, and MF009 are 24-bp oligomers that matched variable regions specific to ab, ac, and ad, respectively. Using primers AM005 and AM006, a PCR product was obtained that corresponds to a 764-bp region within the large structural subunit of the K88 operon common to all 3 antigenic variants. Primer AM005 used with MF007, MF008, or MF009 produced PCR products approximately 500-bp in length from within the large structural subunit of the K88 operon of the 3 respective antigenic variants. Fragments were identified by rates of migration on a 1% agarose gel relative to each other as well as to BstEII-digested lambda fragments. This PCR-based method was comparable to the enzyme-linked immunosorbent assay and western blot test in the ability to differentiate between the antigenic variants. K88+ E. coli were differentiated from among laboratory strains and detected in ileal samples taken from cannulated pigs challenged with a known K88+ variant. K88+ E. coli were also detected from fecal swabs taken from newly weaned pigs, thus confirming that this PCR-based test could provide a convenient clinical assay for the detection of K88+ E. coli. Detection and differentiation of K88+ E. coli using general and specific primers was successful. PCR methods of detection should permit identification of K88+ antigenic variants regardless of the level of expression of the antigen.

Effect of creep feeding on selected microflora and short-chain fatty acids in the ileum of weanling pigs.

Pigs cannulated at the ileum were used to investigate the effect of creep feeding on specific intestinal microflora and VFA and lactate concentrations. In two replicate trials performed 50 d apart, a total of 24 15-d-old nursing pigs from seven sows were cannulated in the terminal ileum and randomly assigned to two test groups. One group was allowed access to a creep diet, whereas the other group was denied access to the diet by removal from the farrowing crate. Ileal samples were collected at 19, 22, 26, and 29 d of age (preweaning) and at 33, 36, and 40 d of age (postweaning). Samples were assayed for total and hemolytic E. coli, total lactobacilli, pH, acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, D(-), and L(+) lactate. Creep feeding did not affect lactobacilli or E. coli concentrations (P > .05), nor did it affect short-chain fatty acid concentrations (P > .05). Most volatile fatty acid concentrations decreased (P < .05), total lactate increased (P < .05), and the proportion of E. coli that were hemolytic increased (P < .05) following weaning. Consumption of creep feed did not affect specific microbial populations or fermentative byproducts in the ileum of the young pig.

Effect of galactan on selected microbial populations and pH and volatile fatty acids in the ileum of the weanling pig.

Studies were conducted to determine the effect of galactan on the colonization of E. coli and lactobacilli and ileal pH and volatile fatty acid production in the digestive tract of the weanling pig. In each of two replicate trials, eight 21-d-old nursing pigs were cannulated in the terminal ileum. After a 7-d recovery period, the pigs were weaned and randomly assigned to two test diets: 1) a corn-soybean meal-based control diet and 2) a similar diet containing 1% galactan. On d 1 after weaning, all pigs were orally subjected to K88+ E. coli (2 x 10(9) colony forming units). Ileal digesta samples were collected on d 0, 2, 4, 6, 8, and 10 after weaning and assayed for total E. coli, K88+ E. coli, lactobacilli, pH, and VFA. At the end of the trials, the pigs were killed and digesta samples were collected from the stomach, duodenum, cecum, and colon. Assays similar to those performed on the digesta samples collected from ileal cannulas were performed. Pigs fed 1% galactan had lower (P < .10) ileal pH, lower (P < .05) total E. coli on d 6 and 8, and lower (P < .05) K88+ E. coli concentrations in the ileum than pigs fed the control diet. There were no differences in ADG or gain:feed ratio between diets. The VFA concentrations were not different in the ileum between diets. The VFA were higher (P < .10) on d 0 than on any other day of the study. Acetate and isobutyrate concentrations were lower (P < .10) in the cecum in pigs fed 1% galactan.(ABSTRACT TRUNCATED AT 250 WORDS)