John C. Galland

Prevalence, Antibiotic Susceptibility, and Diversity of Escherichia coli O157:H7 Isolates from a Longitudinal Study of Beef Cattle Feedlots

ABSTRACT Prevalence, antibiotic susceptibility, and genetic diversity were determined for Escherichia coli O157:H7 isolated over 11 months from four beef cattle feedlots in southwest Kansas. From the fecal pat (17,050) and environmental (7,134) samples collected, 57 isolates of E. coli O157:H7 were identified by use of bacterial culture and latex agglutination (C/LA). PCR showed that 26 isolates were eaeA gene positive. Escherichia coli O157:H7 was identified in at least one of the four feedlots in 14 of the 16 collections by C/LA and in 9 of 16 collections by PCR, but consecutive positive collections at a single feedlot were rare. Overall prevalence in fecal pat samples was low (0.26% by C/LA, and 0.08% by PCR). No detectable differences in prevalence or antibiotic resistance were found between isolates collected from home pens and those from hospital pens, where antibiotic use is high. Resistant isolates were found for six of the eight antibiotics that could be used to treat E. coli infections in food animals, but few isolates were multidrug resistant. The high diversity of isolates as measured by random amplification of polymorphic DNA and other characteristics indicates that the majority of isolates were unique and did not persist at a feedlot, but probably originated from incoming cattle. The most surprising finding was the low frequency of virulence markers among E. coli isolates identified initially by C/LA as E. coli O157:H7. These results demonstrate that better ways of screening and confirming E. coli O157:H7 isolates are required for accurate determination of prevalence.

Comparison of Cultivation and PCR-Hybridization for Detection of Salmonella in Porcine Fecal and Water Samples

ABSTRACT A total of 150 fecal and water samples from four swine farms were tested for the presence of Salmonella enterica using different enrichment techniques as follows: (i) 92 fecal samples from nursery and farrowing barns at three swine farms were preenriched overnight in tryptic soy broth (TSB) at 37°C followed by overnight enrichment in Rappaport-Vassiliadis 10 broth (RV10) at 42°C; (ii) 24 water samples from the third farm were preenriched overnight in 3MC broth at 37°C followed by overnight enrichment in RV10 at 42°C; and (iii) 34 fecal samples from a fourth farm, a finishing farm, were enriched overnight in RV10 at 42°C with no additional enrichment. Following each of the enrichment techniques, samples were subcultured onto modified semisolid Rappaport-Vassiliadis (MSRV) agar prior to transfer to Hektoen Enteric agar plates for the recovery of viableSalmonella bacteria. Presumptive Salmonellaisolates were biochemically and serologically confirmed. For the PCR detection of Salmonella, a 1-ml portion was removed from each sample after the first overnight enrichment and the DNA was extracted using a Sepharose CL-6B spin column. Amplicons (457 bp) derived from primers to the invA and invE genes were confirmed as Salmonella specific on ethidium bromide-stained agarose gels by Southern hybridization with a 20-mer oligonucleotide probe specific for the Salmonella invAgene. Neither the standard microbiological method nor the molecular method detected all of the 65 samples that tested positive by both methods or either method alone. Salmonella bacteria were detected by both cultivation and PCR-hybridization in 68% (17 of 25) of the positive samples that were preenriched in TSB, in 73% (11 of 15) of the positive samples preenriched in 3MC broth, and in 24% (6 of 25) of the positive samples enriched in RV10. Agreement betweenSalmonella detection using cultivation with preenrichment and detection by PCR was 76% using the kappa statistic. However, agreement between Salmonella detection using cultivation without preenrichment and detection by PCR was about 6%; the PCR assay detected 80% (20 of 25) of the 25 positive samples, whileSalmonella bacteria were recovered from only 44% (11 of 25) by cultivation. Our results indicate that the PCR-hybridization approach is equivalent to or better than cultivation for detectingSalmonella in swine feces or water samples from swine farms when using the medium combinations evaluated in this study.

Prevalence of Salmonella in beef feeder steers as determined by bacterial culture and ELISA serology.

Results are presented for monitoring Salmonella infection by bacteriological culture and immune response (enzyme linked immunosorbent assay (ELISA) and haptoglobin) testing of samples collected from beef cattle at a single feedyard sampled over time. A total of 120 beef steers were examined on entry to the feedyard and at days 30, 60, and at time of slaughter (120-150 days). Isolations of Salmonella decreased over time from 40% of the steers sampled at day 0 to 0% at slaughter, whereas serological results varied by serogroup. Seropositivity increased for Salmonella group B up to day 60, and subsequently decreased to about half of the 60-day positivity rate at the time of slaughter. No significant changes in seropositivity were detected during the course of the study for the four other Salmonella serogroups (C1, C3, D1, and E1). Haptoglobin measurements were not a good indicator of Salmonella infection status. Sequential Salmonella testing either by culture, ELISA, or both could be used to monitor pathogen control practices.

Restriction-Site-Specific PCR as a Rapid Test To Detect Enterohemorrhagic Escherichia coli O157:H7 Strains in Environmental Samples

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen in industrialized countries. We developed a rapid and simple test for detecting E. coli O157:H7 using a method based on restriction site polymorphisms. Restriction-site-specific PCR (RSS-PCR) involves the amplification of DNA fragments using primers based on specific restriction enzyme recognition sequences, without the use of endonucleases, to generate a set of amplicons that yield “fingerprint” patterns when resolved electrophoretically on an agarose gel. The method was evaluated in a blinded study of E. coli isolates obtained from environmental samples collected at beef cattle feedyards. The 54 isolates were all initially identified by a commonly used polyclonal antibody test as belonging to O157:H7 serotype. They were retested by anti-O157 and anti-H7 monoclonal antibody enzyme-linked immunosorbent assay (ELISA). The RSS-PCR method identified all 28 isolates that were shown to be E. coli O157:H7 by the monoclonal antibody ELISA as belonging to the O157:H7 serotype. Of the remaining 26 ELISA-confirmed non-O157:H7 strains, the method classified 25 strains as non-O157:H7. The specificity of the RSS-PCR results correlated better with the monoclonal antibody ELISA than with the polyclonal antibody latex agglutination tests. The RSS-PCR method may be a useful test to distinguish E. coli O157:H7 from a large number ofE. coli isolates from environmental samples.

Usefulness of a commercially available enzyme immunoassay for Shiga-like toxins I and II as a presumptive test for the detection of Escherichia coli O157:H7 in cattle feces.

The performance of a commercially available enzyme immunoassay (EIA) for determining the presence of Shiga toxin I and II in human diarrheal stool samples was evaluated for use as a presumptive test for the presence of Escherichia coli O157:H7 in nondiarrheal bovine fecal samples collected from 10 Kansas cow–calf ranches. The prevalence of E. coli O157:H7 in 2,297 samples, as determined by selective bacterial culture, was 1.6%. The sample prevalence of non-E. coli O157:H7 Shiga toxin–producing bacteria, as detected by the Shiga toxin EIA, was 5.8%. Only 2 of 136 samples that tested positive with the Shiga toxin EIA were positive for E. coli O157:H7 by culture. Compared with bacterial culture, the sensitivity of the Shiga toxin EIA was 5.5% and the specificity was 94.1%. Agreement between the 2 tests, as measured by the kappa statistic, was poor (κ = −;0.002). Although the Shiga toxin EIA was not a good presumptive test for the determination of E. coli O157:H7 in bovine fecal samples because of its low sensitivity (5.5%), it might be a useful test for the detection of Shiga toxin producing non-E. coli O157:H7 organisms in bovine feces.

Prevalence of Escherichia coli O157:H7 in cow-calf herds in Kansas

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Effects of tetracycline on shedding of susceptible and resistant salmonella spp. experimentally inoculated into pigs

This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 1997 Kansas State University Agricultural Experiment Station and Cooperative Extension Service.

Effects of antibiotics on shedding of salmonella typhimurium in experimentally inoculated pigs

This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 1996 Kansas State University Agricultural Experiment Station and Cooperative Extension Service.