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James L Bono,
Timothy P L Smith, James E Keen,
Gregory P Harhay,
Tara G McDaneld,
Robert E Mandrell,
Woo Kyung Jung,
Thomas E Besser,
Peter Gerner-Smidt,
Martina Bielaszewska,
Helge Karch,
Michael L Clawson
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ABSTRACT: Cattle are a major reservoir for Shiga toxin-producing Escherichia coli O157 (STEC O157) and harbor multiple genetic subtypes that do not all associate with human disease. STEC O157 evolved from an E. coli O55:H7 progenitor; however, a lack of genome sequence has hindered investigations on the divergence of human- and/or cattle-associated subtypes. Our goals were to 1) identify nucleotide polymorphisms for STEC O157 genetic subtype detection, 2) determine the phylogeny of STEC O157 genetic subtypes using polymorphism-derived genotypes and a phage insertion typing system, and 3) compare polymorphism-derived genotypes identified in this study with pulsed field gel electrophoresis (PFGE), the current gold standard for evaluating STEC O157 diversity. Using 762 nucleotide polymorphisms that were originally identified through whole-genome sequencing of 189 STEC O157 human- and cattle-isolated strains, we genotyped a collection of 426 STEC O157 strains. Concatenated polymorphism alleles defined 175 genotypes that were tagged by a minimal set of 138 polymorphisms. Eight major lineages of STEC O157 were identified, of which cattle are a reservoir for seven. Two lineages regularly harbored by cattle accounted for the majority of human disease in this study, whereas another was rarely represented in humans and may have evolved toward reduced human virulence. Notably, cattle are not a known reservoir for E. coli O55:H7 or STEC O157:H(-) (the first lineage to diverge within the STEC O157 serogroup), which both cause human disease. This result calls into question how cattle may have originally acquired STEC O157. The polymorphism-derived genotypes identified in this study did not surpass PFGE diversity assessed by BlnI and XbaI digestions in a subset of 93 strains. However, our results show that they are highly effective in assessing the evolutionary relatedness of epidemiologically unrelated STEC O157 genetic subtypes, including those associated with the cattle reservoir and human disease.
Molecular Biology and Evolution 02/2012; 29(8):2047-62. · 5.55 Impact Factor
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Michael P Heaton,
Michael L Clawson,
Carol G Chitko-Mckown,
Kreg A Leymaster,
Timothy P L Smith,
Gregory P Harhay,
Stephen N White,
Lynn M Herrmann-Hoesing,
Michelle R Mousel,
Gregory S Lewis,
Theodore S Kalbfleisch, James E Keen,
William W Laegreid
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ABSTRACT: Visna/Maedi, or ovine progressive pneumonia (OPP) as it is known in the United States, is an incurable slow-acting disease of sheep caused by persistent lentivirus infection. This disease affects multiple tissues, including those of the respiratory and central nervous systems. Our aim was to identify ovine genetic risk factors for lentivirus infection. Sixty-nine matched pairs of infected cases and uninfected controls were identified among 736 naturally exposed sheep older than five years of age. These pairs were used in a genome-wide association study with 50,614 markers. A single SNP was identified in the ovine transmembrane protein (TMEM154) that exceeded genome-wide significance (unadjusted p-value 3×10(-9)). Sanger sequencing of the ovine TMEM154 coding region identified six missense and two frameshift deletion mutations in the predicted signal peptide and extracellular domain. Two TMEM154 haplotypes encoding glutamate (E) at position 35 were associated with infection while a third haplotype with lysine (K) at position 35 was not. Haplotypes encoding full-length E35 isoforms were analyzed together as genetic risk factors in a multi-breed, matched case-control design, with 61 pairs of 4-year-old ewes. The odds of infection for ewes with one copy of a full-length TMEM154 E35 allele were 28 times greater than the odds for those without (p-value<0.0001, 95% CI 5-1,100). In a combined analysis of nine cohorts with 2,705 sheep from Nebraska, Idaho, and Iowa, the relative risk of infection was 2.85 times greater for sheep with a full-length TMEM154 E35 allele (p-value<0.0001, 95% CI 2.36-3.43). Although rare, some sheep were homozygous for TMEM154 deletion mutations and remained uninfected despite a lifetime of significant exposure. Together, these findings indicate that TMEM154 may play a central role in ovine lentivirus infection and removing sheep with the most susceptible genotypes may help eradicate OPP and protect flocks from reinfection.
PLoS Genetics 01/2012; 8(1):e1002467. · 8.69 Impact Factor
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ABSTRACT: To compare methods for identification of bulls that were carriers for Tritrichomonas foetus during an outbreak on a large beef ranch and determine whether the percentage of nonpregnant cows was associated with the percentage of bulls infected with T foetus.
Epidemiological study.
121 Angus and Hereford bulls (1.5 to 6 years old) and 2,960 Angus-cross cows (2.5 to 14 years old) managed as 5 herds on a Nebraska beef ranch.
3 sequential preputial scrapings collected from the bulls at 12- to 27-day intervals were cultured, and cultures were examined for live T foetus daily for 5 days. On day 5, aliquots of the culture fluid were tested by means of T foetus-specific gel and real-time PCR assays. Cows were tested for pregnancy by means of rectal palpation.
For 361 preputial scrapings obtained from 121 bulls, results of culture and gel PCR assay were in close agreement. The real-time PCR assay had similar sensitivity to culture and the gel PCR assay but generated more false-positive results. Twenty-four of the 121 (19.8%) bulls were identified as infected with T foetus. For the 5 ranch herds, there was a positive linear correlation between percentage of infected bulls (range, 0% to 40%) and percentage of nonpregnant cows (range, 8.3% to 19.2%).
Results suggested that a combination of culture and the gel PCR assay performed on 3 sequential preputial scrapings was the best method for identifying bulls that were carriers for T foetus during this herd outbreak.
Journal of the American Veterinary Medical Association 11/2010; 237(9):1068-73. · 1.79 Impact Factor
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ABSTRACT: Shiga-toxigenic Escherichia coli (STEC) O157 occurrence was determined along the entire gastrointestinal tract (GIT) of each of four naturally shedding cattle and at three sites in 61 slaughter cattle. STEC O157 was distributed along the entire GIT, though interanimal distribution was variable. Neither feces nor rectoanal-junction samples accurately predicted the STEC O157-negative status of any particular animal.
Applied and environmental microbiology 08/2010; 76(15):5278-81. · 3.69 Impact Factor
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ABSTRACT: The intestinal microbiota of beef cattle are important for animal health, food safety, and methane emissions. This full-length sequencing survey of 11,171 16S rRNA genes reveals animal-to-animal variation in communities that cannot be attributed to breed, gender, diet, age, or weather. Beef communities differ from those of dairy. Core bovine taxa are identified.
Applied and environmental microbiology 07/2010; 76(14):4858-62. · 3.69 Impact Factor
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ABSTRACT: The objectives of the study described here were (i) to investigate the dynamics of Escherichia coli O157:H7 fecal and hide prevalence over a 9-month period in a feedlot setting and (ii) to determine how animals shedding E. coli O157:H7 at high levels affect the prevalence and levels of E. coli O157:H7 on the hides of other animals in the same pen. Cattle (n = 319) were distributed in 10 adjacent pens, and fecal and hide levels of E. coli O157:H7 were monitored. When the fecal pen prevalence exceeded 20%, the hide pen prevalence was usually (25 of 27 pens) greater than 80%. Sixteen of 19 (84.2%) supershedder (>10(4) CFU/g) pens had a fecal prevalence greater than 20%. Significant associations with hide and high-level hide (>/=40 CFU/100 cm(2)) contamination were identified for (i) a fecal prevalence greater than 20%, (ii) the presence of one or more high-density shedders (>/=200 CFU/g) in a pen, and (iii) the presence of one or more supershedders in a pen. The results presented here suggest that the E. coli O157:H7 fecal prevalence should be reduced below 20% and the levels of shedding should be kept below 200 CFU/g to minimize the contamination of cattle hides. Also, large and unpredictable fluctuations within and between pens in both fecal and hide prevalence of E. coli O157:H7 were detected and should be used as a guide when preharvest studies, particularly preharvest intervention studies, are designed.
Applied and environmental microbiology 09/2009; 75(20):6515-23. · 3.69 Impact Factor
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ABSTRACT: To evaluate risk behaviors for transmission of zoonotic diseases at petting zoos during a period without a recognized disease outbreak.
Observational survey with environmental microbiologic sampling.
6 petting zoos in Tennessee.
Attendees were observed for animal and environmental contact, eating or drinking, hand-to-face contact, and use of a hand sanitizer. Hands were examined via bacteriologic culture on some attendees. Environmental samples were collected at three petting zoos.
991 attendees were observed; of these, 74% had direct contact with animals, 87% had contact with potentially contaminated surfaces in animal contact areas, 49% had hand-to-face contact, and 22% ate or drank in animal contact areas. Thirty-eight percent used hand sanitizer; children had better compliance than adults. Results of bacteriologic cultures of hands were negative for Salmonella spp and Escherichia coli O157; Salmonella spp were isolated from 63% and E coli O157 from 6% of the environmental samples.
High risk behaviors were common among petting zoo visitors, and disease prevention guidelines were inconsistently followed. This is an example of the importance of one-medicine, one-health initiatives in protecting the public health. Veterinarians, venue operators, and public health authorities must work together on targeted education to improve implementation of existing disease prevention guidelines.
Journal of the American Veterinary Medical Association 11/2007; 231(7):1036-8. · 1.79 Impact Factor
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ABSTRACT: Accurate Escherichia coli serotyping is critical for pathogen diagnosis and surveillance of non-O157 Shiga-toxigenic strains, however few laboratories have this capacity. The molecular serotyping protocol described in this paper targets the somatic and flagellar antigens of E. coli O111:H8 used in traditional serotyping, and can be performed routinely in the laboratory.
Journal of Microbiological Methods 06/2007; 69(2):381-3. · 2.09 Impact Factor
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ABSTRACT: The fecal prevalence of subclinical Salmonella enterica and Shiga-toxigenic Escherichia coli O157 among animals in human-animal contact exhibits at institutions in the United States accredited by the Association of Zoos and Aquariums was estimated to assess public health risk. The prevalence was less than 0.6% for both zoonotic pathogens among 997 animals sampled at 36 exhibits.
Applied and Environmental Microbiology 02/2007; 73(1):362-5. · 3.83 Impact Factor
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ABSTRACT: Emerging molecular, animal model and epidemiologic evidence suggests that Shiga-toxigenic Escherichia coli O157:H7 (STEC O157) isolates vary in their capacity to cause human infection and disease. The translocated intimin receptor (tir) and intimin (eae) are virulence factors and bacterial receptor-ligand proteins responsible for tight STEC O157 adherence to intestinal epithelial cells. They represent logical genomic targets to investigate the role of sequence variation in STEC O157 pathogenesis and molecular epidemiology. The purposes of this study were (1) to identify tir and eae polymorphisms in diverse STEC O157 isolates derived from clinically ill humans and healthy cattle (the dominant zoonotic reservoir) and (2) to test any observed tir and eae polymorphisms for association with human (vs bovine) isolate source.
Five polymorphisms were identified in a 1,627-bp segment of tir. Alleles of two tir polymorphisms, tir 255 T>A and repeat region 1-repeat unit 3 (RR1-RU3, presence or absence) had dissimilar distributions among human and bovine isolates. More than 99% of 108 human isolates possessed the tir 255 T>A T allele and lacked RR1-RU3. In contrast, the tir 255 T>A T allele and RR1-RU3 absence were found in 55% and 57%, respectively, of 77 bovine isolates. Both polymorphisms associated strongly with isolate source (p < 0.0001), but not by pulsed field gel electrophoresis type or by stx1 and stx2 status (as determined by PCR). Two eae polymorphisms were identified in a 2,755-bp segment of 44 human and bovine isolates; 42 isolates had identical eae sequences. The eae polymorphisms did not associate with isolate source.
Polymorphisms in tir but not eae predict the propensity of STEC O157 isolates to cause human clinical disease. The over-representation of the tir 255 T>A T allele in human-derived isolates vs the tir 255 T>A A allele suggests that these isolates have a higher propensity to cause disease. The high frequency of bovine isolates with the A allele suggests a possible bovine ecological niche for this STEC O157 subset.
BMC Infectious Diseases 01/2007; 7:98. · 3.12 Impact Factor
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ABSTRACT: Agricultural fairs exhibiting livestock are increasingly implicated in human Shiga-toxigenic Escherichia coli O157:H7 (STEC O157:H7) outbreaks. To estimate livestock STEC O157:H7 prevalence at US fairs, we collected 2,919 fecal specimens at 29 county fairs in 2 states and at 3 state fairs in 2002. Fly pools were also collected. STEC O157:H7 was isolated from livestock at 31 (96.9%) of 32 fairs, including 11.4% of 1,407 cattle, 1.2% of 1,102 swine, 3.6% of 364 sheep and goats, and 5.2% of 154 fly pools. Cattle, swine, and flies at some fairs shared indistinguishable STEC O157:H7 isolate subtypes. In 2003, a total of 689 ambient environmental samples were collected at 20 fairgrounds 10-11 months after 2002 livestock sampling while fairgrounds were livestock-free. Four beef barn environmental samples at 3 fairgrounds yielded STEC O157:H7. These data suggest that STEC O157 is common and transmissible among livestock displayed at agricultural fairs and persists in the environment after the fair.
Emerging infectious diseases 06/2006; 12(5):780-6. · 6.17 Impact Factor
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ABSTRACT: To evaluate associations between neonatal serum IgG1 concentration and pre- and postweaning morbidity and mortality rates and average daily gains (ADGs) in beef calves and define a cutoff point for serum IgG1 concentration necessary for optimal health and performance of beef calves.
Nonconcurrent cohort study.
1,568 crossbred beef calves.
Single radial immunodiffusion was used to quantitate IgG1 concentration in sera collected from calves between 24 and 72 hours after birth. Logistic regression, ANCOVA, and likelihood ratios were used to analyze data.
In the preweaning period, lower perinatal IgG1 concentrations were significantly associated with higher morbidity rates, higher mortality rates, and lower ADGs. Calves with serum IgG1 concentration < 2,400 mg/dL were 1.6 times as likely to become ill before weaning and 2.7 times as likely to die before weaning as calves with higher serum IgG1 concentrations. Calves with serum IgG1 concentration of at least 2,700 mg/dL weighed an estimated 3.35 kg (7.38 lb) more at 205 days of age than calves with lower serum IgG1 concentration. No significant association of serum IgG1 concentration with feedlot morbidity, death, or ADG was identified.
By use of likelihood ratios, the threshold of serum IgG1 concentration for optimal health and performance of calves was higher than values reported previously. Implementation and maintenance of management and intervention strategies designed for early detection and treatment of calves at risk for failure of passive transfer will likely result in increases in preweaning health and performance parameters.
Journal of the American Veterinary Medical Association 04/2006; 228(6):914-21. · 1.79 Impact Factor
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ABSTRACT: Serotyping is the foundation of pathogenic Escherichia coli diagnostics; however, few laboratories have this capacity. We developed a molecular serotyping protocol that targets, genetically, the same somatic and flagellar antigens of E. coli O26:H11 used in traditional serotyping. It correctly serotypes strains untypeable by traditional methods, affording primary laboratories serotyping capabilities.
Applied and Environmental Microbiology 09/2005; 71(8):4941-4. · 3.83 Impact Factor
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ABSTRACT: To determine whether a selected set of 20 single nucleotide polymorphism (SNP) markers derived from beef cattle populations can be used to verify sample tracking in a commercial slaughter facility that processes primarily market (ie, culled) dairy cows.
Prospective, blinded validation study.
165 cows and 3 bulls from 18 states (82% Holstein, 8% other dairy breeds, and 10% beef breeds).
Blood was collected by venipuncture from randomly chosen animals just prior to slaughter. The purported corresponding liver samples were collected during beef processing, and genotype profiles were obtained for each sample.
On the basis of SNP allele frequencies in these cattle, the mean probability that 2 randomly selected individuals would possess identical genotypes at all 20 loci was 4.3 x 10(-8). Thus, the chance of a coincidental genotype match between 2 animals was 1 in 23 million. Genotype profiles confirmed appropriate matching for 152 of the 168 (90.5%) purported blood-liver sample pairs and revealed mismatching for 16 (9.5%) pairs. For the 16 mismatched sample pairs, 33% to 76% of the 20 SNP genotypes did not match (mean, 52%). Discordance that could be attributed to genotyping error was estimated to be < 1% on the basis of results for split samples.
Results suggest that this selected set of 20 bovine SNP markers is sufficiently informative to verify accuracy of sample tracking in slaughter plants that process beef or dairy cattle. These or similar SNP markers may facilitate high-throughput, DNA-based, traceback programs designed to detect drug residues in tissues, control of animal diseases, and enhance food safety.
Journal of the American Veterinary Medical Association 04/2005; 226(8):1311-4. · 1.79 Impact Factor
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ABSTRACT: We report an agricultural fair-associated shiga-toxigenic Escherichia coli O157:H7 (STEC O157) outbreak that was unusual in that it affected both livestock exhibitors and visitors. Twenty-five human cases of STEC O157 infection were detected after the Fort Bend County Fair in Rosenberg, Texas, which ran from 9/26/03 to 10/04/03. Seven cases were culture-confirmed. There were four hemolytic uremic syndrome (HUS) cases, and one thrombotic thrombocytopenic purpura (TTP) case. Cases ranged in age from 18 months to 67 years. Twenty-two (88%) cases were female. Analysis of unmatched case-control data linked STEC O157 infection with visiting fair livestock exhibit areas and with multiple fair visits. All outbreak-related isolates were of a single STEC O157 subtype. Fair Ground environmental sampling and culture for STEC O157, conducted 46 days after the end of the Fair, yielded multiple STEC O157 isolates, including the outbreak subtype. Livestock exhibitors and fair visitors should follow guidelines to reduce the risk of transmission of STEC O157 at agricultural fairs.
Vector Borne and Zoonotic Diseases 02/2005; 5(2):193-201. · 2.44 Impact Factor
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ABSTRACT: Escherichia coli O157:H7 (EC O157) is an important cause of foodborne disease. Cattle are reservoirs for the bacteria and are implicated in transmission to humans. Prevalence data in prefeedlot calves are limited. With the use of sensitive methods, a cohort of weaned beef calves (n = 408) was sampled before and after preconditioning to estimate fecal point prevalence and describe changes in EC O157 fecal shedding. EC O157 isolates were confirmed and characterized by PCR and pulsed-field gel electrophoresis. Calves from 29 cow-calf farms were commingled at three preconditioning sites and placed on a transition ration containing oxytetracycline (200 g/ton) for 45 days. Initial animal-level fecal point prevalence was 2.5% (95% confidence interval, 1 to 5) with a herd-level prevalence of 17.2% (95% confidence interval, 6 to 36). Point prevalence following the preconditioning feeding period was 0%. An unexpected finding in our study was EC O157 isolates that were Shiga toxin-deficient. Pulsed-field gel electrophoresis subtypes of EC O157 were unique in epidemiologically unlinked herds, except one herd that had two unique subtypes. We expected, but observed, neither increased fecal shedding in the cohort nor horizontal transmission of unique EC O157 subtypes. The absence of fecal shedding following the 45-day feeding period might be attributable to seasonal influences, inhibitory concentrations of oxytetracycline in the transition ration, or transient colonization that ended before sampling. EC O157 is apparently widely dispersed at low prevalence in U.S. prefeedlot, weaned calves.
Journal of food protection 12/2004; 67(11):2391-6. · 1.94 Impact Factor
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ABSTRACT: The aim of this study was to identify changes in bovine macrophage gene expression in response to treatment with Escherichia coli 0157:H7 lipopolysaccharide (LPS), utilizing a human gene microarray. Bovine cDNA from control and LPS-treated primary macrophages hybridized to greater than 5644 (79.8%) of the non-control gene targets on a commercially available microarray containing greater than 7075 targets (Incyte Genomics, St. Louis, MO). Of these target sequences, 44 were differentially expressed upon exposure to LPS, including 18 genes not previously reported to exist in cattle. These included a pentaxin-related gene, CASP8, TNF-induced genes, interferon-induced genes, and inhibitors of apoptosis. Using the human microarray, cDNA from bovine LPS-treated and control macrophages consistently hybridized to targets known to be expressed constitutively by macrophages, as expected given the predicted cDNA sequence homology. That this human system was accurately estimating levels of bovine transcripts was further verified by real-time quantitative reverse transcriptase polymerase chain reaction (RTQ-PCR) using bovine-specific primers. This first report of bovine-human cross-species expression profiling by microarray hybridization demonstrates the utility of this technique in bovine gene expression and discovery.
Developmental & Comparative Immunology 06/2004; 28(6):635-45. · 3.27 Impact Factor
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ABSTRACT: To describe shiga-toxigenic Escherichia coil O157:H7 (STEC O157:H7) fecal shedding prevalence, seasonal fecal shedding patterns, and site-specific prevalence from the oral cavity, skin, and feces of dairy cattle.
Cross-sectional study.
Adult dairy cattle from 13 herds in Louisiana.
Samples were cultured for STEC O157 by use of sensitive and specific techniques, including selective broth enrichment, immunomagnetic separation, monoclonal antibody-based O:H enzyme immunoassay serotyping, and polymerase chain reaction virulence gene characterization. Point estimates and 95% confidence intervals were calculated for fecal shedding prevalence as well as site-specific prevalence from the oral cavity, skin, and feces. Logistic regression was used to assess seasonal variation and differences at various stages of lactation with respect to fecal shedding of STEC O157 in cattle sampled longitudinally.
Summer prevalence in herds in = 13) was 38.5%, with a cow-level prevalence of 6.5%. Among positive herds, prevalence ranged from 3% to 34.6%. Samples from 3 of 5 herds sampled quarterly over 1 year yielded positive results for STEC O157. In herds with STEC O157, an increase in cow-level prevalence was detected during spring (13.3%) and summer (10.5%), compared with values for fall and winter. Site-specific prevalences of STEC O157:H7 from oral cavity, skin, and fecal samples were 0%, 0.7%, and 25.2%, respectively.
Our data indicated that STEC O157:H7 was commonly isolated from dairy cows in Louisiana, seasonally shed, and isolated from the skin surface but not the oral cavity of cows.
Journal of the American Veterinary Medical Association 05/2004; 224(7):1151-8. · 1.79 Impact Factor
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ABSTRACT: Escherichia coli O157:H7 (EC O157) is an important zoonosis. White-tailed deer (Odocoileus virginianus) have been implicated in transmission of this bacterium to humans and have been suggested as reservoirs that might affect carriage in cattle populations. Our study objectives were to estimate prevalence of EC O157 in feces of hunter-harvested deer and to describe fecal shedding patterns in a captive herd sampled over 1 yr. Prevalence of EC O157 in hunter-harvested deer was 0.3% (n = 338). In August 2001, EC O157 was detected in one of 55 deer (1.8%) from the captive herd. Prevalence over the 1-yr period was 0.4% (n = 226). Escherichia coli O157:H7 was rarely isolated from hunter-harvested deer during the winter. We could not describe a seasonal shedding pattern based on one positive sample in the captive herd. These data do not support a prominent role of deer as a reservoir for EC O157 for cattle or humans.
Journal of wildlife diseases 05/2004; 40(2):361-5. · 1.08 Impact Factor
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ABSTRACT: A commercially available real-time, rapid PCR test was evaluated for its ability to detect Escherichia coli O157. Both the sensitivity and specificity of the assay were 99% for isolates in pure culture. The assay detected 1 CFU of E. coli O157:H7 g(-1) in artificially inoculated bovine feces following enrichment.
Applied and Environmental Microbiology 04/2004; 70(3):1855-7. · 3.83 Impact Factor
