Genetic diversity, isolation frequency, and persistence were determined for Escherichia coli O157 strains from range cattle production environments. Over the 11-month study, analysis of 9,122 cattle fecal samples, 4,083 water source samples, and 521 wildlife fecal samples resulted in 263 isolates from 107 samples presumptively considered E. coli O157 as determined by culture and latex agglutination. Most isolates (90.1%) were confirmed to be E. coli O157 by PCR detection of intimin and Shiga toxin genes. Pulsed-field gel electrophoresis (PFGE) of XbaI-digested preparations revealed 79 unique patterns (XbaI-PFGE subtypes) from 235 typeable isolates confirmed to be E. coli O157. By analyzing up to three isolates per positive sample, we detected an average of 1.80 XbaI-PFGE subtypes per sample. Most XbaI-PFGE subtypes (54 subtypes) were identified only once, yet the seven most frequently isolated subtypes represented over one-half of the E. coli O157 isolates (124 of 235 isolates). Recurring XbaI-PFGE subtypes were recovered from samples on up to 10 sampling occasions and up to 10 months apart. Seven XbaI-PFGE subtypes were isolated from both cattle feces and water sources, and one of these also was isolated from the feces of a wild opossum (Didelphis sp.). The number of XbaI-PFGE subtypes, the variable frequency and persistence of subtypes, and the presence of identical subtypes in cattle feces, free-flowing water sources, and wildlife feces indicate that the complex molecular epidemiology of E. coli O157 previously described for confined cattle operations is also evident in extensively managed range cattle environments.Escherichia coli O157 has become a significant public health concern with a worldwide distribution (3, 9). Although the majority of E. coli O157-related human disease in the United States is estimated to be food borne (27), other forms of transmission (waterborne, animal-to-person, and person-toperson) can occur (3, 9). Cattle feces have been implicated as a main source of contamination in waterborne and food-borne E. coli O157 outbreaks and sporadic infections (3, 9). Therefore, significant resources have been devoted to determining the epidemiology and ecology of E. coli O157 in cattle production environments.Molecular techniques for genotyping or subtyping E. coli O157 and other pathogens have been used to investigate the sources of the organisms in outbreaks of human disease (10). These techniques, particularly pulsed-field gel electrophoresis (PFGE), also have been used in investigations of E. coli O157 in cattle production environments (12,20,25,31,35,39). The ecology and molecular epidemiology of E. coli O157 in cattle operations appear to be complex (21, 32). Several PFGE subtypes can be found in a single cattle operation, but some E. coli O157 subtypes seem to predominate (39). Subtypes can persist in bovine production environments for more than 1 year, and indistinguishable subtypes have been detected in the feces of bovine and nonbovine species, as well as in environmental niches, such a...

To overcome problems associated with application of PCR to clinical samples, we have combined a short cultivation procedure with a Salmonella-specific PCR-hybridization assay to specifically identify Salmonella serovars from clinical samples of various animal species. The technique was investigated by using fecal samples seeded with known numbers of Salmonella organisms and cultivated for different lengths of time in assorted selective and nonselective enrichment media. The ability of PCR to amplify a SalmoneUla-specific DNA product (457-bp sequence covering the Salmonella invE and invA genes) was examined in Southern hybridizations with an internal oligonucleotide probe. Forty-seven Salmonella isolates representing 32 serovars were evaluated, and all Salmonella isolates resulted in a 457-bp product that hybridized with the oligonucleotide probe, whereas no hybridizations were evident with 53 non-Salmonela organisms. The assay detected as few as 9 CFU of Salmonella organisms in pure culture and as little as 300 fg of purified chromosomal DNA. Rappaport-Vassiliadis and tetrathionate broths were inhibitory to PCR, whereas brain heart infusion and selenite-cystine broths were not. The PCR-hybridization assay coupled with a brain heart infusion enrichment culture incubated for 2 h detected as few as 80 CFU of Salmonella organisms in seeded feces. We have successfully identified Salmonella serovars in clinical samples from swine, horses, and cattle more rapidly than with conventional culture techniques. The sensitivity and specificity of this assay were both 100lo compared with culture results. These results indicate that a combined cultivation-PCR-hybridization assay could be applicable and advantageous in the rapid identification of Salmonella serovars in routine diagnostic situations.

Presumptive identification of Escherichia coli O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagic E. coli (EHEC)eaeA gene. In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5′ nuclease assay for presumptively detecting E. coli O157:H7 DNA. The specificity of the eaeA-based 5′ nuclease assay system was sufficient to correctly identify all E. coliO157:H7 strains evaluated, mirroring the previously described specificity of the PCR primers. The SZ-primed,eaeA-targeted 5′ nuclease detection assay was capable of rapid, semiautomated, presumptive detection of E. coliO157:H7 when ≥103 CFU/ml was present in modified tryptic soy broth (mTSB) or modified E. coli broth and when ≥104 CFU/ml was present in ground beef-mTSB mixtures. Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enrichment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen), improved the detection threshold to ≥102 CFU/ml. Surprisingly, immediately after IMS, the sensitivity of culturing on sorbitol MacConkey agar containing cefeximine and tellurite (CT-SMAC) was such that identifiable colonies were demonstrated only when ≥104 CFU/ml was present in the sample. Several factors that might be involved in creating these false-negative CT-SMAC culture results are discussed. The SZ-primed,eaeA-targeted 5′ nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification of E. coli O157:H7 in ground beef and potentially in other food and environmental samples.

Implementing control strategies for E coli O157:H7 at all levels of the cattle industry will decrease the risk of this organism entering the human food chain. Devising effective on-farm strategies to control E coli O157:H7 in cow-calf herds will require an understanding of the epidemiologic characteristics of this pathogen.

Results suggested that vaccination against PCV2 was effective at reducing mortality rate and improving growth performance among pigs in a herd infected with PCV2.

One hundred twelve samples of commercial raw meat used in greyhound diets were collected and cultured for Salmonella using standard procedures. Fifty (44.64%) of these samples were positive for Salmonella. Salmonella typhimurium was the most frequently isolated serovar (48%), followed by S. newport (12.76%), S. agona (8.51%), and S. muenster (6.38%). The remaining 10 serovars recovered in this study represented 27.59% of the total Salmonella isolates. In addition, the meat samples were screened for Salmonella using a commercial DNA probe. Of the 106 samples tested, 70 (66.03%) were positive for Salmonella, which indicated that the DNA probe assay was more sensitive than the culture method for screening of Salmonella in raw meat. Antimicrobial susceptibility testing revealed that most of the Salmonella isolates were sensitive to a variety of antimicrobials, particularly amikacin and apramycin, and resistant to some others, such as clindamycin, erythromycin, penicillin, and sulfadimethoxine. The cumulative percentages of susceptibility (MIC50 and MIC90) of the Salmonella isolates were also determined. Most isolates were susceptible (MIC90) to low concentrations of gentamicin (2.0 micrograms/ml), imipenem (< or = 0.25 microgram/ml), and ciprofloxacin (< or = 0.5 microgram/ml). Marked resistance was found with the other antimicrobial agents. However, the high MIC values found for these isolates would not be achievable in vivo with the normal recommended doses of antimicrobial agents, so their use would not be beneficial. Numerous plasmid patterns were found in 17 randomly selected Salmonella isolates. Eight of the 17 isolates had 2-7 plasmids ranging from 2.4 to 15 kilobases in size. Eight isolates also exhibited large plasmids in the range of 50-60 and 95-105 kilobases.(ABSTRACT TRUNCATED AT 250 WORDS)

During the last 30 years, our concept of cryptosporidiosis has changed from that of a rare, largely asymptomatic disease, to an important cause of diarrhea in animals and humans worldwide. Significant disease first appeared in cattle. Subsequently, the zoonotic danger of the organism was recognized in HIV‐infected persons and young children. Cryptosporidium are now ubiquitous and disease has been described in over 79 host species. Cryptosporidiosis has become a major cause of calfhood diarrhea worldwide. In humans it accounts for up to 20% of all cases of childhood diarrhea in developing countries and is a potentially fatal complication of AIDS. Waterborne contamination is a growing concern as a source of widespread outbreaks of disease. Factors that have contributed to the emergence of cryptosporidiosis in animals include biological features of the organism, the lack of an effective treatment or preventative, increased environmental contamination, and trends in livestock production. In humans the zoonotic nature of infection and an increased at‐risk population have contributed to disease. Genetic characterization of Cryptosporidium, improved detection methods, and a better understanding of the factors that predispose to disease are important contributions to understanding the epidemiology of cryptosporidiosis.

We determined tissue localization, shedding patterns, virus carriage, antibody response, and aerosol transmission of Porcine epidemic diarrhea virus (PEDV) following inoculation of 4-week-old feeder pigs. Thirty-three pigs were randomly assigned to 1 of 3 groups for the 42-day study: inoculated (group A; n = 23), contact transmission (group B; n = 5), and aerosol transmission (group C; n = 5). Contact transmission occurred rapidly to group B pigs whereas productive aerosol transmission failed to occur to group C pigs. Emesis was the first clinical sign noted at 3 days postinoculation (dpi) followed by mild to moderate diarrhea lasting 5 more days. Real-time PCR detected PEDV in fecal and nasal swabs, oral fluids, serum, and gastrointestinal and lymphoid tissues. Shedding occurred primarily during the first 2 weeks postinoculation, peaking at 5-6 dpi; however, some pigs had PEDV nucleic acid detected in swabs collected at 21 and 28 dpi. Antibody titers were measurable between 14 and 42 dpi. Although feces and intestines collected at 42 dpi were PEDV negative by PCR and immunohistochemistry, respectively, small intestines from 70% of group A pigs were PCR positive. Although disease was relatively mild and transient in this age group, the results demonstrate that 4-week-old pigs are productively infected and can sustain virus replication for several weeks. Long-term shedding of PEDV in subclinically affected pigs should be considered an important source for PEDV transmission.