William W. Laegreid

Correlation of enterohemorrhagic Escherichia coli O157 prevalence in feces, hides, and carcasses of beef cattle during processing

A survey was performed to estimate the frequency of enterohemorrhagic Escherichia coli O157:H7 or O157:nonmotile (EHEC O157) in feces and on hides within groups of fed cattle from single sources (lots) presented for slaughter at meat processing plants in the Midwestern United States, as well as frequency of carcass contamination during processing from cattle within the same lots. Of 29 lots sampled, 72% had at least one EHEC O157-positive fecal sample and 38% had positive hide samples. Overall, EHEC O157 prevalence in feces and on hides was 28% (91 of 327) and 11% (38 of 355), respectively. Carcass samples were taken at three points during processing: preevisceration, postevisceration before antimicrobial intervention, and postprocessing after carcasses entered the cooler. Of 30 lots sampled, 87% had at least one EHEC O157-positive preevisceration sample, 57% of lots were positive postevisceration, and 17% had positive postprocessing samples. Prevalence of EHEC O157 in the three postprocessing samples was 43% (148 of 341), 18% (59 of 332) and 2% (6 of 330), respectively. Reduction in carcass prevalence from preevisceration to postprocessing suggests that sanitary procedures were effective within the processing plants. Fecal and hide prevalence were significantly correlated with carcass contamination (P = 0.001), indicating a role for control of EHEC O157 in live cattle.

Porcine Reproductive and Respiratory Syndrome Virus: Description of Persistence in Individual Pigs upon Experimental Infection

ABSTRACT We studied the persistence of porcine reproductive and respiratory syndrome virus (PRRSV) in individual experimentally infected pigs, during a period of up to 150 days postinfection (dpi). The results of this study suggest that the persistence of PRRSV involves continuous viral replication but that it is not a true steady-state persistent infection. The virus eventually clears the body and seems to do it in most of the animals by 150 dpi or shortly thereafter. High genetic stability was seen for several regions of the persistent PRRSVs genome, although some consistent mutations in the genes of envelope glycoproteins and M protein were also observed.

Selection and use of SNP markers for animal identification and paternity analysis in U.S. beef cattle

Abstract. DNA marker technology represents a promising means for determining the genetic identity and kinship of an animal. Compared with other types of DNA markers, single nucleotide polymorphisms (SNPs) are attractive because they are abundant, genetically stable, and amenable to high-throughput automated analysis. In cattle, the challenge has been to identify a minimal set of SNPs with sufficient power for use in a variety of popular breeds and crossbred populations. This report describes a set of 32 highly informative SNP markers distributed among 18 autosomes and both sex chromosomes. Informativity of these SNPs in U.S. beef cattle populations was estimated from the distribution of allele and genotype frequencies in two panels: one consisting of 96 purebred sires representing 17 popular breeds, and another with 154 purebred American Angus from six herds in four Midwestern states. Based on frequency data from these panels, the estimated probability that two randomly selected, unrelated individuals will possess identical genotypes for all 32 loci was 2.0 × 10−13 for multi-breed composite populations and 1.9 × 10−10 for purebred Angus populations. The probability that a randomly chosen candidate sire will be excluded from paternity was estimated to be 99.9% and 99.4% for the same respective populations. The DNA immediately surrounding the 32 target SNPs was sequenced in the 96 sires of the multi-breed panel and found to contain an additional 183 polymorphic sites. Knowledge of these additional sites, together with the 32 target SNPs, allows the design of robust, accurate genotype assays on a variety of high-throughput SNP genotyping platforms.

Prevalence of Escherichia coli O157:H7 in range beef calves at weaning.

This study was designed to determine the prevalence of Escherichia coli O157:H7 infection of beef calves at weaning, prior to arrival at the feedlot or mixing with cattle from other sources. Fifteen range cow-calf herds, which weaned calves in October and November, were sampled in Kansas, Missouri, Montana, Nebraska and South Dakota. Faecal culture for E. coli O157:H7 was performed and anti-O157 serum antibody titres were determined by blocking ELISA. Thirteen of the 15 herds (87%) were found to have at least one positive isolation of E. coli O157:H7 in faecal samples. Within positive herds, prevalence ranged from 1.7-20.0%, with an average of 7.4+/-6.2% S.D. of individual animals shedding E. coli O157:H7 in faeces. All herds had high prevalence of anti-O157 antibodies, ranging 63-100% of individuals within herds seropositive. This study indicates that E. coli O157:H7 infection before weaning, prior to entry into feedlots, is widespread. Furthermore, serologic evidence suggests that most calves (83%) and all herds (100%) have been exposed to E. coli O157.

Mutations in the genome of porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype.

Summary. Although live-attenuated vaccines have been used for some time to control clinical symptoms of the porcine reproductive and respiratory syndrome (PRRS), the molecular bases for the attenuated phenotype remain unclear. We had previously determined the genomic sequence of the pathogenic PRRSV 16244B. Limited comparisons of the structural protein coding sequence of an attenuated vaccine strain have shown 98% homology to the pathogenic 16244B. Here we have confirmed the attenuated phenotype and determined the genomic sequence of that attenuated PRRSV vaccine and compared it to its parental VR-2332 and the 16244B strains. The attenuated vaccine sequence was colinear with that of the strain 16244B sequence containing no gaps and 212 substitutions over 15,374 determined nucleotide sequence. We identified nine amino acid changes distributed in Nsp1β, Nsp2, Nsp10, ORF2, ORF3, ORF5 and ORF6. These changes may provide the molecular bases for the observed attenuated phenotype.

Animal-to-animal variation in fecal microbial diversity among beef cattle.

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.

A highly attenuated host range-restricted vaccinia virus strain, NYVAC, encoding the prM, E, and NS1 genes of Japanese encephalitis virus prevents JEV viremia in swine

A highly attenuated strain of vaccinia virus (NYVAC) was engineered to express the Japanese encephalitis virus (JEV) prM, E, and NS1 genes or the prM and E genes. The recombinant viruses were tested as vaccine candidates in pigs, a natural host of JEV. JEV-neutralizing and hemagglutination-inhibiting antibodies appeared in swine sera 7 days after immunization with 10(8) PFU of the recombinant viruses and increased after a second dose at 28 days. The JEV levels detected in the serum after JEV challenge (d56) of the swine with 2 x 10(5) PFU of JEV were significantly reduced in animals inoculated with the recombinant viruses. These results demonstrate the ability of these NYVAC-vectored recombinants to protect pigs from JEV viremia.

Interferon type I response in porcine reproductive and respiratory syndrome virus-infected MARC-145 cells

Summary.Infection by porcine reproductive and respiratory syndrome virus (PRRSV) results in a weak induction of the innate immune response. There are many genes that collectively comprise this response and the extent to which each gene responds to PRRSV infection is unclear and warrants further investigation. To this end, we have utilized real-time PCR using SYBR Green I dye-based detection to quantify transcript abundance of the type I interferons (IFN-α and -β) and IFN-β transcriptional enhanceasome genes. In MARC-145 cells, both IFN-α and -β transcript abundance were unaffected by PRRSV infection. However, stimulation of MARC-145 cells by exogenous double-stranded RNA, resulted in significant increases in transcript abundance of both IFN-α and -β as well as IFN-β enhanceasome components, indicating that a type I IFN response could be induced in these cells. The double-stranded RNA induction of type I IFN transcription was significantly inhibited by dual-exposure with PRRSV. These results suggest that PRRSV infection directly interferes with type I IFN transcriptional activation early in its pathway, at the level of IFN-β gene transcription.

Genotypic Analyses of Escherichia coli O157:H7 and O157 Nonmotile Isolates Recovered from Beef Cattle and Carcasses at Processing Plants in the Midwestern States of the United States

ABSTRACT Escherichia coli O157:H7 and O157 nonmotile isolates (E. coli O157) previously were recovered from feces, hides, and carcasses at four large Midwestern beef processing plants (R. O. Elder, J. E. Keen, G. R. Siragusa, G. A. Barkocy-Gallagher, M. Koohmaraie, and W. W. Laegreid, Proc. Natl. Acad. Sci. USA 97:2999–3003, 2000). The study implied relationships between cattle infection and carcass contamination within single-source lots as well as between preevisceration and postprocessing carcass contamination, based on prevalence. These relationships now have been verified based on identification of isolates by genomic fingerprinting.E. coli O157 isolates from all positive samples were analyzed by pulsed-field gel electrophoresis of genomic DNA after digestion with XbaI. Seventy-seven individual subtypes (fingerprint patterns) grouping into 47 types were discerned among 343 isolates. Comparison of the fingerprint patterns revealed three clusters of isolates, two of which were closely related to each other. Remarkably, isolates carrying both Shiga toxin genes and nonmotile isolates largely fell into specific clusters. Within lots analyzed, 68.2% of the postharvest (carcass) isolates matched preharvest (animal) isolates. For individual carcasses, 65.3 and 66.7% of the isolates recovered postevisceration and in the cooler, respectively, matched those recovered preevisceration. Multiple isolates were analyzed from some carcass samples and were found to include strains with different genotypes. This study suggests that mostE. coli O157 carcass contamination originates from animals within the same lot and not from cross-contamination between lots. In addition, the data demonstrate that most carcass contamination occurs very early during processing.

Prion gene sequence variation within diverse groups of U.S. sheep, beef cattle, and deer

Prions are proteins that play a central role in transmissible spongiform encephalopathies in a variety of mammals. Among the most notable prion disorders in ungulates are scrapie in sheep, bovine spongiform encephalopathy in cattle, and chronic wasting disease in deer. Single nucleotide polymorphisms in the sheep prion gene (PRNP) have been correlated with susceptibility to natural scrapie in some populations. Similar correlations have not been reported in cattle or deer; however, characterization of PRNP nucleotide diversity in those species is incomplete. This report describes nucleotide sequence variation and frequency estimates for the PRNP locus within diverse groups of U.S. sheep, U.S. beef cattle, and free-ranging deer (Odocoileusvirginianus and O. hemionus from Wyoming). DNA segments corresponding to the complete prion coding sequence and a 596-bp portion of the PRNP promoter region were amplified and sequenced from DNA panels with 90 sheep, 96 cattle, and 94 deer. Each panel was designed to contain the most diverse germplasm available from their respective populations to facilitate polymorphism detection. Sequence comparisons identified a total of 86 polymorphisms. Previously unreported polymorphisms were identified in sheep (9), cattle (13), and deer (32). The number of individuals sampled within each population was sufficient to detect more than 95% of all alleles present at a frequency greater than 0.02. The estimation of PRNP allele and genotype frequencies within these diverse groups of sheep, cattle, and deer provides a framework for designing accurate genotype assays for use in genetic epidemiology, allele management, and disease control.