Scrapie resistance in ARQ sheep.

Variation in the ovine prion protein amino acid sequence influences scrapie progression, with sheep homozygous for A(136)R(154)Q(171) considered susceptible. This study examined the association of survival time of scrapie-exposed ARQ sheep with variation elsewhere in the ovine prion gene. Four single nucleotide polymorphism alleles were associated with prolonged survival. One nonsynonymous allele (T112) was associated with an additional 687 days of survival for scrapie-exposed sheep compared to M112 sheep (odds ratio, 42.5; P = 0.00014). The only two sheep homozygous for T112 (TARQ) did not develop scrapie, suggesting that the allelic effect may be additive. These results provide evidence that TARQ sheep are genetically resistant to development of classical scrapie.

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

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-alpha and -beta) and IFN-beta transcriptional enhanceasome genes. In MARC-145 cells, both IFN-alpha and -beta 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-alpha and -beta as well as IFN-beta 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-beta gene transcription.

Estimation of the basic reproduction ratio (R0) for Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) in beef calves.

To understand the dynamics of transmission of Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) in beef calves, serum samples were obtained from calves in a beef cow-calf herd approximately every 6 weeks from birth until weaning for three consecutive years. The presence of specific anti-O157 antibodies in these serum samples was detected using a blocking ELISA assay incorporating an anti-O157 monoclonal antibody. Using seroconversion data, the basic reproduction ratio (R0) was estimated for each of the three years as well as in aggregate using both deterministic and Martingale methods. R0 for STEC O157 infection in range beef calves by deterministic methods varied from 2.9-5.6, with an average of 4.3 (95% CI 2.8-5.9). Martingale estimates of R0 ranged from 3.5-7.4, or 5.3 (95% CI 3.9-6.6), for data from all three years. Given the above estimate of R0, it is predicted that 65-86% of a herd of calves must be effectively vaccinated, or must be rendered non-susceptible through other means, to eliminate STEC O157 infection from a herd.

Passive transfer of virus-specific antibodies confers protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity.

Immune mechanisms mediating protective immunity against porcine reproductive and respiratory syndrome virus (PRRSV) are not well understood. The PRRSV-specific humoral immune response has been dismissed as being ineffective and perhaps deleterious for the host. The function of PRRSV antibodies in protective immunity against infection with a highly abortifacient strain of this virus was examined by passive transfer experiments in pregnant swine. All of a group of pregnant gilts (n = 6) that received PRRSV immunoglobulin (Ig) from PRRSV-convalescent, hyperimmune animals were fully protected from reproductive failure as judged by 95% viability of offspring at weaning (15 days of age). On the other hand, the totality of animals in a matched control group (n = 6) receiving anti-pseudorabies virus (PRV) Ig exhibited marked reproductive failure with 4% survival at weaning. Besides protecting the pregnant females from clinical reproductive disease, the passive transfer of PRRSV Ig prevented the challenge virus from infecting the dams and precluded its vertical transmission, as evidenced by the complete absence of infectious PRRSV from the tissues of the dams and lack of infection in their offspring. In summary, these results indicate that PRRSV-Igs are capable of conferring protective immunity against PRRSV and furthermore that these Igs can provide sterilizing immunity in vivo.

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.

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 most E. 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.

Sequence evaluation of four pooled-tissue normalized bovine cDNA libraries and construction of a gene index for cattle.

An essential component of functional genomics studies is the sequence of DNA expressed in tissues of interest. To provide a resource of bovine-specific expressed sequence data and facilitate this powerful approach in cattle research, four normalized cDNA libraries were produced and arrayed for high-throughput sequencing. The libraries were made with RNA pooled from multiple tissues to increase efficiency of normalization and maximize the number of independent genes for which sequence data were obtained. Target tissues included those with highest likelihood to have impact on production parameters of animal health, growth, reproductive efficiency, and carcass merit. Success of normalization and inter- and intralibrary redundancy were assessed by collecting 6000-23,000 sequences from each of the libraries (68,520 total sequences deposited in GenBank). Sequence comparison and assembly of these sequences was performed in combination with 56,500 other bovine EST sequences present in the GenBank dbEST database to construct a cattle Gene Index (available from The Institute for Genomic Research at http://www.tigr.org/tdb/tgi.shtml). The 124,381 bovine ESTs present in GenBank at the time of the analysis form 16,740 assemblies that are listed and annotated on the Web site. Analysis of individual library sequence data indicates that the pooled-tissue approach was highly effective in preparing libraries for efficient deep sequencing.

Interleukin-8 haplotype structure from nucleotide sequence variation in commercial populations of U.S. beef cattle.

The aim of the present study was twofold: first, to design a panel of 96 sires that reflects the breadth of genetic diversity in U.S. beef cattle, and second, to use this panel to discover nucleotide sequence diversity and haplotype structures of interleukin (IL)-8 in commercial populations. The latter is a requisite for epidemiological studies designed to test whether IL8 alleles are risk factors for acquiring or maintaining bacterial infections in production environments. IL-8 encodes a proinflammatory cytokine that plays a central role in cell-mediated immunity by attracting and activating neutrophils in the early stages of host defense against bacterial invasion. Seven single-nucleotide polymorphism (SNP) markers were identified by sequencing two IL8 DNA segments amplified from the panel of 17 popular cattle breeds (MARC beef cattle diversity panel, version 2.1). Assays for automated genotype scoring by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) were developed to independently verify the seven SNP alleles in the 96 bulls and 313 cattle from the MARC reference population. Five haplotype structures, spanning the two IL8 DNA segments, were unambiguously defined for the set of seven IL8 SNPs. Based on the breadth of germplasm in bovine diversity panel, the five haplotype structures for IL8 are estimated to represent >98% of those present in these DNA segments in commercial populations of U.S. beef cattle. The frequencies of the five respective haplotypes in the eight Angus sires of the diversity panel (0.75, 0.25, 0.00, 0.00. 0.00) were similar to those scored in 150 purebred Angus cattle from six herds in four Midwestern states (0.82, 0.18, 0.01, 0.00 0.00), suggesting that the diversity panel may also be useful for estimating allele frequencies in commercial populations.

Estimation of DNA sequence diversity in bovine cytokine genes.

DNA sequence variation provides the fundamental material for improving livestock through selection. In cattle, single nucleotide polymorphisms and small insertions/deletions (collectively referred to here as SNPs) have been identified in cytokine genes and scored in a reference population to determine linkage map positions. The aim of the present study was twofold: first, to estimate the SNP frequency in a reference population of beef cattle, and second, to determine cytokine haplotypes in a group of sires from commercial populations. Forty-five SNP markers in DNA segments from nine cytokine gene loci were analyzed in 26 reference parents. Comparison of all 52 haploid genomes at each PCR amplicon locus revealed an average of one SNP per 143 bp of sequence, whereas comparison of any two chromosomes identified heterozygous sites, on average, every 443 bp. The combination of these 45 SNP genotypes was sufficient to uniquely identify each of the 26 animals. The average number of haplotype alleles (4.4) per PCR amplicon (688 bp) and the percentage heterozygosity among founding parents (50%) were similar to those for microsatellite markers in the same population. For 49 sires from seven common breeds of beef cattle, SNP genotypes (1,225 total) were obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) at three amplicon loci. All three of the amplicon haplotypes were correctly deduced for each sire without the use of parent or progeny genotypes. The latter allows a wide range of genetic studies in commercial populations of cattle where genotypic information from relatives may not be available.

Porcine reproductive and respiratory syndrome virus: description of persistence in individual pigs upon experimental infection.

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 PRRSV's genome, although some consistent mutations in the genes of envelope glycoproteins and M protein were also observed.

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.

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.

Single nucleotide polymorphism (SNP) discovery and linkage mapping of bovine cytokine genes.

Polymorphic markers at bovine gene loci facilitate the integration of cattle genetic maps with those of humans and mice. To this end, 31 single nucleotide polymorphism (SNP) markers were developed for seven bovine chemokine genes. Loci were amplified from bovine genomic DNA by the polymerase chain reaction, and candidate amplicons were sequenced to determine their identity. Amplified loci from 24 founding parents and select progeny from a beef cattle reference population were sequenced and analyzed for SNPs. SNP haplotype alleles were determined by examining segregation patterns and used to establish the locus position on the bovine linkage map. Loci for growth-related proteins (GRO3, GRO1, and GROX) were clustered with the related CXC chemokine genes, interleukin (IL) 8, and epithelial cell inflammatory protein 1, at 84 cM from the centromeric end of the bovine chromosome (BTA) 6 linkage group. Bovine loci for a cluster of IL8 receptors, a stromal cell-derived factor 1, interferon-gamma, and tumor necrosis factor-alpha were mapped at 90, 55, 59, and 34 cM, respectively, from the centromeric ends of the BTA 2, 28, 5, and 23 linkage groups. The positions of these bovine loci were compared with those of orthologous loci on the human map to refine the boundaries of conserved synteny. These seven loci provide examples of SNP development in which the efficiency was largely dependent on the availability of bovine genomic or cDNA sequence. The polymorphic nature of these SNP haplotype markers suggests that they will be useful for mapping complex traits in cattle, such as resistance to infectious disease.

Identification and genetic mapping of bovine chemokine genes expressed in epithelial cells.

RNA fingerprinting by arbitrarily primed (RAP)-PCR was used to identify two bovine genes that were differentially expressed in epithelial cells during an inflammatory response. RNA fingerprints revealed two differentially amplified transcripts when monolayers of Madin-Darby bovine kidney (MDBK) cells were stimulated with Escherichia coli O157:H7 lipopolysaccharide (LPS) in combination with cycloheximide (CX). Sequence analysis showed that both transcripts encoded members of the alpha C-X-C chemokine family; one was interleukin 8 (IL-8), and the other was a protein closely related to bovine growth-regulated protein (GRO)-gamma (89% identical). The latter putative epithelial cell inflammatory protein was designated ECIP-1. IL-8 and ECIP-1 genes were placed on the cattle genetic map with single-nucleotide polymorphism (SNP) markers amplified from genomic DNA. Multi-point linkage analysis indicated that the gene locations were indistinguishable from those of serum albumin (ALB) and vitamin D-binding protein (GC) on bovine Chromosome (BTA) 6. In humans, ALB and GC are located near IL-8, GRO-gamma, and seven other alpha chemokines on Chr 4 (HSA 4q11-4q13), suggesting that this gene cluster has been conserved on BTA6. These results provide a starting point for characterizing allelic variation in chemokine genes and their role in the pathogenesis of bacterial infections in cattle.

Use of reverse transcriptase-polymerase chain reaction (RT-PCR) and dot-blot hybridisation for the detection and identification of African horse sickness virus nucleic acids.

A coupled reverse transcriptase-polymerase chain reaction assay (RT-PCR) for the detection of African horse sickness virus (AHSV) dsRNA, has been developed using genome segment 7 as the target template for primers. RNA from isolates of all nine AHSV serotypes were readily detected. The potential inhibitory effects of either ethylene diamine tetra acetic acid (EDTA) or heparin on the RT-PCR were eliminated by washing blood samples before lysis of the red blood cells and storage. There was a close agreement in the sensitivity and the specificity of the RT-PCR and an indirect sandwich ELISA. Confirmation of the presence of AHSV using RT-PCR and dot-blot hybridization on blood samples collected from horses experimentally infected with AHSV serotype 4 (AHSV 4) and AHSV serotype 9 (AHSV 9), was achieved within 24 hours, compared to the period of several days required for virus isolation. The RT-PCR and virus isolation methods showed similar levels of sensitivity when used for the detection of AHSV in 3 horses infected with AHSV 4, and in 2 out of 3 horses infected with a less virulent isolate of AHSV 9. Although viraemia was detected in the third horse by virus isolation, from 6 to 14 days after infection, this animal remained consistently negative by RT-PCR. Conversely, AHSV viral RNA was detected by RT-PCR in the blood of 4 donkeys and 4 mules up to 55 days after their experimental infection despite the absence of any detectable infectious virus. RT-PCR is a sensitive and rapid method for detecting AHSV nucleic acids during either the incubation period at the start of an African horse sickness (AHS) epizootic, or for epidemiological investigations in species where clinical signs may be inapparent.

Actin enhances the haemolytic activity of Escherichia coli.

Actin is a major cytoskeletal protein of mammalian muscle and non-muscle cells. Exposure of cells to soluble factors that damage cell membranes results in the release of actin into the extracellular spaces. The alpha-haemolysin (HlyA) of Escherichia coli is the prototype RTX (repeat in toxin) toxin and is thought to be important in virulence because of its ability to lyse cells by formation of pores in the cell membrane. These studies were conducted to determine if actin influences growth and haemolytic activity of E. coli. Growth of E. coli in the presence of actin resulted in culture supernatant haemolytic activity that was 2.4-, 2.7- and 3.3-fold greater than that of E. coli grown in medium containing BSA, non-supplemented medium, or medium containing heat-denatured actin, respectively. The enhanced haemolytic activity occurred only when actin was present during the growth phase and there was no effect when actin was added to culture supernatants containing haemolysin. The increased haemolytic activity by actin was concentration-dependent, detectable in early-exponential-phase growth, and associated with increased concentrations of secreted HlyA by Western blotting. Actin induced a 2.9-fold increase in alkaline phosphatase activity in E. coli CC118 with a TnphoA insertion in the hlyB determinant of the recombinant haemolysin plasmid pWAM04. These results indicate that extracellular actin enhances haemolysin production by E. coli and may have implications in the pathogenesis of E. coli infections.

Deletion of a CD2-like gene, 8-DR, from African swine fever virus affects viral infection in domestic swine.

An African swine fever virus (ASFV) gene with similarity to the T-lymphocyte surface antigen CD2 has been found in the pathogenic African isolate Malawi Lil-20/1 (open reading frame [ORF] 8-DR) and a cell culture-adapted European virus, BA71V (ORF EP402R) and has been shown to be responsible for the hemadsorption phenomenon observed for ASFV-infected cells. The structural and functional similarities of the ASFV gene product to CD2, a cellular protein involved in cell-cell adhesion and T-cell-mediated immune responses, suggested a possible role for this gene in tissue tropism and/or immune evasion in the swine host. In this study, we constructed an ASFV 8-DR gene deletion mutant (delta8-DR) and its revertant (8-DR.R) from the Malawi Lil-20/1 isolate to examine gene function in vivo. In vitro, delta8-DR, 8-DR.R, and the parental virus exhibited indistinguishable growth characteristics on primary porcine macrophage cell cultures. In vivo, 8-DR had no obvious effect on viral virulence in domestic pigs; disease onset, disease course, and mortality were similar for the mutant delta8-DR, its revertant 8-DR.R, and the parental virus. Altered viral infection was, however, observed for pigs infected with delta8-DR. A delay in spread to and/or replication of delta8-DR in the draining lymph node, a delay in generalization of infection, and a 100- to 1,000-fold reduction in virus titers in lymphoid tissue and bone marrow were observed. Onset of viremia for delta8-DR-infected animals was significantly delayed (by 2 to 5 days), and mean viremia titers were reduced approximately 10,000-fold at 5 days postinfection and 30- to 100-fold at later times; moreover, unlike in 8-DR.R-infected animals, the viremia was no longer predominantly erythrocyte associated but rather was equally distributed among erythrocyte, leukocyte, and plasma fractions. Mitogen-dependent lymphocyte proliferation of swine peripheral blood mononuclear cells in vitro was reduced by 90 to 95% following infection with 8-DR.R but remained unaltered following infection with delta8-DR, suggesting that 8-DR has immunosuppressive activity in vitro. Together, these results suggest an immunosuppressive role for 8-DR in the swine host which facilitates early events in viral infection. This may be of most significance for ASFV infection of its highly adapted natural host, the warthog.

Detection of African horse sickness virus in the blood of experimentally infected horses: comparison of virus isolation and a PCR assay.

A reverse transcription-polymerase chain reaction (RT-PCR) assay followed by dot-blot hybridisation was used to detect African horse sickness virus (AHSV); the primers employed amplified the S7 gene that encodes the VP7 protein. The RT-PCR assay was compared with virus isolation for detecting AHSV in blood samples form horses experimentally infected with AHSV-4 and AHSV-9. The influence of sample storage and transportation and the effects of two anticoagulants (EDTA and heparin) were also studied. RT-PCR results were obtained within 48 hours as opposed to a minimum of 15 days for virus isolation. RT-PCR and virus isolation were equally sensitive for detection of AHSV-4. Viraemia was detected more consistently by RT-PCR than by virus isolation from horses infected with the less virulent AHSV-9 isolate except from one animal in which virus was detected only by virus isolation. The sensitivity of virus isolation was increased by passaging samples five times. This study indicates that RT-PCR is a sensitive and rapid method for use in the face of an outbreak of this serious disease, although it has also some limitations as a diagnostic technique.

Immunization with VP2 is sufficient for protection against lethal challenge with African horsesickness virus Type 4.

Horses were immunized by inoculation with a vaccinia construct containing a full-length cDNA corresponding to the L2 gene segment of African horsesickness virus type 4(AHSV-4). All immunized horses developed serum neutralizing antibodies prior to challenge with virulent AHSV-4. No ELISA-reactive antibodies were present prior to challenge. A group of four seronegative control horses died after developing clinical signs and lesions typical of the pulmonary form of African horsesickness while the immunized horses were clinically normal. Increases in serum neutralizing and ELISA-reactive antibody titers following challenge indicate that at least some replication of challenge virus occurred in immunized horses. These results demonstrate that AHSV VP2 alone is sufficient to induce a protective immune response in horses and indicate the usefulness of ELISA-reactive antibodies for differentiation of vaccinated and naturally exposed horses.

Clinical pathology and hemostatic abnormalities in experimental African horsesickness.

Infection of naive North American horses with 10(4) cell culture infectious doses (CCID50) of virulence variants of African horsesickness virus (AHSV), designated AHSV/4SP, AHSV/9PI, and AHSV/4PI, reproduced three classical forms of African horsesickness: acute (pulmonary), subacute (cardiac), and febrile, respectively. Distinct clinicopathologic and hemostatic abnormalities were associated with each form of disease. Hemostatic abnormalities included increased concentration of fibrin degradation products and prolongation of prothrombin, activated partial thromboplastin, and thrombin clotting times. Hemostatic findings indicated activation of the coagulation and fibrinolytic systems with clotting factor consumption in acute and subacute cases of African horsesickness. Hematologic abnormalities in acute and subacute cases of African horsesickness included leukopenia, decreased platelet counts, elevated hematocrit, and increased erythrocyte counts and hemoglobin concentration. Leukopenia was characterized by lymphopenia, neutropenia, and a left shift. Increased levels of serum creatine kinase, lactate dehydrogenase, aspartate aminotransferase, and alkaline phosphatase, hypocalcemia, hypoalbuminemia, hypoproteinemia, and elevated creatinine, phosphorus, and total bilirubin levels were present in some but not all horses. Metabolic acidosis, indicated by decreased total bicarbonate and increased lactate and anion gap, was present in horses with the acute form of disease. Mild thrombocytopenia and leukopenia were occasionally associated with the febrile form of disease. These results suggest a role for intravascular coagulation in the pathogenesis of African horsesickness.