Embryonating eggs were inoculated with filtered porcine ileal mucosa containing intracellular curved rods (ICR) and incubated for 4 to 6 days. Three of 12 pigs given the eggs per os developed microscopic lesions of proliferative enteritis (PE). Nonchallenge-exposed control pigs did not develop lesions of PE. Four of six positive control pigs given ileal mucosa from pigs with PE also developed microscopic lesions of PE. All of the PE lesions were found in pigs necropsied 10 to 29 days after challenge exposure. None of the swine in the study had clinical signs or gross lesions of PE. Campylobacter spp were isolated from pigs with and without exposure to the ileal mucosa from pigs with PE. There was no relationship between Campylobacter spp isolation and development of lesions. Deoxyribonucleic acids extracted from embryonating chicken eggs injected with the equivalent of 0.5 mg of mucosal lesions and incubated for 4 days hybridized to a DNA probe specific for the ICR whereas DNA extracted from 1.5 mg of mucosal homogenates of the same proliferative tissue did not hybridize with the same probe. Results of these experiments indicated that ICR injected into eggs remained infective for pigs and suggest replication of ICR in the first-passage eggs.

To provide information about oncogenes for molecular biological studies of tumors in domestic animals, theproto-oncogenes homologous to the c-myc, c-erbB-2, c-ros-1, c-yes-1, v-myc, v-Ki-ras, and v-Ha-ras oncogenes of genomic DNA in cattle, horses, pigs, dogs, cats, and chickens were investigated by Southern blot hybridization. High molecular weight genomic DNA in each of the animals contained proto-oncogenes that had a certain homology with the oncogenes used, but the extent of nucleotide homology of the proto-oncogenes differed in number and molecular weight: ie, 1 or 2 bands at 1.6 to 22.0 kilobase (kb) in the c-myc probe, 1 or 2 bands at 1.1 to 16.0 kb in the c-ros-1 probe, 1 to 3 bands at 0.7 to 23.0 kb in the c-erbB-2 probe, 1 to 4 bands at 0.6 to 18.0 kb in the c-yes-1 probe, 1 to 3 bands at 1.6 to 30.0 kb in the v-myc probe, 1 to 7 bands at 1.0 to 36.0 kb in the v-Ki-ras probe, and 1 to 4 bands at 1.0 to 27.0 kb in the v-Ha-ras probe. Furthermore, signal strength of each band, as determined by autoradiography, was not always the same for each probe in the various animals. Our findings indicate that these proto-oncogenes are well conserved with species specificities in each animal.

Pseudorabies virus (PRV), an alpha-herpesvirus, causes substantial economic losses in the swine industry and is currently the focus of eradication and control programs. Some of these programs rely on the ability of veterinarians to differentiate animals exposed to virulent strains of PRV from animals exposed to avirulent vaccine strains of PRV on the basis of a serologic response to nonessential glycoproteins that are deleted in some vaccine strains of PRV. Genetic recombination resulting in the creation of virulent strains of PRV with the same negative immunologic markers as vaccine strains could disrupt these programs. Two strains of PRV were coinoculated either into tissue culture or into sheep to facilitate recombination. Progeny viruses were selected to detect a specific recombinant phenotype. We were able to detect genetic recombination between vaccine strains of PRV following in vitro or in vivo coinoculation of 2 strains of PRV. The selected recombinants had marker-deleted phenotypes in strains with restored virulence genes. Increased virulence was observed in sheep after coinoculation of 2 avirulent vaccine strains of PRV.

Genomic DNA polymorphisms obtained by restriction fragment-length polymorphism from healthy horses and horses with hereditary multiple exostoses were analyzed. These DNA were digested by 12 restriction enzymes and were hybridized against 6 isotopically labeled oncogene probes. Hybridization was not detected with the viral oncogene, v-ras, which indicated this oncogene was absent in the equine genome. Oncogenes (c-raf-1, c-fes, c-myb, c-myc, and c-sis) were present and had similar hybridization patterns and signal intensities in DNA from healthy horses and horses with hereditary multiple exostoses. Unique and distinct restriction fragment-length polymorphisms were detected with the c-raf-1 probe only in BamHI- and PstI-digested equine DNA.

OBJECTIVE To evaluate gene expression and DNA copy number in adipose tissue-derived stromal cells (ADSCs) and in ADSC-derived neurosphere-like cell clusters (ADSC-NSCs) generated from tissues of chronically paraplegic dogs. ANIMALS 14 client-owned paraplegic dogs. PROCEDURES Dorsal subcutaneous adipose tissue (< 1 cm3) was collected under general anesthesia; ADSCs were isolated and cultured. Third-passage ADSCs were cultured in neural cell induction medium to generate ADSC-NSCs. Relative gene expression of mesenchymal cell surface marker CD90 and neural progenitor marker nestin was assessed in ADSCs and ADSC-NSCs from 3 dogs by quantitative real-time PCR assay; expression of these and various neural lineage genes was evaluated for the same dogs by reverse transcription PCR assay. Percentages of cells expressing CD90, nestin, glial fibrillary acidic protein (GFAP), and tubulin β 3 class III (TUJ1) proteins were determined by flow cytometry for all dogs. The DNA copy number stability (in samples from 6 dogs) and neural cell differentiation (14 dogs) were assessed with array-comparative genomic hybridization analysis and immunocytochemical evaluation, respectively. RESULTS ADSCs and ADSC-NSCs expressed neural cell progenitor and differentiation markers; GFAP and microtubule-associated protein 2 were expressed by ADSC-NSCs but not ADSCs. Relative gene expression of CD90 and nestin was subjectively higher in ADSC-NSCs than in ADSCs. Percentages of ADSC-NSCs expressing nestin, GFAP, and TUJ1 proteins were substantially higher than those of ADSCs. Cells expressing neuronal and glial markers were generated from ADSC-NSCs and had no DNA copy number instability detectable by the methods used. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested ADSCs can potentially be a safe and clinically relevant autologous source for canine neural progenitor cells. Further research is needed to verify these findings.

Only a few hybridization experiments have been performed for detection of canine distemper virus (CDV) nucleic acid sequences in tissue cultures and in various tissues. Those published studies used probes derived from tissue culture-adapted CDV, and hybridization signals were not obtained in the CNS tissue, although infective CDV and viral antigen were detectable in this tissue. We developed probes complementary to virulent CDV and were able to detect viral RNA not only in primary brain cell cultures, but also in brain tissues, by use of in situ hybridization. Sensitivity of the test at least equaled that of immunohistochemistry. We applied digoxigenin-labeled, strand-specific RNA probes complementary to the nucleoprotein-coding viral nucleic acid sequence. Our results indicate that to detect CDV nucleic acid sequences in brain tissues, it is essential to use probes derived from the virulent virus.

A method of extracting bacterial DNA from swine feces was developed and used in a molecular assay for the presence of ileal symbiont (IS) intracellularis, formerly known as the Campylobacter-like organism associated with swine with proliferative enteritis. Hybridization with a digoxigenin-labeled, IS intracellularis-specific probe detected the presence of IS intracellularis at a concentration of 10(7) organisms/g of feces. This method was sufficient to detect is intracellularis in the feces of swine with experimentally induced and naturally acquired infection. Results of the hybridization were in agreement with those from histologic postmortem examination.

A slot blot hybridization technique was applied detection of bluetongue virus (BTV) in blood mononuclear cells (BMNC) obtained from cattle with experimentally induced infection. This technique lacked sensitivity to detect the viral nucleic acid directly in clinical specimens. When aliquots of mononuclear cells from these cattle were cultivated in vitro for 10 days to amplify virus titer, only 33.3% of the samples collected during viremia gave a positive signal in the slot blot hybridization format. By contrast results for 34.3% of noncultured and 63.3% of cultured mononuclear cell samples collected during viremia were positive by immunofluorescence. The average number of infected cells, as detected by immunofluorescence in the noncultured mononuclear cell samples, was 1 to 5/300,000, and was usually > 10/300,000 in the cultured cell samples. Virus was isolated from all postinoculation blood samples obtained from 4 heifers that were seronegative at the time of inoculation, but was not isolated from any of the preinoculation samples, or from any of the postinoculation samples obtained from 2 heifers that were seropositive at the time of inoculation. When virus isolation was attempted from separated mononuclear cells in 2 heifers, 43.7% of the noncultured and 87.5% of the cultured samples had positive results.

A recombinant cDNA probe from genome segment 5 obtained from a virulent US bluetongue virus strain (BTV-11 strain UC8) was hybridized to US and Israeli BTV prototypes and field isolates. The cloned genetic probe hybridized with US BTV prototype 10, but not with US prototypes 2, 11, 13, and 17; with the avirulent BTV-11 strain UC2; and with the Israeli prototype 10. When the probe was hybridized to field isolates from the US serotypes, it hybridized to 12 of 14 BTV-10 isolates and 4 of 17 BTV-11 samples, but not to the BTV-13 and BTV-17 samples tested. Hybridization was not observed with the Israeli field isolates studied. Results indicate that a reassortant event occurred between a strain of US BTV-10 and US BTV-11 that originated the BTV-11 strain UC8.

A genomic library to Eperythrozoon suis DNA was constructed in lambda gt11, and from this library, E suis clone KSU-2 was identified as a potential diagnostic probe. In hybridization experiments that used 100-microliter samples of blood collected in chaotropic salt solutions, the KSU-2 probe hybridized strongly with purified E suis organisms and blood samples from splenectomized swine that were parasitized with E suis. However, the probe under stringent conditions did not give radiographic indications of hybridizing with equine blood DNA, bovine blood DNA infected with Anaplasma marginale, canine blood DNA infected with Ehrlichia canis, feline blood DNA infected with Haemobartonella felis, or uninfected swine blood DNA.