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.

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.

In an abattoir-based case-control study, histologic, and macroscopic examination of porcine intestines at slaughter and 2 molecular assays were compared for use as diagnostic tests of proliferative enteritis (PE). Fecal samples and intestinal specimens were collected from pigs with grossly thick ileum and from clinically normal pigs at slaughter. Tissue specimens were fixed in neutral buffered 10% formalin, and sectioned. Sections stained with H&E were examined for proliferative lesions by a pathologist unaware of the group to which the pig had been assigned on the basis of results of gross examination. Adjacent tissue sections, stained with Warthin-Starry (silver) stain, were examined for presence of the intracellular bacterium of PE, ileal symbiont (IS)-intracellularis, in the enterocytes of the intestinal crypts by the senior author, who was unaware either of the group to which the pig had been assigned or diagnosis by the pathologist. Bacterial DNA was extracted from the fecal samples and assayed by dot-blot hybridization and polymerase chain reaction (PCR) for presence of IS-intracellularis DNA, without knowledge of results of the other examinations. The PCR assay for IS-intracellularis was a specific and sensitive diagnostic test for PE, and dot-blot hybridization was sensitive, but was less specific. Macroscopic examination of intestines at slaughter was a sensitive, but not specific, test. Association between IS-intracellularis and proliferative lesions was statistically examined in the same study. There was a highly significant (P = 0.0078) association between presence of naturally acquired proliferative lesions and intracellular infection induced by IS-intracellularis. The odds ratio of greater than or equal to 14 and estimated attributable fraction of greater than or equal to 92% indicate that IS-intracellularis may be a necessary cause of PE.

A sensitive and specific DNA probe for detection and identification of bovine herpesvirus 4 (BHV-4) was developed. Cloned fragments from a library of HindIII fragments of the BHV-4 (DN-599) genome were labeled with 32P or digoxigenin and were tested for sentitivity and specificity in detecting viral DNA by dot-blot hybridization. Two probes were identified that detected 10 pg of purified viral DNA, and detected viral DNA in 0.001 microgram of total DNA extracted from BHV-4-infected cells. Both probes labeled with 32P and 1 labeled with digoxigenin detected viral DNA in samples prepared from cells infected with 2 prototype strains (DN-599 and Movar 33/63) and 4 field isolates of BHV-4. The DNA probes did not hybridize to total DNA prepared from uninfected bovine cells or from cells infected with BHV-1, BHV-2, alcelaphine herpesvirus 1, pseudorabies virus, or equine herpesvirus 1. One probe, labeled with digoxigenin, was tested further by dot-blot hybridization with infected cell lysates that were simply treated with sodium dodecyl sulfate and proteinase K prior to application to the membrane, avoiding extensive DNA purification procedures. This simplified procedure also resulted in specific detection of field isolates of BHV-4 and prototype strains of BHV-4.