Turkey enteric coronavirus (TCV) from intestinal contents of diarrheal poults was isolated and serially propagated in HRT-18 cells, an established cell line derived from a human rectal adenocarcinoma. In these cells, TCV induced cytopathic changes, including polykaryocytosis, which depended on trypsin in the medium and incubation at 41 C. Viral antigens could be demonstrated in the cytoplasm by immunofluorescence, and extracellular virus was detected by an ELISA and negative electron microscopy. The cell-free virus had characteristics of TCV: shape, surface projections, buoyant density of 1.18 to 1.20 g/ml in sucrose, and hemagglutination of rat RBC. The one-step growth curve was complete by postinoculation hours 14 to 16, and maximal titers reached 9 to 9.5 log10 TCID50/ml during 5 passages, after which the titer remained stable. Electron microscopic examination of infected cell monolayers revealed budding of typical coronavirus particles through intracytoplasmic membranes and accumulation of complete virus in cytoplasmic vesicles. Late in the infection, aggregated progeny vial particles were detected near the outer surface of infected cells. One-day-old turkey poults inoculated orally with tissue culture-adapted TCV isolates developed mild to severe diarrhea.

A porcine rotavirus isolate was titrated in neonatal colostrum-fed and colostrum-deprived pigs. The stock rotavirus suspension had a titer of 10 /ml and was in its fifteenth cell culture passage in MA-104 cells. Fourteen colostrum-fed pigs were orally inoculated with dilutions of the stock virus suspension ranging from undiluted to 10-5. These pigs did not develop notable clinical signs during the 7-day experimental trial and no pathologic changes were found in intestine, liver, lung, kidney, spleen, or brain. However, rotavirus was detected in feces of the colostrum-fed pigs, using virus isolation and electron microscopic techniques. Rotavirus was also isolated from lung, brain, or spleen of 4 of 12 of these pigs. Sixteen colostrum-deprived pigs were orally inoculated with dilutions of the stock virus suspension ranging from 10-1 to 10-8. Diarrhea developed in 10 of 12 pigs that were given up to the 10-6 dilution. Seven of these 12 pigs died because of the severity of diarrhea. Pigs that died of rotavirus-induced diarrhea had severe villus loss in the jejunum and ileum. Villi of the small intestine of colostrum-deprived pigs that survived the severe diarrhea were within normal limits at the end of the 7-day trial. The colostrum-deprived pigs that were inoculated with a dilution less than 10-6 and survived past 96 hours underwent seroconversion. Rotavirus was detected by virus isolation and electron microscopy in the feces of all colostrum-deprived pigs that survived beyond 18.5 hours after inoculation. Virus was isolated from lungs, brain, or spleen of 12 of 16 colostrum-deprived pigs.

The use of cultured tissue has not yet become wide-spread in research involving equine disease, and this may be attributable in part to the scarcity of published reports concerning tissue culture methods for this species. We report here the isolation of equine microvascular endothelium (EMVE) from fresh omental tissue of horses and ponies. Fresh donor tissue was minced, subjected to collagenase digestion, and filtered. Cells were layered on 5% bovine serum albumin for gravity sedimentation, the bottom layer was collected, and the cells were plated onto fibronectin-coated flasks. Medium consisted of Dulbecco modified Eagle medium with 10% whole fetal bovine serum (wFBS) and 20 micrograms of endothelial cell growth supplement/ml. The EMVE grew readily in culture, had the cobble-stone morphologic feature at confluence, stained positively for factor VIII-related antigen, and metabolized acetylated low-density lipoprotein. Fibroblast and smooth muscle cell contamination was minimal in primary cell cultures, which were successfully passed and maintained in culture for 3 to 5 serial passages, using various media and substrates. Preliminary studies were undertaken to determine optimal growth conditions with a range of variables: serum concentration, extracellular matrix components, and growth factors. Optimal conditions were achieved with a minimum of 10% wFBS, and with either fibronectin or laminin as extracellular matrix substrates. The EMVE grew adequately in Dulbecco modified Eagle medium plus 10% wFBS, and the added growth factors or serum supplements did not appear necessary for growth of EMVE.

Fecal samples from 131 cattle clinically suspect for paratuberculosis were cultured bacteriologically, using the traditional sedimentation processing method and a processing method that included a centrifugation step. Of 16 samples that were contaminated, 6 were culture-positive on at least 1 medium and by 1 processing method. Ten of 131 (7.6%) fecal samples processed by both methods were lost because of contamination. The number of culture-positive samples (using both processing methods) were 65 of 121 (53.7%) on media without miconazole and 60 of 121 (49.6%) on media with miconazole. Seven of the 121 (5.8%) samples were culture-positive, using centrifugation, after 16 weeks' incubation at 37 C. Thirteen of 60 (21.7%) isolates were obtained only with centrifugation, and 10 of these had low colony counts, suggesting that a centrifugation step may have concentrated microorganisms that would have gone undetected without centrifugation. Six of 60 (10%) isolates positive for M paratuberculosis on the sedimentation method were negative on the centrifugation method. Contamination rates were significantly (P less than 0.001) increased when centrifugation was used. The miconazole significantly (P less 0.001) decreased contamination rates when centrifugation was used.

A bacteriophage for Corynebacterium glutamicum strain LP-6 was isolated from swine waste. It belongs to the Siphoviridae family or Bradley morphologic group B, has a narrow host range, and is sensitive to chloroform and resistant to carbon tetrachloride. The phage is unstable (96% inactivation) in swine waste stored for 4 months at 22 C. The DNA has a molecular weight of approximately 20 Md, cohesive ends, and numerous restriction endonuclease sites. The phage differs from other known C glutamicum phages.