Marvin B. Rhodes’s research while affiliated with University of Nebraska–Lincoln and other places

Seventy-nine diarrheic calf fecal samples were examined by electron microscopy, immunodiffusion and the fluorescent antibody technique for the presence of rotavirus (reovirus-like agent). Thirty-eight (48%) of the samples were positive by electron microscopy, 59% by immunodiffusion and 20% positive by fluorescent antibody technique analyses. Another 9% were suspect-positive by fluorescent antibody technique. Chymotrypsin treatment of the fecal samples increased the ease of observing the viral particles by electron microscopy and also intensified the immunodiffusion arcs obtained. Immunodiffusion analyses using specific antisera to the virus would appear to be a practical method of detecting rotavirus in diarrheic fecal samples.

Six pigs had been immunized with multiple dose of embryonated eggs and an isolated intestinal loop was prepared in each animal. Specific antibodies to Ascaris suum were detected in the soluble protein fraction of washings from the intestinal loops using an indirect fluorescent antibody test. The specific antibodies belonged to the IgA, IgG and IgE classes of immunoglobulins. In contrast, specific antibodies were not detected in the soluble protein fraction from the accumulated fluid from the intestinal loop of one pig. Soluble proteins from the washings of intestinal loops consisted of serum albumin, a large molecular size glycoprotein, and variable amounts of several α-globulins, transferrin, and immunoglobulins. The individual soluble protein solutions were efficiently fractionated using DEAE-cellulose, Sephadex G-200, and Sepharose 6B Chromatographic columns.

Intestinal lesions caused by a virus serologically unrelated to the calf diarrheal rotavirus or coronavirus were studied in gnotobiotic calves. The virion purified from feces from infected calves was a fringed particle with a diameter of about 100 nm. The incubation period from time of inoculation per orum to onset of diarrhea in calves was as short as 8 hours. The viral infection in bacteria-free calves or calves not contaminated with pathogenic bacteria caused severe illness for only 24 hours. When bacteria such as the K99 antigen Escherichia coli were present, the combined infection caused mortality. Lesions occurred only in the small intestinal villous epithelium. Calves euthanatized shortly before or after the onset of diarrhea had developed villous epithelial cell syncytia that contained numberous virions in the cytoplasm. Within 2 to 3 hours after onset of diarrhea, the infected cells were shed and the villi had denuded tips or had cuboidal to squamous epithelial cells.

Larvae of the nematode Ascaris suum were recovered in posterior small intestinal and colonie mucosal scrapings of pigs 4 to 29 hr after inoculation of embryonated eggs. Larvae were also recovered in the mucosal scraping from ligated intestinal segments at 4 to 19 hr after injection of embryonated eggs into the segments. Location of the ligated segment in the small intestine did not appear to affect hatching of eggs or migration of the larvae into the mucosal surface. Embryonated eggs hatched in ligated cecums and larvae were recovered in the livers of pigs after 2.5 and 3.5 days. Larvae were recovered in washings from Thiry-Vella intestinal loops at 5 to 23 hr after embryonated eggs were inoculated into the loop. Hatching of eggs occurred in intestinal loops in pigs unexposed to eggs and in pigs given multiple doses of eggs either orally or into the intestinal loop.

Adult male and female Haemonchus contortus were homogenized and subjected to differential centrifugation. The crude, high-speed, supernatant fraction contained more than 95% of the glutamate dehydrogenase activity. The enzyme was purified through use of DEAE-cellulose columns and sucrose density gradient centrifugation. The enzyme from both crude and purified preparations was detected as a single band of activity following starch or polyacrylamide-gel electrophoresis. The Haemonchus enzyme was compared with ovine and bovine liver glutamate dehydrogenases. The three enzymes were similar in molecular size, Michaelis constants, and pH optimums but differed in electrophoretic mobility in polyacrylamide-gels, activity with NADP as coenzyme, and effect of AMP and ADP on activity. Sheep anti-Haemonchus glutamate dehydrogenase serum inhibited Haemonchus glutamate dehydrogenase, but did not inhibit the ovine or bovine enzymes.

Serum protein alterations in chicks with reticuloendothelial virus disease (RED) were studied by cellulose acetate electrophoresis. The serum electrophoregram of the RE virus inoculated chicks presented profound alterations when compared with the normal control chicks. The changes consisted of a highly increased gamma-1-globulin peak and decreased albumin. Moderate elevations in all the other electrophoretic fractions were also observed, except for prealbumin. There was not a significant difference in the total serum proteins between the RED infected and the control groups of chicks. It was found in previous studies that chicken transferrin migrated in the gamma-1-globulin position of the cellulose acetate electrophoregram. In RED infected chicks the elevation of gamma-1-globulins was caused by an increase in transferrin and not by an increase in immunoglobulins. These findings were substantiated by comparative electrophoretic studies on paper and polyacrylamide-gel, immunodiffusion, gel-filtration on Sephadex G-200, and by an iron-transferrin stain. The marked proliferation of reticuloendothelial cells and the cellular alterations in the thymus and bursa of Fabricius in RED infected chicks appeared to be causally related to the elevated concentration of transferrin in the serum.

A coronavirus like agent isolated in fetal bovine kidney (FBK) cell culture from feces of a calf having diarrhea was adapted to secondary FBK cells. The virus was attenuated by serial passage in consecutively higher passages of FBK cells originating from the fetus used for adaption. The mononucleated cells and syncytia in infected FBK cells and the epithelium of intestine from diarrheal calves inoculated with virulent and cell culture propagated virus (16th passage) fluoresced with the same conjugate. The electron microscopic structural characteristics of cell culture propagated virions were indentical to those of the virus in diarrheal calf feces. In ultrathin sections of detached cells from infected FBK culture, virions were present within membrane bound vesicles of cytoplasm of cells and had an average overall diameter of 78 nm. Calves inoculated orally with attenuated cell culture virus were resistant to oral challenge inoculation with virulent virus of fecal origin.

Lesions induced by a bovine coronavirus like agent were studied in gnotobiotic and colostrum fed calves using gross, histologic and electron microscopic procedures. Lesions in gnotobiotic calves were present in the colon, mesenteric lymph nodes and in all segments of the small intestine. Calves killed 4 hr after the onset of diarrhea had immunofluorescent epithelial cells on the villi of the small intestine and surface of the colon. Calves killed at 44 hr had shortened intestinal villi and cuboidal epithelial cells. The villus to crypt ratio in the lower small intestine averaged 1.0 compared with 5.3 in a control calf. Immunofluorescent cells were present at the tips of the villi, and at the surface and in the crypts of the colon. Colostrum fed calves that had serum neutralizing antibody titers for the coronavirus like agent developed diarrhea when inoculated orally with the agent. There was good correlation between histologic, immunofluorescent and electron microscopic findings.

The pathology induced by a reovirus like agent isolated from diarrheic calves less than 5 days old and by a coronavirus like agent isolated from calves 7 to 21 days old was studied in gnotobiotic calves. Diarrhea induced by the reovirus like agent had an incubation period of 14 to 22 hr. Initially the villous epithelium in sections of upper middle and lower small intestine was composed of tall columnar cells which fluoresced when stained with homologous conjugate. Within 4 to 6 hr after the onset of diarrhea the columnar cells were lost and the epithelium replaced by nonfluorescent cuboidal cells. Diarrhea induced by the coronavirus like agent had an incubation period of 19 to 24 hr. Sections of upper, middle, and lower small intestine from calves killed shortly after the onset of diarrhea had long villi and homologous immunofluorescence of all villous epithelial cells and surface epithelial cells in the colon. Calves killed 48 hr after onset of diarrhea had severe villous atrophy, particularly in the lower small intestine, and more extensive colonic epithelial fluorescence.