key: cord-0005119-wn8o4eju authors: Mebus, C. A. title: Reovirus-like calf enteritis date: 1976 journal: Am J Dig Dis DOI: 10.1007/bf01464768 sha: b726040098769cc29af69e2733001a3f538453a7 doc_id: 5119 cord_uid: wn8o4eju nan REO is an abbreviation for respiratory, enteric orphan. The first viruses in this group were isolated from the respiratory and enteric tracts; however, they could not be associated With disease and thus were called orphans. The calf diarrheal agent belongs to the family Reoviridae (1, (24) (25) (26) (27) on the basis of morphology, cytopathogenicity, resistance to lipid solvents, stability at pH 3, and presence of double-stranded RNA. However, it is serologically unrelated to reoviruses 1, 2, and 3 is thus called a reovirus-like agent. An important practical property of the reovirus-like agent is its stability; feces kept at room temperature for 7 months still contained viable virus (14) . As a result of studies by various groups working on the human reovirus-like agent of infantile gastroenteritis, we now recognize a group of morphologically similar enteric viruses: reovirus-like agent of calf diarrhea, human reovirus-like agent of infantile gastroenteritis, reovirus-like agent of porcine diarrhea (28) , epizootic diarrhea of infant mice (EDIM) (29) , simian SA11 virus, and O agent from Published as Paper Number 4023, Journal Series, Nebraska Agricultural Experiment Station. Research was conducted under Project No. 14-001. sheep and cattle (30, 31) . The calf reovirus-like agent has been shown to be related antigenically to the human reovirus-like agent (17, 32, 33) , porcine reovirus-like agent, and epizootic diarrhea of infant mice (17, 32, 33) , It is serologically unrelated to the reoviruses and to the orbiviruses (33) . The human reovirus-like agent was initially thought to be an orbivirus (6); subsequently the names rotavirus (32) and duovirus (12) were suggested. Two types of calves, hereafter referred to as colostrum-deprived and gnotobiotic, were used in the calf diarrheal reovirus-like agent studies. Colostrum-deprived calves were obtained by hysterotomy, housed in individual isolation rooms which were scrubbed and fumigated with formaldehyde gas between calves, and fed pasteurized, homogenized milk. These calves were contaminated with E. coli and other bacteria. Gnotobiotic calves were delivered by hysterotomy into a sterile plastic surgical isolator glued to the abdominal wall and then transferred to a sterile isolator. The calves were fed autoclaved homogenized milk. Approximately 70% of these calves were bacteriologically sterile during the experimental period and the remainder were contamined with B. subtilis and/or a staphylococcus. The incubation period after oral inoculation was somewhat dependent on the viral titer in the inoculum. When 10 ml of bacteria-free feces containing virulent reovirus-like agent was used, the incubation period was as short as 12 1A-13 hr. Low viral titer inocula caused incubation periods of 20-36 hr. Variations in the incubation period of usually 0nly 1-3 hr occurred among calves inoculated with a constant-size inoculum from the same virulent virus pool. Signs usually progressed in the following order: depression, anorexia, a few-strings of thick saliva hanging from the lips, and diarrhea. Onset of depression was rapid; eg, a calf that appeared normal at 8 AM was SO depressed that it would not stand at 10 AM. The diarrheic period lasted 5-6 hr and the animal passed about 300 ml of liquid yellow feges. Volume of feces was somewhat dependent on the amount of milk consumed. Signs during the postdiarrheic period depended on the type of calf inoculated. Gnotobiotic calves appeared normal, suckled, and had pasty feces 24 hr after the onset of diarrhea. Gnotobiotic calves removed from isolation units and placed in isolation rooms 24 hr after the onset of diarrhea remained normal. Up to 50% of the colostrum-deprived calves died shortly after the onset of diarrhea to 6 days postinoculation. Mortality in natural infections in normally born, colostrum-fed calves on ranches ranges from near 0 to 50% or more depending on the secondary bacterial infection. Changes induced by the calf diarrheal reoviruslike agent were studied using colostrum-deprived calves in isolation rooms and gnotobiotic calves. Infected gnotobiotic calves were killed at two intervals after the onset of diarrhea, 0.5• hr and at 4.5-6 hr. Control gnotobiQt!c calves were killed at approximately the same ~ages as the iinoculated calves (34) . At necropsy the only difference observed between the control gnotobiotic and diarrheic gnotobiotic calves was that the diarrheic calves had more liquid in the small and large intestines. Colostrum- deprived calves that died or were killed when moribund usually had a suppurative arthritis, suppurative menirigitis, and occasionally peritonitis. E. coil was isolated from these fluids and tissue. Sections from the upper, middle, and lower parts of the small intestine from the control gnotobiotic calves were immunofluorescent negative for the reovirus-like agent. Histologically, the epithelium on the distal tWO thirds of the villi in the upper and middle parts of the small intestine was composed of tall columnar cells. The nuclei were located in the apex of the cells next to the lumen, and the basal parts of the cells were vacuolated. In these parts of the small intestine the villous lamina propria consisted mostly of blood vessels, a lacteal and a few reticulum cells (Figure 1 Figure 4) . The viral particles and electron-dense material which was considered to be precursor viroplasm, since it often contained some viral particles in the beginning of a crystalline array, were present within the cisternae of the rough endoplasmic reticulum of the villous epithelial cells (35) . Calves killed approximately 4-6 hr after the onset of diarrhea had no immunofluorescent villous epi-thelial cells, but immunofluorescent cells were present in the intestinal contents in the lower small intestine and colon. No immunofluorescence was observed in other organs. Histologically, the epithelial cells over the villi in the upper and middle parts of the small intestine and on the sides of the villi in the lower part of the small intestine had a cuboidal morphology ( Figure 5 ). Squamous epithelial cells Digestive Diseases, Vol. 21, No. 7(July 1976) sage of lung inoculum. The viral titer in the colonic contents was 106/ml. From tissues of the second calf, virus was isolated from only the jejunal and ileal mesenteric lymph nodes. The viral titer in the colonic contents was 10S/ml (34) . Immunofluorescent, histologic, and transmission of electron microscopic findings suggested the following pathogenesis for the reovirus-like infection. After oral inoculation the columnar epithelial cells over the distal two thirds of the villi in the upper part of the small intestine became infected and the infection rapidly progressed caudally. When the calf became depressed and diarrhea began, the villous epithelial cells were morphologically normal, but the cells contained a large quantity of viral antigen. It is postulated that the viral infection of the villous epithelial cells redirected cell function from absorption to virus production, and thus the digestive fluids and partially digested milk accumulated in the (Figure 6 ). No virions were observed by electron microscopy in the cuboidal or squamous villous epithelial cells (35) . The villi, particularly in the lower part of the small intestine were moderately shortened. Villi in all three levels of the small intestine had an increase in reticulum-like cells in the lamina propria (34) . Two calves inoculated with cell culture-adapted virulent virus and killed 2 and 6 hr after the onset of diarrhea had similar clinical signs and lesions as those inoculated with fecal material. Immunofluorescence was observed only in the small intestinal villous epithelium. In addition, the following fluids and tissues were cultured for the reoviruslike agent: blood plasma, mesenteric lymph nodes draining the ileum and jejunum, spleen, liver, kidney, lung, thymus, and colonic contents. Using tissues from the first calf, virus was detected on the first passage of inoculum prepared from the jejunal mesenteric lymph nodes and on second blind pas- The calf reovirus-like agent has been demonstrated in diarrheic feces using electron microscopy (1, 36) and the fluorescent antibody techniques on fecal smears and culture (37) (38) (39) . Electron microscopic examination of feces for virions having a reovirus morphology has several advantages: (1) a higher percentage of infected feces will be detected, (2) virions can be detected in feces collected several days after the onset of diarrhea, and (3) the method will detect virions in putrefied feces. Immunoelectron microscopy permits a more accurate identification of the particles. Villous epithelial cells which were shed off the ends of the villi can frequently be demonstrated by the immunofluorescent techniques in fecal smears prepared from feces collected early in the diarrheic period. Using electron microscopy, these cells have been shown to contain a large number of virions (31) . Successful clinical implementation of the immunofluorescent technique depends on collecting early fecal samples, examining specimens from 5 to 10 animals in the herd, and prevention of autolysis of the cells in the feces after collection. Some labo-ratories have found this method of diagnosis difficult. Inoculation of bovine kidney cell cultures with fecal filtrate and examination of the culture by the immunofluorescent technique 24--48 hr after inoculation has been successfully used by other (37) (38) (39) for detection of the calf reovirus-like agent. It was observed in the early studies with the virulent calf reovirus-like agent that recovered calves did not develop diarrhea when reinoculated with the virus several days to 4 weeks after the initial infection. Therefore, the possibility of protecting calves by oral inoculation with an attenuated virus seemed feasible. The calf reovirus-like agent was attenuated by passing it approximately 140 times on fetal bovine kidney (FBK) cells at 37~ and an additional 60 times on FBK cells at 29-30~ Safety and potency tests were performed in gnotobiotic calves. Twenty-four 6--7-hr-old calves were inoculated orally with the attenuated virus. 20 of these calves were observed for 48-72 hr and then challenge inoculated orally with l0 ml of gnotobiotic calf feces containing virulent virus. These calves remained normal during the postvaccination observation period, and one developed mild diarrhea after challenge inoculation. 5 nonvaccinated challenge control calves developed severe diarrhea. The remaining 4 calves were not challenge inoculated but were observed for 30 days; they remained normal (40) . Prevaccination serums had neutralizing (SN) antibody titers of less than 2. 30 days postvaccination, the vaccinated-challenge inoculated calf SN titers ranged from 32 to 512; SN titers for the 5 challengecontrol calves ranged from 256 to 512, and the titers in the calves which received only vaccine ranged from 64 to 256 (40) . The calf reovirus-like vaccine was field tested on ranches from which feces collected from diarrheic calves contained immunofluorescent cells. Calves were vaccinated when less than 24 hr old; prevaccination serums were obtained from about 10% of the calves. The reduction in the morbidity and mortality from diarrhea after vaccination in 12 of 14 herds was statistically significant. Most of the calves on these ranches had prevaccination (colostral) SN antibody titers to the reovirus-like agent (40) . On the basis of the above results, protection against infection appears to be dependent on a local cellular resistance and not circulating antibody. Initially this resistance may be due to an interference phenomenon and later to local antibody production. Calf diarrhea (scours): Reproduced with a virus from a field outbreak Cell culture propagation of neonatal calf diarrhea (scours) virus Neonatal calf diarrhea: Purification and electron microscopy of a coronavirus-like agent Neonatal calf diarrhea: Propagation, attenuation and characteristics of a coronavirus-like agent Evidence for viral gastroenteritis Virus particles in epithelial cells of duodenal mucosa from children with acute nonbacterial gastroenteritis Detection of a new virus by electron microscopy of fecal extracts from children with acute gastroenteritis Studies on the etiology of acute infantile diarrhea Rotavirus" in gastroenteritis IT: Occurrence of "viral particles" in diarrhea: Houston, Texas and Guatemala Viruses in gastroenteritis Importance of a new virus in acute sporadic enteritis in children Virus particles in gastroenteritis Epidemic viral enteritis in a long stay children's ward Diagnostic electron microscopy of feces. II. Acute gastroenteritis associated with reovirus-like particles Orbiviruses and gastroenteritis Reovirus-like agent in stools: Association with infantile diarrhea and development of serologic tests Reovirus-like agent in Japanese infants with gastroenteritis Orbivirus acute gastroenteritis of infancy Rotavirus in stored specimens of fecal extracts Viruses in gastroenteritis Virus in faecal extracts from children with gastroenteritis Rotaviruses of man and animals. Lancet I Cell culture adaptation and propagation of a reoviruslike agent of calf diarrhea from a field outbreak in Nebraska Purification, morphology and partial characterizalion of a reovirus-like agent associated with neonatal calf diarrhea Physiochemical characterization of a neonatal calf diarrhea virus Cell culture studies of a neonatal calf diarrhea virus Causes of piglet enteritis Purification and characterization of epizootic diarrhea of infant mice virus Morphological studies on simian virus S.A. I1 and the related "O" agent Electron microscopic and serological studies on simian virus S.A.I1 and the "related" O agent. Onderstepoort Relation between viruses from acute gastroenteritis of children and newborn calves A new complement-fixation test for the human reovirus-like agent of infantile gastroenteritis using the Nebraska calf diarrhea virus as antigen Pathology of neonatal calf diarrhea induced by a reo-like virus Underdah[ NR: Neonatal calf diarrhea: Electron microscopy of intestines infected with a reovirus-Iike agent Laboratory techniques for demonstrating Nebraska calf diarrhea virus Reo-like neonatal calf diarrhea (NCD) virus demonstrated in Denmark Viral enteritis of calves The isolation of a reovirus-like agent associated with diarrhoea in colostrumdeprived calves in Great Britain Immunity to neonatal calf diarrhea virus