Isolation of an atypical rotavirus causing diarrhea in neonatal ferrets.

A rotavirus was isolated from neonatal ferrets (Mustela putorius furo) with diarrhea at a large commercial farm. This virus is classified as an atypical rotavirus, probably belonging to the group C rotaviruses. This classification is based on the lack of the rotavirus group A common antigen and on its distinct dsRNA electropherotype pattern in polyacrylamide gels. The diarrheal disease was reproduced experimentally in neonatal ferrets.

Genetic and antigenic relatedness of human and animal strains of antigenically distinct rotaviruses.

We used nucleic-acid hybridization and enzymatic and immunofluorescence assay to examine the relatedness of human and animal strains of antigenically distinct rotaviruses (ADRVs). ADRVs isolated from rats and humans in Baltimore, Maryland, were shown to be closely related to bovine and porcine strains of group B rotavirus. A human group B rotavirus associated with epidemics of gastroenteritis in China was also found to share antigenic determinants and genome sequence homology with ADRVs passaged in rats in the United States. Closely related strains of group B rotavirus thus appear to infect human and animal populations in widely separated geographic areas.

Rotaviral and coronaviral diarrhea.

A number of different viruses can be primary pathogens in the neonatal calf diarrhea complex. By far the most common viruses causing calfhood diarrhea found throughout the world are rotaviruses and coronaviruses. Primary infection of newborn calves with either one of these viruses can cause severe intestinal alterations and diarrhea. Rotaviruses can produce high-morbidity outbreaks of diarrhea in calves under 10 days of age. Morality is variable mainly owing to secondary bacterial infections and electrolyte imbalances. Rotavirus infection of the small intestinal mucosa leads to loss of enterocytes of the upper third of the intestinal villi with subsequent villous atrophy and malabsorption. There is growing evidence that different rotavirus serotypes of different pathogenicity exist. Coronavirus infections can produce high-morbidity outbreaks of diarrhea in calves under 20 days of age, with variable mortality due to secondary complications. Coronaviruses affect not only the small intestinal mucosa, producing significant villous atrophy, but also the colon, causing a very severe intestinal damage that can lead to death due to subsequent electrolyte disturbances. All coronaviruses associated with neonatal calf diarrhea appear to be of the same serotype. The etiologic diagnosis of viral diarrheas of calves requires the support of the laboratory. One of the most useful diagnostic methods is the examination of fecal extracts for the presence of virus particles by electron microscopy. Other antigen-detection procedures like enzyme immunoassays have been found to be useful in the diagnosis of rotaviral diarrheas. The sample of choice for these diagnostic tests is a fresh fecal sample collected directly from the calf as close as possible to the onset of diarrhea. Samples from more than one calf during the outbreak enhance the laboratory ability to establish a proper viral diagnosis.

Severe respiratory disease in dairy cattle in New York State associated with bovine respiratory syncytial virus infection.

Severe respiratory disease associated with bovine respiratory syncytial virus (BRSV) infection has been identified in dairy cattle in New York State. The cases identified occurred in dairy calves and heifers. The disease was characterized in 4 animals by pathologic changes including interstitial pneumonia, necrotizing bronchiolitis with multinucleated syncytial epithelial cells and interstitial emphysema. BRSV antigen was demonstrated in lung samples or was isolated in tissue culture in all 4 cases. A retrospective survey of 6279 bovine diagnostic accessions between 1977 and 1982 revealed 66 cases of interstitial pneumonia, often with concurrent bronchiolitis. In this 5 year period, only 1 case in 1981 had interstitial pneumonia and bronchiolitis with pathologic features consistent with BRSV infection. It is concluded that pathogenic BRSV has entered New York State and that it is contributing to clinical respiratory disease in dairy cattle.

Effect of combined rotavirus and Escherichia coli in neonatal gnotobiotic calves.

Gnotobiotic calves (24 hours old) were monoinfected with calf rotavirus (CRV) strain NCDV, an enterotoxigenic Escherichia coli (ETEC) strain B44 (K99+), or a nonenterotoxigenic E coli (NETEC) strain 123 (K99-). Calves also were dually infected with CRV and either ETEC or NETEC. Eighteen calves equally allotted between 6 treatment groups were used in these studies: Noninfected controls--group A; CRV--group B; ETEC--group C; NETEC--group D; CRV + ETEC--group E; and CRV + NETEC--group F. Severe diarrhea and villous atrophy were observed in calves of treatment groups B, C, E, and F. Mortality was present only in treatment groups C and E as result of ETEC infection. There were no significant differences in the clinical responses or enteric lesions between treatments B and F, although a significant increase in the concentrations of NETEC was demonstrated in calves dually infected with CRV + NETEC (group F) as compared with calves monoinfected with NETEC (group D). Calves inoculated with ETEC (group C) had severe villous atrophy, neutrophilic infiltration of intestinal lumen, and moderate enterocyte necrosis. Calves dually inoculated with CRV + ETEC (group E) had the most extensive and severe lesions, similar to those in group C, plus a pronounced necrotic fibrino-hemorrhagic enteritis. Infection of enterocytes by CRV did not affect in any way the adherence of ETEC to the intestinal mucosa. Dual viral and bacterial infections of the same enterocytes were evident.

Effect of rotavirus and/or Escherichia coli infection on the aggregated lymphoid follicles in the small intestine of neonatal gnotobiotic calves.

The effect of rotavirus and/or Escherichia coli infections on the follicle-associated epithelium (FAE or M cells) of the domes of the aggregated lymphoid follicles (ALF, or Peyer's patches) of gnotobiotic calves was evaluated by light, scanning electron, transmission electron, and immunofluorescence microscopies. Calf rotavirus (CRV) infection produced loss of FAE cell microvilli, and virions were observed in cytoplasmic vacuoles of FAE cells, as well as in intercellular spaces between FAE cells and lymphoid cells migrating through the dome epithelium. The CRV particles appeared to have entered the FAE cells by phagocytosis, with no subsequent cytoplasmic replication. Enterotoxigenic E coli (ETEC) induced more severe alterations including marked microvilli loss and ballooning in the FAE cells. There was no adhesion to, or colonization of FAE cells by ETEC, but bacteria were observed free or phagocytized within the dome and the germinal centers of the ALF. There were no ETEC observed in the cytoplasm of FAE cells. The presence of nonenterotoxigenic E coli (NETEC) in the intestine of calves had no effect on the intestinal FAE cells. The addition of NETEC to CRV infections did not enhance or modify in any way the response of FAE cells to the viral infection; however, the combination of CRV + ETEC produced severe necrosis of the FAE cells, and loss of dome epithelium of ALF.

Effects of environmental and dietary factors on human rotavirus infection in gnotobiotic piglets.

The addition of proteolytic enzyme to diets fed to newborn gnotobiotic piglets exacerbated their diarrheal response after oral infection with human rotaviruses. Supplementation of diets with proteolytic enzyme and reduced ambient temperature were evaluated for effects upon the clinical response of gnotobiotic piglets infected with human rotavirus Wa strain, type 2. Piglets were divided into four treatment groups combining two variables: ambient temperature of 35 or 26 degrees C, with and without proteolytic enzyme supplementation of the diet. Infected piglets maintained at 26 degrees C with and without enzyme supplementation had 90 and 70% mortality, respectively. No mortality was observed in infected piglets maintained at 35 degrees C. Protease supplementation of diets fed to piglets kept at 35 degrees C resulted in more uniform onset of diarrhea of greater severity than in littermates fed diets without the supplementation.

Effect of Streptococcus faecium C-68 in control of Escherichia coli-induced diarrhea in gnotobiotic pigs.

Streptococcus faecium was fed to prevent colibacillosis in gnotobiotic pigs. Three strains of Escherichia coli were used. With strain O:K103, 987P:NM in pigs fed S faecium before the E coli challenge exposure, the pigs exhibited less severe diarrhea, recovered earlier, and produced better weight gains than did pigs given E coli only. Escherichia coli strains O157:K88ac:H19 and O8:K87, K88ab:H19 were more virulent. Pigs fed S faecium and challenge exposed with these 2 strains of E coli developed mild diarrhea; however, none of the pigs died, and they continued to eat well and gained weight. Pigs given E coli only developed severe diarrhea and lost weight, and 5 of 8 infected pigs died. Bacterial counts of E coli and S faecium from 3 areas of the small intestine and the cecum were all comparable among experimental groups. Histopathologic examinations demonstrated abundant colonization of the intestinal tract with S faecium. Seemingly, S faecium reduced the toxic effects of E coli and prevented generalized infection and death.

Morphologic characteristics of the epithelial surface of aggregated lymphoid follicles (Peyer's patches) in the small intestine of newborn gnotobiotic calves and pigs.

Scanning electron microscopic studies of the dome epithelium of aggregated lymphoid follicles (Peyer's patches) of gnotobiotic newborn calves and pigs revealed distinct characteristics. The dome epithelium in the calves was characterized by a uniform population of lymphoepithelial cells (M cells). These cells were columnar, with a luminal surface that bulged toward the intestinal lumen forming intercellular crevices. Bovine M cells were covered by densely packed blunt microvilli that were irregular, short and thick and that differed from microvilli of absorptive epithelial cells. The dome epithelium in the pigs had 2 distinct cell populations. The majority had surface characteristics indistinguishable from those of ordinary absorptive epithelial cells. Interspersed among the absorptive-like epithelial cells were numerous M cells with distinctive surface morphologic characteristics. Porcine M cells were covered by densely packed, long microvilli that were irregular and thick and that projected above the microvilli of adjacent epithelial cells. The epithelium of aggregated lymphoid follicles in calves differed from that of primates and rodents. The organization of the dome epithelium in pigs resembled that of rodents. However, the surface morphologic features of porcine M cells were unlike that of rodents and human beings.

Scanning electron microscopy of intestine of gnotobiotic piglets infected with porcine rotavirus.

The development of intestinal lesions caused by the porcine rotavirus were studied in six day old gnotobiotic piglets by scanning electron microscopy. The onset of diarrhea followed an incubation period of 17 to 31 hr. The first detectable lesion was observed in the ileum at 12 hr postinfection, a few hours before the onset of diarrhea. At this time enterocytes appeared swollen and began to separate from each other. Seventeen hours after the onset of diarrhea, lesions were quite severe jejunum and ileum. Enterocytes were detaching from the lamina propria leaving denuded areas. Microvilli were sparse on the cell surfaces and there was marked villous atrophy. Regeneration of ileal mucosa was evident at 4.8 days after the onset of diarrhea. Nine days after recovery from diarrhea the intestinal villi had returned to near its normal structure but there remained some evidence of mucosal damage.

Patterns of shedding of human reovirus-like agent in gnotobiotic newborn piglets with experimentally-induced diarrhea.

Virus shedding patterns of neonatal gnotobiotic piglets infected with the reovirus-like agent of human infantile gastroenteritis were studied. Fecal viral counts were highest before or at the onset of diarrhea. In diarrheic piglets, viral particles were usually observed for only 1-2 days after the onset of diarrhea, and total duration of shedding was 2-6 days. One infected piglet shed virus for 4 days but did not develop diarrhea. The presence of virus at or about the time of illness is consistent with the induction of diarrhea in piglets inoculated with the human reovirus-like agent.

Immune response to orally administered calf reovirus-like agent and coronavirus vaccine.

Twenty 6-to 7-hour-old gnotobiotic calves inoculated orally with attenuated calf diarrhea reovirus-like agent and challenge-inoculated with virulent virus 48-72 h post-inoculation (PI) remained clinically normal during the post-vaccination observation period; one developed mild diarrhea after challenge inoculation. Four non-vaccinated challenge control calves developed severe diarrhea. Twenty 6-to 7-hour-old gnotobiotic calves inoculated orally with attenuated calf diarrhea coronavirus and challenge inoculated with virulent virus 96 h later remained clinically normal during the post-vaccination and post-challenge observation period. Four non-vaccinated challenge control calves developed severe diarrhea and 2 of these died. Five-foot long isolated loops prepared in the lower ileum (Thiry-Vella loop) of newborn colostrum-deprived calves were inoculated with attenuated coronavirus. Daily loop washings were cultured for virus and tested for neutralizing antibody. Peak viral titers of 10(6.5) to 10(7) occurred 3-4 days PI and descended rapidly to 0 between 6 and 8 days PI. Neutralizing antibody was first detected in the washings 6-8 days PI and reached a titer of 128-256 7 to 9 days PI. Loop immunoglobulin separated by gel filtration and identified by immunodiffusion were primarily IgM and IgA. Initial resistance to virulent viral infection is thought to be due to an interference phenomenon, with later resistance due to local antibody.

Diarrhea caused in gnotobiotic piglets by the reovirus-like agent of human infantile gastroenteritis.

One- to four-day-old gnotobiotic piglets were inoculated orally with a reovirus-like agent obtained from human infants with acute gastroenteritis. Diarrhea developed in the piglets two to seven days after inoculation and was reproduced for five serial passages in one sequence and for three passages in another. Nineteen of 21 inoculated piglets developed diarrhea; reovirus-like particles were observed in intestinal contents and/or fecal samples from 17 animals with illness and from two inoculated piglets that did not develop diarrhea. One piglet, for which daily fecal samples were examined by electron microscopy, shed the largest number of virus particles at the onset of diarrhea. Immunofluorescent antibody responses to the reovirus-like agent were detected in sera from the seven inoculated animals that were tested.

Recovery of transmissible gastroenteritis virus from chronically infected experimental pigs.

Transmissible gastroenteritis (TGE) virus was reisolated from pulmonary and intestinal tissues from 6 of 9 chronically infected experimental pigs (principals) necropsied 30 to 104 days after inoculation. Tissue homogenates (lung and small intestine) from the principals were prepared and inoculated into 3- to 5-day-old gnotobiotic pigs. The virus reisolated from the tissue homogenates produced a milder disease on 1st passage and a more severe disease on 2nd passage. The chronically infected experimental pigs (principals) developed serum-neutralization titers to TGE of 1:30 to 1:525. There appeared to be no relationship between serum titers and reisolation of TGE virus from the 9 principals. The persistence of virus in lung or intestine to 104 days indicates the recovered (or carrier) pig may be considered the primary source of TGE virus infection.