C A Mebus’s research while affiliated with Center for Disease Analysis and other places

Sera from calves infected in utero or postnatally with bovine rotavirus NCDV or postnatally with human rotavirus D (serotype 1) were tested by plaque reduction neutralization assay for antibody to bovine rotavirus and to three serotypes of human rotavirus. Homologous antibody developed in all animals, but antibody to heterologous rotaviruses developed mainly in animals exposed in utero to bovine rotavirus. The development of heterologous antibody may explain the immunological implications for cross-protection, previously observed between bovine and human rotavirus in experimentally infected calves.

The key word in the assigned topic is “research”. This topic can then be broken down into two subdivisions; (1) comparison of currently available diagnostic techniques to recognize outbreaks of diarrhea due to known agents, e.g. rotavirus, Coronavirus, E. coli, salmonella, etc. and (2) those techniques which can be used to investigate outbreaks of diarrhea for which an etiologic diagnosis cannot be made using procedures in #1.

The possibility of immunizing human infants against rotaviruses, which cause severe dehydrating diarrheal disease, may depend on the use of a related rotavirus, derived from another animal species, as a source of antigen. To test the feasibility of this approach, calves were infected in utero with a bovine rotavirus and challenged with bovine or human type 2 rotavirus shortly after birth. Infection in utero with bovine rotavirus induced resistance to diarrheal disease caused by the human virus as well as the homologous bovine virus. These data suggest that the bovine virus is sufficiently related antigenically to the human type 2 virus to warrant further evaluation of the former as a source of vaccine.

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.

Many rotavirus strains have been detected but few have been grown in vitro and this has hampered the development of serologic tests and antigenic comparison of strains obtained from the same or different host species. Because of this limitation of growth in vitro a different approach for distinguishing rotaviruses was undertaken. The rotavirus genome could be separated into 11 discrete RNA segments by polyacrylamide gel electrophoresis. Differences in RNA migration pattern were observed among human strains as well as between human and animal strains; the number of interspecies differences was greater than the number of intraspecies differences. Three distinct patterns were observed among the eight human rotaviruses obtained from each of four successive annual rotavirus epidemics in the Washington, D.C. area. Each of four animal rotaviruses also had distinct patterns which differed from the human patterns in the mobility of from four to seven RNA segments.

Neonatal calf diarrhea causes severe economic loss in both dairy and beef cattle operations. Reovirus-like agents (Rotavirus) have been demonstrated to be an etiologic agent of, or found to be associated with, diarrhea in mice (1), calves (2, 3), human infants (4-8), foals (9), and lambs (10). Coronavirus causes diarrhea in piglets (11) (transmissible gastroenteritis) and calves (12). Intestinal lesions induced by reovirus-like agents and coronaviruses are similar in the various species affected.

Areas of trachea and right apical and right dorsal diaphragmatic bronchi from gnotobiotic neonatal pigs were examined by scanning electron and transmission electron microscopy at 7, 13, and 21 days after pigs were given intranasal inoculation of Mycoplasma hyopneumoniae. Similar areas were examined by scanning electron microscopy from 2 noninoculated neonatal pigs. Tracheal and bronchial lesions were observed in all inoculated pigs. Severe bronchial lesions were in grossly affected lobes. Early infection consisted of mycoplasmas on ciliated epithelial cells. As the interval between inoculation and euthanasia increased, mycoplasmas and leukocytes on the tracheal and bronchial surfaces became more numerous and fewer cilia remained.