Characteristics and analysis of electropherotypes of avian reovirus field isolates.

Genomic segments of 10 selected isolates of avian reoviruses recovered from the intestine of birds affected with malabsorption syndrome or runting/stunting syndrome were separated by polyacrylamide gel electrophoresis. Different electropherotypes were observed and analysed, depending on the period of recovery and particular geographic locations. The analysis showed great variability in the dsRNA profiles of the isolates and higher mobility of the segments L1, S1, S2, S3 and S4. There was no correlation between electropherotype and geographic origin of the isolate. The analysis also showed the emergence of electropherotypically distinct strains since the introduction of modified live reovirus vaccines.

Effect of sodium diethyldithiocarbamate, Corynebacterium parvum and mycobacterium cell wall extract on in vitro blastogenic responses of bovine blood lymphocytes.

In vivo inoculation of three-month-old calves with sodium diethyldithiocarbamate (DTC), killed Corynebacterium parvum or mycobacterium cell wall extract (MCWE) resulted in an enhancement of in vitro peripheral blood lymphocyte blastogenic responses to mitogens phytohemagglutinin (PHA) and Concanavalin A (Con A) in the first three days after treatment. In a separate experiment, blood lymphocytes isolated from a healthy nontreated calf were incubated in vitro in presence of each of the same immunostimulating agents and tested for their blastogenic responses to PHA and Con A. The results showed that all immunostimulants, excepting DTC, enhanced the in vitro blastogenic responses of lymphocytes to PHA and Con A. Finally, addition of MCWE to cultures of blood lymphocytes isolated from calves vaccinated intramuscularly with bovine rotavirus and adjuvant resulted in an enhancement of the in vitro lymphocyte transformation to rotavirus. Our study demonstrated that DTC, killed Corynebacterium parvum and mycobacterium cell wall extract were able to enhance bovine T cell proliferation in vitro.

Comparison by plaque assay of bovine rotavirus and a bovine enterovirus-like agent.

Enterovirus-like particles from feces of calves are a frequent source of contamination of bovine rotavirus isolates. A study of plaque formation using BSC-1 cells indicated differences in behaviour of the viruses which could be used for differentiation the purification. The enterovirus-like particles produced well-defined plaques earlier and reached their optimal size much more rapidly than did the rotavirus. Furthermore, plaques produced by bovine enterovirus-like particles were significantly larger than those of bovine rotavirus. The viral cytopathic effects on the cells within the plaques were also characteristic for each virus.

Genomic variations and antigenic relationships among cytopathic rotavirus strains isolated in Quebec dairy herds.

Twelve isolates of bovine rotavirus, originating from eight dairy herds in Quebec known to have frequent epizootics of diarrhea in young calves in the last five years, were successfully propagated in cell cultures. The 12 isolates produced clear-cut plaques in BSC-1 cells and, except for one isolate, agglutinated human group "O" erythrocytes to an higher titer than bovine erythrocytes. Antisera to each isolate were produced in rabbits and used to study their antigenic relationships. All the isolates shared the group-specific immunofluorescent antigen and were antigenically related as demonstrated by the seroneutralization and hemagglutination-inhibition tests. However, the relationships to the Nebraska rotavirus was quite weak in cases of two Quebec isolates. When the genomes of the various isolates were compared by polyacrylamide gel electrophoresis, at least three different reproducible fractionation patterns could be identified.

Distinct serotypes of porcine rotavirus associated with diarrhea in suckling piglets in southern Quebec.

Cytopathic rotavirus strains were isolated in cell cultures from the intestinal contents of diarrheic piglets on Quebec pig farms where repeated outbreaks of enteritis occurred. All the isolates shared the common group antigens of rotaviruses as revealed by immunofluorescence and counterimmunoelectrophoresis. A hemagglutinating activity was demonstrated with human group O, porcine and guinea pig erythrocytes. At least one of the isolates was clearly distinguished from the American prototype of porcine rotavirus (strain OSU) by neutralization and hemagglutination inhibition tests; a third serotype was also suspected. By polyacrylamide gel electrophoresis of RNA, it was not possible to differentiate these isolates.

Parvovirus-like particles associated with diarrhea in unweaned piglets.

Numerous parvovirus-like particles, 18 to 26 nm in diameter, were detected by electron microscopy in the intestinal contents of two to three week old piglets with mild to severe diarrhea, in six Quebec pig herds. Hemagglutination of guinea pig and African green monkey red blood cells was obtained with clarified intestinal contents. Two isolates were found to be antigenically related to porcine and canine parvoviruses, while another differed from the porcine parvovirus using the hemagglutination-inhibition test. Three isolates could be cultivated in cell cultures as demonstrated by the development of a cytopathic effect, hemagglutination activity, immunofluorescence and identification of the virions in the cell culture fluids by electron microscopy. The possibility of a primary etiological role for these parvoviruses in diarrhea of unweaned piglets is discussed.

Prevalence of Antibodies to Swine Influenza Virus, Porcine Adenovirus Type 4 and Haemophilus pleuropneumoniae in Quebec Pig Farms with Respiratory Problems.

The prevalence of antibodies to three etiological agents involved in swine pneumonia was determined in Quebec pig farms which had experienced problems of mild to severe respiratory problems. Of the 350 sera collected from adults pigs, 67.0%, 18.3% and 46.6% had antibody titers to Haemophilus pleuropneumoniae type 1 and/or 5, to porcine adenovirus type 4 and to swine influenza virus, respectively. Comparatively, the serological prevalence rates for Haemophilus pleuropneumoniae, porcine adenovirus and swine influenza virus were 43.7%, 0% and 3.3% in farms not experiencing respiratory problems.

Distinct rotaviruses isolated from asymptomatic calves.

Rotaviruses were isolated following cell culture of the intestinal contents of four non-diarrheic calves. The four isolates were serially propagated in MDBK and BSC-1 cells in the presence of trypsin and produced rotavirus particles morphologically similar to those found associated with diarrhea. They were antigenically related to the Nebraska calf rotavirus (Norden strain) as investigated by immunofluorescence. Three isolates could be distinguished from the reference Nebraska rotavirus by their thermal stability and/or their differential responses to intestinal neutralizing antibodies. Two isolates produced on BSC-1 cells plaques significantly different in size from the reference strain, No significant genomic variations were detected among the isolates.

Prevalence of antibodies to bovine respiratory syncytial virus, bovine viral diarrhea virus, bovine herpesvirus-1, and bovine parainfluenza-3 virus in sheep and goats in Quebec.

Serum samples were collected from 1,075 clinically normal sheep and goats from 77 flocks in 7 agricultural regions of Quebec from June to August 1982. Sheep and goats were tested for antibodies to bovine respiratory syncytial virus, bovine viral diarrhea virus, and bovine herpes-virus-1 by the indirect fluorescent antibody technique and for parainfluenza-3 virus by the hemagglutination inhibition test. The prevalence of antibodies in animals to respiratory syncytial virus was 31%; to bovine viral diarrhea virus, 22.2%; to bovine herpesvirus-1, 10.8%; and to parainfluenza-3 virus, 23.2%. Antibodies prevailed in similar proportions in young (less than 1 year) and adult (greater than 1 year) animals.

Comparative study of bovine rotavirus isolates by plaque assay.

Rotaviruses were isolated on BSC-1 cells from counterimmunoelectrophoresis and/or electron microscopy positive intestinal contents from two asymptomatic and six diarrheic calves from Quebec. The plaque assay was performed using these lines and agar overlay medium containing trypsin and DEAE-dextran. This assay was used to compare the Quebec isolates to an attenuated American strain (NCDV) and another strain (TH) obtained from France. The NCDV strain produced plaques that were significantly larger than those produced by the TH strain. Three Quebec isolates produced plaques similar in size to TH strain, one isolate was similar to NCDV strain and another isolate produced larger plaques than those of both NCDV and TH strains. The other isolates induced the production of plaques that were not significantly different from those of NCDV or TH strains.

Cultivation of a porcine adenovirus in porcine thyroid cell cultures.

The porcine adenovirus type 4 was adapted to grow in porcine thyroid cell cultures. A readily recognizable cytopathic effect appeared in these cells as soon as the first passage of the virus and complete degeneration of the monolayers was obtained after only 72 hours post-infection at the fourth passage. A viral yield of 10(6.0) TCID50/ml was calculated after the third passage. The virus was purified by CsCl density gradient centrifugation and was shown to possess a buoyant density of 1.33 g/ml. A specific antiserum was prepared from two specific-pathogen-free piglets and used for indirect immunofluorescent staining. The fluorescence was observed in the nucleus of infected cells at 24 to 72 hours post-inoculation. The use of TP cells is suggested for routine porcine adenovirus diagnosis.

Virus-like particles, 45 to 65 nm, in intestinal contents of neonatal calves.

Enveloped virus particles 45 to 65 nm in diameter, tentatively called minicorona virus, were detected by electron microscopy in the intestinal contents of one normal and seven diarrheic calves in Quebec dairy herds. The agent was shown to be antigenically unrelated to the Nebraska calf diarrhea coronavirus and to the bovine viral diarrhea virus by counterimmunoelectrophoresis and fluorescent-antibody techniques. Antibodies against these particles were demonstrated in the serum of affected calves using immunoelectron microscopy. The agent could not be isolated in cell cultures and its possible role as etiological agent in calf diarrhea is still to be determined.

Detection of infectious bovine rhinotracheitis and bovine viral diarrhea viruses in the nasal epithelial cells by the direct immunofluorescence technique.

Nasal epithelial cells were collected by cotton swabs for the diagnosis in experimental and field cases of infectious bovine rhinotracheitis and field cases of bovine viral diarrhea in calves. A portion of the cells was washed twice in phosphate buffered saline and a 25 microL drop was placed on microscope slides. The cells were dried, fixed and stained according to the direct fluorescent antibody technique. Another portion of the same specimen was inoculated onto primary bovine skin cell cultures for virus isolation. In the experimental studies for infectious bovine rhinotracheitis, 29/35 specimens were positive by fluorescent antibody technique and 32/35 by cell culture and in the field cases, 22/119 were positive by fluorescent antibody technique and 19/119 by cell culture. In the field cases of bovine viral diarrhea, 28/69 samples were positive by fluorescent antibody technique and 14/69 by cell culture. When fluorescent antibody technique was performed on inoculated cell cultures a total of 24/69 specimens were positive for bovine viral diarrhea. The sensitivity of fluorescent antibody technique was thus comparable to that of cell culture method for infectious bovine rhinotracheitis and bovine viral diarrhea.

A natural outbreak of bovine respiratory disease caused by bovine respiratory syncytial virus.

An outbreak of respiratory disease occurred in a herd of 34 calves and 5 cows in a barn. The disease which affected 24 calves and 1 cow, was characterized by coughing, fever, anorexia, nasal discharge and pulmonary rales. Two calves died 5 days after the disease outbreak and were necropsied. Bronchitis, bronchiolitis and alveolitis were among the lesions observed in these calves. Bovine respiratory syncytial virus was detected, in lung sections of the dead calves and also in nasal washings of some of the calves and one cow, by the direct fluorescent antibody technique and/or cell culture method. Seroconversion to the virus was detected in most animals tested.

Coronavirus-like Particles in the Feces of a Cat with Diarrhea.

Coronavirus-like particles were visualized in the feces of a young domestic shorthair female cat with diarrhea. On the surface projections, these particles could be distinguished from the enteric coronavirus-like particles of human, dog, cattle and monkey origin. They appeared morphologically similar to a feline enteric coronavirus recently described by other authors. A precipitin antigen was detected in the cat feces by counterimmunoelectroosmophoresis using a rabbit antibovine coronavirus serum.

Antigenic variations among calf diarrhea coronaviruses by immunodiffusion and counterimmunoelectrophoresis.

The antigenic relationship among five bovine coronavirus isolates was studied using the counterimmunoelectrophoresis (CIE) and immunodiffusion (ID) techniques. By CIE, two precipitating antigens were observed for each coronavirus isolate. One antigen identified as the "M" coronaviral antigen was found to be common to the five isolates while the second antigen identified as the "m" coronaviral antigen differed between the isolates. Considering the later antigen, the isolates studied were placed in two antigenic groups, members of the same group being indistinguishable for the two antigens. By ID, reactions of partial identity were noted between viruses of the different groups.