Detection of bovine coronavirus and type A rotavirus in neonatal calf diarrhea and winter dysentery of cattle in Quebec: evaluation of three diagnostic methods.

The use of direct electron microscopy, enzyme-linked immunosorbent assay, and protein A-gold immunoelectron microscopy for the identification of bovine coronavirus and type A rotavirus were examined. Two hundred and forty-nine samples from diarrheic calves and winter dysenteric cattle from seven geographic areas in Quebec were examined for the presence of viruses by direct electron microscopy of negatively stained preparations. In addition, all the samples were analyzed by enzyme-linked immunosorbent assay, and a random selection of 47 samples were also analyzed by protein A-gold immunoelectron microscopy. Thirty-nine percent of samples examined by direct electron microscopy contained viral particles; bovine coronavirus and type A rotavirus were the most common viruses involved. Overall agreement between any two of the methods used compared favorably with results obtained by others using similar methods. The presence of coronavirus and rotavirus in fecal samples obtained from neonatal calves and the presence of coronavirus in samples from winter dysenteric adult cattle suggested their etiological roles in the respective diseases. Furthermore, results from protein A-gold immunoelectron microscopy of coronavirus-like particles implied that a different coronavirus or some other viruses might be involved in these diseases. Finally, the efficiency of direct electron microscopy, enzyme-linked immunosorbent assay and protein A-gold immunoelectron microscopy as diagnostic tools is discussed.

Protection of mice and swine against infection with Actinobacillus pleuropneumoniae by vaccination.

CaCl2 and LiCl cell extracts and a crude hemolysin preparation were isolated from Actinobacillus pleuropneumoniae serotype 1 strain 4074 and tested for protection against A. pleuropneumoniae serotype 1 and 5 in mice. The LiCl cell extract adsorbed on AlPO4 and the crude hemolysin preparation adsorbed on Al(OH)3 showed a highly significant protection (P < 0.01) against both serotypes. Different vaccine preparations were used to immunize pigs by intra-muscular injection at days 0 and 14; the pigs were then challenged at day 21 by intra-tracheal inoculation of 1 x 10(8) colony forming units (CFU) of a serotype 1 strain 4074. A vaccine which combined the LiCl extract and the crude hemolysin preparation adsorbed on Al(OH)3 gave the best protection with no mortality and no sign of morbidity in the vaccinated pigs. In the other experimental groups which included a group immunized with a commercial bacterin, mortality, respiratory disease and extensive pulmonary lesions were noted. This mixture shows good potential as a vaccine against pleuropneumonia in pigs.

[Serological study of the incidence of murine viruses in a population of small wild rodents (Microtus pennsylvanicus Ord, 1815)]. / Etude sérologique sur l'incidence de virus murins dans une population de petits rongeurs sauvages (Microtus pennsylvanicus Ord, 1815).

The results of a serological survey of a free-living population of meadow voles (Microtus pennsylvanicus) in Pinawa, Manitoba (Canada) showed that these animals possessed antibodies to six of the eleven viruses tested for, namely: reovirus type 3, murine encephalomyelitis agent, ectromelia virus, murine adenovirus, murine hepatitis virus and lymphocytic choriomeningitis virus. The significant increase in the number of individuals possessing specific antibodies suggests that these viruses, or related viruses, may be responsible for the decline in the population studied.

Experimental infection of young rabbits with a rabbit enteric coronavirus.

The clinical signs and lesions caused by the rabbit enteric coronavirus (RECV) were studied in young rabbits orally inoculated with a suspension containing RECV particles. The inoculated animals were observed daily for evidence of diarrhea. Fecal samples and specimens from the small intestine and from the gut associated lymphoid tissue (GALT) were collected from 2 h to 29 days postinoculation (PI) and processed for immune electron microscopy (IEM) and light microscopy. Coronavirus particles were detected in the cecal contents of most inoculated animals from 6 h to 29 days PI. Lesions were first observed 6 h PI and were characterized by a loss of the brush border of mature enterocytes located at the tips of intestinal villi and by necrosis of these cells. At 48 h PI, short intestinal villi and hypertrophic crypts were noted. In the GALT, complete necrosis of the M cells as well as necrosis of the enterocytes lining the villi above the lymphoid follicules with hypertrophy of the corresponding crypts were observed in all the animals. Five inoculated rabbits had diarrhea three days PI. The presence of RECV particles in the feces of the sick animals and the microscopic lesions observed in the small intestine suggested that the virus was responsible for the clinical signs. A few inoculated rabbits remained free of diarrhea. Fecal material collected at postmortem examination contained RECV particles. The results suggest that the virus could also produce a subclinical infection.

Mouse hepatitis virus 3 replication in T and B lymphocytes correlate with viral pathogenicity.

Viral pathogenicity may be regulated by host defense mechanisms at the virus-immune cell interaction level. The immune system plays an important role in the outcome of acute disease induced by the mouse hepatitis virus type 3 (MHV3) virus. The lymphoid cells act as effectors in the virus elimination as well as targets for viral replication. In order to demonstrate a correlation between MHV3 pathogenicity and viral replication in lymphocytes, genetically-determined resistant A/J and susceptible C57BL/6 mice were infected with pathogenic (L2-MHV3) or nonpathogenic (YAC-MHV3) viral strains. Pathogenicity and histopathologic studies have revealed that lymphoid organs such as thymus and spleen, showed injuries or atrophy in susceptible mice infected with L2-MHV3. No histopathologic lesions in the lymphoid organs occurred in C57BL/6 mice infected with YAC-MHV3 or A/J mice infected with both viruses. The mechanisms involved in the lymphoid injuries were studied regarding viral replication in the lymphoid organs and cells in infected mice. Results indicate that cell depletion in lymphoid organs is caused by a complete viral replication in lymphoid cells. Thy1.2+ and surface IgM+ lymphoid cells from susceptible C57BL/6 mice infected with L2-MHV3 were permissive to viral replication and to subsequent cell lysis. No cell lysis, however, occurred in lymphoid cells from C57BL/6 mice infected with YAC-MHV3 and A/J mice infected with both virus strains. In vitro studies, with purified T and B cell populations were performed to determine the mechanism effecting susceptibility or resistance to viral-induced cell lysis occurring in such cells. A blockade, probably occurring at the viral RNA polymerase activity level, prevents viral replication in resistant cells between the stages of fixation of the virus at the cell-surface receptor and the viral protein translation. These experiments indicate that an intrinsic virus-specific resistant mechanism occurs in lymphoid cells that plays a major role in the viral pathogenicity.

Enzootic pneumonia in feeder pigs: Observations on causal factors.

A number of factors were studied in eight feeder pig herds, affected with severe or mild enzootic pneumonia, in order to identify those associated with this disease. Piggeries with poor facilities and management and where procurement of piglets was from sales barns were more severely affected with enzootic pneumonia than were those with good facilities and where pigs originated directly from breeding units. Serological tests and virus isolation revealed that all herds had been exposed to Mycoplasma hyopneumoniae and to many viruses; transmissible gastroenteritis virus infection was the only viral infection that was apparently associated with the severity of enzootic pneumonia and the performance observed in the herds.

Evaluation of mouse thymic virus antibody detection techniques.

Three serologic test methods for detection of serum antibodies to mouse thymic virus (MTV) were compared, including enzyme-linked immunosorbent assay (ELISA), complement fixation (CF) test and indirect fluorescent antibody (IFA) test. Serum was collected at regular intervals from CD-1 mice inoculated intraperitoneally with approximately 200 infectious doses of MTV, and from uninoculated mice placed in cages with the inoculated animals. The inoculated mice became MTV antibody-positive by all three assay methods at 15 days post inoculation, while the cage-contacts were seropositive by all methods at 30 days after contact. Although the incidence of positive results was similar by all methods, titers measured by ELISA were substantially higher than those measured by CF and IFA tests. Because MTV can cause persistent infections that adversely affect the suitability of mice for research, it is recommended that testing for antibodies to this virus be performed routinely.

Experimental sialodacryoadenitis virus infection in athymic CD-1 mice.

The purpose of the study was to investigate the susceptibility of nude mice to sialodacryoadenitis virus. Young adult male CD-1 nude mice were inoculated intranasally with virus, killed at 2, 4, 6, 8, 10 and 20 days postinoculation and examined for virus-induced lesions in tissues including respiratory tract. Inoculated and control mice were examined by virus isolation and serology. In a companion study, male Wistar rats were inoculated intranasally with the same inoculum, and examined by histopathology, immunofluorescence microscopy and serology. In virus-inoculated mice, lesions were minimal in the lower respiratory tract, and were absent in other tissues. Virus was isolated from the lower respiratory tract in animals sampled at six or eight days postinoculation. Antiviral antibody was not detected in sera from inoculated and control mice. Virus-associated lesions and antibodies were readily detected in rats following inoculation. Based on this study, there is no evidence that inadvertent exposure to sialodacryoadenitis virus should pose a threat to CD-1 nude mice, and their susceptibility to the disease appears to be similar to that reported in euthymic CD-1 mice.

Detection of antibodies to mouse thymic virus by enzyme-linked immunosorbent assay.

Enzyme-linked immunosorbent assay was used to detect serum antibodies to mouse thymic virus, a herpesvirus that causes thymic lesions and immunosuppression. Antibodies were detected in mice that had received single or multiple injections of the virus and were also found in mice housed in contact with the experimentally infected animals. By contrast, mice not exposed to mouse thymic virus or those inoculated with an uninfected thymus preparation remained seronegative. A serological survey of eight mouse colonies revealed one positive colony, confirmed by virus isolation. These results show that the test is sufficiently sensitive and specific to be used for routine screening of mice.

Comparison of serological tests for the detection of antibody to natural and experimental murine cytomegalovirus.

Three serological tests, i.e. complement fixation test, indirect immunofluorescent assay, and enzyme-linked immunosorbent assay (ELISA) were compared for sensitivity in the detection and titration of murine cytomegalovirus antibody. The three tests were compared using sera from experimentally inoculated and naturally infected mice bled at intervals from 3 to 140 days postinfection. In the acute infection, complement fixation and indirect immunofluorescent assay tests were of comparable sensitivity for early detection of antibody, whereas the ELISA was less sensitive. In persistent infection, higher titers were recorded with ELISA. Since murine cytomegalovirus has been shown to exert significant effects on the immune response of infected mice, this antigen should be included routinely in viral antibody screening programs.

Serological relationship between mouse adenovirus strains FL and K87.

Three-week-old outbred mice were inoculated intraperitoneally or orally and intranasally with the FL or K87 strains of mouse adenovirus and bled at intervals after infection. Serum was tested by both the complement fixation and indirect immunofluorescence tests for reactivity with either virus antigen. A unilateral relationship was detected between FL and K87 strains. Serum from mice given the FL strain of virus reacted in both tests with FL and K87 antigens. Serum from mice given the K87 strain reacted only with the homologous antigen. Serum antibody titers were generally higher in the immunofluorescence test than in the complement fixation test. These observations stress the need to use both FL and K87 antigens for specific serologic diagnosis of adenovirus infection in mouse colonies.

Serologic study on the prevalence of murine viruses in a population of wild meadow voles (Microtus pennsylvanicus).

The demography and serology of a population of wild meadow voles (Microtus pennsylvanicus) were monitored from 1982 to 1984 near Pinawa, Manitoba, Canada. Serologic tests were performed on 486 samples to detect the presence of viral antibodies to 11 common murine viruses. Meadow voles showed evidence of infection with Theiler's encephalomyelitis, reovirus-type-3, ectromelia, lymphocytic choriomeningitis, adenovirus, and mouse hepatitis viruses. At times of good survival and breeding performance the population was nearly free of evidence of viral infection. During a period of severe mortality in the winter of 1982-1983, evidence of infection by Theiler's encephalomyelitis virus and reovirus-type-3 was obtained. A high prevalence of antibodies and high titers to these two viruses were characteristic of voles that were captured late in the decline in density in the spring of 1983. This association of mortality with a viral outbreak is consistent with the hypothesis that vole population declines are sometimes related to opportunistic pathogens present in the voles' biotic and social environment.

Prevalence of natural virus infections in laboratory mice and rats used in Canada.

Results of serological tests carried out over a period of 6 years to detect the presence of antibodies against 14 indigenous viruses in mice and rats used in 32 Canadian institutions are reported. Close to 20,000 individual sera were tested by the complement fixation or the hemagglutination inhibition technics. In order of mouse colony prevalence the six most common viruses present were pneumonia virus of mice, mouse hepatitis virus, rat virus, minute virus of mice, Sendai, and Theiler's mouse encephalomyelitis viruses. The most common viruses present in rat colonies were minute virus of mice, K virus, coronaviruses (rat coronavirus or sialodacryoadenitis virus), rat virus, H-1, pneumonia virus of mice, Theiler's mouse encephalomyelitis viruses, Sendai, and reovirus 3.

Central nervous system lesions in suckling mice and rats inoculated intranasally with sialodacryoadenitis virus.

Suckling CD-1 and CFW mice and WI Wistar rats were inoculated intranasally with sialodacryoadenitis virus. In animals inoculated during the first week of life, there was an acute necrotizing encephalitis with malacia and minimal inflammatory cell response. In mice and rats which survived for up to 12 days post inoculation, loss of brain substance, gitter cells and occasionally mineralized debris were seen. Viral antigen was readily demonstrated in neurons and nasal epithelium in trypsin-treated, paraffin-embedded sections, and corresponded with the presence of lesions. There was no compelling evidence that sialodacryoadenitis virus invaded the central nervous system directly via the cribriform plate, and hematogenous spread was considered to be a likely route of spread to the brain. An age-related resistance to the encephalitic form was evident beginning at 10 days of age in both species. Thus it is conceivable that encephalitis could occur in young suckling rats exposed during naturally-occurring epizootics of sialodacryoadenitis.

Epizootiological survey of parainfluenza-3, reovirus-3, respiratory syncytial and infectious bovine rhinotracheitis viral antibodies in sheep and goat flocks in Quebec.

A serological survey was conducted in an attempt to detect antibodies against bovine respiratory viruses in sheep and goats from seven geographical areas of Quebec. Sera from 10% of the animals in 182 sheep flocks and 40 goat flocks were collected and specific antibodies against parainfluenza-3, reovirus type 3, respiratory syncytial and infectious bovine rhinotracheitis viruses were detected by hemagglutination-inhibition tests for the former viruses and complement fixation and seroneutralization assays for the latter viruses. Results showed prevalence rates of serological reaction to parainfluenza-3, reovirus type 3 and respiratory syncytial viruses of 28, 72 and 35% in sheep and 26, 64 and 36% in goats, respectively. No antibodies in infectious bovine rhinotracheitis virus were detected in sheep or goats tested. Prevalence rates varied according to the geographical area. No relationships were detected between age, sex, breed, size of flock and prevalence rates of different antibodies except that parainfluenza-3 antibodies were more common in large goat flocks and in sheep flocks with total confinement housing. A relationship between presence of clinical signs in the flocks and prevalence rates of antibodies was only demonstrated for parainfluenza-3 infection in goat flocks.

An enteric coronavirus of the rabbit: detection by immunoelectron microscopy and identification of structural polypeptides.

The immunoelectron microscopy (IEM) technique has been used for the detection of a rabbit enteric coronavirus (RECV). Immune serum was prepared in guinea pigs; the viral antigen used for the immunization procedure was obtained from the caecum of a sick rabbit, concentrated by centrifugation and purified on Percoll gradient. In order to identify the viral particles used in the immunization procedure, the protein pattern of the particles was determined by electrophoresis and compared with the pattern of other known coronaviruses. Analysis of structural polypeptides of the purified viral particles revealed a pattern similar to that reported for other coronaviruses. These polypeptides cross reacted with two other coronavirus specific immune sera (IBV and TGE). IEM assay of fecal samples collected from healthy and sick rabbits showed the presence of immune aggregates in specimens from both sick and healthy rabbits. Those aggregates contained viral particles sharing morphological characteristics with other coronaviruses. Furthermore, IEM assay was shown to be more sensitive than a direct EM procedure to detect coronavirus particles in rabbit feces. This assay also allowed the detection of a larger number of chronic carriers.

Experimental pneumovirus infections. 2. Hydrocephalus of hamsters and mice due to infection with human respiratory syncytial virus (RS).

Respiratory syncytial virus (RS) injected into suckling hamsters and mice via the cerebral route induced hydrocephalus in 35% of the animals. The pathogenesis of the condition has been examined by light microscopy, immunofluorescence, and virus isolation. Infection of leptomeninges and ependyma was revealed by immunofluorescence of virus-specific antigens between Days 3 and 7 after inoculation. Histologically, eosinophilic inclusion bodies were visible within the cytoplasma of ependymal and meningeal cells. A patent aqueduct was observed in all hydrocephalic animals but one. These observations point out the affinity of RS virus for the cerebroventricular system of newborns and the induction of pathologic changes in experimental hydrocephalus without stenosis of the aqueduct.

Comparison of hemagglutination inhibition, serum neutralization and ELISA-PVM tests for the detection of antibodies to pneumonia virus of mice in rat sera.

Recently, the seroneutralization, hemagglutination inhibition, and the ELISA procedures were used to determine the presence of serum antibody to the pneumonia virus of mice in rat sera. This study was initiated to evaluate the reliability of these procedures to detect specific antibodies in our laboratory animal species. The results obtained indicate that: 1) the seroneutralization test is not reliable since numerous false-positive sera were detected; 2) the ELISA is more sensitive than both the seroneutralization and hemagglutination inhibition procedures, and 3) the presence of low hemagglutination inhibition titers should not be considered significant.