Studies on canine parvovirus infection: preparation of challenge virus.

Two techniques, adsorption on to hydroxylapatite and density gradient centrifugation, were investigated as prospective methods for the large scale purification of canine parvovirus from faecal suspensions. Adsorption with hydroxylapatite successfully removed virus from faecal material. However, the resultant virus was contaminated and some virus was left behind in the faecal suspension. Repeated adsorption with hydroxylapatite appeared to result in some damage to the virus particles. In contrast, density gradient centrifugation provided a simple, economical method of purification which yielded uncontaminated, infectious virus. The final method, using both isopyknic and rate zonal centrifugation is described.

Canine parvovirus: interaction between passive immunity and virulent challenge.

Two groups of puppies, one passively immunised by the administration of hyperimmune serum and the other with natural maternally derived antibody, were inoculated orally with virulent canine parvovirus of faecal origin. Serum antibody titres declined more rapidly in both groups after challenge than before. The dogs became clinically affected but the onset of clinical signs, seroconversion and faecal excretion of virus was delayed when compared to controls. It is postulated that this rapid decline of antibody was due to its sequestration by virus after the initial phase of viral replication in the lymphoid tissues. These findings have important implications. The incubation period of the disease is prolonged, making it more difficult to estimate accurately the time of infection in clinically affected animals. Furthermore, the more rapid decline of maternally derived antibody, which could occur in endemically infected premises, may complicate immunisation programmes based on the isolation and segregation of puppies in anticipation of a predicted decline in maternally derived antibody before vaccination.

Studies of parvovirus vaccination in the dog: the performance of live attenuated feline parvovirus vaccines.

The performance of three live attenuated feline parvovirus vaccines licensed for use in the dog was studied. At the end of the primary vaccination course 67 per cent of dogs had inadequate antibody levels (less than or equal to 32) as measured by a haemagglutination inhibition test. Interference by maternal antibody accounted for some of the failures but the fact that there was no significant difference in performance between dogs vaccinated at 12 weeks or 16 weeks of age indicated that maternal antibody was not the only factor.

Characterization of minute virus of canines (MVC) and its pathogenicity for pups.

Minute virus of canines (MVC, canine parvovirus-1), originally isolated in 1970 from the feces of normal dogs, was compared with canine parvovirus type-2 (CPV-2). The two viruses, which differ in their host cell ranges and spectra of hemagglutination, also were found distinct in their antigenic and genomic properties. We demonstrated that the MVC replicates in dogs and is capable of producing pathologic changes that were most prominent in oronasally-exposed neonatal pups. Macroscopic and microscopic lesions were most prominent in the thymus and lymph nodes; minor changes were found in the duodenal crypts. The MVC strain used had been passaged 13 times in cell cultures and it may not represent the true virulence of naturally occurring virus.

Experimental infection of dogs with Campylobacter jejuni.

Three groups of conventional puppies were inoculated orally with Campylobacter jejuni biotype 2 which had been isolated from the small intestine of a dog with enteritis. Mild enteric disease was observed in one group. There was superficial intestinal colonization by the organism but penetration of intestinal epithelial cells was not apparent. C jejuni was isolated from the blood and viscera of inoculated dogs which showed no histological evidence of disease.

Isolation of canine adenovirus-2 from the faeces of dogs with enteric disease and its unambiguous typing by restriction endonuclease mapping.

Fifty-four faecal specimens obtained from kennelled dogs with diarrhoeal disease were used to inoculate a range of cell types in tissue culture. Particles resembling adenovirus virions were seen in three specimens and 22 stools yielded an adenovirus upon culture. Viral DNA from each isolate was digested with the restriction endonucleases Bam H1 and Pst 1. Agarose gel electrophoresis revealed identical restriction patterns for all isolates. One isolate, 9228, was selected as a prototype and was compared with reference strains of canine adenovirus-2 (CAV-2) (Manhattan and Toronto A26/61) and CAV-1. Isolate 9228 was clearly distinct from CAV-1 but identical to both Manhattan and Toronto (A26/61) strains of CAV-2. However, restriction site polymorphism was observed in 9228 following digestion with Hpa II. Isolate 9228 was similarly compared with all commercially available vaccinal isolates of CAV-2 and was shown to be clearly distinct from these.

Encephalitis in dogs associated with a batch of canine distemper (Rockborn) vaccine.

During a period of seven months in 1982-83 cases of postvaccinal encephalitis were recorded in dogs in various parts of Britain after the administration of a particular batch of combined distemper/hepatitis vaccine. Detailed investigations of one of these cases revealed that the distemper component was responsible and the vaccine virus was recovered from the brain of an affected dog.

Canine parvovirus: development of immunofluorescence and immunoperoxidase techniques.

Two methods of immunocytochemistry, immunofluorescence (IFA) and immunoperoxidase (PAP) were used to demonstrate canine parvovirus (CPV) antigens in sections of canine tissue. Specific staining using IFA and PAP was successful only in sections of fresh frozen tissue and formalin fixed/formol sublimate postfixed tissues respectively. A range of tissues was then taken at post mortem examination from a puppy which had been experimentally infected with CPV. Upon comparison, PAP staining gave high resolution, a permanent preparation and clear intracellular localisation of antigen. IFA resulted in less defined localisation of antigen but the technique was simpler and more easily controlled.

Canine parvovirus enteritis 3: Scanning electron microscopical features of experimental infection.

A group of 10-week-old puppies was orally inoculated with canine parvovirus of faecal origin. Scanning electron microscopy was used to study and compare the surface topography in both control and inoculated animals. In control dogs the villi were tall and finger-like in shape and numerous irregular transverse circumferential grooves were present on the surface. At higher magnification, the outlines of individual epithelial cells and depressions, interpreted as goblet cells, could be discerned. In the inoculated dogs, scanning electron microscopy changes were first seen at six days after inoculation. The small intestinal mucosa was covered by a thick layer of mucus. The underlying villi were stunted and had lost their surface features. In some instances there was loss of the luminal epithelium, exposing the lamina propria. In addition, there was dilation of the circumvillar basins and the crypt mouths. There appeared to be regenerative changes by day 7 after inoculation. The surface of the small intestinal mucosa was still covered by a thick layer of mucus. Where villi could be discerned, they were short and pointed and transverse grooves could be seen on their surface. There was some hypertrophy of the intervillus ridges. The changes in the surface topography of the small intestinal mucosa following canine parvovirus infection are compared to those seen in enteric infections in other species and the similarity of the lesion to that seen following sublethal irradiation is discussed.

Canine parvovirus enteritis 2: Pathogenesis.

Two groups of puppies, eight and 10 weeks of age, were inoculated orally with canine parvovirus of faecal origin. The patterns of faecal excretion of virus, antibody production and systemic viral localisation following inoculation were studied. Faecal excretion of virus was first apparent at day 3 after inoculation, was present most frequently and in greatest quantity at days 4 to 7 after inoculation and fell sharply thereafter. Serum antibody was first detected at day 5 after inoculation with high titres in all samples from day 7 onwards. Virus isolation from serum samples revealed a non-cell associated viraemia at days 3 and 4 after inoculation. Immunocytochemical examination, using both immunofluorescence and immunoperoxidase techniques, first revealed antigen in the thymic cortex at day 1 after inoculation and in the germinal centres of the lymph nodes and the splenic white pulp from days 2 and 3. Viral antigen was first detected in the intestines at day 4 in individual cells in the proliferative zone of the crypt epithelium. From day 5 onwards, the amount of antigen present in the lymphoid tissue decreased so that by days 7 and 8, only a trace was present. There was widespread specific staining in the small intestinal mucosa at day 6, but little antigen was present by day 7. Virus was present in the bone marrow of some dogs killed at days 5 and 6.

Canine parvovirus enteritis 1: Clinical, haematological and pathological features of experimental infection.

The effect of oral infection of puppies, eight and 10 weeks old, with canine parvovirus of faecal origin was studied. Clinical signs of enteric disease were first apparent at five days after inoculation and persisted during days 6 and 7 after inoculation. The severity of clinical signs varied from transient dullness and anorexia to emesis, dysentery and death. Changes in haematological parameters were first found at day 3 after inoculation when a relative lymphopenia was observed. A profound neutropenia developed in severely affected dogs after the appearance of clinical enteric disease. Post mortem examination revealed thymic atrophy in all dogs killed on day 4 after inoculation. Macroscopic changes in the small intestine were apparent only in animals examined during the phase of severe enteric disease and consisted of thickening, rigidity and congestion of the small intestines. Microscopically there was lymphocytolysis in the thymic cortex and the germinal centres of the lymph nodes from days 2 and 3 after inoculation respectively and this rapidly resulted in depletion of these tissues. There was repopulation of lymph nodes from day 7 after inoculation but significant thymic regeneration was not apparent during the course of this study. In the small intestine, necrosis of crypt epithelium, atrophy of villi and, in some areas, complete collapse of mucosal architecture were found but the extent of these changes varied along the length of the small intestine and between individuals. Regenerative intestinal changes were observed in those animals surviving the acute phase of enteric dysfunction. The variable severity of clinical and enteric lesions, together with the factors which may affect the expression of clinical disease, are discussed.