Seroprevalence of viral and vector-borne bacterial pathogens in domestic dogs (Canis familiaris) in northern Botswana.

BACKGROUND: Domestic dogs (Canis familiaris) have the potential to act as disease reservoirs for wildlife and are important sentinels for common circulating pathogens. Therefore, the infectious disease seroprevalence among domestic dogs in northern Botswana may be indicative of pathogen exposure of various wildlife species. The objective of this study was to assess the seroprevalence of Ehrlichia spp., Borrelia burgdorferi, Anaplasma spp., Dirofilaria immitis, canine adenovirus, canine parvovirus, and canine distemper virus in domestic dogs as proxies of disease prevalence in the local wildlife in the Okavango Delta region of Botswana. Statistical analysis assessed crude and factor-specific seroprevalence proportions in relation to age, sex, and geographical location as predictors of seropositivity. Logistic regression was used to identify adjusted predictors of seropositivity for each of the pathogens of interest. RESULTS: Samples from 233 dogs in a total of seven locations in Maun, Botswana, and surrounding villages were collected and serologically analyzed. No dogs were seropositive for B. burgdorferi, while low seroprevalence proportions were observed for Anaplasma spp. (2.2%) and D. immitis (0.9%). Higher seroprevalence proportions were observed for the tick-borne pathogen Ehrlichia spp. (21.0%), and 19.7% were seropositive for canine adenovirus (hepatitis). The highest seroprevalence proportions were for canine parvovirus (70.0%) and canine distemper virus (44.8%). The predictors of seropositivity revealed that adults were more likely to be seropositive for canine adenovirus, canine distemper virus, and canine parvovirus than juveniles, and location was a risk factor for canine adenovirus, canine distemper virus, canine parvovirus, and Ehrlichia spp. CONCLUSIONS: Results indicate that increasing tick control and vaccination campaigns for domestic dogs may improve the health of domestic animals, and potentially wildlife and humans in the Okavango Delta since viral and vector-borne bacterial pathogens can be transmitted between them.

Detection of canine distemper virus in bone cells in the metaphyses of distemper-infected dogs.

In the light of recent evidence implicating canine distemper virus (CDV) as a possible etiologic agent in Paget's disease of bone, we thought that it would be of interest to examine distemper-infected bone in the natural host. Samples from the long bones, spleen, and bladder of four distemper-infected and three uninfected dogs were examined for the presence of CDV nucleocapsid and phosphoprotein genes and the measles virus (MV) nucleocapsid gene using the technique of in situ hybridization with radioactively labeled riboprobes. Two of the four distemper-infected dogs showed strongly positive hybridization with both of the CDV probes. The signal was present in marrow cells, in osteoblasts, in osteocytes, and particularly in osteoclasts. No hybridization was seen over the cartilage cells of the growth plate, and there was a clear line of demarcation at the point of invasion of osteoclasts and vascularization. The spleen and bladder samples from infected dogs also showed positive hybridization. There was no hybridization with the MV probe in any of the distemper-infected tissue. Samples from the uninfected dogs showed no evidence of hybridization with either the CDV or MV probes. These results show that CDV can infect bone cells of the natural host and provide further support for the theory that CDV may play a role in human Paget's disease of bone.

Canine distemper virus transcripts detected in the bone cells of dogs with metaphyseal osteopathy.

Using the technique of in situ hybridisation, we have recently extended our observations that canine distemper virus (CDV) is present in the bone cells of patients with Paget's disease, and have shown that CDV is also detectable in the bone cells of dogs that are naturally infected with the virus. Since hybridisation was localised to bone cells within the metaphyses of the affected dogs, we investigated the possibility that CDV might be involved in the canine metaphyseal bone disorder, metaphyseal osteopathy. Bone samples from three cases of metaphyseal osteopathy were examined for the presence of the CDV nucleocapsid (CDV-N) gene and the measles virus nucleocapsid (MV-N) gene, using 35S-labelled sense and antisense riboprobes. As with our previous findings in Paget's disease of bone, only the antisense probe was found to hybridize to the osteoblasts and osteoclasts within the affected metaphyses. No hybridisation was seen with the CDV-N sense and MV-N probes in any of the samples tested. Bone samples were also taken from one of the cases to check for the presence of the CDV-N gene using the polymerase chain reaction (PCR). Our findings with in situ hybridisation were confirmed by PCR and subsequent Southern blotting and probing with a 32P-labelled cDNA probe. The detection of CDV RNA within the bone cells of dogs with metaphyseal osteopathy suggests that this virus may be a cause of the disease and provides further, indirect evidence that CDV might be responsible for the bony abnormalities seen in Paget's disease of bone.

Immunocytochemical methods for demonstrating canine distemper virus antigen in aldehyde-fixed paraffin-embedded tissue.

The effects of enzymatic digestion, sodium borohydride reduction, acids used in decalcification procedures and techniques for inactivation of endogenous peroxidase were sequentially evaluated for their effect on the immunoreactivity of canine distemper virus in aldehyde-fixed paraffin-embedded tissue. Enzyme digestion improved immunoreactivity while sodium borohydride reduced background staining. Paraformaldehyde-glutaraldehyde-fixed tissues required thioglycolic acid treatment prior to enzyme digestion and sodium borohydride reduction to obtain results comparable to results obtained in formalin-fixed tissues. Detailed protocols for indirect immunofluorescence and the avidin-biotin-peroxidase complex procedure are provided.

Identification of negative strand and positive strand RNA of canine distemper virus in animal tissues using single stranded RNA probes.

In this report, we describe a technique for identifying negative strand (genome) and positive strand (messenger) RNA of canine distemper virus (CDV) in dog tissues by using single stranded RNA probes. Plasmids (pSP64-P and pSP65-P) which contain insert DNA corresponding to the P gene of CDV were transcribed by SP6 polymerase in the presence of radioisotope to produce radiolabeled single stranded RNA probes. RNA transcribed from pSP65-P is complementary to the negative strand (genome) and RNA produced from pSP64-P is complementary to the positive strand (message) of CDV. The binding specificity of the single stranded RNA probes was determined on Northern-blots. The use of these RNA probes in hybridization assays resulted in greater sensitivity and specificity than that obtained from double stranded DNA probes (either whole plasmids or purified insert DNA) which were labeled by the nick translation reaction. We also describe the making of single stranded DNA probes by reverse transcription labeling of complementary RNA. The complementary RNA was produced by the transcription of cloned DNA (pSP64-P and pSP65P). Single stranded RNA probes and single stranded DNA probes were similar in sensitivity. The single stranded RNA and DNA probes were applied to ethanolacetic acid fixed tissue sections from dogs infected with CDV-A75/17. We used 32P-labeled probes in tissue hybridizations and 35S-labeled probes in in situ hybridizations to identify negative and positive stranded CDV RNA. In this report we demonstrate that single stranded RNA and DNA probes can be used successfully in tissue hybridization and in situ hybridization assays to study viral expression in this virus-host system.

Canine distemper virus does not infect oligodendrocytes in vitro.

Dissociated canine brain cell cultures were infected with virulent canine distemper virus (CDV). Double immunofluorescent labelling was done to simultaneously demonstrate viral antigen and specific glial cell markers. Virus containing oligodendrocytes were not found at any stage of the infection. A certain proportion of the infected cells were shown to be astrocytes. It was concluded that CDV has no obvious tropism for oligodendrocytes which could explain the mechanism of demyelination in distemper in vivo.

Studies on manifestations of canine distemper virus infection in an urban dog population.

An upsurge of canine distemper was recognized at the beginning of 1991 in the urban dog population of the Copenhagen area. The outbreak had the characteristics of a virulent morbillivirus introduction in a partly immune population, where the disease primarily was manifested in young individuals. Testing of single serum samples for the presence of canine distemper virus (CDV) IgM antibodies using an IgM ELISA confirmed current and recent CDV infections in an urban dog population, where the use of attenuated CDV vaccines was widespread. In 49 out of 66 sera from clinical cases suspected of canine distemper we detected CDV IgM antibodies, as compared to the detection of viral antigen by indirect immunofluorescence in 27 of 65 specimens of conjunctival cells. The antigenic make-up of isolates from acute and subacute clinical cases was investigated with a panel of 51 monoclonal antibodies directed against CDV and the related phocine distemper virus. The isolates exhibited an homogeneous reaction pattern and shared overall antigenic characteristics of the CDV prototype. The majority of cases were diagnosed among unvaccinated dogs and individuals with unknown or obscure vaccination record. However, severe clinical cases were also diagnosed in vaccinated individuals.

Natural infection with canine distemper virus in a Japanese monkey (Macaca fuscata).

A case of encephalitis in a Japanese monkey (Macaca fuscata) was examined histopathologically and serologically. The animal had brain lesions consisting of perivascular cuffs, malacia, inclusion bodies and giant cells. Monoclonal antibody to the nucleoprotein of canine distemper virus (CDV) stained the inclusions, and the distribution of the virus antigen was closely associated with that of the histological lesions. Serologically, all the 22 monkeys in the same group as the diseased monkey had relatively high titers of neutralizing antibody to CDV, but not to measles virus (MV). The pattern of the antibody titers to CDV and MV closely resembled that of cynomolgus monkeys experimentally inoculated with CDV, but differed from that of monkeys inoculated with MV. These findings suggest that an epidemic of CDV occurred in these Japanese monkeys, associated with one case of fatal viral encephalitis. This is believed to be the first report of a natural infection by CDV in non-human primates.

[Diagnostic problems posed by respiratory infections of dogs]. / Les problèmes de diagnostic posés par les infections respiratoires des chiens.

The following viruses as well as bacteria and mycoplasma have been isolated from dogs with contagious respiratory disease: canine distemper virus; Canine adenoviruses (type 1 and 2); Parainfluenza type 2 (SV5); Reovirus type 1; Canine Herpesvirus; Bordetella bronchiseptica, Streptococcus, Pasteurella, Staphylococcus and Mycoplasma. The occurrence of these agents can be in direct relationship with: the evolution of a systemic disease; respiratory disorders being a regular or inconsistant symptom of this disease; the evolution of a disease restricted to the respiratory tract; the tropism of the bacterial or viral agent is exclusively respiratory; secondary bacterial complications to a primary viral infection; saprophyte state or latency without pathologic significance. These various infectious agents are implicated alone or in mixed infections and the wide variety of clinical symptoms don't allow to precise a clinical diagnosis. We will try to bring some bases allowing, by the help of laboratory an etiologic diagnosis. This diagnosis is essential for providing an efficient prevention. We will approach some parameters which we have been confronted with as regards Canine Distemper and Canine Adenovirosis. Our purpose is, through these examples of the canine pathology, to confirm and complete some other similar situations which can appear in other animal species.

Establishment of central nervous system infection by canine distemper virus: breach of the blood-brain barrier and facilitation by antiviral antibody.

Morphologic, immunologic and virologic data implicating antiviral antibody in promoting entry of canine distemper virus (CDV) into brain and reticuloendothelial tissues are reviewed. Infection of central nervous system (CNS) endothelium precedes invasion of virus-positive and -negative leukocytes into Virchow-Robin spaces and central nervous system (CNS) parenchyma by 1-3 days. Platelets are implicated in initiation of endothelial infection in that: CDV-infected dogs are thrombocytopenic; platelets from CDV-infected dogs contain IgG-virus complexes on their plasma membranes; platelet microthrombi were observed adjacent to foci of endothelial infection, and; CDV-susceptible ferrets rendered thrombocytopenic by antiplatelet antibody exhibit delayed viral entry into CNS tissues. Renal glomerular-bound IgG, IgM and occasionally CDV antigen were demonstrated in CDV-infected dogs by immunocytochemical techniques. Distemper-infected dogs with inherited C3 deficiency exhibited enhanced renal glomerular disease associated chiefly with deposition of IgM in mesengial regions vs. their homozygous normal CDV-infected littermates. Direct infusion of virus-positive leukocytes, plasma and platelets into the CNS capillary bed via the right carotid artery should establish the primacy of each in the initiation of CNS vascular endothelial infection by CDV.

Dog lymphocyte cultures facilitate the isolation and growth of virulent canine distemper virus.

Optimal conditions for the isolation and growth of virulent canine distemper virus (CDV) in canine thymic and peripheral blood lymphocyte cultures were determined. Peak virus titers were seen from 3 to 6 days postinoculation of lymphocytes and depended on the multiplicity of infection. Dog lymphocytes were at least as susceptible as canine macrophages to infection with virulent CDV. Virus replication in lymphocytes resulted in higher virus titers than in dog lung macrophages. Peripheral blood lymphocytes (PBL) from CDV-immune dogs were as susceptible to CDV as were PBL from susceptible dogs.

Molecular and serological studies on the recent seal virus epizootics in Europe and Siberia.

The virus epizootics which occurred in seals in both Europe and Siberia during 1987/1988 were caused by two different morbilliviruses, referred to as phocid distemper virus (PDV) 1 and 2, respectively. Molecular and serological studies have shown that the European virus is quite distinct from canine distemper virus (CDV), its closest relative in the morbillivirus group. Analysis of tissues obtained from infected seals from a wide geographical distribution over Northern Europe showed that the infectious agent (PDV 1) was identical in all cases. Nucleotide sequence analysis of one of the virus genes suggested that this virus has evolved away from CDV over a long time period and is most probably an enzootic virus of marine mammals. In contrast, the virus (PDV 2) which caused the deaths of many Siberian seals was indistinguishable, both serologically and at the molecular level, from CDV and must have originated from a land source.

Phocine distemper virus, the agent responsible for the 1988 mass mortality of seals.

The biochemical characterisation of phocine distemper virus (PDV) has shown that PDV is related to but clearly distinct from canine distemper virus (CDV) and relative to its relationship with CDV is only remotely related to the other morbilliviruses, namely measles virus (MV) or rinderpest virus (RPV) and peste-des-petits-ruminants virus (PPRV). Comparative studies with monoclonal antibodies indicate that the virus is serologically closely related to CDV with many conserved epitopes, particularly on the internal proteins of the virus, while the external attachment (H) protein shows the greatest level of variability among the distemper virus isolates. The analysis of the viral proteins by electrophoresis indicates molecular weight differences between CDV and PDV in the fusion (F), phosphoprotein (P), H, nucleocapsid (N) and matrix (M) proteins. The RNA profiles of CDV and PDV are indistinguishable and different from those for RPV and MV. Nucleotide sequence analysis of cDNA clones of the virus show approximately 70% homology between CDV and PDV and approximately 48% with MV. These data prove that PDV is a different virus from CDV and co-circulates with it probably primarily in sea mammals.

Canine distemper virus infection in a masked palm civet (Paguma larvata).

A free-living masked palm civet (Paguma larvata) died after exhibiting signs of canine distemper (CD). The microscopic lesions consisted of cytoplasmic and intranuclear eosinophilic inclusion bodies, bronchointerstitial pneumonia, non-purulent encephalitis accompanied by demyelination and lymphocytic depletion in various lymphoid tissues. CD virus-specific antigens were demonstrated immunohistochemically in intracellular eosinophilic inclusions, which were ultrastructurally confirmed to be viral nucleocapsids. From these findings, the present case was diagnosed as CD virus infection in a masked palm civet.

Viral expression in experimental canine distemper demyelinating encephalitis.

We have characterized the relationship between the expression of canine distemper virus (CDV) and demyelinating lesions in the white matter of the cerebellum of experimentally infected dogs. In animals which had demyelinating lesions, CDV proteins (N, P, F and H) were expressed and infectious virus could be recovered from brain tissue. Viral proteins (N, P, F and H) were detected by monoclonal antibodies and immunocytochemistry within demyelinating lesions as well as in scattered glial cells in areas of the white matter which lacked detectable lesions. Many cell types, including astrocytes, neurons, ependymal cells, choroid plexus cells, meningeal cells and perivascular inflammatory cells were labelled for viral antigen. We conclude from our results that the mechanism of demyelination in canine distemper virus-induced encephalitis involves expression of viral gene products at the lesion site.

Use of B95a cells for isolation of canine distemper virus from clinical cases.

Canine distemper virus (CDV) was readily isolated at high rate with marked cytopathic effect (CPE) in B95a cells, a marmoset lymphoid cell line, from the peripheral blood leukocytes, cerebrospinal fluid cells and brain of dogs. Difference in type of CPE, i.e. syncytium type and round-cell one, among the virus isolates indicate the presence of heterogeneity of virus populations in prevalent CDV. Thus, this cell system is expected to be useful for ecological studies on CDV in the field.

Canine distemper virus: in vivo virulence of in vitro-passaged persistent virus strains.

Groups of ferrets were inoculated intraperitoneally with cell lysates or equivalent doses of whole cells from 9 different cell lines persistently infected with canine distemper virus. Viral persistence in these cell lines was characterized by noncytolytic infection and restricted release of cell-free infectious virus. In vivo replication competency of the various viruses in ferrets ranged from nil to virulent and did not correlate with in vitro titers of inocula. Ferret virulence (cell lysates only) for one cell line (CCL64-RCDV) was associated with morphologic absence of virion assembly, failure to interfere with lytic virus replication after superinfection, and in vitro infectivity restricted to canine macrophage-like tumor cells. Virion protein production in the CCL64-RCDV virulent inoculum and in the CCL64-Ly avirulent inoculum was evaluated by use of the immunoblot technique. All major virion proteins were produced by infected cells. Virulence was not associated with obvious changes in electrophoretic mobility of virion proteins when profiles of ferret-virulent CCL64-RCDV were compared with those of avirulent CCL64-Ly.

Plasma phase viremia in canine distemper virus infection.

Plasma samples from gnotobiotic pups infected with R252 canine distemper virus at 7 days of age contained free infective virus when titrated on canine pulmonary macrophage cultures. Virus was detected 7 days after infection and increased thereafter. Platelets may be involved in leukocyte-free viremia. The present study indicated that the method of dissemination of the virus in vivo involves plasma and canine distemper virus-infected leukocytes.

In situ hybridization of virulent canine distemper virus in brain tissue, using digoxigenin-labeled probes.

Only a few hybridization experiments have been performed for detection of canine distemper virus (CDV) nucleic acid sequences in tissue cultures and in various tissues. Those published studies used probes derived from tissue culture-adapted CDV, and hybridization signals were not obtained in the CNS tissue, although infective CDV and viral antigen were detectable in this tissue. We developed probes complementary to virulent CDV and were able to detect viral RNA not only in primary brain cell cultures, but also in brain tissues, by use of in situ hybridization. Sensitivity of the test at least equaled that of immunohistochemistry. We applied digoxigenin-labeled, strand-specific RNA probes complementary to the nucleoprotein-coding viral nucleic acid sequence. Our results indicate that to detect CDV nucleic acid sequences in brain tissues, it is essential to use probes derived from the virulent virus.