Controversial results of therapy with mesenchymal stem cells in the acute phase of canine distemper disease.

Distemper disease is an infectious disease reported in several species of domestic and wild carnivores. The high mortality rate of animals infected with canine distemper virus (CDV) treated with currently available therapies has driven the study of new efficacious treatments. Mesenchymal stem cell (MSC)-based therapy is a promising therapeutic option for many degenerative, hereditary, and inflammatory diseases. Therefore, the aim of this study was to characterize stem cells derived from the canine fetal olfactory epithelium and to assess the systemic response of animals infected with CDV to symptomatic therapy and treatment with MSCs. Eight domestic mongrel dogs (N = 8) were divided into two groups: support group (SG) (N = 5) and support group + cell therapy (SGCT) (N = 3), which were monitored over 15 days. Blood samples were collected on days 0, 6, 9, 12, and 15 to assess blood count and serum biochemistry (urea, creatinine, alanine transferase, alkaline phosphatase, gamma-glutamyl transferase, total protein, albumin, and globulin), and urine samples were obtained on days 0 and 15 for urinary evaluation (urine I). The results showed a high mortality rate (SG = 4 and SGCT = 2), providing inadequate data on the clinical course of CDV infection. MSC therapy resulted in no significant improvement when administered during the acute phase of canine distemper disease, and a prevalence of animals with high mortality rate was found in both groups due to the severity of symptoms.

CANINE DISTEMPER VIRUS ANTIBODY TITERS IN DOMESTIC CATS AFTER DELIVERY OF A LIVE ATTENUATED VIRUS VACCINE.

Three methods for delivering a live attenuated canine distemper virus (CDV) vaccine to domestic cats ( Felis catus ) were investigated, as models for developing vaccination protocols for tigers (Panthera tigris). Twenty domestic cats were randomly divided into four treatment groups: saline injection (negative controls); and oral, intranasal, and subcutaneous vaccinates. Cats were injected with saline or a CDV vaccine (Nobivac DP, Merck) at wk 0 and 4. Blood and nasal swabs were collected at wk 0 (prior to the initial vaccination) and weekly thereafter for 9 wk. Urine samples were collected on wk 1 to 9 after initial vaccination. Forty-nine weeks following the initial vaccination series, three cats from the subcutaneous group and three cats from the intranasal group were revaccinated. Blood was collected immediately prior, and 7 and 21 days subsequent to revaccination. Nasal swabs and urine samples were collected from each cat prior to wk 49 revaccination and daily for 7 days thereafter. Nasal swabs and urine were analyzed by quantitative PCR for vaccine virus presence. Sera were tested for CDV antibodies by virus neutralization. All cats were sero-negative for CDV antibodies at the beginning of the study, and saline-injected cats remained sero-negative throughout the study. A dramatic anamnestic response was seen following wk 4 subcutaneous vaccinations, with titers peaking at wk 6 (geometric mean = 2,435.5). Following wk 49 revaccination, subcutaneous vaccinates again mounted impressive titers (wk 52 geometric mean = 2,048). Revaccination of the intranasal group cats at wk 49 produced a small increase in titers (wk 52 geometric mean = 203). CDV viral RNA was detected in six nasal swabs but no urine samples, demonstrating low viral shedding postvaccination. The strong antibody response to subcutaneous vaccination and the lack of adverse effects suggest this vaccine is safe and potentially protective against CDV infection in domestic cats.

SENTINEL COYOTE PATHOGEN SURVEY TO ASSESS DECLINING BLACK-FOOTED FERRET (MUSTELA NIGRIPES) POPULATION IN SOUTH DAKOTA, USA.

As part of the national recovery effort, endangered black-footed ferrets (Mustela nigripes) were reintroduced to the Cheyenne River Sioux Reservation in South Dakota, US in 2000. Despite an encouraging start, numbers of ferrets at the site have declined. In an effort to determine possible causes of the population decline, we undertook a pathogen survey in 2012 to detect exposure to West Nile virus (WNV), canine distemper virus (CDV), plague (Yersinia pestis), tularemia (Francisella tularensis), and heartworm (Dirofilaria immitis) using coyotes (Canis latrans) as a sentinel animal. The highest seroprevalence was for WNV with 71% (20/28) of coyotes testing antibody-positive. Seroprevalence of CDV and plague were lower, 27% and 13%, respectively. No evidence of active infection with tularemia or heartworm was seen in the coyotes sampled. As this study did not sample black-footed ferrets themselves, the definitive cause for the decline of this population cannot be determined. However, the presence of coyotes seropositive for two diseases, plague and CDV, lethal to black-footed ferrets, indicated the potential for exposure and infection. The high seroprevalence of WNV in the coyotes indicated a wide exposure to the virus; therefore, exposure of black-footed ferrets to the virus is also likely. Due to the ability of WNV to cause fatal disease in other species, studies may be useful to elucidate the impact that WNV could have on the success of reintroduced black-footed ferrets as well as factors influencing the spread and incidence of the disease in a prairie ecosystem.

Caspase-3/-8/-9, Bax and Bcl-2 expression in the cerebellum, lymph nodes and leukocytes of dogs naturally infected with canine distemper virus.

Canine distemper is an immunosuppressive disease caused by the canine distemper virus (CDV). Pathogenesis mainly involves the central nervous system and immunosuppression. Dogs naturally infected with CDV develop apoptotic cells in lymphoid tissues and the cerebellum, but this apoptotic mechanism is not well characterized. To better understand this process, we evaluated the expression of Bax, Bcl-2, and caspase-3, -8 and -9, by evaluating mRNA levels in the peripheral blood, lymph nodes and cerebellum of CDV-infected (CDV+) and uninfected (CDV-) dogs by real-time polymerase chain reaction (PCR). Blood samples from 12 CDV+ and 8 CDV- dogs, diagnosed by reverse transcription-PCR, were subjected to hematological analysis and apoptotic gene expression was evaluated using real-time-PCR. Tissues from the cerebellum and lymph nodes of four CDV+ and three CDV-dogs were also subjected to real time-PCR. No significant differences were found between CDV+ and CDV- dogs in the hemotological results or in the expression of caspase-3, -8, -9, Bax, and Bcl-2 in the peripheral blood. However, expression of Bax, caspase-3, -8 and -9 was significantly higher in the cerebellum of CDV+ compared to CDV- dogs. Expression of caspase-3 and -8 was significantly higher in the lymph nodes of CDV+ compared to CDV- dogs. We concluded that infection with CDV induces apoptosis in the cerebellum and lymph nodes in different ways. Lymph node apoptosis apparently occurs via caspase-3 activation, through the caspase-8 pathway, and cerebellum apoptosis apparently occurs via caspase-3 activation, through the caspase-8 and mitochondrial pathways.

Prevalence of protective antibody titers for canine distemper virus and canine parvovirus in dogs entering a Florida animal shelter.

OBJECTIVE: To determine the proportion of dogs entering an animal shelter with protective antibody titers (PATs) for canine distemper virus (CDV) and canine parvovirus (CPV) and identify factors associated with having a PAT. DESIGN: Cross-sectional study. ANIMALS: 431 dogs admitted to an open-admission municipal animal shelter in north central Florida with a history of infectious disease outbreaks. PROCEDURES: Blood was collected from dogs on the day of admission to the shelter. Antibody titers for CDV and CPV were measured by virus neutralization and hemagglutination inhibition, respectively. Age, sex, neuter status, address of origin, source (stray or previously owned), health status (healthy or not healthy), and outcome (adoption, euthanasia, or reclaimed by owner) data were also collected. RESULTS: Overall, 64.5% (278/431) of dogs had insufficient titers for antibodies against CDV, CPV, or both. A total of 153 (35.5%) dogs had PATs for both CDV and CPV, 33 (7.7%) had PATs for CDV but not CPV, 136 (31.5%) had PATs for CPV but not CDV, and 109 (25.3%) did not have PATs for either virus. Older dogs were more likely to have PATs for CDV and CPV. Neutered dogs were more likely to have PATs for CDV. Factors not associated with having a PAT included source, health status, and type of community from which the dog originated. CONCLUSIONS AND CLINICAL RELEVANCE: Most dogs had insufficient antibody titers for CDV, CPV, or both at the time of admission to the animal shelter. Findings support current guidelines recommending vaccination of all dogs immediately upon admission to shelters, regardless of source or physical condition.

Neurological signs in juvenile harbour seals (Phoca vitulina) with fatal phocine distemper.

In 2002, the northern European harbour seal (Phoca vitulina) population experienced an epidemic of phocine distemper virus (PDV) in which 22,000 seals died. Clinical signs were recorded in 20 harbour seal pups admitted to the Seal Rehabilitation and Research Centre with clinical disease, and they were diagnosed PDV infection-positive by RT-PCR postmortem. All 20 had respiratory signs, 14 had conjunctivitis and 10 had neurological signs. Severe neurological signs were one of the criteria for euthanasia during the epidemic, and many pups that were euthanased were not included in this study owing to the lack of complete datasets. Neurological signs were therefore among the most prevalent signs of fatal PDV infection in harbour seal pups. The lymphoid depletion reported in dead seals during the epidemic was not reflected in the total mononuclear leucocyte count of the seal pups, but they had an absolute granulocytosis, thrombocytosis, anaemia, and high total white blood cell counts. When first examined, 11 of the pups had a positive serum IgG titre, and four had a positive serum IgM titre. High levels of PDV-specific serum IgG antibodies were not correlated with an absence of clinical signs or longer survival.

Molecular and serological surveys of canine distemper virus: A meta-analysis of cross-sectional studies.

BACKGROUND: Canine morbillivirus (canine distemper virus, CDV) persists as a serious threat to the health of domestic dogs and wildlife. Although studies have been conducted on the frequency and risk factors associated with CDV infection, there are no comprehensive data on the current epidemiological magnitude in the domestic dog population at regional and national levels. Therefore, we conducted a cross-sectional study and included our results in a meta-analysis to summarize and combine available data on the frequency and potential risk factors associated with CDV infection. METHODS: For the cross-sectional study, biological samples from dogs suspected to have canine distemper (CD) were collected and screened for viral RNA. Briefly, the PRISMA protocol was used for the meta-analysis, and data analyses were performed using STATA IC 13.1 software. RESULTS: CDV RNA was detected in 34% (48/141) of dogs suspected to have CD. Following our meta-analysis, 53 studies were selected for a total of 11,527 dogs. Overall, the pooled frequency of CDV positivity based on molecular and serological results were 33% (95% CI: 23-43) and 46% (95% CI: 36-57), respectively. The pooled subgroup analyses of clinical signs, types of biological samples, diagnostic methods and dog lifestyle had a wide range of CDV positivity (range 8-75%). Free-ranging dogs (OR: 1.44, 95% CI: 1.05-1.97), dogs >24 months old (OR: 1.83, 95% CI: 1.1-3) and unvaccinated dogs (OR: 2.92, 95% CI: 1.26-6.77) were found to be positively associated with CDV infection. In contrast, dogs <12 months old (OR: 0.36, 95% CI: 0.20-0.64) and dogs with a complete anti-CDV vaccination (OR: 0.18, 95% CI: 0.05-0.59) had a negative association. CONCLUSION: Considering the high frequency of CDV positivity associated with almost all the variables analyzed in dogs, it is necessary to immediately and continuously plan mitigation strategies to reduce the CDV prevalence, especially in determined endemic localities.

Retrospective serological analysis of spontaneous CDV infection in 192 dogs.

Spontaneous cases of canine distemper virus (CDV) infection were serologically evaluated. The 192 dogs in which CDV antigen was confirmed from tonsil by immunohistological examination were 2- to 4-months old, of various breeds, and unvaccinated. The prognoses were good in 74 dogs with significantly high levels of anti-CDV passive hemolytic aggregation (PHA) titer. In the other 118 dogs with poor prognoses, anti-CDV PHA titer was not detected. Anti-CDV PHA titer had the most significant association with the prognoses of CDV infection, and could be the most reliable and useful indicator for evaluating such prognoses.

Development of recombinant nucleocapsid protein based IgM-ELISA for the early detection of distemper infection in dogs.

An IgM-ELISA based on a 16-kDa recombinant protein produced for the conserved and functional middle region of nucleocapsid protein of Canine distemper virus was developed. Out of 70 serum samples from distemper-suspected and vaccinated dogs analyzed, 34 serum samples (49%) were positive. The specificity of this ELISA was confirmed by blocking and adsorption experiments. The IgM-ELISA based on the recombinant nucleocapsid protein showed a strong correlation (r=0.857, p<0.0001 at 95% CI) and good agreement (kappa=0.714) with the conventional Vero cell culture distemper antigen based IgM-ELISA. The percent positivity was more in dogs with systemic signs (62%) by recombinant nucleocapsid protein IgM-ELISA. Out of 70 clinical serum samples, 69 samples were used along with 4 control sera used in the IgM-ELISA for the detection of viral RNA by Slot blot hybridization and 26 of them (36%) were positive. Fifty-one percent agreement was observed between the recombinant nucleocapsid protein IgM-ELISA and Slot blot hybridization. The analysis of clinical history of the dogs with systemic signs supported the application of IgM-ELISA over Slot blot hybridization in the early detection of distemper infection.

Cerebellum progesterone concentration decreased in canine distemper virus infection.

Progesterone has neuroprotective effects including augmentation of myelination in the central and peripheral nervous system. This study was designed to determine if demyelinating lesions in the cerebellum resulting from canine distemper virus (CDV) infection are associated with progesterone levels. Progesterone was measured using radioimmunoassay in samples of the cerebellum, corpus callosum, medulla oblongata, parietal, frontal, temporal, and occipital cortices as well as cerebrospinal fluid (CSF) and plasma collected from ten CDV infected and six non-infected dogs. The cerebellum progesterone level was significantly different between CDV infected (0.66+/-0.09 ng/g) and control dogs (1.14+/-0.09 ng/g) (p<0.001); however, no difference was observed for the other CNS regions, plasma and CSF (p>0.05). The cerebellum progesterone level was also significantly different between acute (0.71+/-0.0 5 ng/g) and chronic cases (0.61+/-0.09 ng/g) (p<0.05). The CDV infected cerebella were also categorized histopathologically according to the severity of demyelinating lesions as mild (n=5), moderate (n=2), or severe (n=3) among which the cerebellum progesterone level was significantly different (p<0.05). Progesterone concentration was 0.71+/-0.05 ng/g in mild, 0.65+/-0.10 ng/g in moderate, and 0.56+/-0.07 ng/g in severe cases. In conclusion, progesterone concentration decreases in the cerebellum in CDV infection and the severity of demyelinating lesions is the greatest in cerebella with the lowest progesterone concentrations. The results suggest that local impairment of progesterone metabolism may be associated with the initiation and progression of cerebellar lesions in CDV infection.

Prevalence of serum antibody titers against canine distemper virus and canine parvovirus in dogs hospitalized in an intensive care unit.

OBJECTIVE To determine the prevalence of dogs hospitalized in an intensive care unit (ICU) with serum antibody titers against canine distemper virus (CDV) and canine parvovirus (CPV). DESIGN Prospective observational study. ANIMALS 80 dogs. PROCEDURES Dogs hospitalized in an ICU for > 12 hours between February 1 and June 1, 2015, that had at least 0.25 mL of serum left over from diagnostic testing were eligible for study inclusion. Dogs with serum antibody titers > 1:32 (as determined by serum neutralization) and > 1:80 (as determined by hemagglutination inhibition) were considered seropositive for CDV and CPV, respectively. The date of last vaccination was obtained from the medical record of each dog. RESULTS Of the 80 dogs, 40 (50%) and 65 (81%) dogs were seropositive for CDV and CPV, respectively. Of the 40 dogs that were seronegative for CDV, 27 had been vaccinated against CDV within 3 years prior to testing. Of the 15 dogs that were seronegative for CPV, 3 had been vaccinated against CPV within 3 years prior to testing. Ten dogs were seronegative for both CDV and CPV. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated the prevalence of dogs hospitalized in an ICU that were seropositive for CDV and CPV was lower than expected given the high vaccination rate reported for dogs. Although the antibody titer necessary to prevent disease caused by CDV or CPV in critically ill dogs is unknown, adherence to infectious disease control guidelines is warranted when CDV- or CPV-infected dogs are treated in an ICU.

Comparison of tissue and fluid samples for the early detection of canine distemper virus in experimentally infected dogs.

The clinical utility of various specimens was examined for the early diagnosis of canine distemper (CD). Seven healthy dogs at 17 weeks of age were experimentally infected with a field isolate of canine distemper virus. The RT-PCR was carried out to detect CDV NP gene. Dogs showed mild fever and leukopenia, however, typical clinical signs of CD were not seen through the experimental period. CDV amplicons were detected more, earlier and for longer period in the conjunctival swabs than in the other samples employed. These results suggested that conjunctival swab samples, which are easy to obtain and non-invasive, would be the most suitable and practical specimen for the early antemortem diagnosis of CDV infection.

Seroepidemiological survey of morbillivirus infection in Kuril harbor seals (Phoca vitulina stejnegeri) of Hokkaido, Japan.

Serological analysis was performed to detect morbillivirus infection in Kuril harbor seals in Hokkaido, Japan. Serum samples were collected from the seals at Nosappu (231 sera), Akkeshi (16), and Erimo (75) between 1998 and 2005. Antibodies to phocine distemper virus (PDV) were detected by ELISA in seals from Nosappu and Erimo. Antibodies to PDV were found in 56% (5/9) of the sampled seals from Nosappu in 1998, versus only 5% (3/66) for 2003, 1% (1/79) for 2004, and 1% (1/77) for 2005. These suggest epidemic caused by the virus in or before 1998. As antibody-positive seals included juvenile seals in 2003 and 2005, sporadic infections of the virus are thought to have occurred in recent years. In Erimo, antibodies to PDV were found in 50% (14/28) of sampled seals in 2004, versus only 13% (1/8) for 1999, 7% (1/15) for 2003, and 0% (0/24) for 2005. These suggest sporadic infection by the virus before 2003 and the epizootic between after autumn in 2003, when samples of 2003 were collected, and 2004. Since antibodies to canine distemper virus (CDV) were detected in one adult seal from Nosappu in each year from 2003 to 2005, sporadic infections of the virus were suggested. There were no difference in incidence of seals with antibodies to the viruses between males and females and between juveniles and adults.

Phocine distemper virus (PDV) seroprevalence as predictor for future outbreaks in harbour seals.

Phocine distemper virus (PDV) infections caused the two most pronounced mass mortalities in marine mammals documented in the past century. During the two outbreaks, 23,000 and 30,000 harbour seals (Phoca vitulina), died in 1988/1989 and 2002 across populations in the Wadden Sea and adjacent waters, respectively. To follow the mechanism and development of disease spreading, the dynamics of Morbillivirus-specific antibodies in harbour seal populations in German and Danish waters were examined. 522 serum samples of free-ranging harbour seals of different ages were sampled between 1990 and 2014. By standard neutralisation assays, Morbillivirus-specific antibodies were detected, using either the PDV isolate 2558/Han 88 or the related canine distemper virus (CDV) strain Onderstepoort. A total of 159 (30.5%) of the harbour seals were seropositive. Annual seroprevalence rates showed an undulating course: Peaks were seen in the post-epidemic years 1990/1991 and 2002/2003. Following each PDV outbreak, seroprevalence decreased and six to eight years after the epidemics samples were tested seronegative, indicating that the populations are now again susceptible to new PDV outbreak. After the last outbreak in 2002, the populations grew steadily to an estimated maximum (since 1975) of about 39,100 individuals in the Wadden Sea in 2014 and about 23,540 harbour seals in the Kattegat area in 2013. A re-appearence of PDV would presumably result in another epizootic with high mortality rates as encountered in the previous outbreaks. The current high population density renders harbour seals vulnerable to rapid spread of infectious agents including PDV and the recently detected influenza A virus.

Canine distemper virus-induced depletion of uninfected lymphocytes is associated with apoptosis.

Canine distemper virus (CDV), a negative stranded RNA morbillivirus, causes a multisystemic disease in dogs, which is associated with a severe immune suppression. The aim of the study was to examine the influence of early CDV infection on leukocyte depletion, lymphopenia and virus-induced cell death in dogs infected with a virulent CDV strain. From 10 infected dogs, peripheral blood leukocytes were harvested periodically, phenotyped and analyzed for CDV antigen content and apoptosis using Annexin V-FITC and propidium iodide labeling. CDV infection induced a severe CD3+ T cell and CD21+ B cell depletion in all animals at 3 days post-infection (d.p.i.). For dogs with severe distemper, developing virus persistence in the lymphoid tissue and central nervous system, this lymphopenia lasted until the end of the experiment. Increased levels of lymphocyte apoptosis were found at 3 d.p.i., and monocyte apoptosis at 6 d.p.i. This was more prominent in the group of animals with severe distemper. At 3 d.p.i. no leukocyte infection was detectable indicating that the early lymphocyte depletion and apoptosis was not a direct consequence of virus infection. Taken together, our results demonstrate that CDV-induced lymphopenia is an early event and that the degree of lymphocyte depletion correlates with the severity of disease and virus persistence in the lymphoid tissue and central nervous system.

Use of serologic tests to predict resistance to Canine distemper virus-induced disease in vaccinated dogs.

The objective of the current study was to determine whether detection of Canine distemper virus (CDV)-specific serum antibodies correlates with resistance to challenge with virulent virus. Virus neutralization (VN) assay results were compared with resistance to viral challenge in 2 unvaccinated Beagle puppies, 9 unvaccinated Beagle dogs (4.4-7.2 years of age), and 9 vaccinated Beagle dogs (3.7-4.7 years of age). Eight of 9 (89%) unvaccinated adult dogs exhibited clinical signs after virus challenge, and 1 (13%) dog died. As compared to adult dogs, the 2 unvaccinated puppies developed more severe clinical signs and either died or were euthanized after challenge. In contrast, no clinical signs were detected after challenge of the 9 adult vaccinated dogs with post-vaccination intervals of up to 4.4 years. In vaccinated dogs, the positive and negative predictive values of VN assay results for resistance to challenge were 100% and 0%, respectively. Results indicate that dogs vaccinated with modified live CDV can be protected from challenge for ≤4.4 years postvaccination and that detection of virus-specific antibodies is predictive of whether dogs are resistant to challenge with virulent virus. Results also indicate that CDV infection in unvaccinated dogs results in age-dependent morbidity and mortality. Knowledge of age-dependent morbidity and mortality, duration of vaccine-induced immunity, and the positive and negative predictive values of detection of virus-specific serum antibodies are useful in development of rational booster vaccination intervals for the prevention of CDV-mediated disease in adult dogs.

Use of quantitative real-time RT-PCR to investigate the correlation between viremia and viral shedding of canine distemper virus, and infection outcomes in experimentally infected dogs.

We used real-time RT-PCR and virus titration to examine canine distemper virus (CDV) kinetics in peripheral blood and rectal and nasal secretions from 12 experimentally infected dogs. Real-time RT-PCR proved extremely sensitive, and the correlation between the two methods for rectal and nasal (r=0.78, 0.80) samples on the peak day of viral RNA was good. Although the dogs showed diverse symptoms, viral RNA kinetics were similar; the peak of viral RNA in the symptomatic dogs was consistent with the onset of symptoms. These results indicate that real-time RT-PCR is sufficiently sensitive to monitor CDV replication in experimentally infected dogs regardless of the degree of clinical manifestation and suggest that the peak of viral RNA reflects active CDV replication.

Canine distemper virus infectivity of various blood fractions for central nervous system vasculature.

In order to determine the infectivity of various viremic blood fractions for central nervous system (CNS) endothelia, viremic plasma, platelets and mononuclear cells were prepared from canine distemper virus (CDV)-infected dogs and infused into the right carotid arteries of CDV-naive gnotobiotic dogs. All blood fractions were infectious for endothelia as determined by indirect immunofluorescence examination for viral antigen in recipients. Virus-positive platelets, even though possessing only trace amounts (1.0 x 10(1) TCID50/ml) of in vitro titratable virus, were the most effective fraction for infection of vascular endothelium. These data confirm the important role of vascular endothelia in establishing CNS infection in this disease and implicate virus-positive platelets and leukocytes in the initiation of this phenomenon.

Application of N-PCR for diagnosis of distemper in dogs and fur animals.

The immunofluorescence test, routinely used for laboratory diagnosis of canine distemper virus (CDV) in Poland, is not sufficiently sensitive and specific. Therefore, the application of reverse transcriptase polymerase chain reaction (RT-PCR), nested PCR (N-PCR) and Southern blot hybridization for detection of phosphoprotein (P) gene of CDV in peripheral blood mononuclear cells (PBMCs) or internal organs of dogs and fur animals was the aim of these studies. The optimal parameters for two-step PCR were elaborated for reference strains of distemper virus and used for testing biological samples collected from dogs, foxes, ferret and mink with spontaneous distemper. PCR product of 1069bp was obtained in one out of 10 dog blood samples, three out of 14 homogenates of internal organs of dogs and one out of five homogenates of internal organs of fox. Reamplification with the use of CDVa and CDVb primers demonstrated the 429bp fragment in six samples, negative by PCR: two samples collected from dogs, two from foxes, one from mink and one from ferret. The specificity of N-PCR was confirmed by Southern blot hybridization. We conclude that two-step PCR is sensitive and specific method for diagnosis of CDV infection.