A novel linear B cell epitope of the canine coronavirus nucleocapsid protein identified by a monoclonal antibody.

The infection of canine coronavirus (CCoV) causes a highly contagious disease in dogs with acute gastroenteritis. The efficient serological diagnostics is critical for controlling the disease caused by CCoV. Nucleocapsid (N) protein of CCoV is an important target for developing serological approaches. However, little is known about the antigenic sites in the N protein of CCoV. In this study, we generated a monoclonal antibody (mAb) against the N protein of CCoV, designated as 13E8, through the fusion of the sp2/0 cells with the spleen cells from a mouse immunized with the purified recombinant GST-N protein. Epitope mapping revealed that mAb 13E8 recognized a novel linear B cell epitope in N protein at 294-314aa (named as EP-13E8) by using a serial of truncated N protein through Western blot and ELISA. Sequence analysis showed that the sequence of EP-13E8 was highly conserved (100 %) among different CCoV strains analyzed, but exhibited a low similarity (31.8-63.6 %) with the responding sequence in other coronaviruses of the same genus such as FCoV, PEDV and HCoV except for TGEV (95.5 % identity). Structural assay suggested that the epitope of EP-13E8 were located in the close proximity on the surface of the N protein. Overall, the mAb 13E8 against N protein generated and its epitope EP-13E8 identified here paid the way for further developing epitope-based serological diagnostics for CCoV.

Establishment of an indirect ELISA-based method involving the use of a multiepitope recombinant S protein to detect antibodies against canine coronavirus.

Here, we report the development of an indirect enzyme-linked immunosorbent assay (ELISA) method that involves using multiepitope recombinant S protein (rSP) as the coating antigen to detect antibodies against canine coronavirus (CCoV). rSP was designed by arranging its four S fragments (91-135 aa, S1 gene; 377-434 aa, S2 gene; 647-671 aa, S3 gene; 951-971 aa, S4 gene; 207-227 aa) and two T-cell epitopes in tandem: T-E1-E2-E3-E4-T. This multiepitope antigen, which has a molecular weight of approximately 25 kDa and contains a His-tag, was recognized by a CCoV-positive serum in a Western blot assay. The optimal concentration of rSP as a coating antigen in the ELISA was 2 µg/mL, and the optimal dilution of enzyme-labeled secondary antibody was 1:10,000. The cutoff OD450 value was established at 0.2395. No reactivity was observed with antisera against canine distemper virus, canine parvovirus, or feline calicivirus, indicating that this assay is highly specific. We also tested 64 clinical serum samples using our newly established method, and the positive rate was found to be 82.8%. In conclusion, our assay was found to be highly sensitive and specific for the detection of antibodies against CCoV, and it can therefore serve as a new, efficient diagnostic method.

A serological survey of canine respiratory coronavirus in New Zealand.

Aims: To determine the seroprevalence of canine respiratory coronavirus (CRCoV) in New Zealand dogs, and to explore associations with age, sex, breed, month, and geographical region of sampling and reported presence of clinical signs suggestive of respiratory disease.Methods: A total of 1,015 canine serum samples were randomly selected from submissions to a diagnostic laboratory between March and December 2014, and were analysed for CRCoV antibodies using a competitive ELISA. Logistic regression analysis was used to determine associations between seroprevalence of CRCoV and breed category, age, sex, sampling month, region, and reported health status of dogs.Results: Overall, 538/1,015 (53.0%) samples were seropositive for CRCoV, with 492/921 (53.4%) positive dogs in the North Island and 46/94 (49%) in the South Island. Age of dog, sampling month, region, and presence of abnormal respiratory signs were included in the initial logistic regression model. Seroprevalence was higher in dogs aged ≥3 compared with ≤2 years (p < 0.01). The lowest seroprevalence was observed in July (30/105; 28.5%) and August (32/100; 32%), and the highest in June (74/100; 74%). Seroprevalence in dogs from Auckland was higher than in dogs from the Hawkes Bay, Manawatu, Marlborough, and Waikato regions (p < 0.05). Abnormal respiratory signs (coughing, nasal discharge, or sneezing) were reported for 28/1,015 (2.8%) dogs sampled. Seroprevalence for CRCoV tended to be higher among dogs with respiratory signs (67.9 (95% CI = 47.6-83.4)%) than dogs with no reported respiratory signs (52.6 (95% CI = 49.5-55.7)%).Conclusions: Serological evidence of infection with CRCoV was present in more than half of the dogs tested from throughout New Zealand. Differences in CRCoV seroprevalence between regions and lack of seasonal pattern indicate that factors other than external temperatures may be important in the epidemiology of CRCoV in New Zealand.Clinical relevance: Our data suggest that CRCoV should be included in investigations of cases of infectious canine tracheobronchitis, particularly if these occur among dogs vaccinated with current vaccines, which do not include CRCoV antigens.

SURVEILLANCE FOR ANTIBODIES AGAINST SIX CANINE VIRUSES IN WILD RACCOONS (PROCYON LOTOR) IN JAPAN.

Raccoons (Procyon lotor) are found worldwide. They are frequently seen in crowded inner cities as well as in forests or wooded areas, often living in proximity to humans and their pets. We examined sera from 100 wild raccoons in Japan for antibodies to six canine viruses with veterinary significance to assess their potential as reservoirs. We also aimed to understand the distribution of potentially infected wildlife. We found that 7% of samples were seropositive for canine distemper virus (CDV), 10% for canine parvovirus type 2, 2% for canine adenovirus type 1, 6% for canine adenovirus type 2, and 7% for canine coronavirus. No samples were found to be seropositive for canine parainfluenza virus. Seropositivity rates for canine distemper virus and canine parvovirus type 2 were significantly different between areas, and younger raccoons (<1 yr old) were more frequently seropositive than older raccoons. Because raccoons belong to the suborder Caniformia, similar to dogs (Canis lupus familiaris), our results suggest that they can act as reservoirs for some of these important canine viruses and might be involved in viral transmission. Further study should include isolation and analysis of canine viruses in wild raccoons from a wider area.

Genetic and antigenic characterization of recombinant nucleocapsid proteins derived from canine coronavirus and canine respiratory coronavirus in China.

To characterize the antigenicity of nucleocapsid proteins (NP) derived from canine coronavirus (CCoV) and canine respiratory coronavirus (CRCoV) in China, the N genes of CCoV (CCoV-BJ70) and CRCoV (CRCoV-BJ202) were cloned from swabs obtained from diseased pet dogs in Beijing and then sequenced. The recombinant NPs (rNPs) were expressed in Escherichia coli and purified by nickel-affinity column and size exclusion chromatography. Sequencing data indicated that the N genes of CCoV-BJ70 and CRCoV-BJ202 belonging to two distinctly different groups were relatively conserved within each subgroup. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that rNPs of CCoV and CRCoV were expressed efficiently and isolated with a final purity of over 95%. Western blot analysis revealed the rNP from CRCoV could cross-react with mice antisera against human coronavirus (HCoV-229E, NL63, OC43, HKU1), while rNP of CCoV had cross-reactivity with only anti-sera against viruses belonging to the same group (HCoV-229E and NL63). In summary, CCoV and CRCoV rNPs were successfully expressed in E. coli and showed antigenic cross-reactivity with antisera raised against human coronaviruses. These findings indicate that further serologic studies on coronavirus infections at the animal-human interface are needed.

PARASITOLOGY AND SEROLOGY OF FREE-RANGING COYOTES (CANIS LATRANS) IN NORTH CAROLINA, USA.

Coyotes (Canis latrans) have expanded recently into the eastern US and can serve as a source of pathogens to domestic dogs (Canis lupus familiaris), livestock, and humans. We examined free-ranging coyotes from central North Carolina, US, for selected parasites and prevalence of antibodies against viral and bacterial agents. We detected ticks on most (81%) coyotes, with Amblyomma americanum detected on 83% of those with ticks. Fifteen (47%) coyotes were positive for heartworms (Dirofilaria immitis), with a greater detection rate in adults (75%) than juveniles (22%). Serology revealed antibodies against canine adenovirus (71%), canine coronavirus (32%), canine distemper virus (17%), canine parvovirus (96%), and Leptospira spp. (7%). We did not detect antibodies against Brucella abortus/suis or Brucella canis. Our results showed that coyotes harbor many common pathogens that present health risks to humans and domestic animals and suggest that continued monitoring of the coyote's role in pathogen transmission is warranted.

Tropism and pathological findings associated with canine respiratory coronavirus (CRCoV).

Canine infectious respiratory disease (CIRD) occurs frequently in densely housed dog populations. One of the common pathogens involved is canine respiratory coronavirus (CRCoV), however little is known regarding its pathogenesis and the role it plays in the development of CIRD. The pathogenesis of five geographically unrelated canine respiratory coronavirus (CRCoV) isolates was investigated. Following experimental infection in dogs, all five CRCoV isolates gave rise to clinical signs of respiratory disease consistent with that observed during natural infection. The presence of CRCoV was associated with marked histopathological changes in the nares and trachea, with loss and damage to tracheal cilia, accompanied by inflammation. Viral shedding was readily detected from the oropharynx up to 10 days post infection, but there was little or no evidence of rectal shedding. The successful re-isolation of CRCoV from a wide range of respiratory and mucosal associated lymphoid tissues, and lung lavage fluids demonstrates a clear tropism of CRCoV for respiratory tissues and fulfils the final requirement for Koch's postulates. By study day 14 dogs had seroconverted to CRCoV and the antibodies raised were neutralising against both homologous and heterologous strains of CRCoV in vitro, thus demonstrating antigenic homogeneity among CRCoV strains from the two continents. Defining the role that CRCoV and other agents play in CIRD is a considerable, but important, challenge if the disease is to be managed, treated and prevented more successfully. Here we have successfully developed a model for studying the pathogenicity and the role of CRCoV in CIRD.

Immunogenicity and protective efficacy in dogs of an MF59™-adjuvanted vaccine against recombinant canine/porcine coronavirus.

Recently, canine coronavirus (CCoV) strains with putative recombinant origin with porcine transmissible gastroenteritis virus (TGEV) were shown to be widespread in Europe. In this study, a killed vaccine against TGEV-like CCoV strains, included in the new subtype CCoV-IIb, was developed through inactivation with betapropiolactone and emulsification with MF59™ adjuvant. Safety, immunogenicity and efficacy of the developed vaccine were evaluated in vivo. Five 10-week-old beagle pups were administered (three weeks apart) two vaccine doses, whereas two animals served as unvaccinated controls. The vaccine was shown to be safe as no local neither systemic reactions were observed after first and second dose administration. Serum antibodies against CCoV were detected in vaccinates starting from study day 14 (by enzyme-linked immunosorbent assay) or 28 (by virus neutralisation test). Subsequent challenge with virulent CCoV-IIb resulted in the development of mild gastroenteric disease in control pups, whereas vaccinates did not display clinical signs. Faecal shedding of the challenge virus occurred in both treatment groups, but vaccinated dogs were found to shed very low viral titres in comparison to controls. The developed vaccine may help control the CCoV-IIb-induced disease (and active virus circulation) in environments, such as kennels and shelters, where the pathogenic potential of this virus is greater as a consequence of predisposing factors and concurrent infections.

Seroepidemiology of respiratory (group 2) canine coronavirus, canine parainfluenza virus, and Bordetella bronchiseptica infections in urban dogs in a humane shelter and in rural dogs in small communities.

This prospective study evaluated seroepidemiologic features of canine respiratory coronavirus (CRCoV), canine parainfluenza virus (CPIV), and Bordetella bronchiseptica infections in dogs in an urban humane shelter and in rural/small community dog populations in western Canada. Seroprevalence of CRCoV and CPIV was low compared with other countries; seroprevalence of B. bronchiseptica was moderate to high in most populations examined. Rural dogs were 0.421 times (P ≤ 0.0001) less likely to be positive for CRCoV than dogs admitted to the shelter. There were no statistical differences in prevalence of antibodies to B. bronchiseptica and CPIV between urban and rural populations. Dogs from Fort Resolution, NWT were significantly (P < 0.05) less likely to have moderate or high antibody titers to the 3 agents than dogs in the shelter. Seroconversion to CRCoV was common in dogs in the shelter, but was not associated (P = 0.18) with respiratory disease. Antibodies to CRCoV, CPIV, or B. bronchiseptica on arrival were not significantly (P > 0.05) associated with disease-sparing after entry into the shelter.

Prolonged depletion of circulating CD4+ T lymphocytes and acute monocytosis after pantropic canine coronavirus infection in dogs.

A hypervirulent strain (CB/05) of canine coronavirus was employed to infect oronasally 11-week-old pups. Peripheral blood monocytes (CD14(+)), T lymphocytes (CD4(+) and CD8(+)) and B lymphocytes (CD21(+)) were studied by flow cytometry within 5 days post-infection (p.i.) and at later time points. Infection with CB/05 resulted in a profound depletion of T cells and a slight loss of B cells in the first week p.i. In particular, while the CD8(+) and the B lymphocytes returned to baseline levels by day 7 p.i., the CD4(+) T cells remained significantly low until day 30 p.i. and recovered completely only at day 60 p.i. Monocytosis was also observed after CB/05 infection with a peak at day 5 p.i. The prolonged depletion of peripheral CD4(+) T cells did not alter the levels of serum IgG or IgM. The impact of CB/05 infection on the immune performance of infected pups is discussed.

Immunity after natural exposure to enteric canine coronavirus does not provide complete protection against infection with the new pantropic CB/05 strain.

Recently, an outbreak of fatal infection caused by a pantropic variant (strain CB/05) of canine coronavirus (CCoV) has been reported. In this study, evidence is provided that immunity induced by natural exposure to enteric CCoV is not fully protective against strain CB/05. Twenty-two, 10-week-old beagles with a recent natural infection by enteric CCoV were randomly distributed in two experimental groups of eight (groups A and B) and one control group of six (group C) dogs. Dogs in groups A and B were inoculated oronasally with different doses (4 x 10(5) or 4 x 10(3)TCID(50)) of the pantropic strain CB/05, whereas dogs in group C were used as negative controls. Clinical, post-mortem and virological investigations showed that, despite the high serum antibody titres induced by the prior natural infection with enteric CCoV, dogs were susceptible to experimental infection with strain CB/05. This was shown by the occurrence of faecal shedding, and dogs displaying moderate clinical signs, mainly vomiting and diarrhoea. Involvement of the lymphoid tissues was evident as demonstrated by the acute lymphopenia (below 70% of the initial counts), gross lesions in spleen and lymph nodes and detection of CB/05 RNA in thymus, spleen and lymph nodes of some infected dogs. The presence of viral RNA in lymphoid tissues was observed only in dogs euthanised in the early stages of infection and the clinical course of the infection was unrelated to the viral dose administered. The present study demonstrates that strain CB/05 is able to induce infection and disease in dogs seropositive to enteric CCoV, thus highlighting the need for extensive epidemiological investigation and for the possible development of novel antigenically relevant vaccines.

An ELISA based on recombinant spike protein S for the detection of antibodies to transmissible gastroenteritis virus of swine-like canine coronaviruses.

Recombinant canine coronaviruses, resembling the transmissible gastroenteritis virus of swine (TGEV) in a 5' fragment of the S glycoprotein, have been detected recently and showed to be present in canine populations. The 5' fragment of the S protein (S') of a TGEV-like canine coronavirus (CCoV), strain 174/06, was expressed in an Escherichia coli cell-free system. The purified recombinant polypeptide was employed to develop an ELISA test for the detection of TGEV-like CCoV-specific antibodies in dog sera. Four canine sera positive for TGEV-like CCoV, six sera positive to classical CCoV-II strains and 10 negative control sera were examined. The recombinant S' was not recognized by antibodies to classical CCoV-II, as only sera from dogs infected experimentally with TGEV-like CCoV reacted strongly with the recombinant S' polypeptide whereas dog sera with antibodies to classical CCoV-II did not react. As classical CCoV-II and TEGV-like CCoVs are related antigenically, the recombinant S' ELISA is a useful method to investigate serologically the prevalence of TGEV-like CCoVs in dogs.

Effect of recombinant lactobacillus expressing canine GM-CSF on immune function in dogs.

Many Lactobacillus strains have been promoted as good probiotics for the prevention and treatment of diseases. We engineered recombinant Lactobacillus casei, producing biologically active canine granulocyte macrophage colony stimulating factor (cGM-CSF), and investigated its possibility as a good probiotic agent for dogs. Expression of the cGM-CSF protein in the recombinant Lactobacillus was confirmed by SDS-PAGE and Western blotting methods. For the in vivo study, 18 Beagle puppies of 7 weeks of age were divided into three groups; the control group was fed only on a regular diet and the two treatment groups were fed on a diet supplemented with either 1 x 10(9) colony forming units (CFU)/day of L. casei or L. casei expressing cGM-CSF protein for 7 weeks. Body weight was measured, and fecal and blood samples were collected from the dogs during the experiment for the measurement of hematology, fecal immunoglobulin (Ig)A and IgG, circulating IgA and IgG, and canine corona virus (CCV)-specific IgG. There were no differences in body weights among the groups, but monocyte counts in hematology and serum IgA were higher in the group receiving L. casei expressing cGMCSF than in the other two groups. After the administration of CCV vaccine, CCV-specific IgG in serum increased more in the group supplemented with L. casei expressing cGM-CSF than the other two groups. This study shows that a dietary L. casei expressing cGM-CSF enhances specific immune functions at both the mucosal and systemic levels in puppies.

Experimental infection of dogs with a novel strain of canine coronavirus causing systemic disease and lymphopenia.

A pantropic canine coronavirus (CCoV) strain (CB/05) has been recently associated to a fatal outbreak of systemic disease in young dogs. We report the clinical, virological and serological findings in dogs experimentally infected with strain CB/05. The dogs, three 2.5-month-old and two 6-month-old pups, were successfully infected, shedding viral RNA with their faeces for the entire observation period (21 days) and displaying systemic clinical signs resembling those observed during the course of natural infection. Leucopenia (acute lymphopenia) occurred in all infected dogs, with values dropping below 60% of the initial counts. Considering the severity of the CB/05-induced disease, two of the youngest pups were euthanized for ethical reasons at days 8-9 postinfection, whereas the other pups underwent a slow but progressive improvement of their clinical status with complete recovery. At postmortem examination, remarkable lesions were observed in the internal organs of the euthanized pups, that tested positive for CCoV by real-time RT-PCR and virus isolation on cell cultures. All pups seroconverted for CCoV, as shown by the high optical density values and antibody titres detected by ELISA and virusneutralisation tests, respectively. The present study confirms that strain CB/05 is highly pathogenic for dogs, being able to induce a severe disease (and in some cases the death) even in experimental conditions.

High-cell-passage canine coronavirus vaccine providing sterilising immunity.

OBJECTIVES: To evaluate the ability of a high-cell-passage canine coronavirus vaccine to immunise dogs against challenge with a field isolate of the virus. METHODS: Three dogs that had previously tested seronegative and virus-negative for canine coronavirus were inoculated twice, at 21-day intervals, with the vaccine and kept under observation. Two seronegative and virus-negative dogs served as unvaccinated controls. For safety tests, two additional dogs were inoculated oronasally with 10 times the vaccinal dose and no reactions were observed. Faecal samples were collected daily from the vaccinated dogs after the first and second inoculations. Both vaccinated and control dogs were challenged two weeks after the second vaccination with a field canine coronavirus strain. Blood samples were collected for serological tests before vaccination and at weekly intervals after vaccinations and challenge. RESULTS: Virus was not detected in faecal samples after the first or second vaccinations by virus isolation assays and PCR. Significantly, the vaccinated dogs did not have clinical signs after challenge and no virus shedding was observed. The two unvaccinated control dogs had moderate enteritis, and virus was detected in cell cultures starting from three days postchallenge (dog 1) and two days postchallenge (dog 2), and by PCR for 23 median days. CLINICAL SIGNIFICANCE: This study showed the efficacy of a high-cell-passage canine coronavirus vaccine in preventing infection of dogs by virulent virus and, specifically, its ability to induce sterilising immunity.

Serological prevalence of canine respiratory coronavirus.

Canine respiratory coronavirus (CRCoV) has recently been detected in dogs; it is a group 2 coronavirus showing similarity to bovine coronavirus (BCoV) but is distinct from canine enteric coronavirus (CECoV). CRCoV may play an important role in canine infectious respiratory disease (CIRD) either by predisposing to further and potentially more serious viral and bacterial infections or possibly as a primary pathogen. The prevalence of serum antibodies to CRCoV, in a population of dogs in the south east of England, has been shown previously to be 30.1% on the first day of entry to a rehoming kennel [Erles, K., Toomey, C., Brooks, H.W., Brownlie, J., 2003. Detection of a group 2 coronavirus in dogs with canine infectious respiratory disease. Virology 310, 216-223]. The purpose of this study was to establish the prevalence of CRCoV in the general canine population within as well as outside the UK. An ELISA, used to test for the presence of antibodies to CRCoV in canine serum samples, identified seropositive dogs in UK, USA, Canada, Republic of Ireland and Greece. The development of an ELISA based on CRCoV antigen and immunofluorescence assay are described here. 54.7% (547/1000) of North American and 36.0% (297/824) of United Kingdom dogs were seropositive for CRCoV. The age and geographical distribution of seropositive dogs was also assessed. The cross-reactivity demonstrated between CRCoV antibodies from different countries and a UK viral isolate suggests immunological similarity. The overall prevalence of this virus in both North America and the UK suggests that CRCoV has international significance and that further epidemiological studies are required.

[Construction of recombinant canine adenovirus type 2 expressing Canine coronavirus spike glycoprotein and its immunogenicity].

In order to construct a recombinant Canine adenovirus type 2 (CAV-2) expressing the spike glycoprotein of Canine coronavirus (CCV), the S1 gene fragment of CCV strain DXMV, encoding major antigenic region A, B, C and D of S protein, was amplified by RT-PCR and cloned into pVAX1 vector. The complete S1 expression cassette was subcloned into the shuttle vector pVAXE3, then further cloned into the backbone vector pPoly2-CAV2 containing complete genome of CAV-2. To gain the recombinant Canine adenovirus, the recombinant plasmid pCAV-2-CCV-S1 was linearized by Cla I/Asc I to release recombinant genome, and then transfected into MDCK cell. The recombinant virus CAV-2-S1 was gained through 4 passages in MDCK, which showed classical CPE of CAV-2. The expressed S1 protein of CCV, which was identified by RT-PCR and Western blot, can be specifically recognized by polyclonal antibody against CCV. The immunization in dogs indicated that the recombinant CAV-2 could effectively induce the specific antibodies against CCV and CAV.

Investigation into the causes of canine infectious respiratory disease: antibody responses to canine respiratory coronavirus and canine herpesvirus in two kennelled dog populations.

Two training centres for working dogs were monitored for one year to determine the presence of viruses and viral antibodies and their association with canine infectious respiratory disease (CIRD). Tonsillar swabs and serum were obtained from dogs on entry into the kennels and in regular intervals thereafter. Additional samples were collected during outbreaks of CIRD. The swabs were examined by virus culture and PCR for canine parainfluenza virus, canine adenovirus, canine herpesvirus (CHV) and canine respiratory coronavirus (CRCoV). Furthermore the prevalence of antibodies to CHV and CRCoV was determined. During this study CIRD was reported mainly in one of the two kennels investigated. In that kennel antibody responses to CRCoV indicated a seasonal occurrence of the virus, which coincided with two outbreaks of respiratory disease. CHV antibody responses were detected throughout the year. In the other kennel, which reported few cases of CIRD a high prevalence of antibodies to CRCoV was detected on entry but only sporadic seroconversions to CRCoV or CHV. By PCR three dogs were found positive for CRCoV in one kennel whereas all PCR tests for other viruses were negative for both kennels. Virus culture failed to detect any viruses in either kennel.

Safety and efficacy of a modified-live canine coronavirus vaccine in dogs.

The safety and the efficacy of a modified-live (ML) canine coronavirus (CCoV) vaccine strain 257/98-3c was evaluated in 14 dogs seronegative and virus negative for CCoV. For the safety test, four dogs were inoculated, two by intramuscular and two by oronasal route, with 10 times the vaccinal dose. During the observation period (28 days) all dogs did not display any local or systemic reaction. For the efficacy test, eight dogs were vaccinated by intramuscular (four dogs-group A) or by oronasal route (four dogs-group B). Two dogs were maintained as non-vaccinated controls. In the dogs of group A, vaccinal virus was not detected in faecal samples by virus isolation (VI) and by PCR assay, while in the dogs of group B, the virus was revealed for six median days only by PCR. Twenty-eight days later, the vaccinated and control dogs were challenged with a field CCoV strain. After the challenge, the vaccinated dogs did not display clinical signs and the dogs of group A shed virus for 5.5 median days, evaluated by VI, and for 10 median days evaluated by PCR. Virus shedding was not observed, both by VI and PCR assay, in the dogs of group B. The two control dogs displayed moderate clinical signs and the virus was detected by VI for 14.5 median days starting from day 3 post-challenge (dpc 3) and by PCR assay for 23 median days starting from dpc 1.