Serological and virological detection of canine herpesvirus-1 in adult dogs with and without reproductive disorders.

Canine herpesvirus 1 (CaHV-1) is known to cause reproductive disorders in adult dogs and neonatal mortality in puppies. The seroprevalence of CaHV-1 has not been documented in Italy. Sera from 865 dogs were screened for CaHV-1 using a serum neutralization assay (SN). All CaHV-1 positive sera and 100 CaHV-1 negative sera were also tested using an in-house immunofluorescence (IF) test. Thirteen bitches with reproductive disorders and three bitches with no history of reproductive diseases were also examined clinically so that lesions associated with CaHV-1 and CaHV-1 DNA could be identified using PCR analysis of vaginal swabs. An overall seroprevalence of 14.6% was observed using SN, and 18.6% using IF. The correlation between SN and IF was moderate. The SN assay demonstrated a greater sensitivity than IF, with a few exceptions. None of the vaginal swabs tested positive for CaHV-1 DNA. The differences in the seropositivity rates between SN and IF were not statistically significant (P = 0.16). Using the SN test as the reference standard, the sensitivity and specificity of IF were 29% and 95%, respectively. These results suggest that CaHV-1 is common in canine populations and could pose a threat to neonatal survival and canine fertility in breeding kennels in Italy. Vaccination of breeding bitches should be recommended if there is a history of reproductive disorders.

Agreement between the cell culture titrations of canine minute virus determined by two susceptibility-testing methods.

The correct diagnosis of canine minute virus is critical in dog breeding. In this study, the Bland Altman test was used to compare the performance of two susceptibility-testing methods, namely polymerase chain reaction (PCR) and indirect immunofluorescence assay (IFA). The agreement between IFA and PCR in monocytes revealed a mean difference of -1752.16 with 95% confidence and an interval ranging from -3229.80 to -274.53 (SD=2325.62). The agreement between IFA and PCR in Walter Reed canine cells (WRCC) revealed a mean difference of -2396.55 with 95% confidence and an interval ranging from -3774.63 to -1018.48 (SD=2168.93). The Bland Altman test confirmed the overall accuracy of PCR vs IFA and the plot showed that all points were not randomly arranged in the range of average ± 1.96 × SD of the differences.

Action of disinfectants on canine coronavirus replication in vitro.

Canine coronavirus (CCoV) is responsible for enteric disease in pups. Infected dogs generally have a rapid recovery, so the virus is highly contagious and the spread of infection is difficult to control. Chemical disinfectants have been widely used in human disease-control programmes to prevent viral infectious diseases from spreading, but to date, there are no studies in the literature on the sensitivity of CCoV to chemical biocides. The present study investigated the sensitivity of CCoV to disinfectants currently used for prophylaxis in kennel and dog breeding locations. The effects of three agents: alkyl-dimethyl-benzyl-ammonium chloride, benzalkonium chloride and didecyl-dimethyl-ammonium chloride, on the infectivity titre of CCoV in A72 cell lines, were studied at different concentrations. Although they may regard a small number of agents, the findings showed that the sensitivity of CCoV to disinfectants varies and the differences are dose correlated. In general, virus inactivation implies a permanent loss of infectivity which can be evaluated in suspensions and hand disinfection tests.

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.

Canine coronavirus infection in Turkish dog population.

Canine coronavirus (CCoV) is one of the most important viral agents affecting the gastrointestinal system of dogs. In this study virological and serological investigations were performed to demonstrate the existence and prevalence of CCoV infection in a Turkish dog population. A total of 269 animals were subjected to the study. Of 179 dogs tested for CCoV antibodies, 112 (62.5%) were found to be positive by serum neutralization test, while 133 (74.3%) were positive by ELISA. The highest prevalence (94.2%) was detected in kennel dogs. Detection of CCoV genome in faeces was performed in samples from 90 diarrhoeic puppies by reverse transcription-polymerase chain reaction. Fourteen (15.5%) faeces were positive for CCoV RNA, five of which were characterized as CCoV type I. The widespread CCoV infection in the Turkish dog population may be attributed as an important cause of viral diarrhoea in dogs.

Development of a western blotting assay to discriminate Brucella spp. and Yersinia enterocolitica O:9 infections in sheep.

Outer membrane proteins (OMPs) of Rev-1 strain of Brucella melitensis were used in a Western blotting assay for the serological diagnosis of brucellosis in ovine sera. Fifty-four sheep sera were tested and divided into the following groups: Group A) n. 9 samples from one sheep that had been experimentally infected with Y. enterocolitica O:9; Group B) n. 10 samples collected from sheep infected with Brucella melitensis and 1 sample from a sheep vaccinated with the Rev 1 strain; Group C) n. 10 samples collected in "officially brucellosis-free" herds; Group D) n. 12 samples classified as "suspicious"; Group E) n. 12 samples classified as "positive". Antibodies were detected by routine tests performed for the diagnosis of brucellosis in serum samples of the sheep infected with Y. enterocolitica O:9 after the 2nd week post infection. In the WB assay, sera of group B recognised a 17 kDa protein, whereas sera of groups A, and D and 9 out of 12 of group E exhibited no reactivity to this protein. The results obtained encourage the use of the WB assay as a confirmatory test for the diagnosis of brucellosis.

Canine distemper and related diseases: report of a severe outbreak in a kennel.

An outbreak of canine distemper in a kennel of German shepherds in the province of Bari is reported. Six 42-day-old pups developed typical signs of canine distemper (fever, conjunctivitis, respiratory distress and enteritis) and died within 7-10 days. Neurological symptoms were observed only in one pup. Four additional pups, which had shown no sign of illness, were separated and vaccinated, but two of these developed a severe, fatal nervous form 15 days later. Post-mortem examination, carried out on two pups which died without neurological signs, showed pneumonia and enteritis, more severe in one of the two examined pups. Smears from the brain and the conjunctiva of both dogs tested positive for canine distemper virus (CDV) by an immunofluorescent assay, confirmed by the identification of viral RNA using RT-PCR. Bordetella bronchiseptica and a canine adenovirus strain, characterized as canine adenovirus type 2 by a differential PCR assay, were isolated from the lungs of the pup showing the most pronounced lesions. Furthermore, canine coronavirus was detected by PCR in the intestinal content of this pup, suggesting a multifactorial aetiology of the outbreak.

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.

A severe dual infection by feline panleukopenia virus and feline calicivirus in an adult cat.

A dual infection by feline panleukopenia virus (FPV) and feline calicivirus (FCV) in a 7 month-old cat is described. The animal developed a severe illness with depression, anorexia, fever, leucopoenia, nasal and ocular discharge and oral ulcers. Both FPV and FCV were isolated in cell cultures from a rectal swab and the presence of FCV was confimed by polymerase chain reaction. Antibodies to both the viruses were detected in the serum. The severity of the disease induced by the mixed viral infection highlights the need for intensifying FPV vaccination in cats.

Experimental infection of goats at different stages of pregnancy with caprine herpesvirus 1.

Three goats from a group of five caprine herpesvirus 1 (CpHV.1) seronegative pregnant goats were inoculated intranasally with a virulent BA.1 strain of CpHV.1. Goat n.1 was infected on day 45 of pregnancy, goat n.2 on day 92 and goat n.3 on day 127. Each of the three goats produced a single foetus 10-60 days after infection. Foetus n.1 was never found and so it could not be examined for virological findings. Goat n.2 delivered at term of gestation and CpHV.1 was detected by PCR and isolated from most of the foetal organs. Foetus n.3 was partially autolysed and the virus was only detected by PCR but not isolated from foetal organs. The results confirm the damaging effect of CpHV.1 infection on pregnancy, the difficulty in diagnosing the CpHV.1 induced abortion, and the importance developing appropriate prophylactic programmes.

Molecular analysis of the VP7, VP4, VP6, NSP4, and NSP5/6 genes of a buffalo rotavirus strain: identification of the rare P[3] rhesus rotavirus-like VP4 gene allele.

We report the detection and molecular characterization of a rotavirus strain, 10733, isolated from the feces of a buffalo calf affected with diarrhea in Italy. Strain 10733 was classified as a P[3] rotavirus, as the VP8* trypsin cleavage product of the VP4 protein revealed a high amino acid identity (96.2%) with that of rhesus rotavirus strain RRV (P5B[3]), used as the recipient virus in the human-simian reassortant vaccine. Analysis of the VP7 gene product revealed that strain 10733 possessed G6 serotype specificity, a type common in ruminants, with an amino acid identity to G6 rotavirus strains ranging from 88 to 98%, to Venezuelan bovine strain BRV033, and Hungarian human strain Hun4. Phylogenetic analysis based on the VP7 gene of G6 rotaviruses identified at least four lineages and an apparent linkage between each lineage and the VP4 specificity, suggesting the occurrence of repeated interspecies transmissions and genetic reassortment events between ruminant and human rotaviruses. Moreover, strain 10733 displayed a bovine-like NSP4 and NSP5/6 and a subgroup I VP6 specificity, as well as a long electropherotype pattern. The detection of the rare P[3] genotype in ruminants provides additional evidence for the wide genetic and antigenic diversity of group A rotaviruses.

First two confirmed cases of malignant catarrhal fever in Italy.

Two cases of sheep-associated malignant catarrhal fever (SA-MCF) in cattle herds of Southern Italy are reported. The affected animals, a three-year-old cow and a six-month-old calf, developed clinical manifestations resembling those of the "head and eye" form of MCF. Serologically, the calf tested positive in an indirect immunofluorescent (IIF) assay for the detection of MCF viruses antibodies, whereas the cow was found seronegative. One affected animal was from a herd housed together with a flock of sheep, while no contact between the herd of the affected calf and carrier animals was demonstrated. OvHV-2 viral DNA was detected by a PCR test performed on peripheral blood leucocytes (PBL) and tissue samples from both the animals, completing the definitive diagnosis of MCF.

Efficacy of an inactivated canine coronavirus vaccine in pups.

The efficacy of an inactivated CCoV vaccine (Duramune PC) was evaluated in four pups. Two dogs were maintained non-vaccinated. Ten days after the booster shot all the pups were challenged with a field CCoV strain administered by oro-nasal route. The vaccinated pups did not display clinical signs and shed the challenge-virus for 11.25 days, evaluated by virus isolation, and 13.5 days, evaluated by PCR assay. The two non vaccinated pups displayed mild diarrhoea at day post-challenge 4 and shed the challenge-virus for 14 and 15 days respectively, by virus isolation, and for 22 and 24 days respectively, by PCR assay.

Evaluation of antibody response to canine coronavirus infection in dogs by Western Blotting analysis.

We investigated by Western Blotting the antibody responses against the three major structural proteins of Canine coronavirus (CCoV) in dogs naturally infected. A pool of Elisa positive sera were also tested to clearly identify the binding profiles of CCoV proteins. The immune response to S protein was barely detectable in naturally infected dogs, whereas anti-M and anti-N antibodies were detected with a very strong reaction and for a long time post infection. The limited response to S protein may explain the poor protection of dogs and the possibility of persisting infection.

Evaluation of the innate immune response in pups during canine parvovirus type 1 infection.

In two pups (A and B) naturally infected with canine parvovirus type 1 (CPV1) phagocytic responses were evaluated over a period of two weeks (day 0 = T0; day 3 = T1; day 7 = T2; day 14 = T3). CPV1 infection led to a marked reduction of monocyte (MO) phagocytosis in both pups. Also MO killing was impaired and in pup B this function was totally absent. Polymorphonuclear (PMN) phagocytosis values of both pups fluctuated within normal ranges, as well as PMN killing of pup A. In pup B, killing exerted by PMN was absent at T0, then increased but again dropped below normal ranges at T3. The described alterations of phagocytic functions may be regarded as possible viral mechanisms of immune evasion.

Detection and genetic characterization of canine distemper virus (CDV) from free-ranging red foxes in Italy.

Fragments of the genes encoding the haemoagglutinin (H) and the nucleocapsid protein (N) of a canine distemper (CDV)-like virus affecting a red fox (Vulpes vulpes) were sequenced and analysed. The CDV-like virus detected in the fox was found to be not dissimilar, in both the H and N gene, from other CDVs spreading in Italy, as well as all over the world, and phylogenetic analysis on the H protein-encoding gene allowed to include all the Italian CDVs in the H European genotype.

M gene evolution of canine coronavirus in naturally infected dogs.

Two stray pups (A and B), three and five months old, respectively, both naturally infected with canine coronavirus (CCoV), were studied for 180 days. The virus was detected intermittently in the pups' faeces by PCR for periods of 156 and 146 days, respectively. Sequence analysis of a fragment of the gene encoding the M protein revealed that the viruses detected at the onset of the infection were very similar to typical strains of CCoV, whereas from 42 days after infection in pup A and 40 days after infection in pup B the viruses had nucleotide and amino acid mutations resembling sequences in feline coronavirus.

Variation of the sequence in the gene encoding for transmembrane protein M of canine coronavirus (CCV).

A nucleotide variability in the sequence of the gene encoding for the transmembrane protein M of canine coronavirus (CCV) is described. A total of 177 faecal samples from pups with enteritis were analysed by a PCR and n-PCR specific for CCV. Four samples, collected from a dog presenting a long-duration shedding of CCV, and a sample from another diarrhoeic dog, were found positive by PCR but negative by n-PCR. Sequence analysis of the samples revealed silent nucleotide substitutions in the binding site of the internal primer used for the n-PCR. Moreover, the nucleotide substitutions occurring over the whole fragment of the five samples analysed were similar.