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.

Nucleotide sequence variation of the VP7 gene of two G3-type rotaviruses isolated from dogs.

The sequence of the VP7 gene of two rotaviruses isolated from dogs in southern Italy was determined and the inferred amino acid sequence was compared with that of other rotavirus strains. There was very high nucleotide and amino acid identity between canine strain RV198/95 and other canine strains, and to the human strain HCR3A. Strain RV52/96, however, was found to have about 95% identity to the G3 serotype canine strains K9, A79-10 and CU-1 and 96% identity to strain RV198/95 and to the simian strain RRV. Therefore both of the canine strains belong to the G3 serotype. Nevertheless, detailed analysis of the VP7 variable regions revealed that RV52/96 possesses amino acid substitutions uncommon to the other canine isolates. In addition, strain RV52/96 exhibited a nucleotide divergence greater than 16% from all the other canine strains studied; however, it revealed the closest identity (90.4%) to the simian strain RRV. With only a few exceptions, phylogenetic analysis allowed clear differentiation of the G3 rotaviruses on the basis of the species of origin. The nucleotide and amino acid variations observed in strain RV52/96 could account for the existence of a canine rotavirus G3 sub-type.

Development of a nested PCR assay for the detection of canine coronavirus.

A diagnostic test for canine coronavirus (CCV) infection based on a nested polymerase chain reaction (n-PCR) assay was developed and tested using the following coronavirus strains: CCV (USDA strain), CCV (45/93, field strain), feline infectious peritonitis virus (FIPV, field strain), transmissible gastroenteritis virus (TGEV, Purdue strain), bovine coronavirus (BCV, 9WBL-77 strain), infectious bronchitis virus (IBV, M-41 strain) and fecal samples of dogs with CCV enteritis. A 230-bp segment of the gene encoding for transmembrane protein M of CCV is the target sequence of the primer. The test described in the present study was able to amplify both CCV and TGEV strains and also gave positive results on fecal samples from CCV infected dogs. n-PCR has a sensitivity as high as isolation on cell cultures, and can therefore be used for the diagnosis of CCV infection in dogs.

Diagnosis of canine coronavirus infection using nested-PCR.

The results of polymerase chain reaction (PCR) and nested polymerase chain reaction (n-PCR) assays for the diagnosis of canine coronavirus (CCV) infection, and the comparison with other diagnostic techniques, such as electron microscopy (EM) and virus isolation using A-72 cell line are reported. The study was carried out on 71 faecal samples of pups with enteritis. Of 71 samples examined 14 were positive in PCR, whereas 30 samples resulted positive in the n-PCR assay. CCV was detected by EM examination in only four out of 45 samples, and by virus isolation in three out of 30 samples n-PCR positive.

Genomic characterization of pestiviruses isolated from lambs and kids in southern Italy.

A nested polymerase chain reaction was used to identify 13 pestivirus strains isolated from small ruminants in several mixed (sheep and goats) flocks of Southern Italy, and for classification as bovine viral diarrhoea virus (BVDV) type 1, BVDV type 2, and Border disease virus (BDV) genotypes. Of the nine ovine isolates, two were characterized as BVDV type 1, and seven as BVDV type 2. The four pestiviruses isolated from kids belong to BVDV type 1. None of the pestivirus strains tested could be classified as 'true' BDV (genotype 3). Although BVDV type 2 has been described in Europe rarely, the characterization of BD/90-1M strain as BVDV type 2, isolated in Italy in 1990, demonstrates that this genotype has been circulating in Italy since the 1990s.

Isolation and genetic characterization of two G3P5A[3] canine rotavirus strains in Italy.

Two strains of canine rotavirus were isolated from pups with clinical signs of gastroenteritis. Both strains were identified by polymerase chain reaction (PCR) as G3P5A[3], although restriction endonuclease analysis of the PCR amplicons revealed a genetic difference between the two isolates in the VP7 gene. The isolation in Italy of canine rotaviruses displaying the same VP7 and VP4 specificities as in the USA and in Japan, suggests that the G3 and P5A[3] types are highly conserved among canine rotavirus strains.

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 preliminary study on the pathogenicity of a strain of caprine herpesvirus-1.

The strain BA-1 of caprine herpesvirus-1 (CpHV-1), isolated from latently infected goats, was inoculated intranasally into three five-year-old goats. The animals developed fever and leukopenia. The signs began on post-inoculation day (PID) 4 and lasted 7 days. In one goat herpes-like lesions appeared on the vulvar area on PID 7. Virus was consistently recovered from the nasal and the vaginal swabbings obtained from the three goats. Virus was never recovered from the ocular and rectal swabbings nor from any buffy coat samples. However, the buffy coats were positive for viral DNA detected by polymerase chain reaction (PCR). All isolates from the experimental goats were identical in their restriction patterns to the original BA-1 and were similar to the reference E/CH strains of CpHV-1.

The response of pigs inoculated with a thymidine kinase-negative (TK-) pseudorabies virus to challenge infection with virulent virus.

12 Large-White-Landrace piglets were subdivided in four groups of 3 and housed in separate units. The piglets of three groups were inoculated with the 86/27V 6C2 thymidine kinase negative (TK-) mutant of pseudorabies virus (PRV), by different routes. A second inoculation with the same mutant was given to the pigs 21 days later. The animals of a fourth group were left as uninoculated controls. 21 days following the second inoculation with the TK- mutant all pigs were challenge infected with the virulent PRV. On post challenge day (PCD) 30 all pigs were killed and samples for virus detection and histology were taken from several organs. The inoculated TK- mutant of PRV did not induce any ill effects in the pigs except a transient febrile reaction in some animals. Virus was recovered from nasal swabbings from one pig 2 days after the first inoculation of the mutant. After challenge exposure with virulent PRV, the TK- mutant-inoculated pigs were apparently protected, whereas the control pigs all were severely affected and recovered very slowly over 3 weeks. Virus was isolated from the nasal swabbings from the TK- mutant-inoculated pigs on PCDs 2 and 4, whereas the nasal swabbings from the control piglets were all positive for virus from PCD 2 through PCD 10. DNA analysis of the virus recovered showed a pattern identical to that of the virulent PRV. Histologic lesions were found in the respiratory and the central nervous systems, however, the lesions in the TK- mutant-inoculated pigs were much milder compared to those registered for the control pigs. Virus was not isolated from any of the tissue samples that were tested, but viral DNA with sequences typical of PRV genome was detected by PCR in all samples of trigeminal ganglia from either the TK- mutant-inoculated pigs or from the controls.

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.

Natural caprine herpesvirus 1 (CpHV-1) infection in kids.

Twenty-seven kids aged 5-7 days from a flock of 200 goats in which a high rate of abortion occurred died over a 2-month period. All showed hyperthermia, abdominal pain and anorexia. Two of the kids were examined post mortem. Ulcerative and necrotic lesions affected the whole intestine, and macroscopical changes were also observed in the lungs, urinary bladder and liver. Histologically, a severe necrotizing enteritis as well as thickening of the alveolar septa and necrotic bronchiolo-alveolitis were detected. Prominent microscopical lesions were also present in the liver, urinary bladder, spleen, thymus, mesenteric lymph nodes and kidney. Macrophages containing eosinophilic intranuclear inclusion bodies appeared to be the main inflammatory cell in all the organs examined. Ultrastructurally, herpesvirus particles were evident. Characteristic morphological features of type-A capsids (empty), type-C capsids (large core), and type-B capsids were observed. A virus was isolated in cell culture from all organs examined in the two kids. Cytopathogenic effects (rounding cells, syncytia, vacuoles, cell lysis) and acidophilic intranuclear inclusions typical of herpesvirus infection were observed. The virus was identified as caprine herpesvirus 1 (CpHV-1) by the polymerase chain reaction (PCR) technique and by serum neutralization

A study of an experimental infection of sheep with Mycoplasma agalactiae.

The results of an experimental infection of sheep with a field strain of Mycoplasma agalactiae are reported. Six sheep, seronegative to M. agalactiae were used: three sheep were inoculated by the conjunctival route (group A) and three sheep intranasally (group B). The clinical signs were observed 20 days after infection but the shedding of Mycoplasma agalactiae, particularly from the nasal route and with milk, started a few days post infection (d.p.i.) (1st d.p.i. and 9th d.p.i. respectively). Antibody titers were first detected after 28 d.p.i. in group B and after 35 d.p.i. in group A.

Characterization by polymerase chain reaction of ruminant rotaviruses isolated in Italy.

Several group A rotaviruses isolated in Italy from cattle, buffalos and goats were characterized by polymerase chain reaction assay for G- and P-type. G6 and P5 were the types most frequently recovered. The genotypes of buffalo and goat strains were similar to those of cattle isolates.

The polymerase chain reaction for the detection of defective interfering canine parvovirus particles.

The PCR assay was used to amplify a portion of the genome of virulent and vaccinal canine parvovirus strains and of a vaccinal feline panleukopenia virus strain. A DNA fragment corresponding to the gene that encodes the VP1/VP2 proteins was amplified. The size of the PCR products was 2.2 Kbp except for CPV vaccinal 17-80 strain. The PCR product of 17-80 was 1.1 Kbp leading to the hypothesis of the presence of defective particles. All the restriction enzymes digested the 2.2 Kbp amplified products giving restriction fragments of the expected size whereas Hind III, Hpa II and Pvu II did not digest the PCR fragment of 1.1 Kbp.

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.

Isolation and identification of a calicivirus from a dog with diarrhoea.

The isolation and the characterisation of a calicivirus strain (213/95) from a dog with diarrhoea are reported. The virus grows only in cell cultures of feline origin (Crandell Feline Kidney Cells), and in neutralisation test the isolate demonstrated a slight antigenic correlation with the reference feline calicivirus strain (FCV-F9).

Antigenic analysis of canine parvovirus strains isolated in Italy.

Eighty-two canine parvovirus type 2 strains isolated in Italy from pups with severe enteritis were characterizated using four monoclonal antibodies. Sixty-eight isolates resulted CPV-2a, whereas the other fourteen were a CPV-2b variant. The diffusion of CPV-2 variants in the Italian dog population is quite similar to that reported in the United Kingdom and Australia (CPV-2a more prevalent) and different from the epidemiological conditions of the USA and other countries where CPV-2b is more widespread.

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.

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.

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.