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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.

The genes encoding the glycoprotein VP7, the VP8* trypsin-cleavage product of the protein VP4, a fragment of the protein VP6 associated with subgroup (SG) specificity, and the enterotoxin NSP4 of rotavirus strains identified in diarrheic fecal samples of rabbits in Italy were sequenced. The Italian lapine rotavirus (LRV) strains possessed a G3 VP7, SG I VP6, and KUN-like NSP4, a gene constellation typical of LRVs. One LRV strain (30/96), isolated in 1996, shared the closest amino acid (aa) identity (87-96%) with the P[14] genotype, composed of human and LRV strains. Conversely, three LRV strains (160/01, 229/01, and 308/01), identified in 2001, were highly identical (90-95%) among each other, but showed low aa identity (34-77%) to the VP8* genotype-specific sequences of representative rotavirus strains of all remaining P genotypes. This report confirms the worldwide genetic constellations of LRVs and identifies a novel VP4 genotype in rabbits, tentatively proposed as genotype P[22].

The sequence of the S gene of a field canine coronavirus (CCoV), strain Elmo/02, revealed low nucleotide (61%) and amino acid (54%) identity to reference CCoV strains. The highest correlation (77% nt and 81.7% aa) was found with feline coronavirus type I. A PCR assay for the S gene of strain Elmo/02 detected analogous CCoVs of different geographic origin, all which exhibited at least 92-96% nucleotide identity to each other and to strain Elmo/02. The evident genetic divergence between the reference CCoV strains and the newly identified Elmo/02-like CCoVs strongly suggests that a novel genotype of CCoV is widespread in the dog population.

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.

The genetic diversity of 16 canine coronavirus (CCoV) samples is described. Samples were obtained from pups infected naturally living in different areas. Sequence data were obtained from the M gene and pol1a and pol1b regions. The phylogenetic relationships among these sequences and sequences published previously were determined. The canine samples segregated in two separate clusters. Samples of the first cluster were intermingled with reference strains of CCoV genotype and therefore could be assigned to this genotype. The second cluster segregated separately from CCoV and feline coronavirus genotypes and therefore these samples may represent genetic outliers. The reliability of the classification results was confirmed by repeating the phylogenetic analysis with nucleotide and amino acid sequences from multiple genomic regions.

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.

Comparative sequence analysis of the PCR products of the M gene and fragments of the pol1a and pol1b genes of canine coronavirus (CCoV) have demonstrated that two separate clusters of CCoV are present in dogs. This note describes a PCR assay to identify atypical CCoV strains with nucleotide substitutions in the M gene. A total of 177 faecal samples from dogs CCoV positive previously with the PCR assay were analysed. Sixty-two of the 177 samples were amplified with the PCR described in the present study and were thus considered atypical CCoVs. The specificity of the PCR typing assay was confirmed by sequence analysis of the PCR products., (Copyright 2002 Elsevier Science B.V.)

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.

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.

The authors report the sequence analysis of the capsid protein-encoding gene (ORF2) of a calicivirus strain recently isolated in Italy from a pup with enteritis. Sequence comparison and phylogenetic analysis revealed that the isolate is highly similar to field and reference feline caliciviruses (FCVs). The isolation of a feline-like calicivirus and the results of recent serological investigations suggest that FCV infection frequently occurs in dogs, but the consequences of this interspecies infection in the canine host are still unknown.

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.

Two isolates of canine parvovirus (CPV) were obtained from dogs affected with severe haemorrhagic diarrhoea. Type 2b antigenic specificity was predicted by both antigenic analysis with monoclonal antibodies and PCR characterization with type-specific primers. Nevertheless, sequence analysis of the capsid protein-encoding gene revealed two amino acid changes. One of the changes affected position 426 (Asp to Glu), in a major antigenic site of the viral capsid, determining the replacement of a residue unique to CPV type 2b. The failure of established typing methods to distinguish this antigenic variant was overcome by the development of an RFLP assay.

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., (Copyright 2001 Academic Press.)

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.

A CPV-2b strain was detected from an adult vaccinated dog, affected with severe gastroenteritis. The faeces of the dog were positive to canine parvovirus by a hemagglutination assay and gave a CPV-2b-like pattern by a hemagglutination inhibition test using monoclonal antibodies. In vitro-cultivation of the virus was difficult and after a few passages on canine and feline cells, the presence of the virus was detectable only by an immunofluorescence assay on the feline cells, since hemagglutinating activity had disappeared. Characterization of the virus, by an indirect immunofluorescence assay with monoclonal antibodies, confirmed the antigenic CPV-2b-like pattern of the nonhemagglutinating virus.

Canine parvovirus type 2 (CPV2) emerged in 1978 as causative agent of a new disease of dogs. New antigenic variants (biotypes), designated CPV2a and CPV2b, became widespread during 1979 to 1980 and 1984, respectively. At the present time the original CPV2 has disappeared in the dog population and has been replaced by the two new viruses. In the present study the comparison of neutralizing antibody titers in two groups of pups (18 pups in each group) inoculated with CPV2 and CPV2b modified live virus vaccines is reported. Using the hemagglutination inhibition (HI) test, relevant differences between antibody titers, against either the homologous or the heterologous virus, were not constantly observed. Using the neutralization (Nt) test, however, the pups inoculated with CPV2 had antibody titers which were approximately 30 times higher to the homologous virus (mean, 4,732) than to the heterologous virus (CPV2b) (mean, 162). The results of these experiments support two conclusions: (i) the HI test may not always accurately evaluate the true immune status of dogs with respect to CPV, and (ii) dogs inoculated with CPV2 vaccine develop relatively low Nt antibody titers against the heterologous virus (CPV2b). These data may suggest an advantage for new vaccines, considering that most presently licensed vaccines are produced with CPV2, which no longer exists in the dog population.