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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.

A modified (aFT9m) and a degenerate (aFT9d) version of the rotavirus G9-specific primer (aFT9) allowed strains that were previously untypable, because of point mutations accumulating at the primer binding site, to be G typed by reverse transcription-polymerase chain reaction. The strains were collected during 2001-2002 in Italy in hospitals of the Apulia region, from children affected by severe rotavirus-associated enteritis. Using a wide selection of G9 rotaviruses detected worldwide, sequencing of the G9 untypable strains, sequence comparison, and phylogenetic analysis showed that the Italian strains have strong genetic similarity (< or =99.4%) to G9 rotaviruses identified recently in many parts of the world and different from the old G9 strains identified during the 1980s (less than 90%). Genetic variation of G9 rotaviruses explains the constraints encountered in the typing assays and presumably accounts, together with genetic reassortment events, for the emergence on a global scale of the G9 serotype., (Copyright 2004 Wiley-Liss, Inc.)

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.

Fecal secretory immunoglobulin A (IgA) antibodies in dogs infected or vaccinated with canine coronavirus (CCV) were evaluated by an enzyme-linked immunosorbent assay. The study was carried out with 32 fecal samples collected just before inoculation and at 28 days postinoculation. Five groups were studied: naturally infected dogs, experimentally infected dogs, dogs inoculated with a modified live (ML) CCV vaccine by the intramuscular route, dogs inoculated with an ML CCV vaccine by the oronasal route, and dogs given an inactivated CCV vaccine. Both the naturally and the experimentally infected dogs developed high levels of fecal IgAs. Interestingly, dogs inoculated with the ML CCV vaccine by the oronasal route developed levels of fecal IgA that were higher than those observed in the dogs inoculated with the same CCV vaccine by the intramuscular route or those observed in dogs inoculated with the inactivated vaccine. A relationship between the level of fecal IgAs to CCV and the degree of protection against CCV infection was observed.

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.

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].

Group A human rotavirus strains belonging to the unusual serotype G9 were detected at high frequency in stool specimens from infected children with acute diarrhea in Bari, Italy, during a 15-month survey from March 2001 to June 2002. This may signify a local reemergence of the G9 rotaviruses detected in Italy in the early and mid-1990s or may be related to the global emergence of G9 rotaviruses in recent years.

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 membrane (M) protein of canine coronavirus (CCoV) was cloned and expressed in E. coli. The purified recombinant protein was then evaluated for its antigenicity and reliability in an enzyme-linked immunosorbent assay (ELISA) for detection of CCoV antibodies in dog sera. Fifty serum samples, screened previously by whole virus ELISA and Western blotting, were tested. When the performance of the new test was compared with those of whole virus ELISA and Western blotting, an excellent correlation was found with the latter two assays. The ELISA based on recombinant M protein represents an alternative and valid test for detection of antibodies to CCoV in dog sera.

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.

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.

An enzyme-linked immunosorbent assay (Elisa), using as antigen canine coronavirus-infected CrFK cell supernatant, was developed to detect antibodies against canine coronavirus (CCoV). Out of a total of 109 dog serum samples, 80 which were positive by routine virus neutralisation test were also Elisa positive. Seventeen samples which were negative by the virus neutralisation test, were positive by Elisa and by the confirmatory Western blotting test. The Elisa was substantially more sensitive than the virus neutralisation test in detecting antibodies to CCoV and may be used as an alternative technique to virus neutralisation.

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.)

Eight goats were inoculated with an inactivated caprine herpesvirus 1 (CpHV-1) vaccine. Four unvaccinated goats were kept as controls. After 30 days, four of the eight goats were revaccinated. Fifty days after the first vaccination the animals were divided into two groups (A and B) of six goats each (two goats vaccinated twice, two goats vaccinated once and two goats unvaccinated). The two groups were challenged with CpHV-1 intranasally (group A) or intravaginally (group B). After CpHV-1 challenge, the four control goats showed the typical genital lesions, whereas all the vaccinated goats were fully protected against clinical signs. Protection against infection depended on the number of injections of vaccine and the route of challenge.

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.

An outbreak of dual infection in dogs with canine adenovirus type 1 (CAV-1) and canine coronavirus (CCV) infection is reported in an animal shelter that comprised approximately 200 adults stray dogs and 30 puppies. Twenty puppies died 7-8 days after the onset of the clinical signs (severe enteritis, leucopoenia, respiratory distress and dehydration). Both CAV-1 and CCV were isolated from tissue or swab samples. Antibodies to CCV and, at high levels, to CAV-1 also were detected in several puppies. The principal histological findings were atrophy of small intestinal villi, lymphoid depletion, hepatitis and bronchopneumonia. The persistence of CCV in the faeces, observed by the polymerase chain reaction assay, was longer than previously reported. Results demonstrated the serious consequences which may occur with dual infections by CAV-1 and CCV in assembled groups of dogs that are housed in poorly managed kennels with inadequate vaccination programmes.

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.

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.

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.

A multiplex polymerase chain reaction (m-PCR) assay was optimized for the simultaneous detection of several species of small ruminant mycoplasmas. Two sets of oligonucleotide primers specific for Mycoplasma agalactiae (Ma) and Mycoplasma "mycoides" cluster (M.m. cluster) were used in the test. The m-PCR was able to amplify a 375-bp fragment of Ma chromosomal DNA and a 257-260-bp fragment of M.m. cluster chromosomal DNA. Four reference strains (M. agalactiae, M. mycoides subsp. mycoides LC, M. capricolum subsp. capricolum, M. mycoides subsp. capri) and 56 samples (44 milk samples, two nasal swabs, six ocular swabs, three vaginal swabs and one sample of fibrinous exudate from carpal joint), from sheep and goats with clinical signs of contagious agalactia (CA), were examined. The m-PCR confirmed the identification of reference strains and allowed to identify, of the 43 positive samples examined, 35 Ma strains, 12 M. "mycoides" cluster strains; in four samples both Ma and mycoplasmas of M. "mycoides" cluster were revealed. The m-PCR was able to detect 1 pg of mycoplasma DNA. The specificity and sensitivity of the m-PCR suggest its use for the "routine" diagnosis of CA., (Copyright 2001 Academic Press.)

A total of 23 rotavirus strains isolated from pigs were analyzed. Twenty strains had been isolated from diarrheic piglets from an outbreak that occurred in northern Italy in 1983. Three strains had been isolated in 1984 from swine herds located in distinct areas of northern Italy. All 23 strains were characterized as type G6P[5] by PCR. The isolation from piglets of rotaviruses displaying typical bovine G- and P-type specificities points out the high frequency of rotavirus transmission between cattle and pigs.

The results of vaccination trials carried out on pups with maternally derived antibodies (MDA) to canine parvovirus (CPV), using a modified-live CPV-2b variant vaccine (29-97/40 strain), are reported. The vaccine was able to overcome the obstacle of MDA, and to elicit protective immunity in 100% of the pups whose antibody titres were 1:10-1:40, 83% of the pups with titres of 1:80, 57% of the pups with titres of 1:160, and even in 60% of the pups with antibody titres of 1:320.

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.

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.

Pestivirus infection was detected in several flocks of sheep and goats located in the south of Italy by means of serological, virological and histopathological investigations. From four animals, two lambs and two kids, showing enteric symptoms which died during the first week of life, four pestivirus strains were isolated and typed as BVDV-like (three isolates) and "tipic" BDV strains (one isolate). The histopathological lesions consisted of areas of hypomyelination in the brain, and a slight depletion of thymic medullary lymphocytes associated with an increase in reticular cells.

Six group A rotaviruses were isolated in Italy from buffaloes during different outbreaks of calf diarrhoea occurring in the same dairy herd over a 5-year period of observation. The isolates were characterized by polymerase chain reaction assay for G- and P-type antigens. G8 and P1 were the types most frequently isolated.

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.

A study of the latency of caprine herpesvirus 1 (CpHV.1) was carried out with four latently infected goats. Three goats were treated with dexamethasone and euthanized after 4 and 6 days. PCR and virus isolation allowed us to detect CpHV.1 only in the third and fourth sacral ganglia of the two animals euthanized 6 days after the start of treatment.

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.