Maria Stella Lucente

Atypical Pestivirus and Severe Respiratory Disease in Calves, Europe

In 2010, a HoBi-like pestivirus was isolated from clinically affected calves in Italy. This European virus reproduced a milder form of disease under experimental conditions and was genetically related to previously reported HoBi-like strains. Isolation of this novel virus from a clinical outbreak may have implications for cattle health and prophylactic programs.

Identification of Group A Porcine Rotavirus Strains Bearing a Novel VP4 (P) Genotype in Italian Swine Herds

ABSTRACT The VP4 gene of a G5 Italian porcine rotavirus strain, 344/04-1, was nontypeable by PCR genotyping. The amino acid sequence of the full-length VP4 protein had low identity (≤76.6%) with the homologous sequences of representative strains of the remaining P genotypes, providing evidence for a novel P genotype.

Genetic Heterogeneity and Recombination in Canine Noroviruses

ABSTRACT Alphatronlike (genogroup IV [GIV]) noroviruses (NoVs) have been recently identified in carnivores. By screening a collection of 183 fecal samples collected during 2007 from dogs with enteric signs, the overall NoV prevalence was found to be 2.2% (4/183). A unique strain, Bari/91/07/ITA, resembled GIV.2 NoVs in its ORF1 (polymerase complex), while it was genetically unrelated in its full-length ORF2 (capsid gene) to GIV animal and human NoVs (54.0 to 54.4% amino acid identity) and to any other NoV genogroup (<54.7% amino acid identity). It displayed the highest identity (58.1% amino acid identity) to unclassified human strain Chiba/040502/04/Jp. Interestingly, the very 5′ end of ORF2 of the canine virus matched short noroviral sequences (88.9% nucleotide identity and 98.9% amino acid identity) identified from oysters in Japan, indicating that similar viruses may be common environmental contaminants.

Hobi-Like Pestivirus in Aborted Bovine Fetuses

ABSTRACT An outbreak of abortion affecting multiparous cows was associated with Hobi-like pestivirus infection. Viral RNA and antigens were detected in the tissues of two aborted fetuses. Molecular assays for other common abortogenic agents tested negative. At the genetic level, the Hobi-like pestivirus displayed the closest relatedness to Italian, Australian, and South American viruses, whereas it diverged from the prototype Thai isolate. These findings may have important implications for the pestivirus control/eradication programs in cattle herds.

Serological and molecular evidence that canine respiratory coronavirus is circulating in Italy.

Abstract Canine respiratory coronavirus (CRCoV) is a group II coronavirus that was firstly identified in lung samples of dogs with canine infectious respiratory disease (CIRD) in UK in 2003. We report for the first time the identification of CRCoV in Italy, together with serological evidence that the virus has been circulating in the Italian dog population as from 2005. Serological investigations on 216 dog sera, carried out by an ELISA test using the strictly related bovine coronavirus (BCoV) as antigen, revealed an overall CRCoV seroprevalence of 32.06% in the last 2 years. RT-PCR targeting the S-gene of CRCoV was carried out on 109 lung samples collected from carcasses of dogs submitted for diagnostic investigations. Positive results were obtained from the lungs of a dog of the Apulia region that was co-infected with canine parvovirus type 2. Sequence analysis of the S-gene fragment amplified by RT-PCR (595bp) showed similarity to group II coronaviruses, with the highest nucleotide identity (98%) to the only CRCoV strain currently available in the GenBank database (strain T101). The results of the present study show that CRCoV is present also in continental Europe, although further studies are required to determine the real pathogenic potential of the virus.

Experimental infection of cattle, sheep and pigs with 'Hobi'-like pestivirus.

Abstract To date, limited information is available on the ability of ‘Hobi’-like pestiviruses (putative bovine viral diarrhoea 3) to infect and cause disease in animal species traditionally affected by pestiviruses. In order to obtain new insights into host range and pathogenic potential of this atypical pestivirus, BVDV-seronegative calves (n =5), lambs (n =5) and piglets (n =5) were experimentally infected with the European ‘Hobi’-like strain Italy-1/10-1, whereas two animals per species served as uninfected controls. Appearance of clinical signs, leukopenia, viremia, viral shedding and seroconversion were monitored for 28 days post-infection. Calves and lambs were successfully infected, displaying respiratory signs (nasal discharge), moderate hyperthermia and leukopenia, viremia and viral shedding through the nasal and faecal routes. Antibody responses were observed in both animal species by ELISA and virus neutralisation assays. In contrast, inoculated piglets did not display any clinical signs nor leukopenia and viral RNA was not detected in any biological samples. Nevertheless, the presence of detectable antibodies by virus neutralisation accounted for a successful, albeit limited infection of these animals.

Recombinant Canine Coronaviruses in Dogs, Europe

Subtype IIb originates from recombination with porcine transmissible gastroenteritis virus.

Characterisation of canine parvovirus strains isolated from cats with feline panleukopenia

Abstract Unlike the original canine parvovirus type 2 (CPV-2), CPV-2 variants have gained the ability to replicate in vivo in cats but there is limited information on the disease patterns induced by these variants in the feline host. During 2008, two distinct cases of parvoviral infection were diagnosed in our laboratories. A CPV-2a variant was identified in a 3-month-old Persian kitten displaying clinical sign of feline panleukopenia (FPL) (acute gastroenteritis and marked leukopenia) and oral ulcerations, that died eight days after the onset of the disease. Two pups living in the same pet shop as the cat were found to shed a CPV-2a strain genetically identical to the feline virus and were likely the source of infection. Also, non-fatal infection by a CPV-2c strain occurred in a 2.5-month-old European shorthair kitten displaying non-haemorrhagic diarrhoea and normal white blood cell counts. By sequence analysis of the major capsid protein (VP2) gene, the feline CPV-2c strain showed 100% identity to a recent canine type-2c isolate. Both kittens had been administered multivalent vaccines against common feline pathogens including FPL virus. Whether and to which extent the FPL vaccines can protect cats adequately from the antigenic variants of CPV-2 should be assessed.

Genetic analysis of feline panleukopenia viruses from cats with gastroenteritis.

Thirty-nine parvovirus strains contained in faecal samples collected in Italy (n=34) and UK (n=5) from cats with feline panleukopenia were characterized at the molecular level. All viruses were proven to be true feline panleukopenia virus (FPLV) strains by a minor groove binder probe assay, which is able to discriminate between FPLV and the closely related canine parvovirus type 2. By using sequence analysis of the VP2 gene, it was found that the FPLV strains detected in Italy and UK were highly related to each other, with a nucleotide identity of 99.1-100 and 99.4-99.8% among Italian and British strains, respectively, whereas the similarities between all the sequences analysed were 98.6-100%. Eighty-eight variable positions were detected in the VP2 gene of the field and reference FPLV strains, most of which were singletons. Synonymous substitutions (n=57) predominated over non-synonymous substitutions (n=31), and the ratio between synonymous and non-synonymous substitutions (dN/dS) was 0.10, thus confirming that evolution of FPLV is driven by random genetic drift rather than by positive selection pressure. Some amino acid mutations in the VP2 protein affected sites that are thought to be responsible for antigenic and biological properties of the virus, but no clear patterns of segregation and genetic markers, were identified, confirming that FPLV is in evolutionary stasis.

Duplex real-time polymerase chain reaction for simultaneous detection and quantification of Anaplasma marginale and Anaplasma centrale.

Anaplasma marginale and Anaplasma centrale are rickettsial pathogens responsible for acute disease and mild infections, respectively, in cattle herds. A duplex real-time polymerase chain reaction (PCR) assay with probes labeled with different fluorophores was developed for simultaneous detection and quantification of A. marginale and A. centrale DNA in bovine blood samples. The assay was able to detect as few as 101 and 102 DNA copies for A. marginale and A. centrale, respectively, with optimal specificity and reproducibility. Analysis by real-time and nested PCR carried out on 54 samples previously tested by reverse line blot hybridization showed that the established duplex real-time PCR assay can detect and quantify the 2 Anaplasma spp., even if present simultaneously in the same blood samples. Such an assay could be used in pathogenesis studies on bovine acute anaplasmosis.