Francesco Cirone

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.

Genetic diversity of a canine coronavirus detected in pups with diarrhoea in Italy

Abstract 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.

Two Genotypes of Canine Coronavirus Simultaneously Detected in the Fecal Samples of Dogs with Diarrhea

ABSTRACT Sixty-nine fecal samples from diarrheic puppies were examined by reverse transcription-PCR assays for the M and the S genes of canine coronaviruses (CCoVs). The isolates in 10 samples were recognized as CCoV type I, and the isolates in 6 samples were recognized as CCoV type II, while isolates of both genotypes were simultaneously detected in 53 samples.

Molecular characterisation of the virulent canine coronavirus CB/05 strain ☆

Abstract This paper characterises a virulent strain (CB/05) of canine coronavirus (CCoV) isolated from the internal organs of pups that had died of a systemic disease without evidence of other common canine pathogens. High viral RNA titres were detected in the internal organs by a real-time RT-PCR assay specific for CCoV type II. Sequence analysis of the 3′ end (8.7kb) of the genomic RNA of strain CB/05 revealed conserved structural as well as non-structural proteins, with the exception of a truncated form of non-structural protein 3b. The exceptional form was due to a 38-nucleotide deletion and a frame shift in ORF3b that introduced an early stop codon. By phylogenetic analysis of the structural proteins, the spike (S) protein was found to cluster with feline coronavirus type II strain 79-1683, whereas, the envelope (E), membrane (M) and nucleocapsid (N) proteins segregated together with the reference strain Purdue of transmissible gastroenteritis virus of swine.

Prevalence of canine coronavirus antibodies by an enzyme-linked immunosorbent assay in dogs in the south of Italy

Abstract 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.

Antigenic characterization of canine parvovirus strains isolated in Italy

28 isolates of canine parvovirus type-2 (CPV-2) were obtained from dogs with hemorrhagic gastroenteritis in Italy. The antigenic structure of CPV-2 isolates was characterized, using four discriminating monoclonal antibodies. In addition, four vaccinal strains were examined. Similar to reports from Australia and the United Kingdom, a much higher prevalence of CPV-2a (25/28 isolates) was observed than the other variant type, CPV-2b (3/28 isolates). DNA fragments (2.2 kbp) of representative strains of CPV-2, CPV-2a and CPV-2b were amplified by the polymerase chain reaction (PCR) and the products were digested by the restriction enzymes (RE) RsaI, HpaII, HindIII and PvuII. The RvaI enzyme allows the differentiation of CPV-2 from CPV-2a and CPV-2b.

Detection of bovine coronavirus using a TaqMan-based real-time RT-PCR assay

Abstract A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of bovine coronavirus (BCoV) RNA in clinical samples is described. The assay is based on TaqMan technology, consisting of two primers and one probe labeled with the reporter dye 6-carboxyfluorescein that binds selectively to the transmembrane-protein gene of BCoV. The BCoV real-time RT-PCR assay was able to detect the tested BCoV and BCoV-like viruses (canine respiratory coronavirus and bubaline coronavirus), whereas other common viral pathogens of cattle were not recognised by the established oligonucleotide set, thus showing that the test was specific for bovine-like CoVs. The detection limit of the assay was 20 BCoV RNA copies (1-log higher with respect to traditional gel-based RT-PCR) and the reproducibility was satisfactory, thus allowing for a sensitive and accurate measurement of the viral RNA load in clinical samples. Two hundred and twenty clinical specimens (92 rectal, 82 nasal and 46 ocular swabs) were subjected to gel-based and real-time RT-PCR. By conventional amplification, 43 rectal, 54 nasal and 34 ocular samples tested positive, whereas the TaqMan assay was able to detect the BCoV nucleic acid in 49 rectal, 60 nasal and 37 ocular swabs. The rapidity and high throughput of the BCoV TaqMan assay makes this method a powerful tool for a sensitive and specific diagnosis of BCoV infection in cattle.

Respiratory Disease Associated with Bovine Coronavirus Infection in Cattle Herds in Southern Italy

Four outbreaks of bovine respiratory disease (BRD) associated with bovine Coronavirus (BCoV) infection in Italian cattle herds were reported. In 3 outbreaks, BRD was observed only in 2–3-month-old feedlot calves, whereas in the remaining outbreak, lactating cows, heifers, and calves were simultaneously affected. By using reverse transcription polymerase chain reaction (RT-PCR), BCoV RNA was detected in all outbreaks without evidence of concurrent viral pathogens (i.e., bovine respiratory syncytial virus, bovine herpesvirus type 1, bovine viral diarrhea virus, bovine parainfluenza virus). Common bacteria of cattle were recovered only from 2 outbreaks of BRD: Staphylococcus spp. and Proteus mirabilis (outbreak 1) and Mannheimia haemolytica (outbreak 4). A recently established real-time RT-PCR assay showed that viral RNA loads in nasal secretions ranged between 3.10 × 10 2 and 7.50 × 10 7 RNA copies/μl of template. Bovine Coronavirus was isolated from respiratory specimens from all outbreaks except outbreak 1, in which real-time RT-PCR found very low viral titers in nasal swabs.

Severe outbreak of bovine coronavirus infection in dairy cattle during the warmer season.

Abstract A severe outbreak of enteric and respiratory disease associated with bovine coronavirus (BCoV) infection is described. The outbreak occurred in a dairy herd of southern Italy in the first decade of September 2006, when summer temperatures were still recorded, affecting calves, heifers and adult cows, with a marked decrease in milk production. By virus isolation and RT-PCR targeting the S gene, BCoV was identified as the etiological agent of the outbreak, whereas bacteriological, parasitological and toxicological investigations failed to detect other causes of disease. BCoV strains with 99–100% nucleotide identity in the S gene were isolated from nasal, ocular and rectal swabs, thus proving the absence of separate clusters of virus on the basis of tissue tropism. Sequence analysis of the haemagglutination-esterase and spike proteins of the strain detected in one rectal sample (339/06) showed a high genetic relatedness with recent BCoV isolates (98–99% amino acid identity), with several unique amino acid substitutions in the S protein. The BCoV outbreak described in this paper presents interesting aspects: (i) the occurrence of a severe form of disease in the warmer season; (ii) the simultaneous presence of respiratory and enteric disease; (iii) the involvement of young as well as adult cattle.

Lights and shades on an historical vaccine canine distemper virus, the Rockborn strain

Both egg- and cell-adapted canine distemper virus (CDV) vaccines are suspected to retain residual virulence, especially if administered to immuno-suppressed animals, very young pups or to highly susceptible animal species. In the early 1980s, post-vaccine encephalitis was reported in dogs from various parts of Britain after administration of a particular batch of combined CDV Rockborn strain/canine adenovirus type-1 vaccine, although incrimination of the Rockborn strain was subsequently retracted. Notwithstanding, this, and other reports, led to the view that the Rockborn strain is less attenuated and less safe than other CDV vaccines, and the Rockborn strain was officially withdrawn from the markets in the mid 1990s. By sequencing the H gene of the strain Rockborn from the 46th laboratory passage, and a commercial vaccine (Candur(®) SH+P, Hoechst Rousell Vet GmbH), the virus was found to differ from the commonly used vaccine strain, Onderstepoort (93.0% nt and 91.7% aa), and to resemble more closely (99.6% nt and 99.3% aa) a CDV strain detected in China from a Lesser Panda (Ailurus fulgens). An additional four CDV strains matching (>99% nt identity) the Rockborn virus were identified in the sequence databases. Also, Rockborn-like strains were identified in two vaccines currently in the market. These findings indicate that Rockborn-like viruses may be recovered from dogs or other carnivores with distemper, suggesting cases of residual virulence of vaccines, or circulation of vaccine-derived Rockborn-like viruses in the field.