Outbreak of canine norovirus infection in young dogs.

An outbreak of norovirus (NoV) infection was identified in a kennel. Sequence analysis of a short fragment in the polymerase complex indicated the clonal origin of the strains, which were similar to the prototype canine NoV strain GIV.2/Bari/170/07-4/ITA (94.7% nucleotide identity). The findings demonstrate that canine NoV circulates in dogs in Greece and that it can spread easily across a group of animals.

Genetic heterogeneity and recombination in canine noroviruses.

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.

Detection and molecular characterization of a canine norovirus.

We identified a novel calicivirus in a pup with enteritis. The isolate was related genetically (90.1% aa identity in the capsid protein) to a lion norovirus strain.

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

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.

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 10(1) and 10(2) 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.