Grazia Greco

Norovirus in Captive Lion Cub (Panthera leo)

African lions (Panthera leo) are susceptible to viral diseases of domestic carnivores, including feline calicivirus infection. We report the identification of a novel enteric calicivirus, genetically related to human noroviruses of genogroup IV, in a lion cub that died of severe hemorrhagic enteritis.

Molecular Characterization of the VP4, VP6, VP7 and NSP4 genes of lapine rotaviruses identified in Italy: emergence of a novel VP4 genotype

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].

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.

Development of a nested PCR assay for the detection of canine coronavirus

Abstract 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), trasmissible 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.

Isolation of Salmonella strains from reptile faeces and comparison of different culture media

Aims:  To provide information on epidemiology and isolation of Salmonella strains from reptiles.

Genomic Characterization of Porcine Rotaviruses in Italy

ABSTRACT 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.

Molecular Analysis of the VP7, VP4, VP6, NSP4, and NSP5/6 Genes of a Buffalo Rotavirus Strain: Identification of the Rare P[3] Rhesus Rotavirus-Like VP4 Gene Allele

ABSTRACT 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.

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.

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.