Skin lesion-associated pathogens from Octopus vulgaris: first detection of Photobacterium swingsii, Lactococcus garvieae and betanodavirus.

The common octopus Octopus vulgaris Cuvier, 1798 is extremely important in fisheries and is a useful protein source in most Mediterranean countries. Here we investigated pathogens associated with skin lesions in 9 naturally deceased specimens that included both cultured and wild common octopus. Within 30 min after death, each octopus was stored at 4°C and microbiologically examined within 24 h. Bacterial colonies, cultured from swabs taken from the lesions, were examined using taxonomical and biochemical analyses. Vibrio alginolyticus and V. parahaemolyticus were only isolated from cultured animals. A conventional PCR targeting the 16S ribosomal RNA (rRNA) gene and sequencing were performed on 2 bacterial isolates that remained unidentified after taxonomical and biochemical analysis. The sequence results indicated that the bacteria had a 99% identity with Lactococcus garvieae and Photobacterium swingsii. L. garvieae was confirmed using a specific PCR based on the 16S-23S rRNA internal transcribed spacer region, while P. swingsii was confirmed by phylogenetic analyses. Although all animals examined were found to be infected by the protozoan species Aggregata octopiana localised in the intestines, it was also present in skin lesions of 2 of the animals. Betanodavirus was detected in both cultured and wild individuals by cell culture, PCR and electron microscopy. These findings are the first report of L. garvieae and betanodavirus from skin lesions of common octopus and the first identification of P. swingsii both in octopus skin lesions and in marine invertebrates in Italy.

Detection of Cyprinid herpesvirus 2 in association with an Aeromonas sobria infection of Carassius carassius (L.), in Italy.

Sixteen specimens of female crucian carp, Carassius carassius (L.), during the breeding season, were investigated for post-mortem and full diagnostic examination during a mortality outbreak in a tributary stream of the Arno River in Tuscany in 2011. Necropsy highlighted the presence of a swollen anus and widespread haemorrhages in the body, fins, gills and eyes. Haemorrhages in internal organs and spleen granulomas were also observed. Bacteria isolated from the brain, kidney and spleen of affected fish were identified as A. sobria. Microscopic lesions observed in gills were characterized by necrosis of the secondary lamellae, congestion and multifocal lamellar fusion. The kidney showed necrosis, oedema, fibrin exudation and areas of haemorrhages, while in the spleen the main lesions were by multifocal necrosis of the lymphoid tissue. In the gills, transmission electron microscopy revealed herpesvirus-like particles, subsequently identified as Cyprinid herpesvirus-2 (CyHV-2) with a nested PCR protocol. Although it was not possible to attribute a pathogenic role to CyHV-2 in this mortality event, the identification of this herpesvirus in crucian carp increases the concern about its potential role in this species.

Genetic characterization of equine influenza viruses isolated in Italy between 1999 and 2005.

During local respiratory disease outbreaks, occurring in 2003 and 2004 in horse training stables within race-tracks in Rome, and on a stud horse farm in Bari in 2005, four strains of equine influenza (EI) virus were isolated. All outbreaks occurred in flu-vaccinated horses. Here, we are reporting the results of the genetic characterization of these isolates, together with that of another EI virus strain isolated in 1999 from a dead foal presenting pulmonary lesions. Alignment and phylogenetic analyses were carried out using the haemagglutinin amino acid sequences. The Rome and Bari isolates were identified as members of the American lineage, closely related to other recent strains isolated in America as well as in Europe, including the latest recommended American lineage vaccine prototype A/eq/SouthAfrica/4/2003. In contrast, the Italian 1999 isolate was clustered within the European lineage. In Italy, the most recent outbreaks of EI have been caused by the currently circulating American-like strains, even in vaccinated populations, confirming that vaccines should contain an updated representative strain of this lineage. Presently, companies are still in the process of registering updated vaccines but no product is yet available on the market.

Felis catus papillomavirus type-2 but not type-1 is detectable and transcriptionally active in the blood of healthy cats.

Papillomaviruses (PVs) are small DNA viruses that induce benign and/or malignant epithelial tumours in different species, including the domestic cat (Felis catus). To date, five F. catus papillomavirus genotypes have been identified (FcaPV-1 to FcaPV-5). FcaPV-1 is associated with skin and oral benign lesions, while FcaPV-2 infection is widely associated with feline squamous cell carcinomas. Several human and animal PVs have been found in body fluids such as peripheral blood; however, the presence of FcaPVs in non-epithelial tissues has not previously been investigated. The aim of this study was to assess the presence and gene expression of FcaPV-1 and FcaPV-2 in the blood of healthy cats. We detected FcaPV-2 DNA in 26 of 103 (25%) blood samples. Importantly, FcaPV-2 L1, E2, E6 and E7 genes were found to be expressed in 3 (25%), 11 (92%), 6 (50%) and 5 (42%) of the samples available for mRNA analysis, respectively. FcaPV-1 was not detected in any of the blood samples analysed here. The data obtained in this work suggest active and eventually productive infection of FcaPV-2 in the blood of healthy cats, implying a possible role in intra-individual spreading as well as in vertical and horizontal transmission.

A real time PCR assay for the detection and quantification of orf virus.

A real time quantitative PCR assay based on TaqMan technology was developed for orf virus (ORFV) DNA quantification in clinical samples, infected cells and organotypic cultures. This method was based on the amplification of a 70 bp fragment from the ORFV B2L gene (orthologue of the Vaccinia virus Copenhagen F13L gene) that encodes the major envelope protein. Both intra- and inter-assay variability were well within +/-0.25 log(10) S.D. showing the high efficiency and reproducibility of the assay. The TaqMan PCR was subsequently used to determine the titre of several batches of the ORFV strain NZ-2, with it being possible to quantify virus solutions in the range of 1 x 10(1) to 1 x 10(6) TCID(50)/ml. A good correlation between the titre determined by the TaqMan PCR and by conventional endpoint dilution was found. The PCR assay is reproducible and can be used for a rapid quantification of ORFV in vitro and ex vivo, being readily achievable within 1h.

Bacterial and viral pathogens detected in sea turtles stranded along the coast of Tuscany, Italy.

During 2014, six loggerhead turtles, Caretta caretta and one green turtle, Chelonia mydas, found stranded on the Tuscany coast of Italy, were examined for the presence of specific bacterial and viral agents, along with their role as carriers of fish and human pathogens. Thirteen different species of bacteria, 10 Gram negative and 3 Gram positive, were identified. Among them, two strains of Vibrio parahaemolyticus and one strain of Lactococcus garviae were recovered and confirmed by specific PCR protocols. No trh and tdh genes were detected in V. parahaemolyticus. The first isolation of L. garviae and the first detection of Betanodavirus in sea turtles indicate the possibility for sea turtles to act as carriers of fish pathogens. Furthermore, the isolation of two strains of V. parahaemolyticus highlights the possible role of these animals in human pathogens' diffusion.

Active circulation of bluetongue vaccine virus serotype-2 among unvaccinated cattle in central Italy.

Several seroconversions occurring in 2002 among sentinel cattle during the bluetongue-vaccination campaign in Lazio and Tuscany (central Italy) led to the suspicion of vaccine-virus circulation. Therefore in 2003, 17 seroconverting sentinel herds were investigated for the characteristics of the virus involved. From these farms, 91 unvaccinated animals and 57 Culicoides pools were tested for the presence of the bluetongue vaccine virus (serotype-2) or other strains. The presence of vaccine virus serotype-2 was confirmed by PCR followed by restriction analysis in the whole blood of 17 unvaccinated sentinel cattle and 12 pools of Culicoides imicola or C. obsoletus. Of the 17 herds, five were positive only for vaccine virus serotype-2, four were positive for other strains and two for both the vaccine and other strains; the remaining premises were virologicaly negative. The vaccine virus serotype-2 also was detected in areas not included in the vaccination campaign.

Identification of Culicoides obsoletus (Diptera: Ceratopogonidae) as a vector of bluetongue virus in central Italy.

In 2001 and 2002, 235 outbreaks of bluetongue were observed in the Lazio and Tuscany regions of central Italy. During entomological surveillance Culicoides imicola, the main vector of bluetongue virus in the Mediterranean region, was detected in only 14 of 28 municipalities affected by outbreaks; Culicoides obsoletus was the most abundant species, contributing 83 per cent of individuals in catches, whereas C. imicola contributed only 2 per cent. In affected municipalities the maximum catch of C. obsoletus was 18,000 specimens, compared with 54 of C. imicola. In October 2002 bluetongue virus serotype 2 was isolated from a single pool of wild-caught, non-blood-engorged parous C. obsoletus inoculated on to BHK-21 cells. Its identity was confirmed by reverse transcriptase-PCR.

Isolation of encephalomyocarditis virus from dormice (Myoxus glis) in Italy.

Two isolates of encephalomyocarditis (EMC) virus (ZRC 276RA/90 and ZRC 292RA/90) were isolated from two dormice (Myoxus glis) in Tuscany, Italy. The two isolates were lethal for laboratory mice and caused a rapid cytopathic effect characterized by rounded and wrinkled cells in both baby hamster kidney cells (BHK21) and African green monkey kidney cells (Vero). We found neutralizing antibodies against EMC virus in 408 (77%) of 529 domestic pigs (Sus scrofa scrofa) and in 165 (49%) of 338 wild boars (S. scrofa ferus majori) in Tuscany.

Post mortem investigations on cetaceans found stranded on the coasts of Italy between 1990 and 1993.

Detailed pathological and virological examinations were carried out on 25 cetaceans found stranded between 1990 and 1993 on the coasts of six Italian regions (Latium, Tuscany, Apulia, Abruzzo, Veneto and Sicily). There were 16 striped dolphins (Stenella coeruleoalba), three bottlenosed dolphins (Tursiops truncatus), three Risso's dolphins (Grampus griseus), one rough-toothed dolphin (Steno bredanensis), one fin whale pup (Balaenoptera physalus), and one minke whale (Balaenoptera acutorostrata). Apart from parasitic diseases (44 per cent), the most frequently detected lesions were pneumonia (68 per cent), enteritis (44 per cent), non-purulent hepatitis (40 per cent), interstitial nephritis (32 per cent) and encephalitis (32 per cent). Morbilivirus infection was diagnosed by immunocytochemistry in four striped dolphins, two stranded on the coasts of Latium in 1991 and two on the coasts of Tuscany in 1993. Despite the presence of lesions consistent with morbilliviral pneumonia in two other striped dolphins stranded on the coast of Apulia in 1991, no morbillivirus antigen was demonstrated in the tissues of these animals. Anticanine distemper virus antibodies were detected in the serum of the adult minke whale found stranded on the coast of Tuscany in 1993. However, no viruses were isolated from the tissues of any of the 25 cetaceans.

Genetic detection and characterization of atypical bovine pestiviruses in foetal bovine sera claimed to be of Australian origin.

Two European laboratories independently detected atypical bovine pestiviral nucleic acids in three commercial batches of foetal bovine serum (FBS) that was claimed by the producers to be of Australian origin. Additional batches of FBS were obtained directly from Australia to exclude possible contamination of the Australian FBS with that of South American origin during manufacturing/packaging in European countries. RT-PCR amplification of partial 5'untranslated region and the complete N(pro) gene yielded a specific band with expected size, which was confirmed by DNA sequencing. Bayesian analysis of sequence data demonstrated a closer phylogenetic relation of the newly detected atypical bovine pestiviruses to those of South American origin, which were related to the recognized bovine pestivirus species, bovine viral diarrhoea virus. Taken together, the results indicated the presence of atypical bovine pestiviruses in the Australian FBS, and most likely in Australian Continent.

A serologic survey of encephalomyocarditis virus infection in pigs in Italy.

Neutralizing antibodies to encephalomyocarditis virus (EMCV) were found in 69.13% of the serum samples obtained from pigs from representative regions of Italy. The antibodies are distributed fairly uniformly throughout the swine populations with all age groups being equally involved.

Protection tests in pigs vaccinated with the lapinized Chinese strain of hog cholera virus (HCV) previously adapted in a minipig kidney (MPK) cell line, to challenge infection with virulent HCV.

Pigs which had been vaccinated with the Lapinized Chinese strain of Hog Cholera Virus previously adapted in a minipig cell line cultures (MPK-LC-HCV), resultet to be protected when they were subjected to challenge infection with virulent Hog Cholera Virus (HCV) 6 or 11 months later. The challenge virus was never isolated from any of the vaccinated pigs. The MPK-LC-HCV vaccine induced a significant rise of the antibody titer to the HCV in pigs kept under field conditions.

The response of pregnant gilts previously given an inactivated preparation of porcine parvovirus (PPV) to challenge infection with a fully virulent PPV.

Eight 40-day pregnant gilts, previously treated with an adjuvanted-inactivated viral preparation (AIVP) obtained with a field strain of porcine parvovirus (PPV) together with 4 pregnant untreated controls, were subjected to challenge infection with a virulent strain of PPV at the 40th day of gestation. After challenge, all controls became febrile for 2 to 8 days, whereas only one gilt among those which had been treated with the AIVP experienced fever which lasted 4 days. Virus was consistently recovered from fecal swabs obtained from the controls and only sporadically from feces of AIVP-treated gilts. When the gilts were killed 53 days after challenge infection, no macroscopic lesions were found in any of the gilts in either groups, but fetal death was observed in the two groups of animals. However, the rate of dead fetuses was much higher among the control (70.5%) than among those from the AIVP-treated gilts (10.1%). Virus was recovered from 23 of the 24 dead fetuses in the control group and only from 3 among the 8 dead fetuses which were reported for the AIVP-treated gilts.