Search

Organic acids (OA) and nature-identical compounds (NIC) such as monoterpenes and aldehydes are well-known growth and health promoters in terrestrial livestock while their application for fish production is recent and their mechanisms of action require further study. Hence, this study tested the increasing dietary level (D0, D250, D500, D1000; 0, 250, 500 and 1000 mg kg feed−1 respectively) of a microencapsulated blend containing citric and sorbic acid, thymol and vanillin over 82 days on rainbow trout to assess the effects on growth, feed utilization, intestine cytokine gene expression and gut microbiota (GM). Furthermore, the effects on intestinal cytokine gene expression and GM were also explored after one week at high water temperature (23 °C). OA and NIC improved specific growth rate (SGR) and feed conversion rate (FCR) during the second half (day 40–82) of the feeding trial, while at the end of the trial protein (PER) and lipid efficiency (LER) increased with increasing dietary level. GM diversity and composition and cytokine gene expression analysis showed no significant differences in fish fed with increasing doses of OA and NIC (82 days) demonstrating the absence of inflammatory activity in the intestinal mucosa. Although there were no statistical differences, GM structure showed a tendency in clustering D0 group separately from the other dietary groups and a trend towards reduction of Streptococcus spp. was observed in the D250 and D1000 groups. After exposure to high water temperature, lower GM diversity and increased gene expression of inflammatory intestinal cytokines were observed for both inclusions (D0 vs. D1000) compared to groups in standard condition. However, the gene up-regulation involved a limited number of cytokines showing the absence of a substantial inflammation process able to compromise the functional activity of the intestine. Despite further study should be conducted to fully clarify this mechanism, cytokines up-regulation seems to be concomitant to the reduction of the GM diversity and, particularly, to the reduction of specific lactic acid bacteria such as Leuconostoc. The application of the microencapsulate blend tested can be a useful strategy to improve growth and feed utilization in rainbow trout under normal temperature conditions. According to the results organic acids and nature-identical compounds did not revert the effects triggered by the increased temperature of water.

Aims: This study aims to investigate the presence and spatial-seasonal variability of human and fish viruses in coastal marine systems using Ravenna’s harbour area (Adriatic Sea, Italy) as a model. Methods and Results: Human viruses (noroviruses and hepatitis A virus) and one of the most threatening finfish pathogens, the nervous necrosis virus (NNV), were investigated in mussels living inside and offshore Ravenna’s harbour. Thirty-three and 36·7% of tested mussel samples resulted contaminated by human and fish viruses respectively. A different spatial-seasonal distribution was observed. Human viruses were detected mainly in inner port sites during colder months, while NNV was detected in both inside and offshore of Ravenna’s harbour, mainly during warmer months. Conclusions: The presence of human viruses in the inner port close to the city centre could be attributed to wastewaters carrying pathogens in the port environment and this arises public health concerns, however, the presence of these viruses limited to the canal port during the winter can greatly reduce the risk to human health. Regarding NNV, the accumulation and release of viable virus by mussels, could represent a viral source for susceptible finfish. These findings reflect the different epidemiological features of these infections and indicate the importance to choose the correct indicator to monitor viral contaminations. Significance and Impact of the Study: The high frequency of viral contamination pointed out in the study stresses the imperative to monitor the viral presence in all coastal habitats where the high natural value meets several recreational and commercial activities such as the Ravenna’s harbour area. Particularly, this study could represent a novel starting point for the development of a more structured bio-monitoring program, in order to ensure improved environmental management and safety of coastal areas.

Hypomyelination is a rare consequence of in utero bovine viral diarrhoea virus (BVDV) infection. We describe a BVDV outbreak in a naïve Holstein dairy herd in northern Italy, with an unusually high prevalence of calves with neurological signs, generalised tremors and ataxia. Histological analysis showed that hypomyelination was the predominant lesion and that the most typical BVDV neuropathological findings (e.g. cerebellar hypoplasia) were absent. Virological and molecular analyses showed that non-cytopathic BVDV genotype 1b was associated with the calves’ neurological signs and excluded other viruses responsible for congenital infection or neurological disorders. Whole-genome sequencing of BVDVs from the brain of a calf with neurological signs and the whole blood of a persistently infected herd-mate with no such sign showed >99.7 % sequence identity. Analysis of the quasispecies distribution revealed the greatest variation rates in regions coding for the structural proteins E1 and E2. Variation was slightly greater in the brain- than in the blood-derived sequence and occurred at different sites, suggesting the occurrence of distinct evolutionary processes in the two persistently infected calves. Molecular characterisation of BVDV genomes from five other calves with neurological signs from the same farm confirmed that the E1 and E2 regions were the most variable. Several factors, including genetic variability and host factors, appear to have contributed to the observed unique BVDV disease phenotype, characterised by hypomyelination and neurological signs.

Diseased outbreaks with high mortality in farmed sturgeon are a limiting factor to the success of this emerging aquaculture sector in Europe. Thorough investigations of outbreaks can determine the aetiological agents, identify important pathological and epidemiological pathways of infections and pave the way for effective control strategies. A thorough investigation of a mortality outbreak in Russian ( Acipenser gueldenstaedtii ) and Siberian ( Acipenser baerii ) sturgeons in Italy, demonstrated the primary involvement of a sturgeon nucleo-cytoplasmic large DNA virus (NCLDV). While, the taxonomy classification of this new virus is still uncertain, its involvement in sturgeon mortality outbreaks in Europe is, for the first time, fully investigated and described. Furthermore, the coinfection of bacteria such as motile Aeromonas spp. and Acinetobacter spp. was reported. Genetic characterisation showed the close relationship between the European sturgeon NCLDV with North American sturgeon NCLDVs. Similarly to the latter, the European sturgeon NCLDV persists in survivors. Furthermore, a systemic distribution of the European sturgeon NCLDV was evident in diseased A. baerii and A. gueldenstaedtii and in recovered A. gueldenstaedtii . These epidemiological and pathological findings will help in the identification of effective control strategies for sturgeon NCLDV infection, which afflicts an important and emerging European aquaculture sector.

The genus Pestivirus, which belongs to the Flaviviridae family, includes ssRNA+ viruses responsible for infectious diseases in pigs, cattle, sheep, goats and other domestic and wild ruminants. Like most of the RNA viruses, pestivirus has high genome variability with practical consequences on disease epidemiology, diagnosis and control. In addition to the officially recognized species in the genus Pestivirus, such as BVDV-1, BVDV-2, BDV and CSFV, other pestiviruses have been detected. Furthermore, most of the ruminant pestiviruses show low or absent species specificity observed in serological tests and are able to infect multiple species. Particularly, small ruminants are receptive hosts of the most heterogeneous group of pestiviruses. The aim of this study was to carry out the molecular characterization of pestiviruses isolated from sheep and goats in Sicily, Italy. Phylogenetic analysis of two viral genomic regions (a fragment of 5'-UTR and the whole Npro regions) revealed the presence of different pestivirus genotypes in the analysed goat and sheep herds. Two of five viral isolates were clustered with BVDV-1d viruses, a strain widespread in Italy, but never reported in Sicily. The other three isolates formed a distinct cluster with high similarity to Tunisian isolates, recently proposed as a new pestivirus species. This represents the first evidence for Tunisian-like pestivirus presence in small ruminants in Italy. Furthermore, one of the isolates was collected from a goat, representing the first isolation of Tunisian-like pestivirus from this species.

Quasispecies composition and tissue distribution of feline coronaviruses (FCoVs) were studied in naturally infected cats. The genomic complexity of FCoVs was investigated using single-strand conformational polymorphism (SSCP) analysis of N and ORF7b amplicons, and the evolutionary process was investigated by sequence-based phylogenetic analysis. SSCP analysis showed high heterogeneity of the FCoV genome which was correlated with the seriousness of the clinical form. The two genomic regions analysed showed different levels of variation; the N region demonstrated significant heterogeneity as compared to ORF7b. Phylogenetic analysis of the nucleotide sequences showed the clear separation of sequences analysed on the basis of virulence and geographical origin. A maximum likelihood analysis of N and ORF7b data sets showed a situation of strong heterogeneity for the N region.

Proliferative orf virus infections in adult sheep have increased in Italy in the past few years: these extreme cases are frequently fatal and difficult to differentiate from other infectious diseases of sheep such as blue tongue. A probable explanation for the proliferative and highly vascularized nature of the lesions was found in the expression of the VEGF-E gene encoded by the orf virus. To investigate a possible role of the viral VEGF in the pathogenesis of severe persistent orf virus lesions, the activity of four VEGF-E variants was compared by an angiogenesis in vitro model. Similar angiogenic activity was found between strains isolated from the classical and the proliferative forms of the disease, even if the latter was able to develop a higher number of vessels during the first 24 h of infection. Our in vitro findings seems to exclude that the VEGF variants encoded by the strain isolated from the atypical form of the disease could be the responsible for the histopathological aspect of the proliferative lesions.

A rotor-gene SYBR Green base real time system was developed for the detection and quantification of Betanodavirus. Two primers set were designed to target conserved sequence in the nodavirus capsid protein gene, yielding an amplification product of 122 and 116 nucleotides. A plasmid was cloned and quantify for producing standard curve. The method was then apply to cDNA retrotranscripted from RNA extracted from cell-supernatants and directly from tissues of infected animals. The assay allowed analitycal enumeration of 300 copies of plasmid and 500 target cDNA copies for primer set 1 and 10 copies of plasmid and 20 target cDNA copies for primer set 2. The reproducibility of the assay was evaluated by intra-assay variability of standard control that showed a coefficient of variation (CV) ranging from 1.5 to 4.6 and from 1.5 to 4.9 respectively for the two assaysas. Intra-assay and inter-assay variability were further assessed using high and low amounts of cDNA showing a high reproducibility. The method revealed to be very sensitive and quick, reducing post-PCR processing and suitable for both diagnostic and pathogenesis studies.

The full-length F1L gene was amplified by PCR and cloned into pCR T7/CT-TOPO vector fused with a six-histidine tag at the C terminus. Full-length F1L protein was expressed in Escherichia coli BL21 and was purified using a metal-chelate affinity chromatography. Two rabbits were immunized with the purified recombinant full-length F1L protein in Gerbu adjuvant and the immune response of the animals was checked by Western immunoblot analysis and virus neutralization. The results showed that F1L protein is able to stimulate the production of neutralizing antibodies .

Betanodaviruses are small ssRNA viruses responsible of Viral Nervous Necrosis in marine fishes worldwide. Genetic analysis of a fragment of the coat protein gene sequences permit to divide Betanodavirus in four genotypes. Preview studies evidenced Betanodavirus infection in several wild asymptomatic fish species, also in the Mediterranean Sea (1) and in invertebrates from Korea, mostly crustaceans and in a mussel (2). Both fish and invertebrates could be a potential source of virus for farmed fish. In this study, we examined bivalve mollusks for the presence of Betanodavirus and genetically analyzed the detected viruses to find out whether these animals could be a source of genetically closely related Betanodaviruses transmissible horizontally to wild and farmed fish. A total of 46 samples were analyzed including species reared on the seabed (Tapes philippinarum; Crassostrea gigas) and in the water column (Mytilus galloprovincialis). Betanodavirus presence was investigated by RT-nested/PCR (3). Positive samples were sequenced to study the variable region of the viral RNA2 coding for the capside protein. No viruses were detected in mussels (M. galloprovincialis), whereas clams (T. philippinarum) and oysters (C. gigas), both species reared on the seabed, resulted highly positive (26.6%). Phylogenetic analysis showed the belonging of these viruses to the RGNNV genotype. Betanodaviruses from clams clustered with viruses detected in wild and farmed fish collected from the same region (Adriatic Sea). Oyster viruses clustered separately, however inside the same genotype; considering the foreign origin of oysters (France), they could act as vectors of the infection between different areas. This is the first study focusing on Betanodavirus in bivalve mollusks; it evidences important presence of Betanodaviruses in bivalve mollusks; in some cases viral strains are highly correlated to those found in fish. These results highlight the possible role of mollusks in the spread of Betanodavirus to wild and farmed fish.