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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.

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.

Sea bass were experimentally infected with Listonella anguillarum or Photobacterium damselae subsp. piscicida (Phdp). At 24 and 72h post-infection, the expression analysis of immune-relevant genes (IL-1β, IL-6, IL-8, Hepcidin), the transcriptional level and detection of HSP70, and the quantification of serum iron were investigated in association with the histological analysis and the bacterial recognition in tissues by immunohistochemistry. At 15 days post-infection, the specific antibody response was detected in surviving fish, as well as the transcriptional levels of TcR and BcR sequences. Both experimental infections were characterized by a similar acute response, whereas different histological and immunohistochemistry evidences were observed. In particular, the early reaction appeared suitable for the clearance of L. anguillarum, thus limiting the histological lesions, the bacterial dissemination and the further development of acquired immunity in surviving fish. On the contrary, the innate response appeared not enough to resolve the Phdp infection, which was characterized by tissue damage, bacterial widespread and substantial detection of specific humoral immunity in surviving fish, also associated to lymphocytes clonal expansion. Besides the opportunistic conditions involved in fish vibriosis and pasteurellosis, the comparison between these experimental infection models seems to suggest that the rate of development of the acquired immunity is strictly linked to the activation of the host innate response combined to the degree of bacterial virulence.

This study reports the detection of co-infection by multiple CPV variants and the high genetic complexity of a CPV-2 strain detected in a domestic cat. The CPV variants selected by cloning the VP2 gene were sequenced, and genetic diversity and selection pressure were investigated. Comparison of the nucleotide sequences has evidenced 10 different viral populations, and, in the same animal, more CPV variants coexist. Our analysis excludes the possibility that the recombination events took place during infection and that negative selection acted on the VP2 gene. These findings confirm that CPV-2 shows high genetic heterogeneity resembling the quasispecies found in RNA viruses.

The orf virus is the type species of the Parapoxvirus genus and is the causative agent of contagious echtyma, a debilitating skin disease of sheep and goats, which can also affect man. The virus exhibits a restricted host range, even if it has been shown to bind to a wide range of tissues of non-permissive species. This ability is an argument for its potential use as an expression vector. Since most mammalian cell types express heparan sulfate (HS) surface receptors, we assumed that HS could serve as receptors to mediate orf virus binding. In this study, we showed that orf virus is inhibited by the addition of soluble heparin in cell cultures. Affinity chomatography using heparin agarose demonstrated that orf virus F1L is the major heparin binding protein. Furthermore, the recombinant F1L protein was visualised on the cell surface by confocal microscopy, and rabbits immunised with recombinant F1L protein produced virus neutralising antibodies. These results confirm that the F1L immunodominant protein is also involved in virus binding to cells as for the vaccinia homologue H3L protein. Heparin also inhibited the binding of the F1L protein to cells showing that this protein has a role in the early stages of infection.

Genetic and antigenic properties of 62 field isolates of canine parvovirus (CPV-2) collected from 1994 to 2001 in Italy were investigated. Antigenic characterisation was conducted using specific monoclonal antibodies (Mabs). The VP1\VP2 gene was amplified by PCR and characterised with restriction endonucleases to detect the 297 and 265 variant. The VP2 gene of 16 isolates was sequenced and molecular genetic analysis was conducted. The antigenic type prevalent among our isolates is type 2a as well as the 297 variant, which is also prevalent in the rest of Europe. Only the 9.7% of the isolates have the T265P mutation. The VP2 sequences of CPV-2 isolates were very similar to recent Asian isolates. In the threefold spike of CPV-699 a coding change was detected in the 440 residue where threonine was substituted by alanine: the same mutation has been found in two Asian CPV-2 isolates from leopard cats [Virology 278 (2000) 13]. Phylogenetic analysis revealed that the Italian CPV-2 strains followed the same evolution as observed in other countries and they gave no indication of a separate lineage.

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.