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.

Red mark syndrome (RMS) is a non-lethal inflammatory skin disorder spreading in farmed adult rainbow trout (Oncorhynchus mykiss) and reported worldwide. The aetiology is still uncertain, but positive correlation was found between Midichloria-like organism and RMS-affected fish. Here, we describe the first cases of RMS in Bosnia and Herzegovina. The outbreaks under study occurred in two intensive farms during the late winter and spring of 2020. Affected fish showed signs of disease ascribable to RMS, confirmed by pathological and molecular examination.

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.

A 71-day study was conducted to explore the effect of increasing dietary levels (0, 250, 500, 1000 mg kg feed−1; D0, D250, D500 and D1000, respectively) of a blend of microencapsulated organic acids (OA, specifically citric and sorbic acid) and nature identical compounds (NIC, specifically thymol and vanillin), on growth, intestinal immune parameters and gut microbiota (GM) of European sea bass juveniles reared under normal and subsequently suboptimal environmental conditions (high temperature, 30.0 ± 0.4 °C and low oxygen, 4.6 ± 0.6 mg L−1). OA and NIC did not promote growth, feed utilisation and feed intake at the inclusion tested but induced a significantly upregulation of IL-8, IL-10 and TGFβ. GM analyzed by next-generation sequencing showed that OA and NIC were able to exert prebiotic properties stimulating the development of beneficial bacteria taxa such as Lactobacillus, Leuconostoc, and Bacillus sp. Picrust analyses displayed a significant potential functional reconfiguration of GM promoting a decrease in inflammation-promoting and homeostatic functions at increasing OA and NIC administration. For the first time on this species the exposure to suboptimal rearing conditions was able to modify GM structure reducing LAB and increasing Proteobacteria, findings which were consistent with the inflammatory process observed at mRNA level.

Apart from the established pestivirus species Pestivirus A to Pestivirus K novel species emerged. Pigs represent not only hosts for porcine pestiviruses, but are also susceptible to bovine viral diarrhea virus, border disease virus (BDV) and other ruminant pestiviruses. The present study focused on the characterization of the ovine Tunisian sheep-like virus (TSV) as well as Bungowannah virus (BuPV) and BDV strain Frijters, which were isolated from pigs. For this purpose, we performed genetic characterization based on complete coding sequences, studies on virus replication in cell culture and in domestic pigs, and cross-neutralization assays using experimentally derived sera. TSV forms a distinct phylogenetic group more closely related to Pestivirus C (classical swine fever virus, CSFV) than to Pestivirus D (BDV). In contrast to BDV and BuPV, TSV replicates by far more efficiently on ovine than on porcine cells. Nevertheless, pigs were susceptible to TSV. As a consequence of close antigenic relatedness of TSV to CSFV, cross-reactivity was detected in CSFV-specific antibody assays. In conclusion, TSV is genetically closely related to CSFV and can replicate in domestic pigs. Due to close antigenic relatedness, field infections of pigs with TSV and other ruminant pestiviruses can interfere with serological diagnosis of classical swine fever.

Redspotted grouper nervous necrosis virus (RGNNV), genus Betanodavirus, family Nodaviridae, is the causative agent of viral encephalopathy and retinopathy (otherwise known as viral nervous necrosis) and can infect several fish species worldwide. Betanodaviruses, including RGNNV, are very resilient in the aquatic environment, and their presence has already been reported in several wild marine species including invertebrates. In order to investigate the interaction between a bivalve mollusc (Manila clam Ruditapes philippinarum) and RGNNV, we optimised a culture-based method. The bioaccumulation of the pathogenic RGNNV by R. philippinarum and the potential shedding of viable RGNNV from RGNNV-exposed clams were evaluated through a culture-based method. R. philippinarum clearly accumulated viable RGNNV in their hepatopancreatic tissue and were able to release viable RGNNV via faecal matter and filtered water into the surrounding environment. The role of clams as bioaccumulators and shedders of viable RGGNV could put susceptible cohabiting cultured fish at risk. RGNNV-contaminated molluscs could behave as reservoirs for this virus and may modify the virus epidemiology.

Doctor fish (Garra rufa, Heckel, 1843) are increasingly used for cosmetic treatment raising particular concerns regarding the potential transmission of infections to clients. Investigations of microbial causes undertaken in two outbreaks of mortality among G.rufa used for cosmetic treatment revealed the presence of multiple bacteria, including both fish and human pathogens such as Aeromonas veronii, A.hydrophila, Vibrio cholerae, Shewanella putrefaciens, Mycobacterium marinum and M.goodii. This range of bacteria indicates an intense microbial proliferation involving multiple pathogens, most likely induced by the poor health condition of the fish. Most of the detected pathogens are well-known agents of zoonosis. Indeed, M.goodii is an emerging nosocomial human pathogen that has never been detected in fish to date, nor in other animals. This first detection of M.goodii associated with fish infection points out a new zoonotic potential for this pathogen. These findings point out that handling, poor environmental conditions and the presence of fish pathogens, that can compromise the immune system of fish, can result in a mixed microbial proliferation and increase the spread of waterborne bacteria, including zoonosis agents. Accordingly, the microbiological surveillance of fish used for cosmetic treatment is extremely important, particularly in association with mortality outbreaks.

The objective of the present study was to evaluate the occurrence of pathogens and diseases in laboratory fish over a 10-year period at the Centre for Experimental Fish Pathology of Sicily, University of Messina. This report also emphasizes the adverse effects of subclinical infections on research endpoints, as well as the importance of animal health with respect to welfare. Infections in fish used for research can alter experimental outcomes, increase the variability of data, and impede experimental reproducibility. For this purpose, 411 diseased fish of different species (out of a total of 2820 fish) that belonged to four marine species ( Dicentrarchus labrax, Sparus aurata, Argyrosomus regius and Mugil cephalus) and to four fresh water species ( Danio rerio, Carassius auratus, Xiphophorus variatus and Poecilia reticulata) were examined in this study. Our results showed that mycobacteriosis and myxosporidiosis were the most important diseases found in our research fish, and the results represent a useful tool to obtain wider knowledge on the incidence of various diseases in different fish species. Further studies in this field are necessary to improve knowledge on the state of the health of fish used for research.

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.

Betanodaviruses are small ssRNA viruses responsible for viral encephalopathy and retinopathy, otherwise known as viral nervous necrosis, in marine fish worldwide. These viruses can be either horizontally or vertically transmitted and have been sporadically detected in invertebrates, which seem to be one of the possible viral sources. Twenty-eight new betanodavirus strains were retrieved in three molluscs species collected from different European countries between 2008 and 2015. The phylogenetic analyses revealed that strains retrieved from bivalve molluscs are closely related to viruses detected in finfish in Southern Europe in the period 2000-2009. Nevertheless, a new betanodavirus strain, markedly different from the other members of the RGNNV genotype, was detected. Such a massive and varied presence of betanodaviruses in bivalve molluscs greatly stresses the risks of transmission previously feared for other invertebrates. Bivalve molluscs reared in the same area as farmed and wild finfish could act as a reservoir of the virus. Furthermore, current European regulations allow relaying activities and the sale of live bivalve molluscs, which could pose a real risk of spreading betanodaviruses across different geographic regions. To our knowledge, this is the first study, which focuses on the detection and genetic characterization of betanodaviruses in bivalve molluscs. Copyright

Background Cutaneous squamous cell carcinoma (cSCC) is an uncommon disease affecting koi carp (Cyprinus carpio). Cutaneous papilloma (carp pox) is a benign epidermal proliferation reported in koi and has been shown to be caused by Cyprinid Herpesvirus 1 (CyHV1). Hypothesis/Objectives Histological, ultrastructural and molecular investigations were carried out aiming to investigate the aetiology of cSCC within archived tissue samples. Animals Surgical samples of masses located on the integument, fins and lips of 13 koi carp belonging to different private owners were included in this retrospective study. Methods CyHV1 DNA and RNA presence were investigated in five cSCC formalin-fixed paraffin-embedded tissue samples to recognize CyHV1 presence and its replication activity. Results All cases were histologically diagnosed as cSCC. The ultrastructural observations confirmed the squamous differentiation of neoplastic epithelial cells, which showed abundant tonofilament bundles and desmosomes. Although no virus particles were revealed ultrastructurally, the molecular investigation detected viral DNA in five epidemiologically unrelated cSCC. Viral transcript analysis revealed no evidence for viral replication in the tested cSCC, which could be consistent with latent infection. Conclusions and clinical importance These findings illustrate the frequent association of carp cSCC with CyHV1, although a direct cause–effect relationship cannot be established at this time. Therefore, surveillance programmes should take into account the suspected viral origin of cSCC to better inform prevention and control of CyHV1 in the future.

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.

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.

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.

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.

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.

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.

This study examined the in vitro effects of temperature on Betanodavirus infection in the SSN-1 cell line. A Betanodavirus isolated from moribund sea bass fry (Dicentrarchus labrax) farmed in the Adriatic Sea and characterised as a RGNNV (Redspotted grouper nervous necrosis virus) genotype was used in our study. Virus infected SSN-1 cells were incubated at temperatures between 10° and 30°C and observed for cytopathic effects daily for 15 days. Cell-free and cell-associated viral growth were evaluated by 50% tissue culture infectious dose (TCID50) titration at 0, 24, 48, 72, 96, 144, 192, 240, 312 and 360 hours post-infection. Virus replication was observed at all temperatures from 15°C to 30°C. The optimal temperature for virus growth was 25°C. A temperature of 10°C was detrimental to the growth of the SSN-1 cells and cell death interfered with interpretations of viral growth. The isolate of Betanodavirus from Italian sea bass in this study demonstrates a different temperature range for growth compared to previous reports for related Betanodavirus strains and most likely this is due to an adaptation to the normal environmental temperatures of the host fish species of origin.

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.

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 .

Nervous Necrosis Virus (NNV) represents one of the most threatening pathogens for Mediterranean aquaculture. Several NNV strains are currently co-circulating in the Mediterranean Basin with a high prevalence of the RGNNV genotype and the RGNNV/SJNNV reassortant strain and a more limited diffusion of the SJNNV genotype and the SJNNV/RGNNV reassortant. In the present study, a one-step multiplex RT-PCR (mRT-PCR) assay was developed as an easy, cost-effective and rapid diagnostic technique to detect RGNNV and the reassortant RGNNV/SJNNV strain and to distinguish them from SJNNV and the reassortant SJNNV/RGNNV strain in a single RT-PCR reaction. A unique amplification profile was obtained for each genotype/reassortant enabling their rapid identification from cell culture lysates or directly from brain tissues of suspected fish. The method’s detection limit varied between 102.3 and 103.4 TCID50 ml-1 depending on viral strains. No cross-reacitivty with viruses and bacteria frequently associated with gilthead seabream, European seabass and marine environment was observed. The mRT-PCR was shown to be an accurate, rapid and affordable method to support traditional diagnostic techniques in the diagnosis of VNN, being able to reduce considerably the time to identify the viral genotype or the involvement of reassortant strains.