30 research outputs found
Development and validation of a real-time TaqMan PCR assay for the detection of betanodavirus in clinical specimens
No full textDevelopment and validation of a real-time TaqMan PCR assay for the detection of betanodavirus in clinical specimens
Panzarin V, Patarnello P, Mori A, Rampazzo E, Cappellozza E, Bovo G, Cattoli G.
Betanodaviruses are the causal agents of viral encephalo-retinopathy, an infectious disease affecting more than 40 marine fish species, characterized by high morbidity and mortality. Because of its severe impact, robust diagnostic tools are required. The aim of this work was to develop and validate a real-time TaqMan PCR assay to detect betanodaviruses in clinical specimens by amplifying a conserved region of the RNA2 strand. The method proved to be specific and sensitive, being capable of detecting as low as 10 TCID(50)/ml. For clinical validation, samples from 100 marine fish were collected during a natural outbreak of disease and tested by three distinct laboratory methods, namely real-time TaqMan PCR, RT-seminested PCR and virus isolation. The results indicated optimal agreement between tests. The assay that was developed is capable of detecting members of all of the betanodavirus genetic groups currently described and can be considered a valid alternative to the time-consuming and contamination-prone nested PCR
Isolation and molecular characterization of viral encephalopathy and retinopathy virus from gilthead seabream larvae (Sparus aurata) showing mass mortalities
No full textGilthead seabream (Sparus aurata, L.1578) has been reared since several years in close connection with the European seabass (Dicentrarchus labrax) often affected by severe losses caused by viral encepahalopathy and retinopathy (VER) without showing any clinical sign or mortality. This observation led to consider Sparus aurata a species completely resistant to betanodavirus infection. In 2001, Castric et al., during a VER outbreak affecting a farmed seabass population, succeeded to obtain a VER isolate from asymptomatic seabream, collected in the same farm. In the following years several rumours about seabream larvae mortalities caused by betanodavirus infections have circulated among farmers and in the scientific world but very few investigations have been performed. In 2007, Beraldo et al. described the occurrence of a betanodavirus outbreak in sea bream larvae showing mass mortalities. Clinical signs, histological and immunohistochemistry findings were described. Frozen samples belonging to the same outbreak were accidentally made available later on by the farmer and processed for virus isolation and characterization. In the present study we describe the isolation of the causative agent in SSN-1 cells, its identification by PCR and molecular characterization as reassortant virus containing the genome from RGNNV (RNA1) and SJNNV(RNA2) genotypes. Our findings corroborate the preliminary results described by Beraldo et al. and definitively demonstrate that gilthead seabream must be included in the list of VER susceptible species at least during larval stage.
References
Beraldo P., G. De Nigris, F. Rogato and M. Galeotti (2007). Histological and immunohistochemistry findings of viral encephalopathy-retinopathy in gilthead seabream larvae (Sparus aurata, L. 1578) reared in Italy. Book of abstract 13th international conference of the EAFP, diseases of fish and shellfish . Grado, 17-22 September 2007
Detection and molecular characterization of betanodaviruses retrieved from bivalve molluscs
No full textBetanodaviruses 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. Copyrigh
Viral encephalopathy and retinopathy outbreak in freshwater fish farmed in Italy.
No full textViral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a neuropathological condition affecting > 40 species of fish. Although VER affects mainly marine fish, the disease has also been detected in certain species reared in freshwater environments. There are relatively few reports concerning the disease in freshwater species, and there is not much information on clinical signs. Nevertheless, the most common clinical findings reported from affected freshwater species are consistent with the typical signs observed in marine species. In this paper we describe the main clinical signs and the laboratory results associated with the detection of a betanodavirus in hybrid striped bass x white bass (Morone saxatilis x Morone chrysops) and largemouth bass Micropterus salmoides, reared in a freshwater environment. We also detected the virus by real-time PCR and isolated it in cell culture from a batch of pike-perch Sander lucioperca farmed in the same system
Accumulation of trace elements in feathers of the Kentish plover Charadrius alexandrinus
No full textA non-invasive study of trace element accumulation in tail feathers of the Kentish plover (Charadrius alexandrinus) was performed along the coastline of the northern littoral strip of the Venice Lagoon, with the aim to verify whether contamination may be a factor affecting conservation status of Kentish plover populations.Body burdens in feathers of 11 trace elements including toxic metals/metalloids and essential elements (As, Cd, Co, Cr, Cu, Hg, Ni, Pb, Se, V, Zn) were quantified by ICP-MS, then concentrations were normalized to feather's age calculated using ptilochronology in order to obtain daily deposition rates.Mercury emerged as a major threat to the conservation of the species, since average feather concentration was clearly above the adverse-effect threshold associated with impairment in the reproductive success in a number of bird species. Also Cd and Se occurred at levels that may impact on the conservation status of the studied species at local scale, even if to a lesser extent than Hg.Gender-related differences in trace element accumulation emerged only for As, although for this element the risks associated to environmental exposure seem to be negligible.A non-invasive study of trace element accumulation in tail feathers of the Kentish plover (Charadrius alexandrinus) was performed along the coastline of the northern littoral strip of the Venice Lagoon, with the aim to verify whether contamination may be a factor affecting conservation status of Kentish plover populations.Body burdens in feathers of 11 trace elements including toxic metals/metalloids and essential elements (As, Cd, Co, Cr, Cu, Hg, Ni, Pb, Se, V, Zn) were quantified by ICP-MS, then concentrations were normalized to feather's age calculated using ptilochronology in order to obtain daily deposition rates.Mercury emerged as a major threat to the conservation of the species, since average feather concentration was clearly above the adverse-effect threshold associated with impairment in the reproductive success in a number of bird species. Also Cd and Se occurred at levels that may impact on the conservation status of the studied species at local scale, even if to a lesser extent than Hg.Gender-related differences in trace element accumulation emerged only for As, although for this element the risks associated to environmental exposure seem to be negligible
Viral encephalopathy and retinopathy in freshwater fish farmed in Italy.
No full textViral encephalopathy and retinopathy (VER), also known as viral
nervous necrosis (VNN), is a
neuropathological condition affecting more than forty species fish species, mainly in the marine environment. In a few occasions, the disease has also been reported in certain freshwater species, but limited information is available particularly as regard on clinical signs, characterization of causative agents and epidemiological links with the marine environment
Transcriptome analysis reveals a complex response to the RGNNV/SJNNV reassortant Nervous Necrosis Virus strain in sea bream larvae
No full textThe gilthead sea bream (Sparus aurata) is a marine fish of great importance for Mediterranean aquaculture. This species has long been considered resistant to Nervous Necrosis Virus (NNV), an RNA virus that causes massive mortalities in several farmed fish animals. However, the recent appearance of RGNNV/SJNNV reassortant strains started to pose a serious threat to sea bream hatcheries, as it is able to infect larvae and juveniles of this species. While host response to NNV has been extensively studied in adult fish, little attention has been devoted to early life history stages, which are generally the most sensitive ones. Here we report for the first time a time-course RNA-seq analysis on 21-day old fish gilthead sea bream larvae experimentally infected with a RGNNV/SJNNV strain. NNV-infected and mock-infected samples were collected at four time points (6 h, 12 h, 24 h, and 48 h post infection). Four biological replicates, each consisting of five pooled larvae, were analysed for each time point and group. A large set of genes were found to be significantly regulated, especially at early time points (6 h and 12 h), with several heat shock protein encoding transcripts being up-regulated (e.g. hspa5, dnaj4, hspa9, hsc70), while many immune genes were down-regulated (e.g. myd88 and irf5 at T06, pik3r1, stat3, jak1, il12b and il6st at T12). A gene set enrichment analysis (GSEA) identified several altered pathways/processes. For instance, the formation of peroxisomes, which are important anti-viral components as well as essential for nervous system homeostasis, and the autophagy pathway were down-regulated at 6 h and 24 h post infection (hpi). Finally, two custom "reactomes" (i.e. significant gene sets observed in other studies) were defined and used. The first reactome integrated the transcriptomic response to NNV in different fish species, while the second one included all genes found to be stimulated either by interferon (IFN) or by IFN and Chikungunya virus in zebrafish. Genes in both reactomes showed predominant up-regulation at 6hpi and 12hpi and a general down-regulation at 24hpi. Such evidence suggest a certain degree of similarity between the response of sea bream and that of other fish species to NNV, while the observed down-regulation of IFN- and viral-stimulated pathways argues for a possible interference of NNV against the host response
Development of a Novel Assay Based on Plant-Produced Infectious Bursal Disease Virus VP3 for the Differentiation of Infected From Vaccinated Animals
No full textInfectious bursal disease virus is the causative agent of Gumboro disease, a severe infection that affects young chickens and is associated with lymphoid depletion in the bursa of Fabricius. Traditional containment strategies are based either on inactivated or live-attenuated vaccines. These approaches have several limitations such as residual virulence or low efficacy in the presence of maternally derived antibodies (MDA) but, most importantly, the impossibility to detect the occurrence of natural infections in vaccinated flocks. Therefore, the development of novel vaccination strategies allowing the differentiation of infected from vaccinated animals (DIVA) is a priority. Recently, commercial vectored and experimental subunit vaccines based on VP2 have been proved effective in protecting from clinical disease and posed the basis for the development of novel DIVA strategies. In this study, an engineered version of the VP3 protein of IBDV (His-VP3) was produced in plants, successfully purified from Nicotiana benthamiana leaves, and used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-VP3 antibodies. The His-VP3 ELISA was validated with a panel of 180 reference sera and demonstrated to have 100% sensitivity (95% CI: 94.7–100.0) and 94.17% specificity (95% CI: 88.4–97.6). To evaluate the application of His-VP3 ELISA as a DIVA test, the novel assay was used to monitor, in combination with a commercial kit, detecting anti-VP2 antibodies, the immune response of chickens previously immunized with an inactivated IBDV vaccine, a recombinant Turkey herpes virus carrying the VP2 of IBDV (HVT-ND-IBD) or with plant-produced VP2 particles. The combined tests correctly identified the immune status of the vaccinated specific pathogen free white-leghorn chickens. Moreover, the His-VP3 ELISA correctly detected MDA against VP3 in commercial broiler chicks and showed that antibody titers fade with time, consistent with the natural decrease of maternally derived immunity. Finally, the novel assay, in combination with a VP2-specific ELISA, demonstrated its potential application as a DIVA test in chickens inoculated with VP2-based vaccines, being able to detect the seroconversion after challenge with a very virulent IBDV strain
Age dependency of RGNNV/SJNNV viral encephalo-retinopathy in Gilthead Sea Bream (Sparus aurata)
No full textGilthead sea bream (GSB) (Sparus aurata) have been generally considered to be resistant to viral encephalo-retinopathy. However, the recent increase of the number of outbreaks in sea bream hatcheries caused by ner -vous necrosis virus (NNV) called RGNNV/SJNNV, a reassortant virus, has encouraged researchers to investigate the disease pathogenesis in this species, with particular emphasis on fish age. For this purpose, experimental challenges of juveniles and larvae were carried out.Four experimental trials were performed by infecting GSB of different ages with the RGNNV/SJNNV virus: juveniles of 7 g of weight and larvae of 70, 35 and 21 days post hatch (dph).Virological, histological and immunohistochemical analysis, as well as qualitative and quantitative real time PCR at different time points post infection (pi) were performed. ELISA test for antibody detection was also implemented, where applicable.Overall results showed that the gilthead seabream larvae are susceptible to RGNNV/SJNNV infection irre-spective to the age, as proved by the massive viral replication detected by quantitative RT-PCR and the massive presence of immunoprecipitates in the nervous tissues evidenced by immunohistochemistry. However, clear clinical signs and mortality were observed only in the youngest group of 21-dph larvae. The viral kinetics in-vestigations led to think that the efficiency of RGNNV/SJNNV replication was higher in 21dph larvae compared to the older ones, which in turn correlated with the disease outcome. Most likely the latter was related to the developmental stage of the immune system. Noteworthy survivors were always tested negative for the presence of specific antibodies. The identification of the factors governing the host ability to control viral replication, or conversely those inhibiting the virus in older fish will be crucial to better understand the disease pathogenesis in this species. Notably, despite the age and the disease outcome of the infection, GSB remained persistently infected for a long time, even up to one year, becoming asymptomatic carriers
Bottlenecks in diagnostics of Mediterranean fish diseases
No full textThree independent Horizon 2020 projects are prioritising a common task of solving diagnostic issues using different approaches and strategies. PerformFISH focuses its research objectives on diagnostic methods, MedAID has prepared a diagnostic manual for the most devastating fish diseases of the Mediterranean and ParaFishControl has prepared a diagnostic manual on parasitic diseases of European aquaculture species. Currently, the projects are investing large collaborative efforts to reinforce their tasks and to deliver mutual and cutting-edge material on the subject. Nevertheless, further efforts for the harmonisation, standardisation and validation of diagnostic methods for infectious diseases in Mediterranean aquaculture are needed, involving as many relevant stakeholders and experts as possible. The main goal of the workshop (WS) “Bottlenecks in diagnostics of Mediterranean fish diseases”, organised during the 19th International EAFP conference on Diseases of Fish and Shellfish, was to present results already obtained from the projects and discuss the bottlenecks in diagnostic procedures of target groups of pathogens. The WS provided an ideal environment and opportunity to exchange and discuss experiences and data with international experts in the diagnostic field attending the conference. Interactive discussions on diagnostics for a specific group of pathogens have resulted in highlighting new initiatives needed in the management of diseases in Mediterranean aquaculture. Furthermore, the WS emphasised a need for the establishment of this strong interactive network which will collaborate to receive, collect, compile and analyse all information gathered from different parts of the region
