Advances in Viral Aquatic Animal Disease Knowledge: The Molecular Methods' Contribution.

Aquaculture is the fastest-growing food-producing sector, with a global production of 122.6 million tonnes in 2020. Nonetheless, aquatic animal production can be hampered by the occurrence of viral diseases. Furthermore, intensive farming conditions and an increasing number of reared fish species have boosted the number of aquatic animals' pathogens that researchers have to deal with, requiring the quick development of new detection and study methods for novel unknown pathogens. In this respect, the molecular tools have significantly contributed to investigating thoroughly the structural constituents of fish viruses and providing efficient detection methods. For instance, next-generation sequencing has been crucial in reassignment to the correct taxonomic family, the sturgeon nucleo-cytoplasmic large DNA viruses, a group of viruses historically known, but mistakenly considered as iridoviruses. Further methods such as in situ hybridisation allowed objectifying the role played by the pathogen in the determinism of disease, as the cyprinid herpesvirus 2, ostreid herpesvirus 1 and betanodaviruses. Often, a combination of molecular techniques is crucial to understanding the viral role, especially when the virus is detected in a new aquatic animal species. With this paper, the authors would critically revise the scientific literature, dealing with the molecular techniques employed hitherto to study the most relevant finfish and shellfish viral pathogens.

Cutaneous angiomatosis-like presentation in koi carp (Cyprinus carpio koi): Clinical-pathological investigations.

The skin represents an indicator of an animal's health status. Causes of cutaneous diseases in fish most often trace back to biological agents. However, fish skin diseases can also arise from a complex interaction of infectious and non-infectious causes, making it more difficult to identify a specific aetiology. In the period between April and September of the years 2019-2022, four koi carp (Cyprinus carpio koi) from two European countries presented with multifocal, irregularly round, few mm to 1 cm, variably raised cutaneous reddened areas. The fish displayed good general condition. Cutaneous samples, investigated by microbiological and molecular methods and microscopy, did not indicate a primary pathogenic agent. Gross and histological findings of the cutaneous biopsies were consistent with a multifocal/reactive process centred on dermal vessels. The histological features were reminiscent of angiomatosis, a benign proliferative condition affecting the dermal vessels of mammals, including human patients. The clinical-pathological presentation and the dermatologic condition that affected the koi carp are discussed and compared with the veterinary and human literature.

Redspotted Grouper Nervous Necrosis Virus and the Reassortant RGNNV/SJNNV In Vitro Susceptibility against a Commercial Peroxy-Acid Biocide under Different Conditions of Use.

Aquaculture is a constantly growing sector. The intensification of fish production and the movement of aquatic animals could cause the spread of infectious diseases. Remarkably, the diffusion of viral agents represents the major bottleneck for finfish production, and viral encephalopathy and retinopathy (VER) is considered the most impacting disease for Mediterranean aquaculture. No effective therapies are available to contrast VER, and vaccination can be applied only in grow-out facilities. Hence, programs to minimize the sanitary risks in farms are paramount to implementing hygienic standards and biosecurity. This study aimed to evaluate the in vitro virucidal activity of a peroxy-acid disinfectant (Virkon® S, DuPont, Sudbury, UK) towards the two NNV strains most widespread in the Mediterranean Sea. Remarkably, two protocols were applied to assess the virucidal activity under different conditions of use: the suspension test and the net test. The latter has been applied to evaluate the efficacy of the biocide on instruments, simulating the in-field application. The obtained results demonstrated the suitability of the tested biocide for NNV inactivation, being effective under some of the tested conditions. However, the presence of organic matter, the concentration of the product, and the application conditions can significantly affect the result of the disinfection procedure.

Development and diagnostic validation of a one-step multiplex RT-PCR assay as a rapid method to detect and identify Nervous Necrosis Virus (NNV) and its variants circulating in the Mediterranean.

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.

Detection of Human and Fish Viruses in Marine Gastropods.

Marine gastropods represent a major food source for higher trophic levels and an important source of animal protein for humans. Like bivalve molluscs, gastropods can accumulate several types of contaminants; however, the bioaccumulation of microorganisms, particularly viruses, has been poorly investigated in these animals. This study focused on gastropods (Tritia mutabilis, Bolinus brandaris and Rapana venosa) collected during the fishing season from 2017 to 2021 in the north-western Adriatic Sea, and on clams (Ruditapes philippinarum) harvested in the same geographical area, in order to evaluate the presence of human and fish viruses in their tissues. A virological investigation was carried out on the digestive gland using molecular methods. The presence of hepatitis A virus was detected in one sample, whereas noroviruses were not present in the investigated specimens. Regarding fish viruses, it was possible to detect the presence of nervous necrosis virus (NNV) in 26.5% of the analyzed gastropods; however, the histological examination did not show any pathological changes in the nervous tissue in both NNV-positive and -negative batches. As a whole, the investigated gastropods showed the ability to bioaccumulate viruses; however, lower contamination by human viruses compared to bivalve molluscs was pointed out, posing a minor concern to human health.

Pathological and Tissue-Based Molecular Investigation of Granulomas in Cichlids Reared as Ornamental Fish.

Cichlids include hundreds of species with a high economic value for aquaculture. These fish are subjected to intensive trade and farming that expose them to the risk of infectious diseases. This work focuses on ornamental cichlids held in an aquarium commercial facility presenting emaciation, in order to evaluate the presence of lesions in fish skin and organs. The fish were sampled during routine management activities and subjected to pathological and molecular investigations. The presence of lymphocystis disease virus, typically associated with cutaneous nodular disease, was ruled out. Histologically, they presented granulomas in the spleen, sometimes extending to the other visceral organs. Bacterial heat-shock protein 65 PCR products were detected in tissues associated, in the majority of cases, with granulomas; molecular investigation identified Mycobacterium spp. in two cases and Cutibacterium acnes in seven cases. Immunoreactivity to anti-Mycobacterium and anti-C. acnes antibodies was detected within granulomas. The presence of C. acnes within granuloma is elucidated for the first time in fish; however, similarly to what is found in humans, this bacterium could be harmless in normal conditions, whereas other contributing factors would be required to trigger a granulomatogenous response. Further confirmation by bacterial culture, as well as using large-scale studies in more controlled situations, is needed.

Integrated Management Strategies for Viral Nervous Necrosis (VNN) Disease Control in Marine Fish Farming in the Mediterranean.

Viral nervous necrosis (VNN) is the most important viral disease affecting farmed fish in the Mediterranean. VNN can affect multiple fish species in all production phases (broodstock, hatchery, nursery and ongrowing) and sizes, but it is especially severe in larvae and juvenile stages, where can it cause up to 100% mortalities. European sea bass has been and is still the most affected species, and VNN in gilthead sea bream has become an emerging problem in recent years affecting larvae and juveniles and associated to the presence of new nervous necrosis virus (NNV) reassortants. The relevance of this disease as one of the main biological hazards for Mediterranean finfish farming has been particularly addressed in two recent H2020 projects: PerformFISH and MedAID. The presence of the virus in the environment and in the farming systems poses a serious menace for the development of the Mediterranean finfish aquaculture. Several risks associated to the VNN development in farms have been identified in the different phases of the farming system. The main risks concerning VNN affecting gilthead seabream and European seabass have been identified as restocking from wild fish in broodstock facilities, the origin of eggs and juveniles, quality water supply and live food in hatcheries and nurseries, and infected juveniles and location of farms in endemic areas for on-growing sites. Due to the potential severe impact, a holistic integrated management approach is the best strategy to control VNN in marine fish farms. This approach should include continuous surveillance and early and accurate diagnosis, essential for an early intervention when an outbreak occurs, the implementation of biosecurity and disinfection procedures in the production sites and systematic vaccination with effective vaccines. Outbreak management practices, clinical aspects, diagnostic techniques, and disinfections methods are reviewed in detail in this paper. Additionally, new strategies are becoming more relevant, such as the use of genetic resistant lines and boosting the fish immune system though nutrition.

Comparative Analysis of Tunisian Sheep-like Virus, Bungowannah Virus and Border Disease Virus Infection in the Porcine Host.

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.

Bovine viral diarrhoea virus 1b infection associated with congenital tremor and hypomyelination in Holstein calves.

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.

Red mark syndrome (RMS) in farmed rainbow trout: First report of outbreaks in Bosnia and Herzegovina.

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.

Effects of dietary organic acids and nature identical compounds on growth, immune parameters and gut microbiota of European sea bass.

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 TGFß. 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.

Multifactorial Causes of Chronic Mortality in Juvenile Sturgeon (Huso huso).

This investigation focused on an episode of chronic mortality observed in juvenile Huso huso sturgeons. The examined subjects underwent pathological, microbiological, molecular, and chemical investigations. Grossly severe body shape deformities, epaxial muscle softening, and multifocal ulcerative dermatitis were the main observed findings. The more constant histopathologic findings were moderate to severe rarefaction and disorganization of the lymphohematopoietic lymphoid tissues, myofiber degeneration, atrophy and interstitial edema of skeletal epaxial muscles, and degeneration and atrophy of the gangliar neurons close to the myofibers. Chemical investigations showed a lower selenium concentration in affected animals, suggesting nutritional myopathy. Other manifestations were nephrocalcinosis and splenic vessel wall hyalinosis. Septicemia due to bacteria such as Aeromonas veronii, Shewanella putrefaciens, Citrobacter freundii, Chryseobacterium sp., and pigmented hyphae were found. No major sturgeon viral pathogens were detected by classical methods. Next-generation sequencing (NGS) analysis confirmed the absence of viral pathogens, with the exception of herpesvirus, at the order level; also, the presence of Aeromonas veronii and Shewanella putrefaciens was confirmed at the family level by the metagenomic classification of NGS data. In the absence of a primary yet undetected biological cause, it is supposed that environmental stressors, including nutritional imbalances, may have led to immune system impairment, facilitating the entry of opportunistic bacteria and mycotic hyphae.

Swim Bladder Disorders in Koi Carp (Cyprinus carpio).

Swim bladder disorders and consequent buoyancy problems are encountered in ornamental fish, including koi carp. Nevertheless, beyond clinical and pharmacological management, they are largely underdiagnosed. In this study, nine koi carp showing abdominal swelling and abnormal swimming behavior were investigated. Clinical approach, varying from case to case, included ultrasonographic and X-ray investigations, bacteriological analysis of the collected fluid, antimicrobial susceptibility pattern, and possibly histological analysis. Diagnostic imaging, corroborating gross examination, documented swim bladder deformation/dislocation and serous fluid within the swim bladder chambers of most animals. Bacteria belonging to the Aeromonas hydrophila/caviae group and Shewanella xiamenensis were identified. S. xiamenensis strains showed a sensibility to all tested molecules except for one strain, which was resistant to tetracycline and cyprofloxacin. Antibiotic treatment succeeded in the full recovery of three cases in which S. xiamemensis infection was detected. Chronic aerocystitis was histologically documented where tissue was available. The swim bladder histopathological findings highlighted a chronic process that had compromised the quality of life of the animals. A multidisciplinary clinical-pathological and microbiological approach is highly suggested to recognize swim bladder conditions as early as possible, aiming to drive medical intervention and raising the chances of fish survival.

Effects of increasing dietary level of organic acids and nature-identical compounds on growth, intestinal cytokine gene expression and gut microbiota of rainbow trout (Oncorhynchus mykiss) reared at normal and high temperature.

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.

Evidence of fish and human pathogens associated with doctor fish (Garra rufa, Heckel, 1843) used for cosmetic treatment.

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.

Occurrence of diseases in fish used for experimental research.

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.

Detection of Cyprinid Herpesvirus 1 DNA in cutaneous squamous cell carcinoma of koi carp (Cyprinus carpio).

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.

Determination of Salmonella spp., E. coli VTEC, Vibrio spp., and Norovirus GI-GII in Bivalve Molluscs Collected from Growing Natural Beds in Sardinia (Italy).

The aim of the present study was to evaluate the presence of Salmonella spp., verotoxigenic E. coli (VTEC), Vibrio spp., and Norovirus GI-GII in bivalve molluscs, cockles, and European grooved carpet shells (Cerastoderma spp. and Ruditapes decussatus) collected from a class B growing natural bed in Sardinia (Italy). All of the samples were analysed for Salmonella spp. detection according to European Commission Regulation (EC) 2285/2015. Detection and enumeration of Vibrio spp. were performed according to previously published methods. Presumptive identification of Vibrio spp. isolates was performed by means of conventional biochemical tests. E. coli VTEC was isolated following a direct multiplex polymerase chain reaction (PCR) screening test. Norovirus GI and GII were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). No Salmonella spp. were detected. The prevalence of Vibrio spp. was 90%, and the average contamination levels were 3.19 ± 1.07 and 2.84 ± 0.31 Log10 cfu/g in cockles and European grooved carpet shells, respectively. The prevalence of E. coli VTEC was 6.6%. All of the isolates showed a complete pathogenicity profile. The presence of Norovirus was highlighted in 25% of European grooved carpet shells samples. Results showed the typical microbiological profile of bivalve molluscs collected from backwaters and confirmed the capability of shellfish to accumulate E. coli VTEC, pathogenic vibrios, and Norovirus. The presence of such pathogens in shellfish is of major concern for the safety of consumers.

Development and application of a real-time PCR assay for the detection and quantitation of lymphocystis disease virus.

Lymphocystis disease virus (LCDV) is responsible for a chronic self-limiting disease that affects more than 125 teleosts. Viral isolation of LCDV is difficult, time-consuming and often ineffective; the development of a rapid and specific tool to detect and quantify LCDV is desirable for both diagnosis and pathogenic studies. In this study, a quantitative real-time PCR (qPCR) assay was developed using a Sybr-Green-based assay targeting a highly conserved region of the MCP gene. Primers were designed on a multiple alignment that included all known LCDV genotypes. The viral DNA segment was cloned within a plasmid to generate a standard curve. The limit of detection was as low as 2.6DNA copies/µl of plasmid and the qPCR was able to detect viral DNA from cell culture lysates and tissues at levels ten-times lower than conventional PCR. Both gilthead seabream and olive flounder LCDV has been amplified, and an in silico assay showed that LCDV of all genotypes can be amplified. LCDV was detected in target and non-target tissues of both diseased and asymptomatic fish. The LCDV qPCR assay developed in this study is highly sensitive, specific, reproducible and versatile for the detection and quantitation of Lymphocystivirus, and may also be used for asymptomatic carrier detection or pathogenesis studies of different LCDV strains.

Defensive response of European sea bass (Dicentrarchus labrax) against Listonella anguillarum or Photobacterium damselae subsp. piscicida experimental infection.

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-1ß, 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.