Sviluppo di metodiche innovative per la diagnosi e lo studio di malattie ad eziologia virale dei pesci

The increase in aquaculture operations worldwide has provided new opportunities for the transmission of aquatic viruses. The occurrence of viral diseases remains a significant limiting factor in aquaculture production and for the sustainability. The ability to identify quickly the presence/absence of a pathogenic organism in fish would have significant advantages for the aquaculture systems. Several molecular methods have found successful application in fish pathology both for confirmatory diagnosis of overt diseases and for detection of asymptomatic infections. However, a lot of different variants occur among fish host species and virus strains and consequently specific methods need to be developed and optimized for each pathogen and often also for each host species. The first chapter of this PhD thesis presents a complete description of the major viruses that infect fish and provides a relevant information regarding the most common methods and emerging technologies for the molecular diagnosis of viral diseases of fish. The development and application of a real time PCR assay for the detection and quantification of lymphocystivirus was described in the second chapter. It showed to be highly sensitive, specific, reproducible and versatile for the detection and quantitation of lymphocystivirus. The use of this technique can find multiple application such as asymptomatic carrier detection or pathogenesis studies of different LCDV strains. The third chapter, a multiplex RT-PCR (mRT-PCR) assay was developed for the simultaneous detection of viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN), infectious pancreatic necrosis (IPN) and sleeping disease (SD) in a single assay. This method was able to efficiently detect the viral RNA in tissue samples, showing the presence of single infections and co-infections in rainbow trout samples. The mRT-PCR method was revealed to be an accurate and fast method to support traditional diagnostic techniques in the diagnosis of major viral diseases of rainbow trout

Efficacy of fungoid chitosans from Aspergillus niger and Agaricus bisporus in controlling the oxidative browning of model white wines

The efficacy of two water-insoluble chitosans from Aspergillus niger and Agaricus bisporus, in controlling the browning of model white wine solutions was assessed and compared with respect to sulfite addition (70 mg/L). A water-soluble oligomeric preparation from Agaricus bisporus was also included to test the effect of solubility and reduced molecular weight on the antibrowning capability of the polysaccharide. Chitosans were added at 0.5 g/L and 1 g/L. Color development, iron oxidoreductive equilibrium and generation of phenolic intermediates were monitored. Results demonstrated a significant and comparable anti-browning efficacy of both insoluble formu-lations (up to 85% reduction in browning development with respect to control samples), which mainly acted by chelating iron (up to around 4.4 mg/g of chitosan) and shifting its oxidoreductive equilibrium toward the reduced form. Oligomeric chitosan was ineffective for this purpose as it completely lacked chelating activity, which it is proposed, depended on its negligible interaction with tartaric acid. Data on browning and oxidation-related phenolic intermediates also revealed that sulfite promotes browning once it is completely oxidized.Industrial relevance: Following its very recent European authorization as novel food, chitosan from Agaricus bisporus has been evaluated for the first time and compared in wine-like conditions with the already known water-insoluble chitosan from Aspergillus niger. A further novelty are the data on water-soluble chitosan prepa-rations, not yet permitted in wine but potentially interesting due to the potentially higher specific surface once in solution. The results, apart from providing information on a recently introduced source for enological chitosan, can be useful to producers and winemakers in deciding among fungoid preparations aimed to control the browning of products

Quality Changes during Frozen Storage of Mechanical-Separated Flesh Obtained from an Underutilized Crustacean

Despite their high nutritional value, high quantities of fish caught in the Adriatic Sea are underused or discarded for their insignificant economic value. Mechanical separation of flesh represents an opportunity for developing innovative semi-finished products, even if it can promote an increased quality degradation rate. The aim of this study was to evaluate physico-chemical modifications of mechanically separated mantis shrimp flesh during deep-freezing storage. Flesh samples obtained using a belt-drum separator, frozen and vacuum-packed, were stored at 3 temperatures (industrial: -26 \ub0C; domestic: -18 \ub0C and abuse: -10 \ub0C) for 12 months. During storage, qualitative (color, water content, pH, fatty acids (FA) and lipid oxidation) were evaluated. Fish freshness parameters (e.g., trimethylamine (TMA), dimethylamine (DMA) and amino acids) were assessed using nuclear magnetic resonance (1H-NMR). The mechanical separation process accelerated the initial oxidation phenomena, promoting color alterations, compared to manual separation. The main degradation phenomena during storage were significantly affected by temperature and were related to changes in luminosity, oxidation of n-3 polyunsaturated fatty acids (PUFA), increased lipolysis with release of free FA, production of TMA and DMA by residual enzymatic activity, and changes in amino acids due to proteolysis. The inter-disciplinary approach permitted important findings to be made, in terms of the extent of different degradative phenomena, bound to processing and storage conditions of mechanically separated mantis flesh

Investigação dos vírus da síndrome de Taura e da Mionecrose Infecciosa em cultivos de camarão marinho Litopenaeus vannamei em Pernambuco

A carcinicultura cresceu rapidamente em todo o mundo e hoje constitui uma grande indústria, proporcionando emprego para milhares de trabalhadores e milhões em receita. Como toda atividade de aqüicultura, a passagem do ambiente selvagem para o cativeiro, é acompanhada de mudanças, que propiciam o surgimento de doenças. Surtos de diversas doenças foram reconhecidos como uma ameaça a sustentabilidade da indústria do camarão, e alguns deles causaram sérias perdas econômicas para os produtores de vários países. O vírus da Síndrome de Taura é considerado um dos mais perigosos, com mortalidade cumulativa de 60 a 90%, sendo responsável por grandes prejuízos na América Latina. O vírus da mionecrose infecciosa - IMNV (Infectious Myonecrosis Virus), ainda não registrado em outros países, foi identificado no Nordeste brasileiro e apontado como uma das causas para a diminuição da produção nacional em 2004. O objetivo deste trabalho foi investigar a ocorrência dos vírus Taura e IMN, a partir do diagnóstico molecular de Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), no estado de Pernambuco a fim de subsidiar informações importantes para a construção de um plano de biosseguridade. Onze fazendas pertencentes a seis municípios diferentes, localizados no litoral norte e sul do Estado dePernambuco, foram amostradas aleatoriamente no período de agosto a dezembro de 2004. Dos 530 camarões analisados, nenhum apresentou diagnóstico positivo para o TSV, o que significa que a prevalência deste patógeno no estado de Pernambuco é inferior a uma prevalência mínima por fazenda de 0.06%, mesmo diante dos surtos recentes em países próximos. Em nove das onze fazendas analisadas foram encontrados casos positivos para o IMNV. O vírus IMN apresentou uma prevalência que variou de 0,00% a 35,56%, por fazenda, o que é considerado preocupante, e desperta a necessidade de um monitoramento emnível nacional. Este resultado constitui o primeiro levantamento de vírus de camarão realizado no país, conduzido em uma amostragem não dirigida.Shrimp culture has grown rapidly worldwide and nowadays constitutes a solid industry generating jobs for thousand people. In any aquatic species, the transition from life in a wild environment to aquaculture systems is usually followed by several changes, such as culture densities, frequent degradation of environmental quality, mixing of populations of different origin and manipulations, etc. These facts increase the probability of outbreaks of serious diseases. Several virus diseases have occurred, threatening the sustainability of shrimp industry worldwide. Taura virus is considered one of the most harmful pathogens in the Americas, with cumulative mortality between 40 and 95%. Another virus, named Infectious Myonecrosis Virus (IMNV) was first identified in Litopenaeus vannamei reared in Northern Brazil and it is pointed as one of the main causes of the crash in shrimp production in 2004. The objective of this study was to investigate the prevalence of TSV and IMNV by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) in Pernambuco state, in order to provide important information for the construction of a biosecurity plan. Eleven commercial farms, located in six different cities of the north and south coast, were randomly sampled betweenAugust and December of 2004. Among the 530 animals analyzed, none were vírus positive for Taura, suggesting that the prevalence of this pathogen is lower than 0,06%, even with recent outbreaks in nearby countries. The IMN virus prevalence lied between 0 and 35,56% among the farms, which is considered alarming and requires monitoring mechanisms at the national level. These results constitute the first survey of these shrimp virus carried out in the Americas using a total randomized sample

Optimization of emerging treatments for seafood products and by-products valorization

The development of emerging technologies in seafood processing can contribute significantly to meet consumer needs toward safe, healthy, and minimally processed foods. This PhD thesis is focused on the qualitative and quantitative evaluation of the effects of emerging treatments applied to extend shelf-life of seafood processing and to preserve the qualitative characteristics of raw materials. The application of innovative non-thermal technologies to recover bioactive substances from crustacean by-products were also studied. Several experimental procedures were evaluated individually by the following studies: i) study of the effect of modified atmosphere packaging (MAP) with unconventional gas mixtures on the modification of the main qualitative parameters of sardine fillets during refrigerated storage; ii) valorization of an underutilized crustaceans by the application of mechanical flesh separation and freezing iii) application and evaluation of the main effects of high pressure processing (HPP) treatments on the quality and stability of different seafood products; iv) study and application of innovative non-thermal technologies, such as accelerated solvent extraction (ASE) and pulsed electric fields (PEF) for the recovery and valorization of value-added products from crustacean processing by-products. Based on the obtained results, the use of argon for MAP of sardine fillets showed an inhibitory effect on bacterial spoilage increasing their shelf-life. The effect of HPP treatment on the different types of considered seafood products, intended for raw consumption, highlighted a significant microbiological shelf-life increase at the highest applied pressure levels for all the considered species. The application of ASE and PEF showed high yield of astaxanthin and seemed to be an effective tool to recover high antioxidant compounds from crustacean by-products. The overall results of this PhD thesis highlighted the great advantages in the application of emerging technologies for both seafood products and crustacean by-product valorization, therefore contributing to the potential increase of the sustainability of the seafood sector

Development of a multiplex RT-PCR assay for simultaneous detection of the major viruses that affect rainbow trout (Oncorhynchus mykiss)

The major viral diseases that affect rainbow trout (Oncorhynchus mykiss) are viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN), infectious pancreatic necrosis (IPN) and sleeping disease (SD). In the presented study, we developed a multiplex RT-PCR (mRT-PCR) assay for the simultaneous detection of these four rainbow trout viruses in a single assay. The choice of primers was carried out based on the expected size of the fragments, the temperature and time required for the amplification, and the specificity for the target sequence. Firstly, the method was optimised using reference strains of viral haemorrhagic septicaemia virus (VHSV), infectious haematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV) and sleeping disease virus (SDV) cultivated with permissive cell culture lines; subsequently, the method was used for the identification of these viral infections in rainbow trout samples. Twenty-two samples of rainbow trout, clinically suspected of having viruses were analysed by the developed method to detect the presence of the four viruses, by directly analysing the animal tissues. The mRT-PCR method was able to efficiently detect the viral RNA in infected cell culture supernatants and in tissue samples, highlighting the presence of single infections as well as co-infections in rainbow trout samples. VHSV/SDV and IHNV/SDV co-infections were demonstrated for the first time in rainbow trout. The mRT-PCR method was revealed to be an accurate and fast method to support traditional diagnostic techniques in the diagnosis of major viral diseases of rainbow trout

Quantitation of infectious myonecrosis virus in different tissues of naturally infected Pacific white shrimp, Litopenaeus vannamei, using real-time PCR with SYBR Green chemistry

The Pacific white shrimp, Litopenaeus vannamei, is the most important shrimp species in volume in world aquaculture. However, in recent decades, outbreaks of diseases, especially viral diseases, have led to significant economic losses, threatening the sustainability of shrimp farming worldwide. In 2004, Brazilian shrimp farming was seriously affected by a new disease caused by the Infectious myonecrosis virus (IMNV). Thus, disease control based on rapid and sensitive pathogen detection methods has become a priority. In this study, a specific quantitation method for IMNV was developed using real-time PCR with SYBR Green chemistry and viral load of the principal target tissues of chronically infected animals was quantified. The quantitative analysis revealed that mean viral load ranged from 5.08 7 108 to 1.33 7 106 copies/g of total RNA in the hemolymph, 5.096 7 105 to 1.26 7 103 copies/g in the pleopods, 6.85 7 108 to 3.09 7 104 copies/g in muscle and 8.15 7 106 to 3.90 7 103 copies/g in gills. Different viral loads of IMNV were found with greater values in the hemolymph and muscle, followed by the pleopods and gills

Distribuzione di Lymphocystivirus in organi target e non target di orate (Sparus aurata) naturalmente infette

Lymphocystis is a cutaneous viral disease, caused by a virus (LCDV) of genus Lymphocystivirus, family Iridoviridae. The disease is frequently benign and in the Mediterranean area it affects mainly Gilthead seabream (Sparus aurata). This disease, in seabream farms, causes significant economic losses related to poor growth rate, non-marketability of fish with lesions and mortality due to secondary infections. LCDV infection causes single or clustered tumour-like cutaneous nodules. Histology, generally, does not evidence lesions in internal organs, however, recent studies have revealed the systemic spread of the virus. The in vitro isolation of LCDV is difficult, reducing the possibilities of pathogenetical investigations on lymphocystis. Recently, our research team has developed a real time PCR (qPCR) assay able to detect and quantify the LCDV DNA both in diseased and recovered fish. In this study, the qPCR assay has been applied to detect the LCDV DNA in target (pectoral and caudal fins) and not target (spleen, brain and eyes) organs of Gilthead seabream at different clinical stage (diseased/in regression) and asymptomatic. This study has revealed the viral DNA presence in both target and not target organs with statistically higher values in pectoral and caudal fins than in internal organs. Diseased, in regression and asyntomatic fish have shown similar viral DNA distribution