Participating organizations (WP leader in bold): P1. HCMR, P2. FCPCT, P3. IRTA, P5. UNIABDN and P20. SARC
This WP will address a number of identified bottlenecks relating to meagre health. The tasks include (a) studies of key disease states, (b) development of appropriate treatments, and (c) a first characterisation of the meagre immune system/ immune responses required for future immune intervention.
Task 24.1. Systemic Granulomatosis. The most important disease of cultured meagre is a newly described condition under this provisional name (Katharios et al., 2011). It is characterized by multiple systemic visceral granulomas and is manifested progressively by calcified and necrotic liver and kidney. The aetiology of the disease is unknown however there is evidence that it may be a metabolic disorder. To investigate the hypothesis that the disease is related to nutrition we will run several feeding trials to assess the effect of vitamin D and Ca/P levels in feeds, the effect of low/high protein levels in feeds and Mg inclusion, the effect of plant ingredients in the feeds, as well as the effect of minerals and vitamins levels.
Sub-task 24.1.1. Feeding trials
Sub-task 24.1.2. Health and pathological assessment (HCMR). Depending on size, samples will include whole fish (fish <2 g) or dissected organs and tissues, which will be preserved in various fixatives. Analyses will include histology for the description of the development of SG using standard and special staining techniques (Bennett et al., 1976; McDowell and Trump, 1976), Transmission Electron Microscopy with X-ray microanalysis (Suzuki et al., 1997), Scanning Electron Microscopy (HCMR) and tissue biochemical analysis.
Task 24.2. Chronic Ulcerative Dermatopathy. This is a newly described condition affecting the lateral line canals of many cultured fishes. From previous studies, it is established that Chronic Ulcerative Dermatopathy (CUD) is directly associated with the use of borehole water (Katharios et al 2011). Two parallel rearing trials of meagre in borehole and natural seawater will be conducted in order to study the development of CUD. The development of the disease in the different environments will be studied using histological and molecular techniques. The study will also involve physicochemical analysis and comparison of the two water sources, in order to identify the aetiological agent of the condition. The analysis includes monitoring of pH, carbon dioxide, salinity and selected heavy metals
Task 24.3. Anti-parasitic treatments. According to Colorni (2004), the major diseases which confront fish farmers vary in prevalence according to the geographic latitude of each country around the Mediterranean basin. “Old” pathogens (Lymphocystis and encephalitis viruses, “Pasteurella” piscicida, Amyloodinium ocellatum) still cause serious economic losses, and diseases once considered sporadic (caused by Mycobacterium marinum, Lactococcus garvieae, Enteromyxum [formerly Myxidium] leei, Kudoa sp.) are emerging. In the case of meagre, the presence of several parasites has been reported and some bacteria have been associated with outbreaks of disease in meagre. The management and control of parasitic infections in aquaculture are a constant challenge, complicated by the current limited availability of efficacious licensed products. In addition, parasite control in aquaculture requires awareness of environmental, water quality and host parameters. Therefore, treatment with anti-parasitic drugs remains the most widely used tool for managing parasite infections in cultured aquatic species, since efficacious options for intervention other than anti-parasitic drugs remain limited. Here we propose to develop appropriate treatments which can be used in meagre by studying the host parameters in relation to chemicals already avalaible in the market and with antiparasitic properties. The study will use histological and biochemical techniques in order to study these parameters, whilst performing experiments under controlled conditions.
Task 24.4. Nocardia infection in meagre
Sub-task 24.4.1. Isolation and characterization of the pathogen. Nocardia infection has been reported recently in large cultured meagre and may pose a significant threat and bottleneck for its production, since Nocardiosis is a chronic disease which is hard to eradicate and apart from the direct mortalities may also adversely affect the final product quality. Since there is only one report in the literature for Nocardiosis in meagre and in the Mediterranean aquaculture in general, in this WP we will monitor fish from various geographic localities (Greece and Spain) for the presence of the bacterial pathogen. The screening involves isolation using standard microbiological techniques and physiological characterization based on colony morphology, bacterial morphology, Gram and Ziehl-Neelsen staining, antibiotic sensitivity, biochemical profiling using API ZYM and BIOLOG Microsystems. Isolated strains will be also genetically characterized based on DGREA analysis and sequencing of 16s Rrna following PCR with universal or Nocardia-specific primers, along with the putative virulence of the chosen strains, based on clinical data (mortalities, morbidity). With this task we will assess the existence of Nocardia in meagre and its genetic and phenotypic variability that will be important for future vaccine design.
Sub-task 24.4.2. Preparation of an autogenous vaccine. Based on the results of Sub-task 24.4.1, a monovalent or polyvalent vaccine will be prepared using formalin inactivated bacterin. The vaccine will be tested for sterility and toxicity and evaluated in vaccination/challenge experiments. Nocardia challenge will be initially optimised with the isolated strain, and subsequently this method will be used to determine the effectiveness of the pilot vaccine after prior immunisation of the fish.
Task 24.5. First characterisation of the immune system. Anticipating that future management of disease issues in meagre will require vaccines as part of the arsenal of approaches used, we will undertake a characterization of the immune system to identify key immune molecules, as potential markers of immune system development and induction of antiviral and antibacterial responses.
For markers of the adaptive immune system, we will clone RAG1/2, Ig and TcR genes from meagre, and determine their expression during development, to allow an analysis of when to vaccinate as the immune system matures.
In addition, we will clone marker genes of inflammation, antibacterial responses and antiviral response. The latter will require prior stimulation of fish/cells, since these genes show low constitutive expression but can be markedly induced upon infection. Pathogen Associated Molecular Patterns (PAMPS) will be used for this purpose and will include bacterial LPS, polyI:C. In each case qPCR assays will be established for future gene expression profiling following vaccination or immunostimulant treatment.
Task 24.6. Monitor specific immune responses. In this task we will monitor meagre specific immune responses following vaccination with a bacterin. Classically antibody responses are measured post-vaccination, and we will develop anti-meagre IgM and anti-meagre IgT antibodies for this purpose. In addition, a range cytokines associated with helper-T cell responses will be cloned to allow analysis of cell-mediated responses. Meagre will be vaccinated with a vaccine to Vibrio anguillarum, a common Gram-negative bacterium found in the marine environment, and blood/mucus samples (for antibody analysis) and tissue samples (gill/kidney) for cytokine analysis will be collected at a range of times post-vaccination and post-exposure to the pathogen.
Task 24.7. Description, diagnosis and treatment of other bacterial/viral infectious diseases occurring in meagre
Increase in meagre production, as it occurs in other warm water species, may lead to the emergence of bacterial diseases such as vibriosis or photobacteriosis and some viral diseases such as nodavirus and lymphocystis disease. In this task we will monitor meagre throughout the project to establish the seasonality of the potential diseases. External morphology, necropsies and samples of liver, spleen, kidney, gut and nervous system will be studied to determine the presence of pathogenic organisms and histopathological damage to the different organs. These samples will be processed for determination of bacterial diseases by classical microbiological techniques, and by PCR.
For viral diseases, cell cultures suited to each disease will be used for virus propagation, following by RT-PCR or PCR verification depending on the specific virus. Susceptibility of meagre will be set against field and reference strains of the following pathogens: Vibrio anguillarum, Photobacterium damselae subsp. piscicida, and betanodavirus. Susceptibility values will be set by challenge test using intraperitoneal or intramuscular injections for bacteria or nodavirus respectively, in studies conducted in a biosecurity facility. When outbreaks of disease occur, methods will be established to determine the causal agent of the disease and attempt to find appropriate preventive and therapeutic measures for control. Specific diagnosis protocols will range from molecular (PCR, RT-PCR) to antibody based (ELISA, Immunohistochemistry, Immunoflourescence) methods. Treatment protocols will be established with selected antibiotics (depending on the limitation of their use) for each pathogen isolated from natural outbreaks of disease, by studying the MICs of the different pathogens, as well as the best routes of administration, depending on the type of fish (age, type, etc.).
Task 24.8 Diagnostic-recommendation manual for meager health. This will be a practical diagnostic manual and recommendation guide for meager health issues targeted to fish health specialists and aquaculture scientists and producers. The manual will be the synopsis of major findings of WP24. It will be published in electronic format (pdf file) and uploaded in the project website, and will be freely available for the public. The manual will be organized in chapters describing the major diseases of the species with original photographic material, epidemiological and pathological data.
This WP will address a number of identified bottlenecks relating to meagre health. The tasks include (a) studies of key disease states, (b) development of appropriate treatments, and (c) a first characterisation of the meagre immune system/ immune responses required for future immune intervention.
Task 24.1. Systemic Granulomatosis. The most important disease of cultured meagre is a newly described condition under this provisional name (Katharios et al., 2011). It is characterized by multiple systemic visceral granulomas and is manifested progressively by calcified and necrotic liver and kidney. The aetiology of the disease is unknown however there is evidence that it may be a metabolic disorder. To investigate the hypothesis that the disease is related to nutrition we will run several feeding trials to assess the effect of vitamin D and Ca/P levels in feeds, the effect of low/high protein levels in feeds and Mg inclusion, the effect of plant ingredients in the feeds, as well as the effect of minerals and vitamins levels.
Sub-task 24.1.1. Feeding trials
- Trial 1. The effect of vitamin D (3 levels) inclusions in diets will be examined in the development of Systemic Granulomatosis (SG). Meagre fry (1-5 g) will be used for this trial. Growth, feed efficiency, body composition, vitamin D determination in target tissue and specific biomarkers will be evaluated. Samples will be provided for pathological assessment at HCMR (Sub-task 24.1.2). Together with the health and pathological assessment in Sub-task 24.1.2.
- Trial 2. The effect of Ca/P ratio in the diet will be examined, as well as the Low/High protein content of the diet will be investigated.
- Trial 3. The effect of high plant protein diets will be examined against a control fishmeal based diet on gut morphology and the potential appearance of SG. Three to five fish per tank in a non-fasting state will be sampled from each treatment for histology of intestine. Sampling will be done at the beginning of the experiment and after 30 and 60 days.
- Trial 4. The combined effect of vitamins E, C and carotenoids will be examined in SG prevention. Diets will contain several combinations of these antioxidants to determine optimum levels required to reduce or completely prevent the occurrence of SG. The effect of these nutrients on survival and growth parameters and on several biomarkers of fish metabolism in relation to these nutrients, will be also studied.
- Trial 5. The effect of Se, Mn and Fe will be examined in SG prevention. Diets will contain several combinations of these minerals in relation to the polyunsaturated fatty acid contents of the diets to determine optimum levels required to reduce or completely prevent the occurrence of SG. The effect of these nutrients on survival and growth parameters and on several biomarkers of fish metabolism in relation to these nutrients, such as oxidative stress enzymes and health, will be also studied.
Sub-task 24.1.2. Health and pathological assessment (HCMR). Depending on size, samples will include whole fish (fish <2 g) or dissected organs and tissues, which will be preserved in various fixatives. Analyses will include histology for the description of the development of SG using standard and special staining techniques (Bennett et al., 1976; McDowell and Trump, 1976), Transmission Electron Microscopy with X-ray microanalysis (Suzuki et al., 1997), Scanning Electron Microscopy (HCMR) and tissue biochemical analysis.
Task 24.2. Chronic Ulcerative Dermatopathy. This is a newly described condition affecting the lateral line canals of many cultured fishes. From previous studies, it is established that Chronic Ulcerative Dermatopathy (CUD) is directly associated with the use of borehole water (Katharios et al 2011). Two parallel rearing trials of meagre in borehole and natural seawater will be conducted in order to study the development of CUD. The development of the disease in the different environments will be studied using histological and molecular techniques. The study will also involve physicochemical analysis and comparison of the two water sources, in order to identify the aetiological agent of the condition. The analysis includes monitoring of pH, carbon dioxide, salinity and selected heavy metals
Task 24.3. Anti-parasitic treatments. According to Colorni (2004), the major diseases which confront fish farmers vary in prevalence according to the geographic latitude of each country around the Mediterranean basin. “Old” pathogens (Lymphocystis and encephalitis viruses, “Pasteurella” piscicida, Amyloodinium ocellatum) still cause serious economic losses, and diseases once considered sporadic (caused by Mycobacterium marinum, Lactococcus garvieae, Enteromyxum [formerly Myxidium] leei, Kudoa sp.) are emerging. In the case of meagre, the presence of several parasites has been reported and some bacteria have been associated with outbreaks of disease in meagre. The management and control of parasitic infections in aquaculture are a constant challenge, complicated by the current limited availability of efficacious licensed products. In addition, parasite control in aquaculture requires awareness of environmental, water quality and host parameters. Therefore, treatment with anti-parasitic drugs remains the most widely used tool for managing parasite infections in cultured aquatic species, since efficacious options for intervention other than anti-parasitic drugs remain limited. Here we propose to develop appropriate treatments which can be used in meagre by studying the host parameters in relation to chemicals already avalaible in the market and with antiparasitic properties. The study will use histological and biochemical techniques in order to study these parameters, whilst performing experiments under controlled conditions.
Task 24.4. Nocardia infection in meagre
Sub-task 24.4.1. Isolation and characterization of the pathogen. Nocardia infection has been reported recently in large cultured meagre and may pose a significant threat and bottleneck for its production, since Nocardiosis is a chronic disease which is hard to eradicate and apart from the direct mortalities may also adversely affect the final product quality. Since there is only one report in the literature for Nocardiosis in meagre and in the Mediterranean aquaculture in general, in this WP we will monitor fish from various geographic localities (Greece and Spain) for the presence of the bacterial pathogen. The screening involves isolation using standard microbiological techniques and physiological characterization based on colony morphology, bacterial morphology, Gram and Ziehl-Neelsen staining, antibiotic sensitivity, biochemical profiling using API ZYM and BIOLOG Microsystems. Isolated strains will be also genetically characterized based on DGREA analysis and sequencing of 16s Rrna following PCR with universal or Nocardia-specific primers, along with the putative virulence of the chosen strains, based on clinical data (mortalities, morbidity). With this task we will assess the existence of Nocardia in meagre and its genetic and phenotypic variability that will be important for future vaccine design.
Sub-task 24.4.2. Preparation of an autogenous vaccine. Based on the results of Sub-task 24.4.1, a monovalent or polyvalent vaccine will be prepared using formalin inactivated bacterin. The vaccine will be tested for sterility and toxicity and evaluated in vaccination/challenge experiments. Nocardia challenge will be initially optimised with the isolated strain, and subsequently this method will be used to determine the effectiveness of the pilot vaccine after prior immunisation of the fish.
Task 24.5. First characterisation of the immune system. Anticipating that future management of disease issues in meagre will require vaccines as part of the arsenal of approaches used, we will undertake a characterization of the immune system to identify key immune molecules, as potential markers of immune system development and induction of antiviral and antibacterial responses.
For markers of the adaptive immune system, we will clone RAG1/2, Ig and TcR genes from meagre, and determine their expression during development, to allow an analysis of when to vaccinate as the immune system matures.
In addition, we will clone marker genes of inflammation, antibacterial responses and antiviral response. The latter will require prior stimulation of fish/cells, since these genes show low constitutive expression but can be markedly induced upon infection. Pathogen Associated Molecular Patterns (PAMPS) will be used for this purpose and will include bacterial LPS, polyI:C. In each case qPCR assays will be established for future gene expression profiling following vaccination or immunostimulant treatment.
Task 24.6. Monitor specific immune responses. In this task we will monitor meagre specific immune responses following vaccination with a bacterin. Classically antibody responses are measured post-vaccination, and we will develop anti-meagre IgM and anti-meagre IgT antibodies for this purpose. In addition, a range cytokines associated with helper-T cell responses will be cloned to allow analysis of cell-mediated responses. Meagre will be vaccinated with a vaccine to Vibrio anguillarum, a common Gram-negative bacterium found in the marine environment, and blood/mucus samples (for antibody analysis) and tissue samples (gill/kidney) for cytokine analysis will be collected at a range of times post-vaccination and post-exposure to the pathogen.
Task 24.7. Description, diagnosis and treatment of other bacterial/viral infectious diseases occurring in meagre
Increase in meagre production, as it occurs in other warm water species, may lead to the emergence of bacterial diseases such as vibriosis or photobacteriosis and some viral diseases such as nodavirus and lymphocystis disease. In this task we will monitor meagre throughout the project to establish the seasonality of the potential diseases. External morphology, necropsies and samples of liver, spleen, kidney, gut and nervous system will be studied to determine the presence of pathogenic organisms and histopathological damage to the different organs. These samples will be processed for determination of bacterial diseases by classical microbiological techniques, and by PCR.
For viral diseases, cell cultures suited to each disease will be used for virus propagation, following by RT-PCR or PCR verification depending on the specific virus. Susceptibility of meagre will be set against field and reference strains of the following pathogens: Vibrio anguillarum, Photobacterium damselae subsp. piscicida, and betanodavirus. Susceptibility values will be set by challenge test using intraperitoneal or intramuscular injections for bacteria or nodavirus respectively, in studies conducted in a biosecurity facility. When outbreaks of disease occur, methods will be established to determine the causal agent of the disease and attempt to find appropriate preventive and therapeutic measures for control. Specific diagnosis protocols will range from molecular (PCR, RT-PCR) to antibody based (ELISA, Immunohistochemistry, Immunoflourescence) methods. Treatment protocols will be established with selected antibiotics (depending on the limitation of their use) for each pathogen isolated from natural outbreaks of disease, by studying the MICs of the different pathogens, as well as the best routes of administration, depending on the type of fish (age, type, etc.).
Task 24.8 Diagnostic-recommendation manual for meager health. This will be a practical diagnostic manual and recommendation guide for meager health issues targeted to fish health specialists and aquaculture scientists and producers. The manual will be the synopsis of major findings of WP24. It will be published in electronic format (pdf file) and uploaded in the project website, and will be freely available for the public. The manual will be organized in chapters describing the major diseases of the species with original photographic material, epidemiological and pathological data.
