justification of the fish health work planned under diversify
The most important, but also interesting disease of meagre is a condition called Systemic Granulomatosis, which affects almost 100% of cultured populations. The disease is characterized by multiple systemic visceral granulomas that manifest progressively as calcified and necrotic organs (Katharios et al., 2011a). The aetiology is unknown, but there is evidence it may be a metabolic disorder, and similar to systemic granulomas observed in cultured fish species such as gilthead sea bream (Paperna, 1987), rainbow trout (Dunbar & Herman, 1971) and turbot (Messager et al., 1986). In all cases the development of the disease has been associated with a nutritional imbalance (phosphorus levels, vitamin C deficiency, etc) or inadequacy (plant ingredients, tyrosine, etc). Systemic Granulomatosis is not associated with high mortalities, however, both prevalence and intensity are so high that this disease ranks as one of the major bottlenecks of further expansion of meagre production. The disease may lead to reduced growth and physiological performance during grow-out and, in addition, it affects the final product, making it unacceptable to the consumer. DIVERSIFY will study the development of the disease under different feeding/nutritional regimes, in order to investigate the underlying causes and to reduce its incidence in aquaculture. Since there is evidence that the cause is related to nutrition, the variety of feeding trials foreseen in these studies will provide a thorough insight on the aetiology of the disease.
Chronic Ulcerative Dermatopathy (CUD) is a newly described disease affecting the lateral line organ of freshwater and marine cultured fish, including meagre. The disease is directly associated with the use of borehole water, although the aetiological factor is still unknown. High mortalities are not associated with CUD, although it results in severe disfigurement of the fish, especially around the head where the lateral line canals are located, making the fish unacceptable for marketing. The lesions resolve when fish are transferred to surface water, either fresh or marine depending on the species. Chronic Ulcerative Dermatopathy has been reported in several marine fish (Katharios et al., 2011b), amongst which meagre seems to be one of the most vulnerable (Rigos & Katharios, 2010). Here, we propose the study of the development of the disease using two parallel rearing systems with different water sources; natural seawater and borehole water. Lesion resolution will be studied following transfer of CUD-affected fish in natural seawater. The study will involve advanced histological techniques and measurements of the abiotic factors in order to identify the causes of the disease.
A number of parasite infections have also been seen during meagre rearing, including Sciaenacotyle panceri, a monogenean found on the gills (Merella et al., 2009). These diseases are also known to cause mortality in fish farmed in the Mediterranean and require development of appropriate treatments. Here we will use a range finding test to evaluate the tolerance of fish to a variety of chemical treatments, with a view to optimize treatment via a medicated feed or medicated bath, as outlined in the call.
Very little is known about the meagre immune system and responses. Currently there are some 85 nucleotide and 19 protein sequences in NCBI for meagre, and none of these entries are for immune genes (they are mostly relatively conserved genes used for evolutionary analysis and/or microsatellites). Hence there have been no studies on the development of the immune system in this species, or on responses post-vaccination. DIVERSIFY will use information from related farmed fish species to target key immune genes for cloning, for use as markers of immunity. Anticipating that future vaccines will be required, initially the focus will be on molecules of adaptive immunity, and development of antisera to the main immunoglobulins present. Expression of these molecules will be monitored during development, and post-vaccination with a common bacterial vaccine. In addition, key antimicrobial and antiviral genes will be cloned and studied in the context of how they are regulated and whether they can be used as markers for increased resistance by dietary (e.g., immunostimulants) or other means. Health problems in meagre may be related to the use of inadequate feeds, as it occurs in other aquaculture produced species (Cooke and Sneddon, 2007, Montero and Izquierdo, 2010). Farmers are also aware of the harmful consequences of nutritionaly-unbalanced diets on fish welfare (Conte, 2004). In turn, poor welfare conditions could not only markedly reduce meagre growth as it has been seen in a closely related species (Pirozzi et al., 2009), but also negatively affect immune system and disease resistance. Imbalances in nutrition negatively affect fish welfare and health in many fish species (Lall, 2000). The importance of nutrition on welfare and health involve almost all physiological functions related to health, including a direct effect on modulation of the immune system, stress response, mechanisms of defense against infection or tissue integrity (Waagbø, 2006). Among different nutrients, deficiency in essential fatty acids or antioxidants including vitamin E and vitamin C, negatively affect fish welfare, increasing plasma cortisol levels and affecting fish behaviour and stress responses (Montero et al., 1998; Montero et al., 1999). Besides unbalanced levels of these nutrients cause inmune deficiencies, pathological features in several tissues and reduce disease resistance in several fish species (Montero and Izquierdo, 2010; Betancor et al., 2012a, 2012b) including some Sciaenids (Sealey and Gatlin, 2002). Amino acids and their metabolites have been characterized as important regulators of main physiological pathways that are required for fish maintenance, growth performance, feed utilization, protection from oxidative stress and resistance to environmental stressors and pathogenic organisms (Wilson and Halver, 1986; Li et al., 2009). Among them, lysine has a role in calcium absorption and formation of collagen, a substance important for bones and connective tissues including skin and cartilage (Civitelli et al., 1992), being essential for the health status of these tissues. Despite the importance of those nutrients on fish health, the nutritional requirements of meagre have not been yet defined particularly in relation to welfare and health issues.
In greater amberjack, the most important disease during early stages (post-larva until juvenile) is epitheliocystis, caused by intracellular chlamydia or clamydia-like microorganisms. Mortalities up to 100% have been observed in mesocosm rearings (Katharios et al., 2008). The disease has been described as acute in the literature causing mass mortalities in young cultured greater amberjack (Crespo et al., 1990) or as a chronic, non-pathogenic condition in larger wild greater amberjack (Grau & Crespo, 1991). Since treatment of the disease (oxytetracycline) is based on early diagnosis, new methods for detecting the pathogen in water are required. Chlamydia-like organisms are obligate intracellular parasites and it is speculated that they enter rearing water using an invertebrate host as a vehicle. Since the production of greater amberjack will be based on mesocosm technologies where water at certain stages is not treated or disinfected, epitheliocystis will constitute a major bottleneck for juvenile production. Here we propose the development of molecular probes for the detection and early diagnosis of pathogenic Chlamydia and Chlamydia-like organisms in the rearing water.
A variety of other bacterial diseases has also been noted during greater amberjack larval culture and may also be considered potential bottlenecks for production. For example, high mortality of Seriola spp. has been reported to be associated with bacterial pathogens in Australia (Stephen & Savage, 2010), and different bacterial species such as Streptococcus dysgalactiae (Nishiki et al., 2010) or Lactoccocus garvieae (Kawanishi et al., 2005) have been isolated from greater amberjack and yellowtail (Seriola quinqueradiata) in Japan. To better characterise this problem, larvae will be reared under different dietary regimes and the incidence of different bacterial diseases will be determined. The most important diseases will be typed and cultures stored. Diets (from WP 9 Nutrition – greater amberjack and WP 21 Grow out husbandry – greater amberjack) that promote better larval health will be identified.
Greater amberjack juveniles suffer from a number of parasitic diseases during grow out, which require the development of treatments. Here, we will modulate juvenile resistance to parasite infection through dietary means, where fish will be given diets enriched with nucleotides, vitamins or immunostimulants. The impact on mucosal defences and on parasite resistance will be determined, by biomarker analysis and morphological studies of gills, skin and intestine.
In relation to the development of treatments for increased disease resistance, it is essential to have some immune markers that can aid selection of the best treatments and to optimise their delivery. As with meagre, there are very few known immune markers currently in greater amberjack, although a partial sequence for RAG-1 exists and in yellowtail nearly 4,000 ESTs are in GenBank, with many used in a recent microarray experiment by Darawiroj et al. (2008). The latter include some immune genes, such as TcRα, Ig heavy and light chain, MHC class II beta chain, and two cytokines (IL-1β and a CC chemokine), which will aid in cloning the equivalent greater amberjack genes. DIVERSIFY will expand the immune gene markers to include those useful for mucosal immunity and study their expression at mucosal sites following the different antiparasite and antibacterial treatments, at different developmental stages.
Chronic Ulcerative Dermatopathy (CUD) is a newly described disease affecting the lateral line organ of freshwater and marine cultured fish, including meagre. The disease is directly associated with the use of borehole water, although the aetiological factor is still unknown. High mortalities are not associated with CUD, although it results in severe disfigurement of the fish, especially around the head where the lateral line canals are located, making the fish unacceptable for marketing. The lesions resolve when fish are transferred to surface water, either fresh or marine depending on the species. Chronic Ulcerative Dermatopathy has been reported in several marine fish (Katharios et al., 2011b), amongst which meagre seems to be one of the most vulnerable (Rigos & Katharios, 2010). Here, we propose the study of the development of the disease using two parallel rearing systems with different water sources; natural seawater and borehole water. Lesion resolution will be studied following transfer of CUD-affected fish in natural seawater. The study will involve advanced histological techniques and measurements of the abiotic factors in order to identify the causes of the disease.
A number of parasite infections have also been seen during meagre rearing, including Sciaenacotyle panceri, a monogenean found on the gills (Merella et al., 2009). These diseases are also known to cause mortality in fish farmed in the Mediterranean and require development of appropriate treatments. Here we will use a range finding test to evaluate the tolerance of fish to a variety of chemical treatments, with a view to optimize treatment via a medicated feed or medicated bath, as outlined in the call.
Very little is known about the meagre immune system and responses. Currently there are some 85 nucleotide and 19 protein sequences in NCBI for meagre, and none of these entries are for immune genes (they are mostly relatively conserved genes used for evolutionary analysis and/or microsatellites). Hence there have been no studies on the development of the immune system in this species, or on responses post-vaccination. DIVERSIFY will use information from related farmed fish species to target key immune genes for cloning, for use as markers of immunity. Anticipating that future vaccines will be required, initially the focus will be on molecules of adaptive immunity, and development of antisera to the main immunoglobulins present. Expression of these molecules will be monitored during development, and post-vaccination with a common bacterial vaccine. In addition, key antimicrobial and antiviral genes will be cloned and studied in the context of how they are regulated and whether they can be used as markers for increased resistance by dietary (e.g., immunostimulants) or other means. Health problems in meagre may be related to the use of inadequate feeds, as it occurs in other aquaculture produced species (Cooke and Sneddon, 2007, Montero and Izquierdo, 2010). Farmers are also aware of the harmful consequences of nutritionaly-unbalanced diets on fish welfare (Conte, 2004). In turn, poor welfare conditions could not only markedly reduce meagre growth as it has been seen in a closely related species (Pirozzi et al., 2009), but also negatively affect immune system and disease resistance. Imbalances in nutrition negatively affect fish welfare and health in many fish species (Lall, 2000). The importance of nutrition on welfare and health involve almost all physiological functions related to health, including a direct effect on modulation of the immune system, stress response, mechanisms of defense against infection or tissue integrity (Waagbø, 2006). Among different nutrients, deficiency in essential fatty acids or antioxidants including vitamin E and vitamin C, negatively affect fish welfare, increasing plasma cortisol levels and affecting fish behaviour and stress responses (Montero et al., 1998; Montero et al., 1999). Besides unbalanced levels of these nutrients cause inmune deficiencies, pathological features in several tissues and reduce disease resistance in several fish species (Montero and Izquierdo, 2010; Betancor et al., 2012a, 2012b) including some Sciaenids (Sealey and Gatlin, 2002). Amino acids and their metabolites have been characterized as important regulators of main physiological pathways that are required for fish maintenance, growth performance, feed utilization, protection from oxidative stress and resistance to environmental stressors and pathogenic organisms (Wilson and Halver, 1986; Li et al., 2009). Among them, lysine has a role in calcium absorption and formation of collagen, a substance important for bones and connective tissues including skin and cartilage (Civitelli et al., 1992), being essential for the health status of these tissues. Despite the importance of those nutrients on fish health, the nutritional requirements of meagre have not been yet defined particularly in relation to welfare and health issues.
In greater amberjack, the most important disease during early stages (post-larva until juvenile) is epitheliocystis, caused by intracellular chlamydia or clamydia-like microorganisms. Mortalities up to 100% have been observed in mesocosm rearings (Katharios et al., 2008). The disease has been described as acute in the literature causing mass mortalities in young cultured greater amberjack (Crespo et al., 1990) or as a chronic, non-pathogenic condition in larger wild greater amberjack (Grau & Crespo, 1991). Since treatment of the disease (oxytetracycline) is based on early diagnosis, new methods for detecting the pathogen in water are required. Chlamydia-like organisms are obligate intracellular parasites and it is speculated that they enter rearing water using an invertebrate host as a vehicle. Since the production of greater amberjack will be based on mesocosm technologies where water at certain stages is not treated or disinfected, epitheliocystis will constitute a major bottleneck for juvenile production. Here we propose the development of molecular probes for the detection and early diagnosis of pathogenic Chlamydia and Chlamydia-like organisms in the rearing water.
A variety of other bacterial diseases has also been noted during greater amberjack larval culture and may also be considered potential bottlenecks for production. For example, high mortality of Seriola spp. has been reported to be associated with bacterial pathogens in Australia (Stephen & Savage, 2010), and different bacterial species such as Streptococcus dysgalactiae (Nishiki et al., 2010) or Lactoccocus garvieae (Kawanishi et al., 2005) have been isolated from greater amberjack and yellowtail (Seriola quinqueradiata) in Japan. To better characterise this problem, larvae will be reared under different dietary regimes and the incidence of different bacterial diseases will be determined. The most important diseases will be typed and cultures stored. Diets (from WP 9 Nutrition – greater amberjack and WP 21 Grow out husbandry – greater amberjack) that promote better larval health will be identified.
Greater amberjack juveniles suffer from a number of parasitic diseases during grow out, which require the development of treatments. Here, we will modulate juvenile resistance to parasite infection through dietary means, where fish will be given diets enriched with nucleotides, vitamins or immunostimulants. The impact on mucosal defences and on parasite resistance will be determined, by biomarker analysis and morphological studies of gills, skin and intestine.
In relation to the development of treatments for increased disease resistance, it is essential to have some immune markers that can aid selection of the best treatments and to optimise their delivery. As with meagre, there are very few known immune markers currently in greater amberjack, although a partial sequence for RAG-1 exists and in yellowtail nearly 4,000 ESTs are in GenBank, with many used in a recent microarray experiment by Darawiroj et al. (2008). The latter include some immune genes, such as TcRα, Ig heavy and light chain, MHC class II beta chain, and two cytokines (IL-1β and a CC chemokine), which will aid in cloning the equivalent greater amberjack genes. DIVERSIFY will expand the immune gene markers to include those useful for mucosal immunity and study their expression at mucosal sites following the different antiparasite and antibacterial treatments, at different developmental stages.
