Aetiologic Agents of Fry Mortality Syndrome in the Rainbow Trout (Oncorhynchus Mykiss) in Iran

An investigation was conducted in order to find out the etiological factors of Fry Mortality Syndrome (FMS) that causes serious economical loss in rainbow trout farms in Iran. In recent years obscure fry mortalities have been observed in many hatchery farms in Iran. It was reported that the rate of fry and juvenile mortality increased dramatically in some provinces e.g. 23 million fry were produced in hatchery centers of Chahar Mohal Bakhtiary province in 2002 but nearly 21 million fry (91.3%) in different stages of growth died before distribution to farmers. Also close to 23 million fry were produced in Mazandaran province, but 12 million fry equivalent to 52.12% of total fry production died mysteriously.This investigation was carried out with objectives of detecting and confirming the main causative agent that contribute to the occurrence of Fry Mortality Syndrome in Iran. During 32 months, from October of 2001 until May of 2004, 52 different hatchery centers and rearing farms of rainbow trout (Oncorhynchus mykiss) which were located in Tehran, Mazandaran, Guilan, Fras, Markazi, Kerman and Kohkiloyeh Boyerahmad provinces, were visited and various samples from affected farms were collected. Collected samples consisted of ovarian fluid, milts, eggs, eyed-eggs, larvae, fry < 1 g and 1-3 g as well as internal organs from adult fishes. A total of 2,107 samples were collected from farms in six provinces and were examined by five methods such as virology (410 samples), bacteriology (899 samples), serology (consisted of IFAT: 392 samples and ELISA: 44 samples), histopathology (160 samples) and hematology (202 samples). Some of the mentioned approaches such as fish cell culture, ELISA and IFAT techniques were set-up and optimized for the first time in Iran. The clinical signs of suspected fishes were darkening, exophthalmia, ascites, abnormal swimming and whirling. From 410 samples that of tissues inoculated on to cell cultures two samples showed CPE in EPC and BF-2 cell lines which were inoculated with ovarian fluid from broodstock obtained from hatchery farms in Mazandaran province. The CPE was similar to IHN virus induced. The CPE foci revealed dying cells congegrated as grape-like clusters (ballony performance with cytolysis).TEM findings in infected cells showed bullet-shaped particles having sizes of 130-180 nm in length and 65-70 nm in diameter. From the virion morphology it was suggested that observed particles were similar to Rhabdovirus. FAT examination revealed that all samples were examined with MAbs and PAbs against IPNV and VHSV were negative. On the other hand, two samples were positive when examined with MAbs and PAbs against IHNV. These smears were originated from samples that had showed CPE in EPC and BF-2 cell lines and bullet shaped particles in electron microscopy. ELISA findings (cut-off value, optical density and detectionlevel percentage) showed that IHNV had higher percentage of detection with 23.25% in comparison with other relevant viral diseases i.e. IPNV with 7.31% and VHSV with 14.29%. Results of histopathological study on the sampled fry revealed that the target tissues in the kidney, liver, spleen, hepatopancreas, intestine and gills showed different degree of tissue changes beginning from cell degeneration to complete necrosis. There were also renal blood vessels congestion, marked degenerative changes in posterior kidney with tubular necrosis and interstitial hematopoeitic tissue degeneration. In addition, interstitial degeneration and oedema in anterior portion of kidney, focal necrosis in the tubular area and several stages of cell necrosis in the hematopoeitic tissue were the most important histopathological changes seen in kidney tissues examined. Hepatopancreatic tissues also revealed marked changes such as congestion, atrophy and necrosis of pancreatic acinar cells and Islets of Langerhans. Spleen samples revealed spleenic congestion, severe necrosis, hemosiderosis and increased presence of melanomacrophage centers (MMC). Gills tissue in sampled fry showed hyperplasia, clubbing and fusion of lamellae. Hematological findings revealed that total white blood cell count, i.e. lymphocyte and neutrophil in investigated fish showed significant increased compared with the control fish (p< 0.05). On the contrary, all the samples showed a decreased in RBC, Hb and HCT values. In addition, MCHC and total protein plasma showed a marked decreased (p<0.05). In the blood serum components analysis, similarly it was revealed LDH and AST showed a significant decreased (p<0.05). In conclusion, with marked clinical signs, cell culture observation and TEM findings, ELISA and IFAT results, histopathology and hematological findings (blood and biochemical parameters) seen in the current investigation lead to possibility of a viral disease agent infection as the cause of fry mortality syndrome in the hatchery and rearing trout farms in Iran. From findings of the current study, it is concluded that IHN-like virus could be most probable etiologic of fry mortality syndrome in Iran. Key words: Fry Mortality Syndrome, Rainbow trout, Cell culture, TEM, ELISA, IFAT, Histopathology, Hematology, IHNV, IPNV, VHSV, Ira

Conservation and restoration of Caspian salmon (Salmo trutta caspius) stocks in the southern Caspian

The project is funded by the CASPECO Project which is the Third Phase of the GEF supported project in the Caspian Sea region titled “The Caspian Sea: Restoring Depleted Fisheries and Consolidation of a Permanent Regional Environmental Governance Framework”, and implemented by the Iranian Fisheries Research Organization (IFRO) in partnership with four Coastal Research Centers affiliated to IFRO, Coldwater Fishes Research Centre (CFRC).The project's objective is to improve the conservation and restoration of Caspian Salmon (Salmo trutta caspius) stocks. In accordance with IUCN criteria Caspian Salmon is placed in the red list of endangered fish. In the recent decades the destruction of habitats for juveniles, reduction of genetic diversity and fish immune system were main causes of the Caspian salmon decline. Numbers of public meetings were held with participation of fishing communities’ representatives, experts, relevant local authorities and experts of the IFRO and Iranian Fisheries Organization (IFO), Iran Environment Protection Organization (IEPO), Water Resource Management (Ministry of Energy) and University senior professors, in order to create interaction and understanding of the project objectives. Educational awareness and extension program were conducted with the involvement of fisheries communities, private sector and NGO associations. The results of the project suggests that cross-sectoral coordination for sustainable fisheries can be achieved through building partnerships and enhancing communication between the environment, private, fisheries sectors and academia. Cross-sectoral cooperation through this project improved public awareness on growing risks facing Caspian Salmon from different sources including local sources (illegal fishing and over-fishing, urban wastewater, environment destruction, industrial pollutants, pollution of air and water resources), new emerging and invasive organisms, climate change, implications of the Caspian salmon population decline on economic and social affairs of the people who depend on them for their livelihood and food security and their role in fish stock protection. The project, through its capacity building, extension and educational programs and efforts to link science to management, could translate scientific findings into an action agenda for managers and policy makers that can bring an improvement in the future of the Caspian Sea salmon stock

A survey on health status of coldwater rearing and hatcheries fish farms in Iran (Mazandarn, Gilan, Ardebil, West Azerbaiejan, East Azerbaiejan and Kordestan provinces)

The national research plan with title of "Study on health status of Iran Coldwater hatchery and rearing farms" was done in order to identification and tracking of main causative agents of recent mortality in Iran Coldwater hatchery and rearing farms and recognize of infected zones and design of landscape distribution of Epizootic Coldwater fish diseases in the country. This plan was conducted parallel and in same time in some polar provinces of Coldwater fish production such as Mazandaran, Gilan, Ardebil, West Azerbaijan, East Azerbaijan and Kurdistan in three years from 2006-2008. In this regard, about 23,21 and 20 farms in Mazandaran province, 30 farms in West and East Azerbaijan and Kurdistan provinces and 19 farms in Gilan and Ardebil were selected as randomly and tracking were done yearly from 2006-2008. This research was conducted according to regular planning consists of farms visit that was done according to statistical plan and completion of Questionnaires and sampling. The findings in Mazandaran province revealed that fish infections background and infectious diseases were increased in period of 2006-2008. In fact, 56% of all fish farms in 2006, 71% in 2007 and 85% in 2008 were recorded as infected farms. In other side, average weight of fish final products was more 500gr in consume market. So, regarding to long period of fish culture in mentioned fish occurrence of more morbidity could be expected. The streptococcus infections were most important fish bacterial diseases that have more incidence and pathogenicity in collected questionnaires. Also it revealed more occurrences in summer season in above 15oC temperature in affected fish farms. Also, Enteric Redmouth Disease (ERM) and Saprolegniasis were reported as second and third degree in examined farms. Meanwhile, findings of control, prevention and treatment of our survey revealed that using of antibiotics and detergent materials were increased in mentioned province. Indeed, 34.7% of all fish farms in 2006, 71.4% in 2007 and 75% in 2008 have applied treatment operation and using of detergent materials in affected farms. In other side, frequent using of Erythromycin antibiotic was reported several times in 2008 so fish farmer faced to Bacterial resistance and should be using from Florfenicol as alternative antibacterial. Also, in feed sampling from all fish farms about 3 fish farms in 2006 and 5 fish farms in 2008 were faced with food poisoning that originated from high TVN and peroxidase in fish food consumed. These farms revealed mass mortality that stopped after change feeding regime. In conclusion absence of fish health management could be considered as main agent of mentioned mortality in Mazandaran province. The findings of our survey in West and East Azerbaijan and Kurdistan revealed that absence of sufficient experiences in fish farmers and their neglect from water quality concepts, farm cleanness, using of suitable detergent materials for fish ponds and instruments and disability in continuous monitoring of physic-chemical factors of consumed water could be considered as most important problems. So, fish morbidity and mortality, growth decrement and low production rate were expected. Unfortunately, despite the occurrence of infectious and non- infectious diseases in examined fish farms, there were no documents and information about diseases history, clinical signs in dead fish, feeding regime, fish mortality (rate of daily mortality, age and weight of moribund fish), treatment operation, drugs usage, growth rate, physico-chemical factors and fish density. So planning for control and prevention of mentioned diseases were unsuccessful in affected fish farms. Also, occurrence of some epizootic fish viral diseases such as VHS, IPN and IHN were observed frequently with mass mortality about 40100% in some examined fish farms that produced some tragic economic lost in the mentioned provinces. Similarly, some bacterial, fungal and parasitic diseases were observed repeatedly in some inspected farms such as Flavobacterium, streptococcus infections. Also, some unknown causative agents in parasitic diseases were observed several times such as Dactylogyrus, Gyrodactylus, Ichthyophthirius multifiliis, Trichodina, Chilodonella, Diplostomum and fungal diseases such as Saprolegniasis. In conclusion it seems that absence of fish health management, inadequate rate of hygienic technical services, absence of quarantine programs for transfer of new eyed-eggs, juvenile and live broodstocks packages without health certificates from authorized organizations, uncontrolled entry of foreign eyed-eggs (France, Denmark, Australia and Armenia) and neglect in using of suitable detergent materials for fish ponds, instruments, consumed water and infected eggs, not screening of broodstocks in hatcheries could be considered as most important causative agents in occurrence of infectious diseases and main problems in affected farms in mentioned provinces. Similarly, our findings in Gilan and Ardebil provinces revealed that their problems were similar to other provinces. In fact, neglect in establish of necessary Infrastructures in fish farms and dereliction in health management concepts could be considered as main reasons of occurrence of fish infectious and non-infectious diseases in studied fish farms in mentioned provinces. In fact, often fish farms visited hadn't suitable structures without control and prevention approach. There were not observed detergent using, quarantine programs and prevention methods. Entrance of unknown persons, birds and wild animals were ordinary in mentioned farms. Unfortunately level of farmer’s knowledge was low and no training courses and extension programs were planned by authorized organizations. Meanwhile, food storages were unsuitable and food packages were stored in inappropriate situation so poisoning conditions were increased in examined farms. In conclusion it could be finalized that Coldwater fish farms in Gilan and Ardebil provinces situated in insufficient position and correction of current situation are needed urgently. In final elicitation, it would be mentioned that lack of basic infrastructures could be introduced as most important reasonfor spread of diseases, mortality and related economic losses in studied provinces. Therefore, attention to environmental affairs and access to Sustainable Development are recommended. Also consideration of biosecurity regulations and health management concepts would be important requirements for modification and reformation of Coldwater fish farms in examined provinces towards A Better Tomorrow

Study on viral nervouse necrosis (isolation, characterisation and pathogenesis) in golden grey mullet in the Caspian Sea and study of pathogenecity and possibility of transmission to the other fish species (Sturgeon fishes, Rutilus frisii kutum and reared Rainbow trout and Carp)

Study on Viral Nervouse necrosis (isolation, characterisation and pathogenesis) in Golden grey mullet in the Caspian Sea and study of pathogenecity and possibility of transmission to the other fish species (Sturgeon fishes, Rutilus frisii kutum and reared Rainbow trout and Carp) Viral Nervous Necrosis (VNN) is a worldwide disease affecting several species of cultured marine fish. For the past two decades, betanodavirus infections that cause Viral Nervous Necrosis (VNN) have emerged as major constraints on the culture and sea ranching of marine fish in almost all parts of the world. More than forty species mainly of marine origin have been so far affected and this number is likely to rise in future following the introduction of new species and the increase of aquaculture trade. Unknown acute mortality occurred in wild golden grey mullet Lisa auratus and Liza saliens in Iranian waters of Caspian Sea in recent years. In order to isolation and confirmation of causative agents of golden grey mullet mortality in the Caspian Sea, a complementary research investigation project was designed in 2005 and approved immediately in Iranian Fisheries Research Organization (IFRO). Many diagnostic aspects such as Virology (Cell culture and Elctereone Microscopy), Hemathology, Bacteriology, Histopathology, Molecular biology (NestedRT-PCR), Heavy metals measuerment and Serology (IFAT and IHC) were employed in mentioned multidiciplinary project. About 322 moribund fish samples which revealed skin darkening, erratic swimming behavior such as spiral and belly-up at rest and high distention of swimming bladder. Suspected samples were collected from coastal capture sites in iranian north proviences in 2006 till 2009. Targets tissue such as brain and eye were removed in strile condition and then kept in -80oC frezzer for cell culture and Nested-RT-PCR. Other tissue samples from liver, kidney, intestine, stomach, gill, skin and muscle, gall bladder and gonads were taken and fixed in 10% buffer formalin and same parts fixed in glutaraldehyde 3% for histopathology, IHC and EM respectively. Cytopathic effect (CPE) was observed in those cell cultures just six days after inoculation with the dilutions of the tested 312 homogenate supernatants. CPE in monolayers of cells cultured (SSN-1 cell line) was characterised by thin or rounded, refractile, granular cells with vacuoles. Nine samples were positive in virology assay. Nested- RT-PCR was done on suspected tissue samples and supernatant of CPE positive samples and 21 tissue samples and all CPE positive samples were positive. IFAT was selected as a confirmatory method for identifying viral strains replicating on cell cultures and carried out with rabbit anti-betanodavirus serum on suspected tissue samples and some smears of CPE positive samples. Some bright points approved betanodavirus antigen and confirmed cell culture and Nested-RT-PCR findings. In fixed tissue samples widespread and massive vacuolation were observed in brain, spinal cord, retina and optical nerve. In order to confirmation of diagnostic findings , IHC was done with monoclonal antibody antibetanodavirus and some red-brown points were observed. Theses findings revealed expected viral antigens and confirmed previous results. Moreover, virus particles with 25-30 nm in diameter were visualized in infected brain and retina using positive staining in TEM. Also pathogenicity test was employed to confirm the obtained results. So Guppy fish Poecilia reticulata and sturgeon fry were used instead of the experimental host due to ease of handling and susceptibility. After 15 days post infection, guppy bathed in VNN-infected tissue culture with 104 TCID50 showed clinical signs similar to naturally infected Golden grey mullet, and the mortality rate reached up to 100% in 75 dpi. When target organs were examined by cell culture isolation, serology, and histopathology, all revealed the presence of virus in the Guppy. Suspected supernatant injected to sturgeon fry through intravitreous injection and widespread vacuolation were observed in brain and spinal cord buy IHC and Real time PCR were negative. In conclusion, with attntion to obtained results in this investigation such as ecological factors, clinical signs, histopathological, virological and bacteriological results, molecular analysis, (IHC, IFAT, PCR), TEM demonstration, serological and hematological findings, it could be confirmed that VNNV was the main causative agent for disease outbreak in Golden grey mullet in Southern coastline of Caspian Sea