UV-B impacts on morphology and retina of Oncorhynchus mykiss larvae

The harmful effects of ultraviolet radiation on aquatic animals, due to ozone layer reduction, have been long studied in recent years. Exposure of Rainbow Trout larvae (Oncorhynchus mykiss) to Ultraviolet-B radiation (UV-B) at different doses (68.75 µw/cm^2 and 94.83 µw/cm^2 as the minimum and maximum dose of UV-B in natural environment respectively) for 15 minutes once a day in dark condition in comparison to control group (without any solar or UV) showed a wide variety of body abnormalities and eye damages. Body curvature, yolk sac edema, fin blistering, dwarfism, eye and head abnormalities as morphological malformations were revealed during the experiment, none of the malformations were observed in control group. Histopathological changes in retina such as irregular and discontinues pigmented epithelium, necrosis of photoreceptors and degeneration of nucleus layers confirmed the destructive effects of UV-B radiation in the eyes of Rainbow Trout. Such changes in larvae can be valid as bio-indicator for pollution and UV radiation and also introduce fishes as model for toxicological studies

Isolation and confirmation of viral nervous necrosis (VNN) disease in golden grey mullet (Liza aurata) and leaping mullet (Liza saliens) in the Iranian waters of the Caspian Sea

The present study was conducted on 428 moribund mullet fish samples to isolate and identify the causative agent of a mysterious acute mortality which recently occurred in wild mullets in Iranian waters of Caspian Sea, suspected to be due to viral nervous necrosis (VNN) disease. Disease investigation was carried out employing various diagnostic procedures such as virology, bacteriology, parasitology, haematology, histopathology, IFAT, IHC and nested RT-PCR. Brain and eye samples of affected fishes were collected in sterile conditions and then kept at -80 °C for cell culture isolation and nested RT-PCR detection of the causative agent. Other tissue samples were also collected and fixed for histopathology, IHC and EM examinations. CPE was observed in cell cultures at 6 days after inoculation. Nine samples were found positive with virological assay. Nested RT-PCR, performed on suspected tissues and CPE positive samples, showed that about 21 tissue samples and all the CPE positive samples were positive for VNN virus (VNNV). IFAT was selected as a confirmatory method for detecting the presence of Betanodavirus antigen, cell culture isolation results and nested RT-PCR findings. Moreover, VNNV particles with 25-30 nm in diameter were also visualized in the infected brain and retina. In pathogenicity studies, guppy fishes bathed in VNNV-infected tissue culture (10-4 TCID50) showed clinical signs similar to naturally infected mullet after 15 days post infection (dpi), with mortality rates reaching up to 100% at 30 dpi. Affected organ samples as examined by cell culture isolation, IFAT, IHC and histopathology, revealed the presence of VNNV in the guppy fishes. In conclusion, it was confirmed that VNNV was the main causative agent for the disease outbreak in mullet fish in the Caspian Sea, and this is such first official report of VNN disease from Iran

Hygienic monitoring of freshwater crayfish (Astacus leptodactylus) on Aras Lake reservoir

Aras dam reservoir situated in the northwest of Iran, west Azarbaijan province, is the only water resource of Astacus leptodactylus harvest in the country that more than 250tons of this species were exported to different countries all over the world, annually. On the other hand, one of the polices of Iranian Science Fisheries Institute is the release of this species into other water resources in the country and for this purpose, the study of risky diseases such as Crayfish pest (Aphanomysis astasi) and other zoonotic diseases are considered as the research priorities of aquaculture development of the country. This study was carried out to health screening of Astacus leptodactylus at Aras dam reservoir from winter 2013 to fall 2014. In this regard, A total of 394 harvested livefreshwater crayfish Astacus leptodactylus (255males, 139females) weretested. 9 epibionts and parasites peritrich protozoans were identified. From Metazoan parasites group, Branchiobdella kozarovi with incidence rate of (100%) in obtained samples was the only isolated organism from this group that identified up to species level. There was a heavy damage in gills of samples with Aeolosoma hemprichi (Annelid) in winter with90% prevalence. Furthermore, Other Epibiont fouling organisms such as Rotatoria; free living nematods and suctoria were observed in this survey. The fungi study of the lesions and melanized spots of mentioned samples revealed their infection to Penicillium expansum; Aspergillus flavus; Alternaria sp. ; Fusarium sp. and Saprolegnia sp. The results of bacterial study confirmed the presence of pathogen bacteria in Astacus leptodactylus. The most frequency percentage (15.16%) in hepatopancrease were related to Aeromonas hydrophila and the least one (1.37%) were due to Yersinia bacteria. Also, only Aeromonas hydrophila and Staphylococcus aureus were isolated and identified from heamolymph, respectively. The results revealed that the combination of Salmonella typhi, Escherichia coli and Staphylococcus sp. has caused the most infection rate while. Yersinia ruckeri and Salmonella typhi has caused the least infections in Astacus leptodactylus. According to the isolation of 6 bacteria species from hepatopancreas and 2 species from heamplymph , it can be concluded that hepatopancreas enjoyed the higher infection rate compared to haemolymph in the obtained samples

Investigation on probable viral agents associated with grass carp and silver carp mortality in Guilan province

Warm water aquaculture development along with import of eyed eggs, Broodstocks and ornamental fish also transferring of fingerlings and broodstocks from one province to another one has led to spreading of some unwanted diseases. According to the Gilan Fisheries Department report, mortality rate of silver carp(Hypophthalmichthys molitrix) in the summer of 2010 caused losing of silver carp production more than 1162 tons which was valued over than twenty-three milion rials that was increased to about 40 million rials in 1391. Also grass carp had a less mortality rate in some farms. Observed symptoms and severity of losses increased the probability of viral diseases such as spring viremia of carp, koi herpes virus and grass carp reovirus. In this research, totally 411 silver carp and grass carp with 5 to 2300 gram weight from 83 farms were sampled during 2011-2012. Clinical symptoms was different in various farms and subcutaneous hemorrhages on the flanks, red fin base or total fin and Red belly and operculum, mild exophthalmia, hemorrhaging in the abdominal cavity (yellow, transparent or red fluid) and petechial haemorrhages of skin and viscera and Ascites in abdominal cavity and some silver carps and grass carps were observed with palied liver and gill. In some cases swelling muscle was observed in Silver carp. Several fries with the severe emaciation were observed. Difference between clinical symptoms can indicate the presence of different causes of mortality in different farms. Various water colors from completely transparent to dark green, yellow or slightly brown observed in different pool, which shows the difference in overall culture management including water quality management. The minimum Water turbidity using secchi disk was 40 centimeters to one meter page in the fields. Source semi-deep wells or deep pools that were used as water source. Average water temperature, oxygen and pH in fileds 26/5-31 °C , 4/3-5/7 mg / L and 7/4-9/38. In order to virological surveys, sampling of internal organs, including kidneys, and spleen were done.and Homogeneous target tissues after spending 0/45 micron filter on EPC and BF2 cell line were inoculated to observe cell damage(CPE) in case of confirmation of antibody detection methods brilliant, RT-PCR and PCR done. In cell culture examination, a kind of cell damage (CPE-like changes) was observed after inoculating of 3 samples of Silver carp those were suspected to SVC, but in the second and third passages there were no sign of cell damage. It may be because of toxic effect of tissue filtrate on the cell monolayer. Concerning sample of Grass carp on two EPC and BF2 cells no evidence of cell damage was found. In indirect fluorescent antibody test, 72 hours after inoculation of tissues filtrates of of silver carp and grass carp samples on EPC, no positive reaction was observed. PCR and RT-PCR tests using specific primer pairs were done to test all of the silver carp and grass carp samples for SVC and KHV diseases Also for GCRV in grass carp samples. In addition, simultaneously commercial PCR kits applied for testing of SVC and KHV (IQ2000. Taiwan). The results of PCR and RT-PCR tests showed no evidence of robdovirus Carpio and grass carp reovirus RNA and no sign of koi herpesvirus DNA in tested samples. In conventional PCR and RT-PCR tests Using specific primer pairs none of positive bands related to SVCV(470 bp) and GCRV(292bp, 697bp and 320bp) were not confirmed in the tested samples. Also in electrophoresis of PCR products using IQ2000 kit revealed 471 bp and 640 bp negative bands in all samples and the positive bands were not observed. For confirmation of the results, three suspected samples were sent to Europe Union Reference Laboratory and none of mentioned diseases were confirmed. Simultaneously bacteriolocical examination applied for 26 using blood agar. In bacterial culture and related analyzes 6 isolates of Pseudomonas and 21isolates of Aeromonas hydrophila were detected from 26 farms while no virus was confirmed in the same samples. Based on the results of cell culture, PCR, RT-PCR and IFAT tests no one of SVCV, KHV and GCRV viruses were confirmed in tested samples. Therefore, the etiological reason of the mortalities in the tested farms was not viral diseases and should be prevent and control by biosecurity and health management in the farms

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

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

Diversity and community composition of ichthyofauna at Konhaye Stream, district Dir Lower, Pakistan

This study was undertaken to investigate the diversity, abundance ratio, and distribution of the fish species and to record the water quality of Konhaye Stream, district Dir Lower, Pakistan. A total of 16 fish species were recorded, belonging to 4 orders (Cypriniformes, Channiformes, Siluriformes and Mastacebilformes), and 5 families including Cyprinidae (Schizopyge esocinus, Racoma labiata, Cyprinion watsoni, Cyprinus carpio, Barilius pakistanicus, B. vagra, B. modestus, Crossocheilus diplocheilus, Garra gotyla, Puntius ticto, and P. sophore), Channidae (Channa punctatus and Channa gachua), Nemacheilidae (Schistura macrolepis), Sisoidae (Glyptothorax punjabensis), and Mastacembelidae (Mastacembelus armatus). Different ichthyo-diversity indices [Simpson's biodiversity index (D=0.918), Simpson's reciprocal index (1/D=1.088), Simpson's evenness index (E-1/D=0.068), species richness (S), Shannon-Weiner's index (H'=3.775), Menhinick's index (D-mn=0.804), and Margalef's index (D-mg=2.510)] were calculated for the stream. The physicochemical parameters [temperature (23.125 +/- 3.514 degrees C), dissolved Oxygen (9.003 +/- 0.627 mg L-1), pH (7.333 +/- 0.201), turbidity (76.5 +/- 6.403 NTU), electric conductivity (201.68 +/- 11.31 mu s cm(-1)), free CO2 (124.75 +/- 9.912 ppm), total dissolved solids (126.1 +/- 9.477 ppm), total alkalinity (4.325 +/- 0.171 mg L-1), total suspended solids (127.1 +/- 6.864 ppm), total hardness (5.225 +/- 0.341 mg L-1), salinity (42.25 +/- 6.292 ppt), calcium hardness (2.975 +/- 0.670 mg L-1), magnesium hardness (105 +/- 9.954 mg L-1), potassium (0.0145 +/- 0.001 mg L-1), sodium (16.55 +/- 3.861 mg L-1), chloride (1.825 +/- 0.727 mg L-1), and nitrate (0.053 +/- 0.006 mg L-1) level] were monitored and found to be in varying but permissible ranges. The stream was found to be harboring a number of economically valuable fish species. An exotic fish species, C. carpio, was found well flourished, indicating the potential of the stream to be used for mitigating the declining ichthyofaunal diversity in the main nearby rivers. Two species, P. sophore and P. ticto, were recorded for the first time from the district. Proper management, appropriate fish stocking, and implementing environmental/fishing laws are suggested for the maintenance of the diversity and alleviating anthropogenic stress/threats