Assessment of some specific and nonspecific immune responses of beluga (Huso huso) following exposure to organophsphte diazinon

Nowadays, the offspring of sturgeon species in the Caspian Sea is under danger because of biological and non-biological impacts such as pollution and illegal catching as well as dams obstacles of fish migration to the upstream of the rivers for the spawning. One of the most practical way to protect and maintain the natural stocking of these valuable species in the sea is artificial propagation and releasing of the produced larvae into the sea and the entering rivers as Iran Fishery Organization is currently producing about 22 million larvae per year according to the forth national plan of the country. However, protecting these natural resources of these species from the toxic chemicals is a critical issue because of high level pollution of their natural environment. In this research work an attempt was made to evaluate the toxicity of diazinon and its effect on some specific and nonspecific immune parameters of these sturgeon species in particular great sturgeon in order to give some recommendations for improving of their natural environment. A number of 300 fish weighing 12±2 g from great sturgeon obtained from sturgeon farms in Golestan states were used. Fish were transported to the Caspian Sea institute of ecology and were kept in 2000 L tanks with well aeration. Fish were fed commercial feed containing Kilka meal. The water quality parameters consisting of NO2, NH3, pH, DO and hardness were <0.1, 0 mg.l, 0.02mg/l, 7.9, 7-9/1 mg/l, and 145 mg/l, respectively. The acute toxicity of diazinon was assessed in beluga (Huso huso) and some of hematological and biochemical parameters of the survival fish examined. The obtained LC50-96 hours was 5.63 mg/l for Huso huso. Also, the obtained results showed that RBC, HB and PCV were significantly lower in the survival fish than the control fish. However, level of MCV in test groups was higher than control groups (P<0.05). Also, leucocytes count level was different between the test and control groups. Also, no difference was observed in level of enzymes of ALD, AST, LDH and ALP between test and control groups, while levels of total protein and glucose in test groups were lower and higher than control fish, respectively. IgM of these species were purified and partially characterized using affinity chromatography and SDSPAGE methods, the obtained results showed than all fish species possess and IgM like protein with a molecular weight of 870KDa. This IgM showed to have heavy chains with 77-84 KDa and light chains with 28-30 KDa. The chronic/sublethal effect of this diazinon was assessed in great sturgeon juvenile and hematological and biochemical changes, leucocyte population size, lysozyme activity, chemiluminuscence (CL) response, antibody titration and histhopathological change were studied in beluga (Huso huso) weighing 450±50 g in the present of Antigen of Aeromonas Hydrophila as a single intraperitoneal injection at 1x107 cell per fish and thereafter a chronic exposure to organophosphate, diazinon at concentrations of 1,5 and 10 mg/L as a 48 hours bath and sample collected on day one and every week interval upto 22 days provided at 22±1?C and acceptable water quality condition. Results of erythrocyte profile in fish treated with diazinon generally showed significantly decrease of RBC, HCT (P<0.05) compare to control fishes. While there was no significant difference (P>0.05) in the values of MCH, MCV and MCHC between these groups. Compare to control group (group A) the values of white blood cells and lymphocyte were significantly lower in the exposed fish to diazinon (groups of C, D, E, F, G and H) while, the level of neutrophile and eosinophil was higher than control one (P<0.05). No significant differences was found in values of monocytes and immature neutrophiles between control group and these groups (P>0.05).Furthermore, fish treated with diazinon showed a higher levels of asparate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) in the early stage of post-exposure, compare to control fishes, while the level of these enzymes was lower in this group for the rest of experiment. Also, fish treated with Antigen-diazinon showed generally lower and higher levels of total protein and glucose concentrations in blood plasma, respectively compared to fish untreated(control) (P<0.05).The level of cholinesterase in fish treated with diazinon bath generally lower than control group(P<0.05).The level of lysozyme in liver of groups B,D,F,G and H were significantly higher (P<0.05) than group A up to 2 weeks post-treatment and then it insignificantly reduced to lower levels until week 3 post-exposure. Also, kidney lysozyme contents were significantly higher (P>0.05) in groups of B,F,G and H than group A up to 2 weeks post-treatment and then was lower for the rest of the experiment. The level of spleen lysozyme in groups of B,G and H was also, higher than group A for the first 2 weeks post-treatment and, then it reduced to below levels measured during the rest of experiment (P>0.05). Lysozyme level of sera samples were significantly higher (P<0.05) in groups of B, D, E, F, G and H than group A in the one week post-treatment. There was significant difference in the lysozyme contents of tissues of liver, spleen and serum between groups of A and E,F,G,H(P<0.05). Mean spontaneous CL response in groups of diazinon bath were significantly lower than group A throughout the experiment (P<0.05). Maximum peak was found in group D one day post-exposure, while the minimum peak was found in group E throughout the experiment. The antibody titration in groups of treated with diazinon bath generally lower than control group (P<0.05).but the antibody titer in group B that treated with Antigen without diazinon bath was higher than the other groups. The histopathological effects of diazinon on the liver, kidney, spleen, gills, nostril and barbels of gain sturgeon examined under light and electron microscope, showed that diazinon caused severe damage to the cell structure such as congestion of blood vessels, hemorrhage, cellular infiltration, pyknosis of cells nuclei, vacuolar degeneration and general necrosis in the tissues of kidney, spleen and liver. There were also degenerative changes of interstitial tissue, detachment of tubular basement membrane in kidney. In the gills, hyperplasia and fusion of secondary lamellae, separation and sloughing of epithelium from the underlying basement membrane were also observed In conclusion, diazinon at toxic and sub lethal concentrations is able to seriously affect the sturgeon immunity resulting in suppression of fish immune system and making fish susceptible to both non-infectious and infectious diseases.

Iranian Fisheries Science Research Institute

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