The possible identification and introduction of metallothionein in Persian Sturgeon (Acipenser persicus)

Due to anthropogenic activities, toxic metals still represent a threat for various marine organisms. Metallothionein (MT) and cadmium concentration in gills, liver, and kidney tissues and cadmium partitioning in soluble (cytosol) and insoluble fractions of mentioned tissues of Persian sturgeon (Acipenser persicus) were determined following exposure to sub-lethal levels of waterborne cadmium (Cd) (50, 400 and 1000 μg L-1) after 1, 2, 4 and 14 days. The increases of MT from background levels in comparison to controls were 4.6-, 3-, and 2.8-fold for kidney, liver, and gills, respectively after 14 days. The matallothionein concentration in liver was in the range of 56.89-168.44 μgL-1 and for kidney and gills, 39.78-189.30 and 28.15-91.20 μgL-1, respectively. The results showed that MT level change in the kidney is time and concentration dependent. Also, cortisol measurement revealed elevation at the day 1 of exposure and that followed by MT increase in the liver. Cd concentrations in the cytosol of experimental tissues were measured and the results indicated that Cd levels in the cytosol of liver, kidney, and gills increased 240.71-, 32.05-, and 40.16-fold, respectively 14 days after exposure to 1000 μgL-1 Cd. The accumulation of Cd in cytosol of tissues is in the order of liver > gills > kidney. Spearman correlation coefficients showed the MT content in kidney is correlated with Cd concentration, the value of which is more than in liver and gills. Thus, kidney can be considered as a tissue indicator in Acipenser persicus for waterborne Cd contamination. Also, tissue metal accumulations (gills, liver, kidney and muscle) in Persian sturgeon (Acipenser persicus) were compared following exposure to sublethal levels of waterborne Cd (50, 400 and 1000 μg L-1) after periods of 1, 2, 4 and 14 days. Meanwhile, the trends of Cd concentration increase in different tissues during the exposure periods and concentrations were modelled as equations. The obtained results indicate that at the end of 4 and 14 days of exposure, total tissue cadmium concentration followed the pattern: liver> gill> kidney> muscle. Calculation of bioconcentration factor (BCF) after 14 days exposure showed that at low and high concentrations, highest BCFs were found in kidney and liver, respectively. According to the results, the accumulation capacity of muscle was the lowest at all exposure concentrations. The hematological parameters including osmolarity, total protein, cortisol and glucose of plasma were measured, too. Total protein of plasma was in the range of 416.90-1068.10 mg dl-1 plasma.Total protein decreased not significantly (P≥0.05) after exposure to Cd. Cortisol increased after 1 day exposure that followed by significant (P≤0.05) elevation of glucose. The range of cortisol was very vast and it was determined between 0.03 to 16.21 ng mL-1. The content of plasma osmolarity was in the range of 282.33-294.20 mOsmol L-1.Osmolarity of treated fish plasma showed no significant decrease (P≥0.05). Total protein in gills, liver, and kidney showed that at high concentrations of metal, protein content decreased significantly (P≤0.05) in the liver after 4 and 14 days exposure. Thus, total protein of liver and glucose of plasma can be used as general biomarkers of exposure to Cd. Also, the metallothionein and cadmium were measured in gills, kidney and liver of 8 wild Persian sturgeon caught in coast of Guilan Province. According to the results, the concentration of metallothionein was in the range of 45.87-154.66 microgram per liter with the maximum and minimum concentrations in liver and gills, respectively. The trend of cadmium concentration in cytosol of tissues was: liver> kidney> gills. The results of Spearman correlation test showed that there was a significant positive correlation between metallothionein and cadmium in cytosol of liver (r2= 0.850, p≤ 0.01). In the kidney, the correlation between cadmium and metallothionein was significantly positive (r2= 0.731, p≥ 0.05). But there was not such significant correlation in the gills (p≥ 0.05).

Evaluation and comparison of durability of polycyclic aromatic hydrocarbon compounds (PAHs) and its relation to the lipid content in smoked fishes, Silver carp (Hypophthalmichthys molitrix), Rutilus frisii kutum and Herring (Alosa caspia)

Cold smoking method is one of the commonest ways for fish smoking. It is done by the smoke that is the result of burning hard and soft woods is smoking rooms. Smoke includes a number of chemical constructs and its main part is poly aromatic hydrocarbons. More than one hundred kinds of these constructs are recognized in smoke that is produced from saturated hydrocarbons resulted from the solution of the woods Ligno cellulose in high temperature and lack of oxygen conditions. The high poisoning potentials and carcinogenic features sixteen constructs among them are proved and observed on humans. In this research, the PAH compounds were identified and observed in a three month period after smoking and during storing among three types of smoked fishes Silver carp and Caspian sea Sefid and herring. They are the most produced and consumed smoked fish in Iran. To find the relationship between the concentrations of PAH constructs and the amount of lipid in fish, first, the amount of lipid were determined separately in the skin and flesh of 30 samples of each type. The method used was Bligh and Dyer (1959). PAH compounds derivation were made for all skin and flesh samples smoked fish using organic solvents with Soxeleh and the derived samples were injected to gas chromatography (GC) by Hamilton injectors for determining their components quality and their quantity. The height of the used column was 25 meters and its diameter was 0.32 mm with the silica filler, nitrogen gas as carrier and flame ionization detector (FID) that are special for these constructs. For data analysis, Statistical tests were used by computer soft ware identified that the difference in the amount of lipid within the flesh and skin of each species and also among each other is significant. The largest amount was in Herrings flesh and skin, 18.74% in skin and 14.47% in flesh. The least amount in the skin 4.19% and the flesh 3.10% of Sefid. The amount in Silver carp was 13.28%in skin and 8.16% in flesh. The examination of the PAH compounds in smoked fish showed that is carcinogenic compounds; exist in these in these fish with different quantities in each. It seems that its amount is directly related to the amount of their lipid. The amount is different in flesh and skin. One of the most important reasons is the direct content of smoke and the concentration of lipid in tissues of all three types. The maintenance of the smoked fish for three months showed that most of PAH compounds were solved and their density decreased. The changes in density within time in different in each type and in flesh and skin. The amount of their receiving in human through the consumption of the smoked fish depends on the resulted density, the way and the amount of consumption and now we can determine and execute standards for the maximum dosage per day and per month regarding effective factors.

Development and evaluation of the polymerase chain reaction (PCR) method for diagnosis of spring viremia of carp virus (SVCV)

Aquaculture has been expanded rapidly to become a major commercial and food-producing sector worldwide in recent decade. In parallel, viral diseases rapidly spread among farms causing enormous economic losses. The accurate detection of pathogens at early stages of infection is a key point for disease control in aquaculture. Spring Viraemia of Carp Virus (SVCV) is a very severe pathogen of carp fishes in different parts of the world and is categorized as a reportable listed disease in the annual published list of World Organization for animal Health (OIE). The objective of this study was to develop and evaluate RT- PCR test for detecting SVC virus and also the sensitivity and specificity of this test. A semi nested RT- PCR was designed using combination of three primers: two external (SVCF , SVCR) and one internal (SVCS) primers which based on conserved region of G gen. The specificity of designed primers (only external ones) by examination on Viral Hemorrhagic Septicemia Virus (VHSV) and Infectious Hematopoietic Necrosis Virus (IHNV) was confirmed. For optimizing of the PCR test, primer concentration, primer annealing temperature, cycle number and Mgcl2 concentration were surveyed. Also for validity test, prevention of false negative and Assurance of its accuracy, a competitive internal control (mimic) designed and its suitable concentration was defined. Evaluation of the sensitivity of designed test were conducted first by comparing the different commercially available RNA isolation guidelines, two guidelines: isotiocyanate phenol–chloroform based protocols (RNX–Plus Iran, Iq2000 kit Taiwan ) and two column based protocols (Cinna pure RNA Iran , high pure viral RNA kit, Roche Germany ). The results indicated that the column based protocols (Roche method and Cinna pure), yield 36.77 ng/μl and 16/47 ng/μl RNA concentration respectively, which were significantly higher than other protocols(P<0.05). Then for evaluation of extracted RNA sensitivity, Serial dilution of SVCV strain 56.70 grown in EPC (1.9×105 TCID50/ml) was examined To compare sensitivity. Extracted RNA from serial dilution with stone's primers and commercial IQ-2000 kit were examined simultaneously. The result indicated that designed semi- nested RT- PCR was able to recognize SVC virus to 10-4 dilution and stone's primer recognize to 10-3 dilution whereas Iq-2000 commercial kit did not recognized in any dilution. In high virus titer in designed test two DNA band (462 bp and 266 bp) produced, and by decreasing virus titer 462 bp was omitted. In low virus titer or lack of virus, just DNA band (mimic) 729 bp can propagate. After designing and optimizing PCR test, a total of 400 suspected cultured Cyprinus carpio with high mortality from 4 aquaculture zone of Khuzestan province were collected and tested for SVCV during 2012- 2013 using developed PCR method and IQ- 2000. The results indicated that SVC virus was not observed in samples using both methods.