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According to FAO statistics in 2018, Iran has produced more than 30% of world production of this fish with a production of 180000 tons. Unfortunately, Streptococcus and Yersiniosis are two important infectious diseases in rainbow trout that cause severe losses (depending on fish age and bacteria strain, 30 – 80% mortality) and severe using of antibiotics (with environmental and health hazardous effects) causing economic and public health damages. Therefore, using immunized fish can be the best solution. This project was carried out with the aim of preventing and minimizing the losses caused by these two diseases and increasing the resistance of fish. At the first, the fish infected were sampled from Mazandaran (Haraz) and Tehran (Firozkoh) and finally 7 bacterial isolates, 5 Yersinia ruckeri and 2 suspected to streptococcus iniae respectively, were isolated. After PCR and sequencing, the bacteria were confirmed and registered in the Gen Bank. To selection bacteria for the vaccine production, log 8 of the each bacteria in a 0.1 mL were injected to the rainbow trout and based on the mortality during 96h, two isolate of Y. ruckeri (MT968736) and S. iniae (MT968734) were choosen. To prepare the vaccine, at the first the bacterin were prepared of the two isolates. The prepared bacterin was mixed with Vax-Orient IPA50 (oil base vaccine) and alum (water base vaccine) at ratio of 50:50. The final volume of the injectable vaccine was 0.2 ml containing 0.1 bactrin (including 1 × 10 8 each of the bacteria and 0.1 ml of the adjuvant (in the bacterin + adjuvant groups) and saline in the bacterin group. In this study, 360 fish with an average weight of 110.22 ± 7.55 g in 4 groups treatments (each treatment including three replications and each replicate including 30 fish) were injected intraperitoneally (0.2 ml). Bactrin alone, bactrin + montanide, bactrin + alum and control group (physiological serum). To determine the effectiveness of the vaccine one month after receiving the vaccine, 60 fish (from each treatment) were intraperitoneally exposed with Yersinia rockeri (MT968736) (30 fish) and Streptococcus iniae (MT968734) (30 fish) in double dose (2 × 10 8). The other fish were kept and blood sampling were gathered at the first, second and third months after vaccination. The blood and serum samples were use to determine of the production of free radical oxygen, lysozyme, total serum IgM, total serum protein, albumin and specific antibody titers were determined by microagglutination method.
The results of this study showed that the survival rate of fish in the presence of Yersinia rocker in the bacterial + Adjuvant group was more than 80% and in the bacterial group was more than 60% and in the presence of Streptococcus inia in the bacterial + Adjuvant group more than 86% and in the group. Bactrin more than 80% compared to control groups with zero% survival. During the three months of the study, it was found that the amount of oxygen free radical in the first month in the groups containing Adjuvant + bactrin and bactrin was significantly higher than the control. In the second and third months, the rate of this index was significantly higher in the groups containing adolescents than the control. Comparing this index in each group during the quarter, only in the group containing montanide, there was no significant difference in the amount of this index during the three-month period, but in the group of bactrin and bactrin + alum gradually from the second month, the amount of this index was significantly decreased. Lysozyme levels in the first month in treatment groups increased significantly compared to the control. In the second month, there was still a significant increase in the bactrin + adjuvant group compared to the control, but in the third month, there was only a significant increase in the adjuvant + bactrin treatments compared to the control and bactrin. The changes in this index in each group did not show a significant difference from the first to third month after vaccination in the bacterin and control groups, but in the bacterin + montanide group a significant increase in this index from the first to the third month and in the bacterin + alum group This index was seen in the third month compared to the first and second months. In the first and second months after vaccination, the level of total serum IgM in the treatment groups was significantly increased compared to the control, but in the third month and only the bacterin + adolescent treatments had a significant increase compared to the control and bactrin. In comparison with the treatments during different months, the amount of antibodies in the second and third months compared to the first month in bacterial + adolescent treatments was significantly different from the first month and in bactrin treatment only a significant increase in the second month compared to the first month and The third was observed. Total serum protein in this study in the first, second and third months after vaccination in the groups receiving bacterin + Adjuvant had a significant increase with the control. However, in the bacterial group, this significant increase was observed only in the first and second months after vaccination. There was no significant difference in the evaluation of this index in each group and during different months. Regarding albumin, no significant difference was observed between different treatments and also in each treatment in different months. The level of specific antibody titer against Yersinia Rocky and Streptococcus inia obtained by microagglutination (MAT) method showed that the specific antibody titer in the groups receiving bactrin + Adjuvant in the first, second and third months after Vaccination is significantly higher than the bacterial group. Also, in comparison with each group during different months, in the group exposed to Yersina Rocky, the specific antibody titer in the third month had a significant decrease compared to the second month, while in the groups receiving Adjuvan, the specific titer increased significantly. Antibodies were seen in the second month compared to the first and third months. In the same condition, in the second month, the highest antibody titer was observed in all treatments against Streptococcus iniae in the second and first months and the lowest in the third month, respectively. According to the results of this study, the decrease in cumulative mortality and the increase in survival percentage in the groups receiving Adjuvant + Bactrin compared to the bacterial and control groups after exposure to pathogenic bacteria is quite evident. Also, the improvement and increase in the performance of intrinsic safety indices (production of oxygen and lysozyme free radicals) in the groups receiving bactrin + adjuven compared to the bactrin and control groups up to three months after injection shows good intrinsic safety performance of fish. In addition, an increase in the shelf life of specific antibody titers in the bacterial + adjuvant groups compared to the bacterial and control groups up to three months after injection indicates good performance of acquired humoral immunity. Based on what has been said, it seems that the Yersiniosis-Streptococcus bivalent killed vaccine, in which the bacteria used were selected based on the virulence test, had good immunogenicity up to 3 months after injection, and the adjuvants used in this study were sufficient. They have greatly enhanced the performance of this vaccine.
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