Uimmunogenic Potential of Different Adjuvanted Vaccines of Aeromanas Hydrophila in Culturable Fish Species of Pakistan

Abstract

Aeromonas hydrophila is a key bacterial pathogen that causes infectious diseases in freshwater fish species cultured in Pakistan. The present research is aimed at studying immunogenic potential of indigenously prepared adjuvanted vaccines against A. hydrophila and observing immune response in cultured fish species of Pakistan. The procedure was carried out on diseased fish samples to isolate and molecularly characterize the A. hydrophila strains. Suspected fish samples were collected from fish farms in Punjab, Pakistan. The samples were processed and A. hydrophila strains were isolated. The primary identification of isolates was verified at species level by colony morphology, microscopy and phenotypic characterization with biochemical reactions. The A. hydrophila strains of representative isolates were molecularly characterized by polymerase chain reaction (PCR) using 16S rRNA gene at a desired size of 356 bp. The PCR amplified product was subjected to DNA sequencing and phylogenetic analysis showed close similarity with related strains of Aeromonas spp. in neighboring countries. This study also includes detection of virulence associated genes Aerolysin (aerA) and Hemolysin (Ahh1) of A. hydrophila using type-specific polymerase chain reaction (PCR) at a desired size of 309 bp for aerolysin and 130 bp for hemolysin respectively. Seventy five percent of the isolates harbored aerolysin and hemolysin virulent genes. Results of genetic analysis confirmed strains as A. hydrophila. By antibiotic sensitivity test, the isolates were checked for nine antibiotics in which the pathogen was sensitive to three (Colistin sulphate, Gentamicin and Trimethoprim) and resistant to four drugs (Penicillin, Novobiocin, Amoxicillin and Tetracycline) whereas partially resistant results were obtained for two antibiotics (Norfloxacin and Erythromycin). The antibiotic resistance pattern of local A. hydrophila strains suggests alternative ways of disease prevention such as vaccines. The bacterial isolates were cultured and inactivated vaccines were prepared at 3 different concentrations of 1×108 , 1×109 and 1×1010 Colony Forming Units (CFU)/mL respectively by following standard protocols. Montanide oil and alum-precipitate were separately used as adjuvants. The vaccines were evaluated by injecting through intraperitoneal route (I.P.) to healthy Rohu (Labeo rohita), Mori (Cirrhinus mrigala) and Grass carp (Ctenopharyngodon idella) in two separate experiments lasting for 60 days. The 1st dose of 0.1 mL was injected on 1 st day of vaccination, while booster dose was delivered on 15th day post-vaccination. The fishes in control group were injected with equal volume of 0.85% saline at the same time. On 14, 28, 35, 48 and 60 days post-vaccination, blood samples were collected. The growth parameters of fish and physiochemical parameters of tank water were also observed. Antibody titers in serum samples were determined using complement fixation test (CFT). The statistical differences between immune response fish species to all vaccines and growth performance were analyzed. In alum-precipitated vaccination experiment, significantly high antibody GMT (P<0.05) were observed in all the vaccinated groups as compared to control group at 28 day post-vaccination which remained significant till 60 day post-vaccination. No significant difference in antibody GMT (P>0.05) was observed among three vaccine potencies at 14 day post-vaccination. However, significantly higher antibody GMT values (P<0.05) were observed in 108 and 109 CFU/mL potencies as compared to 1010 CFU/mL potency at later stage of the experiment. The immunokinetics show highest antibody GMT values at 35 day post-vaccination and subsiding gradually afterwards. The overall immunogenic potential of L. rohita and C. idella were found to be better than C. mrigala for alum-precipitated inactivated vaccines during experiment. The statistical analysis shows that there is no significant difference (P>0.05) among the growth performance of all vaccinated groups and unvaccinated control at 0, 14 and 28 day post-vaccination. However, there is significant suppression in growth rate of fish vaccinated with 1010 CFU/mL. This reveals that there is no growth related side effect of 108 and 109 CFU/mL but 1010 CFU/mL concentration presented retarded growth. The Tukey‘s multiple comparisons test shows no significant variations (P>0.05) in weight gain among species (L. rohita, C. mrigala and C. idella) in xiii vaccinated and non-vaccinated groups. In case of oil-based vaccination experiment, no significant difference in antibody GMT (P>0.05) was observed among three vaccine potencies at 14 and 28 days post-vaccination. However, significantly higher antibody GMT values (P<0.05) were observed in 1010 and 109 CFU/mL potencies as compared to 108 CFU/mL at later stage of the experiment. The immunokinetics show highest antibody GMT values at 35 day post-vaccination subsiding gradually afterwards. There is no significant difference (P>0.05) among the growth performances of all vaccinated groups and unvaccinated control at 0, 14, 28, 35 and 48 day post-vaccination. This indicates that there are no growth related side effects on fish due to vaccine injection during first six weeks of trial. However, significant increase in growth rate (P<0.05) of fish is observed in all vaccinated groups as compared to control group at 60 day post-vaccination. This reveals that there is eventually a positive impact on growth performance due to oil-based vaccine injections. The Tukey‘s multiple comparisons test shows no significant variations in weight gain among species (L. rohita, C. mrigala and C. idella) in vaccinated and control group. Higher antibody titer values in case of oil-based vaccines as compared to alum-based vaccines were observed. The 109 and 1010 CFU/mL potencies for oil-based and 108 CFU/mL potency for alum-precipitated vaccines were found to be the most suitable concentrations for a fixed dose of 0.1mL/fish. At 60 day post-vaccination, the challenge studies were conducted for both vaccination experiments in which infectious dose of 1×108 CFU/mL A. hydrophila was exposed through immersion method. For alum-precipitated vaccination experiment the average RPS (relative percentage survivability) was 71% for groups vaccinated with 109 and 1010 CFU/mL potencies while 86% for 108 CFU/mL potency for all species. In case of oil-based vaccination experiment, the average RPS was 89% for fish groups vaccinated with 109 and 1010 CFU/mL potencies while 78% for 108 CFU/mL potency. Therefore, more and long protection against infectious abdominal dropsy caused by A. hydrophila was developed in oil based vaccine with concentration of 109 CFU/mL and 1010 CFU/mL. Normal histology was observed in different organs of fish in all vaccinated groups whereas minor deteriorative changes and degenerations were found in control group and in fishes vaccinated with high concentration of alum-precipitated vaccine. No significant histopathological changes were observed in fish immunized with oil-based inactivated vaccines. The results of vaccination trials and challenge study show that mainly cultured carp species of Pakistan attain reasonable immunogenic potential against A. hydrophila when vaccinated with inactivated vaccines. The montanide oil and alum based adjuvants are safe and have demonstrated beneficial effects in vaccines of present study. The vaccines have no adverse effect on growth performance of fish. Some histopathology results show minor deteriorative changes in organs which suggest adjustment in dose and concentration of vaccines as a future work. Keeping in view the impact of dropsy disease in cultured fish market, and non-availability of effective disease prevention and control mechanism in the country, the present study paves a way forward for indigenous development of vaccines for better fish health management in aquaculture sector of Pakistan. Key words: Aeromonas hydrophila, Cultured fish, Isolation, Abdominal dropsy, Molecular characterization, Polymerase chain reaction (PCR), Antibiotic sensitivity, Phylogenetic analysis, Aerolysin, Hemolysin, Antibody titer, vaccine, Adjuvant, Complement fixation test (CFT), Histopathology