Immunomodulation with probiotics against aeromonas veronii in grass carp (Ctenopharyngodon idellus)

Abstract

Aeromonas comprise a wide range of opportunistic bacteria that cause septicemia (blood poisoning) in fish. With expanding aquaculture, myriad methods have been used to combat these pathologies including probiotics and plant extracts which can potentially address these diseases without harming the environment. In this study, four strains of opportunistic pathogens were isolated from diseased fish, then molecular identification of 16SrDNA was performed. Results identified the four strains as Aeromonas veronii, Shewanella sp., Bacillus cereus, and Aeromonas salmonicida. Experimental trials revealed that A. veronii was the primary strain. Multiple virulence-related factors were discovered, including aerolysin (Aer), cytotoxic enterotoxin (Act), and the outer membrane protein (OmpAII). These were found only in A. veronii. LC50 in grass carp was 3.6×104 CFU/mL over 96 h, whereas the safe concentration was 9.11×103 CFU/mL. Grass carp were challenged with 3 different concentrations of A. veronii. Following a 48 h incubation period they were treated for 24 h, 48 h, and 72 h with 8.1×106 CFU/mL concentrations. The expression of Interleukin-8 (IL-8) and Tumor Necrosis Factor-α (TNF-α) was up-regulated in the kidney, liver, spleen, and heart using qRT-PCR. Simultaneously, enzyme activity involving Pyruvate Kinase (PK), Alkaline Phosphatase (AKP), Alanine aminotransferase (ALT), Succinate Dehydrogenase (SDH), and Lysozyme (Lsz) in serum and liver tissues was significantly up-regulated. We found that probiotics enhanced growth of grass carp, improved health status, and was beneficial in reducing the prevalence of this disease. Subsequent to feeding mixed probiotics, IL-8 and TNF-α gene expression was significantly lower (p>0.05) than in the infected group, at 16 and 31 days. Expression of these genes in the liver, spleen, and intestines of healthy grass carp fed probiotics was significantly up-regulated in contrast to grass carp fed a basal diet (p<0.05). Our results suggest that use of probiotics as immune stimulator may improve the control of fish diseases in aquaculture.