Isolation and identification of bacterial pathogens from Procambarus clarkii and study on variations in serum immune factors

In China, Procambarus clarkii is the most widely cultivated freshwater crayfish now, the breeding area and output are increasing every year and the culture models have also been constantly innovated. In recent years, the area of rice-P. clarkii co-culture has been increasing explosively, and its output has accounted for more than 80% of the national total output, so the rice-P. clarkii co-culture has become the basis for the rapid development of crayfish industry. With the expansion of aquaculture scale and deterioration of aquaculture environment, various diseases occur frequently which seriously affect the yield and quality of P. clarkii. In order to find out the death cause of P. clarkii cultured in paddy field, the pathogenic bacteria were isolated from stomach and intestine of these dying crayfish with obvious pathological characteristics, and were identified through physiological and biochemical testing, sequence alignment of 16S rDNA gene and phylogenetic analysis. At the same time the virulence and drug sensitivity of pathogenic bacteria were evaluated by artificial infection and drug sensitivity test, the pathogenic mechanism of pathogenic bacteria was studied by analyzing the variations of serum immune related enzymes activities of P. clarkii injected with pathogenic bacteria. Research results showed that a dominant strain named PCW-01 was obtained and identified as Aeromonas veronii which could cause the healthy P. clarkii to have these same pathological symptoms as the natural disease, such as less exercise, effusion in stomach, excessive intestinal mucus in the regression experiment, and its median lethal dose was 0.60×10⁵ CFU/g to P. clarkii. The analysis of antimicrobial susceptibility test showed that A. veronii (PCW-01) was highly sensitive to furantoin, tobramycin, ceftazidime and other seven antibiotics. The results of serum immune enzyme test showed that in the body of P. clarkii injected with pathogenic bacteria, the activity of lysozyme (LZM) and alanine arninotransferase (ALT) increased considerably (P<0.01), the activity of total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) decreased notably (P<0.01), and the concentration of MDA increased slightly (P>0.05). The conclusion was that the strain PCW-01 was the pathogen of the death of P. clarkii, which could inactivate antioxidant factors, destroy cell membrane structure and function, subsequently the pathological phenomena of tissues and organs or death appeared. In the study, these results revealed immune response law of P. clarkii to pathogenic bacteria and will provide scientific support for the prevention and control of the bacterial diseases of P. clarkii in future.