Effects Of Clostridium Butyricum On Resistance Of Procambarus Clarkii To Aeromonas Hydrophila Infection

The severity of the Procambarus clarkii disease has increased over the past few years as a result of the aquaculture industry’s quick development and scale expansion.The P.clarkii disease outbreak brought on by Aeromonas hydrophila and the efforts taken to avoid and control it have garnered considerable attention.In aquaculture,probiotics are added to prevent and treat disease and reduce the cost of disease outbreaks.There has been a lot of research and application of Clostridium butyricum as probiotics in animal husbandry,but comparatively little in P.clarkii culture.In this research,the effect of A.hydrophila on P.clarkii’s hemocytes and non-specific immunological components was studied,and the mechanism underlying its toxicological impact on P.clarkii was also investigated.In order to investigate the protective effects of C.butyricum on P.clarkii infected with A.hydrophila,C.butyricum was added to the meal for a 14-day feeding experiment.1.Healthy P.clarkii were divided into four treatment groups,and each treatment group set up three repeat tanks with 20 P.clarkii in each tank.P.clarkii in the treatment group were injected with A.hydrophila suspensions of 0 CFU/ml（control group）,1×10⁴CFU/ml（L-AH group）,1×10⁶CFU/ml（M-AH group）and 1×10⁸CFU/ml（H-AH group）,respectively.Hemolymph was collected 36 h after injection for detection.The results showed that the total hemocytes count（THC）of P.clarkii decreased significantly with the injected doses of A.hydrophila（P<0.05）;and the THC in the H-AH group was only 8.15%of that in the control group.The proportion of hyaline cell（HC）was significantly reduced with the injection concentration of A.hydrophila（P<0.05）,while the change trend of semi-granule cell（SGC）and granular cell（GC）was opposite（P<0.05）.A.hydrophila significantly lower plasma total antioxidant capacity（T-AOC）and the activity of total superoxide dismutase（T-SOD）in P.clarkii（P<0.05）,and a dramatic rise of reactive oxygen species（ROS）concentration in hemocyte was observed in the M-AH and H-AH groups（P<0.05）.Particularly,the m RNA expressions of pro-apoptotic genes,including Bax,AIF,Cytc,and Caspase-3 significantly up-regulated under the injection of A.hydrophila（P<0.05）,whereas the m RNA expressions of anti-apoptotic genes（Bcl-2 and Bi-1）significantly down-regulated in hemocytes（P<0.05）.In addition,the m RNA expression levels of TLR,CTL,Crustin-3,Crustin-4,ALF-6,and PPO in the H-AH group were lower than those in the control group（P<0.05）.2.C.butyricum was added to the commercial feed by spraying,and the content of C.butyricum in the feed reached 1×10¹¹CFU/kg.The healthy P.clarkii were separated into two treatment groups,and each treatment group set up three repeat tanks with 20 P.clarkii in each tank.They were fed common commercial diet（control group）,and C.butyricum diet（CB group）,respectively.A.hydrophila was injected after being fed for 14days.At 0 h,12 h,24 h,36 h,48 h,and 96 h following injection,hemolymph of P.clarkii was collected for detection.The results showed that the survival rate of P.clarkii was increased from 30%to 50%by adding C.butyricum in the diet.Compared with the control group,the THC of P.clarkii in the CB group was substantially higher at 24-48 h（P<0.05）,and the apoptosis rate of hemocytes also showed the same trend（P<0.05）.Compared with the control group,T-AOC and T-SOD in plasma in the CB group were considerably higher than those in the control group between 24 h and 48 h,and the content of malonaldehyde（MDA）showed the opposite trend（P<0.05）.At 36 h and 48 h,the ROS content of P.clarkii hemocytes in the CB group was dramatically lower than that in the control group（P<0.05）.Compared with the control group,the relative expression level of Bcl-2 in hemocytes of P.clarkii in the CB group was significantly up-regulated at36-96 h（P<0.05）,and at 24-96 h,the m RNA expression levels of Bax,Cytc,and Caspase-3 in hemocytes of P.Clarkii in the CB group showed the opposite trend（P<0.05）.From 24 to 96 hours,adding C.butyricum to the diet can significantly up-regulated the relative expression levels of TLR and CLT in hemocytes of P.clarkii（P<0.05）.However,adding C.butyricum to the diet can significantly down-regulated the relative expression of NF-κB at the later stage of infection（48-96 h）（P<0.05）.From 24 to 96hours after infection with A.hydrophila,adding C.butyricum to the diet can significantly up-regulated the relative expression levels of Crustin-4,ALF-6 and PPO（P<0.05）.In conclusion,when the concentration of A.hydrophila reaches above 1×10⁶CFU/ml,can destroy the antioxidant capacity of P.clarkii,induce apoptosis of hemocytes,lead to a large reduction of THC,collapse the immune system,and eventually cause the death of P.clarkii.C.butyricum can protect the antioxidant capacity of P.clarkii,reduce the apoptosis of its hemocytes,stimulate the production of immune-active substances in P.clarkii,and have a certain protective effect on P.clarkii in the infection of A.hydrophila.