Isolation Of Pathogenic Bacteria From Rana Chensinensis In Jilin Province And Preliminary Application Of Traditional Chinese Herb

Rana dybowskii,as an economic farmed animal,is mainly farmed in northern my country.In recent years,with the substantial increase in people’s living needs,the intensive breeding of forest frogs has developed rapidly,and the breeding area has rapidly expanded.Its related diseases have also frequently outbroken,especially the increasing bacterial diseases,which have had a huge impact on the long-term stable development of the forest frog breeding industry.Among them,Aeromonas hydrophila,as a common pathogen in aquaculture,seriously harms the aquaculture industry,but also has strong pathogenicity to amphibians.The prevention and control measures were mainly concentrated on the feeding of antibiotics,which can easily cause the production of drug-resistant strains and the residue of antibiotics in the body.New immune enhancers can effectively improve the body’s immunity and alleviate the occurrence and development of bacterial diseases.Therefore,the development of new immune enhancers would show considerable development prospects as an effective way to replace antibiotics.In this study,diseased forest frogs in farms were collected,pathogenic bacteria were isolated from the diseased tissues.We conducted tests on hemolytic activity,pathogenicity,physiology and biochemistry,drug sensitivity,growth characteristics,median lethal dose,virulence gene carrying and molecular identification to explore the characteristic of pathogenic bacteria.Through isolation and culture,three pathogenic bacteria were successfully isolated from diseased Rana dybowskii.Among them,Proteus suggests that there are problems in the breeding environment and the quality of the bait.Pathogenicity and drug susceptibility tests showed that A.hydrophila has strong pathogenicity,lower than ATCC 7966,and sensitive to drugs such as ciprofloxacin,kanamycin and chloramphenicol.The growth rate of this strain is slightly faster than ATCC 7966;and it carries six virulence genes: act,aer,ser,lip,aha and Luxs.To develop a new type of immune enhancer to prevent this bacterial disease,the A.hydrophila isolated in Chapter 1 was used as the test strain,and through the in vitro antibacterial test,the Chinese medicine MIC and MBC test,test on the ability of traditional Chinese medicine to inhibit the formation of R5 biofilm,to screen the Chinese medicine extract that can effectively inhibit the bacteria.Subsequently,the forest frogs were fed with mixed feed for 56 days.On the 0d,14 d,28d,42 d and 56 d,3 R.dybowskii were randomly selected to collect serum and the heart,liver and spleen to determine the relevant immune indicators.The protective effect of traditional Chinese medicine was evaluated by intraperitoneal injection of R5 suspension.The results of the in vitro antibacterial test showed that both gallnut and safflower could significantly inhibit the growth of R5,and the MIC and MBC of gallnut were 0.05g/m L and 0.1g/m L,respectively,and the MIC and MBC of safflower were 0.1g/m L and 0.15,respectively,which are significantly better than other traditional Chinese medicines.The AKP,SOD and LZM in the serum were detected by ELISA,and the results showed that gallnut can significantly increase the serum AKP,SOD and LZM levels,and safflower can significantly increase the serum AKP and SOD levels.The q PCR results showed that gallnut and safflower can up-regulate the expression levels of IL-10,IL-1β,TNF-α and IFN-γ in the heart,liver,and spleen,and improve the immunity of the host.Acute intraperitoneal challenge test showed that gallnut and safflower could alleviate the invasion of A.hydrophila to R.dybowskii,indicating that the two Chinese medicines have a certain protective effect.In summary,this experiment successfully isolated Proteus,Shewanella and A.hydrophila(R5)from diseased forest frogs,and R5 has strong pathogenicity against forest frogs.In addition,gallnut and safflower can significantly inhibit the growth of R5 and the formation of biofilms.Oral feeding with mixed bait can significantly improve the immune response of forest frogs,increase the expression levels of various enzymes and pro-inflammatory factors,and significantly reduce the mortality of R.dybowskii after infection with A.hydrophila.The research results provide a theoretical basis for the further development of new immune enhancers.