Epidemiological Investigation, Isolation, Identification And Drug Resistance Of Aeromonas Hydrophila From P. Bream In Nanjing Area

Aeromonas hydrophila is a model bacterium belonging to the genus Vibriocetes and Aeromonas.It is a conditional pathogenic bacterium for human-livestock and aquatic animals.A.hydrophila is a gram-negative bacterium,short rod-shaped,with extreme solitary flagella but without spores and decidua.It can grow at 0-41℃,and grows vigorously on nutrient agar and blood agar plates.It is generally believed that the pathogenicity of A.hydrophila is closely related to the toxins it secretes.In aquaculture,it can cause bacterial septicemia in some fish.In recent years,with the development of intensive aquaculture technology in the aquaculture industry,and higher and higher breeding density,the ecological environment has deteriorated,various diseases have emerged leading to large-scale outbreaks and epidemics as well as serious economic losses.So far,antibiotic therapy has been used for the diseases caused by this bacterium.The use of quinolones has shown good results.But the use of large amounts of antibiotics has led to the emergence of multidrug-resistant strains.This thesis carried out an epidemiological survey of fish Megalobrama amblycephala with hemorrhagic diseases in the cultured areas of Nanjing,and proved that A.hydrophila was the main pathogen causing the outbreak of the disease through pathogen isolation and artificial infection by A.hydrophila.Isolation,identification,and susceptibility testing experiments,as well as the study of resistance to A.hydrophila,provided a valuable theoretical basis for scientific drug use in grass-roots aquaculture,ensuring the quality and safety of aquatic products.1.Epidemiological investigation of Hemorrhoids disease in M.amblycephala.After investigation,it was found that bacterial diseases caused by A.hydrophila occur in all parts of Nanjing,but the hazards formed were different.The disease in key breeding areas in Jiangning and Pukou are relatively serious.And the disease had the characteristics of aggressiveness,rapid epidemic,wide range,and great harm.The survey data showed that A.hydrophila was detected from April to November,and the bacterial disease caused by A.hydrophila began to appear in June,and gradually increased with the increase of water temperature.It is popular from late July to the end of September,and August is the peak season.In shallow ponds,high-density ponds,and single-pool ponds,the onset was often acute,and the attack was rapid.A large number of deaths occurred within 2-3 days after onset,and death peaks occur around 10 days.In the large-scale ponds or polyculture ponds,the disease often develops into a chronic type.The development of the disease was moderate,and the number of deaths per day was not obvious.However,the course of disease was longer and often lasts until October.Early onset had no effect on fish eating.In later stages,the diseased fish was anorectic or non-feeding,motionless,or mad swimming in the pond,and eventually died until failure.Diseased fish,upper and lower jaw,mouth,lid,eyes,fin base and fish on both sides of congestion,anal swelling,abdominal enlargement,intraperitoneal accumulation of light yellow or red turbid ascites.The diseased fish was severely anemic,with a pale white appearance,and the liver and kidneys were pale in color and showed a patchy appearance.Some diseased fish scales were erected,and muscles and diaphragm walls were congested.Due to the length of the disease and the development of the disease,the age of the diseased fish,the symptoms of diseased fish were diverse.2.Isolation,identification and artificial infection experiments of hemorrhagic disease caused by A.hydrophila.Under sterile conditions,the diseased fish were taken and the liver was streaked and inoculated on LB solid medium to separate and culture the bacteria.The colonies on the medium were yellow,round,opaque,centrally raised,and smooth.Identification by API strips,the results showed that N03,TRP,GLU,ADH,ESC,GEL,PNPG,GLU,ARA,MNE,NAG,MAL,GNT,CAP,MLT,OX were all positive,and were judged as A.hydrophila.A single colony on LB solid medium was picked and subjected to an artificial infection experiment.As a result,strain JNYK1702 had a strong pathogenicity.The fish mortality in the experimental group was 100%when the bacterial fluid density was 1×106.When the density of bacteria was 1×103,only partial fish died in the experimental group.The fish in the experimental group showed typical symptoms consistent with the natural pathogenesis.The strain JNYK1702-1 identified by bacteriological isolation was the same strain as the tested strain JNYK1702.3.Pathogenicity of A.hydrophila drug sensitivity test and drug resistance test.The drug susceptibility test was performed using the KB disk method,ie,the density of the broth was adjusted to 1×107 to 1×108.The sterilized cotton swab was used to dip the bacterial suspension and the bactericidal liquid to be tested was uniformly applied to the LB in the medium plate.The susceptibility test piece(Hangzhou Tianhe Microbial Reagent Co.,Ltd.)was taken with a sterile camera,and tightly attached to the surface of the LB agar medium.The plate was placed in an incubator at 28 0C for 24 hours and recorded.From the test results,the strain showed resistance to penicillin,ampicillin,tetracycline,amoxicillin and oxacillin and sensitive to ofloxacin,gentamycin,streptomycin,norfloxacin,roxithromycin,florfenicol and ceftriaxone.The drug was tested by two-fold dilution with streptomycin sulfate,ampicillin,oxytetracycline,chlortetracycline hydrochloride,enrofloxacin,florfenicol,and norfloxacin.The measurement was performed according to the method of the Japan Society of Chemotherapy(1975).The ready-to-prepare liquid stock solution is filtered through the contaminated membrane,and then the diluted drug is diluted with the sterilized BHI medium.Finally,the test tube was diluted by the culture bacteria as an antibacterial drug.The results showed that chlortetracycline hydrochloride,florfenicol,streptomycin sulfate,and enrofloxacin had the best antibacterial effects.The minimum inhibitory concentrations were 2,4,4,5μg/mL respectively,followed by oxytetracycline and norfloxacin with a minimum inhibitory concentration of 15μg/mL.Among 7 drugs,ampicillin was the worst,with a minimum inhibitory concentration higher than 40μg/mL.