| The Hoplobatrachus occipitalis has been introduced to China by Thailand since 1996 and has been promoted on a large scale in Hainan,Guangdong and Guangxi,and is one of the special economic aquatic animals in China.Streptococcus agalactiae,also known as group B streptococcus(GBS),is a human-animal and aquatic commensal pathogen that can have serious effects on human health,dairy cattle and aquaculture.There are still many deficiencies in the technology related to the breeding and disease control of Hoplobatrachus occipitalis in China,and once large-scale bacterial diseases occur,the treatment means are still based on external disinfectants and internal antibiotic drugs.Therefore,in this study,the outbreak of infectious diseases in Hoplobatrachus occipitalis farms in Guangxi area was used as a background to isolate and identify the pathogens of diseased frogs through clinical symptoms observation,pathological dissection,physiological and biochemical detection and PCR detection,and to carry out preliminary research on relevant immune control methods to provide a certain theoretical basis for immune control of infectious diseases in frogs.The infected diseased frog carcasses were dissected to observe the pathological changes.Gastrointestinal bleeding could be seen;there was more ascites in the abdominal cavity,enlarged liver,fluid in the gastrointestinal tract,and some diseased frogs showed overlapping at the gastrointestinal junction;there were white attachments on the surface of the heart.Bacterial strains were isolated from heart,liver,spleen,kidney and some brain tissues and cultured on BHI plates and blood plates,all of which were found to produce the same strains with the same colony status.Representative strains isolated from each organ were selected for morphological,physiological,biochemical and 16S r RNA identification,and the selected strains were determined to be Streptococcus agalactiae.The representative strain isolated from the brain tissue of the diseased frog was named WN202110-001,and this strain was used for subsequent experiments.The 16S r RNA sequencing results of this strain were used to construct a phylogenetic tree and found to be 99%homologous to S.agalasia strain GXYL7;drug sensitivity tests were performed on this strain,and it was found to be resistant to lomefloxacin,streptomycin,gentamicin,kanamycin and tetracycline;the genomic DNA of the strain was extracted and its podocyte serotype,multilocus sequence typing and virulence genes were detected by PCR and found to be podocyte serotype III,multilocus sequence typing ST-739,and all 14 virulence genes detected were expressed except for bac gene;the animal regression test results showed that the test frogs showed the same symptoms as the sick frogs in the frog farm,and the bacterium was identified as the causative agent of the case frog.The LD50 of strain WN202110-001 was measured to be2.34×10~8cfu/m L.Samples of frog heart,liver,spleen,kidney and brain were prepared into paraffin sections and stained with HE for comparative observation,all of which were observed for the presence of Streptococcus agalactiae.The inactivated vaccine was made by inactivating Streptococcus agalactiae with 0.4%formaldehyde,and the experimental frogs were immunized by intramuscular injection.The experimental results of serum-specific antibody assay and relative immune protection rate determination showed that the prepared inactivated Streptococcus agalactiae vaccine could produce a good immunization effect with the highest agglutinating antibody potency of 1:256.The vaccine was attacked 1 week after the second vaccination and was found to provide good protection to the immunized Pelophylax nigromaculatus with an immune protection rate of84.7%.In conclusion,this study identified the causative agent causing the disease of sick frogs in Guangxi tiger frog farms as Streptococcus agalactiae,and the established inactivated vaccine against Streptococcus agalactiae could produce good protection for artificially infected test frogs. |
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