Isolation And Identification Of Pathogenic Bacteria From Hybrid Snakehead(Channa Maculate♀×Channa Argus♂) With Ulcer Disease And Screening Of Chinese Herbal Medicine For Prevention And Treatment

Hybrid snakehead(Channa maculate♀×Channa argus♂), which is delicious and nutritious, has always been favorable for consumers. However, with the increasing stocking density, ulcer disease occurs frequently. This has resulted in large amounts of hybrid snakeheads’ death and loss of commercial value, and strongly limits the development of hybrid snakehead aquaculture. In the present study, we took samples of hybrid snakehead, which suffered from ulcer disease in Sanjiao town Zhongshan city, Guangdong province in 2014. We isolated and characterized the pathogenic bacteria by traditional and molecular methods. Then drug susceptibility test, herbal antibacterial and bactericidal test were conducted to screen antibiotics and herbs with potent antibacterial activity.A fungal strain, named as G1, was isolated from the lesion of infected hybrid snakehead. G1 colony grew fast and had loose structure and grey-green surface. Optical microscope observation showed that the cell consisted of branched mycelia. The vegetative mycelium had intervals, while part of aerial mycelium formed long and rough conidiophores. There was a spherical vesicle at the top of the conidiophore, which had many cannulas with spherical conidia attached. G1 seemed to be Aspergillus flavus according to its morphological characteristics. Genomic DNA of G1 was amplified using primers ITS1 and ITS4. After comparing ITS sequences on NCBI using Blast, G1 was confirmed to belong to genus Aspergillus. Phylogenetic trees were reconstructed using MAGA 5.0 via neighbor-joining methods, with Aphanomyces invadans(AY283642.1, EU422990.1, AF396684.1) being out group. The results showed that G1 was clustered with Aspergillus flavus. G1 was not so genetically related to pathogenic Aphanomyces invadans reported in other studies.Three bacterial strains were isolated from liver, spleen and muscle of infected hybrid snakeheads. They were named as L9, S8, M7, respectively. According to artificial infection test, dosage of bacterial strains S8 over 1.0×107 CFU/tail could killed all of hybrid snakeheads, which indicated the pathogenicity of bacterial strains S8. This strain could form off-white, semi-transparent, round convex colonies with smooth and neat edges on Brian Heart Infusion(BHI) plates. This strain was Gram-negative, motile, positive for oxidase test, arginine dihydrolase test and lysine decarboxylase test, able to utilize maltose, galactose and d- mannitol, Indole-positive, and negative for rhamnose test, hydrogen sulfide test, arabinose test, tartaric acid test, cellobiose test, sorbitol test, VP test, salicin test, simon’s citrate test.PCR amplification was conducted using bacterial 16 S rRNA gene universal primers as primers and bacterial genomic DNA as template. A 1440 bp sequence was obtained and was submitted to NCBI to get an accession number, KT036666. The sequence was compared on NCBI using Blast. Phylogenetic trees were reconstructed using MAGA 5.0 via neighbor-joining methods. The results showed that S8 was clustered with Aeromonas sobria KF761305.1. Since gyrB is more specific than 16 S rRNA gene in terms of identification of bacteria, specific primers for gyrB were used to amplify the gene. A 1048 bp sequence was obtained and submitted to NCBI to get an GeneBank accession number, KT036666. The result of multiple sequence alignments and phylogenetic tree was in accordance with that of 16 S rRNA analysis, where S8 was clustered with Aeromonas sobria. The strain was confirmed to be Aeromonas sobria in combination with morphological physiological and biochemical characteristics.Drug sensitivity test on Aeromonas sobria S8 showed that amikacin, cephradine, levofloxacin, enoxacin, ofloxacin, tetracycline nitrofurantoin, and norfloxacin had strong inhibitory effect, followed by kanamycin, streptomycin, gentamicin, neomycin and ceftriaxone. Ceftazidime, ampicillin, cotrimoxazole had no inhibitory effect on bacterial strains S8. The antibiotic activities of 18 kinds of Chinese herb against Aeromonas sobria were tested. The results showed that Punica granatum, Rhus chinensis and Scutellaria baicalensis had significant inhibitory effect on Aeromonas sobria, with minimum inhibitory concentration(MIC) and minimum bactericidal concentration(MBC) being 3.91 mg/mL and 7.81 mg/mL, respectively. Cornus officinalis, Aloe and Sanguisorba officinalis also exerted inhibitory effect with the MIC and MBC being 7.81 mg/mL and 15.63 mg/mL, respectively. The inhibitory effect of Citrus reticulata Blanco, Forsythia suspensa, Caesalpinia sappan, Artemisia argyi and Crataegus pinnatifida was weak, while the other seven herbs including Isatis tinctoria had no inhibitory effect against Aeromonas sobria. To sum up, Punica granatum, Rhus chinensis and Scutellaria baicalensis on Aeromonas sobria showed good antibacterial effect and can be used for the control and prevention of ulcer disease of hybrid snakehead.