The Effects Of Chinese Herbal Medicine Effective Components On Aeromonas Veronii Virulence Gene Expression

In recent years, aquatic animals diseases caused by bacteria to the fishery development have caused serious economic losses. And Aeromonas veronii as pathogen have presented ascendant trend in recent cases. With the antibiotics frequently using, most pathogenic bacteria have drug resistance. At present, more and more traditional Chinese medicine (TCM) treatment have been recognized to prevent bacteria diseases. However, TCM have many ingredients complex, and there is no complete study can explain which one have the real active function to prevent or kill bacterias. Therefore, we select several Chinese herbal medicine effective components to test on A.veronii. We hope to explain the antibacterial mechanism of the Chinese herbal medicine effective components from expression level changes of the virulence gene, and provide a scientific basis for application of aquatic animal disease prevention and controlling.Test Ⅰ:Isolation and identification of pathogenic Aeromonas veronii from snakeheadA pathogenic bacterial strain swurc was isolated from diseased snakehead in Neijiang, SiChuan. The healthy Crucian carp showed the same pathogenic symptoms by the artificial infection test with isolated strain. The strain swurc was identified as Aeromonas veronii based on the determination of the16S rRNA sequence given priority to, and combined with the physiology and biochemistry results. Constructing phylogenetic trees, the results showed that the strain swurc16S rRNA sequence have high homology with top40strains of Aeromonas veronii (A.veronii), the homology is above99%.Test Ⅱ:The virulence Genes Aero and OMP Cloning and sequence analysis of Aeromonas veronii According to related literature references on virulence genes aerolysin Aero and outer membrane protein OMP primer sequences of Aeromonas. With Aeromonas veronii genome DNA as template, after PCR amplification, the gene fragments were cloned into pMD19-T vector to construct the recombinant plasmid and identified positive clones and sequenced the virulence gene. Then we compared and analyzed the amplified gene sequence homology. The results showed that PCR amplified Aerolysin Aero gene417bp and outer membrane protein gene OMP1017bp. The cloned Aero gene protein sequence compared with related GenBank Aero gene sequence had homology of99%; OMP gene protein sequence related with GenBank reported OMP gene sequence had homology of99%. The primers were designed based on the conservative region of Genbank reported Aero and OMP genes sequence of Aeromonas. We can amplify the violence gene fragments of the strain swurc. It indicated that Aeromonas virulence genes had the high homology.Test Ⅲ:Drug sensitive test on Aeromonas veroniiSelecting12kinds of Chinese herbal medicine effective components (quercetin, matrine, baicalin, giant knotweed, galangal, berbamine hydrochloride, gallic acid, hesperidin, emodin, mignonette, emodin methyl ether,rutin reference substance) adopted plate digging method for Aeromonas drug sensitivity test. And using double dilution method to determine minimum suppression bacteria concentration (MIC) and minimum bactericidal concentration (MBC) of the Chinese herbal medicine effective components on the Aeromonas veronii. The results showed that gallic acid had high sensitivity. Quercetin, emodin, baicalin and kaempferol had middle sensitivity, and the rest of the Chinese herbal medicine effective components showed low sensitivity. The MIC of quercetin on the strain swurc was0.125mg/mL, MBC was0.25mg/mL; MIC and MBC of gallic acid and baicalin were0.25mg/mL; the MIC of emodin was0.25mg/mL, MBC was0.5mg/mL.TestIV:The effects of Chinese herbal medicine effective components on Aeromonas veronii virulence gene expression outside the bodySelecting better four kinds of Chinese herbal medicine effective components including gallic acid, quercetin, baicalin and emodin. Taking the MIC as the test middle concentration, set three concentrations affecting the Aeromonas veronii. Extracted Aeromonas veronii total RNA and served16S rRNA as the internal reference, after reverse transcription PCR (RT-PCR) amplification of the Aeromonas veronii virulence gene. We analyzed the effects of Chinese herbal medicine effective components on the Aeromonas veronii virulence gene expression levels. The results showed that gallic acid and baicalin testing on A.veronii, the0.25mg/mL group and0.5mg/mL of virulence genes Aero and OMP had significant difference with blank group(P<0.05). Quercetin testing on A.veronii, the0.25mg/mL group of virulence genes Aero and OMP had significant difference with blank group、0.0625mg/mL group and0.125mg/mL group (P <0.05). And emodin testing on A.veronii,0.5mg/mL group of virulence gene Aero had significant difference with blank group、0.125mg/mL group and0.25mg/mL group (P<0.05). Comparing the virulence gene OMP between0.25mg/mL group and0.5mg/mL group with blank group showed the significant difference (P<0.05). That is with the concentration of Chinese herbal medicine effective components increasing, the expression of virulence genes showed the different degrees of decline. It indicated the Chinese herbal medicine effective components could inhibit the expression levels of Aeromonas veronii virulence genes Aero and OMP.Test V:The effects of Chinese herbal medicine effective components on Aeromonas veronii virulence gene expression inside the bodySelecting four kinds of Chinese herbal medicine effective components including gallic acid, quercetin, baicalin and emodin. Mixing the four kinds of Chinese herbal medicine effective components into the feed. And taking the MIC as the test middle concentration, set three concentrations to feed the Crucian carp. The Crucian carp were injected with0.2mL of Aeromonas veronii. One week later. The concentration of Aeromonas veronii. was10cfu/mL. After another week, we selected the dying Crucian carp to extract the RNA of Aeromonas veronii from hepatopancreas. After reverse transcription PCR (RT-PCR) amplification of the Aeromonas veronii virulence genes Aero and OMP. The result showed that we could not amplify the virulence gene fragments.