Isolation Identification And Biological Characterization Of Pathogenic Neisseria Isolated From Ducks

An acute infectious disease outbroke in some scale duckeries in April2012,Anhui Province. The disease made ducks sick and even blind with pathological changes of eyes--wollen eyes, increasing number of eyelid secretions, glued eye conjunctivas. The disease spreads very fast, once a few ducks suffer, all the ducks would fall ill in1-2days. The morbidity rate of the disease is high,but the mortality of the disease is only about5%.Besides,the rejection rate is high because of their incapacity of eating with monocular blindness or blindness in both eyes. This situation has caused serious economic losses to local duck industry.The two strains of Neisseria are isolated from ducks in Anhui Area. We did the morphological observation,the analyses and identify of cultural characters, biochemical tests and16S rRNA sequence for the two strains. In the observations of scanning electron microscope, every coccus looks like an egg or a kidney. The thallus is about0.5-0.7μm long,0.5μm wide, and it has the model morphological characteristics of the Neisseria. The isolated strains inoculated in the Chocolate Agar Plate grew well, and their bacterial colonies are gray, smooth and round in the Chocolate Agar Plate.Phylogentic tree constructed by16S rRNA sequence of strains from different Neisseria can prove that the isolated strain is a bacteria of Neisseria. The16S rRNA sequence of the strains is99%homologous identity to the Neisseria. So it can be preliminary proved hat the isolated strains are the bacteria of Neisseria. We respectively named the two strains as AH-N10and XC. For the fast detection of the Neisseria isolated from ducks, we constructed the PCR diagnostic method. A pair of primer was designed and synthesized based on asd gene sequence from Neisseria strain for detecting Neisseria isolated from ducks. The PCR method developed in this study is proved steady effective and highly sensitive after specificity test and sensitivity test. And it is practically useful and fast in detecting Neisseria isolated from ducks.For the further understanding of the biological characterization of the Neisseria isolated from ducks, we do research on their phylogenetics, culture characteristics, ways of saving and pathogenicity. Phylogentic tree constructed by16S rRNA sequence of strains from different Neisseria can prove that the isolated strains are the bacteria of Neisseria and they have the highest homologous identity with N. Flavescens and N. mucosa.When the strains were cultured in TSB medium supplemented with5%fetal bovine serum, they can reach the stable phase with the bacterial content of109cfu/mL in8hours and begin to enter the decline phase in20hours.We use3different methods--glycerol preservation method, skim milk preservation method and freeze drying preservation method for the preserving effect test of the isolated strains. The results showed that glycerol preservation method isn’t suitable for the Neisseria isolated from ducks, the bacteria will lose their activity and can’t get recoveries. Skim milk preservation method and freeze drying preservation method can achieve good preservation effect. And the morphological characteristics of the bacteria did not change after recovery.The infection test of animals made it clear that this isolated strain has pathogenicity to ducklings and mice. After injecting the leg muscles108cfu the isolated bacteria, The ducklings were consistent with clinical ill ducks in clinical and autopsy lesions, the mortality rate was around30%. The mice under intraperitoneal injection also appeared obvious signs of eye disease. According to the Karber’s method,LD50of the two isolated strains are3.47×108,3.40×108.To resolve duck source neisseria fungus infection in clinical treatment, screening of the isolated bacteria sensitive antibacterial drugs and traditional Chinese medicine compound, drug sensitive test indicates that strains of Neisseria isolated are saitivity for Benzylpenicillin and sulfanilamide drugs. Using Chinese drug pulsatilla, coptis chinensis, phellodendron chinense et.. Chinese drug compound recipe is active for strains of Neisseria isolated duck.The MIC of Chinese drug compound for Chinese drug Chinese pulsatilla is0.25g/mL,using the Methods of solid medium, least concentration of Chinese drug pulsatilla is0.005g/mL and zone diameter is16mm.Chinese drug pulsatilla is useful to release duck’s sign, decrease death rate recovery rate reach90%. Clarificat that use Chinese drug pulsatilla has satisfactory clarification.