| The snake is a reptile that has high economic value in medicine and food industry. Pseudomonas aeruginosa(P. aeruginosa) is one of the opportunistic pathogens. P. aeruginosa produced endotoxin, cytotoxin, leukocidin and other pathogenic factors. These pathogenic factors could cause extensively damage to the organs, futhermore, cause various disease and inflammation. With the development of intensive animal husbandry sysytem, the P. aeruginosa infection rate growed rapidly. It brought great threat to the development of the snake breeding industry. By studying the biological characteristics and drug resistance of P. aeruginosa, our results provide theoretical basis for diagnosis and treatment of P. aeruginosa disease. In this study two strains of bacteria with common lesions were isolated from the incidence of death of the snake in a snake farm in Shangluo District,Shaanxi province. The biological characteristics, the drug resistance of bacteria, pathogenicity and 16 SrRNA amplification and sequences of these two strains s were investigated. The results were summarized in the following points:1. The bacteria was detected from the tissue samples of two sick snakes in a snake farm. Two suspected bacteria, named as B2 and 2G2, with similar characteristics of P. aeruginosa were isolated. They were both gram-negative, medium-sized and coccobacillus, arranged alone or in pairs and occasionally into short chain. Both of them could the growth in general media, with round colonies, medium-sized, smooth and moist ones with neat edge. Colonies produced a green pigment which spread to the surrounding and dyed the medium. The large, raised, wet, dark grey colonies with neat edge appeared on the blood medium and medium-sized, moist, round, colorless colonies appeared on MacConkey medium after 24 h incubation.2. The ability of B2 and 2G2 to ferment sugars and alcohol was not strong. They could ferment arabinose, glucose and sorbose, but could not ferment raffinose, lactose, fructose, sorbitol, mannitol, xylose and sucrose. Oxidase and catalase tests were positive, and MR-VP test was negative. The strains of bacterium were initially identified as P. aeruginosa.3. The 16 S r RNA sequences of the two strains B2 and 2G2 were amplified with a pair of specific primers by PCR. The 16 S rRNA sequences of the Two strains were 100% identical. And the sequence data has been deposited in GenBank. The BLAST analysis result showed that the identity of 16 S r RNA sequences of two strains to that of typical strains of P. aeruginosa, were higher than 99%, indicating that these two strains belonged to P. aeruginosa. In order to determine the relationship among strains B2, 2G2 and other strains of P. aeruginosa, a phylogenetic tree was constructed. The strains B2 and 2G2 showed close relation with MW3 A, which was isolated from marine environment.4. The antimicrobial susceptibility of two strains B2 and 2G2 was analysis using the antimicrobial assay. The result showed that the bacteria had strong resistance to erythromycin, tetracycline, chloramphenicol, oxacillin, ampicillin and cotrimoxazole.5. In order to determine whether the isolated bacteria are pathogenicity in animals, mice were infected with two strains B2 and 2G2 at different dosage. All the infected mice were dead within 72 h after intraperitoneal injection, except for the control. Obvious lesions and pathological changes were observed in hearts, livers, kidneys and lungs. The tissues were collected and fixed in 10 % formaldehyde solution to make paraffin sections. The following pathological changes were observed: a small amount of bleeding between myocardial fibers, liver and central venous congestion between leaf expansion, renal tubular epithelial cell necrosis leaded to some pathological changes such as Lumen contraction.In conclusion, two strains of bacterium were identified as P. aeruginosa. They were not only multidrug-resistant but also high pathogenesis. |
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