大鲵“腹水病”病原的鉴定及其组织病理学研究
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摘要
为了研究大鲵腹水病的主要病原菌及其组织病理,于2009～2010年间在陕西省汉中市多家大鲵养殖场进行腹水病大鲵样品采集,采集的患病大鲵经活体解剖,在无菌条件下从腹腔、肝脏、肌肉等处分离出单个菌落,纯化后进行生理、生化鉴定和分子生物学鉴定;对致病菌采用纸片法进行药敏实验;对主要病灶组织肝脏、心脏和脾脏等采用10%福尔马林溶液固定,常规石蜡切片制片, H·E染色,在光学显微镜下观察不同病原导致的大鲵腹水病的病理变化。取得以下结果:
1、大鲵“腹水病”病原菌的分离鉴定:通过对4株致病菌的16S rRNA序列测定,并与GenBank数据库中的16S rRNA序列进行比对,同时进行系统进化树分析;结合致病菌的形态特征、培养特征及生理生化特性,将4株致病菌确定为腐败斯瓦尼菌(Shewanella putrefaciens)、荧光假单胞菌(Pseudomonas fluorescens)、布鲁氏杆菌(Brucellaceae bacterium)和嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia)。
2、大鲵“腹水病”病原菌的药敏试验:(1)腐败斯瓦尼菌对硫酸链霉素、硫酸卡那霉素、氟苯尼考、乳酸环丙沙星、盐酸恩诺沙星、盐酸诺氟沙星、盐酸左氧氟沙星等高度敏感;(2)荧光假单胞菌对硫酸卡那霉素、氟苯尼考、盐酸恩诺沙星、盐酸左氧氟沙星等高度敏感;(3)布鲁氏杆菌对硫酸链霉素、硫酸卡那霉素、氟苯尼考、乳酸环丙沙星、盐酸恩诺沙星、酒石酸吉他霉素、阿奇霉素等高度敏感;(4)嗜麦芽窄食单胞菌对硫酸链霉素、硫酸卡那霉素、氟苯尼考、盐酸恩诺沙星、酒石酸吉他霉素、盐酸左氧氟沙星等高度敏感。4株菌对磺胺对甲胺嘧啶钠、红霉素不敏感。
3、大鲵“腹水病”主要病灶组织的病理变化:4种病原菌均可造成大鲵肝脏、心脏等组织发生不同程度的病理变化。其主要表现为心肌细胞和肝细胞肿胀,胞浆浑浊,胞浆中可见微细的蛋白质颗粒。肝组织中见大量炎性细胞浸润。荧光假单胞菌、布鲁氏杆菌、嗜麦芽窄食单胞菌都引起大鲵脾脏病变,致使脾脏淋巴细胞增生,但是腐败斯瓦尼菌没有引起大鲵脾脏的病理变化。
本试验鉴定了4株导致大鲵“腹水病”的致病菌,同时进行了药敏试验和组织病理的观察,对于大鲵“腹水病”的防治具有重要指导意义。
During 2009 and 2010, many samples of giant salamander infecting ascites disease were collected to investigate the pathogen of ascites disease and its histopathology. The collected giant salamanders were vivisected to isolate the pathogen from the peritoneal cavity, liver, muscle, et al. And then the isolated pathogens were identified by physiology, biochemistry and moleculer biology trials. Drug sensitive tests were performed on pathogenic bacteria by paper disk method, and the mainly focus tissues of a disease, such as liver, heart and spleen, were immobilizated in 10% formol, prepared paraffin section and H.E staining to observe the different pathological changes of giant salamander ascites disease resulted by different pathogens under the optical microscope. The main results are as follows:
1. Isolation and identification of pathogen of ascites disease of giant salamander: 4 pathogenic bacteria strains were identified as Shewanella putrefaciens, Pseudomonas fluorescens, Brucellaceae bacterium and Stenotrophomonas maltophilia by 16S rRNA analysis, morphological, Physiology and biochemistry characteristics.
2. The drug sensitive tests of pathogen of ascites disease of giant salamander: (1) Shewanella putrefaciens was very sensitive to the streptomycin sulfate, kanamycin sulphate, florfenicol, ciprofloxacin lactate, Enrofloxacin HCL, Norfloxacin Hydrochloride, Levofloxacin Hydrochloride. (2) Pseudomonas fluorescens was very sensitive to the kanamycin sulphate, florfenicol, Enrofloxacin HCL, Levofloxacin Hydrochloride.(3)Brucellaceae bacterium was very sensitive to the streptomycin sulfate, kanamycin sulphate, florfenicol, ciprofloxacin lactate, Enrofloxacin HCL, kitasamycin tartrate, Azithromycin. (4) Stenotrophomonas maltophilia was very sensitive to the streptomycin sulfate, kanamycin sulphate, florfenicol, Enrofloxacin HCL, kitasamycin tartrate, Levofloxacin Hydrochloride. But the four pathogenic bacteria strains were not sensitive to p- Sulfanilamide-p- methylamine pyrimidine sodium and erythromycin.
3. The mainly pathological changes of focus tissues of a disease: different pathological changes can be occurred in the tissue of liver and heart. The pathological characteristics are the cellular swelling of heart and liver, kytoplasm muddy and some minute protein particle in kytoplasm. And many inflammatory cell infiltrates can be seen in the tissue of liver. Pseudomonas fluorescens, Brucellaceae bacterium, Stenotrophomonas maltophilia can cause the pathological changes of the tissue and lymphocyte proliferation of spleen, but the Shewanella putrefaciens did not cause any pathological changes of spleen.
In this study, four pathogenic bacteria strains were isolated and identified. At the mean time, drug sensitive tests were performed and pathological changes of the tissue were observed, which were significance for the prevention and cure of the ascites disease of giant salamander.
1、大鲵“腹水病”病原菌的分离鉴定:通过对4株致病菌的16S rRNA序列测定,并与GenBank数据库中的16S rRNA序列进行比对,同时进行系统进化树分析;结合致病菌的形态特征、培养特征及生理生化特性,将4株致病菌确定为腐败斯瓦尼菌(Shewanella putrefaciens)、荧光假单胞菌(Pseudomonas fluorescens)、布鲁氏杆菌(Brucellaceae bacterium)和嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia)。
2、大鲵“腹水病”病原菌的药敏试验:(1)腐败斯瓦尼菌对硫酸链霉素、硫酸卡那霉素、氟苯尼考、乳酸环丙沙星、盐酸恩诺沙星、盐酸诺氟沙星、盐酸左氧氟沙星等高度敏感;(2)荧光假单胞菌对硫酸卡那霉素、氟苯尼考、盐酸恩诺沙星、盐酸左氧氟沙星等高度敏感;(3)布鲁氏杆菌对硫酸链霉素、硫酸卡那霉素、氟苯尼考、乳酸环丙沙星、盐酸恩诺沙星、酒石酸吉他霉素、阿奇霉素等高度敏感;(4)嗜麦芽窄食单胞菌对硫酸链霉素、硫酸卡那霉素、氟苯尼考、盐酸恩诺沙星、酒石酸吉他霉素、盐酸左氧氟沙星等高度敏感。4株菌对磺胺对甲胺嘧啶钠、红霉素不敏感。
3、大鲵“腹水病”主要病灶组织的病理变化:4种病原菌均可造成大鲵肝脏、心脏等组织发生不同程度的病理变化。其主要表现为心肌细胞和肝细胞肿胀,胞浆浑浊,胞浆中可见微细的蛋白质颗粒。肝组织中见大量炎性细胞浸润。荧光假单胞菌、布鲁氏杆菌、嗜麦芽窄食单胞菌都引起大鲵脾脏病变,致使脾脏淋巴细胞增生,但是腐败斯瓦尼菌没有引起大鲵脾脏的病理变化。
本试验鉴定了4株导致大鲵“腹水病”的致病菌,同时进行了药敏试验和组织病理的观察,对于大鲵“腹水病”的防治具有重要指导意义。
During 2009 and 2010, many samples of giant salamander infecting ascites disease were collected to investigate the pathogen of ascites disease and its histopathology. The collected giant salamanders were vivisected to isolate the pathogen from the peritoneal cavity, liver, muscle, et al. And then the isolated pathogens were identified by physiology, biochemistry and moleculer biology trials. Drug sensitive tests were performed on pathogenic bacteria by paper disk method, and the mainly focus tissues of a disease, such as liver, heart and spleen, were immobilizated in 10% formol, prepared paraffin section and H.E staining to observe the different pathological changes of giant salamander ascites disease resulted by different pathogens under the optical microscope. The main results are as follows:
1. Isolation and identification of pathogen of ascites disease of giant salamander: 4 pathogenic bacteria strains were identified as Shewanella putrefaciens, Pseudomonas fluorescens, Brucellaceae bacterium and Stenotrophomonas maltophilia by 16S rRNA analysis, morphological, Physiology and biochemistry characteristics.
2. The drug sensitive tests of pathogen of ascites disease of giant salamander: (1) Shewanella putrefaciens was very sensitive to the streptomycin sulfate, kanamycin sulphate, florfenicol, ciprofloxacin lactate, Enrofloxacin HCL, Norfloxacin Hydrochloride, Levofloxacin Hydrochloride. (2) Pseudomonas fluorescens was very sensitive to the kanamycin sulphate, florfenicol, Enrofloxacin HCL, Levofloxacin Hydrochloride.(3)Brucellaceae bacterium was very sensitive to the streptomycin sulfate, kanamycin sulphate, florfenicol, ciprofloxacin lactate, Enrofloxacin HCL, kitasamycin tartrate, Azithromycin. (4) Stenotrophomonas maltophilia was very sensitive to the streptomycin sulfate, kanamycin sulphate, florfenicol, Enrofloxacin HCL, kitasamycin tartrate, Levofloxacin Hydrochloride. But the four pathogenic bacteria strains were not sensitive to p- Sulfanilamide-p- methylamine pyrimidine sodium and erythromycin.
3. The mainly pathological changes of focus tissues of a disease: different pathological changes can be occurred in the tissue of liver and heart. The pathological characteristics are the cellular swelling of heart and liver, kytoplasm muddy and some minute protein particle in kytoplasm. And many inflammatory cell infiltrates can be seen in the tissue of liver. Pseudomonas fluorescens, Brucellaceae bacterium, Stenotrophomonas maltophilia can cause the pathological changes of the tissue and lymphocyte proliferation of spleen, but the Shewanella putrefaciens did not cause any pathological changes of spleen.
In this study, four pathogenic bacteria strains were isolated and identified. At the mean time, drug sensitive tests were performed and pathological changes of the tissue were observed, which were significance for the prevention and cure of the ascites disease of giant salamander.
引文
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