鲤源嗜水气单胞菌分离鉴定、耐药性及毒力基因检测

为查明贵州某鲤鱼养殖厂鲤鱼死亡的病因,本试验进行了细菌分离培养、菌体形态观察、细菌生理生化鉴定、16S rRNA基因测序分析、药敏试验、耐药基因检测、动物感染试验、毒力基因检测等。结果显示,从死亡鲤鱼病变组织中分离到的细菌呈圆形凸起、表面光滑的灰白色菌落,为革兰氏阴性短杆菌;生化鉴定结果显示,分离菌具有运动性,能发酵葡萄糖产酸产气,精氨酸双水解酶、赖氨酸脱羧酶、M-R和V-P试验阳性;应用细菌16S rRNA基因通用引物进行PCR扩增获得大小为1 421 bp的片段,测序发现,该分离菌与16株嗜水气单胞菌相应序列同源性达75.3%~100.0%;药敏试验结果显示,该分离菌对氟苯尼考、头孢曲松、妥布霉素、氧氟沙星、恩诺沙星5种药物呈现高度敏感,对复方新诺明、磺胺异噁唑、羧苄西林等药物呈现耐药,经耐药基因PCR检测显示,该分离菌携带Sul1、Sul2和Intl1 3种耐药基因,与药敏表型相符;人工感染试验显示,该分离菌有致病力,经毒力基因检测显示,该分离菌携带aer、hly和act 3种毒力基因。上述试验结果表明,本研究分离得到了一株耐磺胺类药物的鲤源嗜水气单胞菌,为该鲤鱼养殖厂的鲤鱼疾病防治与合理用药提供了科学依据。     

Aeromonas hydrophila as cause of hemorrhagic septicemia in a ground-hornbill (Bucorvus abyssinicus)

An acute hemorrhagic septicemia in a captive ground-hornbill (Bucorvus abyssinicus) is reported. Aeromonas hydrophila was isolated from lungs, liver, kidney, and intestines. Postmortem lesions were characterized by hemorrhages in the internal organs. Microscopically, necrotic lesions were found in the liver, lungs, and kidney. Factors that might have contributed to the clinical disease condition are discussed.     

锦鲤致病性嗜水气单胞菌分离和药敏试验

从北京某渔场发病锦鲤中分离出8株细菌。经形态学、生化特性鉴定有凝集试验，证明其中3株为嗜水气单胞菌。通过溶血试验、脱脂奶琼脂平板法、Dot-ELISA法及动物试验，显示这3株菌均能产生溶血毒素和胞外蛋白酶，并能致死小鼠。药敏试验表明它们对常用抗生素呈不同程度的耐药性。     

Disseminated infection due to Mycobacterium avium subsp. avium in an Asian elephant (Elephas maximus)

A disseminated infection caused by Mycobacterium avium subspecies avium (MAA) was diagnosed in a 57-yr-old male Asian elephant (Elephas maximus) housed at the Seoul Zoo, Gyeonggi, Republic of Korea. An apparent granulomatous inflammation with central caseous necrosis was evident in the lung sections. To confirm mycobacterial infection, polymerase chain reaction-restriction enzyme polymorphism analysis (PCR-RFLP) of the rpoB and hsp65 genes was performed from multiple organs and cultured bacteria. The PCR-RFLP revealed a M. avium subspecies. MAA was identified by multiplex PCR for detection of IS901 and IS1311. Thus, it is believed that MAA caused the disseminated infection in this case. Although the source of infection was not determined, the elephant may have become infected through contamination of soil and feed by free-living birds infected with MAA. This is the first reported case of disseminated infection due to MAA in a captive elephant in the Republic of Korea.     

嗜水气单胞菌对黑斑蛙和花背蟾蜍的致病性研究

利用经分类鉴定并保存于固定培养基中的ＡＨ９８０４嗜水气单胞菌株对黑斑蛙和花背蟾蜍进行动物接种感染试验（腹腔接种剂量２ｍＬ、含菌数１４４×１０８个）,结果显示,该菌对上述２种动物具有明显的致病作用。同时,使用所制备高免血清作了同种动物免疫保护试验（血清抗体效价１２８０、肌肉接种剂量３０ｍＬ）,对其结果进行了验证。     

Class 1 integrons in Aeromonas hydrophila isolates from farmed Nile tilapia (Oreochromis nilotica)

The aim of this study was to determine antimicrobial resistance of Aeromonas hydrophila isolated from farmed Nile Tilapia. A total of 50 A. hydrophila isolates from clinical cases were screened for the presence of class 1, 2 and 3 integrons and all the strains resistant to enrofloxacin and/or ciprofloxacin (n=19) examined for mutation in the quinolone resistance-determining regions (QRDRs) of gyrA and parC. The intI1 gene was detected in 23 A. hydrophila strains (46%) but no intl2 and intl3 were detected. Among these, 14 isolates (60.8%) carried gene cassettes inserted in variable regions i.e., partial aadA2, aadA2, dfrA1-orfC and dfrA12-aadA2, of which the most common gene cassette array was dfrA12-aadA2 (26.09%). Conjugal transfer of class 1 integrons with resistance gene array was detected. All the A. hydrophila strains resistant to enrofloxacin and/or ciprofloxacin possessed mutations in the QRDRs of gyrA and parC. Only a Ser-83-Ile substitution was identified in GyrA and only a Ser-80-Ile amino change was found in ParC. The data confirms that A. hydrophila from farm-raised Nile Telapia serve as a reservoir for antimicrobial resistance determinants.     

Isolation and Identification of Aeromonas hydrophila from an Outbreak of Haemorrhagic Septicemia in Snakes

Aeromonas hydrophila was isolated during an outbreak of a fatal disease among snakes kept in the serpentarium of the Razi Institute. The organism was identified on the basis of morphological and biochemical properties, as well as pathogenicity for both warm blood and poikilothermic animals.     

1株锦鲤致病性嗜水气单胞菌的分离鉴定及生物学特性研究

从患病锦鲤体内分离得到1株细菌ML1,经人工感染后发现ML1具有较高的致病性,临床症状与自然发病症状一致,96 h的LD50为5.0×107cfu/m L。ML1菌株革兰染色呈阴性,杆状。16S rRNA基因序列分析结果显示,序列片段长度为1 419 bp,ML1菌株在系统发育树上与嗜水气单胞菌聚类,相似性达到100%。综合VITEK 2 Compact全自动微生物鉴定仪的生理生化特征分析、形态学观察、人工感染以及16S rRNA基因序列分析,将ML1菌株鉴定为嗜水气单胞菌。ML1菌株的最适生长p H为7~9,最适生长温度为28℃。在28℃200 r/min条件下的生长周期是:0~4 h为生长延迟期,4~10 h为对数生长期,10~24 h为稳定期,24~48 h为衰亡期。药敏试验表明,ML1菌株对多黏霉素B、环丙沙星、诺氟沙星等高度敏感,对磺胺异恶唑中度敏感,对麦迪霉素、氨苄西林、林可霉素等不敏感。研究结果为指导临床合理用药提供了科学依据。     

Effect of an immunopotentiator on Aeromonas salmonicida infection in rainbow trout (Salmo gairdneri)

The immunoactive peptide FK-565 (heptanoyl-y-D-glutamyl-(L)-mesodiaminopimelyl-(D)-alanine) was found to induce protection against intraperitoneal Aeromonas salmonicida infection in rainbow trout (Salmo gairdneri Richardson). The survival rate was as high as 60% when FK-565 was given intraperitoneally as a single dose (1mg/kg) one day before bacterial challenge. A non-specific stimulation of phagocytic cells by FK-565 at an early stage of the bacterial infection may contribute to the resistance observed. The phagocytic activity of peritoneal phagocytic cells as well as phagocytic cells of the pronephros were stimulated by FK-565 in vivo and in vitro, respectively, as compared to untreated control fish. Furthermore, decreased activity of phagocytic cells previously immunosuppressed with cyclophosphamide was rapidly restored by application of FK-565.     

嗜水气单胞菌J-1株外膜蛋白Omp38的抗原性分析

根据GenBank已发表的嗜水气单胞菌(Aeromonas hydrophila)外膜蛋白Omp38的基因序列设计引物,以Ah J-1株基因组为模板,扩增omp38基因片段,定向克隆至表达质粒pET32 a中,将重组原核表达质粒pET32 a-omp38转化至大肠杆菌BL21,诱导表达出分子量约35 ku的重组蛋白。用纯化的重组蛋白免疫新西兰兔制备抗血清,ELISA检测抗体效价为1∶25 600,表明该蛋白有良好的免疫原性。PCR检测表明,omp38基因可在73%(22/30)的嗜水气单胞菌中发现。重组蛋白抗血清与30株嗜水气单胞菌分离株进行凝集试验,所有分离株均可在不同程度上发生反应,表明该重组蛋白是一种潜在的共同保护性抗原,可作为基因工程亚单位疫苗的候选成分。     

6株嗜水气单胞菌的毒力因子及其对小鼠的致死性

根据嗜水气单胞菌毒素、蛋白酶及Ｓ蛋白３种毒力因子的检测结果,从６６株嗜水气单胞菌中选出３种毒力因子分布情况各异的有代表性的６个菌株。分别用含菌量均为１０８ＣＦＵ／ｍＬ的６株嗜水气单胞菌肉汤培养物注射小鼠,结果,毒素、蛋白酶及Ｓ蛋白均有的菌株以及具有前两者而无Ｓ蛋白的菌株对小鼠的致死率均达１００％;仅有蛋白酶的菌株对小鼠致死率达６０％;而只有Ｓ蛋白的,或既有毒素又有Ｓ蛋白的,或３种毒力因子均无的菌株对小鼠无致死性。     

PEG对嗜水气单胞菌疫苗制备与免疫效果影响的研究

为评价聚乙二醇(PEG)作为超声介质及疫苗佐剂对嗜水气单胞菌疫苗免疫效果的影响,本研究将该疫苗与常规疫苗分别经腹腔注射免疫接种锦鲤成鱼,检测血细胞吞噬百分率、吞噬指数、抗菌活力、抗体效价及免疫保护力等指标,评价疫苗效果.结果表明PEG6000作为佐剂制备的疫苗,其各指标均高于对照组及其他试验组.PEG6000疫苗免疫保护力达到77.8％,高于常规灭活疫苗22.2％,表明PEG6000作为佐剂制备的疫苗效果最好.     

温和气单胞菌毒力基因的检测及其对鲫鱼致病性试验

为探究温和气单胞菌菌株A.S1的致病性,采用PCR方法扩增溶血素基因(hly)、细胞兴奋性肠毒素基因(alt)、黏附素基因(aha1)和气溶素基因(aerA)4种毒力基因,并运用动物回归试验探究菌株A.S1对鲫鱼的致病性。结果表明,菌株A.S1可扩增出hly、aha1和alt 3种毒力基因,未扩增出aerA基因。动物回归试验显示,试验组鲫鱼在腹腔注射温和气单胞菌0.3mL(浓度为1.5×108 CFU/mL)后,5d内死亡率达100%。表明毒力基因型为hly+、aha1+、alt+和aerA-的菌株A.S1为高毒力菌株,且菌株的毒力基因型与致病性呈正相关。     

Correlation between production of acyl homoserine lactones and proteases in an Aeromonas hydrophila aroA live vaccine

Aeromonas hydrophila is a pathogen that causes disease in a wide range of homeothermic and poikilothermic hosts due to its multifactorial virulence. We have previously described the characterisation and use of an auxotrophic aroA mutant of the A. hydrophila AG2 strain as a live attenuated vaccine against A. hydrophila infections in rainbow trout (Oncorhynchus mykiss). In this study we report the expression of extracellular proteolytic activities and of quorum-sensing molecules by this mutant grown under different culture conditions, and in vaccine inocula. The aroA strain expresses extracellular proteases efficiently during in vitro growth and this ability is retained in vaccine inocula that were prepared by washing the bacterial cultures and resuspending the cells in phosphate-buffered saline. Since proteases are considered to be major bacterial antigens, the expression of these enzymes in the live attenuated vaccine may contribute to the superior protection afforded by these kind of vaccines. On the other hand, the production of serine- and metalloprotease activities in A. hydrophila has been described as controlled in a cell density-dependent fashion, through a mechanism known as quorum sensing. A microtiter method was developed that allowed correlation of the production of quorum-sensing molecules and of proteases produced by the aroA strain during in vitro growth and in the vaccine inocula. The production of both products was related to the type of culture medium and conditions used to grow the aroA mutant, whereas there was no correlation between the concentration of acyl homoserine lactones and protease production.