野生胡子鲶“腹水病”病原分离和鉴定

从发生“腹水病”的野生胡子鲶体内分离出１４株致病菌，经生化鉴定，有１２株细菌（８７％）定为豚鼠气单胞菌，１株定为大肠埃希氏菌。动物回归试验也证实了细菌的致病性。因此，本次野生胡子鲶发生“腹水病”主要病原是豚鼠气单胞菌，药敏试验结果对细菌有效的药物有头孢类抗生素、丁胺卡那霉素，卡那霉素，新霉素，氯霉素，庆大霉素。     

中华鳖几种常见疾病病原的分离鉴定及药敏试验

从患“穿孔病”、“腐皮病”、“溶血性腹水病”等濒死鳖的肝、心血和腹水中分离到20株细菌，其中温和气单胞菌（Aeromonas sobria）5株，豚鼠气单胞菌（A.caviae）3株、凡隆气单胞菌（A.veronii）2株、奇异变形杆菌（Proteus mirabilis）2株、产碱假单胞菌（Pseudomonas alcaligene）2株、无色杆菌（Achro sp.）3株、不动杆菌（Acinetobacter sp.）2株、黄杆菌（Flavobacterium sp.）1株。人工感染试验证实，运动型气单胞菌的温和气单胞菌和豚鼠气单胞菌是其主要病原菌。药敏试验结果表明，分离菌株对新霉素、菌必治、丁胺卡那、庆大霉素、环丙沙星高度敏感。     

草鱼致病性豚鼠气单胞菌的分离鉴定

为鉴定一株引起草鱼(Grass carp)发病的致病菌,并评估其对健康草鱼的致病性和相关药物的敏感性,本研究从患病草鱼病变器官中分离到一株优势菌,对该分离株的形态学、生长特性、生理生化特性、16S rRNA序列等进行鉴定。结果显示:该菌为革兰氏阴性菌,具有β溶血特性,在LB培养基中生长14 h可达7.41×10~9 cfu/mL,其理化特性与豚鼠气单胞菌基本符合,16S rRNA序列与参考菌株ATCC15468序列同源性在99.5%以上。基于16S rRNA序列进化分析显示,该分离株与豚鼠气单胞菌同属一个分支,亲缘关系较近。该分离菌株对草鱼的半数致死量(LD_(50))为4.928×10~6 cfu,对恩诺沙星、卡那霉素、磺胺甲氧嘧啶等药物比较敏感。综合判断该致病菌为豚鼠气单胞菌,命名为JS分离株,对草鱼具有较强致病性,对部分药物较为敏感。     

豚鼠气单胞菌引起山羊猝死的鉴定

2020年5月,江苏省某养殖场饲养的220只山羊疑似感染产气荚膜梭菌,共死亡25只。为鉴定感染的产气荚膜梭菌复合群类型,采集大肠、小肠内容物以及肠系膜淋巴结、肝、脾、肾等组织病料进行细菌分离鉴定,并对分离菌株进行药敏试验及致病性试验。结果显示：分离菌株为革兰氏阴性短杆菌,经生化试验和16S rRNA试验鉴定为豚鼠气单胞菌;该分离株对多种常用抗菌药物耐药,仅对呋喃唑酮敏感;该菌能造成小鼠发病死亡。此次从临床发病羊病料中分离到致病性豚鼠气单胞菌,在国内较为少见,截至目前未见类似报道,本研究为避免此类疫病的临床误诊以及做好该病防控提供了帮助。     

鸭源致病性嗜水气单胞菌的分离鉴定与生物学特性分析

为了解嗜水气单胞菌的肉鸭健康带毒、致病与耐药情况,对贵州省三穗县某肉鸭屠宰场临床健康肉鸭随机取样,进行嗜水气单胞菌的分离鉴定、毒力基因检测及耐药性分析。结果显示,分离菌具有嗜水气单胞菌典型的培养特征,菌落形态、菌体形态和生化特性均与嗜水气单胞菌相符;16 S rRNA基因序列系统进化树显示,该分离菌与嗜水气单胞菌聚为一支,同源性均>99%;动物回归试验显示,该分离菌对小鼠有较强的致病性;毒力基因PCR检测显示,该分离菌携带aer、hly、epa、act、alt和ahp等6种毒力基因;药敏试验结果显示,该分离菌对复方新诺明、磺胺嘧啶、环丙沙星和诺氟沙星等15种药物耐药;耐药基因PCR检测显示,该分离菌携带qnrB、Sul1和IntI1等3种耐药基因,与药敏试验表型相符。研究结果为嗜水气单胞菌的生物学特性研究及防控提供参考依据。     

猪源杀鲑气单胞菌分离鉴定及毒力因子检测

为了确定河南省某猪场导致保育仔猪死亡的病原菌,从病死猪的肺脏、肝脏和脾脏分离得到一株优势菌。结果显示,经16S rDNA测序确定该优势菌为杀鲑气单胞菌;动物致病试验显示,分离菌株对小鼠具有致病性;药敏试验显示,该分离菌对磺胺异噁唑、四环素、土霉素、阿莫西林/克拉维酸、卡那霉素、氨苄西林等耐药;毒力基因PCR检测显示,act、ast、fla、lipase毒力基因为阳性,alt、elastase为阴性。本研究首次从发病猪体内分离到杀鲑气单胞菌,并为该条件致病菌的诊疗提供参考依据。     

斑点叉尾鮰源致病性维氏气单胞菌的分离与生物学特性鉴定

为鉴定一起致斑点叉尾鮰突然发病死亡的病原，本研究从发病斑点叉尾鮰中分离到1株致病菌GZTL2017，通过临床解剖观察、细菌分离培养、革兰氏染色镜检、动物回归试验、生化试验、16S rDNA序列分析、部分毒力基因检测和药敏试验进行鉴定。临床解剖观察结果显示，患病斑点叉尾鮰呈现体表溃疡、鳍根部出血、烂腮、肠管充血等症状；分离菌在培养基中呈现表面湿润凸起、边缘光滑半透明、形态均一的乳白色菌落；革兰氏染色镜检显示，分离菌为单个或成双存在、两端钝圆的短直阴性杆菌；动物回归试验显示，该分离菌有较强的致病性；生化试验结果显示，分离菌具有运动性，且氧化酶、V-P、赖氨酸脱氢酶等反应阳性，精氨酸双水解酶、硫化氢等反应阴性；16S rDNA基因序列系统进化树显示，该菌与维氏气单胞菌聚为一支，同源性均>99%；毒力基因检测结果显示，该菌能检出气溶素基因（Aer）、黏附素基因（Aha）、外膜蛋白基因（OmpA）3种毒力基因；药敏试验结果显示，该菌对氟苯尼考、强力霉素、氧氟沙星等14种药物敏感；对诺氟沙星、新霉素、万古霉素等7种药物中度敏感；对麦迪霉素、苯唑西林、头孢拉定等8种药物耐药，且对氟苯尼考、强力霉素的药物最小抑菌浓度分别为1和2 μg/mL。本试验成功分离到1株维氏气单胞菌，为斑点叉尾鮰维氏气单胞菌病防治提供参考依据。     

改良DHL培养基分离嗜温有动力气单胞菌的实验研究与评价

嗜温有动力气单胞菌是指嗜水气单胞菌（Ａｅｒｏｍｏｎａｈｙｄｒｏｐｈｉｌａ）、豚鼠气单胞菌（Ａ．ｃａｖｉａｅ）和温和气单胞菌（Ａ．ｓｏｂｒｉａ）,后两种的性状与嗜水气单胞菌基本相同。该菌对水产养殖业的危害主要是引起各种鱼类及鳖等的败血症,给水产养殖业带...     

中国豹源豚鼠气单胞菌的分子鉴定与毒力基因分析

从表现为急性出血性败血症的中国豹分离到一株细菌,为了鉴定这株细菌,并对其主要毒力基因进行检测分析,实验中进行了细菌的分离培养、生化鉴定及动物毒力试验;PCR扩增gyrB基因并结合系统发育树的构建和分析进行了菌种的分子鉴定;对分离菌进行了气溶素(aerolysin,aer)、热敏感细胞肠毒素(heat-Labile cytotonic enterotoxin,alt)、细胞毒性肠毒素(cytotoxic enterotoxin,act)、热敏蛋白酶(temperature-sensitive protease,eprCAI)、丝氨酸蛋白酶(serine protease,ahp)5种毒力基因的PCR扩增和测序。结果显示:根据培养特性、生化试验、小白鼠毒力试验鉴定分离菌为豚鼠气单胞菌;中国豹源细菌gyrB基因与豚鼠气单胞菌(JQ815381)的同源性为98.9%,应为豚鼠气单胞菌;具有act、alt、eprACI 3种毒力基因。该研究可为快捷、准确检测气单胞菌引起的疾病提供参考。     

匙吻鲟致病性豚鼠气单胞菌的分离鉴定和致病性研究

为了确定匙吻鲟发病的原因,从患病匙吻鲟的表皮组织中分离病原菌,对其进行形态学观察、生理生化试验和16S r DNA基因的扩增并经序列分析构建系统发育树,同时测定该菌株的致病能力和对药物的敏感性,结果表明,该菌株为豚鼠气单胞菌(Aeromonas caviae)。人工感染试验结果显示,分离菌株对匙吻鲟具有很强的致病性,并同时对禾花鲤和罗非鱼也有较强的致病性。药物敏感结果显示,分离菌株对磺胺二甲嘧啶、多黏菌素B、氟苯尼考等5种药物完全耐药,对左氧沙星、链霉素、恩若沙星等10种药物的敏感性较强。     

Identification and Pathogenicity Analysis of Aeromonas caviae M12 from Fox

We tried to identify the bacteria and explore the mechanism of the bacteria's pathogenicity via housekeeping gene gyrB and in vitro organ culture (IVOC) of ileum and intramuscular injection. Microscope, electron microscope and scanning electron microscope were also used to observe and the structure, pathogen of M12 and changes of infected tissues. The results showed that M12 was short gram-negative bacteria, and both ends of it were obtuse and the size without flagellum was (0.6 to 1.6) μm×(0.6 to 0.7)μm while length of flagellum was about 2 to 3 times of the length of bacteria.It had 100.0% similarity with Aeromonas caviae from GenBank. IVOC test observed M12' adhesion in intestinal epithelial cell, resulting in formulation of biofilm structure and damage in intestinal epithelial cell. Histopathologic examination showed that M12 could cause damage in intestine, liver, lung, kidney, muscle tissue of rabbit.The assay would offer references for researching the pathogenic mechanism of Aeromonas caviae.     

A comparison of Shiga-toxin negative Escherichia coli O157 aflagellate and intimin deficient mutants in porcine in vitro and in vivo models of infection

Isolation of Shiga-toxin (Stx) positive Escherichia coli O157:H7 from commercially grown pigs has been reported. Furthermore, experimental infection studies have demonstrated that Stx-positive E. coli O157:H7 can persist in 12-week-old experimentally orally inoculated conventional pigs for up to 2 months and that persistence was not dependent upon intimin. We have shown that the flagellum of Stx-negative E. coli O157:H7 does not have a role to play in pathogenesis in ruminant models whereas, in poultry, the flagellum of E. coli O157:H7 was important for long-term persistent infection. The contribution of the flagellum of Stx-negative E. coli O157 in the colonisation of pigs was investigated by adherence assays on a porcine (IPI-21) cell line, porcine in vitro organ culture (IVOC) and experimental oral inoculation of conventional 14-week-old pigs. E. coli O157:H7 NCTC12900nal(r) and isogenic aflagellate and intimin deficient mutants adhered equally well to IPI-21 cells. In porcine IVOC association assays, E. coli O157:H7 NCTC12900nal(r) was associated in significantly higher numbers to tissues from the caecum and the terminal rectum than other sites. The aflagellate and intimin deficient mutants significantly adhered in greater numbers to more IVOC gastrointestinal tissues than the parent. Groups of 14-week-old pigs were dosed orally with 10(10)CFU/10ml of either E. coli O157:H7 NCTC12900nal(r) or isogenic aflagellate and intimin deficient mutants and recovery of each test strain was similar. Histological analysis of pig tissues at post mortem examination revealed that E. coli O157 specifically stained bacteria were associated with the mucosa of the ascending and spiral colon. These data suggest that colonisation and persistence of Stx-negative E. coli O157:H7 in pigs, involves mechanisms that do not require the flagellum or intimin.     

五倍子对鳗鲡致病性气单胞菌的体外抗菌后效应

采用试管液体二倍稀释法测定中药五倍子对鳗鲡养殖中致病性气单胞菌(威隆气单胞菌K17、肠棕气单胞菌G03、豚鼠气单胞菌R37和嗜水气单胞菌G06)的最小抑菌浓度(MIC).然后再采用菌落计数法分别测定五倍子在0.25、0.5、1、2、4倍于各菌MIC时对鳗鲡4株致病性气单胞菌K17、G03、R37、G06的体外抗菌后效应(PAE).结果显示:五倍子在0.25、0.5、1、2、4倍MIC值时对鳗鲡4株致病性气单胞菌均具有一定的PAE,且PAE与药液浓度在一定范围内(0.25～4倍MIC)呈剂量依赖性,当药液浓度达4倍MIC时,PAE明显延长(P＜0.05);五倍子对各致病菌株K17、G03、R37、G06的PAE值在1倍MIC浓度以上差异显著(P＜0.05),在4倍MIC时对于致病菌株K17和R37的PAE之间差异较小.结论:试验结果提示,在养殖鳗鲡病害防治中设计投药方案,选用中药五倍子治疗由致病性气单胞菌感染所造成的烂鳃、肝胆肿大、败血症等疾病时,除了考虑药代动力学和MIC指标外,还应考虑PAE因素,可适当延长给药间隔时间,降低药物对鳗鲡的副作用及对其摄食量和生长的负面影响.     

益生菌对肠黏膜屏障的保护及作用机制研究进展

益生菌可以通过调节肠上皮细胞间紧密连接蛋白的表达,增加黏液分泌,抑制肠上皮细胞异常凋亡,激发肠黏膜免疫应答,从而保护肠黏膜屏障。对肠黏膜屏障的结构、病原菌对肠黏膜的损伤以及益生菌在受损肠黏膜保护及修复中的作用机制进行了综述,旨在阐明益生菌对维护机体健康的重要意义。     

鲎素对嗜水气单胞菌的抑菌活性及作用机制研究

为探讨鲎素对嗜水气单胞菌的抑杀作用,采用最小抑菌浓度、AB染料、扫描电镜、流式细胞仪和琼脂糖凝胶电泳等方法研究鲎素对嗜水气单胞菌的抗菌活性、杀菌率、胞内紫外物质泄露、膜负电荷数、外部形态结构及胞内基因组DNA的影响。结果表明,鲎素对嗜水气单胞菌的抗菌活性较其他细菌弱;2倍最低杀菌浓度(MBC)鲎素能在短时间内迅速杀死嗜水气单胞菌并导致胞内生物大分子泄漏;扫描电镜发现1倍MBC鲎素能导致轻微的壁膜破损及一些胞内物质渗出和粘连;流式细胞仪结果表明,1倍MBC鲎素能破坏细胞膜的完整性;AB染料测定表明鲎素处理后能改变膜负电荷数;琼脂糖凝胶电泳表明,鲎素能够与嗜水气单胞菌的基因组DNA发生作用,并呈浓度依赖关系,浓度越高对细菌基因组DNA损伤越大。鲎素对嗜水气单胞菌的抑菌活性弱,只有高浓度时才能短时间内导致胞内紫外吸收物质泄露、膜负电荷数发生改变及基因组DNA损伤。     

Research Advance on Intestinal Microbial-epithelial Cell Barrier Interactions

Intestinal epithelial cells (IECs) are the first line of defense against pathogenic microorganisms of animal organism, which are important component of mucosal mechanical barrier, immune barrier and chemical barrier, they have absorption and barrier double function. In the intestine, there are many kinds of microorganisms. According to its relationship with the host, it is divided into three types of commensal bacteria, conditional pathogenic bacteria and pathogenic bacteria, it plays an important role in the construction of intestinal barrier. Firstly, IECs identify the intestinal microbes by direct or indirect ways, and distinguish their own and non-self, it is immune tolerance to their own substances (such as, commensal bacteria), and produce specific immune response to non-self-substances (pathogenic bacteria). Both of IECs and intestinal commensal bacteria together against pathogens maintain intestinal health. When the pathogenic microorganisms invade the intestine, IECs defense pathogenic microorganisms mainly through extracellular secretions and cell surface mucus layer, and the former largely include mucin, antibacterial molecular and antimicrobial immunoglobulin. The intestinal symbiotic bacteria can resist the pathogenic microorganisms and maintain the normal intestinal mucosal barrier function through the competitive identification sites, the secretion of antimicrobial substances, the increase of mucus secretion, the induction of IECs renewal, proliferation and repair. In the process of resisting invasion of gut microbes, pathogenic microorganisms through their own movement, secretion of toxins and enzymes to destroy the intestinal epithelial barrier, and directly contact with IECs to damage them. So the interaction between IECs and intestinal bacteria maintain the intestinal homeostasis. In this paper, a review is made of the IECs and intestinal microbial structure and functional adaptations, and hope to elaborate the mechanism of intestinal microbial-epithelial cell barrier interaction.     

肠道微生物-上皮细胞屏障互作的研究进展

肠道上皮细胞（intestinal epithelial cells,IECs）是动物机体抵御病原微生物的第一道防线,是黏膜机械屏障、免疫屏障和化学屏障的重要组成部分,具有吸收和屏障双层功能。肠道中微生物数量庞大、种类繁多,根据其与宿主的关系,主要分为共生菌、条件致病菌和病原菌3类,在肠道屏障的构建中发挥重要作用。IECs首先通过直接或间接方式对肠道微生物进行识别,区别自身与非自身,对自身物质（即共生菌）免疫耐受,对非自身物质（即病原菌）产生特异性免疫反应。IECs与肠道共生菌共同抵御肠道病原微生物,维持肠道健康,病原微生物侵入肠道,IECs主要通过胞外分泌物和细胞表面黏液层双重屏障发挥作用,其中胞外分泌物主要包括黏蛋白、抗菌分子和抗微生物免疫球蛋白。肠道共生菌可以通过竞争识别位点,分泌抗菌物质,增加黏液分泌,诱导IECs更新、增殖和修复等方式抵御病原微生物,维护正常的肠黏膜屏障功能。在IECs抵御肠道病原微生物入侵过程中,病原微生物通过自身运动、分泌毒素和酶等破坏肠上皮屏障,直接接触IECs,对其进行损伤。因此IECs和肠道菌群间相互作用,共同维持肠道内环境稳态。作者就IECs和肠道微生物结构、功能的适应性变化作一综述,以期阐述肠道微生物-上皮细胞屏障互作的机制。     

五倍子对鳗鲡4株常见病原菌的体外抗菌后效应

采用试管液体二倍稀释法测定中药五倍子对鳗鲡养殖中常见主要致病菌(肠杆菌B01、迟缓爱德华氏菌B09、豚鼠气单胞菌B18和嗜水气单胞菌B27)的最小抑菌浓度(MIC).然后再采用菌落计数法分别测定五倍子在0.25、0.5、1、2、4倍于各菌MIC时对鳗鲡病原菌株B01、B09、B18、B27的体外抗菌后效应(PAE).结...     

嗜水气单胞菌体外粘附Caco-2细胞对细胞膜生物学特性的影响

采用Caco-2细胞培养模型，研究了嗜水气单胞菌粘附Caco-2肠上皮细胞后时其活力、细胞膜磷脂酶A2（PLA2）、胞浆游离钙浓度、膜通透性和膜流动性的影响。结果显示，细菌粘附后1h，肠上皮细胞活力显著下降，PLA2活性和胞浆游离钙浓度显著增加，细胞外LDH含量和细胞损伤率显著升高（膜通透性异常增加），肠上皮细胞膜荧光，偏振度、膜脂微粘度显著增加（膜流动性降低），且上述指标均随着粘附时间的延长而持续升高或降低。