霍乱弧菌及肠出血性大肠杆菌多重PCR检测方法的建立

针对霍乱弧菌肠毒素和肠出血性大肠杆菌志贺毒素基因（ctx、stx1和stx2）设计引物,扩增大小不同的特异性片段,优化反应条件,建立多重PCR方法,对人工污染的水样品进行模拟检测。结果显示,多重PCR扩增系统针对目的菌具有高度的特异性。敏感性试验证实,当多重PCR反应体系中模板含量在10CFU时仍能检出。模拟水样品多重PCR检测的敏感性试验证明,人工污染的河水直接集菌检测敏感性为100～1 000CFU/mL,增菌培养后多重PCR检测敏感性可达1CFU/mL。本试验于同一PCR体系检测霍乱弧菌和肠出血性大肠杆菌的毒素基因,可用于这2种致腹泻性病原菌的快速检测。     

南美白对虾白便综合征病原霍乱弧菌的分离与药敏试验

为确定南美白对虾白便综合征的病原菌及其药物敏感性,采用传统方法从患白便综合征的南美白对虾肝胰腺中分离到病原菌菌株BB31,结合API 20E革兰阴性菌鉴定系统、16SrDNA序列及聚类分析对菌株BB31进行了鉴定,并通过纸片琼脂扩散法对菌株BB31的药物敏感性进行了测定。结果表明,菌株BB31为霍乱弧菌(GenBank登录号:KF446244),其16SrDNA序列与GenBank数据库中弧菌菌株的16S rDNA序列有99%~100%的同源性,而且与霍乱弧菌菌株RD1(GenBank登录号:KF307775)的亲缘性最近。菌株BB31对四环素、庆大霉素、恩诺沙星、氧氟沙星、诺氟沙星等抗生素的敏感性高,对磺胺甲氧嘧啶、呋喃唑酮、复方新诺明等抗生素不敏感。本研究证实霍乱弧菌是南美白对虾白便综合征的病原菌,庆大霉素、诺氟沙星等常规渔药可作为防控霍乱弧菌引起的南美白对虾白便综合征的用药参考。     

霍乱弧菌DPO-PCR特异性检测方法的建立与应用

采用一种新型的引物设计方法——双启动寡核苷酸引物（dual-priming oligonucleotide,DPO）,以霍乱弧菌mdh基因为靶序列,建立了霍乱弧菌DPO-PCR特异性检测方法,分析了DPO引物退火温度不敏感性、检测灵敏度及特异性,并对检测方法进行了初步应用。灵敏度结果显示,DPO-PCR方法对霍乱弧菌的最低检出限为1.07×10^2 CFU/mL。退火温度不敏感性试验中,与常规PCR引物相比,DPO引物在45～65℃退火温度均能够高效扩增出靶基因片段。特异性结果显示,DPO-PCR方法的特异性比常规PCR方法强,不产生任何非特异性扩增。利用建立的霍乱弧菌DPO-PCR检测方法对采集的550份样本进行检测,检出43份霍乱弧菌阳性样本,经行业标准法（SN/T2425-2010）复检,检测结果相同,表明所建立的DPO-PCR检测方法具有良好的实用性,为霍乱弧菌的快速准确检测提供了新途径。     

1株鸭源非O1/O139群霍乱弧菌的分离鉴定与致病性分析

为探明引起贵州省某鸭场雏鸭发病的病原及其致病性和耐药情况，本研究对该鸭场送的疑似细菌感染病鸭进行剖检，取鼻黏膜、心脏和肝脏等组织器官接种于培养基中进行细菌分离鉴定，通过对分离菌进行药敏试验、动物回归试验和毒力基因检测研究其耐药情况和致病性。结果显示，分离菌在血琼脂培养基上生长16 h后呈现为边缘整齐、有光泽的乳白色菌落，伴有β-溶血现象，经革兰氏染色后在生物显微镜下呈两端钝圆、弧状、排列无规则的革兰氏阴性短小杆菌，与霍乱弧菌相符；16S rDNA基因序列同源性及系统进化树显示，该分离菌与霍乱弧菌同源性高达99.6%~99.7%聚为一支；药敏试验结果显示，分离菌对大部分药物都表现为耐药，其中对氨苄西林、克林霉素、复方新诺明、苯唑西林和克林霉素等抗菌药耐药性较强，对头孢哌酮和头孢曲松敏感；动物回归试验显示，分离菌可导致试验组雏鸭5 d内全部发病死亡，表明该分离菌对雏鸭具有较强的致病性；毒力基因PCR检测结果显示，检测的霍乱弧菌相关毒力基因hlyA、ompW和chxA为阳性，而检测的O1群rfb、O139群rfb、tcpA及ctxA基因为阴性，表明本次分离的霍乱弧菌携带有致病基因，但不属于O1和O139血清群。结果表明，该鸭场雏鸭发病的疫情病原为非O1/O139血清群霍乱弧菌，该菌致病性强且对多种抗菌药物耐药。本试验结果为贵州省鸭霍乱弧菌病的防控提供了参考依据。     

台湾牛蛙感染霍乱弧菌的检验报告

本文报告从台湾进境的食用牛蛙中多次检出霍乱弧菌，其中O1群霍乱弧菌6批次，非O1和非O139群霍乱弧菌2批次，经、血清学和噬菌体试验确定这些O1群霍乱弧菌均为稻叶型非流行株，动物实验表明这些菌株对小鼠有较强的致病力和致死率。     

Prevalence and diversity of Aeromonas and Vibrio spp. in coastal waters of Southern Italy

A survey was undertaken to examine sea water and sediment for the presence of Vibrio and Aeromonas spp. along approximately 900 km of coast in Southern Italy during early and late summer. A quantitative analysis was also done to evaluate the water fecal contamination at the stations examined. The results indicate that all the investigated areas were submitted to a wide spatial fluctuation of fecal contamination and that Vibrio and Aeromonas spp. were present in both high and low fecal-contaminated stations. Sixty two percent of the investigated samples were positive for Aeromonas spp., while 42% of samples were positive for Vibrio spp. It was interesting to note that 38% of the positive stations for both Aeromonas and Vibrio spp. showed a fecal coliform contamination of water at < 10(2) cells 100 ml(-1). Thus, these findings support the hypothesis that the bacterial indicators (such as fecal coliforms) do not always satisfactorily reflect the hygienic quality of water. The presence of Vibrionaceae on copepods was also investigated. Copepods were sampled at a station located inside the harbour of the city of Naples and were found contaminated by V. cholerae non-O1, V. alginolyticus, V. fluvialis and A. caviae. Furthermore, the antibiotic resistance patterns of isolated bacteria showed the presence of a number of resistant strains among the isolates. In order to discriminate the isolates on the basis of their biochemical profiles and/or antibiotic resistance patterns, cluster analysis was carried out which showed that no unique assay could fully discern these isolates. However, the best discrimination resulted from complete pattern profile based on both biochemical profiles and antibiotic resistance patterns.     

Isolation of Vibrio vulnificus and atypical Vibrio from surface water of the Baltic Sea in Germany

From 1995 to 1997 several defined species like V. alginolyticus, V. anguillarum, V. cholerae (non O1 and non O139), V. mimicus, V. parahaemolyticus and V. vulnificus were isolated during a survey to determine the presence of V. vulnificus in the brackish water of the Baltic Sea in Germany. Moreover atypical Vibrio isolates were detected. Four isolates belonging to a group of atypical Vibrio and possibly representing a new species in the genus Vibrio were characterized in detail. All four strains were isolated from surface costal waters. Based on 16S rDNA sequence analysis they showed the highest relatedness to the species V. navarrensis and V. vulnificus. The strains did not harbor the species specific hemolysin gene vvhA from V. vulnificus as shown by PCR and hybridization experiments. Moreover, they differed in at least two biochemical parameters tested from the hitherto described Vibrio species. All these strains induced hemolysis on washed blood agar dishes and showed phase variations on Luria Bertani agar dishes. Because of the similarity to the eel pathogen V. vulnificus, we infected eels with one of the four atypical strains (CH-291), but no pathogenicity for eels could be detected. Furthermore, Vero cell tests with supernatants of bacterial cultures did not reveal secreted Vero cell cytotoxic compounds. This indicates a nonpathogenic nature of these strains.     

Isolation and identification of Vibrio metschnicovii from domestic ducks and geese.

Seven Vibrio-like field strains of German origin were isolated culturally from diseased domesticated ducks, muscovy ducks and geese, and were compared with reference strains NCTC 8443 (type strain) and NCTC 11170 of Vibrio metschnicovii using classical phenotypic and chemotaxonomic tests. Some V. cholerae strains were included in the chemotaxonomic tests for comparative purposes. On the basis of the classical phenotypic characteristics studied and the numerical analysis of the whole-cell fatty acid patterns, the Vibrio-like field strains were identified as Vibrio metschnicovii. The identification tables and the database of the computer software of two commercial micro-identification kits (API-20 NE, ID-32 E) did not identify the field strains. Of the reference strains used, only NCTC 8443 was correctly identified by the ID-32 E software.     

IFN-γ renders human intestinal epithelial cells responsive to lipopolysaccharide of Vibrio cholerae by down-regulation of DMBT1

Although intestinal epithelial cells (IECs) are continuously exposed to high densities of enteric bacteria, they are not highly responsive to microbe-associated molecular patterns (MAMPs). However, inflammatory cytokines such as interferon-γ (IFN-γ) are potentially capable of priming IECs to enhance responsiveness to MAMPs. In this study, we observed that heat-killed Vibrio cholerae (HKVC) and its lipopolysaccharide (LPS) poorly induced IL-8 production in a human IEC line, HT-29. However, both HKVC and the LPS showed a substantial induction of IL-8 production in IFN-γ-primed HT-29 cells. LPS-induced IL-8 production was proportional to the IFN-γ-priming period and LPS could not induce IL-8 production in the presence of polymyxin B. Moreover, LPS-induced IL-8 production in the IFN-γ-primed HT-29 cells was mediated through signaling pathways requiring p38 kinase and ERK, but not the JNK/SAPK pathway. Since deleted in malignant brain tumor 1 (DMBT1) is known to interact with and antagonize the action of LPS, we hypothesized that IFN-γ enhanced the responsiveness to LPS in HT-29 through down-regulation of DMBT1. We found that IFN-γ indeed attenuated DMBT1 expression at both the mRNA and protein levels in HT-29 cells. Conversely, when the cells were transfected with small interfering RNA to specifically silence DMBT1, IL-8 expression was augmented even in the absence of IFN-γ and the augmentation was further enhanced by treatment with V. cholerae LPS. Since IFN-γ is known to increase IFN-β expression in the IECs, we examined if IFN-β functioned similar to IFN-γ. Although IFN-β alone was able to induce IL-8 expression, it failed to render HT-29 cells responsive to V. cholerae LPS. In conclusion, our study suggests that IFN-γ primes IECs to become responsive to V. cholerae and its LPS by suppressing the expression of DMBT1.     

天津市水产养殖动物霍乱弧菌携带情况调查

从2008年4月至11月,对天津市11种水产养殖动物进行霍乱弧菌检验,共取得样品211份,其中携带霍乱弧菌的样品36份,检出率为17.1%,经血清凝集试验证明都为非O1霍乱弧菌。在11种水产养殖动物中,以南美白对虾霍乱弧菌的检出率最高,为25.0%;鲤鱼和金鱼次之,分别为7.7%和5.3%;其他8种水产养殖动物中未检出霍乱弧菌。     

二温式PCR检测对虾白斑综合征病毒

本研究设计了一对能扩增大小为306bp对虾白斑综合征病毒(WSSV)某段基因的特异性引物，优化建立了能快速检测WSSV的二温式PCR，在对包括105份临床样品、10份SPF南美白对虾组织样品和其他对虾病害病原在内的样品检测结果中，有65份临床样品呈现WSSV阳性，而10份SPF南美白对虾组织样品和其他对虾病害病原的PCR结果为阴性。该二温式PCR最低能检测到1pg的WSSV感染对虾组织样品总DNA。这些结果表明，该PCR具有高度的特异性和敏染性。     

Spread of zoonotic agents by foods of animal origin

Due to the expansion of the international trade with food, there is a great and increasing danger of transmitting zoonotic agents with these foods over long distances from one region to another. However, in concrete cases it is difficult to determine which zoonotic agents are transmitted through which foodstuffs and how great the emergency is. The worldwide distribution of most of these agents, the easing up of import controls for goods coming from certain export countries, and the abstinence from evaluating epidemiological data in cases of detection of agents in foods are responsible for this. Merely, the transmission path of Vibrio spp. (V. cholerae, V. parahaemolyticus, V. vulnificus) can be significantly traced from the East Asian countries to Europe via fish products.     

Visceral nodular cartilaginous metaplasia in rockfishes (Sebastes spp.) in the eastern North Pacific Ocean.

Multiple, discrete, nodular foci of cartilaginous metaplasia were found in the spleens and kidneys of rockfishes taken from the northeastern Pacific Ocean during a survey to determine the incidence and the nature of diseases in these animals. These nodules sometimes occurred in association with granulomatous inflammation and distinct granulomas. Many of these fish were infected by Ichythophonus spp. or acid-fact bacteria (presumably Mycobacteria spp.). Some of the metaplastic foci contained encapsulated accumulations of eosinophilic vesicles and basophilic granular debris, described by other authors as "cysts of unknown etiology," which have been observed at different sites in a variety of temperate and tropical fish species.     

Entérite nécrotique chez le poulet de gril I. Aspect clinico-pathologique

This study represents an analysis of the principal clinical factors and pathological lesions of 150 cases of necrotic enteritis encountered during 1968 and 1969 in Quebec. Following multiple investigations no common factor was observed which might have explained the pathogenesis of the condition which was observed during every month of the year, especially from May to November. Birds two to four weeks of age were the most susceptible. The main lesion was a fibrino-necrotic enteritis always localized in the small intestine, either in its entire length or just a segment and characterized by the disappearance of the surface epithelium and necrosis of the villi. The lumen was filled with desquamated epithelial cells and bacteria. Foci of coagulation necrosis were observed in the liver and foci of nephrosis in the kidneys. A Gram+ bacillus, strictly anaerobic, was always isolated from the viscera.     

Porcine salmonellosis. 3. Production of fibrinous colitis by intravenous injections of a mixture of viable cells of salmonella cholerae-suis and disintegrated cells of the same agent, or hemolytic Escherichia coli

Hemorrhagic necrosis of the gastric and colonic mucosa with capillary thrombosis were induced in pigs experimentally infected with Salmonella cholerae-suis when an additional intravenous supply of disintegrated cells of Salmonella cholerae suis or hemolytic Escherichia coli was administered. Except for the gastrointestinal lesions, the necropsy findings were almost identical with the picture found in acute septicemic salmonellosis. The gastrointestinal changes were interpreted as a special gastroenteric effect of endotoxins of Gramnegative bacteria during the development of septicemic salmonellosis.     

Immunohistopathological findings in the lungs of calves naturally infected with Mycoplasma bovis

Histology and immunohistochemistry were used to analyse the lesions and distribution of Mycoplasma bovis antigen in the lungs of 18 naturally infected calves. Microscopic examination of pneumonic lungs revealed two distinct patterns of necrosis and inflammation. The first pattern was observed in six of 18 (33.3%) calves in which microscopic lesions were characterized by large irregular areas of coagulative necrosis surrounded by a dense zone of degenerated neutrophils. Moderate amounts of mycoplasmal antigen were in the centre and periphery of these necrotic foci and, to a lesser extent, in mononuclear cells of the peribronchial lymphoid tissue. The second pattern was observed in 18 of 18 (100%) calves and consisted of rounded foci of caseous necrosis composed by granular eosinophilic material surrounded by a rim of granulation tissue. Large amounts of M. bovis antigen were detected in the centre and periphery of these necrotic foci and, to a lesser extent, in the peribronchial lymphoid tissue, and alveolar and interstitial macrophages. It was concluded that both caseous and coagulative necrosis of the lung parenchyma was primarily caused by M. bovis. Infection with M. bovis should be suspected in bovine necrotic bronchopneumonia, particularly in cases in which the pulmonary necrosis is part of a pyogranulomatous inflammation centred around airways. The pattern of caseous necrosis with pyogranulomatous inflammation is characteristic of M. bovis infection while the pattern of coagulative necrosis is similar to and must be differentiated from Mannheimia haemolytica and Haemophilus somnus infection.     

副溶血弧菌和溶藻弧菌双重PCR检测方法的建立与初步应用

为建立同时快速检测海产品中的副溶血弧菌(VP)和溶藻弧菌(VA)的双重PCR方法,本研究根据VP和VA的toxR基因序列设计针对这两种细菌的两对特异性引物,建立能够快速同时检测这两种细菌的双重PCR方法,并对该反应体系的特异性和灵敏度进行检测。结果显示纯培养细菌VP和VA的检测灵敏度分别为2.32×103cfu/mL和2.56×103cfu/mL,临床病料检测灵敏度分别为2 cfu和3 cfu;与枸橼酸杆菌、沙门氏菌、美人鱼弧菌、霍乱弧菌、麦氏弧菌无交叉反应。研究表明本实验方法操作简便快速、特异性强、灵敏度高、稳定性好,并且经济实惠,值得推广应用。     

Bacteria flora and heavy metals in cultivated oysters <Emphasis Type="Italic">Crassostrea iredalei</Emphasis> of Setiu Wetland,East Coast Peninsular Malaysia

Slipper oyster Crassostrea iredalei is a species of good demand for its sweet flavor and white coloured flesh. The filter feeding nature predisposes oysters to accumulation of pathogenic and heavy metals in waters impacted by sewage pollutions and may thus render the oysters unfit for human consumption. A study was undertaken to investigate the presence of bacteria flora and heavy metal concentrations in cultivated oysters Crassostrea iredalei at Setiu Wetland, Terengganu, the only source of cultivated oysters in East Coast of Malaysia. A total of 200 slipper oyster samples were analyzed. The bacteria were isolated using non selective agar such as TSA agar and selective agars before they were then identified using conventional methods in combination with BBL Crystal identification kit. Heavy metals such as zinc (Zn), copper (Cu), cadmium (Cd), and lead (Pb) concentrations were determined using atomic absorption spectrophotometry. Results showed that the oysters harbor predominantly Shewanella putrifaciens followed by Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio cholerae, Enterobacter cloacae, Escherichia coli and Chromobacterium violaceum. They also contain high concentration of Zn (785.68 ± 285.88 μg/g) with the lowest heavy metal was Pb (0.17 ± 0.15 μg/g), whilst the concentrations of other heavy metals were Cu (38.9 ± 13.2 μg/g) and Cd (1.60 ± 0.28 μg/g). The study is very useful to evaluate the type of bacteria and heavy metal present in oyster meat for human consumption.     

用PCR检测副溶血性弧菌耐热溶血素基因

根据副溶血性弧菌的耐热溶血素基因（ｔｄｈ）设计了２对引物，经聚合酶链式反应（ＰＣＲ）检测，所有１８株神奈川试验阳性副溶血性弧菌均呈阳性反应，而６株神奈川试验阴性副溶血性弧菌呈阴性反应，其他９种（３４株）弧菌和８株其他革兰氏阴性菌也呈阴性反应。检测敏感性可达２ＣＦＵ的副溶血性弧菌。     

Vibrios as causal agents of zoonoses

Vibrios are Gram-negative rod-shaped bacteria that are widespread in the coastal and estuarine environments. Some species, e.g. Vibrio anguillarum and V. tapetis, comprise serious pathogens of aquatic vertebrates or invertebrates. Other groups, including Grimontia (=Vibrio) hollisae, Photobacterium (=Vibrio) damselae subsp. damselae, V. alginolyticus, V. harveyi (=V. carchariae), V. cholerae, V. fluvialis, V. furnissii, V. metschnikovii, V. mimicus, V. parahaemolyticus and V. vulnificus, may cause disease in both aquatic animals and humans. The human outbreaks, although low in number, typically involve wound infections and gastro-intestinal disease often with watery diarrhoea. In a minority of cases, for example V. vulnificus, there is good evidence to actually associate human infections with diseased animals. In other cases, the link is certainly feasible but hard evidence is mostly lacking.