牙鲆病原秦皇岛弧菌主要生物学性状研究

从秦皇岛某海水养殖场牙鲆(ParalichthysolivaceusL.)细菌性败血感染症的病例中,分离到了相应的病原细菌。经对12株纯培养菌进行形态特征、培养特性、理化特性等方面较系统的表观分类学指征及代表菌株DNA中G Cmol%的测定,表明为弧菌属(VibrioPacini1854)细菌的一个新种,并定名为秦皇岛弧菌(Vibrioqinhuangdaorasp.nov.)。同时对该菌的血清型进行了检定,表明12株菌具有同种的K抗原和同种的O抗原(血清同源);另外,经以37种抗菌类药物做敏感性测定,结果显示对供试的头孢唑啉等32种药物敏感、对青霉素G等5种耐药,在不同菌株间无明显的敏感与耐药差异。     

鮰爱德华氏菌外膜微孔蛋白N基因PCR快速检测方法的建立

根据Gen Bank中鮰爱德华氏菌Edwardsiella ictaluri外膜微孔蛋白N(porin N)基因序列(Gen Bank No:NC_012779.2)设计了1对引物,预计目的片段大小为381 bp。通过对反应体系和条件的优化,并进行特异性试验、敏感性试验及人工感染组织样品检测,建立了一种快速检测鮰爱德华氏菌的PCR方法。结果表明,在所检测的鮰爱德华氏菌、迟缓爱德华氏菌、嗜水气单胞菌、温和气单胞菌、杀鲑气单胞菌、豚鼠气单胞菌、嗜麦芽寡养单胞菌、鲁氏耶尔森氏菌、海豚链球菌、不动杆菌、产气肠杆菌、大肠杆菌、拟态弧菌、荧光假单胞菌、弗氏柠檬酸杆菌15种细菌中仅鮰爱德华氏菌扩增出特异性条带;敏感性试验结果显示,该方法最小核酸检出量为9.35×10-3ng·μL-1;同时对人工感染的病料肝脏、细菌基因组DNA、细菌菌液及菌落进行扩增,结果显示4种材料均能检测出大小为381 bp的基因片段。本研究所建立的方法特异强、灵敏度高,适用于鮰爱德华氏菌感染病例的高效、快速检测。     

患病大鲵中弗氏柠檬酸杆菌的分离与鉴定

【目的】确定导致大鲵(Andrias davidianus)细菌性感染死亡的病原。【方法】从大鲵肝脏中分离细菌,通过Biolog微生物自动鉴定系统及分子生物学方法对纯培养的细菌进行鉴定,再用大鲵和鲫鱼分别进行人工感染试验,以确定分离菌的致病性,同时对分离到的病原菌进行药物敏感试验。【结果】从患病大鲵肝脏中分离到一株致病菌JZ01,经人工感染健康大鲵,可复制与自然发病相同的症状,且从人工感染病鲵体内再次分离到相同的病原菌。该致病菌对健康鲫鱼也有致病性。经Biolog微生物自动鉴定系统的鉴定,以及进一步的16S rDNA基因序列和系统发育分析都表明,此致病菌为弗氏柠檬酸杆菌。药物敏感性试验表明,该菌株对氨曲南、头孢三嗪、先锋噻肟等9种药物高度敏感。【结论】弗氏柠檬酸杆菌是大鲵的一种致病菌。本文在国内外首次报道了该菌对大鲵具有致病性。     

■爱德华氏菌变异株C9605及对鳖的致病性研究

从患白板综合症的病鳖分离到一株细菌（C9605）,该菌为革兰氏阴性,直杆状,周生鞭毛。接触酶阳性,氧化酶阴性,还原硝酸盐,对多粘菌素不敏感,不利用柠檬酸盐和丙二酸盐作唯一碳源,不从甘露醇、蔗糖、海藻糖、L-阿拉伯糖产酸。根据这些特性,菌株可归于爱德华氏菌。但是该菌发酵木糖产酸,产生H2S,耐青霉素,故鉴定为爱德华氏菌变异株（Edwardsiellaictalurivariationstrain）。人工感染实验证实,该菌株是鳖白板综合症的病原菌。     

Hui爱德华氏菌变异株C9605及对弊的致病性研究

从患白板综合症的病鳖分离到一株细菌（Ｃ９６０５）,该菌为革兰氏阴性,直杆状,周生鞭毛。接触酶阳性,氧化酶阴性,还原硝酸盐,对多粘菌素不敏感,不利用柠檬酸盐和丙二酸盐作唯一碳源,不从甘露醇、蔗糖、海藻糖、Ｌ－阿拉伯糖产酸。根据这些特性,菌株可归于Ｈｕｉ爱德华氏菌。但是该菌发酵木糖产酸,产生Ｈ２Ｓ,耐青霉素,故鉴定为Ｈｕｉ爱德华氏菌变异株。人工感染实验证实,该菌株是鳖白板综合症的病原菌。     

宽体金线蛭嗜水气单胞菌感染的病原检验

对河北某宽体金线蛭（Whitmania pigra Whitman）养殖场所养殖的宽体会线蛭发生的病害,进行了发病情况、临床表现、病理变化等方面的枪验。同时,择代表菌株进行了16S rRNA基因的分子鉴定,测定了16S rRNA基因序列、分析了相关细菌相应序列的同源性、构建了系统发生树。结果表明所检病例为由嗜水气单胞菌（Aeronmnas hydrophila）所引起的感染。分离后做纯培养的10株嗜水气申胞菌均为同种血清型菌株;代表菌株对健康宽体金线蛭的人工感染试验表明了相应的原发病原学意义;药敏试验结果显示,对供试37种抗菌药物中的头孢噻肟等高度敏感、对链霉素等敏感、对苯唑青霉素等耐药。     

黄颡鱼“裂头病”病原二重PCR检测方法的建立及应用

"裂头病"是黄颡鱼养殖业的主要病害之一,其病原为鮰爱德华氏菌或迟钝爱德华氏菌。以GenBank所收录鮰爱德华氏菌与迟钝爱德华氏菌16S rRNA基因为模板,优化设计两对特异性引物,经多重PCR反应体系优化及特异性与敏感性检测,建立了检测鮰爱德华氏菌和迟钝爱德华氏菌的二重PCR检测方法。结果显示,阳性对照样品的琼脂糖凝胶电泳条带同时检测到鮰爱德华氏菌和迟钝爱德华氏菌,扩增产物大小分别为470 bp及268 bp,灵敏度为1.38 ng/μL。此法用于检测江西南昌地区多个养殖场所患"裂头病"黄颡鱼脑部DNA,11份患病黄颡鱼的脑部组织均检出鮰爱德华氏菌,表明该地区黄颡鱼所患"裂头病"的病原菌为鮰爱德华氏菌,此结果与常规细菌分离鉴定的检测结果一致。所建二重PCR检测法敏感度高,特异性强,检测成本低,对黄颡鱼"裂头病"的快速诊断与流行病学调查有较好的应用价值。     

爱德华氏菌变异株C9605及对鳖的致病性研究

从患白板综合症的病鳖分离到一株细菌（C9605）,该菌为革兰氏阴性,直杆状,周生鞭毛。接触酶阳性,氧化酶阴性,还原硝酸盐,对多粘菌素不敏感,不利用柠檬酸盐和丙二酸盐作唯一碳源,不从甘露醇、蔗糖、海藻糖、L-阿拉伯糖产酸。根据这些特性,菌株可归于爱德华氏菌。但是该菌发酵木糖产酸,产生H₂S,耐青霉素,故鉴定为爱德华氏菌变异株（Edwardsiellaictalurivariationstrain）。人工感染实验证实,该菌株是鳖白板综合症的病原菌。     

牛蛙腐皮——红腿病并发症致病菌研究

对牛蛙腐皮－红腿病并发症病菌进行了分离。鉴定和人工感染试验,从病蛙分离出４株细菌,通过对健康幼蛙的人工感染试验,奇异变形杆菌和克氏耶尔森氏菌独立致发牛蛙腐皮病;嗜水气单胞菌荧光单胞菌独立致发牛蛙红腿病,且都是通过伤口感染,初步牛蛙患腐皮－－红腿并发症,是由于奇异变形杆菌、嗜水气单胞菌或克氏耶尔森氏蓖与芝假单胞菌合并感染所致。     

牛蛙腐皮-红腿病并发症致病菌研究Ⅱ.荧光假单胞菌的分离和鉴定

对牛蛙腐皮 -红腿病并发症致病菌进行了分离、鉴定和人工感染试验 .从病蛙分离出 4株细菌 ,通过对健康幼蛙的人工感染试验 ,奇异变形杆菌和克氏耶尔森氏菌独立致发牛蛙腐皮病 ;嗜水气单胞菌和荧光单胞菌独立致发牛蛙红腿病 ,且都是通过伤口感染 .初步认为牛蛙患腐皮—红腿病并发症 ,是由于奇异变形杆菌、嗜水气单胞菌或克氏耶尔森氏菌与荧假单胞菌合并感染所致     

Glyceraldehyde-3-phosphate dehydrogenase of Edwardsiella tarda has protective antigenicity against Vibrio anguillarum in Japanese flounder

Edwardsiella tarda glyceraldehyde-3-phosphate dehydrogenase (GAPDH) may be an effective vaccine candidate against infection by E. tarda in Japanese flounder Paralichthys olivaceus. The GAPDH of E. tarda is highly homologous to that of Vibrio cholerae (91%), and therefore E. tarda GAPDH may have protective antigenicity against Vibrio species. In this study, we immunized Japanese flounder with GAPDH of E. tarda and infected the fish with V anguillarum. The result showed that GAPDH prepared from E. tarda protected Japanese flounder effectively in a challenge of V anguillarum. Therefore, E. tarda GAPDH should be considered as a multi-purpose vaccine candidate against several kinds of pathogenic bacteria.     

Identification of novel putative virulence factors, adhesin AIDA and type VI secretion system, in atypical strains of fish pathogenic Edwardsiella tarda by genomic subtractive hybridization

Edwardsiella tarda, which is known to be the causative agent of edwardsiellosis in freshwater and marine fish, has two motility phenotypes. Typical strains exhibiting motility are isolated mainly from freshwater fish and Japanese flounder. Atypical strains exhibiting non-motility are isolated mainly from marine fish, with the exception of Japanese flounder. Subtractive hybridization was performed to identify genomic differences between these two phenotypes. Two fragments which showed homology to potential virulence factors were isolated from atypical strains: the autotransporter adhesin AIDA and a component of T6SS. We analysed DNA sequences of about 5 kbp containing these fragments and identified two partial ORF, and ORF encoding for other components of T6SS. The predicted amino acid sequences showed remarkably low homology to components of T6SS reported in the typical E. tarda strain PPD130/91. Furthermore, the organization of these ORF was different from the gene cluster of the typical E. tarda strain. AIDA and T6SS may therefore be associated with different pathogenicity in typical and atypical E. tarda hosts.     

Vaccine efficacy of recombinant GAPDH of Edwardsiella tarda against Edwardsiellosis

Thirty-seven kilodalton glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Edwardsiella tarda was suggested to be an effective vaccine candidate against E. tarda infection in previous research. For developing a vaccine, obtaining GAPDH in large quantities is necessary. In this study, we determined the complete nucleotide sequence of the gene that encodes GAPDH of E. tarda, and overexpressed the GAPDH of E. tarda by using the Escherichia coli expression system. We immunized Japanese flounder with recombinant GAPDH (rGAPDH) and evaluated its vaccine efficacy. Our results showed that rGAPDH effectively protected Japanese flounder from experimental E. tarda infection, and will contribute to the development of a vaccine against E. tarda.     

Sensitive and rapid detection of edwardsiellosis in fish by a loop-mediated isothermal amplification method

Here we report a rapid and sensitive method (using loop-mediated isothermal amplification [LAMP]) for the diagnosis of edwardsiellosis, a fish disease caused by Edwardsiella tarda, in Japanese flounder. A set of four primers was designed, and conditions for the detection were optimized for the detection of E. tarda in 45 min at 65 degrees C. No amplification of the target hemolysin gene was detected in other related bacteria. When the LAMP primers were used, detection of edwardsiellosis in infected Japanese flounder kidney, and spleen and seawater cultures was possible. We have developed a rapid and sensitive diagnostic protocol for edwardsiellosis detection in fish. This is the first report of the application of LAMP for the diagnosis of a fish pathogen.     

Edwardsiella tarda sialidase: Pathogenicity involvement and vaccine potential

Bacterial sialidases are a group of glycohydrolases that are known to play an important role in invasion of host cells and tissues. In this study, we examined in a model of Japanese flounder (Paralichthys olivaceus) the potential function of NanA, a sialidase from the fish pathogen Edwardsiella tarda. NanA is composed of 670 residues and shares low sequence identities with known bacterial sialidases. In silico analysis indicated that NanA possesses a sialidase domain and an autotransporter domain, the former containing five Asp-boxes, a RIP motif, and the conserved catalytic site of bacterial sialidases. Purified recombinant NanA (rNanA) corresponding to the sialidase domain exhibited glycohydrolase activity against sialic acid substrate in a manner that is pH and temperature dependent. Immunofluorescence microscopy showed binding of anti-rNanA antibodies to E.?tarda, suggesting that NanA was localized on cell surface. Mutation of nanA caused drastic attenuation in the ability of E.?tarda to disseminate into and colonize fish tissues and to induce mortality in infected fish. Likewise, cellular study showed that the nanA mutant was significantly impaired in the infectivity against cultured flounder cells. Immunoprotective analysis showed that rNanA in the form of a subunit vaccine conferred effective protection upon flounder against lethal E.?tarda challenge. rNanA vaccination induced the production of specific serum antibodies, which enhanced complement-mediated bactericidal activity and reduced infection of E.?tarda into flounder cells. Together these results indicate that NanA plays an important role in the pathogenesis of E.?tarda and may be exploited for the control of E.?tarda infection in aquaculture.     

Infection with Edwardsiella tarda causes hypertrophy of liver cells in the Japanese flounder Paralichthys olivaceus

To study the direct cause of liver enlargement in the Japanese flounder Paralichthys olivaceus infected with Edwardsiella tarda, the fish were challenged with E. tarda and reared without feeding. The liver of fish exposed to the bacteria was markedly enlarged compared to that of the controls while no severe histopathological change appeared in the organ during the experiments. No notable difference was observed in the crude fat, glycogen, and water content of the liver between challenged and control fish. The size of liver cells and nuclei of the challenged fish was apparently larger than that of the controls. Analysis of crude DNA in the liver suggested that the number of liver cells of starved control fish significantly decreased during the experiment while that of the challenged fish was maintained at a level of the initial control. RNA/DNA ratio of the liver of challenged fish clearly increased while it decreased in the control fish during the experiment. These observations suggest that liver enlargement of flounder infected with E. tarda, at least in the early stage of infection, is not a result of any readily observable histopathological changes and that E. tarda infection causes hypertrophy of the cells, as well as preventing decrease in liver cell number.     

应用绿色荧光蛋白标记迟缓爱德华菌感染斑马鱼

目的建立斑马鱼模型研究迟缓爱德华菌的致病性及感染途径。方法应用绿色荧光蛋白标记迟缓爱德华菌,追踪观察其感染斑马鱼的动力学过程及病理组织学变化。结果病理组织学检查以肝脏水肿变性,肝细胞萎缩、坏死、脱落,脾脏散在增生性结节、充血、水肿、淋巴细胞大量缺失等病变为主;感染后,该菌先后在斑马鱼肠道、鳃和皮肤中定植。结论斑马鱼可作为研究迟缓爱德华菌致病性的动物模型。肠道、鳃和皮肤可能是迟缓爱德华菌先后感染斑马鱼的主要途径。     

石鲽病原琼氏不动杆菌形态型Ⅰ的鉴定

从由杀鲑气单胞菌(Aeromonas salmonicida)所引起的石鲽(Stone flounder,Kareius bicoloratus L)细菌性败血感染症病(死)鱼肝、脾、肾及肠内容物中,同时检出了作为继发感染的病原菌。经对6株纯培养菌在形态特征、理化特性等方面较系统的表观分类学指征鉴定及代表菌株DNA中G C mol％的测定,表明为琼氏不动杆菌的一个新形态型并定名为琼氏不动杆菌形态型Ⅰ(Acinetobacter junii morphovar Ⅰ);同时,对该菌进行了血清型、对抗菌类药物的敏感性及致病作用等方面的试验,初步表明此6株菌具有同种的表面(K)及同种的菌体(O)抗原,对供试37种抗菌类药物在不同菌株间的敏感及耐药无明显差异,对供试石鲽及牙鲆均具有较强的致病作用。