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1.
Aeromonas hydrophila is a broad-host-range pathogen and its pathogenesis is multifactorial. A regulatory mechanism known as quorum sensing has been found to be involved in the regulation of virulence in many bacteria. In A. hydrophila the ahyR gene encodes LuxR-type response regulator. Here we describe the inactivation of the ahyR gene of A. hydrophila J-1 by the insertion of a DNA fragment containing a kanamycin resistance determinant and reintroduced by allelic exchange into the chromosome of A. hydrophila J-1 by means of the suicide plasmid pJP5603. Cytotoxic effects on EPC cells assay and LD(50) determinations in fish demonstrated that the ahyR mutant was highly attenuated relative to the wild-type strain. Compared with the parent strain, some characteristics, such as biochemical characters and outer membrane protein profiles, had changed. Some main virulent determinants could not be detected, including proteases, amylase, Dnase, hemolysin and S layer. This article confirmed the important function of AhyR in the pathogenesis of A. hydrophila J-1. 相似文献
2.
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。 相似文献
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4.
Chu WH Lu CP 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2005,52(4):180-182
The influence of the cytoskeleton on the invasion of Aeromonas hydrophila strain AhJ-1, isolated from diseased fish, in the monolayer cell of epithelioma papillosum cells of carp (EPC) was evaluated by the recovery of gentamicin-resistant bacteria from Triton X-100 cell lysates. The depolymerization of microfilaments (MF) by cytochalasin B and D inhibited the uptake of A. hydrophila in a dose-dependent manner and that of microtubules (MT) by colchicines and nocodazole did not affect the invasion of A. hydrophila in EPC cells significantly. The invasion frequency decreased approximately 62% with the addition of 0.1 microg/ml cytochalasin D and nearly 86% by the addition of 5.0 microg/ml. Invasion decreased approximately 49% and 83% by addition of cytochalasin B in a concentration of 2.5 microg/ml and 10.0 microg/ml. Colchicine and nocodazole, inhibitors of MT formation appears to have little effect on the invasion of EPC cells by strain Ah J-1. Thus MF formation, but not MT formation seems to play an important role in the internalization of A. hydrophila J-1. 相似文献
5.
Protozoan Ichthyophthirius multifiliis Fouquet (Ich) and bacterium Aeromonas hydrophila are two common pathogens of cultured fish, which cause high fish mortality. Currently there is no information available for the effect of parasitism by Ich on survival of channel catfish and invasion of A. hydrophila in fish tissues following exposure to A. hydrophila. A trial was conducted in this study to: (1) determine whether A. hydrophila increased fish mortality in Ich-parasitized channel catfish; and (2) compare the bacterial quantity in different tissues between non-parasitized and Ich-parasitized catfish by real-time polymerase chain reaction (qPCR). The results demonstrated that the Ich-parasitized catfish showed significantly (P<0.05) higher mortality (80%) when exposed to A. hydrophila by immersion than non-parasitized fish (22%). Low mortality was observed in catfish exposed to Ich alone (35%) or A. hydrophila alone (22%). A. hydrophila in fish tissues were quantified by qPCR using a pair of gene-specific primers and reported as genome equivalents per mg of tissue (GEs/mg). Skin, gill, kidney, liver and spleen in Ich-parasitized fish showed significantly higher load of A. hydrophila (9400-188,300 GEs/mg) than non-parasitized fish (4700-42,100 GEs/mg) after exposure to A. hydrophila. This study provides evidence that parasite infections enhance bacterial invasion and cause high fish mortality. 相似文献
6.
为了研究嗜水气单胞菌重组弹性蛋白酶的酶学性质,试验根据GenBank中的嗜水气单胞菌弹性蛋白酶基因ahyB设计1对含酶切位点的特异引物,以嗜水气单胞菌J-1(AhJ-1)株为模板,经PCR扩增得到不含信号肽的成熟弹性蛋白酶基因片段(787 bp),并与pMD18-T载体连接、测序,再用DNAStar软件分析。结果表明:该基因片段与豚鼠气单胞菌胞外蛋白酶同源性高达95%,与嗜水气单胞菌AG2株弹性蛋白酶ahyB基因同源性为92%,与铜绿假单胞菌LasB基因同源性为82%;将PCR产物连入表达载体pET-32a,转化至大肠杆菌BL21菌株中进行诱导表达,出现50 ku的融合表达蛋白,该表达产物纯化复性后表现出酶的活性。 相似文献
7.
Combined effects of beta-glucan and lipopolysaccharide (LPS) on survival and immune response were studied in Cyprinus carpio that were challenged with the pathogen Aeromonas hydrophila. beta-Glucan from Saccharomyces cervisiae and LPS from a virulent strain of A. hydrophila were used in this study. Different concentrations of beta-glucan+LPS mixture were administered on days 1, 7, and 14 through different routes (intraperitoneal injection, bathing, and oral administration). Control and test fish were challenged by intraperitoneal injection of LD50 concentration of A. hydrophila on day 16 and subsequently, mortality and relative percent survival (RPS) were recorded. Intraperitoneal injection elicited 100% RPS even at the lowest concentration (100 microg beta-glucan+10 microg LPS); whereas, oral administration improved RPS rate of carps at higher concentration (1% beta-glucan+0.25% LPS). Bathing did not improve the RPS. Test animals injected with even the minimum dose of the immunomodulators (100 microg beta-glucan+10 microg LPS/fish) had a significant increase in total blood leukocyte counts and an increase in the proportion of neutrophils and monocytes. Superoxide anion production by macrophages was also elevated, which presumably aided the efficient killing of bacterial pathogen. Lower concentration of beta-glucan+LPS had an adjuvant effect on antibody production as pretreatment by injection of 100 microg beta-glucan+10 microg LPS/fish resulted in higher antibody titer against A. hydrophila following vaccination. RT-PCR analyses showed that the expression of interleukin-1beta mRNA did not increase in test fish when compared with the control. Classical and alternative complement pathways were not affected by either the dose or the route of administration of the compounds. It may be concluded that intraperitoneal injection and oral administration, and not the bathing, of beta-glucan+LPS mixture in carp could enhance resistance to challenge by A. hydrophila through changes in several non-specific and specific immune responses. 相似文献
8.
根据GenBank上登陆的嗜水气单胞菌AH-1株Ⅲ型分泌系统aopN基因序列,设计1对特异性引物,以J-1株的基因组为模板,PCR扩增得到aopN基因,连入pET-32a(+),转化至大肠杆菌表达,同时PCR检测aopN基因在66株嗜水气单胞菌中的分布情况。aopN基因测序分析发现,片段长度为874 bp,与嗜水气单胞菌AH-1、SSU、AH-3的同源性分别为97%、81%、82%,与杀鲑气单胞菌杀鲑亚种A449的同源性为81%,与温和气单胞菌的同源性为82%。PCR检测结果表明,57株能检测到aopN基因的目的片段。SDS-PAGE分析表明重组蛋白分子量为54.5ku,用兔抗J-1株的全菌抗血清进行Western blot分析表明,该蛋白具有较好的免疫反应性。由于多数菌株都含有aopN基因,提示AopN可能是嗜水气单胞菌的共同保护性抗原。 相似文献
9.
M E Olson D W Morck H Ceri 《The Canadian veterinary journal. La revue veterinaire canadienne》1997,38(12):777-779
Twenty puppies were vaccinated with a trophozoite-derived Giardia vaccine on day 0 and boosted on day 21 (Group 1); 10 control puppies received only saline (Group 2). Both groups were experimentally infected on day 35 with 1 x 10(6) Giardia duodenalis trophozoites by intraduodenal injection. Immunization provided protection to experimental Giardia infection. 相似文献
10.
根据已发表的嗜水气单胞菌Ⅲ型分泌系统（TTSS）的ascV基因保守区核苷酸序列设计合成1对引物,以国内疫苗菌株J-1的基因组为模板,通过PCR扩增得到331bp保守基因片段,将目的片段进行测序。在此基础上,分4段克隆J-1株的Ⅱscv基因并进一步拼接,全长为2166bp,同时PCR检测ascV基因在66株嗜水气单胞菌中的分布情况。测序分析发现,扩增出的J-1株ascV全长基因与嗜水气单胞菌AH-1、SSU、AH-3的同源性分别为97％、86％、86％,与杀鲑气单胞菌杀鲑亚种A449的同源性为86％,与温和气单胞菌的同源性为87％。PCR检测表明,64株能扩增出n5fV基因的目的片段,包括2株无毒菌株,而在2株有毒菌株中却未能检测到,说明TTSS在嗜水气单胞菌致病机制上的作用值得进一步探讨。 相似文献
11.
An equine antiserum to core lipopolysaccharide was produced by inoculation of 6 horses with a boiled cell bacterin made from the J-5 mutant of Escherichia coli O111:B4. The antiserum immunoglobulin G titer to J-5 mutant E coli, as determined by enzyme-linked immunosorbent assay, was 1:15,006. Pooled serum prepared before inoculation (preimmune serum) had a J-5 immunoglobulin G titer of 1:350. The J-5 antiserum was tested for its protective efficacy in sublethal endotoxemia in 14 horses. Four horses served as nontreated controls and were given nothing before endotoxin challenge exposure (10 micrograms/kg of body weight, IV). Pooled preimmune serum (3 ml/kg, IV) was administered to 5 horses and J-5 antiserum (3 ml/kg, IV) was administered to 5 other horses 2 to 15 hours before endotoxin challenge exposure. During the 24 hours postendotoxin challenge exposure, endotoxemia was accompanied by significant (P less than 0.05) time-related changes in temperature, heart rate, pulse character, respiratory rate and character, capillary refill time, mucous membrane color, fecal composition, attitude, PCV, total plasma protein, WBC count, platelet count, plasma fibrinogen, prothrombin time, activated partial thromboplastin time, fibrinolytic degradation products, plasma glucose, and plasma lactate in all horses. There were no apparent treatment vs time interactions (P greater than 0.05). Two horses (1 control and 1 given J-5 antiserum) died suddenly from unknown causes immediately after endotoxin challenge exposure. Seemingly, equine antiserum to core lipopolysaccharide did not provide protection from the adverse effects of experimental endotoxemia produced by bolus IV infusion of 10 micrograms of endotoxin/kg. 相似文献
12.
The immune response to mixed whole cell antigens of Aeromonas hydrophila, Edwardsiella tarda and Pseudomonas fluorescens, the common Gram negative bacterial pathogens associated with diseases of Indian major carps were evaluated for their efficacy in triggering antibody responses in rohu, Labeo rohita (Ham.). The rohu yearlings were either immunized with antigens from single bacterial strain, A. hydrophila, E. tarda and P. fluorescens or a combination of all three. An antibody response was detected at 1st week post immunization that rose significantly (p<0.05) at 4th week post immunization in all the immunized groups. The antibody level started declining after 8th week but persisted up to 10th week post immunization in all the immunized groups. Similarly, no significant difference (p>0.05) in the antibody level was found between groups immunized with single and mixed bacterial antigens. Moreover, the use of mixed bacterial antigens did not jeopardize the specific immune response to the vaccine components. Upon challenge with single pathogen, a high relative percent survival was recorded in the group immunized with mixed bacterial antigens and was comparable to those fish immunized with the single bacteria. 相似文献
13.
已构建的能表达产气荚膜梭菌α毒素保护性抗原基因工程菌株Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３）（ｐＸＥＴＡ１）经动物试验证实没有毒性。从ＩＰＴＧ诱导后的工程菌中提取包涵体,再辅以氢氧化铝胶制成抗原,免疫小鼠３０ｄ后,用产气荚膜梭菌强毒株培养物上清及培养菌体攻击,结果免疫鼠能抵抗至少２ＬＤ１００的攻击,证明Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３）（ｐＸＥＴＡ１）工程菌株表达产物具有良好的免疫原性。 相似文献
14.
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. 相似文献
15.
化学修饰对嗜水气单胞菌弹性蛋白酶活性及稳定性的影响 总被引：2，自引：0，他引：2
用10 ku的截流膜超滤、30%~60%的硫酸铵分级沉淀、凝胶过滤层析和阴离子交换层析,获得纯化的嗜水气单胞菌胞外弹性蛋白酶。SDS-PAGE显示,该酶是分子量约为33 ku的单体蛋白。用活化的右旋糖酐及单甲氧聚乙二醇(MPEG)分别对纯化的嗜水气单胞菌弹性蛋白酶进行化学修饰。修饰后的酶与原酶相比,修饰酶保留了天然酶的活性,两种修饰酶活性均能保留在60%以上,用MPEG修饰酶的保留活性更高(85.5%),而且两种修饰酶在耐热性、耐酸性等方面都优于天然酶,但修饰后酶的最适pH没变。修饰酶较天然酶稳定,具有较高的实用意义。 相似文献
16.
《Journal of aquatic animal health》2013,25(4):328-337
Abstract A neutralizing monoclonal antibody against infectious hematopoietic necrosis virus (IHNV) was used to select neutralization-resistant mutants from isolates of virus obtained from adult steelhead Oncorhynchus mykiss returning to the Round Butte Hatchery (RB mutants) on the Deschutes River in Oregon, USA, and from rainbow trout (nonanadromous O. mykiss) at a commercial hatchery in the Hagerman Valley of Idaho, USA (193-110 mutants). Two of the mutants, RB-1 and 193-110-4, were significantly (P < 0.001) attenuated compared with parental strains. Vaccination of rainbow trout by waterborne exposure to the mutants conferred solid protection against challenge with wild-type virus. In some trials, fish vaccinated with the RB-1 mutant at 50% tissue culture infectious doses (TCID50) of 1 × 104–1 × 105 TCID50/mL or with the 193-110-4 mutant at 1 × 102–1 × 103 TCID50/mL, held for 14 d, then challenged with the homologous wild-type strain at 1 × 105 TCID50/mL showed relative percent survival of 95–100% (P < 0.005). There was no significant difference (P > 0.05) in protection among fish exposed to the RB-1 vaccine strain at a dose of 1 × 105 TCID50/mL for periods of either 1, 12, or 24 h, held for 14 d, and then challenged with the wild-type RB isolate, although the 1-h exposure seemed to be somewhat less effective. Fish were vaccinated with the RB-1 strain at 1 × 103–1 × 105 TCID50/mL for 24 h then challenged after 1, 7, 14, or 21 d with the wild-type RB isolate. No significant (P > 0.1) protection was observed at 1 d postvaccination, but the relative percent survival increased progressively at each subsequent challenge period, becoming statistically significant by day 7 (P < 0.001) and beyond. These results suggested that resistance to challenge with wild-type virus resulted from development of IHNV-specific immunity and not from viral interference or interferon induction, and they reinforce the potential of an attenuated vaccine to control this important disease. 相似文献
17.
The objective of this study was to evaluate of the growth performance, serum biochemical indices, liver antioxidant parameters and ability of anti-infection with Aeromonas hydrophila by adding the fermented Chinese medicine by Bacillus Subtilis to the carp fish feed, and to find out the appropriate supplemental level of fermented Chinese medicine in diets. Carp fingerling with an initial body weight of (35±0.5) g were set up to 6 experimental diets supplemented with 0 (control group), 0.1‰, 0.5‰, 1‰, 2‰ and 3‰ fermented Chinese medicine for 56 days, respectively. Each group had 30 fish and set up 3 repetitions. After the feeding trail,the fish were challenged with Aeromonas hydrophila to calculate the relative percentage survival (RPS).The results showed as follows:Supplementing fermented traditional Chinese medicine by Bacillus subtilis, the weight gain rate (WGR), specific growth rate (SGR), condition factor (CF), the activities of lysozyme (LSZ), complement 3 (C3),total protein (TP),total anti-oxidation capacity (T-AOC),total superoxide dismutase (T-SOD) and catalase (CAT) in serum of common carp were enhanced at different degrees, while the feed coefficient (FCR)and malondialdehyde (MDA) in serum were declined at different degrees.When the fermented Chinese medicine supplemental levels were 2‰ and 3‰,the effects were significant (P<0.05)(except for C3, ALP and Cr).The experimental results of the virulence of common carp infected with pathogen Aeromonas hydrophila showed that,the fermented herbal could obviously improve the ability of anti-infection, the immune protective rate of the group with 2‰, 3‰ doses of the fermented Chinese medicine could reach 77.23% and 74.63%, respectively. From what had been discussed above, the group with 2‰ and 3‰ doses of the fermented Chinese medicine had significant effect on the growth performance, serum biochemical indices, liver antioxidant parameters and Aeromonas hydrophila anti-infection ability. Considering the cost of adding fermented Chinese medicine, the recommendations for the best additive quantity was 2‰. 相似文献
18.
本试验旨在研究饲料中添加枯草芽孢杆菌发酵中药制剂对鲤鱼生长性能、血清生化指标、抗氧化指标及对嗜水气单胞菌抗感染能力的影响,并确定其适宜的添加水平。将枯草芽孢杆菌发酵中药制剂投喂初始体重为（35±0.5）g的鲤鱼,试验设6个组,添加水平分别为0（对照组）、0.1‰、0.5‰、1‰、2‰、3‰,每组30尾,设3个重复,试验周期56 d。养殖试验结束后,腹腔注射嗜水气单胞菌进行感染试验,计算免疫保护率（RPS）。结果显示,饲料中添加枯草芽孢杆菌发酵中药制剂,鲤鱼增重率（WGR）、特定生长率（SGR）、肥满度（CF）、溶菌酶（LSZ）、补体3（C3）、总蛋白（TP）、总抗氧化能力（T-AOC）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）均不同程度提高,饲料系数（FCR）、丙二醛（MDA）含量不同程度降低,且当饲料中发酵中药添加量为2‰、3‰时,与对照组相比各指标（C3、碱性磷酸酶（ALP）和肌酐（Cr）除外）均差异显著（P<0.05）。用嗜水气单胞菌对鲤鱼感染试验结果表明,发酵中药可显著提高鲤鱼的抗感染能力,添加发酵中药2‰、3‰组免疫保护率可达77.23%和74.63%。可见,饲料中添加枯草芽孢杆菌发酵中药2‰、3‰能显著提高鲤鱼的生长性能、血清生化指标、肝脏抗氧化能力及抗嗜水气单胞菌感染能力。基于对添加成本的考虑,建议发酵中药的最佳添加剂量为2‰。 相似文献
19.
嗜水气单胞菌粘附特性的研究 总被引：7，自引：1，他引：6
不同来源的６株嗜水气单胞菌（Ａｅｒｏｍｏｎａｓｈｙｄｒｏｐｈｉｌａ）对１０种不同红细胞的血凝试验表明，Ｊ－１株能凝集所有的１０种红细胞，而与其他菌株的血凝谱有所不同。血凝图式有２种：人、鸡、麻雀的红细胞凝集快，呈大小不等的絮状；而鲫鱼、绵羊、猪、小鼠、兔、鸭、犬的红细胞凝集慢，且呈均匀颗粒状。这两种血凝均能被Ｄ－甘露糖抑制，但不能被Ｊ－１株Ｒ菌毛抑制。组织粘附试验显示，Ｊ－１株能粘附小鼠和鲫鱼的肠组织和肠绒毛。将提纯的Ｒ菌毛预处理肠组织，或用Ｄ－甘露糖或木瓜蛋白酶消化的Ｒ菌毛抗血清Ｆａｂ片段预处理菌体，都不能抑制上述的粘附作用。Ｊ－１株对ＨＥｐ－２细胞显示强粘附，菌体随机粘附在细胞上，有少数侵入细胞浆内。其余５株菌的粘附特性与Ｊ－１株不尽相同。所有６株菌对草鱼肠组织及肠绒毛、ＥＰＣ细胞（鲤鱼乳头状上皮瘤细胞系）均无粘附性。 相似文献
20.
C Bogni M Segura J Giraudo A Giraudo A Calzolari R Nagel 《Canadian journal of veterinary research》1998,62(4):293-298
An avirulent mutant, designated RC122, was derived from Staphylococcus aureus bovine mastitis strain RC108 after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. Mutant RC122, which was isolated on the basis of reduced colony size, showed diminished virulence in mice (LD50 of RC122: 3.1 x 10(10) cfu vs LD50 of RC108: 2.3 x 10(7) cfu). Mutant RC122 grew more slowly than its parental strain and showed decreased production of several exoproteins, such as alpha- and beta-hemolysin, DNAse and coagulase. The production of its capsule was induced only under in vivo growth conditions. Clearance studies performed in the mouse kidney revealed that the kinetics of disappearance of the mutant was similar to that of its parental strain. Protection experiments carried out by intraperitoneal administration in mice showed that mutant RC122 conferred a good degree of protection from challenge with homologous and heterologous strains. 相似文献