Isolation,Identification and in vitro Antimicrobial Susceptibility of Pathogenic Aeromonas veronii from Soft-shelled Turtles

In order to effectively control diseases caused by Aeromonas veronii,pathogenic bacteria were isolated and incubated from infected soft-shelled turtles with traditional bacterial isolation method. Four strains of pathogenic bacteria were isolated and identified with traditional biochemical identification method and modern molecular biological identification techniques. According to the results, four strains of pathogenic bacteria were identified as A. veronii biovar sobria. Drug sensitivity test and in vitro antimicrobial test against Bacillus amyloliquefaciens were performed with agar diffusion method. The results showed that B. amyloliquefaciens and several antibiotics such as ofloxacin and gentamicin exerted strong antimicrobial effects on four isolates. B. amyloliquefaciens could be used for the prevention and control of diseases caused by A. veronii in aquaculture.

Characterization and Expression of Outer Membrane Protein A I Gene of Aeromonas veronii

The outer membrane protein, ompA, ofAeromonas veronii has a role in the virulence of the organism and is a potential candidate for vaccine development. In this study, ompA I ofAeromonas veronii strain WA106 was cloned and sequenced, then, it was expressed in Escherichia coli BL21. The nucleotide sequence of ompA I gene was 1 023 base pairs （GenBank Accession NO.KC748024）, which showed 100% homology with that of A. veronii （NO.AB290200.1）. This predicted protein was composed of 340 amino acid residues. Its molecular weight was 35.78 ku and isoelectric point was 5.18. The protein was a hydrophilic protein containing alpha helix and random coil with percentage of 35.0% and 49.7%, respectively. The tertiary structure, quaternary structure prediction showed that ompA I protein contained two peptide chains. SDS-PAGE showed that the actual value of the fusion protein was consistent with the expected result. It will facilitate further study of the role of ompA I protein.

Pichia pastoris composition expressed aerolysin mutant of Aeromonas veronii as an oral vaccine evaluated in zebrafish(Danio rerio)

Vaccines are one of the most practical means to stop the spreading of Aeromonas veronii in aquaculture.In this study,virulence factor aerolysin mutant NTaer which has lost its hemolytic activity was used as a target antigen.Pichia pastoris constitutive secretory expression NTaer(GS115-NTaer)was used as a potential safe oral vaccine to evaluate its effectiveness on zebrafish immunity.The result shows that vaccination of GS115-NTaer for four weeks did not affect the growth performance of the host,while eliciting an effective immune protective response.Compared with the control group,the GS115-NTaer could significantly up-regulate the relative expression level of the intestinal tight junction protein 1α(TJP1α)gene,and significantly increased the contents of lysozyme(LYZ),complement C3 and C4 in the gut,indicating that the innate immune response of the fish was activated.The relative gene expression levels of macrophage-expressed gene 1(MPEG1)and T cell receptor(TCR-α)in the gut,and MPEG1,CD4,CD8,TCR-α,GATA3,and T-bet in the spleen were all increased significantly,indicating that the cellular immune response of the fish was activated.Furthermore,the contents of serum IgM and intestinal mucosa IgZ antibodies were significantly increased,which showed that humoral immunity was also activated.Moreover,inoculation with GS115-NTaer significantly changed the structure of gut microbiota.In particular,the relative ratio of(Firmicutes+Fusobacteriota+Bacteroidota)/Proteobacteria was significantly higher than that of the control and GS115 groups.Lastly,the vaccinated fish were challenged with A.veronii,and the relative percent survival of GS115 and the GS115-NTear groups was 14.28%and 33.43%.This improvement of immunity was not only due to the specific immune response but also attributed to the improvement of innate immunity and the gut microbiota which was demonstrated by the germ-free zebrafish model.Collectively,this study provides information on the effectiveness of GS115-NTear as an oral vaccine for the green prevention and control of A.veronii infection in fish aquaculture.

Pathogenic infection and microbial composition of yellow catfish(Pelteobagrus fulvidraco)challenged by Aeromonas veronii and Proteus mirabilis

The catfishes are a group of economically important freshwater fish in China,which in recent years have suffered heavy losses as a result of bacterial outbreaks.In this study,we examined the diversity of the microbiome of infected skin mucus of yellow catfish(Pelteobagrus fulvidraco)and channel catfish(Ictalurus punctatus)and analyzed the bacterial pathogens.We found several common pathogenic bacteria,such as Aeromonas spp.,Vibrio spp.,Moraxella spp.and Proteus spp.present in both fish species,but with significantly different bacterial community structures.We isolated and cultured Aeromonas veronii and Proteus mirabilis and validated their infectivity in zebrafish(Danio rerio)and yellow catfish.Intraperitoneal injection of either bacteria into zebrafish,but not immersion,caused 100%mortality and ovary fragmentation.Yellow catfish was more sensitive than zebrafish with 100%mortality in the immersion challenges with A.veronii or P.mirabilis,and with a higher abundance of A.veronii in the P.mirabilis-challenged group compared to control.To our knowledge,this is the first study on the pathogenicity of P.mirabilis in yellow catfish,and the results will help to develop effective strategies for the disease prevention and control in catfish farming.

Aeromonas veronii isolated from climbing perch(Anabas testudineus)suffering from epizootic ulcerative syndrome(EUS)

Motile Aeromonads are responsible for various diseases in fishes that cause significant economic losses in aquaculture.Aeromonas spp.are also frequently isolated from fish suffering from Epizootic Ulcerative Syndrome(EUS).The present study was conducted to isolate and identify A.veronii from Vietnam climbing perch(Anabas testudineus)suffering from EUS,evaluate the pathogenicity of the isolates,and find out antibiogram profile and susceptibility of the isolates to medicinal plant extracts.Vietnam climbing perch(A.testudineus)suffering from EUS was collected from fish farms located at Savar,Dhaka and bacteria were isolated from the external ulcerative lesion,and kidney and liver of the infected fish on nutrient agar plates.Preliminary phenotypic identification of the bacterial isolates was performed following morphological,physiological and biochemical characterization tests.The 16S rRNA gene sequencing was accomplished for further molecular identification of five selected isolates.To evaluate the pathogenicity of the isolates in vivo artificial infection challenge test was conducted through immersion method.Another in vivo pathogenicity test was done at three different temperatures(18◦C,28◦C,and 37◦C)to examine the effects of temperature on the pathogenicity of the isolates.Ten commercial antibiotics were used to find out the antibiogram profile of the isolates.Finally,susceptibility of the isolates was screened against 19 medicinal plants extracts.A total of 38 out of 46 isolates isolated from Vietnam climbing perch(A.testudineus)suffering from EUS were phenotypically identified as Aeromonas sp.Five randomly selected isolates were further confirmed as A.veronii by analyzing the 16S rRNA gene sequence homology.The pathogenicity of the isolates was found moderate to high(41%-80%mortality)in the artificially challenged fish.The highest and lowest mortality was obtained in the fish challenged with the isolates BFKA33 and BFKA12,respectively.The isolates were found resistant to several antibiotics,including amoxicillin,ampicillin,penicillin-G,and vancomycin.Nevertheless,extracts of three medicinal plant(Allium sativum,Syzygium aromaticum,and Tamarindus indica)demonstrated antibacterial activity to the isolates(BFKA12,BFKA15,BFKA18,BFKA29,and BFKA33).

Mucosal immune responses and protective efficacy in yellow catfish after immersion vaccination with bivalent inactivated Aeromonas veronii and Edwardsiella ictaluri vaccine

Mucosal vaccination,which has the potential to induce both mucosal and systemic immune responses,is considered the most suitable method of preventing infectious diseases in farmed fish.Aeromonas veronii and Edwardsiella ictaluri are two pathogenic bacteria found in yellow catfish and often infect the fish through mucosal surfaces.Delivery of a bivalent inactivated vaccine by injection has been shown to induce a strong systemic immune response against both bacterial infections.However,mucosal immune responses and protective efficiency induced by this inactivated vaccine administrated via immersion are yet to be investigated.We developed a bivalent vaccine containing formalin-inactivated A.veronii and E.ictaluri and evaluated the immune response in yellow catfish after immersion vaccination using body fluids biochemistry indices,agglutinating antibody titers,and the expression level of immune-related genes in the skin,gills,spleen,and head kidney.The activities of innate immune-related enzymes and specific agglutination antibody titers in body fluids,as well as the expression of innate and adaptive immune-related genes in both the mucosal and systemic tissues of vaccinated fish,were significantly higher than that in control fish.Next,we assessed the protective efficacy by a challenge model of virulent strains of E.ictaluri and A.veronii.The relative survival percent of vaccinated fish was 80%and 87%after challenging fish with E.ictaluri and A.veronii,respectively,which was higher than unvaccinated control fish(43%and 57%).These results confirm that the bivalent inactivated vaccine administered via immersion induces a strong mucosal immune response and confers good protection against both E.ictaluri and A.veronii.Our results also reinforce the notion that immersion vaccination could stimulate both mucosal and systemic immunity contributing to protection against pathogens.

Comparative transcriptomic analysis of Macro brachium nippon ense in response to Aerom onas veronii or Staphylococcus au reus infection

Macrobrachium nipponense is an economically important freshwater prawn that is often threatened by many aquatic pathogens.In this study,comparative transcriptomic analysis was fi rstly used to explore the transcriptional response of M.nipponense to Aeromonas veronii or Staphylococcus aureus stimulation.A total of 400.19 million clean reads were obtained and assembled into 56944 unigenes with an average length of 1253 bp.A total of 1857 diff erentially expressed genes were found after A.veronii infection,including 677 genes that were up-regulated and 1180 genes that were down-regulated,while 1061 signifi cant diff erentially expressed genes were identifi ed after S.aureus infection,including 390 up-regulated and 671 down-regulated genes.Many immune-related genes including Spaetzle,prophenoloxidase activating factor,C-type lectin,anti-lipopolysaccharide factor,and inhibitor of apoptosis 2 protein were commonly up-regulated after A.veronii or S.aureus infection.This study will enrich our understanding of the immune response to gram-positive and gram-negative bacteria infection in crustaceans.

维氏气单胞菌拮抗菌株J2-2的筛选、鉴定与效果评价

为筛选对维氏气单胞菌(Aeromonas veronii)有潜在抑菌作用的菌株,本研究从湖北公安县泥鳅养殖池塘底泥中分离一株细菌J2-2,对其特性进行了研究,并对抑菌活性物质进行了初步分析。结果显示:菌株J2-2为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),具有抑菌广谱性,对米尔伊丽莎白菌(Elizabethella milieri)、嗜水气单胞菌(A.hydrophila)等6种水产病原菌的抑菌直径在9.6~29.5 mm。菌株J2-2的抑菌物质具有较好的酸碱稳定性、热稳定性和蛋白酶K处理的稳定性。电镜结果显示菌株J2-2的发酵产物对维氏气单胞菌的细胞膜结构具有破坏作用。通过LC-MC分析该拮抗细菌可产生至少15种不同的脂肽类化合物如杆菌霉素等。药敏试验结果显示,菌株J2-2对氨苄西林、青霉素具有耐药性,对克林霉素中度敏感,对大多数抗生素高度敏感。

黄颡鱼暴发性疾病病因分析及防治措施

为探究湖北省部分地区黄颡鱼(Pelteobagrus fulvidraco)暴发性疾病发生原因,实地调查了生产情况,并从患病鱼体内分离鉴定出维氏气单胞菌(Aeromonas veronii)和类志贺邻单胞菌(Plesiomonas shigelloides)。药敏试验结果表明,维氏气单胞菌对硫酸新霉素和氟甲喹较为敏感,对甲砜霉素、氟苯尼考、磺胺间甲氧嘧啶钠均具有较大程度的耐药性。类志贺邻单胞菌仅对氟甲喹中度敏感,对其他7种国标渔药都具有较强的耐药性。综合气温、水质、喂养情况等因素分析了黄颡鱼暴发性疾病的发生原因,认为此次暴发性疾病主要由细菌感染引起,与气温的剧烈波动、过量投喂、水质恶化等因素密切相关。

31种中草药及其复方对维氏气单胞菌体外抑菌研究

为筛选对昆明裂腹鱼源维氏气单胞菌(Aeromonas veronii)有抑菌作用的中草药及复方,本研究通过煎煮法制备31种常见中草药水提取物,以牛津杯法测定各味中草药对该菌的抑菌圈直径,采用二倍稀释法测定最小抑菌浓度(MIC)和最小杀菌浓度(MBC);选取体外抗菌活性较好的6种中草药,应用棋盘法进行15组二联药方的部分抑菌浓度指数(FIC)试验;按正交设计表配伍25组复方,同样进行药敏试验。结果显示,31种中草药中,五倍子、地榆、诃子、苏木、黄芩、黄连等6种中草药抑菌效果最好;15组联用药方均未发生拮抗作用,其中以五倍子+地榆、诃子+黄芩联用效果最好,FIC分别为0.31、0.50,表现为协同作用;综合复方中草药的抑菌作用,复方F16(五倍子∶地榆∶诃子∶苏木∶黄芩∶黄连=4∶4∶2∶5∶3∶1)的抑菌效果最佳,抑菌圈直径达23.2 mm,MIC与MBC分别为0.98 mg/mL、1.95 mg/mL,可作为昆明裂腹鱼源维氏气单胞菌抑菌药物的首选配伍组合。

青虾源维氏气单胞菌双重PCR检测方法的建立

根据NCBI已发表的维氏气单胞菌(Aeromonas veronii)促旋酶B亚单位基因gyrB和编码细菌RNA聚合酶σ70因子基因rpoD的保守序列设计引物,建立了一种快速检测青虾源维氏气单胞菌的双重PCR检测方法。结果显示:青虾源维氏气单胞菌gyrB和rpoD基因PCR扩增产物大小分别为815 bp、554 bp,而对嗜水气单胞菌(Aeromonas hydrophila)、弗氏柠檬酸杆菌(Citrobacter freundii)、哈维弧菌(Vibrio harveyi)、副溶血性弧菌(Vibrio parahemolyticus)、需钠弧菌(Vibrio natriegens)、非O1霍乱弧菌(non-O1 Vibrio cholerae)、阴沟肠杆菌(Enterobacter cloacae)、产气肠杆菌(Enterobacter aerogenes)扩增结果皆为阴性;灵敏性试验结果显示该方法检测到的最低菌体DNA量为1.8×10-2 ng/μL,且10份送检样本检测结果与传统细菌分离鉴定结果相一致。结果表明,双重PCR检测方法特异性好、灵敏性高,适用于青虾源维氏气单胞菌的快速检测。

昆明裂腹鱼溃疡病病原菌分离鉴定及药物敏感性

为探明昆明裂腹鱼(Schizothorax grahami)患"溃疡病"的致病原理,本研究在无菌条件下从患病昆明裂腹鱼体表溃烂处、肝脏与肾脏组织中分离、纯化病菌,采用形态学观察、生理生化特性鉴定及16S rDNA、dnaJ、cpn60等基因的序列分析进行细菌种类鉴定,通过回归感染确定其致病性,并以K-B纸片扩散法进行药敏试验。结果显示:自患病昆明裂腹鱼分离纯化获得形态大小均一的细菌菌落,随机选取肾脏分离细菌(BL61C)作为试验菌;形态学观察、生理生化特性鉴定及基因序列分析结果显示菌株BL61C为维氏气单胞菌(Aeromonas veronii);健康昆明裂腹鱼回归感染BL61C后的发病特征与自然病例一致,并从组织中再次分离到同种致病菌,表明维氏气单胞菌BL61C是引起本次昆明裂腹鱼溃疡病的致病菌,半数致死浓度(LC50)为5.09×107 CFU/mL;药敏试验结果表明分离菌对大观霉素、阿奇霉素、氧氟沙星、氯霉素、氟苯尼考、头孢他啶、头孢克肟和头孢噻肟等8种药物敏感,对克拉霉素、制霉菌素、四环素等20种药物耐药。

泗洪地区克氏原螯虾病原维氏气单胞菌鉴定及致病性分析

为了探究江苏省泗洪县多家养殖场克氏原螯虾(Procambarus clarkii)出现暴发性死亡的病因,本研究以濒死的克氏原螯虾进行病原检测分析,从濒死虾肝胰腺中分离到优势生长菌(菌株编号CPA2020),经对分离菌进行致病性、形态与生理生化特征及gyrB基因同源性与系统发育分析,结果表明引起克氏原螯虾暴发性死亡的病原为维氏气单胞菌(Aeromonas veronii),人工感染实验结果表明分离菌CPA2020对克氏原螯虾具有较强的致病性,LD 50为6.31×10^(5) CFU/mL。组织病理学观察发现克氏原螯虾的肝胰腺与肠道组织出现严重的病理损伤,肝细胞裂解,空泡化严重,部分肝细胞坏死,肠壁结缔组织萎缩,皱嵴减少,肠上皮细胞坏死,与自然患病虾的病理变化相同。毒力基因检测表明分离菌CPA2020携带lip、hly、flgN、ompA、flgM、flaH、aha和flgA 8种毒力基因。此外分离菌CPA2020可分泌卵磷脂酶、明胶酶和溶血素。耐药谱测定结果显示,该菌对链霉素、万古霉素、环丙沙星等12种药物耐药,对头孢唑林、克拉霉素等9种药物中介,对头孢呋辛、左氟沙星、氨曲南等13种药物敏感。

一株维氏气单胞菌的分离鉴定及药敏特性研究

从邯郸魏县患病锦鲤(Cyprinus carpio koi)体内分离获得一株革兰氏阴性菌,对其形态特征、生理生化特性及16S rDNA序列进行分析,鉴定其为维氏气单胞菌(Aeromonas veronii);经人工感染试验,发现菌液浓度3.0×10^(10)CFU/mL下感染锦鲤发病,对发病锦鲤进行病原菌分离、观察、生理生化特性鉴定及16S rDNA序列分析,与感染试验菌株为同一种菌。采用微量肉汤稀释法对该菌株进行药敏试验,发现其对盐酸多西环素、恩诺沙星、氟甲喹、氟苯尼考、甲砜霉素、磺胺间甲氧嘧啶钠、磺胺甲噁唑+甲氧苄啶敏感,对硫酸新霉素中度敏感。

泥鳅白头病病原菌的分离鉴定、药敏试验及拮抗菌的筛选

为确定连云港市墩尚镇泥鳅(Misgurnus anguillicaudatus)白头病的病原菌,本研究采用常规微生物分离鉴定技术和分子生物学相关方法,对患病泥鳅进行病原菌的分离、鉴定并分析其药物敏感性,采用滤纸片抑菌圈法筛选了该病原菌的拮抗菌。从患病泥鳅病灶部位分离到1株病原菌并命名为W1,经人工感染发现体腔注射浓度为1.6×10^(7)CFU/mL 0.1 mL菌液时泥鳅死亡率为80%,在1.6×10^(7)CFU/mL浓度下创伤和浸泡感染死亡率为100%。通过形态特征观察、生理生化实验和16S rRNA基因序列分析,将该病原菌初步鉴定为维氏气单胞菌(Aeromonas veronii)。药敏试验结果显示,该菌株对头孢克肟高度敏感,对呋喃唑酮、链霉素、庆大霉素和恩诺沙星中度敏感,对红霉素、环丙沙星、四环素等不敏感。筛选该病原菌的拮抗菌发现,解淀粉芽孢杆菌(Bacillus amyloliquefaciens)X8对维氏气单胞菌W1具有较强的抑制作用。