细鳞鱼杀鲑气单胞菌杀鲑亚种的分离鉴定

为确定吉林省某渔场细鳞鱼发病死亡的病因,本研究由濒死的细鳞鱼体内分离出一株优势菌,经动物回归试验,该菌株对细鳞鱼具有致病性,将其命名为BRL-15。经菌体、菌落形态,理化特性,16S rRNA基因序列分析,鉴定菌株BRL-15为杀鲑气单胞菌杀鲑亚种。药敏试验结果显示,该菌株对所选抗菌药物中绝大多数高度敏感。试验结果为细鳞鱼杀鲑气单胞菌病的防治提供一定理论依据。     

大鲵脾脏结节症病原菌的分离鉴定及药敏试验

为探究重庆地区中国大鲵(Andrias davidianus)脾脏结节症的病因及防控措施,本研究对发病大鲵进行了临床剖检及病理组织学观察,并采用微生物学方法从其肝脏、脾脏中分离病原菌,通过人工感染试验确定分离菌株的致病性,并对菌株的基本形态、理化特性、分子特征及药物敏感性等进行了系统研究。结果显示,患病大鲵脾脏有大量白色结节,质脆,病理切片发现脾组织有出血及丝网状纤维素分布,淋巴细胞部分消失;肝细胞肿胀、变性。从肝脏中分离到1 株病原菌,命名为KNL-1。人工感染试验发现,该菌对试验鲫鱼和大鲵均有较强的致病力,试验动物死亡率高达100%,大鲵发病症状与临床自然发病症状一致,确认为该病的致病菌。综合分离菌的16SrDNA 序列测定、培养特性、生化试验及BIOFOSUN-GN 系统鉴定结果,确定KNL-1 为杀鲑气单胞菌杀日本鲑亚种(Aeromonas salmonicida ssp. masoucida)。药敏试验结果显示,该菌对头孢氨苄、卡那霉素、头孢他啶、头孢曲松、先锋霉素、新霉素、庆大霉素、左氧氟沙星、米诺环素、丁胺卡那霉素和氟苯尼考高度敏感;对氨苄西林、麦迪霉素和羧苄西林耐药。     

猪源气单胞菌分离鉴定及耐药谱检测

为调查气单胞菌在我国猪粪便中流行情况及耐药谱,2017年3~7月从河南省4个地区,共采集370份猪粪样品,通过缓冲液蛋白胨水增菌培养后,使用添加有氨苄青霉素的气单胞菌基础培养基选择培养,分离气单胞菌。可疑菌株通过形态特征、生化试验和16SrDNA序列比对进一步鉴定。微量稀释法测定分离菌株对17种抗菌药物耐药谱。结果显示,共分离到11株气单胞菌,分离率为2.97%,其中豚鼠气单胞菌5株(占45.45%),杀鲑气单胞菌2株(占18.18%),中间气单胞菌、嗜水气单胞菌、水生气单胞菌和贝斯特润气单胞菌各1株(各占9.09%);阿莫西林/克拉维酸菌耐药率为100%,磺胺甲恶唑、氟苯尼考耐药率81.82%,阿米卡星、美罗培南敏感率均为100%,除了其中1株气单胞菌,其他菌株对头孢噻呋、头孢曲松、庆大霉素、强力霉素、磷霉素、恩诺沙星均敏感,各菌株均呈多重耐药性。表明我国健康猪粪便中携带气单胞菌,如果造成水源或者食物污染,可能有引起人类或者动物感染的风险,应引起重视。     

细鳞鲑疖疮病的病原鉴定及防治药剂筛选

自然发病的细鳞鲑(Brachymystax Lenok)出现鳃盖边缘、鳍条基部充血,鳍条溃烂,肛门红肿等症状。从病灶处分离得到的优势菌株,定名为BJSY-1、BJSY-2、BJSY-3。通过回归感染试验证明BJSY-1为致病菌,其LD_(50)为6.97×10~5CFU/mL。经生理生化鉴定和16S rDNA序列分析最终确定BJSY-1菌株为杀鲑气单胞菌杀鲑亚种(Aeromonas salmonicida subsp.Salmonicida)。该菌对四环素类、喹诺酮类等药物高度敏感。选择氟苯尼考作为治疗药物,按20 mg/kg·bw的用药量拌饵投喂,每天投喂1次,连续投喂有较好的治疗效果,为杀鲑气单胞菌的治疗和防控提供了参考。     

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。     

大鲵源杀鲑气单胞菌的分离鉴定及药敏试验

为了查明2016年3月份河南省洛阳市某大鲵(Andrias davidianus)养殖厂发生大规模疫病的原因,试验采用平板划线法从患病大鲵中分离细菌,结合革兰氏染色镜检、透射电镜观察、16S rDNA序列分析及遗传进化树构建对其进行鉴定,并对代表株进行生长曲线的测定及人工感染试验以鉴定其致病性,最后进行药敏试验测定水产常用药物的抑菌效果。结果表明:从大鲵组织中共分离出5株细菌,编号为HN1～HN5,均为革兰氏阴性短杆菌,且有一根鞭毛;5株细菌与杀鲑气单胞菌(Aeromonas salmonicida)序列的相似度高达99%,为同种菌株,鉴定其均为杀鲑气单胞菌;代表株HN3菌株与杀鲑气单胞菌LMG 22214株(NR_115351.1)同源性最高; HN3菌株在培养6 h后进入对数生长期,42 h后进入稳定期;HN3菌株为大鲵的病原菌,且对甲砜霉素和恩诺沙星极度敏感,对氟苯尼考、复方新诺明、多西环素高度敏感,对磺胺二甲氧嘧啶钠等4种药物中度敏感,对磺胺间甲氧嘧啶钠等3种药物有耐药性。     

1株犬类志贺邻单胞菌的分离鉴定及序列分析

从病死犬病料中分离到1株类志贺邻单胞菌,并对该菌做了生理生化鉴定、药敏试验,致病性试验。结果表明,本菌对多种抗生素耐药,其庆大霉素耐药机制与所携带的质粒相关;本菌对小白鼠有强致病性,其LD50为1．6×10^7.4CFU。用PCR方法扩增分离菌株16SrDNA基因并测序,并将其与GenBank上其他细菌16SrDNA核苷酸序列进行同源性分析。结果表明,分离株的16SrDNA核苷酸序列与类志贺邻单胞菌（GQ359962．1）的同源性为98％,因此将该分离菌株鉴定为致病性肠球菌,命名为YN-1株（云南-1株）。     

丁鲑鱼维氏气单胞菌的鉴定和生物学特性研究

对引起丁鲑鱼发病的菌株——XN602进行鉴定和生物学特性研究,经形态特征与生理生化鉴定,结果表明:该菌为革兰氏阴性杆菌,其16SrDNA序列与GenBank中多株维氏气单胞菌相似性高达99%,系统发育树显示该菌与维氏气单胞菌聚为一簇。因此,分离菌株XN602在分类学上应属于维氏气单胞菌。     

中华绒螯蟹点状产气单胞菌的分离鉴定与药敏试验

为确定诱发中华绒螯蟹肠炎的病原菌,筛选潜在的防控药物,采用传统方法从患肠炎的中华绒螯蟹体内分离到一株病原菌(DD5),通过API 20E细菌鉴定系统和16SrDNA序列分析对菌株DD5进行了鉴定,并采用KB纸片扩散法对菌株DD5的抗菌药物敏感性进行了测定。结果表明,菌株DD5为点状产气单胞菌(GenBank登录号:KP260655),其16SrDNA序列与基因库中气单胞菌属菌株的16SrDNA序列有99%~100%的同源性,而且与点状产气单胞菌136c株(GenBank登录号:EU488688)的亲缘关系最近。此外,菌株DD5对四环素、链霉素、卡那霉素、庆大霉素、恩诺沙星、氧氟沙星、左氟沙星、复方新诺明等抗菌药物高度敏感。本研究证实点状产气单胞菌是中华绒螯蟹肠炎的病原菌,恩诺沙星、庆大霉素等常规渔药可作为点状产气单胞菌引起的中华绒螯蟹肠炎的防控用药。     

杀鲑气单胞菌对鲟的杂交致病性及病理变化分析

为初探虹鳟鱼源杀鲑气单胞菌对杂交鲟是否具有感染性及人工感染后的主要临床症状及病理变化.本研究使用虹鳟鱼源杀鲑气单胞菌对杂交鲟进行人工感染,通过对半数致死量的测定、临床症状观察、发病杂交鲟解剖学变化观察及组织病理切片观察(HE染色)来确定杀鲑气单胞菌对杂交鲟是否具有致病性及致死性.结果 显示:虹鳟鱼源杀鲑气单胞菌对杂交鲟...     

家养观赏地图鱼肺炎克雷伯氏菌、维氏气单胞菌与黏质沙雷氏菌的分离鉴定及耐药性分析

本试验旨在通过细菌分离鉴定,明确家养观赏地图鱼的死因,筛选敏感药物。采用常规方法分离纯化细菌后,进行细菌形态学观察,并通过小白鼠致病性试验、细菌主要生化鉴定、16SrDNA序列测定分析、药敏试验及耐药基因检测等方法对分离的细菌进行鉴定及耐药分析。分离出3株革兰氏阴性短杆菌,根据细菌形态特征及理化特性,结合16SrDNA序列测定与系统发育分析结果,判定其分别为肺炎克雷伯氏菌(Klebsiella pneumoniae)、维氏气单胞菌(Aeromonas veronii)和黏质沙雷氏菌(Serratia marcescens),其中肺炎克雷伯氏菌具有较强致病性。3株细菌均对洛美沙星、氧氟沙星、阿米卡星、卡那霉素敏感,对阿莫西林、氟苯尼考、克林霉素、甲硝唑等具有较强耐药性。结果表明,肺炎克雷伯氏菌、维氏气单胞菌、黏质沙雷氏菌的混合感染是家养观赏地图鱼的死亡原因。     

Host factors associated with the detection of Aeromonas salmonicida and Yersinia ruckeri in Ontario, Canada government fish hatcheries

This paper presents an epidemiological investigation of Ontario Ministry of Natural Resources Fish Health Laboratory data from 1981 to 1997, to determine whether fish species and age were associated with lot-level detection of Aeromonas salmonicida and Yersinia ruckeri in hatchery fish. In stepwise logistic regression, the species brook trout and back-cross (lake trout crossed with the hybrid “splake”) were more likely to test A. salmonicida-positive compared to all other species reared in the hatcheries. Similarly, the species brook trout was significantly more likely to test Y. ruckeri-positive compared to all other species. For both pathogens, the 1–5-month age group was associated significantly with detection. These findings suggest that purposive sampling of higher-risk fish lots could increase the likelihood of detecting both study pathogens.     

肺炎克雷伯氏菌强毒株的分离鉴定及16-23SrRNAITS序列分析

为确诊疑似仔猪肺炎克雷伯氏菌(K.pneumonia)感染,并研究其病原的致病性、耐药性、16-23SrRNA ITS系统进化特征,本研究从云南因肺炎、腹泻而大量死亡的仔猪中分离到1株革兰氏阴性短粗杆菌,命名为KP14013,对其进行生化鉴定、16SrRNA鉴定,研究其对小白鼠和仔猪的致病性,并对其16-23SrRNA ITS基因进行测序和遗传进化分析。结果显示,KP14013分离株生化特征与肺炎克雷伯氏菌相符,其16SrRNA与GenBank中23株肺炎克雷伯氏菌代表株之间的同源性均为99%,将KP14013鉴定为肺炎克雷伯氏菌。KP14013对小白鼠半数致死量(LD50)为3×101.8 CFU,腹腔注射3×108 CFU可使仔猪100%致死。16-23SrRNA ITS系统进化关系结果表明,KP14013与GenBank中收录的15株肺炎克雷伯氏菌形成进化树的一个分支,属于同一个亚群,它们之间的核苷酸同源性为98.4%~99.2%。本研究证实了肺炎克雷伯氏菌是该起仔猪腹泻大量死亡的病原;KP14013分离株为毒力极强菌株,具有多重耐药性,其16-23SrRNA ITS与GenBank中收录的肺炎克雷伯氏菌代表株之间核苷酸存在差异,可用于肺炎克雷伯氏菌菌株间的鉴别。     

Detection of the causative agent of furunculusis, Aeromonas salmonicida in salmonids of the Krka River

In this paper we describe the bacterial community associated with salmonids from the Krka River. Diversity analysis demonstrated that majority of the recovered bacteria were related to Aeromonadaceae group. Bacterial analysis also revealed the presence of Shigella spp. and Pseudomonas fluorescens. Isolation of Aeromonas salmonicida from trout, presents first isolation of this bacteria Croatian rivers.     

In Vitro Antimicrobial Activity of Sarafloxacin against Clinical Isolates of Bacteria Pathogenic to Fish

Abstract

An in vitro susceptibility assay of sarafloxacin (A-56620), a new 4-quinolone, was performed against five important bacterial species that are pathogenic to fish. A collection of 44 clinical isolates and five corresponding type strains were included in the study. The objectives were to determine the minimal inhibitory concentrations (MICs) of sarafloxacin by a drug microdilution method and to compare the MIC values at two different temperatures, 4 and 15°C. Sarafloxacin was active against all species tested and showed the following mean MIC values at 15 and 4°C, respectively, against the bacterial pathogens investigated: Aeromonas salmonicida subspecies salmonicida, 0.029 and 0.045 μg/mL; atypical A. salmonicida, 0.053 and 0.041 μg/mL; Vibrio anguillarum, 0.085 and 0.054 μg/mL; V. salmonicida, 0.125 and 0.123 μg/mL; and Yersinia ruckeri, 0.023 and 0.031 μg/mL. The MICs ranged from 0.0025 μg/mL (or less) for two strains of A. salmonicida salmonicida to 0.32 μg/mL for one strain of atypical A. salmonicida and one strain of V anguillarum. A decrease in antimicrobial activity was observed as the incubation temperature was lowered from 15 to 4°C; however, no significant statistical difference between the measured values was demonstrated.     

水貂奇异变形杆菌的分离鉴定及16S rRNA基因序列分析

从辽宁某貂场发病水貂中分离到1株致病菌,命名为PMSD株,通过形态学观察和生化试验等常规鉴定发现符合奇异变形杆菌特性,进一步经VITEK 2Compact 30全自动细菌鉴定及药敏分析系统鉴定该株细菌为奇异变形杆菌。药敏试验结果显示PMSD株对氨基糖苷类药物、喹诺酮类药物等敏感,而对β-内酰胺类药物和磺胺类药物等不敏感。以细菌16SrRNA基因为模板应用通用引物进行PCR扩增,得到PMSD株的16SrRNA基因序列,长约1 504bp,提交到GenBank中,登录号:KM229530。将该序列与GenBank中序列进行BLAST比对,结果发现与其匹配度最高的均是奇异变形杆菌各株系的16SrRNA序列,均高达99%以上。选取其中前20株作为参考序列,运用生物学软件构建系统发育树并进行同源性比对,结果表明,分离菌PMSD株与20个代表菌株的同源性为98.9%~99.7%,其中与BB2000株、HI4320株、B1株和FCC141株同源性最高,为99.7%。本研究为预防和控制奇异变形杆菌引起的水貂疾病奠定了一定的基础。     

Development and evaluation of a multiplex PCR assay for simultaneous detection of Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida in culture fisheries

Bacterial cold water disease, enteric red mouth disease and frunculosis are the common bacterial diseases of fish worldwide. The etiologic agents of these diseases are Flavobacterium (F.) psychrophilum, Yersinia (Y.) ruckeri and Aeromonas (A.) salmonicida subsp. salmonicida, respectively. In this study, a multiplex polymerase chain reaction (m-PCR) method with YER8/10-Fer3/4-FP1/3 primer pairs which can identify these fish pathogens simultaneously was developed and optimized. In optimized conditions, neither false specific nor nonspecific amplification occurred. The detection limits of the m-PCR method using DNA extracts from dilutions of pure cultures of bacteria were 35 pg for Y. ruckeri and F. psychrophilum and 70 pg for A. salmonicida subsp. salmonicida. It was determined that 15 CFU Y. ruckeri and F. psychrophilum and 30 CFU A. salmonicida subsp. salmonicida could be detected by m-PCR developed using genomic DNA extracted from dilutions of the suspensions. The detection limits in the presence of tissue debris were 125 CFU for Y. ruckeri and F. psychrophilum and 250 CFU for A. salmonicida subsp. salmonicida. In conclusion, we submit that the m-PCR method developed and optimized in this study can be used for accurate and rapid identification of these bacteria.     

Journal of Aquatic Animal Health: Guide for Authors 2014

Abstract

Brook trout Salvelinus fontinalis were treated with single 60-min static baths of 250 mg formalin/L, 3% NaCl, and 15 mg Chloramine-T/L to evaluate the efficacy of these compounds against external infections of Aeromonas salmonicida. Prevalence of A. salmonicida was significantly lower in brook trout treated with Chloramine-T than among those treated with formalin or salt. Further laboratory tests substantiated the therapeutic value of a single treatment of ChloramineT (15 mg/L) against A. salmonicida. In two experiments, viable counts of A. salmonicida in mucus did not vary among replicate groups of treated brook trout, but the counts for treated fish were significantly (P < 0.05) lower than those for untreated controls. In vitro tube dilution assays indicated that mean minimum inhibitory concentrations of Chloramine-T for 10 isolates of A. salmonicida were 9.0 mg/L for 1 h and 2.25 mg/L, for 24 h. In field trials at the White River National Fish Hatchery (Bethel, Vermont), the pathogen was detected principally as an external infection of juvenile Atlantic salmon Salmo solar maintained in two culture ponds. In one pond, the bacterium accounted for 100% of the total distribution of tnicroflora isolated from mucus. Seven days after treatment with Chloramine-T, A. sahnonicida accounted for 11% of the total bacterial counts identified from these fish. In the second pond, A. salmonicida composed 3% of the counts of bacteria isolated from the mucus of fish before treatment but was not isolated after treatment.     

Kinetics of substrate oxidation and hydrogen peroxide production by Mycoplasma mycoides subsp. mycoides Large Colony (LC) type and Mycoplasma mycoides subsp. capri

Mycoplasma mycoides subsp. mycoides Large Colony (LC) type is a pathogen of goats causing contagious agalactia and respiratory disease, found on all continents where small ruminants are kept. It shares close genetic characteristics with M. mycoides subsp. capri. Substrate oxidation by 22 strains of M. mycoides subsp. mycoides LC from nine countries was compared with that of eight strains of M. mycoides subsp. capri from five countries. There was considerable similarity in the substrates used, but substrate saturation coefficients (K_s) varied for different substrates. Substrate utilization patterns and K_s values did not (1) significantly differentiate the LC strains from each other, (2) show any correlation with geographical origin, or (3) distinguish the LC strains from the capri strains. These results support previous studies justifying the reclassification of these subspecies as a single species.