斑点叉尾鮰冬季大规模死亡病原分离与鉴定

2016年1—2月湖南安化地区网箱养殖的斑点叉尾鮰暴发大规模死亡,死亡率接近80%,发病鱼体重为1.5~2.0 kg。本研究从患病濒死鱼肝、脾、肾分离到1株致病菌,对其进行常规理化特性、分子生物学、人工感染实验以及药敏性检测。结果显示,分离菌株理化特性与鲁氏耶尔森氏菌(Yersinia ruckeri)特征相符,与NCBI中登录的鲁氏耶尔森氏菌的16S rDNA基因同源性达99%以上,在系统发育树上与鲁氏耶尔森氏菌聚为一族。人工感染鱼出现与自然发病相似症状,并能够从发病鱼组织中再次分离到相同病原菌,表明鲁氏耶尔森氏菌为斑点叉尾鮰该病病原菌。该分离菌株对氟苯尼考、多西环素及新霉素等药物高度敏感,本研究为斑点叉尾鮰暴发性出血病防控提供了理论依据。     

鲟源致病性鲁氏耶尔森菌的分离、鉴定及药敏研究

2017年,新疆阜康市某鲟鱼养殖场的西伯利亚鲟出现大批死亡,主要症状为生殖孔红肿外突,腹部有点状出血。自患病西伯利亚鲟肝脏、脾脏分离到一株优势菌株XB2,通过革兰氏染色、生理生化试验、药敏试验,并对其16S rRNA基因序列进行测定,用Mega 6.0对序列进行分析。生理生化试验结果显示,菌株XB2为革兰氏阴性短杆菌,单独或成对出现;产酸不产气,V-P反应呈阳性,能利用蔗糖、果糖、葡萄糖、明胶、枸橼酸;在含1%(m/V)氯化钠中能生长,高于3%(m/V)氯化钠中不生长;在4℃不生长,20～30℃均能生长,但在37℃不生长;不利用乳糖、水杨苷、纤维二糖、硫化氢、酒石酸、阿拉伯糖。进化树显示,菌株XB2与鲁氏耶尔森菌KJ812974同源性达99%。通过回感试验确定其半致死密度为2.37×104 cfu/mL。结合生理生化鉴定结果和16S rRNA基因序列分析结果,确定菌株XB2为鲁氏耶尔森菌。药敏结果显示,鲁氏耶尔森菌XB2对氟苯尼考、左氧氟沙星、哌拉西林、阿洛西林等9种药物敏感,对麦迪霉素、大观霉素、链霉素、氨苄西林等15种药物耐药。     

鲟源鲁氏耶尔森菌多位点序列分型及耐药谱测定

2019年7—8月,甘肃省刘家峡某鲟鱼养殖场杂交鲟[体质量(160±20) g]大批死亡,死鱼泄殖孔红肿出血,偶见吻部出血。自60尾病鱼肝脏分离出的60株优势菌株的16S rRNA基因序列检测结果显示,分离菌株均为鲁氏耶尔森菌。生物型检测和多位点序列分型结果表明,60株鲁氏耶尔森菌为生物2型,获得ST1、ST2、ST3、ST5、ST8和ST13 6个已知ST型;在此基础上,对鲁氏耶尔森菌6个管家基因串联的系统发育分析显示,同一来源地分离株存在差异。对60株鲁氏耶尔森菌进行了10种抗生素药物的耐药谱测定,共获得9种耐药谱。本试验结果可为刘家峡养殖杂交鲟鲁氏耶尔森菌的耐药状况提供数据支撑,并为鲟鱼鲁氏耶尔森菌病的防治和疫苗研制提供理论依据。     

耶尔森氏菌对水产养殖的危害及防治

耶尔森氏菌是肠杆菌科、耶尔森氏菌属的总称,属兼性厌氧革兰氏阴性杆菌群。该属包含鼠疫耶尔森氏菌、假结核耶尔森氏菌、小肠结肠炎耶尔森氏菌等共11个种。其中鲁氏耶尔森氏菌是引起冷水性鲑鳟负类红嘴病的主要病原菌,是淡水养殖鱼类暴发性败血症的常见致病菌（气单胞菌、河弧菌、鲁氏耶尔森氏菌）之一。     

一株鳙源鲁氏耶尔森氏菌的分离鉴定及其感染的肠道病理损伤

为确定患病鳙鱼致病菌及筛选敏感药物,通过平皿划线法从患病鳙鱼血液及体表溃烂组织中分离到1株革兰氏阴性的短杆菌JL9510,该菌株在电镜下呈短杆状,在LB固体培养基上呈透明状菌落。VITEK^?MS全自动快速微生物质谱检测结果证明,该菌株为鲁氏耶尔森氏菌的置信度为99.9。通过16S rRNA及系统发育树构建分析,发现菌株JL9510与已知菌株JQ657818.1 Yersinia ruckeri聚为一支,确定其是一株鲁氏耶尔森菌。人工回归感染试验结果证明,该菌株为患病鳙鱼的致病菌。VITEK 2 Compact自动化细菌鉴定和药物敏感性分析系统结果显示,鲁氏耶尔森氏菌JL9510有较高的致病力,且对氟苯尼考敏感,对环丙沙星耐药。试验的抗生素表现出良好的治疗效果,为鲁氏耶尔森氏菌的防治奠定了基础。     

大口黑鲈鲁氏耶尔森氏菌的分离鉴定、主要毒力基因及致病性研究

为探明2021年3月福建某水库养殖大口黑鲈疾病暴发的原因,实验从患病大口黑鲈肝脏、肾脏、脾脏中分离优势菌,通过人工感染实验确定病原菌,并综合16S rDNA基因序列分析、生理生化和质谱特征等技术对该病原菌进行种属鉴定,同时,进行毒力基因检测、药物敏感性实验以及组织病理学观察。结果显示,从病鱼脾脏中分离获得1株优势菌并鉴定为鲁氏耶尔森氏菌;该菌株对大口黑鲈的半致死剂量为3.8×10⁵ CFU/尾;该菌株携带yrP1、yhl A、yhl B等毒力基因,对恩诺沙星、盐酸多西环素、氟甲喹、硫酸新霉素、氟苯尼考等5种药物相对敏感。组织病理学观察发现,鲁氏耶尔森氏菌的感染造成大口黑鲈肝脏、肾脏、脾脏不同程度的损伤,表现为明显的变性、坏死及炎症细胞的浸润等。本研究首次报道了鲁氏耶尔森氏菌对养殖大口黑鲈的致病性,可为养殖大口黑鲈鲁氏耶尔森氏菌病的诊断和药物防治提供参考依据。     

氟苯尼考对鲁氏耶尔森氏菌体外药效研究

本文研究了氟苯尼考对鲁氏耶尔森氏菌(Yersinia ruckeri)体外药效学,测定了最小抑菌浓度(MIC)、最小杀菌浓度(MBC)、生长动力学曲线和杀菌动力学曲线和抗菌后效应(PAE)及四种培养因子对氟苯尼考体外抑制鲁氏菌活性的影响。结果表明:MIC、MBC和MBC/MIC分别为0.5μg.mL-1、1μg.mL-1和2;鲁氏菌在液体培养基中1h后进入对数生长,大约持续7h;在用药4～6 h达到最大药效。由杀菌曲线可知,氟苯尼考的杀菌功效具有浓度依赖性;在2 MIC、4 MIC和8 MIC时,PAE分别为3.71±0.11、4.54±0.27和5.52±0.23;氟苯尼考对鲁氏菌作用最适pH值为6～8,且二价阳离子(Mg2+)、血清含量及细菌数量小于108时对药效无显著影响。因此,保证药物浓度和作用时间,并配合最适培养条件,是氟苯尼考发挥最高药效的前提条件。     

诺氟沙星在西伯利亚鲟体内蓄积规律及其对组织的病理损伤

为了确定诺氟沙星在鲟养殖中的科学使用方法,对不同剂量、不同给药次数情况下诺氟沙星在西伯利亚鲟(Acipenser baerii)体内蓄积规律及毒性进行了研究。分别以0、30 mg/kg、50 mg/kg、100 mg/kg的剂量对西伯利亚鲟连续口灌诺氟沙星3~5 d,每天1次,并于停药后24 h及240 h采集实验鱼血、肝、肾及软骨组织,对4种组织中药物蓄积量进行测定,并对肝、肾和软骨组织进行切片观察。实验结果表明:随着给药剂量增加、给药次数增多,实验鱼4种组织中诺氟沙星浓度逐渐升高,浓度由高到低依次为为:肾、软骨、肝、血清,且血清中药物浓度远远低于另外3种组织。相同给药剂量、不同给药次数时血清中药物浓度无显著差异;而肝、肾和软骨中均差异显著,停药240 h后,显著性消失。切片结果表明,50 mg/kg剂量连续给药5 d后,实验鲟肝、肾组织开始结构损伤变化,100 mg/kg剂量连续给药3~5 d后,损伤情况持续加重,停药240 h后,肝组织损伤情况有逐渐恢复趋势,而肾组织未见明显恢复趋势;而100 mg/kg连续给药3~5 d后,软骨组织出现软骨膜内层细胞减少;停药240 h后有恢复趋势。研究结果表明,诺氟沙星在鲟中使用剂量应小于50 mg/kg,连续给药次数低于5次,以避免对鲟肝组织造成损伤,引起肾不可逆损伤及影响软骨细胞发育和再生,对软骨组织造成潜在影响。     

鲁氏耶尔森菌qPCR快速检测方法的建立和应用

鲁氏耶尔森菌(Yersinia ruckeri)是导致虹鳟(Oncorhynchus mykiss)肠炎红嘴病的病原。本研究以鲁氏耶尔森菌毒力因子rup A基因为靶标设计1对特异性引物,以含有rup A基因部分序列的重组质粒为标准品构建标准曲线,优化建立检测鲁氏耶尔森菌的SYBR Green I实时荧光定量PCR检测方法,并对腹腔注射鲁氏耶尔森菌菌悬液的虹鳟肝、肾、脾、血液样品进行检测。结果显示,设计的引物具有良好的种间特异性,标准曲线线性方程为y=–3.3766x+40.012(R~2=0.9958);最低检测限为57 copy/μL,较常规PCR的灵敏度高出约100倍。应用检测结果表明,本方法可准确检测被鲁氏耶尔森菌感染的虹鳟样品。研究表明,所建立的qPCR方法具有特异、灵敏、快速、定量的优点,可用于快速诊断虹鳟肠炎红嘴病早期病症及定量检测鲁氏耶尔森菌。     

中草药添加剂对鲁氏耶尔森菌感染虹鳟血液生化指标的影响

为研究复方中草药对鲁氏耶尔森菌(Yersinia ruckeri)感染虹鳟(Oncorhynchus mykiss)后其血液生化指标的影响,在虹鳟饲料中分别添加两种复方中草药(复方1组、复方2组),持续投喂28 d。在第28天感染鲁氏耶尔森菌,检测感染后72 h的血清生化指标,并统计各组存活率。结果显示:复方中草药组对虹鳟增重无抑制作用。添加复方中草药可提高虹鳟感染鲁氏耶尔森菌后的存活率,其中复方2组的保护效果最好。感染72 h后,复方2组总蛋白、白蛋白、球蛋白、甘油三酯、总胆固醇含量和碱性磷酸酶活性均高于对照组。复方2组谷丙转氨酶、谷草转氨酶活性低于对照组,乳酸脱氢酶活性显著低于对照组。复方1组和复方2组尿酸含量显著低于对照组。复方中草药组总胆红素、尿素氮、肌酐和血糖含量与对照组相比无显著性差异。结果表明,添加复方中草药能提高鲁氏耶尔森菌感染后虹鳟的存活率,其中复方2组的保护效果最好。     

Immunosuppression in rainbow trout, Salmo gairdneri Richardson, caused by the haemoflagellate Cryptobia salmositica Katz, 1951

Abstract. An experimental Cryptobia salmositica infection in rainbow trout, Salmo gairdneri Richardson, produced suppression of the humoral response against sheep red blood cells as measured by direct haemagglutination. Two-month and 5-month infections produced equal suppression. The parasite also produced suppression of the humoral response against a bacterial pathogen, Yersinia ruckeri . Anti- Y. ritckeri titres were significantly lower in most fish infected with C. salmositica than in non-infected fish. Immunosuppression became evident when C. salmositica first appeared in the blood (first 2 weeks of infection), Immunosuppression was confirmed by challenge with Y. ruckeri . Mortality at challenge occurred in 64·3% to 83·3% of the fish already infected with C. salmositica at the time of initial Y. ruckeri exposure. There was no mortality at challenge if fish were not infected with C. salmositica at initial bacterial exposure, nor in those concurrently infected with both pathogens. Antigenic competition may have caused the immunosuppression.     

Stress-induced transmission of Yersinia ruckeri infection from carriers to recipient steelhead trout Salmo gairdneri Richardson

Abstract. The transmission of Yersinia ruckeri has been investigated in steelhead trout using asymptomatic carriers of the causative bacterium of enteric redmouth disease. It was found that unstressed carrier fish did not transmit the bacterium to recipient fish to cause either an epizootic or produce new carrier fish. However, when the carriers were stressed with heat, the bacterium was transmitted from the carrier to recipient fish producing a lower intestinal carrier state but no deaths. Examination of experimentally infected fish to determine the number of carriers among the survivors indicated that the frequency varied as a function of time following infection. When immunized fish, were challenged with Y. ruckeri they became temporary carriers of the bacterium for up to 3 days; but were not able to transmit the infection to healthy recipient fish.     

中草药与复方对鲁氏耶尔森菌的体外抑菌作用

采用试管二倍稀释法测定了16种中草药单方及5种复方制剂对鲁氏耶尔森菌（Yersinia ruckeri）的最小抑菌浓度（MIC）。结果表明,黄芩（Radix Scutellariae）和诃子（Fructus Chebulae）对鲁氏耶尔森菌的抑菌作用最强,MIC均为0.028g·mL^-1,可作为防治水产养殖动物耶尔森菌病害的主要备选中草药;大黄（Radix et Rhizoma Rhei）的抑菌作用较强,其MIC值为0.227g·mL^-1,也可作为候选药物;青蒿（Herba Artemisiae）、金樱子（Fructus Rosae Laevigatae）、连翘（Fructus Forsythiae）、茵陈（Herba Artemisiae Scopariae）、五倍子（Galla Chinensis）及金银花（Flos Lonicerae）的抑菌作用较弱,其MIC值均为0.455g·mL^-1。5种复方制剂中,大黄、黄柏（Cortex Phel-lodendri）和黄芩3种中草药以5：3：2的比例配伍对鲁氏耶尔森菌具有最佳抑菌作用,其MIC值为0.057g·mL^-1,抑菌效果弱于黄芩,但远强于大黄单独使用,可为该细菌性疾病的防治提供复方配伍参考。     

Determination of Dose Rate of Florfenicol in Feed for Control of Mortality in Channel Catfish Ictalurus punctatus (Rafinesque) Infected with Edwardsiella ictaluri, Etiological Agent of Enteric Septicemia

–A dose titration study was conducted to determine the appropriate dosage of florfenicol in feed to control mortality in channel catfish Ictalurus punctatus associated with enteric septicemia of catfish caused by Edwardsiella ictaluri. Six tanks (20 fish/ tank) were assigned to each of the following treatment: 1) not challenged with E. ictaluri and fed unmedicated feed; 2) challenged with E. ictaluri and fed unmedicated feed; 3) challenged and fed 5-mg florfenicol/kg body weight (kg bw); 4) challenged and fed 10-mg florfenicol/kg bw; or 5) challenged and fed 15-mg florfenicol/kg bw. Treatment was initiated the day after inoculation, and feed was administered by hand at 2.5% body weight for 10 consecutive days. Feeding activity was scored for all groups and was noted to be significantly less than the challenged, unmedicated group. Cumulative mortality in the challenged untreated group was 60%. The mortality in the unchallenged untreated group was 0%, and in die 5-, 10-, 15-mg florfenicol/kg bw group was 2.5%, 0.8%, and 2.5%, respectively. The mortality in each challenged, treated group and the non-challenged control group was significantly less than the challenged, unmedicated controls (P < 0.0001 for each contrast). There were no pairwise statistically significant contrasts among the florfenicol treated groups and the non-challenged control group. All 600 fish in the study were necropsied, cultured for bacteria, and examined by gross pathology. No specific lesions that could be associated with the antibiotic were observed. The efficacy of the 10 mg/kg dosage was confirmed in a separate dose confirmation study. In this study, fish in 30 tanks (20 fish/ tank) were infected with E. icraluri by immersion. Two days post-inoculation, fish in 15 tanks were hand-fed unmedicated feed, and 15 tanks were hand-fed medicated feed at a dosage of 10-mg florfenicol/kg bw at 2.5% body weight for 10 d. Feeding activity was scored and was noted to be significantly less than the challenged, unmedicated group. Cumulative mortality in the florfenicol group (14%) was significantly less than cumulative mortality in the untreated group (87.3%) (P < 0.0001). All 600 fish were submitted for bacterial culture, necropsied. and examined for gross pathology, and once again, no specific lesions that could be associated with the antibiotic were observed. The minimum inhibitory concentration of florfenicol against E. ictaluri in both studies was 0.25 ug/mL. Florfenicol was palatable, safe, and efficacious for control of mortality due to infection by E. ictaluri in catfish.     

The use of a real-time PCR primer/probe set to observe infectivity of Yersinia ruckeri in Chinook salmon, Oncorhynchus tshawytscha (Walbaum), and steelhead trout, Oncorhynchus mykiss (Walbaum)

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM), a common pathogen affecting aquaculture facilities and implicated in large losses of cultured fish. Fisheries scientists continue to gain a greater understanding of the disease and the pathogen by investigating methods of identification and pre- and post-infection treatment. In this study, a real-time PCR probe set for Y. ruckeri was developed to detect daily changes in the bacterial load during pathogen challenges. Two species of fish, Chinook salmon, Oncorhynchus tshawytscha, and steelhead trout, Oncorhynchus mykiss, were exposed to two strains of Y. ruckeri (Hag and SC) during bath challenges. A subset of fish was killed daily for 14 days, and the kidney tissue was biopsied to enumerate copies of pathogen DNA per gram of tissue. While Chinook exposed to either the Hag or SC strains exhibited similar pathogen loads, those exposed to the Hag strain displayed higher mortality (～66%) than fish exposed to the SC strain (～24% mortality). Steelhead exposed to the Hag strain exhibited a greater pathogen load and higher mortality (～42%) than those exposed to the SC strain (<1% mortality). Steelhead challenged with either strain showed lower pathogen loads than Chinook. The study illustrates the efficacy of the probe set to enumerate Y. ruckeri bacterial growth in the kidneys of fish. Also, strains of Y. ruckeri display species-specific growth patterns that result in differential mortality and pathogen load.     

Efficacy of florfenicol for control of mortality associated with Francisella noatunensis subsp. orientalis in Nile tilapia,Oreochromis niloticus (L.)

Francisella noatunensis subsp. orientalis (Fno) (syn. F. asiatica) is an emergent Gram‐negative facultative intracellular bacterium. Although it is considered one of the most pathogenic bacteria in fish, there are no commercially available treatments or vaccines. The objective of this project was to determine the most efficacious concentration of florfenicol (FFC) [10, 15 or 20 mg FFC kg^?1 body weight (bw) per days for 10 days] administered in feed to control experimentally induced infections of Fno in Nile tilapia, Oreochromis niloticus (L.), reared in a recirculating aquaculture system. The cumulative mortality of fish that received 0, 10, 15 or 20 mg FFC kg^?1 bw per day was 60, 37, 14 and 16%, respectively. Francisella noatunensis subsp. orientalis genome equivalents were detected in water from all challenged groups with slight reduction in the concentration in the florfenicol‐treated groups 4 days after treatment. The mean LOG of CFU Fno mg^?1 spleen was 3–5 and was present in all challenged groups at necropsy 11 days after treatment (21 days after challenge). Results show that florfenicol administered at doses of 15 and 20 mg FFC kg^?1 bw per days for 10 days significantly reduced mortality associated with francisellosis in Nile tilapia.     

Development and validation of real-time PCR for the detection of Yersinia ruckeri

Yersiniosis (enteric red mouth disease) is a contagious bacterial disease caused by Yersinia ruckeri, which primarily affects salmonids. A real-time PCR assay using a molecular beacon has been developed and validated to improve the detection of the causative biotypes of Y. ruckeri. The assay, which targets the glnA (glutamine synthetase) gene, proved to have 100% analytical specificity and analytical sensitivities of 5 fg and 3 × 10(3) CFU g(-1) for DNA and seeded kidney tissue, respectively. The assay was highly repeatable with low % CV for intra- and inter-run experiments, and the optimized parameters transferred easily between different real-time PCR platforms. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon (n = 750) from 10 farms during 2007/08. The real-time PCR was run in parallel with the bacterial culture detection method, and all fish tested were found to be negative by both methods for Y. ruckeri, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real-time PCR system is specific, sensitive, reproducible and a rapid method for the detection of Y. ruckeri and has the potential to be used for routine diagnostic testing, health certification and active surveillance programmes.     

The disease status of Australian salmonids: bacteria and bacterial diseases

Abstract. Eleven freshwater salmonid hatcheries in southern Australia were surveyed for bacterial pathogens and diseases between 1981 and 1985, Twenty-five populations of fish were examined in the study, representing a total of 2755 fish, from which kidney, liver, spleen, and in some cases peritoneum, blood and faeces were cultured. Bacteria of pathogenic significance isolated included Aeromonas hvdrophila, Streptococcus sp., Lactobacillus piscicola, Yersinia ruckeri, Mycobacterium fortuitum, M. chelonei and a filamentous acid-fast organism of uncertain taxonomic position. Lacto-bacillus piscicola and Streptococcus sp. were associated with clinical and subclinical peritonitis. Mycobacterium chelonei was isolated from visceral granulomas in an externally normal fish. Aeromonas salmonicida, Renibacterium salmoninarum and Edwardsiella tarda were not isolated, indicating that the diseases furunculosis, bacterial kidney disease and edwardsiellosis are exotic to Australian salmonids. Similarly, while Y. ruckeri was isolated, enteric redmouth disease had not been recorded and is considered an exotic disease.     

饱食投喂频率对子二代中华鲟稚鱼生长及胃肠排空的影响

为探计不同饱食投喂频率对子二代中华鲟(Acipenser sinensis)的影响,选择子二代中华鲟(0.15±0.02)g稚鱼在水温(18.0±0.5)℃的不同饱食投喂频率(2、3、4次/d)条件下,测定其生长及胃肠排空特征,并用线性、指数和平方根模型对其胃肠排空数据进行拟合。结果显示:(1)饱食投喂频率的增加显著降低了实验鱼的摄食率(FR),但对增重率(WGR)、特定生长率(SGR)、肥满度(CF)、肝体比(HSI)、脏体比(VSI)等指标未产生显著影响;(2)随着饱食投喂频率的增加,实验鱼的体长、体质量变异系数显著降低,生长离散程度明显减小;(3)饱食投喂频率对子二代中华鲟稚鱼的胃、肠排空特征未产生影响,但对胃、肠排空最优模型的选择产生了影响;2、3和4次/d组胃排空最优模型依次为平方根模型(Y 0.5=2.253-0.077t)、平方根模型(Y 0.5=2.12-0.081t)和指数模型(Y=5.001 e-0.225t);(4)3种模型均不适于拟合实验鱼肠排空过程的全部数据,但可拟合t≥T后的肠排空数据(T为肠内容物比率达到最大值的时间);2、3和4次/d组肠排空的最优拟合模型分别为平方根模型(Y 0.5=1.904-0.056t,t≥10)、指数模型(Y=3.383 e-0.076t,t≥8)和指数模型(Y=3.090 e-0.077t,t≥6);(5)随着饱食投喂频率的增大,子二代中华鲟稚鱼的胃、肠排空速度也逐渐递增,2、3和4次/d组胃排空率依次为0.077%/h、0.081%/h和0.225%/h,其肠排空率依次为0.056%/h、0.076%/h和0.077%/h。