Model prediction of inactivation of Aeromonas salmonicida grown on poultry in situ by intense pulsed light

The aim of this study was to evaluate the factors influencing the inactivation effect of intense pulsed light(IPL)on Aeromonas salmonicida grown on chicken meat and skin,and to further develop prediction models of inactivation.In this work,chicken meat and skin inoculated with meat-borne A.salmonicida isolates were subjected to IPL treatments under different conditions.The results showed that IPL had obvious bactericidal effect in the chicken skin and thickness groups when the treatment voltage and time were 7 V combined with 5 s.In addition,the lethality curves of A.salmonicida were fitted under IPL conditions of 3.5-7.5 V.The comparison of statistical parameters revealed that the Weibull model could best fit the mortality curves and could accurately predict the mortality dynamic of A.salmonicida grown on chicken skin.And further a secondary model between the scale factor b and the treatment voltage in Weibull model was established using linear equations,which determined that the secondary model could accurately predict the inactivation of A.salmonicida.This study provides a theoretical basis for future prediction models of Aeromonas,and also provides new ideas for sterilization approaches of meat-borne Aeromonas.

鱼类致病杀鲑诺卡氏菌(Nocardia salmonicida)特异性PCR检测方法的建立

鱼类诺卡氏菌病是全球性的鱼病,杀鲑诺卡氏菌(Nocardia salmonicida)是引起鱼类诺卡氏菌病的主要病原之一。根据杀鲑诺卡氏菌16S-23S转录间隔区(ITS)序列设计引物,建立特异性PCR检测杀鲑诺卡氏菌的方法。结果表明,筛选的PCR引物可特异性地扩增出杀鲑诺卡氏菌的ITS片段,检测灵敏度(DNA浓度)为28.3 pg·μL-1。检测人工感染杀鲑诺卡氏菌的鱼组织,结果显示,该方法可从未分离到病原菌的病鱼组织中检出阳性片段,较传统细菌分离鉴定方法更为高效灵敏。

Immuno-Protective Efficiency of the Bivalent Inactivated Vaccine Against Vibrio scophthalmi and Aeromonas salmonicida Infections in Turbot(Scophthalmus maximus L.)

Vibrio scophthalmi and Aeromonas salmonicida can cause high turbot mortality and huge economic losses.Presently,vaccination is the most promising method for preventing communicable diseases.In this study,we used formalin to kill V.scophthalmi and A.salmonicida cells,and mixed with the mineralized oil adjuvant(Montanide^(TM)ISA 763 AVG)to prepare the bivalent inactivated vaccine.The results showed that turbot inoculated with the bivalent inactivated vaccine exhibited strong tolerance to the infection of V.scophthalmi and A.salmonicida,and no obvious clinical symptoms and pathological changes were observed.The activities of enzymes lysozyme,acid phosphatase and complement C3 had significantly increased after the vaccination.The antibody titer response of vaccinated turbot was greatly boosted,which was positively connected with the immunological impact according to ELISA results.Simultaneously,the expression levels of immune-related genes such as MHC-IIα,MHC-IIβ,CD4,CD8,TNF-αand IL^(-1)βwere up-regulated,demonstrating that it might stimulate humoral and cellular immunological response in turbot.These findings highlight the potential of the bivalent inactivated vaccine for controlling V.scophthalmi and A.salmonicida infections in turbot.

Concentration of Enrofloxacin Residue from Tilapia （Oreochromis niloticus） Muscular That Infected by Aeromonas salmonicida

Aim of this research was to find out the concentration of enrofloxacin residue in tilapia meat for several weeks after antibiotic treatment. Twenty seven tilapia fishes were divided into three groups. The first group was not infected and treated, the second group was infected with A. salmonicida subsp, smithia and the third group was infected with A. salmonicida subsp. achromogenes intramuscularly. Six days after infection, treatment was carried out using Baytril administered orally for the second group and intramuscularly for the third group during five days. At the 1 st, 4th and 8th week after the treatment, Three fish were taken from each group to be analyzed for its concentration of enrofloxacin residue by diffusion on Mueller Hinton Agar （MHA） method and quantitatively using high performance liquid chromatography （HPLC） method. The MHA test showed the formation of inhibition zone, at the 1 st week and 4th week after the treatment, while at 8th week after treatment did not show inhibition zone. The HPLC test on enrofloxacin residual concentration in tilapia infected with A. salmonicida subsp, smithia （second group） at the 1st, 4th and 8th week after treatment showed the average of 33.0, 6.10 and 0.0021 μg/g of enrofloxacin residue level. While in tilapia infected with A. salmonicida subsp, achromogenes and treated with enrofloxacin intramuscularly （third group） showed the average of residue level 35.79, 2.18 and 0.00065 μg/g. In conclusion, the residue of enrofloxacin was still high concentration until the fourth week after treatment in the second and third groups. Based on Indonesian National Standards and Rules, the maximum limit of enrofloxacin residue is 0.01 μg/g. The concentration of enrofloxacine residue was very low and the concentration of enrofloxacin residue collected from tilapia using orally and intramuscularly method of treatment was not different.

A recombinase polymerase amplification with lateral flow assay for rapid on-the-spot detection of Aeromonas salmonicida

Aeromonas salmonicida is a common pathogen of salmonid fishes that poses a significant threat to the fresh water and marine culture industry,potentially resulting in huge economic losses.To prevent and control fish diseases caused by A.salmonicida,rapid and effective diagnostic approaches must be developed,and which are important for routine monitoring and clinical care.By combining recombinase polymerase amplification(RPA)technology with a visible lateral flow strip(RPA-LF),we have enhanced both the precision of RPA detection and the convenience of real-time monitoring.In this study,we introduce a robust method for detecting A.salmonicida using RPA-LF.This assay specifically targets the ASA_1441 gene of A.salmonicida,ensuring high specificity,without cross-reactivity with other prevalent fresh water or marine pathogens.The optimal amplification temperature of the RPA assay was 39℃.Its sensitivity extends to as low as 100 fg of purified DNA,representing more than 1000-fold higher sensitivity than conventional PCR methods.Furthermore,to enhance the usability of the RPA-LF assay,we developed a rapid sample preparation method using cellulose dipsticks for nucleic acid extraction.This method achieves a limit of detection(LOD)as low as 1.67 CFU/μL and completes the entire process within 20 min.In conclusion,our findings present a rapid and precise tool for monitoring A.salmonicida infection in aquaculture and marine culture.This advancement offers valuable insights for effective disease prevention and control strategies.

Identification and histopathological and pathogenicity analysis of Aeromonas salmonicida salmonicida from goldfish (Carassius auratus) in North China

In this study,bacterial pathogens were isolated from the liver,spleen,kidney,heart,and muscle of diseased goldfish(Carassius auratus)after a massive outbreak of disease in an aquaculture farm in Anshan,Liaoning Province,North China(N41°05′58.42″E122°53′31.89″).Based on physiological and biochemical characterization of the bacteria and 16S rDNA sequence analysis,the bacterial isolates were identified as Aeromonas salmonicida salmonicida.Virulence gene multilocus sequence typing analysis showed that the bacterial isolates from all the organs collected were of the same type,and clustered in the same phylogenetic group as A.salmonicida salmonicida.The pathogen,was named AS.17 and was resistant to rifampicin,bacitracin,vancomycin,penicillin and compound sulfamethoxazole,but was highly sensitive to nalidixic acid,lomefloxacin,spectinomycin,ofloxacin,and ciprofloxacin.Goldfish experimentally infected with AS.17 had similar symptoms to naturally infected fish,including gill filament anemia,intestinal bleeding,intra-abdominal fluid,and jaw bleeding.Histological analyses showed that the liver,the spleen,the kidney and the intestines of fish infected with AS.17 had severe pathological alterations.In summary,this study reports the isolation of A.salmonicida salmonicida from C.auratus in North China and provides a reference strain and a research foundation for further studies on A.salmonicida disease control and epidemiology.

河川沙塘鳢病原杀鲑气单胞菌致病性及感染后宿主免疫相关基因差异表达分析

为探明2021年3月常熟某养殖场河川沙塘鳢(Odontobutis potamophilus)疾病暴发的原因,本研究从患病鱼体内分离到优势菌株,通过形态观察、理化特性测定及gyrB基因同源性分析鉴定优势菌,同时通过人工感染试验、组织病理观察、毒力因子及毒力基因检测确定其致病性,并测定分离菌的耐药性和感染后河川沙塘鳢免疫相关基因表达变化。结果显示:引起河川沙塘鳢大量死亡的病原菌为杀鲑气单胞菌(Aeromonas salmonicida)。代表菌株G2-4-1对河川沙塘鳢的LD 50为1.8×106 CFU/mL;该病原菌感染可引起河川沙塘鳢肝脏、脾脏、肾脏和鳃组织出现明显的变性、坏死和炎性细胞浸润;该菌株携带aer、hly、exu等毒力基因,且具有蛋白酶、卵磷脂酶、淀粉酶、脂酶、明胶酶和溶血素活性,但不具有DNA酶活性;耐药性分析发现,分离菌G2-4-1对青霉素G、氨苄西林、苯唑西林等8种药物耐药,对克拉霉素和大观霉素2种药物中介,对恩诺沙星、氟苯尼考等24类药物敏感;免疫相关基因表达分析显示,在感染初期MHCⅡB、MyD88、TLR和SOD在鳃、脾脏和肾脏组织中都显著上调表达。

杀鲑气单胞菌对亚东鲑血液免疫指标的影响

为预防杀鲑气单胞菌在亚东鲑养殖过程中造成的危害,以杀鲑气单胞菌为研究对象,通过感染亚东鲑,检测其对亚东鲑免疫指标的影响。结果表明:GR组亚东鲑血清中白细胞介素1β(IL-1β)含量随感染时间增加显著性上升(p<0.05),免疫球蛋白A(Ig A)含量也呈现上升趋势,而转化生长因子β1(TGF-β1)含量则表现出相反趋势,其含量显著性降低(p<0.05);GR组血常规指标中除血小板分布宽度(PDW)外,其它指标均在48h呈现显著性降低(p<0.05),除红细胞压积(HCT)外,其它指标均在96 h呈现上升趋势。杀鲑气单胞菌能够引发亚东鲑炎症反应,导致IL-1β和IgA含量升高,TGF-β1含量降低;在感染亚东鲑48 h能够破坏其免疫系统,导致白细胞、红细胞及血小板等数目显著性减少。

大西洋鲑杀鲑气单胞菌的分离鉴定

从皮肤溃烂的大西洋鲑(Salmon salar)肌肉、肝、肾分离到一致病性的菌株(AB080226),经人工感染实验证实其为该病的病原菌,研究了该菌的形态、生理生化特征并对其16SrDNA序列进行了在线Classifer和Blast分析。结果显示:该菌为革兰氏阴性杆菌,葡萄糖氧化发酵阳性,氧化酶检测阳性,0%NaCl生长;能利用甘露醇,不具运动性,能发酵麦芽糖,葡萄糖产酸阳性,精氨酸双水解酶阳性,苯丙氨酸脱氨酶阴性;该菌属于气单胞菌属的细菌,在系统发育树上与杀鲑气单胞菌形成一个簇群,同源性高达99.9%以上。综合形态学、生理生化特征及16SrDNA序列鉴定其为杀鲑气单胞菌(Aeromonas salmonicida)。

患病细鳞鱼杀鲑气单胞菌的分离与鉴定

从患病细鳞鱼(Brachymystax lenok)的病变组织处分离到1株致病菌,经过分离培养,生化鉴定,16 S rRNA序列分析和人工感染实验确定该病原菌为杀鲑气单胞菌无色亚种(Aeromonas salmonicida subsp.achromogenes)。采用20种药物进行药敏分析,结果显示:分离菌株对阿米卡星、哌拉西林、恩诺沙星等9种抗生素敏感;对环丙沙星、诺氟沙星等4种抗生素中度敏感;对卡那霉素、青霉素、氨苄西林等7种抗生素耐药。

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LD50为6.97×10^5CFU/mL。经生理生化鉴定和16SrDNA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20mg/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。

绿色荧光蛋白标记杀鲑气单胞菌的构建及其初步应用

杀鲑气单胞菌(Aeromonas salmonicida)是引起疖疮病的典型致病细菌,几乎可以侵染所有的鲑科(Aeromonas)鱼类,为水产养殖业带来巨大的经济损失。本研究通过电击转化法将原核表达载体p GFPuv成功导入杀鲑气单胞菌C4菌株(AS-C4)中,获得了绿色荧光蛋白标记的杀鲑气单胞菌菌株AS-C4^(GFP)。AS-C4^(GFP)菌株在氨苄青霉素抗性平板上生长良好,并在紫外光下激发出较强的绿色荧光。通过荧光显微镜观察菌体和PCR检测gfp和杀鲑气单胞菌vap A基因,表明gfp基因已经成功导入AS-C4中。用AS-C4^(GFP)人工感染大西洋鲑(Salmo salar),从死亡鱼的脾脏、肝脏、肾脏中均能分离出AS-C4^(GFP),AS-C4^(GFP)在脾脏中最多。AS-C4^(GFP)具有特异性和可视性,为研究杀鲑气单胞菌对大西洋鲑的侵染途径提供了一种良好的生物材料。

虹鳟杀鲑气单胞菌耐药性研究与盐酸多西环素给药方案制定

为科学制定虹鳟(Oncorhynchus mykiss)杀鲑气单胞菌(Aeromonas salmonicida)病的给药方案,对3株分离自虹鳟的杀鲑气单胞菌采用纸片扩散法(K-B法)进行24种抗生素耐药性测定,并对其中4种敏感且批准使用的抗生素进行体外药效学实验,研究20、30、40和60 mg/kg盐酸多西环素在染病虹鳟体内的药代动力学特征。结果显示:3株杀鲑气单胞菌对复方新诺明、磺胺异恶唑、青霉素、阿莫西林、头孢唑林耐药,对盐酸多西环素最敏感,其最小抑菌浓度(MIC)为0.5μg/mL,最小杀菌浓度(MBC)在2~4μg/mL之间,防耐药突变浓度(MPC)为1μg/mL;恩诺沙星的MIC在0.25~1μg/mL之间,MBC为4μg/mL,MPC为1.5μg/mL;氟苯尼考的MIC为1μg/mL,MBC为8μg/mL,MPC为2μg/mL;硫酸新霉素MIC为4μg/mL,MBC为8μg/mL,MPC为12μg/mL。40 mg/kg剂量单次给药,盐酸多西环素在患病虹鳟血浆中的峰浓度为1.19μg/mL,达峰时间为4 h,且血药浓度维持在MIC以上16 h,可以满足虹鳟杀鲑气单胞菌病治疗需要。60 mg/kg剂量单次给药,盐酸多西环素在患病虹鳟血浆、肌肉、肾脏和肝脏中药物浓度维持在MIC以上的时间分别为23、18、23.5和23.5 h,在患病虹鳟血浆、肾脏和肝脏中药物浓度维持在MPC之上的时间分别为6、23.5和23.5 h,此使用剂量既能达到虹鳟杀鲑气单胞菌治疗作用,又能有效防止产生耐药性。

斑点叉尾■溃烂症的病原鉴定和致病特性

为探明斑点叉尾[鱼回](Ictalunes punctatus)溃烂症的病因,从4尾患鱼肝脾中分离纯化出4株优势菌株,并进行病原鉴定、毒力基因检测、动物回归感染和药敏试验。4株优势菌经鉴定并命名为杀鲑气单胞菌无色亚种(Aeromonas salmonicida subsp achromogenes)X-G1,杀鲑气单胞菌杀鲑亚种(A.s subsp salmonicida)X-P2、X-P3和嗜水气单胞菌(A.hydrophila)X-P4。15℃时,杀鲑气单胞菌X-G1、X-P2和X-P3的世代时间(约14 min)均小于嗜水气单胞菌X-P4(约20 min);25℃时,杀鲑气单胞菌X-G1、X-P2和X-P3株的世代时间(约20 min)均大于嗜水气单胞菌X-P4株(约16 min)。X-G1株可检到弹性蛋白酶、溶血素和甘油磷脂胆固醇酰基转移酶等3种毒力基因;X-P2株仅可检到弹性蛋白酶1种毒力基因;X-P3株可检测到弹性蛋白酶、溶血素、细胞毒性肠毒素、丝氨酸蛋白酶、酯酶、气溶素和甘油磷脂胆固醇酰基转移酶等7种毒力基因;X-P4株可检测到鞭毛、弹性蛋白酶、气溶素、细胞毒性肠毒素、热不稳定性肠毒素、丝氨酸蛋白酶和溶血素等7种毒力基因。分离株X-G1、X-P2、X-P3和X-P4在15~17℃水温下腹腔注射攻毒的半数致死浓度(LD 50)依次为0.49×10^4、0.78×10^4、0.53×10^4、3.84×10^4 CFU/g;而在23~26℃水温下测得的LD 50依次为1.48×10^4、1.80×10^4、0.82×10^4、0.68×10^4 CFU/g。分离株混合感染比单一株感染均表现出更强的致死能力。分离菌株对多西环素、恩诺沙星、氟苯尼考均敏感,但因患病鱼不能摄食药饵而导致治疗失败。

太湖激浪鱼内脏中藻毒素降解菌的筛选及其特性研究

针对蓝藻分泌的微囊藻毒素引起的水污染问题,从太湖激浪鱼内脏中筛选出1株能够降解藻毒素的菌株,命名为JZ-4。采用实验室分离纯化的藻毒素作为惟一碳源、氮源,考察了菌株降解MC-LR的特性及其动力学模型;并通过生理生化、16S r DNA基因序列分析及其系统发育树分析对菌种进行鉴定。降解试验表明,菌株JZ-4能够在分离提纯的藻毒素混合液中生长,并且具有较强的降解藻毒素的能力,7 d内能把初始浓度为13.98μg/L的MC-LR降解到1.43μg/L,降解率为89.77%;菌株JZ-4对MC-LR降解符合一级反应动力学模型,其方程式为Se=S0exp(-0.301 6t)。经16S r DNA基因序列及其系统发育分析表明,该菌与杀鲑气单胞菌(Aeromonas salmonicida)的相似性最高,达98%。

养殖刺参溃疡病杀鲑气单胞菌的分离、致病性及胞外产物特性分析

从患溃疡病的养殖刺参(Apostichopus japonicus)病灶处分离出1株优势菌H1,以浸浴、创伤浸浴、体腔注射和体壁肌肉注射等方式进行感染实验,证实菌株H1为养殖刺参溃疡病病原菌,并证明该菌通过体表创伤侵入的方式感染刺参,以创伤浸浴和体壁肌肉注射感染的LD50(半数致死量)分别为2.26×107CFU/尾和1.80×107CFU/尾。经形态学观察、生理生化特性分析和mini API系统鉴定,确定菌株H1为杀鲑气单胞菌杀日本鲑亚种(Aeromonas salmonicida ma-soucida)。提取菌株H1的胞外产物(ECP)进行致病性实验,结果表明ECP可导致刺参死亡,其对刺参的LD50为5.24μg蛋白/g体质量。H1-ECP具有酪蛋白酶、明胶蛋白酶、几丁质酶和淀粉酶活性,并具有溶血素活性;对底物偶氮酪蛋白(Azocasin)作用的酶比活力可达到674.5活力单位/mg蛋白,最适作用温度为50℃;对热不稳定,70℃作用30 min时,酪蛋白酶活性降到0;100℃作用30 min,ECP对刺参的毒性消失;ECP酶活可被10 mmol/L EDTA完全抑制,可被5 mmol/L PMSF抑制98.8%,Ca2+和Mg2+可使酶活性分别提高约9%和4%。结论认为,该病原菌通过体表创伤侵入方式感染宿主刺参,菌株H1胞外产物是其对刺参致病的因子之一。

大西洋鲑杀鲑气单胞菌无色亚种的分离鉴定和致病性研究

杀鲑气单胞菌为气单胞菌属(Aeromonas)革兰阴性短杆菌,可导致鲑鳟鱼类发生疖疮病或溃疡病。患病鱼的典型症状表现为体侧或尾部形成特征性脓肿,严重时脓肿部位溃烂形成溃疡,剖检可见肝脏、脂肪组织出现点状出血[1]。已有研究表明,杀鲑气单胞菌包括多个亚种如无色亚种(Achromogenes)、杀鲑亚种(Salmonicida)、杀日本鲑亚种(Masoucida)、史氏亚种(Smith)和溶果胶亚种(Pectinolytica)等[2],

养殖大菱鲆病原菌——杀鲑气单胞菌无色亚种的分离鉴定和组织病理学研究

自然发病的养殖大菱鲆幼苗出现口部周围皮下出血,肝脏灰白色并萎缩坏死的症状。从肝脏中分离纯化得到优势菌株,定名为db14。该菌株在LB培养基上生长较缓慢,早期菌落稍隆起,白色,质地较硬;人工感染试验证实对大菱鲆有较强的致病性,其半数致死剂量LD50为2.75×103cfu/g;生理生化特征测定显示其与杀鲑气单胞菌无色亚种(Aeromonas salmonicidasubsp.achromogenes)相似度最高;细菌16S rDNA序列测定后在GenBank中进行序列比对分析,结果与杀鲑气单胞菌无色亚种的序列同源性最高,为100%。综合上述结果,确定该菌株为杀鲑气单胞菌无色亚种。该菌株对复达欣、菌必治、丁胺卡那霉素和氯霉素等较为敏感。通过对人工感染菌株db14的大菱鲆组织切片观察,发现病鱼的肝脏、肾脏、脾脏、肠道等器官的组织均有明显的病理变化,而心脏和脑未发现明显异常。

温度及pH对杀鲑气单胞菌生长的影响研究

利用 A 值法与活菌平板计数法,测定了杀鲑气单胞菌在不同条件下的生长曲线,研究了不同温度及pH对杀鲑气单胞菌（Aeromonas salmonicha）生长的影响。结果表明,活菌计数法更能真实的反映细菌数量的动态变化。在28℃,15℃,10℃,5℃条件下,随着温度降低,杀鲑气单胞菌生长速率明显下降；在pH6.0-8.0时,酸性条件能明显抑制杀鲑气单胞菌的生长；在28℃、pH7.5条件下,杀鲑气单胞菌生长速率最快,达到稳定期时细菌数量也最多。在养殖生产过程中,为了抑制杀鲑气单胞菌的生长繁殖,建议在不影响鱼类正常生长的条件下,尽量降低养殖水体温度并合理控制水体pH,以降低养殖鱼类发病率。

12种中草药提取物对杀鲑气单胞菌的抑制效果

为观察中草药对杀鲑气单胞菌(Aeromonas salmonicida)的体外抑菌效果,采用水煎煮法提取黄柏、黄芩等12种中草药活性成分,首先采用纸片法判断中草药有无抑菌作用,然后使用试管二倍稀释法测定具有抑菌效果的2种中药的最小抑菌浓度。结果表明,黄芩和连翘抑菌效果较好,黄芩的最小抑菌浓度为10 mg/mL,连翘的最小抑菌浓度为20 mg/mL,其余10种中草药抑制杀鲑气单胞菌效果不佳。