迟缓爱德华菌(Edwardasiella tarda)对小鼠和剑尾鱼的免疫保护试验

为了评价迟缓爱德华菌 (Edwardasiella tarda)的免疫保护效果 ,用致病性 E.tarda JEL4的全菌灭活物 (FKC)和胞外产物 (ECP)分别 2次免疫小鼠 15 0只和 1次免疫剑尾鱼 30 0尾 ,7周后用同源菌株和异源菌株进行攻击。结果显示 ,除 ECP组对某一异源菌株为 4/ 5保护外 ,免疫小鼠 FKC组和 ECP组全部获得保护 (5 / 5 )。在免疫的剑尾鱼中 ,FKC和 ECP组对同源菌株保护率分别为 6 0 %和 10 0 % ;对异源菌株的攻击 ,均不能全部保护 ,但 ECP组可明显延缓剑尾鱼死亡时间     

Occurrence of multiple antibiotic resistance and genotypic characterization in Edwardsiella tarda isolated from cage‐cultured hybrid red tilapia (Oreochromis sp.) in the Ping River,Northern Thailand

Edwardsiella tarda is a pathogen that causes edwardsiellosis in aquatic animals. The emergence of multiple antibiotic‐resistant strains makes antibiotic treatment difficult. This study aimed to investigate the antibiotic susceptibility patterns and the genotypic characterization of E. tarda isolated from cage‐cultured red tilapia in Thailand. A total of 30 isolates were identified as E. tarda using biochemical and molecular analysis. The disc diffusion method for testing antibiotic susceptibility showed all the isolates were resistant to colistin sulphate and oxolinic acid. High levels of resistance to amoxicillin, ampicillin, ceftazidime, oxytetracycline and sulphamethoxazole/trimethoprim were observed as well. The multiple antibiotic resistance index ranged from 0.25 to 0.92, indicating that these isolates had been exposed to high risk sources of contamination where antibiotics were commonly used. All the isolates carried the bla_TEM gene based on polymerase chain reaction (PCR). The tetA and sul3 genes were detected in 90% (27/30) and 26.7% (8/30) of the isolates respectively. Nine different genetic groups of isolates were obtained using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC‐PCR). A correlation between genetic types and multiple antibiotic‐resistant patterns was found. These results highlight the potential risks of multiple antibiotic‐resistant isolates for humans and the environment.     

迟缓爱德华氏菌检验程序的研究

本试验研究了迟缓德华氏菌（Edwardsiellatrada,Et）的细菌学及致病因子检测,确定了相应检测指标,制定了检验程序。对不同来源的Et作分离及生化鉴定,确定10项生化鉴定指标。用三种方法即平板法（PlateAssay,PA）、接触法（ContactHemolysis,CH）和上清法（SunernatantAs－say,SA）检测Et溶血素,阳性率依次为75％、75％、57．14％。28株Et中,18／26的胞外产物（Extra－cellularProducts,ECP）对HEP－2细胞有细胞毒性,17／28菌株有侵袭力。用ATCC15947株ECP的抗血清进行Det－ELISA,检测阳性率为19／26（73．08％）。     

Surface-exposed expression of Edwardsiella tarda EseB in live attenuated Vibrio anguillarum based on novel surface display systems

Live, attenuated Vibrio anguillarum strains can serve as vectors for the delivery of heterologous antigens for development of multivalent recombinant vaccines. Based on the outer membrane anchoring elements of V. anguillarum , we have previously constructed several efficient surface display systems Lpp-Omporf1, Lpp-OmpU, Lpp-Omp26La, Wza-Omporf1, Wza-OmpU and Wza-Omp26La. In this study, with these constructed surface display systems, a putative antigen protein EseB from pathogenic Edwardsiella tarda was successfully expressed on the surface of an attenuated V. anguillarum strain to get multivalent vaccine candidates. Further immune protection evaluation in zebra fish ( Danio rerio ) demonstrated that the V. anguillarum EseB-display strain AV/pW-26La-B could trigger full protection against V. anguillarum infection and early protection against E. tarda infection in the immunized fish. These results suggest that surface display of heterologous protective antigens in attenuated V. anguillarum could be used as a tool to develop potential V. anguillarum vector vaccine.     

用低龄动物制备高效价Et免疫血清的探讨

[目的]建立一种用低龄日本大耳白兔制备高效价迟钝爱德华氏菌（Edwardsiella tarda,Et）免疫血清的方法。[方法]先制备迟钝爱德华氏菌（Et）抗原。用日本大耳白兔作免疫动物,分2组试验,一组是2月龄组（T组）,另一组是6月龄（S组）。采取耳缘静脉注射的方法,小剂量连续多次（第1、7、14、21、27、33、41天时免疫剂量分别为O．2、0．2、0．25、0．2、0．25、0．3、0．4ml）免疫日本大耳白兔。免疫后,收集免疫血清,然后用微量凝集反应法分2次试血（第1次是免疫第33天后的第7天,采用1：20抗血清;第2次是免疫第41天时,采用1：40抗血清）,测定免疫血清抗体效价。[结果]凝集反应显示,在免疫剂量相同的情况下,第1次试血测＆免疫血清效价,2月龄组T：血清效价最高为1：640,6月龄组S1、S2和S4血清效价均达1：1280,显示6月龄组的体液免疫应答反应强于2月龄组;经继续免疫8d后,第2次试血测凸免疫血清效价可见,2月龄组的T1、T2、T3和6月龄组的S1、S2、S4免疫血清效价一样,均高达1：5120。表明此时在接受相同剂量助抗原刺激后,2月龄组和6月龄组的体液免疫应答反应已趋相同。[结论]试验中所采用的T组动物（日本大耳白兔）月龄较小,免疫后仍产生了与S组一致的高效价抗血清,说明用低龄日本大耳白兔制备高效价凰免疫血清的方法可行。     

用低龄动物制备高效价Et免疫血清的探讨

［目的］建立一种用低龄日本大耳白兔制备高效价迟钝爱德华氏菌（Edwardsiellatarda,Et）免疫血清的方法。［方法］先制备迟钝爱德华氏菌（Et）抗原。用日本大耳白兔作免疫动物,分2组试验,一组是2月龄组（T组）,另一组是6月龄（S组）。采取耳缘静脉注射的方法,小剂量连续多次免疫日本大耳白兔,免疫后,收集免疫血清,然后用微量凝集反应法测定免疫血清抗体效价。［结果］凝集反应显示,在免疫剂量相同的情况下,第1次试血测Et免疫血清效价,S组的血清效价比T组的血清效价高,但在第2次试血测Et免疫血清效价时T组的血清效价与S组的血清效价一致。［结论］试验中所采用的T组动物（日本大耳白免）月龄较小,免疫后仍产生了与S组一致的高效价抗血清,说明用低龄日本大耳白兔制备高效价Et免疫血清的方法可行。     

迟钝爱德华氏菌黏附及侵袭特性的研究

将引发养殖大菱鲆Scophthalmus maximus出血性败血症的病原菌迟钝爱德华氏菌Edwardsiella tarda（L-49231）接种于体外培养细胞—人上皮角质层形成细胞（HaCaT—cell）,观察其对细胞的黏附性和侵袭性,研究了不同作用时间下以及用不同因素处理菌体后,细菌对HaCaT细胞黏附性及侵袭性的影响。结果表明：L-49231菌株能黏附HaCaT细胞,2h后即可侵入细胞;随着作用时间的延长,细菌黏附细胞与侵入细胞的数量增加。分别用甲醛、戊二醛、盐酸、胰蛋白酶处理L-49231菌体,细菌的黏附力及侵袭力均有所下降;用蔗糖、甘露糖处理L-49231菌体时,细菌的黏附力变化不大;用葡萄糖处理时,能够促进细菌对细胞的黏附作用。用甘露糖和葡萄糖处理菌体时,能促进细菌对细胞的侵袭;用蔗糖处理菌体时,能部分抑制细菌对细胞的侵袭作用。用抗体处理L-49231菌体后,细菌的黏附力及侵袭力均明显下降。说明L-49231菌株对HaCaT细胞具明显的侵袭性,其主要黏附因子为胞外蛋白质成分,无确切的糖受体。     

地图鱼迟钝爱德华氏菌病病原菌的鉴定及毒力基因的检测

从患病地图鱼Astronotus ocellatus肝脏组织中分离到菌株AO1,经人工感染试验证实其为致病菌。通过ATB Expression半自动细菌鉴定仪和常规生理生化特性的分析,初步鉴定该菌株为迟钝爱德华氏菌Ed-wardsiella tarda。测定菌株AO1的16S rRNA基因序列并进行分析,结果显示,该菌株与迟钝爱德华氏菌的同源性最高,达99.9%。因此,可鉴定该菌株为迟钝爱德华氏菌。根据迟钝爱德华氏菌的毒力基因菌毛亚基(fimA)基因序列设计引物,进行PCR扩增,得到383 bp序列,该序列与迟钝爱德华氏菌的fimA基因序列相似性达99.0%,进一步说明菌株AO1具有fimA基因,有一定的致病力。药物敏感试验结果表明,菌株AO1对阿米卡星、氧氟沙星、庆大霉素、新生霉素、氨苄西林、氨曲南、卡那霉素、头孢咪啉、诺氟沙星、环丙沙星、呋喃妥因、妥布霉素、氨苄青霉素13种抗生素较为敏感。     

河鲈细菌性肠炎致病菌的分离、鉴定及药敏实验

河鲈(Perca fluviatilis Linnaeus)是新疆额尔齐斯河特有的土著鱼类,已开展人工养殖,但其病害相关研究较少。2016年,五家渠某水产推广站河鲈突然大批死亡,作者从发病的河鲈肝脏和肠道分离病原菌,对分离菌进行了传代纯化培养。结果显示,在20℃培养24 h后,ZL1能形成边缘整齐、中间微隆起、表面光滑、浅黄色、有特殊芳香气味、大小为1.5~2 mm的菌落,在血琼脂平板上能形成明显的β-溶血圈;ZL3和ZL5可形成圆形、微隆起、表面湿润光滑、灰白色、半透明、菌落大小为0.5~ 1 mm的菌落,但在血琼脂平板上不能形成溶血圈。经革兰氏染色、生理生化、16S rRNA序列比对和基因序列进化树分析,鉴定ZL1株为温和气单胞菌(Aeromonas sobria),ZL3和ZL5株为迟缓爱德华菌(Edwardisella tarda)。药敏实验结果显示,ZL1株对庆大霉素、米诺环素、头孢他啶等药物高度敏感,对链霉素、青霉素、克拉霉素等耐药;ZL3和ZL5株对左氧氟沙星、链霉素和恩诺沙星敏感,对庆大霉素、氨苄西林、青霉素、复方新诺明等药物耐药。人工感染结果显示,分离菌均具有明显致病性。