Administration of probiotic Bacillus licheniformis induces growth,immune and antioxidant enzyme activities,gut microbiota assembly and resistance to Vibrio parahaemolyticus in Litopenaeus vannamei

This study aimed to elucidate the effects of dietary Bacillus licheniformis ATCC 11946 (BL) on the growth, immune and antioxidant activities, intestinal morphology and microbiota, and susceptibility to Vibrio parahaemolyticus (VP_AHPND) in juvenile pacific white shrimp, Litopenaeus vannamei (LV). Juvenile LV (initial weight = 0.63 ± 0.001) were fed diets containing varying BL concentrations (0 (BL0), 10⁶ (BL1), 10⁷ (BL2) and 10⁸ (BL3) CFU/g feed) for 8 weeks. Growth performance, immune and antioxidant enzyme activities, and intestinal morphology significantly improved in the probiotic‐treated groups than the untreated. Regardless of the treatment group, the two dominant phyla were Proteobacteria and Bacteroidetes, whereas the two dominant genera were Ruegeria and Vibrio. Increasing inclusion of probiotics in diets led to significant increase in beneficial bacterial genera (Ruegeria, and Pseudoalteromonas) and a significant decrease in some known opportunistic pathogens (Vibrio, Tenacibaculum, Photobacterium, Kangiella and Spongiimonas) with the BL3 group witnessing the best. A 7‐day challenge study with VP_AHPND showed significantly high protection in the probiotic‐treated groups, with the BL3, BL2 and BL1 obtaining 51%, 34% and 22% relative percentage survival, respectively. In conclusion, BL at 10⁸ CFU/g feed should be used to help in shrimp production since it attained the most significant improvement.     

半滑舌鳎创伤弧菌分离鉴定及其荧光定量PCR方法的建立

为确定患病半滑舌鳎的病原,从其体内分离到3株革兰氏阴性菌,将其分别命名为ST-1、ST-3和ST-6,进行细菌理化特性、药敏试验、16S rDNA测序以及组织病理学观察等方面的研究,并建立了外膜蛋白(OMP)基因的SYBR Green I荧光定量PCR检测方法。结果显示,3株分离菌株的理化特性符合创伤弧菌特征,16S rDNA与创伤弧菌标准菌株ATCC29307序列相似性达99%以上,系统发育树分析显示,3株分离株与创伤弧菌自然聚为一支,最终鉴定为创伤弧菌;药敏试验显示,3株菌对头孢克肟、头孢哌酮、链霉素、亚胺培南和氟苯尼考等药物高度敏感。人工回归感染试验表明,分离株具有致病性,且半滑舌鳎、斑马鱼的临床症状与自然患病鱼症状相似;组织病理学观察显示,鳃丝上皮细胞增生,鳃小片肿胀黏连、颗粒细胞和黏液细胞增多,肝脏中央静脉淤血,肾脏出血、坏死,肠道黏膜固有层萎缩、黏液细胞增多等。进一步建立OMP基因的荧光定量PCR方法,结果显示,标准曲线的相关系数为0.995,检测下限为1.88×10~2 CFU/mL。以上结果可为半滑舌鳎弧菌病的致病机理、分子诊断和防治提供科学参考。     

水生动物源嗜芳香环弧菌的鉴定及耐药性分析

本研究从患病的半滑舌鳎分离得到5株弧菌,对其进行种属鉴定,并研究其耐药性及分子水平耐药机制。采用生化检测方法和分子生物学方法(基因16S rRNA、HSP60和mreB)进行鉴定,结果显示,5株弧菌均为嗜芳香环弧菌。通过肉汤稀释法检测菌株对抗菌药物的耐药性,通过PCR扩增和测序技术检测菌株中耐药基因和4类整合子并对其携带基因盒进行分析。结果显示,5株嗜芳香环弧菌均对环丙沙星、链霉素、红霉素、氟苯尼考、氨苄西林、磺胺甲恶唑、复方新诺明和利福平耐药,且均携带qnr VC、bla CARB-17、strA、strB和sul耐药基因。此外,菌株JS291和JS295携带Ⅰ类整合子、qacEΔ1和sul1耐药基因;可变区携带基因盒分别为dfr16-aad A2和arr-3-dfr A27。5株嗜芳香环弧菌为多重耐药菌株,且多重耐药表型与耐药基因密切相关。     

基于核酸适配体的PCR法检测溶藻弧菌及其灭活菌

溶藻弧菌(Vibrio alginolyticus)分布广,数量多,发病率高,是水产养殖中常见的条件致病菌,而对溶藻弧菌进行快速准确的识别鉴定是其病害防治的前提和基础。核酸适配体,因为具有较高的亲和特异性,在微生物的识别鉴定方面展现出了巨大的优势。本文利用核酸适配体和适配体筛选产物,通过结合、洗涤、加热分离、PCR扩增以及电泳检测等步骤,对溶藻弧菌进行了检测鉴定。结果表明,适配体和筛选产物都能对溶藻弧菌及其灭活菌进行较好的识别鉴定,适配体筛选产物对溶藻弧菌的检测下限为10~3cfu/mL,而对其灭活菌的检测下限为10~2cfu/mL,适配体对溶藻弧菌及其灭活菌的检测下限都可达到10 cfu/mL。该方法对溶藻弧菌有较好的亲和特异性,并能较好地区分溶藻弧菌与哈维氏弧菌等水产常见病原菌,在水产病害的检测中显示了较好的应用前景。     

Vibrio harveyi (VirB11) recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides)

Despite significant improvements in aquaculture to compensate wild catch, disease organisms have thrived in limiting its national and global potential. Using antibiotics, in a bid to remedy the havoc, has given rise to complications, attracting attention to disease prevention by immune enhancement against diseases. Grouper production has been inhibited for the threats of bacterial infection, particularly of Vibrio origin. Considering the rise in vibriosis cases, improved vaccines are necessary; moreover, recombinant vaccines, the choice for trial in the present experiment have been effective and more specific in improving immunity. The current work deals with grouper immune system enhancement with a recombinant vaccine developed from VirB11 gene in Vibrio harveyi. VirB11 was cloned in V. harveyi for recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides). As indicated by the results, recombinant VirB11 protein showed effectiveness in conferring protection against vibriosis with observable specific antibody response in enzyme‐linked immunosorbent assay (ELISA) analysis; a significant increase (p < 0.05) in antibody levels was observed after a week and after 8 weeks post‐vaccination. From the weeks post‐vaccination, log2 (antibody titres) in the sera of vaccinated groups reached a peak of 14.2 at week 5 in the vaccinated group in comparison with a peak of approximately 5 and 2 in adjuvant and PBS controls. As indicated by the challenge results, 90% relative survival was observed in vaccinated group and 13% relative survival in control group I (adjuvant control). The cumulative performance of protein concludes VirB11 commendable for recombinant vaccine development.     

麻醉剂丁香酚对鳗弧菌抑菌效果初步研究

水产麻醉剂丁香酚具有良好的抑菌和杀菌作用,不仅对食源性致病菌具有良好的抑制作用,也对水产致病菌具有潜在的抑菌效力。为探讨丁香酚对水产养殖业主要致病菌的抑菌作用,文章初步开展了丁香酚对鳗弧菌(Vibrio anguillarum)的抑菌效果研究。丁香酚质量浓度为6 400μg·mL^-1时,鳗弧菌培养平板中抑菌圈直径为(21.13±0.74) mm,表明鳗弧菌对此浓度的丁香酚表现为极敏。吸光度测定结果显示微孔中丁香酚质量浓度≥400μg·mL^-1时弧菌没有生长,表明丁香酚对鳗弧菌的最低抑菌浓度(MIC)为400μg·mL^-1。取400μg·mL^-1、800μg·mL^-1和1 600μg·mL^-1微孔中溶液涂抹于培养平板继续培养24 h,质量浓度≥800μg·mL^-1时鳗弧菌没有生长,表明最小杀菌浓度(MBC)为800μg·mL^-1。研究结果表明丁香酚对鳗弧菌具有良好的抑菌作用。     

一株感染深水网箱养殖许氏平鲉的病原菌分离与鉴定

2016年7月,山东省长岛县深水网箱养殖许氏平鲉(Sebastes schlegeli)暴发严重皮肤溃疡症。作者对病鱼进行病原菌分离。通过形态学观察、常规生理生化试验、gyrB和16S rDNA基因克隆测序等方法对分离菌株进行鉴定。在病灶溃疡处分离到一株绝对优势菌BZ01,该菌株在TSB固体培养基上呈半透明菌落,在TCBS选择性培养基上菌落呈绿色。透射电镜观察为短棒状,具有单根极生鞭毛。人工回接感染证明,该菌株对许氏平鲉具有较强的致病力,可以引起皮肤溃疡等症状,且与自然感染症状一致,其LD₅₀为2.07×10~6 CFU/ml。通过gyrB和16S rDNA基因序列测定并构建系统发育树显示,菌株BZ01与弧菌属同源性最高,并在系统发育树中与轮虫弧菌(Vibrio rotiferianus)聚为一枝,结合形态及生理生化表型测定结果,将该菌株鉴定为轮虫弧菌(V.rotiferianus)。药敏试验结果显示,该菌株对四环素类、喹诺酮类、香豆素类、肽酰转移酶类高度敏感,而对大环内酯类、多肽类、磺胺类、β-内酰胺类、氨基糖苷类中度敏感或不敏感。     

对虾养殖池副溶血弧菌的分离鉴定及其耐药特征、毒力基因分析

为了解对虾养殖池中副溶血弧菌(Vibrio parahaemolyticus)的耐药性和毒力基因的携带情况,2018年从山东4个地区的对虾养殖池收集分离副溶血弧菌,采用Kirby-Bauer纸片法检测其对12种抗生素的耐药性,用PCR方法检测其携带耐热直接溶血素基因(tdh)和耐热相关溶血素基因(trh)的情况。从对虾养殖池共分离副溶血弧菌50株。药敏实验结果显示,副溶血弧菌对庆大霉素、硫酸新霉素和氨苄西林的耐药情况最为严重,耐药率分别高达98%、90%和86%,对氟苯尼考、氯霉素、头孢他啶等敏感性较高,耐药率分别为10%、10%和20%。88%的菌株具有多重耐药性。毒力基因检测结果显示,所有菌株均不携带tdh基因,4%的菌株表现为trh阳性。本研究表明,对虾养殖水环境中的副溶血弧菌对抗生素的耐药性较为严重,应加强副溶血弧菌的病原学监测,在养殖过程中合理使用抗生素,以实现水产养殖业健康发展。     

一株大黄鱼致病性溶藻弧菌的耐药性及耐药基因分析

对一株分离自大黄鱼的致病性溶藻弧菌进行耐药性分析,分别用纸片扩散法、倍比稀释法检测了该菌对磺胺甲噁唑、氟苯尼考、盐酸多西环素、红霉素、盐酸环丙沙星、土霉素、恩诺沙星、硫酸新霉素、氨苄西林钠共9种药物的耐药性,同时用PCR法检测了该菌β-内酰胺酶基因bla CTX-M,喹诺酮类耐药基因(qnrVC,qnrVC5),四环素耐药基因(tetS、tetM)等耐药基因的携带情况。结果表明该菌对氨苄西林钠等药物耐药。对氟苯尼考、磺胺甲噁唑高度敏感。同时该菌携带bla CTX-M,qnrVC 2种耐药基因。本研究的结果为溶藻弧菌病的精准用药提供了数据支持。     

常用中草药对4种病原弧菌体外抗菌效果的研究

采用琼脂扩散法(打孔法)测定了五倍子等18种中草药对鳗弧菌、副溶血弧菌、河口弧菌及霍乱弧菌等4种病原弧菌的抑制作用。结果表明,五倍子、苏木、地锦草、石榴皮、黄芩、虎杖和山楂等7种中草药对病原弧菌具有较好的抑菌效果,而金银花等11种中草药抑菌作用较弱;同时采用试管连续稀释法,测定了五倍子等抑菌效果较强的7种中草药对4种病原弧菌的抑菌和杀菌浓度。结果表明,苏木、五倍子、地锦草和石榴皮4种中药具有较好的抑菌和杀菌作用,此4种中药对鳗弧菌、副溶血弧菌、河口弧菌及霍乱弧菌等4种病原弧菌的最小抑菌浓度(MIC)为0.78～6.25mg/ml,最小杀菌浓度(MBC)为3.12～12.5mg/ml。