凡纳滨对虾中发光坎氏弧菌的分离、鉴定及致病性

Isolation, identification and pathogenicity analysis of luminous Vibrio campbellii from Litopenaeus vannamei

  • 摘要: 为研究凡纳滨对虾死亡的致病机理,实验采用TCBS培养基从濒死凡纳滨对虾肝胰腺分离到浅绿色、直径3~7 mm的发荧光菌落;革兰氏染色为阴性短杆菌;经API 20E鉴定,分离株PvL-1与坎氏弧菌参考菌株CAIM249相似度为90%;fitZ序列与坎氏弧菌CP020076相似度为99.31%;gapA序列与坎氏弧菌EF596552相似度为98.89%;采用坎氏弧菌种特异性引物对PvL-1进行PCR分析,可扩增出坎氏弧菌种特异性片段,不能扩增出轮虫弧菌和哈维氏弧菌特异性片段,上述结果表明PvL-1为坎氏弧菌成员。分离菌株在10%脱纤维羊血平板呈β溶血,脱脂奶粉平板出现明显透明圈,表明存在溶血性和胞外蛋白酶活性。分析了PvL-1的急性肝胰腺坏死病、溶血素基因、菌毛基因和其他毒力基因,表明PvL-1具有AP4、TLHvcahHlyflaCmukFgloBsodBesrB等毒力基因。PvL-1可使健康凡纳滨对虾发病死亡,濒死凡纳滨对虾与自然发病虾呈现类似症状,对凡纳滨对虾半数致死浓度(LD50)2.94×104 CFU/尾;病理组织观察发现,人工感染发病凡纳滨对虾肝胰腺小管细胞脱落、崩解、血细胞浸润等,与自然发病凡纳滨对虾病理特征相同。研究表明,本次实验分离到可发光的坎氏弧菌PvL-1,对凡纳滨对虾有较强致病性,拓展了对凡纳滨对虾发光坎氏弧菌的认知。

     

    Abstract: A serious disease occurred in cultured white-leg shrimp, Litopenaeus vannamei, in greenhouse pond of mariculture farm in Taizhou, Zhejiang Province, in 2019. The diseased L. vannamei showed corkscrew swimming, anorexia, slow growth, hepatopancreas discoloured, with a mortality rate of more than 80% in 2-3 days. To study the pathogenic mechanism of PvL-1 in L. vannamei, the bacteria were isolated from morbid L. vannamei with typical symptoms by using thiosulfate citrate bile salts sucrose (TCBS) agar, with the light Green colony of 3-7 mm in diameter, luminous in dark and Gram negative rods. PvL-1 was identified as Vibrio campbellii with API20E biochemical bacteria identification, with similarity of 90% to reference strains CAIM249. PvL-1 belonged to the same cluster with V. campbellii CP020076 on phylogenetic analysis for fitZ, with 99.31% similarity to reference strains, and same cluster as V. campbellii EF596552, with 98.89% similarity to gapA. V. campbellii species-specific primers were used for PCR test, with 328 bp band for PvL-1 and negative for V. rotiferianus and V. harveyi. These results indicating that PvL-1 was a member of V. campbellii. PvL-1 can form typical β hemolyisis on sheep blood agar and transparent circle on the milk agar, indicating the existence of hemolytic and extracelllar protease toxin. Acute hepatopanergic necrosis, hemolysin gene, flagellate gene and other virulence genes of PvL-1 were analyzed, indicating that PvL-1 has virulence genes such as AP4, TLH, vcahHly, flaC, mukF, gloB, sodB and esrB. The healthy L. vannamei, infected with V. campbellii PvL-1, died with the similar symptoms to naturally morbid L. vannamei, with LD50 of 2.94×104 CFU/ind. Histopathologic examination showed that PvL-1 challenged L. vannamei hepatopancreas showed tubular epithelial cells massive shed and sloughing, hemocytic infiltration etc., which was similar to the naturally morbid L. vannamei. In summary, the luminescent V. campbellii PvL-1 was isolated, with high pathogenicity, which expanded our cognition of the luminescent V. campbellii of L. vannamei.

     

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