Identification and Drug Resistance of Pathogenic Bacteria HY3 in Acute Hepatopancreatic Necrosis Disease from Pacific White Shrimp Litopenaeus vannamei
YU Dayong1,2, CHEN Bixiu1,2, ZHONG Yongjun1,2, LIANG Xiuyan1,2, HE Xinwei1,2, XIA Haiyang1, BAO Jiawei3
1. Institute of Biopharmaceuticals, Taizhou University, Taizhou 318000, China; 2. School of Life Sciences, Taizhou University, Taizhou 318000, China; 3. Zhejiang Hongye Seafood Co., Ltd., Linhai 317000, China
Abstract：A pathogenic bacterium strain HY3 was isolated from Pacific white shrimp Litopenaeus vannamei with acute hepatopancreatic necrosis disease (AHPND) symptom collected in Taizhou fishery farm by streak plate method, and identified as Vibrio parahaemolyticus by the characteristics of colony on TCBS plate and the sequence analysis of 16S rRNA and fur gene, aiming to investigate the pathogen of AHPND of Pacific white shrimp and to propose prevention strategy. The HY3 strain had tlh (thermolabile hemolysin), pirAVP and pirBVP genes which caused shrimp AHPND. The pathogenic challenge test showed that the strain had strong pathogenicity, with cumulative mortality rate of nearly 100% for the shrimp on the seventh day after infection. Drug susceptibility test revealed that the strain was resistant to streptomycin and ampicillin. The resistance gene test indicated that related resistance genes blaTEM, strA and strB were detected in the strain by PCR and DNA sequencing. Potassium permanganate, a fishery disinfectant, showed strong bactericidal activity to the strain HY3, with bactericidal rate of 99.99% at 10 mg/L potassium permanganate for 2 h. V. parahaemolyticus was proposed as the main pathogen for the AHPND of Pacific white shrimp in Taizhou, with resistant to streptomycin and ampicillin which is not used in control AHPND in Taizhou. However, potassium permanganate is recommended to be applied as disinfectant to prevent AHPND in Taizhou.
[1]蒋葛,沈辉,万夕和,等.凡纳滨对虾急性肝胰腺坏死综合征研究进展[J].动物医学进展,2018,39(4):87-91. [2]Dong X, Wang H, Xie G, et al. An isolate of Vibrio campbellii carrying the pirVP gene causes acute hepatopancreatic necrosis disease [J]. Emerging Microbes & Infections,2017,6(1):e2. [3]Joshi J, Srisala J, Truong V H, et al. Variation in Vibrio parahaemolyticus isolates from a single Thai shrimp farm experiencing an outbreak of acute hepatopancreatic necrosis disease (AHPND)[J]. Aquaculture,2014,428/429:297-302. [4]Nunan L, Lightner D, Pantoja C, et al. Detection of acute hepatopancreatic necrosis disease (AHPND) in Mexico [J]. Diseases of Aquatic Organisms,2014,111(1):81-86. [5]Dabu I M, Lim J J, Arabit P M T, et al. The first record of acute hepatopancreatic necrosis disease in the Philippines [J]. Aquaculture Research,2017,48(3):792-799. [6]Lee C T, Chen I T, Yang Y T, et al. The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin [J]. Proceedings of the National Academy of Sciences of the United States of America,2015,112(34):10798-10803. [7]Soto-Rodriguez S A, Gomez-Gil B, Lozano-Olvera R, et al. Field and experimental evidence of Vibrio parahaemolyticus as the causative agent of acute hepatopancreatic necrosis disease of cultured shrimp (Litopenaeus vannamei) in Northwestern Mexico [J]. Applied and Environmental Microbiology,2015,81(5):1689-1699. [8]张宝存,刘飞,边慧慧,等.一株凡纳滨对虾病原菌的分离、鉴定及其致病力分析[J].渔业科学进展,2012,33(2):56-62. [9]孙明玉,冯博,张昭寰,等.引起凡纳滨对虾急性肝胰腺坏死病的副溶血弧菌MLST新序列型[J].水产学报,2018,42(3):410-418. [10]Kondo H, Van P T, Dang L T, et al. Draft genome sequence of non-Vibrio parahaemolyticus acute hepatopancreatic necrosis disease strain KC13.17.5, isolated from diseased shrimp in Vietnam [J]. Genome Announcements,2015,3(5):e00978-15. [11]Xiao J, Liu L, Ke Y, et al. Shrimp AHPND-causing plasmids encoding the PirAB toxins as mediated by pirAB-Tn903 are prevalent in various Vibrio species [J]. Scientific Reports,2017,7:42177. [12]Liu L, Xiao J, Zhang M, et al. A Vibrio owensii strain as the causative agent of AHPND in cultured shrimp, Litopenaeus vannamei [J]. Journal of Invertebrate Pathology,2018,153:156-164. [13]Theethakaew C, Nakamura S, Motooka D, et al. Plasmid dynamics in Vibrio parahaemolyticus strains related to shrimp Acute Hepatopancreatic Necrosis Syndrome (AHPNS) [J]. Infection, Genetics and Evolution,2017,51:211-218. [14]Flegel T W, Lo C F. Free release of primers for specific detection of bacterial isolates that cause acute hepatopancreatic necrosis disease (AHPND)[EB/OL].(2014-01-13)[2019-07-18]https://enaca.org/?id=88&title=primers-for-detection-of-bacterial-isolates-that-cause-ahpnd. [15]Sirikharin R, Taengchaiyaphum S, Sritunyalucksana K, et al. A new and improved PCR method for detection of AHPND bacteria [EB/OL].(2014-06-18)[2019-07-18]https://enaca.org/?id=96&title=new-pcr-detection-method-for-ahpnd. [16]Dangtip S, Sirikharin R, Sanguanrut P, et al. AP4 method for two-tube nested PCR detection of AHPND isolates of Vibrio parahaemolyticus [J]. Aquaculture Reports,2015,2:158-162. [17]贾丹,史成银,黄倢,等.凡纳滨对虾急性肝胰腺坏死病(AHPND)病原分离鉴定及其致病性分析[J].渔业科学进展,2018,39(3):103-111. [18]Machado H, Gram L. The fur gene as a new phylogenetic marker for Vibrionaceae species identification [J]. Applied and Environmental Microbiology,2015,81(8):2745-2752. [19]Machado H, Cardoso J, Giubergia S, et al. FurIOS: a web-based tool for identification of Vibrionaceae species using the fur gene [J]. Frontiers in Microbiology,2017,8:414. [20]Kim H J, Ryu J O, Lee S Y, et al. Multiplex PCR for detection of the Vibrio genus and five pathogenic Vibrio species with primer sets designed using comparative genomics [J]. BMC Microbiology,2015,15:239. [21]Kim Y B, Okuda J, Matsumoto C, et al. Identification of Vibrio parahaemolyticus strains at the species level by PCR targeted to the toxR gene [J]. Journal of Clinical Microbiology,1999,37(4):1173-1177. [22]Taniguchi H, Ohta H, Ogawa M, et al. Cloning and expression in Escherichia coli of Vibrio parahaemolyticus thermostable direct hemolysin and thermolabile hemolysin genes [J]. Journal of Bacteriology,1985,162(2):510-515. [23]Nishibuchi M, Ishibashi M, Takeda Y, et al. Detection of the thermostable direct hemolysin gene and related DNA sequences in Vibrio parahaemolyticus and other Vibrio species by the DNA colony hybridization test [J]. Infection and Immunity,1985,49(3):481-486. [24]Honda T, Iida T. The pathogenicity of Vibrio parahaemolyticus and the role of the thermostable direct haemolysin and related haemolysins [J]. Reviews in Medical Microbiology,1993,4(2):106-113. [25]Phiwsaiya K, Charoensapsri W, Taengphu S, et al. A natural Vibrio parahaemolyticus ΔpirAVp pirBVp+ mutant kills shrimp but produces neither PirVp toxins nor acute hepatopancreatic necrosis disease lesions [J]. Applied and Environmental Microbiology,2017,83(16):e00680-17. [26]Lane D J. 16S/23S rRNA sequencing [G]// Stackebrant E, Goodfellow M.Nucleic Acid Techniques in Bacterial Systematics. New York:John F. Kennedy International,1991:125-175. [27]Dallenne C, Da Costa A, Decré D, et al. Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae [J]. Journal of Antimicrobial Chemotherapy,2010,65(3):490-495. [28]Lanz R, Kuhnert P, Boerlin P. Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland [J]. Veterinary Microbiology,2003,91(1):73-84. [29]Tran L, Nunan L, Redman R M, et al. Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp [J]. Diseases of Aquatic Organisms,2013,105(1):45-55. [30]Lai H C, Ng T H, Ando M, et al. Pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in shrimp [J]. Fish & Shellfish Immunology,2015,47(2):1006-1014. [31]Dong X, Bi D, Wang H, et al. pirABvp-bearing Vibrio parahaemolyticus and Vibrio campbellii pathogens isolated from the same AHPND-affected pond possess highly similar pathogenic plasmids [J]. Frontiers in Microbiology,2017,8:1859. [32]Han J E, Mohney L L, Tang K F, et al. Plasmid mediated tetracycline resistance of Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease (AHPND) in shrimps [J]. Aquaculture Reports,2015,2:17-21. [33]甄晓然,沈辉,万夕和,等.对虾源副溶血弧菌拮抗菌的筛选与评价[J].水产科学,2019,38(6):767-773. [34]张继平,郭照良,杨权立.稳定性二氧化氯在南美白对虾疾病防治中的应用[J]. 黑龙江畜牧兽医,2004(8):64-65. [35]刘志轩,王印庚,张正,等.几种消毒剂对凡纳滨对虾致病性弧菌的杀灭作用[J]. 渔业科学进展,2018,39(3):112-119.
2020, Vol. 39 
