副溶血弧菌攻毒过程中文蛤肝胰腺弧菌载量变化的分析
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摘要
文蛤(Meretrixpetechialis)是我国一种重要的海水养殖贝类,其养殖过程中病害导致的规模性死亡时有发生,已有的工作证实致病性弧菌是导致文蛤大规模死亡最为常见的病原。在本研究中,我们通过浸泡感染实验模拟了文蛤在自然条件下的弧菌胁迫环境,明确了攻毒水体中弧菌生长变化规律;攻毒过程中宿主载菌量分析显示,文蛤肝胰腺组织弧菌载量在攻毒后呈第1天急剧上升,第3天迅速下降的变化趋势,而且不同个体间载菌量差异较大;通过不同弧菌含量攻毒实验,发现在攻毒早期文蛤体内弧菌载量与水体环境的弧菌含量之间呈显著的正相关(Spearman’sρ=0.899,P=0.000),而在感染中后期不同攻毒强度组之间无显著差异,且呈现较低水平,为0～205CFU/mg,预示着宿主免疫系统和肝胰腺微生物群落可将致病菌的数量控制在一定的范围。上述研究为开展文蛤感染发病过程中弧菌载量和免疫抗性评价相关研究提供了参考。
The clam Meretrix petechialis is an important mollusk cultured in China. Massive deaths frequently occur in the major clam producing areas. Previous research has indicated that several diseases are caused by the genus Vibrio in mollusks, which is also the primary pathogenic bacteria causing deaths in M. petechialis. In the present study, we analyzed the bacterial growth and reproduction in seawater under Vibrio challenge for a period of 24 h. Vibrio immersion experiments were conducted to simulate Vibrio challenge in the natural environment, and we observed that bacterial load in the hepatopancreas of the clam abruptly increased on day 1 and decreased rapidly on day 3 after the challenge, with significant differences in the bacterial load among different clam individuals. In the challenge experiments conducted using different Vibrio doses, a significant positive correlation was observed between the bacterial load and the Vibrio dose in the seawater(Spearman's ρ = 0.899, P = 0.000) in the initial phase; however, there were no significant differences in the bacterial load among the different groups in the middle–late phases with a lower Vibrio dose of 0–205 CFU/mg. These findings indicate that the host immune system and the microbial community in the host hepatopancreas can regulate the bacterial number to a certain extent. Our results provide a reference for the correlational research of Vibrio load during infection as well as for the evaluation of resistance in the clam M. petechialis.
The clam Meretrix petechialis is an important mollusk cultured in China. Massive deaths frequently occur in the major clam producing areas. Previous research has indicated that several diseases are caused by the genus Vibrio in mollusks, which is also the primary pathogenic bacteria causing deaths in M. petechialis. In the present study, we analyzed the bacterial growth and reproduction in seawater under Vibrio challenge for a period of 24 h. Vibrio immersion experiments were conducted to simulate Vibrio challenge in the natural environment, and we observed that bacterial load in the hepatopancreas of the clam abruptly increased on day 1 and decreased rapidly on day 3 after the challenge, with significant differences in the bacterial load among different clam individuals. In the challenge experiments conducted using different Vibrio doses, a significant positive correlation was observed between the bacterial load and the Vibrio dose in the seawater(Spearman's ρ = 0.899, P = 0.000) in the initial phase; however, there were no significant differences in the bacterial load among the different groups in the middle–late phases with a lower Vibrio dose of 0–205 CFU/mg. These findings indicate that the host immune system and the microbial community in the host hepatopancreas can regulate the bacterial number to a certain extent. Our results provide a reference for the correlational research of Vibrio load during infection as well as for the evaluation of resistance in the clam M. petechialis.
引文
[1]刘连生,闫茂仓,林志华,等.引起文蛤暴发性死亡病原菌的分离和鉴定[J].微生物学通报, 2009, 36(1):71-77.Liu Liansheng, Yan Maocang, Lin Zhihua, et al. Pathogens isolation and identification of eruptive epidemic disease in Meretrix meretrix[J]. Acta microbiologica Sinica, 2009, 36(1):71-77.
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[4]李国,闫茂仓,常维山,等.我国海水养殖贝类弧菌病研究进展[J].浙江海洋学院学报(自然科学版),2008, 27(3):327-334.Li Guo, Yan Maocang, Chang Weishan, et al. Review on studing of Vibriosis of shellfish farming in China[J].Journal of Zhejiang Ocean University(Natural Science),2008, 27(3):327-334.
[5] Howick V, Lazzaro B. The genetic architecture of defense as resistance to and tolerance of bacterial infection in Drosophila melanogaster[J]. Molecular Ecology,2017, 26(6):1533.
[6] Boots M, Bowers R G. Three mechanisms of host resistance to microparasites-avoidance, recovery and tolerance-show different evolutionary dynamics[J]. Journal of Theoretical Biology, 1999, 201(1):13-23.
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[9] Yue X, Liu B, Xiang J, et al. Identification and characterization of the pathogenic effect of a Vibrio parahaemolyticus-related bacterium isolated from clam Meretrix meretrix with mass mortality[J]. Journal of Invertebrate Pathology, 2010, 103(2):0-115.
[10]王段香,余志坚,朱学春,等.鱼害粘球菌对兴国红鲤和彭泽鲫致病性[J].南昌大学学报(理科版), 2003,27(3):288-291.Wang Duanxiang, Yu Jianzhi, Zhu Xuechun, et al. The pathogenic ability of Mayxococcus pisicola to xingguo red carp(Cyprinus carpio var. singuonensis)and pengzecrucian carp(Carassius auratus var. pengzenensis)[J].Journal of Nanchang University(Natural Science), 2003,27(3):288-291.
[11] Elston R, Leibovitz L. Pathogenesis of experimental Vibriosis in larval American oysters, Crassostrea virginica[J]. Canadian Journal of Fisheries and Aquatic Sciences, 1980, 37(6):964-978.
[12] Tubiash H S, Colwell R R, Sakazaki R. Marine Vibrios associated with bacillary necrosis a disease of larval and juvenile bivalve mollusks[J]. Journal of Bacteriology,1970, 103(1):271-272.
[13] Brown C, Losee E. Observations on natural and induced epizootics of vibriosis in Crassostrea virginica larvae[J].Journal of Invertebrate Pathology, 1978, 31(1):41-47.
[14]丁丹,潘宝平,王玉梅,等.青蛤(Cyclina sinensis)AP-1基因的克隆及在鳗弧菌(Vibrio anguillarum)侵染下的表达分析[J].海洋与湖沼, 2018, 49(1).Ding Dan, Pan Baoping, Wang Yumei, et al, Cloning and expression of Ap-1 gene from Cyclina sinensis infection by Vibrio anguillarum[J]. Oceanologia et Limnologia Sinica, 2018, 49(1).
[15] Taylor S W, Tincu J A. Antimicrobial peptides from marine invertebrates[J]. Antimicrobial Agents&Chemotherapy, 2004, 48(10):3645.
[16] Mitta G, Hubert F, Noel T, et al. Myticin, a novel cystein-rich antimicrobial peptide isolated from haemocytes and plasma of the mussel Mytilus galloprovincialis[J]. European Journal of Biochemistry, 2010,265(1):71-78.
[17] Wang X W, Wang J X. Crustacean hemolymph microbiota:Endemic, tightly controlled, and utilization expectable[J]. Molecular Immunology, 2015, 68(2):404-411.
[18] Rungrassamee W, Klanchui A, Maibunkaew S, et al.Bacterial dynamics in intestines of the black tiger shrimp and the Pacific white shrimp during Vibrio harveyi exposure[J]. Journal of Invertebrate Pathology, 2016,133:12-19.
[19] Phuoc L H, Corteel M, Thanh N C, et al. Effect of dose and challenge routes of Vibrio spp. on co-infection with white spot syndrome virus in Penaeus vannamei[J].Aquaculture, 2009, 290(1-2):0-68.
[20] Kim B M, Jeong C B, Rhee J S, et al. Transcriptional profiles of Rel/NF-κB, inhibitor of NF-κB(IκB), and lipopolysaccharide-induced TNF-αfactor(LITAF)in the lipopolysaccharide(LPS)and two Vibrio sp.-exposed intertidal copepod, Tigriopus japonicus[J]. Developmental&Comparative Immunology, 2014, 42(2):229-239.
[21] Yu B P. Cellular defenses against damage from reactive oxygen species[J]. Physiological Reviews, 1994, 74(1):139-162.
[2]张彬,黄婷,熊建华等.文蛤主要弧菌性病害研究进展[J].广东农业科学, 2012, 39(17):128-130.Zhang Bin, Huang Ting, Xiong Jianhua et al. Research progress of vibriosis in hard calm, Meretrix meretrix Linnaeus[J]. Guangdong Agricultural Sciences, 2012,39(17):128-130.
[3]王凤青,孙玉增,任利华,等.海水养殖中水产动物主要致病弧菌研究进展[J].中国渔业质量与标准,2018, 8(2):49-56.Wang Fengqing, Sun Yuzeng, Ren Lihua, et al. Research progress on the main pathogenic Vibrio affecting aquatic animals in mariculture[J]. China Fishery Quality And Standards, 2018, 8(2):49-56.
[4]李国,闫茂仓,常维山,等.我国海水养殖贝类弧菌病研究进展[J].浙江海洋学院学报(自然科学版),2008, 27(3):327-334.Li Guo, Yan Maocang, Chang Weishan, et al. Review on studing of Vibriosis of shellfish farming in China[J].Journal of Zhejiang Ocean University(Natural Science),2008, 27(3):327-334.
[5] Howick V, Lazzaro B. The genetic architecture of defense as resistance to and tolerance of bacterial infection in Drosophila melanogaster[J]. Molecular Ecology,2017, 26(6):1533.
[6] Boots M, Bowers R G. Three mechanisms of host resistance to microparasites-avoidance, recovery and tolerance-show different evolutionary dynamics[J]. Journal of Theoretical Biology, 1999, 201(1):13-23.
[7] Roy B A, Kirchner J W. Evolutionary dynamics of pathogen resistance and tolerance[J]. Evolution, 2000, 54(1):51-63.
[8] Malo D, Skamene E. Genetic control of host resistance to infection[J]. Trends in Genetics, 1994, 10(10):365-371.
[9] Yue X, Liu B, Xiang J, et al. Identification and characterization of the pathogenic effect of a Vibrio parahaemolyticus-related bacterium isolated from clam Meretrix meretrix with mass mortality[J]. Journal of Invertebrate Pathology, 2010, 103(2):0-115.
[10]王段香,余志坚,朱学春,等.鱼害粘球菌对兴国红鲤和彭泽鲫致病性[J].南昌大学学报(理科版), 2003,27(3):288-291.Wang Duanxiang, Yu Jianzhi, Zhu Xuechun, et al. The pathogenic ability of Mayxococcus pisicola to xingguo red carp(Cyprinus carpio var. singuonensis)and pengzecrucian carp(Carassius auratus var. pengzenensis)[J].Journal of Nanchang University(Natural Science), 2003,27(3):288-291.
[11] Elston R, Leibovitz L. Pathogenesis of experimental Vibriosis in larval American oysters, Crassostrea virginica[J]. Canadian Journal of Fisheries and Aquatic Sciences, 1980, 37(6):964-978.
[12] Tubiash H S, Colwell R R, Sakazaki R. Marine Vibrios associated with bacillary necrosis a disease of larval and juvenile bivalve mollusks[J]. Journal of Bacteriology,1970, 103(1):271-272.
[13] Brown C, Losee E. Observations on natural and induced epizootics of vibriosis in Crassostrea virginica larvae[J].Journal of Invertebrate Pathology, 1978, 31(1):41-47.
[14]丁丹,潘宝平,王玉梅,等.青蛤(Cyclina sinensis)AP-1基因的克隆及在鳗弧菌(Vibrio anguillarum)侵染下的表达分析[J].海洋与湖沼, 2018, 49(1).Ding Dan, Pan Baoping, Wang Yumei, et al, Cloning and expression of Ap-1 gene from Cyclina sinensis infection by Vibrio anguillarum[J]. Oceanologia et Limnologia Sinica, 2018, 49(1).
[15] Taylor S W, Tincu J A. Antimicrobial peptides from marine invertebrates[J]. Antimicrobial Agents&Chemotherapy, 2004, 48(10):3645.
[16] Mitta G, Hubert F, Noel T, et al. Myticin, a novel cystein-rich antimicrobial peptide isolated from haemocytes and plasma of the mussel Mytilus galloprovincialis[J]. European Journal of Biochemistry, 2010,265(1):71-78.
[17] Wang X W, Wang J X. Crustacean hemolymph microbiota:Endemic, tightly controlled, and utilization expectable[J]. Molecular Immunology, 2015, 68(2):404-411.
[18] Rungrassamee W, Klanchui A, Maibunkaew S, et al.Bacterial dynamics in intestines of the black tiger shrimp and the Pacific white shrimp during Vibrio harveyi exposure[J]. Journal of Invertebrate Pathology, 2016,133:12-19.
[19] Phuoc L H, Corteel M, Thanh N C, et al. Effect of dose and challenge routes of Vibrio spp. on co-infection with white spot syndrome virus in Penaeus vannamei[J].Aquaculture, 2009, 290(1-2):0-68.
[20] Kim B M, Jeong C B, Rhee J S, et al. Transcriptional profiles of Rel/NF-κB, inhibitor of NF-κB(IκB), and lipopolysaccharide-induced TNF-αfactor(LITAF)in the lipopolysaccharide(LPS)and two Vibrio sp.-exposed intertidal copepod, Tigriopus japonicus[J]. Developmental&Comparative Immunology, 2014, 42(2):229-239.
[21] Yu B P. Cellular defenses against damage from reactive oxygen species[J]. Physiological Reviews, 1994, 74(1):139-162.
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