刺激隐核虫对大黄鱼致病力及中草药抑制其世代繁殖的研究
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摘要
由刺激隐核虫寄生感染而引起的"白点病"成为大黄鱼养殖生产中危害最为严重的病害之一。为研究刺激隐核虫对大黄鱼的致病力及复方中草药HD-2对刺激隐核虫世代繁殖的抑制和阻断其感染的效果,本研究在测定了患病大黄鱼产生刺激隐核虫包囊数量、大小、孵化率和幼虫产率的基础上,测定了幼虫体外存活时间和感染力。同时,研究了HD-2中草药液对刺激隐核虫幼虫、滋养体和包囊的杀除效果以及口服复方中草药HD-2对患病大黄鱼的治疗效果。结果显示,自然患病大黄鱼体型规格与产生包囊总数呈正相关,包囊直径为(340.8±64.9)μm,平均孵化率为76.4%,每个包囊可产生280±42.5个幼虫;幼虫体外最长存活时间超过24 h,幼虫孵化20 h后便丧失感染力。幼虫感染大黄鱼7 d的LD_(90)为9 712个幼虫/尾,LD_(50)为4 366个幼虫/尾。HD-2中草药液可有效杀灭刺激隐核虫幼虫,分别口服含复方中草药5、10和15 g/kg饲料30 d的实验组大黄鱼在LD50攻毒剂量下其相对感染强度较对照组分别下降32.0%、44.5%和51.8%,包囊体积较对照组分别减少35.4%、36.1%和37.3%,孵化率分别下降16.3%、23.3%和27.9%;在LD90攻毒剂量下3个实验组大黄鱼的存活率分别达到40.0%、55.0%和58.3%,显著高于对照组(8.3%),且鳃丝、胸鳍上滋养体数量较对照组显著下降。口服复方中草药HD-2对自然患病大黄鱼的治疗效果表明,采集口服15 d的3组大黄鱼的包囊数量分别较对照组下降73.1%、87.7%和93.8%,鱼体死亡率分别为58.3%、36.7%和21.7%,较对照组83.3%的死亡率明显降低,且用药组大黄鱼的生理状态明显优于对照组。由此可见,口服复方中草药HD-2能有效阻断刺激隐核虫的二次感染,抑制包囊的形成和发育,降低包囊孵化率和幼虫孵化数量,从而有效抑制了虫体的世代繁殖。
The "white spot disease" caused by the parasitic infection of Cryptocaryon irritans has become one of the most serious diseases in the production of Larimichthgs crocea. This study aims to investigate the pathogenicity of C. irritans to L. crocea, as well as the effect of Chinese herbal compound HD-2 on inhibiting the generation reproduction of parasite C. irritans. The number, size, the hatching rate and number of hatched theronts of tomonts produced by natural sick L. crocea were investigated. Then the survival time and infectivity of theront was determined in vitro. The effects of Chinese herbal compound HD-2 on theront, trophont, and tomont, and the therapeutic effects of oral HD-2 on diseased L. crocea were studied. The results showed that the total number of tomonts produced by L. crocea was positively correlated to the fish body weight; the average diameter of tomonts were(340.8±64.9) μm and the hatching rate was 76.4%, and each tomont could produce(280±42.5) theronts; the theront could survive for more than 24 h in vitro and lost their infectivity in 20 h after hatching. The 90% lethal dose(LD90) of L. crocea infected with C. irritans in 7 days was 9 712 theronts/fish and the median lethal dose for50%(LD50) was 4 366 theronts/fish. HD-2 could kill theront effectively. After oral administration with different doses of HD-2(control, 0; G1, 5 g/kg; G2, 10 g/kg; G3, 15 g/kg) for 30 days, the relative infection intensity of the L. crocea groups challenged with LD50 decreased by 32.0%, 44.5% and 51.8%, respectively. Meanwhile, the volume of tomonts reduced by 35.4%, 36.1% and 37.3% and the hatching rate reduced by 16.3%, 23.3% and27.9%, respectively. The fish survival rate of G1, G2 and G3 challenged with LD90 was 40.0%, 55.0% and 58.3%,which was significantly higher than the control group(only 8.3%). Using HD-2 oral treatment for 15 d, the tomonts produced from diseased fish reduced 73.1%, 87.7% and 93.8%, and the fish mortality rate of the three groups were 58.3%, 36.7% and 21.7%, which were significantly lower than the control group(83.3%). The physiological status of L. crocea in the treated group was significantly better than that of the control group.Therefore, HD-2 oral treatment could effectively interrupt the secondary infection of C. irritans, suppress the formation and development of tomonts, reduce the tomont hatching rate and the number of hatching theronts, and then inhibit the generation reproduction of parasite C. irritans.
The "white spot disease" caused by the parasitic infection of Cryptocaryon irritans has become one of the most serious diseases in the production of Larimichthgs crocea. This study aims to investigate the pathogenicity of C. irritans to L. crocea, as well as the effect of Chinese herbal compound HD-2 on inhibiting the generation reproduction of parasite C. irritans. The number, size, the hatching rate and number of hatched theronts of tomonts produced by natural sick L. crocea were investigated. Then the survival time and infectivity of theront was determined in vitro. The effects of Chinese herbal compound HD-2 on theront, trophont, and tomont, and the therapeutic effects of oral HD-2 on diseased L. crocea were studied. The results showed that the total number of tomonts produced by L. crocea was positively correlated to the fish body weight; the average diameter of tomonts were(340.8±64.9) μm and the hatching rate was 76.4%, and each tomont could produce(280±42.5) theronts; the theront could survive for more than 24 h in vitro and lost their infectivity in 20 h after hatching. The 90% lethal dose(LD90) of L. crocea infected with C. irritans in 7 days was 9 712 theronts/fish and the median lethal dose for50%(LD50) was 4 366 theronts/fish. HD-2 could kill theront effectively. After oral administration with different doses of HD-2(control, 0; G1, 5 g/kg; G2, 10 g/kg; G3, 15 g/kg) for 30 days, the relative infection intensity of the L. crocea groups challenged with LD50 decreased by 32.0%, 44.5% and 51.8%, respectively. Meanwhile, the volume of tomonts reduced by 35.4%, 36.1% and 37.3% and the hatching rate reduced by 16.3%, 23.3% and27.9%, respectively. The fish survival rate of G1, G2 and G3 challenged with LD90 was 40.0%, 55.0% and 58.3%,which was significantly higher than the control group(only 8.3%). Using HD-2 oral treatment for 15 d, the tomonts produced from diseased fish reduced 73.1%, 87.7% and 93.8%, and the fish mortality rate of the three groups were 58.3%, 36.7% and 21.7%, which were significantly lower than the control group(83.3%). The physiological status of L. crocea in the treated group was significantly better than that of the control group.Therefore, HD-2 oral treatment could effectively interrupt the secondary infection of C. irritans, suppress the formation and development of tomonts, reduce the tomont hatching rate and the number of hatching theronts, and then inhibit the generation reproduction of parasite C. irritans.
引文
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[5]崔晓翠,王印庚,陈霞,等.槟榔、川楝子复方中草药对大黄鱼4种酶活性的影响及对刺激隐核虫的杀灭效果分析[J].中国水产科学,2017, 24(2):362-373.Cui X C, Wang Y G, Chen X, et al. Effects of a Chinese herbal compound on non-specific immune activities and extermination of parasite Cryptocaryon irritans in Larimichthys crocea[J]. Journal of Fishery Sciences of China,2017, 24(2):362-373(in Chinese).
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[9]Pironet F N, Jones J B. Treatments for ectoparasites and diseases in captive Western Australian dhufish[J]. Aquaculture International, 2000, 8(4):349-361.
[10] Yoshinaga T, Im H J, Nishida S, et al. In vitro and in vivo efficacies of ionophores against Cryptocaryon irritans[J]. Aquaculture, 2011, 321(3-4):167-172.
[11] Hirazawa N, Goto T, Shirasu K. Killing effect of various treatments on the monogenean Heterobothrium okamotoi eggs and oncomiracidia and the ciliate Cryptocaryon irritans cysts and theronts[J]. Aquaculture, 2003, 223(1-4):1-13.
[12]郑晓叶,刘顺,周旻曦,等.几种理化因子对刺激隐核虫包囊及幼虫活力的影响[J].宁波大学学报(理工版),2015,28(1):15-20.Zheng X Y, Liu S, Zhou M X, et al. Influence of environmental factors and chemotherapeutants on Cryptocaryon irritans tomont and theront[J]. Journal of Ningbo University(NSEE), 2015, 28(1):15-20(in Chinese).
[13] Kawano F, Hirazawa N, Gravningen K, et al. Antiparasitic effect of dietary Romet.30(SDMX-OMP)against ciliate Cryptocaryon irritans infection in the red sea bream Pagrus major and tiger puffer Takifugu rubripes[J]. Aquaculture,2012, 344-349:35-39.
[14]邬阳,牛素芳,金媛,等.闽东海域刺激隐核虫的生活史观察及乙酸杀虫效果[J].厦门大学学报(自然科学版),2013, 52(2):281-288.Wu Y, Niu S F, Jin Y, et al. Observation on the life history of Cryptocaryon irritans in eastern sea of Fujian and research on antiparasitic effect of acetic acid[J].Journal of Xiamen University(Natural Science), 2013,52(2):281-288(in Chinese).
[15]胡梁及,朱盛山,张雄飞,等.中草药防治寄生性鱼病的研究进展[J].中国兽药杂志,2014, 48(9):66-69.Hu L J, Zhu S S, Zhang X F, et al. Research progress of Chinese herbal medicine prevention and control of parasitic diseases[J]. Chinese Journal of Veterinary Drug,2014, 48(9):66-69(in Chinese).
[16]王印庚,刘志伟,林春媛,等.养殖大菱鲆隐核虫病及其治疗[J].水产学报,2011,35(7):1105-1112.Wang Y G, Liu Z W, Lin C Y, et al. Cryptocaryoniosis in cultured turbot and its treatment[J]. Journal of Fisheries of China, 2011, 35(7):1105-1112(in Chinese).
[17]但学明.刺激隐核虫(Cryptocaryon irritans)的传代、保存及刺激隐核虫病的防治研究[D].广州:暨南大学,2006.Dan X M. The propagation, preservation of Cryptocaryon irritans Brown 1951, and control of cryptocaryonosis[D]. Guangzhou:Jinan University, 2006(in Chinese).
[18]周旻曦.大黄鱼刺激隐核虫病病原生物学特性的研究[D].宁波:宁波大学,2012.Zhou M X. Study on the biological characteristics of Cryptocaryon irritans on Pseudosciaena crocea[D]. Ningbo:Ningbo University, 2012(in Chinese).
[19] Burgess P J, Matthews R A. A standardized method for the in vivo maintenance of Cryptocaryon irritans(Ciliophora)using the grey mullet Chelon labrosus as an experimental host[J]. Journal of Parasitology, 1994,80(2):288-292.
[20] Nigrelli R F, Ruggieri G D S J. Enzootics in the New York Aquarium caused by Cryptocaryon irritans Brown,1951(=Ichthyophthirius marinus Sikama, 1961), ahistophagous ciliate in the skin, eyes and gills of marine fishes[J]. Zoologica,1966, 51(9):97-102.
[21] Colorni A. Aspects of the biology of Cryptocaryon irritans, and hyposalinity as a control measure in cultured gilt-head sea bream Sparus aurata[J]. Diseases of Aquatic Organisms,1985, 1:19-22.
[22]江飚.大黄鱼刺激隐核虫病的防治措施研究[D].广州:华南农业大学,2016.Jiang B. Study on prevention and treatment of Cryptocaryon irritans infection of cultured large yellow croaker(Pseudosciaena crocea)[D]. Guangzhou:South China Agricultural University, 2016(in Chinese).
[23]刘婷婷,唐小千,周丽.15种中草药对刺激隐核虫(Cryptocaryon irritans)的杀灭效果及包囊破裂的条件[J].渔业科学进展,2015, 36(6):113-120.Liu T T, Tang X Q, Zhou L. The pesticidal effects ofChinese herbal medicine on the trophonts and the theronts of ciliate Cryptocaryon irritans and the influence of temperature and salinity on the cyst rupture[J]. Progress in Fishery Sciences,2015, 36(6):113-120(in Chinese).
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[2]农业部渔业渔政管理局.2011中国渔业统计年鉴[M].北京:中国农业出版社,2011:28.Fisheries and Fisheries Administration Bureau of the Ministry of Agriculture. China Fishery Statistical Yearbook[M]. Beijing:China Agriculture Press, 2011:28.
[3]农业部渔业渔政管理局.2017中国渔业统计年鉴[M].北京:中国农业出版社,2017:26.Fisheries and Fisheries Administration Bureau of the Ministry of Agriculture. China Fishery Statistical Yearbook[M]. Beijing:China Agriculture Press, 2017:26(in Chinese).
[4]樊海平,林煜,钟全福,等.大黄鱼口服硝唑尼特后抗刺激隐核虫感染试验[J].福建农业学报,2015, 30(6):558-561.Fan H P, Lin Y, Zhong Q F, et al. Efficacy of orally administrated nitazoxanide in treating Cryptocaryon irritans infection on Pseudosciaena crocea[J]. Fujian Journal of Agricultural Sciences, 2015, 30(6):558-561(in Chinese).
[5]崔晓翠,王印庚,陈霞,等.槟榔、川楝子复方中草药对大黄鱼4种酶活性的影响及对刺激隐核虫的杀灭效果分析[J].中国水产科学,2017, 24(2):362-373.Cui X C, Wang Y G, Chen X, et al. Effects of a Chinese herbal compound on non-specific immune activities and extermination of parasite Cryptocaryon irritans in Larimichthys crocea[J]. Journal of Fishery Sciences of China,2017, 24(2):362-373(in Chinese).
[6] Colorni A, Burgess P. Cryptocaryon irritans brown1951,the cause of'white spot disease'in marine fish:an update[J]. Aquarium Sciences and Conservation, 1997,1(4):217-238.
[7]尹飞,龚颀杨,施兆鸿,等.刺激隐核虫感染对褐菖鲉的胁迫及鱼体的免疫应答[J].水生生物学报,2014,38(4):681-688.Yin F, Gong X Y, Shi Z H, et al. Stress and immune response of the marbled rockfish Sebastiscus marmoratus infected with Cryptocaryon irritans[J]. Acta Hydrobiologica Sinica,2014, 38(4):681-688(in Chinese).
[8]刘振勇,谢友佺,林小金,等.感染刺激隐核虫的大黄鱼对低溶氧量的耐受力研究[J].福建水产,2012,34(6):471-475.Liu Z Y, Xie Y Q, Lin X J, et al. Research on tolerance of Pseudosiaena crocea suffered from Cryptocaryon irritans disease to low dissolved oxygen[J]. Journal of Fujian Fisheries, 2012, 34(6):471-475(in Chinese).
[9]Pironet F N, Jones J B. Treatments for ectoparasites and diseases in captive Western Australian dhufish[J]. Aquaculture International, 2000, 8(4):349-361.
[10] Yoshinaga T, Im H J, Nishida S, et al. In vitro and in vivo efficacies of ionophores against Cryptocaryon irritans[J]. Aquaculture, 2011, 321(3-4):167-172.
[11] Hirazawa N, Goto T, Shirasu K. Killing effect of various treatments on the monogenean Heterobothrium okamotoi eggs and oncomiracidia and the ciliate Cryptocaryon irritans cysts and theronts[J]. Aquaculture, 2003, 223(1-4):1-13.
[12]郑晓叶,刘顺,周旻曦,等.几种理化因子对刺激隐核虫包囊及幼虫活力的影响[J].宁波大学学报(理工版),2015,28(1):15-20.Zheng X Y, Liu S, Zhou M X, et al. Influence of environmental factors and chemotherapeutants on Cryptocaryon irritans tomont and theront[J]. Journal of Ningbo University(NSEE), 2015, 28(1):15-20(in Chinese).
[13] Kawano F, Hirazawa N, Gravningen K, et al. Antiparasitic effect of dietary Romet.30(SDMX-OMP)against ciliate Cryptocaryon irritans infection in the red sea bream Pagrus major and tiger puffer Takifugu rubripes[J]. Aquaculture,2012, 344-349:35-39.
[14]邬阳,牛素芳,金媛,等.闽东海域刺激隐核虫的生活史观察及乙酸杀虫效果[J].厦门大学学报(自然科学版),2013, 52(2):281-288.Wu Y, Niu S F, Jin Y, et al. Observation on the life history of Cryptocaryon irritans in eastern sea of Fujian and research on antiparasitic effect of acetic acid[J].Journal of Xiamen University(Natural Science), 2013,52(2):281-288(in Chinese).
[15]胡梁及,朱盛山,张雄飞,等.中草药防治寄生性鱼病的研究进展[J].中国兽药杂志,2014, 48(9):66-69.Hu L J, Zhu S S, Zhang X F, et al. Research progress of Chinese herbal medicine prevention and control of parasitic diseases[J]. Chinese Journal of Veterinary Drug,2014, 48(9):66-69(in Chinese).
[16]王印庚,刘志伟,林春媛,等.养殖大菱鲆隐核虫病及其治疗[J].水产学报,2011,35(7):1105-1112.Wang Y G, Liu Z W, Lin C Y, et al. Cryptocaryoniosis in cultured turbot and its treatment[J]. Journal of Fisheries of China, 2011, 35(7):1105-1112(in Chinese).
[17]但学明.刺激隐核虫(Cryptocaryon irritans)的传代、保存及刺激隐核虫病的防治研究[D].广州:暨南大学,2006.Dan X M. The propagation, preservation of Cryptocaryon irritans Brown 1951, and control of cryptocaryonosis[D]. Guangzhou:Jinan University, 2006(in Chinese).
[18]周旻曦.大黄鱼刺激隐核虫病病原生物学特性的研究[D].宁波:宁波大学,2012.Zhou M X. Study on the biological characteristics of Cryptocaryon irritans on Pseudosciaena crocea[D]. Ningbo:Ningbo University, 2012(in Chinese).
[19] Burgess P J, Matthews R A. A standardized method for the in vivo maintenance of Cryptocaryon irritans(Ciliophora)using the grey mullet Chelon labrosus as an experimental host[J]. Journal of Parasitology, 1994,80(2):288-292.
[20] Nigrelli R F, Ruggieri G D S J. Enzootics in the New York Aquarium caused by Cryptocaryon irritans Brown,1951(=Ichthyophthirius marinus Sikama, 1961), ahistophagous ciliate in the skin, eyes and gills of marine fishes[J]. Zoologica,1966, 51(9):97-102.
[21] Colorni A. Aspects of the biology of Cryptocaryon irritans, and hyposalinity as a control measure in cultured gilt-head sea bream Sparus aurata[J]. Diseases of Aquatic Organisms,1985, 1:19-22.
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