刺激隐核虫与多子小瓜虫免疫学特性比较研究综述

刺激隐核虫(Cryptocaryon irritans)与多子小瓜虫(Ichthyophthirius multifiliis)是影响渔业生产重要寄生虫,本文主要阐述了刺激隐核虫与多子小瓜虫种内和种间的免疫学相似性和差异性,及其两种寄生虫抑动抗原的免疫学特性以及克隆表达研究。为免疫学方法预防两种寄生虫病提供理论依据。     

Rainbow trout (Oncorhynchus mykiss) immune response towards a recombinant vaccine targeting the parasitic ciliate Ichthyophthirius multifiliis

The protective effect in rainbow trout (Oncorhynchus mykiss) of an experimental subunit vaccine targeting antigens in the parasite Ichthyophthirius multifiliis has been evaluated and compared to effects elicited by a classical parasite homogenate vaccine. Three recombinant parasite proteins (two produced in E. coli and one in insect cells) were combined and injected i.p., and subsequently, protection and antibody responses were analysed. Both the experimental and the benchmark vaccine induced partial but significant protection against I. multifiliis when compared to control fish. Specific antibody responses of vaccinated trout (subunit vaccine) were raised against one neurohypophysial n‐terminal domain protein #10 of three recombinant proteins, whereas the benchmark vaccine group showed specific antibody production against all three recombinant proteins. The immunogenic parasite protein #10 may be a potential vaccine candidate supplementing the protective I‐antigen in future vaccine trials.     

四氢异喹啉衍生物的合成及体外抗多子小瓜虫活性的研究

合成四氢异喹啉衍生物并进行杀多子小瓜虫(Ichthyophthirius multifiliis)药效评价。以1-甲基-1,2,3,4-四氢异喹啉为起始原料,在2位的胺基上引入与环己甲酰氯、苯甲酰氯、噻吩甲酰氯、乙酰氯以及氯乙酰氯等不同的酰基进而合成5种四氢异喹啉衍生物(化合物1~化合物5),研究其对小瓜虫掠食体和包囊的杀虫活性,并对杀虫活性物质进行安全性评价。结果显示,5种化合物均具有一定的杀虫活性,其中,化合物1 [(1-甲基-3,4-二氢异喹啉-2(1H)-基)-苯基-甲酮]的杀虫活性最强,其对多子小瓜虫掠食体4 h的100%杀灭浓度为24.0 mg/L,对包囊6 h的100%杀灭浓度为60.0 mg/L,对掠食体的半数致死浓度(LD₅₀)为16.4mg/L。急性毒性实验结果显示,化合物1对翘嘴红鲌(Erythroculter ilishaeformi)的48 h LD₅₀为234.3 mg/L,其安全浓度为64.1 mg/L。研究表明,化合物1[(1-甲基-3,4-二氢异喹啉-2(1H)-基)-苯基-甲酮]是一种具有较好开发前景的杀小瓜虫药物。     

四氢异喹啉衍生物的合成及体外 抗多子小瓜虫活性的研究

合成四氢异喹啉衍生物并进行杀多子小瓜虫(Ichthyophthirius multifiliis)药效评价。以1-甲基-1,2,3,4-四氢异喹啉为起始原料,在2位的胺基上引入与环己甲酰氯、苯甲酰氯、噻吩甲酰氯、乙酰氯以及氯乙酰氯等不同的酰基进而合成5种四氢异喹啉衍生物(化合物1~化合物5),研究其对小瓜虫掠食体和包囊的杀虫活性,并对杀虫活性物质进行安全性评价。结果显示,5种化合物均具有一定的杀虫活性,其中,化合物1 [(1-甲基-3,4-二氢异喹啉-2(1H)-基)-苯基-甲酮]的杀虫活性最强,其对多子小瓜虫掠食体4 h的100%杀灭浓度为24.0 mg/L,对包囊6 h的100%杀灭浓度为60.0 mg/L,对掠食体的半数致死浓度(LD50)为16.4 mg/L。急性毒性实验结果显示,化合物1对翘嘴红鲌(Erythroculter ilishaeformi)的48 h LD50为234.3 mg/L,其安全浓度为64.1 mg/L。研究表明,化合物1 [(1-甲基-3,4-二氢异喹啉-2(1H)-基)-苯基-甲酮]是一种具有较好开发前景的杀小瓜虫药物。     

MH II‐DAB gene expression in grass carp Ctenopharyngodon idella (Valenciennes) after infection with the ciliate parasite,Ichthyophthirius multifiliis

The grass carp, Ctenopharyngodon idella (Valenciennes), is one of the most extensively aquacultured freshwater fish in China. However, because of the lack of effective control measures and the high‐density culture environment, considerable economic losses are caused by infection of C. idella with the parasitic ciliate, Ichthyophthirius multifiliis. The major histocompatibility (MH) DAB gene belongs to antigen‐presented genes in the class II genomic region, which is associated with parasite resistance. To understand the relationship of the DAB gene with I. multifiliis infection in grass carp, the expression profiles of MH II‐DAB were studied in tissues using real‐time quantitative polymerase chain reaction. The results showed that expression of the MH II‐DAB gene was up‐regulated in head kidney after I. multifiliis infection, and the expression peak appeared earlier in the study (case) group than in the control group. The obvious up‐regulation peak of MH II‐DAB gene was found at days 2 and 4 in skin; at 12 h to day 4 in spleen; at 12 h and days 1 and 6 in gill; and at day 10 in blood, whereas the MH II‐DAB gene was down‐regulated in liver and intestines after I. multifiliis infection. These results have implications for better understanding C. idella resistance to I. multifiliis infection.     

Susceptibility of three strains of blue catfish,Ictalurus furcatus (Valenciennes), to Ichthyophthirius multifiliis

This study compared the susceptibility of three blue catfish strains (D&B, USDA 101 and USDA 102) to the parasite Ichthyophthirius multifiliis (Ich). In Trial I, a cohabitation study (all strains stocked communally) was conducted and fish were exposed to theronts at 0, 200, 1000, 5000 or 25 000 theronts fish^?1, respectively. All fish died when exposed to theronts at 5000 or 25 000 theronts fish^?1. When exposed to 1000 theronts fish^?1, USDA 102 strain of blue catfish showed significantly lower mortality (78.5%) compared to USDA 101 and D&B strains (92.7% and 100%). In Trial II, the same three strains of blue fish were evaluated for their susceptibility to Ich with strains challenged in separate tanks by adding Ich theronts at 0, 200 and 1000 theronts fish^?1, respectively. All D&B and USDA 101 blue catfish died; however, 42.3% of USDA 102 strain survived the infection when exposed to 1000 theronts per fish. The results indicate that there are differences among strains of blue catfish for susceptibility to Ich, and these differences will be useful in the development of improved catfish germplasm for commercial aquaculture.     

小瓜虫抑动抗原研究综述

小瓜虫是重要的淡水鱼类寄生虫.小瓜虫抑动抗原是重要的保护性抗原,它在防治小瓜虫这类体表寄生虫方面有着巨大应用潜力.本文阐述了小瓜虫抑动抗原的发现过程及其生物学意义,并介绍了抑动抗原免疫学研究进展.本综述旨为抑动抗原在小瓜虫疫苗研制中的应用提供理论依据,也为其他体表寄生虫保护性抗原的研究提供参考.     

滇池金线鲃、西畴金线鲃及其杂交F1代抵抗小瓜虫与车轮虫能力的探究

用小瓜虫(Ichthyophthirius multifiliis)和车轮虫(Trichodina spp.)对滇池金线鲃(Sinocyclocheilus grahami)和西畴金线鲃(S.xichouensis)及其杂交F1代分别进行了两次人工感染实验。18月龄的杂交F1代、滇池金线鲃(Sinocyclocheilus grahami)和西畴金线鲃(S.xichouensis)感染小瓜虫后平均死亡率分别为(50.00±11)%、(56.70±23)%、(80.0±18)%。经方差检验杂交F1代死亡率显著低于父本西畴金线鲃。6月龄杂交F1代、滇池金线鲃、西畴金线鲃感染车轮虫后的平均死亡率分别为(40.56±13)%、(56.11±5)%、(42.78±13)%。经方差检验杂交F1代死亡率显著低于母本滇池金线鲃。在感染小瓜虫和车轮虫后,三种鱼的死亡量均呈现激增后又显著回落的趋势,死亡高峰在第4天至第7天。本次感染实验的小瓜虫和车轮虫主要分布于病鱼体表:躯干分布最多57%~79%,其次为鳍12%~28%,头部分布较少4%~15%。与本研究中车轮虫几乎于鳃丝无分布(0%~3%)不同,有大约10%~20%的小瓜虫分布于鳃丝上。     

20种中草药杀灭离体小瓜虫的药效研究

选用20种不同中草药,研究其不同浓度的水提物对多子小瓜虫(Ichthyophthirius multifiliis)成虫、包囊和幼虫的离体杀灭实验效果。结果显示:0.1 g/L槟榔和乌梅对各个阶段的虫体均有杀灭效果,0.1 g/L大黄和黄芩1 h内对幼虫有杀灭效果,1.0 g/L浓度能杀灭成虫和包囊,表明槟榔、乌梅、大黄、黄芩的杀虫效果相对较好;贯众、乌药、枳壳等13种中药浓度达到10 g/L、50 g/L或100 g/L才具有一定杀虫作用,但效果不很显著;熟地黄、茯苓和黄芪即使浓度达到100 g/L时,也不能杀灭虫体。     

Induced cross-protection in channel catfish, Ictalurus punctatus (Rafinesque), against different immobilization serotypes of Ichthyophthirius multifiliis

Abstract Channel catfish, Ictalurus punctatus (Rafinesque), were immunized with Ichthyophthirius multifiliis (Ich) theronts and trophonts, and the immune response and host protection against both homologous and heterologous serotypes of Ich were evaluated. Immunizations were done with two immobilization serotypes (ARS4 and ARS6) of live theronts by bath immersion (trial I) and with sonicated trophonts by intraperitoneal (i.p.) injection (trial II). Cutaneous and serum antibody titres against Ich following immunization were measured and survival of catfish was determined after theront challenge. Theronts were immobilized by the antiserum from fish immunized with homologous theronts or trophonts, but not by the serum of fish immunized with the heterologous serotype. Serum from fish immunized by immersion with live theronts showed higher enzyme-linked immunosorbent assay titres against both homologous and heterologous serotypes than fish immunized by i.p. injection of trophonts. Channel catfish immunized by immersion with live theronts or by i.p. injection with sonicated trophonts developed an immune response against Ich and provided cross-protection against challenge from both serotypes (ARS4 and ARS6) of the parasite. Sonicated trophont antigens in aqueous solution by i.p. injection could stimulate an immune response in fish, but the immunity was of short duration.     

Experimental evidence for direct in situ binding of IgM and IgT to early trophonts of Ichthyophthirius multifiliis (Fouquet) in the gills of rainbow trout, Oncorhynchus mykiss (Walbaum)

Freshwater fish are able to mount a protective immune response against the parasite Ichthyophthirius multifiliis (Ich) following a non‐lethal exposure. Factors involved in immunity comprise cellular and humoral factors, but antibodies have been suggested to play a prominent role in protection. However, host antibodies have not yet been demonstrated to bind to the parasite in situ. By the use of immunohistochemical techniques, this study demonstrated that IgT and IgM bind to surface structures, including cilia, on the early feeding stage of the parasite in the gills of immune rainbow trout, Oncorhynchus mykiss, shortly (2 h) after invasion. No binding of IgT and no or only a weak binding of IgM was observed on the parasites in the gills of similarly exposed but naïve rainbow trout. This study indicates that antibodies play an important part in the protection of immune fish against Ich although additional humoral and cellular factors may contribute to this reaction.     

Heavy infection of Ichthyophthirius multifiliis in striped catfish (Pangasianodon hypophthalmus,Sauvage 1878) and its treatment trial by different therapeutic agents in a control environment

ABSTRACT

Striped catfish (Pangasianodon hypophthalmus) fingerlings were stocked (60 fishes/m³) in cemented square tanks for ornamental fishery purposes at the College Fish Farm in Mangalore. A total of 400 fishes with a mean weight (g) ranging from 3.24 ± 1.21 to 6.70 ± 1.13 and a mean length (cm) of 7.50 ± 0.94 to 9.50 ± 1.10 were examined. A severe outbreak of salt-like granule white spots was found on the body surface. Ichthyophthirius multifiliis, a ciliate pathogen, was identified as the causative agent by clinical signs, wet mount, and histopathological observations. Infected fishes were transferred and equally distributed to the 0.45 m³ glass aquaria and treated with three treatments: (T1) methylene blue + salt; (T2) raising temperature with salt; and (T3) formalin + malachite green. The best fingerling survival (55 ± 9.36%) was obtained by elevated water temperature with salt in T2.     

Efficacy of Different Salt (NaCl) Concentrations in the Treatment of Ichthyophthirius multifiliis-Infected Silver Catfish,Rhamdia quelen,Fingerlings

ABSTRACT

The objective of this study was to evaluate different salt concentrations in the treatment of Ichthyophthirius multifiliis. Silver catfish, Rhamdia quelen, fingerlings infected with I. multifiliis were submitted to four sodium chloride concentrations (g/L): 0,1,2, and 4 for 23 days. In a second experiment, fingerlings were maintained with 4 g/L salt, but for 45 days. Treatments with 2 and 4 g/L salt significantly increased fingerling survival compared with 0 and 1 g/L, and survival was significantly correlated with salt concentrations. In the second experiment, fish maintained at 4 g/L salt showed a gradual reduction of “white spots” and survival was 100%. Therefore, salt offers an alternative for treatment of silver catfish fingerlings infected with I. multifiliis.     

Apoptosis in Ichthyophthirius multifiliis is associated with expression of the Fas receptor of theronts

The expression of type I membrane Fas receptors on the surface of Ichthyophthirius multifiliis (Ich) theronts and the possible association between Fas expression and theront apoptosis induced by the immune antibody was examined. Fas receptors were detected on the theront surface using fluorescein isothiocyanate-conjugated mouse monoclonal antibody against Fas. Fas-positive theronts significantly increased with time during in vitro incubation and with increasing theront concentration. Furthermore, the immune cutaneous antibody induced theront apoptosis; however, Fas ligand did not. A highly significant correlation was noted between theront Fas expression and immune cutaneous antibody-induced theront apoptosis. Numbers of apoptotic theronts increased with increasing number of Fas-positive theronts. The data indicated that theront apoptosis induced by immune cutaneous antibody appears to be positively correlated with the expression of Fas on the surface of Ich theronts.     

Impact of Pseudomonas H6 surfactant on all external life cycle stages of the fish parasitic ciliate Ichthyophthirius multifiliis

A bacterial biosurfactant isolated from Pseudomonas (strain H6) has previously been shown to have a lethal effect on the oomycete Saprolegnia diclina infecting fish eggs. The present work demonstrates that the same biosurfactant has a strong in vitro antiparasitic effect on the fish pathogenic ciliate Ichthyophthirius multifiliis. Three life cycle stages (the infective theront stage, the tomont and the tomocyst containing tomites) were all susceptible to the surfactant. Theronts were the most sensitive showing 100% mortality in as low concentrations as 10 and 13 μg/ml within 30 min. Tomonts were the most resistant but were killed in concentrations of 100 μg/ml. Tomocysts, which generally are considered resistant to chemical and medical treatment, due to the surrounding protective cyst wall, were also sensitive. The surfactant, in concentrations of 10 and 13 μg/ml, penetrated the cyst wall and killed the enclosed tomites within 60 min. Rainbow trout fingerlings exposed to the biosurfactant showed no adverse immediate or late signs following several hours incubation in concentrations effective for killing the parasite. This bacterial surfactant may be further developed for application as an antiparasitic control agent in aquaculture.     

Molecular cloning and functional characterization of the fatty acid delta 6 desaturase (FAD6) gene in the sea cucumber Apostichopus japonicus

The sea cucumber (Apostichopus japonicus), an important echinodermata, had high value in nutrition and medicine for its rich collagen, sulphated polysaccharide, glycosides and polyunsaturated fatty acids (PUFA). The cDNA of the fatty acid desaturase gene in A. japonicus (AJFAD6) was cloned and was found to encode a desaturase with delta 6 FAD activity. Sequence analysis indicated that AJFAD6 included an open reading frame of 1392 bp, encoding 463 amino acids. AJFAD6 has all the conserved motifs found in other members of the FAD6 family, including an N‐terminal cytochrome b5 domain and three histidine‐rich regions. qRT‐PCR showed that AJFAD6 was expressed in all tissues tested during juvenile development and was mainly expressed in the respiratory tree at 150 days after adherence (150 days) and in the intestine at 100 days. Furthermore, AJFAD6 mRNA was also detected in the analysed adult tissues, with higher expression in the intestine and testis. Functional characterization of AJFAD6 in a recombinant yeast, Pichia pastoris, showed that AJFAD6 could catalyse exogenous linoleic acid (LA) and α‐linolenic acid (ALA) to produce γ‐linoleic acid (GLA) and stearidonic acid (STA), respectively, at conversion rates of 11.1% for LA to GLA and 3.4% for ALA to STA. Our results suggested that the biosynthetic pathway of PUFA existed in the sea cucumber, but endogenous production of eicosapentaenoic acid, arachidonic acid and docosahexaenoic acid from either LA or ALA precursor appeared to be limited.     

斑点叉尾鮰源海豚链球菌DGX07株simA基因克隆、鉴定及分子特性分析

以Primer p14(5’-GATCAAGTCC-3’)为引物对分离自患病斑点叉尾海豚链球菌强毒株DGX07进行RAPD分析。同时参照GenBank中海豚链球菌simA基因序列设计特异性引物,以DGX07基因组DNA为模板,扩增出约1 500 bp的simA基因,并将其克隆到pMD19-T载体上,之后对重组质粒进行PCR和双酶切(BamHⅠ+XhoⅠ)鉴定,鉴定正确后送测序公司测序。RAPD分析结果显示,DGX07和标准菌株均能扩增出750 bp大小的条带。通过生物信息学软件对测序结果分析显示,simA基因全长1 566 bp,由521个氨基酸组成,与海豚链球菌simA和simB亲缘性达100%,存在1个由41个氨基酸组成的信号肽,具有Bap31和Gram_pos_anchor两个超家族的保守结构域;具有与蛋白翻译后修饰功能相关的磷酸化位点22个和N-糖基化位点2个,编码多肽链中亲水区大于疏水区,是一种膜外蛋白,并具有多个抗原优势位点区域。密码子偏爱性分析表明,斑点叉尾源海豚链球菌simA基因密码子使用频率差异较大,密码子偏爱性与酵母较为接近。获得GenBank登录号为JF330100。     

Cloning and expression of the gene encoding an extracellular alkaline serine protease from Vibrio alginolyticus strain HY9901, the causative agent of vibriosis in Lutjanus erythopterus (Bloch)

A 750-bp internal fragment of the alkaline serine protease gene (asp) from the Vibrio alginolyticus strain HY9901 was amplified by polymerase chain reaction (PCR). The flanking sequences of the 5'- and 3'- ends of the asp gene were characterized by reverse and nested PCR. Sequence analysis showed that the asp gene contained an 1893-bp ORF encoding 630 amino acids. The deduced amino acid sequence of the ASP (alkaline serine protease) precursor showed significant homology with several bacterial alkaline serine proteases. Expression of the asp gene in Escherichia coli and activity tests of the ASP indicated that the N-signal peptide of the ASP precursor was essential to autocatalyse and fold correctly the enzyme to obtain activity. The purified ASP was lethal for Lutjanus erythopterus with an LD(50) of 0.25 microg protein g(-1) body weight.