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

比较分析了15种中草药对刺激隐核虫(Cryptocaryon irritans)滋养体和幼虫的离体杀灭效果,并探讨了刺激隐核虫包囊破裂产生幼虫的最适温度和盐度条件。结果显示,中草药药物浓度为4.55 g/L时,槟榔对滋养体和幼虫有杀灭效果,大黄和野菊花仅对幼虫有杀灭效果;药物浓度为9.09 g/L时,苦参、贯众对滋养体和幼虫具有杀灭效果。药物浓度为18.18 g/L时,黄芩、川楝子、枳壳对滋养体和幼虫都具有杀灭效果。野菊花对滋养体也具有杀灭效果;在45.45、90.09 g/L较高药物浓度时,黄芪、鱼腥草、板蓝根、白头翁、金银花、熟地黄对幼虫和滋养体具有杀灭效果。研究表明,槟榔、苦参、大黄、贯众、黄芩、枳壳、川楝子、野菊花杀虫效果显著;黄芪、鱼腥草、板蓝根、白头翁、金银花、熟地黄、黄连的杀虫效果不显著。不同培养温度和盐度对刺激隐核虫包囊破裂产出幼虫所需时间比较结果显示,包囊破裂产出幼虫的最适温度为26℃、最适盐度为20–30。     

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

比较分析了15种中草药对刺激隐核虫(Cryptocaryon irritans)滋养体和幼虫的离体杀灭效果，并探讨了刺激隐核虫包囊破裂产生幼虫的最适温度和盐度条件。结果显示，中草药药物浓度为4.55 g/L时，槟榔对滋养体和幼虫有杀灭效果，大黄和野菊花仅对幼虫有杀灭效果；药物浓度为9.09 g/L时，苦参、贯众对滋养体和幼虫具有杀灭效果。药物浓度为18.18 g/L时，黄芩、川楝子、枳壳对滋养体和幼虫都具有杀灭效果。野菊花对滋养体也具有杀灭效果；在45.45、90.09 g/L较高药物浓度时，黄芪、鱼腥草、板蓝根、白头翁、金银花、熟地黄对幼虫和滋养体具有杀灭效果。研究表明，槟榔、苦参、大黄、贯众、黄芩、枳壳、川楝子、野菊花杀虫效果显著；黄芪、鱼腥草、板蓝根、白头翁、金银花、熟地黄、黄连的杀虫效果不显著。不同培养温度和盐度对刺激隐核虫包囊破裂产出幼虫所需时间比较结果显示，包囊破裂产出幼虫的最适温度为26℃、最适盐度为20–30。     

青蒿末水浸提液对多子小瓜虫杀灭效果的研究

采用0.05、0.1、0.5、1.0以及5.0 g/L 5个浓度梯度的青蒿末水浸提液对多子小瓜虫成虫、幼虫及孢囊进行离体杀灭试验。结果表明:0.5 g/L以上浓度的青蒿末水浸提液可完全杀灭多子小瓜虫的幼虫,0.1～1.0 g/L浓度可部分杀灭成虫,1.0 g/L浓度可部分抑制孢囊分裂,5.0 g/L浓度可完全杀灭成虫和孢囊。     

丁香酚的分离及其杀多子小瓜虫活性研究

利用不同极性大小的溶剂石油醚、乙酸乙酯、氯仿、甲醇回流提取丁香制备粗提物,进行杀灭多子小瓜虫幼虫和包囊的药效活性追踪,确定丁香杀虫的活性部位为石油醚提取部分,进而利用硅胶柱层析和中压制备液相等分离技术结合活性追逐试验追踪分离丁香石油醚提取物(对多子小瓜虫幼虫和包囊的100%致死质量浓度分别为50mg/L和80mg/L),最终分离获得一种杀虫活性物质,经质谱、核磁氢谱、核磁碳谱等多种波谱分析技术确定该活性物质为丁香酚。体外杀虫结果表明,丁香酚对多子小瓜虫具有较强的杀灭作用,其对幼虫15min、1h、2h、3h和4h的半数致死质量浓度分别为6.21(5.85~6.65)mg/L、3.50(3.24~3.84)mg/L、2.90(2.62~3.22)mg/L、2.33(2.10~2.50)mg/L和2.15(1.98~2.32)mg/L,对幼虫4h的100%杀灭的质量浓度为3.5mg/L,对包囊6h的100%杀灭质量浓度为3.5mg/L。结果表明,丁香酚是一种具有较好开发前景的杀多子小瓜虫药物。     

环烷酸铜对离体小瓜虫杀灭效果的研究

采用0.05、0.1、0.2、0.4、0.8mg/L 5个浓度梯度的环烷酸铜溶液对小瓜虫成虫、幼虫以及孢囊进行离体杀灭试验,结果表明:0.1~0.2mg/L环烷酸铜溶液对小瓜虫幼虫及成虫都有较强杀灭作用,0.4~0.8 mg/L环烷酸铜能控制小瓜虫包囊分裂。     

肉桂活性成分肉桂醛杀灭离体多子小瓜虫效果

研究从肉桂中分离纯化出具有抑杀多子小瓜虫活性的化合物。以乙醇为提取剂,用索氏超声提取法从肉桂中提取浸膏,再用石油醚、乙酸乙酯、甲醇为萃取剂,萃取不同有效组分,以不同浓度分别进行杀灭离体多子小瓜虫实验,发现石油醚萃取物的杀虫效果最优。然后对石油醚萃取物采用硅胶层析柱和制备型高效液相色谱进行分离纯化,利用质谱和核磁波谱分析,最终鉴定其杀虫活性成分为肉桂醛;将肉桂醛溶于二甲基亚砜并用二倍梯度稀释法配成不同浓度的药液测试其对离体小瓜虫的杀灭活性。结果表明,100%杀灭滋养体和感染性幼虫的剂量分别为50和8 mg/L,半数有效浓度分别为13.9和1.8 mg/L;使用剂量在50 mg/L可完全抑制小瓜虫包囊孵化。     

杀灭离体小瓜虫的药物试验

研究不同的药物及同一药物不同浓度对小瓜虫的成虫、幼虫、包囊的杀灭作用。 0 2～ 0 5mL/L甲醛、 2 %食盐水、 0 2～ 0 2 5mL/L冰乙酸、 0 2mL/L双氧水、辣椒老姜混合熬煮液、苦楝果熬煮液对离体小瓜虫均有明显的杀灭作用。而 1%食盐水、辣椒熬煮液、老姜熬煮液、苦楝树皮熬煮液、黄荆粗枝及细枝熬煮液、 0 7mg/L铜铁合剂对生活史中任何阶段小瓜虫均无杀灭效果。     

复方中草药防治锦鲤指环虫病试验研究

用黄柏、茯苓、百部、苦参、苦楝、贯众、青蒿、槟榔等中草药组成复方,将感染有指环虫的锦鲤在不同浓度的复方中草药煎熬物的水体中浸泡,控制一定的水体条件,显微镜下定期观察鱼鳃上的指环虫数量,统计杀虫率。结果表明:复方中草药中8mg/L的药液杀灭指环虫效果最好,其6h内杀虫率为20%～50%;24h时的杀灭指环虫效果最好,最高杀灭率为100%。     

6种中草药体外杀灭刺激隐核虫及乌梅预防效果研究

研究了6种中草药水提取物对刺激隐核虫(Crytocaryon irritans)的体外杀灭效果,发现用7.14 mg·m L–1的乌梅(Fructus mume)、槟榔(Areca catechu)、贯众(Dryopteris setosa)和石榴皮(Punica granatum)的水提取物分别处理幼虫5 min,幼虫死亡率达100%,用28.57 mg·m L–1处理包囊4 h,包囊死亡率达84.1%以上。为评估喂服乌梅水提物对珍珠石斑鱼(Epinephelus lanceolatus♀×E.fuscoguttatus♂)预防刺激隐核虫病的效果,投喂第14天后对石斑鱼的增重、溶菌酶活性、补体旁路途径溶血活性和鱼体滋养体数量及死亡率进行测试和评估。结果显示,药物组石斑鱼的增重和旁路补体溶血活性与对照组没有显著性差异(P0.05),溶菌酶活性均显著高于对照组(P0.05),感染刺激隐核虫后滋养体数量均显著低于对照组(P0.05);致死剂量攻毒后,药物组(11.4 g·kg–1、6.84 g·kg–1、3.42 g·kg–1)和对照组鱼的成活率分别为40%、20%、46.67%和0%。结果表明乌梅对预防刺激隐核虫病具有一定的开发前景。     

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

海水小瓜虫病是养殖大黄鱼(Larimichthys crocea)和数十种海水鱼类较难控制的主要疾病,它是由刺激隐核虫(Cryptography irritans)感染引发,严重威胁鱼类养殖产业的健康发展。本研究利用复方中草药"HD-2"(槟榔、川楝子、绵马贯众、大青叶、穿心莲等以一定比例混合,超微粉碎经过200目过筛后收集粉末)投喂大黄鱼,研究大黄鱼溶菌酶(LZM)、总超氧化物歧化酶(T-SOD)、碱性磷酸酶(AKP)、酸性磷酸酶(ACP)等非特异性免疫指标变化和不同用药方式对刺激隐核虫的杀灭效果。结果显示,口服"HD-2"可以促进鱼体LZM、T-SOD、AKP、ACP活性升高,以添加2%中草药组在提高4种酶活性方面作用显著,投喂后第14~21天酶活性最高。"HD-2"对体外刺激隐核虫幼虫的杀灭实验证实:4 h内在50 mg/L浓度下对幼虫没有杀伤作用,80 mg/L、100 mg/L、200 mg/L浓度分别可以杀死20%、54%、89%的幼虫,400 mg/L浓度下幼虫被全部杀死,幼虫4 h半致死浓度为109 mg/L。在室内为期15 d的大黄鱼小瓜虫病治疗实验中,A组按20 g/kg添加"HD-2"药物口服,同时按100 mg/L浓度药浴,存活率为50%;B组按20 g/kg添加药物口服,同时按50 mg/L浓度药浴,存活率50.67%;C组,在无药浴情况下,仅按20 g/kg添加药物口服,存活率为42.67%;A、B、C组存活率均显著高于对照组存活率2.67%(P0.05)。此外,A、B、C 3组中,大黄鱼胸鳍上白点数量明显少于对照组;同时,3个组的大黄鱼鳃丝上隐核虫数量均呈显著下降趋势(P0.05),实验结束时几乎观察不到虫体;而对照组大黄鱼鳃丝上隐核虫数量明显上升。实验过程中A、B、C 3组大黄鱼释放到体外的包囊数量明显下降,第12天时其包囊数量分别下降100%、91.1%、77.7%,与对照组差异极显著(P0.01)。综上所述,口服和药浴同时用药具有最佳效果,仅口服中草药亦能达到良好治疗效果。从实用性和成本考虑,建议仅进行口服处理,即能保障大黄鱼较高成活率,中草药添加量为2%,投喂时间为6~14 d。该结果为防控鱼类小瓜虫病提供了理论参考和技术支撑,在养殖生产中将具有良好的应用前景。     

In vitro assessment of the antiparasitic effect of caprylic acid against several fish parasites

The antiparasitic effect of caprylic acid against several fish parasites, i.e., the ciliate Cryptocaryon irritans (theronts), monogenean Benedenia seriolae (oncomiracidia and adults), copepod Pseudocaligus fugu (copepodids and adults) and myxosporean Kudoa shiomitsui (spores), was examined by in vitro trials.

Caprylic acid at a concentration of 1 mM had a parasiticidal effect against C. irritans theronts, B. seriolae oncomiracidia and K. shiomitsui spores and a contractile effect against B. seriolae adults, but had no clear effect against P. fugu copepodids and adults. These results suggest that caprylic acid may have an antiparasitic effect against various fish parasites, including parasites classified as monogenea, ciliates and myxosporea.     

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。     

Activity of the antimicrobial polypeptide piscidin 2 against fish ectoparasites

Abstract The antiparasitic effects of piscidin 2, an antimicrobial polypeptide (AMPP) first isolated from mast cells of hybrid striped bass, were tested against three protistan ectoparasites of marine fish (the ciliates Cryptocaryon irritans and Trichodina sp., and the dinoflagellate Amyloodinium ocellatum) and one ciliate ectoparasite of freshwater fish (Ichthyophthirius multifiliis). I. multifiliis was the most susceptible parasite, with all theronts killed at 6.3 microg mL(-1) piscidin 2. The most resistant parasite was Trichodina, where a few cells were killed at 12.5 microg mL(-1), but several were still alive even at 100 microg mL(-1). C. irritans was of intermediate sensitivity, with some theronts killed at 12.5 microg mL(-1) and all killed at 25 microg mL(-1). High parasite density apparently exhausted the piscidin 2 before it could attain its maximal effect, but surviving parasites were often visibly damaged. The lower efficacy of piscidin 2 against marine parasites compared with the freshwater ciliate might be related to the inhibitory effects of high sea water cation levels. The tissue concentration of piscidins estimated in healthy hybrid striped bass gill (40 microg mL(-1)) suggests that piscidin 2 is lethal to the parasites tested at physiological concentrations and is thus an important component of innate defence in fish expressing this type of AMPP.     

Immunization of grouper, Epinephelus coioides, confers protection against a protozoan parasite, Cryptocaryon irritans

The present study aimed to determine whether protection is conferred by immunization of grouper, Epinephelus coioides, against a protozoan parasite, Cryptocaryon irritans. The immunization of E. coioides was carried out by a low level exposure of fish to live C. irritans theronts from predetermined number of tomonts and by an intraperitoneal injection of a vaccine consisting of formalin-killed C. irritans theronts.

Mucus titers detected by ELISA were significantly higher in fingerling and adult grouper subjected to the low level of exposure to C. irritans theronts at 3-week post-exposure compared to fish that had no previous exposure. In addition, significantly smaller tomonts were produced from adult grouper after three successive exposures than the tomonts produced after a single exposure to the parasite.

In the vaccine-immunization experiment, no mortality was monitored in fish that received high dose vaccine (100 μg/fish), while 40% cumulative mortality and 100% cumulative mortality were recorded in low dose group (10 μg/fish) and control group (PBS-injected), respectively. In the succeeding replicate, the vaccine-immunized group (high dose) had 37.5% cumulative mortality and 100% cumulative mortality for the control. In addition, a total of 1830 tomonts were collected at 5-day post-challenge from the control group while none from the vaccine-immunized group. Significantly fewer trophonts and tomonts were enumerated at 5-day and 7-day post-challenge, respectively, in the vaccine-immunized group than the control.

Results suggest that a protective immunity has been conferred on the immunized grouper as indicated by high antibody titers in the mucus of C. irritans-exposed fish and higher survival and fewer parasites in vaccine-immunized fish than the control groups. The conferred immunity played a major role in preventing or limiting the adhesion, invasion, and development of C. irritans theronts on the skin of the immunized grouper.     

Ichthyophthiriasis in carp, Cyprinus carpio L.: fate of parasites in immunized fish

Abstract. The host-parasite relationship between O-group carp and the ciliatc Ichthyophthirius multifiliis Fouquet was investigated, with the specific aim of characterizing the fate of parasites encountering immunized fish. Carp were immunized by repeated controlled infections; immunized fish and control fish naive to I. multifiliis were then infected in the caudal fin epidermis by a single controlled exposure to theronts, which were applied in a droplet suspension to the tail surface. The number of parasites present within the caudal fin was monitored over a subsequent 5-day period by means of in situ parasite mapping. Results indicated that, contrary to previous reports, theronts penetrated the skin of immunized fish in numbers comparable to those of fish receiving a primary infection. However, the majority of parasites which penetrated immune skin did not complete normal development; 79% of the parasites which had initially penetrated the immune skin were not relocated within 2h of exposure, and since no parasite material was detected at penetration sites, it was concluded that these parasites had prematurely exited the skin rather than been killed in situ. Subsequently, these sites became populated by leucocytes, predominantly macrophages, and the infiltrations continued for up to 5 days after the initial exposure. In contrast, at sites where mature trophonts had exited the skin of fish following a primary infection, more diffuse leucocytic infiltrations were recorded, and these were predominated by neutrophils. Differences in the response to parasite exit from immunized and previously unexposed control fish skin are discussed, with particular reference to the mode of protection and the fate of parasites encountering immune fish.     

气相色谱法同时测定水产品中氯霉素、氟甲砜霉素和甲砜霉素残留量

以尼罗罗非鱼(Oreochromis niloticus)和南美白对虾(Penaeus vannamei)为实验材料,建立了测定水产品中氯霉素、氟甲砜霉素和甲砜霉素残留量的毛细管电子捕获气相色谱(GC-ECD)法。用乙酸乙酯同时提取水产品中的待测物,提取液浓缩至干后溶于甲醇/氯化钠溶液,正己烷脱脂,过C18柱净化,乙腈洗脱,加BSTFA-TMCS在65℃下衍生反应30min,正己烷定容后进样分析,外标法定量。本方法氯霉素在1.0~500.0μg/L,氟甲砜霉素和甲砜霉素在5.0~500.0μg/L浓度范围内呈线性相关,相关系数r≥0.9983,加标水平在0.2~10.0μg/kg时,回收率为83.30%~101.22%,相对标准偏差为1.36%~12.57%。氯霉素、氟甲砜霉素和甲砜霉素的检测限分别为0.1、0.2、0.2μg/kg。     

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.     

颤藻浓度和水温对凡纳滨对虾响应颤藻粗提液毒性的影响

文章研究了绿色颤藻(Oscillatoria chlorina)不同成分对凡纳滨对虾(Litopenaeus vannamei)的毒性,分析了颤藻浓度与水温对对虾响应粗提液毒性的影响,并初步检测了颤藻粗提液所含的毒素。结果显示,湿藻质量浓度为5.04 mg·m L~(-1)的颤藻细胞及其粗提液可导致93.72%以上的对虾死亡,藻细胞培养液对对虾无致死效应。用湿藻质量浓度高于2.52 mg·m L~(-1)的粗提液注射对虾,对虾死亡率超过86.67%,随质量浓度低至0.504 mg·m L~(-1),对虾死亡率降至2.22%,差异显著(P0.05);水温显著影响了粗提液对对虾的致死效应(P0.05),湿藻质量浓度为2.52 mg·m L~(-1)的粗提液在22℃水温条件下可引起20.0%对虾死亡,当温度升至34℃,对虾死亡率增加至97.8%;以微囊藻毒素(Microcystin-RR/YR/LR,MC-RR/YR/LR)作标准品,使用高效液相色谱法检测颤藻粗提液毒素,结果在粗提液中均未检出RR、YR、LR等3种常见的微囊藻毒素。结果表明,绿色颤藻可致对虾急性死亡,其主要有害成分是颤藻粗提液所含的非MC-RR/YR/LR等藻毒素,对虾对颤藻粗提液毒性的响应与颤藻浓度和水温显著相关(P0.05)。