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。     

N-取代四氢异喹啉衍生物的合成及杀多子小瓜虫研究

以苯乙醇为起始原料,对醇基的α位碳上的氢进行取代,反应合成3种N-取代四氢异喹啉衍生物(化合物1～3),研究其对多子小瓜虫掠食体和包囊的杀虫活性,评价杀虫活性物质的安全性。试验结果表明,合成的3种四氢异喹啉衍生物对多子小瓜虫的掠食体和包囊均具有一定的杀灭作用,其中化合物1(2-甲胺基-1,2,3,4-四氢异喹啉-1-甲腈)的杀虫活性最强,其对多子小瓜虫掠食体4 h的100%灭杀质量浓度为20.0 mg/L,对包囊6 h的100%灭杀质量浓度为40.0 mg/L,对掠食体的半数致死质量浓度为11.6 mg/L。急性毒性试验结果显示,化合物1对翘嘴红鲌的48 h半致死质量浓度为62.78 mg/L。2-甲胺基-1,2,3,4-四氢异喹啉-1-甲腈具有较好的体外杀多子小瓜虫活性,且毒性较低,具有较好的开发潜力。     

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。     

黄姜杀灭鱼类指环虫活性部位的初步研究

以寄生于金鱼鳃部的指环虫为指示寄生虫,采用活体感染、活体杀虫的方法,通过不同极性的溶剂回流提取黄姜,制备粗提物,进行杀灭金鱼指环虫的药效活性追踪试验,确定黄姜杀灭鱼类指环虫的活性部位,并对活性部位进行安全性评价.试验结果表明,黄姜的杀虫活性部位是70%乙醇部位,其质量浓度为20.0 mg/L时,平均最高杀虫率为100%.70%乙醇提取物经过有机溶剂进一步萃取后,杀虫药效试验表明,石油醚和最终水相萃取部位对指环虫具有杀灭作用,但石油醚萃取部位作用较佳,其质量浓度为5.0 mg/L时,平均最高杀虫率为100%.活性部位对草鱼急性毒性试验结果显示,石油醚萃取部位对草鱼的48 h半致死质量浓度(LC50)为33.54 mg/L,其安全质量浓度为9.64 mg/L,表明黄姜是一种比较安全,杀虫效果良好的水产用中草药.     

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。     

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。     

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。     

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。     

过氧乙酸对神仙鱼小瓜虫病的治疗实验

为了减轻小瓜虫病对观赏鱼养殖的严重危害,试验选择过氧乙酸对七彩神仙鱼进行杀灭小瓜虫的有效性试验,在此基础上再进行过氧乙酸对神仙鱼急性毒性试验,并在安全浓度下对患有小瓜虫病的神仙鱼进行治疗试验。结果显示:过氧乙酸对七彩神仙鱼的安全浓度为8.55mg/L,过氧乙酸在神仙鱼安全浓度内对小瓜虫的幼虫和包囊都具有较好的杀灭效果,过氧乙酸浓度在4.26～8.55mg/L的治愈率达70%。     

Immune responses and host protection of channel catfish, Ictalurus punctatus (Rafinesque), against Ichthyophthirius multifiliis after immunization with live theronts and sonicated trophonts

The humoral immune responses and host protection of channel catfish, Ictalurus punctatus (Rafinesque), against Ichthyophthirius multifiliis (Ich) were determined after immunization with live theronts and sonicated trophonts. Immunizations with live theronts or sonicated trophonts were carried out by both bath immersion and intraperitoneal (i.p.) injection. Cutaneous and serum immunoglobulin (Ig) levels and anti-Ich antibodies were measured 12 and 21 days post-immunization. The level of Ich infection and survival of catfish were determined after theront challenge. Cutaneous and serum anti-Ich antibodies were significantly higher (P < 0.05) in fish immunized with live theronts by immersion or i.p. injection, or with sonicated trophonts administered by i.p. injection, than in fish immunized with sonicated trophonts by immersion, with bovine serum albumin by i.p. injection, or non-immunized controls. Host protection was noted only in fish immunized with live theronts by immersion or i.p. injection or with sonicated trophonts by i.p. injection. There was a positive correlation between higher levels of anti-Ich antibodies and host survival in the immunized fish.     

Protective Immunity of Goldfish Against Ichthyophthirius multifiliis Infection Induced by Different Trophont Vaccine Preparations

Ichthyophthirius multifiliis, commonly called “Ich,” is a protozoan parasite that infects the epidermis and gills of freshwater fish. Here, we used goldfish (Carassius auratus) to determine whether the four vaccine preparations (live trophonts, formalin‐fixed trophonts, freeze‐thawed trophont lysates, and trophont cilial and cell cortical fractions) of I. multifiliis (Fouquet) can elicit resistance of immunized fish against subsequent theront challenge, and whether a relationship exists between in vitro immobilization of theronts in sera or mucus from immunized fish and host protective immunity against the parasite. Experimental goldfish were randomly divided into five groups with five parallel controls, with each group or subgroup consisting of 20 individuals. Each test goldfish was immunized with one of the four Ich vaccine preparations administered by one of the three routes: live trophonts by gavage (Group 1), formalin‐fixed trophonts by injection (Group 2) or by immersion (Group 3), freeze‐thawed trophont lysates by injection (Group 4), and trophont cilial and cell cortical fractions by injection (Group 5). The fish were then challenged by a standard Ich theront challenge on Day 21 postimmunization. For every 10 d following immunization, we tested the in vitro immobilization of Ich theronts by sera or mucus from immunized and control fish. We found that although all control (nonimmunized) and Group 3‐immunized goldfish died following theront challenge, more than half (55–65%) of the immunized fish from Groups 1, 2, 4, and 5 survived. Furthermore, except for the fish from Groups 3 and 4, the number of mean days to death in each test group was significantly higher than that in the respective control. These results indicate that goldfish immunized by injection or by gavage with any of the four vaccine preparations gained resistance to Ich infection. Further analysis indicated that this protective immunity was closely associated with the in vitro immobilization of Ich theronts by fish sera or mucus.     

Two in vitro methods for screening potential parasiticides against Ichthyophthirius multifiliis using Tetrahymena thermophila

Ichthyophthirius multifiliis (Ich) is a ciliate parasite that infects many species of freshwater fishes worldwide and causes heavy economic losses in aquaculture. Currently, parasiticides for controlling this parasite are limited, and few pond‐practical chemical therapies exist. Hence, the search for new parasiticides is urgently needed. One challenge confronting the screening of potential parasiticides is the difficulty in raising enough parasite for efficacy testing as Ich is an obligate parasite. This study used species of Tetrahymena, Ich‐related and cultivable ciliate protozoa, to evaluate two in vitro methods to test parasiticides. Plate counting and MTS assays (CellTiter 96^® AQ_ueous Non‐Radioactive Cell Proliferation Assay) were used to compare lethal concentrations or median lethal concentrations (LC₅₀) of copper sulphate, formalin and malachite green between T. thermophila and Ich theronts or between T. thermophila and Ich tomonts. The parasiticides that killed T. thermophila have been demonstrated to kill theronts or tomonts. These in vitro methods using T. thermophila can be used to screen novel parasiticides against Ich.     

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.     

Antibody mediated immune response against Ichthyophthirius multifiliis using excised skin from channel catfish, Ictalurus punctatus (Rafinesque), immune to Ichthyophthirius

This study investigated antibody mediated immune response against Ichthyophthirius multifiliis (Ich) by determining whether theronts would retain the potential for reinfection, both in vitro and in vivo, after treatment with the culture fluid of excised skin from channel catfish, Ictalurus punctatus , immune to Ich. The invasion was reduced significantly ( P  < 0.05) for theronts treated with the immune culture fluid compared with those treated with the culture fluid from naive fish. The treatment of theronts with the immune culture fluid greatly reduced the size and survival of trophonts compared with those treated with the culture fluid from naive fish. Fewer fish were infected and the infection density was less for fish exposed to theronts treated with immune culture fluid. The infection was severe for fish invaded by theronts treated with the culture fluid from naive fish, with a high number of infected fish and heavy density of trophonts per fish. All fish were infected by Ich when exposed to the theronts treated with the immunoadsorbed culture fluid. In summary, results of this study show that cutaneous antibodies in the culture fluid of excised skin from immune fish significantly reduces theront infectivity by immobilizing or weakening theronts.     

Use of formalin and copper to control ichthyophthiriosis in the Australian freshwater fish silver perch (Bidyanus bidyanus Mitchell)

Infestations of the protozoan parasite, Ichthyophthirius multifiliis, cause the serious disease ichthyophthiriosis in freshwater fish throughout the world. Formalin is a recommended treatment for ichthyophthiriosis in the Australian fish silver perch (Bidyanus bidyanus Mitchell), but the disease is difficult to control in ponds, particularly at low water temperatures. Experiments were carried out to develop an improved treatment regime for formalin and to evaluate copper as a therapeutant. Silver perch fingerlings infested with I. multifiliis were stocked into 55 L aquaria at temperatures of 14.8–17.6 °C and alkalinities of 70–110 mg L^?1. Formalin (34–38% formaldehyde) or copper (24.5% copper sulphate) were added to the aquaria and then monitored and readjusted to nominal concentrations daily. A concentration of 30 mg L^?1 formalin controlled ichthyophthiriosis, but fish treated with 20 mg L^?1 remained infested with theronts and trophonts on day 17; survival at both concentrations was 100%. A concentration of 10 mg L^?1 formalin did not control ichthyophthiriosis and all fish were dead from the infestation by day 17. Fish treated with 0.1 or 0.2 mg L^?1 copper were free of theronts and trophonts by days 17 and 14, respectively, and survival was 100%. Survival at 0.05 mg L^?1 copper was 100%, but fish remained infested. At 0.25 mg L^?1 copper, survival was 82.5% and there were no theronts or trophonts on gill and skin tissues of fingerlings by day 14. There was total mortality of fish treated with 0.5 or 1.0 mg L^?1 copper suggesting these concentrations are toxic to silver perch. All fish in infested‐control treatments died. In earthen ponds containing silver perch, 0.2 mg L^?1 copper was depleted to below 0.1 mg L^?1 within 24 h, and concentrations of 25–38 mg L^?1 formalin were depleted to below 15 mg L^?1 within 48 h. Treatment regimes involving daily applications of formalin or copper controlled ichthyophthiriosis in silver perch in earthen ponds at costs of $US466.37 and $US65.58 hectare^?1 day^?1 respectively. This study has developed a new formalin‐treatment regime for the control of ichthyophthiriosis, and demonstrated that copper sulphate is a potential therapeutant for this serious disease of silver perch.     

Protective effect of cutaneous antibody produced by channel catfish,Ictalurus punctatus (Rafinesque), immune to Ichthyophthirius multifiliis Fouquet on cohabited non-immune catfish

Fish which survive a sublethal ichthyophthiriasis acquire protective immunity against Ichthyophthirius multifiliis Fouquet (Ich). This study evaluated the protective effect of cutaneous antibody secreted by channel catfish, Ictalurus punctatus (Rafinesque), immune to Ich on cohabited non-immune catfish. Non-immune and immune fish controls were separately maintained and infected with theronts. The Ich infection was assessed by scoring 0, < 50, 50-100, and > 100 trophonts fish(-1) at 5 days post-infection. The results of infection showed that cohabited fish at the ratio of 15 non-immune to two immune fish had < 50 trophonts fish(-1). Eighty per cent of the cohabited fish at the ratio of 10 non-immune to two immune fish showed 0 or < 50 trophonts fish(-1). The 76% of control non-immune fish had more than 100 trophonts fish(-1). The control immune fish had 0 trophonts fish(-1). Anti-Ich antibody was detected using enzyme-linked immunosorbent assay in water samples taken from tanks containing immune fish after the water samples were concentrated 40-fold. The study suggests that immune fish cohabited with non-immune fish may protect non-immune fish against Ich 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.     

Effects of Water pH and Hardness on Infection of Silver Catfish,Rhamdia quelen,Fingerlings by Ichthyophthirius multifiliis

The aim of this study is to determine the effect of (1) different pH levels (5, 6, 7, 8, and 9) with water hardness 20 mg/L CaCO₃ and (2) pH 5 and 7 with varying water hardnesses (20, 60, and 120 mg/L CaCO₃) on the intensity of infection by Ichthyophthirius multifiliis in fingerlings of the silver catfish, Rhamdia quelen. In Experiment 1, the lowest and highest mortality rates were observed in fingerlings exposed to pH 5 and 9, respectively; in Experiment 2, fish kept at pH 5 and 7 with hardness 20 mg/L CaCO₃ showed significantly lowest and highest cumulative mortality, respectively, than those maintained in other conditions. The lowest intensity of trophonts/fingerling was observed at pH 5 and hardness 20 mg/L CaCO₃ in both experiments. We conclude that infection by I. multifiliis is less severe in silver catfish maintained at pH 5 and hardness 20 mg/L CaCO₃. High water hardness led to an increased intensity of trophonts and decreased survival in silver catfish kept at pH 5, but decreased intensity of trophonts and improved survival when the fish were kept at pH 7.