苏云金芽孢杆菌晶体蛋白对钉螺体内日本血吸虫幼虫的作用

将不同菌株、不同浓度的苏云金芽孢杆菌伴胞晶体蛋白作用于钉螺体内的日本血吸虫幼虫阶段，经24h和48h后，通过显微镜观察，结果发现4种苏云金芽孢杆菌伴胞晶体蛋白均对钉螺体内血吸虫幼虫有毒杀作用，其中Bt-02的毒杀作用最强。     

苏云金芽胞杆菌伴胞晶体毒素对小鼠体内猪蛔虫三期幼虫的作用及其免疫组织化学定位

将培养好的感染性猪蛔虫卵以每只5×103个虫卵感染小鼠,于次日以不同方式(灌胃、肌肉注射、静脉注射和腹腔注射)注入苏云金芽胞杆菌伴胞晶体蛋白(insecticidal crystal proteins,Icps),对照组静脉注射BSA(小牛血清白蛋白),每只0.5mL,连续3d。小鼠于第6天剖杀,肝脏分离幼虫,计算减虫率。取肝脏,以4%多聚甲醛固定48h,制作石蜡切片,应用免疫组织化学SABC法定位蛔虫幼虫体内的Icps。研究结果显示,静脉注射晶体蛋白毒素效果最好,减虫率达到72.7%;肌肉注射、灌胃、腹腔注射效果次之,分别为19.1%、14.6%、14.36%。免疫组织化学研究显示,Icps结合到虫体的体表及肠道。     

苏云金芽孢杆菌毒素对羊捻转血矛线虫第三期幼虫毒力研究

从人工感染捻转血矛线虫山羊的直肠内取出粪便，在２５℃温箱中培养７ｄ，用贝尔曼法分离出第三期幼虫，将它们加入含苏云金芽孢杆菌以色列亚种伴孢晶体毒素的培养液中，置２５℃下培养。当伴孢晶体蛋白含量为４００ｇ／ｍｌ时，幼虫的死亡率２４ｈ后为９４．７％，４８ｈ后为９７．３％。毒素对幼虫作用２４ｈ与４８ｈ，其毒力无显著差异（Ｐ〉０．０５），并且作用２４ｈ与４８ｈ，其ＬＤ５０值伴孢晶体蛋白含量分别为８４．０５μ     

涂林吉（苏云金）芽胞杆菌伴胞晶体毒素对捻转血矛线虫虫卵的致死作用

从感染捻转血矛线虫山羊的直肠内取粪便，分离出虫卵，置单克隆板孔中，再往各孔中加入系列浓度的涂林吉（苏云金）芽胞杆菌（ＹＢＴ－１９５３）伴胞晶体毒素溶液，置２５℃培养２４ｈ。结果表明，当伴胞晶体毒素总蛋白为１２０μｇ／ｍＬ时，虫卵死亡率为９９．４％，０．６％未死的虫卵孵出的第１期幼虫也因毒素的作用而全部死亡。本研究表明，涂林吉芽胞杆菌（ＹＢＴ－１９５３）伴胞晶体蛋白对捻转血矛线虫虫卵具有较强的毒力。     

产气荚膜梭菌和腐败梭菌在厌氧菌基础培养基中的生长特性

测定了产气荚膜梭菌B型、D型和腐败梭菌在新研制的厌氧菌基础培养基中的生长曲线和产毒能力等特性。结果表明,这些细菌可以在新培养基中良好生长和产毒。产气荚膜梭菌B 型经2 h 的适应期后,很快进入对数生长期,培养到8 h细菌密度接近最高值,OD630为4.329;经检测,培养4 h~24 h的细菌所产生的毒素(培养物上清)1μL~2μL可致死KM小鼠。产气荚膜梭菌D型的生长速度也较快,无适应期就直接进入对数生长期,培养到12 h 时细菌密度最高, OD630 为1.75;细菌最佳产毒期在6 h~14 h,培养物上清0.5μL~0.65μL可致死KM小鼠。腐败梭菌生长速度较慢,经6 h的适应期后,才进入对数生长期,培养到14 h时细菌密度最高,OD630为1.335;细菌毒力在培养后的6 h~24 h内10μL菌液可达到在24 h内致死KM小鼠的效果。     

印楝油对兔疥螨幼虫的体外杀螨活性

将兔疥螨幼虫置于聚苯乙烯小平皿中，分别加入不同浓度的印楝油液体名蜡溶液，以天然除虫菊酯和阿维菌素为阳性对照，蒸馏水和液体石蜡为阴性对照，观察记录不同时间段的螨虫死亡数，以死亡率、半数致死时间（LT50）和半数致死浓度（LC50）为指标评价了印楝油对兔疥螨幼虫的离体杀螨活性。结果显示，未稀释的印楝油能在25min内杀死所有幼螨，其杀螨活性显著强于500g／L的天然除虫菊酯（825min，P〈0．01），而与25g／L的阿维菌素无显著差异（19min，P〉0．05）；500、250和125mL／L印楝油对兔疥螨幼虫的LT50分别为1、2、5h；24h的LC50和LC95分别为2．908和12．018mL／L。结果证实，印楝油对兔疥螨幼虫具有较好的离体杀螨活性。     

3株少孢节丛孢菌分离株通过绵羊消化道试验的评估

本研究旨在评估3株少孢节丛孢菌(Arthrobotrys oligospora)通过绵羊消化道后对粪便中三期幼虫(L3)的捕杀活性。利用实验室保存的当地分离的3株A. oligospora,用液固双相法培养分生孢子,每株菌制成5×105的分生孢子悬液,按照5×105个孢子/kg(活重)经口给3～4月龄人工感染有捻转血矛线虫的绵羊灌服,真菌灌服0 h灌服前4 h(对照组)及灌服后24 h(试验组)收集绵羊的粪便样品,然后进行粪便培养分离和计数三期幼虫(L3),统计不同时点取样时三期幼虫的平均数,计算杀虫率。3株少孢节丛孢菌中,2个菌株对照组和试验组粪便中获得的L3平均数,差异显著,一株不显著,通过绵羊消化道后对粪便中三期幼虫的捕杀率分别为17.84%,46.00%,99.47%。结果证明,3株A. oligospora中1株具有通过绵羊消化道的能力并具有良好的杀虫活性,1株通过绵羊消化道的能力较弱,1株不能通过而被灭活。     

农药灭多威不同剂型产品对家蚕的毒性评价

为了评估农药灭多威不同剂型产品应用于桑园害虫防治对家蚕安全性的影响,选用目前生产上常用的20%灭多威乳油、24%灭多威可溶性液剂和40%灭多威乳油3种不同剂型产品,稀释成不同质量浓度药液浸泡桑叶后给家蚕3龄幼虫添食,测定其对家蚕的急性毒性。结果表明:3种剂型产品对家蚕3龄幼虫的24 h食下LC50在14.626 4～17.617 9 mg/L之间,48 h食下LC50在10.760 3～11.498 7 mg/L之间,72 h食下LC50在9.450 2～10.213 7 mg/L之间,差异均不显著(P>0.05);3种剂型产品对家蚕3龄幼虫的24 h毒力和72 h毒力之间差异显著(P<0.05);3种剂型产品对家蚕3龄幼虫的毒力回归方程的坡度均在48 h最大,说明灭多威对3龄幼虫的毒力发挥最大时间在食下后48 h。将灭多威农药的3种剂型产品稀释成含灭多威质量浓度为66.67、133.33、266.67 mg/L的3种药液,分别喷施桑树后检测对家蚕的残毒期长短略有差异,以24%灭多威可溶性液剂3 600倍稀释液处理组的残毒期最短(8 d),结合毒力测定,确定灭多威3种剂型产品对家蚕的安全性间隔期为8～12 d。     

蜜蜂球囊菌胞外蛋白酶活性与菌株毒力关系的研究

为了探讨蜜蜂球囊菌(Ascosphaera apis)胞外蛋白酶活性(PU)与其毒力的关系,评价胞外蛋白酶活力作为毒力指标的可靠性。本研究测定了9个不同来源地的蜜蜂球囊菌菌株的胞外蛋白酶活性及接种后各日累积蜜蜂幼虫死亡率和致死中时(LT50),将胞外蛋白酶活性与接种后各日累积死亡率及致死中时(LT50)进行回归分析,以确定胞外蛋白酶活性与其对蜜蜂幼虫的毒力间的相关性。结果表明各菌株胞外蛋白酶活性与其毒力间的存在显著相关性。因此,蜜蜂球囊菌胞外蛋白酶活性可作为大量菌株筛选的参考毒力指标,但不能作为测定相关性的唯一指标。     

不同时期旋毛虫抗原对肥大细胞的作用

观察旋毛虫不同时期抗原对肥大细胞释放组胺和β-氨基己糖苷酶的影响。用质量浓度为1、5、25、50、100μg/L的肌幼虫抗原、成虫抗原、新生幼虫抗原分别与2.5×104的肥大细胞37℃条件下作用10min。采用荧光检测系统检测在上清液面及下层颗粒组分中的组胺水平,计算组胺释放率;加入20μL相同质量浓度的旋毛虫不同时期抗原分别与50μL 5mol/L对硝基酚-N-乙酰-β-D-葡糖胺和肥大细胞37℃孵育2h,酶标仪测定D值,计算β-氨基己糖苷酶释放率。结果显示,25μg/L肌幼虫抗原刺激肥大细胞释放组胺率最高,50μg/L最低;成虫抗原50μg/L最高,1μg/L最低,但均高于阴性对照组(P<0.05);而新生幼虫刺激肥大细胞不释放组胺,旋毛虫各期抗原不刺激肥大细胞释放β-氨基己糖苷酶。结果表明,肥大细胞在抗寄生虫感染的最初阶段扮演重要角色。     

伊维菌素浇泼剂治疗猪蛔虫及血虱的驱除效果

应用0.5%伊维菌素浇泼剂进行了驱除猪血虱和猪蛔虫的试验。结果:给药后第7d对猪血虱的转阴率达100%;第15d对猪蛔虫的驱虫率达到100%,表明0.5%伊维菌素浇泼剂对驱除猪蛔虫和血虱是高效安全的。     

使君子提取物对驱除猪蛔虫效果研究及对猪主要生理生化指标的影响

云南使君子提取物作为实验用药,对感染猪蛔虫的动物进行了药效实验研究。结果使君子提取物给药最佳剂量为0．05mg／kg。粪检虫卵数计数表明,每克粪便虫卵数与用药前相比存在差异显著性（P〈0．05）。所检测的血液生化指标在给药前后均没有出现显著性的变化（P〉0．05）。从而可初步推断使君子提取物对猪的肝脏、肾脏、心脏和肌肉等组织器官没有产生明显的影响。使君子提取物对猪蛔虫有较好的驱虫效果,且毒副作用小。     

烟焦油及被动吸烟对小鼠肺芳香烃羟化酶活性的影响

为研究烟焦油和被动吸烟对小鼠肺芳香烃羟化酶(AHH)活性的影响,同时观察烟焦油对AHH活性影响的剂量反应关系和时间反应关系,以1%LD50(5.29 mg/kg),2%LD50(10.58 mg/kg)和3%LD50(15.87mg/kg)的烟焦油分别使小鼠染毒24,48 h和72 h,每日1次,然后测定肺AHH的活性。被动吸烟组置于体积50 L自制染毒柜中,烟雾浓度为69.41 g/m3,每日2次。对照组不吸烟,但处于相同条件。2周后测定肺AHH的活性。结果表明,2%LD50烟焦油组在72h后和3%LD50烟焦油组在48 h和72 h后均可以提高小鼠肺AHH的活性。2%LD50和3%LD50烟焦油作用小鼠72 h和48 h比较,对AHH活性的影响差异显著(P<0.05)。被动吸烟也可提高小鼠肺AHH的活性。表明烟焦油在一定的浓度和时间内可使AHH的活性上调,这种上调作用表现为剂量依赖性和时间依赖性,被动吸烟也可提高小鼠肺AHH的活性。     

Identification of larval-stage antigens of ascaris suum recognized with immune sera from pigs.

Antigens were identified from the third-stage larvae (L3) and lung-stage larvae of Ascaris suum by two-dimensional immunoblot method with antisera obtained from pigs that received chemically abbreviated Ascaris suum larval infections. Forty-seven and 13 spots were recognized as antigens from the L3 and lung-stage larvae, respectively. Their apparent molecular weight ranged from 20 to 101 kDa and their isoelectric point from 3.6 to 8.0. The present study provides a framework for further molecular cloning of those antigens and consequently leads to the development of recombinant peptide vaccines against A. suum.     

苏云金芽胞杆菌伴胞晶体蛋白对猪体内蛔虫的作用及其血液生理生化指标的分析

感染性猪蛔虫卵以每头份3000个卵的量感染断奶仔猪，于第3天开始肌肉注射不同剂量苏云金芽胞杆菌晶体蛋白（insecticidal crystal proteins，ICPs），每天1次，连续4d。同时测定猪血液生理生化指标，饲喂2个月后收集猪粪便计算虫卵数。结果显示，感染猪出现咳嗽、发热等临床症状，GOT、GPT、ALP、LDH等指标明显升高，经ICPs治疗后又恢复正常。粪便检查，ICPs高剂量组（16．08mg）的EPG（每克粪便虫卵数）为0，而ICPs低剂量组（9．45mg）的EPG为394，未经ICPs作用对照组EPG为2113，差异极显著（P〈0．01），证明ICPs对猪体内猪蛔虫具有较好的杀灭作用。     

Comparative studies of experimental oral inoculation of pigs with Ascaris suum eggs or third stage larvae

This experimental study was designed to compare the acquired resistance in pigs to Ascaris suum eggs following 4-weekly oral immunizations with either 200 A. suum infective eggs or 50 A. suum third stage larvae (L3). The two immunized groups (n = 7) together with an unimmunized control group (n = 7) of pigs were challenged orally with 50 infective A. suum eggs per kilogram bodyweight on day 19 after the last immunization. Seven days post-challenge the group immunized with eggs showed signs of resistance as evidenced by reduced lung larval counts compared with the challenge control group. Such significant resistance was not observed in the L3-immunized group. However, a markedly increased inflammatory liver reaction and white spot formation was demonstrated in the L3-immunized pigs after challenge compared with both control animals and egg-immunized pigs. On the day of challenge only the egg-immunized pigs mounted an anti-Ascaris antibody response both in serum and in lung lavage fluid. Ascaris-antigen induced increased histamine release from peripheral leucocytes following both immunization and challenge could only be demonstrated in the egg-immunized pigs. On day 7 post-challenge local IgA-anti-Ascaris antibodies were further demonstrated in bile of the egg-immunized group and in the small intestine of both immunized groups. In conclusion, oral A. suum egg immunization of pigs induced a significant reduction in lung larval counts upon challenge. In contrast, oral L3 immunization seemed to prime the pigs as observed by the presence of stunted lung larval growth and increased liver reaction post-challenge with A. suum eggs.     

猪蛔虫性别特异基因在第三、四期幼虫的表达谱研究

为研究猪蛔虫性别特异基因在第三期、第四期幼虫的表达情况，本研究采用表达谱基因芯片技术，从所构建的猪蛔虫雌、雄成虫cDNA消减文库中分别挑取克隆，制备成cDNA微阵列芯片、标记有荧光素Cy5-dUTP和Cy3-dUTP的各组探针（L3＋♀；L3＋♂；L4＋♀；L4＋♂）分别与制备好的芯片进行杂交、扫描，原始信号值经均一化处理后，获取每个点的Ratio值（Ratio=Cy5／Cy3）．根据该值筛选出表达差异最明显的841个克隆，进行测序分析，在获得的707个有效序列中有61个是猪蛔虫新的ESTs、将在第三、四期幼虫以及成虫中显著表达的克隆进行排列组合比较，获得各期特有和各期之间共有的表达克隆一在第三期幼虫中，雌、雄性别特异基因分别有21和9个，编码的主要蛋白有卵黄原前导蛋白、睾丸幼胚蛋白等；在第四期幼虫中特异表达的雌、雄性别基因分别有22和6个，其中免疫抑制卵巢信息蛋白、主要精子纤维蛋白（MFP）等表达明显本项研究结果不仅初步阐明了猪蛔虫性别特异基因在第三、四期幼虫的表达情况，而且亦为筛选控制猪蛔虫病的“关键”基因并阐明其功能奠定了基础。     

猪蛔虫感染期幼虫抑制消减cDNA文库的构建

为筛选与线虫感染性相关的基因,本研究以猪蛔虫为对象,构建猪蛔虫感染期幼虫差异表达消减cDNA文库,为研究线虫期特异性发育的分子机制奠定基础。分别提取感染期幼虫和其它各期幼虫及成虫的总RNA,纯化mRNA后,采用Clontech公司PCR-selectTM试剂盒进行反转录合成cDNA并进行抑制消减杂交(SSH),构建猪蛔虫感染期幼虫差异表达的消减cDNA文库,并采用Southern斑点杂交进行消减效率的检测。随机从文库中抽取45个克隆进行测序及在线BLAST分析。试验结果表明,感染期幼虫差异表达的消减cDNA文库具有较强的特异性;在得到的41个表达序列标签(ESTs)中,有40个ESTs与已报道的基因有较高的相似性,主要代表猪蛔虫第三期幼虫基因和成虫头部基因,有1个cDNA片段可能代表新基因。猪蛔虫感染期幼虫差异表达消减cDNA文库的成功构建,为进一步研究幼虫发育差异表达基因的功能奠定了基础。     

Immunohistochemical distribution of antigens in liver of infected and immunized pigs with Ascaris suum

In the present work, we carry out an immunopathological study of the swine ascariosis, under different conditions (control, infection and immunization). Twenty-one Iberian pigs were used and divided in seven groups. Groups 1 and 2 were the uninfected and challenged controls, respectively. Groups 3 and 4 were weakly infected with increasing doses of Ascaris suum eggs and treated with pyrantel (Group 4). Groups 5-7 were immunized with 14, 42 and 97 kDa proteins from the parasite, respectively. Groups 2-7 were challenged with 10,000 infective eggs 7 days before the sacrifice. The focal parasitic granulomata with eosinophils and lymphocytes were the main histopathological lesions in the liver of reinfected pigs, while more marked cellular infiltrate and abundant connective tissue were seen in the livers of immunized animals. There were important deposits of antigens in the livers of immunized and infected pigs. Antigens were mainly located in the connective tissue, with positive staining detection of the somatic larvae antigen, the body wall from the adult worms and the 14-, 42- and 97-kDa proteins. However, cholangiols, biliary ducts and macrophages presented an immunohistochemical positive stain against excretory-secretory and somatic antigens from the larvae and the body fluid antigen from the adult parasite. The detection of A. suum antigens in the liver of infected pigs improves the diagnosis of swine ascariosis. It may be possible to apply these procedures for diagnosis of human ascariosis in liver biopsies since A. suum from swine have been previously used as a substitute for the study of the human parasite Ascaris lumbricoides.     

Anthelmintic paraherquamides are cholinergic antagonists in gastrointestinal nematodes and mammals

Oxindole alkaloids in the paraherquamide/marcfortine family exhibit broad-spectrum anthelmintic activity that includes drug-resistant strains of nematodes. Paraherquamide (PHQ), 2-deoxoparaherquamide (2DPHQ), and close structural analogs of these compounds rapidly induce flaccid paralysis in parasitic nematodes in vitro, without affecting adenosine triphosphate (ATP) levels. The mechanism of action of this anthelmintic class was investigated using muscle tension and microelectrode recording techniques in isolated body wall segments of Ascaris suum. None of the compounds altered A. suum muscle tension or membrane potential. However, PHQ blocked (when applied before) or reversed (when applied after) depolarizing contractions induced by acetylcholine (ACh) and the nicotinic agonists levamisole and morantel. These effects were mimicked by the nicotinic ganglionic blocker mecamylamine, suggesting that the anthelmintic activity of PHQ and marcfortines is due to blockade of cholinergic neuromuscular transmission. The effects of these compounds were also examined on subtypes of human nicotinic ACh receptors expressed in mammalian cells with a Ca2+ flux assay. 2DPHQ blocked nicotinic stimulation of cells expressing alpha3 ganglionic (IC50 approximately 9 microm) and muscle-type (IC50 approximately 3 microm) nicotinic cholinergic receptors, but was inactive at 100 microm vs. the alpha7 CNS subtype. PHQ anthelmintics are nicotinic cholinergic antagonists in both nematodes and mammals, and this mechanism appears to underlie both their efficacy and toxicity.