Hepatic lesions caused by migrating larvae of Ascaris suum in chickens

Group A consisted of chickens infected with a single dose of Ascaris suum and group B of chickens infected with two successive doses. At days 1, 3, 7, 14 and 21 after the first or second infection dose, six chickens from each group were sacrificed. In both groups, larvae were recovered from the livers on days 1, 3, and 7 and lungs on days 3 and 7. No larvae were detected in chickens on day 14. Clear white lesions were noticed only on the livers from chickens of group B at day 7 but had disappeared at day 14. A comparison with group B showed mild histological changes that developed relative to the livers from group A.     

Screen for anthelmintics, using larvae of Ascaris suum

A multiwell culture system was used to assay the effects of 12 known anthelmintic compounds on Ascaris suum larval development from 2nd-stage (L2; hatched from eggs) to early 3rd-stage (L3) and from in vivo-derived late L3 to early 4th-stage (L4). Larval survival, development, and motility were monitored for drug effects. Development of L2 to L3 was sensitive to thiabendazole, albendazole (ABZ), ABZ/sulfoxide, ABZ/sulfone (SO), mebendazole, L-tetramisole, D-tetramisole, piperazine, or closantel at a concentration of 0.01 microgram/ml; however, the effects of these drugs on larval development did not correlate well with known effects in vivo. The development of L3 to L4 was blocked by ABZ or mebendazole at 0.01 microgram/ml, by thiabendazole or ABZ/sulfoxide at 0.1 microgram/ml, and by ABZ/SO at 1.0 microgram/ml; however, except for ABZ/SO, most larvae were viable at these concentrations. In contrast, L-tetramisole or morantel appeared to inhibit development of L3 to L4 and to reduce survival at concentrations of greater than or equal to 1 microgram/ml; however, D-tetramisole was at least 10 times less effective. Haloxon, ivermectin, and closantel blocked development of L3 to L4 at 0.1, 1, and 10 micrograms/ml, respectively, in the absence of serum, but their activity was reduced by the presence of serum. Seemingly, in vitro development of A suum larvae was a convenient and sensitive bioassay for anthelmintic activity and could serve as a screen for anthelmintic residues in edible tissues.     

Optimization of the agar-gel method for isolation of migrating Ascaris suum larvae from the liver and lungs of pigs

Experiments on use of an agar-gel method for recovery of migrating Ascaris suum larvae from the liver and lungs of pigs were conducted to obtain fast standardized methods. Subsamples of blended tissues of pig liver and lungs were mixed with agar to a final concentration of 1% agar and the larvae allowed to migrate out of the agar-gel into 0.9% NaCl at 38 degrees C. The results showed that within 3 h more than 88% of the recoverable larvae migrated out of the liver agar-gel and more than 83% of the obtained larvae migrated out of the lung agar-gel. The larvae were subsequently available in a very clean suspension which reduced the sample counting time. Blending the liver for 60 sec in a commercial blender showed significantly higher larvae recovery than blending for 30 sec. Addition of gentamycin to reduce bacterial growth during incubation, glucose to increase larval motility during migration or ice to increase sedimentation of migrated larvae did not influence larvae recovery significantly.     

猪蛔虫幼虫琼脂胶移行法的建立

本实验首次在国内建立起了线虫的琼脂胶移行法 (Agar- gel Migration Assay,AMA)。通过对影响幼虫活力的某些理化因素 ,如 :琼脂胶的温度、每孔加入的琼脂胶和幼虫混合液的体积、琼脂胶的浓度及幼虫在琼脂胶内移行的时间等研究发现 :加胶的温度和加胶的量对幼虫的移出率有较大的影响。幼虫的移出率随加胶温度的升高而降低 ,当温度达到 70℃时 ,则几乎无虫体从琼脂胶内移出 (移出率仅为 0 .5 % ) ;幼虫的移出率随加入琼脂胶量的增多而减小 ,当每孔加入 10 0 0μl时 ,幼虫的移出率为 16 .6 8% ,当加入 4 0 0μl时幼虫的移出率则能达到2 5 .17%。琼脂胶的浓度对幼虫移出的影响并不明显 ,采用 SAS软件 (for Windows V6 .12 )分析发现 1.2 %、1.0 %、0 .8%、0 .4 %的结果间无显著差异 (P>0 .0 5 ) ,但琼脂胶的浓度能影响胶液凝固的时间和凝固后的韧性。实验表明 :最适宜的加胶量为 4 0 0μl /孔 ,最适宜的加胶温度为 5 3℃ (Agar- gel,sigm a) ,在琼脂胶中最佳培养移行时间为 2 4 h,琼脂胶液的最适实验浓度为 1.5 %琼脂胶     

猪蛔虫幼虫琼脂胶移行法的建立

本实验首次在国内建立起了线虫的琼脂胶移行法(Agar-gelMigrationAssay,AMA).通过对影响幼虫活力的某些理化因素,如琼脂胶的温度、每孔加入的琼脂胶和幼虫混合液的体积、琼脂胶的浓度及幼虫在琼脂胶内移行的时间等研究发现加胶的温度和加胶的量对幼虫的移行率有较大的影响.幼虫的移行率随加胶温度的升高而降低,当温度达到70℃时,则几乎无虫体从琼脂胶内移出(移行率仅为0.5%);幼虫的移行率随加入琼脂胶量的增多而减小,当每孔加入1000μl时,幼虫的移行率为16.68%,当加入400μl时幼虫的移行率则能达到25.17%.琼脂胶的浓度对幼虫移出的影响并不明显,采用SAS软件(forWindowsV6.12)分析发现1.2%、1.0%、0.8%、0.4%的结果间无显著差异(P＞0.05),但琼脂胶的浓度能影响胶液凝固的时间和凝固后的韧性.实验表明最适宜的加胶量为400μl/孔,最适宜的加胶温度为53℃(Agar-gel,Sigma),在琼脂胶中最佳培养移行时间为24h,琼脂胶液的最适实验浓度为1.5%琼脂胶.     

Susceptibility of fourth-stage Ascaris suum larvae to fenbendazole and to host response in the pig

Fenbendazole given at the rate of 2.5 g/kg of feed for 3 days had 100% efficacy against 4th-stage Ascaris suum larvae in 8 pigs. Eight control pigs had a total of 108 A suum. In 6 pigs infected 3 times with 3rd-stage A suum larvae and treated with fenbendazole after the larvae molted to the 4th stage, the challenge exposure-derived population was decreased by 64%. Similar sequential infections in 6 pigs similarly infected, but not treated with fenbendazole, decreased the challenge exposure-derived population by 98%; however, developing and/or adult worms from the vaccinating infections were present.     

Immunity of swine to Ascaris suum

Swine were hyperimmunized to Ascaris suum by giving multiple oral inoculations of embryonated eggs. Sera and lymphocyte lysate from these pigs were administered parenterally to 4-week-old pigs. The latter animals were no more resistant to larval migration than control pigs receiving sera or lymphocyte lysate from non-immunized pigs. Other pigs were infected with transmissible gastroenteritis (TGE) virus, allowed to recover and challenged with embryonated ascarid eggs. They likewise were no more resistant to ascarid larval migration than control pigs.     

Blocking of nonspecific IgG in indirect radioimmunoassay for detecting Ascaris suum antigens

The sensitivity of an indirect radioimmunoassay (IRIA) used for detecting larval body wall (LBW) antigens of Ascaris suum was enhanced by using various blocking agents which prevented nonspecific binding of immunoglobulin G (IgG) or free ¹²⁵I without preventing binding of specific antibodies to the antigen. The use of blocking agents reduced counts for both positive and negative sera, resulting in an increase in calculated binding ratios (BR) and deltas and, thus, in the sensitivity of the assay. The relative effectiveness of blocking agents, in decreasing order, were turkey serum (TS), rabbit gamma globulin (RGG), rabbit whole serum (RS), bovine serum albumin (BSA), rabbit IgG (RIgG), bovine alpha globulin (BAG), bovine gamma globulin (BGG) and Tween-20.     

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。     

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.     

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.     

Attempts to separate female Ascaris suum antigen and to investigate its partial characterization

The location and separation of Ascaris suum antigen for serological testing was investigated. The antigenic constituent was rich in the ovary of the adult worm and was obtained by dialysis with 50% ammonium sulphate saturated solution. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting analysis demonstrated that the heat labile antigenic preparation showed one major and seven faint bands. The major band seemed also to be a glycoprotein. The sera from pigs with/without hepatic milk spot showed relatively high precipitation titres, while, those from the specific pathogen free pigs manifested low titres.