Examination of murine antibody response to secondary hydatidosis using ELISA and immunoelectrophoresis

Summary Antibody responses in mice with up to 64 weeks of secondary Echinococcus granulosus hydatidosis were examined by ELISA using hydatid protoscolex antigen (Px), Antigen 5 (Ag5) and Antigen B (AgB), and by immunoelectrophoresis (IEP) using sheep hydatid cyst fluid (SHCF). Anti-Px IgG antibodies, evident from 3–5 days post infection (p.i.), increased steadily until 16 weeks and maintained a high level afterwards. Anti-Ag5 IgG antibodies were negligible up to two weeks, but they showed a small increase around 2–3 weeks which was followed by a big increase around 16 weeks p.i. The high level of anti-Ag5 IgG antibodies persisted to the end of experiment. The level of anti-AgB IgG antibodies remained relatively low throughout infection. Anti-Px IgM antibodies appeared in the early period of infection, but became insignificant as the infection proceeded. Specific IgM antibodies to Ag5 and AgB showed two waves of increase, one between 3 days to 4 weeks p.i. and the other between 16 weeks to 46 weeks p.i. The level of IgA antibodies to Ag5 and AgB was low and only a moderate amount of anti-Px IgA antibodies was detected. Generally, a higher level of serum antibodies are associated with a larger number of mature cysts. Serum samples from 5 of 8 mice harbouring hydatid cysts formed 1–3 bands with SHCF in IEP, including Arc 5, but a precipitation arc with AgB was not observed. Analysis of hydatid cyst fluid from the infected mice (MHCF) in IEP also failed to demonstrate AgB. Despite the high levels of antihydatid antibodies generated in the infected mice, protoscoleces appeared to be unhindered in their growth to mature cysts.     

Antigenic polypeptides of Echinococcus granulosus oncospheres and definition of protective molecules

Immunoblotting and in vitro oncosphere-killing were used to identify a putative protective molecule in Echinococcus granulosus mature oncospheres. A range of sera from sheep that had been shown to be protected against E. granulosus , and from those that were not, were tested. The sera used were obtained from sheep hyperimmunized with E. granulosus oncospheres, or immunized with oncosphere non-denatured extract, with immature oncosphere extract or with denatured extracts of oncospheres. Results indicated the involvement of native antigens of 23, 25, 30, 34 and 40 kDa in the protective response to E. granulosus infection. The rapid appearance of antibodies to the 23, 25 kDa antigens, their association with early onset of protection and in vitro oncosphere lysis by affinity-purified antibodies obtained from these fractions, indicated that these antigens contained protective epitopes. Final confirmation was provided by immunization of sheep with fractions prepared by preparative SDS/PAGE, and challenge infection. Only the fraction containing the 23 and 25 kDa molecules was able to stimulate protection. Antisera against this pair of molecules should provide a useful probe for screening an E. granulosus oncosphere cDNA library to identify clones expressing protective molecules.     

细粒棘球蚴重组抗原rEgferritin诱导宿主抗感染免疫应答的初步研究

目的 初步探讨细粒棘球蚴重组铁蛋白rEgferritin免疫小鼠产生的抗细粒棘球蚴感染的免疫机制.方法 rEgferritin免疫ICR小鼠3次、同时设立佐剂组和对照组,第12w用细粒棘球蚴原头蚴对3组小鼠进行攻击感染,5个月后剖杀小鼠,检查各组小鼠腹腔内包囊个数,根据公式计算保护力.分别于0w,12w,32w取血,制...     

Analysis of host components in hydatid cyst fluid and immunoblot diagnosis of human Echinococcus granulosus infection.

To improve serodiagnosis of cystic hydatidosis, immunoblotting studies were performed to look for a highly specific parasite antigen(s). First, commercially available hydatid cyst fluid antigen preparations were characterized by SDS-PAGE and by immunoblotting with sera specific for parasite and host animal proteins. One preparation, designed for use in complement fixation tests, did not appear to be suitable for immunoblotting because of the low concentrations of parasite antigens. Several host proteins, including serum albumin and IgG, were detected in the cyst fluid. Sera from patients with Echinococcus granulosus infections and other parasitic diseases were examined by immunoblotting for antibodies against specific cyst fluid parasite antigens. Several parasite antigens were variably recognized. Only one antigen, a 40 kDa protein, was recognized by all E. granulosus-infected patients. Reactivity against this antigen was also observed in all sera from E. multilocularis, cysticercosis, and schistosomiasis patients as well as in some filariasis cases. Two E. granulosus antigens, molecules of 12.5 and approximately 17 kDa, were only recognized by antibodies from some E. granulosus patients.     

细粒棘球绦虫(中国大陆株)亲肌肉抗原重组蛋白的免疫保护性研究

目的探讨细粒棘球蚴(Echinococcus granulosus,Eg)亲肌肉抗原重组蛋白的免疫保护性及其作为候选疫苗的潜在价值。方法ICR小鼠随机分为蛋白免疫组和佐剂对照组,每隔2w皮下免疫1次,在第3次免疫后2周,用Eg原头蚴进行攻击感染,感染后20w剖杀小鼠,检获棘球蚴包囊,计算免疫保护力,并用ELISA法测定血清中IgG及其亚型和IgE水平。结果与佐剂对照组比较,蛋白免疫组小鼠的免疫保护力为94.46%;与免疫前比较,免疫后和攻击感染后蛋白免疫组小鼠血清IgG、IgG1、IgG3和IgE水平均明显升高(P<0.05),IgG2b降低(P<0.05)。结论细粒棘球绦虫亲肌肉抗原重组蛋白能诱导小鼠产生一定的保护性免疫,是潜在的疫苗候选抗原分子。     

''Arc 5'' antibodies in sera of sheep infected with Echinococcus granulosus, Taenia hydatigena and Taenia ovis

The 'arc 5' precipitin band, formed when test human serum is reacted against immunoelectophoresed hydatid cyst fluid antigen, has provided a positive diagnosis of Echinococcus granulosus infection. These antibodies to 'arc 5' antigen have now been found in the sera of sheep. They appear 2 weeks after infection with Taenia ovis, after 3 weeks with T. hydatigena and after 16 weeks with E. granulosus. An antigen similar to the 'arc 5' antigen of E. granulosus cyst fluid was also demonstrated in cyst fluid from T. hydatigena, but it would not be positively identified in T. ovis cyst fluid. The presence of 'arc 5' in immunoelectrophoresis tests is not suitable for specific immunodiagnosis of E. granulosus infections in sheep in New Zealand. 'Arc 5' antibodies were only associated with living E. granulosus cysts and were not present if cysts were dead. The location of the cysts in either liver or lungs and the onset of brood capsule production did not influence the presence of 'arc 5' antibodies.     

Antibody response of Echinococcus granulosus infected mice: recognition of glucidic and peptidic epitopes and lack of avidity maturation

The antibody response was followed weekly during 68 weeks in 17 Balb/c mice intraperitoneally (i.p.) infected with 2000 Echinococcus granulosus protoscoleces (PSC) and in three mice i.p. immunized with 2000 dead PSC. Antibodies against hydatid cyst fluid (HCFA) and its peptidic (periodate-resistant) and carbohydrate (periodate-sensitive) epitopes were titrated by ELISA. Avidity and the antigen recognition pattern of antibodies were also analysed during infection and immunization by ELISA and immunoblot, respectively.
The antibody response of infected mice showed quantitative and qualitative variations during infection, since both titre as well as recognition of peptide and carbohydrate epitopes in HCFA depended on time post infection. No avidity maturation was evident during the course of infection. Sera from infected mice recognized the 38 kDa subunit of Ag5 but did not react with the 8 kDa subunit of AgB. On the contrary, the antibody response of immunized mice showed only one peak of antibodies that recognized both peptidic and carbohydrate epitopes of HCFA. In addition, sera from these mice recognized mainly 60 and 110 kDa bands. Our results suggest that: a) avidity and antigen recognition patterns of antibodies in mice treated with live PSC are different from those treated with dead PSC; b) antibodies against HCFA glucidic or peptidic epitopes appear at different times post infection.     

Serum antibody response following parenteral immunization with hydatid cyst fluid in sheep infected with Echinococcus granulosus

Production of specific serum antibodies following immunization with hydatid cyst fluid antigens was investigated in sheep with Echinococcus granulosus infection and in noninfected controls. Six of 10 infected animals responded to intramuscular injection of antigen by rapid production of antibodies detected in indirect hemagglutination assays. Similar responses did not occur in any of 10 noninfected controls. It is suggested that differences in the rate of response to immunization with cyst fluid antigens between groups of sheep could be exploited in serodiagnosis of E. granulosus infection in sheep. The results also suggest that low levels of antibody found in the serum of sheep infected with E. granulosus are not the result of immunosuppression or immunological tolerance, but are due to sequestration of antigen from the immune system of the host.     

绵羊细粒棘球蚴囊液抗原诱发小鼠免疫反应及保护性免疫的研究

本文用绵羊细粒棘球蚴囊液抗原制剂,给小鼠3次腹腔注射诱发初次免疫,然后腹腔移植泡球蚴组织作攻击感染,观察保护性免疫。实验提示,注射免疫原制剂后1月能诱发小鼠免疫反应,间接血凝试验(IHA)可测出血清抗体;攻击感染后23周,IHA阳性率达94.4％,而未诱发初次免疫的对照鼠IHA阳性率为90.0％,表明无论是初次免疫或攻击感染,均能诱发小鼠产生抗体,二者无显著差别(P>0.05);但IHA血清滴度1:256百分率分别为94.1％和55.5％,有显著差别(P<0.01),表明攻击感染有强化初次免疫的作用。攻击感染后23周,对比免疫鼠和对照鼠的腹腔泡球蚴湿重均值和病理分级率均无显著差别(P>0.05),提示本文小鼠初次免疫未显保护性免疫之效。     

Echinococcus granulosus human infection stimulates low avidity anticarbohydrate IgG2 and high avidity antipeptide IgG4 antibodies

Total IgG and IgG subclasses recognizing carbohydrate and peptidic epitopes from native and periodate treated partially purified hydatid cyst fluid (ppHCFA) and protoscolex somatic antigens (PSA) were tested by ELISA in hydatid patients sera. Binding of the major cross-reactive antiphosphorylcholine antibodies was inhibited with free reagent. A predominant anticarbohydrate antibody response against ppHCFA and PSA is shown. Although the main contributing IgG subclass to the antipeptide response against both antigens was IgG4, IgG1 also significantly contributed to the anti-PSA peptidic epitopes response. Western blot showed that IgG1 antibodies strongly recognized in ppHCFA a periodate susceptible 38 kDa antigen. The IgG4 antibodies mainly recognized the periodate-resistant 12, 16 and 24 kDa antigens. In addition, IgG2 antibodies recognized three strongly periodate-susceptible broad bands (116, 55 and 24 kDa antigens). PSA-specific IgG1 and IgG4 antibodies showed similar patterns of antigen recognition as well as no significant reduction of reactivity after periodate treatment while the IgG2 antibody recognition was strongly affected by this treatment. Furthermore, IgG2 showed significantly lower avidities than IgG1 and IgG4 antibodies recognizing both antigens. In conclusion, hydatid patients showed an enhanced production of low avidity anticarbohydrate IgG2 as well as high avidity antipeptide IgG4 antibodies.     

Detection of specific circulating antigen, immune complexes and antibodies in human hydatidosis from Turkana (Kenya) and Great Britain, by enzyme-immunoassay

Circulating antigen, specific immune complexes (IgG and IgM) and specific antibodies (IgG, IgM, IgE and IgA) were detected by enzyme-linked immunosorbent assay (ELISA) in the sera of hydatid (Echinococcus granulosus) patients from Turkana (Kenya) and the UK. Specific IgG and IgM antibodies predominated in current UK hydatid infections, while all classes of specific antibodies were lower in the Turkana patients. Circulating antigen, detected in 3% polyethylene glycol (PEG) precipitated complexes, using peroxidase conjugated hyperimmune human hydatid IgG (Fab) was more specific in ELISA than either antibody or immune complex assays where peroxidase conjugated anti-human IgG was used. Anti-human immunoglobulin ('rheumatoid' factor) was not detected in hydatid sera. Serum antigen, specific IgM immune complexes and specific IgM antibodies were associated with UK cases of current hydatid infection in contrast to patients with previous histories of hydatidosis. In 3 hydatid patients (from UK) levels of circulating antigen and specific IgM immune complexes rapidly declined within 1-4 months after surgical cyst removal. The detection of specific IgG and antigen in PEG precipitated immune complexes from false-negative/low responder Turkana hydatid sera, suggests that antibody 'mopping' by specific antigen may be occurring. After SDS-PAGE/immunoblotting analysis, antigen of mol. wt 67 000, present in hydatid cyst fluid and protoscoleces, was identified as putative circulating antigen in 3% PEG precipitates of sera from albendazole treated hydatid patients.     

pIL-12质粒DNA对细粒棘球蚴感染小鼠免疫应答的影响

目的探讨pIL-12质粒DNA免疫对细粒棘球蚴感染小鼠免疫应答的影响。方法将pIL-12质粒DNA肌肉注射免疫BALB/c鼠,免疫后8w用Eg原头节进行攻击感染,感染后18w剖杀小鼠,计算减蚴率;取脾并分离脾细胞,流式细胞仪检测脾CD4+和CD8+T淋巴细胞亚群的百分比;用EgAg或ConA刺激培养脾细胞,收集脾细胞培养上清液,用试剂盒检测脾细胞培养上清液的IL-2、IFN-γ、TNF-α和IL-4水平;流式细胞仪检测脾细胞的凋亡发生率,同时设有BCG和PBS对照。结果pIL-12质粒DNA免疫组的减蚴率为44.85%,脾CD4+和CD8+T细胞亚群显著增加,IL-2、IFN-γ和TNF-α水平升高,IL-4水平降低,脾细胞凋亡发生率明显低于PBS对照组。结论pIL-12质粒DNA可诱导细粒棘球蚴感染小鼠产生一个保护性的免疫反应。     

Antibody response of Echinococcus granulosus infected mice: Protoscolex specific response during infection is associated with decreasing specific IgG1/IgG3 ratio as well as decreasing avidity

The antibody response was followed during 68 weeks in 17 Balb/c mice intraperitoneally (i.p.) infected with Echinococcus granulosus protoscoleces (PSC) and in three mice i.p. immunized with dead PSC. Titres of antibodies recognizing peptidic and glucidic PSC epitopes, as well as their isotypic and avidity profiles were followed by ELISA. In addition, antigen recognition patterns were analysed by immunoblot. The response against carbohydrate epitopes was dominant in infected and immunized mice but stronger in the first group. Infected mice showed similar profiles of specific IgG and IgM with maximum titres from week 38 to 53. Although IgG1 and IgG3 were the predominant antibody subclasses, the ratio of IgG1/IgG3 antibody titres as well as antibody avidity decreased during the experiment, encompassing a decrease in recognition of peptidic epitopes. Immunized mice did not show significant levels of specific IgM and, after week 15, showed IgG titres lower than the infected mice. IgG1 was the predominant IgG subclass during all the experiment with background levels of IgG3. The mean Ab avidity was high and showed no significant changes during immunization. Different patterns of response were thus produced by dead and developing live parasites. Although high avidity IgG1 antibodies were early found in both cases, lower avidity IgG3 antibodies were increasingly produced afterwards only in infected animals. The isotype switch and avidity decrease observed only during infection are consistent with a possible parasitic mechanism to evade host immunity .     

细粒棘球绦虫重组BCG-Eg95疫苗诱导小鼠脾细胞淋巴因子变化的研究

目的 探讨细粒棘球绦虫（Eg）重组BCG-Eg95疫苗免疫后对受攻击小鼠的包囊减重率及脾细胞淋巴因子的影响。方法 细粒棘球绦虫重组BCG-Eg95疫苗分别采用皮下注射、鼻腔内接种、口服灌胃和肌肉注射4种途径免疫BALB/C小鼠，免疫后8周用Eg原头节攻击感染，50个Eg原头节/每只小鼠，感染后18周剖杀小鼠，分离并称重细粒棘球蚴，计算包囊减重率；取脾，分离脾细胞，用Eg粗抗原（EgAg）或伴刀豆球蛋白A（ConA）刺激培养，收集脾细胞培养上清液，检测脾细胞培养上清液的白介素-2（IL-2）、γ干扰素（IFN-γ）、肿瘤坏死因子α（TNF-α）和白介素-4（IL-4）水平。同时设卡介苗（BCG）和磷酸盐缓冲液（PBS）对照。 结果 以上4种疫苗接种组的包囊减重率分别为45.77%、18.20%、88.05%和92.46%；疫苗肌肉接种组的IL-2、IFN-γ和TNF-α均较PBS对照为高，分别为（30.0±0）pg/ml、（65.0±0）pg/ml和（425.0±10.7） pg/ml， IL-4低于PBS对照组， 为（10.0±0） pg/ml。 结论 细粒棘球绦虫重组BCG-Eg95疫苗诱导小鼠产生辅助性T细胞1型（Th1）反应，从而对抗Eg原头节攻击感染。     

Further characterization of the 38 kDa antigen from Echinococcus granulosus (hydatid disease) cyst fluid: evidence for antigenic heterogeneity and reactivity with anti-P1 antibodies

A panel of 5 IgM and 6 IgG1 monoclonal antibodies (MoAbs) was produced against a band, eluted from a reducing SDS-PAGEgel, containing the 38 kDa subunit of antigen 5 (Ag5) from Echinococcus granulosus cyst fluid; seven of the MoAbs were shown subsequently to bind epitopes on Ag5 but none recognizedphosphorylcho-line or periodate-sensitive carbohydrate epitopes. Differences in the fine specificity of the MoAbs were apparent and, upon reduction, heterogeneity in 38 kDa components from hydatid cyst fluids of different intermediate host origin was revealed by peptide fingerprinting and immuno-blotting using the MoAbs. One of the IgGl MoAbs (ED9) was able to distinguish a reduced 38 kDa molecule in cyst fluids from two distinct genotypes–the horse/dog and sheep/dog strains–of E. granulosus and this may have implications for hydatid serology, immunoepidemiology and strain typing. Furthermore, epitopes on this 38 kDa component or on a different molecule with the same or similar M_r are reactive with anti-P1 blood group antigen antibodies and this could result in false-positive reactions where sera from P2 patients with suspected hydatid disease are tested by immunoblot or immunoprecipitation analysis.,     

棘球蚴感染绵羊并诱发休克期间特异性IgG、IgE抗体对特异性抗原的识别

目的 探讨细粒棘球蚴(E.g.)囊液粗制抗原和B抗原(EgB)识别绵羊感染E.g.后及诱发过敏性休克期间特异性IgG和IgE抗体的特异性反应,并对抗原特性及分子量进行描述。 方法 制备E.g.囊液粗制抗原及EgB,应用免疫印迹技术检测经剖杀证实感染E.g.并诱发过敏性休克的20只绵羊血清特异性IgG和IgE抗体对两种抗原的抗体反应性,并对抗原特性和分子量进行描述。 结果 特异性IgE抗体与耐热、低分子量的EgB(8、12和16 kDa)抗原未见明显的反应条带,而与E.g.囊液粗制抗原在43 kDa处可见明显的反应条带。IgG抗体与E.g.囊液粗制抗原未见明显的反应条带,但与EgB抗原反应后在31、43和66.2 kDa处可见较为明显的反应条带。 结论 EgB可能不是特异性IgE抗体的特异性抗原,而是IgG抗体的特异性抗原。E.g.粗制囊液抗原中含有特异性IgE抗体的特异性抗原组分,其分子量大于43 kDa,可能是导致棘球蚴病患者过敏性休克的主要致敏原。     

细粒棘球绦虫重组Bb-Eg95-EgA31疫苗诱导小鼠脾细胞亚群变化的研究

目的探讨细粒棘球绦虫(Eg)重组双歧杆菌(Bb)-Eg95-EgA31疫苗免疫和Eg原头节攻击后小鼠的囊重抑制率及脾细胞亚群的变化。方法将细粒棘球绦虫重组Bb-Eg95-EgA31疫苗分别采用皮下注射、肌肉注射、鼻腔内接种和口服灌胃4种途径免疫BALB/c小鼠,免疫后8周每鼠用50个Eg原头节攻击感染,感染后25周剖杀小鼠,分离细粒棘球蚴包囊并称重,计算囊重抑制率;取脾,分离脾细胞,流式细胞仪检测脾CD4+和CD8+T细胞亚群的百分比,同时设有空载体、Bb和MRS对照。结果以上4种疫苗接种组小鼠的囊重抑制率分别为45.33%、41.33%、70.67%和62.67%;疫苗接种组的脾CD4+和CD8+T细胞亚群显著增加,鼻腔内接种和口服免疫组的的CD4+T细胞亚群和CD4+/CD8+比值显著高于皮下和肌肉注射组。结论CD4+和CD8+T细胞亚群在疫苗诱导的保护力中起重要作用。疫苗鼻腔内接种和口服免疫是两种较好的免疫途径。     

Immunodiagnosis of human hydatid disease

A study was undertaken to evaluate the efficacy of Enzyme Linked Immunosorbent Assay using locally prepared antigens for immunodiagnosis of human hydatid disease. A total of 90 cases clinically suspected to be suffering from hydatid disease and 100 controls matched for age and sex were included in the study. Two types of ELISA were performed on detected specific antihydatid antibodies belonging to IgG/IgM/IgA classes and other type detected IgE class of antibodies. Antigen prepared from the human hydatid fluid was found to be unsuitable for diagnosis as it contained host proteins i.e. IgG. Sheep hydatid fluid obtained from the fertile hydatid cyst was used to prepare and standardize the antigen. ELISA test to detect anti hydatid antibodies belonging to either IgG, IgM and or IgA was found to be highly specific (98 per cent) in surgically confirmed hydatid disease and was negative in all the controls. The results of the study indicate that ELISA along with casoni test may provide the best results in diagnosis of hydatid disease.     

感染细粒棘球蚴绵羊诱发过敏性休克期间IgG、IgG1和IgE水平的探讨

目的　测定感染细粒棘球蚴(E.g)绵羊诱发过敏性休克期间特异性IgG、IgG1和IgE抗体水平。了解抗原B对人工感染E.g绵羊IgG抗体的反应性。　方法　从绵羊E.g囊液中制备抗原B和E.g囊液粗制抗原,ELISA测定感染E.g绵羊诱发休克期间特异性IgG、IgG1和IgE抗体的动态变化。　结果　感染E.g绵羊6个月,特异性IgG、IgG1和IgE抗体水平较正常绵羊显著升高;诱发休克后特异性IgE抗体水平显著下降,尤其因休克致死的绵羊下降更为明显;IgG及IgG1抗体的衰减时间不同,趋势各不相同;抗原B和E.g囊液粗制抗原与血清IgG抗体反应阳性率分别为91%、32%。　结论　特异性IgE是导致棘球蚴病所致过敏性休克的主要抗体,而IgG和IgG1抗体也起着重要作用。抗原B与感染E.g绵羊IgG抗体的血清反应性较好,可作为一种血清免疫学诊断方法监测绵羊感染E.g的状况。     

左旋咪唑对细粒棘球蚴感染小鼠不同时期T细胞亚群的影响

目的探讨左旋咪唑（LMS）对细粒棘球蚴感染鼠的免疫调节作用和对病程转归的影响。方法昆明种小鼠腹腔接种细粒棘球蚴，建立感染动物模型。在感染后2、4、8、12、16、20周，未治疗组和实验组分别给予生理盐水和左旋咪唑（25mg／kg），连续7d，测定各组小鼠脾指数、胸腺指数及囊重，运用流式细胞术（FCM）测定小鼠脾CD4^＋和CD8^＋T细胞百分率及CD4^＋／CD8^＋比值的动态变化。另设8只未接种小鼠为健康对照。结果生理盐水组小鼠感染2周时CD4^＋、CD8^＋T细胞百分率显著高于健康对照组（P〈0．01）），随后CD4^＋T细胞的百分率逐渐降低，CD8^＋T细胞的百分率逐渐增高，至感染后16周和20周，CD4^＋T细胞、CD8^＋T细胞的百分率及CD4^＋／CD8^＋比值与健康对照组比较差异仍具有显著性（P〈0．01）。与生理盐水组小鼠相比，左旋咪唑组小鼠在感染2周时CD4^＋、CD8^＋T细胞的百分率明显增高，与健康组小鼠相比有统计学意义（P〈0．01）；在感染16周和20周时脾指数升高，CD4^＋T细胞百分率上升，CD8^＋T细胞的百分率下降，CD4^＋／CD8^＋比值升高，平均囊重下降，与生理盐水组比较差异均有显著性（P〈0．01）。结论左旋咪唑对细粒棘球蚴感染小鼠有免疫调节作用，在感染后期可升高小鼠脾指数，使CD4^＋T细胞及CD8^＋T细胞比例恢复正常，机体的细胞免疫功能增强，从而减缓细粒棘球蚴的增殖。