抗体依赖细胞介导对马来丝虫L_3细胞毒作用的体外研究

在体外小鼠特异性抗周期型马来丝虫感染期幼虫(L_3)血清(AIMS)或正常小鼠血清(fNMS)存在时,中性粒细胞(Neu)、巨噬细胞(Mφ)和嗜酸性粒细胞(Eos)均可粘附于虫体表面,Neu和Mφ对L_3的杀伤作用明显高于Eos。Eos只是在AIMS存在时,才表现出弱的细胞毒作用。通过用羊抗鼠IgG血清或葡萄球菌A蛋白处理AIMS后发现细胞毒作用消失,提示参与该作用的抗体分子主要是IgG。IFAT表明L_3表面确实存在IgG分子。AIMS经56℃,30min可降低细胞毒作用,说明补体能增强该作用。该作用还可因加入EDTA而不是EGTA受到明显抑制,表明补体是通过激活替代途径参与了该反应。扫描电镜观察被细胞粘附的虫体表面出现损伤现象。     

中华按蚊感染马来丝虫幼虫后生殖,生殖营养周期...

Under the experimental conditions of 26 +/- 10 degrees C, relative humidity 70-80%, 100 +/- 20 Lux and photoperiod 16 hours/day and using a membrane feeding method, the development of filarial larvae and the variations in reproductive capacity, gonotrophic cycle and longevity of An. sinensis infected with microfilariae of Brugia malayi were observed. The infection rate and infection intensity of filarial larvae in An. sinensis increased with the microfilarial density in the blood meal from 50 to 150 mf/20 microliters. The mature rate and IEI decreased when the density rose to 200 mg/20 microliters. The concentration ratio of mf in blood by An. sinensis was 1.2-1.4. In the 3rd gonotrophic cycle, the feeding ratio of infected mosquitoes became lower when the mf density rose to 150 mf/20 microliters, but the infection of filarial did not affect the number of oviposition, the regularity of egg-production activity and the hatching rate of eggs, while the quantity of egg-production increased when the mf density was 150 mf/20 microliters. The egg-production rate, and gonotrophic cycle were not basically influenced by filarial larvae infection. The multifeeding ratio was 16.7-48.4% in An. sinensis. The longevity of An. sinensis was extended by infection with mf density of 100 mf/20 microliters and shortened in infection with mf density of 200 mf/20 microliters. The authors conclude that "Brugia malayi-Anopheles sinensis" is a highly adapted "pathogen-vector" system.     

应用单克隆抗体检测蚊体内幼丝虫的实验观察

以马来丝虫感染期幼虫为靶抗原制备多株单克隆抗体从中筛选出１Ｇ１，１Ｈ１单克隆抗体，检测人工感染马来丝虫的中华按蚊。当中华按蚊吸食马来微丝蚴的兔血后，分别解剖同时以ＥＬＩＳＡ及Ｄｏｔ－ＥＬＩＳＡ检测饲养不同时间的中华按蚊。结果显示，饲养９ｄ的按蚊马来丝虫幼虫阳性率，人工解剖为８６．６％，ＥＬＩＳＡ和Ｄｏｔ－ＥＬＳＩＡ法分别为８２．２％和７７．８％。     

应用DNA探针检测蚊体内丝虫幼虫的研究

本文用人工合成的寡核苷酸探针，经斑点杂交法检测蚊体内马来丝虫幼虫。可测到丝虫和微丝蚴２ｎｇ的ＤＮＡ量，不与其它动物丝虫标本发生交叉反应。将单个蚊直接压于硝酸纤维素膜上进行检测，感染蚊中含有１条感染期幼虫不可出现了性反应。将一组蚊虫在裂液中研磨集体检测时，在２０只蚊虫中有１只感染蚊即可检出。显示该探针可用于马来丝虫地区的蚊媒监测。     

马来丝虫福建株成虫与幼虫氨基酸组成及含量的测定分析

材料与方法一、成虫（adn）取自我室丝虫库福建建阳株长爪沙鼠腹腔液中，25条，35条，用生理盐水冲洗后，冷冻备用。二、微丝蚴（mf）取自上述阳性长爪沙鼠腹腔液，用程文芳[5]法分离约1ml，冷冻备用。三、感染期蚴（L3）取自我室实验感染的阳性中华按蚊体内，采用贝氏分离改进     

应用单克隆抗体检测蚊体内幼丝虫的实验观察

以马来丝虫感染期幼虫为靶抗原制备多株单克隆抗体（ＭｃＡｂ）从中筛选出１Ｇ１、１Ｈ１单克隆抗体，检测人工感染马来丝虫的中华按蚊。当中华按蚊吸食含马来微丝蚴的兔血后，分别解剖，同时以ＥＬＩＳＡ及Ｄｏｔ－ＥＬＩＳＡ检测饲养不同时间的中华按蚊。结果显示，饲养９ｄ的按蚊马来丝虫幼虫阳性率，人工解剖为８６．６％，ＥＬＩＳＡ和Ｄｏｔ－ＥＬＩＳＡ法分别为８２．２％和７７．８％。经统计学处理，人工解剖与单抗检测无显著性差异，其灵敏度为每只蚊０．５条幼虫。实验证明，此ＭｃＡｂ与牛腹腔指状丝虫、猪肺丝虫抗原无交叉反应，显示了一定的特异性。     

应用DNA探针检测蚊体内丝虫幼虫的研究

本文用人工合成的寡核苷酸探针，经斑点杂交法检测蚊体内马来丝虫幼虫。可测到丝虫和微丝蚴２ｎｇ的ＤＮＡ量，不与其它动物丝虫标本发生交叉反应。将单个蚊直接压于硝酸纤维素膜上进行检测，感染蚊中含有１条感染期幼虫就可出现阳性反应。将一组蚊虫在裂解液中研磨集体检测时，在２０只蚊虫中有１只感染蚊即可检出。显示该探针可用于马来丝虫地区的蚊媒监测。     

Differentiation of infective larvae of Brugia malayi and Wuchereria bancrofti by scanning electron microscopy

A comparative scanning electron microscopy of the infective (third stage) larva of Wuchereria bancrofti and Brugia malayi revealed the following features: The lateral caudal papillae in both species are rounded and the terminal papilla is relatively pointed and smaller. The caudal papillae in W. bancrofti are more protruding while in B. malayi they are flattened. The lateral caudal papillae in B. malayi have an indentation around their bases which is absent in W. bancrofti. This is the most useful distinguishing feature and easily recognizable. The oral opening and the cephalic papillae in both the species are similar and not useful for differentiation.     

Combined detection of Brugia malayi and Wuchereria bancrofti using single PCR

A single step PCR method has been developed for the combined detection of the human filarial parasites, Brugia malayi and Wuchereria bancrofti. Parasites' DNA were isolated from filaria positive blood samples that were collected from endemic areas. The primers used were Hha1 and Ssp I, which amplified the DNA fragments of 322 bp and 188 bp specific to B. malayi and W. bancrofti, respectively. The sensitivity of the assay was tested with blood and mosquito samples having one W. bancrofti in a pool of 10 B. malayi. The assay was further evaluated on field collected blood and mosquito samples. Use of this assay as a diagnostic tool for the detection of filariasis being the most promising aspect of this study, offers scope for detection of both the parasites even at low levels of infection.     

Cryopreservation of third-stage larvae of Brugia malayi and Dipetalonema viteae

Methods are described for the cryopreservation of third-stage larvae of Brugia malayi. Optimum conditions utilized larvae free from the mosquito host frozen at the rate of -1 degree or -0.8 degrees C per min in medium containing 9% dimethyl sulfoxide and 0.004 M polyvinylpyrrolidone. Nonfrozen or thawed larvae were inoculated intraperitoneally into jirds (Meriones unguiculatus), the thawed larvae after cryogenic storage for 5-378 days. In general, the percentage of adult worms recovered at necropsy was comparable between the two groups and ranged from a mean of 6-9% of the larval inoculum. In addition, three of four patas monkeys (Erythrocebus patas) inoculated with thawed B. malayi larvae developed patent infections. The cryopreservation of third-stage larvae of Dipetalonema viteae also is discussed.     

Development of Brugia malayi in Mongolian gerbils previously exposed to Wuchereria bancrofti

Twelve Mongolian gerbils, Meriones unguiculatus, were infected with 100 third-stage larvae of Wuchereria bancrofti. One month later these animals, along with 4 control animals, were given 100 third-stage larvae of Brugia malayi. Eleven of the 12 experimental animals and the 4 controls survived, and 8 of the experimental animals and all of the controls demonstrated microfilaremia after 3 months. The animals were killed at 6-months post-infection and examined for parasites. One W. bancrofti larva was found in one of the experimental animals, and 15% of the B. malayi given were recovered as adults from the testes, viscera, and carcass. Thirty-eight percent of the worms given to the controls were recovered from the testes, viscera, and pelt. The worms from the experimental animals also appeared to be smaller. This study suggests that gerbils are able to develop partial resistance to Brugia malayi following a previous infection with Wuchereria bancrofti.     

Release of prostaglandin E2 by microfilariae of Wuchereria bancrofti and Brugia malayi.

To elucidate the local release of immunomodulatory prostaglandins by intravascular filarial parasites, the formation of prostaglandin E2 (PGE2) was examined in individual microfilariae of Wuchereria bancrofti and Brugia malayi. Following incubation of living microfilariae immobilized in an agar matrix, prostaglandins released by the parasites were fixed by carbodiimide and localized by indirect immunofluorescence. Prostaglandin E2 was specifically detected around the entire surface of microfilariae with anti-PGE2 antiserum, but not with control nonimmune or PGE2 affinity-immunoadsorbed antiserum. These results provide direct evidence that individual microfilariae of W. bancrofti as well as B. malayi release prostaglandins into their microenvironment. The release of PGE2 by these intravascular parasites may modulate host leukocyte responses, and thereby contribute to the immune defects observed in infected humans with peripheral microfilaremia.     

Attempt to culture Wuchereria bancrofti in vitro

Third-stage larvae of Wuchereria bancrofti recovered from laboratory raised Aedes togoi and Anopheles maculatus fed on a human volunteer were recovered by mass dissection methods and introduced into in vitro culture. LLC-MK2 cells were used as feeder cells, and the culture medium consisted of RPMI-1640 buffered with Hepes and sodium bicarbonate and supplemented with human AB serum. The third-stage larvae molted as early as 12 days and those surviving had all molted by 16 days. The fourth-stage parasites averaged in length from 1.4 mm to a maximum of 1.8 mm. Some larvae remained alive in culture as long as 40 days and while the worms were distorted in fixation, possible primodial cells of a spicule could be visualized in the rectal region. The cuticle also appeared to be separating in the posterior end. Although complete development was not achieved, it seems that with a continuing effort, success could be obtained using this culture system with feeder cells.     

Cryopreservation of microfilariae and third-stage larvae of Brugia malayi and subsequent development in the hosts

Methods are studied for the cryopreservation of microfilariae (mf) and third-stage larvae (L3) of periodic Brugia malayi. RPMI-1640 tissue culture medium containing 6% dimethyl sulfoxide (DMSO) and 15% newborn calf serum were used as cryoprotectant. The larvae survived best when specimens were frozen at the rate of -0.5 degrees C to -1.0 degrees C per minute using the vapor phase of liquid nitrogen, when the temperature reached -70 degrees C to -90 degrees C the specimens were placed directly into the liquid nitrogen (-196 degrees C). After the thawing of the mf which had been stored for 6,212 and 375 days in cryogen, 96.2% of the mf were shown to be viable and developed in Aedes togoi. It was also shown that the survival rate of L3 cryopreserved for 28-321 days was also 96.2% and that, when 107 L3 frozen for 321 days were inoculated after being thawed into one jird, one live female adult was recovered at autopsy 71 days after inoculation, its morphology being the same as the unfrozen specimens. There was no correlation between the time of cryopreservation and the survival rate of the larvae.     

Interaction of monoclonal antibodies with cuticular antigens of filarial parasites, Brugia malayi and Wuchereria bancrofti

Monoclonal antibodies (mAbs) have been prepared against excretory-secretory-metabolic (ESM) antigens of microfilariae (mf) of Wuchereria bancrofti (WbmfESM) and against third stage larvae (L3) of Brugia malayi (BmL3), and purified from ascites fluids with ammonium sulphate. Both antibodies were of the IgM type and did not react with phosphorycholine. The mAb against BmL3 (F46) reacted in ELISA with antigens of L3 of B. malayi, B. pahangi and W. bancrofti and of adults of B. malayi. The mAb raised against wbmfESM (F32) resembled F46 in this respect, though with a lower titer towards the antigens, and in addition reacted with the ESM-antigens of mf and of L3 of W. bancrofti. F46 was able to detect L3 antigens of filarial parasites in spiked serum samples with a detection limit of 8-16 ng in absolute amount. The antibody was found to label the cuticular portion of L3 and adults of the lymphatic parasites, and not the epicuticular surface, in immunoelectron microscopic studies. The antibody recognized a 36 kDa component of the beta-mercaptoethanol extracts of B. pahangi-adults in Western blot analysis.     

The influence of the gene sb in Culex pipiens on the development of sub-periodic Brugia malayi and Wuchereria bancrofti

The gene sb (filarial susceptibility, Brugia pahangi) in Culex pipiens controls the development also of sub-periodic B. malayi, but has no influence on the development of periodic Wuchereria bancrofti (Ceylon strain). C.p. fatigans (Kuala Lumpur), C.p. molestus (London) and Aedes aegypti (re fm strain) were all susceptible to the Ceylon strain of W. bancrofti, with susceptibility rate of 90.3%, 92.9% and 52.6% respectively. However, a low proportion of the larvae in A. aegypti developed to maturity, and this mosquito is less well adapted to W. bancrofti than is C. pipiens.