周期型马来丝虫感染期幼虫在人卵巢癌细胞中的体外培养

1985年我室成功地建立了我国第一株人卵巢粘液性囊腺癌细胞系(OMC_685).1986年7～10月我们用OMC_685细胞系作营养层的培养系统,体外培养周期型马来丝虫感染期幼虫(L_3),L_3可在体外蜕皮发育为第四期幼虫(L_4),幼虫最长可存活66d。     

十二指肠钩虫14日龄童虫体外培养至成虫的研究

用十二指肠钩虫幼虫人工感染幼犬后14d,从犬肠道获得体长约2mm、生殖器官尚未发育的十二指肠钩虫童虫共106条,经犬血清和等量的犬血清RPMI 1640培养基培养后,童虫生长发育良好,存活率达90％,生殖器官逐渐显现并发育成熟,雌虫在培养后24d开始排卵,最高日产卵数7000个。50％的童虫存活54d以上,最长可存活154d,体长达8～9mm。有两对童虫在体外发育成熟后出现交配,雌虫排出受精虫卵,将该虫卵培养后,并获得感染期钩虫幼虫,此种幼虫再感染幼犬,首次检获十二指肠钩虫成虫436条。     

周期型马来丝虫感染期幼虫体外培养发育的观察

马来丝虫感染期幼虫(L_3)在沙鼠睾丸细胞系培养液中发育良好,一般在10～14天开始蜕皮进入第四期幼虫(L_4),成双地扭集在睾丸细胞系中最长可活38天,幼虫长度增加32.6％,宽度增加36.8％,可初步分辨雌雄;在鼠肾细胞系培养液中L_3于10～20天开始蜕皮,少数进入L_4,最长可活23天,幼虫长度增加25.9％,宽度增加34.5％,L_3在单纯Earle、Earle加水解乳蛋白及RPMI-1640中,仅能活2～4天。在培养过程中应防止细菌污染,合适的温度和pH亦系培养成功的重要因素。     

马来丝虫长爪沙鼠动物模型保种衰退期的观察

目的　观察马来丝虫经长爪沙鼠传代的衰退情况。　方法　用马来丝虫微丝蚴感染中华按蚊 ,收集感染期幼虫 (L3) ,通过腹腔接种感染长爪沙鼠 ,连续观察 3 3代长爪沙鼠体内马来丝虫微丝蚴的发育情况。　结果　从 1974年建立的马来丝虫长爪沙鼠动物模型至今 ,通过 3 3代传代 ,发现随着转种代数增加 ,从第 2 8代起长瓜沙鼠的阳性率逐年下降 ,由 2 8代的 80 %下降至 3 2代的 16% ,到 3 3代时阳性率降为 0。　结论　经过较长期的传代 ,马来丝虫幼虫难以在长爪沙鼠体内发育繁殖。     

PCR及PCR-ELISA法检测蚊体内马来丝虫幼虫

目的：建立一种检测蚊体内马来丝虫幼虫的灵敏、快速、特异的方法。方法：选择应用PCR及PCR－ELISA法检测马来丝虫幼虫DNA的最佳反应条件，并在该条件下分别以马来丝虫Ⅰ期、Ⅱ期、Ⅲ期幼虫各1条作模板，测定检测的灵敏度及检测实验室人工感染中华按蚊体内的马来丝虫幼虫。结果：PCR及PCR－ELISA法均能检测出1条Ⅰ期幼虫（L1），而PCR的检测下限为1／10条L1，PCR－ELISA检测下限为1／100条L1；将分离的感染期幼虫加阴性蚊媒进行粗提、扩增及电泳，结果未见明显的扩增条带，扩增产物作ELISA检测，全部为阴 性；个体解剖人工感染的中华按蚊120只，分别收集113只阳性蚊体内的幼丝虫，用两种方法检测，结果均为阳性。结论：初步建立了PCR及PCR－ELISA法检测蚊体内马来丝虫幼虫的方法。     

马来丝虫微丝蚴体外培养的研究——在三种培养基中发育到腊肠期蚴

本文报告周期型马来丝虫微丝蚴经含20mM CaCl_2的HBSS缓冲液内人工脱鞘后,在28±1℃下,分别培养于添加有5％小牛血清、2mg/ml葡萄糖和600μg/ml谷胺酰氨三种成份的HamF12、TC199和RPMI 1640培养基中,培养至第10天,其发育至腊肠期蚴的比率,分别为38.9％、37.1％和29.2％。     

马来丝虫贵州株传代的实验观察

目的 观察马来微丝蚴贵州虫株传代现象。方法 分别用周期型马来丝虫贵州虫株f31和f25微丝蚴人工感染中华按蚊，在温度、相对湿度相同的条件下，观察幼丝虫在蚊体内发育情况。结果 贵州虫株f31在感染中华按蚊后24h解剖，仅在按蚊腹部和胃内可见脱鞘和未脱鞘的微丝蚴，第2～9d未发现各期幼丝虫，经6批次实验，结果相同。贵州虫株f25感染中华按蚊后24h解剖，蚊胃内未发现微丝蚴，在胸部可见早期腊肠期(L1)幼丝虫，第2～9d解剖可见各期发育中的幼丝虫。结论 贵州虫株f31马来微丝蚴经6批次传代失败，可能与同一地方单一虫株的多次传代发生遗传突变有关。     

海群生抑制彭亨丝虫第三期幼虫发育的体外试验

本实验观察了海群生在体外对彭亨丝虫第三期幼虫的作用。采自感染14天的埃及伊蚊体内的彭亨丝虫第三期幼虫,以80～100条为一组接种于盛有10ml RPMI 1640培养液(内加10％灭活胎牛血清与恒河猴肾细胞)的培养瓶内,置37℃培养。接种后每隔一天更换一次培养液,每6天转种一次,至第22天终止培养。存活幼虫经固定后测定大小,并区分发育阶段。结果表明,培养至22天,未加海群生的对照组中幼虫存活率为51.1％～74.2％,其中77.8％的幼虫发育至第四期,幼虫的平均长     

彭亨丝虫第三期幼虫体外培养条件的改进及其用于抗丝虫药物测定

作者报道了狗肾细胞及不同浓度的胎牛血清对彭亨丝虫第3期幼虫生长、蜕皮期、蜕皮率及存活的影响,并将这种双相培养系统用于体外药物测定。用灌洗法从感染沙鼠体腔获取发育4天的彭亨丝虫第3期幼虫,用每毫升含100U青霉素和1,000μg链霉素的MEM液洗涤2次,将初步融合的单层狗肾细胞以每孔40、30、20及10×10~4的浓度加入组织培养多孔碟,每孔含1.8ml培养基,表面面积约为2cm~2,每种细胞浓度分别以11.1,4.4或2.2%灭活胎牛血清的MEM液混悬,用前置37℃通有5%CO_2气体培养24小时,细     

在不同体外培养条件下彭亨丝虫幼虫的生长发育

作者研究了不同培养条件促进彭亨丝虫第三期幼虫(L3)生长、分化和蜕皮的作用,评价了培养基中不同补充成分和血清、细胞饲养层和以往在其它虫培养中的一些确定成分对幼虫生长的影响,确定体外培养发育的L4幼虫接种入沙鼠后能存活并继续发育成熟,并对沙鼠体内获得的L4在体外培养下     

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.     

Survival and infectivity of Brugia malayi microfilariae after cryopreservation

Methods were studied for the cryopreservation of microfilariae of periodic Brugia malayi. RPMI-1640 tissue culture medium containing 6% dimethyl sulfoxide (DMSO) and 15% newborn calf serum was used as cryoprotectant. Samples were frozen slowly in the vapor phase of liquid nitrogen prior to emersion in liquid nitrogen (-196 degrees C). The freezing rate was -0.5 to -1.0 degrees C per minute, microfilariae remained viable for as long as, 212 and 375 days, survival rates were 94 to 98% and they were infective to Aedes togoi mosquitos. The infective larvae (L3) were obtained for 10-11 days after feeding at 28 degrees C room-temperature and the infection rate of L3 in test mosquitos was 22.4-30.6%. All DMSO should be removed from the freezing medium to restore microfilariae activity after freezing.     

Cryopreservation of infective-stage larvae of Brugia malayi

Third-stage larvae of Brugia malayi (Guizhou, China strain), in RPMI-1640, 6% DMSO and 15% calf serum, frozen slowly in the vapor phase of liquid nitrogen prior to emersion in liquid nitrogen, remained viable for as long as as 321 days and were infective to Mongolian jirds. All DMSO should be removed from the freezing medium to restore larval activity after freezing.     

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.     

A simple technique for the in vitro cultivation of nocturnally subperiodic Brugia malayi infective larvae

A simple system for the in vitro cultivation of nocturnally subperiodic Brugia malayi was developed. The manner of cultivation consisted of a 1:1 (v/v) mixture of Iscove's Modified Dulbecco's medium and NCTC-135 medium supplemented with 20% fetal bovine serum by using candle jar incubation at 37 degrees C instead of CO2 incubator. Changing the media: every 2 days, 3 days and changing media on day 7, then every 2 days produced a larval survival rate of 50% (70/140) on day 10, 49% (82/166) on day 6, and 53% (105/200) on day 9. With this technique, up to 50% of the infective stage larvae (L3) survived for up to 10 days and had long life for at least 27 days in all experiments with low larval survival rate in the fourth week. In addition, the culture system promoted molting L3 to fourth stage larvae (L4) after 7 days, as shown by light microscope.     

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.     

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.     

Dipetalonema viteae (Filarioidea): development of the infective larvae in vitro

The development of Dipetalonema viteae third stage larvae was attempted in vitro. A monophasic culture system consisting of BHK-21 medium supplemented with 10% tryptose-phosphate broth and 15% fresh jird serum allowed the growth of previously in vivo triggered larvae, but not of those isolated from ticks. The larvae could complete their third moult and grew on a fourth stage larvae up to 5 mm. This development was comparable to that observed in vivo. The presence of an irradiated hamster kidney cell feeder layer could replace an in vivo trigger. The development of the infective larvae took place up to the third moult. Some larvae completed the moult to the fourth stage and reached a maximum length of 3 mm after 14 days. These culture systems offer the possibility to study in vitro the complete development of the infective larvae to fourth stage larvae.     

Preliminary observations on the development of larval filariae in Toxorhynchites species

Brugia malayi and B. Pahangi microfilariae from gerbil intraperitoneal infections were inoculated into the thorax of male and female Toxorhynchites amboinesis and developed into third-stage larvae as early as 11 days. In a comparative study with Aedes togoi fed on microfilaremic gerbils, third-stage larvae were found at 10 days. Some third-stage larvae of B. malayi inoculated into gerbils developed to advanced stages. Third-stage larvae of Wuchereria bancrofti were recovered in low numbers from Tx. amboinesis and Tx. aurifluus inoculated with microfilariae recovered from human blood by membrane filtration. Development of all filarial species was similar in both male and female mosquitoes. Toxorhynchites species are plant feeders and therefore reduce the hazards of laboratory transmission of pathogenic agents. Because of their large size, manipulations with this mosquito species are easy and the size allows for a larger inoculum to be used. This group of mosquitoes should develop into useful laboratory vectors for the transmission of arthropod-borne diseases.