体外培养周期型马来丝虫感染期幼虫

关于体外培养周期型马来丝虫(periodic Brugia malayi)感染期幼虫(L_3),近年来国内外有一些报道,系应用含哺乳动物组织细胞系或原代细胞的培养系统培养彭亨丝虫和马来丝虫L_3,可使其幼虫发育至Ⅳ期蚴(L-4)或早期成虫(Chen and Howells,1979; Wong et al.,1982;Mak et al.,1983;郑惠君等,1984)。但未见应用含人体组织细胞系的体外培养系统培养布鲁属丝虫L_3的报道。本研究     

周期型马来丝虫实验感染小白鼠体内发育的观察

本实验用长爪沙鼠体内周期型马来丝虫微丝蚴,感染东乡伊蚊收集感染期幼虫,经腹腔内注射５５只小白鼠（昆明种）后,定期抽查小白鼠腹腔液和解剖小白鼠观察丝虫发育情况。在１４天、３０天、６０天、１４０天和１６０天时,分别检获了Ⅲ期、Ⅳ期幼虫、童虫和成虫。成虫感染率为３４．６％（９／２６）。９１天还查出微丝蚴。实验结果表明：周期型马来丝虫可以在昆明种小白鼠体内发育到成虫并产生微丝蚴。     

周期型马来丝虫成虫和第三期幼虫扫描电镜的观察

近年来,不少学者应用扫描电镜观察恶丝虫(Wong和Brummer,1978)、盘尾丝虫(Franz,1980;Franz和Renz,1980)、彭亨丝虫(Aoki et al., 1980)、成虫和/或童虫、幼虫、亚周期型马来丝虫(Franz,1982)和周期型马来丝虫(程富川等,1982)成虫的形态。我们在1978年成功地用周期型马来丝虫感染长爪沙鼠之后,应用扫描电镜观察马来丝虫成虫和第三期幼虫的形态。现将结果报告如后。     

周期型马来丝虫人工感染长爪沙鼠所致淋巴系病变的观察

有关周期型马来丝虫(Wuchereria malayi)引起的肉眼可见病理变化,至今未见报告。现将我们用周期型马来丝虫人工感染长爪沙鼠后在淋巴系出现的肉眼可见的病理变化简报于下。 每鼠经右侧腹股沟皮下注射周期型马来丝虫感染期幼虫89—169条,共注射53只沙鼠。分别在注射后240、330、543天处死沙鼠,在未用染料作皮内注射的情况下,对有关淋巴管、结进行解剖观察。 在解剖观察的53只(在接种后225天内眶窦血内微丝蚴阳性者45只)沙鼠中有25只出现病理变化。病变率为47.2％。     

马来丝虫幼虫低温保存的初步研究

以5％二甲基亚砜为冷冻保护剂,将马来丝虫微丝蚴置于含20％小牛血清的RPMI-1640保存液内,在低温下(-196℃)保存。经过30—270天,微丝蚴解冻后的存活率为93—98％,大部均可使蚊虫宿主感染,并在终宿主长爪沙鼠体内发育为成虫,在其腹腔液内检获微丝蚴。 同法冻存含马来丝虫感染期幼虫的蚊体,经30、90、及181天后,解冻的蚊体内幼虫,其存活率为46.2—54.4％,90天前大部幼虫可在沙鼠体内发育并产出子代,但冻存180天组似已失去感染性。     

昆虫蜕皮的分子机理及应用

昆虫蜕皮是一个由PTTH启始的、激素介导的基因序列表达和相互作用的级联反应过程。阐明昆虫蜕皮的分子机理,不仅可以解释发育生物学的科学问题,为害虫控制提供新的思路,还可以从中发现新的可资生产应用的分子。作者通过蛋白质组学方法从棉铃虫Helicoverpa armigera Hubner蜕皮幼虫鉴定到30个差异表达的蛋白质。通过抑制性消减杂交技术,从棉铃虫蜕皮幼虫、变态决定幼虫和5龄取食幼虫鉴定到100个表达序列标签(EST)。证明其中的11个EST在蜕皮或变态时差异表达。通过RT-PCR方法克隆棉铃虫激素接受子3基因,研究该基因在发育中的表达模式。用该基因构建具有绿色荧光蛋白标记和多角体蛋白的基因重组病毒(AcMNPV-GFP-HHR3-Polh)。实验结果表明,AcMNPV-GFPHHR3-Polh病毒可以通过注射或口服感染棉铃虫,导致棉铃虫幼虫非正常蜕皮、生长延缓、半数存活时间下降。该研究显示昆虫蜕皮功能基因在害虫控制中有很好的应用前景。蜕皮功能基因的表达与调控、蜕皮激素介导的信号转导通路、变态过程中组织解体和重建的分子机理、激素调控基因顺序表达的分子机理、变态起始因子、JH受体等是本领域今后的主要研究方向。     

马来丝虫Brugia malayi(Brug,1927)Buckley,1960成虫形态的观察

Rao与Maplestone(1940及Bonne等(1941)先后报告得自人体的马来丝虫成虫形态之后,至1956年Buckley与Edeson以得自动物的(猴、猫)马来丝虫(W.malayi?)成虫形态为依据,作了一些补充和改正;1961年Buckley与Wharton认为此种动物寄生的马来丝虫与人体寄生者相同,只是在生物学性质上可能不一样。但是,陈子达等曾于1953—1954年在我国浙江沿海岛屿上研究人体的马来丝虫形态,1957年发表论文,根据他们所记载的与Buckley等(1956)所报告的并不完全相同;再则,在1957—1961年之间,虽曾有人以马来亚地区人体寄生的周期出现型与半周期出现型马来丝虫感染动物成功     

周期型马来丝虫在长爪沙鼠体内发育情况的观察

马来丝虫(Brugia malayi Brug,1927)是我国重要人体寄生虫之一,对劳动人民健康危害较大。该种丝虫在蚊体内的发育情况过去已有详细的描述(Feng,1936),但它在终末宿主体内的发育却甚少了解。我们自1976年以来,在建立丝虫动物模型的过程中,对马来丝虫在长爪沙鼠体内的发育情况作了一些实验观察。现将观察结果报告如下。     

八字地老虎核型多角体病毒对宿主的弱化作用

八字地老虎核型多角体病毒(Amathes c-nigrum nucleopolyhedrovirus,简称AnNPV)接种八字地老虎3龄初幼虫,感染3d-6d,体重明显小于对照幼虫,发育历期显著延长,幼虫蜕皮和化蛹严重受阻.4龄幼虫感染病毒后1-2天取食量未减少,从第3天开始食量显著减少,从感染至第6天,单头幼虫总取食量为696.1mg,比对照幼虫总取食量的1313.2mg减少47.0%.     

嘴壶夜蛾的形态、生活史及昼夜节律

嘴壶夜蛾Oraesia emarginata (Fabricius)是危害水果果实的重要害虫之一, 其成虫和幼虫取食不同的寄主植物, 可以作为理想的嗅觉研究模式昆虫。为了全面地了解嘴壶夜蛾的形态特性和生物学特性, 本实验通过室内饲喂和红外摄像机观察, 对嘴壶夜蛾各虫态的外部形态、 发育以及昼夜活动节律进行了系统研究。结果表明： 嘴壶夜蛾的各龄幼虫可以通过体色, 体表色斑的类型、 位置和数量, 以及腹足的数量进行区别。通过蛹的生殖孔和成虫触角能够很好地区别雌雄。在室内饲养条件下, 嘴壶夜蛾的寿命为53.18±1.70 d, 存活率为63.62%±2.15%, 其中幼虫的发育历期最长, 存活率最低, 卵的发育历期最短, 存活率最高, 雄成虫的存活时间显著长于雌成虫（P=0.008）。6龄幼虫的发育历期（5.29±0.15 d）显著长于其余各龄幼虫（P<0.001）。同一龄发育中期幼虫的体长和体重显著大于将蜕皮幼虫和刚蜕皮幼虫（P≤0.037）; 第2-6龄刚蜕皮幼虫的体重和体长与前一龄发育中期幼虫之间没有显著差异（P≥0.106）。幼虫在光期的孵化、 蜕皮和化蛹比例高于暗期, 而成虫在暗期的飞行、 产卵比例高于光期, 成虫的飞行随着暗期时间的增加而逐渐变少, 光期成虫的飞行主要在开灯之后1 h。成虫交配集中在暗期的第3-5 小时。本研究结果有助于制定有效的嘴壶夜蛾防治措施, 而且为嘴壶夜蛾作为嗅觉研究模式奠定基础。     

Brugia malayi and B. pahangi: Cultivation in vitro of infective larvae to the fourth and fifth stages

Cultivation of fourth stage Brugia pahangi and B. malayi larvae from infective larvae (stage 3) were obtained in culture medium RPMI 1640 supplemented with 10% human AB serum and an LCC-MK₂ rhesus monkey kidney continuous cell line feeder layer. This culture system kept larvae alive in excess of 7 weeks, and served as a source for collection of the worms' secretory, excretory, and moulting antigens.     

Effects of medium alterations on in vitro development of Brugia malayi larvae in cultured mosquito thoraces.

A recent study showed that 1-day-old, intracellularly lodged larvae of Brugia species develop in vitro to the infective third-stage larvae (L3) in excised thoraces of susceptible mosquitoes in the diphasic insect tissue culture medium containing a nutrient agar base overlaid with a 1:1 mixture of Schneider's Drosophila medium and Grace's insect cell culture medium supplemented with 20% fetal bovine serum (FBS) and antimicrobial agents. In the present investigation, the diphasic culture medium was used to evaluate the effects of medium alterations on the development of 1-day-old, intracellularly lodged larvae of subperiodic Brugia malayi in excised thoraces of Aedes aegypti to the L3. One-day-old larvae developed to the L3 in medium without nutrient agar base, at pH 7.0 and pH 7.5, in Hanks' balanced salt solution (HBSS) and in HBSS supplemented with bovine albumin fraction-V (BAF-V). These larvae also developed in the absence of FBS in the overlay medium, in overlay medium containing 5-20% FBS, in medium components obtained from different sources, in serum free Sf-900 (GIBCO) medium, and when FBS is replaced by BAF-V in the overlay medium. The percentage of L3 was not increased substantially in infected excised thoraces of mosquitoes when nutrient supplements, such as folic acid, p-aminobenzoic acid, glucose, lipid concentrate, hemin, or reduced glutathione, were added to the overlay medium containing BAF-V. These results suggested that 1-day-old, intracellularly lodged larvae developed to the L3 in infected excised thoraces of mosquitoes at almost the same rate as in intact mosquito, when excised thoraces were maintained alive under optimal conditions in a culture medium.     

Development of a serum-free system for the in vitro cultivation of Brugia malayi infective-stage larvae

Over the past several years, numerous attempts have been made to culture the infective-stage (L3) larvae of the human filarial parasite Brugia malayi in an in vitro system that promotes molting to the fourth larval stage (L4). Although there have been reports in the literature of successful L3 to L4 development in vitro, all of these systems have required serum supplementation. The complexity of serum as a culture supplement has made reproducibility of results and identification of specific factors necessary for L3 development problematic. We have developed a serum-free in vitro system consisting of RPMI 1640 supplemented with one of three fatty acids (arachidonic, linoleic, or linolenic) that supports consistent and reproducible molting to the fourth larval stage in the presence of a basidiomycetous yeast, Rhodotorula minuta. Coculture with this yeast, initially isolated as a culture contaminant, is required for successful molting. In serum-free cultures lacking R. minuta, L3 larvae survive for upward of 2 weeks, but do not molt successfully. The L4 larvae generated in cultures containing R. minuta are well formed, as seen by light and electron microscopy, and are capable of further development upon transfer to a permissive host. This culture system is inexpensive and easily reproducible, thus making it a useful tool for studying the requirements for the development of B. malayi L3.     

Establishment of an in vitro culture system to study the developmental biology of Onchocerca volvulus with implications for anti-Onchocerca drug discovery and screening

BackgroundInfections with Onchocerca volvulus nematodes remain a threat in Sub-Saharan Africa after three decades of ivermectin mass drug administration. Despite this effort, there is still an urgent need for understanding the parasite biology especially the mating behaviour and nodule formation as well as the development of more potent drugs that can clear the developmental (L3, L4, L5) and adult stages of the parasite and inhibit parasite reproduction and behaviour.Methodology/Principal findingsPrior to culture, freshly harvested O. volvulus L3 larvae from dissected Simulium damnosum flies were purified by centrifugation using a 30% Percoll solution to eliminate fly tissue debris and contaminants. Parasites were cultured in both cell-free and cell-based co-culture systems and monitored daily by microscopic visual inspection. Exhausted culture medium was replenished every 2–3 days. The cell-free culture system (DMEM supplemented with 10% NCS) supported the viability and motility of O. volvulus larvae for up to 84 days, while the co-culture system (DMEM supplemented with 10% FBS and seeded on LLC-MK₂ feeder cells) extended worm survival for up to 315 days. Co-culture systems alone promoted two consecutive parasite moults (L3 to L4 and L4 to L5) with highest moulting rates (69.2±30%) observed in DMEM supplemented with 10% FBS and seeded on LLC-MK₂ feeder cells, while no moult was observed in DMEM supplemented with 10% NCS and seeded on LEC feeder cells. In DMEM supplemented with 10% FBS and seeded on LLC-MK₂ feeder cells, O. volvulus adult male worms attached to the vulva region of adult female worms and may have mated in vitro. Apparent early initiation of nodulogenesis was observed in both DMEM supplemented with 10% FBS and seeded on LLC-MK₂ and DMEM supplemented with 10% NCS and seeded on LLC-MK₂ systems.Conclusions/SignificanceThe present study describes an in vitro system in which O. volvulus L3 larvae can be maintained in culture leading to the development of adult stages. Thus, this in vitro system may provide a platform to investigate mating behaviour and early stage of nodulogenesis of O. volvulus adult worms that can be used as additional targets for macrofilaricidal drug screening.     

Brugia malayi: effects of antibacterial agents on larval viability and development in vitro

Recent studies have suggested that intracellular Wolbachia bacteria are necessary for reproduction and survival of adult filarial worms. We now report results of in vitro studies of effects of antibacterial antibiotics (tetracycline, rifampicin, chloramphenicol, azithromycin, and doxycycline) on Brugia malayi infective larvae (L3) motility and molting. All of the antibiotics tested except chloramphenicol decreased L3 motility by 50% or more at 10 days, with minimal effective concentrations (MECs) of 20-100 microg/ml. Tetracyclines, rifampicin, and chloramphenicol inhibited L3 to L4 molting by 12 days in a concentration- and time-dependent manner, with MECs in the range of 1-20 microg/ml. These studies show that antibiotics active against Rickettsiaceae inhibit B. malayi L3 molting at low concentrations in vitro; higher concentrations kill the larvae. While it is possible that antibiotics directly affect filarial L3, we believe it is more likely that the effects seen are indirect effects related to bacterial killing.     

Inhibitors of the lipoxygenase pathway block development of Brugia malayi L3 in vitro

Brugia malayi L3 molt to the L4 stage in serum-free cultures supplemented with arachidonic, linoleic, or linolenic acids and the basidiomycetous yeast Rhodotorula minuta. These fatty acids are capable of entering the eicosanoid pathway of arachidonate metabolism, the pathway responsible for generating a number of biologically active mediators, including prostaglandins, leukotrienes, and lipoxins. To determine whether this pathway was required for L3 development, we added dual inhibitors of cyclooxygenase and lipoxygenase to in vitro cultures containing B. malayi L3. These compounds significantly inhibited L3 molting. To evaluate whether 1 or both of these pathways of arachidonate metabolism were involved in molting, we tested drugs inhibiting either cyclooxygenase or lipoxygenase. Lipoxygenase inhibitors blocked L3 molting, whereas cyclooxygenase inhibitors did not. To assess whether enzymes operating downstream of lipoxygenase were also involved in L3 molting, we added inhibitors of enzymes involved in leukotriene synthesis and found they were also capable of preventing development. We tested the same inhibitor panel on Dirofilaria immitis L3. A single lipoxygenase inhibitor and inhibitors of 2 different enzymes operating downstream of lipoxygenase disrupted D. immitis development. These results demonstrate that a lipoxygenase pathway product is required for molting of the infective stage larvae of filarial parasites.     

In vitro cultivation of Dipetalonema viteae third-stage larvae: evaluation of culture media, serum, and other supplements

Third-stage larvae of Dipetalonema viteae obtained from the tick vector developed to the fourth stage in several cell-free culture systems. Survival and development of larvae in a number of commercially available cell culture media, supplemented with serum and other defined and undefined components, were compared. All cultures were gassed with 5% carbon dioxide in nitrogen. Best survival, growth and development were obtained in stationary cultures containing 1:1 (v/v) mixtures of NCTC 135, either RPMI 1640 or Iscove's Modified Dulbecco's Medium, and a supplement of 20% non-heat-inactivated fetal bovine serum. The importance of the medium composition and physical environment of the culture system, for the survival, growth and development of D. viteae was demonstrated.     

Support of Xenopus laevis spermatogenesis in vitro by different energy substrates

The support of Xenopus laevis spermatogenesis in vitro by different energy-yielding substrates has been investigated. Isolated spermatogenic cells maintained their levels of adenosine-triphosphate for 24 h in serum-free medium containing only amino acids as energy substrates. DL-Aminocarnitine, an inhibitor of carnitine palmitoyltransferase, reduced cell viability 87% during a 15-h culture in the same medium, indicating that beta oxidation of endogenous fatty acids is a significant source of energy when exogenous substrates are unavailable. Isolated spermatocytes developed into spermatids for 7 days in medium supplemented with either pyruvate, oxaloacetate, or lactate, with maximal survival and development at 0.5 mM pyruvate, 2.0 mM oxaloacetate, and 4.0 mM lactate. Few spermatocytes survived more than 3 days in serum-free medium supplemented with only glucose and amino acids as energy substrates. In contrast, glucose-supplemented medium supported spermatocyte differentiation for 14 days in testis fragment culture and 7 days in spermatocyte-Sertoli cell cocultures due to the excretion of lactate and pyruvate by Xenopus Sertoli cells during culture in glucose-supplemented medium. Glucose also enhanced spermatocyte development in medium containing dialyzed, heat-inactivated fetal calf serum. Spermatogenic cells oxidized glucose to CO2 with C1 oxidized 6- to 7-fold more than C6, suggesting that glucose may be metabolized in the hexose monophosphate shunt. The results are discussed in comparison to energy metabolism in mammalian testes and spermatogenic cells.     

球形红杆菌积累聚β-羟基链烷酸(PHA)的研究

通过对球形红杆菌(Rhodobacter sphaeroides)生长和积累聚卢-羟基链烷酸(PHA)条件的研究,确定采用两段培养法提高PHA的产量。第一阶段提供适合菌体生长的条件:以葡萄糖作碳源,尿素为氮源,光照微好氧培养。第二阶段则提供使菌体积累PHA的条件:补加乙酸钠厌氧光照培养。经两段培养后菌体PHA含量可占细胞干重的45％,PHA产量每升发酵液可达1.7g。     

A cell-free culture system for development of large numbers of Brugia pahangi larvae.

Techniques used in the in vitro culture of massive numbers of Brugia pahangi third-stage larvae (L3) are described. Procedures for larval preparation and four culture conditions, with or without animal cell co-cultures, were studied, resulting in the adoption of a relatively simple cell-free culture system for routine harvesting of larval moulting excretory/secretory product.