Functional and antigenic maturation of Brugia malayi microfilariae

Brugia malayi microfilariae of specified ages were obtained from gerbils implanted with fertile adult worms. Such microfilariae were tested for their capacity to infect mosquitoes. A strong age dependence was found for the microfilariae's capacity to: penetrate the mosquito midgut, exsheath in response to 20 mM calcium, and develop to third stage larvae in the mosquito. In addition, differences were found between 2-day-old microfilariae and controls (from larva-infected gerbils) in their reactivities with a series of monoclonal antibodies. Thus, defined immunochemical changes occur in microfilariae as they assume functional maturity.     

Characterization of immunosuppressive proteins of Brugia malayi microfilariae

Inhibition of Concanavalin A-induced lymphocyte proliferation was used to monitor the partial purification and characterization of suppressor molecules from microfilariae of Brugia malayi. Suppressor activity was present in high molecular weight fractions of microfilarial extracts (Mr greater than 50 kd on SDS-PAGE) and was protease-sensitive but resisted treatment with sodium periodate, indicating that it is associated with parasite proteins. Suppressor activity was released by microfilariae cultured in vitro and could be detected in peritoneal exudates of intraperitoneally-infected jirds and in lymph and sera from athymic C3H/HeN mice with patent B. malayi infections. These findings indicate that immune unresponsiveness during patent filarial infections may result from the in vivo release by microfilariae of high molecular weight proteins that suppress host immune responses.     

Incorporation of arachidonic acid by microfilariae of Brugia malayi

To initiate studies on the significance of filarial arachidonic acid metabolism in the immunopathogenesis of human filariasis, we evaluated the ability of microfilariae of the human filarial parasite Brugia malayi to take up and incorporate exogenous arachidonate. When 4 X 10(5) microfilariae were incubated in vitro with 3 nM [3H]arachidonic acid for 0.2, 24, and 48 hr, 23%, 70%, and 75% of tritium activity were associated with microfilariae, respectively. [3H]arachidonic acid was taken up by viable but not by killed microfilariae. Electron microscopic autoradiographic examination of living microfilariae incubated with [3H]arachidonic acid demonstrated numerous tritium-induced silver grains over sectioned parasites. Chromatographic resolution and quantitation of classes of neutral lipids and phospholipids of parasites established that incorporated [3H]arachidonic acid was rapidly and almost completely esterified into these lipids, predominantly into phosphatidylinositol and phosphatidylcholine. Microfilariae, the blood-borne stage of B. malayi, possess the requisite biochemical pathways to rapidly take up and incorporate exogenous arachidonate.     

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.     

Brugia malayi: serum dependent cell-mediated reactions to microfilariae

Sheathed and exsheathed microfilariae of Brugia malayi are killed by normal rat cells in the presence of immune serum in vitro. Immune serum heated at 56 degrees C for 1 hour lost this activity which was largely restored by the addition of fresh normal rat serum. EDTA but not EGTA abolished this activity indicating the operation of complement by alternate pathway. Fresh normal rat serum alone promoted cellular adherence without exerting cytotoxicity to the microfilariae. The activity in the immune serum could be removed with Staphylococcus aureus cells containing Protein A or anti-IgG antiserum. The activity could also be absorbed to and eluted from Protein A--sepharose CL-4B suggesting the involvement of IgG. Neutrophils and macrophages participate in the antibody dependent cell-mediated cytotoxicity phenomenon. Eosinophils while adhering to the microfilariae exert cytotoxicity only to the exsheathed parasites.     

Brugia malayi: ivermectin inhibits the exsheathment of microfilariae.

Brugia malayi-infected microfilaremic jirds (Meriones unguiculatus) were treated with ivermectin at a single dose of 200 micrograms/kg of body weight injected subcutaneously. Susceptible Aedes aegypti mosquitoes were fed on treated jirds 24 hours later. Mosquitoes fed on untreated jirds served as controls. Infected mosquitoes were dissected at 1, 3, 24, 48, 72, and 96 hr after the blood meal, and differential counts of sheathed microfilariae, exsheathed microfilariae, and cast sheaths were performed using fluoresceinated wheat germ agglutinin. Microfilariae failed to exsheath in mosquitoes fed on ivermectin-treated jirds. Microfilariae from ivermectin-treated jirds also did not exsheath in vitro in the presence of 10 mM CaCl2, whereas 85-90% of sheathed microfilariae from untreated jirds exsheathed in vitro. In addition, sheathed microfilariae from untreated jirds, when pretreated in vitro with ivermectin at 0.25, 0.5, or 1 microgram/ml, lost their ability to exsheath in vitro in the presence of 10 mM CaCl2. However, ivermectin treatment had no effect on exsheathing of microfilariae when incubated with papaya protease. Thus, ivermectin appears to inhibit the intrinsic exsheathing process of microfilariae in the mosquito host, thereby blocking their development and further transmission of infection.     

Susceptibility of Mansonia uniformis to Brugia malayi microfilariae from infected domestic cat

Microfilariae of Brugia malayi is transmitted to man and other susceptible hosts via mosquito. The transmission of B. malayi from cat to man by Ma. uniformis bite has never been reported. The Ma. uniformis mosquito is the normal vector for Wuchereria bancrofti but has never been reported as a vector for B. malayi, or a susceptible host for the growth and development of the microfilariae of B. malayi. The purpose of this study was to examine the development of B. malayi in Mansonia uniformis after feeding on the blood of an infected cat in the laboratory. The B. malayi infected cat was identified using PCR with the primers Bm-1/Bm-2 on DNA (at 10 ng/50 microl) extracted from the WBC of the cat. W. bancrofti was employed as a negative control. The sensitivity of the B. malayi DNA detection by PCR was 0.0001 ng. Adult Ma. uniformis mosquitos at the ages of 5, 10, and 15 days, 100 mosquitos in each group, were fed on the infected cat blood. Recovery of third stage microfilariae was found to be the highest in the 5-day old mosquito group (48%), followed by the 10- and 15-day old mosquito groups (32% and 18%, respectively). The mean number of B. malayi microfilariae found in thorax, head, and abdomen of the mosquitos were composed. The 5-day old (40.3%) and 10-day old (41.9%) mosquitos were significantly more susceptible to microfilariae than the 15-day old mosquitos (17.8%) (p-values using the Scheffe method: 0.027 and 0.039, respectively). There was no significant difference in the mean number of microfilariae in the thorax (p = 0.482) by age, but the mean numbers of microfilariae in the heads, and abdomens were significantly different by age between the 5- and10-, and the 15-day old mosquitos (p < 0.001 and p = 0.004, respectively).     

Ivermectin disrupts the function of the excretory-secretory apparatus in microfilariae of Brugia malayi

Ivermectin (IVM) is a broad-spectrum anthelmintic used in filariasis control programs. By binding to nematode glutamate-gated chloride channels (GluCls), IVM disrupts neurotransmission processes regulated by GluCl activity. IVM treatment of filarial infections is characterized by an initial dramatic drop in the levels of circulating microfilariae, followed by long-term suppression of their production, but the drug has little direct effect on microfilariae in culture at pharmacologically relevant concentrations. We localized Brugia malayi GluCl expression solely in a muscle structure that surrounds the microfilarial excretory-secretory (ES) vesicle, which suggests that protein release from the ES vesicle is regulated by GluCl activity. Consistent with this hypothesis, exposure to IVM in vitro decreased the amount of protein released from microfilariae. To better understand the scope of IVM effects on protein release by the parasite, three different expression patterns were identified from immunolocalization assays on a representative group of five microfilarial ES products. Patterns of expression suggest that the ES apparatus is the main source of regulated ES product release from microfilariae, as it is the only compartment that appears to be under neuromuscular control. Our results show that IVM treatment of microfilariae results in a marked reduction of protein release from the ES apparatus. Under in vivo conditions, the rapid microfilarial clearance induced by IVM treatment is proposed to result from suppression of the ability of the parasite to secrete proteins that enable evasion of the host immune system.     

注射感染马来丝虫微丝蚴在蚊虫体内早期...

This paper reports the early development and variation of inoculated Brugia malayi microfilariae in 3 groups of mosquitoes, i. e. young Anopheles sinensis, young Culex quinquefasciatus and old Cx. quinquefasciatus. Significant differences were observed among these groups. Sixty hours after inoculation, the percentage of normally developing filarial larvae in young An. sinensis was 72.3%; the percentage of melanized microfilariae in young Cx. quinquefasciatus was 75.4% and the percentage of filarial larvae which developed abnormally or remained in diapause status in old Cx. quinquefasciatus was 67.1%, each being much higher than that in the other groups. It was suggested that the immune competence of the mosquitoes had influence upon the filarial development. The intensity of the immune response not only varied with the species, but also diminished with the aging of mosquitoes.     

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.     

细胞毒作用对周期型马来丝虫微丝蚴化学元素含量的影响

目的　通过观察细胞毒作用对周期型马来丝虫微丝蚴 (mf)化学元素含量的影响 ,进一步探讨宿主体内杀伤虫体的机制。方法　应用原子吸收光谱分析法对细胞毒作用前后的周期型马来丝虫mf7种微量元素和 2种常量元素的含量进行检测分析。结果　所测的周期型马来丝虫mf7种微量元素中铁 (Fe)含量最高 ;镉 (Cd)含量最低。常量元素钙 (Ca)高于镁(Mg)。经细胞毒作用后虫体各微量元素和常量元素含量均有所下降 ,尤其是微量元素中的铁 (Fe)、铜 (Cu)、锰 (Mn)、铬 (Cr)、镉 (Cd)铝 (Al)和常量元素中的镁 (Mg)下降显著 (P <0 0 1 )。结论　经细胞毒作用后虫体内的微量元素和常量元素大量丢失 ,造成其营养代谢和生理功能的障碍 ,可能是宿主体内杀伤丝虫mf,导致虫体死亡的因素之一     

马来丝虫幼虫经细胞毒作用后氨基酸的变化

目的观察马来丝虫微丝蚴(mf)经细胞毒作用后氨基酸含量的变化,进一步探讨宿主体内杀虫机制.方法应用高速氨基酸分析仪对细胞毒作用前后的周期型马来丝虫mf进行氨基酸组成及含量的分析比较.结果马来丝虫mf含17种氨基酸,缺少色氨酸.经宿主细胞毒作用后的虫体氨基酸绝对含量明显低于细胞毒作用前(P＜0.05).其中,精氨酸、脯氨酸、甲硫氨酸和赖氨酸下降显著(P＜0.01).结论氨基酸含量下降,尤其是碱性氨基酸与酸性氨基酸、芳香族氨基酸之间比率下降可能是宿主体内杀伤丝虫mf的后果之一.     

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.     

Mechanism of destruction of Brugia malayi microfilariae in vitro: the role of antibody and leucocytes

Human and monkey leucocytes in serum from previously patent Brugia malayi infected monkeys adhered to and caused cytotoxicity to microfilariae of B. malayi in vitro. The phenomenon also occurred in human serum from confirmed cases of bancroftian and malayan elephantiasis. Adherence of leucocytes was not seen in serum from uninfected monkeys, monkeys with patent B. malayi infection and serum from rats infected with Breinlia booliati but now amicrofilaraemic. Immune serum failed to promote adherence of leucocytes to L3 of B. malayi. The activity was lost when the serum was either incubated with Protein A-bearing Staphylococcus aureus or heat inactivated at 56 degrees C for 30 min. Light and electron microscopic observations regarding the type of adhering cells and sequence of events following adherence are described.     

Quantitative studies on the development of inoculated Brugia malayi microfilariae in Anopheles sinensis and Culex quinquefasciatus

Definite numbers of B. malayi mf were inoculated into An. sinensis and Cx. quinquefasciatus, and their subsequent development was observed. The relationships between the dosage and the L3 positive rate on the one hand and the average filarial maturity rate on the other were defined. At the same dosages of 4 and 10 mf/mosquito, the L3 positive rates and the average filarial maturity rates in An. sinensis were much higher than those in Cx. quinquefasciatus (p less than 0.01). It was demonstrated that the immune response to filariae of the mosquitos was the main factor resulting in these differences. The intensity of the immune response not only varied with the species, but also declined with the aging of mosquitos. The results of our experiment might be useful to studies on the dynamics of filariasis transmission and the interaction between filariae and their insect hosts.     

细胞毒作用杀伤周期型马来丝虫幼虫的超微结构研究

本文在特异性免疫血清存在下，就嗜中性粒细胞和巨噬细胞对周期型马来丝虫幼虫的细胞毒作用进行了超微结构观察。结果显示，两种效应细胞对虫体都具有强大的杀伤作用。该作用开始于效应细胞伸出伪足包围并粘附于虫体，然后分泌颗粒性物质、释放各种对虫体有毒性作用的酶和其它生物活性成分于虫体表面。由于微丝蚴鞘膜和角皮层的溶解和脱落，导致了虫体内部组织迅速变性坏死。电镜下可见虫体肿胀变形、核溶解和空泡样坏死以及最终崩解剥落成絮状等连续变化。本试验还发现效应细胞伪足插入脱鞘微丝蚴内部的现象，说明微丝蚴脱鞘后更易受到效应细胞的攻击。扫描电镜观察到被效应细胞粘附的感染期幼虫表面出现裂痕。