Monoclonal antibody to a unique surface epitope of the human filaria Brugia malayi identifies infective larvae in mosquito vectors.

We describe properties of an IgM monoclonal antibody (NEB-D1E5) raised against the human filarial parasite Brugia malayi. The antibody reacts with a stage- and species-specific determinant located on the surface of the infective-stage larva, as determined by indirect immunofluorescence. To use this reagent in epidemiological field studies, we developed an enzyme-linked immunoassay with which B. malayi larvae can be differentiated from other filarial parasites in mosquito vectors, including the morphologically indistinguishable parasite of animals Brugia pahangi. The immunoenzyme assay was 91-94% specific and 90-97% sensitive when performed on infected mosquitoes. In the absence of mosquito tissue, the levels of specificity and sensitivity increased to 100% and 97.5-100%, respectively. Binding of antibody to the surface of living larvae was abrogated by treatment of the worms with the enzymes pronase and proteinase K and with the detergents Triton X-100, octyl beta-D-glucopyranoside, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS). In contrast, treatment with trypsin, endoglycosidase-F, O-Glycanase, N-Glycanase, lipase, various phospholipases, boiling, 2-mercaptoethanol at 37 degrees C, or periodate did not reduce the antigenicity of the larval surface to antibody NEB-D1E5. These results suggest that the species-specific epitope is a peptide domain attached to a hydrophobic anchoring residue.     

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.     

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.     

Protective immunity against Brugia malayi infective larvae in mice. I. Parameters of active and passive immunity

Protective immunity against infective larvae of Brugia malayi was studied in different strains of mice using various sources of antigens. The following strains of mice were susceptible to infective larvae development for 2 weeks after primary ip challenge: BALB/c, C3H/HeJ, C3H/NeN, C3H/HeJms, C57BL/6Jms, and DDD. In comparison to gerbils, BALB/c mice developed stronger resistance to infective larvae after immunization with irradiation attenuated larvae or with killed microfilariae (mf). However, killed mf failed to enhance resistance in C3H/HeJ mice, although C3H/HeN mice were strongly protected and C3H/HeJms mice were protected to a lesser degree by this antigen. Extracts of mf with phosphate buffered saline and sodium dodecyl sulfate both induced high levels of resistance in BALB/c mice. Transfer of resistance from BALB/c mice immunized with attenuated infective larvae to naive mice was accomplished at a high level at protection with nylon wool nonadherent spleen cells (T cells) but not with adherent cells treated with anti-Thy 1.2 serum and complement. In contrast, sera from immunized mice were much less protective.     

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.     

Brugia malayi: vaccination of jirds with 60cobalt-attenuated infective stage larvae protects against homologous challenge

Vaccination of inbred jirds (Meriones unguiculatus) with 60cobalt radiation-attenuated Brugia malayi infective stage larvae (L3) protected against homologous challenge given either subcutaneously (sc) or by the intraperitoneal (ip) route. Groups of jirds vaccinated once sc with 75, 15 Krad L3 showed from 69% to 91% reduction in recovered worms after ip challenge infection compared to infection in non-vaccinated control jirds, while 75% reduction in mean worm burden was seen in jirds receiving sc challenge infection. A single sc vaccination with 75, 10 or 20 Krad L3 produced no protection (10 Krad) and 64% reduction in recovered worms (20 Krad). Therefore the 15 Krad dose appeared to be best. A marked increase in anti-B. malayi antibody in vaccinated jirds was seen (by ELISA) immediately after challenge infection and an immunofluorescence assay showed that L3 incubated in serum from vaccinated jirds were completely and uniformly covered with specific antibody. Eosinophil-rich granulomas containing dead and moribund L3 were recovered from vaccinated jirds. This model of protective immunity in a Brugia-susceptible small rodent may provide a useful system for identification of molecularly defined filarial-protective immunogens.     

Brugia malayi infective larvae fail to activate Langerhans cells and dermal dendritic cells in human skin

Filarial infection in humans is initiated when a mosquito deposits third‐stage parasite larvae (L3) in the skin. Langerhans cells (LCs) and dermal dendritic cells (DDCs) are the first cells that the parasite encounters, and L3s must evade these highly effective antigen‐presenting cells to establish infection. To assess LC and DDC responses to L3 in human skin, we employed three models of increasing physiologic relevance: in vitro‐generated LCs, epidermal blister explants and full‐thickness human skin sections. In vitro‐generated LCs expressed TLR1‐10 and robustly produced IL‐6 and TNF‐α in response to PolyI:C, but pre‐exposure to L3s did not alter inflammatory cytokine production or TLR expression. L3s did not modulate expression of LC markers CDH1, CD207, or CD1a, or the regulatory products TSLP or IDO in epidermal explants or in vitro‐generated LC. LC, CD14+ DDC, CD1c+ DC and CD141+ DC from human skin sections were analysed by flow cytometry. While PolyI:C potently induced CCL22 production in LC, CD1c+ DC, and CD141+ DC, and IL‐10 production in LC, L3s did not modulate the numbers of or cytokine production by any skin DC subset. L3s broadly failed to activate or modulate LCs or DDCs, suggesting filarial larvae expertly evade APC detection in human skin.     

Effects of serum from treated patients on antibody-dependent cell adherence to the infective larvae of Brugia malayi

Adherence assays were used to demonstrate the in vitro effect of serum-dependent cellular adherence of human buffy coat cells to infective larvae of Brugia malayi in filariasis patients treated with antifilarial drugs. In this study, microfilaraemic patients were treated with either diethylcarbamazine citrate (DEC), mebendazole or levamisole hydrochloride. It was found that DEC and mebendazole decreased the motility of infective larvae due to a direct action of the drugs. Sera of levamisole-treated patients caused increased adherence of human buffy coat cells to infective larvae, leading to a decrease in motility and cuticular damage as confirmed by scanning electron microscopic studies. However, serum of levamisole-treated patients alone could cause a similar lethal effect on infective larvae. Studies with the indirect fluorescent antibody test suggested that IgM was involved in this phenomenon. Complement did not appear to be important.     

Ultrastructural observations on the fate of Brugia malayi in jirds previously vaccinated with irradiated infective stage larvae

Vaccination of inbred jirds (Meriones unguiculatus) with 60cobalt radiation-attenuated Brugia malayi infective stage larvae (L3) protected against homologous challenge given either subcutaneously (sc) or by the intraperitoneal route (ip). At necropsy numerous nodules were recovered from the peritoneal cavities of jirds which had been vaccinated sc and challenged ip. Histopathologic analysis showed these to be granulomas containing dead and dying larvae and transmission electron microscopy showed that eosinophils were present in high numbers around and within the larvae. Structural damage to the L3 cuticle was apparent in discrete areas and eosinophils actively entering the breached cuticle at the time of fixation were observed. Coalescence of eosinophil secretion granules and the formation of degranulation vacuoles were seen in eosinophils throughout the granulomas. Degranulation resulted in the deposition of electron-dense material on the surface of the larval cuticle. The jird vaccine model for B. malayi thus appears to be a potentially useful tool for investigation of immune mechanisms in filariasis.     

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.     

Activation of the alternative complement pathway in normal human serum by Loa loa and Brugia malayi infective stage larvae

Infective stage larvae (L3) of Loa loa and Brugia malayi upon in vitro incubation with normal human serum activated the alternative complement pathway. C3 conversion products were detected on larval cuticles by eosinophil adherence and by immunofluorescence with C3c antiserum. No evidence for cuticle binding of IgG, IgA, IgM, Clq, or C4 was found by immunofluorescence. L3-induced C3 activation was inhibited by 10 mM EDTA but unaffected by 10 mM Mg++-EGTA. Human sera deficient in C2, C4, or C6 incubated with L3 resulted in C3 activation. However, sera treated with zymosan or heated for 1 h, 56 degrees C were unreactive with L3. Immunoelectrophoresis of fresh serum exposed to L3 for 1 h at 37 degrees C showed C3 cleavage products. The results indicate that these nematode L3 activate the alternative complement cascade via cuticular surface components. Larval viability was unaffected by complement activation or by adherence of eosinophils.     

Humoral immune response to infective larval antigen in Brugia malayi infected Mastomys natalensis.

The sequential changes in the humoral immune response against infective larval antigens during the course of Brugia malayi infection in Mastomys natalensis have been studied using enzyme linked immunosorbent assay. IgM antibody against B. malayi infective larval excretory secretory (ES) antigen was detected in the peripheral circulation within a week of infection, whereas IgM antibody against B. malayi infective larval somatic antigen and IgG antibody against both somatic and ES antigens were detected on day 20 post-inoculation. Thereafter, the antibody levels showed a steady increase until day 150. A gradual decrease of IgM antibody level was observed upto day 360, whereas IgG antibody level was decreased upto day 250 and then maintained almost the same level upto day 360. Wuchereria bancrofti cross reactive antigen as well as B. malayi infective larval ES antigen were detected in blood circulation on day 20, the level increased upto day 150 and then remained almost the same upto day 360 with slight variations. Studies of antigen and antibody levels in microfilaraemic and amicrofilaraemic animals show that there is no significant difference in antibody level whereas elevated antigen titre was observed in active infection with microfilaraemia.     

The effect of p-aminobenzoic acid and folic acid on the development of infective larvae of Brugia malayi in Aedes aegypti

Adult Aedes aegypti mosquitoes, infected with the subperiodic Brugia malayi, were found to enhance the development of the filarial parasites to the infective stage when they were exposed to a cotton pad soaked in 10% sucrose solution containing p-aminobenzoic acid (PABA) in 0.001, 0.005, 0.01, 0.05 and 0.1% concentrations. Similarly, larval development increased when the mosquitoes were fed with folic acid at 0.001, 0.01 and 0.1% concentrations. This stimulation was more when PABA or folic acid was given prior to the infected blood meal through the developmental period of the larvae. The data thus suggest that PABA and folic acid are nutrients for the development of B. malayi-microfilariae to the infective stage in A. aegypti.     

Radiolabeling of Brugia malayi infective larvae in mosquitoes with 75Se-methionine and detection of these larvae in tissues of the Mongolian jird by autoradiography

Through preliminary experiments, an effective method for radiolabeling Brugia malayi-infected mosquitoes in order to produce labeled infective Brugia larvae was developed. Starting on the 6th day after the infective blood meal, mosquitoes were fed a 7% sucrose solution containing 100 microCi/ml 75Se-L-methionine for 5 days. Infective larvae, retrieved 2 days after this labeling period, averaged 381 +/- 136 counts/min. Jirds were infected with these infective, labeled larvae either by allowing infected mosquitoes to feed on uninfected jirds for 30 min or by inoculating jirds subcutaneously in the groin with washed larvae recovered from mosquitoes. Jirds were killed at various times after infection and were sliced into approximately 0.5 mm thick sagittal sections, which were dried and placed on X-ray film. Autoradiograms were developed after 30-60 days at 5 degrees C. In a sample of 26 inoculated jirds, approximately 30% of the infecting larvae could subsequently be accounted for as Ag degrees foci on autoradiograms. The Ag degrees foci representing larvae were apparent up to 2.5 weeks after infection. In jirds infected by mosquito feeding, the Ag degrees associated with the feeding site persisted for more than 6 weeks after infection.     

Detection of serum IgG antibodies specific for Wolbachia surface protein in rhesus monkeys infected with Brugia malayi.

The mechanism of lymphedema development in individuals with lymphatic filariasis is presently poorly understood. To investigate whether Wolbachia, symbiotic bacteria living within filarial nematodes, may be involved in disease progression, Wolbachia-specific immune responses were assayed in a group of Brugia malayi-infected rhesus monkeys. Serum IgG antibodies specific for a major Wolbachia surface protein (WSP) were detected in 2 of 12 infected monkeys. It is interesting that both of these monkeys developed lymphedema after becoming amicrofilaremic. WSP-specific antibody responses were temporally associated with increases in antifilarial IgG1 antibodies as well as lymphedema development. These findings suggest that Wolbachia may be important in understanding disease caused by filarial worms.     

Phosphocholine-containing antigens of Brugia malayi nonspecifically suppress lymphocyte function

The immunosuppressive effect of Brugia malayi antigen (BmA) on phytohemagglutinin (PHA) driven T cell proliferation was evaluated in patients with filariasis (n = 14) and compared to control individuals (n = 12). When peripheral blood lymphocytes were co-cultured with BmA and PHA, BmA markedly suppressed the T cell proliferative response to PHA in both filarial patients and control individuals in a dose-dependent manner. The suppression resulted neither from any direct toxicity of BmA nor from nonspecific absorption of the PHA mitogenic activity by BmA. The major suppressive component appears to be phosphocholine (PC), an immunodominant molecule present in abundance on filarial parasites and on circulating filarial antigen. Both purified PC as well as PC-containing antigens affinity purified from BmA were capable of suppressing the proliferative responses of co-cultured autologous lymphocytes to PHA. The suppressive activity was not abolished by mitomycin-C treatment and was greater in patients with filariasis than in normal controls, suggesting that levels of PC-containing antigens determines the magnitude of the suppressive effect of PC-antigen. Further, as induction of the suppressive activity was completely abrogated when antigen pre-treated cells were T cell-depleted, the suppressive effect appears to be mediated primarily by T cells.     

Comparative assessment of the in vitro sensitivity of Brugia malayi infective larvae to albendazole, diethylcarbamazine and ivermectin alone and in combination

The antifilaricidal drugs ivermectin (IVM), diethylcarbamazine (DEC), and albendazole (ALB), used alone or in combinations against infective third-stage larvae (L3) of nocturnally subperiodic (NSP) Brugia malayi (Narathiwat strain), were tested in vitro for sensitivity, for 7 days. IVM alone reduced larval motility at concentrations of 10(-7), 10(-6), and 10(-5) M on day 3. DEC alone also had this effect at concentrations of 10(-6). 10(-5), and 10(-4) M on day 2. ALB alone did not have this effect throughout the experiment, at various concentrations. However, it had greater effect when used in combination with either DEC or IVM. The result also indicated that DEC or IVM, when used in combination with ALB at concentrations of 10(-6) M/10(-6) M, and 10(-5) M/10(-5) M was effectively better than each drug used alone at those concentrations. When both drug combinations were compared, ALB/DEC seemed to be more effective than ALB/IVM at a concentration of 10(-6) M/10(-6) M on day 3. Although IVM and DEC can reduce larval motility when used alone or in combination with ALB, they cannot kill these larvae in an in vitro cultivation, even at a high concentration (10(-5) M).