In vitro effects of antibiotics on Brugia malayi worm survival and reproduction

Recent studies have suggested that intracellular Wolbachia spp. endobacteria are necessary for the reproduction and survival of filarial nematodes. The effects of antibiotics that are active against related bacteria on adult worms and microfilariae (Mf) of Brugia malayi in vitro were investigated. Antibiotics tested were doxycycline (Doxy), tetracycline (Tet), rifampicin (Rif), azithromycin (Azith), and chloramphenicol (Chlor). Doxy, Tet, Rif, and Azith reduced release of Mf by adult female worms. The minimum effective concentrations that reduced Mf release by 50% were 5 microg/ml for Doxy, 20 microg/ml for Tet, 40 microg/ml for Rif, and 100 microg/ml for Azith. The same drugs (at higher concentrations) killed adult worms and Mf. Embryograms showed that Tets blocked embryogenesis in female worms. Electron microscopy (EM) showed that the Tets, Rif, and Azith cleared Wolbachia spp. from adult worms and damaged developing embryos. These studies show that antibiotics active against Rickettsiaceae affect adult B. malayi worms and Mf in vitro. Additional studies will be needed to elucidate the mechanisms of action of these antibiotics on Wolbachia and filarial worms.     

Tetracycline inhibits development of the infective-stage larvae of filarial nematodes in vitro

In recent years, studies have linked tetracycline treatment of filaria-infected animals with reduced adult worm burdens and decreased levels of microfilaremia. These observations are believed to be attributable to clearance of Wolbachia, intracellular rickettsial-like organisms found within filarial tissues. Although maximal worm reductions were observed when treatment was initiated early in infection, it is not known whether tetracycline inhibits development of infective-stage larvae. To address this issue, we studied the effect of tetracycline on three different species of filarial nematodes, Brugia malayi, Brugia pahangi, and Dirofilaria immitis, in a serumfree in vitro system supporting molting to the fourth larval stage. Tetracycline was capable of inhibiting L3 to L4 molting within a dosage range similar to that reported for susceptible rickettsial organisms. However, Wolbachia DNA could still be detected in nematodes from tetracycline-treated cultures. In addition, three other antibiotics with anti-rickettsial and anti-chlamydial activity (chloramphenicol, erythromycin, and ciprofloxacin) failed to inhibit L3 to L4 molting. Although tetracycline is capable of completely blocking molting of infective-stage larvae, it remains possible that this effect is due to pharmacological activities unrelated to its anti-rickettsial functions.     

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.     

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.     

Macrofilaricidal and microfilaricidal effects of Neurolaena lobata, a Guatemalan medicinal plant, on Brugia pahangi

Twelve extracts of 11 Guatemalan medicinal plants were initially screened in vitro for potential macrofilaricidal activity against Brugia pahangi, a lymphatic dwelling filarial worm, using concentrations from 125 to 1000 microg ml(-1) of each extract that could be dissolved in the culture medium. Of 12 extracts used, the ethanol extract of leaves of Neurolaena lobata showed the strongest activity against the motility of adult worms. Subsequently, the extract of N. lobata was extensively examined in vitro for macro- and micro-filaricidal effects using a series of concentrations of 500, 250, 100, 50 and 10 microg ml(-1). The effects were assessed by worm motility, microfilarial release by female worms and a MTT assay. The effect on the motility of adult worms was observed in a concentration- and time-dependent manner. The time required to stop motility of both sexes of adult worms was 6 h at 500 microg ml(-1), 24 h at 250 microg ml(-1), and 3 days for females and 4 days for males at 100 microg ml(-1). The movement of females ceased at 4 days at a concentration of 50 microg ml(-1) whereas the motility of males was only reduced. The loss of worm's viability was confirmed by the MTT assay and was similar to the motility results. These concentrations, including 10 microg ml(-1), prevented microfilarial release by females in a concentration- and time-dependent manner. Concentrations higher than 100 microg ml(-1) even induced mortality of the microfilariae. The present study suggested that the ethanol extract of Neurolaena lobata has potential macro- and micro-filaricidal activities.     

Studies of the growth-regulating effects of ivermectin on larval Onchocerca lienalis in vitro

At concentrations of 0.1-100 ng/ml ivermectin inhibited L3-L4 molting by Onchocerca lienalis in vitro. The degree of inhibition was dose-dependent with a significant effect apparent at 0.1 ng/ml and complete inhibition occurring at 100 ng/ml. The ED50 for molt inhibition was 0.19 ng/ml. Molt-inhibiting levels of the drug were not acutely toxic to the worms. In the presence of 10 ng/ml, a concentration giving 95% molt inhibition, motility at day 7 postinoculation was 71% of that seen in nontreated controls. A more pronounced effect on motility was apparent in larvae under long-term cultivation in the presence of ivermectin. Kinetic studies indicated that the majority of the larvae respond irreversibly to the drug within the first 2 hr of exposure. Twenty-four hours of exposure were required for a maximal response. The inhibitory effects of ivermectin were less pronounced if larvae were allowed to develop under normal culture conditions for 24 or more hours prior to the initiation of drug treatment.     

Suppression of Brugia malayi (sub-periodic) larval development in Aedes aegypti (Liverpool strain) fed on blood of animals immunized with microfilariae

Preliminary studies were carried out to investigate the role of filarial specific antibodies, raised in an animal model against the filarial parasite, Brugia malayi (sub-periodic), in blocking their early development in an experimental mosquito host, Aedes aegypti (Liverpool strain). In order to generate filarial specific antibodies, Mongolian gerbils, Meriones unguiculatus, were immunized either with live microfilariae (mf) of B. malayi or their homogenate. Mf were harvested from the peritoneal cavity of Mongolian gerbils with patent infection of B. malayi and fed to A. aegypti along with the blood from immunized animals. Development of the parasite in infected mosquitoes was monitored until they reached infective stage larvae (L3). Fewer number of parasites developed to first stage (L1) and subsequently to L2 and L3 in mosquitoes fed with blood of immunized animals, when compared to those fed with blood of control animals. The results thus indicated that filarial parasite specific antibodies present in the blood of the immunized animals resulted in the reduction of number of larvae of B. malayi developing in the mosquito host.     

Ivermectin inhibits molting of Wuchereria bancrofti third stage larvae in vitro

The effect of ivermectin or diethylcarbamazine (DEC) on Wuchereria bancrofti molting from the third to the fourth larval stage (L3 to L4) was evaluated in vitro. L3 larvae were harvested from laboratory-reared Aedes togoi 2 wk after feeding upon a microfilaremic human volunteer. The larvae were kept in an artificial medium (Franke's NI medium) with 10% human serum under an atmosphere of 5% CO2 for 20 days. Experimental tubes also contained ivermectin (0.1-1,000 ng/ml) or DEC (0.1-10,000 ng/ml). An estimated concentration of 50 ng/ml ivermectin inhibited molting in 50% of the larvae expected to molt. For DEC, this value was roughly 1,000 ng/ml. In this in vitro culture system, ivermectin inhibited the L3 to L4 molt of W. bancrofti and was roughly 20-fold more potent in this activity than DEC.     

Effects of environmentally relevant concentrations of nonylphenol on growth and 20-hydroxyecdysone levels in mysid crustacean, Americamysis bahia

We investigated the effects of environmentally relevant concentrations of nonylphenol (NP) on survival, growth, sexual development and molting of the mysid Americamysis bahia (Crustacea: Mysidacea), including assay of 20-hydroxyecdysone (20E) levels. Seven-day-old mysids were exposed to NP concentrations of 0.3, 1, 3, 10 and 30 microg/L for 14 days. Among growth-related traits we assayed, an effect on body length was the most prominent, with the effects observed at concentrations as low as 1 microg/L NP. In addition, the total number of molting exposed to NP for 14 days was significantly decreased in the treatment groups at 10 or 30 microg/L relative to the controls. These results clearly indicate that subchronic exposure to environmentally relevant concentrations of NP affects growth of mysids. Furthermore, we investigated the effect of NP on production of 20E in female mysids during the molting period. Production of 20E in pre-molting mysids exposed to 30 microg/L of NP was lower than those in control groups, and exposure of female mysids to NP disrupted molting cycles. Taken together, the results suggest that environmentally relevant concentrations of NP can disrupt growth of A. bahia via inhibition of 20E levels and an associated delay in molting.     

Setaria cervi: immunoprophylactic potential of glutathione-S-transferase against filarial parasite Brugia malayi

Glutathione-S-transferase (GST) has been detected in the adult female Setaria cervi, a bovine filarial parasite. The role of S. cervi GST antigen in inducing immunity in the host against Brugia malayi microfilariae and infective larvae was studied by in vitro antibody dependent cell mediated reaction as well as in situ inoculation of filarial parasites within a microchamber in Mastomys. The immune sera from glutathione-S-transferase immunized Mastomys promoted the adherence of peritoneal exudate cells to B. malayi microfilariae and infective larvae in vitro inducing 80.7 and 77.6% cytotoxicity, respectively in 72 h. In the microchambers implanted in the immunized Mastomys host cells could migrate and adhere to the microfilariae and infective larvae and induced 77.8 and 75% cytotoxicity to B. malayi microfilariae and infective larvae in 72 h, respectively. These results suggest that native GST from S. cervi is effective in inducing protection against heterologous B. malayi filarial parasite and thus has potential in immunoprophylaxis.     

Effects of condensed tannins and crude sesquiterpene lactones extracted from chicory on the motility of larvae of deer lungworm and gastrointestinal nematodes

The objective of this study was to determine the effects of condensed tannins (CT) and an extract containing crude sesquiterpene lactones (CSL) from chicory (Cichorium intybus) on the motility of the first-(L1) and third-stage (L3) larvae of deer lungworm Dictyocaulus viviparus and the L3 larvae of gastrointestinal nematodes in vitro, using the larval migration inhibition (LMI) assay. The CT and CSL had a profound effect on the motility of the larvae displayed by their ability to inhibit larval passage through nylon mesh sieves. Incubation of lungworm L1 larvae in rumen fluid (collected from deer fed pasture) containing 100, 400 and 1000 microg CT/ml, inhibited 12, 28 and 41% of the larvae from passing through the sieves, respectively, while the incubation of L3 larvae with rumen fluid (pH 6.6) containing the same concentrations inhibited 26, 37 and 67% of L3 larvae from passing through the sieves, respectively. Gastrointestinal larvae seem more susceptible to CT than lungworm larvae especially at higher concentrations. CT inhibited 27, 56 and 73% of gastrointestinal larvae from passing through the sieves when used at a concentration of 100, 400 and 1000 microg/ml, respectively. CT were more effective (P<0.001) at reducing the motility of lungworm L1 and L3 larvae when added to the rumen fluid than when added to the abomasal fluid (pH 3.0). Addition of 2 microg polyethylene glycol/microg CT eliminated the inhibitory effect of CT against L1 and L3 larvae especially during incubation in rumen fluid, confirming the effect as due to CT. The CSL extract also showed similar inhibitory activity against L1 and L3 lungworm and L3 gastrointestinal larvae in both fluids, indicating that this extract was not affected by the pH of the fluid, and was more effective against L3 than L1 lungworm larvae. Condensed tannins appeared to be more effective than CSL at inactivating L1 and L3 lungworm and L3 gastrointestinal larvae in rumen fluid, but CSL were particularly effective against L3 lungworm larvae in abomasal fluid. Activity of these secondary compounds explains the reduced parasite problem of young deer grazing chicory.     

Evaluation of five antibiotics on larval gut bacterial diversity of Plutella xylostella (Lepidoptera: Plutellidae)

Larvae of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), have rich microbial communities inhabiting the gut, and these bacteria contribute to the fitness of the pest. In this study we evaluated the effects of five antibiotics (rifampicin, ampicillin, tetracycline, streptomycin sulfate and chloramphenicol) on the gut bacterial diversity of P. xylostella larvae. We screened five different concentrations for each antibiotic in a leaf disc assay, and found that rifampicin and streptomycin sulfate at 3 mg/mL significantly reduced the diversity of the bacterial community, and some bacterial species could be rapidly eliminated. The number of gut bacteria in the rifampicin group and streptomycin sulfate group decreased more rapidly than the others. With the increase of antibiotic concentration, the removal efficiency was improved, whereas toxic effects became more apparent. All antibiotics reduced larval growth and development, and eventually caused high mortality, malformation of the prepupae, and hindered pupation and adult emergence. Among the five antibiotics, tetracycline was the most toxic and streptomycin sulfate was a relatively mild one. Some dominant bacteria were not affected by feeding antibiotics alone. Denaturing gradient gel electrophoresis graph showed that the most abundant and diverse bacteria in P. xylostella larval gut appeared in the cabbage feeding group, and diet change and antibiotics intake influenced gut flora abundance. Species diversity was significantly reduced in the artificial diet and antibiotics treatment groups. After feeding on the artificial diet with rifampicin, streptomycin sulfate and their mixture for 10 days, larval gut bacteria could not be completely removed as detected with the agarose gel electrophoresis method.     

The in vitro effect of albendazole, ivermectin, diethylcarbamazine, and their combinations against infective third-stage larvae of nocturnally subperiodic Brugia malayi (Narathiwat strain): scanning electron microscopy.

Scanning electron microscopy (SEM) was employed to observe the effects of ivermectin (IVM), diethylcarbamazine (DEC), and albendazole (ALB) alone, and the drugs in combination (ALB+IVM and ALB+DEC) against infective third stage larvae (L3) of nocturnally subperiodic (NSP) Brugia malayi (Narathiwat strain) in vitro. IVM, at a concentration of 10(-4) M, killed L3 within 1-2 h. The SEM data showed damage to the L3 surface and loss of regular cuticular annulations. The cuticles were grooved in the middle region of the body. In comparison with normal L3 before treatment with IVM, the cuticular surface showed transversed striations with periodic annulations. The result demonstrated that IVM showed a larvicidal activity against L3 of NSP B. malayi cultivated in vitro. Compared with those larvae in the control group, the treated larvae had no morphological changes in the cuticular surface at the head, body, and tail regions after cultivation with all drugs alone, and in their combinations at a concentration of 10(-5) M for 7 d. In this system, and at that concentration, only the larvae cultured with ALB alone remained highly motile. Although no morphological changes had been observed by SEM, those drugs used alone (IVM and DEC) and in combinations (ALB+IVM and ALB+DEC), reduced larval motility throughout the experiments at a concentration of 10(-5) M. The minimum lethal concentration (MIC) of IVM against NSP B. malayi was 10(-4) M.     

Effectiveness of the antibiotics chloramphenicol and rifampin in the treatment of Streptococcus pneumoniae-induced meningitis and systemic infections

Streptococcus pneumoniae, a common pathogen in pediatric infections, has become resistant to penicillin and make these infections difficult to treat. Rifampin and chloramphenicol have been recommended as alternative therapies, since they are less costly and more accessible to communities with limited resources. However, their use may be restricted by the differing levels of resistance found in target populations. The objective was to determine minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) for chloramphenicol and rifampin in strains of S. pneumoniae. These strains were newly isolated from children under age 5 that had demonstrated systemic infections and meningitis. A subgroup of 107 isolates of S. pneumoniae was selected from 324 strains isolated during a period of 2 years (1994-1996). Among these isolates, 60 were penicillin-resistant and 47 were susceptible; 53 isolates were from children with meningitis. MIC and MBC for chloramphenicol and rifampicin were obtained by standard methods recommended by the National Committee for Clinical Laboratory Standards (NCCLS). S. pneumoniae ATCC strain 49619 served as the control. An isolate was considered susceptible to chloramphenicol when MIC = 4 microg/ml and resistant when MIC = 8 microg/ml. A strain was considered susceptible to rifampin when MIC = 1 microg/ml and resistant when MIC = 4 microg/ml. MBC was determined by recording the lower concentration of the antibiotic that inhibited 99.9% of the initial inoculum. Chloramphenicol resistance was found in 21% of the 107 isolates. In the group susceptible to penicillin, 11% were resistant to chloramphenicol and in the group resistant to penicillin 28% was resistant to chloramphenicol as well. MBC was found > 4 microg/ml in 28% of the isolates susceptible to penicillin and in 60% of the resistant isolates. No isolates were found resistant to rifampin. However, 2 penicillin resistant isolates showed CBM > 1 microg/ml to rifampin, and one with CIM = 1 microg/ml had a MBC to rifampicin of 16 microg/ml. Meningitis isolates showed higher CIM and CBM than the group of total isolates. These data suggest that chloramphenicol is not recommended for invasive infections caused by S. pneumoniae in Colombia. Rifampin is a more effective therapy in combination with other antibiotics for treatment of this kind of infections. Further studies are necessary to clarify the significance of low levels of MBC to rifampin found in some strains, since this may affect the efficacy of therapies that include this antibiotic.     

Brugia malayi and Brugia pahangi: transmission blocking activity of ivermectin and brugian filarial infections in Aedes aegypti

Brugia malayi- or Brugia pahangi-infected, microfilaremic jirds (Meriones unguiculatus) were treated with ivermectin at a single dose of 200 micrograms/kg body weight, administered subcutaneously. After different time intervals, Aedes aegypti mosquitoes were fed on treated or untreated jirds. Sausage stage, L2, and L3 larvae failed to develop in mosquitoes that fed on jirds from 15 to 30 days post-treatment. After 1 month, the numbers of L3 larvae recovered from mosquitoes fed on treated B. pahangi jirds were comparable to controls. However, the number of L3's recovered from mosquitoes fed on B. malayi jirds remained significantly lower than controls, 2 and 3 months after treatment. This reduction suggests that ivermectin may be more effective in blocking transmission of B. malayi than B. pahangi. Ivermectin treatment had no effect on the mean number of circulating microfilariae in treated jirds. Therefore, mosquitoes ingested comparable numbers of microfilariae when compared to those mosquitoes fed on untreated controls. Only in the case of jirds infected with B. malayi did the circulating microfilarial counts fall 30 days after treatment. The failure of microfilariae to develop to the L3 stage in mosquitoes fed on jirds within 30 days of treatment was not due to failure of mosquitoes to ingest microfilariae. Brugia malayi microfilariae also failed to develop to L3 in mosquitoes that were allowed to feed on microfilaremic jird blood treated with ivermectin (50 ng/ml) in vitro, indicating its efficacy at low concentrations. In addition to N-acetyl glucosamine, microfilariae obtained for a period of 15 days from ivermectin-treated but not control jirds showed D-mannose, N-acetyl galactosamine, and L-fucose moieties on the surface of the sheath.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro culture of Dirofilaria immitis third- and fourth-stage larvae under defined conditions

The objective of the present study was to define culture conditions under which larval Dirofilaria immitis would molt, grow, and survive. Third-stage larvae (L3) survived for over 3 wk with a molt rate of up to 95% in a variety of media supplemented with fetal calf serum. Bovine albumin, added to several media at concentrations of 10-30 mg/ml, also proved to be an effective culture supplement for the induction of molting and for supporting larval survival. Two gas phases were tested, 5% CO2/95% N2 and 5% CO2/air; no differences were noted in larval development based on gas phase. Larvae, maintained in media with FCS or albumin for 48 hr, were capable of completing the molting process and growing in length in unsupplemented media. If the temperature at which cultures were maintained was changed from 37 C to 27 C, L3 did not molt but did survive for several weeks. Two factors required for larval D. immitis molting and growth have been identified, temperature of approximately 37 C and the presence of albumin in the culture medium. The defined culture system developed for D. immitis L3 may provide a source for collection of excretory-secretory antigens, which could prove useful in immunodiagnosis or immunoprophylaxis as well as provide a means of studying the process and requirements of filarial larval molting.     

Adult 175 kDa collagenase antigen of Setaria cervi in immunoprophylaxis against Brugia malayi in jirds

A 175 kDa antigen fraction with collagenase activity was isolated and purified from somatic extracts of adult Setaria cervi females using column chromatography involving consecutive steps of DEAE-Sepharose CL6B and Sephadex G-100. The optimum pH for 175 kDa collagenase was found to be pH 7.0. Sensitivities to a variety of inhibitors and activators indicated that the 175 kDa coIlagenolytic enzyme was metalloserine in nature. The enzyme hydrolysed a variety of protein substrates such as haemoglobin, casein, azocasein (general substrates) and collagen, FALGPA (furanoyl-acryloyl-leu-gly-pro-ala), the specific substrate of collagenase. The enzyme showed 57% inhibition by jird anti-somatic collagenase antibodies and reacted insignificantly with normal jird sera. Further analysis was undertaken on the immunoprophylactic potential of 175 kDa collagenase in inducing immunity against Brugia malayi (a human filarial parasite) in jirds (Meriones unguiculatus) in vitro and in situ. Immune sera of jirds raised against this antigen promoted partial adherence of peritoneal exudate cells to B. malayi microfilariae (mf) and infective larvae (L3) in vitro and induced partial cytotoxicity to the parasites within 48 h. The anti-S. cervi 175 kDa antigen serum was more effective in inducing cytotoxicity to B. malayi L3, than mf. In the microchambers implanted inside immune jirds, host cells could migrate and adhere to the mf and infective larvae thereby killing them partially within 48 h.     

Antifilarial activity of marine sponge Haliclona oculata against experimental Brugia malayi infection

The present study incorporates the findings on in vitro and in vivo antifilarial activity in the marine sponge, Haliclona oculata using an experimental rodent infection of human lymphatic filarial parasite, Brugia malayi. The in vitro antifilarial action was determined on both adult female worms as well as microfilariae using two parameters viz. adverse effect on motility and inhibition in MTT reduction by the treated adult parasite over control worm. The antifilarial activity could be located in the methanol extract and one of its four fractions (chloroform). Bioactivity guided fractionation of chloroform fraction led to localization of in vitro activity in one of its eight chromatographic fractions. Methanol extract, chloroform fraction and one of the chromatographic fractions revealed IC(50) values of 5.00, 1.80, and 1.62μg/ml, respectively when adult B. malayi were exposed to these test samples for 72h at 37°C. Under similar exposure conditions, the IC(50) values for microfilariae were 1.88, 1.72 and 1.19μg/ml, respectively. The active test samples were found to be safe revealing >10 selectivity indices (SI) on the basis of cytotoxicity to Vero cells (monkey kidney cells) and therefore selected for in vivo evaluation against primary (adult B. malayi intraperitoneal transplanted jird) and secondary (subcutaneous infective larvae induced mastomys) screens. In primary jird model, the three test samples at 100mg/kg for five consecutive days by subcutaneous route demonstrated significant macrofilaricidal efficacy to the tune of 51.3%, 64% and 70.7% by methanol extract, chloroform and chromatographic fraction, respectively. The three samples demonstrated 45-50% macrofilaricidal activity with moderate embryostatic effect in secondary model at 5×500, 5×250 and 5×125mg/kg by oral route. Chromatographic fraction possessing highest antifilarial action was primarily found to be a mixture of four alkaloids Mimosamycin, Xestospongin-C, Xestospongin-D and Araguspongin-C in addition to few minor compounds.     

Expansion of Foxp3+ regulatory T cells in mice infected with the filarial parasite Brugia malayi

Many helminths, including Brugia malayi, are able to establish long-lived infections in immunocompetent hosts. Growing evidence suggests that the immune system's failure to eliminate parasites is at least partially due to the effects of regulatory T cells (Tregs). To test whether parasites may directly stimulate host regulatory activity, we infected mice with two key stages of B. malayi. Both mosquito-borne infective larvae and mature adults i.p. introduced were found to preferentially expand the proportion of CD25(+)Foxp3(+) cells within the CD4(+) T cell population. The induction of Foxp3 was accompanied by raised CD25, CD103, and CTLA-4 expression, and was shown to be an active process, which accompanied the introduction of live, but not dead parasites. CTLA-4 expression was also markedly higher on Foxp3(-) cells, suggesting anergized effector populations. Peritoneal lavage CD4(+)CD25(+) cells from infected mice showed similar suppressive activity in vitro to normal splenic "natural" Tregs. Both B. malayi larvae and adults were also able to induce Foxp3 expression in adoptively transferred DO11.10 T cells, demonstrating that filarial infection can influence the development of T cells specific to a third party Ag. In addition, we showed that induction was intact in IL-4R-deficient animals, in the absence of a Th2 or alternatively activated macrophage response. We conclude that filarial infections significantly skew the balance of the host immune system toward Treg expansion and activation, in a manner dependent on live parasites but independent of a concomitant Th2 response.