Antibody-mediated cytotoxic effects in vitro and in vivo of rat cells on infective larvae of Brugia malayi

Albino rat macrophages and neutrophils in the presence of immune serum adhered to and promoted killing of Brugia malayi infective larvae in vitro. At a similar cell-target ratio, macrophages were more potent than neutrophils in inducing cytotoxic response to the larvae. Eosinophils were also effective in killing but only at a high cell-target ratio. The activity in the immune serum could be absorbed to and eluted from a Protein A-Sepharose column suggesting involvement of IgG antibody in the reaction. An indirect fluorescent antibody test confirmed the presence of IgG on the surface of larvae incubated in immune serum. Infective larvae were attacked by host cells within micropore chambers 16-24 h after implantation into immunized rats. Further, a strong cytotoxic response to the larvae was seen when they were introduced intraperitoneally into immune rats indicating the role of antibody and cells in vivo. We suggest that antibody-dependent cellular cytotoxicity may represent an important mechanism of parasite killing in an immune host.     

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.     

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.     

Immunoprophylaxis against filarial parasite,Brugia malayi: potential of excretory-secretory antigens in inducing immunity

The role of excretory-secretory antigens in inducing immunity in the host againstBrugia malayi microfilariae and infective larvae was studied byin vitro antibody dependent cell-mediated reaction as well asin vivo inoculation of filarial parasites within a microchamber in the host. The immune sera of jirds raised againstBrugia malayi microfilarial and infective larval excretory-secretory antigens(Bm Mf ESA andBm L₃ ESA) promoted the adherence of peritoneal exudate cells toBrugia malayi microfilariae and infective larvaein vitro and induced cytotoxicity to the parasites within 48 h. The antiBm Mf ESA serum was more effective than antiBm L3 ESA serum in inducing cytotoxicity to microfilariae and both antisera had a similar cytotoxic effect on infective larvae. In the microchambers implanted in the immune jirds, host cells could migrate and adhere to the microfilariae and infective larvae and kill them within 48–72 h. Further,Mastomys natalensis immunized againstBm Mf ESA and L₃ ESA generated a high degree of protective response against circulating microfilariae. These results suggest that excretory-secretory antigens are effective in inducing resistance against filarial parasites and thus have potential in immunoprophylaxis.     

A combination of two Brugia malayi filarial vaccine candidate antigens (BmALT-2 and BmVAH) enhances immune responses and protection in jirds

In this study filarial recombinant protein or DNA vaccine constructs encoding BmALT-2 and BmVAH as single or as cocktail antigens were evaluated. Male jirds were immunized intramuscularly with DNA vaccine constructs or were immunized intraperitoneally with protein vaccine. The single and bicistronic DNA constructs induced substantial interferon-γ responses in spleen cells; antigen-specific responses were higher following immunization with the bicistronic cocktail construct and evoked a significant protective response of 57% in jirds challenged with Brugia malayi that was similar in the antibody-dependent cellular cytotoxicity (ADCC) assay and micropore chamber experiment. The cocktail protein vaccines induced a mixture of IgG2a (Th1) and IgG1 (Th2) responses with 80% protective response when challenged with B. malayi infective larvae. However, the single protein vaccine rALT-2 induced Th2 (IgG1/IgG3) with a 70% protective response and rVAH induced Th1 (IgG2a) with a lower proliferative response with 60% protection following challenge with B. malayi infective larvae. These results suggest that filarial cocktail protein vaccines are able to elicit substantial immune and protective responses when compared with single antigen vaccination in suitably vaccinated jirds.     

Induction of protection against Brugia malayi infection in jirds by microfilarial antigens

The development of immunologic methods to reduce transmission of human lymphatic filariasis depends on measures that will enhance the host's ability to eliminate infective larvae, adult worms, or blood-borne microfilariae (mf). The present study was designed to assess the capacity of a crude extract of Brugia malayi mf to decrease the level of microfilaremia and adult worm burden in jirds inoculated with infective larvae, and to identify the filarial antigens that elicit antibody responses in these animals. Thirty weeks after subcutaneous inoculation with 75 infective larvae, 100% of control jirds were patent (i.e., had microfilaremia) compared with 60% of the group immunized with 10 micrograms of crude microfilarial extract (p less than 0.05). In addition, microfilaremia was lower in patent immunized animals compared with controls (p less than 0.05). The mean total number of adult female B. malayi per jird recovered at necropsy in control animals was 16.0 vs 7.0 in immunized jirds (p less than 0.05). Serum of immunized jirds contained anti-mf antibodies with an end titer of 1:8000, a value similar to that of animals with chronic B. malayi infection. Microfilarial antigens of Mr approximately 150,000, 75,000, 42,000, and 25,000 were identified in immunoblotting studies by reactivity with antibodies in sera of immunized jirds. Antibodies induced by immunization with microfilarial extract were not specific for this stage of the parasite life cycle, as jird anti-mf antibodies reacted with a Mr approximately 150,000 and several Mr 50,000 to 110,000 antigens derived from immature and mature adult parasites of both sexes. These data indicate that immunization of jirds with a water soluble microfilarial extract enhances the host's ability to eliminate adult worms and blood-borne mf. The filarial antigens that induce antibodies in immunized jirds have been identified.     

Brugia pahangi: stage-specific antigens on microfilariae detected by serum from infected jirds or by monoclonal antibodies

Experiments were carried out to determine whether there are stage-specific antigens on microfilariae of Brugia pahangi, using sera from Mongolian jirds infected with B. pahangi and monoclonal antibodies against microfilariae of B. pahangi. These studies showed that microfilariae have both stage-specific and nonspecific antigens. The nonspecific antigens were also present on adult worms and on infective larvae. Among monoclonal antibodies, 6 out of 14 clones produced antibodies against the microfilarial stage-specific antigens, and 8 clones produced antibodies against nonspecific antigens. These monoclonal antibodies could not distinguish between adults, microfilariae, or infective larvae of B. malayi and B. pahangi.     

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)     

The effect of sera from cats infected with Brugia pahangi and subsequently treated with levamisole on the infectivity of third-stage larvae of B. pahangi

Cats were treated with levamisole and the infective (L3) stage of Brugia pahangi. Serum from infected cats was subsequently tested for its ability to infect jirds. Jirds autopsied at 33 days postmortem showed significant levels of parasitemia. This is contrary to reports of a previous study wherein serum from humans infected with B. malayi was found to induce cell adherence and death of the L3.     

Variable infectivity of third-stage larvae of Brugia malayi

Infectivity of third-stage larvae of Brugia malayi was assessed following intraperitoneal inoculation into jirds, Meriones unguiculatus. Larvae were of two ages and were derived from two sites in Aedes aegypti mosquitoes, i.e., specimens collected from the thorax 11 days after infection and from the head on day 14. Larvae from the thorax had just completed the second moult and measured 990 to 1100 micron in length. Only 6% of these specimens developed to adult worms in jirds. Larvae that migrated to the head were 1400 to 1700 micron long on day 14 and, in contrast, 23% of the inocula developed to adult worms. This study establishes that all third-stage larvae, regardless of their age or location in the arthropod host, are potentially infective. However, pronounced physical maturation does seem to be accompanied by a marked increase in infectivity.     

Brugia malayi: detection of parasite antigen in sera from infected jirds

Sera from Brugia malayi-infected jirds were demonstrated to contain a heat-stable, 95- to 105-kDa parasite antigen by immunoblot with rabbit antibody to the parasite and with a monoclonal antibody that binds to phosphorylcholine. This antigen is a major component of B. malayi adult worm excretory/secretory antigen, and it is present in lavage fluid obtained from ip-infected animals. The antigen was detected by enzyme immunoassay in all sera collected from jirds 9-54 weeks after sc injection with 100 or 300 infective larvae (L3). Parasite antigen titers were higher in animals infected with the higher L3 dose. Antiphosphorylcholine antibodies were present in jird sera for the first 12 weeks after larval injection, but thereafter, antibody titers decreased to undetectable levels. Parasite antigen was not detected by immunoblot or enzyme immunoassay in sera from 21 human subjects with B. malayi microfilaremia. Antigen may be cleared from human sera by antiphosphorylcholine antibodies, which were present in all sera tested. The practical significance of B. malayi antigen detection in the jird is that it provides a sensitive means of noninvasively monitoring the status of infection in this important experimental filariasis model.     

Identification of 38kDa Brugia malayi microfilarial protease as a vaccine candidate for lymphatic filariasis

A FPLC purified 38kDa protease (Bm mf S-7) isolated from B. malayi microfilarial soluble antigen was identified. It showed pronounced reactivity with sera collected from 'putatively immune' asymptomatic and amicrofilaraemic individuals residing in an endemic area for bancroftian filariasis. Further the immune protective activity of Bm mf S-7 antigen was evaluated in susceptible hosts, jirds (Meriones unguiculatus) against B. malayi filarial infection. The antigen showed 89% cytotoxicity against mf and 87-89% against infective (L3) larvae in in vitro antibody dependent cellular cytotoxicity Assay (ADCC) and in situ micropore chamber methods. Bm mf S-7 immunized jirds after challenge infection showed 81.5% reduction in the adult worm burden. The present study has shown that, the 38kDa microfilarial proteases (Bm mf S-7) could stimulate a strong protective immune response against microfilariae and infective larvae in jird model to block the transmission of filariasis. Analysis of IgG subclasses against Bm mf S-7 revealed a significant increase in IgG2 and IgG3 antibodies in endemic normals. Lymphocyte proliferation to Bm mf S-7 was significantly high in endemic normal group as compared to that in clinical and microfilarial carriers. Significantly enhanced levels of IFN-gamma in the culture supernatant of PBMC of endemic normals followed by stimulation with Bm mf S-7 suggest that the cellular response in this group is skewed towards Th 1 type.     

Microfilarial periodicity in Mastomys natalensis

The microfilarial level in the peripheral blood of Mastomys natalensis infected with the filarial parasites, Litomosoides carinii, Brugia pahangi, B. malayi and Dipetalonema viteae was monitored at two-hour intervals for 24 hours. The microfilariae of B. pahangi and B. malayi exhibited nocturnal and diurnal subperiodicity, respectively; no such periodicity was seen in L. carinii and D. viteae infections. The level of B. pahangi and D. viteae microfilariae in peripheral blood was significantly increased when the host was anaesthetized with diethylether or pentothal sodium. Ether-induced anaesthesia had no effect on the level of B. malayi microfilariae but pentothal was most effective. The peripheral blood count of L. carinii microfilariae tended to decrease in the anaesthetized animals but the reduction was not statistically significant.     

Induction of host resistance to Brugia pahangi in jirds (Meriones unguiculatus) protected by chemoprophylaxis

Jirds were given a chemoprophylactic inoculation of flubendazole (FMBZ) and then five injections of infective larvae of Brugia pahangi whilst still protected by the FMBZ. When the drug was thought to be non-effective the jirds (and controls) were given a challenge infection of B. pahangi larvae. By comparison with control jirds the treated-infected-challenged jirds had 40% fewer adult worms. The control treated-challenged jirds contained mostly sterile female worms showing that they were still partially protected by FMBZ but worms numbers were not significantly reduced as compared with untreated controls.     

Studies with Brugia pahangi 17. The anthelmintic effects of diethylcarbamazine.

Diethylcarbamazine (DEC) was active in vitro against infective larvae and microfilariae of Brugia pahangi but only at high concentrations. When fed to mosquitoes which were infected with B. pahangi it had little or no activity. In jirds it was inactive against B. pahangi microfilariae and adults when administered at 300 mg/kg for 5 days either by the intraperitoneal or oral route. In cats given 25 or 50 mg DEC/kg intraperitoneally on 3 or 5 occasions it was not microfilaricidal, but most of the adult worms died within 30 days of the end of treatment. Although most microfilariae disappeared from the blood of cats immediately (i.e., within an hour) after treatment, they reappeared within a few hours in the same numbers. Microfilarial levels were reduced after treatment but there was no precipitate decline as occurs in human B. malayi patients.     

Possible direct effect of diethylcarbamazine on the infective larvae of Brugia pahangi

The direct action of diethylcarbamazine (DEC) on the infective larvae of Brugia pahangi was studied. The larvae were cultured in RPMI 1640 supplemented with foetal bovine serum and antibiotics for 22 days. Most of the larvae remained alive for 8 days, but survival rate of larvae decreased rapidly from day 10 onwards. The larvae did not grow in the culture system. The addition of DEC did not affect the morbidity of the larvae and no difference was observed in the morphological characteristics between the larvae cultured in the presence or absence of DEC. The infective larvae were cultured in vitro for 5 days in the presence or absence of DEC, and inoculated into jirds. The animals were necropsied at intervals, and developing larvae and adult worms were recovered. When the larvae were cultured without DEC and then inoculated subcutaneously into jirds, 29.8% of the inoculum was recovered 3-15 days, and 25% 19-22 weeks, post-inoculation. However, when the larvae were exposed to DEC in vitro and inoculated into jirds, the rate of recovery was reduced to 25% 3-15 days post-inoculation and 2% after 19-22 weeks. When the control larvae cultured in vitro were inoculated intraperitoneally into jirds, 41.3% of inoculum was recovered 3-15 days, and 42.8% 19-22 weeks, post-inoculation. Again the corresponding value for larvae exposed to DEC in vitro was reduced to 19.8% 3-15 days, and 8% 19-22 weeks, post-inoculation. It was observed that the larvae exposed to DEC in vitro were retarded in their development in jirds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protective immunity against Brugia malayi infective larvae in mice. II. Induction by a T cell-dependent antigen isolated by monoclonal antibody affinity chromatography and SDS-PAGE

A mAb directed against filarial worm secretory/excretory product and reactive with Brugia malayi larval worm surface was used in conjunction with preparative SDS-PAGE to isolate protective Ag from extracts of adult B. malayi. The IgM mAb OVH bound to a repeating carbohydrate epitope present in adult, infective, and fourth stage larvae and microfilariae of B. malayi, and on the surface of fourth stage larvae. Ag bearing this epitope were also present in the sera of hosts infected with a variety of helminths, including Brugia, Onchocerca, Dirofilaria, and Paragonimus. Affinity chromatography of SDS extract of adult Brugia, using mAb OVH immobilized on agarose beads, isolated several Ag that separated into multiple protein staining bands on SDS-PAGE. In comparing SDS-PAGE-fractionated Ag from the crude SDS extract with fractionated mAb OVH-isolated Ag for the ability to protect BALB/c mice from challenge with B. malayi-infective larvae, it was found that of the mAb OVH-isolated Ag only those at a molecular mass of 26 to 32 kDa were protective while the original SDS extract yielded protective Ag at the following molecular mass: greater than 200, 170 to 200, 40 to 44, 33 to 36, 23 to 28, 20 to 22, and 17 to 19 kDa. Although Ag isolated by mAb OVH were highly protective, they failed to induce high antibody levels against the immunogen or SDS extracts compared to crude SDS extract immunized mouse sera, as determined by immunoblot and ELISA. Transfer of nylon wool non-adherent T cells from BALB/c mice immunized with the 26- to 28-kDa fraction of mAb OVH-isolated Ag to naive mice just before challenge with infective larvae of B. malayi resulted in a 70% reduction in larvae recovered 14 days after challenge.     

Oral transmission of Brugia pahangi and Dipetalonema viteae to adult and neonatal jirds

Sullivan J. J. and Chernin E. 1976. Oral transmission of Brugia pahangi and Dipetalonema viteae to adult and neonatal jirds. International Journal for Parasitology6: 75–78. Confirming previous studies, anaesthetized adult jirds became infected after oral doses of 100 infective larvae of B. pahangi; 4 of 5 jirds became microfilaria-positive and all 5 harbored adult worms. Among 7 unanaesthetized adult jirds similarly exposed, none developed microfilaraemia although 5 each harbored a few adult worms. In these unanaesthetized jirds, presumably, rapid passage of the inoculum through the mouth permitted fewer larvae to penetrate the mucosa, and the rest were probably killed in the stomach. Unanaesthetized 4-day old jirds proved highly and equally susceptible to oral or subcutaneous infection with B. pahangi as indicated by microfilaraemias and large worm-burdens. Direct and indirect evidence suggest that the baby's stomach and small intestine are not inimical to swallowed larvae, thus accounting for the relatively numerous mature worms in the peritoneal cavity. Third-stage larvae of D. viteae, readily infective subcutaneously, succeeded relatively infrequently in maturing when given orally to anaesthetized adult or to unanaesthetized baby jirds. Consistent oral infectivity may thus be a feature of filariae more closely related to B. pahangi.     

Brugia pahangi: granulomatous lesion development in jirds following single and multiple infections

The development of adult worm burdens and microfilaremias were determined in jirds which received 2, 3, or 4 subcutaneous inoculations of 50 Brugia pahangi infective larvae. Parasite burdens in multiply inoculated jirds were compared to those in four different groups of jirds which received single inoculations of 50 infective larvae. One of each of these singly inoculated groups was infected on the same day that one of the inoculations was given to the multiply infected jirds. Thus, the duration of the infections in the four groups of jirds receiving one inoculation was 54, 118, 189, and 254 days. The development of lymphatic lesions and granulomatous hypersensitivity to B. pahangi antigen was assessed in all jirds at necropsy. The percentage recoveries of adult worms and their locations did not differ in the singly inoculated jirds with infections of different durations. A protective resistance to reinfection, as measured by adult worm recovery in multiply infected jirds, did not occur. The lymphatic lesion scores and numbers of intralymphatic thrombi was greatest in singly inoculated jirds examined 54 days after infection. Pulmonary granuloma areas around adult filarial antigen coated beads embolized in the lungs of jirds 3 days prior to necropsy were also greatest in singly inoculated jirds examined 54 days after infection. Using criteria of lesion scores and lymph thrombi numbers to assess lymphatic lesion severity, a decrease in lesion severity as well as pulmonary granuloma size around antigen coupled beads was seen by 118 days after infection in singly inoculated jirds.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.