A Brugia species infecting rabbits in the northeastern United States

Brugia sp. microfilariae were observed in more than 60% of wild rabbits collected on Nantucket Island, Massachusetts. The microfilariae measured 294-344 microns in length and had the characteristic subterminal and terminal nuclei observed in other Brugia microfilariae. The microfilaria is similar to those described for Brugia leporis in rabbits in Louisiana. This may be the Brugia species responsible for 21 documented cases of human infection in the northeastern United States.     

Sensitivity of the nested-polymerase chain reaction (PCR) assay for Brugia malayi and significance of 'free' DNA in PCR-based assays

The blood filtration method was used as the gold standard to determine the detection level of simple blood-spot sampling and nested-polymerase chain reaction (PCR) for Brugia malayi. Of 100 samples, 48 were filtration-positive. Of these, 26 had microfilaria counts that were low enough (<1-29 microfilariae/ml) to accurately assess the limit of detection by nested-PCR. Nested-PCR consistently detected B. malayi DNA in samples with > or = 10 microfilariae/ml. Post-filtration, microfilaria-depleted, blood-spots from microfilaria-positive samples were screened by nested-PCR and B. malayi specific 'free' DNA was detected in 51.7% of these samples. There was no evidence for 'free' DNA in microfilaria-negative individuals from this endemic community.     

Lymphatic filariasis and Brugia timori: prospects for elimination

Brugia timori is a pathogenic filarial nematode of humans, replacing the closely related species Brugia malayi on some islands in eastern Indonesia. Recent studies on Alor island show that, locally, B. timori is still of great public health importance, causing mainly acute filarial fever and chronic lymphedema. PCR-based assays to detect parasite DNA, in addition to assays for detecting specific antibodies that have been originally developed for B. malayi, can be used efficiently as diagnostic tools for B. timori. In the framework of the Global Program to Eliminate Lymphatic Filariasis, a single annual dose of diethylcarbamazine, in combination with albendazole, was found to reduce the prevalence and density of microfilaraemia persistently. Therefore, elimination of B. timori appears to be achievable.     

Differentiation of species and life cycle stages of Brugia spp. by isoenzyme analysis

The isoenzyme patterns of glucose phosphate isomerase and phosphoglucomutase of 3 species of Brugia, B. pahangi, subperiodic B. malayi, and B. patei, and 3 life cycle stages, adult, third-stage larva, and microfilaria were compared using the technique of isoelectricfocusing on polyacrylamide gels. The results demonstrated that the adults of all 3 species could be identified from one another and that differences existed between the sexes of any one species. Hybridization between B. pahangi and B. patei could be detected in the progeny of the cross. Both the third-stage larvae and microfilariae of B. malayi and B. pahangi were differentiated and the epidemiological significance and the application of these findings to arthropod-borne filarial infections were discussed.     

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)     

A direct fluorescence technique for the rapid detection of sheathed microfilariae in blood smears

Thick and thin blood smears containing microfilariae of Wuchereria bancrofti, Loa loa, Brugia malayi, Brugia pahangi, Brugia patei or Acanthocheilonema vileae were prepared from either cryopreserved blood samples or from freshly collected blood, fixed in methanoi and treated with a fluoresceinated lectin wheat germ agglutinin. Sheathed microfilariae of W. bancrofti, L. loa, B. malayi, B. pahangi and B. patei in the blood smears could be easily detected and counted using a fluorescence assay. The unsheathed microfilaria of Acanthocheilonema viteae did not fluoresce. The possibility of adapting this technique, which does not require the use of parasite specific antibody for the sensitive, parasitological detection offilarial infections, is discussed.     

The ultrastructure of the microfilaria of Brugia, Nematoda: Filarioidea

The microfilaria of Brugia pahangi is a differentiated nematode larva. The basic nematode body plan is present showing cuticle, hypodermis, dorsal, ventral, and lateral cords, muscle cells, longitudinal nerves, papillary nerves, amphids and phasmids. Secretory granules are present in ganglionic cells and in axons in the nerve ring. There is no differentiated pseudocoelom. There is only a single row of muscle cells between each pair of cords. The excretory cell complex is similar in structure to the hypodermal gland cells of other nematodes. The alimentary canal of the microfilaria is very much modified. The pharyngeal cells are attached to the pharyngeal thread which is circular in cross section and there is no pharyngeal musculature. The intestine is represented by the solid mass of the inner body within paired intestinal cells. The intestine is separated from the rectum. The three rectal cells form a syncytium of villi in the anal vesicle. The structure in Brugia is related to the ultrastructure of other microfilariae and it is concluded that the evolution of the modifications of the basic larval structure is due to the small size of these nematodes as a consequence of their adaptation to a parasitic mode of life in the capillaries of the vertebrate host with transmission through an intermediate arthropod vector.     

Ascorbic acid is a requirement for the morphogenesis of the human filarial parasite Brugia malayi

The nematode parasites Wuchereria bancrofti, Brugia malayi, and B. timori cause a disease in humans known as lymphatic filariasis, which afflicts approximately 120 million people worldwide. The parasites enter the human host from the mosquito either as L3 or as infective larvae and subsequently differentiate through 2 molts. In this article, we show that B. malayi depends on an exogenous source of vitamin C to complete the L3 to L4 molt, a critical morphogenic step in its life cycle. Brugia malayi apparently belongs to a small group of living organisms that depend on an exogenous source of vitamin C. This group includes only primates (including man) and guinea pigs among mammals.     

Brugia malayi and Brugia pahangi: inherent difference in immune activation in the mosquitoes Armigeres subalbatus and Aedes aegypti

The inherent ability of Brugia malayi and Brugia pahangi (Nematoda) to establish successful relationships with the mosquitoes Armigeres subalbatus and Aedes aegypti Liverpool strain was evaluated. Brugia pahangi microfilariae (mff) avoided the immune response and developed normally in A. subalbatus exposed to the parasite by an infective bloodmeal, whereas nearly 85% of B. malayi were destroyed by the immune response. Because A. aegypti supports the development of both filarial worm species but destroys intrathoracically inoculated B. pahangi isolated from jird blood, blood-isolated B. malayi were inoculated into A. aegypti, and the immune response was compared with that observed against B. pahangi. The response against B. malayi was significantly more rapid and effective than the response against B. pahangi. Similar results were obtained when blood-isolated B. pahangi or B. malayi were inoculated into A. subalbatus. Microfilariae of both species were able to avoid immune destruction in A. aegypti if they were allowed to penetrate the Liverpool midgut in vitro prior to inoculation. Most B. pahangi that had first penetrated an Armigeres midgut prior to inoculation into A. subalbatus were able to avoid the immune response, but by day 3 postinoculation, less than 40% of the B. malayi, treated in the same manner, were able to escape the immune response. Genetic susceptibility of mosquitoes to infection by filarial worms and potential mechanisms of immune evasion/suppression are discussed regarding B. malayi and B. pahangi.     

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.     

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.     

Exogenous nucleosides are required for the morphogenesis of the human filarial parasite Brugia malayi

The nematode parasites Wuchereria bancrofti, Brugia malayi, and B. timori cause a human disease known as lymphatic filariasis, which afflicts approximately 120 million people worldwide. The parasites enter the human host from the mosquito as L3 or infective larvae and subsequently differentiate through 2 molts. In this communication, I report that B. malayi and B. pahangi depend on an exogenous source of at least 1 purine and 1 pyrimidine nucleoside to complete the L3 to L4 molt. The requirement for exogenous nucleosides opens the door for possible chemotherapeutic intervention.     

Brugia malayi: microfilarial polyunsaturated fatty acid composition and synthesis

The polyunsaturated fatty acid composition of Brugia malayi microfilariae was analyzed by gas chromatography and compared to that of sera from B. malayi-infected jirds. The essential fatty acid, linoleic acid (18:2 omega 6), was the most abundant fatty acid present in both microfilarial total lipids and phospholipids as well as in jird sera. In contrast, arachidonic acid (20:4 omega 6), as well as the 18:3 omega 6, 20:2 omega 6, and 20:3 omega 6 intermediates that are formed in the enzymatic conversion of linoleic acid to arachidonic acid, were proportionally more abundant in microfilariae than in jird sera. To assess the capacity of microfilariae to transform linoleic acid into arachidonic acid, B. malayi microfilariae were incubated with [14C]linoleic acid. Microfilarial lipids were extracted and resolved by high-pressure liquid chromatography and thin-layer chromatography. A portion of [14C]linoleic acid incorporated by microfilariae was converted to [14C]arachidonic acid. Thus, microfilariae can not only incorporate exogenous arachidonic acid, as previously demonstrated, but can also synthesize arachidonic acid from exogenous linoleic acid. The capacity of microfilariae to utilize specific host polyunsaturated fatty acids raises the possibility that intravascular filarial parasites may synthesize eicosanoid metabolites of arachidonic acid that could mediate filarial-host cell interactions.     

Cloning and characterisation of mmc-1, a microfilarial-specific gene, from Brugia pahangi

Nine differentially expressed genes were cloned from Brugia pahangi in a screen which sought to identify cDNAs that were differentially expressed between the microfilariae from the mammalian host and the mosquito vector. One gene (mmc-1), that was up-regulated in mammalian-derived microfilariae, was characterised in detail. RT-PCR analysis demonstrated that mmc-1 was specific to the microfilarial stage of the life cycle and was not transcribed by developing microfilariae in utero, but only following the release of the microfilariae from the adult female. Analysis of DNA from other filarial worms suggested that mmc-1 may be a Brugia-specific gene. Using serum samples from individuals exposed to Brugia malayi infection, it was shown that MMC-1 was specifically recognised by antibodies of the IgG3 subclass. mmc-1 has no homologues in the data bases and its function in the parasite is unknown.     

Brugia malayi: intravenous injection of microfilariae in ferrets as an experimental method for occult filariasis

Microfilaremia, immune responses, and pathology were compared in ferrets infected with 100 third-stage larvae of Brugia malayi (subperiodic strain) or injected intravenously with 10(6) microfilariae. Ferrets (Mustela putorius furo) inoculated with third-stage larvae typically became patent during the third month after infection, with a mean patency of 123 +/- 25 (SE) days. Ferrets injected intravenously with microfilariae exhibited a relatively constant microfilaremia for 3-4 weeks and usually cleared microfilariae before the fourth month. Ferrets that cleared microfilariae after intravenous injection of microfilariae or after infection with third-stage larvae failed to become patent or became amicrofilaremic within 3 weeks after a challenge intravenous injection of 10(6) microfilariae. Clearance of circulating microfilariae was associated with eosinophilia and serum antibody specific for the microfilarial sheath in ferrets injected with microfilariae and in most ferrets infected with third-stage larvae. Ferrets infected with third-stage larvae and necropsied after clearance of microfilariae had tissue inflammatory reactions to microfilariae characteristic of occult filariasis (tropical eosinophilia) in man; these ferrets exhibited immediate cutaneous hypersensitivity and circulating reaginic antibody to antigens of microfilariae. In ferrets necropsied following two intravenous injections of microfilariae, the majority of ferrets examined within 10 days after clearance of microfilariae had visible liver lesions to microfilariae identical to those of the ferrets infected with third-stage larvae; immediate cutaneous hypersensitivity and reaginic antibody were not consistently detected in ferrets injected with microfilariae. Sera from ferrets that had cleared circulating microfilariae were transferred passively into ferrets made microfilaremic by intravenous injection of microfilariae. Sera with microfilarial sheath-reactive IgG antibody titers (greater than or equal to 1:200) and microfilarial agglutination titers (greater than or equal to 1:40) rapidly cleared injected microfilariae (less than 24 hr); this serum also cleared or greatly reduced circulating microfilariae established by an infection with third-stage larvae; only the IgG-containing fraction of the sera was active in immune clearance. Sera that cleared microfilariae of B. malayi did not clear circulating microfilariae of Dirofilaria immitis or prevent recurrence of circulating microfilariae of B. malayi in ferrets infected with adult filariae.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Protective efficacy of a cloned Brugia malayi antigen in a mouse model of microfilaremia

Immunization of mice with irradiated Brugia larvae or parasite extracts has been shown to induce partial resistance to microfilaremia and enhance clearance of infective larvae. We recently reported the cloning of a 548 amino acid 62-kDa Brugia malayi Ag identified on the basis of reactivity with antisera to a subset of protective microfilarial Ag. Our study describes the protective efficacy against microfilaremia in mice, immunogenicity, and parasite stage-specificity of this candidate vaccine molecule. Immunization of Swiss or BALB/c mice with 1 to 3 micrograms of a 92-kDa trpE fusion protein encoding amino acids 1-479 reduced the intensity of microfilaremia by 40 to 60% compared to control animals given buffer or bacterial trpE (p less than 0.01 to 0.001). Mice immunized with the 92-kDa fusion protein developed delayed-type hypersensitivity reactivity to B. malayi as assessed by enhanced footpad swelling 24 and 48 h after intradermal injection of adult worm extract and in vitro lymph node mononuclear cell proliferation (3H-thymidine uptake) in response to the fusion protein (mean +/- SD stimulation index 4.7 +/- 0.8 vs 2.0 +/- 1.4 for trpE, p less than 0.05). Proliferative responses of lymph node cells coincubated with three other fusion proteins corresponding to the filarial protein truncated from its carboxyl-terminus suggest that dominant T cell epitopes of the 62-kDa Ag are encompassed by amino acids 437-479. Rabbit antibody to the 92-kDa trpE fusion protein immunoprecipitated a 62-kDa polypeptide from [35S] methionine biosynthetically labeled B. malayi microfilariae, adult female, and adult male worms. These data indicate that a recombinant Ag expressed in several developmental stages of B. malayi is capable of inducing partial resistance against microfilariae and Ag-specific T cell responses in mice.     

Stage-specific and common antigens of Brugia malayi identified with monoclonal antibodies

To identify parasite antigens that trigger protective, pathogenic, and allergic immune responses during filarial infections, we generated a series of monoclonal antibodies to infective larvae, adult worms, and microfilariae of Brugia malayi, a human pathogen. Quantitative and qualitative analysis of the reaction patterns of these monoclonal antibodies indicates the existence of stage-specific antigens of B. malayi, as well as of antigens shared by different stages of this parasite and by other related and unrelated helminths. These antibodies should provide invaluable tools for the analysis of host-parasite interactions in filariasis at the molecular level.