Lymphatic filariasis: perspectives on lymphatic remodeling and contractile dysfunction in filarial disease pathogenesis.

Lymphatic filariasis, one of the most debilitating diseases associated with the lymphatic system, affects over a hundred million people worldwide and manifests itself in a variety of severe clinical pathologies. The filarial parasites specifically target the lymphatics and impair lymph flow, which is critical for the normal functions of the lymphatic system in maintenance of body fluid balance and physiological interstitial fluid transport. The resultant contractile dysfunction of the lymphatics causes fluid accumulation and lymphedema, one of the major pathologies associated with filarial infection. In this review, we take a closer look at the contractile mechanisms of the lymphatics, its altered functions, and remodeling during an inflammatory state and how it relates to the severe pathogenesis underlying a filarial infection. We further elaborate on the complex host-parasite interactions, and molecular mechanisms contributing to the disease pathogenesis. The overall emphasis is on elucidating some of the emerging concepts and new directions that aim to harness the process of lymphangiogenesis or enhance contractility in a dysfunctional lymphatics, thereby restoring the fluid imbalance and mitigating the pathological conditions of lymphatic filariasis.

Parasite-specific anergy in human filariasis. Insights after analysis of parasite antigen-driven lymphokine production.

The antigen-specific immune unresponsiveness seen in bancroftian filariasis was studied by examining lymphokine production in peripheral blood mononuclear cells (PBMC) or PBMC subpopulations from 10 patients with asymptomatic microfilaremia, 13 patients with elephantiasis and 6 normal North Americans. In each group of patients, the kinetics of the lymphokine response and the response to mitogens and nonparasite antigens did not differ significantly. In marked contrast, when antigen-induced lymphokine production was examined, most patients with microfilaremia were unable to produce either interleukin 2 (IL-2) or gamma-interferon (i.e., were nonresponders), and the few who could (hyporesponders, generally with quite low microfilaremia levels) did so at levels significantly less than those of patients with elephantiasis, all of whom showed strong responses to parasite antigen. Removal of neither adherent cells or T8+ cells affected the parasite-specific anergy seen in those with microfilaremia, suggesting a state of T cell tolerance to the parasite in patients with this most common clinical manifestation of bancroftian filariasis.

Morphological and molecular characteristics of Malayfilaria sofiani Uni, Mat Udin & Takaoka n. g., n. sp. (Nematoda: Filarioidea) from the common treeshrew Tupaia glis Diard & Duvaucel (Mammalia: Scandentia) in Peninsular Malaysia.

BACKGROUND: The filarial nematodes Wuchereria bancrofti (Cobbold, 1877), Brugia malayi (Brug, 1927) and B. timori Partono, Purnomo, Dennis, Atmosoedjono, Oemijati & Cross, 1977 cause lymphatic diseases in humans in the tropics, while B. pahangi (Buckley & Edeson, 1956) infects carnivores and causes zoonotic diseases in humans in Malaysia. Wuchereria bancrofti, W. kalimantani Palmieri, Pulnomo, Dennis & Marwoto, 1980 and six out of ten Brugia spp. have been described from Australia, Southeast Asia, Sri Lanka and India. However, the origin and evolution of the species in the Wuchereria-Brugia clade remain unclear. While investigating the diversity of filarial parasites in Malaysia, we discovered an undescribed species in the common treeshrew Tupaia glis Diard & Duvaucel (Mammalia: Scandentia). METHODS: We examined 81 common treeshrews from 14 areas in nine states and the Federal Territory of Peninsular Malaysia for filarial parasites. Once any filariae that were found had been isolated, we examined their morphological characteristics and determined the partial sequences of their mitochondrial cytochrome c oxidase subunit 1 (cox1) and 12S rRNA genes. Polymerase chain reaction (PCR) products of the internal transcribed spacer 1 (ITS1) region were then cloned into the pGEM-T vector, and the recombinant plasmids were used as templates for sequencing. RESULTS: Malayfilaria sofiani Uni, Mat Udin & Takaoka, n. g., n. sp. is described based on the morphological characteristics of adults and microfilariae found in common treeshrews from Jeram Pasu, Kelantan, Malaysia. The Kimura 2-parameter distance between the cox1 gene sequences of the new species and W. bancrofti was 11.8%. Based on the three gene sequences, the new species forms a monophyletic clade with W. bancrofti and Brugia spp. The adult parasites were found in tissues surrounding the lymph nodes of the neck of common treeshrews. CONCLUSIONS: The newly described species appears most closely related to Wuchereria spp. and Brugia spp., but differs from these in several morphological characteristics. Molecular analyses based on the cox1 and 12S rRNA genes and the ITS1 region indicated that this species differs from both W. bancrofti and Brugia spp. at the genus level. We thus propose a new genus, Malayfilaria, along with the new species M. sofiani.

Ancient horizontal transfers of retrotransposons between birds and ancestors of human pathogenic nematodes.

Parasite host switches may trigger disease emergence, but prehistoric host ranges are often unknowable. Lymphatic filariasis and loiasis are major human diseases caused by the insect-borne filarial nematodes Brugia, Wuchereria and Loa. Here we show that the genomes of these nematodes and seven tropical bird lineages exclusively share a novel retrotransposon, AviRTE, resulting from horizontal transfer (HT). AviRTE subfamilies exhibit 83-99% nucleotide identity between genomes, and their phylogenetic distribution, paleobiogeography and invasion times suggest that HTs involved filarial nematodes. The HTs between bird and nematode genomes took place in two pantropical waves, >25-22 million years ago (Myr ago) involving the Brugia/Wuchereria lineage and >20-17 Myr ago involving the Loa lineage. Contrary to the expectation from the mammal-dominated host range of filarial nematodes, we hypothesize that these major human pathogens may have independently evolved from bird endoparasites that formerly infected the global breadth of avian biodiversity.

Case report and brief commentary: Wuchereria bancrofti and Onchocerca volvulus co-infection in a refugee from Sierra Leone.

Filarial infection is endemic in the tropics and is a public health problem in Africa, Asia, South and Central America, and the Pacific Islands. Co-infection with filarial nematodes, if unrecognized, can result in untoward therapeutic consequences. We report a case of co-infection of Wuchereria bancrofti and Onchocerca volvulus that was diagnosed by direct blood smear (W. bancrofti ) and serology (O. volvulus) in a native of Sierra Leone. We comment briefly on the therapeutic implications of the co-infection.

Chronic wuchereriasis presenting as a vaginoperineal fistula: report of a case with aspiration cytologic diagnosis.

BACKGROUND: Filariasis is often an occult disease with myriad presentations. Cytology has an established role in diagnosing clinically unsuspected cases. CASE: A 20-year-old female presented with recurring perineal ulcers, vaginal discharge and inguinal lymphadenopathy. Radiology revealed a vaginoperineal fistula. Fine needle aspiration (FNA) of the ulcer bed and smears prepared from the ulcer discharge showed an adult, gravid, female filarial worm and unsheathed larvae. Similar microfilariae were also seen in FNA smears from inguinal lymph nodes. The atypical clinical presentation and unusual parasite morphology posed initial difficulties in characterization of the microfilarial species. CONCLUSION: This case report highlights the morphologic clues to the dif ferential diagnosis offilarial species on cytologic specimens. Chronic wuchereriasis presenting as a vaginoperineal fistula has not been reported previously.

Analysis of salivary gland proteins of the mosquito Armigeres subalbatus.

Quantitative studies of total salivary gland protein of Armigeres subalbatus mosquito revealed that the total salivary gland protein increased dramatically during the five days after emergence as adults. The amount of salivary gland protein of female and male mosquitos at day five after adult emergence were on the average 11.55 and 1.32 microg/pair gland respectively. SDS-PAGE studies showed that salivary gland protein profiles of Armigeres subalbatus demonstrated 9 major polypeptide bands of 68, 65, 60, 55, 40, 30, 28, 21, and 15 kDa. The 21 and 65 kDa bands were found only in the distal lateral region of the mosquito salivary gland and were depleted after the female mosquito took a blood meal.

Immunodiagnostic Properties of Wucheraria bancrofti SXP-1, a Potential Filarial Diagnostic Candidate Expressed in Tobacco Plant, Nicotiana tabacum.

Transgenic tobacco plants were developed expressing WbSXP-1, a diagnostic antigen isolated from the cDNA library of L3 stage larvae of Wucheraria bancrofti. This antigen produced by recombinant Escherichia coli has been demonstrated by to be successful as potential diagnostic candidate against lymphatic filariasis. A rapid format simple and qualitative flow through immune-filtration diagnostic kit has been developed for the identification of IgG antibodies to the recombinant WbSXP-1 and is being marketed by M/S Span Diagnostics Ltd in India and Africa. Here, we present the results of experiments on the transformation and expression of the same filarial antigen, WbSXP-1, in tobacco plant, Nicotiana tabacum, to produce plant-based diagnostic antigen. It was possible to successfully transform the tobacco plant with WbSXP-1, the integration of the parasite-specific gene in plants was confirmed by PCR amplification and the expression of the filarial protein by Western blotting. The immunoreactivity of the plant-produced WbSXP-1 was assessed based on its reaction with the monoclonal antibodies developed against the E. coli-produced protein. Immunological screening using clinical sera from patients indicates that the plant-produced protein is comparable to E. coli-produced diagnostic antigen. The result demonstrated that plants can be used as suitable expression systems for the production of diagnostic proteins against lymphatic filariasis, a neglected tropical infectious disease which has a negative impact on socioeconomic development. This is the first report of the integration, expression and efficacy of a diagnostic candidate of lymphatic filariasis in plants.Key MessageTransgenic tobacco plants with WbSXP-1, a filarial diagnostic candidate, were developed. The plant-produced protein showed immunoreactivity on par with the E. coli product.

Trans-spliced leader RNA, 5S-rRNA genes and novel variant orphan spliced-leader of the lymphatic filarial nematode Wuchereria bancrofti, and a sensitive polymerase chain reaction based detection assay.

A genomic library of Wuchereria bancrofti was examined for the presence of the 22 nucleotide spliced leader (SL) which plays a vital role in the maturation of the 5' end of certain mRNAs through the addition of a small spliced leader (SL) exon and also in the generation of monocistronic mRNA from initial polycistronic transcripts in nematodes. Here, we report the characterization of three SL RNA genes (SLG1, SLG2 and SLG3), an internal copy of a novel variant SL1 sequence (SL1v) with 23 nucleotides within an open reading frame of 75 amino acid residues of an unknown gene and two 5S-rRNA genes (5SR2 and 5SR3) from two genomic clones (TZP/11, TZP/91) of W. bancrofti. Our results revealed that the genes for the spliced leader RNA of W. bancrofti (SL RNA) is reiterated within the 5S-rRNA gene cluster and are in the same orientation. The genes SLG1, SLG2 and SLG3 were identical in nucleotide sequence except for an additional nucleotide at position 43 on SLG2. Sequence analysis of the three genes indicated that the 22-nt sequence is invariably adjacent to the dinucleotide GT, characteristic of a potential spliced donor site. The Sm-binding sequence AATTTTGG was conserved in SLG1, SLG2 and SLG3. Further, both 5' and 3' flanking regions of genes SLG1, SLG2 and SLG3 shared considerable sequence similarity. Two 5S-rRNA genes characterized from the genomic clone TZP 11 were shown to have sequence heterogeneity. Genomic southern showed that the spliced leader sequence is multicopy within the W. bancrofti genome and is also encoded in the region of DNA unlinked to the 5S rRNA gene cluster. Primers designed to amplify intergenic regions between 5S-rRNA and SL RNA genes in a PCR assay were found to be specific for W. bancrofti and was sensitive enough to detect 1 pg of W. bancrofti DNA or 1/8th of a microfilariae in infected blood samples. The high specificity and sensitivity of the optimised PCR assay makes it an ideal diagnostic tool for the identification of W. bancrofti in both the host and the vector.

Secreted and surface antigens from larval stages of Wuchereria bancrofti, the major human lymphatic filarial parasite.

Antigenic proteins of microfilariae and infective larvae of Wuchereria bancrofti have been identified by intrinsic and extrinsic radiolabelling, and specific immunoprecipitation with sera from filarial patients. From 125I surface-labelling experiments, the most prominent antigen on both stages is of relative molecular mass (Mr) 17 000, while a molecule of similar size is both synthesized and released in vitro following labelling with [35S]methionine. A second similarity between the two stages is the production and secretion of a Mr 21 000 component, which is, however, not detected on the worm surfaces. A series of additional proteins from larval W. bancrofti are described from each parasite compartment (secreted, surface and somatic) and the antigenicity and specificity of these components explored with serum from patients with filariasis due to W. bancrofti or Brugia species, and with onchocerciasis. Among additional molecules released in vitro we have found a Mr 51 000 antigen from both stages, and also several proteins which are not recognised by antibody from human filarial patients.

Secreted and circulating antigens of the filarial parasite Brugia pahangi: analysis of in vitro released components and detection of parasite products in vivo.

A range of excretory-secretory (ES) antigens have been characterised following in vitro culture of adult Brugia pahangi filarial nematodes in serum-free medium. Analysis by radioiodination, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and immunoprecipitation of purified macromolecules with antibodies from human and experimental animal infections reveals both host and parasite components. Two host molecules appear by molecular weight and immunoprecipitation analysis to be immunoglobulin and serum albumin, presumed to be taken up from the jird host from which adult worms were recovered. A further prominent component, of 19 kDa, reacts with neither anti-host nor anti-filarial antibodies, and may represent a non-immunogenic parasite product. Three additional bands, although less intensely radiolabelled, did prove to be consistently antigenic, with apparent molecular weights of 15, 29 and 40 kDa. A further ES antigen, which does not take up radio-iodine or lend itself to electrophoretic analysis, has also been detected. This molecule reacts in a immunoradiometric assay in which monoclonal antibody directed against a repetitive epitope acts both to capture and indicate antigen presence. The same antibody, Bp-1, may also be employed to detect circulating antigen in the serum of animals experimentally infected with Brugia pahangi, and in the serum of patients with each of the three species of human lymphatic filariasis, Brugia malayi, Brugia timori and Wuchereria bancrofti.

Differential recognition of two cloned Brugia malayi antigens by antibody class.

The humoral and cellular immune response to filarial parasites is complex. Numerous studies have shown that antibodies to a large number of protein and non-protein antigens may be produced over the course of infection and that immune recognition of any given antigen may vary by disease manifestation and by immunoglobulin class. We have used the techniques of molecular cloning to attempt to dissect this complex interaction, and describe here two clones, isolated from an expression library constructed from Brugia malayi genomic DNA, whose products are recognized by distinct immunoglobulin classes. A lambda gt11 fusion protein containing part of the B. malayi myosin tail region is recognized by antibodies of the IgG class from a high percentage of bancroftian filariasis patients. A fusion protein containing a collagen-like sequence is less frequently and weakly recognized under the same experimental conditions, but is almost universally recognized when the developing reagent is specific for IgE. We thus identify specific filarial proteins against which the infected human host responds preferentially with antibodies of a specific immunoglobulin class.

Filarial parasites exhibit unusually high levels of choline acetyltransferase activity.

The presence of unusually high levels of choline acetyltransferase (ChAT, EC 2.3.1.6) in human and animal filarial parasites has been demonstrated. The levels of ChAT were highest in male worms of Brugia malayi and Brugia pahangi, with specific activities in crude extracts of about 2.27 and 1.26 mumol min-1 (mg protein)-1, respectively. The enzyme levels in these worms were over 10-20 times higher than in male worms of Litomosoides carinii. The ChAT levels were about 2-5 times higher in male than in female worms. The enzyme was also present in appreciably high levels in microfilariae of Brugia species, L. carinii and Wuchereria bancrofti. The levels of ChAT in male worms of Brugia species were several thousand-fold higher than in the intestinal nematodes Trichuris muris and Necator americanus, and were over three orders of magnitude higher than in mammalian brain. Unlike the mammalian ChAT, the parasite enzyme was extremely stable. The parasite enzyme was not inhibited by any of the antifilarial agents except suramin. The filarial ChAT was strongly inhibited by sulphydryl reagents and diethylpyrocarbonate. Ethacrynic acid (EA), a diuretic and a sulphydryl reagent, irreversibly inhibited the filarial ChAT activity at low concentrations. In contrast, EA inhibited the activity of mammalian brain ChAT at much higher concentrations. The motility of adult worms and microfilariae was irreversibly inhibited by low concentrations of EA. Furthermore, the inhibition of motility was paralleled by the inactivation of ChAT in these parasites. These studies indicate that ChAT activity appears to be vital for parasite's survival and that acetylcholine might play a key role in the control of worm motility.

Antibodies to diethylcarbamazine cross-react with microfilariae of Wuchereria bancrofti.

Antibodies directed against the microfilarial sheath have been instrumental in the immune elimination of circulating microfilariae in human lymphatic filariasis. We report here that antibodies to diethylcarbamazine (DEC, the most commonly used anti-filarial drug) cross-react with the sheath of Wuchereria bancrofti microfilariae. Antibodies with reactivity to DEC were raised in rabbits by immunization with a conjugate of methylpiperazine carboxylic acid (MPCA, an acid hydrolysis product of DEC) coupled to bovine serum albumin. The reactivity of these antibodies with microfilarial sheath of W. bancrofti was demonstrated by indirect immunofluorescent assay and indirect immunoperoxidase assay. This reactivity could be effectively inhibited by pre-incubation of the antisera with different haptens such as DEC, MPCA or piperazine citrate.

Studies on the laboratory diagnosis of human filariasis: preliminary communication.

Five laboratory methods used for the recovery of microfilariae from the blood were compared for efficiency of recovery and time involved. The methods used were thin blood films, thick blood films, wet preparations, the Polyvidone technique, and the microhaematocrit technique. The last proved superior in both efficiency and saving time.

Microfilaria of Wuchereria bancrofti in bone marrow smear.

Microfilariae of Wuchereria bancrofti were detected in seven cases of anemia in persons with asymptomatic filariasis. In three cases microfilariae could not be detected in the blood. Their presence in bone marrow may indicate the ability of microfilariae to cross the vessel wall.

Cryopreservation of the microfilariae of Brugia malayi, Dirofilaria corynodes, and Wuchereria bancrofti.

Methods are presented for the cryopreservation of a sheathed microfilaria, Brugia malayi, and an unsheathed species, Dirofilaria corynodes. The former survived best when frozen at the rate of -0.8 degree or -0.5 degree C per minute using 9% dimethyl sulfoxide (DMSO) as the cryopreservative. Approximately 52-79% of the thawed microfilariae developed to the third stage in Aedes aegypti mosquitoes versus 79% of the unfrozen specimens. For D. corynodes the optimum freezing rate was -2 degrees or -5 degrees C per minute, and 6% DMSO combined with 0.004 M polyvinylpyrrolidone (PVP) afforded the best cryoprotective effect. The development of thawed microfilariae in mosquitoes ranged from 22-32% versus 29% for unfrozen specimens. In general, the viability of both species of microfilaria was retained best when stored in liquid nitrogen (-196 degrees C). The entire life cycle of B. malayi was completed in the laboratory using cryopreserved microfilariae as the initial source. The cryopreservation of Wuchereria bancrofti also is discussed.

Bancroftian filariasis in Puerto Limón, Costa Rica.

In four neighborhoods of Puerto Limón, a Caribbean coastal city in a tropical rainforest area of Costa Rica, examination of 1-ml samples of night blood from 1,142 randomly selected, and 1,196 associated, persons by the Knott and filter-chamber techniques revealed microfilariae of Wuchereria bancrofti in about 3% of the 2,338 samples. The frequency of infection was higher in males (3.9%) than in females (1.9%), higher in persons of black (4.1%) than of white (1.0%) race, and highest in persons aged 10-19 (4.0%) and over 50 years (4.8%), lowest in those under 10 years (1.1%). The median microfilaria density was 3.5, the highest 45, per 20 lambdas of blood. Microfilaremia was distinctly periodic. Dissection of 663 female Culex pipiens fatigans from 42 houses of infected persons revealed filarial larvae in 25; only 1 larva was third (infective) stage. Of 64 infected persons, 11 had clinical findings suggestive of filariasis. Elephantiasis was seen in 21 others. Other forms of symptomatic filariasis without microfilaremia, though presumed to be present, were not assessed specifically.

Serological diagnosis of bancroftian and malayan filariasis.

A study was undertaken to define the sensitivity and specificity of serological tests in bancroftian and malayan filariasis and correlate the findings with clinical disease. Sera were collected from subjects on three different islands in the Philippines: one endemic for bancroftian filariasis, another endemic for malayan filariasis and the third without endemic filariasis. Antibodies were measured, using rugia malayi as the source of antigen. Antibodies against adult worms measured by indirect immunofluorescence were found at a titer of 1:8 or greater in all patients with bancroftian or malayan filariasis but not in the control subjects. There was no relationship of antibody titer to clinical status. Antibodies against microfilariae were measured by indirect immunofluorescence and microfilarial agglutination. A high correlation was observed between the two methods. These antibodies were found in only one quarter, approximately, of patients with filariasis. Microfilarial antibodies were found more commonly in those patients with chronic lymphatic obstruction. It is concluded that measurement of antibodies to adult worms is a useful indicator of infection while microfilarial antibodies are correlated with disease.

Comparison of Dot-ELISA with Sandwich-ELISA for the detection of circulating antigens in patients with bancroftian filariasis.

We compared the performance of a newly developed Dot-ELISA with that of a previously described Sandwich-ELISA to detect parasite antigens in sera from patients with bancroftian filariasis. The same monoclonal antibody and the same sera were used in both tests. In the Dot-ELISA, 67 of 70 sera from microfilaremic donors were deemed to contain filarial antigens when screened at a dilution of 1:50. End titers were 1:80-1:1280. With the Sandwich-ELISA, 64 of the same sera were positive at a dilution of 1:10 and 42 were positive at a dilution of 1:50. End titers were 1:10-1:320. The specificity of both assays was greater than 95%, but their sensitivity was remarkably different. The Dot-ELISA could detect as little as 0.055 ng/ml microfilarial antigen added to normal human sera, whereas the lower limit with the Sandwich-ELISA was 10 ng/ml parasite antigen. Additionally, the Dot-ELISA does not require radioactivity or sophisticated equipment and, therefore, can be performed in virtually all filariasis-endemic areas.