Comparison of the Impact of Annual and Semiannual Mass Drug Administration on Lymphatic Filariasis Prevalence in Flores Island, Indonesia.

We compared the impact of annual and semiannual mass drug administration (MDA) on the prevalence of Brugia timori and Wuchereria bancrofti in Flores Island. Two villages (Paga, B. timori only; Lewomada, co-endemic) received annual MDA with diethylcarbamazine/albendazole and a larger village (Pruda, co-endemic) received semiannual MDA. Infection parameters (microfilariae [Mf], antibodies to recombinant filarial antigen BmR1 [Brugia Rapid (BR)], and a test for W. bancrofti antigenemia [immunochromatographic test (ICT)]) were assessed before and after treatment. The crude Mf prevalence in Pruda decreased after five semiannual treatments from 14.2% to 1.2%, whereas the Mf prevalence in the other two villages decreased after three annual treatments from 3.9% to 0% and from 5% to 0.3%, respectively. ICT positivity prevalence in Pruda and Lewomada decreased from 22.9% and 6.5% to 7% and 0.8%, respectively, whereas BR antibody prevalence in Pruda, Lewomada, and Paga decreased from 28.9%, 31.7%, and 12.5% to 3.6%, 4.1%, and 1.8%, respectively. Logistic regression analysis indicated that that Mf, BR, and ICT prevalence decreased significantly over time and that for the Mf and ICT outcomes the semiannual treatment had higher odds of positivity. Model-adjusted prevalence estimates revealed that apparent differences in treatment effectiveness were driven by differences in baseline prevalence and that adjusted prevalence declined more rapidly in the semiannual treatment group. We conclude that in this setting, annual MDA was sufficient to reduce Mf prevalence to less than 1% in areas with low to moderate baseline prevalence. Semiannual MDA was useful for rapidly reducing Mf prevalence in an area with higher baseline endemicity.

Association of a PD-L2 Gene Polymorphism with Chronic Lymphatic Filariasis in a South Indian Cohort.

Lymphatic filariasis (LF) is a parasitic infection, caused by three closely related nematodes, namely Wuchereria bancrofti, Brugia malayi, and Brugia timori. Previously, we have shown that lysate from B. malayi microfilariae induces the expression of interleukin (IL)-10 and programmed death-ligand (PD-L) 1 on monocytes, which lead to inhibition of CD4+ T-cell responses. In this study, we investigated associations of IL-10 and programmed cell death (PD)-1 pathway gene polymorphisms with clinical manifestation in LF. We evaluated the frequency of alleles and genotypes of IL-10 (rs3024496, rs1800872), IL-10RA (rs3135932), IL-10RB (rs2834167), PD-1 (rs2227982, rs10204525), PD-L1 (rs4143815), PD-L2 (rs7854413), and single-nucleotide polymorphisms (SNPs) in 103 patients with chronic pathology (CP), such as elephantiasis or hydrocele and 106 endemic normal (EN) individuals from a South Indian population living in an area endemic for LF. Deviations from the Hardy-Weinberg equilibrium were tested, and we found a significant difference between the frequency of polymorphisms in PD-L2 (rs7854413; P < 0.001) and IL-10RB (rs2834167; P = 0.012) between the CP and the EN group, whereas there were no significant differences found among IL-10, IL-10RA, PD-1, and PD-L1 SNPs. A multivariate analysis showed that the existence of a CC genotype in PD-L2 SNP rs7854413 is associated with a higher risk of developing CP (OR: 2.942; 95% confidence interval [CI]: 0.957-9.046; P = 0.06). Altogether, these data indicate that a genetically determined individual difference in a non-synonymous missense SNP of PD-L2 might influence the susceptibility to CP.

Vector transmission heterogeneity and the population dynamics and control of lymphatic filariasis.

A long-standing gap in lymphatic filariasis epidemiology is quantifying the potential effect that heterogeneous infection processes occurring in the major mosquito vector genera may have on parasite transmission and control. Although previous studies have focussed on examining the forms of the density dependent mechanisms regulating larval infection in various mosquito genera, there has been little work done thus far in investigating how such differential processes might interact with density-dependent processes occurring in other stages of the parasite life cycle to influence overall transmission dynamics between areas exposed to different transmitting vector populations. Here, we explore the impact that differences in vector genus-related larval infection dynamics may have on filariasis transmission and control using newly derived parasite transmission models incorporating the forms of the density-dependent processes regulating larval infection in the two major vectors transmitting filariasis, viz. culicine and anopheline mosquitoes. The key finding in this work is that filarial infection thresholds, system resilience, transmission dynamics and parasite response to control efforts, can all be influenced by the prevailing transmitting mosquito genus. In particular, we show that infection thresholds may be raised, system resilience to perturbations lowered and effects of repeated mass treatments in eliminating infection enhanced in anopheline filariasis compared to culicine filariasis, as a direct result of the occurrence and action of multiple positive density-dependent mechanisms influencing infection in this vector-parasite system, such as the "facilitation" function regulating larval infection dynamics in the vector and the inverse probability function governing adult worm mating in the host. These findings indicate that anopheline filariasis may be easier to eradicate than culicine filariasis for a given precontrol infection level, although the actual intensity of interventions required to achieve eradication may in fact be similar to that for culicine filariasis because of the higher infection levels generated as a result of the "facilitation" process in Anopheles transmission areas.

Determinants of the eradicability of filarial infections: a conceptual approach.

Lymphatic filariasis and onchocerciasis are subject to major intervention programs by the WHO. The Onchocerciasis Control Programme in West Africa was launched 30 years ago and has led to considerable insights into the control of this infection. The Global Alliance to Eliminate Lymphatic Filariasis is a relatively recent control program with ambitious targets concerning its efficacy and its schedule. These expectations, however, are based on certain assumptions about the density-dependent processes of limitation and facilitation which determine eradicability: the levels of transmission thresholds and breakpoints. Here, we review these processes operating in filarial infections and show their impact on the persistence of the parasite, as well as pointing out those issues where more information is required to develop sound predictions about the eradicability of these infections.

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.

Interleukin-4 influences the production of microfilariae in a mouse model of Brugia infection.

Sub-cutaneous infection of interleukin (IL)-4-/- mice on the BALB/c background with third stage larva (L3) of Brugia pahangi revealed an altered cytokine profile consistent with the absence of the Th2 promoting cytokine IL-4. Splenocytes from IL-4-/- mice secreted significantly more antigen (Ag)-specific IL-2 and interferon-gamma and significantly less Ag-specific IL-5, compared to those from L3-infected wild-type mice. However, levels of Ag-specific IL-13 were similar between groups. Despite the alteration in immune responses, there was no significant difference in recovery of developing worms from the peritoneal cavity of the two strains of mice at any time postinfection. However, at later time points of infection, the IL-4-/- mice contained large numbers of microfilariae (Mf) in the peritoneal cavity while the wild-type mice contained comparatively few Mf. The differences in Mf levels appear to relate to differences in worm fecundity in the two strains of mice, with adult female worms from the wild-type mice containing few developing Mf. Moreover, implantation of sexually mature adult female worms into the peritoneal cavity of both strains of mice resulted in equal levels of Mf, confirming that the primary role of IL-4 is to limit fecundity during the maturation phase of infection.

The third-stage larva (L3) of Brugia: its role in immune modulation and protective immunity.

In this review, we focus on the role of the L3 (third-stage larva) of lymphatic filarial nematodes in immunomodulation and in the development of protective immunity. Studies in the mouse models of Brugia have been fundamental to our understanding of the mechanisms by which infection with L3 results in Th2 responses and the active suppression of Th1 responses. The relevance of these phenomena to the human infection is discussed.

Discrete transcripts encode multiple chitinase isoforms in Brugian microfilariae.

The blood-borne microfilariae of the Brugian nematodes produce multiple isoforms of chitinase, whose expression is coincident with the onset of microfilarial infectivity for mosquitoes. A single cDNA sequence was previously obtained by screening a Brugia malayi microfilarial cDNA library, yet two chitinase isozymes are readily distinguished in this species. In this paper, we present evidence for the existence of multiple transcripts encoding Brugian microfilarial chitinases. Using primers based on the previously-sequenced cDNA clone, we amplified and sequenced two discrete products from B. malayi microfilarial RNA by RT-PCR. While the shorter fragment was nearly identical to the previously sequenced cDNA, the larger fragment contained an extra copy of a serine/threonine-rich repeat. RNAse protection assays were used to demonstrate that both sequences represent true transcripts, and not PCR artifacts. Using primers based on the B.malayi sequence, two novel sequences were generated by RT-PCR from B. pahangi microfilariae. Homologous and cross-species RNAse protection assays verified that multiple transcripts also encode chitinase isozymes in B. pahangi microfilariae.

Brugia beaveri: microscopic morphology in host tissues and observations on its life history.

The filaria Brugia beaveri is a parasite of raccoons (Procyon lotor) in Louisiana. Its microfilariae, which circulate in the peripheral blood without any periodicity, develop to the infective stage in mosquitoes. The filaria can be transmitted in the laboratory to other raccoons, the domestic cat, and jirds (Meriones unguiculatus). The prepatent period is 70-107 days depending on the definitive host. Adult worms are found in lymphatics and associated subcutaneous tissues of raccoons and in the heart, lungs, and testes of jirds. In host tissues, the parasite is recognized by its small diameter and the morphology of the body wall. There is a thin cuticle, which is characteristically thickened in the lateral fields; in males, a lateral, internal cuticular ridge is sometimes present. The hypodermis forms large lateral chords and less conspicuous dorsal and ventral chords. Muscle cells are coelomyarian; in females there is an average of 4 cells per body quadrant and in males about 4-6. Internal organs are easily identified as to type, but do not provide any clues to species identification.

Brugia pahangi: production of a monoclonal antibody reactive with the surface of infective larvae.

Monoclonal antibodies against infective third-stage larvae (L3) of Brugia pahangi were generated from mice immunized with L3 antigens. The monoclonal antibodies were L3 stage-specific or stage-nonspecific. A BpG1 monoclonal antibody (IgG1 subclass) showing L3 stage-specificity was examined in detail. BpG1 recognized the surface of B. pahangi L3 and also reacted with the surface of Brugia malayi L3 but not with the surface of filarial worms of other genera, such as Acanthocheilonema viteae and Litomosoides carinii. BpG1 promoted cellular adhesion to the surface of B. pahangi L3. BpG1 bound on living L3 was shed but the shedding rate was relatively slow. The surface antigen recognized by BpG1 had a molecular weight of 58 kDa. It was stable to heat and periodate treatments but sensitive to trypsin digestion and was released from living L3 by SDS but not by Triton X-100 or CTAB. Preincubation of L3 with BpG1 significantly reduced the recovery rate of worms compared with the preincubation with a monoclonal antibody (IgG1 subclass) against the inner tissues of B. pahangi L3 or control supernatant of P3U1 myeloma cells. This result suggests that the antigen containing the BpG1 epitope may be one of the targets of a protective immune response against Brugia infection.

Induced polypeptides associated with filarial worm refractoriness in Aedes aegypti.

Brugia malayi and Wuchereria bancrofti are mosquito-borne parasitic nematodes responsible for lymphatic filariasis in approximately 90 million people. The genetic control of the susceptibility of Aedes aegypti mosquitoes to B. malayi was well defined 30 years ago, but no data have since been provided regarding the gene products responsible for susceptibility or refractoriness or both. We addressed this problem by assessing polypeptide synthesis in thoracic tissue, the developmental site of this parasite, in susceptible and refractory strains of A. aegypti. Polyacrylamide gel electrophoresis of radiolabeled polypeptides synthesized in vivo were compared between (i) established susceptible and refractory strains and (ii) a refractory strain newly isolated from the established susceptible strain. Six polypeptide differences recognized by SDS/PAGE and two-dimensional gel electrophoresis were seen only in the refractory strains after they took a blood meal. A seventh polypeptide was present in those refractory mosquitoes that had ingested sucrose but increased in intensity after blood-feeding. The presence of parasites in the blood meal was not necessary to stimulate the synthesis of these polypeptides. These refractory strain-associated molecules may mediate genetically determined variation in susceptibility.

Identification of a novel transglutaminase from the filarial parasite Brugia malayi and its role in growth and development.

Recently, we reported the presence of a putative transglutaminase in adult female worms of Brugia malayi [1]. The enzyme activity was shown to be essential for in utero growth and development of microfilariae. Here, we demonstrate that adult worms of B. malayi have a large amount of epsilon-(gamma-glutamyl)lysine isopeptide bonds, a product of physiologically active transglutaminase. A 25-kDa immunoreactive band detected in female worm extracts by a monospecific monoclonal antibody (CUB 7401) against guinea pig liver transglutaminase was associated with the enzymatic activity. Unlike the mammalian enzyme, the parasite enzyme did not require Ca2+ for its catalytic activity. Furthermore, in utero developing embryos, especially during early stages of development, contained very high amounts of this enzyme. Adult female worms contained several proteins that could serve as suitable substrates for the enzyme. Inhibition of the enzyme activity by an enzyme-specific pseudosubstrate, monodansylcadaverine, led to a time- and dose-dependent inhibition of microfilariae production and release by gravid female worms. The inhibition of microfilariae production was due to the inhibition of transglutaminase-catalyzed crosslinking of parasite proteins that in turn seemed to be essential for in utero growth and development of the embryos. The results suggest that transglutaminase-catalyzed reactions may play an important role during early development of embryos to mature microfilariae inside the adult female worms of filarial parasites.

Repeated infection of cats with Brugia pahangi: parasitological observations.

Cats were repeatedly inoculated with infective larvae of Brugia pahangi. On parasitological grounds they could be divided into 5 groups. Group I--most cats (some 70%) became microfilaraemic (mf+) and retained high levels of microfilariae (mf) in their blood for over 2 years. In some Group I cats mf counts stabilized at high levels whilst in others mf counts continued to increase. Large numbers of fecund adult worms were recovered from their lymphatics. Adult counts were not made on the cats in the current experiments but over 100 adults have been recovered from 'super-susceptible' cats. Large amounts of B. pahangi adult antigen were consistently present in the serum of all Group I cats. About 30% of cats became amicrofilaraemic (mf-). In these cats the peak mf levels were seldom above 10,000 mf/ml. Group II--these cats had less than 10,000 mf/ml and low antigen levels. After more than 1 year of being repeatedly infected B. pahangi adult antigen slowly declined and eventually could no longer be detected in their serum and the number of mf declined very slowly after the fall in antigen levels. This shows that in Group II cats the adult worms die and as the cats are resistant to the development of the continuing weekly inoculation of L3 no new adults can develop. Group III--these cats became mf--during the first year of infection but remained B. pahangi antigen-positive for many weeks after this and, at autopsy, had living adults in their lymphatics.(ABSTRACT TRUNCATED AT 250 WORDS)

Surface-associated antigens of Brugia malayi L2 and L3 parasites during vector-stage development.

Surface and metabolic labeling procedures were used to characterize the composition and the time of expression of Brugia malayi L2 and L3 surface-associated molecules as the larvae develop within the mosquito vector. Larvae were harvested from mosquito tissues at 5 (early L2), 8 (late L2) and 11 (L3) days post-infection and labeled with 125I-Iodo-Gen. The results of one-dimensional analysis showed that there is a progressive increase in the complexity of peptides associated with the surface of developing larvae, culminating in the expression of 7 major labeled components on L3s. Both L2 and L3 parasites have surface-associated components of 42, 35, 33, 19 and 17 kDa. Between days 8 and 11 of development in the insect vector, Brugia malayi undergoes the L2 to L3 molt and acquires additional major immunogenic peptides of 40 and 22 kDa. Two-dimensional analyses of extracts from 125I-labeled L2s and L3s revealed that the major 35-, 33-, 19- and 17-kDa molecules are part of a peptide complex that forms a 'ladder' between 17 and 150 kDa. To gain information on the times during which the major surface-associated molecules are produced by the parasite, larvae were labeled with [35S]methionine either in situ as they developed within the mosquito or during culture after exiting the vector. For in situ labeling, [35S]methionine was introduced into the hemolymph of infected mosquitoes by micro-injection at days 2, 5 and 8 post-infection and the larvae were allowed to develop for an additional 3 days. The results of 1- and 2-dimensional analyses of [35S]methionine-labeled extracts from vector-stage or post-vector-stage larvae indicate that the molecules associated with the surface of B. malayi L3s are synthesized between day 5 and day 11 of development in the insect host. Immediately after the larvae exit the vector, the synthesis of the 40 and 22-kDa peptides is drastically reduced or terminated.

Identification of a novel Brugia pahangi beta-tubulin gene (beta 2) and a 22-nucleotide spliced leader sequence on beta 1-tubulin mRNA.

We have examined the expression of beta-tubulin genes in the parasitic nematode, Brugia pahangi. A genomic library was constructed and screened by hybridization with a Haemonchus contortus beta-tubulin cDNA fragment which recognizes several B. pahangi beta-tubulin sequences, including sequences which correspond to the previously characterized beta 1-tubulin gene. The B. pahangi beta 2-tubulin gene was isolated by selecting clones which hybridize to the H. contortus beta-tubulin gene but which do not hybridize to the beta 1-tubulin gene. A partial sequence of the beta 2-tubulin gene confirms that it codes for a distinct beta-tubulin. Southern hybridization analyses show that the beta 2-tubulin sequence exists as a single copy gene within the B. pahangi genome. Expression of the beta 2-tubulin gene is developmentally regulated and the message is found predominantly in adult male worms, whereas the beta 1-tubulin gene is expressed in microfilariae and approximately equal levels of the transcript are found in male and female adult worms. During mRNA maturation the beta 1-tubulin mRNA of microfilariae and adult worms acquires a trans-spliced leader identical to the SL1 of Caenorhabditis elegans.

Brugia malayi: patterns of expression of surface-associated antigens.

Patterns of expression of surface-associated antigens were analyzed in the filarial nematode Brugia malayi immediately prior, and during development in the vertebrate host. Two surface-associated protein molecules, i.e., accessible to surface radioiodination and soluble in aqueous buffers, were investigated: Mrs 29-30,000 and 16,000, both of which are antigenic in infected animals. The Mr 29-30,000 glycoprotein is expressed in a surface-associated manner by adult worms and by fourth-stage larvae, but is not detectable in preparasitic third-stage larvae. The 16,000 component, which appears not to be glycosylated, is surface-associated in adult worms and fourth-stage larvae. In contrast to the 29-30,000 glycoprotein, the 16,000 protein is also expressed both by pre- and postparastic third-stage larvae. However, it becomes surface-associated only after infection. Thus, immediately prior, and during development within the vertebrate host, B. malayi displays at least two different patterns of expression of surface-associated antigens: (i) de novo, intiated either immediately after infection (phase specific) or during genesis of the fourth-stage larva (stage specific); (ii) continuous, but with phase-dependent surface exposure of previously cryptic antigens, during the transition from intermediate to definitive host.

A cell-free culture system for development of large numbers of Brugia pahangi larvae.

Techniques used in the in vitro culture of massive numbers of Brugia pahangi third-stage larvae (L3) are described. Procedures for larval preparation and four culture conditions, with or without animal cell co-cultures, were studied, resulting in the adoption of a relatively simple cell-free culture system for routine harvesting of larval moulting excretory/secretory product.

The dynamics of microfilaraemia and its relation with development of disease in periodic Brugia malayi infection in south India.

Rates of acquisition and loss of Brugia malayi microfilaraemia were estimated using the parasitological data of a cohort of population in Shertallai, South India. The rate of acquisition of microfilaraemia was found to be dependent on age but not gender. The decline in the rate of acquisition of microfilaraemia in adults above 35 years could be due to the development of acquired immunity. The mean reproductive lifespan for the periodic Brugia malayi adult female worm was estimated to be 3.4 years and it was independent of host age and gender. The age-specific estimated proportion of population at risk (microfilaria carriers who lost their microfilaria in course of time) of developing lymphoedema approximately mirrored the observed age specific prevalence of lymphoedema in the study population. On an average, 99% of population at risk developed disease in different endemic areas is compared and its epidemiological significance is discussed.