Brugia pahangi: immunization with early L3 ES alters parasite migration, and reduces microfilaremia and lymphatic lesion formation in gerbils (Meriones unguiculatus).

Previous studies have shown that intradermally (ID) injected Brugia pahangi L3 s migrate through various tissues and into the lymphatics of gerbils in a distinct pattern. Excretory/secretory products (ES) produced at the time of invasion of B. pahangi are likely to be important in this early migration phase of the parasite life cycle in their rodent host. Hence, early L3 ES was collected from 24h in vitro cultures of B. pahangi L3 larvae and used in immunization experiments to investigate the effect of immunity to early L3 ES on worm migration, survival and development of B. pahangi. Immunization of gerbils with ES in RIBI adjuvant produced antibodies to numerous ES proteins eliciting a strong humoral response to ES and indirect fluorescent antibody (IFA) assay using anti-ES serum recognized the ES proteins on the surface of B. pahangi L3 larvae. Following ES immunization, gerbils were challenged either ID or intraperitoneally (IP) with 100 L3 s of B. pahangi and euthanized at 3 or 106 days post inoculation (DPI). Immunization with early ES slowed the migration of ID inoculated L3 at 3 DPI and significantly altered the locations of adult worms at 106 DPI. Immunization did not induce protection in any treatment group. However, immunized animals had significantly fewer microfilariae per female worm suggesting the antigens in ES are important in microfilariae development or survival in the host. The number of lymphatic granulomas was also significantly reduced in ES immunized animals. It is important to note that microfilariae serve as a nidus in these granulomas. Our results shows immunization with early Brugia malayi L3 ES alters the worm migration, affects circulating microfilarial numbers and reduces lymphatic granulomas associated with B. pahangi infection in gerbils.

In vitro chemotactic responses of Brugia pahangi infective larvae to sodium ions.

In vitro chemotactic responses of infective third-stage larvae (L3) of Brugia pahangi to NaCl, Na2HPO4, KCl, K2HPO4, MgCl2 and CaCl2 were assessed. Compared to deionized water as a control, 200 mm NaCl and 100 mm Na2HPO4 significantly attracted L3 (P < 0.01 and P < 0.01), whereas L3 were likely to avoid 200 mm KCl and 100 mm K2HPO4 (P < 0.05 and P < 0.05). L3 showed no significant tendency to avoid or to be attracted to 200 mm CaCl2 and 200 mm MgCl2. Furthermore, NaCl exhibited a significant chemoattractant activity for L3 at a low concentration of 100 mm.

Immune activation by life-shortening Wolbachia and reduced filarial competence in mosquitoes.

Wolbachia strain wMelPop reduces the longevity of its Drosophila melanogaster host and, when introduced into the mosquito Aedes aegypti, halves its life span. We show that wMelPop induces up-regulation of the mosquito's innate immune system and that its presence inhibits the development of filarial nematodes in the mosquito. These data suggest that wMelPop could be used in the global effort to eliminate lymphatic filariasis and possibly for the control of other mosquito-borne parasites where immune preactivation inhibits their development. The cost of constitutive immune up-regulation may contribute to the life-shortening phenotype.

Evaluation of the function of a type I peritrophic matrix as a physical barrier for midgut epithelium invasion by mosquito-borne pathogens in Aedes aegypti.

In addition to modulating blood meal digestion and protecting the midgut epithelial cells from mechanical and chemical damage, a biological function attributed to the mosquito type I peritrophic matrix (PM) is preventing or reducing pathogen invasion, especially from Plasmodium spp. Previously, we demonstrated that chitin is an essential component of the PM and is synthesized de novo in response to blood feeding in Aedes aegypti. Therefore, knocking down chitin synthase expression by RNA interference severely disrupts formation of the PM. Utilizing this artificial manipulation, we determined that the absence of the PM has no effect on the development of Brugia pahangi or on the dissemination of dengue virus. However, infectivity of Plasmodium gallinaceum is lower, as measured by oocyst intensity, when the PM is absent. Our findings also suggest that the PM seems to localize proteolytic enzymes along the periphery of the blood bolus during the first 24 hours after blood feeding. Finally, the absence of the PM does not affect reproductive fitness, as measured by the number and viability of eggs oviposited.

In vitro chemotaxis of Brugia pahangi infective larvae to the sera and hemolymph of mammals and lower animals.

The jird (Mongolian gerbil) is a highly susceptible experimental host for the lymphatic filarial nematode, Brugia pahangi. The chemotactic activity of serum from this host for B. pahangi infective larvae was compared in vitro to that of sera or hemolymph of a wide variety of other organisms including mammals, reptiles, fishes and invertebrates. The range of the Chemotactic Index (CI) was from 96.0 for the jird to 56.2 for a snail. An average of CI of saline control was 4.5. Significant chemotactic activity was present in many organisms, especially mammals, but was not closely related to either the phylogenetic position of the organism and to its known susceptibility as definitive host for B. pahangi. Migratory response was diminished in a consistent way by serial dilution of sera of humans, jirds and fetal bovine serum. Pre-incubation of larvae in fetal bovine serum inhibited migration, especially towards the sera of humans. Inhibition could be reversed by rinsing larvae in saline, longer rinse periods resulting in greater recovery of CI. These results are the first to suggest the activity of the specific amphid chemoreceptors in the chemotaxis of the infective larvae of B. pahangi.

Brugia pahangi: in vivo tissue migration of early L3 alters gene expression.

Events occurring during early filarial nematode migrations are central to parasite establishment but rarely studied. Brugia pahangi larvae injected intradermal (ID) into the hind limb of the gerbil (Meriones unguiculatus) can be recovered from the popliteal lymph node (POP) at 3 days post-infection (DPI). They have been designated migrating larvae (IDL3). Alternatively, L3 recovered at 3DPI from the peritoneal cavity (IPL3) do not migrate. Subtracted cDNA libraries using IDL3 and IPL3 revealed distinct gene profiles between IDL3 and IPL3. Troponin-c was significantly upregulated in IDL3, while Cathepsin L was significantly increased in IPL3. Differences in mRNA levels were also observed with these and other genes between IDL3, IPL3 and L3 isolated from mosquitoes (VL3). These data suggest that migratory activity, exposure to potentially different host environments and/or host location may be important external factors in influencing larval gene expression.

Mosquito transcriptome changes and filarial worm resistance in Armigeres subalbatus.

BACKGROUND: Armigeres subalbatus is a natural vector of the filarial worm Brugia pahangi, but it rapidly and proficiently kills Brugia malayi microfilariae by melanotic encapsulation. Because B. malayi and B. pahangi are morphologically and biologically similar, the Armigeres-Brugia system serves as a valuable model for studying the resistance mechanisms in mosquito vectors. We have initiated transcriptome profiling studies in Ar. subalbatus to identify molecular components involved in B. malayi refractoriness. RESULTS: These initial studies assessed the transcriptional response of Ar. subalbatus to B. malayi at 1, 3, 6, 12, 24, 48, and 72 hrs after an infective blood feed. In this investigation, we initiated the first holistic study conducted on the anti-filarial worm immune response in order to effectively explore the functional roles of immune-response genes following a natural exposure to the parasite. Studies assessing the transcriptional response revealed the involvement of unknown and conserved unknowns, cytoskeletal and structural components, and stress and immune responsive factors. The data show that the anti-filarial worm immune response by Ar. subalbatus to be a highly complex, tissue-specific process involving varied effector responses working in concert with blood cell-mediated melanization. CONCLUSION: This initial study provides a foundation and direction for future studies, which will more fully dissect the nature of the anti-filarial worm immune response in this mosquito-parasite system. The study also argues for continued studies with RNA generated from both hemocytes and whole bodies to fully expound the nature of the anti-filarial worm immune response.

Penetration of the mosquito midgut is not required for Brugia pahangi microfilariae to avoid the melanotic encapsulation response of Armigeres subalbatus.

Insect vectors of disease have the capacity to respond to, and prevent further development of, parasites and pathogens using a response known as melanotic encapsulation. The naturally-occurring Armigeres subalbatus-Brugia spp. system provides an excellent way to investigate melanotic encapsulation and immune recognition in a mosquito host, because Brugia malayi microfilariae (mf) acquired via a blood meal are rapidly melanized in the body cavity of Ar. subalbatus, but Brugia pahangi mf evade or suppress the immune response and develop normally into infective stage larvae. Previous studies have suggested that B. pahangi mf are changed in some manner in the process of exiting the mosquito gut, thereby facilitating escape from, or suppression of, the melanotic encapsulation response. By inoculating mosquitoes with parasites, thus circumventing the midgut, we show that approximately 88% of B. pahangi mf escape the melanotic encapsulation response while approximately 90% of inoculated B. malayi mf are melanized. Methods to isolate parasites for this procedure are described. These results mimic those observed in Ar. subalbatus against Brugia spp. mf that are ingested following blood feeding, and demonstrate that midgut penetration is not required for B. pahangi mf to avoid the melanotic encapsulation response of Ar. subalbatus.

Temporary shift of microfilariae of Brugia pahangi from the lungs to muscles in Mongolian jirds, Meriones unguiculatus, after a single injection of diethylcarbamazine.

A single-dose treatment with diethylcarbamazine (DEC) reduced microfilaria (mf) counts of Brugia pahangi by >90% at 30 min post-treatment in Mongolian jirds (Meriones unguiculatus). The reduction was followed by a rapid increase in microfilaremia, with the count reaching pretreatment level in 3 hr. The mechanisms behind this temporary reduction of mf were investigated. Without treatment, mf accumulated in the lungs. At 30 min post-treatment, they had moved from the lungs and accumulated in the muscle. At the same time, electron microscopy revealed many mf in the muscle interstitium. DEC concentrations at 30 min were much lower in the muscle (12.2 microg/g of tissue) than in the lungs, liver, and kidneys (19.8-40.7 microg/g), all of which declined to < 0.6 microg/g by 3 hr. The presence of mf in the muscle would be advantageous for avoiding high DEC concentrations, and their extravascular location could prevent attack by host effector cells.

Tissue migration capability of larval and adult Brugia pahangi.

Infection with mosquito-born filarial nematodes occurs when hosts are bitten by a vector carrying the infective third stage larvae (L3) of the parasites. These larvae, deposited on the skin by the feeding mosquito, are presumed to enter the skin via the vector-induced puncture wound. Larvae of Brugia spp. must then migrate from the entry site, penetrate various skin layers, and locate a lymphatic vessel that leads to their lymphatic predilection site. We have recently established an intradermal (ID) infection model using B. pahangi and the Mongolian gerbil, allowing us to investigate the migratory capability ofB. pahangi. Larval and adult parasites recovered from the peritoneal cavities of gerbils were capable of establishing an infection following ID (larvae) or subcutaneous (adult) injection. Third and fourth stage larvae both migrated away from the injection site within hours, although data suggest they localize to different lymphatic tissues at 3 days postinfection (DPI). Immature adult (28 day) B. pahangi also migrated away from their SC inoculation site within 7 DPI. Mature (45 day) adult B. pahangi displayed little migration away from the SC infection site, suggesting tissue migration may be limited to developing stages of the parasite.

The observation of microfilarial rate and density in cats inoculated with increasing numbers of Brugia pahangi infective larvae.

Having close kinship to Brugia malayi, B. pahangi is a member of the family Filariidae, which causes lymphatic filariasis in dogs and cats. Although this nematode is unlikely to cause a zoonotic disease in humans, study of the B. pahangi life cycle may help control human filariasis. The objective of this study was to examine microfilarial rates and densities of B. pahangi in experimentally induced infections in cats as a relative measurement. Cats were infected with 3 different amounts of 3rd-stage larvae (L3); 100, 300 and 500. Cats infected with 100 L3 became patent for microfilariae longer than the other groups (mean100 = 99+/-44 days). In comparison, the pre-patent period of B. pahangi was somewhat shorter in cats with 300 and 500 L3 infections (mean300 = 76+/-13 and mean500 = 63+/-5 days). The microfilarial densities of these cats were also determined; the density of microfilariae (mf/1 ml blood) increased relative to the duration of infection. One-way ANOVA tests were used to compare the microfilarial densities of the cats with varying numbers of L3. We found that the microfilarial density of cats with 500 L3 exhibited significant differences (p < 0.05) from cats with 300 and 100 L3. However, we concluded that the amount of microfilariae produced in the blood circulation of these cats were not increasing relative to the numbers of L3 taken by the host.

Differential effects of diethylcarbamazine, tetracycline and the combination on Brugia pahangi adult females in vitro.

Anti-filarial effects of diethylcarbamazine (DEC), tetracycline (TC) and the combination on Brugia pahangi adult females were studied in 7-day cell-free culture, in terms of microfilaria release, parasite motility, MTT assay for parasite viability and embryogram. TC 50 microg/ml (TC50) effectively reduced microfilaria release from day 1 of culture. Combined with DEC 100 microg/ml (DEC100) or DEC 500 microg/ml (DEC500), microfilaria release reduced further and synergistically. TC50 also reduced motility, but DEC100 and DEC500 did not. The combination of TC50 and DEC500 reduced motility synergistically. The MTT assay supported the results of motility study in general. The embryogram showed that only DEC500 reduced the total number of intrauterine embryos, especially ova, indicating that DEC500 inhibited early embryogenesis. TC50 did not affect the total number of embryos, but resulted in apparent accumulation of microfilariae in the uterus, suggesting that the drug inhibited release of microfilariae in this in vitro system. These results clarified different anti-female mechanisms between DEC and TC. A PCR-based study showed that endosymbiont bacteria, Wolbachia, in B. pahangi females decreased significantly after TC treatment. However, this study could not determine whether the effects of TC were direct or Wolbachia-mediated.

Filarial susceptibility and effects of Wolbachia in Aedes pseudoscutellaris mosquitoes.

The mosquito Aedes pseudoscutellaris (Theobald), a member of the Aedes (Stegomyia) scutellaris complex (Diptera: Culicidae), is an important vector of subperiodic Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae), causing human lymphatic filariasis, on South Pacific islands. Maternal inheritance of filarial susceptibility in the complex has previously been asserted, and larval tetracycline treatment reduced susceptibility; the maternally inherited Wolbachia in these mosquitoes were suggested to be responsible. To investigate the relationship of these two factors, we eliminated Wolbachia from a strain of Ae. pseudoscutellaris by tetracycline treatment, and tested filarial susceptibility of the adult female mosquitoes using Brugia pahangi (Edeson & Buckley). Filarial susceptibility was not significantly different in Wolbachia-free and infected lines of Ae. pseudoscutellaris, suggesting that the Wolbachia in these mosquitoes do not influence vector competence. Crosses between Wolbachia-infected males and uninfected females of Ae. pseudoscutellaris showed cytoplasmic incompatibility (CI), i.e. no eggs hatched, unaffected by larval crowding or restricted nutrient availability, whereas these factors are known to affect CI in Drosophila simulans. Reciprocal crosses between Ae. pseudoscutellaris and Ae. katherinensis Woodhill produced no progeny, even when both parents were Wolbachia-free, suggesting that nuclear factors are responsible for this interspecific sterility.

Robust parameter estimation techniques for stochastic within-host macroparasite models.

We present a stochastic model of the within-host population dynamics of lymphatic filariasis, and use a simulated goodness-of-fit (GOF) method to estimate immunological parameters and their confidence intervals from experimental data. A variety of deterministic moment closure approximations to the stochastic system are explored and compared with simulation results. For the maximum GOF parameter estimates, none of the methods of closure accurately reproduce the behaviour of the stochastic model. However, direct analysis of the stochastic model demonstrates that the high levels of variation observed in the data can be reproduced without requiring parameters to vary between hosts. This indicates that the observed aggregation of parasite load may be dynamically generated by random variation in the development of an effective immune response against parasite larvae.

Removal of Wolbachia from Brugia pahangi is closely linked to worm death and fecundity but does not result in altered lymphatic lesion formation in Mongolian gerbils (Meriones unguiculatus).

Approximately 30 years ago, researchers reported intracellular bacteria in filarial nematodes. These bacteria are relatives of the arthropod symbiont Wolbachia and occur in many filarial nematodes, including Brugia pahangi and Brugia malayi. Wolbachia bacteria have been implicated in a variety of roles, including filaria development and fecundity and the pathogenesis of lymphatic lesions associated with filarial infections. However, the role of the bacteria in worm biology or filarial disease is still not clear. The present experiments support previous data showing that tetracycline eliminates or reduces Wolbachia bacteria in B. pahangi in vivo. The elimination of Wolbachia was closely linked to a reduction in female fecundity and the viability of both sexes, suggesting that the killing of Wolbachia is detrimental to B. pahangi. The gerbils treated with tetracycline showed reduced levels of interleukin-4 (IL-4) and IL-5 mRNA in renal lymph nodes and spleens compared with the levels in B. pahangi-infected gerbils not treated with tetracycline. However, similar findings were noted in B. pahangi-infected gerbils treated with ivermectin, suggesting that the loss of circulating microfilariae, not the reduction of Wolbachia bacteria, was associated with the altered cytokine profile. Despite the change in T-cell cytokines, there was no difference in the sizes of renal lymph nodes isolated from gerbils in each treatment group. Furthermore, the numbers, sizes, or cellular compositions of granulomas examined in the lymphatics or renal lymph nodes did not differ with treatment. These data suggest that Wolbachia may not play a primary role in the formation of lymphatic lesions in gerbils chronically infected with B. pahangi.

Brugia pahangi and Wolbachia: the kinetics of bacteria elimination, worm viability, and host responses following tetracycline treatment.

Wolbachia spp., first reported from filariae nearly 30 years ago, have been suggested to contribute to the pathogenesis associated with human filarial infection. Tetracycline has been used to cure filariae of Wolbachia, as a novel means of chemotherapeutic treatment for both ocular and lymphatic filariasis. Tetracycline treatment of L4 or adult Brugia pahangi in vivo resulted in Wolbachia clearance. Less tetracycline was required to clear Wolbachia when treatment began at the L4 stage, compared with adults. Female worms died earlier than male worms when tetracycline was administered at the L4 stage. In all cases, Wolbachia clearance was closely associated with worm death. Worm recoveries decreased following the L4-L5 molt, suggesting tetracycline does not interrupt molting in this model system. Despite worm death and the assumed release of both bacterial- and worm-derived molecules, differences in inflammatory cell population and T cell cytokine mRNA profiles were negligible between tetracycline-treated and non-treated B. pahangi infected gerbils. These data suggest the contribution of Wolbachia to the in vivo induction of the gerbil immune response to B. pahangi may be small.

Chemotactic response of Brugia pahangi infective larvae to jird serum in vitro.

The Brugia pahangi infective larval response to jird serum was studied using an agar plate assay. Larvae placed onto the agar remained at the same place for 60 min. Once the larvae were stimulated by serum, more than 95% oriented towards the serum and reached it within few minutes. This larval response was inhibited by an activator of phosphodiesterase (imidazole), adenylate cyclase inhibitors (SQ22536 and MDL-12330A) and protein kinase A inhibitor. An inhibitor of phosphodiesterase (IBMX), an activator of adenylate cyclase (forskolin) and an membrane permeant analogue of cAMP (8-bromo-cAMP), caused a number of larvae to move out from the inoculation area towards the other zones. To our knowledge, this is the first report of a chemotactic response by B. pahangi larvae to host serum. We conclude that B. pahangi larvae show a chemotaxic response to host serum, and that cAMP and cAMP dependent protein kinase are involved in the signal transduction.

Brugian infections in the peritoneal cavities of laboratory mice: kinetics of infection and cellular responses.

Standard, immunocompetent, inbred strains of mice are non-permissive for infection with the human filarial nematode, Brugia malayi or the closely related Brugia pahangi. This non-permissiveness allows one to address the mechanism(s) that might be used by mammalian hosts to eliminate large, multicellular, metazoan, extracellular invertebrate pathogens. We describe here the time course of intraperitoneal Brugian infections in naïve and primed +/+ mice from two commonly used, inbred laboratory strains (C57BL/6J and BALB/cByJ). We believe that this documentation of the course of infection in normal mice will serve as a reference for future studies using mice with gene-targeted immunological deficits or which have been pharmacologically or immunologically manipulated to manifest such deficits. Our data show that even though both strains of mice eliminate the parasite before the onset of patency, there are significant differences in the time course of infection and in the fractions of input larvae that can be recovered at any time after infection. In a secondary infection, the time course of elimination is accelerated. We examined the cells in the peritoneal cavity, the site of infection, by flow microfluorimetry using forward and side scatter properties and cell surface antigen expression using fluorescent antibodies. These studies reveal a complex cellular pattern, predominated by B lymphocytes, macrophages, and eosinophils. The most notable gross morphological findings at necropsy during the phase of elimination of the parasite are nodules of tissue containing larvae, which appear viable in some cases and undergoing various stages of disintegration in others. These nodules, which are histologically granulomas, are primarily composed of macrophages and eosinophils, with few if any lymphocytes. Transmission electron micrographs reveal that eosinophils can penetrate under the cuticles of the larvae and be seen in close approximation with internal structures. These granulomas may represent an important mechanism by which worms are eliminated.

Infection with Brugia microfilariae induces apoptosis of CD4(+) T lymphocytes: a mechanism of immune unresponsiveness in filariasis.

In humans infected with lymphatic filariasis, microfilaraemia [the presence of microfilariae (Mf) in the blood] is generally associated with both poor antigen (Ag)-specific proliferative responses and with protection from severe disease. Clonal deletion has been suggested as one possible mechanism by which parasite-reactive lymphocytes, that may be capable of mediating resistance and/or immunopathology, are silenced in asymptomatic carriers. In this study we demonstrate that splenic lymphocytes from mice infected with microfilariae of Brugia pahangi display an Ag-specific proliferative defect. However, these cells were not completely unresponsive since they produced high levels of Ag-specific IFN-gamma. Using TdT-mediated dUTP-biotin nick end labeling for flow cytometry, CD4(+) lymphocytes from Mf-infected mice cultured with Ag showed high levels of apoptosis when compared to those from L3-infected mice which proliferated well in response to Ag. Treatment of Ag-stimulated cultures with aminoguanidine (AMG), an inhibitor of inducible nitric oxide synthase, rescued the CD4(+) T cells from apoptosis and reversed the proliferative defect. Furthermore, carboxyfluorescein diacetate succinimidyl ester labeling allowed the visualization of dividing CD4(+) T cells in cultures from Mf-infected animals only in the presence of AMG. We hypothesize that CD4(+) T cells indirectly trigger their own apoptosis by secreting significant quantities of IFN-gamma resulting in the induction of high levels of nitric oxide, and the subsequent elimination of effector T cells. Our findings are the first direct evidence that infection with Brugia Mf can selectively induce lymphocyte apoptosis, a phenomenon that could contribute to the proliferative defect and parasite persistence associated with the microfilaraemic state in the infected human.

Electrophysiological analysis of responses of adult females of Brugia pahangi to some chemicals.

Electrophysiological techniques were used to obtain recordings of extracellular electrical activity from the anterior end of live adult females of Brugia pahangi. Stimulation with 100 mM, 10 mM and 1 mM acetylcholine resulted in an increase in spike activity which was concentration dependent, whereas stimulation with phosphate-buffered saline and 0.1 mM acetylcholine gave no increase in activity. The delay in response was not concentration dependent. The action of possible host cues was investigated. Stimulation with heat-inactivated foetal calf serum (IFCS) and 10 mM glutathione gave an increase in spike activity but exposure to 5 mg/dl haemoglobin elicited no response. The response to IFCS was found to be suppressed completely by pre-incubation for 30 min in ivermectin.