What does not kill them makes them stronger: larval environment and infectious dose alter mosquito potential to transmit filarial worms.

For organisms with complex life cycles, larval environments can modify adult phenotypes. For mosquitoes and other vectors, when physiological impacts of stressors acting on larvae carry over into the adult stage they may interact with infectious dose of a vector-borne pathogen, producing a range of phenotypes for vector potential. Investigation of impacts of a common source of stress, larval crowding and intraspecific competition, on adult vector interactions with pathogens may increase our understanding of the dynamics of pathogen transmission by mosquito vectors. Using Aedes aegypti and the nematode parasite Brugia pahangi, we demonstrate dose dependency of fitness effects of B. pahangi infection on the mosquito, as well as interactions between competitive stress among larvae and infectious dose for resulting adults that affect the physiological and functional ability of mosquitoes to act as vectors. Contrary to results from studies on mosquito-arbovirus interactions, our results suggest that adults from crowded larvae may limit infection better than do adults from uncrowded controls, and that mosquitoes from high-quality larval environments are more physiologically and functionally capable vectors of B. pahangi. Our results provide another example of how the larval environment can have profound effects on vector potential of resulting adults.

Analysis of nematode motion using an improved light-scatter based system.

BACKGROUND: The detailed assessment of nematode activity and viability still remains a relatively undeveloped area of biological and medical research. Computer-based approaches to assessing the motility of larger nematode stages have been developed, yet these lack the capability to detect and analyze the more subtle and important characteristics of the motion of nematodes. There is currently a need to improved methods of assessing the viability and health of parasitic worms. METHODS: We describe here a system that converts the motion of nematodes through a light-scattering system into an electrical waveform, and allows for reproducible, and wholly non-subjective, assessment of alterations in motion, as well as estimation of the number of nematode worms of different forms and sizes. Here we have used Brugia sp. microfilariae (L1), infective larvae (L3) and adults, together with the free-living nematode Caenorhabditis elegans. RESULTS: The motion of worms in a small (200 ul) volume can be detected, with the presence of immotile worms not interfering with the readings at practical levels (up to at least 500 L1 /200 ul). Alterations in the frequency of parasite movement following the application of the anti-parasitic drugs, (chloroquine and imatinib); the anti-filarial effect of the latter agent is the first demonstrated here for the first time. This system can also be used to estimate the number of parasites, and shortens the time required to estimate parasites numbers, and eliminates the need for microscopes and trained technicians to provide an estimate of microfilarial sample sizes up to 1000 parasites/ml. Alterations in the form of motion of the worms can also be depicted. CONCLUSIONS: This new instrument, named a "WiggleTron", offers exciting opportunities to further study nematode biology and to aid drug discovery, as well as contributing to a rapid estimate of parasite numbers in various biological samples.

Effects of intermediary metabolites and electron transport inhibitors on action of chloroquine on Brugia pahangi and Onchocerca volvulus.

We examined the possibility that chloroquine is interfering with aerobic energy-generating processes in the adult filarial parasites, Brugia pahangi and Onchocerca volvulus. Using motility of these parasites as an assay of drug effect, we found that micromolar concentrations of chloroquine caused significant paralysis, but only in alkaline medium (pH 8.4). The addition of 12 mM glutamine or 10 mM albizziin to the medium completely antagonized drug-induced paralysis. In addition, in B. pahangi, all of the tricarboxylic acid cycle intermediates (10 mM) except citrate and pyruvate antagonized the effect of chloroquine on motility; in O. volvulus, oxaloacetate as well as glutamine inhibited the effect of the drug. The effect of chloroquine on both parasites was enhanced when it was used in combination with 10 microM acivicin, a glutamine antimetabolite. Here motility of B. pahangi was reduced significantly within 24-48 hr at acidic (6.8) neutral (7.4) and alkaline (8.4) pH. This effect was partially reversible by glutamine (12 mM). Motility of O. volvulus was reduced to near zero within 4 hr with this drug combination. Antimycin A and rotenone, both electron transport inhibitors, also synergized with chloroquine at any pH to produce paralysis in B. pahangi. The effects of the rotenone and chloroquine combination were reversed in the presence of 10 mM succinate. However, glutamine (12 mM) was unable to antagonize the effects of chloroquine plus antimycin A on the motility of B. pahangi. These findings suggest that chloroquine may be inhibiting aerobic energy metabolism in the filariae, possibly at the level of electron transport. Furthermore, since chloroquine is well-tolerated but only weakly filaricidal in vivo, the data indicate that use of this drug in combination with other inhibitors of aerobic energy metabolism may be a chemotherapeutically useful approach to the treatment of filariases.

Haemolymph monophenol oxidase activity in Armigeres subalbatus infected with Brugia pahangi.

The activity of monophenol oxidase can be elicited in the haemolymph of Armigeres subalbatus by both blood and filaria-infected blood feeding. Haemolymph collected from both blood-fed and filaria-infected mosquitoes was investigated using a quantitative radiometric assay that measured the amount of tritiated water formed during the hydroxylation of L-[3,5-3H]tyrosine to dopa. Enzyme activity in filaria-infected mosquitoes was found to be significantly lower than that found in the blood-fed mosquitoes within 3 days post-ingestion, but still remained measurable 72 h post-ingestion. The decreased enzyme activity coincided in time with the development of capsules around the microfilariae. The consumption of monophenol oxidase by the melanization of migrating microfilariae in the haemocoel of filaria-infected mosquitoes and the effects of excretory and secretory products of developing larvae on monophenol oxidase activity are suggested.

Lymphatic filariasis: new insights into an old disease.

Lymphatic filariasis has afflicted people in the tropical areas of the world for thousands of years but even up to comparatively recent times it has been poorly understood and its importance under recognised. In the last 2 decades or so there has been a flurry of activity in filariasis research, which has provided new insights into the global problem of filariasis, the pathogenesis of filarial disease, diagnosis and control.

Cultivation of sexually mature Brugia malayi in vitro.

Sexually mature male and female Brugia malayi were developed from third stage larvae after 60 days in the in vitro culture system described by Franke and others in 1987 (Am J Trop Med Hyg 37: 370-375). Between 75 and 100 days in culture, many worms produced living microfilariae. Each gravid female produced 200-1,500 microfilariae/day.

Increased dissemination of dengue 2 virus in Aedes aegypti associated with concurrent ingestion of microfilariae of Brugia malayi.

We investigated whether concurrent ingestion of dengue 2 virus and microfilariae of Brugia malayi would increase viral infection and dissemination rates in Aedes aegypti. Infection rates were similar in mosquitoes that ingested virus alone or both virus and microfilariae concurrently. However, viral dissemination rates, as determined by recovery of dengue virus from both legs and bodies separately, were significantly greater in mosquitoes that ingested both agents concurrently than in those that ingested virus alone. This study confirms that vectorial capacity of a natural vector of an arbovirus may be enhanced by the concurrent ingestion of microfilariae.

Radiolabeling of Brugia malayi infective larvae in mosquitoes with 75Se-methionine and detection of these larvae in tissues of the Mongolian jird by autoradiography.

Through preliminary experiments, an effective method for radiolabeling Brugia malayi-infected mosquitoes in order to produce labeled infective Brugia larvae was developed. Starting on the 6th day after the infective blood meal, mosquitoes were fed a 7% sucrose solution containing 100 microCi/ml 75Se-L-methionine for 5 days. Infective larvae, retrieved 2 days after this labeling period, averaged 381 +/- 136 counts/min. Jirds were infected with these infective, labeled larvae either by allowing infected mosquitoes to feed on uninfected jirds for 30 min or by inoculating jirds subcutaneously in the groin with washed larvae recovered from mosquitoes. Jirds were killed at various times after infection and were sliced into approximately 0.5 mm thick sagittal sections, which were dried and placed on X-ray film. Autoradiograms were developed after 30-60 days at 5 degrees C. In a sample of 26 inoculated jirds, approximately 30% of the infecting larvae could subsequently be accounted for as Ag degrees foci on autoradiograms. The Ag degrees foci representing larvae were apparent up to 2.5 weeks after infection. In jirds infected by mosquito feeding, the Ag degrees associated with the feeding site persisted for more than 6 weeks after infection.

Brugia malayi microfilariae from the peritoneal cavity of jirds vary in their ability to penetrate the mosquito midgut.

Development in mosquitoes of Brugia malayi microfilariae obtained from the blood of jirds was compared to that of microfilariae from the peritoneal cavity. Penetration of the mosquito midgut wall as well as development into third-stage larvae was assessed. About 70% of blood-borne microfilariae penetrated the midgut wall whether ingested directly from a microfilaremic jird or from a membrane feeder containing blood from the same donor. In contrast, less than 30% of microfilariae from the peritoneal cavity penetrated the midgut wall. Microfilariae in the peritoneal cavity of jirds vary in ability to penetrate the midgut of mosquitoes; some penetrate as rapidly as do blood-borne microfilariae, others penetrate more slowly, and most fail to penetrate the midgut. Regardless of origin, microfilariae that penetrated the midgut wall developed into third-stage larvae.

Effects of serum from treated patients on antibody-dependent cell adherence to the infective larvae of Brugia malayi.

Adherence assays were used to demonstrate the in vitro effect of serum-dependent cellular adherence of human buffy coat cells to infective larvae of Brugia malayi in filariasis patients treated with antifilarial drugs. In this study, microfilaraemic patients were treated with either diethylcarbamazine citrate (DEC), mebendazole or levamisole hydrochloride. It was found that DEC and mebendazole decreased the motility of infective larvae due to a direct action of the drugs. Sera of levamisole-treated patients caused increased adherence of human buffy coat cells to infective larvae, leading to a decrease in motility and cuticular damage as confirmed by scanning electron microscopic studies. However, serum of levamisole-treated patients alone could cause a similar lethal effect on infective larvae. Studies with the indirect fluorescent antibody test suggested that IgM was involved in this phenomenon. Complement did not appear to be important.

A comparison of host responses of the Mongolian jird to infections of Brugia malayi and B. pahangi.

Host responses of jirds receiving a single subcutaneous inoculation of subperiodic Brugia malayi were compared with those of jirds similarly infected with B. pahangi. Parasite burdens, lymphatic lesion severity, granulomatous reactivity, antibody responses to parasite antigens, and complete blood cell counts were assessed at 60 and 150 days post-inoculation. At 60 days post-inoculation, percentages of adults recovered at necropsy and lymphatic lesion severity were greater in B. pahangi-infected jirds. At 150 days post-inoculation, lesion severity and percentages of worms recovered were similar in both infections. No significant differences were noted in either infection in reactivity to homologous or heterologous parasite antigens in any parameter measured. Similarities in the kinetics of the inflammatory reactivities of the 2 infections suggest that previous observations made in the jird-B. pahangi model could be utilized in designing studies using B. malayi. Further, the more marked lesion severity observed in B. pahangi-infected jirds and the relative ease of maintaining B. pahangi in the laboratory support the continued use of this system as a conceptual model for the study of lymphatic lesion pathogenesis.

Successful cryopreservation of Brugia pahangi third-stage larvae in liquid nitrogen.

Experiments are described which lead to the retention of infectivity of Brugia pahangi third-stage larvae after cooling to -196 degrees C. Methanol, a well documented cryoprotectant was used at a concentration of 20% (v/v). The schedule consisted of a 5 degrees C min-1 cool to an intermediate temperature of -21 degrees C and a subsequent rapid cool into liquid nitrogen. A rapid thaw of the parasites led to approximately 34% of motile cryopreserved larvae developing in multimammate rats (Mastomys natalensis) compared to unfrozen control larvae. Cooling rate and intermediate temperature were both found to be crucial variables affecting survival levels of the larvae.

On the longevity and behaviour of microfilariae of Wuchereria bancrofti, Brugia pahangi and Dirofilaria immitis transfused to laboratory rodents.

Microfilariae of Wuchereria bancrofti and Brugia pahangi, which were inoculated into Meriones unguiculatus and Mastomys natalensis respectively, were found in the peripheral blood of the rodents until the ninth day after infection, but no microfilarial periodicity was observed. Microfilariae of W. bancrofti disappeared much faster from the peripheral blood of jirds after reinfection than after the first inoculation. Microfilariae of Dirofilaria immitis inoculated into M. natalensis and laboratory mice were recovered in the peripheral blood until the 17th day after infection and remained infective to mosquitoes. For the first few days the microfilarial periodicity observed in the rodents was similar to that observed in the donor dog, but then reversed.

The number and distribution of Brugia pahangi in cats at different times after a primary infection.

The number of larvae and adults of Brugia pahangi and their distribution throughout the lymphatics and extra-lymphatic tissue were studied in cats infected by subcutaneous injection of larvae into their hind feet. For the first 20 days approximately 55% of the inoculum is recovered as living worms. After 25 days the recovery falls by a half. It is suggested that this loss of worms may be due to either the developing immunological response or the moult from the 4th to the 5th stage. Larvae penetrate the lymphatics rapidly (50% within 3 h) and migrate to the popliteal lymph node after about 20 days they migrate back down into the afferent lymphatic.

Intracellular melanization in the mosquito Anopheles quadrimaculatus (Diptera: Culicidae) against the filarial nematodes, Brugia spp. (Nematoda: Filarioidea).

Intracellular melanization responses to developing larvae of Brugia species (B. malayi (Buckley), B. pahangi (Buckley and Edeson), and B. patei (Buckley, Nelson, and Heisch] in the thoracic muscle fibers of eight strains of Anopheles quadrimaculatus Say were first observed 48 to 72 h after an infective blood meal. Three to 4 d later, large numbers of melanized first-stage larvae were found within the thoracic muscle fibers. These intracellular responses were in addition to the extracellular responses to microfilariae and microfilarial sheaths of B. pahangi in the abdominal hemocoel of An. quadrimaculatus described in literature. Simultaneously, normal development of larvae of the three Brugia species also was observed in all eight strains of An. quadrimaculatus. Comparisons of melanized first-stage larvae and normally developing larvae of the three Brugia species in the thoracic muscle fibers of the eight strains of An. quadrimaculatus showed that there were distinct variations in numbers of melanized and developing larvae and percentage of females with melanized and developing larvae in different strains. Numbers of melanized first-stage larvae reflected the extent of refractoriness of An. quadrimaculatus strains. Fully melanized larvae showed no abnormalities in parasite organelles, indicating that refractoriness is due to an enhanced ability of the host to recognize the living parasite. Further comparison among the strains suggested that the mutants, Yellow Larvae and Vero Beach Colony were significantly more susceptible, and Red Stripe was the most refractory to all three Brugia species. Thus, the gene(s) controlling susceptibility and refractoriness to all three Brugia species probably occurs on the same autosomal chromosome as the mutations in these strains. The significance of intracellular melanization of filarial larvae is discussed with reference to the melanization responses to different parasites in other mosquitoes.

Susceptibility of mansonia indiana (Diptera: Culicidae) to nocturnally subperiodic Brugia malayi (Spirurida: Filariodea).

Mosquitoes, Mansonia indiana Edwards, 1930, were collected from non-endemic area of human lymphatic filariasis and tested for their susceptibility of infection using nocturnally subperiodic Brugia malayai Buckley & Edeson, 1956. Three cats naturally infected with B. malayi were used in the experiment for mosquitoes feeding. The data revealed that the susceptibility of mosquito infection ranged from 30 to 70%. The results also revealed that the susceptibility rates were not linearly correlated to the microfilarial densities in the cat at the time of feeding. The microfilarial density in cats ranged from 15 to 27 per 10 microl of blood whereas the mean number of third stage larvae in the infective mosqiitoes ranged from 21.6 to 26.8. In addition, statistical analysis showed no significant difference (P > 0.05) between the mean number of third-stage larvae in mosquitoes and the density of microfilaria in cats. The study indicated that Ma. indiana, collected from non-endemic areas, is capable for transmitting the nocturnally subperiodic B. malayi.

Microfilarial perforation of the midgut of a mosquito.

To determine whether the midgut envelope of mosquitoes is disrupted by the passage of microfilariae, ultrastructural changes induced by microfilariae of Brugia malayi were observed in midguts of Aedes aegypti mosquitoes. Basal and apical plasma membranes were destroyed, disrupting the full depth of the midgut wall. Ingested ferritin lay against the gut wall, suggesting absence of the peritrophic membrane during penetration. Exsheathment of microfilariae appears to be enhanced by movement against the constricting midgut wall. It was concluded that particles present in the lumen of the gut may be disseminated passively to the hemocoel.

Rapid morphological transformations of spermatozoa in the uterus of Brugia pahangi (Nematoda, Filarioidea).

Mature, umated male and female Brugia pahangi worms were implanted into the peritoneal cavity of gerbils, allowed to mate, and then recovered and examined by means of electron microscopy. The proximal portion of the uterus of female worms recovered in copula contained a morphologically heterogenous population of sperm ranging from a rigid, nonmotile form to the mature ameboid sperm. The immotile sperm are identical in morphology to sperm found within the seminal vesicle of the male. The in utero transformations occur in less than 1 hr, and probably represent a final maturation event preceding competency to fertilize eggs. First in the sequence of events leading to the mature spermatozoon is a decrease in the density of the cytoplasmic matrix concomitant with the elaboration of an abundant tubular smooth endoplasmic reticulum. The position and appearance of the sperm in the uterus suggest that the formation of this membranous system occurs within minutes and involves de novo assembly from cytoplasmic precursors. Following the appearance of the tubular membranous system, certain other membranous organelles become spherical, fuse with the plasmalemma, and release their electron-dense contents into the lumen of the uterus. Filamentous rodlike elements, previously extending the length of the peripheral cytoplasm, begin to disintegrate and cytoplasmic projections form at the margins of the cell. Polarization of the sperm results from the aggregation of mitochondria, nuclear material, endoplasmic reticulum, and membranous organelles at one pole of the cell leaving an organelle-free filamentous pseudopod at the other.

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

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

In vitro antifilarial effects of three plant species against adult worms of subperiodic Brugia malayi.

Five aqueous extracts from three plant species, i.e., dried husks (HX), dried seeds (SX) and dried leaves (LX) of Xylocarpus granatum (Meliaceae), dried stems (ST) of Tinospora crispa (Menispermaceae) and dried leaves (LA) of Andrographis paniculata (Acanthaceae) were tested in vitro against adult worms of subperiodic Brugia malayi. The relative movability (RM) value of the adult worms over the 24-h observation period was used as a measure of the antifilarial activity of the aqueous extracts. SX extract of X. granatum demonstrated the strongest activity, followed by the LA extract of A. paniculata, ST extract of T. crispa, HX extract and LX extract of X. granatum.