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.

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.

Nanocurcumin: a novel antifilarial agent with DNA topoisomerase II inhibitory activity.

OBJECTIVE: The aim of this study is to evaluate the antifilarial, antiwolbachial and DNA topoisomerase II inhibitory activity of nanocurcumin (nano-CUR). METHODS: Nano-CUR formulations (F1-F6) were prepared using free radical polymerization and were characterized by particle size, morphology, encapsulation efficiency and in vitro release kinetics. Antifilarial potential was evaluated in vivo against Brugian filariasis in an experimental rodent model, Mastomys coucha, by selecting the formulation that maximized parasite elimination characteristics. Wolbachial status was determined by PCR and a relaxation assay was used to estimate DNA topoisomerase II inhibitory activity. RESULTS: Nano-CUR (F3) having a 60 nm diameter and 89.78% entrapment efficiency showed the most favorable characteristics for the elimination of filarial parasites. In vivo pharmacokinetic and organ distribution studies demonstrate significantly greater C(max) (86.6 ± 2.56 ng ml(-1)), AUC0-∞ (796 ± 89.8 ng d ml(-1)), MRT (19.5 ± 7.82 days) and bioavailability of CUR (70.02%) in the organs from which the adult parasites were recovered. The optimized nano-CUR (F3) (5 × 5 mg/kg, orally) significantly augmented the microfilariciadal and adulticidal action of CUR over free CUR (5 × 50 mg/kg, orally) or Diethylcarbamizine (50 mg/kg, orally) against the Brugia malayi Mastomys coucha rodent model. The PCR results showed complete elimination of wolbachia from the recovered female parasites. Interestingly, nano-CUR was also found to be a novel inhibitor of filarial worm DNA topoisomerase II, Setaria Cervi in vitro. CONCLUSION: This study recognizes the beforehand antimicrofilarial, antimacrofilarial, anti-wolbachial activity of nano-CUR (F3) over free forms and additionally its strong inhibitory action against the major target filarial parasite enzyme DNA topoisomerase II in vitro.

Midgut barrier imparts selective resistance to filarial worm infection in Culex pipiens pipiens.

Mosquitoes in the Culex pipiens complex thrive in temperate and tropical regions worldwide, and serve as efficient vectors of Bancroftian lymphatic filariasis (LF) caused by Wuchereria bancrofti in Asia, Africa, the West Indies, South America, and Micronesia. However, members of this mosquito complex do not act as natural vectors for Brugian LF caused by Brugia malayi, or for the cat parasite B. pahangi, despite their presence in South Asia where these parasites are endemic. Previous work with the Iowa strain of Culex pipiens pipiens demonstrates that it is equally susceptible to W. bancrofti as is the natural Cx. p. pipiens vector in the Nile Delta, however it is refractory to infection with Brugia spp. Here we report that the infectivity barrier for Brugia spp. in Cx. p. pipiens is the mosquito midgut, which inflicts internal and lethal damage to ingested microfilariae. Following per os Brugia exposures, the prevalence of infection is significantly lower in Cx. p. pipiens compared to susceptible mosquito controls, and differs between parasite species with <50% and <5% of Cx. p. pipiens becoming infected with B. pahangi and B. malayi, respectively. When Brugia spp. mf were inoculated intrathoracically to bypass the midgut, larvae developed equally well as in controls, indicating that, beyond the midgut, Cx. p. pipiens is physiologically compatible with Brugia spp. Mf isolated from Cx. p. pipiens midguts exhibited compromised motility, and unlike mf derived from blood or isolated from the midguts of Ae. aegypti, failed to develop when inoculated intrathoracically into susceptible mosquitoes. Together these data strongly support the role of the midgut as the primary infection barrier for Brugia spp. in Cx. p. pipiens. Examination of parasites recovered from the Cx. p. pipiens midgut by vital staining, and those exsheathed with papain, suggest that the damage inflicted by the midgut is subcuticular and disrupts internal tissues. Microscopic studies of these worms reveal compromised motility and sharp bends in the body; and ultrastructurally the presence of many fluid or carbohydrate-filled vacuoles in the hypodermis, body wall, and nuclear column. Incubation of Brugia mf with Cx. p. pipiens midgut extracts produces similar internal damage phenotypes; indicating that the Cx. p. pipiens midgut factor(s) that damage mf in vivo are soluble and stable in physiological buffer, and inflict damage on mf in vitro.

Role of vector control in the global program to eliminate lymphatic filariasis.

Lymphatic filariasis (LF) is a major cause of acute and chronic morbidity in the tropical and subtropical parts of the world. The availability of safe, single-dose, drug treatment regimens capable of suppressing microfilaremia to very low levels, along with improvements in techniques for diagnosing infection, has resulted in the targeting of this major mosquito-borne disease for global elimination. The Global Program to Eliminate Lymphatic Filariasis (GPELF) was launched in 2000 with the principal objective of breaking the cycles of transmission of Wuchereria bancrofti and Brugia spp. through the application of annual mass drug administrations (MDAs) to entire at-risk populations. Although significant progress in initiating MDA programs in endemic countries has been made, emerging challenges to this approach have raised questions regarding the effectiveness of using MDA alone to eliminate LF without the inclusion of supplementary vector control. Here, we review advances in knowledge of vector ecology, vector-parasite relationships, and both empirical and theoretical evidence regarding vector management to assess the feasibility and strategic value of including vector control in the GPELF initiative to achieve the global elimination of LF.

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.

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.

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.

External stimuli and intracellular signalling in the modification of the nematode surface during transition to the mammalian host environment.

Previous work has shown that the surface of infective larvae of parasitic nematodes will not bind the fluorescent lipid analogue 5-N-(octadecanoyl)aminofluorescein (AF18) until after exposure of the parasite to mammalian tissue-culture conditions. In this study, culture media which are permissive or non-permissive for the acquisition of lipophilicity for AF18 were altered in order to examine possible stimuli involved. This showed that external alkaline pH and high sodium ion concentration were highly stimulatory. The internal signalling pathways which may be involved in the surface alteration were then examined using agents which are known to affect intracellular signalling in mammalian cells. The results indicated that elevation of cGMP levels was stimulatory whereas inhibition of a putative Na+/H+ antiporter or calcium mobilization was inhibitory, and it is argued that high intracellular levels of cAMP may be inhibitory. Whilst the precise effects of the agents used on nematode cells remain to be established, these results provide a framework for the examination of the processes involved in the modification of the nematode surface which takes place immediately after the infection event.

Current status of filariasis in Indonesia.

Filariasis in Indonesia is widely distributed. Three species consisting of 5 ecologically different types have been identified infecting man. Compared to older data, infection rates are much lower, partly due to environmental change and partly as a result of control programs. Various dosage treatments have given good results. The higher dosage treatment gave severe reactions especially in brugian filariasis. Pockets of high endemicity can still be found in remote rural areas. Therefore a weekly low dosage treatment of 40 weeks through the Primary Health Care approach has been adopted. Filariasis research in Indonesia at present is concentrating on the use of biotechnological tools, especially for diagnostic and vector identification purposes, and to understand better the pathophysiology. Treatment trials with new drugs such as Ivermectin and DEC are being conducted both in man and experimental animals.

Recent advances in epidemiological field techniques in filariasis.

There have been very few advances in epidemiological field techniques for filariasis in recent years. Advances in other areas of study of filariasis have been published and a summary of some of those which have some application to field technology will be given. Studies on vectors involving host influences on the development of filariae and the use of DNA probes for vector speciation were reported as well as the development of techniques that yielded species-specific identification of larvae in mosquito vectors. Experiments on animal transmission potentials also have been described. Mathematical models are becoming popular for various biological systems. It is postulated that models will enable workers to make predictions on prevalence of diseases and effect of control measures. A model has been proposed for bancroftian filariasis transmission dynamics and frequency distribution of microfilariae in blood samples. Other statistical methodologies have been published relating to age and sex distribution and frequency distribution and one suggested that females have increased resistance to filariasis, especially in the reproductive years. Advances in diagnostic technologies are highly relevant to the epidemiology of filariasis. The development of antibody and especially antigen detection systems using monoclonal antibodies, DNA probes, and polymerase chain reactions, etc, will make a significant impact on the identification of infected persons. Findings along other lines may also have application to field epidemiology. The quantitative blood count (QBC) acridine orange hematocrit tube has also been found to be of value in diagnosis. Elevated levels of ecdysteroids found in other filarial infections should be tested in cases of lymphatic filariasis.

Comparative susceptibility of species A, B and C of Anopheles quadrimaculatus complex to infection with subperiodic Brugia malayi and Brugia pahangi (Nematoda: Filarioidea).

Susceptibilities of natural populations of sibling species A, B and C of the Anopheles quadrimaculatus complex and the colonized strain A to subperiodic Brugia malayi and Brugia pahangi were compared. All 3 sibling species showed varying degrees of susceptibility to both B. pahangi and B. malayi, and they were considerably more susceptible to B. pahangi than to B. malayi. The rate and intensity of infection to B. pahangi were highest for species A (66.2% and 7.4 L3/female, respectively) and lowest for species B (21.3% and 1.7 L3/female). For B. malayi these values were higher for species A (29.7% and 1.84 L3/female) than for species B (13.3% and 0.86 L3/female) and C (12.6% and 0.75 L3/female). The colonized strain A of An. quadrimaculatus was significantly more susceptible to both Brugia species than the natural populations of sibling species A, B and C.

On the infectivity of early third-stage Brugia larvae isolated from the abdomen of Aedes aegypti.

Third-stage larvae (L3) of Wuchereria bancrofti, Brugia malayi and B. pahangi are known to be recovered not only from the head and thorax but also from the abdomen of dissected vector mosquitoes. For epidemiological reasons, was of interest to determine whether L3 larvae from the abdomen of the vector would be infectious for the final host. Early abdominal L3 larvae of B. pahangi isolated as early as on day 7 post-infection (p.i.) from Aedes aegypti were injected s.c. into five male Meriones unguiculatus. Four of the five jirds were microfilaria-positive after 67 days.

Effect of N-acetyl-D-glucosamine on the migration of Brugia pahangi microfilariae into the haemocoel of Aedes aegypti.

Two strains of Aedes aegypti (L.), differing in their susceptibility to Brugia pahangi (Buckley & Edeson), were examined with regard to the effect on the proportion of microfilariae migrating from the mid-gut, of specific carbohydrate supplements in the infecting bloodmeal. N-Acetyl-D-glucosamine (GlcNAc), a sugar also present on the microfilarial sheath, significantly increased the migration rate. This enhancement is greater for the refractory strain of Ae.aegypti. The use of sucrose as a control sugar results in no enhancement of microfilariae migration. It is postulated that the GlcNAc is acting by blocking endogenous gut/peritrophic membrane carbohydrate binding proteins, which would normally inhibit microfilariae migration. Furthermore, there is a significant correlation whereby increasing loads of microfilariae ingested result in decreasing proportions migrating across the mid-gut.

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.

A proline-rich structural protein of the surface sheath of larval Brugia filarial nematode parasites.

Both cDNA and genomic DNA sequences have been isolated which encode a proline-rich precursor protein of the sheath from microfilariae, the first stage larvae of the filarial nematode parasites Brugia pahangi and Brugia malayi. This 22-kDa protein is soluble only under reducing conditions and is extensively cross-linked by both disulfide and nonreducible bonds. Immunogold electron microscopy shows that the protein is localized exclusively in the sheath, a vestigial remnant of the eggshell, which is retained by and encloses the mature microfilaria. Analysis by Western blotting confirms that the protein is expressed only in microfilariae and adult female worms, although transcripts are detectable only in adult females. The deduced amino acid sequence contains a short N-terminal hydrophobic putative leader sequence, a central repetitive domain that contains 14 copies of a degenerate 5-amino acid repeat with the consensus sequence Met-Pro-Pro-Gln-Gly, and a C-terminal proline-rich domain flanked by clusters of cysteine residues. These clusters can be aligned with cysteine residues implicated in cross-linking of a family of cuticular collagens originally identified in Caenorhabditis elegans but which extends to other nematodes.

Filariasis and its control in Fujian, China.

Epidemiological survey of filariasis in Fujian Province, China showed that malayan filariasis, transmitted by Anopheles lesteri anthropophagus was mainly distributed in the northwest part and bancroftian filariasis with Culex quinquefasciatus as vector, in middle and south coastal regions. Both species of filariae showed typical nocturnal periodicity. Involvement of the extremities was not uncommon in malayan filariasis. In contrast, hydrocele was often present in bancroftian filariasis, in which limb impairment did not appear so frequently as in the former. Hetrazan treatment was administered to the microfilaremia cases identified during blood examination surveys, which were integrated with indoor residual spraying of insecticides in endemic areas of malayan filariasis when the vector mosquito was discovered and with mass treatment with hetrazan medicated salt in endemic areas of bancroftian filariasis. At the same time the habitation condition was improved. These factors facilitated the decrease in incidence. As a result malayan and bancroftian filariasis were proclaimed to have reached the criterion of basic elimination in 1985 and 1987 respectively. Surveillance was pursued thereafter and no signs of resurgence appeared.