Actions of acetylcholine and GABA on spontaneous contractions of the filariid, Dipetalonema viteae.

1. Isotonic contractions were recorded from the filarial nematode, Dipetalonema viteae (Acanthocheilonema viteae), in an isolated tissue chamber. 2. Nicotine (10(-6) M) and pilocarpine (10(-5) M) increased the spontaneous contractions in the intact filariid, but acetylcholine (ACh, 10(-4) M) and muscarine (10(-5) M) were inactive. 3. When ACh was applied to an opened D. viteae, it was 10,000 times more potent. This indicates that the cuticle is an effective barrier to the penetration of ACh to the muscle cells. 4. The effects of ACh on the opened D. viteae were not affected by hexamethonium (10(-3) M) or atropine (10(-5) M) and were only partially reduced by (+)-tubocurarine (10(-4) M). 5. gamma-Aminobutyric acid (GABA, 10(-3) M) reduced the spontaneous activity of the intact D. viteae; however, the effect of GABA had a slow onset and recovery. Muscimol (10(-5) M) was more potent than GABA and had a more rapid onset and recovery. 6. GABA was 1,000 times more potent on the opened D. viteae than on the intact D. viteae. Baclofen (10(-3) M) was inactive on both preparations. 7. The effect of GABA was not antagonized by bicuculline (10(-4) M), picrotoxin (10(-5) M or penicillin G (10(-3) M). 8. It is concluded that the filariid cuticle acts like a lipid structure and blocks the penetration of polar substances, such as ACh and GABA. Also, due to the lack of efficacy of the ACh and GABA antagonists, it was concluded that the nematode receptors are somewhat different from the mammalian ACh and GABA receptors.

Potential of a soluble tetrazolium/formazan assay for the evaluation of filarial viability.

Using female Acanthocheilonema viteae we have investigated the bioreduction of the tetrazolium reagent XTT (2,3-bis(2-methoxy-4-nitro-sulphonyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide). Unlike the formazan formed by other tetrazolium salts, that derived from XTT readily diffuses out of A. viteae in vitro. Formazan formation can therefore be quantified by direct absorbance reading of the incubation medium, eliminating the need for a DMSO solubilization step. Optimum assay conditions involved a 4 h incubation, in the presence of the electron coupling agent phenazine methosulphate (PMS). Repeat 4 h incubations with XTT-PMS were well tolerated by worms for 5 consecutive days. This confirmed the low toxicity of XTT formazan and its usefulness in the semi-continuous assessment of filarial viability. In comparison to our previously reported MTT (3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide)-reduction assay XTT-PMS reduction showed comparable drug sensitivity and accuracy, however XTT-PMS appears to be at least 10-15 times less efficiently reduced by A. viteae females. A possible application of the XTT assay using female Onchocerca volvulus is discussed.

Development of in vitro screening system for assessment of antifilarial activity of compounds.

Evaluation of antifilarial activity of new potential agents in vivo is extremely time consuming and uneconomic. In the present study effort has been made to develop an in vitro screening method using Acanthocheilonema viteae, a subcutaneously dwelling rodent filariid with anaerobic metabolic characteristics like human filariids, W. Bancrofti/Brugia malayi as test parasite. Motility test and tetrazolium (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT) based colorimetric assay were used as parameters in in vitro assay. Results showed that 92.3% of compounds (in vivo active) could be picked up in the in vitro assay when both adults and microfilarae (mf) were used simultaneously. Mf and adult stages separately detected, respectively, 84.6 and 69.2% of in vivo active compounds. The adults and mf separately and both the life stages together exhibited, respectively, 80.0, 50.0 and 80.0% false positive results in the in vitro test with in vivo inactive compounds. It is felt that mf stage when used in in vitro test using motility and MTT assays as parameters would be useful in primary screening of new potential filaricides.

Dipetalonema vitae: survival of adult females and microfilarial release in both a chemically defined and serum-supplemented medium.

Studies were conducted on survival and microfilarial release of afult Dipetalonema viteae in culture, using worms of various ages derived from jirds. In chemically defined NI medium (a 1:1 mixture of NCTC 135 and Iscove's modified Dulbecco's medium) under a gas phase of 5% CO2 in nitrogen (pO2 of medium approximately 40 mm Hg), the peak of microfilarial release of several thougsand microfilariae per female per 24 hr occurred at approximately day 10. Thereafter, microfilarial release declined and generally ended about 1 mo after the start of culture. The adult females moved actively for about 50 days or more and survived up to 82 days in NI medium alone. The females in NI medium supplemented with fetal bovine serum showed serpentine movement for approximately 2 mo. Some of the worms survived more than 83 days. The total number of microfilariae deposited in culture by D. viteae increased as adult females grew in size (volume) over time. Microfilarial deposition continued to increase after worms reached maximum size, deposition reaching a plateau between approximately 300 and 400 days of age. Thereafter, microfilarial deposition decreased as females continued to age. Addition of fetal bovine serum to the NI medium increased the number of microfilariae released and extended the period of release.

Transplantation into jirds as a method of assessing the viability and reproductive integrity of adult Acanthocheilonema viteae from culture.

The reproductive integrity and viability of adult female Acanthocheilonema viteae (syn. Dipetalonema viteae) maintained in culture for relatively long periods were assessed by transplantation into jirds. Worms cultured in chemically defined NI medium for approximately 3-4 wk remained active, but microfilarial release declined to barely detectable levels. Microfilarial production, however, was restored when the worms were transplanted subcutaneously into jirds. When cultured in NI medium beyond 4 wk no restoration of microfilarial production occurred on transplantation, presumably due to irreversible injury to the reproductive system. However, when NI medium was supplemented with fetal bovine serum resumption of microfilarial production occurred in transplanted females that had been in culture for as long as 2 1/2 mo. The addition of serum to NI medium played an important role in maintaining and protecting the functional integrity of the reproductive system.

Transplanted Dipetalonema viteae in the jird: effect of worm burden on parturition rates and microfilaremia.

Dipetalonema viteae was studied in the jird, Meriones unguiculatus, to determine the mechanism controlling the level of peripheral microfilaremia. Jirds killed 40 days after infection served as donors of female worms of known age and reproductive status. These worms were transplanted into uninfected jirds and the resultant microfilaremias were monitored. After approximately 100 days, the recipient jirds were killed and 58% of the transplanted worms were recovered alive but depleted of sperm and microfilariae, regardless of the total number implanted in a given host. A direct linear relationship between microfilaremia and the number of recovered adult worms was found. Based on the uniform absence of sperm and microfilariae in the recovered worms it was concluded that female worms, under the conditions of the present study, do not control the peripheral microfilaremia in multi-worm infections through a reduced parturition rate.

Distribution and movement of infective-stage larvae of Dipetalonema viteae (Filarioidea) in the vector tick, Ornithodoros tartakowskyi (Argasidae).

Histological sections and dissection of infected ticks, Ornithodoros tartakowskyi, showed that in the resting tick the 3rd-stage larvae of Dipetalonema viteae were distributed in clumps throughout the hemocoel. In the biting tick, larvae moved anteriorly and congregated especially in the capitulum; and the forward migration occurred even though no blood was ingested. This suggests that the act of biting and not the ingestion of blood is the critical factor in migration. The larvae may reach the buccal cavity through 4 possible avenues: 1) the junction of the pharynx with the buccal cavity; 2) the esophagus; 3) the salivary ducts; and 4) the roof of the hypostome. Developing forms produce direct injury to muscle fibers, and the migrating larvae further disorganize the muscles, affecting to some extent the normal activities of the ticks.

Behavior of Dipetalonema viteae (Filarioidea) during escape from the vector tick, Ornithodoros tartakowskyi (Argasidae).

To determine the behavior of Dipetalonema viteae in its tick vector, Ornithodoros tartakowskyi, the ticks were fed on jirds at successive intervals of 30 to 35 days after a single infective blood meal, and the number of larvae passing from the tick during each bite was determined by recovery of: 1) adult worms from the jirds' tissues; 2) larvae from skin snips taken at the feeding site immediately after the bite; and 3) larvae from serum and tissue after artificial feeding through a skin-membrane. All methods gave similar results. Ticks harboring few larvae released most of them (82%) during the first bite, and required only 2 bites to transmit all. Ticks with moderate or heavy infections required 3 or 4 bites to transfer all larvae. Factors which may explain this are: 1) relatively short duration of the bite of heavily infected ticks due to irritation and damage to the muscles of mouthparts and pharynx by the larvae; 2) resistance of the anterior alimentary tract to penetration by the larvae; and 3) retarding effects of crowding on development and migration of larvae. Aging of infection in the tick apparently did not influence the rate of transfer of larvae. Infection adversely affected the feeding and retarded the molting of young nymphs, but with the loss of larvae in successive bites the ability to suck blood was regained.

Glutamine-supported motility of adult filarial parasites in vitro and the effect of glutamine antimetabolites.

The survival in culture of adult female Brugia pahangi, Acanthocheilonema viteae, and Onchocerca volvulus and adult male Onchocerca gibsoni was assessed by measuring parasite motility. Survival of all species was maximal in a nutritionally complex medium (RPMI-1640). All species survived for up to 48 hr in a simpler medium in which the only energy source was 10 mM glutamine; motility in this medium was dependent upon pH. For the species of Onchocerca, motility was maintained better in the presence of glutamine as the sole energy source than in glucose-only medium. Motility of B. pahangi incubated in 10 mM succinate was equivalent to that seen with 10 mM glutamine, but no other tricarboxylic acid intermediate supported this parasite in vitro. Antimycin A (1 microM) and potassium cyanide (KCN, 100 microM) paralyzed B. pahangi incubated in 10 mM glutamine, an effect antagonized by glucose. KCN at 10 or 100 microM was effective also against Onchocerca gutturosa in glutamine-only medium. Several glutamine antimetabolites reduced motility of B. pahangi by 72 hr. This inhibition was prevented by 2 mM glutamine. However, the inhibition of motility in the species of Onchocerca caused by these compounds was attenuated only partially by glutamine. These data demonstrate that, under certain conditions, filarial nematodes can utilize non-sugar substrates as energy sources. The differential sensitivity seen among these organisms to mitochondrial toxins and glutamine antimetabolites may be related to the extent to which they can use these alternative substrates to generate energy.

Optimization of test conditions for development of MTT as in vitro screen.

The quick and easy method of tetrazolium based colorimetric assay with MTT [3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] was used to test the viability of the adult parasites of a rodent filariid Acanthocheilonema viteae in vitro. The ideal conditions required for antifilarial screening were determined by correlating the MTT reduction ability of worms with their size and age in the vertebrate host, also the duration of incubation and temperature of the in vitro culture. It was observed that the worms collected from the host after 90 days of L3 (infective larvae) exposure were not suitable for in vitro screen as they could not reduce MTT to that extent as the worms of early infection. Healthy and full grown worms and also those incubated at 37 degrees C for 16 hr or more caused maximum MTT reduction. Thus, it is recommended to select healthy adult filariids of proper age and size (male > 3.5 cm; female > 7.0 cm). The incubation temperature of the in vitro culture system needs to be adjusted to 37 degrees C and parasites might be exposed to drugs upto 24 hr without much alteration in MTT reduction of untreated controls.

Impact of surface modifications of Acanthocheilonema viteae microfilariae on cell adhesion.

Exposure of A. viteae microfilariae to various lectins reduced their capacity to react with the peritoneal exudate cells of the host, Mastomys natalensis. Sugars corresponding to these lectins with the exception of N-acetyl glucosamine, did not affect the adhesion per se. They however, protected the parasite against the adverse effect of lectins. Neuraminidase and chitinase also suppressed adhesion capacity of the microfilariae. Except sodium dodecylsulphate which enhanced cell attachment, other surfactants inhibited this reaction considerably. The results indicate that antibody dependent adhesion of the microfilariae with the macrophages involves surface moieties of the parasite, where N-acetylglucosamine acts as the principal sugar residue. Participation of -SH groups also is inferred from the observations that p-chloromercuribenzoate and dithiobis-(2-nitrobenzoic acid) inhibited cell attachment and dithiothreitol provided protection against these agents.

Lack of immunological cross-reactivity between parasite-derived and recombinant forms of ES-62, a secreted protein of Acanthocheilonema viteae.

The longevity of filarial nematodes is dependent on secreted immunomodulatory products. Previous investigation of one such product, ES-62, has suggested a critical role for post-translationally attached phosphorylcholine (PC) moieties. In order to further investigate this, ES-62 lacking PC was produced, using the Pichia pastoris recombinant gene expression system. Unlike parasite-derived ES-62, which is tetrameric the recombinant material was found to consist of a mixture of apparently stable tetramers, dimers and monomers. Nevertheless, the recombinant protein was considered to be an adequate PC-free ES-62 as it was recognized by existing antisera against the parasite-derived protein. However, subsequent to this, recognition of parasite-derived ES-62 by antibodies produced against the recombinant protein was found to be absent. In an attempt to explain this, recombinant ES-62 was subjected to structural analysis and was found to (i) contain 3 changes in amino acid composition; (ii) demonstrate significant alterations in glycosylation; (iii) show major differences in protein secondary structure. The effects of these alterations in relation to the observed change in immunogenicity were investigated and are discussed. The data presented clearly show that recognition by existing antibodies is insufficient proof that recombinant proteins can be used to mimic parasite-derived material in studies on nematode immunology and vaccination.

[The mechanism controlling the crossing of the vector's stomach wall by microfilariae (Dipetalonema dessetae-Aedes aegypti)]. / Le mécanisme assurant la régulation de la traversée de la paroi stomacale du vecteur par les microfilaires (Dipetalonema dessetae-Aedes aegypti)

In some of the human filariasis, the number of microfiliariae which succeed in crossing the vector's stomach wall is smaller when the number of ingested microfilariae is larger (limitation). In the couple Dipetalonema dessetae-Aedes aegypti, this phenomenon appears to be due to a specific lysis of the stomach cells invaded by the microfilariae. This reaction is started when the microfilariae are very numberous. There is "information" transmitted to the whole of the vector's stomach.

Turnover of microfilariae in small mammals 2. Disintegration of microfilariae (Acanthocheilonema viteae) (Filarioidea: Nematoda) after intravenous injection into the jird, Meriones unguiculatus.

After i.v. injection of 305 x 10(3) microfilariae (mf) per animal (50 g) into naive jirds, 50.8% of them could be recovered at autopsy 15 min later. Of these, 65.8% were calculated to be in the peripheral circulating blood (PCB) and were completely intact; 18.6% were recovered by perfusion of the lungs and 13.6% from the liver. In both organs about half the mf were associated with adherent lymphocytes and neutrophils but a few were partly disintegrated. Only 2.6% were recovered from the kidneys and the spleen. In long-term injection experiments using the same inoculum size the autopsy was done 15 min and 1, 3 and 6 weeks post-injection (p.i.) of mf into naive jirds. Throughout the experimental period the density of mf remained more or less constant in the PCB, but 3 weeks p.i. the density in the lungs increased up to 14 times to that in the PCB, whereas in the liver it decreased at the same time to a density similar to that in the PCB. In patent animals with adult worms delivering mf these were distributed as follows: 34.7% were calculated to be in the PCB; 24.4% were obtained by perfusion from the lungs and 22.0% from the liver; the rest were found in the kidneys (16.6%) and spleen (2.3%). In the lungs and the liver about 5/6 were associated with adherent cells, partly disintegrated or as fragments. In view of the fact that very few mf become disintegrated immediately after i.v. injection and also from their extremely long sojourn in the PCB, a low turnover rate of mf is presumed.(ABSTRACT TRUNCATED AT 250 WORDS)