Essential role of chitinase in the development of the filarial nematode Acanthocheilonema viteae.

Chitinases of pathogens have been proposed as potential targets of vaccines or specific inhibitors. We studied the genomic organization, transcript levels, developmental expression, and biological function of chitinases in the rodent filarial nematode Acanthocheilonema viteae, a model organism for human-pathogenic filarial worms. Characterization of nine genomic clones from an A. viteae phage library and Southern blot experiments revealed the existence of three different chitinase genes, two of which could theoretically yield functional transcripts. The deduced proteins of these genes had the common modular organization of family 18 chitinases. Northern blot experiments and rapid amplification of cDNA ends-PCR with adult worms and larval stages showed that only one gene is expressed, with high variation in transcript levels, as determined by real-time PCR. Chitinase transcript levels were lowest in the late male stage 4 larva (L4) and peaked in the stage 3 larva (L3), which was corroborated by Western blotting. RNA interference (RNAi) experiments showed that treatment of L3 and adult female worms with double-stranded RNA of chitinase inhibited molting of L3 worms and hatching of microfilariae. RNAi also led to the death of 50% of female worms, revealing the essential role of chitinase in the life cycle of filarial nematodes.

Acanthocheilonema viteae: octopamine and its physiological role.

Octopamine acts as an important neurotransmitter and neuromodulator in arthropods, mollusks, and nematodes. In mammals, however, no definite function for this amine has yet been described. By virtue of this difference in the neurophysiological requirement of the mammalian host and nematodes, octopamine offers good opportunity for exploring this area deeply with a view to identify a unique target for filarial chemotherapy. Results of the present study indicated that Acanthocheilonema viteae, the rodent filarial parasite, utilized tyrosine as a precursor for producing octopamine and some other biogenic amines. Octopamine exhibited specific saturable binding with the membrane prepared from the anterior portion of the filariid. This amine induced concentration dependent increase in the membrane potential which possibly caused tonic paralysis of the filariid. The rate of micro filarial release by the female worms also declined in the presence of this amine. The study thus provided preliminary evidences for the presence of an octopamine neurotransmitter system and also about some of the roles it plays in A. viteae.

Expression of the filarial nematode phosphorylcholine-containing glycoprotein, ES62, is stage specific.

ES62, an immunomodulatory phosphorylcholine-containing glycoprotein secreted by the rodent filarial nematode Acanthocheilonema viteae, has previously been shown to be produced by L4 larvae and adult worms only. However, homologous sequences to ES62 have recently been found in L1 and L3 cDNA libraries of certain human filarial nematodes. Therefore, the various stages of A. viteae were re-examined and it was again found that only the post-L3 stages secreted ES62. Synthesis but not secretion by earlier stages was ruled out by examination of the protein content of whole worm extracts and by immunoelectron microscopy. However, examination by PCR of the mRNA for ES62 revealed that it was found in the L1 and L3 larvae. This may explain why homologous sequences to ES62 have been found in Brugia malayi and Onchocerca volvulus larval cDNA libraries. It also suggests that filarial nematodes, in general, may secrete ES62. To obtain evidence for this, we investigated production by Brugia pahangi, a close relation of B. malayi. We found that ES62 was indeed secreted but, as with A. viteae, only by the post-L3 stages, although again the mRNA for ES62 could be detected in the earlier stages. Overall our results suggest that production of ES62 is not species specific, that it is indeed stage specific, and that this may be due to post-transcriptional control of expression.

Protective immunity induced by irradiated third-stage larvae of the filaria Acanthocheilonema viteae is directed against challenge third-stage larvae before molting.

Jirds (Meriones unguiculatus) were vaccinated with irradiated L3 third-stage larvae (L3) of Acanthocheilonema viteae, and the time required for killing of the challenge L3 was determined. The number of parasites recovered from vaccinated jirds was reduced to about 10% of the control values on the second day after challenge infection and later on. Histological studies revealed an eosinophil-rich infiltrate containing macrophages, neutrophils, and mast cells in the vicinity of the L3 on day 2 after challenge and destruction of the worms by day 4 after challenge. Ultrastructural studies confirmed these data and showed that eosinophils, macrophages, and mast cells were close to the L3 on day 2 after challenge. Flattening of the eosinophils onto the surface of the worms, degranulation of electron-dense material, and rupture of the L3 surface was observed on day 4 after challenge, followed by invasion of the inner of the worms by phagocytic cells. These data show that immune attack against the challenge L3 in vaccinated jirds is initiated between the first and the second day after challenge and that killing occurs around the fourth day after challenge, before the worms undergo their first molt.

Mechanisms underlying the transfer of phosphorylcholine to filarial nematode glycoproteins--a possible role for choline kinase.

Phosphorylcholine (PC) is a common constituent of proteins secreted by filarial nematodes. As this substance has been shown to interfere with immune responses, we are interested in designing strategies for blocking its attachment. Towards this end, we are investigating the mechanism of incorporation of PC into filarial molecules and in the present manuscript we describe experiments relating to elucidating the source of PC for attachment. Synthesis of phosphatidylcholine in eukaryotic organisms can occur by a mechanism involving the transfer of PC from CDP-choline to diacylglycerol (the Kennedy pathway). By (i) measuring transfer of radio-isotope labelled PC from CDP-choline to parasite molecules and (ii) employing inhibitors of CDP-choline synthesis, we have investigated whether CDP-choline can act as a source of PC for transfer to ES-62, a major secreted glycoprotein of the rodent filarial nematode Acanthocheilonema viteae. Although we can find no evidence of this, we show that attachment of PC is blocked by hemicholinium-3, an inhibitor of choline kinase, the first enzyme in the Kennedy pathway. Thus, at least the first step in this pathway--phosphorylation of choline, would appear to be necessary for attachment of PC to ES-62.

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.

PCR-based amplification of total cDNA with high fidelity and high yield from minute amounts of parasite RNA.

cDNA, synthesised from total RNA from Acanthocheilonema viteae, was amplified by PCR with a primer derived from the spliced leader 1 sequence of nematodes and oligo-dT. Due to the great number of side products observed in the reaction, a biotinylated oligo-dT primer was used for cDNA-synthesis and the first cycles of PCR. After binding of the PCR products to streptavidin/paramagnetic particles, the (+)strands of the cDNAs were recovered and reamplified. Analysis of the PCR products obtained revealed the presence of full-length cDNAs of at least 1.7 kbp in size in amplified total cDNA from microfilariae, postinfective L3, and adult worms. The total cDNA, from only 20 ex vivo recovered postinfective L3, was efficiently amplified.

Characterization of heterotrimeric G-proteins in adult Acanthocheilonema viteae.

Heterotrimeric G-proteins have been found in eukaryotic cells, from yeast to humans, but have received little attention, to date, with respect to parasitic organisms. We now present the first report of the characterization of heterotrimeric G-proteins expressed in a filarial nematode, Acanthocheilonema viteae. Using a combination of (i) affinity labelling with [alpha-32P]GTP; (ii) ADP-ribosylation with cholera toxin and pertussis toxin; (iii) Western blotting with a panel of anti-G-protein antibodies; and (iv) reverse transcriptase-PCR with degenerate G-protein oligonucleotide primers followed by hybridization analysis using oligonucleotides specific for individual G-protein subunits, we demonstrate that adult A. viteae expresses homologues of the beta 1- and/or beta 2-like subunits and alpha-subunits of the Gs, G1, Gq and G12 subfamilies found in mammals. The role which these G-proteins may play in the biology of the organism is discussed.

Chitinase genes expressed by infective larvae of the filarial nematodes, Acanthocheilonema viteae and Onchocerca volvulus.

State-specific products of 220 and 75 kDa were identified by metabolic labelling of infective larvae of the filarial nematode Acanthocheilonema viteae in ticks. Synthesis was temperature sensitive, occurring at 27 degrees C but not at 37 degrees C. These products were secreted 3-6 days after leaving the vector during post-infective development, but subsequent expression was not detected. The smaller protein with a pI of 6.2, was purified by two-dimensional electrophoresis and the N-terminal amino acid sequence was derived. This provisionally identified the protein as a chitinase, which was confirmed biochemically by glycol-chitin substrate gel electrophoresis. The polymerase chain reaction was used to amplify a product from a cDNA library of A. viteae infective larvae. The nucleotide sequence codes for a putative signal peptide of 20 amino acids and a mature protein of 504 residues (Mr 56 kDa), exhibiting 69% identity (81% similarity allowing for conservative substitutions) with the MF1 chitinase described from microfilariae of Brugia malayi. N-linked glycosylation may account for some, or all, of the discrepancy in Mr between the predicted polypeptide and the native parasite product (75 kDa). Primers based on the A. viteae sequence were used to amplify a related sequence from a cDNA library of Onchocerca volvulus infective larvae. The O. volvulus cDNA codes for a 20-amino acid signal peptide followed by 477 residues with an Mr of 54 kDa, and shares 67% identity with the A. viteae chitinase (80% similarity allowing for conservative substitutions) and 69% identity with the B. malayi MF1 molecule. It is proposed that chitinases expressed by infective stages of these filarial nematodes may play a role in ecdysis during post-infective development.

Acanthocheilonema viteae: rational design of the life cycle to increase production of parasite material using less experimental animals.

The maintenance of the life cycle of Acanthocheilonema viteae is described with the aim to increase the production of parasite material using less experimental animals. The filaria was maintained in jirds (Meriones unguiculatus) and in soft ticks (Ornithodoros moubata). The optimal infection dosis for jirds was 80 infective larvae (L3). The mean worm number in groups of animals varied between 18 and 30 adult worms. A stable microfilaremia developed and only few animals developed pathological alterations as a consequence of the infection. A simple membrane feeding apparatus allowed mass feeding of ticks. Infection of ticks with microfilariae (mf) using this technique resulted in a mean no. of 594 +/- 527.2 L3/tick. L3 and mf were cryopreserved in liquid N2 with a simple technique. The described maintenance of the life cycle reduced the amount of required experimental animals to 30-40% of the originally needed numbers.

Stage-specific development of a filarial nematode (Dipetalonema dracunculoides) in vector ticks.

This paper reports the development of the canid filarial worm, Dipetalonema dracunculoides, in the brown dog tick, Rhipicephalus sanguineus by determining whether development is similar within larval, nymphal and adult stages of infected ticks. This study demonstrates that only infected nymphal ticks can support the complete development of the filarial worm. Infected larval ticks are not suitable intermediate hosts, nor are infected adults. Development depends on some stage-specific property of the vector, and the maturation to the infective stage is stimulated during the course of the nymphal-adult moult.

Protective immunity linked with a distinct developmental stage of a filarial parasite.

Repeated low dose infections of the jird Meriones unguiculatus, with the filarial parasite Acanthocheilonema viteae cause a substantial reduction of the total worm burden, suggesting a parasite-driven immune mechanism that controls super-infections. Quantitative recovery of parasites from tissues of triple infected jirds reveals that the larvae derived from a subsequent challenge infection are inactivated or severely impaired several days after transmission, precisely during their molt from the L3 to the L4 stage. Moreover, only larvae undergoing the molt from L3 to L4 stages are capable of stimulating an immune response directed against the challenge infection, indicating that protective Ag are produced during the molting period. Consistent with this, inactivated L3 or live L4 do not produce the same effect. In contrast to susceptible animals, immune jirds elicit high serum antibody titers against molting Ag. Indirect fluorescence antibody-binding tests with sera from protected jirds reveal specific labeling of the surface of molting L3 and not other larval stages, implying a stage-specific elimination process. The identification of molting L3 as a natural target for host immune mechanisms, emphasizes the central importance of this larval stage for future efforts aimed toward the development of a filarial vaccine.

Experimental transmission of Dipetalonema dracunculoides (Cobbold 1870) by Rhipicephalus sanguineus (Latreille 1806).

A dog naturally infected with Dipetalonema dracunculoides and having a microfilaremia of 6050 microfilariae per mm3 of blood was used as source of infection. Experimentally cultivated nymphs of Rhipicephalus sanguineus were fed on the donor dog. Once engorged, ninety-three nymphs were removed and kept at 30 degrees C and RH 90% until they moulted to the adult stage. To study the development of microfilariae in the vector, ten ticks were dissected at Day 37 post-infection. Adult infected ticks were fed on two uninfected dogs. The observed pre-patent periods were 69 and 76 days. Trans-stadial transmission of Dipetalonema dracunculoides by Rhipicephalus sanguineus was demonstrated.

Changes in the surface composition after transmission of Acanthocheilonema viteae third stage larvae into the jird.

This study describes the dynamics and the biochemical nature of changes in the surface of the filarial nematode Acanthocheilonema viteae after its transmission into the vertebrate host. Vector-derived third-stage larvae (mL3) were inoculated into naive Meriones unguiculatus and recovered from the tissues at different times post-infection until their moult to fourth-stage larvae (L4). Surface-specific labelling with fluoresceinated lectins revealed that the larvae are covered by a carbohydrate envelope. Although the mL3 envelope was strongly reduced one day after transmission, new surface carbohydrates appeared until the onset of moulting, some of which could also be identified on the surface of L4. In general, surface carbohydrates were partially shed by moving larvae, suggesting a loose association of these components in the epicuticle. The fate of cuticular lipids and proteins of L3 and L4 was monitored by external 125I-labelling and differential extraction of the components. Thin-layer chromatography of surface-labelled lipids revealed only minor changes 1 day after parasite transmission. Afterwards the number of lipids accessible to label decreased further until moulting was complete. Two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis of surface-labelled proteins showed a consistent surface exposure of mL3 specific proteins until 1 day post-infection. Thereafter, the composition of surface-labelled proteins changed rapidly, resembling that of the L4 as early as several days before moulting. During this period individual differences in the composition of surface proteins were evident.

Dose-dependent recovery of adult Acanthocheilonema viteae (Nematoda: Filarioidea) after single and trickle inoculations in jirds.

Increasing single doses of 5, 15, 30, 60 and 90 third-stage Acanthocheilonema viteae larvae per animal were inoculated into jirds. The adult worm load increased steadily, whereas the recovery rate decreased significantly, i.e. the correlation between dose and recovery was negative (rs, -0.90; n = 5; alpha, 0.05). The same inoculation doses were given as trickle inoculations of 5 L3 each (3 x 5, 6 x 5, 12 x 5, 18 x 5) at intervals of 2-6 days throughout the prepatency period. Irrespective of the number of repeated inoculations, a rather constant but low load of 7-10 worms/animal was reached. The recovery rate decreased drastically (rs, -1.0; n = 5). When trickle inoculations were carried out in animals exhibiting patient infections, the superinoculated larvae seemed to be destroyed almost completely; thus, a parasite-host equilibrium was guaranteed in all cases. The immunological background is discussed.

Recovery, distribution, and development of Acanthocheilonema viteae third- and early fourth-stage larvae in adult jirds.

Living third- and fourth-stage larvae (L3 and L4) of Acanthocheilonema viteae were recovered quantitatively from adult Meriones unguiculatus within the first 10 days after subcutaneous inoculation of 60 arthropod-derived larvae (mL3). The average recovery of the inoculated larvae was about one third (28.5%), and the majority (87.7%) were found in muscular tissues. Seventy-two hours after inoculation, larvae could be isolated from all body locations, although the majority still was found near the site of inoculation. Morphological and biometrical data indicated that, at least until molting, the development of the larval population was not synchronous, with molting occurring over a period of 48 hr on days 7 and 8 postinoculation. The stomatal rings of postinvasive L3's and L4's were distinguishable structurally and could be used as stage-specific determinants. Immediately after infection, L3's showed a linear growth in diameter; rapid longitudinal growth started after the molt, leading to a doubling in the length of L4's within 4 days. The time course of shedding was reconstructed in detail using isolated L3/L4 intermediates.

Apodemus sylvaticus, a new host for Acanthocheilonema viteae (Nematoda: filarioidea).

Susceptibility of Apodemus sylvaticus and A. agrarius to infection with Acanthocheilonema viteae was compared with that of hamsters and jirds. Microfilaremia in A. sylvaticus was first noted on day 52 post-infection (p.i.) and lasted during the course of the study (up to day 150 p.i.). Maximum microfilaremic levels (female worm basis) of A. sylvaticus [mean +/- S.D. (n) = 690 +/- 1288(6)] were considerably higher than those of hamsters [16 +/- 18(6)] and jirds [51 +/- 25(5)]. Adult worm recovery in A. sylvaticus ranged from 2 to 40% of the number of infective larvae inoculated. Worm development in A. sylvaticus resembled that in hamsters and jirds. In contrast, microfilaremia was not detected in, nor adult worms recovered from A. agrarius throughout the study.

Association between circulating antigen and parasite load in a model filarial system, Acanthocheilonema viteae in jirds.

Jirds (Meriones libycus) were infected with various numbers of Acanthocheilonema viteae L3 stage parasites. During the course of the ensuing 16 weeks, blood samples were collected at 2 weekly intervals and the amount of the major parasite excretory-secretory product (E-S 62) and antibodies directed against it measured. After 16 weeks, animals were sacrificed and the size of the mature worm burden established. In spite of interaction between E-S 62 and host antibody, a statistically significant relationship was found to exist between the amount of E-S 62 present in the bloodstream and the size of the parasite load. It is suggested that the detectable antigen level is more influenced by the size of the worm burden than the presence of antibody and that antibody is only likely to affect adversely antigen measurement in situations where the amount released is relatively low. Examples of this are early in infection and in low-level infections. These ideas are discussed in relation to the development and assessment of serological assays which attempt to predict parasite burden in human filarial infections.