The sheaths of Brugia microfilariae: isolation and composition.

Burgia malayi and B. pahangi microfilariae were isolated from the blood of infected Mastomys natalensis, and were exsheathed by freezing, thawing and agitation. Pure sheaths were obtained by a filtration procedure. The sheaths were found to contain about 95 mol% of amino acids, with proline, glutamic acid/glutamine, alanine, cysteine/cystine and glycine being the major components, and 5 mol% of carbohydrates, notably (N-acetyl)galactosamine, but no (N-acetyl)glucosamine.

[Advances in filariasis control and research in China during 1986-1990. National Technical Steering Group for Filariasis Control and Research, MOPH].

Based upon the statistical data during 1986-1990 from provincial, regional or municipal institutions involved in the control and research of filariasis, important achievement in filariasis control has been achieved since 1986. During the 5 years, 22,350,772 person were blood-examined, among them, 268,034 were found to be microfilaria positive; a total of 78,550,945 man/times of treatment with DEC were given (including administration of DEC-medicated salt). Till the end of 1990, filariasis has been basically eliminated in 823 counties/cities out of the 864 endemic counties/cities, and has been basically eliminated in 12 endemic provinces, one autonomous region and one municipality. Thus far, filariasis is still prevalent in 41 counties/cities of Anhui province. Parasitological, serological and entomological monitoring on filariasis is being executed according to the "Technical scheme for filariasis surveillance in areas where the disease has been basically eliminated" formulated by the Ministry of Public Health. The preliminary results of longitudinal surveillance indicated that the decline of microfilaremia rate and the absence of resurgence coincided with the decrease in antibody titre in the population by IFAT. The progress of research work dealing with the transmission potential in areas where filariasis had been under control, the methods of immunodiagnosis and the treatment of patients with clinical signs was also reviewed.

Ecdysteroid-like compounds in the serum and urine of African patients infected with Loa loa and Mansonella perstans microfilariae.

Ecdysteroids are compounds related to 20-hydroxyecdysone, the insect moulting hormone. Surprisingly, they have been found in serum and urine of patients infected with helminths. In these cases, the substances are assumed to be produced by the parasites and, therefore, might be used as a marker of parasitic infection. Thus, we need to know exactly which species, at which developmental stage, can release ecdysteroids in such large quantities that they could be detected in the biological fluids of the host. Large-scale investigations must, accordingly, be devoted to the major species of helminths. In the present study, we examined 100 African patients with Loa loa and/or Mansonella perstans microfilaraemia. About 70 of them had high levels of ecdysteroid-like materials in serum or urine. In contrast, uninfected patients and European controls had much lower concentrations. However, the ecdysteroid titres did not reflect the concentration of microfilariae actually present in the blood, and some heavily infected patients were even negative. Nevertheless, the most important point was that high ecdysteroid levels in man were always associated with a pathological condition. The precise significance of the phenomenon should be determined.

Localization and immunogenicity of tubulin in the filarial nematodes Brugia malayi and B. pahangi.

Tubulin was identified in the filarial nematodes Brugia malayi and B. pahangi by several approaches. Initially, a monoclonal antibody (6D8) was selected for its unusual binding to B. malayi microfilariae in indirect immunofluorescence assays: 6D8 showed granular, heterogeneously dispersed fluorescence on fixed parasites but did not bind to unfixed microfilariae. The microfilarial sheath did not bind 6D8, although it did bind fluoresceinated wheatgerm agglutinin. By Western blotting against microfilarial sonicate, 6D8 reacted with a 50,000-55,000 mol. wt protein, and also bound to purified chicken brain beta-tubulin. Additionally, this monoclonal antibody reacted with a recombinant fusion protein expressed by a clone (Bpa-7) originally isolated from an adult B. pahangi cDNA expression library by its reaction with chronic human filariasis serum. This clone encodes a small 40 amino acid C-terminal segment corresponding to residues 409-449 of beta-tubulin, and shows complete amino acid sequence homology with vertebrate beta-tubulin from 409 to 430 but 55% divergence (six amino acid substitutions, four insertions and one deletion) from human and chicken beta-tubulin over positions 431-449 at the C terminus. Antibody to both parasite and vertebrate (chicken) tubulin was found in filarial infection sera, with higher levels of autoreactive antibody apparent in amicrofilaraemic individuals. Immunogold electron microscopy was then used to localize beta-tubulin in B. malayi microfilariae and adult worms. Tubulin was shown not to be exposed on the microfilarial sheath or in the cuticle of either stage, but was found to be abundant in the somatic tissues. In microfilariae, 6D8 bound myofibril structures under the hypodermal layer, and also bound within cell nuclei. In the adult stage, tubulin was associated with muscle blocks, as well as the intestinal brush border and the embryonic uterine microfilariae.

Comparative analysis of surface components of adult, micro-filariae and infective larvae of Brugia malayi.

A comparative analysis of surface proteins of adult, microfilariae and infective larvae of Brugia malayi, the human filarial parasite, has been carried out using IODOGEN (1,3,4,6-tetrachloro-3,alpha 6 alpha-diphenyl-glycoluril) and lactoperoxidase methods. SDS-polyacrylamide gel electrophoretic and autoradiographic analyses revealed the presence of 9 proteins (15-200 kDa) in adults, while microfilariae and infective larvae showed 8 and 6 proteins (15-120 kDa), respectively. The pattern of proteins radiolabelled by IODOGEN method was very similar to that of proteins labelled by the lactoperoxidase method. Since these proteins are released by the protease treatment of whole parasites, they are likely to be present on the surface of the parasite.

Litomosoides carinii: characterization of surface carbohydrates of microfilariae and infective larvae.

The carbohydrate moieties of microfilariae (Mf) and infective larvae (L3) have been investigated by lectin-binding technique. Mf derived from three sources, namely, uteri (in utero), released in vitro from adults and from blood of rodents infected with Litomosoides carinii were examined by using fluoresceinated lectins. Wheat germ agglutinin (WGA) bound to these Mf and the binding was inhibited by N-acetyl glucosamine. In addition to WGA, Concanavalin A (Con A) and lentil lectin (LCH) bound to in vitro-released and in utero-derived Mf showing the presence of mannose moieties on their surface. In utero-derived Mf also showed binding with the agglutinins of Limulus polyphemus (LPA), peanut (PNA), Ricinus communis (RCA), Helix pomatia (HPA), Soyabean (SBA) and Dolichos biflorus (DBA) but not to that of Ulex europaeus (UEA) indicating the presence of additional carbohydrate molecules like sialic acid, galactose and N-acetyl galactosamine on their sheath. None of the lectins bound to the cuticle of exsheathed Mf. Treatment of blood-derived and in vitro-released Mf with certain proteases exposed additional binding sites for SBA, HPA, Con A and LCH. In case of L3, only PNA bound to the larvae isolated from infective mites Bdellonyssus bacoti, and the binding was inhibited by D-galactose. No such binding of the lectins was seen to the larvae that migrated to the pleural cavity of jirds indicating that there is considerable change on the parasite surface during their migration in the vertebrate host. Sheathed Mf and mite-derived L3 when incubated with immune rat sera, bind Con A and LCH lectins possibly due to the mannose components of the specific immunoglobulins that coat onto the Mf and L3.

Lectin binding studies on adult filariae, intrauterine developing stages and microfilariae of Brugia malayi and Litomosoides carinii.

Sections of macrofilariae of Brugia malayi and Litomosoides carinii revealed binding of the gold-labelled lectins WGA, DBA and PNA. Specificity of binding was controlled by competitive inhibition with the respective sugars. N-acetyl-glucosamine, N-acetylgalactosamine and galactose residues seem to be present in the respective tissues. The lectins were bound preferentially to parts of the reproductive organs and to the fluid contents of their lumina. The results of the chitosan test and binding experiments with WGA-gold conjugate suggest the presence of chitin in the sheath of oocytes or zygotes. Binding of WGA could not be inhibited with 0.5 M N-acetylglucosamine, but only with 10 mM triacetyl chitotriose. In older stages, binding of WGA to the sheath could be inhibited by 0.5 M N-acetylglucosamine. In mature microfilariae, the outer surface of the sheath did not show affinity for WGA, but small amounts were bound to the inner surface. Therefore, the sheath of later developmental stages and microfilariae does not contain chitin but only N-acetylglucosamine residues. The degradation of the chitin content might enable the elongation and flexibility of the sheath of microfilariae.

Calcification in adult Onchocerca volvulus.

Calcified worm fragments of adult Onchocerca volvulus from patients in West Africa were collected for a chemical analysis. The material contained predominantly calcium carbonate. Some results of investigations on the occurrence of calcified worms in untreated populations are reported on. Relations between the numbers of old parasites and those of calcified worms are discussed. The significance of possible resorption of calcified worm fragments for the interpretation of the effects of drug trials or other control measures are mentioned.

Biochemical surface components of Brugia pahangi microfilariae.

The sheath and cuticle of microfilariae of Brugia pahangi were examined by electron microscopy and the presence of various proteins, carbohydrate and enzymes sought. The epicuticle of microfilariae consists of a pentalaminate structure (24.0 +/- 1.4 nm), a cortex (13.7 +/- 3.6 nm) and a basal zone (27.8 +/- 4.8 nm) which is often banded in appearance. The pentalaminate layers are not continuous at the base of the interannular grooves. The sheath and the epicuticle of B. pahangi stained positively with concanavalin A and saccharated iron oxide. The sheath of approximately 50% of microfilariae showed activity for acid phosphatase, 5' nucleotidase and peroxidase, but not for ATPases, alkaline phosphatase or esterase. No enzymes were detected in the epicuticle although the cortex and basal layers of the cuticle did show enzymic activity. Structures beneath the cuticle in the main body of the worms contained considerable enzymic activity. Microfilariae directly isolated from the blood of infected cats were found by immunochemical means to carry serum proteins on their sheaths but not on their cuticles. These studies extend the definition of the outer structures of microfilariae and confirm that they significantly differ in morphology and enzyme content from typical mammalian cell membranes.

Brugia malayi: stage-specific expression of carbohydrates containing N-acetyl-D-glucosamine on the sheathed surfaces of microfilariae.

Microfilariae, infective larvae, and adult worms of Brugia malayi were incubated with a panel of seven lectins in order to study the expression of surface carbohydrates. Infective larvae and adult worms did not bind any of the lectins utilized. Microfilariae, on the other hand, bound wheat germ agglutinin. The binding of this lectin was saturable and specific, and attributed to the presence of N-acetyl-D-glucosamine. In addition, microfilariae derived in vitro bound concanavalin A, indicating the presence of glucose and/or mannose on this stage of the parasite. The fact that similar concanavalin A binding was not seen on microfilariae recovered directly from the infected host implies that there is masking or loss of parasite surface antigens as microfilariae mature in vivo.

Comparison of activities of mononuclear phagocyte system between the cotton rat, Sigmodon hispidus, and the jird, Meriones unguiculatus, in relation to the treatment of filariasis.

The cotton rat and jird, matched according to age and the period after infection with Litomosoides carinii, were administered intraperitoneally with diethylcarbamazine (DEC) at a single dose of 100 mg/kg. Microfilaria counts were conducted 0.5, 1, and 6 hrs and 1, 4, and 7 days after treatment with DEC. The average of percent microfilaria density was significantly higher in the jird than in the cotton rat at any given time except at 6 hrs, when a slight increase was observed in the cotton rat. To clarify the cause of different microfilaricidal activities of DEC between the cotton rat and jird, function of the mononuclear phagocyte system (MPS) was compared in the two animals by observing clearance of the carbon particles introduced into blood vessels. The amount of remaining carbon in the vessels was always lower in the cotton rat than in the jird from 30 min. on after carbon injection. The clearance index K, which is thought to reflect the phagocytic capability of MPS, was higher in the cotton rat than in the jird. Microscopic examination also showed differences in the amount of carbon phagocytosed by the Kupffer cells of the liver in the two animals. The results indicated that the difference in microfilaricidal effect of DEC may be due to different phagocytic activity of MPS.

Characterization of the exposed carbohydrates on the sheath surface of in vitro-derived Brugia pahangi microfilariae by analysis of lectin binding.

Adult female Brugia pahangi were maintained metabolically active in vitro for up to 35 days in Click's medium supplemented with 10% horse serum. For the first 14 to 18 days microfilariae were released into culture. Although these in vitro-derived microfilariae were morphologically identical to in vivo-derived microfilariae, they could be differentiated by their characteristic of binding to a panel of fluorescein-conjugated lectins. The results suggest that maturation and release of microfilariae are correlated with glycosidic alterations on the sheath surface.

Ocular and systemic complications of diethylcarbamazine therapy for onchocerciasis: association with circulating immune complexes.

Diethylcarbamazine (DEC) therapy for Onchocerca volvulus infection results in frequent ocular and systemic complications, but the pathogenesis of these complications is unclear. Twenty men with O. volvulus infection were treated over a period of six months with DEC given daily for one week and weekly thereafter. Major systemic and ocular complications included proteinuria, severe pruritus, visual field constriction, optic nerve pallor, chorioretinitis, anterior uveitis, and punctate keratitis. Levels of circulating immune complexes (CICs) were increased (greater than 11% [125I]C1q binding) in 14 of the 20 men prior to treatment. Persons with pretreatment C1q binding activity of greater than 30% were at increased risk to develop constriction of visual fields (P less than 0.05) and proteinuria (P less than 0.015). Linear regression analysis revealed a striking correlation between pretreatment levels of CICs and the total number of both systemic and ocular complications (P less than 0.001) and ocular complications alone (P less than 0.005). These results suggest that CICs may be important in the pathogenesis of the delayed systemic and ocular complications following DEC therapy for O. volvulus infection.

Protein composition of various developmental stages of dipetalonema viteae (Filarioidea).

The protein composition of various developmental stages of Dipetalonema viteae was analysed on polyacrylamide slab gels in the presence of sodium-dodecylsulphate. When the total proteins of adult male and female parasites, microfilariae, eggs, and third-stage larvae were compared, apparent qualitative similarities between mature and immature filariae were observed. However, several stage specific components were also identified.

Identification of radioiodinated cuticular proteins and antigens of Onchocerca gibsoni microfilariae.

The filarial parasite of cattle, Onchocerca gibsoni, has been used to establish procedures of antigen identification with a view of applying these techniques to studies on human filarial parasites. Emphasis has been placed on methods suitable for use with small numbers of parasites. Microfilariae (mf) of O. gibsoni were extracted from nodular worms, purified and 125I-labeled using IODO-GEN in solid-phase. Radioactivity was shown to be confined to the cuticle of sectioned mf using the technique of electronmicroscope autoradiography. Radiolabeled mf were analysed by two-dimensional gel electrophoresis. Autoradiographs of 125I-labeled proteins of O. gibsoni mf were relatively complex, there being at least 32 proteins ranging in molecular weights from 20,000 to 120,000 and displaying considerable charge heterogeneity. Evidence was obtained that at least the major serum proteins of the host, albumin or immunoglobulin, were not absorbed on the surface of these uterine mf and detectable in the labeled surface protein patterns. Sera from infected cattle immunoprecipitated 5 labeled proteins from a Triton X-100 extract of 125I-labeled mf. Sera from either of two calves which had been given multiple injections of mf subcutaneously, and which had no detectable skin mf, recognised 6 additional proteins in this extract as well as 3 of the proteins recognised by sera from infected cattle.

Biochemical composition of microfiliarial and adult stages of Setaria cervi.

The microfilariae and adults of Setaria cervi, a filarial parasite of Indian water-buffalo (Bubalus bubalis Linn.) and of other ruminants were analysed for various biochemical constituents. Both stages of the parasite were rich in protein, carbohydrate, and lipids while nucleic acids were detectable in only small amounts. Microfilariae and adults contained 10 and 25% dry matter, 57.4 and 63.0% protein, 8.9 and 14.8% carbohydrate, and 12.8 and 9.2% lipid, respectively. Reducing sugars accounted for 52 and 67% respectively of the total carbohydrate in these two stages. Glycogen accounted for 95% of total carbohydrate in the cold PCA soluble fraction of adult worms while its concentration in microfilariae was very low. Glucose was the main constituent of reducing sugars while concentrations of fructose were very low. Microfilariae contained more lipids, nucleic acids, mucopolysaccharides, and total phosphorus than adults. The phospholipid percentage was higher in microfilariae than in adults. The amino acid pattern of both stages was very similar.

Preliminary studies on the histochemical differentiation of strains of Onchocerca volvulus microfilariae in Togo.

Skin snips were taken from 75 people living in four villages of northern Togo. The 7824 microfilariae that emerged were examined by staining for the presence of acid phosphatase. Four distinct patterns of enzyme staining were observed, and descriptions of the stained microfilariae are given. The study confirms the view that a number of biological strains or variants of Onchocerca volvulus coexist in West Africa, and suggestions are made for further research that could result in the practical application of these observations in onchocerciasis control programmes.