Biochemical analyses of secretory and excretory products of adult Dipetalonema viteae in culture.

Radioisotopically labeled glucose and pyruvate were employed to elucidate biochemical mechanisms utilized by the filariid Dipetalonema viteae during cultivation. Adults isolated from amicrofilaremic hamsters were incubated at 37 C in a mixture of NCTC135:IMDM (NI), with either D-[14C-(U)]glucose or [1-14C]pyruvate, under a gas phase of 5% CO2/N2 for 3 days. Labeled organic acids were separated and quantified by ion exchange chromatography. High performance liquid chromatography (HPLC) was used for separation and quantification of the 23 free amino acids in the NI medium. Ion exchange chromatography revealed that lactate was the major glycolytic end product, accounting for 90-97% of the original carbon utilized. Small amounts of radioactivity were recovered in succinate and variably in acetate fractions. HPLC analysis demonstrated that some amino acids increased, some decreased, and some remained at the initial concentration. Alanine exhibited the greatest change, consistently increasing from 2 to 4 times the original concentration. Analyses of purified amino acid peaks revealed radioactivity only in the alanine peak, accounting for 2-4% of the original carbon utilized.

Origin, kinetics of circulation and fate in vivo of the major excretory-secretory product of Acanthocheilonema viteae.

The excretions-secretions (E-S) of Acanthocheilonema viteae consist mainly of one product, molecular weight 62kDa. This molecule is synthesized during the vertebrate phase of the parasite life-cycle and is first detectable in the E-S of L4 parasites. It is cross-reactive with E-S of human filarial parasites as a consequence of possessing a phosphorylcholine (PC) moiety. The 62 kDa molecule has been employed as a model for the study of the origin and fate of filarial E-S. Immunohistological analysis has shown the molecule to be located predominantly in the parasite gut. Transplantation of adult female [35S] methionine pulsed worms into uninfected jirds resulted in the radio-labelled secreted 62 kDa antigen being detected in the bloodstream within 4 h by SDS-PAGE/immunoprecipitation analysis. The systemic half-life of the molecule as estimated by clearance of injected, purified 125I-labelled material was measured in naive and infected jird hosts. It was reduced from 2-7 h in naive animals to less than 30 min in 4-10 week infected rodents, a finding which correlated with clearance of antigen by antibody in the infected group. In animals infected for longer time periods the serum half-life returned to the values observed in naive jirds. The idea that this change in half-life may reflect differences in the nature of 62 kDa antigen containing circulating immune complexes as infection progresses is discussed. The 125I-labelled antigen is predominantly removed from the circulation via the liver and ultimately excreted in the urine in a non-antigenic form. This work provides the first description of the origin, kinetics of circulation and fate of a defined filarial E-S product and may aid in determining the function and assessing the diagnostic utility of PC-bearing E-S components.

Cytochemical demonstration of lectin binding-sites in the cuticle and tissues of Acanthocheilonema viteae (Filarioidea).

The lectin-gold technique was used for the ultrastructural localization of lectin binding sites on thin sections of Lowicryl K4M embedded adult females, infective larvae and SDS-2-mercaptoethanol-insoluble cuticle components of Acanthocheilonema (Dipetalonema) viteae. Helix pomatia lectin (HPL) coupled to 14 nm gold particles, was used for the demonstration of N-acetyl-D-galactosamine-containing glycoconjugates. Triticum vulgaris (wheat germ) agglutinin (WGA) coupled to 10 nm gold particles after cross-linking to BSA or ovomucoid-gold after application of unlabeled WGA, demonstrated WGA binding sites (N-acetyl-D-glucosamine). With both lectins no surface labelling of the cuticle was observed, but subcuticular layers reacted positively. HPL-gold was bound to cuticular fibers, the matrix and to the electron dense layer within the cortical zone of the cuticle of female worms. WGA-gold complexes were bound mainly to the cuticle matrix and somatic tissues. The results support the hypothesis that tissue-dwelling parasitic nematodes have reduced their surface carbohydrates perhaps as a consequence of their parasitic life.

Distribution of iodinated proteins in Dipetalonema viteae after surface labelling.

Adult females of the filarial parasite Dipetalonema viteae were radiolabelled using chloroglycoluril and different concentrations of iodine with and without carrier iodide. A detailed quantitative analysis of the distribution of the labelled proteins were carried out using sodium dodecylsulfate and beta-mercaptoethanol to isolate the cuticle after different iodination periods. The highest specific activity was found in the pellet, which comprised the cuticular cortical zone with the highly insoluble epicuticle. However, 50% of the radiolabelled proteins were recovered in the extracts, which contained solubilized material from the somatic compartments and the basal and median zones of the cuticle. The data indicate that the isolation of surface-iodinated antigens of filariae is hampered by the presence of a detergent-insoluble epicuticle. Radiolabelled antigens solubilized by detergents are either proteins from internal somatic or cuticular regions or proteins adsorbed onto the epicuticle.

Putative neurotransmitters in three experimental filariasis models.

Some putative neurotransmitters in three experimental filariasis models were investigated by a new relevant chromatographic method, sensitive and specific. No catecholaminergic compounds have been detected, but serotonin was found in Dipetalonema vitae. However, further investigations revealed very high levels of gamma amino butyric acid (GABA) in the macro-filariae. These data allow us to foresee new fields in filariasis therapeutics.

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.

Fractionation of a soluble somatic extract and solubilized cuticular extracts of Dipetalonema viteae adult worms.

A soluble somatic preparation (SSP) of adult Dipetalonema viteae was prepared. Aliquots of the aqueous insoluble debris (cuticles and membranes) left after the extraction of SSP were solubilized separately with Triton X-100 and lithium diiodosalicylate to yield a Triton solubilized preparation (TSP) and lithium diiodosalicylate solubilized preparation (LSP), respectively. SSP was sequentially chromatographed on a series of Sephadex columns, G50; then fraction 1 from G50 on G100 and finally fraction 1 from G100 on G200 yielding 5, 4, and 5 fractions on G50, G100, and G200, respectively. TSP and LSP were each sequentially chromatographed on Sephadex G50 and G200 to yield 5 and 4 fractions for TSP and 6 and 3 fractions for LSP, respectively. Each Sephadex fraction from each separation was further resolved by polyacrylamide gel electrophoresis (PAGE). Immunodiffusion studies with a rabbit antiserum to SSP gave 4 precipitin bands with SSP, 1 with TSP, and none with LSP. The single precipitin arc produced with TSP showed partial identity with an SSP arc. Disc immunoelectrophoresis allowed the identification of the proteins separated by PAGE, which precipitated with the rabbit anti-SSP serum.

Infections with Dipetalonema perstans and Mansonella ozzardi in the Aboriginal Indians of Guyana

Blood surveys conducted among the aboriginal Indians (Amerinds) in the interior of Guyana revealed that Dipetalonema perstans or Mansonella ozzardi, or both, occurred in approximately 12 percent of the persons examined, D. perstans being more common and widely distributed. It was found in Amerinds in all of the districts surveyed and in persons of other than aboriginal stock living in the interior of the country. M. ozzardi, in contrast to D. perstans, had a low prevalence and a very limited geographic distribution. It was found among Amerinds in the Pakaraima Mountains of the Mazaruni-Potaro and Rupununi Districts and in the North West District, but was absent from many areas in which it had been found at the turn of the century. M. ozzardi occurred more frequently mixed with D. perstans than as a pure infection. The geographic distribution of each species in the various administrative districts of the country is presented. (AU)