Inhibition of NADP-linked malic enzyme from Onchocerca volvulus and Dirofilaria immitis by suramin.

NADP-linked malic enzyme (malate dehydrogenase (oxaloacetate-decarboxylating) NADP+, EC 1.1.1.40) has been partially purified from adult Onchocerca volvulus and Dirofilaria immitis. Suramin was found to inhibit the activity of malic enzyme from both filarial worms. The inhibition constants for suramin were calculated to be 0.011 microM and 0.015 microM for the enzymes from O. volvulus and D. immitis, respectively. In the case of NADP-linked malic enzyme from Trypanosoma brucei and chicken liver the inhibition by suramin was less pronounced. The inhibition constants were found to be 0.8 microM and 2.5 microM for the protozoan and vertebrate enzymes, respectively. The type of inhibition was competitive with respect to malate. The Michaelis constants for malate and pyruvate were determined to be 0.9 and 4.5 mM for O. volvulus and 0.85 and 5.0 mM for D. immitis, respectively. The low Km values for malate compared to those for pyruvate and the about 15-fold greater turnover in the direction of decarboxylation compared to carboxylation indicated that malic enzyme from both filarial sources might be involved in an alternative pathway leading from phosphoenolpyruvate via oxaleacetate, malate and pyruvate to lactate. It is suggested, that the inhibition of malic enzyme activity from O. volvulus by suramin might interfere with the generation of NADPH for biosynthetic reactions.

Parasite-specific inhibition of 5'-nucleotidase from Onchocerca volvulus and Dirofilaria immitis by the amoscanate-derivative CGP 8065.

The presence of 5'-nucleotidase was demonstrated in Onchocerca volvulus and Dirofilaria immitis; the bulk of activity was found in the particulate fraction. The enzyme of filarial worms exhibited a broad pH-optimum between 6.4 and 8.0 and substrate specificity for nucleotides compared to glucose-6-phosphate and p-nitrophenyl phosphate. The apparent Km-values for AMP were found to be 0.15 mM and 0.22 mM for the enzyme from O. volvulus and D. immitis, respectively. The activity of 5'-nucleotidase from both filarial worms was effectively inhibited by the filaricidal compound CGP 8065, a dithiocarbamate-derivative of amoscanate, whereas the 5'-nucleotidase from rat liver was not affected. The parasite-specific inhibition by CGP 8065 was found to be reversible and to be competitive with respect to the substrate AMP. The inhibition constants were calculated to be 24 microM and 8 microM for the enzyme from O. volvulus and D. immitis, respectively.

Dolichol kinase in Ascaris suum and Onchocerca volvulus.

Dolichol kinase was demonstrated in the microsomal fraction of Ascaris suum and Onchocerca volvulus. The enzyme from nematodes exhibited specificity for CTP as phosphoryl donor and was found to be inhibited by the reaction product CDP. Enzyme activity was optimal at pH 7.4, in the temperature range between 30 degrees and 37 degrees C, and in the presence of 0.5% Triton X-100. In addition, the enzyme was found to depend on divalent cations for activity; Mg2+ being more effective than Mn2+ and Ca2+. The dolichol kinase from both nematodes was shown to be independent of Ca2+-calmodulin for activity. The apparent Km values for dolichol were determined to be 7.5 and 9.0 micrograms ml-1 for the enzyme from A. suum and O. volvulus, respectively. Those for CTP were estimated to be 0.85 and 0.75 mM, respectively.

Prevalence of Paragonimus uterobilateralis infection in children in a Liberian village.

Stool and sputum samples from 127 children in an endemic paragonimiasis area in Liberia, West Africa, were examined for the presence of lung fluke eggs. Samples from nine children (7%) were positive. The eggs were identified as those of Paragonimus uterobilateralis.

Onchocerca volvulus: enzyme polymorphism in relation to the differentiation of forest and savannah strains of this parasite.

Isozyme analysis was carried out on Onchocerca volvulus worms collected from Liberia, Ivory Coast, Burkina Faso and Sudan to see whether this technique could detect differences between forest and savannah populations of this parasite. A total of 243 forest and 189 savannah individual female worms were electrophoresed and stained for seven enzymes. Four showed some polymorphism, LDH, MDH, PGM and MPI and the other three, GAPDH, PEP and GPI were invariant. Statistical analysis of the results showed that the relative proportions of genotypes from within the different countries conformed to Hardy-Weinberg expectations. Pairwise comparisons of allele frequencies between countries showed that populations from Liberia and Ivory Coast had a very similar composition; there was some divergence between all the other pairs of populations and the genetic distance was calculated to summarize the degree of divergence. The number of loci examined was small and the genetic distances were within the range expected for separate geographical populations of the same species. The usefulness of this technique in worm identification is discussed.

Detection of serum antibodies and circulating antigens in a chimpanzee experimentally infected with Onchocerca volvulus.

The course of the humoral immune response was followed in a chimpanzee experimentally infected over 27 weeks with a total of 168 Onchocerca volvulus 3rd-stage larvae obtained from naturally infected wild-caught blackflies. Antibodies against an adult worm extract could be detected by ELISA from week 16 onwards (after the inoculation of 44 larvae). Peak antibody levels were observed between weeks 66 and 74 (about one year after the last larval injection). Thereafter, antibody levels markedly decreased but rose again after week 120. First microfilariae could be detected from week 124 onwards. Microfilarial counts remained low (not more than two microfilariae per skin snip) until the end of the observation period. High levels of IgM antibodies against adult O. volvulus antigens were detectable between weeks 26 and 80 by ELISA. Total IgE levels were found to be only marginally elevated during the course of the infection. Circulating parasite antigens were only detectable for a short time (weeks 34 to 44) of the prepatent period by immuno-radiometric assays (IRMAs) using monoclonal antibodies which were raised against O. gibsoni eggs. Competitive radio-immuno-assays detected host antibodies inhibiting binding of 125I-labelled monoclonal antibodies to parasite antigens from week 28 onwards. Host antibodies clearly interfere later in infection with the detection of circulating antigens.

Studies on nodules and adult Onchocerca volvulus during a nodulectomy trial in hyperendemic villages in Liberia and Upper Volta. I. Palpable and impalpable onchocercomata.

In hyperendemic onchocerciasis areas of West Africa nodulectomy trials were performed. 163 nodule carriers from four villages in the Liberian rain-forest and 52 nodule carriers from a savanna village in Upper Volta were operated on. During the surveys in the villages in Liberia and Upper Volta, an average of three and five nodules respectively per patient were palpated. Before each operation a further palpation was done and two more nodules per patient from both countries were found. The nodulectomies revealed 75% more nodules in the Liberian patients and 53% more in the patients from Upper Volta than were palpated immediately before. Half of these additional subcutaneous nodules extirpated in Liberians were superficially located, the other half deeper and impalpable through the skin. In the patients from Upper Volta two thirds of the additional nodules were seated in the depth of the subcutis. In both countries an average of three deep subcutaneous nodules per patient were found and this was equivalent to 20% of all extirpated onchocercomata. Most of these deep subcutaneous nodules were at those sites where most of the superficial nodules were located.

Long-term treatment of onchocerciasis patients from the Liberian rain-forest with low doses of diethylcarbamazine citrate after complete nodulectomy.

60 completely nodulectomized volunteers from the Liberian rain-forest were given an initial treatment with diethylcarbamazine citrate (DEC-C) for one week with gradually increasing doses up to a total of 1.1 g per patient. A long-term treatment followed with 50 mg DEC-C per week for 12 months. Immediately after the initial treatment the mean microfilarial density decreased to 4% to 6% of the pre-treatment level. After one year the microfilarial density had again increased to 12% to 29% of the pre-treatment level. Although the intake of the weekly 50 mg DEC-C was in no case regular, the microfilarial densities could be kept at a low level throughout the trial. However, even with the combined treatment--preceding nodulectomy and long-term trial with DEC-C--it was not possible to get the patients free from 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.

Effects of a single complete nodulectomy on nodule burden and microfilarial density two years later.

In hyperendemic onchocerciasis areas of the Liberian rain-forest it was possible to re-examine 210 microfilaria carriers two years after a first examination. At the first examination of these patients 143 had presented with palpable nodules, and 110 nodule carriers had been completely nodulectomized. Two-thirds of the operated patients had an average of one newly detected nodule two years later, and their microfilarial densities were slightly increased. In the remaining third of the operated patients no nodules could be palpated two years later, and the average microfilarial densities were slightly decreased. The 33 nodule carriers who had not undergone nodulectomy had, however, significantly higher numbers of nodules and microfilariae in the skin two years after the first examination. The possible benefit of a single, complete nodulectomy is discussed.

Phosphoenolpyruvate carboxykinase from Onchocerca volvulus and O. gibsoni.

Phosphoenolpyruvate carboxykinase, a rate-limiting enzyme at the branchpoint of phosphoenolpyruvate (PEP), was demonstrated in Onchocerca volvulus and O. gibsoni. The activity of PEP-carboxykinase from both filarial worms depends absolutely on the presence of divalent cations; in addition to Mg2+ the enzyme activity was strongly activated by Mn2+. The Michaelis constants for PEP, GDP and KHCO3 of the PEP-carboxykinase from O. volvulus were determined to be 0.16 mM, 0.15 mM and 20 mM, respectively; those of the enzyme from O. gibsoni were 0.16 mM, 0.13 mM and 12 mM. Quinolinate was found to be a potent inhibitor of the enzyme from both filarial worms. The inhibition constants were determined to be 11 microM and 15 microM for the enzyme from O. volvulus and O. gibsoni. It is suggested that the activity of PEP-carboxykinase, the initial enzyme of the alternate route from PEP to succinate, may be regulated by ATP-levels. The inhibition constants for ATP were determined to be 0.26 mM and 0.13 mM for the enzyme from O. volvulus and O. gibsoni.

The Testryp CATT (Card Agglutination Test for Trypanosomiasis): a field study on gambiense sleeping sickness in Liberia.

The inhabitants of three Liberian villages in a known gambiense sleeping sickness area with a low prevalence were examined comparatively with the haematocrit centrifugation technique (HCT), the miniature anion exchange centrifugation technique (m-AECT), the indirect fluorescent antibody test (IFAT) and the Testryp CATT (Card Agglutination Test for Trypanosomiasis). Among 418 persons examined was one individual from whom trypanosomes could be isolated with the HCT and m-AECT. This positive case and another 21 individuals had positive titres of 1:40 and more in the IFAT. The same number, but not the same individuals, showed positive reactions in the CATT in the 1:5 serum dilution. The 1:25 dilution of blood and serum was inferior to the corresponding 1:5 dilution and could therefore be omitted. The 'false positive' rate in the CATT was 5.0% in the serum, 10.1% in the blood, and 11.5% in blood or serum. The combination of HCT and m-AECT in the field did not achieve better results than the CATT or IFAT. Thus the CATT can be recommended as the preferable technique for screening of gambiense trypanosomiasis in the field.

Interaction of amoscanate with the cyclic AMP-phosphodiesterases from Schistosoma mansoni, Onchocerca volvulus and bovine heart.

The anthelmintic compounds amoscanate (4-isothiocyanato-4'-nitrodiphenylamine) and its derivatives CGP 6140 and CGP 8045 are potent inhibitors of the activities of cyclic AMP-phosphodiesterases from schistosomal and filarial worms as well as from both the calmodulin-dependent and the -independent enzyme of bovine heart. As shown for CGP 8045 the type of inhibition is non-competitive with respect to the substrate and the inhibition constants were determined to be in the range of 50 micrograms/ml for the enzymes of parasites and mammalian. It is proposed that the inhibition of cyclic AMP-phosphodiesterase-activity by amoscanate and derivatives - resulting in accumulation of cyclic AMP - might subsequently lead to disturbances in the regulation of metabolic areas like glycogen metabolism which are controlled by cyclic AMP-dependent protein kinases.

Worm burden of onchocerca volvulus in a hyperendemic village of the rain-forest in West Africa.

During a study on the effects of nodulectomy on onchocerciasis in a village in the Liberian rain-forest 400 Onchocerca-nodules were excised from 34 inhabitants. The analysis of the nodules revealed a worm burden of 718 complete live male and 1062 complete live female worms as well as 9 male and 117 female worms which were mostly calcified and degenerating. 73% of the live females contained developing embryos or released microfilariae, the other females either contained eggs or showed empty uteri. Some observations were made on degenerating worms. The male:female sex ratio was 1:1.5 and possible reasons for this are discussed.

Diagnosis and extirpation of nodules in human onchocerciasis.

The clinical search for onchocercal nodules, and their preferred sites on the body in different geographic areas are described and illustrated. The differential diagnosis of onchocercomata is discussed and figures on the frequency of false diagnoses are reported. The prevalence of adult Onchocerca volvulus in relation to the endemicity of infection, the age and sex of the patients, the localisation of nodules on the body, and the measures used to control onchocerciasis are discussed. Recommendations are made on the selection of patients for different medical studies. The technique of nodulectomy and the equipment needed are described.

Ocular onchocerciasis in a hyperendemic village in the rain forest of Liberia.

A sample of 76 persons in a hyperendemic village of onchocerciasis in the rain forest of Liberia was examined ophthalmologically. A reduced visual acuity of less than 6/18 was found in nearly 10%. Punctate keratitis was observed in 36%, microfilariae in the anterior chamber in 46%, proliferative chorioretinitis in 4% and optic atrophy in one person. The overall frequency of ocular onchocerciasis was 62%, but the severity of findings in most individuals was very minimal. The occurrence of microfilariae in the anterior chamber was positively correlated with microfilarial densities in the skin of the hip, outer canthus and blood and the burden of adult O. volvulus isolated from extirpated onchocercomata.

[Lymphographic and morphological studies of the conjunctiva in onchocerciasis patients in Liberia]. / Lymphographische und morphologische Untersuchung der Bindehaut von Onchozerkosepatienten in Liberia.

Conjunctival lymphographies in patients infected with Onchocerca volvulus in Liberia showed normal or only slightly dilated lymphatics. The light-microscopical studies of conjunctival specimens revealed microfilariae predominantly in edematous dilated interstitial channels. Microfilariae in lymphatics and interstitial channels without endothelium of the conjunctivae were demonstrated by scanning and transmission electron microscopy. No signs of inflammatory cellular reaction were found in the vicinity of the parasites. In conclusion, the dilatation of lymphatics and interstitial channels as well as the pathways along which the microfilarial penetrated the cornea and the anterior segments of the eye are discussed.

Parasitological studies on "Onchocerca volvulus" eight years after interruption of the transmission in Upper Volta.

The macrofilaria population was analysed in a hyperendemic focus in western Upper Volta to determine the effect of eight years of Simulium control on the adult Onchocerca volvulus. During the first months of 1983 in three neighbouring villages 629 out of 786 inhabitants were examined for onchocerciasis and from 76 patients as far as possible all onchocercomata were extirpated. The macrofilariae were isolated from the nodules after collagenase digestion and examined microscopically. There were no major differences between men and women or different age groups. The average worm burden was 9.8 live macrofilariae per patient. All worms were several years old but developing sperms were found in 94% of the male and embryos in 55% of the female filariae. 36% of all worms were dead and 23% were calcified. Compared with the findings in a Liberian focus with continuous transmission it is concluded that the interruption of the transmission has lead to a reduced worm burden of entirely old worms but that a few more years of control will be needed until the production of microfitariae will end.