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Comparison of methods for the detection of microfilariae of Wuchereria bancrofti in Western Samoa.

The 1 ml nuclepore filtration technique (NP method), 60 c.mm finger-prick or venous blood smear method and the counting chamber method were compared for their effectiveness in detecting microfilariae. The loss of microfilariae by the NP method was small enough to be disregarded. The 60 c.mm blood smear method was shown to be as efficient as the 60 c.mm NP method, when using finger-prick blood. The finger-prick blood contained 32% more microfilariae than venous blood when compared by the 60 c.mm blood smear method. Probably reflecting this difference of mf count between finger-prick and venous blood, 1 ml NP method using venous blood detected only 12.4 times more microfilariae than 60 c.mm NP method using finger-prick blood, despite the fact that the former requires 16.7 times more blood than the latter. No difference of mf count was shown between the 60 c.mm blood smear and 60 c.mm counting chamber methods, when using finger-prick blood.

Animals↗

Microfilariae of Wuchereria bancrofti in cytologic smears.

Microfilariae of Wuchereria bancrofti were observed in cytologic material in 35 cases. The material included cervicovaginal smears (17 cases), effusions (14), urine (2), bronchial washings (1) and ovarian cyst fluid (1). The initial diagnosis was made from the cytologic smear in all cases; none had clinical filariasis. Symptomatic vaginal bleeding in 9 of the 17 cases with microfilaria-positive cervicovaginal smears was reflected in the large numbers of red blood cells found in the smear. Blood eosinophilia was present in 11 of 19 cases investigated. Eosinophils were seen in the smears in 20 cases. In the majority of the cases of effusions with microfilariae the effusions were malignant. Significant adherence of inflammatory cells and macrophages to microfilariae was present in 7 of the 35 cases. The significance of these findings is discussed.

Adhesiveness↗

Efficacy of ivermectin against Dirofilaria immitis microfilariae in naturally infected dogs.

Ivermectin, given in a single subcutaneous dose of 200 micrograms/kg, was evaluated for its efficacy against Dirofilaria immitis microfilariae in a trial with naturally infected dogs. Sodium thiacetarsamide (2.2 mg/kg) was administered twice daily for 2 days to 2 groups of 10 dogs. Two weeks later, 1 group was treated with ivermectin, and the other group was given the vehicle. Microfilariae were not detected (by blood sample concentration techniques) in 90% of the dogs 21 days after they were treated with ivermectin. Microfilariae were not detected in samples of any dog at days 28 and 35. However, 1 dog (not the one positive at day 21) had a detectable microfilaria at day 42. One dog with pretreatment ascites, exercise-induced cough, and high microfilaremial count was anorectic and depressed 2 days after treatment with ivermectin; the clinical signs were resolved 4 days later.

Animals↗

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.

Animals↗

Survival and behavior of Onchocerca gutturosa microfilariae in vitro.

Microfilariae of Onchocerca gutturosa were extracted from the skin of the bovine naval by teasing in saline and then suspending the skin in a gauze bag overnight in Tyrode's solution with 20% human serum. They survived for ten days in this medium, with antibiotics, in plastic microtitration plates at 11 C. After dispersal in flat containers, microfilariae slowly assumed clumped dispersion patterns. They also accumulated in glass wool, suggesting that aggregation was a tactile phenomenon. Analysis of videotapes of microfilarial movement indicated that activity, expressed as the number of undulations present on the body and the rate of knot formation (where the microfilaria held itself tightly coiled), increased up to 40 C and thereafter declined. The rate of headwave formation, however, appeared constant up to 40 C after which it declined. Where the greatest number of undulations were found on the body the wave amplitude was minimal. Because greater apparent activity was not correlated with increased forward progression, microfilariae at lower temperatures may travel farther than those at 40 C. This may relate to site preference in the host and to uptake by the vector Simulium ornatum.

Animals↗

[Intake of microfilariae by the vector in the case of a low microfilareamia (author's transl)].

The study of the intake of microfilariae of Dipetalonema dessetae by Aedes aegypti leads to two conclusions with respect to the microfilaraemia: --there is no concentration of microfilariae in the uptake of blood by Aedes. --the microfilariae are nearly homogeneously distributed in the vertebrate host blood available to the vector for feeding. Such a distribution of microfilariae in the cutaneous blood supply of the host gives the maximum chance for a mosquito to become infected when taking a blood meal. In the pair Wuchereria bancrofti--Aedes polynesiensis the capacity of individuals with a low microfilaraemia to transmit infections seems to be related to a limitation phenomenon.

Aedes↗

Brugia pahangi: stage-specific antigens on microfilariae detected by serum from infected jirds or by monoclonal antibodies.

Experiments were carried out to determine whether there are stage-specific antigens on microfilariae of Brugia pahangi, using sera from Mongolian jirds infected with B. pahangi and monoclonal antibodies against microfilariae of B. pahangi. These studies showed that microfilariae have both stage-specific and nonspecific antigens. The nonspecific antigens were also present on adult worms and on infective larvae. Among monoclonal antibodies, 6 out of 14 clones produced antibodies against the microfilarial stage-specific antigens, and 8 clones produced antibodies against nonspecific antigens. These monoclonal antibodies could not distinguish between adults, microfilariae, or infective larvae of B. malayi and B. pahangi.

Animals↗

Rapid uptake and esterification of arachidonic acid and other fatty acids by microfilariae of Brugia malayi.

We have examined the differential incorporation and esterification of exogenous fatty acids by microfilariae of the human filarial parasite Brugia malayi. Microfilariae incubated with 2 nM [3H]arachidonic acid over 1 h rapidly took up this fatty acid. Palmitic, oleic and linoleic acids were also incorporated by parasites. In contrast to these other fatty acids, little incorporated arachidonic acid remained as free fatty acid within microfilariae. Arachidonate was rapidly esterified into phospholipids, with 66% of incorporated arachidonate esterified into phospholipids at 1 min. Esterification of other fatty acids into phospholipids was quantitatively lesser and occurred into phosphatidylcholine and phosphatidylethanolamine. Arachidonate was preferentially esterified into phosphatidylinositol, which constituted only 10% of the total parasite phospholipid pool, and into phosphatidylcholine. By 1 min these two phospholipid classes, respectively, comprised 53% and 43% of [3H]arachidonyl-phospholipids. Neither the microfilarial incorporation of arachidonate nor its esterification into parasite phospholipids could be saturated by noncytotoxic concentrations of up to 600 microM. Microfilariae, which in vivo are exposed to arachidonate in blood, can rapidly, avidly and with high capacity incorporate exogenous arachidonate and esterify it preferentially into specific classes of phospholipids, including phosphatidylinositol. Like many mammalian cells, these phylogenetically distinct metazoan parasites possess efficient means for utilizing host-derived arachidonic acid.

Animals↗

Active release of surface proteins: a mechanism associated with the immune escape of Acanthocheilonema viteae microfilariae.

Living Acanthocheilonema viteae microfilariae obtained from peripheral blood of parasitised Meriones unguiculatus were surface-labelled with 125I. Four major surface exposed proteins of approximately 14.50, 14.55, 17.5, 19 kDa and one less abundant protein of 40 kDa were identified. Under non-reducing conditions the low-molecular-weight (LMW) proteins were isolated as multimers suggesting the presence of intermolecular disulphide linkages. In gels containing Triton X-100 the labelled epicuticular proteins behaved lipophilically. By cultivation of surface-labelled and metabolically labelled microfilaria in vitro, a continuous shedding of two LMW proteins was demonstrated. These proteins were produced in large amounts and released into the culture supernatant as monomeric and pentameric molecules. Concomitant with this release, one of the proteins appeared to lose its lipophilic character, giving rise to a hydrophilic 14.50-kDa entity. Although most of the extracted surface proteins reacted with sera from patent jirds, these sera failed to recognise the surface of living microfilariae. However, microfilariae pretreated with glutaraldehyde or attenuated with Na-azide could be labelled with surface specific antibodies.

Acanthocephala↗

203Hg and other gamma-emitting radio-isotopes as labels for Dirofilaria immitis microfilariae.

The in vitro uptake of gamma-emitting radionuclides by microfilariae of Dirofilaria immitis was investigated. Radionuclides tested were 133Ba, 207Bi, 82Br, 109Cd, 51Cr, 60Co, 59Fe, 203Hg, 125I, 54Mn, 32P, 125Sb, 46Sc, 75Se and 65Zn. Only 207Bi, 59Fe, 203Hg, 54Mn and 46Sc showed more than 2% of the available radioactivity to bind to the microfilariae. When tested for retention in vitro only 203Hg showed retention levels approaching 90%. Moreover, when dimethyl-sulphoxide was incorporated into the medium at levels of 1% (v/v) the uptake of 203Hg could be increased by 3-5 times; no other radio-isotope tested responded in this manner. The uptake of 203Hg was directly related to temperature and time of incubation. Mercury, as mercuric chloride, was toxic to the microfilariae and represents an impediment to the incorporation of high levels of 203Hg in microfilariae.

Animals↗

Integrin-like RGD-dependent cell adhesion mechanism is involved in the rapid killing of Onchocerca microfilariae during early infection of Simulium damnosum s.l.

Injection trials with compatible and non-compatible Onchocerca species into S. damnosum s.l., the vector of human and bovine onchocerciasis, demonstrated that the rapid killing of microfilariae within the blackfly's haemocoel is species specific. In the presence of the peptide RGDS as a blocking agent for integrin-like receptors of haemocytes, the survival of O. ochengi microfilariae in its natural intermediate host was significantly increased. This increased survival 24 h p.i. correlated with a significant decrease of apoptosis levels in the microfilariae following a 2 h exposure to the haemolymph in vivo. These findings suggest that haemocytes are directly involved in the killing of Onchocerca microfilariae in the blackfly.

Animals↗

Co-infection with Onchocerca volvulus and Loa loa microfilariae in central Cameroon: are these two species interacting?

Ivermectin treatment may induce severe adverse reactions in some individuals heavily infected with Loa loa. This hampers the implementation of mass ivermectin treatment against onchocerciasis in areas where Onchocerca volvulus and L. loa are co-endemic. In order to identify factors, including co-infections, which may explain the presence of high L. loa microfilaraemia in some individuals, we analysed data collected in 19 villages of central Cameroon. Two standardized skin snips and 30 mul of blood were obtained from each of 3190 participants and the microfilarial (mf) loads of both O. volvulus and L. loa were quantified. The data were analysed using multivariate hierarchical models. Individual-level variables were: age, sex, mf presence, and mf load; village-related variables included the endemicity levels for each infection. The two species show a certain degree of ecological separation in the study area. However, for a given individual host, the presence of microfilariae of one species was positively associated with the presence of microfilariae of the other (OR=1.79, 95% CI [1.43-2.24]). Among individuals harbouring Loa microfilariae, there was a slight positive relationship between the L. loa and O. volvulus mf loads which corresponded to an 11% increase in L. loa mf load per 100 O. volvulus microfilariae. Co-infection with O. volvulus is not sufficient to explain the very high L. loa mf loads harboured by some individuals.

Adolescent↗

Diethylcarbamazine enhances antibody-mediated cellular adherence to Brugia malayi microfilariae.

Treatment with the antifilarial drug diethylcarbamazine (DEC) results in a rapid decline in the number of microfilariae circulating in the blood of infected hosts. DEC induces morphological changes in the surface layers of microfilariae, but these alterations alone are probably insufficient to cause the death of the parasite, because the drug fails to reduce microfilaraemia in animals lacking filarial antibodies, and also does not shorten the survival of microfilariae in vitro. The effect of DEC in vivo is thought to result from the trapping of microfilariae in the liver, where they undergo lysis.

Animals↗

Microfilariae in association with neoplastic lesions: report of five cases.

Microfilariae and adult filarial worms have occasionally been detected in association with neoplastic lesions in cytological smears. The presence of microfilariae along with neoplasms is generally regarded as a chance association, yet some authors suggest that such parasitic infestations may be a causative factor for tumourigenesis. There are only a few reported cases in cytology literature documenting this association. We report the presence of microfilariae in routine cytology smears from one benign and four malignant tumours. Microfilariae could not be identified on histopathology available in four of these cases.

Adult↗

Cellular hemolymph response of Simulium vittatum (Diptera:Simuliidae) to intrathoracic injection of Onchocerca lienalis (Filarioidea:Onchocercidae) microfilariae.

Hemolymph cellular changes in Simulium vittatum Zetterstedt in response to intrathoracic injection with Onchocerca lienalis Stiles were characterized by increased numbers of tissue fragments that contained cells embedded within a noncellular matrix. Cell numbers within the matrix fragments increased both for sham and microfilariae-injected files, indicating a blastogenic response to injection alone. Morphological characteristics of fixed, Giemsa-stained cells within these tissue fragments were most similar to those previously described for prohemocytes. The total population of freely circulating hemocytes collected 24 h after injection also responded to injection alone. Differential cell counts showed a complex pattern of changes that was influenced strongly by increased numbers of prohemocytes at 24 h in microfilariae-injected flies. Fat body fragments collected in hemocoel perfusates at 24 h were fewer when flies were maintained at 27 degrees C than at 21 degrees C. More fat body fragments were collected from microfilariae-injected flies than from control and sham-injected flies held at 27 degrees C. Injection of 1.25-5 micrograms of lipopolysaccharide per fly did not elicit similar hemolymph-associated matrix tissue and fat body changes, indicating that S. vittatum response to microfilaria infection and bacteria infection are likely to involve different mechanisms.

Animals↗

Increased activity of macrophages from mice infected with Brugia pahangi: in vitro adherence to microfilariae.

After the inoculation of infective larvae of Brugia pahangi into the peritoneal cavities of CBA/Ca mice adult worms developed, but by 12 weeks post-infection the parasites were usually dead and surrounded by granulomatous tissue. Macrophages were the most common cell type in these granulomas and were also found in increasing numbers free in the peritoneal cavity. We have investigated the ability of macrophages to damage microfilariae in vitro, in a system where microfilariae were cultured together with cells and serum taken from either uninfected female CBA/Ca mice or at various intervals after infection. Macrophages adhered to and killed microfilariae in this system and there was an increase in vitro adherence of these cells during the course of infection. The peak level of in vitro activity of these cells coincided with the phase of parasite killing in vivo. The presence of serum also affected the degree of macrophage adherence and subsequent death of the parasite. Analysis of serum components using EGTA, zymosan or heat inactivation suggested that complement and possibly heat labile antibody were involved. Immunoglobulins were shown by immunofluorescence to be present on the surface of microfilariae cultured in serum from infected mice. It is concluded from this study that macrophages are actively involved in the termination of murine filarisis.

Animals↗

The morphology of O. volvulus microfilaria in the northwestern onchocerciasis endemic foci, Ethiopia.

Microfilariae obtained by corneoscleral biopsy machines from persons in the Humera area along the Sudan boarder in Northwestern Ethiopia were stained using hematoxylin. Morphological measurements were made using a microscope fitted with micrometer and camera, as well as map curvimeter. The mean size of microfilariae was 189 microns by 5.8 microns. The percentage of nucleifree cephalic space as percent of total body length was 3.4 and is close to reports in the literature. The measurement of the distance of the anterior end nerve ring as a percent of the total body length (22.1%) is also close to measurements obtained elsewhere. Although the microfilaria encountered in this study are of the short variety, morphological measurements and the absence of sheath suggests that the microfilaria observed belong to O. volvulus.

Animals↗

Inhibition of the activation of Hageman factor (factor XII) and of platelet aggregation by extracts of Brugia malayi microfilariae.

In human filariasis, large numbers of blood-borne microfilariae circulate unimpeded through the blood stream. How intravascular filarial parasites avoid precipitating thrombosis has not been studied in detail. We hypothesized that extracts of Brugia malayi microfilariae would contain factors that inhibit activation of hemostatic mechanisms. Initial studies demonstrated an inhibitor specific for the intrinsic coagulation cascade. The addition of microfilarial extracts to human plasma prolonged the activated partial thromboplastin time in a dose-dependent fashion but did not prolong the prothrombin, thrombin, or Russell's viper venom times. Microfilarial extracts (0.1 mg/ml) completely inhibited activation of Hageman factor (factor XII, at 0.05 U/ml) as measured in an amidolytic assay. Hageman factor previously activated by ellagic acid (factor XIIa) retained full enzymatic activity in the presence of microfilarial extract (0.1 mg/ml). The presence of inhibitory activity in the culture medium of live parasites raises the possibility that microfilariae secrete an inhibitory protein into their local environment. Microfilarial extracts at a final concentration of 0.1 mg/ml also inhibited collagen- and adenosine diphosphate-induced platelet aggregation. Arachidonic acid-induced platelet aggregation was inhibited by microfilarial extracts at a final concentration of 0.6 mg/ml. These results suggest that microfilariae of Brugia malayi, a human filarial parasite, may avoid initiating thrombosis through inhibition of the intrinsic coagulation pathway and platelet aggregation.

Adenosine Diphosphate↗