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Lectin-binding characteristics of Brugia pahangi microfilariae.

The binding of FITC-CON-A, SBA, DBA, WGA, PNA and RCA120 to microfilariae of B. pahangi and D. immitis was investigated. Sheathed uterine, peritoneal or blood microfilariae of B. pahangi bound only WGA on the surface of the sheath. The cuticle of D. immitis microfilariae and of artificially exsheathed B. pahangi microfilariae failed to bind any of the lectins tested. Binding of WGA to the sheath surface was specific and was inhibited in the presence of the complementary saccharide, N-acetyl glucosamine. WGA also penetrated the sheath of B. pahangi microfilariae. Treatment of peritoneal B. pahangi microfilariae with certain proteases exposed additional binding sites for CON-A, SBA, DBA, PNA and RCA120 but not for UEA.

Animals↗

Comparative morphology of Mansonella ozzardi microfilariae from Colombia and Haiti. A light microscope study.

Comparative morphological studies were conducted at the light microscope level to determine whether any difference could be detected in thin smears, thick smears, or Knott samples, between Colombian (simuliid-transmitted) and Haitian (culicoid-transmitted) Mansonella ozzardi microfilaria. Results indicated that the microfilariae from these two geographical areas are essentially identical. The small differences observed in the dimensions of microfilariae in thick and in thin smears can be attributed to different preparative procedures. The consistent morphological differences, noted in PAS-hematoxylin-stained microfilariae, from Knott smears, was that the central body was thinner and stained less intensely with the Schiff reagent in the Haitian than in the Colombian microfilariae. Although these observations suggest a physiological difference between these two forms, additional studies are needed to exclude the possibility that these differences are not indications of the age or a state of physiological activity of the microfilariae.

Animals↗

Recovery and viability of Dirofilaria immitis microfilariae.

The viability of Dirofilaria immitis microfilariae recovered from canine blood by different methods was determined. Microfilaria recovery techniques included saponin lysis, saponin lysis with a trypsin treatment, dextran sedimentation and phytohemagglutinin treatment. Criteria for evaluating viability were microfilarial motility in vitro at 37 degrees C, microfilarial development in mosquitoes and the ability of microfilariae to circulate in mice. Although each method produced motile microfilariae, differences among groups of microfilariae recovered by different techniques were apparent by each of the criteria for viability. Saponin lysis gave superior yields of viable microfilariae.

Aedes↗

Effect of diethylcarbamazine on serum-dependent cell-mediated immune reactions to microfilariae in vitro.

The effect of diethylcarbamazine citrate (DEC) on serum-dependent cellular adherence and cytotoxicity to sheathed and exsheathed microfilariae of Litomosoides carinii and Brugia pahangi in vitro has been investigated. DEC at 5 micrograms/ml promoted antibody-mediated neutrophil adherence and cytotoxicity with sheathed L. carinii microfilariae. With exsheathed parasites, the drug promoted significantly complement-and antibody-mediated cellular adherence and cytotoxicity. In the case of B. pahangi, antibody-dependent cell-mediated cytotoxicity (ADCC) of neutrophils to sheathed and exsheathed microfilariae were similarly stimulated by DEC. Pre-incubation of microfilariae with DEC promoted the cellular adherence and cytotoxicity whereas pre-incubation of cells had no such effect. DEC stimulated the adherence of eosinophils to both sheathed and exsheathed microfilariae of the two species but the adherence resulted in cytotoxic effect only on the L. carinii microfilariae. DEC at higher concentrations (100 or 500 micrograms/ml) inhibits these processes. Data on the role of different serum factors involved in this phenomenon have been presented and discussed.

Animals↗

Neutrophil-mediated killing of Dipetalonema viteae microfilariae: simultaneous presence of IgE, IgG antibodies and complement is required.

Neutrophils from the peripheral washings of normal rats in the presence of sera obtained from rats immune to circulating microfilariae adhered to and killed the microfilariae of Dipetalonema viteae in vitro within 16-24 hr. No significant adherence or cytotoxicity was mediated by sera collected from animals with a high microfilaraemia or from normal rats. Ultrastructural studies show that neutrophils, which are bigger than microfilariae, can easily internalize the small larvae resulting in the disintegration of the parasite. Immunoadsorption and inhibition experiments showed that the adherence-promoting activity resides both in IgG and IgE classes of antibody. However, the mere participation of these two antibodies is not sufficient to effect neutrophil adherence towards microfilariae, the presence of complement is also required. Samples of fresh immune rat serum (fIRS) depleted in alternative pathway components of complement by treatment with zymosan A failed to mediate cell adherence to the parasite. fIRS inactivated for the classical pathway of complement by the chelating agent EGTA partially retains its activity in mediating cytotoxicity to microfilariae. The striking antigenic specificity of D. viteae antibodies was shown by their ability to mediate cytotoxicity only to D. viteae but not towards Brugia malayi microfilariae.

Animals↗

The action of diethylcarbamazine on the skin-dwelling microfilariae of Monanema globulosa (Nematoda: Filarioidea) in rodents.

Diethylcarbamazine (DEC) administered orally to jirds and striped mice (300 mg/kg for five days) was effective against the microfilariae of Monanema globulosa in experimental infections. Based on the small numbers of animals used in this trial, it is questionable whether the drug had significant activity against the adult worms in pulmonary arteries. Densities of microfilariae in the ears (mf/mg) fell by 80-100% of pretreatment levels over a four week period following treatment. There was no apparent action of the developing embryos in utero, and in some rodents densities of microfilariae in the skin returned to 73-88% of pretreatment levels within 70 days. No microfilariae were detected in the blood or urine during treatment. Twenty-four hours after the first dose of DEC, many of the microfilariae in the dermis became surrounded by a local infiltration of neutrophils, eosinophils and histiocytes. Cellular reactions were accompanied by an acute pruritus in rodents, as indicated by scratching and distress exhibited soon after treatment: No reactions to microfilariae were seen in untreated rodents. Similarities between this response and the Mazzotti reaction induced by DEC in human onchocerciasis indicate that M. globulosa may be of value as a rodent model to study adverse drug reactions in Onchocerca volvulus infections.

Animals↗

Litomosoides carinii: effect of diethylcarbamazine on microfilaremias of Mastomys natalensis harboring old infections, new infections, and transfused microfilariae.

Mastomys natalensis, matched according to age and microfilaremia, were divided into Experimental and Control groups. The former were given diethylcarbamazine (Caricide, 500 mg/kg) per os. Microfilariae counts were made at 15, 30, 60, and 120 min and at 1, 5, and 12 days. This protocol was performed on rats with: old infections (patent for 7 mo), new infections (patent for 10 days), and no infection but made microfilaremic by injection of blood or culture-derived microfilariae. In all treated groups, the microfilaremia dropped precipitously within the first 15 min. Microfilaremias of animals with old infections remained low until day 12 after treatment when they began to rise, whereas those of animals with new infections began to rise immediately, i.e., within minutes after the initial decline. Microfilaremias of animals with no adult worms present remained depressed for the duration of the experiment. Thus, the resurgence of microfilariae after treatment with diethylcarbamazine is the result of release of new microfilariae by the female worms rather than release of trapped microfilariae by the host. This investigation also demonstrates that elimination of microfilariae by a nonsensitized host is possible upon treatment with diethylcarbamazine.

Animals↗

[The larval development of Dipetalonema rugosicauda (syn. Wehrdikmansia rugosicauda) in the tick Ixodes ricinus. II. The development of Dipetalonema rugosicauda in Ixodes ricinus and investigations about the occurrence of the microfilariae in the roe deer (C. capreolus) (author's transl)].

Microfilariae of D. rugosicauda, which were taken up by nymphs of I. ricinus during their attachment on roe deers, developed in those ticks to infective larvae after two moults. The larval development lasted 56-67 days at 20-22 degrees C and occurred similar to other species of Dipetalonema inclusive a sausage stage. During this time the nymphs moulted to adult ticks. Adult females and males of I. ricinus collected in the area of Biberach/Riss, Southern-Germany, contained up to 12 infective larvae of D. rugosicauda per tick. The occurrence of those infective larvae and of microfilariae in roe deers depended on the season. The highest rate of infected ticks was 4.3% in spring, whereas the maximum rate of infected roe deers was 46.8% in autumn. Based on investigations of fawns of roe deers the prepatent period of D. rugosicauda can be estimated as approximately 6 months. Compared with microfilariae of Onchocerca tarsicola from red deers the microfilariae of D. rugosicauda prefered deeper layers of the ear skin of their hosts. Besides D. rugosicauda microfilariae of a further hitherto unknown species were detected in the ear skin of roe deers additionally. They resembled microfilariae of O. tarsicola.

Animals↗

IgE antibody-mediated cytotoxicity of rat macrophages against microfilaria of Dipetalonema citeae in vitro.

The Fischer rat develops an acquired resistance against circulating microfilariae. Macrophages from the peritoneal washings of normal rats preincubated at 37 degrees C with the sera obtained from rats immune to circulating microfilariae adhered to and kill the microfilaria of Dipetalonema viteae in vitro within 16 to 24 hr. No significant adherence and cytotoxicity was mediated by sera collected from animals with microfilaraemia or from normal rats. Adherence of macrophages to microfilaria was associated with damage to the surface of the larva as revealed by ultrastructural studies. Neither adherence nor cytotoxicity was induced by preincubation of microfilariae, instead of macrophages with immune serum. The serum factor which mediated adherence and cytotoxicity was heat-labile, but was not a complement component. Immune absorption experiments showed that the relevant serum factor resided in the IgE class of antibody. The immune adherence to D. viteae by macrophages is stage-specific because adherence to infective larvae was not observed whether rate macrophages were preincubated in sera obtained from rats immune to microfilariae or in sera collected from animals after exposure to infective larvae.

Animals↗

Life span and distribution of Onchocerca volvulus microfilariae in mice.

Microfilariae of Onchocerca volvulus obtained from onchocercomas were inoculated subcutaneously into the inguinal region of mice. Microfilariae disseminated rapidly, invading the tail and lungs within 1 hr and the ears within 3 hr postinoculation. During the early period of infection microfilariae appeared in the eyes, ears, viscera, pelt, carcass, and tail; later, the microfilariae tended to accumulate in the tail. Liver microfilariae were recovered up to 12 wk postinoculation. Intraperitoneal inoculation and subcutaneous inoculation into the inguinal region resulted in the concentration of microfilariae in the tail, whereas inoculation into the scalp caused microfilarial accumulation in the ears.

Animals↗

Isolation and concentration of microfilariae from peripheral blood of Wuchereria bancrofti infected patients by density gradient centrifugation.

A Ficoll-Hypaque gradient centrifugation technique was used for isolation and concentration of microfilariae from peripheral blood of 30 subjects with clinically and parasitologically diagnosed Wuchereria bancrofti infections. 86% of the microfilariae were found in the Ficoll-Hypaque layer. None were detected in the plasma, leucocyte layer or lower erythrocyte layer. 14% of microfilariae were identified on the top part of the erythrocyte layer. A 35 fold concentration and 88% quantitative recovery of parasites was achieved by conventional centrifugation of microfilariae-rich Ficoll-Hypaque layer. Following the centrifugation procedures, living motile microfilariae were separated. These results indicate that Ficoll-Hypaque centrifugation technique could be an effective method for the detection of low levels of microfilaraemia, and for obtaining relatively pure suspensions of living microfilariae for metabolic studies, production of antigen-rich excretory-secretory products and antigen analysis.

Animals↗

External monitoring of 203Hg-labelled Dirofilaria immitis microfilariae in mice and dogs.

Microfilariae of Dirofilaria immitis were labelled with 203Hg2+ in vitro and injected into irradiated mice and Beagle dogs. With irradiated mice it was possible to demonstrate microfilariae present in the blood and to detect 203Hg by external counting as long as 28 days after dosing. The 203Hg2+ label had a half-time of 4-5 days; the amount of stable mercury in the labelling medium strongly influenced the survival of microfilariae in vivo. In dogs, external counting showed the lungs to be a major location of the microfilariae soon after reinjection into the host. Evidence was obtained that labelled microfilariae can circulate; however, the detection of dispersed microfilariae is difficult because of the relative insensitivity of the detecting system. For radiomercury the accumulation of the inorganic form in the liver and kidneys limits the long-term usefulness of 203Hg2+ as a label if the organism being studied also accumulates in these organs.

Animals↗

Neutrophil granule proteins: evidence for the participation in the host reaction to skin microfilariae of Onchocerca volvulus after diethylcarbamazine administration.

The participation of neutrophil granulocytes in the cellular reaction to skin microfilariae of Onchocerca volvulus was studied by immunohistochemistry. Skin biopsies were obtained from adult Liberian and Ugandan patients with generalized onchocerciasis after exposure to topically applied diethylcarbamazine (DEC) and from untreated patients. After DEC many damaged microfilariae were observed either in dermal infiltrates or in epidermal microabscesses consisting both of neutrophils and eosinophils. Infiltrates and microabscesses contained some intact granulocytes and many neutrophils releasing myeloperoxidase, elastase, lactoferrin, defensin, lysozyme, alpha 1-antitrypsin and alpha 1-antichymotrypsin. Eosinophils discharged peroxidase and cationic proteins. Released granule proteins and remnants of disrupted granulocytes were found on the surface and in close proximity of damaged microfilariae in dermal infiltrates and epidermal microabscesses. In larger microabscesses neutrophils were predominant. These observations show that neutrophils and not only eosinophils recruit, accumulate, localize around and release their helminthotoxic granule proteins such as myeloperoxidase onto or closely around skin microfilariae of O. volvulus after topical DEC administration. The association between these processes and the damage of the microfilariae indicated that neutrophils together with eosinophils attack and damage microfilariae of O. volvulus after DEC treatment in the skin.

Animals↗

Interleukin-4 is essential for the control of microfilariae in murine infection with the filaria Litomosoides sigmodontis.

Litomosoides sigmodontis is the only filaria which develops from infective larvae into microfilaria-producing adults in immunocompetent laboratory mice. In this study we report that interleukin-4 knockout (IL-4 KO) mice have an up to 100-fold-higher and a significantly prolonged microfilaremia compared to wild-type BALB/c mice, as well as 20 times more microfilariae in the thoracic cavity, the site of infection. While worm development and adult worm persistence were equivalent in IL-4 KO and wild-type mice, the fertility and length of adult female worms in IL-4 KO mice was clearly enhanced. The high susceptibility to microfilariae in IL-4 KO mice required the presence of adult worms in a full infection cycle since microfilariae loads did not differ much between IL-4 KO and wild-type mice when purified microfilariae were injected into mice. In addition, we found that eosinophilia was diminished and immunoglobulin E (IgE) was absent in IL-4 KO mice. IgE, however, does not seem to be the essential factor for microfilarial containment since microfilaremia was not elevated in B-cell KO mice. In conclusion, IL-4 is shown for the first time to be essential for the control of microfilarial loads but not of adult worm loads in a fully permissive murine filarial infection. IL-4 dependent effector pathways seem to operate on adult worms rather than directly on microfilariae.

Animals↗

Diethylcarbamazine activity against Brugia malayi microfilariae is dependent on inducible nitric-oxide synthase and the cyclooxygenase pathway.

BACKGROUND: Diethylcarbamazine (DEC) has been used for many years in the treatment of human lymphatic filariasis. Its mode of action is not well understood, but it is known to interact with the arachidonic acid pathway. Here we have investigated the contribution of the nitric oxide and cyclooxygenase (COX) pathways to the activity of DEC against B. malayi microfilariae in mice. METHODS: B. malayi microfilariae were injected intravenously into mice and parasitaemia was measured 24 hours later. DEC was then administered to BALB/c mice with and without pre-treatment with indomethacin or dexamethasone and the parasitaemia monitored. To investigate a role for inducible nitric oxide in DEC's activity, DEC and ivermectin were administered to microfilaraemic iNOS-/- mice and their background strain (129/SV). Western blot analysis was used to determine any effect of DEC on the production of COX and inducible nitric-oxide synthase (iNOS) proteins. RESULTS: DEC administered alone to BALB/c mice resulted in a rapid and profound reduction in circulating microfilariae within five minutes of treatment. Microfilarial levels began to recover after 24 hours and returned to near pre-treatment levels two weeks later, suggesting that the sequestration of microfilariae occurs independently of parasite killing. Pre-treatment of animals with dexamethasone or indomethacin reduced DEC's efficacy by almost 90% or 56%, respectively, supporting a role for the arachidonic acid and cyclooxygenase pathways in vivo. Furthermore, experiments showed that treatment with DEC results in a reduction in the amount of COX-1 protein in peritoneal exudate cells. Additionally, in iNOS-/- mice infected with B. malayi microfilariae, DEC showed no activity, whereas the efficacy of another antifilarial drug, ivermectin, was unaffected. CONCLUSION: These results confirm the important role of the arachidonic acid metabolic pathway in DEC's mechanism of action in vivo and show that in addition to its effects on the 5-lipoxygenase pathway, it targets the cyclooxygenase pathway and COX-1. Moreover, we show for the first time that inducible nitric oxide is essential for the rapid sequestration of microfilariae by DEC.

Journal Article↗

Studies on Brugia pahangi 9. The longevity of microfilariae transfused from cat to cat.

Microfilariae in whole uncentrifuged cat blood were transfused from infected to normal cats. The recipient cats were bled frequently and the life-span of the microfilariae in the peripheral circulation was determined; The life-span on microfilariae in different cats was ver variable. Microfilariae were detected fro 2-136 days after inoculation. In immunized cats microfilariae could not be detected 18 hours after inoculation. Any attempt to concentrate or clean the microfilariae caused them to disappear from the circulation more quickly.

Animals↗

Daytime variation in the density of Onchocerca volvulus microfilariae in human skin.

One aim of this study was to determine whether a corneal-scleral punch biopsy instrument provides skin specimens of sufficiently uniform size so as to eliminate the need to weigh or measure the specimens in order to provide a reproducible index of the density of Onchocerca volvulus microfilariae in the skin. A second aim was to determine whether the density of microfilariae in the skin varies with the time of day. The reports of others on the latter are contradictory. Duplicate skin specimens were taken from 10 people four times during one day with a corneal-scleral punch biopsy instrument. Each specimen was weighed and measured, and the number of microfilariae were counted. Although the specimens were not of uniform weight or surface area, the number of microfilariae per specimen was as good an index of the density of larvae in the skin as the number of microfilariae per mg, or per mm(2) of skin. This may not be true when other methods of procuring skin specimens are utilized. A universally acceptable standard method is advocated. The density of microfilariae in the skin approximately doubled from morning to midday, and began to decline by early evening.

Adult↗

The intake and transmissibility of Onchocerca volvulus microfilariae by Simulium damnosum fed on patients treated with Diethylcarbamazine, suramin or Mel W.

The best hope for the control of transmission of Onchocerca volvulus over much of Africa lies in combining larvicidal measures directed against the Simulium vectors with chemotherapeutic measures designed to reduce the reservoir of transmissible microfilariae in man.There are few drugs effective against O. volvulus. Diethylcarbamazine kills the microfilariae but has virtually no effect on the adult worms. Suramin kills adult worms and many, but not all, of the microfilariae. Mel W kills adult worms but has little or no action on microfilariae. All these drugs suffer at present from disadvantages of toxicity, which tend to limit their use on a mass scale in the field. Nevertheless, before they, or indeed any new drugs with similar actions on the parasites, can be used intelligently for the control of onchocerciasis transmission, it is necessary to have accurate quantitative information on the effect that each of them has on the microfilarial population available for intake by Simulium, as well as on their actions on the developmental potential of those microfilariae ingested by the flies.The present paper describes the effects of treatment with various courses of diethylcarbamazine, suramin or Mel W on the numbers of microfilariae ingested by groups of S. damnosum and on the numbers of infective larvae developing therefrom.

Arsenicals↗