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Effect of diethylcarbamazine on the concentration of Onchocerca volvulus microfilariae in hydrocoele fluid and urine.

The effect of graded doses of diethylcarbamazine on the concentration of microfilariae in the hydrocoele fluid, urine and skin of patients suffering from onchocerciasis was studied. The results showed that a significant number of microfilariae migrated into hydrocoele fluid and urine immediately after the drug was given and thereafter returned to pre-treatment levels rather more quickly in the former than the latter. The increase was found to be roughly directly proportional to the intensity of infection in the skin. By contrast the skin concentration of microfilariae fell sharply after treatment and remained significantly lower than pre-treatment levels for over four weeks. The potential use to which these observations could be put with respect to clinical screening of drugs in onchocerciasis is discussed.

Adult↗

Morphological differences between Venezuelan and African microfilariae of Onchocerca volvulus.

Comparative morphological and biometric characteristics of microfilariae of Onchocerca gutturosa and O. volvulus from different geographical areas (Upper Orinoco, Venezuela; Togo; Liberia) were assessed. "Stepwise" discriminant analysis and Mahalanobis estimators were applied to measure distance between populations. The results indicate a strong similarity between the two strains from the Upper Orinoco (Venezuela) and the Togo strain, as well as a clear separation between these strains and that of O. gutturosa. The Liberian strain was easily distinguishable from microfilariae from Togo and Venezuela. Discriminant analysis showed the Liberian deme to be as different from the Venezuelan and Togo demes as these demes were from microfilariae of the reference species, O. gutturosa. Although it is necessary to confirm these data using formalin-fixed specimens obtained from the skin, the present findings suggest the existence of geographically-different strains of O. volvulus in America and Africa.

Animals↗

Uptake of diethylcarbamazine by microfilariae and adults of Litomosoides carinii and Dipetalonema viteae.

Microfilariae of Litomosoides carinii and Dipetalonema viteae absorbed about ten times as much diethylcarbamazine (DEC) as did their adults, but bound much less. The higher uptake of DEC by the microfilariae might be related to the fact that DEC is microfilaricidal. Binding of DEC with the parasites supports the view that the microfilaricidal action begins with the fixation of the drug to the microfilariae.

Animals↗

The infectivity of microfilariae of Brugia pahangi of different ages to Aedes aegypti.

By transferring microfilariae of Brugia pahangi which had been born over a 24-hour period in the peritoneal cavities of jirds (Meriones unguiculatus) to the blood circulation of other jirds, infections of known age were tested for their ability to develop into third-stage larvae (L3) in mosquitoes Aedes aegypti. Microfilariae less than three days old were not able to develop to L3. Microfilariae which had been in circulation for three days to six months were capable of developing if ingested.

Aedes↗

An improved technique for the cryopreservation of Onchocerca microfilariae.

Experimental describe the use of ethanediol to store Onchocerca microfilariae in liquid nitrogen. The technique involves a 2-stage incubation of the parasites at 37 degrees C and 0 degrees C in ethanediol, before rapid cooling to -196 degrees C. Viability has been assessed by motility, by migration in a proxy host and by development to the infective stage in the insect vector. A total of 71-79% of the cryopreserved microfilariae was shown to be viable compared to unfrozen controls. The technique is simple, inexpensive and very effective when compared to previous cryopreservation procedures for microfilariae and should be particularly suited for use under field conditions.

Animals↗

Resistance to Onchocerca lienalis microfilariae in mice conferred by egg antigens of homologous and heterologous Onchocerca species.

Embryonic stages of various Onchocerca species have been used to stimulate resistance in CBA mice to challenge injections with the microfilariae of Onchocerca lienalis. Comparable levels of resistance to challenge (29-37% reductions) were conferred by living, freeze-killed, or sonicated organisms administered with Freunds' Complete Adjuvant (FCA). Antigens extracted in saline, or with the detergent sodium deoxycholate, were also protective. Adjuvants enhanced the protective effect, particularly FCA (78% reduction), Freunds' Incomplete Adjuvant (74% reduction), aluminum hydroxide (70% reduction) and Bordetella pertussis (70% reduction). Detergent extracts prepared from intact embryos with n-octyl glucoside also stimulated significant levels of protection against microfilarial challenge when given with FCA (37-45% reductions). Levels of resistance induced by immunizations with intact organisms were greatest following subcutaneous (s.c.) injection over the neck or by intramuscular inoculation. Soluble extracts were also particially effective given by s.c. inguinal or intraperitoneal injection. A time-interval of greater than 3 weeks between the completion of immunization and challenge was required for the expression of immunity. Cross-protection against challenge with O. lienalis microfilariae was also afforded to mice by immunization with intact embryos or detergent extracts of Onchocerca gutturosa (45 and 34% reductions), Onchocerca gibsoni (66 and 47% reductions) or Onchocerca volvulus (58 and 41% reductions). It is concluded that the embryonic stages of both human and animal parasites provide a source of cross-protective antigens of value in studies on resistance to Onchocerca microfilariae in experimental hosts.

Animals↗

Density-dependent processes in the transmission of human onchocerciasis: intensity of microfilariae in the skin and their uptake by the simuliid host.

The transmission success of Onchocerca volvulus is thought to be influenced by a variety of regulatory or density-dependent processes that act at various points in the two-host life-cycle. This paper examines one component of the life-cycle, namely, the ingestion of microfilariae by the simuliid vector, to assess the relationship between intake of larvae and the density of parasites in the skin of the human host. Analysis is based on data from three areas in which onchocerciasis is endemic and includes published information as well as new data collected in field studies. The three areas are: Guatemala (Simulium ochraceum s.l.), West and Central Africa (savanna members of the S. damnosum complex), and South Venezuela (S. guianense). The data record experimental studies of parasite uptake by flies captured in the field and fed to repletion on locally infected subjects who harboured varying intensities of dermal microfilarial infection. Regression analyses of log transformed counts of parasite burdens ingested by the flies plotted against log transformed counts of microfilariae per mg of skin revealed little evidence for saturation in parasite uptake by the flies as the intensity in the human host increased. There was a positive and highly significant rank correlation between both variables for the three blackfly species. In an alternative analysis a model was fitted to data on prevalence of flies with ingested microfilariae (mff) versus dermal mean intensities. The model assumed an overdispersed distribution of the number of mff/fly and a given functional relationship between intake and skin load. The results of both approaches were consistent. It is concluded that parasite ingestion by the vector host is not strongly density dependent in the three geographical areas and ranges of dermal loads examined. It therefore appears that this transmission process is of reduced importance as a regulatory mechanism in the dynamics of parasite population growth.

Africa, Central↗

The morphological discrimination of microfilariae of Onchocerca volvulus from Mansonella ozzardi.

There is no published account which allows the morphological discrimination of microfilariae of Onchocerca volvulus and M. ozzardi from each other. However, they occur together in parts of Brazil and Venezuela, and presumably there is always the possibility that migration could establish new sympatric populations in the future. The objective of this study was to evaluate simple morphological characters that might be used for species-diagnosis of microfilariae. The conclusions were that the location of microfilariae in the blood or skin, the body size and the nucleation of the nerve ring are expected to be useful first indications of species identity, but cannot be used for confident diagnosis. The structure of the cephalic armature (stained with alcian blue) seems to be species specific, but is of limited application because it is often difficult to see. However, the pattern of nucleation of the tail (as expressed by the ratio of the length of the terminal nucleus compared with the length of the tail space) is distinctive and is expected to be diagnostic.

Animals↗

Effects of inhibitors of serine protease, phenoloxidase and dopa decarboxylase on the melanization of Dirofilaria immitis microfilariae with Armigeres subalbatus haemolymph in vitro.

The melanization of Dirofilaria immitis microfilariae in Armigeres subalbatus haemolymph in vitro is a two-step process. Firstly, the microfilariae are encased in a transparent capsule, then the capsule material is melanized later. Benzamadine HC1 and p-amidinophenylmethanesulfonyl fluoride, both serine protease inhibitors, inhibited the deposition of the transparent capsule material and melanization. Diethyldithiocarbamate, a phenoloxidase inhibitor, did not prevent the deposition of the transparent capsule material but it did inhibit melanization. m-Hydroxybenzylhydrazine, a dopa decarboxylase inhibitor, had no inhibitory action on the deposition of the transparent capsule material. However, the time for the transparent capsule to become melanized was delayed due to the presence of m-hydroxybenzylhydrazine in the test system. Immmunocytochemical localization showed that propheoloxidase and/or phenoloxidase was present in the transparent capsule material. These results suggest (1) that prophenoloxidase cascade is essential in the melanization of microfilariae in mosquitoes and its putative activation behaviour is similar to that of other insects and (2) that the deposition of transparent capsule material is the result of the activation of prophenoloxidase by serine proteases and the melanization of the transparent capsule material is due to the presence of active phenoloxidase in capsule.

Animals↗

Setaria digitata adult 14- to 20-kDa antigens induce differential Th1/Th2 cytokine responses in the lymphocytes of endemic normals and asymptomatic microfilariae carriers in bancroftian filariasis.

High titers of parasite antigen-specific IgG4 antibodies have been found to be circulating in the peripheral blood of chronic patients, asymptomatic microfilariae carriers, and endemic normals in bancroftian filariasis. But in contrast to this, the titers of antigen-specific IgG1, IgG2, and IgG3 isotype antibodies are much lower. Using soluble antigens of adult Setaria digitata, a cattle parasite which shows strong antigenic reactivity with filaria sera, we have identified, by immunoblot, 14- to 20-kDa antigens which are recognized only by the IgG4 isotype antibodies present in the sera of asymptomatic microfilariae carriers. These 14- to 20-kDa antigens, after fractionation by SDS-PAGE and transfer to nitrocellulose paper, when solubilized and tested in vitro, induced secretion of a higher quantity of IFN-gamma and a lower quantity of IL-4, IL-5, and IL-10 (differential Th1 and Th2 response) in the lymphocytes of endemic normals in comparison to what they induced in the lymphocytes of asymptomatic microfilariae carriers.

Adolescent↗

Transplacental transfer of rodent microfilariae induces antigen-specific tolerance in rats.

Microfilariae are the smallest form in the life-cycle of filarial nematode parasites. They are released by the adult female worms and migrate through the blood and extracellular fluids where they can be transmitted by vectors. A few reports have indicated the possibility of the transmission of microfilarial infection from mother to offspring. We have infected rats with adult females of the rodent filaria, Dipetalonema viteae, and report here that the transfer to D. viteae microfilaria does indeed occur through the placenta. Exposure to specific antigens early in development can readily induce immune tolerance. We observed that a state of reversible immune unresponsiveness occurred in rats as a result of pre- and post-natal exposure to microfilariae and this was associated with impairment of T-cell responses. The induction of tolerance allowed D. viteae infective larvae to reach maturity in the Fischer rat which is otherwise innately resistant to this practice.

Animals↗

Secretory acetylcholinesterase of Setaria cervi microfilariae and its antigenic cross-reactivity with Wuchereria bancrofti.

Setaria cervi, a bovine filarial parasite, secretes acetylcholinesterase during in vitro cultivation. A significant amount of enzyme activity was detected both in culture media and somatic extracts of different developmental stages of the parasite. The microfilarial stage showed a higher level of AChE activity than adult worms, with females being considerably more active than males. The secretory enzyme from microfilariae preferentially utilized acetylthiocholine iodide as substrate and showed two electrophoretically distinct isoforms in native PAGE. Secretory enzyme was purified from the excretory/secretory products of microfilariae using edrophonium chloride linked to epoxy-activated sepharose. Analysis of purified acetylcholinesterase by SDS-PAGE revealed the existence of two proteins of 75kD and 45kD under nonreducing conditions. These secretory enzymes are antigenic and cross-reactive with Wuchereria bancrofti-infected asymptomatic microfilaraemic human sera when tested by enzyme linked immunosorbent assay and immunoblotting. The secretory AChE(s) from S. cervi microfilariae may be utilized for diagnosis of early filarial infections.

Acetylcholinesterase↗

Loa loa microfilariae aspirated during oocyte retrieval.

Loa loa is a filarial parasite which is mainly confined to West and Central Africa. Although considered as mildly pathogenic for people living in endemic areas, its appearance in other places might be a cause for concern. We report here the unusual finding of Loa loa microfilariae in the follicular aspirate of a 35-year-old black women during oocyte retrieval for in-vitro fertilization. Most likely these microfilariae were aspirated, along with follicular fluid, from the small blood vessels around the ovarian follicles, which are frequently lacerated during ovarian puncture. Despite the spectacular microscopic view of vigorous moving worms trapped on the cumulus cells, the presence of Loa loa microfilariae does not seem to prevent fertilization and cleavage completely.

Adult↗

Onchocercidae (Nematoda: Filarioidea) Infecting Colombian Avian Hosts: Insight From Morphotypes of Microfilariae and Molecular Lineages.

Members of the Onchocercidae family are parasites that infect a wide range of tetrapods, including wild birds and other animals. In Colombia, studies on avian blood parasites have diagnosed infections with microfilariae in several species using light microscopy. However, no morphological or molecular analyses of these nematodes have been conducted to date. The present study examined samples from 3820 wild birds with material deposited in the biological collection of the Host-Parasite Relationship Study Group since 1999. Of these individuals, 142 (3.7%) were infected with microfilaria and were analyzed using morphological and morphometric measurements. Additionally, 55 samples with blood or tissue preserved in ethanol were analyzed for sequencing of nuclear marker 18S rDNA and mitochondrial markers 12S rDNA and COI. Morphological analyses showed infection by at least one of 13 designed morphotypes of microfilariae. Twenty-nine sequences were obtained (10 of COI, 6 of 12S, and 13 of 18S), corresponding to 21 molecular lineages associated with Onchocercidae. These sequences were associated with the subfamilies Lemdaninae and Splendidofilariinae, the genera Aproctella Cram, 1931, Splendidofilaria Skrjabin, 1923, and Eufilaria Seurat, 1921, and other sequences did not cluster within any genus for which sequences were available for a particular molecular marker. These findings establish the first framework of morphological and molecular diversity of avian Onchocercidae in the megadiverse Neotropical country Colombia, expanding the known distribution of several genera and highlighting the need for further sampling of adult filariids to refine taxonomic resolution.

Animals↗

Transovarial transmission of chikungunya virus by Aedes albopictus mosquitoes ingesting microfilariae of Dirofilaria immitis under laboratory conditions.

Female Aedes albopictus mosquitoes of the Miki strain were experimentally fed on defibrinated sheep blood containing 5 x 10(7) PFU of chikungunya virus and 20,000 microfilariae of Dirofilaria immitis per milliliter. Fully engorged mosquitoes transmitted the virus to a small percentage of the F1 progeny, but females of the F1 generation did not transmit the virus to the F2 progeny. The control mosquitoes that ingested the virus without microfilariae did not transmit the virus to their eggs, larvae, or pupae in the F1 or F2 generations. These results showed that A. albopictus of this strain that concurrently ingested the virus and microfilariae transmitted the virus by the transovarial route under experimental conditions.

Aedes↗

Immunological and ultrastructural aspects of the cell-mediated killing of Dirofilaria immitis microfilariae.

Neutrophils in the presence of serum from dogs with occult Dirofilaria immitis infections were shown to be cytotoxic to D. immitis microfilariae recovered from the blood of microfilaraemic dogs. This cytotoxicity was correlated with the presence of IgM and IgG antibodies on the cuticular surface of microfilariae incubated in the sera from occult dogs. Such antibodies were not observed on the surface of microfilariae incubated in sera from microfilaraemic or normal dogs. The neutrophil attack was directed at the cuticular crypts, at which sites the worms appeared to be structurally most vulnerable because of the absence of the outer layer of the cuticle. The IgM antibodies were shown to be bound preferentially to these sites. Our data suggested that the neutrophil-mediated toxicity involved both hydrogen peroxide release and degranulation.

Animals↗

Roles for both CD4+ and CD8+ T cells in protective immunity against Onchocerca lienalis microfilariae in the mouse.

Mice inoculated with microfilariae of the filarial nematode Onchocerca lienalis clear their parasites from the skin over a period of 3 to 4 months and are highly resistant to a challenge infection. The adoptive transfer of spleen cells at various time points following primary and secondary infections of mice shows that exposures of 50 days or greater are required for the generation of lymphocytes capable of transferring protection to naive recipients. This adoptive transfer of protection with spleen cells from infection-primed mice partitions with the T lymphocyte population. In contrast, the passive transfer of protection with spleen-derived B cells, or sera taken at various time points following infection was not achieved. Moreover, there was no detectable synergistic effect when B and T cells were co-administered to recipient animals. Depletion of CD4+ and CD8+ T cells with monoclonal antibodies shows that CD8+ T cells have some regulatory effect on parasite establishment early in primary infection, but this is later superseded by CD4+ T cell reactivity that is predominant both when primary infection microfilariae are cleared and also during resistance to reinfection. Measurement of cytokines in the sera of mice undergoing primary and secondary infections support a microfilariae-induced Th2 activity, with high levels of IL-5 that are sustained upon reinfection, and low levels of IFN-gamma that are negligible at the time when mice are most strongly immune.

Animals↗

The effects of H2 and non-H2 genes on the survival of Onchocerca lienalis microfilariae in the mouse.

The effect of H2 and non-H2 genes in a mouse model of protective immunity against Onchocerca lienalis microfilariae have been investigated. Non-H2 effects were determined using CBA, BALB/c, B10, SJL and TO strains. All were permissive for establishment of a primary infection with microfilariae, although significant differences in parasite recoveries were evident amongst the various strains. The effect of H2 genes upon a primary infection was investigated using H2 congenic B10 and BALB strains, B10, B10.S, B10.BR, B10.D2/n, BALB/c, BALB.B, and BALB.K. Significant H2 effects were seen among the relatively weak responder B10 strains, but were not present among the relatively strong responder BALB strains. These results support a dominant effect of non-H2 genes following primary exposure to microfilariae, and a 'fine tuning' effect of H2 genes that is apparent only in weaker responding strains. Upon reinfection of all the strains investigated, a gradation of protection was detected that appeared to be exclusively dependent upon H2.

Animals↗