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PubMed1986

Comparative studies on the melanization response of male and female mosquitoes against microfilariae.

The melanization response of adult male and female Aedes trivittatus and the black-eyed Liverpool strain of Aedes aegypti against intrathoracically inoculated Dirofilaria immitis microfilariae (mff) was assessed at 1, 3, and 5 days postinoculation (PI). The melanization reaction of males is significantly less effective than the response elicited by female mosquitoes. No mff in male A. aegypti and only 17% of mff recovered from A. trivittatus were fully melanized by day 5 PI compared with 80% and 100% complete melanization of recovered mff from A. aegypti and A. trivittatus females, respectively. A significantly greater percentage of mff retained their viability in males, and inoculation of heat-killed mff did not significantly increase the melanization response as compared with female mosquitoes. Males have significantly lower total hemocyte populations and hemolymph volumes than females, and the possible relationship of hemocyte numbers and reduced melanization capabilities in males is discussed.

Aedes↗
PubMedDate not supplied

Phenol oxidase activity in hemolymph compartments of Aedes aegypti during melanotic encapsulation reactions against microfilariae.

Monophenol oxidase (MPO) and diphenol oxidase (DPO) activity was assessed in hemocytes, cell-free plasma and complete hemolymph collected from Aedes aegypti Liverpool strain, intrathoracically inoculated with saline alone, immune activated by the inoculation of Dirofilaria immitis microfilariae (mff), and uninoculated. Enzyme activities between groups were compared using a radiometric hydroxylation assay (MPO) and a high pressure liquid chromatography with electrochemical detection assay (DPO). There were no significant differences in enzyme activity in hemocytes, cell-free plasma, and complete hemolymph between uninoculated and saline-inoculated controls. Both MPO and DPO activity of mosquito hemocytes and complete hemolymph from immune-activated mosquitoes were significantly increased at 12 and 24 h postinoculation as compared with the enzyme activity from saline-inoculated mosquitoes, but no significant increase in enzyme activity was detected in cell-free plasma from immune-activated mosquitoes. Increases of MPO and DPO activity in hemocytes and hemolymph following immune activation were proportional, thereby suggesting that a single enzyme might react with both monophenols and o-diphenols within the hemolymph of A. aegypti. Results also suggest that augmented phenol oxidase activity associated with melanotic encapsulation reactions is associated primarily with hemocytes.

Aedes↗
PubMed1989

The efficacy of 22,23-dihydroavermectin B1 (Ivermectin) acting singly or in combination with a benzodiazepine on microfilariae of Onchocerca species and Brugia pahangi (an in vitro study).

An in vitro study of the antinematodal action of two groups of compounds which act on the receptor complex of the inhibitory neurotransmitter, Gamma-aminobutyric acid (GABA) in mammalian systems is described. The compounds, Ivermectin and two benzodiazepines, Diazepam and a water soluble Midazolam were tested singly or in combination against two microfilarial parasites Onchocerca lienalis (closely related to Onchocerca volvulus) and Brugia pahangi. The combination of ivermectin and diazepam at a concentration of 0.1 microgram/ml and 33 micrograms/ml respectively achieved the same effect on microfilarial motility as when ivermectin was given at 1 microgram/ml alone or diazepam at 66 micrograms/ml alone. Similarly when the combination of ivermectin at 0.1 microgram/ml and midazolam at 10 micrograms/ml was used it achieved the same effect as ivermectin at 1 microgram/ml alone or midazolam at 33 micrograms/ml alone. This showed that both benzodiazepines had a synergistic effect on the activity of ivermectin. The microfilariae of B. pahangi were insensitive to both groups of compounds at all concentrations used.

Animals↗
PubMed1991

Onchocerca lienalis: comparison of techniques for the cryopreservation of microfilariae within skin-snips or free of host tissues.

Onchocera lienalis microfilariae (mf) were cryopreserved in liquid nitrogen within skin-snips using methanol as a cryoprotectant and their viability evaluated and compared to mf cryopreserved free of host tissues using ethanediol as a cryoprotectant. Despite an initial delay in emergence, the methanol technique did not significantly affect the total numbers of mf emerging from skin-snips of various sizes (3.3-59.81 mg) compared to untreated controls over a 6 h period. Following thawing, the initial motility index (MI) scores of mf cryopreserved by either method were not significantly different from untreated controls; however, over a period of 15 days in culture the MI scores of both cryopreserved groups showed a small but significant overall decline, with the methanol technique producing the lowest scores. These changes in motility levels correlated with the numbers of mf which developed to the infective stage following intrathoracic injection into Simulium ornatum, although this ability to develop was a much more sensitive measure of parasite viability; compared to untreated control recoveries of 3rd-stage larvae, 63.9-71.7% (ethanediol technique) and 34.2-36.9% (methanol technique) of this number were recovered from cryopreserved groups. There were no significant differences in the lengths of infective larvae recovered from the insect heads from each treatment group, nevertheless there were higher numbers of 2nd-stage larvae recovered from the cryopreserved groups compared to the untreated controls. The methanol technique has the advantage of being easier to carry out under field conditions, while parasite viability is significantly better using the ethanediol technique.

Animals↗
PubMed1988

Chemotherapy of Onchocerca lienalis microfilariae in mice: a model for the evaluation of novel compounds for the treatment of onchocerciasis.

The model of Onchocerca lienalis microfilariae (mf) injected into inbred CBA/Ca mice was studied for its usefulness as an additional primary/secondary drug screen for onchocerciasis. Invermectin, DEC, suramin, flubendazole, mebendazole, levamisole, Mel W, furapyrimidone, metrifonate, amoscanate and the new Ciba-Geigy compounds CGP 6140, CGP 20'376 and CGI 17658 all significantly reduced levels of mf at a dose of 5 X 100 mg/kg or less. An early dosing protocol, on days 3-7 after infection, was found to be generally more effective than dosing on days 11-15, followed by necropsy on day 18. In some cases there were important differences in levels of drug activity depending on whether the drug was administered by the subcutaneous or oral route, indicating that new compounds should be tested via both routes. Ivermectin was by far the most active compound examined, virtually clearing mf from the skin at a dose of 5 X 0.0063 mg/kg and producing a significant mf reduction (63.5%) at 5 X 0.0008 mg/kg following subcutaneous administration. In comparison, DEC was much less active, producing a 32.4% mf reduction at 5 X 25 mg/kg ranging up to a maximum of 72% reduction at 5 X 100 mg/kg. CGI 17658 was the most active compound examined next to ivermectin, almost 100% effective against skin mf at a dose of 5 X 6.25 mg/kg via the oral route while being less effective via subcutaneous administration (65% reduction). The lowest effective dose examined was 5 X 3.13 mg/kg (per os) which reduced mf levels by 64%. CGP 20'376 was also very active, resulting in a 46% (subcutaneous) and 62% (per os) reduction at a dose of 5 X 6.25 mg/kg. This mouse model has clearly identified all the known microfilaricides examined and also, to a lesser extent, those compounds considered to be principally macrofilaricides. We believe it has value as an additional drug screen for onchocerciasis, which will enable the evaluation of novel compounds against skin-dwelling Onchocerca mf at the primary/secondary level, providing complementary information to new in vitro screens using adult Onchocerca.

Administration, Oral↗
PubMed1988

Microfilaria density distribution in the human population and its infectivity index for the mosquito population.

A new method of computing the infectivity index of microfilariae (mf) for the mosquito population is proposed using the estimated mf density distribution in the human population. the observed density distribution is considered a compound of the Poisson and the gamma distributions. The former distribution describes the probability of a specimen containing a specified number of mf and the latter describes the density distribution of mf in the host population. The mf infectivity index is the probability that a blood meal will include at least 1 mf, conditional on the population-density distribution of mf as specified by the gamma distribution. Actual data indicate that this population-density-based infectivity index can be considerably different from the conventional index based on the survey-density distribution. The level of the carrier rate of mf in a survey is greatly influenced, apart from the sample variation, by the average volume of blood taken from each person. The rate computed on the estimated population-density distribution of mf is convertible to any base amount of blood.

Animals↗
PubMed1988

Brugia malayi microfilariae share epitopes with Aedes aegypti.

Shared antigens between Brugia malayi and Aedes aegypti were studied. The experiments carried out with sera from infected Mastomys natalensis indicated that an immunological response against A. aegypti antigens (Mr 185, 35, 32 kDa) appeared often when animals became microfilaraemic and increased progressively in intensity during the time-course of infection. Sera of animals immunized with B. malayi reacted with the crude extract of mosquitoes and conversely, antibodies from animals immunized with A. aegypti reacted with the surface of B. malayi microfilariae. The implications of these findings of the natural history of B. malayi infection are discussed.

Aedes↗
PubMed1990

Frequency distribution of lymphatic filariasis microfilariae in human populations: population processes and statistical estimation.

This paper uses simple mathematical models and statistical estimation techniques to analyse the frequency distribution of microfilariae (mf) in blood samples from human populations which are endemic for lymphatic filariasis. The theoretical analysis examines the relationship between microfilarial burdens and the prevalence of adult (macrofilarial) worms in the human host population. The main finding is that a large proportion of observed mf-negatives may be 'true' zeros, arising from the absence of macrofilarial infections or unmated adult worms, rather than being attributable to the blood sampling process. The corresponding mf distribution should then follow a Poisson mixture, arising from the sampling of mf positives, with an additional proportion of 'true' mf-zeros. This hypothesis is supported by analysis of observed Wuchereria bancrofti mf distributions from Southern India, Japan and Fiji, in which zero-truncated Poisson mixtures fit mf-positive counts more effectively than distributions including the observed zeros. The fits of two Poisson mixtures, the negative binomial and the Sichel distribution, are compared. The Sichel provides a slightly better empirical description of the mf density distribution; reasons for this improvement, and a discussion of the relative merits of the two distributions, are presented. The impact on observed mf distributions of increasing blood sampling volume and extraction efficiency are illustrated via a simple model, and directions for future work are identified.

Animals↗
PubMed1992

Age-related differences in parasitosis may indicate acquired immunity against microfilariae in cattle naturally infected with Onchocerca ochengi.

Onchocerca ochengi is a common parasite of cattle in savanna areas of West Africa and its adult stages inhabit intradermal nodules. We have exploited the accessibility of the macrofilariae to examine quantitative and qualitative differences in both the macro- and microfilariae (mff) in relation to age in naturally infected cattle. Autochthonous cattle at a site on the Adamawa plateau, North Cameroon, situated near a perennial Simulium damnosum s.l. breeding site, were examined in three age groups (1.5-2.5, n = 34; 3-5, n = 39 and greater than or equal to 8 years old, n = 21). Skin mff densities were assessed from ventral skin biopsies, total body counts of nodules were done by palpation and 1-4 nodules were excised from positive animals, collagenase digested and the numbers and condition of macrofilariae determined. Embryogenesis profiles (embryograms) were done on a representative number of female worms in each age group. The overall prevalence of infection in the study population was 71% for mff and 85% for nodules. Nodule prevalence increased significantly from the 1.5-2.5 to 3-5 years old groups, but no other inter-age prevalences were significantly different. While the mean number of nodules per animal increased with age, geometric means (gm) 3.7, 15.5 and 23.1, the skin mff density maximized in the middle age group and decreased significantly in the old animals (gm 15.8, 44.7 and 11.4 mff/g, difference between latter two parameters significant, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1995

Density-dependent processes in the transmission of human onchocerciasis: relationship between the numbers of microfilariae ingested and successful larval development in the simuliid vector.

A previous paper reported that the intake of Onchocerca volvulus microfilariae (mff) by different species of Simulium is essentially proportional to the parasite load in the skin of infected carriers. This paper examines the fate of the ingested mff in susceptible vectors to assess the relationship between parasite intake and infective larval output in blackfly species with and without well-developed cibarial armatures. Analysis is based on data from 3 onchocerciasis endemic areas: Guatemala (S. ochraceum s.l.), West Africa (S. damnosum s.l./S. sirbanum) and the Amazonian focus between South Venezuela and Northern Brazil (S. guianense and S. oyapockense s.l.). The data, which include published and unedited information collected in the field, record experimental studies of parasite uptake by wild flies maintained in captivity until the completion of the extrinsic incubation period. The relationship between L3 output (measured as the mean number of successful larvae/fly or, as the proportion of flies with infective larvae) and average microfilarial intake, was strongly non-linear. This non-linearity was best represented by a sigmoid function in case of armed simuliids (S. ochraceum s.l., S. oyapockense s.l.), or by a hyperbolic expression in that of unarmed flies (S. damnosum s.l., S. guianense). These results are compatible, respectively, with the patterns of 'initial facilitation' and 'limitation' described in culicid vectors of lymphatic filariases. A maximum mean number of 1-3 L3/fly was observed in all 4 vectors. It is concluded that O. volvulus larval development to the infective stage is regulated by density-dependent mechanisms acting at the early phase of microfilarial migration out of the blackfly's bloodmeal. Damage by the bucco-pharyngeal armature may also be density dependent. A hypothesis, based on this density dependence is forwarded to explain initial facilitation, so far only recorded in vectors with well-developed cibarial teeth. Our results provide quantitative support for the conjecture that chemotherapy alone is likely to have a greater impact on reducing onchocerciasis transmission in endemic areas where the main vector has a toothed fore-gut than in foci where the vectors have unarmed cibaria.

Africa, Western↗
PubMed1990

Hydrogen peroxide is the most toxic oxygen species for Onchocerca cervicalis microfilariae.

The toxicity of the active oxygen species hydrogen peroxide, superoxide radical, hydroxyl radical and singlet oxygen to microfilariae (mf) has been studied in vitro, using active oxygen-generating systems and scavengers/inhibitors. Mf viability was monitored by uptake of the radiolabel, [3H]2-deoxy-D-glucose. Hydrogen peroxide and singlet oxygen, but not superoxide radical or hydroxyl radical, are toxic for mf. Hydrogen peroxide was toxic for mf within 2 h at concentrations as low as 5 microM, an amount eosinophils have been shown to release in vitro (Weiss et al. 1986). Catalase and thiourea, but not inactivated catalase, superoxide dismutase (SOD), singlet oxygen scavengers, or hydroxyl radical scavengers, protected mf. Mf have relatively high levels of endogenous SOD but no measurable glutathione peroxidase and low levels of catalase when compared with other parasites (Callahan, Crouch & James, 1988). The low levels of hydrogen peroxide-scavenging enzymes correlate well with mf sensitivity to hydrogen peroxide and the protective effect of exogenous catalase.

Animals↗
PubMed2002

Bancroftian filariasis: a 13-year follow-up study of asymptomatic microfilariae carriers and endemic normals in Orissa, India.

The natural history of human filarial infections leading to development of disease has been a subject of intense debate. The models proposed so far have largely been based on cross-sectional data on microfilariae (Mf) and disease prevalence in filariasis endemic areas. In an attempt to study the parasitological and clinical consequences of filarial infection in Beldal (Orissa, India), an area endemic for Bancroftian filariasis, cohorts of 59 asymptomatic Mf carriers (AS) and 187 asymptomatic and amicrofilaraemic subjects or 'endemic normals' ('EN'), were followed-up and a fraction (73% and 46% respectively) re-examined after 13 years to monitor (a) Mf prevalence, (b) Mf density, (c) circulating filarial antigen (CFA) and (d) chronic disease manifestations. The Mf prevalence and density were also monitored in Mf carriers after 1 and 4 years. Both Mf prevalence and density decreased progressively in the cohort of Mf carriers over a period of 13 years in Beldal. Only 37% of them continued to be microfilaraemic and the Mf density in these subjects was only 10% of the original level. However, loss of circulating Mf in this cohort did not result in loss of CFA and 95% remained CFA positive regardless of Mf status. About 23% of males in the 'EN' cohort developed hydrocoele while only 5.7% of male Mf carriers, who were not treated with DEC, had developed hydrocoele after 13 years. A cohort of Mf carriers in another area, Jatni, was also examined after 10 years to study the parasitological and clinical outcome. In this area, about 59% of the Mf carriers continued to be microfilaraemic after 10 years. These results reveal that in Mf carriers adult filarial worms persist for several years and that loss of circulating Mf with or without chemotherapy with DEC (single 12-day course) does not influence adult worm survival. The findings have been discussed in the context of 'static' and 'dynamic' models describing the relationship between infection and disease in human filariasis.

Adolescent↗
PubMed1998

The relationship between microfilarial load in the human host and uptake and development of Wuchereria bancrofti microfilariae by Culex quinquefasciatus: a study under natural conditions.

The uptake of Wuchereria bancrofti microfilariae (Mf) by Culex quinquefasciatus and their development in relation to human Mf density were quantified by allowing a total of 1096 wild mosquitoes to feed on 13 volunteers sleeping under partially open bed-nets. For each volunteer, each hour between 18.00 and 06.00 h the Mf density in finger-prick blood was determined and engorged mosquitoes collected. Each hourly collection of mosquitoes was kept separately. Half of them was dissected within 18 h post-feeding for the presence of ingested Mf, the other half was reared for 12 days to allow for the development of L3 larvae. About 20% of the latter mosquitoes died during these 12 days and these harboured significantly more larvae than the surviving ones, which could be an indication of excess-mortality among heavily infected mosquitoes. Assuming that variability in Mf uptake and in the number of developed L3 larvae can be described by a negative binomial distribution, a maximum-likelihood procedure was applied to estimate the relationship between human Mf density and both the arithmetic mean Mf uptake and L3 development. Both were adequately described by a saturating hyperbolic function that significantly differed from linearity. The saturation level for Mf was estimated at 29 (CI: 20-54) and for L3 larvae at 6.6 (CI: 4.3-17.0). Next, the L3 yield was related to Mf uptake indicating that the W. bancrofti-C. quinquefasciatus complex shows 'limitation', i.e. a decreasing yield for an increasing uptake. Both the number of Mf ingested and the number of L3 larvae developing per mosquito were found to be highly aggregated, with the level of aggregation decreasing in a non-linear way with human Mf density.

Adolescent↗
PubMed1986

Stimulation of neutrophil respiratory burst and iodination reaction by opsonized microfilariae of Dirofilaria immitis.

It has been shown that the interaction of Dirofilaria immitis microfilariae (Mf) opsonized with sera from infected but amicrofilaraemic dogs (occult dogs) stimulated the respiratory burst and degranulation of neutrophils as measured by chemiluminescence and iodination. Sera from normal and microfilaraemic dogs gave either low level or non-significant reactions. Since the sera required were also those required for neutrophil-mediated cytotoxicity to D. immitis Mf in vitro, the results suggested that the products of oxygen reduction as well as the myeloperoxidase system could be involved in the killing of Mf by neutrophils. However, whether these pathways have a major role to play in neutrophil-mediated cytotoxicity to Mf is uncertain, as various chemical and enzymatic inhibitors of the products of the respiratory burst were unable to prevent or reduce cytotoxicity. Azide, which is a known inhibitor of the iodination reaction, also failed to reduce cytotoxicity.

Animals↗
PubMed1987

Complement activation by eggs and microfilariae of filarial parasites.

The complement of fresh normal rat serum was activated by filarial eggs and microfilariae (mf). C3 was deposited on the surface of Litomosoides carinii, Brugia pahangi, Brugia malayi and Dipetalonema viteae as seen by immunofluorescence. Intra-uterine and in vitro-derived mf did not bind C3. In contrast, C3 bound to the blood-derived mf of B. pahangi and B. malayi as well as exsheathed mf of L. carinii and B. malayi. Significant consumption of complement was observed with eggs of all filarial species, as well as sheathed mf of B. pahangi, B. malayi and exsheathed mf of L. carinii and B. malayi. These experiments indicated that complement was activated by filarial parasites via the alternative pathway. The bound complement promoted neutrophil-mediated adherence and cytotoxicity.

Animals↗
PubMedDate not supplied

Clearance of Brugia pahangi microfilariae in immunized mice.

Active Brugia pahangi microfilariae (Mf) were injected into naive male BALB/c mice intraperitoneally. Microfilaraemia was studied for 28 days; and Mf circulated in blood in optimum numbers from 3 to 14 days. Anti-Mf response was assessed by the rate of disappearance of Mf from blood as well as their absence from visceral organs. Sonicated antigens of Mf (MfE) and whole worm extract of adults (WWE) induced absolute protection against the establishment of Mf in mice. This potent anti-Mf response elicited by sonicated antigens was intense, rapid and withstood repeated challenge infection. In comparison, immunization with in vitro excretory, secretory and metabolic antigens of Mf (MfESM) produced a partial but significant level of protection. Sera collected periodically from immunized animals showed antibody by micro-ELISA compared to sham-vaccinated controls. When Mf were used as targets along with the peritoneal exudate cells in vitro, sera from mice immunized with MfE and MfESM showed about four-fold cellular adherence to (Mf-ADCA) and 10-fold killing (Mf-ADCC) of Mf which was antibody-dependent. Some degree of correlation was apparent when the antibody levels, Mf-ADCA/Mf-ADCC activity, and Mf clearance were compared. This murine microfilaraemia model was therefore shown to be suitable for studying the host-protective immune response against filarial parasites.

Animals↗
PubMed2002

Interleukin-4 influences the production of microfilariae in a mouse model of Brugia infection.

Sub-cutaneous infection of interleukin (IL)-4-/- mice on the BALB/c background with third stage larva (L3) of Brugia pahangi revealed an altered cytokine profile consistent with the absence of the Th2 promoting cytokine IL-4. Splenocytes from IL-4-/- mice secreted significantly more antigen (Ag)-specific IL-2 and interferon-gamma and significantly less Ag-specific IL-5, compared to those from L3-infected wild-type mice. However, levels of Ag-specific IL-13 were similar between groups. Despite the alteration in immune responses, there was no significant difference in recovery of developing worms from the peritoneal cavity of the two strains of mice at any time postinfection. However, at later time points of infection, the IL-4-/- mice contained large numbers of microfilariae (Mf) in the peritoneal cavity while the wild-type mice contained comparatively few Mf. The differences in Mf levels appear to relate to differences in worm fecundity in the two strains of mice, with adult female worms from the wild-type mice containing few developing Mf. Moreover, implantation of sexually mature adult female worms into the peritoneal cavity of both strains of mice resulted in equal levels of Mf, confirming that the primary role of IL-4 is to limit fecundity during the maturation phase of infection.

Animals↗
PubMed2001

Molecular cloning of a second prophenoloxidase cDNA from the mosquito Armigeres subalbatus: prophenoloxidase expression in blood-fed and microfilariae-inoculated mosquitoes.

Melanization constitutes an important component in various aspects of insect life, including cuticular sclerotization, egg-shell tanning, melanization of parasites and wound healing. Recently, a cDNA encoding prophenoloxidase (pro-PO), a key enzyme in the biosynthesis of melanotic material in insects, was cloned from microfilariae (mf)-inoculated mosquitoes, Armigeres subalbatus. However, results of Northern blot analyses indicated that two pro-POs might be present in Ar. subalbatus and these pro-POs might be responsible for two distinct physiological functions, egg-shell tanning and melanization of parasites. Subsequently, the second pro-PO cDNA (As-pro-PO II) was cloned from blood-fed Ar. subalbatus by rapid amplification of cDNA ends polymerase chain reaction. The 2210 bp As-pro-PO II cDNA contains a 41 bp 5'-non-coding region, a 2064 bp open reading frame and a 105 bp 3'-non-coding region. A hydrophobic signal peptide for endoplasmic reticulum targeting is not found in the N-terminal region. The deduced amino acid sequence of As-pro-PO II shares a high degree of identity (81.5%) with that of the As-pro-PO I obtained from mf-inoculated Ar. subalbatus. Both Northern blot and reverse transcription polymerase chain reaction analysis demonstrated that these two mosquito pro-POs are persistently expressed in mosquito haemocytes and not in fat body, midgut, or ovaries. The expression of As-pro-PO I and As-pro-PO II in mosquitoes is associated with melanization of mf and blood feeding, respectively.

Amino Acid Sequence↗