Malayi-infected Jirds Research Articles

Real-time PCR detection of the HhaI tandem DNA repeat in pre- and post-patent Brugia malayi Infections: a study in Indonesian transmigrants.

BackgroundLymphatic filariasis caused by Wuchereria bancrofti or Brugia spp. is a public health problem in developing countries. To monitor bancroftian filariasis infections, Circulating Filarial Antigen (CFA) test is commonly used, but for brugian infections only microfilariae (Mf) microscopy and indirect IgG4 antibody analyses are available. Improved diagnostics for detecting latent infections are required.MethodsAn optimized real-time PCR targeting the brugian HhaI repeat was validated with plasma from microfilariae negative Mongolian gerbils (jirds) infected with B. malayi. Plasma samples from microfilaremic patients infected with B. malayi or W. bancrofti were used as positive and negative controls, respectively. PCR results of plasma samples from a transmigrant population in a B. malayi endemic area were compared to those of life-long residents in the same endemic area; and to IgG4 serology results from the same population. To discriminate between active infections and larval exposure a threshold was determined by correlation and Receiver-Operating Characteristics (ROC) curve analyses.ResultsThe PCR detected HhaI in pre-patent (56 dpi) B. malayi infected jirds and B. malayi Mf-positive patients from Central Sulawesi, Indonesia. HhaI was also detected in 9/9 elephantiasis patients. In South Sulawesi 87.4% of the transmigrants and life-long residents (94% Mf-negative) were HhaI PCR positive. Based on ROC-curve analysis a threshold for active infections was set to >53 HhaI copies/μl (AUC: 0.854).ConclusionsThe results demonstrate that the HhaI PCR detects brugian infections with greater sensitivity than the IgG4 test, most notably in Mf-negative patients (i.e. pre-patent or latent infections).

Tetracycline treatment targeting Wolbachia affects expression of an array of proteins in Brugia malayi parasite

Wolbachia is an intracellular endosymbiont of Brugia malayi parasite whose presence is essential for the survival of the parasite. Treatment of B. malayi-infected jirds with tetracycline eliminates Wolbachia, which affects parasite survival and fitness. In the present study we have tried to identify parasite proteins that are affected when Wolbachia is targeted by tetracycline. For this Wolbachia depleted parasites (B. malayi) were obtained by tetracycline treatment of infected Mongolian jirds (Meriones unguiculatus) and their protein profile after 2-DE separation was compared with that of untreated parasites harboring Wolbachia. Approximately 100 protein spots could be visualized followed by CBB staining of 2-D gel and included for comparative analysis. Of these, 54 showed differential expressions, while two new protein spots emerged (of 90.3 and 64.4 kDa). These proteins were subjected to further analysis by MALDI-TOF for their identification using Brugia coding sequence database composed of both genomic and EST sequences. Our study unravels two crucial findings: (i) the parasite or Wolbachia proteins, which disappeared/down-regulated appear be essential for parasite survival and may be used as drug targets and (ii) tetracycline treatment interferes with the regulatory machinery vital for parasites cellular integrity and defense and thus could possibly be a molecular mechanism for the killing of filarial parasite. This is the first proteomic study substantiating the wolbachial genome integrity with its nematode host and providing functional genomic data of human lymphatic filarial parasite B. malayi.

Comparative study of hemolymph phenoloxidase activity in Aedes aegypti and Anopheles quadrimaculatus and its role in encapsulation of Brugia malayi microfilariae

Hemolymph phenoloxidase activity of sugar-fed and blood-fed females of Anopheles quadrimaculatus and Aedes aegypti showed similar characteristics. Phenoloxidase was present as an inactive proenzyme in both mosquito species and was partially activated during collection of the hemolymph. In both mosquito species, phenoloxidase activity was modulated by different buffers and activated phenoloxidase did not need Ca 2+. Enzymatic activity was higher in the hemocytes than in the plasma in both mosquito species. Trypsin, laminarin, and blood-feeding on uninfected and Brugia malayi-infected jirds enhanced hemolymph phenoloxidase activity in both mosquito species. The appearance of hemolymph phenoloxidase activity was inhibited by p-nitrophenyl p′-guanidinobenzoate HCl, soybean trypsin inhibitor, ethylenediaminetetraacetic acid, diethyldithiocarbamic acid, saturated 1-phenyl-2-thiourea and reduced glutathione, but not by benzamidine in A. quadrimaculatus. The appearance of hemolymph phenoloxidase activity was inhibited by benzamidine, diethyldithiocarbamic acid, saturated 1-phenyl-2-thiourea, reduced glutathione, β-nitrophenyl p′-guanidinobenzoate and soybean trypsin inhibitor, but not by ethylenediaminetetraacetic acid in A. aegypti. It is suggested that in both mosquito species, blood-feeding and migration of sheathed microfilariae in the homocoel activated the prophenoloxidase in the hemolymph and caused the encapsulation and melanization of microfilarial sheaths and microfilariae of B. malayi.

Characterization of the Intracellular Melanization Response in Anopheles quadrimaculatus against Subperiodic Brugia malayi Larvae

Intracellular melanization, a defense or an immune response in the thoracic muscle cells, was investigated in a refractory strain of Anopheles quadrimaculatus infected with larvae of Brugia malayi. In mosquitoes fed on B. malayi-infected jirds, intracellular melanization against first-stage larvae (L1) was better expressed when fewer than 40 microfilariae reached the thoracic muscle cells than when more than 40 microfilariae reached the thoracic muscle cells. This result suggests that when large numbers of microfilariae invade the thoracic muscle cells, the immune response of the mosquito may become overloaded. Intracellular melanization response against L1 in the thoracic muscle cells also showed a significant decrease in older females (14-16-day-old) as compared to the younger ones (4-9-day-old). A comparison is made between intracellular and extracellular responses of mosquitoes to filarial larvae. It is significant that in both cases high rate of infection can reduce both the number and percentage of larvae melanized.

Nutritional Factors and Antimicrobials on Development of Infective Larvae of Subperiodic Brugia malayi (Nematoda: Filarioidea) in Anopheles quadrimaculatus and Aedes aegypti (Diptera: Culicidae)

The effects of nutritional factors and antimicrobials on the development of infective larvae of subperiodic Brugia malayi in susceptible Anopheles quadrimaculatus Say and Aedes aegypti (L.) were investigated. Larvae of both species of mosquitoes were reared on brewers yeast or a 1:1 brewers yeast-liver powder mixture. After emergence, one-half of the adults from each rearing condition were maintained on a 10% sucrose solution and the other half on a 10% sucrose solution containing 0.1% p-aminobenzoic acid (PABA). Females of both species of mosquitoes that fed on B. malayi-infected jirds showed a significant increase in the ineffective rate and intensity of infectiveness when the mosquito larvae were reared on the brewers yeast-liver powder diet. The addition of 0.1% PABA to the adult diet increased numbers of infective larvae of B. malayi that developed in Ae. aegypti but not in An. quadrimaculatus. The intensity of infectiveness of B. malayi was significantly greater when An. quadrimaculatus females were provided with a second blood meal from an uninfected jird and when females of both species were maintained on different concentrations of an antimicrobial solution.

In vitro development of Brugia pahangi and Brugia malayi in cultured mosquito thoraces

In vitro cultivation of Brugia pahangi and subperiodic Brugia malayi one-day old larvae to infective stage larvae (L 3) within thoraces excised from Aedes aegypti (Black Eye, Liverpool) and Anopheles quadrimaculatus was attempted. The mosquito thoraces were excised under aseptic conditions, 24 h after a blood meal on either B. pahangi- or B. malayi-infected jirds. The excised thoraces were washed aseptically and inoculated into a diphasic media. A nutrient agar base was overlaid with either Grace's insect cell culture medium or Schneider's Drosophila medium or a mixture (1:1) of these two media. Each overlay medium contained a 1 × concentration of antibiotic/antimycotic mixture plus 20% fetal bovine serum. The excised thoraces provided the intracellular milieu for development of Brugia larvae. In Grace's or Schneider's insect tissue culture medium alone, the filaria larvae of both species developed only to the second larval stage after 12 days; whereas, in a mixture (1:1) of Grace's and Schneider's media, some one-day old larvae of both Brugia species developed to the infective larval (L 3) stage after 12 days. However, large numbers of both species of larvae developed to the infective larval stage when, prior to providing an infective blood meal, the mosquitoes of both species were fed 1 × concentration of antibiotic/antimycotic mixture in a 10% sucrose solution containing 0.1% p-aminobenzoic acid for 6 days. These results showed for the first time that if one-day old Brugia larvae are confined intracellularly in excised thoraces, they can then develop in insect tissue culture media without adding a feeder layer of mosquito cells or conditioning the media with mosquito cell lines.

A rapid DNA assay for the species-specific detection and quantification of Brugia in blood samples

This report describes a new assay for detecting filarial parasites of the genus Brugia in blood samples using labeled DNA probes. The sequences of these DNA probes are based on the HhaI repeat DNA family found in the genus Brugia. These DNA probes are species-specific and can detect the DNA from a single microfilaria in hybridization assays. To adapt this test for use on blood samples collected in the field, complex steps to separate microfilariae from blood cells and to purify parasite DNA were eliminated. We found that the most effective method was to filter blood samples through 5.0 μm pore nitrocellulose membranes, lyse the microfilariae on the membranes by proteinase K digestion, denature the parasite DNA with sodium hydroxide, and hybridize with the DNA probe. With this method, individual microfilarie can be visualized and counted on autoradiograms. The assay was evaluated in a mock field study using Brugia malayi-infected jirds and was found to be an efficient and accurate method for quantifying microfilariae in blood samples.

Use of Parasite Antigen Detection to Monitor Macrofilaricidal Therapy in Brugia malayi-Infected Jirds

Improved methods are needed to evaluate new treatments for filarial infections. We have recently developed a monoclonal antibody-based enzyme immunoassay to detect circulating parasite antigen in sera from Brugia malayi-infected jirds. In the present study, parasite antigen levels were compared to parasitological parameters after treatment of B. malayi-infected jirds with CGP 20376 that has been reported to be active against both microfilariae and adult worms of this parasite. Microfilariae were cleared promptly and permanently after CGP 20376 treatment, and no adult worm was recovered in jirds at necropsy 20 wk after treatment. In contrast, untreated animals had sustained microfilaremia throughout the course of the study, and adult worms were recovered in all control animals (mean worm recovery; 24.3 +/- 7.8 SE). Parasite antigen was present in sera from all infected animals before treatment. Parasite antigen titers in sera were unchanged 5 wk after treatment but fell to undetectable levels in 4 of 6 animals by 20 wk after treatment. Low-level antigenemia was detected in 2 of 6 animals at 20 wk, perhaps suggesting incomplete killing of parasites or incomplete clearance of antigen. Parasite antigen levels were stable throughout the study in control animals. These preliminary results suggest that parasite antigen detection is useful as a means of noninvasively monitoring the efficacy of anti-filarial drug therapy.

Brugia malayi: Microfilarial polyunsaturated fatty acid composition and synthesis

The polyunsaturated fatty acid composition of Brugia malayi microfilariae was analyzed by gas chromatography and compared to that of sera from B. malayi-infected jirds. The essential fatty acid, linoleic acid (18:2ω6), was the most abundant fatty acid present in both microfilarial total lipids and phospholipids as well as in jird sera. In contrast, arachidonic acid (20:4ω6), as well as the 18:3ω6, 20:2ω6, and 20:3ω6 intermediates that are formed in the enzymatic conversion of linoleic acid to arachidonic acid, were proportionally more abundant in microfilariae than in jird sera. To assess the capacity of microfilariae to transform linoleic acid into arachidonic acid, B. malayi microfilariae were incubated with [ 14C]linoleic acid. Microfilarial lipids were extracted and resolved by high-pressure liquid chromatography and thin-layer chromatography. A portion of [ 14C]linoleic acid incorporated by microfilariae was converted to [ 14C]arachidonic acid. Thus, microfilariae can not only incorporate exogenous arachidonic acid, as previously demonstrated, but can also synthesize arachidonic acid from exogenous linoleic acid. The capacity of microfilariae to utilize specific host polyunsaturated fatty acids raises the possibility that intravascular filarial parasites may synthesize eicosanoid metabolites of arachidonic acid that could mediate filarial-host cell interactions.

Brugia malayi: Detection of parasite antigen in sera from infected jirds

Sera from Brugia malayi-infected jirds were demonstrated to contain a heat-stable, 95- to 105-kDa parasite antigen by immunoblot with rabbit antibody to the parasite and with a monoclonal antibody that binds to phosphorylcholine. This antigen is a major component of B. malayi adult worm excretory/secretory antigen, and it is present in lavage fluid obtained from ip-infected animals. The antigen was detected by enzyme immunoassay in all sera collected from jirds 9–54 weeks after sc injection with 100 or 300 infective larvae (L 3). Parasite antigen titers were higher in animals infected with the higher L 3 dose. Antiphosphorylcholine antibodies were present in jird sera for the first 12 weeks after larval injection, but thereafter, antibody titers decreased to undetectable levels. Parasite antigen was not detected by immunoblot or enzyme immunoassay in sera from 21 human subjects with B. malayi microfilaremia. Antigen may be cleared from human sera by antiphosphorylcholine antibodies, which were present in all sera tested. The practical significance of B. malayi antigen detection in the jird is that it provides a sensitive means of noninvasively monitoring the status of infection in this important experimental filariasis model.

Identification of Phosphorylcholine Epitope-Containing Antigens in Brugia Malayi and Relation of Serum Epitope Levels to Infection Status of Jirds with Brugian Filariasis

The Gib 13 monoclonal antibody was raised against eggs of Onchocerca gibsoni and subsequently found to react with a phosphorylcholine epitope designated as the T15 idiotype. Since an immunoradiometric assay based on the Gib 13 monoclonal antibody holds promise for serodiagnosis of filariasis, the goals of the current study were to evaluate phosphorylcholine epitope production and release by various parasite stages and to assess changes in serum epitope levels during different phases of Brugia malayi infection in jirds. Extracts of B. malayi adult male worms, female worms, and microfilariae contained Gib 13 monoclonal antibody-reactive antigens of Mr 25-30,000, 57-90,000, and approximately equal to 200,000. Adult female worms secreted ten-fold more epitope than microfilariae on a weight basis. Phosphorylcholine-containing antigens were localized in female and male worms, respectively, in egg-bearing regions and the intestines. Assessment of the relationship between serum levels of Gib 13 antibody-binding epitope and parasitologic status of B. malayi-infected jirds showed that the immunoradiometric assay distinguishes patent infected from uninfected control animals, detects a significant rise in epitope level during the prepatent phase of infection, and is unaffected by diethylcarbamazine-induced reduction in the intensity of microfilaremia. There was a direct positive correlation between serum epitope level and female adult worm load. Quantification of serum phosphorylcholine epitope of the T15 idiotype may be useful as an indirect measure of parasite burden in humans with lymphatic filariasis that is independent of microfilaremia.

Antibody response to heterologous and homologous antigens in Brugia malayi- and B. pahangi-infected Mongolian jirds as measured by the enzyme-linked immunosorbent assay (ELISA).

Sequentially collected sera from Mongolian jirds (Meriones unguiculatus) infected with Brugia malayi and B. pahangi were tested for antibodies to homologous and heterologous antigens by the enzyme-linked immunosorbent assay (ELISA). Titers were less than 1:100 prior to infection and rose rapidly (within 2 weeks). Peak titers were observed prior to patent microfilaremia, and high titers persisted during infection. Use of the homologous antigen did not increase sensitivity or specificity of the ELISA. In fact, B. malayi-infected jirds demonstrated higher titers to the heterologous antigen, B. pahangi, than to the homologous antigen. Fractionation of B. malayi antigen over a wide pH range using isoelectric focusing did not eliminate cross-reactions, but the reactions of 20 B. malayi sera and 20 B. pahangi sera tested more strongly to specific fractions, particularly in the lower pH range.