Separation of viable microfilariae free of blood cells on Percoll gradients

A consistent and reproducible method is described for isolating pure populations of microfilariae of Litomosoides carinii, Brugia pahangi, B. malayi and Dipetalonema viteae, free of cells, from blood, by density gradient centrifugation on Percoll in 0.25 M sucrose. The recovery of the microfilariae was 85 to 97%.     

Time courses of antibody levels in Mastomys natalensis after infections with Litomosoides carinii, Dipetalonema viteae, Brugia malayi or B. pahangi, Determined by ELISA

Using a Litomosoides carinii adult antigen, time courses of antibody levels were followed by an ELISA in L. carinii, Dipetalonema viteae, Brugia malayi and B. pahangi infected Mastomys natalensis. Using various groups of infected animals, periods up to 400 days after infection were covered. In L. carinii infected Mastomys, antibodies were first detected 11 days p.i. and levels increased rapidly until day 40. Temporarily reduced levels about the beginning of patency were followed by increasing values until about 100 days p.i. Then the antibody content of the sera remained more or less constant until about 250 days p.i. although maximum levels were found at day 170. Thereafter, the antibody concentration in the sera declined slowly but high levels were still observed 390 days p.i. The antibody content was usually higher in animals with high microfilariae densities than in those with low microfilariae counts but relations could not be proven statistically. In D. viteae infected Mastomys, maximum antibody values were reached within the beginning of patency. Levels were not altered markedly until about 110 days p.i. Thereafter they decreased slightly but then remained constant until the end of the investigation period 350 days p.i. B. malayi infected animals showed a rapid increase of the antibody content in the sera; a maximum was reached by 20 days after the infection. Thereafter, somewhat constant levels were found for 4--5 months. After 300 days p.i. the antibody levels declined progressively, accompanied with increasing parasitaemia densities; after 380 days the levels reached about 2/3 of the maximum. However, despite this, no relation was found between the levels of parasitaemia and antibody in individual animals. In B. pahangi infections the main prepatent antibody increase occurred during week 5 p.i., when maximum values were observed. The beginning of patency and the early patency were accompanied with slightly declining antibody levels. From 150 days p.i. until the end of the investigation 400 days p.i., the antibody content of the sera was fairly constant.     

Comparative utilization of pyruvate by Brugia pahangi, Dipetalonema viteae, and Litomosoides carinii

The metabolism of pyruvate by the adult filarial parasites Brugia pahangi, Dipetalonema viteae, and Litomosoides carinii has been compared. Istopic carbon-balance studies indicate the presence of significant pyruvate dehydrogenase activity in L. carinii but little or no activity in either B. pahangi or D. viteae. In all 3 helminths, the quantities of pyruvate that were completely oxidized to CO2 and water were very small. The activities of some of the tricarboxylic acid cycle enzymes of B. pahangi also were determined. In particular, a relatively low level of isocitrate dehydrogenase was noted in the mitochondria of B. pahangi. It is suggested that the tricarboxylic acid energy generating pathway is of doubtful importance as an energy yielding pathway in any of these parasites.     

Studies of potential filaricides: Part 14--Activity of 1-iso-butoxycarbonyl-4-methylpiperazine against experimental filariasis

The synthesis and filaricidal activity of 1-iso-butoxycarbonyl-4-methylpiperazine against Litomosoides carinii in Sigmodon hispidus and Dipetalonema viteae in Mastomys natalensis is reported. At an intraperitoneal or oral dose of 3 mg/kg given for 6 days, the compound removed 91% of the circulating microfilariae but had no effect on adult L. carinii. However, it killed all microfilariae and adults of D. viteae at a subcutaneous dose of 50 mg/kg given for 6 days. The compound also possessed chemoprophylactic activity against the larvae of L. carinii and D. viteae at a dose of 30 and 50 mg/kg respectively.     

Detection of antibodies in Brugia pahangi-infected cats by counter immunoelectrophoresis, indirect fluorescent antibody test and enzyme-linked immunosorbent assay

In cats infected with Brugia pahangi, antibodies first appeared against the larvae (L3), then against the adults (L5) and the microfilariae (mf). Homologous antigens were better than antigens prepared from heterologous species (Dirofilaria immitis, Dipetalonema viteae, Litomosoides carinii and Onchocerca gutturosa) in detecting antibodies to B. pahangi in the infected cats by indirect fluorescent antibody test (IFAT). Metabolic products of L5, but not L3 or mf, of B. pahangi were antigenic and were used in the enzyme-linked immunosorbent assay (ELISA) for detection of antibodies. Using various homologous antigens, IFAT was found to be more sensitive than counter immunoelectrophoresis and ELISA in the detection of antibodies in the infected cats. The best antigen was cryosections of L3, with a positivity rate of 81%. However, using L3, L5 and mf antigens in IFAT, a total positivity of 97% was obtained.     

The carbohydrate metabolism of Brugia pahangi microfilariae.

Evidence is presented that the microfilariae of Litomosoides carinii, Dipetalonema viteae and Brugia pahangi have an aerobic requirement for motility, but possibly not for survival. In addition, the data suggest that in an in vitro anaerobic environment, B. pahangi microfilariae ferment glucose only as far as lactate. In an aerobic environment, however, the data are consistent with a portion of glucose being dissimilated via a one step oxidative decarboxylation of pyruvate formed from glycolysis to acetate and CO2. In addition, a low level of complete oxidation, possibly via a tricarboxylic acid cycle pathway, may be occurring. Finally, if B. pahangi microfilariae are immobilized with levamisole in an aerobic atmosphere, the drug appears to alter the aerobic glucose metabolism of the parasite both qualitatively and quantitatively. A decreased glucose utilization occurs, together with a shift to a more nearly homolactate fermentation. It is suggested that the effects of levamisole on the metabolism of the microfilariid are secondary to the observed paralysis.     

Brugia malayi and Brugia pahangi: transmission blocking activity of ivermectin and brugian filarial infections in Aedes aegypti

Brugia malayi- or Brugia pahangi-infected, microfilaremic jirds (Meriones unguiculatus) were treated with ivermectin at a single dose of 200 micrograms/kg body weight, administered subcutaneously. After different time intervals, Aedes aegypti mosquitoes were fed on treated or untreated jirds. Sausage stage, L2, and L3 larvae failed to develop in mosquitoes that fed on jirds from 15 to 30 days post-treatment. After 1 month, the numbers of L3 larvae recovered from mosquitoes fed on treated B. pahangi jirds were comparable to controls. However, the number of L3's recovered from mosquitoes fed on B. malayi jirds remained significantly lower than controls, 2 and 3 months after treatment. This reduction suggests that ivermectin may be more effective in blocking transmission of B. malayi than B. pahangi. Ivermectin treatment had no effect on the mean number of circulating microfilariae in treated jirds. Therefore, mosquitoes ingested comparable numbers of microfilariae when compared to those mosquitoes fed on untreated controls. Only in the case of jirds infected with B. malayi did the circulating microfilarial counts fall 30 days after treatment. The failure of microfilariae to develop to the L3 stage in mosquitoes fed on jirds within 30 days of treatment was not due to failure of mosquitoes to ingest microfilariae. Brugia malayi microfilariae also failed to develop to L3 in mosquitoes that were allowed to feed on microfilaremic jird blood treated with ivermectin (50 ng/ml) in vitro, indicating its efficacy at low concentrations. In addition to N-acetyl glucosamine, microfilariae obtained for a period of 15 days from ivermectin-treated but not control jirds showed D-mannose, N-acetyl galactosamine, and L-fucose moieties on the surface of the sheath.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies with Brugia pahangi 17. The anthelmintic effects of diethylcarbamazine.

Diethylcarbamazine (DEC) was active in vitro against infective larvae and microfilariae of Brugia pahangi but only at high concentrations. When fed to mosquitoes which were infected with B. pahangi it had little or no activity. In jirds it was inactive against B. pahangi microfilariae and adults when administered at 300 mg/kg for 5 days either by the intraperitoneal or oral route. In cats given 25 or 50 mg DEC/kg intraperitoneally on 3 or 5 occasions it was not microfilaricidal, but most of the adult worms died within 30 days of the end of treatment. Although most microfilariae disappeared from the blood of cats immediately (i.e., within an hour) after treatment, they reappeared within a few hours in the same numbers. Microfilarial levels were reduced after treatment but there was no precipitate decline as occurs in human B. malayi patients.     

Brugia malayi and Brugia pahangi: inherent difference in immune activation in the mosquitoes Armigeres subalbatus and Aedes aegypti

The inherent ability of Brugia malayi and Brugia pahangi (Nematoda) to establish successful relationships with the mosquitoes Armigeres subalbatus and Aedes aegypti Liverpool strain was evaluated. Brugia pahangi microfilariae (mff) avoided the immune response and developed normally in A. subalbatus exposed to the parasite by an infective bloodmeal, whereas nearly 85% of B. malayi were destroyed by the immune response. Because A. aegypti supports the development of both filarial worm species but destroys intrathoracically inoculated B. pahangi isolated from jird blood, blood-isolated B. malayi were inoculated into A. aegypti, and the immune response was compared with that observed against B. pahangi. The response against B. malayi was significantly more rapid and effective than the response against B. pahangi. Similar results were obtained when blood-isolated B. pahangi or B. malayi were inoculated into A. subalbatus. Microfilariae of both species were able to avoid immune destruction in A. aegypti if they were allowed to penetrate the Liverpool midgut in vitro prior to inoculation. Most B. pahangi that had first penetrated an Armigeres midgut prior to inoculation into A. subalbatus were able to avoid the immune response, but by day 3 postinoculation, less than 40% of the B. malayi, treated in the same manner, were able to escape the immune response. Genetic susceptibility of mosquitoes to infection by filarial worms and potential mechanisms of immune evasion/suppression are discussed regarding B. malayi and B. pahangi.     

A direct fluorescence technique for the rapid detection of sheathed microfilariae in blood smears

Thick and thin blood smears containing microfilariae of Wuchereria bancrofti, Loa loa, Brugia malayi, Brugia pahangi, Brugia patei or Acanthocheilonema vileae were prepared from either cryopreserved blood samples or from freshly collected blood, fixed in methanoi and treated with a fluoresceinated lectin wheat germ agglutinin. Sheathed microfilariae of W. bancrofti, L. loa, B. malayi, B. pahangi and B. patei in the blood smears could be easily detected and counted using a fluorescence assay. The unsheathed microfilaria of Acanthocheilonema viteae did not fluoresce. The possibility of adapting this technique, which does not require the use of parasite specific antibody for the sensitive, parasitological detection offilarial infections, is discussed.     

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.     

Brugia pahangi: production of a monoclonal antibody reactive with the surface of infective larvae.

Monoclonal antibodies against infective third-stage larvae (L3) of Brugia pahangi were generated from mice immunized with L3 antigens. The monoclonal antibodies were L3 stage-specific or stage-nonspecific. A BpG1 monoclonal antibody (IgG1 subclass) showing L3 stage-specificity was examined in detail. BpG1 recognized the surface of B. pahangi L3 and also reacted with the surface of Brugia malayi L3 but not with the surface of filarial worms of other genera, such as Acanthocheilonema viteae and Litomosoides carinii. BpG1 promoted cellular adhesion to the surface of B. pahangi L3. BpG1 bound on living L3 was shed but the shedding rate was relatively slow. The surface antigen recognized by BpG1 had a molecular weight of 58 kDa. It was stable to heat and periodate treatments but sensitive to trypsin digestion and was released from living L3 by SDS but not by Triton X-100 or CTAB. Preincubation of L3 with BpG1 significantly reduced the recovery rate of worms compared with the preincubation with a monoclonal antibody (IgG1 subclass) against the inner tissues of B. pahangi L3 or control supernatant of P3U1 myeloma cells. This result suggests that the antigen containing the BpG1 epitope may be one of the targets of a protective immune response against Brugia infection.     

Dipetalonema viteae and Brugia pahangi transplant infections in gerbils for use in antifilarial screening

Transplanted infections of Dipetalonema viteae and Brugia pahangi have been evaluated as tools for experimental chemotherapy. Attempts were made to establish these filariae in similar pharmacokinetic sites within the same host, so that direct comparisons of in vivo drug susceptibilities could be made. Unfortunately, it was not possible to establish B. pahangi in the subcutaneous tissues, the preferred site of D. viteae. Therefore, intraperitoneal B. pahangi and subcutaneously implanted D. viteae in gerbils were used for the study. D. viteae infections were significantly enhanced by concomitant infections with B. pahangi, while B. pahangi infection rates were unaffected by the presence of D. viteae. Experiments with amoscanate, CGP6140 and Mel W demonstrated the importance of employing both B. pahangi and D. viteae for antifilarial discovery work and the fundamental effect of parasite location on drug efficacy. D. viteae rapidly migrate from the peritoneal cavity of gerbils following implantation; twenty one hours after infection 73% of transplanted worms were found in the subcutaneous tissues. It was shown that the migration response could be used as a stringent parameter for demonstrating antifilarial activity. D. viteae were exposed to antifilarial drugs for 24 hours in vitro, washed and implanted into the peritoneal cavity of gerbils. At autopsy, 5 days later, 10(-8)M ivermectin and milbemycin D had prevented migration; CGP6140, amoscanate, suramin, flubendazole and furapyrimidone were also detected at less than 10(-6)M using this parameter. In all cases the migration response was more sensitive to drugs than parasite kill. Ivermectin's ability to inhibit worm migration through the tissues is discussed, with respect to the role of itinerant males in the reproductive cycle of Onchocerca volvulus.     

Filaricidal efficacy of anthelmintically active cyclodepsipeptides

PF 1022A, a novel anthelmintically active cyclodepsipeptide, and Bay 44-4400, a semisynthetic derivative of PF 1022A were tested for filaricidal efficacy in Mastomys coucha infected with Litomosoides sigmodontis, Acanthocheilonema viteae and Brugia malayi. The parent compound PF 1022A showed limited anti-filarial efficacy in L. sigmodontis and B. malayi infected animals. Oral doses of 5 x 100 mg/kg on consecutive days caused only a temporary decrease of microfilariaemia levels. By contrast, Bay 44-4400 was highly effective against microfilariae of all three species in single oral, subcutaneous and cutaneously applied (spot on) doses. Minimum effective doses (MED, reducing parasitaemia density by > or =95%) determined 3 and 7 days after treatment were 3.125-6.25 and 6.25-12.5mg/kg, respectively. Using the spot on formulation, doses of 6.25mg/kg (L. sigmodontis), 12.5mg/kg (A. viteae) and 25mg/kg (B. malayi) were required to cause reductions of microfilaraemia levels by > or =95% until day 56. Adulticidal effects, determined as minimum curative doses (MCD, eliminating adult parasites within 56 days by >95%) after single dose treatment were limited to A. viteae (MCD, 100mg/kg independent of the route of administration). Repeated oral treatment (100mg/kg on 5 consecutive days) killed all adult L. sigmodontis but did not affect B. malayi. However, single doses of 6.25 and 25mg/kg resulted in severe pathological alterations of intrauterine stages of L. sigmodontis and B. malayi, respectively. These alterations may be responsible for long-lasting reductions of microfilaraemia even when curative effects could not be achieved.     

Differentiation of species and life cycle stages of Brugia spp. by isoenzyme analysis

The isoenzyme patterns of glucose phosphate isomerase and phosphoglucomutase of 3 species of Brugia, B. pahangi, subperiodic B. malayi, and B. patei, and 3 life cycle stages, adult, third-stage larva, and microfilaria were compared using the technique of isoelectricfocusing on polyacrylamide gels. The results demonstrated that the adults of all 3 species could be identified from one another and that differences existed between the sexes of any one species. Hybridization between B. pahangi and B. patei could be detected in the progeny of the cross. Both the third-stage larvae and microfilariae of B. malayi and B. pahangi were differentiated and the epidemiological significance and the application of these findings to arthropod-borne filarial infections were discussed.     

Nitric oxide synthase in filariae: demonstration of nitric oxide production by embryos in Brugia malayi and Acanthocheilonema viteae

The radical gas nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) from l-arginine and molecular oxygen. Nitric oxide is an important signaling molecule in invertebrate and vertebrate systems. Previously we have shown that NOS is localized to more tissues in Brugia malayi than has been reported in Ascaris suum. In this paper, we analyze the distribution of NOS in Acanthocheilonema viteae, a filarial nematode that differs from B. malayi in that A. viteae females release microfilariae without a sheath. A. viteae is also one of a few filarial parasites without the Wolbachia intracellular endosymbiont. By use of a specific antibody, NOS was demonstrated in extracts of A. viteae and Dirofilaria immitis. The localization pattern of NOS in A. viteae was similar to that seen in B. malayi, with the enzyme localized to the body wall muscles of both sexes, developing spermatozoa, intrauterine sperm, and early embryos. By use of DAF-2, a fluorescent indicator specific for nitric oxide, the embryos of B. malayi and A. viteae were demonstrated to produce NO ex utero. The near identical staining patterns seen in A. viteae and B. malayi argue that NO is not produced by Wolbachia, nor is it produced by the nematodes in response to the infection. Localization of NOS to the sperm of filarial nematodes suggests a role for NO during fertilization as has been described for sea urchin and ascidian fertilization. Demonstration of the activity of embryonic NOS supports our earlier hypothesis that NO is a signaling molecule during embryogenesis in filarial nematodes.     

Zoonotic Brugia pahangi filariasis in a suburbia of Kuala Lumpur City, Malaysia

Five local Malaysian patients with clinical manifestations consistent with lymphatic filariasis were referred to our medical centre between 2003 and 2006. Although no microfilariae (mf) were detected in their nocturnal blood samples, all were diagnosed to have lymphatic filariasis on the basis of clinical findings and positive serology results. PCR on their blood samples revealed that two of the patients were infected with Brugia pahangi, an animal filarial worm hitherto not known to cause human disease in the natural environment. All the patients were successfully treated with anti-filarial drugs: four patients were treated with a combination of diethylcarbamazine (DEC) and albendazole, and one with doxycycline. Four of them were residents of Petaling Jaya, a residential suburbia located 10 km southwest of Kuala Lumpur city, Malaysia. The fifth patient was a frequent visitor of the suburbia. This suburbia has no history or record of B. malayi infection. The most likely vector of the worm was Armigeres subalbatus as extensive entomological surveys within the suburbia revealed only adult females of this mosquito species were infected with B. pahangi larvae. Wild monkeys caught in the suburbia were free from B. pahangi mf, but domestic cats were mf positive. This suggests that infected cats might be the source of the zoonotic infection in the suburbia.     

Brugia malayi microfilaraemia in mice: a model for the study of the host response to microfilariae

Microfilariae of Brugia malayi were obtained from the peritoneal cavities of infected gerbils and were then injected intravenously into mice. A sub-periodic, nocturnal microfilaraemia was produced. The level of microfilaraemia was proportional to the number of parasites injected, with approximately 1-3% of microfilariae being found in the peripheral circulation. The duration of microfilaraemia was proportional to the number of parasites injected; it subsided by 30 days after injection of 104 microfilariae but was still present at a low level 120 days after injection of 2 x 105 microfilariae. A transient splenomegaly developed after injection of microfilariae. Histopathological examination revealed large numbers of microfilariae free in the lumens of pulmonary small blood vessels and without any accompanying inflammatory reaction. Lesser numbers of microfilariae were seen in the cardiac blood and hepatic and renal blood vessels for the first few days after injection. There was cellular proliferation in the splenic white pulp and vascular congestion of the red pulp. Microfilariae labelled with 51Cr were injected intravenously; 57% of radioactivity was found in the lungs, 8.5% in the liver and 2.9% in the spleen. Mice developed immediate hypersensitivity reactions to B. malayi antigen by 4 weeks after injection, but Arthus and delayed hypersensitivity reactions were not seen at any time. when mice which had been injected 5 months previously were challenged with a 2nd injection of microfilariae, there was an accelerated clearance of parasites over 2 weeks and a marked peripheral blood eosinophilia developed. In contrast with natural infections, in which the continuous production of microfilariae complicates assessment, this model provides a system in which factors controlling the circulation of microfilariae in the bloodstream can be studied independently.     

Sensitivity of the nested-polymerase chain reaction (PCR) assay for Brugia malayi and significance of 'free' DNA in PCR-based assays

The blood filtration method was used as the gold standard to determine the detection level of simple blood-spot sampling and nested-polymerase chain reaction (PCR) for Brugia malayi. Of 100 samples, 48 were filtration-positive. Of these, 26 had microfilaria counts that were low enough (<1-29 microfilariae/ml) to accurately assess the limit of detection by nested-PCR. Nested-PCR consistently detected B. malayi DNA in samples with > or = 10 microfilariae/ml. Post-filtration, microfilaria-depleted, blood-spots from microfilaria-positive samples were screened by nested-PCR and B. malayi specific 'free' DNA was detected in 51.7% of these samples. There was no evidence for 'free' DNA in microfilaria-negative individuals from this endemic community.