Antifilarial and Antibiotic Activities of Methanolic Extracts of Melaleuca cajuputi Flowers

We evaluated the activity of methanolic extracts of Melaleuca cajuputi flowers against the filarial worm Brugia pahangi and its bacterial endosymbiont Wolbachia. Anti-Wolbachia activity was measured in worms and in Aedes albopictus Aa23 cells by PCR, electron microscopy, and other biological assays. In particular, microfilarial release, worm motility, and viability were determined. M. cajuputi flower extracts were found to significantly reduce Wolbachia endosymbionts in Aa23 cells, Wolbachia surface protein, and microfilarial release, as well as the viability and motility of adult worms. Anti-Wolbachia activity was further confirmed by observation of degraded and phagocytized Wolbachia in worms treated with the flower extracts. The data provided in vitro and in vivo evidence that M. cajuputi flower extracts inhibit Wolbachia, an activity that may be exploited as an alternative strategy to treat human lymphatic filariasis.

Successful Treatment of Brugia pahangi in Naturally Infected Cats with Ivermectin

Lymphatic filariasis is a common parasitic disease of cats in tropical regions including Thailand. The objective of this study was to determine the efficacy of ivermectin against microfilariae of Brugia pahangi in naturally infected cats. Eight cats naturally infected with B. pahangi were divided into control (untreated) and treated groups. Cats in the latter group were given ivermectin injection at 400 microg/kg weekly for 2 months. Microfilariae were counted every week until 48 weeks. Microfilaremia was significantly decreased in the treated group 4 weeks after starting the treatment and become zero at week 9 and afterwards. On the other hand, cats in the control group had high microfilaremia throughout the study. It was successful to treat and control B. pahangi infection in naturally infected cats using ivermectin.

The observation of microfilarial rate and density in cats inoculated with increasing numbers of Brugia pahangi infective larvae.

Having close kinship to Brugia malayi, B. pahangi is a member of the family Filariidae, which causes lymphatic filariasis in dogs and cats. Although this nematode is unlikely to cause a zoonotic disease in humans, study of the B. pahangi life cycle may help control human filariasis. The objective of this study was to examine microfilarial rates and densities of B. pahangi in experimentally induced infections in cats as a relative measurement. Cats were infected with 3 different amounts of 3rd-stage larvae (L3); 100, 300 and 500. Cats infected with 100 L3 became patent for microfilariae longer than the other groups (mean100 = 99+/-44 days). In comparison, the pre-patent period of B. pahangi was somewhat shorter in cats with 300 and 500 L3 infections (mean300 = 76+/-13 and mean500 = 63+/-5 days). The microfilarial densities of these cats were also determined; the density of microfilariae (mf/1 ml blood) increased relative to the duration of infection. One-way ANOVA tests were used to compare the microfilarial densities of the cats with varying numbers of L3. We found that the microfilarial density of cats with 500 L3 exhibited significant differences (p < 0.05) from cats with 300 and 100 L3. However, we concluded that the amount of microfilariae produced in the blood circulation of these cats were not increasing relative to the numbers of L3 taken by the host.

The ultrastructure of the gravid uterus of Brugia pahangi, another rich source of antigen of the filarial parasite.

The gravid uterus with zygotes and microfilariae in utero of Brugia pahangi, a rich source of antigen as revealed by a recent immunofluorescent technique, were studied ultrastructurally. The epithelial cells of uterus show ultrastructural features of synthetically active cells. Their secretions may provide nutrients for the egg in utero. On the basal side, the uterine epithelial cells may also secrete substances to form the basal lamina of the uterus which is rather thick and irregularly fused with the basal lamina lining the body wall where the pseudocoelomic cavity is obliterated. For the most part, the uterine basal lamina contains uniform granular material of moderate electron density. There are also elongated visceral muscle cells embeded in it, and which surround the uterus, with adjacent cells overlapping. The gravid uterus contains several stages of developing microfilariae within its lumen, the cleaving zygotes are also present at another level. The morula of zygotes are composed of several closely packed cells surrounded loosely by their own egg shell membranes. The egg shell becomes more convoluted as development proceeds. The egg shell surrounding the developing microfilariae in utero is secreted by the uterine epithelium. This structure later becomes the sheath of circulating microfilariae, and is highly antigenic as indicated by intense labeling with fluorescent antibodies.

Differentiation of Brugia malayi and Brugia pahangi by PCR-RFLP of ITS1 and ITS2.

Lymphatic filariasis has been targeted by the World Health Organization for elimination by the year 2020. Malayan filariasis, caused by Brugia malayi, is endemic in southern Thailand where domestic cats serve as a major reservoir host. However, in nature, domestic cats also carry B. pahangi infection. In addition to chemotherapy and vector control, control in reservoir hosts is necessary to achieve the elimination of the disease. Therefore, differentiation between B. malayi and B. pahangi in the cat reservoir will help the lymphatic control program to monitor and evaluate the real disease situation. It is difficult to differentiate these two Brugia species by microscopic examination. The technique is also time-consuming and requires expertise. We employed the polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) technique of internal transcribed spacer regions, ITS1 and ITS2, of ribosomal DNA (rDNA) to differentiate B. malayi from B. pahangi species. Among the restriction enzymes tested, only the PCR product of ITS1 digested with Ase I could differentiate B. malayi from B. pahangi. This PCR-RFLP technique will be useful for lymphatic filariasis control programs for monitoring and evaluating animal reservoirs.

Antibody responses of Wuchereria bancrofti patients to recombinant Brugia pahangi superoxide dismutase.

Lymphatic filarial parasite Brugia malayi contains significant amount of Cu/Zn superoxide dismutase (SOD) activity in the extract of different life stages and in the excretory-secretory product of adults. In the present study recombinant SOD from B. pahangi has been used to see the antibody response in Wuchereria bancrofti infected patients. The recombinant SOD from B. pahangi reacted specifically with W. bancrofti infected sera in ELISA and immunoblotting. The reactivity of IgM subclass was more as compared to IgG subclass both in the asymptomatic microfilaraemic and symptomatic amicrofilaraemic when tested by ELISA. Serum from other helminthic infection was very low and found to be insignificant. The antibody response to rec SOD was directly proportional to the number of microfilariae in infected patients. The circulating filarial SOD was detected in filarial patients using polyclonal antibodies raised against recombinant Cu/Zn SOD in rabbits. The apparent molecular masses as determined by immunoblotting were 29 and 22 kDa. The specificity of recombinant SOD could be explored for its use in immunodiagnosis of lymphatic filariasis.

Depressed specific and nonspecific immune responses in secondary Brugia pahangi infection in jirds.

The immune responsiveness to specific antigens or mitogens was examined in jirds after primary and secondary infections with Brugia pahangi. When spleen cells were obtained from secondarily infected jirds, their proliferative responses to mitogens such as Con A or LPS, or to specific antigens prepared from infective larvae or adult worms were significantly lower than those of spleen cells obtained from primarily infected jirds. The proliferative responses of the peripheral blood mononuclear cells obtained from animals undergoing primary and secondary infections also showed a similar tendency. The depressed proliferative responses of the secondary infected spleen cells to Con A or LPS was partially restored by removing adherent/phagocytic cells from the original cell populations. After deletion of the adherent cells, however, antigen-specific proliferative responses were not altered and remained at low level. These results suggest that at least two different mechanisms of depression, namely adherent cell-mediated antigen-nonspecific suppression and unresponsiveness of lymphocytes to filarial antigens, are induced in jirds in the secondary infection.

Recent advances in the application of molecular biology in filariasis.

Monitoring of filarial parasites in the host and vector has traditionally depended on morphological identification. Recently, species-specific DNA probes have been developed for Brugia malayi, Brugia pahangi and Wuchereria bancrofti. Repeated DNA sequences are useful in developing DNA probes because they evolve more rapidly then coding sequences and their high copy number increases the sensitivity of detection. The Hhal repeated DNA family represents 12% of the total B. malayi DNA. This DNA family is present in species of Brugia (B. malayi, B. timori and B. pahangi) but not W. bancrofti. Sequence analysis of the repeated DNA in B. malayi and B. pahangi has allowed construction of two species-specific DNA probes. These probes were used in a double blind field study in Indonesia. Microfilariae (mf) from infected cats and humans were identified by classical morphological methods and DNA probes. Agreement was found in 98.6% of the 642 samples tested by the two different techniques. Besides mf identification DNA probes can be used to determine the species of infective larvae (L3s) in infected mosquitos. This is useful because the L3s have similar morphology. DNA probes for the identification of W. bancrofti have recently been developed and are in the initial stages of testing in China (Piessens, personal communication) and Egypt (Williams, personal communication). An alternative approach for identification of infected individuals is to detect specific parasite antigens in circulation. A WHO initiative to use either an antigen or antibody assay to replace night blood is presently underway. This approach, if successful would not require the presence of microfilariae, but could detect occult infections.

Detection of circulating antigens and parasite specific antibodies in filariasis.

In Peninsular Malaysia, only Wuchereria bancrofti and Brugia malayi are reported to cause human filariasis. Brugia pahangi infects many of the same animal hosts as the zoonotically transmitted subperiodic B. malayi. There is a well-recognized need for improved diagnostic techniques for lymphatic filariasis. Parasite antigen detection is a promising new approach, and it will probably prove to be more sensitive and specific than clinical, microscopic and antibody-based serological methods. We recently generated monoclonal antibodies (MAb XC3) from in vitro culture products of adult B. pahangi (B.p. IVP). Filarial antigenemia was quantitated in various hosts including the sera from 6 Malaysian Aborigines with acute lymphatic filariasis. In hosts infected with brugian filariasis and dirofilariasis, antigenemia was scored ranging from 90 ng/ml to 960 ng/ml. None of the control animal and human sera had antigenemia above 90 ng/ml. In addition, MAb XC3 and B.p. IVP were applied in several seroepidemiological surveys among household cats in Kuala Selangor in order to correlate information gathered for future studies of possible cases of human infection. Out of the 81 cats surveyed, 10 (12.35%) and 5 (6.17%) were parasitologically positive for B. pahangi and B. malayi, respectively. However, 21 (25.92%) were antigenemia positive when serologically investigated with MAb XC3. Antifilarial antibodies to B.p. IVP by direct ELISA showed very high cross-reactivity with non-filarial gut worm infections. 16 (19.75%) cats had reciprocal titers ranging from 320 to 2,560. Only 1 (1.23%) cat from this group was antigenemic.