Suppression of Brugia malayi (sub-periodic) larval development in Aedes aegypti (Liverpool strain) fed on blood of animals immunized with microfilariae

Abstract

(Ottesen et al. 1997). However, in order to attain the goal of filariasis elimination, it is desirable to have more than one control tool. Recently, great deal of interest has been generated in the possibility of blocking the development and transmission of parasites by vectors of diseases such as leishmaniasis (Tonui 1999), schistosomiasis (Capron et al. 1992), and Lyme disease (de Silva et al.1996). In the case of malaria, transmission-blocking vaccines have reached advanced stage of development (Kaslow 1997, Carter 2001. But there is only one such study on the antibodies regulating the development of filarial parasites (Fuhrman et al. 1992), which reports that a monoclonal antibody, MF1, could degrade the chitin containing structures of B. malayi mf in the vector mosquito. In the present paper, results of a preliminary study on the role of anti-filarial polyclonal antibodies in suppressing the development of early larval stages of the parasite in the mosquito host are presented.Mongolian gerbils, Meriones unguiculatus, were immunized by intra-peritoneal (i/p) inoculation of microfilariae (mf) of B. malayi (sub-periodic strain). obtained by lavaging the peritoneal cavity of B. malayi infected Mongolian gerbils. The animals were 6-8 weeks old and the mf were harvested on 120 days post-inoculation of infective larvae to ensure that the mf were identical with respect to their age and viability. The harvested mf were purified by Percoll gradient centrifugation (Chandrashekar et al.1984), and were used for immunization as well as feeding the mosquitoes. Either single dose of 1000 live mf or their homogenate (20 µg of protein) per animal was given in sterile phosphate buffered saline (PBS, pH 7.4). Homogenate of mf was prepared in PBS containing a cock-tail of protease inhibitors (Maizles et al. 1999) such as, EDTA-1M, EGTA-1M, NEM-1M, Pepstatin-1 mM, PMSF-0.33 Mm, and TPCK-0.1 M at 5 µl level/ml of the parasite extract. The animals were immunized in duplicate for each antigen type and an equal numbers were left as controls with only PBS inoculation. Considering the ethical issues involved in the usage of laboratory animals and the duration involved, the number of animals used in this experiment was restricted to only two in each group. The antibody titre in each animal was determined two weeks after immunization by direct enzyme linked immunosorbent assay (ELISA) as per Voller et al. (1976), using mf homogenate antigen. Briefly, crude antigen of B. malayi was coated (1 µg/well) onto 96 well polysterene plates (Nunc-US) and incubated overnight at 4 o C. Sera samples from the experimental and control animals diluted to 1:100 with 0.75% bovine serum albumin (BSA) in PBS/ T were reacted for 2 h after blocking the wells with 1% BSA in PBS/T. Anti-mouse peroxidase conjugate (Sigma Chem. Co., US) was allowed to react with the antigenantibody complex, followed by the addition of θ-Phenylenediamine dihydrochloride (Sigma Chem. Co., US) as substrate in phosphate citrate buffer (pH 5.0). The reac- tion was arrested with 5 N ...