OBJECTIVE To determine mitogenic and antigen-specific cellular immune responses of two species of rodents, viz. Meriones ungruiculatus and Mastomys coucha to assess the usefulness of the A. viteae/Mastomys model for cellular immune studies in experimental filariasis. METHODS Lymphocyte blast transformation test (LTT) using spleen cells of normal and A. viteae infected animals. RESULTS The proliferative response of gerbils was much higher than that of Mastomys to both ConA and filarial antigens. Cells of both species of rodents did not respond to microfilarial (mf) antigen, however, their mitogenic response differed during infection. Some degree of nonspecific suppression was observed in gerbils during prepatent and patent stages of infection, while Mastomys revealed highest proliferation during patent microfilaraemia. Mastomys cells did not respond to adult or mf antigen, while adult-specific proliferation was detected in the case of gerbils. CONCLUSION The A. viteae/gerbil model shows more similarity to human filarial infection regarding cellular immune response. Markedly low responsiveness of a high percentage of Mastomys and wide variations in the cellular response to nonspecific mitogen limit the usefulness of Mastomys coucha in immunological studies, especially cellular immunity.

Antigen derived from three major life-stages of human lymphatic filariid, Brugia malayi was fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and reactivity of filarial antibodies, present in sera of human bancroftian patients belonging to different categories of the disease, to immunoreactive molecules was evaluated by Western blotting and immune recognition. Antigen molecules of >180, ~180, ~116, ~66, 58, 33 and <20 kDa were strongly identified in blots especially by symptomatic and endemic normal sera. Although mf positive serum reacted with the majority of these molecules, the intensity of bands was poor. These functional molecules along with ~43 kDa were later isolated from a preparative SDS-polyacrylamide gel by elution. The diagnostic utility of these purified molecules was then assessed by enzyme linked immunosorbent assay (ELISA) for detecting the IgG antibodies in patients' sera of various categories. Tropical pulmonary eosinophilic subjects revealed highest reactivity with <20 and ~116 kDa molecules while mf positive ones reacted feebly with the majority of the molecules except ~43 and ~66 kDa. Sera of endemic normals revealed high IgG levels but antibodies to ~ 66 kDa were highest. Symptomatic patients showed moderate reactivity. Non-endemic sera neither reacted in blots nor in ELISA. The study demonstrates the usefulness of ~43 kDa in detecting IgG antibodies of mf positive asymptomatic patients. High IgG levels to ~66 kDa followed by ~116 kDa and <20 kDa in sera of endemic normals warrants their immunoprophylactic evaluation.

For optimal solubilization of immunoreactive proteins of filarial parasites, adult worms of Acanthocheilonema viteae were extracted in different detergents including anionic, cationic, nonionic and zwitterionic agents under varying incubation periods. Each preparation was then analysed by SDS-PAGE and immunoblotting using pooled sera from Mastomys natalensis infected with A. viteae. Amongst the detergents used, maximum immunoreactive proteins were exposed by sodium dodecylsulphate (SDS), closely followed by sodium deoxycholate (DOC). Nevertheless, a few additional protein bands were recognized by infected sera in DOC preparations, but not in SDS, and vice versa. Most of the proteins were completely or partially dissolved within 2 hrs extraction time. It is felt that DOC may be used in place of SDS because of the strong denaturing character of the latter.

The efficacy of microfilarial antigens derived from Acanthocheilonema viteae has been evaluated in normal as well as exposed animals simulating human population in filaria-endemic area. The crude antigen (CAg) or one of its Sephadex fractions (Fr. I) offered only microfilarial-stage specific resistance against challenge made with either infective larvae, microfilariae or adult parasites. Subsequent study with CAg showed better protection, including significant effect on adult parasite population, if the recipients were already carrying a prepatent infection, or displaying patent microfilaraemia or even challenged with a second exposure. SDS-PAGE and Western blot analysis of microfilarial antigen revealed certain low-molecular-weight antigens to be responsible in the development of resistance against infection. The resistant sera that induced strong in vitro cellular adherence and cytotoxicity to microfilariae, transferred resistance to naive animals on passive transfer.

An approach has been made to develop an in vitro screening system to evaluate antifilarial efficacy of compounds and an effort has been made to establish correlation between in vivo and in vitro screening technique. The in vitro experiments were conducted simultaneously using three life-forms (adult, microfilaria and infective larva) of Acanthocheilonema viteae using five antifilarial agents representing four chemical groups. All the selected antifilarials were known to be active against one or more life-stages of human lymphatic or animal filariids. Diethylcarbamazine and Centperazine showed 100% microfilaricidal and infective larvicidal actions at concentrations of 0.5 and 0.25 mg/ml and 0.5 and 0.0313 mg/ml respectively with no effect on adult worms even at 1 mg/ml. Levamisole was effective against all the three life-stages killing 100% adult worms at 1 mg/ml, infective larvae at 0.0625 mg/ml and microfilariae at 0.0125 mg/ml, while mebendazole exhibited activity only against adult worms (100% at 0.5 mg/ml). Ivermectin killed adult females and microfilariae at 0.063 and 0.5 mg/ml respectively but did not affect infective larvae even up to 1 mg/ml concentration. The study indicated that in vitro screening system can be used for primary screening of potential antifilarial agents provided three life-forms of A. viteae are used simultaneously to avoid false negative results. It would however be more appropriate if a few compounds of a particular chemical class are initially assessed both in vivo and in vitro for validity of subsequent test results in vitro.

The susceptibility of hosts to filarial infection during pregnancy and lactation was studied using Dipetalonema viteae in Mastomys natalensis as working model. Though no difference in prepatency could be observed yet significantly higher density of microfilaraemia than controls was detected in both pregnant and lactating groups. Extended duration of patency was also found in both pregnant and lactating animals. On autopsy, animals exposed at the time of pregnancy or lactation revealed significantly higher recovery of adult worms as compared to normal controls. Thus pregnancy and/or lactation may lead to higher density and duration of microfilaraemia and enhanced establishment of adult worms in female mastomys which are otherwise less susceptible to filarial infection.

Stage-specific haemagglutinating (HA) and precipitin antibodies (PA) to infective larvae (L3), adult worms and microfilariae (mf) have been demonstrated in sera of Mastomys natalensis during the course of Dipetalonema viteae infection using indirect haemagglutination (IHA) and Ouchterlony's gel-diffusion tests. L3-specific HA antibody titre was shown to be quite low and lasted for shorter period whereas adult-specific antibody response was significantly higher and persisted for longer duration (beyond day 240 post exposure). No precipitin antibody to L3 stage could be detected, however, significant adult-specific antibody was detected which ultimately tapered off by day 210 post exposure (p.e.). In contrast, mf-specific PA which appeared at the beginning of patency, never disappeared even at the late stage of infection. Mf-specific HA antibody appeared at early incubation period (day 15 p.e.) and after exhibiting two peaks, one in the midst of prepatent period (day 30 p.e.) and the other on day 180 p.e. persisted at low level during the late stage of infection when amicrofilaraemia developed.

The mechanism of the adverse reaction in microfilaraemic subjects following diethylcarbamazine (DEC) therapy has remained an enigma. To study the mechanisms involved Mastomys natalensis infected with Dipetalonema viteae have been used. Animals showing up to 50 mf/5 cmm of blood did not show any perceptible reactions when treated with the minimum effective dose of 350 mg/kg of DEC citrate, while those having more than 80 mf/5 cmm died, suggesting an intensity dependent severity of reactions. Infective larvae contained maximum amounts of biogenic amines followed by mf and adults in that order. 16 mg of histamine or 24 mg of 5-hydroxytryptamine caused death when injected into healthy animals, whereas sonicated mf (5 million) and adult somatic products (4 mg protein) did not, signifying that the biogenic amine contents of the parasites were less than the critical level to cause death. Adult somatic products did not produce any visible reactions when injected into animals with microfilariaemia, but sonicated mf (5 million) caused death. It is concluded that the adverse reactions seen after DEC treatment are due to the release of biogenic amines in an anaphylactic type of reaction subsequent to massive release of parasite antigen in a sensitised host, which is directly proportional to the intensity of microfilaraemia and that the adverse reactions are not due to liberation of endogenous biogenic amines from dead mf.