R. Chandrashekar

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%.

Serum dependent cell-mediated immune reactions to Brugia pahangi infective larvae.

Summary Fresh normal rat serum (fNRS) promoted adherence and cytotoxicity of albino rat neutrophils and macrophages to Brugia pahangi infective larvae (L3) in vitro. EDTA and not EGTA abolished the adherence activity suggesting the involvement of complement components via the alternate pathway. C3 molecules were detected on the surface of the parasite by immunofluorescence. fNRS depleted of complement by treatment with Zymosan A or of factor B by heating at 50°C for 20 min, failed to promote cell adherence to the parasite. fNRS and cells from albino rat were more potent in inducing cytotoxicity to L3 than those from jird or Mastomys which may reflect the greater resistance offered by the albino rat to B. pahangi infection. In the presence of IgG and a heat labile factor, possibly complement, of immune serum, neutrophils and macrophages and to a lesser extent eosinophils adhered to and killed the larvae. Immune sera raised against microfilariae of different filarial parasites promoted cell‐mediated cytotoxicity to B. pahangi L3 suggesting sharing of antigens between the two stages.

Antibody-mediated cytotoxic effects in vitro and in vivo of rat cells on infective larvae of Brugia malayi.

Albino rat macrophages and neutrophils in the presence of immune serum adhered to and promoted killing of Brugia malayi infective larvae in vitro. At a similar cell-target ratio, macrophages were more potent than neutrophils in inducing cytotoxic response to the larvae. Eosinophils were also effective in killing but only at a high cell-target ratio. The activity in the immune serum could be absorbed to and eluted from a Protein A-Sepharose column suggesting involvement of IgG antibody in the reaction. An indirect fluorescent antibody test confirmed the presence of IgG on the surface of larvae incubated in immune serum. Infective larvae were attacked by host cells within micropore chambers 16-24 h after implantation into immunized rats. Further, a strong cytotoxic response to the larvae was seen when they were introduced intraperitoneally into immune rats indicating the role of antibody and cells in vivo. We suggest that antibody-dependent cellular cytotoxicity may represent an important mechanism of parasite killing in an immune host.

Brugia malayi: Rat cell interactions with infective larvae mediated by complement

Albino rat macrophages and neutrophils, in the presence of fresh normal rat serum as a source of complement, adhered to and promoted killing of Brugia malayi infective larvae in vitro. Eosinophils, by themselves, were marginally cytotoxic at a high cell-target ratio but promoted cytotoxicity when mixed with macrophages. Eosinophil culture supernatants enhanced the macrophage mediated killing of infective larvae. The complement of fresh normal rat serum was found to act by the alternate pathway. Fresh normal rat serum depleted of alternate pathway complement activity by treatment with zymosan A, or of Factor B by heating at 50 C for 20 min, or of Factor D by passing through Sephadex G75 column, failed to promote cell adherence to the parasite. C3 molecules were detected on the surface of infective larvae by immunofluorescence. There was a significant consumption of complement when Brugia malayi infective larvae were incubated in fresh normal rat serum. Albino rat cells were more potent in inducing cytotoxicity to infective larvae in vitro than those from jird or Mastomys natalensis, which may reflect the greater resistance offered by the rat to B. malayi infection. There was much less cellular infiltration on introduction of Brugia malayi infective larvae into the peritoneal cavity of jirds compared to rats and Mastomys natalensis indicating the greater susceptibility of jirds to intraperitoneally induced infections.

IgG response of rats to the excretory-secretory products of Litomosoides carinii.

The nature of antibody responses to the excretory-secretory (ES) products of adult worms and microfilariae (Mf) ofLitomosoides carinii in albino rats and their possible role in protection have been studied. Rats were immunized with ES products derived from in vitro incubation of adults or Mf. The sera from these rats promoted neutrophil-mediated killing of Mf in vitro. The antibody responsible for the cytocidal activity was identified as IgG isotype. Indirect fluorescent antibody test showed the presence of IgG on the surface of Mf incubated in either of the immune sera. The immune sera were effective in clearing the circulating Mf inMastomys natalensis, indicating the protective nature of the antibody. Thus,L. carinii in culture liberate functional antigens that seem to have protective potential.

Effect of ivermectin on filariae of Mastomys natalensis.

SummaryThe efficacy of ivermectin (Iv) was evaluated against four species of filariae,Litomosoides carinii, Acanthocheilonema viteae, Brugia pahangi andBrugia malayi inMastomys natalensis. Animals with patent infections, induced with L3 larvae, by intravenous (iv) infusion of the respective microfilariae (Mf) (5×104 Mf per animal) or by intraperitoneal (ip) route (2×104 Mf per animal) were used in this study. A single dose of Iv (100 μg·kg−1) given subcutaneously (sc) toMastomys infected withL. carinii orA. viteae resulted in the disappearance of microfilaremia within 2 h of treatment. Iv treatment of sc-infected animals withBrugia spp. had no immediate effect on the circulating Mf 60 days posttreatment. In contrast, such treatment of animals infected with Mf by intravenous infusion completely eliminated the larvae of all four species from the circulation. Iv treatment had no significant effect on the Mf ofL. carinii, B. pahangi andB. malayi in animals infected by the ip route. However, the drug had dramatic effect in killing the Mf ofA. viteae in the peritoneal cavity. Sera from Iv-treated normal or fromL. carinii- orA. viteae-infectedMastomys were effective in clearing the circulating Mf of the species when administered to animals with the respective infections. Similar rapid clearance of Mf was seen when the sera were administered to animals infected iv with these larvae. Furthermore, adult females ofL. carinii andA. viteae recovered fromMastomys on different days after Iv treatment released smaller numbers of Mf in vitro. Thus, Iv effect was most pronounced on the Mf ofA. viteae and ofL. carinii and less so on the Mf ofBrugia. Iv kills the adults ofA. viteae and to a lesser extent those ofL. carinii and arrests the release of Mf from them. Iv had no effect on adults ofBrugia parasites.

Complement activation by eggs and microfilariae of filarial parasites

The complement of fresh normal rat serum was activated by filarial eggs and microfilariae (mf). C3 was deposited on the surface of Litomosoides carinii, Brugia pahangi, Brugia malayi and Dipetalonema viteae as seen by immunofluorescence. Intra‐uterine and in vitro‐derived mf did not bind C3. In contrast, C3 bound to the blood‐derived mf of B. pahangi and B. malayi as well as exsheathed mf of L. carinii and B. malayi. Significant consumption of complement was observed with eggs of all filarial species, as well as sheathed mf of B. pahangi, B. malayi and exsheathed mf of L. carinii and B. malayi. These experiments indicated that complement was activated by filarial parasites via the alternative pathway. The bound complement promoted neutrophil‐mediated adherence and cytotoxicity.

Sharing of antigens among filarial species revealed by antibody dependent cell-mediated reactions †

Antisera raised in albino rats against microfilariae ofLitomosoides carinii, Brugia pahangi, Brugia malayi and sera fromBancroftian elephantiasis patients promoted rat neutrophil-mediated adherence and cytotoxicity to the microfilariae. Pre-treatment of the immune sera, with microfilarial antigen at a final concentration of 5 and 25μg per ml blocked cellular adherence and cytotoxicity to the microfilariae indicating the presence of cross-reactive antibodies. The heterologous immune sera were effective in eliminating the circulatingLitomosoides carinii microfilariae inMastomys natalensis.

Immune response to Acanthocheilonema viteae infection in multimammate rats (Mastomys natalensis).

The multimammate rat Mastomys natalensis, when infected with the filarial parasite Acanthocheilonema viteae, develops amicrofilaraemia. Worm recovery and the duration and intensity of microfilaraemia were analysed and related to the humoral and cellular immune responses of the host by using an antibody‐dependent cell‐mediated cytotoxicity (ADCC) assay towards microfilariae (Mf). Mf were detected in the peripheral blood at 7 weeks post‐infection (p.i.), reaching maximum levels by 20 weeks p.i., and then gradually decreasing to undetectable levels during the next 36 weeks. The cytotoxic antibodies appeared around 15–18 weeks p.i., and the serum at 36 weeks p.i. induced 70% cytotoxicity to the Mf in vitro in the presence of host cells. The IgM fraction of the immune serum from amicrofilaraemic Mastomys promoted ADCC to Mf both in vitro and in vivo. Macrophages were more potent in inducing cytotoxic effect than eosinophils and neutrophils. Platelets were ineffective in killing the Mf in the presence of immune serum. IgM antibody cleared the circulating Mf from the blood when given passively to infected Mastomys. The average recovery of adult worms was about 20% of the inoculated larvae. No live females could be recovered 56 weeks p.i. Thus protective immune responses built up over an extended period of time are elicited against the Mf and perhaps even to adults in Mastomys infected with A. viteae.