Circulatory excretory-secretory antigen levels and IgM and IgG responses to larval antigens were monitored in the serum of 20 BALB/c mice that had been given approximately 500 infective eggs of Toxocara canis by stomach tube. Other groups of mice received different doses of infective eggs, ranging from 5 to 1,250 eggs. Excretory-secretory antigens were collected from culture fluid in which mechanically hatched larvae of T. canis were maintained. An indirect enzyme-linked immunosorbent assay was used to monitor specific antibody responses. Circulating antigen levels were monitored using a direct ELISA which incorporated an IgG fraction of a rabbit antiserum to the excretory-secretory antigens as a capture antibody and a biotin-conjugated form of the same rabbit IgG as the second antibody. The antigen-specific IgM response was evident the first week of infection and peaked 3 to 6 weeks post-infection. The antigen-specific IgG response first appeared the second week of infection and peaked at 6 to 8 weeks post-infection. Both isotype levels stayed near their peak values for the remainder of the study. In the untreated sera, circulating antigen was initially evident and highest the first week of infection; the antigen concentrations dropped by the third month of infection to low, but significant, levels that persisted for the duration of the study. The administration of greater than 25 eggs produced antigenemias. There appeared to be a positive linear trend between the number of eggs given and the amount of antigen in the circulation.
The objective of this study was to determine if Dirofilaria immitis larvae would survive in diffusion chambers implanted in dogs and mice and secondly to determine if mice could be immunized against infection with D. immitis. Dirofilaria immitis third-stage larvae (L3) survived and grew in diffusion chambers implanted in dogs and mice for at least 3 wk. BALB/c mice, which were repeatedly infected with live L3, showed resistance to challenge infections. Dead L3, with or without adjuvants elicited no protective immunity. A correlation was found between the degree of immune protection seen in mice and antibody levels to soluble larval antigen but not to antibody levels to surface antigens. A monoclonal antibody was prepared that reacted with the surface of D. immitis and Onchocerca lienalis L3, but not to the surfaces of other stages and species of various filarial worms. When this antibody was administered to mice prior to challenge no significant reduction in larval survival was observed.
Microfilariae of Dirofilaria immitis retained their infectivity for susceptible mosquitoes after cooling to −196°C in the presence of 5% dimethylsulphoxide (Me2SO) using a two-step cooling sequence. Motility and in vitro development of cryopreserved microfilariae also compared favourably with unfrozen controls. Third-stage larvae frozen by the same cooling sequence in the presence of either 5% Me2SO or 16% hydroxyethyl starch were motile upon thawing. Thawed larvae completed the third- to fourth-stage moult in vitro at a frequency approximately 5 to 10% of that seen in unfrozen controls.
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