Abstract
ABSTRACT This studyaimed to establish and evaluate the sensitivity and specificity of Toxocaracanis excretory-secretory (TES) antigens and recombinantTES-120 as antigen for the diagnosis oftoxocariasis in animal models by using enzyme-linked immunosorbent assays (ELISA). In vitro culture methods for T. canislarvae have been established for the production of antigens.Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the protein components of whole adult worms and the larvae of T. caniswere more complex than those of TES antigens. At least 11 prominent bandsranging between14 and 185kDa appeared in crude somaticadultantigens and 9 similar bands between 20 and 185 kDawere present in crudelarvae somatic antigens.At least 7 prominent bands between 5 and 194 kDa were detected in TES antigens concentrated by freeze-drying, whereas only 3 bands between 19 and 43 kDa appeared in TES antigens which were concentrated by Millipore filtration and precipitation using trichloroacetic acid. Anexperimental model of toxocariasis has been developed in BALB/c mice followingoral administration of embryonatedT. canis eggs.At day 3 post-infection, 54% of inoculated eggs were recovered as viable larvae from liver, lungs and brain with most of them in liver (34%) and lungs (19%).Thereafter, larvaemigrated from liver and lungs to other tissues including brain which reached a maximum larval recovery of 15% by day 14. Optical conditions were established for the detection of anti-TES antibodies in mouse serum; IgG and IgM were most effective for the diagnosis of toxocariasis in an ELISA format and IgG subtypes did not offer any advantages in terms of sensitivity. Adminstration of mebendazole or albendazole to mice between days 3-9 post-infection reduced by 60%the number of larvae recovered from brain whilst fenbendazole-treated mice showed no significant affect.Among all groups of micetreated between days 14-20 post-infection, only albendazole-treated mice showed a significant reduction in larvae recovered in the brain, and no effect was found for all studied drugs given between days 35-41 post-infection. The time course of anti-TES IgG and IgM was broadly similar after inoculation and all three anthelmintic drugs did not significantly affect serum anti-TES IgG or IgM concentrations, irrespective of time of administration. ELISA using crude lysates from induced BL21 cells harbouring a pET21/TES-120 plasmidgave good differentiation between sera from infected and non-infected mice. The time course of antibody appearance in infected mice was compared by ELISA using expressed proteins from induced BL21cells and TES antigens.Whilst the time courses of IgG and IgM appearance were similar, ELISA using TES-antigens gave approximately 4-fold higher ODs (sensitivity) than using crude lysates from induced BL21 cells. Attempts were made to purify the recombinant TES-120 antigen by targeting the histidine-tag using QiagenNi-NTA Spin Kits. Whilst the purified recombinant TES-120 performed better than crude lysates in an anIgG-ELISA, OD values were still only 50% of those obtained using TES antigens.