DETECTION AND DIFFERENTIATION BETWEEN TRYPANOSOME SPECIES IN EXPERIMENTALLY INFECTED TSETSE-FLIES (GLOSSINA SPP) USING DOT-ELISA

A modified NC membrane-based dot-ELISA was used to detect and differen tiate between Trypanosoma brucei, T. congolense and T. simiae procycli cs in the midguts of experimentally infected tsetse flies. The modific ation of the assay consisted of(a) the lysis of T. congolense or T. si miae in NC membrane applied sample dots using Triton X-114, and (b) tr eatment of sample applied NC membrane strips with hydrogen peroxide to remove non-specific stains. Also, T. brucei was detected in the saliv ary glands, and T. congolense and T. vivax were detected in the mouthp arts, however, in dot-ELISA without modification. In all the assays, T . brucei and T. congolense parasites were detected directly using MoAb s specific to each of them, whereas T. simiae parasites were detected by exclusion using a T. congolense specific and Nannomonas subgenus-sp ecific MoAbs. The sensitivity of the assay for detecting midgut infect ions was 90.5%, 84.6% and 94.4% in detecting T. brucei, T. congolense and T. simiae, respectively. Sample dots stored at room temperature (1 9-26 degrees C) under desiccated conditions did not show any loss in a ctivity in 90 days. However, after 7 days of storage, a ring-pattern r eaction appeared on some sample dots that were tested with T. brucei s pecific MoAb, irrespective of whether T. brucei antigens were present or not. These ring reactions, however, did not interfere with correct interpretation of the assay results. The specificity of the assay for detection of T. brucei in the salivary glands was 100% and the sensiti vity was 90%. Also, T. vivax and T. congolense organisms were each det ected in the mouthparts of infected tsetse flies, with 100% specificit y. The sensitivity was, however, lower, 43.8% for T. vivax and 55.6% f or T. congolense.