The chromosome profiles of Trypanosoma congolense isolates from Kilifi, Kenya and their relationship to serodeme identity.

Chromosomal DNA from 117 Trypanosoma congolense clones from 54 stocks, isolated from cattle introduced onto a ranch in Kilifi in the coastal area of Kenya, was fractionated by the orthogonal field alternation gel electrophoresis technique. The technique resolved chromosomes in the size range of 100 kb-1 Mb. The chromosome profile for cloned trypanosome populations was relatively stable with regard to number and size of the chromosome bands following transmission in mice, cattle, goats or tsetse flies. Only in one clone was a shift observed in the position of one medium-sized chromosome band following cyclical development in tsetse. On the basis of their chromosome profiles, the 117 clones could be divided into 18 distinct groups. Representative clones, randomly selected from 7 of the 18 chromosome profile groups were inoculated into steers and goats in order to raise variable antigen type (VAT) repertoire-specific infection sera. Cross-neutralization assays demonstrated that recovery sera from animals infected with a clone neutralized all the clones with an identical chromosome profile. This suggests that clones having an identical chromosome profile also express an identical VAT-repertoire (serodeme).

A species-specific antigen of Trypanosoma (Duttonella) vivax detectable in the course of infection is encoded by a differentially expressed tandemly reiterated gene.

A monoclonal antibody that is used as a Trypanosoma vivax species-specific diagnostic reagent on antigen-trapping enzyme-linked immunosorbent assay recognized an 8-kDa peptide on western blots. The 8-kDa species-specific antigen was isolated and employed in raising rabbit polyclonal antibodies, which were used in the immunoscreening of a T. vivax cDNA library in lambda gt11.2. A clone containing a 0.8-kb insert was isolated. The cloned gene is tandemly repeated, with a monomeric unit length of 900 bp, in the genomes of all T. vivax isolates from diverse geographic locations in Africa and South America. The gene is differentially expressed, since both the transcript and antigen are present in bloodstream-stage parasites, but not in the epimastigotes of T. vivax. Although the gene is found in all T. vivax isolates so far tested, it either exists in low copy number or in a divergent form in one isolate from Kilifi at the Kenya Coast. Sequence translation revealed a remarkable degree of bias in codon usage with preference for G and C (82%) in the wobble position. Using the deduced amino acid sequence to search the databases for any structurally related peptides, revealed no significant identity with any known proteins. The function of the species-specific antigen of T. vivax is thus unknown. Nevertheless the identification and characterization of proteins released into the circulation of protozoan parasite-infected animals is important and should allow the determination of what role such molecules may play in the modulation of disease pathology.

Differences between N'Dama and Boran cattle in the ability of their peripheral blood leucocytes to bind antibody-coated trypanosomes.

Investigations were undertaken to evaluate the immune response of trypanotolerant N'Dama (Bos taurus) and susceptible Boran (Bos indicus) cattle to two Trypanosoma congolense variable antigen types (VATs) expressed in both breeds following tsetse-transmitted challenge. The VAT-specific antibodies of both IgM and IgG1 isotypes produced by both breeds had similar neutralizing titres. The interaction between immune sera, trypanosomes and freshly isolated peripheral blood leucocytes (PBL) from uninfected N'Dama and Boran animals was studied. It was found that both N'Dama and Boran immune sera were able to induce adherence of trypanosomes to the N'Dama PBL, but not to Boran PBL. The adherence-inducing activity was exhibited by both IgM and IgG1 antibodies, but IgG1 antibodies were more efficient in this respect. These results suggest that there are qualitative and/or quantitative differences in the immunoglobulin receptor function of PBL between the two breeds of cattle.

Cerebral trypanosomiasis in cattle with mixed Trypanosoma congolense and T. brucei brucei infections.

Six Boran steers were infected simultaneously with Trypanosoma congolense and T. brucei brucei while another group of 3 was inoculated with T. b. brucei one year after infection with T. congolense. Three further steers were infected with T. b. brucei alone. Whereas, the six animals which received simultaneous infections developed clinical signs of cerebral trypanosomiasis as evidenced by depression, ataxia and occasional circling, those infected with T. b. brucei alone did not. At necropsy, 4 out of the 6 simultaneously infected animals had a mild to severe disseminated non-suppurative meningoencephalitis. Trypanosoma b. brucei was isolated from the cerebrospinal fluid (CSF) of three out of the four animals with histological lesions. Two of the cattle superinfected with T. b. brucei one year after infection with T. congolense also developed both clinical and histological evidence of cerebral trypanosomiasis. Trypanosoma congolense was isolated from the CSF of one of these 2 animals. Specific antibodies to the variable surface glycoproteins (VSGs) of the infecting T. b. brucei and T. congolense clones were found in the CSF of the 8 animals that developed cerebral trypanosomiasis. In these animals however, there was neither temporal nor quantitative correlation between VSG-specific antibodies in serum and in CSF, implying a de novo synthesis of antibodies to the infecting trypanosomes in the CSF.

Comparative sensitivity of dot-ELISA, PCR and dissection method for the detection of trypanosome infections in tsetse flies (Diptera: glossinidae).

A visually read dot-enzyme linked immunosorbent assay (dot-ELISA) developed for the detection of trypanosomes in tsetse flies (Glossina spp.) was evaluated in the laboratory and under field conditions. In the evaluation, the fly dissection method was used as a standard technique and compared to the polymerase chain reaction (PCR). In laboratory studies, 133 and 126 tsetse flies were experimentally infected with different stocks of Trypanosoma brucei and T. congolense, respectively. Twenty-five days after infection, the flies were dissected and tested for the presence of trypanosomes using dot-ELISA and PCR. Dot-ELISA detected 98.4% of T. brucei and 94% of T. congolense infections in tsetse midguts, while PCR detected 97.6% of T. brucei and 96% of T. congolense tsetse midgut samples. For field evaluation of dot-ELISA, 700 tsetse flies were caught and screened for trypanosome infections by dissection. Seven of these (1%) had trypomastigotes in the midgut, 23 (3.3%) in the proboscis and none had trypanosomes in the salivary glands. All the flies with midgut infections also had trypanosomes in their proboscides. Five of the seven flies (71.4%) with midgut infections revealed by dissection, were also positive for T. congolense by the dot-ELISA and PCR techniques. Dot-ELISA detected T. congolense infections in an additional 86 (12.4%) of the 700 flies dissected. Of the 23 infections in the proboscis, 16 were T. vivax. Dot-ELISA detected 13 of the 16 (81%) while PCR detected 15 of 16 (94%) T. vivax infections. No T. brucei infection was detected by any of the methods in all the 700 tsetse flies examined. The results obtained from both the laboratory and field studies indicate that the dot-ELISA and PCR techniques are sensitive and species-specific in revealing trypanosome infections in tsetse flies. While dot-ELISA required a single test to detect T. congolense, several primer pairs were needed for PCR. The potential use of dot-ELISA as a tool for studying the epidemiology of trypanosomosis, while considering its field applicability and relatively lower cost is discussed.

Characterization of trypanosome isolates from naturally infected horses on a farm in Kenya.

Following an outbreak of trypanosomosis in horses on a farm in Kenya, 18 trypanosome isolates were collected from the infected animals over a period of one and a half years and cryopreserved for characterization. The characterization was done on the basis of morphology using Giemsa-stained blood and buffy coat smears, infectivity to mice, recombinant DNA hybridization, and chromosome separation by orthogonal field alternation gel electrophoresis (OFAGE). Morphologically, all the trypanosome isolates were identified as belonging to the subgenus Nannomonas, and a total of 16 out of the 18 isolates grew in mice. Using the recombinant DNA hybridization technique, the isolates were further classified as the 'savannah' type of Trypanosoma congolense. Furthermore, chromosome separation by OFAGE, carried out on six clones derived from different isolates, exhibited a profile characteristic of T. congolense, 'savannah' type. However, there were differences in the number and positions of the medium-sized and minichromosomes indicating a diversity of serodemes within the isolates. Hence the infecting trypanosomes in this disease outbreak were T. congolense, 'savannah' type, and comprised several serodemes or strains.

The application of PCR-ELISA to the detection of Trypanosoma brucei and T. vivax infections in livestock.

Teneral tsetse flies infected with either Trypanosoma brucei or T. vivax were fed on healthy cattle. Blood samples collected daily from the cattle were examined by microscopy for the presence of trypanosomes, in thick smear, thin smear and in the buffy coat (BC). All the cattle fed upon by infected tsetse developed a fluctuating parasitaemia. DNA was extracted from the blood of these cattle and subjected to polymerase chain reaction (PCR) using oligonucleotide primers specific for T. brucei or T. vivax. The PCR products unique to either T. brucei or T. vivax were identified following amplification of DNA from the blood samples of infected cattle, whereas none was detectable in the DNA from the blood of the cattle exposed to non-infected teneral tsetse. In a concurrent set of experiments, one of the oligonucleotide primers in each pair was biotinylated for use in PCR-ELISA to examine all the blood samples with this assay. Both the PCR and the PCR-ELISA revealed trypanosome DNA in 85% of blood samples serially collected from the cattle experimentally infected with T. brucei. In contrast, the parasitological assays showed trypanosomes in only 21% of the samples. In the blood samples from cattle experimentally infected with T. vivax, PCR and PCR-ELISA revealed trypanosome DNA in 93 and 94%, respectively. Microscopy revealed parasites in only 63% of the BCs prepared from these cattle. Neither PCR nor PCR-ELISA detected any trypanosome DNA in blood samples collected from the animals in the trypanosome-free areas. However, both assays revealed the presence of trypanosome DNA in a number of blood samples from cattle in trypanosomosis-endemic areas.

Sensitivity and specificity of antigen-capture ELISAs for diagnosis of Trypanosoma congolense and Trypanosoma vivax infections in cattle.

Sensitivity and specificity of the FAO/IAEA antigen-ELISA kits for diagnosis of bovine trypanosomosis were investigated using sera from experimental cattle infected by tsetse challenge with cloned populations of Trypanosoma congolense (three populations) or T. vivax (one population). The kits are based on monoclonal antibodies that recognise internal antigens of tsetse-transmitted trypanosomes. Ten cattle were infected with each trypanosome population for at least 60 days, and in combination with uninfected cohorts (n = 16) were used in a double-blind study design. Sensitivity and specificity of the tests depended on the choice of positive-negative thresholds expressed as percent positivity with respect to the median OD of four replicates of the strong positive reference serum provided with the kit. In general, while overall specificities were high, sensitivities of the antigen-ELISAs were poor. For example, at a cut-off of 5% positivity, the sensitivities of the antigen-ELISAs were 11% for samples (n = 1162) from T. congolense infected cattle (n = 30), and 24% for samples (n = 283) from T. vivax infected cattle (n = 10). The corresponding specificity values were 95% and 79%, respectively. At a cut-off of 2.5% positivity sensitivity for T. congolense was 25%, and for T. vivax 35%; corresponding specificity values were 85% and 63% respectively. There were no values of the positive-negative threshold at which both sensitivity and specificity were satisfactory. Restricting the analyses to samples taken more than 2 weeks after tsetse challenge did little to improve sensitivity estimates. Trypanosome species specificities of the antigen-ELISAs were also poor. Sensitivity and species specificity of the antigen-ELISA for Trypanosoma brucei infections were not investigated. In contrast to the antigen-ELISA, the sensitivity of the buffy-coat technique when applied to the same experimental animals was fairly high at 67% for T. congolense infections and 60% for T. vivax infections. For samples taken more than 2 weeks after tsetse challenge, high sensitivity estimates of 96% for T. congolense and 76% for T. vivax infections were obtained.

Study on the sequential tsetse-transmitted Trypanosoma congolense, T. brucei brucei and T. vivax infections to African buffalo, eland, waterbuck, N'Dama and Boran cattle.

Susceptibility of African buffalo, eland, waterbuck, N'Dama and Boran cattle to sequential Glossina morsitans centralis-transmitted infections of Trypanosoma congolense, T. brucei brucei and T. vivax was compared, and their possible role as reservoirs of these parasites for G. moristans centralis, G. pallidipes, G. austeni, G. brevipalpis and G. longipennis determined. The buffalo, eland, waterbuck and N'Dama controlled T. congolense parasitaemias and were able to prevent anaemia. By contrast, one Boran became severely anaemic whilst the other controlled parasitaemia and anaemia. When the above five species of Bovidae were rechallenged with T. brucei brucei they showed persistent parasitaemias but did not develop anaemia. The buffalo died of other causes. When the remaining four bovids were rechallenged with T. vivax they became infected with mixed T. vivax/T. b. brucei parasites. Eland, waterbuck and N'Dama controlled parasitaemias and anaemia whereas the Boran became anaemic. Cyclical development of T. congolense occurred in G. moristans centralis when fed on the bovid hosts, with buffalo being infective for tsetse flies for a much longer period. There was no relationship between the levels of T. congolense parasitaemia in the bovid hosts and the resultant infection rates in tsetse flies. Glossina m. centralis was more susceptible than G. pallidipes to T. brucei brucei whilst G. austeni the least; G. brevipalpis and G. longipennis were refractory to the mature infection. Again there was no relationship between T. brucei brucei parasitaemia levels in the hosts and infection rates in the flies. Glossina m. centralis and G. pallidipes showed mixed T. brucei brucei/T. vivax infections whilst G. austeni, G. brevipalpis and G. longipennis became infected with T. vivax alone. Tsetse flies showed higher T. vivax infection rates when fed on the hosts with high parasitaemias than thosewith low parasitaemias. Thus trypanotolerant African buffalo, eland, waterbuck, N'Dama as well as some trypanosusceptible Boran cattle can serve as reservoirs of single or mixed trypanosome infections for tsetse flies. This study has also shown that the Ag-ELISA on the sera from the five bovid hosts had low sensitivity and species-specificity. Examinations of thin wet blood films and buffy coats with a phase-contrast microscope were not sensitive enough to detect the parasites on all occasions. Xenodiagnosis using mice for T. brucei brucei and T. congolense infections, and tsetse flies for all the three trypanosome species were most sensitive for the detection of trypanosome infections in the bovid hosts.

Comparative sensitivity of antigen-detection enzyme immunosorbent assay and the microhaematocrit centrifugation technique in the diagnosis of Trypanosoma brucei infections in cattle.

Four Boran cattle were infected with Trypanosoma brucei using Glossina morsitans centralis and were left untreated throughout the experimental period of 18 months. During this period, sequential blood samples were collected and examined for the presence of antitrypanosome antibodies and their antigens. Using the buffy coat technique (BCT), trypanosomes were detected in 38 (16.3%) of the 233 blood samples. Unlike the BCT, antigen-detection enzyme-linked immunosorbent assay (Ag-ELISA) diagnosed infections in 189 (81.1%) of the blood samples. These results were supported by the presence of antitrypanosome antibodies in the same samples. Thus Ag-ELISA was 5.5 times more sensitive than the BCT. Towards the end of the observation period, G.m. centralis tsetse were fed on the aparasitaemic cattle to determine whether they still harboured the infection as the persistent antigenaemia seemed to suggest. Bloodmeals from the four cattle were infective to tsetse, thus emphasising the importance of Ag-ELISA in diagnosis of sub-patent infections.

Sensitivity of an antigen detection enzyme immunoassay for diagnosis of Trypanosoma congolense infections in goats and cattle.

The sensitivity of a monoclonal antibody-based antigen-detection enzyme immunoassay (antigen-ELISA) for the diagnosis of Trypanosoma congolense was evaluated using sera from experimentally infected goats and cattle. Ten goats (Galla x East African Masai) and 7 steers (Bos indicus) were infected with different clones of T. congolense and left to run a chronic course for 46 and 24 mo, respectively. During this period, monthly blood samples were collected and analyzed for the presence of trypanosomes and antigens in peripheral blood. Of 383 caprine blood samples, 361 (94.3%) were positive for circulating antigens whereas only 42 (10.9%) had demonstrable trypanosomes as revealed by the microhematocrit centrifugation technique. In cattle, 570 (82.5%) of 691 blood samples were antigen-ELISA positive compared to 136 (19.7%) samples with detectable trypanosomes. In an analysis of serum samples from goats in an area known to be endemic for trypanosomiasis, 106 (80.9%) of 131 were positive for T. congolense antigens whereas none of the corresponding blood samples had detectable trypanosomes. Control sera from 24 goats in a trypanosomiasis-free region were all antigen-ELISA negative. Hence, the antigen-ELISA was at least 4 times more sensitive than the microhematocrit centrifugation technique in monitoring T. congolense infections in goats and cattle.

Evaluation of the structural and functional changes in the lymphoid organs of Boran cattle infected with Trypanosoma vivax.

Experimental infection of Boran cattle with Trypanosoma vivax resulted in fluctuating parasitemia detectable in peripheral blood which, as the infection progressed, diminished to undetectable levels. In animals necropsied during the initial stages of the infection, trypanosomes were detected in lymph and in the sinuses of the lymph nodes. The lymph nodes and spleen exhibited marked proliferative response which was maximal in animals killed on days 10 and 17 of infection. The response was characterized by an increase in the size and activity of the germinal centers accompanied by increased numbers of large proliferating lymphocytes in the medullary cords and peripheral part of the paracortex of the lymph nodes and in the periarteriolar regions and peripheral follicular areas of the spleen. This was associated with the production of large numbers of plasma cells which were present in the red pulp of the spleen, in the medullary cords of the lymph nodes, and focally in the peripheral paracortical areas of the lymph nodes. By immunofluorescence, many of these plasma cells were found to contain immunoglobulin (Ig) M. There was also increased number of follicles which exhibited granular deposits of IgM and reduction in the areas containing small lymphocytes bearing IgM. During the remainder of the infection and paralleling the decrease in parasitemia, there was gradual reduction in the reactivity of the lymphoid organs. Accompanying the initial proliferative response was a marked increase in plasma concentration of IgM; this decreased to base line during the 3rd week of the infection. The response to phytohemagglutinin, pokeweed mitogen, and lipopolysaccharide of leukocytes obtained from peripheral blood lymph nodes and spleen at intervals during the infection was not significantly different from controls.

The pathogenesis of infection with Trypanosoma vivax in goats and cattle.

The pathology of Trypanosoma vivax (stock Y-486) in East African goats (Galla cross Masai) and Friesian cattle was studied sequentially. In both goats and cattle parasitaemia was detectable six to seven days after inoculation with 1 X 10(5) trypanosomes. The goats were more susceptible than cattle as indicated by higher parasitaemias, more severe anaemia and death in 25 per cent of the goats. The major pathological changes occurred in the lymph nodes, spleen, eyes, pituitary gland, testicles and heart. There was marked, generalised lymph node enlargement associated with an increase in the number and size of lymphocytic follicles accompanied by germinal centre formation. The medullary cords of the lymph nodes were distended and distorted by the high population of plasma cells. Similar changes were noted in the spleen. However there was no evidence for extramedullary erythropoiesis in both organs. Inflammatory changes were noted in the eyes, pituitary glands, testicles and heart.

Specific antibody responses to the variable surface glycoproteins of Trypanosoma congolense in infected cattle.

Sera from cattle infected with three Trypanosoma congolense clones (ILNat 2.1, ILNat 3.1 and ILRAD 588) derived from different stocks were analysed for the presence of specific antibodies against the surface glycoproteins (VSGs) of the infecting trypanosomes using the solid and liquid-phase radioimmunoassays and the neutralization of infectivity test. High levels of IgM, IgG1 and IgG2 antibodies against the VSGs of the infecting variable antigen types (VATs) as well as other VATs that arose during the course of infection were detected. In addition, 11 out of 12 infected animals showed recurrent peaks of antibody activity against the infecting trypanosomes. The recurrent peaks of antibody activity against the VSGs of the infecting organisms would suggest either a reappearance of trypanosomes of the infecting types or emergence of organisms that bear similar surface determinants. In contrast to the studies on murine trypanosomiasis, there was little or no antibody activity against 2,4,6-trinitrophenylated bovine serum albumin (TNP-BSA) in sera from these cattle.

Trypanosoma (Nannomonas) congolense: identification of two karyotypic groups.

Orthogonal-field-alternation gel electrophoresis and DNA blot hybridizations have been used to investigate the genomic relationships among trypanosome clones of subgenus Nannomonas. The results indicate that Trypanosoma (Nannomonas) congolense comprises at least two distinct groups of parasites that differ in both molecular karyotype and repetitive DNA sequences. A description of these two groups and their distinction from Trypanosoma (Nannomonas) simiae is presented.

Characterization of Trypanosoma congolense serodemes in stocks isolated from cattle introduced onto a ranch in Kilifi, Kenya.

A herd of 20 cattle was introduced on a ranch in Kilifi, Coast Province of Kenya, where they were in contact with Glossina austeni for 6 months. In total, 65 trypanosome isolates were made from these animals. Examination of the isolates revealed that 61 were Trypanosoma congolense and 4 were T. theileri. Out of the 61 T. congolense isolates, 55 were successfully passaged and cloned in mice to provide trypanosome populations for further analyses. The stocks and their clones were inoculated into goats on which teneral G. morsitans centralis were later fed in order to provide metacyclics for use in serodeme analysis. Identification of serodemes was carried out by indirect immunofluorescence and neutralization using antimetacyclic hyperimmune sera prepared in mice against metacyclics of cloned trypanosome populations. So far 4 serodemes have been identified in 8 stocks and 7 clones. Each of the 9 stocks contained a mixture of at least 2 of the 4 serodemes identified. Furthermore, stocks isolated sequentially from individual animals contained the same serodemes despite repeated treatment with a curative dose (6 mg/kg body weight) of Berenil between isolations. From the latter finding, it can be inferred that the 4 serodemes were present on the ranch throughout the study period.

Bovine immune response to Theileria parva: neutralizing antibodies to sporozoites.

The role of antibodies in the neutralization of sporozoites of Theileria parva was investigated. It was found that serum obtained from cattle recovered from East Coast fever (ECF) and rabbits immunized with sporozoites was capable of neutralizing the parasites. The serum was then fractionated by gel filtration and anion exchange chromatography to identify which antibody class or subclass was responsible for the neutralization. In bovine hyperimmune serum the neutralizing capacity was in the fraction containing IgG2 immunoglobulins. In rabbit anti-sporozoite serum the neutralizing capacity also resided in the IgG2 containing fraction. These results suggest that humoral antibodies may play a role in resistance to reinfection with T. parva. A mechanism for this acquired resistance is proposed based upon the recently established biological properties of bovine IgG2 immunoglobulins.

The in vitro response to mitogens of leucocytes from cattle infected with Trypanosoma congolense.

The in vitro proliferative response of bovine leucocytes from peripheral blood to LPS, PWM and Con A was monitored during the course of infection with T. congolense. The stimulatory effect of LPS and PWM on PBL was significantly decreased in infected cattle while the reduction of stimulatory response to Con A was less marked. There was a high background proliferation of unstimulated leucocytes from infected cattle. This increase in background counts correlated with the parasitaemia in that both features were abolished by treatment with the trypanocidal drug Berenil (4,4'-diamidino-diazoaminobenzene-diacetamide acetone). Co-cultivation of trypanosomes with PBL from normal cattle did not depress the responses of the latter to mitogens, and trypanosomes maintained in a cell-free medium failed to incorporate 14C-thymidine under the conditions used.

The pathogenesis of primary and secondary infection with Fasciola hepatica in mice.

Primary and secondary infections of F. hepatica in mice were compared to determine how prior exposure to the parasite affected host response. Mice with primary parenchymal Fasciola infections initially had hemorrhagic tunnels filled with inflammatory cells and connective tissue. These lesions were progressive and became most severe 30 days after exposure as the parasites entered the bile ducts. At this time there was much hyperplasia and thickening of all layers of the duct system near the parasites and occasionally severe periportal fibrosis. By 2 months after exposure regeneration of the damaged liver cells was complete although hyperplasia persisted in bile ducts containing flukes. In mice with secondary infections (mice exposed 40 to 50 days after first infection), the inflammatory response was faster and shorter. Most lesions were resolved by 30 days after the second exposure. There was little difference in histology of primary and secondary infections during the chronic phase of the disease.

Field evaluation of a dot-ELISA for the detection and differentiation of trypanosome species in infected tsetse flies (Glossina spp.).

A rapid, visually read, dot-ELISA developed for the detection and differentiation of trypanosome species in tsetse flies (Glossina spp.), was field tested alongside the standard fly dissection method on a range in south eastern Kenya. Of 104 G. pallidipes dissected, 2 were found to be infected with trypanosomes in their midguts. By the dissection method the infecting trypanosome species could not be identified, as both flies had no salivary gland infections. However, using the dot-ELISA, the 2 flies were shown to be infected with Trypanosoma congolense in their midguts. The midguts of an additional 6 (5.8%) of the 104 G. pallidipes tested positive for T. congolense int he dot-ELISA, even though no trypanosomes were seen on dissection. The infection rate for this fly species as determined using the dot-ELISA, therefore, was 7.7% for T. congolense in midgut infections compared to 1.9% identified by fly dissection. The salivary glands and mouthparts of the 6 additional tsetse flies identified by dot-ELISA were all negative as determined by the 2 techniques. None of 390 G. longipennis flies dissected and examined for trypanosomes in the midgut, salivary glands and mouthparts was shown, by this method, to be infected. Using the dot-ELISA, however, 17 (4.4%) of the flies tested positve for T. congolense in the midgut, whilst the salivary glands and mouthparts of the same flies were negative. Thus, the dot-ELISA appears to be more sensitive than the fly dissection method under field conditions. Moreover, the dot-ELISA can be performed in the field without electricity. It is simple to perform, and was not affected by high ambient temperatures (22-32 degrees C), or by contamination of reactants with dust.