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.

Sensitive and specific detection of Trypanosoma vivax using the polymerase chain reaction.

The nucleic acid probes that are currently in use detect and distinguish Trypanosoma vivax parasites according to their geographic origin. To eliminate the need for using multiple DNA probes, a study was conducted to evaluate the suitability of a tandemly reiterated sequence which encodes a T. vivax diagnostic antigen as a single probe for detection of this parasite. The antigen is recognized by monoclonal antibody Tv27 currently employed in antigen detection ELISA (Ag-ELISA). A genomic clone which contained a tetramer of the 832-bp cDNA sequence was isolated and shown to be more sensitive than the monomer. Oligonucleotide primers were designed based on the nucleotide sequence of the 832-bp cDNA insert and used in amplifying DNA sequences from the blood of cattle infected with T. vivax isolates from West Africa, Kenya, and South America. The polymerase chain reaction (PCR) product of approximately 400 bp was obtained by amplification of DNA from all the isolates studied. The oligonucleotide primers also amplified DNA sequences in T. vivax-infected tsetse flies. Subsequently, PCR was evaluated for its capacity to detect T. vivax DNA in the blood of three animals experimentally infected with the parasite. T. vivax DNA was detectable in the blood of infected animals as early as 5 days post-infection. Blood and serum samples from the three cattle and from six other infected animals were also examined for the presence of trypanosomes and T. vivax-specific diagnostic antigen. Trypanosomes appeared in the blood 7-12 days post-challenge, while the antigenemia was evident on Days 5-20 of infection. Analysis of the data obtained in the three animals during the course of infection revealed that the buffy coat technique, Ag-ELISA, and PCR revealed infection in 42, 55, and 75% of the blood samples, respectively. PCR amplification of genomic DNA of T. vivax is thus superior to the Ag-ELISA in the detection of T. vivax. More importantly, both the T. vivax diagnostic antigen and the gene encoding it are detectable in all the T. vivax isolates examined from diverse areas of Africa and South America.

TrypTect CIATT--a card indirect agglutination trypanosomiasis test for diagnosis of Trypanosoma brucei gambiense and T. b. rhodesiense infections.

A simple and rapid test, the card indirect agglutination trypanosomiasis test (TrypTect CIATT) is described, for detecting circulating antigens in persons suffering from Trypanosoma brucei gambiense and T. b. rhodesiense infection by latex agglutination. The sensitivity of the test (95.8% for T. b. gambiense and 97.7% for T. b. rhodesiense) was significantly higher than that of lymph node puncture, microhaematocrit centrifugation and cerebrospinal fluid examination after single and double centrifugation. The specificity of the test was also high: 106 blood donor sera as well as sera from 37 patients with malaria, 25 with visceral leishmaniasis, 10 with schistosomiasis, 5 with filariasis and 10 with hydatid disease, from trypanosomiasis-free areas, gave negative results. Eighteen clinical suspects from active disease transmission foci, without microscopically detectable parasitaemia but with a positive test result, were further examined by lumbar puncture and inoculation of blood into mice; 11 (61%) were found to be infected, suggesting that the test had a high positive predictive value. This study showed that TrypTect CIATT is a useful test for rapid diagnosis of both patent and non-patent T. b. gambiense and T. b. rhodesiense infections.

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.

Differentiation between culture-derived insect stages of T. brucei, T. vivax, T. congolense and T. simiae using a monoclonal antibody-based dot-ELISA.

A sensitive and specific nitrocellulose (NC) membrane-based dot-ELISA, utilizing a panel of monoclonal antibodies (mAbs), was developed for differentiation between in vitro-derived procyclic forms of Trypanosoma brucei, T. congolense and T. simiae, and epimastigotes of T. vivax. Trypanosomes in suspension were applied onto NC membrane in dots and probed with unlabelled trypanosome species-specific mAbs. Bound mAb was revealed by enzyme labelled anti-mouse IgG and precipitable chromogenic substrate. The assay detected the aforementioned trypanosome species in both single and artificially mixed preparations. Ten T. brucei, 4 T. vivax, 7 T. congolense and 3 T. simiae procyclic stocks and clones from different geographical areas were tested and identified using the specific mAbs in the dot-ELISA which had a specificity of 100%. Some of the T. brucei, T. congolense and Nannomonas-specific mAbs could detect as few as 10 trypanosomes/dot, whilst 1 T. vivax mAb was able to detect a minimum of 100 trypanosomes/dot in monospecies preparations. A concentration of 1 x 10(4) trypanosomes/microliters/dot was eventually determined as ideal for testing in the dot-ELISA. Antigen dots stored at 4 degrees C under desiccated conditions did not show any loss in activity for up to 90 days. However, when stored under similar conditions at room temperature (17-26 degrees C), the T. congolense-specific antigen remained unaffected up to 60 days, and then showed decreased activity when tested on day 90.

Production and characterization of a monoclonal antibody specific for Trypanosoma simiae.

Monoclonal antibodies (MoAb) were produced against invariant antigens of vector forms of Trypanosoma simiae. X63/AG8.653, NSI/1AG401 and Sp20Ag14 myeloma cells were fused with splenic lymphocytes from BALB/c mice that had been immunized with various preparations of T. simiae procyclics. A T. simiae-specific MoAb [KNS7/14.X (IgG1)] was detected in the hybridoma culture supernatants, which were screened for antibody activity by micro-plate and dot ELISA. Immunofluorescence studies showed that KNS7/14.X stained cytoplasmic antigens in T. simiae procyclics. Proteinase-K digestion and periodate oxidation studies revealed that KNS7/14.X binds to a carbohydrate antigenic determinant in glycoprotein or glycolipid. Cross-reactivity studies using vector forms of T. brucei, T. vivax, T. congolense, T. simiae and T. grayi showed that KNS7/14.X only reacted with T. simiae. Attempts to generate other T. simiae-specific MoAb, using 107-, 75- or 41.7-43.6-kDa peptides selected by western blotting analysis, did not yield positive results.

Immunological characterization and expression in Escherichia coli and baculovirus systems of a Trypanosoma vivax antigen detected in the blood of infected animals.

A monoclonal antibody (MAb)Tv27 employed in an antigen-detection enzyme immunosorbent assay (Ag-ELISA) for diagnosis of Trypanosoma vivax infection was shown to react with a T. vivax-specific protein of an approximate molecular weight of 10 kDa. This protein is diffusely distributed throughout the cytosol and nucleus of metacyclic forms, bloodstream forms, and procyclic-like elongated trypomastigotes, but is not detectable in epimastigotes of T. vivax. The T. vivax-specific antigen prepared from parasite lysates appeared to be of lower molecular mass than the form expressed in either Escherichia coli or in baculovirus-infected silkworm insect cells. In the recombinant baculovirus-infected cells, the protein was expressed mostly as an 18-kDa peptide with less abundant forms of 13 and 12 kDa, while the protein expressed in E. coli was approximately 14 kDa. Both the low- and higher-molecular-weight proteins are recognized by the MAb Tv27 in Western blots and in Ag-ELISA. Although the crude preparations of the protein produced by the insect cells are labile when kept for more than 2 hr at 24 degrees C, they retained reactivity at temperatures below 4 degrees C for several weeks. The proteins expressed in both the insect cells and E. coli captured anti-T. vivax antibodies in sera prepared from trypanosome-infected animals. Since the recombinant protein expressed in the baculovirus-infected cells is available in large homogeneous quantities, it would serve as a positive control in Ag-ELISA and is also usable for antibody detection assays.

Hydrogen peroxide destaining: a new method for removing non-specific stains in nitrocellulose membrane-based dot-ELISA for the detection of trypanosomes in tsetse flies (Glossina spp.).

Gut samples prepared from laboratory-reared tsetse flies and applied in dots onto nitrocellulose (NC) membrane were found to stain the membrane with differing coloration and intensity. The stains were, predominantly, either reddish to brown or blackish-brown to black and occasionally greenish to almost colourless, depending on the stage of digestion of the bloodmeal in the fly. NC membrane strips applied with tsetse gut samples from T. brucei infected and uninfected control flies were tested with the standard antigen detection dot enzyme-linked immunoassay (dot-ELISA), using a T. brucei specific monoclonal antibody (MoAb) and horseradish peroxidase goat anti-mouse conjugate. The stains in both infected and uninfected sample dots persisted through the assay. Furthermore, the staining intensity of some assayed uninfected sample dots were enhanced as a result of non-specific reactivity, making it difficult to distinguish between the infected and uninfected flies. This necessitated the development of a simple technique by which the non-specific stains and reactions could be removed. Sample 'dotted' NC membrane strips were destained by incubation with 5% hydrogen peroxide (H2O2) diluted in 5% skimmed milk in Tris buffer, pH 8.0. After washing, the destained strips were tested in the dot-ELISA. This method gave satisfactory reproducible results, since the most intense stains could be removed, and it had no effect on trypanosome antigens detected by a panel of four T. brucei species-specific, three T. vivax species-specific, four T. congolense species-specific and four Nannomonas subgenus-specific MoAbs. Using the destaining process in a modified dot-ELISA, 86 out of 95 (90.5%) of Glossina morsitans centralis flies experimentally infected with T. brucei, were identified. The destaining method was also used successfully to decolorize NC membrane bound tsetse faecal material.

Sensitivity of antigen ELISA test for detecting Trypanosoma evansi antigen in horses in the subtropical area of Argentina.

The sensitivity of an antigen detection enzyme immunoassay (Ag-ELISA) based on a Trypanosoma brucei group-specific monoclonal antibody was evaluated to detect circulating Trypanosoma evansi antigen in horse sera. Three horses and 2 mules were experimentally infected with T. evansi. Circulating antigens were detected on 7 and 21 days postinfection. Antigen levels increased during the course of the illness and remained high even when parasitemia was low or when parasites could not be detected. Antigens were cleared from serum when drug treatment was effective but persisted when it was not. In 6 outbreaks of "mal de caderas" involving 125 horses, T. evansi was found in 78 horses using standard parasite detection methods and antigenemia was detected in 58 of them (74%). The Ag-ELISA sensitivity rate varied between 63% and 100% for the 6 different outbreaks. A combination of Ag-ELISA and parasitologic methods diagnosed a total of 93 infected animals. These results show that the Ag-ELISA test is useful both to diagnose T. evansi and to assess the efficacy of drug treatment in horses.

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 differentiate between Trypanosoma brucei, T. congolense and T. simiae procyclics in the midguts of experimentally infected tsetse flies. The modification of the assay consisted of (a) the lysis of T. congolense or T. simiae in NC membrane applied sample dots using Triton X-114, and (b) treatment of sample applied NC membrane strips with hydrogen peroxide to remove non-specific stains. Also, T. brucei was detected in the salivary glands, and T. congolense and T. vivax were detected in the mouthparts, however, in dot-ELISA without modification. In all the assays, T. brucei and T. congolense parasites were detected directly using MoAbs specific to each of them, whereas T. simiae parasites were detected by exclusion using a T. congolense specific and Nannomonas subgenus-specific MoAbs. The sensitivity of the assay for detecting midgut infections was 90.5%, 84.6% and 94.4% in detecting T. brucei, T. congolense and T. simiae, respectively. Sample dots stored at room temperature (19-26 degrees C) under desiccated conditions did not show any loss in activity in 90 days. However, after 7 days of storage, a ring-pattern reaction appeared on some sample dots that were tested with T. brucei specific 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 sensitivity was 90%. Also, T. vivax and T. congolense organisms were each detected in the mouthparts of infected tsetse flies, with 100% specificity. The sensitivity was, however, lower, 43.8% for T. vivax and 55.6% for T. congolense.

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.

Trypanosoma brucei rhodesiense: use of an antigen detection enzyme immunoassay for evaluation of response to chemotherapy in infected vervet monkeys (Cercopithecus aethiops).

Thirty eight Trypanosoma brucei rhodesiense-infected vervet monkeys (Cercopithecus aethiops) in the late (meningoencephalitic) stage of disease, treated with various trypanocidal drugs, were monitored for a period of more than 600 days to assess the rate of clearance of trypanosome antigens from serum and cerebrospinal fluid (CSF). There was a complete but gradual reduction in antigen titres, as assessed by ELISA, in animals treated intravenously with melarsoprol, the standard drug for the late stage disease. In 8 of the 9 monkeys treated with melarsoprol, the antigen titres, as assessed by optical density values, dropped by 50% within 252 days (mean value 68 days for antigens in CSF and 116 for serum) following treatment. The remaining animal in this group, that displayed persistent antigenaemia, had been treated with a sub-curative drug dosage level. Thus, if time to 50% reduction in antigen levels were to be taken as an index to predict cure, the follow-up period after melarsoprol treatment could have been reduced from 600 to 252 days for 8 of the 9 animals, leaving only one animal for further follow up. The animals treated with experimental drug combinations displayed a variable picture. Five monkeys showed a persistence of antigens in both serum and CSF throughout the observation period, suggesting failure of the drugs to cure the infection. Parasitologically confirmed relapse of the infection was indeed observed in all the five monkeys. In some monkeys, the parasite antigens eventually cleared from serum and CSF completely, but this took a longer time duration than in the melarsoprol treated animals; others showed persistence of parasite antigens in serum, but the parasites were not detected in blood or CSF throughout the entire follow-up period. These results suggest that the experimental drug combinations used were not effective in clearing the parasites from cryptic foci and hence the persistence of antigens in serum and/or CSF.(ABSTRACT TRUNCATED AT 250 WORDS)

Trypanosoma brucei, T. congolense and T. vivax infections in horses on a farm in Kenya.

Equines are particularly susceptible to infection with Trypanosoma evansi and T. brucei, but rarely is natural T. congolense and T. vivax infection seen in horses. An outbreak of trypanosomosis occurred in a herd of horses used for patrolling the pineapple fields on the Del Monte Farm, Thika, Kenya initially involving 6 horses. On subsequent screening of the entire group, T. brucei, T. congolense and T. vivax infections were detected in 16 of the 35 horses. The tests used for diagnosis included microscopic examination of stained blood smears, buffy coat technique, mouse inoculation and antigen detection enzyme immunoassay (antigen ELISA).

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.

Suratex: a simple latex agglutination antigen test for diagnosis of Trypanosoma evansi infections (surra).

A simple and rapid, field-oriented latex agglutination test for the detection of circulating invariant trypanosomal antigens in Trypanosoma evansi infections (surra) is described. In an initial evaluation, the test, Suratex, detected the antigens in 53 (88.3%) of 60 serial blood samples collected over a period of 11 weeks from 3 experimentally infected rabbits. By comparison, the buffy coat technique and wet blood film examination diagnosed the infection in 22 (36.7%) and 2 (3.3%) of the blood samples, respectively. In experimental infections in camels, Suratex diagnosed the infection in 10 of the 12 animals by day 7 post-infection, compared to 7 animals detected by the buffy coat techniques. The 5 animals misdiagnosed by the buffy coat technique remained parasite negative throughout the study period of 127 days, yet they had persistent antigenaemia. Analysis of field sera from two camel herds which were experiencing a T. evansi outbreak, confirmed the superior sensitivity of Suratex. Whereas only 5 (15.6%) animals in a herd of 32 were diagnosed by the buffy coat technique, 30 (94.0%) tested positive for antigens. This high prevalence of T. evansi infections in the herd was confirmed by the high antibody titres detected in sera from all the 32 animals. The second herd, consisting of 60 camels, was investigated using both mouse inoculation and Suratex. Twenty-eight (46.7%) of the animals were diagnosed to be infected with T. evansi by mouse inoculation, while Suratex showed all the 60 animals to be infected. Four of the 5 camels diagnosed using the BCT and all the 28 detected by mouse inoculation (32/33; 97.0%) were Suratex positive, thus showing a high degree of correlation between parasitological results and those obtained by Suratex. All the control sera from 30 camels from a T. evansi-free herd were negative.

A comparison of the antigen detection ELISA and parasite detection for diagnosis of Trypanosoma evansi infections in camels.

Two herds of 60 camels each, living in Trypanosoma evansi endemic areas, were selected and studied for a period of 18 months. Animals in one herd were treated prophylactically with quinapyramine prosalt (May and Baker, Dagenham, UK), while those in the other herd were treated individually with quinapyramine dimethylsulphate (May and Baker, Dagenham, UK) when proven parasitaemic. The herd on prophylaxis was sampled for antigen and patent infection monthly. The other herd was sampled weekly for patent infection and fortnightly for antigen. The results obtained could be divided into four categories. The first category comprised cases (52 out of 61) in which the presence of trypanosome antigens could be correlated with parasitological diagnosis. In 80% of these animals the antigens disappeared from the circulation within a period of 30 days following chemotherapy. The second category comprised those animals with parasitologically proven infections but which did not have antigens in their sera. This was observed in nine camels, seven of which were from the herd that was being examined weekly for the presence of trypanosomes. These were considered to be animals in early infection, as the subsequent sera were also negative for anti-trypanosome antibodies and immune complexes. The third category comprised camels which were antigen-positive but aparasitaemic. Sera from these animals were also positive for anti-trypanosome antibodies, indicating that antigen-positivity was a true reflection of trypanosome infections in these animals. The last category comprised pre-weaned camel calves which appeared to have some form of protection against trypanosomiasis, as evidenced by the absence of trypanosomes, antigens and antibodies throughout the early period of their lives. Only occasional antigenaemia was found in a few calves. It is concluded that trypanosome antigen detection may give a more accurate idea of the prevalence of T. evansi infections than does whole parasite detection.

Use of antigen capture tube enzyme-linked immunosorbent assay for the diagnosis of Trypanosoma evansi infections in dromedary camels (Camelus dromedarius).

Parasitological diagnosis of Trypanosoma evansi infection in camels is hampered by the small number of parasites in blood circulation, coupled with the tendency of this trypanosome to invade tissues. To overcome this, a more sensitive assay, an antigen enzyme-linked immunosorbent assay (ELISA), was developed, capable of detecting trypanosome antigens released into the bloodstream by dying parasites. To evaluate the usefulness of this assay in assisting chemotherapy, an experiment was designed to compare the ability of a Trypanozoon subgenus-specific monoclonal antibody (TR7) to capture antigens in whole blood and serum of camels in a T. evansi-endemic area of the Marsabit district in northern Kenya. The tests were performed in polystyrene tubes coated with TR7. Antigen ELISA using whole blood was performed in the field, while serum collected on the same day from the same animals was stored at -20 degrees C and tested in the laboratory at a later date. A total of 100 camels were examined. Twelve per cent of the camels were found to be antigenaemic when whole blood was tested, compared to thirteen per cent detected using serum. Thus, the results obtained so far do not show a significant difference in the sensitivity of tube ELISA when detecting antigens either in whole blood or serum.

Application of a monoclonal antibody-based antigen detection enzyme-linked immunosorbent assay (antigen ELISA) for field diagnosis of bovine trypanosomiasis at Nguruman, Kenya.

A monoclonal antibody-based, enzyme immunoassay (antigen ELISA) for the detection of species-specific invariant antigens of Trypanosoma congolense, T. vivax or T. brucei in the serum of infected animals was evaluated as a means of diagnosis using bovine field sera from a trypanosomiasis endemic area, Nguruman, Kenya. Circulating trypanosome antigens were detected in 126 (96.2%) of 131 serum samples from animals with parasitologically confirmed diagnosis: 74.8% were positive for antigens of two or three trypanosome species, while 21.4% tested positive for one trypanosome species. When 70 sera from animals (at Nguruman), which had tested negative for trypanosomes by the buffy coat technique, were tested, 35 (50.0%) of them were shown to be antigen-ELISA positive: 24 (34.3%) showing infection with a single species and 11 (15.7%) with mixed infections. The predominant trypanosome species diagnosed in the two herds by antigen ELISA was T. vivax, which was detected in 133 (82.6%) of the 161 sera that tested positive for antigens, followed by T. congolense in 122 (75.8%) sera, with 109 (67.7%) showing evidence of mixed infections with two or three trypanosome species. In single infections, T. vivax exceeded T. congolense by a ratio of 2:1, with T. brucei accounting for less than 1.0%. Evidence for the specificity of the test was provided by analysis of field sera from 100 cattle, from a trypanosomiasis-free area, infected with other haemoparasites (anaplasmosis, babesiosis and theileriosis), which all tested negative in the assay.

Relationships between trypanosome infection measured by antigen detection enzyme immunoassays, anaemia and growth in trypanotolerant N'Dama cattle.

Relationships were evaluated between trypanosome infection as measured by antigen detection enzyme immunoassays (antigen ELISA), anaemia as determined by average packed red cell volume (PCV), and animal performance as assessed by daily weight gain in 99 N'Dama cattle in Gabon exposed to natural tsetse challenge at 11.5 months of age and recorded 14 times over a 13 week period. Approximately half the animals were found to be infected for an average of five of the 14 times that they were examined: 38% with Trypanosoma congolense, 13% with Trypanosoma vivax and 49% with a mixed infection. Trypanosoma congolense infections had significant deleterious effects on animal growth, while T. vivax infections did not. Animals found on several occasions to be infected with T. congolense had significantly lower PCV values than those demonstrated to be infected on fewer occasions. No relationship was found between mean optical density (OD) values in antigen ELISA and PCV values. Animals capable of maintaining PCV values, even when antigen ELISA positive on a high number of occasions, grew at the same rate as uninfected animals. Animals that could not maintain PCV values when infected had poorer growth. Antigen ELISA has the potential to increase the efficiency of selection of trypanotolerant N'Dama cattle under tsetse challenge in the field, in three main ways. (1) Accurate identification of trypanosome species, especially in mixed species infections, clarifies relations between infection, anaemia and animal performance. (2) Detection of animals antigenaemic without patent parasitaemia could allow individuals with superior ability to control trypanosome infection to be identified. (3) More accurate measurement of the proportion of time an animal is infected allows more accurate evaluation of its anaemia control capability.

Multicentre evaluation of an antigen-detection ELISA for the diagnosis of Trypanosoma brucei rhodesiense sleeping sickness.

The performance of an enzyme-linked immunosorbent assay (antigen ELISA) for the detection, in serum or cerebrospinal fluid, of an invariant trypanosome antigen to diagnose Trypanosoma brucei rhodesiense sleeping sickness was evaluated in four clinical treatment centres. The test, which was carried out in polystyrene test-tubes, was positive in 88 (88.9%) of 99 parasitologically confirmed cases that were tested at the National Institute for Medical Research, Tabora, United Republic of Tanzania; 99 (94.3%) of 105 cases tested at the National Sleeping Sickness Control Programme, Jinja, Uganda; 86 (87.8%) of 98 cases tested at the Uganda Trypanosomiasis Research Organisation, Tororo, Uganda; and 59 (96.7%) of 61 cases tested at the Tropical Diseases Research Centre, Ndola, Zambia. The overall detection rate was 91.5%. There was no cross-reactivity with the agents of the common bacterial, viral, or parasitic diseases prevalent in the areas where the studies were conducted. The only false-positive result involved a blood donor from a trypanosomiasis endemic focus. The test was simple to perform, was read visually, and is therefore a potential tool for diagnosing human African trypanosomiasis.