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.

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.

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.

Diagnosis of Trypanosoma brucei gambiense sleeping sickness using an antigen detection enzyme-linked immunosorbent assay.

A monoclonal antibody-based enzyme-linked immunosorbent assay (antigen ELISA) developed for detection of trypanosome antigens in the serum and cerebrospinal fluid (CSF) of patients as a means for diagnosis of Trypanosoma brucei gambiense sleeping sickness was evaluated at the Bureau Central de la Trypanosomiase, Kinshasa, Zaire. Sixty-nine (89.6%) of 77 parasitologically confirmed cases examined at the Daloa clinic had antigens in serum; 35 (45.5%) had antigens in CSF and, in 4 of these, the antigens were detected in CSF only. Taking the serum and CSF results together, 73 (94.8%) of the 77 patients were positive in the assay. In the Kinshasa series, 168 (89.4%) of 188 parasitologically confirmed cases were positive by antigen ELISA. The controls, who included 165 blood donors and 40 patients with malaria, 2 with hydatidosis and 12 with leishmaniasis, were negative by antigen ELISA. Analysis of CSF results for 35 patients who had antigens in CSF revealed that 34 (97.1%) had elevated CSF white cell counts, 29 (82.9%) had elevated protein levels, and 23 (65.7%) had trypanosomes in their CSF. Moreover, analysis of results for 34 patients whose CSF had been shown to harbour trypanosomes by the double centrifugation technique showed that 24 (70.6%) had antigens in CSF, 28 (82.6%) had elevated protein levels, and 33 (97.1%) had elevated CSF white cell counts. Antigens were rapidly cleared from peripheral circulation following institution of treatment. Antigen clearance was accompanied by a rapid fall in CSF protein levels and white cell counts. These results demonstrate the potential of antigen ELISA, not only as a tool for diagnosis, but also for clinical staging and treatment follow-up of patients with T. b. gambiense sleeping sickness.

Comparison between bloodstream and procyclic form trypanosomes for serological diagnosis of African human trypanosomiasis.

Trypanosoma brucei brucei MiTat 1.2 bloodstream and corresponding procyclic forms, as well as procyclics of T. b. gambiense and T. b. rhodesiense, were fixed, in suspension, using a mixture of 80% acetone and 0.25% (v/v) formalin in saline and used as antigens for diagnosis of African human trypanosomiasis by the indirect immunofluorescent antibody test. T. b. brucei bloodstream forms detected 41/42 and 37/41 parasitologically diagnosed cases of T. b. rhodesiense and T. b. gambiense trypanosomiasis, respectively, while the procyclic stages detected the same number (41/42) of T. b. rhodesiense, but fewer (29/41) T. b. gambiense infections.

Suppression of cyclical development of Trypanosoma brucei brucei in Glossina morsitans centralis by an anti-procyclics monoclonal antibody.

Five hundred and sixty teneral male Glossina morsitans centralis were fed, at the height of parasitaemia, on a goat infected with Trypanosoma brucei brucei. Thereafter, the tsetse were divided into 4 equal groups. Group I was fed in vitro once weekly for 4 weeks and Group II twice weekly for 4 weeks on fresh defibrinated ox blood containing 2 mg/ml purified monoclonal antibody against T. b. brucei procyclics, while Group III was fed twice a week for 4 weeks on blood containing 2 mg/ml anti-T. vivax monoclonal antibody. The last group was fed on a rabbit. The tsetse were dissected on day 31 and the percent salivary gland infection rates observed were 18.2, 18.6, 39.8 and 40.8, respectively. In another experiment, 2 groups of tsetse, 120 per group, were fed on fresh defibrinated ox blood containing 2 mg/ml anti-T. b. brucei (test group) or anti-T. vivax (control group), on days 3, 6 and 9 following the infected feed. Dissection of the tsetse on day 31 revealed salivary gland infection rates of 0% in the test group and 6.5% in the control group. Thus the monoclonal antibody had a marked, specific suppression of the cyclical development of T. b. brucei in the tsetse vector.

Stabilization and preservation of the antigenic specificity of Trypanosoma (Trypanozoon) brucei variant specific surface antigens by mild fixation techniques.

Living bloodstream trypansomes fixed by suspension in a 1% formalin solution maintain both their morphology and the immunological specificity of their variant specific surface glycoprotein, so allowing precise identification of the variant types present in a trypanosome population by direct or indirect immunofluorescence combined with phase microscopy. The technique is simple, adaptable to the study of low parasitaemias and should facilitate analysis of the phenomenon of antigenic variation both in the field and the laboratory.

Studies on Trypanosoma (nannomonas) congolense. I. On the morphological appearance of the parasite in the mouse.

The pleomorphism of bloodstream Trypanosoma (Nannomonas) congolense was studied during the course of the first parasitaemic wave in mice using cloned and uncloned derivatives of three recent field isolates. The different morphological types were identified using the criteria described by Godfrey (1960). It was found that at any point of parasitaemia there were several morphological types of the parasite present, ranging from short to long forms. In the rising phase of parasitaemia, the short forms predominated, while at peak parasitaemia the parasites were highly pleomorphic, with significant proportions of short and "intermediate" forms although the long forms predominated. Pleomorphism was observed both in normal and in lethally irradiated (900 R) mice, even when the infection was initiated using a single organism. Such pleomorphism may result from physiological differences between the different forms of this parasite since these morphological types of T. congolense also differed in their ability to infect a new mammalian host.

Studies on Trypanosoma (nannomonas) congolense II. Observations on the cyclical transmission of three field isolates by Glossina morsitans morsitans.

Teneral flies of Glossina morsitans morsitans were fed on mice infected with cloned and uncloned derivatives of three recent field isolates of Trypanosoma (Nannomonas) congolense. Flies with mature infections were identified by the warm-slide probe method and phase-contrast microscopy. High infection rates were achieved when such flies were fed on mice at peak parasitaemia. The infection rates were low when flies were fed on mice prior to or late after peak parasitaemia. The duration of the developmental cycle of T. congolense in the tsetse fly varied from 7 to 40 days: in 45% of the infective flies the developmental cycle was completed within 12 days; and in 76%, within 18 days.

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.

Use of antigen-detection enzyme immunoassays in assessment of trypanotolerance in N'Dama cattle.

Antigen-detection enzyme immunoassays (ELISA) were used for the diagnosis of Trypanosoma vivax, T. congolense and T. brucei in N'Dama cattle in Gabon, Central Africa. The assays are based on monoclonal antibodies which recognise trypanosome antigens specific for each of the three species and animals were termed 'antigenaemic' when found positive by this technique but not found parasitaemic by the buffy coat technique. 148 one-year-old animals were exposed to a medium natural tsetse challenge and an average of 6 assays per animal were carried out over a 92-day period. Blood samples were routinely examined 11 times over this period and 28% of animals were detected as parasitaemic by the buffy coat technique. 90% of these were antigen-ELISA positive. More importantly, 40% of the animals with negative parasitological findings were also found to be antigenaemic. Parasitaemic animals with above-average packed-red-cell volume percent (PCV) values had 32% higher daily weight gains than those with below average, while antigenaemic animals showed no significant linkage between PCV values and weight gain. Thus only the 28% of animals with detectable parasitaemias could have been used for selection decisions based on PCV values. Antigenaemic animals grew at the same rate as negative animals and had 22% superior growth rates to parasitaemic animals. When antigenaemic animals were classified as having more ability to control parasite growth than parasitaemic animals, a significant sire effect suggested some possibility of a degree of genetic control being involved. Thus the ELISA could offer a practical possibility for selection of trypanotolerant animals based on infection criteria.

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.

Analysis of the variable antigen composition of Trypanosoma brucei brucei metacyclic trypanosomes using monoclonal antibodies.

The metacyclic trypanosomes of a Trypanosoma brucei brucei clone (ILTat 2.1) were analysed with regard to their variable antigen (VAT) composition using monoclonal antibodies. The metacyclic population was antigenically heterogeneous. Despite the heterogeneity, however, the overall VAT composition of the metacyclic population appeared to be limited in number. A similar pattern of reactivity was observed when the monoclonal antibodies were tested on metacyclics of another clone (IL Tat 2.2) derived from a rabbit 30 days after infection with IL Tat 2.1 as well as those of the parent stock (STIB 247). The VAT characteristics of the metacyclics of this serodeme were consistent regardless of whether they were transmitted by Glossina morsitans morsitans or G.m. centralis. The monoclonal antibodies also reacted with some of the bloodstream VATs isolated within 72 h from cyclically infected mice. None of the monoclonal antibodies, however, reacted with metacyclics of a different stock (LUMP 227).

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.

Production and evaluation of specific antisera against sera of various vertebrate species for identification of bloodmeals of Glossina morsitans centralis.

Specific antisera against sera of 46 species of vertebrates were prepared. The antisera to 21 Bovidae species were raised in goats except the antiserum to goat serum which was raised in sheep. The antisera to 3 Suidae species were produced either in domestic pigs or warthogs, while antisera to most of the other vertebrate species were raised in rabbits. The antisera were used in an enzyme-linked immunosorbent assay (ELISA) to identify the source of bloodmeals ingested by teneral and non-teneral tsetse at different time intervals after feeding. The bloodmeal donors were identifiable in 100% of the teneral tsetse up to 40 h post-feeding and in 87.5% in those tested up to 74 h post-feeding. Non-teneral tsetse digested the species-distinguishing bloodmeal components faster than the tenerals. Bloodmeals could be identified in 100% non-tenerals at 20 h post-feeding but only 67.5% and 50% of the bloodmeals could be identified 40 h and 74 h post-feeding, respectively. The antisera were also able to identify mixed bloodmeals from closely related species.

Role of the chancre in induction of immunity to tsetse-transmitted Trypanosoma (Nannomonas) congolense in goats.

Local skin reactions (chancres) developed in goats at the sites of deposition, by tsetse flies, of metacyclics of Trypanosoma congolense. The chancres developed much faster and were more pronounced when ten infected tsetse were allowed to feed on a spot as compared to only one fly per spot. The initial host cellular reaction in the chancre was predominantly polymorphonuclear, followed at the peak of development of the chancre by a predominantly lymphoblastic and plasmacytic reaction. Trypanosomes were found in various stages of division as well as degeneration in chancre biopsies taken at various days post-infection (p.i.). Most of the trypanosomes recovered from the chancre tissue fluid were found to bear the same variable surface glycoprotein (VSG) epitopes as the corresponding metacyclics for as long as 13 days p.i., as revealed by indirect immunofluorescence using mouse anti-metacyclic VSG hyperimmune sera and monoclonal antibodies. Immunization of goats with metacyclic trypanosomes, by exposure to infected tsetse bites followed by treatment of the infected goats on day 13 p.i., gave rise to the development of protection to homologous tsetse-transmitted challenge, whilst immunization by intravenous inoculation of the metacyclics did not induce such protection. Chancre formation would thus appear to be vital for the induction of comprehensive immune recognition of the metacyclic variable antigen repertoire deposited in the skin by infected tsetse, and hence development of protective immunity.

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.

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.

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.