The involvement of endotoxins in Trypanosoma brucei brucei infection in CD-1 mice was investigated by the Limulus amoebocyte lysate (LAL) test. At 7 days post-infection mean serum endotoxin level was elevated by 2·5 times (36·4 pg/ml cf. control 14·25 pg/ml, P < 0·001) and a similar increase was maintained throughout the infection (survival 28–35 days). Purified disrupted parasites contained significant endotoxin activity (mean value 280 pg/mg protein). The mouse infections were also associated with progressive Gram-negative bacteraemia (present in 4 Out of 5 infected animals by day 28 p.i.). The increased endotoxin levels may be due to parasite products, the products of intercurrent bacterial infections, other unidentified sources (e.g. from the gut), or a combination of these. It is concluded that the raised endotoxins may be important contributive factors in the pathogenesis of experimental murine trypanosomiasis.

Four histones, a, b, c, d from procyclic Trypanosoma brucei brucei, which show similarities with the amino acid composition of the core histones H3, H2A, H2B and H4, were isolated and cleaved with Endoproteinase Glu-C. The fragments were separated by FPLC reversed phase chromatography and a subset of the fragments (a5, a9, b6, c8, d3, d9, d11) was subjected to sequence analysis. A 54–71% identity was found in the sequences of the fragment c8 and the C-terminal half of H2B and of three fragments of protein d covering the N-terminal half as well as the C-terminal region of H4. The amino acid sequence of the fragment a9 showed a 57 and 54% identity with H3 sequences of Saccharomyces cerevisiae and Xenopus laevis. Neither the a5 nor the b6 sequence could be aligned with histone sequences of other eukaryotes. The significant differences of 21–48% between the T. b. brucei, histone sequences and those of calf thymus histones, which are more pronounced than the differences of Tetrahymena pyriformis and the higher eukaryote, resulted partially from replacements of amino acids with different properties and indicate specific patterns of histone–histone and/or histone–DNA contact sites in the nucleosome of T. b. brucei. These differences, together with the lack of a functional histone H1, may be sufficient to explain the lack of a salt-dependent formation of the nucleosome filament into the 30 nm fibre, which reflects alternative methods of organizing and processing the genetic information in the nucleus of the protozoan parasite and which may be of chemotherapeutic significance.

Two trypanosome clones, representing East and West African homozygotes at 2 isoenzyme loci (T. b. rhodesiense MHOM/ZM/74/58 [CLONE B] and T. b. brucei MSUS/CI/78/TSW 196 [CLONE A]), were cotransmitted through tsetse flies and the resulting trypanosome populations checked for the presence of non-parental karyotypes by pulsed-field gel electrophoresis. Ten clones isolated from these populations proved to have 5 different recombinant genotypes by analysis of nuclear and kinetoplast DNA (kDNA) polymorphisms. It is inferred that genetic exchange occurred between the 2 trypanosome clones in the fly, as previously reported for 2 other T. brucei spp. clones by Jenni and colleagues. For the most part, the hybrid clones shared many characteristics with both parents and their genotypes were consistent with segregation and reassortment of parental alleles. The least amount of genetic material exchanged was kDNA alone. Regarding the mechanism of genetic exchange, several hybrid clones had identical and unique nuclear DNA polymorphisms, but different kDNA type. Assuming that the same reassortment of nuclear markers is unlikely to occur by chance, these clones most probably arose from a predecessor carrying both types of kDNA.

Trypanosome infection of mammalian hosts leads, within days, to a strong early response against a small, distinct number of parasite proteins. One of these proteins is the variable surface glycoprotein (VSG). Most of the others are apparently non-variable, intracellular trypanosome proteins. Two of these antigens I2 and I17 are now characterized at the molecular level. Both exhibit a highly repetitive amino acid sequence organization, but they show no sequence similarity either to each other or to any other proteins known to date. Preliminary serological analyses indicate that both allow the early, sensitive and specific detection of infections with different species of trypanosomatids, making them interesting candidates for the development of diagnostic tools for trypanosomiasis detection.

Nucleosome filaments of two stages of the life-cycle of Trypanosoma brucei brucei, namely bloodstream forms and procyclic culture forms, were investigated by electron microscopy. Chromatin of bloodstream forms showed a salt-dependent condensation. The level of condensation was higher than that shown by chromatin from procyclic culture forms, but 30 nm fibres as formed in rat liver chromatin preparations were not found. Analysis of histones provided new evidence for the existence of H1-like proteins, which comigrated in the region of the core histones in SDS–PAGE and in front of the core histones in Triton acid urea gels. Differences were found between the H1-like proteins of the two trypanosome stages as well as between the core histones in their amount, number of bands and banding pattern. It can be concluded that T. b. brucei contains a full set of histones, including H1-like proteins, and that the poor condensation of its chromatin is not due to the absence of H1, but most probably due to histone–DNA interaction being weak. It is obvious that structural and functional differences of the chromatin exist not only between T. b. brucei and higher eukaryotes, but also between various stages of the life-cycle of the parasite. It is therefore not adequate to investigate the chromatin only of the procyclic culture forms as a model for all stages of the life-cycle of T. b. brucei.

Procyclic forms of Trypanosoma brucei brucei remain and propagate in the midgut of tsetse fly where iron is rich. Additional iron is also required for their growth in in vitro culture. However, little is known about the genes involved in iron metabolism and the mechanism of iron utilization in procyclic-form cells. Therefore, we surveyed the genes involved in iron metabolism in the T. b. brucei genome sequence database. We found a potential homologue of vacuole protein sorting 41 (VPS41), a gene that is required for high-affinity iron transport in Saccharomyces cerevisiae and cloned the full-length gene (TbVPS41). Complementation analysis of TbVPS41 in ΔScvps41 yeast cells showed that TbVPS41 could partially suppress the inability of ΔScvps41 yeast cells to grow on low-iron medium, but it could not suppress the fragmented vacuole phenotype. Further RNA interference (RNAi)-mediated gene knock-down in procyclic-form cells resulted in a significant reduction of growth in low-iron medium; however, no change in growth was observed in normal culture medium. Transmission electron microscopy showed that RNAi caused T. b. brucei cells to have larger numbers of small intracellular vesicles, similar to the fragmented vacuoles observed in ΔScvps41 yeast cells. The present study demonstrates that TbVPS41 plays an important role in the intracellular iron utilization system as well as in the maintenance of normal cellular morphology.

Efficient, cyclical transmission of trypanosomes through tsetse flies is central to maintenance of human sleeping sickness and nagana across sub-Saharan Africa. Infection rates in tsetse are normally very low as most parasites ingested with the fly bloodmeal die in the fly gut, displaying the characteristics of apoptotic cells. Here we show that a range of antioxidants (glutathione, cysteine, N-acetyl-cysteine, ascorbic acid and uric acid), when added to the insect bloodmeal, can dramatically inhibit cell death of Trypanosoma brucei brucei in tsetse. Both L- and D-cysteine invoked similar effects suggesting that inhibition of trypanosome death is not dependent on protein synthesis. The present work suggests that antioxidants reduce the midgut environment protecting trypanosomes from cell death induced by reactive oxygen species.

Trypanosomosis is a major cause of mortality for dogs in Nigeria and treatment with diminazene aceturate has steadily become less effective, either as a result of low quality of the locally available diminazene preparations or of drug resistance. To investigate these alternatives, samples of locally obtained drugs were analysed for diminazene aceturate content and a strain of Trypanosoma brucei brucei was isolated from a diminazene-refractory dog in Nsukka, south-eastern Nigeria, and used to infect albino rats. The quality of diminazene aceturate-based preparations was variable, with two preparations containing less than 95% of the stated active compound. Rats infected with T. brucei isolated from the dog were treated 7 and 10 days after infection either with 7 mg/kg diminazene aceturate (intraperitoneally, once) or with 4 mg/kg pentamidine isethionate (intramuscularly, 7 consecutive days). Relapse rates were 100% for both trypanocides in the groups of rat treated 10 days post-infection, and 83% and 50% of rats treated 7 days after infection relapsed to diminazene aceturate and pentamidine isethionate, respectively. Careful consideration of physiological parameters showed that pentamidine was only marginally superior to diminazene aceturate as applied in this study. It was concluded that dogs in Nigeria are infected with genuinely diminazene aceturate-resistant trypanosomes that appear to be cross-resistant to pentamidine isethionate.

The involvement of intestinal damage in experimental African trypanosomiasis was investigated in rats infected with Trypanosoma brucei brucei by measuring the urinary excretion of the previously administered non-metabolizable sugar probes, D-mannitol and lactulose, and the flux of FITC-dextran across isolated, everted gut segments. There was increased urinary recovery and flux of the sugar probes across the intestine which were significant (P<0·05) and maximum at day 21 of the infection, but subsequently reduced, in the terminal stages of infection (day 33 p.i.). In the case of the everted sac studies the reductions were to less than 25% control values (P<0·001). Levels of circulating endotoxin were increased approximately 3-fold at day 21 p.i., 4-fold at day 33 p.i., compared to controls. At day 21 there was a significant correlation (r = 0·63, P<0·01) between the log endotoxin levels and the increased sugar excretion expressed as the lactulose/mannitol ratios. Histological studies showed damage to the villi, wall thinning and marked cellular infiltrations, which were very prominent in the proximal jejunum and duodenum. These results demonstrate that during trypanosome infections in rats, increased intestinal leakage and increased circulating endotoxins are significant pathological features.

The effects of infection by Trypanosoma brucei and DNA virus on the ultrastructure of the salivary gland cells in Glossina pallidipes Austen were investigated. Cytoplasm of uninfected cells contains a dense ribosomal population and rough endopiasmic reticulum, scattered Golgi areas and mitochondria. In infected ceils of salivary glands the ultrastructural integrity of the cytoplasm is profoundly changed due to cellular proliferation giving rise to a stratified epithelium and gland enlargement; cell degeneration characterized by formation of cytoplasmic vacuoles, chromatin margination, disorganization and elimination of cell organelles and gland hypertrophy. The virus particles are found in the nucleoplasm as well as the cytoplasm of cells. The ultrastructural evidence indicates that the virus are largely assembled in nuclei of cells and virions pass through the nuclear membrane. The trypanosomes are found within the degenerating cytoplasm and lumen of the cell.

The implications of the features observed in the infected cells are discussed, and possible suggestions are made regarding alteration of the metabolic functions due to infection.

Haemocoelic injections of live Escherichia coli and oral administration of three tetracycline compounds, namely Reverin, Maxitet and Terramycin to female Glossina morsilans morsitans prior to their first larviposition, caused a decrease in fecundity, but had no effect on longevity. Trypanosoma brucei brucei and T. congolense administered by haemocoelic injections, however, caused no change in fecundity or longevity, but tsetse infected with T. b. brucei and T. congolense in their mouthparts had decreased fecundity and longevity as compared to controls.

Animal-infective forms (metacyclics) of Trypanosoma brucei brucei were grown in continuous culture at 29°C from the salivary glands of infected tsetse. The parasites were found by immunofluorescent staining to be antigenically stable in the course of cultivation. When the antigenically stable parasites were incubated at 37°C they transformed from long and thin forms to short and stumpy ones and finally to long and slender parasites. The latter generated antigenic variants starting from the second, third or fourth week of incubation at 37°C. Once antigenic variants appeared at 37°C it was not possible to restore the parasites back to antigenically stable populations when they were transferred back to 29°C. These findings may have far-reaching implications in the study of antigenic variation in pathogenic African trypanosomes and the possible development of immunizing reagents against tsetseborne trypanosomiasis.

In the course of the in vitro cultivation of Trypanosoma brucei as midgut forms, immature metatrypomastigotes, and metacyclic forms, a phenomenon was observed that suggested possible existence of sexual forms consisting of two parasites of unequal size which were seen to come close to each other with their posterior ends apposed. The smaller parasite which was more active than the larger one travelled along the side of the latter so that its posterior end came to lie in the region of the kinetoplast of the larger one. At the latter site, the smaller parasite displayed vigorous twisting movements and later came to lie parallel to its partner. Electron-microscopic examination of the parasites revealed that the points of contact were the kinetoplast and the flagellar pocket. These observations point to the possible existence of sexual forms, and the transfer of genetic material in the 3 types of T. brucei cultured from the vector, G. m. morsitans.