Immunological control of chronic Trypanosoma brucei gambiense in outbred rodents

Recent human isolates of Trypanosoma brucei gambiense generally fail to become or remain patent in laboratory rodents. The purpose of this study was to determine if this was due to acquired immunity and if so which immunosuppressive method was the most efficient in raising parasitemia levels. Prior to infection, rats and mice were immunosuppressed by treatments with cobra venom factor, anti-lymphocyte sera, hydrocortisone acetate, cyclophosphamide; by splenectomy; or by lethal X-irradiation. While no parasites were detected in the blood of most of the untreated rodents for 30 days postinfection, all immunosuppressive procedures resulted in patent parasitemias in at least fifty percent of the treated animals. The most effective method, lethal X-irradiation, consistently caused fulminating infections typical of acute African trypanosomiasis. Cyclophosphamide had the same effect as X-irradiation in rats but was less effective in mice. Splenectomy allowed fulminating first peak parasitemias in two-thirds of the rodents while cobra venom factor and anti-lymphocyte sera in general allowed only low first peaks of parasitemia that were resolved within 10 days of infection. Hydrocortisone acetate allowed low grade and sporadically patent infections throughout the 30-day study. To determine if in untreated rodents, the parasites were eliminated or maintained in a subpatent state, rodents infected for 30-45 days were immunosuppressed with cyclophosphamide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemotherapeutic effects of Annona senegalensis in Trypanosoma brucei brucei

Mice infected with Trypanosoma brucei brucei 8/18 strain were treated orally and intramuscularly (im) with aqueous root extracts of Annona senegalensis, in doses of 27.8 mg kg-1 and 9.5 mg kg-1 respectively, for four consecutive days commencing 72 hours after the mice were infected. At these dosages the parasites were cleared from the circulation and no relapse was recorded over 60 days. The plant extract, however, had no effect on the trypanosomes when therapy was initiated at the late stages of infection, that is, about the sixth day when the parasitaemia level was 0.9 x 10(6); and all the animals died a day or two later. The herbal extracts also did not show any prophylactic action when given prior to infection. The root extract possesses different margins of safety in the mice depending on the route of administration. The therapeutic index for oral administration was 5.13, and that for im administration was 1.8. Chemical tests revealed that the plant extract contains alkaloids, saponins and tannins. Adverse reactions, especially to doses of 2.3-5.76 mg kg-1, were noted in animals that received the drug parenterally, but not when the drug was administered orally. However, A. senegalensis is shown to be therapeutically effective against T. b. brucei in mice, which agrees with the claims of Nigerian practitioners of Traditional Medicine that it is effective against trypanosomiasis in man.     

Survival of Trypanosoma brucei brucei in cerebrospinal fluid.

Different compositions of artificial cerebrospinal fluid (CSF) and the CSF from sleeping sickness patients both with and without late-stage [i.e. central nervous system (CNS)] involvement were evaluated for their abilities to support survival of Trypanosoma brucei brucei. Artificial CSF, containing the major electrolytes, amino acids and carbohydrate components of healthy fluid, was equivalent to the CSF from patients without CNS involvement (survival time 20 hours). Normal CSF does not therefore contain substances which deter the parasite. The CSF from the late-stage patients did not support survival for longer than 10 hours, probably because it contained increased numbers of lymphocytes and antibodies.     

Gonadotropic dysfunction produced by Trypanosoma brucei brucei in the rat

Hormonal disorders have been frequently observed in humans and animals infected with tsetse-transmitted (African) trypanosomes. We studied the pituitary gonadal axis (plasma concentrations of testosterone, luteinizing hormone (LH), follicle stimulating hormone (FSH) and the pituitary gonadotropin (LH, FSH) concentrations) in rats as an experimental model infected with an acute stock of Trypanosoma brucei brucei (AnTat 1.1A). The same investigations in vivo were carried out with rats inoculated by trypanosomal preparations: surface coat components slowly released at pH 5.5 and the parasitic cellular pellet. The releasing procedure as firstly described by Baltz et al. (1976) was performed in the presence or the absence of protease inhibitors. We noted a testicular hypogonadism produced by the acute infection with the decrease of the testosterone level and an increase of the pituitary LH concentration, although the other circulating FSH and LH hormone levels were stable. The injection of the trypanosomal pellet, obtained in the presence of antiproteases, generated a similar clinical hormonal picture: decrease of testosterone level; increase in pituitary LH, FSH content; absence of significant variation of circulating FSH and LH rates. When the trypanosomal pellet was prepared in absence of antiproteases the circulating gonadostimuline levels were significantly decreased. In the same conditions (absence of antiproteases) the trypanosomal supernatant pH 5.5 induced the decrease of the testosterone and plasma LH levels. These results suggested that component(s) from trypanosomes generated hormonal perturbations.     

Parasite-driven pathogenesis in Trypanosoma brucei infections

Trypanosomes are protozoan parasites of medical and veterinary importance. It is well established that different species, subspecies and strains of trypanosome can cause very different disease in the mammalian host, exemplified by the two human-infective subspecies of Trypanosoma brucei that cause either acute or chronic disease. We are beginning to understand how the host response shapes the course of the disease and how genetic variation in the host can be a factor in disease severity, particularly in the mouse model, but until recently the role of parasite genetic variation that determines differential disease outcome has been a neglected area. This review will discuss the recent advances in this field, covering both our current knowledge of the T.?brucei genes involved and the approaches that are leading towards the identification of T.?brucei virulence genes. Finally, the potential for using parasite genotype variation to examine the evolutionary context of virulence will be discussed.     

The relapse of Trypanosoma brucei brucei infections after chemotherapy in rabbits

Infections of T. brucei in the rabbit were found to relapse after chemotherapy. The results indicated that 25 mg/kg diminazene aceturate given 3 days after infection resulted in a complete cure but if given 7 days after infection relapses frequently occurred. However, treatment was apparently successful if delayed until 14 or 21 days. Six of the rabbits originally treated with diminazene aceturate on day 7 were treated with suramin 21 days later; in 3 rabbits the infections relapsed. In all rabbits in which drug treatment was not curative, the clinical condition nevertheless improved. An attempt to locate a cryptic focus of infection in rabbits was unsuccessful.     

Global metabolic responses of mice to Trypanosoma brucei brucei infection

Human African trypanosomiasis (HAT) is transmitted by tsetse flies and, if untreated, is fatal. Treatment depends on infection stage, and early diagnosis is crucial for effective disease management. The systemic host biochemical changes induced by HAT that enable biomarker discovery or relate to therapeutic outcome are largely unknown. We have characterized the multivariate temporal responses of mice to Trypanosoma brucei brucei infection, using (1)H nuclear magnetic resonance (NMR) spectroscopic metabolic phenotyping of urine and plasma. Marked alterations in plasma metabolic profiles were detected already 1 day postinfection. Elevated plasma concentrations of lactate, branched chain amino acids, and acetylglycoprotein fragments were noted. T. brucei brucei-infected mice also had an imbalance of plasma alanine and valine, consistent with differential gluconeogenesis (parasite)-ketogenesis (host) pathway counterflux, involving stimulated host glycolysis, ketogenesis, and enhanced lipid oxidation in the host. Histopathologic evidence of T. brucei brucei-induced extramedullary hepatic hemopoiesis, renal interstitial nephritis, and a provoked inflammatory response was also noted. Metabolic disturbance of gut microbiotal activity was associated with infection, as indicated by changes in the urinary concentrations of the microbial co-metabolites, including hippurate. Concluding, parasite infection results in multiple systemic biochemical effects in the host and disturbance of the symbiotic gut microbial metabolic interactions. Investigation of these transgenomic metabolic alterations may underpin the development of new diagnostic criteria and metrics of therapeutic efficacy.     

Erythrina abyssinica prevents meningoencephalitis in chronic Trypanosoma brucei brucei mouse model

Human African trypanosomiasis is prevalent in Sub-sahara African countries that lie between 14° North and 29° south of the equator. Sixty million people are at risk of infection. Trypanosoma brucei gambesience occurs in West and Central Africa while Trypanosoma brucei rhodesience occurs in East and Southern Africa. The neurological stage of the disease is characterized by neuroinflammation. About 10 % of patients treated with the recommended drug, melarsoprol develop post treatment reactive encephalopathy, which is fatal in 50 % of these patients, thus melarsoprol is fatal in 5 % of all treated patients. This study was aimed at establishing the potential activity of Erythrina abyssinica in reducing neuroinflammation following infection with Trypanosoma brucei brucei. Swiss white mice were divided into ten groups, two control groups and eight infected groups. Infected mice received either methanol or water extract of Erythrina abyssinica at 12.5, 25, 50 or 100 mg/kg body weight. Parasite counts were monitored in peripheral circulation from the third day post infection up to the end of the study. Brains were processed for histology, immunohistochemistry scanning and transmission electron microscopy. Following infection, trypanosomes were observed in circulation 3 days post-infection, with the parasitaemia occurring in waves. In the cerebrum, typical brain pathology of chronic trypanosomiasis was reproduced. This was exhibited as astrocytosis, perivascular cuffing and infiltration of inflammatory cells into the neuropil. However, mice treated with Erythrina abyssinica water extract exhibited significant reduction in perivascular cuffing, lymphocytic infiltration and astrocytosis in the cerebrum. The methanol extract did not have a significant difference compared to the non-treated group. This study provides evidence of anti-inflammatory properties of Erythrina abyssinica and may support its wide use as a medicinal plant by various communities in Kenya.     

Studies of hyperalgesia induced by Trypanosoma brucei brucei infection in rats.

Sprague-Dawley rats were infected by intraperitoneal injections of a strain of Trypanosoma brucei brucei. This parasite spreads early to areas in the nervous system which lack a so-called blood-brain or blood-nerve barrier including spinal ganglia. Signs of sensory disturbance in the form of hyperalgesia were observed already seven days post infection, using a hot plate analgesimeter. The sensory disturbances progressed and the rats started to lose weight about 3 weeks post infection, and died about one week later. Immunohistochemistry showed no reduction in density of calcitonin gene-related peptide or substance P immunoreactive fibres in the skin or spinal cord. It is suggested that the sensory disturbances are mediated via release of kinin or interleukin-1 which are known as potent hyperalgesic agents. The hot plate analgesimeter is a simple and reliable method and may be useful in experimental trypanosomiasis to test drugs developed to combat this disease.     

Biochemical properties of histone-like proteins of procyclic Trypanosoma brucei brucei.

Four histone-like proteins a, b, c, d were extracted with 0.2 M H2SO4 from soluble nuclear chromatin of Trypanosoma brucei brucei procyclic culture forms and purified by FPLC reversed phase chromatography. The amino acid composition of these proteins and their electrophoretic mobilities in three different gel systems strongly indicated their core histone nature. Similarities were found between a, b, c and d with the core histones H3, H2A, H2B and H4 of higher eukaryotes, respectively. On the other hand, these proteins also showed differences as compared to higher eukaryotes; proteins a and d clearly differed from their counterparts H3 and H4 on the basis of their hydrophobic properties. The results indicate the occurrence of core histone variants in T.b. brucei which may influence DNA-histone and histone-histone interactions as well as the chromatin compaction in the nucleus of this protozoan parasite.     

Evaluation of DL-alpha-difluoromethylornithine against susceptible and drug-resistant Trypanosoma brucei brucei.

The antitrypanosomal activity of the ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine (DFMO, eflornithine) was tested in ten stocks and one clone of the hemoflagellate Trypanosoma brucei brucei in an in vitro system. They showed varying levels of susceptibility to DFMO, their IC50 (the concentration which inhibited growth by 50%) values ranging from 81-691 microM. Differences in DFMO susceptibility were also demonstrated in mice. Combinations of melarsonyl potassium (mel W; trimelarsan) and DFMO showed an additive effect in vitro in a mel W-susceptible and a mel W-resistant stock, but an antagonistic effect in a mel W- and DFMO-susceptible clone. Combinations of suramin and DFMO showed an antagonistic effect in vitro in a suramin-susceptible clone, but a potentiation in a suramin-resistant stock.     

Failure of trypanosomal membrane antigens to induce protection against tsetse-transmitted Trypanosoma vivax or T. brucei in goats and rabbits

A purified protein, relative molecular weight 83 kilodalton (kD), and plasma membranes from Trypanosoma brucei were tested as potential vaccines against tsetse-transmitted T. vivax and T. brucei in goats and rabbits. The 83 kD protein was found in lysates of all clones of T. brucei examined, as well as in lysates of T. vivax, T. congolense and T. rhodesiense. Rabbits and goats were immunized with various amounts of antigen in Freund's complete adjuvant and boosted twice with antigen in Freund's incomplete adjuvant. Two weeks after the last inoculation, the goats were challenged with T. vivax-infected and the rabbits with T. brucei-infected Glossina morsitans morsitans. Although high antibody levels were detected in all the animals immunized with either antigen as measured by radioimmunoassay and immunodiffusion, they became infected and the course of disease was the same as that in unimmunized controls.