Mathematical and statistical analysis of the Trypanosoma brucei slender to stumpy transition.

We propose a new model for the Stumpy Induction Factor-induced slender to stumpy transformation of Trypanosoma brucei gambiense cells in immunosuppressed mice. The model is a set of delay differential equations that describe the time-course of the infection. We fit the model, using a maximum-likelihood method, to previously published data on parasitaemia in four mice. The model is shown to be a good fit and parameter estimates and confidence intervals are derived. Our estimated parameter values are consistent with estimates from previous experimental studies. The model predicts the following. Slender cells can be classified as uncommitted, committed and dividing, and committed and non-dividing. A committed slender cell undergoes about 5 divisions before exiting the cell-cycle. Committed slender cells must produce SIF, and stumpy cells must not produce SIF. There are two mechanisms for differentiation, a background differentiation rate, and a SIF-concentration-dependent differentiation rate, which is proportional to SIF concentration. SIF has a half-life of about 1.4 h in mice. We also show, with suitable changes in the parameter values, that the model reflects behaviours seen in other host species and trypanosome strains.

African trypanosomiasis research: 100 years of progress, but questions and problems still remain.

Past and present progress in our understanding of African trypanosomiasis is briefly reviewed. Although tremendous scientific strides have been achieved, an epidemic of the disease is currently underway. Three areas of research which are believed necessary for the control of African trypanosomiasis are discussed. It is suggested that a better understanding of the host-parasite relationship is essential; more emphasis and a broader approach to drug development is required; and finally, further research into the socio-economic aspects of African trypanosomiasis is urgently needed before the human disease can again be controlled.

Innate and acquired resistance to African trypanosomiasis.

The review discusses the current field status of human and bovine trypanosomiases, and focuses on the molecular basis of innate and acquired control of African trypanosomes in people, cattle, and Cape buffalo.

A revised arithmetic model of long slender to short stumpy transformation in the African trypanosomes.

An arithmetic model that closely approximates an African trypanosome infection in immunosuppressed mice is presented. The final model was based on an examination of the following parameters: the rate of long slender to short stumpy transition, the maximum percentage of long slender to short stumpy stages that can be induced, the survival time or half life of the short stumpy stage in vivo, and the rate (%) of long slender to short stumpy stage transition following the peak in transformation. The model is based on the assumption that the long slender to short stumpy transition is parasite population dependent and that in mice the long slender to short stumpy transition only begins when the trypanosome population reaches a density of 1 x 10(7) trypanosomes/ml. The model predicts that the parasitemia during the first several days of an infection is controlled solely by the kinetics of the transition of the dividing long slender stage to the nondividing short stumpy stage. It was not necessary to include in the model the host's immune response in order to simulate the early growth kinetics of pleomorphic trypanosomes in infected mice.

Anti-Trypanosoma brucei activity of nonprimate zoo sera.

Constitutive anti-Trypanosoma brucei subsp. brucei S 427 clone 1 and 22 activities were evaluated in sera from 22 species of nonprimate mammals. The sera fell into 5 categories. Sera from Cape buffalo, giraffe, and greater kudu showed a concentration-dependent inhibition of replication of the 2 clones of organisms, which was dependent on the presence of xanthine oxidase. Sera from warthog and springbok also severely limited trypanosome replication but lacked xanthine oxidase. Their antitrypanosome activity was inactivated by heating at 56 C for 30 min but not affected by absorbing with trypanosomes at 4 C. Sera from lion and leopard showed a concentration-dependent inhibition of the growth of T. brucei S427 clone 1 organisms, but not clone 22 organisms. These sera lacked xanthine oxidase. Their anti-T. brucei S 427 clone 1 activity was inactivated by heating at 56 C for 30 min but not removed by absorbing with trypanosomes. Serum from Grant's gazelle prevented replication of both T. brucei clones, lacked xanthine oxidase, and was not affected by heating at 56 C. Sera from waterbuck, Thompson's gazelle, sitatunga, Cape hartebeeste, gerenuk, Grant's zebra, cow, several cat, cougar, bobcat, and domestic cat were fully supportive of trypanosome replication irrespective of concentration tested up to a maximum of 48% v/v in culture medium. Sera from different individuals of the same mammal species had similar effects on trypanosomes, and samples collected from the same individual at different times also had similar activities indicating species-specific stable expression, or lack thereof, of constitutive serum antitrypanosome components.

Increased excretion of aromatic amino acid catabolites in animals infected with Trypanosoma brucei evansi.

Aromatic amino acid catabolism by Trypanosoma brucei evansi was investigated in vivo using C3HeB/FeJ mice. The major catabolites detected by gas chromatography in the urines of infected animals were phenylpyruvic acid, 4-hydroxyphenylpyruvic acid, and indole-3-pyruvic acid. Identity of each compound was confirmed by gas chromatography-mass spectrometry. Concentrations of catabolites in urine of infected mice were correlated with parasitemia and returned to normal following suramin treatment. Other aromatic amino acid metabolites, including indole-3-acetic acid, indole-3-lactic acid, and 4-hydroxyphenyllactic acid, were detected in urine from infected animals by gas chromatography mass spectrometry, although quantities were too low to be quantified reproducibly. Both phenylpyruvic acid and 4-hydroxyphenylpyruvic acid were also detected in urine of dogs and donkeys experimentally infected in Egypt with a recent field isolate of T. b. evansi. Tryptophan metabolites could not be assayed in dog and urine samples because formalin, which degraded the indole acids, had to be added before the samples could be imported into the U.S. Finally, concentrations of urinary catabolites during infection were correlated with the tyrosine aminotransferase activity in infected mouse sera.

Ascaris lumbricoides intensity in relation to environmental, socioeconomic, and behavioral determinants of exposure to infection in children from southeast Madagascar.

Ascaris lumbricoides worm counts were examined as the outcome products of exposure proxy variables. A survey of 663 children, 4-10 yr old, living in southeastern Madagascar revealed prevalences of 93% for A. lumbricoides, 55% for Trichuris trichiura, and 27% for hookworm. Worm expulsions were conducted on 428 of these children; the data revealed an overdispersed distribution of A. lumbricoides, with an arithmetic mean of 19.2 worms per child. A concurrent socioeconomic household survey was conducted by visitation and interview. Exposure to infection was assessed by environmental, demographic, behavioral, and socioeconomic indicators. Ascaris lumbricoides aggregations were associated with gender, housing style, ethnicity, and agricultural factors. The results suggest that exposure and infection are ubiquitous in this child population, and that A. lumbricoides intensity is influenced by gender-related behavioral and environmental factors that contribute to exposure.

Diagnosis of trypanosomiasis in experimental mice and field-infected camels by detection of antibody to trypanosome tyrosine aminotransferase.

Sera from animals with acute and chronic Trypansoma evansi infections were examined directly for trypanosome tyrosine aminotransferase activity and indirectly for their ability to inhibit tyrosine aminotransferase activity. It was shown that sera from acutely infected mice and camels with high parasitemias contained significant levels of trypanosome tyrosine aminotransferase activity. In contrast, the sera from chronically infected mice and camels did not contain significant tyrosine aminotransferase activity, but they were able to neutralize the enzyme activity in trypanosome homogenates. The sera from camels with other pathological conditions did not neutralize this enzyme activity. It is suggested that the inhibitory factor in the chronic sera is antibody. The potential use of the direct enzyme assay and the indirect neutralization assay as diagnostic tools are discussed. Finally, the use of these assays to distinguish between early (acute) and late (chronic) infections are also suggested.

A proposed density-dependent model of long slender to short stumpy transformation in the African trypanosomes.

A simple arithmetic model is developed that is based upon the assumption: (1) that transformation of replicating long slender Trypanosoma brucei to nonreplicating short stumpy forms is parasite population density dependent; (2) that as the slender population increases there is a change in the external environment that triggers the slender to stumpy transformation; and (3) that stumpy forms of T. brucei do not induce the change in external environment that triggers slender to stumpy transformation or do so to a lesser extent than slender forms, thus preventing the proportion of stumpy forms in a population from reaching 100%. A simulation based on these assumptions shared many features with curves on numbers of long slender, intermediate, and short-stumpy forms of T. brucei during the first parasitemic wave of the 3 T. brucei subspecies in intact and immunosuppressed inbred mice.

The individual host, a unique evolutionary island for rapidly dividing parasites: a theoretical approach.

It is hypothesized that rapidly dividing parasites producing high parasitemias within an individual host are in different environmental settings. It is further suggested that these infrapopulations experience the drastic environmental changes of free-living forms in an island environment and, that in chronically infected animals, the environmental conditions will over time select the parasites best suited to grow in their changing habitat. Evidence is presented to demonstrate that the host environment does change during an infection with the African trypanosomes and, that with time, each host becomes environmentally unique. Data are also provided to show that parasites cloned from different hosts are phenotypically different and are assumed to be genetically different as well. The evidence provided is consistent with the hypothesis that each individual host provides a unique habitat in which selection occurs, and that the rapidly dividing protozoans, such as the African trypanosomes and plasmodia are continuously evolving in the individual host.

Ascaris lumbricoides aggregation in relation to child growth status, delayed cutaneous hypersensitivity, and plant anthelmintic use in Madagascar.

The relationships between Ascaris lumbricoides worm burden, growth status, general delayed cutaneous hypersensitivity (DCH) response, and plant anthelmintic use were investigated in a 12-mo prospective survey of 663 children, 4-10 yr old, living in the Ranomafana rainforest, Madagascar. Initial fecal examinations revealed prevalences of 93% for A. lumbricoides, 55% for Trichuris trichuria, and 27% for hookworm. After anthelmintic treatment and a 12-mo reinfection period, 428 of the children participated in worm expulsion studies using pyrantel pamoate, revealing an overdispersed A. lumbricoides worm population, mean = 19.2 (SD = 20.4). Malnutrition was common with 72% of the children growth stunted, 61% underweight, and 6% wasted. The children were also skin tested to recall antigens for DCH, with 94% reacting. The DCH immune response was significantly decreased in the malnourished children; however, DCH was not reduced in relation to increasing worm intensity. Growth status, growth velocity, and triceps skinfold did not vary significantly in relation to A. lumbricoides worm burden. Traditional plant anthelmintic treatment was effective in significantly reducing worm intensity. This study indicates that, in human communities where the children are predominately stunted, A. lumbricoides does not aggregate in the most malnourished or immunodepressed children.

The in vitro HL-60 cell--Trypanosoma brucei rhodesiense culture system: a rapid in vitro drug screen.

The in vitro culture system is described in which Trypanosoma brucei rhodesiense (LOUTat.1) was grown with the human feed layer cell HL-60. The use of this system in determining the 50% growth Inhibitory Concentration (IC50) of unknown compounds for both the trypanosomes and the host cell was demonstrated. The data shows that several analogues of pentamidine have significantly reduced host cell toxicity but maintain or have increased typanocidal activity. The value of the trypanosome/HL-60 in vitro culture system as a rapid primary in vitro drug screen is discussed. Based upon the ability of this primary screen to predict potential drug efficacy, several analogues screened in vitro were then tested in vivo. The results of the in vivo tests confirmed the ability of the in vitro screen to predict drug efficacy, and also suggests that better analogues of pentamidine (less host toxicity and greater trypanocidal activity) can be obtained to treat human trypanosomiasis.

Catabolism of tryptophan by Trypanosoma evansi.

Trypanosoma brucei gambiense, which causes human African trypanosomiasis, catabolizes the aromatic amino acid tryptophan via an initial aminotransferase catalyzed reaction to form several indole end products, which have been suggested to contribute to the pathogenesis of trypanosomiasis. To determine if this same pathway exists in T. evansi, the closely related trypanosome pathogen of domestic animals, tryptophan catabolism was examined in vitro and in vivo. As is the case with human African trypanosomes, T. evansi catabolized tryptophan to form indole-3-pyruvic acid and smaller amounts of indole-3-acetic acid and indole-3-lactic acid. Large concentrations of indole-3-pyruvic acid are excreted in urine of trypanosome-infected mice. However, indole-3-ethanol could not be detected in incubates of T. evansi or T. b. gambiense, even though the latter species had previously been reported to form this neutral metabolite. A new, previously unreported tryptophan metabolite was isolated and partially characterized from incubates of T. evansi and T. b. gambiense. Although the functional significance of tryptophan catabolism to trypanosomatids remains obscure, the pathway is quantitatively significant in all species examined thus far.

Parasitology year 2000.

We predict that in order for parasitology to thrive by the year 2000 the various subdisciplines of evolution, ecology, biosystematics, and genetics must develop holistic approaches and use parasite models to answer basic biological questions. The students of tomorrow must work as part of a multidisciplinary team; and their questions and answers must be conceptually integrated into the broader biological framework of evolution and ecology.

The removal of trypanolytic activity from human serum by Trypanosoma brucei gambiense and its subsequent recovery in trypanosome lysates.

Susceptible African trypanosomes are lysed by a factor in human serum (HS), which presumably binds to their surface and is then internalized. It has been suggested that internalization of the factor is required for lysis. The hypothesis predicted that if the trypanolytic factor (TLF) binds and is endocytosed by trypanosomes, the lytic activity in HS should be removed by them. The experiments in this report have demonstrated that the lytic activity in HS can be almost completely removed. This was shown using both human serum sensitive (HSS) and resistant clones. As it might have been expected, HSS cells remove a greater percentage of the trypanolytic activity. In addition, the hypothesis also predicts that if the TLF is processed and activated from inside the trypanosome, its activity should be detected in the lysates of thoroughly washed trypanosomes previously incubated with HS. The results showed that the lysates consistently contained a soluble active form of the TLF that has been internalized by the trypanosomes. Antiserum specific to human high-density lipoprotein was found to neutralize the trypanolytic activity present in the lysates but failed to prevent the lysis of trypanosomes already exposed to HS.

The inheritance of factors controlling resistance in mice infected with Trypanosoma brucei rhodesiense.

In crosses between 2 recombinant inbred strains of mice (B x H-2 and B x H-14), resistance to infection with Trypanosoma brucei rhodesiense as measured by survival time is suggested to be controlled by a dominant gene(s). In prior studies using the same clone of trypanosomes, but a different set of inbred mouse strains, it was demonstrated that resistance in H-2 congenic mice was a recessive trait. This work suggests that in mouse trypanosomiasis, the number of genes involved in resistance and their dominant or recessive nature will vary between different inbred mouse strains. There was a statistically significant difference between the survival times of animals with high or low antibody anti-trypanosome titers. Differences in survival time were not correlated with the height of the first parasitemia. There was, however, a strong negative correlation between the number of trypanosomes at the second peak in parasitemia and survival time. It is also suggested that the extent to which the host is immunosuppressed early in infection determines the ability to control the later peaks in parasitemia, and, therefore, survival time.

The epidemiology of Ascaris lumbricoides, Trichuris trichiura, and hookworm in children in the Ranomafana rainforest, Madagascar.

An epidemiological study of intestinal nematodes was conducted with 1,292 children, ranging from birth through 11 yr old, living in the Ranomafana rainforest of southeast Madagascar. Fecal examinations revealed prevalences of 78% for Ascaris lumbricoides, 38% for Trichuris trichiura, 16% for hookworm, and 0.4% for Schistosoma mansoni. Infection intensity was measured indirectly by fecal egg counts and directly by A. lumbricoides expulsion following treatment with pyrantel pamoate. The mean A. lumbricoides worm burden for children, 5-11 yr old, was 19.2 (SD 20.4) worms per child, with a median of 13 worms (n = 428). The distributions were overdispersed for all 3 nematodes. The age profiles showed a rapid acquisition of A. lumbricoides during infancy, increasing to 100% prevalence by age 10. After mebendazole anthelmintic treatment and a 12-mo reinfection period, the nematodes had rebounded to pretreatment prevalence and intensity levels. There was evidence for age-dependent predisposition of the children to infection intensity for each of the 3 nematodes. Dual species intensity correlation was consistently strong for A. lumbricoides and T. trichiura. The significantly higher prevalence and intensity of ascariasis in girls were thought to be related to exposure.

Mechanism of lysis of Trypanosoma brucei gambiense by human serum.

Resistance to lysis by human serum (HS) is an important parameter used to distinguish Trypanosoma brucei brucei from both Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Neither the exact nature of the trypanolytic factor (TLF) nor the mechanism of action by which HS lyses susceptible trypanosomes is well understood. This report tries to elucidate the role played by the variable surface glycoprotein (VSG) coat and trypanosome surface-related processes in the mechanism of HS lysis of HS-sensitive (HSS) and HS-resistant (HSR) trypanosomes. Procyclic forms of T. brucei gambiense transformed from either HSS or HSR bloodstream stages were found to be HSR. These procyclic forms were shown to have lost their VSG coat. However, the addition of excess soluble VSG from HSS trypanosomes did not block lysis of HSS trypanosomes. Human serum lysis was significantly inhibited if the trypanosomes were incubated with membrane stabilizers, i.e., including cytochalasins (B, D, and E specifically), zinc acetate, vinblastine, and benzyl alcohol, or with the lysosomotropic agents ammonium chloride and chloroquine. The inhibition exerted by these compounds was always reversible. The results in this report, taken together, strengthen the hypothesis that the lytic factor interacts with and moves along the trypanosome surface to be internalized eventually.

Characterization of human serum-resistant and serum-sensitive clones from a single Trypanosoma brucei gambiense parental clone.

A protocol was developed to select clones of Trypanosoma brucei gambiense having different levels of resistance to normal human serum. Human serum-resistant clones were selected from a single parental clone by continuous serum treatment of infected immunosuppressed mice. Human serum-sensitive revertant clones were also obtained by continuous passage of resistant clones in immunosuppressed mice but without human serum pressure. It has been demonstrated that our trypanosome clones express distinct but stable levels of resistance. The variant antigenic type of each clone was characterized serologically and by 1-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. After selective pressure with human serum, variant antigen-type differences always occurred among clones in which different human serum susceptibilities were found. The work reported here demonstrates that in our T. brucei gambiense immunosuppressed mouse model there is a predictable association between variant antigen type and human serum resistance.