Hepatic mixed-function oxidase activity in mice infected with Trypanosoma brucei gambiense or treated with trypanocides.

Three days after mice were inoculated with Trypanosoma brucei gambiense, total hepatic cytochrome P-450 levels were decreased 14% from control values, while the specific mixed-function oxidase activity (per nmole of cytochrome P-450) was inhibited almost 40% in infected animals. Furthermore, drugs used to treat trypanosomiasis (Suramin and Melarsoprol B) are themselves potent in vitro inhibitors of hepatic mixed-function oxidase activity. These two trypanocides also produced a decrease in mixed-function oxidations and an increase in pentobarbital sleeping time when administered intraperitoneally in vivo. These results demonstrate that mice with trypanosomiasis or undergoing trypanosome chemotherapy have a significantly impaired capacity to metabolize foreign compounds.

Hepatic microsomal alterations during chronic trypanosomiasis in the field vole, Microtus montanus.

The field vole, Microtus montanus, was used as a model system to evaluate the chronic effects of infection by Trypanosoma brucei gambiense on hepatic mixed-function oxidase activity. At day 28 post inoculation there was a 97% increase in liver wet weight per g body weight. A portion of the increase (21%) was accounted for by tissue edema which occurred after day 14 of infection. Total hepatic cytochrome P-450 content and related total tissue mixed-function oxidase activities were decreased to about 60% of control levels at day 28 post inoculation. The decrease in total tissue mixed-function oxidase activity was partly due to a small decrease in microsomal protein per cell, and partly to a large decrease in cytochrome P-450 concentration in the endoplasmic reticulum. Although the decrease in total liver monooxygenase activity in several substrates roughly paralleled the loss in cytochrome P-450 content, several other microsomal enzyme markers not related to cytochrome P-450 monooxygenation were elevated in proportion to total liver microsomal protein content. The results suggest that in M. montanus during trypanosomiasis, there is proliferation of hepatic cells with normal content of endoplasmic reticulum. Furthermore, there appears to be selective toxicity for hepatic cytochrome P-450 and related monooxygenase activities. This may compromise the animals' ability to metabolize and dispose of other drugs to which the animal may be exposed in the course of infection.

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.

Further biochemical characterization of chronic Trypanosoma brucei gambiense-Microtus montanus infection.

Tyrosine aminotransferase (TAT), glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), and alkaline phosphatase (ALP) were measured in the serum and livers of Microtus montanus infected with Trypanosoma brucei gambiense. Only liver TAT and serum ALP showed significant changes. In addition, blood glucose, pyruvate and lactate, and liver glycogen levels were assayed. All four compounds showed significant changes, strongly suggesting increased glycogen mobilization and increased catabolic activity. Interestingly, the serum ketone levels were very low and no significant changes were observed. These chronically infected animals had an organic aciduria in which pyruvate, lactate, beta-hydroxybutyrate, alpha-ketoglutarate, phenylpyruvate, and p-hydroxyphenylpyruvate were significantly increased. The possible significance of these observations is discussed.

Colonization of Trypanosoma brucei gambiense within transplanted Ehrlich's tumors of Microtus montanus.

Trypanosoma brucei gambiense has been observed growing in extravascular sites throughout a solid Ehrlich ascites tumor (EAT) in Microtus montanus. These trypanosomes appear in clusters in the tumor. The possible importance of this observation to the host-parasite relationship is discussed.

The seroprevalence of cysticercosis, malaria, and Trypanosoma cruzi among North Carolina migrant farmworkers.

A seroprevalence study of cysticercosis, Trypanosoma cruzi, and plasmodia species and screening for active malaria was conducted among a randomly selected group of 138 Hispanic and Haitian migrant farmworkers. A random sample of labor camps in eastern North Carolina was selected. Blood samples were tested by Indirect Fluorescent Antibody techniques for plasmodial antibody and by enzyme-linked immunosorbent assay (ELISA) for cysticerci and T. cruzi antibodies. Questionnaires collected demographic data and medical history of the workers and family. Blood films stained with Leukostat stain were examined for plasmodia species. The seroprevalence of cysticercosis was 10 percent, T. cruzi 2 percent, and plasmodia species 4.4 percent. One case of active malaria (Plasmodium vivax) was demonstrated. The clinical significance of seropositivity was not determined, but these results suggest that a small but significant number of farmworkers are infected with cysticercosis, T. cruzi, and malaria. Migrant health clinicians should be aware of the possible presence of these infections. Greater observance and enforcement of sanitation regulations in farmwork is needed to prevent transmission of cysticercosis.

Growth of pleomorphic Trypanosoma brucei rhodesiense in irradiated inbred mice.

It was shown that irradiation (650 rad) of 7 inbred strains of mice did not block the ability of Trypanosoma brucei rhodesiense to transform from the long slender (LS) to the short stumpy (SS) form or alter the plateau in parasitemia. In addition, it was observed that significant differences in parasitemia levels, in the rate of transformation from the LS to the SS form, as well as in the survival times occurred between the irradiated C3HeB/FeJ and several of the other strains. These differences in the nonspecific ability to control parasitemia appeared to be characteristic for each inbred strain of mice. The resistant strains generally had lower parasitemia than the susceptible strains. However, it was also shown that there is not a one-to-one correlation between the innate ability of a mouse strain to control its initial parasitemia, and the strain's ability to clear the parasitemia or increase its survival time. It was therefore concluded that the hypothesis which states that the ability of an animal to increase nonspecifically the rate of transformation, and therefore to lower the parasitemia, allowing intact animals to respond immunologically and survive longer is either incorrect or incomplete. The results further show that the ability of mice to clear their initial parasitemia by an antibody response is not necessarily correlated with their survival time. Therefore, this study suggests that factors other than an antibody response and the nonspecific control of parasitemia are important in resistance.

Nature of the trypanocidal factor in human serum.

The chemical nature of the trypanocidal factor in human serum was investigated. The results show that although the trypanocidal factor is contained within the high density lipoprotein (HDL) fraction of human serum, it is apparently not one of the major apolipoproteins of the HDL complex such as apolipoprotein A-I, A-II, or apolipoprotein B. The factor would appear to be a minor component of the HDL fraction whose chemical nature is still uncertain.

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.

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.

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 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.

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.

Changes in albumin levels in blood and urine of Microtus montanus chronically infected with Trypanosoma brucei gambiense.

Serum albumin and glucose concentrations and urinary excretion of alpha-keto acids and proteins were determined in samples obtained throughout a chronic Trypanosoma brucei gambiense infection in Microtus montanus. An increase in urinary excretion of alpha-keto acids and proteins during the terminal stage of disease was accompanied by a decrease in serum glucose concentration. This terminal hypoglycemia reflected a depletion of liver glycogen in most animals. In contrast (and the major focus of this study) serum albumin concentration was decreased by the second week of infection and in the final sample obtained was less than 50% of that measured in preinfection samples. Female animals survived approximately 1 wk longer than males and were less susceptible during the acute phase of disease. This relative resistance was most likely due to the fact that female animals were relatively more efficient in limiting parasitemia during the first week of infection. The similarity between humans and voles in terms of protein and alpha-keto acid excretion and changes in serum concentrations of glucose and albumin during trypanosome infection further validate the use of Microtus as an experimental model for trypanosomiasis in humans.

Tissue alterations in Microtus montanus chronically infected with Trypanosoma brucei gambiense.

Changes in liver, spleen, kidneys, heart, and brain are reported for Microtus montanus chronically infected with Trypanosoma brucei gambiense. An increase in body weight of infected animals was attributable to a significant increase in total mass of spleen, liver and kidney. Cellular infiltrate consisting primarily of lymphocytes and plasma cells was observed in all organs and was particularly evident in intralobular connective tissue of the liver, adipose tissue of the hilum, and adjacent medullary region of the kidney, spleen, and the meninges. Disruption of normal metabolism and the pathological changes observed in liver and kidney suggest that the survival of trypanosome-infected voles is dependent largely on the physiological response occurring in these organs.

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.

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.

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.

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.

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.