Domestic and wild mammals infection by Trypanosoma evansi in a pristine area of the Brazilian Pantanal region

The objective of this study was to estimate the Trypanosoma evansi infection rate and epizootical status of wild and domestic animals from the Brazilian Pantanal region using a standardized polymerase chain reaction (PCR). We used a simple DNA extraction method based on Chelex resin (BioRad, USA) on blood eluted from filter paper confetti. Primers directed to repetitive nuclear DNA sequences were used in the PCR, and could detect 30 fg of T. evansi DNA. The analytical specificity of the assay was evaluated using T. evansi, T. rangeli, T. cruzi, Leishmania braziliensis, Crithidia fasciculata and Herpetomonas muscarum DNAs as templates and the technique showed the expected 164 bp specific band solely for Trypanozoon trypanosomes. The application of the standardized PCR protocol in 274 field samples from domestic and wild mammals from the Rio Negro (Brazilian Pantanal region), showed a general infection rate of 10.2% while the traditional parasitological technique (direct search of the protozoan by the microematocrit centrifugue technique) was able to determine infection in only 1.1% of the animals. The peccaries and feral pigs were found to be the animals most frequently infected with T. evansi (24.4% and 30.7%, respectively). Both sampling and extraction methods used herein, showed to be simple and efficient to be applied in epidemiological surveys using PCR.     

Immunization with recombinant actin from Trypanosoma evansi induces protective immunity against T. evansi, T. equiperdum and T. b. brucei infection

Actin gene of Trypanosoma evansi (STIB 806) was cloned and expressed in Escherichia coli. The predicted amino acid sequence of T. evansi actin shows 100%, 98.7%, and 93.1%, homology with Trypanosoma equiperdum, Trypanosoma brucei brucei, and Trypanosoma cruzi. Recombinant actin was expressed as inclusion bodies in E. coli. It was purified and renatured for immunological studies. Mice immunized with the renatured recombinant actin were protected from lethal challenge with T. evansi STIB 806, T. equiperdum STIB 818, and T. b. brucei STIB 940, showing 63.3%, 56.7%, and 53.3% protection, respectively. Serum collected from the rabbit immunized with recombinant actin inhibited the growth of T. evansi, T. equiperdum, and T. b. brucei in vitro cultivation. Serum from mice and rabbits immunized with recombinant actin only recognized T. evansi actin but not mouse actin. The results of this study suggest that the recombinant T. evansi actin induces protective immunity against T. evansi, T. equiperdum, and T. b. brucei infection and may be useful in the development of a vaccine with other cytoskeletal proteins to prevent animal trypanosomiasis caused by these three trypanosome species.     

Expression of fluorescent genes in Trypanosoma cruzi and Trypanosoma rangeli (Kinetoplastida: Trypanosomatidae): its application to parasite-vector biology

Two Trypanosoma cruzi-derived cloning vectors, pTREX-n and pBs:CalB1/CUB01, were used to drive the expression of green fluorescent protein (GFP) and DsRed in Trypanosoma rangeli Tejera, 1920, and Trypanosoma cruzi Chagas, 1909, isolates, respectively. Regardless of the species, group, or strain, parasites harboring the transfected constructs as either episomes or stable chromosomal integrations showed high-level expression of fluorescent proteins. Tagged flagellates of both species were used to experimentally infect Rhodnius prolixus Stal, 1953. In infected bugs, single or mixed infections of T. cruzi and T. rangeli displayed the typical cycle of each species, with no apparent interspecies interactions. In addition, infection of kidney monkey cells (LLC-MK2) with GFP-T. cruzi showed that the parasite retained its fluorescent tag while carrying out its life cycle within cultured cells. The use of GFP-tagged parasites as a tool for biological studies in experimental hosts is discussed, as is the application of this method for copopulation studies of same-host parasites.     

Detection of Trypanosoma cruzi and Trypanosoma rangeli infection by duplex PCR assay based on telomeric sequences

We used the species specificity and repetitious nature of subtelomeric kinetoplastida sequences to generate a duplex PCR assay for the simultaneous detection of Trypanosoma cruzi and Trypanosoma rangeli in experimentally and naturally infected triatomine (Reduviid) bugs and in infected human subjects. The assay was species specific and was capable of detecting 1/20th of T. cruzi and 1/4th of T. rangeli cell equivalents without complementary hybridization. In addition, the PCR-based assay was robust enough for direct application to difficult biological samples such as Reduviid feces or guts and was capable of recognizing all T. cruzi and T. rangeli strains and lineages. Because the assay primers amplify entirely different target sequences, no reaction interference was observed, facilitating future adaptation of this assay to an automated format.     

Absolute and allometric relationships between internal morphology and body mass in the adult collared peccary, Tayassu tajacu (Tayassuidae)

Selected morphological features of 8 adult male and 8 adult female collared peccaries (Tayassu tajacu) shot from southern Texas during March 1983 are described. A total of 16 adult peccaries with an average body mass of 18.68 +/- 0.61 (SE) Kg was examined. Significant differences between males and females were observed for absolute and relative mass of liver and lungs, and relative heart mass. These visceral organs were heavier among females than males. Significant sex effects were also found for absolute and relative mass of the dorsal scent gland. The dorsal scent gland contributed twice as much to total body mass in males as in females. No sexual dimorphisms of the gastrointestinal tract were noted. Females had a significantly greater portion of total visceral fat deposited around the kidneys than did males. Relative mass of the mandible was significantly greater in males than in females. Adult males had extremely large accessory sex glands. The bulbourethral and seminal vesicle glands comprised 0.27 per cent of the total body mass. Allometric growth coefficients (b) varied among the various organs and glands examined, ranging from below (eyes, b = 0.34) to well above (seminal vesicles, b = 1.87) unity. Growth coefficients of lungs, kidneys, pituitary gland, and thyroid gland during adulthood greatly exceeded respective values in developing nurslings.     

Restriction fragment length polymorphisms in the ribosomal gene spacers of Trypanosoma cruzi and Trypanosoma conorhini

The ribosomal RNA genes of two species of Trypanosoma, Trypanosoma cruzi, the etiological agent of Chagas' disease, and Trypanosoma conorhini, a non-pathogenic rodent trypanosome, were cloned and partially characterized. The physical maps derived for their rRNA genes were similar throughout the region that encompasses the SSU-and LSU-rRNA coding sequences. However, the non-transcribed spacer DNA of both T. cruzi and T. conorhini was found to be polymorphic for several restriction enzyme sites. We show that strains of T. cruzi can be typed according to the characteristic restriction fragment length polymorphism of their NTS DNAs.     

Relationships between internal morphology and body mass in the developing, nursling collared peccary, Tayassu tajacu (Tayassuidae)

Morphological characteristics that reflect size differences due to dietary conditions are in wide use to assess nutritional status of many wild ungulates. This study was designed to provide baseline information on the development of internal visceral and endocrine structures of nursling collared peccaries (Tayassu tajacu) from birth to six weeks of age (weaning). A high energy-high protein ration was fed ad libitum to lactating females, and absolute and relative mass of selected visceral organ, endocrine, and fat depots were measured in various aged nurslings. Linear measurements were also obtained on components of the gastrointestinal tract. Allometric growth coefficients (b) varied considerably among the various organs and glands examined, ranging from below (brain, b = 0.26) to well above (thymus, b = 1.61) unity.     

Lack of manipulation of Rhodnius prolixus (Hemiptera: Reduviidae) vector competence by Trypanosoma cruzi

Behavioral implications of Trypanosoma cruzi Chagas infection in Rhodnius prolixus St?l were observed. Feeding and defecation behaviors of infected versus uninfected insects were assessed on an artificial membrane-feeding system and on live guinea pigs. Based on a defecation index, fifth instars were the most efficient vectors, followed by adult females, fourth instars, and adult males. Bugs fasted for longer periods (5-6 mo) took smaller blood meals but defecated significantly earlier than bugs fasted for shorter periods (2-3 mo). Multiple blood feeding, degree of fasting, life stage, T cruzi infection, and gender affected the vector potential of R. prolixus. Our data indicate that T. cruzi and R. prolixus have not coevolved to facilitate the transmission of T. cruzi, which suggests that this parasite-host relationship may be relatively recent.     

Trypanosoma cruzi isolates from Argentina and Chile grouped with the aid of DNA probes

Fifty-two isolates and several clones from Trypanosoma cruzi, the agent of Chagas' disease, were analyzed using cloned minicircles or total kinetoplast DNA as probes. Isolates were obtained from triatomines, guinea pigs and infected humans in the Central and Northern regions of Argentina and the North of Chile. 35% of all the randomly selected isolates could be identified with one cloned minicircle probe. This widely distributed T. cruzi group was detected on both sides of the Andes mountain range (Argentina and Chile) in Triatoma infestans as well as in human infections. Most of the other isolates could be grouped with four kinetoplast DNAs as probes, but their geographical distribution seems to be restricted as compared with the one mentioned above. These results confirm the heterogeneity of T. cruzi subspecies in nature and the usefulness of DNA probes to group them.     

Human Trypanosoma evansi infection linked to a lack of apolipoprotein L-I

Humans have innate immunity against Trypanosoma brucei brucei that is known to involve apolipoprotein L-I (APOL1). Recently, a case of T. evansi infection in a human was identified in India. We investigated whether the APOL1 pathway was involved in this occurrence. The serum of the infected patient was found to have no trypanolytic activity, and the finding was linked to the lack of APOL1, which was due to frameshift mutations in both APOL1 alleles. Trypanolytic activity was restored by the addition of recombinant APOL1. The lack of APOL1 explained the patient's infection with T. evansi.     

Biochemical characterization of stage-specific isoforms of aspartate aminotransferases from Trypanosoma cruzi and Trypanosoma brucei

Three genes encoding putative aspartate aminotransferases (ASATs) were identified in the Trypanosoma cruzi genome. Two of these ASAT genes, presumably corresponding to a cytosolic and mitochondrial isoform, were cloned and expressed as soluble His-tagged proteins in Escherichia coli. The specific activities determined for both T. cruzi isozymes were notably higher than the values previously reported for Trypanosoma brucei orthologues. To confirm these differences, T. brucei mASAT and cASAT were also expressed as His-tagged enzymes. The kinetic analysis showed that the catalytic parameters of the new recombinant T. brucei ASATs were very similar to those determined for T. cruzi orthologues. The cASATs from both parasites displayed equally broad substrate specificities, while mASATs were highly specific towards aspartate/2-oxoglutarate. The subcellular localization of the mASAT was confirmed by digitonin extraction of intact epimastigotes. At the protein level, cASAT is constitutively expressed in T. brucei, whereas mASAT is down-regulated in the bloodstream forms. By contrast in T. cruzi, mASAT is expressed along the whole life cycle, whereas cASAT is specifically induced in the mammalian stages. Similarly, the expression of malate dehydrogenases (MDHs) is developmentally regulated in T. cruzi: while glycosomal MDH is only expressed in epimastigotes and mitochondrial MDH is present in the insect and mammalian stages. Taken together, these findings provide evidence for a metabolically active mitochondrion in the mammalian stages of T. cruzi, and suggest that the succinate excreted by amastigotes more likely represents a side product of an at least partially operative Krebs cycle, than an end product of glycosomal catabolism.     

Curcumin treatment provides protection against Trypanosoma cruzi infection

Trypanosoma cruzi, the etiologic agent of Chagas disease, causes an acute myocarditis and chronic cardiomyopathy. The current therapeutic agents for this disease are not always effective and often have severe side effects. Curcumin, a plant polyphenol, has demonstrated a wide range of potential therapeutic effects. In this study, we examined the effect of curcumin on T. cruzi infection in vitro and in vivo. Curcumin pretreatment of fibroblasts inhibited parasite invasion. Treatment reduced the expression of the low density lipoprotein receptor, which is involved in T. cruzi host cell invasion. Curcumin treatment of T. cruzi-infected CD1 mice reduced parasitemia and decreased the parasitism of infected heart tissue. This was associated with a significant reduction in macrophage infiltration and inflammation in both the heart and liver; moreover, curcumin-treated infected mice displayed a 100% survival rate in contrast to the 60% survival rate commonly observed in untreated infected mice. These data are consistent with curcumin modulating infection-induced changes in signaling pathways involved in inflammation, oxidative stress, and apoptosis. These data suggest that curcumin and its derivatives could be a suitable drug for the amelioration of chagasic heart disease.     

Differential susceptibilities of mice genomically deleted of CD4 and CD8 to infections with Trypanosoma cruzi or Trypanosoma brucei.

The role of CD4+ and CD8+ T cells in the surveillance of Trypanosoma cruzi or Trypanosoma brucei brucei was studied in mice which lacked CD4 or CD8 molecules and which were generated by embryonic stem cell technology. Whereas wild-type mice infected with T. cruzi (Tulahuén strain) displayed low levels of parasitemia and no mortality, striking increases in parasite growth and mortality occurred in both CD8- and CD4- mice. On the contrary, CD8- and, to a lesser degree, CD4- mice showed enhanced resistance to T. b. brucei. T-cell-dependent immunoglobulin G-specific responses were produced in CD8- but not CD4- mice. Normal T-cell proliferative responses were measured in both CD4- and CD8- mice. Interleukin-4 production after concanavalin A or anti-CD3 monoclonal antibody stimulation was strikingly enhanced in CD8- but not CD4- spleen cells, whereas gamma interferon production was normal in both CD4- and CD8- spleen cells. Spleen and lymph node cells from CD8- (but not CD4-) mice at 20 days postinfection with T. cruzi had higher levels of interleukin-4 mRNA than the wild-type controls, as shown in a competitive polymerase chain reaction assay. On the other hand, CD4- (but not CD8-) mice at 20 days postinfection with T. cruzi had lower levels of gamma interferon mRNA than the wild-type mice.     

Ultrastructural alterations in the adrenal gland cortex of mice experimentally infected with a Venezuelan isolate of Trypanosoma evansi

The ultrastructural study of adrenal gland from mice experimentally infected with Trypanosoma evansi, in addition to intravascular and intracellular trypanosomes, showed different degrees of cortical cell alterations and capillary wall modifications. Beside its biological scope, these results suggest a role for the adrenal cortex to partake in Surra's etiopathogenesis and describe for the very first time a T. evansi intracellular stage.     

Identification and analysis of epimastigote surface and metabolic proteins in Trypanosoma cruzi

Metabolic and surface membrane proteins of four epimastigote-stage Trypanosoma cruzi clones were analyzed by one and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No major inter-clonal differences were observed in the metabolic protein patterns, indicating that these proteins are highly conserved. However, marked quantitative and qualitative differences were observed in surface-labeled protein patterns following both one and two-dimensional electrophoretic analyses. Inter and intra-clonal differences in antigenic properties also were demonstrated by immunoprecipitation of the surface proteins with sera from animals immunized or infected with various T. cruzi stocks. Thus, a wide spectrum of both phenotypic and antigenic diversity exists in T. cruzi which may be relevant to problems of the diagnosis and immunotherapy of Chagas' disease.     

Immunopathology of experimental Chagas'' disease: binding of T cells to Trypanosoma cruzi-infected heart tissue.

The immunopathology of Chagas' disease was studied in the experimental model of chronic infection in C57BL/10JT or mice. Sublethal infection with Trypanosoma cruzi, Y strain, induced specific antibodies and a delayed hypersensitivity response to parasite antigens. Mice developed chronic chagasic myocarditis but not skeletal muscle myositis. Binding of T cells to infected heart tissue was investigated during short-term cocultivation of lymphocytes with heart cryostat sections. T cells from infected mice and from normal controls bound equally to myocardium and liver sections from both infected and normal mice. A search in depth was attempted with cells heavily tagged with 99mTc. Labeled T cells from chagasic mice bound to both normal and infected myocardium slices. 99mTc-labeled T cells from controls gave the same binding values. Glass-adherent spleen cells behaved identically to T cells. Prior treatment of the tissue with serum from chronically infected mice did not increase the number of binding cells. Peritoneal macrophages tagged with 99mTc-sulfur colloid also bound to infected myocardium slices. The binding of macrophages was not changed by pretreatment of infected tissue with anti-T, cruzi antibodies. In short, this work did not detect any population of T cells or macrophages which could bind specifically to infected heart tissue to initiate an autoreactive process.     

Glycosyl-sn-1,2-dimyristylphosphatidylinositol is the membrane anchor for Trypanosoma equiperdum and T. (Nannomonas) congolense variant surface glycoproteins

We have analysed the structures of the Trypanosoma (Nannomonas) congolense and T. equiperdum variant surface glycoprotein (VSG) membrane anchors. Myristic acid uptake, phospholipase treatment, and nitrous acid deamination showed that, for each species, the anchor is glycosyl-sn-1,2-dimyristylphosphatidylinositol, as has been previously described for T. brucei. Osmotic lysis of these trypanosomes resulted in the release of soluble VSG, lacking fatty acid. In both species and in T. evansi, an endogenous phospholipase C, which cleaved diacylglycerol from membrane form VSG, was identified.     

Use of full-length recombinant calflagin and its c fragment for improvement of diagnosis of Trypanosoma cruzi infection

Serological diagnosis of Trypanosoma cruzi infection is hampered by issues related to test specificity due to the cross-reactivity of most antigens with proteins of related parasites such as Leishmania spp. The recombinant calflagins are considered relevant antigens for the diagnosis of infection by Trypanosoma cruzi. In the present work, we describe two genes coding for putative calflagins in Leishmania major with the N-terminal moieties presenting high similarity with T. cruzi genes. This fact raised questions about their role in some cross-recognition of this antigen by sera from Leishmania spp.-infected individuals. The complete T. cruzi calflagin and two fragments of the protein, consisting of 146 amino acids of the N-terminal and 65 amino acids of the C-terminal regions, were expressed and evaluated against a panel of sera, which included well-characterized samples from T. cruzi, and Leishmania-infected patients. We were able to show that sera from Leishmania (Viannia) braziliensis-infected individuals recognized the recombinant full-length calflagin. Both the N-terminal and the complete protein presented the same high sensitivity (98.5% of sera from T. cruzi-infected patients was detected) but different specificities (94% and 98%, respectively, when evaluated against sera from people not infected by T. cruzi, including 15 sera from people infected with L. braziliensis). The C-terminal fragment presented low sensitivity (70%) but 100% specificity. We propose the use of these antigens in two sequential assays to optimize the serological diagnosis of T. cruzi infection in humans in geographic areas where Leishmania spp. infection is coendemic.     

Trypanosoma cruzi and Chagas' Disease in the United States

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi and causes potentially life-threatening disease of the heart and gastrointestinal tract. The southern half of the United States contains enzootic cycles of T. cruzi, involving 11 recognized triatomine vector species. The greatest vector diversity and density occur in the western United States, where woodrats are the most common reservoir; other rodents, raccoons, skunks, and coyotes are also infected with T. cruzi. In the eastern United States, the prevalence of T. cruzi is highest in raccoons, opossums, armadillos, and skunks. A total of 7 autochthonous vector-borne human infections have been reported in Texas, California, Tennessee, and Louisiana; many others are thought to go unrecognized. Nevertheless, most T. cruzi-infected individuals in the United States are immigrants from areas of endemicity in Latin America. Seven transfusion-associated and 6 organ donor-derived T. cruzi infections have been documented in the United States and Canada. As improved control of vector- and blood-borne T. cruzi transmission decreases the burden in countries where the disease is historically endemic and imported Chagas' disease is increasingly recognized outside Latin America, the United States can play an important role in addressing the altered epidemiology of Chagas' disease in the 21st century.     

75Se-methionine labelled Trypanosoma cruzi blood trypomastigotes: opsonization by chronic infection serum facilitates killing in spleen and liver.

Blood trypomastigote forms of Trypanosoma cruzi have been labelled with 75Se-methionine. Opsonization by sera from mice chronically infected with T. cruzi promoted their uptake by both liver and spleen. Opsonized parasites within spleens and livers were less infectious when transferred to normal recipients demonstrating in situ parasite killing by these organs.