Ancestral genomes, sex, and the population structure of Trypanosoma cruzi

Acquisition of detailed knowledge of the structure and evolution of Trypanosoma cruzi populations is essential for control of Chagas disease. We profiled 75 strains of the parasite with five nuclear microsatellite loci, 24Salpha RNA genes, and sequence polymorphisms in the mitochondrial cytochrome oxidase subunit II gene. We also used sequences available in GenBank for the mitochondrial genes cytochrome B and NADH dehydrogenase subunit 1. A multidimensional scaling plot (MDS) based in microsatellite data divided the parasites into four clusters corresponding to T. cruzi I (MDS-cluster A), T. cruzi II (MDS-cluster C), a third group of T. cruzi strains (MDS-cluster B), and hybrid strains (MDS-cluster BH). The first two clusters matched respectively mitochondrial clades A and C, while the other two belonged to mitochondrial clade B. The 24Salpha rDNA and microsatellite profiling data were combined into multilocus genotypes that were analyzed by the haplotype reconstruction program PHASE. We identified 141 haplotypes that were clearly distributed into three haplogroups (X, Y, and Z). All strains belonging to T. cruzi I (MDS-cluster A) were Z/Z, the T. cruzi II strains (MDS-cluster C) were Y/Y, and those belonging to MDS-cluster B (unclassified T. cruzi) had X/X haplogroup genotypes. The strains grouped in the MDS-cluster BH were X/Y, confirming their hybrid character. Based on these results we propose the following minimal scenario for T. cruzi evolution. In a distant past there were at a minimum three ancestral lineages that we may call, respectively, T. cruzi I, T. cruzi II, and T. cruzi III. At least two hybridization events involving T. cruzi II and T. cruzi III produced evolutionarily viable progeny. In both events, the mitochondrial recipient (as identified by the mitochondrial clade of the hybrid strains) was T. cruzi II and the mitochondrial donor was T. cruzi III.     

Characterisation of large and small subunit rRNA and mini-exon genes further supports the distinction of six Trypanosoma cruzi lineages

It has been proposed that isolates of Trypanosoma cruzi, the agent of American trypanosomiasis, can be ordered into two primary phylogenetic lineages, first based on multilocus enzyme electrophoresis and random amplified polymorphic DNA, and subsequently based on the 24Salpha rRNA and mini-exon genes. Recent multilocus enzyme electrophoresis and random amplified polymorphic DNA data have additionally shown that the major multilocus enzyme electrophoresis/random amplified polymorphic DNA lineage II is further subdivided into five smaller lineages, designated IIa-IIe. In this study, the precise correspondence between the multilocus enzyme electrophoresis/random amplified polymorphic DNA and rRNA/mini-exon lineages was investigated. Using the 24Salpha rRNA and mini-exon markers in combination, five sets of strains were distinguished, corresponding to the multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages I, IIa, IIc, IId and to lineages IIb/IIe together, respectively. The previous categorisation into only two primary lineages based on 24Salpha rRNA and mini-exon characterisation is explained, in part, by the lack of representativeness of the breadth of T. cruzi diversity in earlier study samples. Additionally, a PCR assay based on a length-variable region of the 18S rRNA gene distinguished lineage IIe from lineage IIb. Thus, the six multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages could be readily identified by combining data from the 24Salpha rRNA, mini-exon and 18S rRNA characterisation assays, further supporting the relevance of these genetic units for T. cruzi strain classification and subspecific nomenclature. The recently proposed groups T. cruzi I and T. cruzi II correspond to multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages I and IIb, respectively. Our findings show that T. cruzi lineage characterisation based on a single marker (either mini-exon or 24Salpha rRNA) has insufficient resolution, and leads to important reinterpretations of recent epidemiological and evolutionary studies based on the oversimplified rRNA/mini-exon dichotomic classification of T. cruzi isolates.     

Maximum-likelihood divergence date estimates based on rRNA gene sequences suggest two scenarios of Trypanosoma cruzi intraspecific evolution.

The phylogenetic relationships of Trypanosoma cruzi strains were inferred using maximum-likelihood from complete 18S rDNA sequences and D7-24Salpha rDNA regions from 20 representative strains of T. cruzi. For this we sequenced the 18S rDNA of 14 strains and the D7-24Salpha rDNA of four strains and aligned them to previously published sequences. Phylogenies inferred from these data sets identified four groups, named Riboclades 1, 2, 3, and 4, and a basal dichotomy that separated Riboclade 1 from Riboclades 2, 3, and 4. Substitution models and other parameters were optimized by hierarchical likelihood tests, and our analysis of the 18S rDNA molecular clock by the likelihood ratio test suggests that a taxa subset encompassing all 2,150 positions in the alignment supports rate constancy among lineages. The present analysis supports the notion that divergence dates of T. cruzi Riboclades can be estimated from 18S rDNA sequences and therefore, we present alternative evolutionary scenarios based on two different views of T. cruzi intraspecific divergence. The first assumes a faster evolutionary rate, which suggests that the divergence between T. cruzi I and II and the extant strains occurred in the Tertiary period (37-18 MYA). The other, which supports the hypothesis that the divergence between T. cruzi I and II occurred in the Cretaceous period (144-65 MYA) and the divergence of the extant strains occurred in the Tertiary period of the Cenozoic era (65-1.8 MYA), is consistent with our previously proposed hypothesis of divergence by geographical isolation and mammalian host coevolution.     

Trypanosoma cruzi discrete typing units in Chagas disease patients from endemic and non-endemic regions of Argentina

Genetic diversity of Trypanosoma cruzi may play a role in pathogenesis of Chagas disease forms. Natural populations are classified into 6 Discrete Typing Units (DTUs) Tc I-VI with taxonomical status. This study aimed to identify T. cruzi DTUs in bloodstream and tissue samples of Argentinean patients with Chagas disease. PCR-based strategies allowed DTU identification in 256 clinical samples from 239 Argentinean patients. Tc V prevailed in blood from both asymptomatic and symptomatic cases and Tc I was more frequent in bloodstream, cardiac tissues and chagoma samples from immunosuppressed patients. Tc II and VI were identified in a minority of cases, while Tc III and Tc IV were not detected in the studied population. Interestingly, Tc I and Tc II/VI sequences were amplified from the same skin biopsy slice from a kidney transplant patient suffering Chagas disease reactivation. Further data also revealed the occurrence of mixed DTU populations in the human chronic infection. In conclusion, our findings provide evidence of the complexity of the dynamics of T. cruzi diversity in the natural history of human Chagas disease and allege the pathogenic role of DTUs I, II, V and VI in the studied population.     

Origins of Chagas disease: Didelphis species are natural hosts of Trypanosoma cruzi I and armadillos hosts of Trypanosoma cruzi II, including hybrids

Trypanosoma cruzi, the causative agent of Chagas disease, has at least two principal intraspecific subdivisions, T. cruzi I (TCI) and T. cruzi II (TCII), the latter containing up to five subgroups (a-e). Whilst it is known that TCI predominates from the Amazon basin northwards and TCII to the South, where the disease is considered to be clinically more severe, the precise clinical and evolutionary significance of these divisions remains enigmatic. Here, we present compelling evidence of an association between TCI and opossums (Didelphis), and TCII and armadillos, on the basis of key new findings from the Paraguayan Chaco region, together with a comprehensive analysis of historical data. We suggest that the distinct arboreal and terrestrial ecologies, respectively, of these mammal hosts provide a persuasive explanation for the extant T. cruzi intraspecific diversity in South America, and for separate origins of Chagas disease in northern South America and in the southern cone countries.     

Evidence for multiple hybrid groups in Trypanosoma cruzi

A role for parasite genetic variability in the spectrum of Chagas disease is emerging but not yet evident, in part due to an incomplete understanding of the population structure of Trypanosoma cruzi. To investigate further the observed genotypic variation at the sequence and chromosomal levels in strains of standard and field-isolated T. cruzi we have undertaken a comparative analysis of 10 regions of the genome from two isolates representing T. cruzi I (Dm28c and Silvio X10) and two from T. cruzi II (CL Brener and Esmeraldo). Amplified regions contained intergenic (non-coding) sequences from tandemly repeated genes. Multiple nucleotide polymorphisms correlated with the T. cruzi I/T. cruzi II classification. Two intergenic regions had useful polymorphisms for the design of classification probes to test on genomic DNA from other known isolates. Two adjacent nucleotide polymorphisms in HSP 60 correlated with the T. cruzi I and T. cruzi II distinction. 1F8 nucleotide polymorphisms revealed multiple subdivisions of T. cruzi II: subgroups IIa and IIc displayed the T. cruzi I pattern; subgroups IId and IIe possessed both the I and II patterns. Furthermore, isolates from subgroups IId and IIe contained the 1F8 polymorphic markers on different chromosome bands supporting a genetic exchange event that resulted in chromosomes V and IX of T. cruzi strain CL Brener. Based on these analyses, T. cruzi I and subgroup IIb appear to be pure lines, while subgroups IIa/IIc and IId/IIe are hybrid lines. These data demonstrate for the first time that IIa/IIc are hybrid, consistent with the hypothesis that genetic recombination has occurred more than once within the T. cruzi lines.     

Chagas cardiomyopathy manifestations and Trypanosoma cruzi genotypes circulating in chronic Chagasic patients

Chagas disease caused by Trypanosoma cruzi is a complex disease that is endemic and an important problem in public health in Latin America. The T. cruzi parasite is classified into six discrete taxonomic units (DTUs) based on the recently proposed nomenclature (TcI, TcII, TcIII, TcIV, TcV and TcVI). The discovery of genetic variability within TcI showed the presence of five genotypes (Ia, Ib, Ic, Id and Ie) related to the transmission cycle of Chagas disease. In Colombia, TcI is more prevalent but TcII has also been reported, as has mixed infection by both TcI and TcII in the same Chagasic patient. The objectives of this study were to determine the T. cruzi DTUs that are circulating in Colombian chronic Chagasic patients and to obtain more information about the molecular epidemiology of Chagas disease in Colombia. We also assessed the presence of electrocardiographic, radiologic and echocardiographic abnormalities with the purpose of correlating T. cruzi genetic variability and cardiac disease. Molecular characterization was performed in Colombian adult chronic Chagasic patients based on the intergenic region of the mini-exon gene, the 24Sα and 18S regions of rDNA and the variable region of satellite DNA, whereby the presence of T.cruzi I, II, III and IV was detected. In our population, mixed infections also occurred, with TcI-TcII, TcI-TcIII and TcI-TcIV, as well as the existence of the TcI genotypes showing the presence of genotypes Ia and Id. Patients infected with TcI demonstrated a higher prevalence of cardiac alterations than those infected with TcII. These results corroborate the predominance of TcI in Colombia and show the first report of TcIII and TcIV in Colombian Chagasic patients. Findings also indicate that Chagas cardiomyopathy manifestations are more correlated with TcI than with TcII in Colombia.     

Trypanosoma cruzi: Multiplex PCR to detect and classify strains according to groups I and II

A multiplex PCR was developed for simultaneous detection of Trypanosoma cruzi DNA and classification of the parasite strain into groups I and II. As little as 10 fg of T. cruzi DNA could be detected by multiplex PCR. The technique was shown to be specific for T. cruzi DNA, since no PCR amplification products were obtained with DNA from other tripanosomatid species. Multiplex PCR was validated by assaying genomic DNA from 34 strains of T. cruzi that had been previously characterized; 24 blood samples from experimentally-infected mice and non-infected controls; 20 buffy coat samples from patients in the acute phase of Chagas disease and non-infected individuals, and 15 samples of feces from naturally-infected Triatoma infestans. T. cruzi samples from patients and from Y strain-infected mice were classified by multiplex PCR as T. cruzi II and samples from T. infestans and Colombiana strain-infected mice as T. cruzi I.     

Pre-Columbian Chagas disease in Brazil: Trypanosoma cruzi I in the archaeological remains of a human in Peruaçu Valley, Minas Gerais, Brazil

We evaluated the presence and distribution of Trypanosoma cruzi DNA in a mummy presenting with megacolon that was dated as approximately 560 +/- 40 years old. The mummy was from the Perua?u Valley in the state of Minas Gerais, Brazil. All samples were positive for T. cruzi minicircle DNA, demonstrating the presence and broad dissemination of the parasite in this body. From one sample, a mini-exon gene fragment was recovered and characterized by sequencing and was found to belong to the T. cruzi I genotype. This finding suggests that T. cruzi I infected humans during the pre-Columbian times and that, in addition to T. cruzi infection, Chagas disease in Brazil most likely preceded European colonization.     

Risk of Trypanosoma cruzi I (Kinetoplastida: Trypanosomatidae) transmission by Panstrongylus geniculatus (Hemiptera: Reduviidae) in Caracas (Metropolitan District) and neighboring States, Venezuela

The collection of Panstrongylus geniculatus bugs by inhabitants of dwellings in Caracas city (Metropolitan District) and in the neighboring Miranda and Vargas Sates, Venezuela, allowed for the gathering of data on the potential role of this sylvatic triatomine bug as a vector of Chagas disease in this area. The natural infection by Trypanosoma cruzi was recorded by examining fresh and stained faeces of the bugs. Additionally, a random amplification of polymorphic DNA technique for parasite identification and group typing was employed. A dot-ELISA test was used to identify the gut content of the triatomine bugs with the aim of assessing and quantifying the vector-human contact. Sixty-seven specimens (76.1%) were positive to T. cruzi (identified as T. cruzi I) and 60.2% (53/88) gave a positive reaction to the human antiserum. The human blood-positive samples included mixed blood meals with domestic animals (dog, pig and cow) (9.4%) and with mouse (3.8%). The overall Human Blood Index, measured as the percentage of bugs whose gut contents reacted with human antiserum on the total numbers of bugs that reacted with all the antisera tested, was 98.1%. Almost 41% of the bugs that had fed on humans were also positive for T. cruzi. These data show that the feeding of P. geniculatus on humans does not seem to be accidental and that its rate of infection by T. cruzi is high in this area which is not regarded as endemic for Chagas disease by the National Control Programme. This situation is particularly striking because it occurs in and around Caracas, the capital city, where 20% of the whole population of Venezuela live, human migrations from endemic areas are continuous, people in the crowded shantytown as well as people living in high-quality country houses are equally at risk and the epidemiological cycle Didelphis marsupialis/Rattus rattus-P. geniculatus-human does appear to occur successfully.     

Trypanosoma cruzi: exploring the nuclear genome of zymodeme 3 stocks by chromosome size polymorphism

Chagas disease is emerging in the Brazilian Amazon. We evaluated the position of eight zymodeme 3 isolates from Amazonian sylvatic vectors and one human case in relation to Trypanosoma cruzi I and II major groups and hybrid strains by chromosome size polymorphism. Nineteen isolates were analyzed by mapping nine coding sequences on chromosomal bands (0.6-3.3Mbp). Numerical analysis was based on the absolute chromosomal size difference index (aCSDI). A dendrogram was obtained applying the minimum evolution criterion and considering the aCSDI values to estimate the branch lengths. The isolates were distributed in four groups. Group A clustered hybrid isolates; Groups B and C, T. cruzi II and T. cruzi I isolates, respectively. Seven Z3 stocks were clustered in Group D, which showed low intra-group diversity and was the most divergent. The proportion of two different-sized homologous chromosomes was determined. Wild vectors harboring Z3 stocks constitute a potential reservoir of human infection in the Amazon.     

Genetic Heterogeneity in Trypanosoma cruzi Strains From Naturally Infected Triatomine Vectors in Northeastern Brazil: Epidemiological Implications

Eighteen Trypanosoma cruzi strains isolated from naturally infected triatomines were studied genetically. The majority of the strains were from Triatoma brasiliensis, the principal vector of Chagas disease in the northeast of Brazil. Multilocus enzyme electrophoresis (MLEE) and randomly amplified polymorphic DNA (RAPD) analyses were used to investigate the genotypic diversity and the spread of the T. cruzi genotypes in different environments. MLEE clearly distinguished two distinct isoenzyme profiles, and RAPD analysis revealed 10 different genotypes circulating in rural areas. The strains could be typed as isoenzyme variants of the T. cruzi principal zymodeme Z1 (T. cruzi I). An effective program of epidemiological vigilance is required to prevent the spread of T. cruzi I strains into human dwellings.     

The identification of two Trypanosoma cruzi I genotypes from domestic and sylvatic transmission cycles in Colombia based on a single polymerase chain reaction amplification of the spliced-leader intergenic region

A single polymerase chain reaction (PCR) reaction targeting the spliced-leader intergenic region of Trypanosoma cruzi I was standardised by amplifying a 231 bp fragment in domestic (TcI_DOM) strains or clones and 450 and 550 bp fragments in sylvatic strains or clones. This reaction was validated using 44 blind coded samples and 184 non-coded T. cruzi I clones isolated from sylvatic triatomines and the correspondence between the amplified fragments and their domestic or sylvatic origin was determined. Six of the nine strains isolated from acute cases suspected of oral infection had the sylvatic T. cruzi I profile. These results confirmed that the sylvatic T. cruzi I genotype is linked to cases of oral Chagas disease in Colombia. We therefore propose the use of this novel PCR reaction in strains or clones previously characterised as T. cruzi I to distinguish TcI_DOMfrom sylvatic genotypes in studies of transmission dynamics, including the verification of population selection within hosts or detection of the frequency of mixed infections by both T. cruzi I genotypes in Colombia.     

DNA damage and nitric oxide synthesis in experimentally infected Balb/c mice with Trypanosoma cruzi

This study aimed to evaluate whether experimental Chagas disease in acute phase under benznidazole therapy can cause DNA damage in peripheral blood, liver, heart, and spleen cells or induce nitric oxide synthesis in spleen cells. Twenty Balb/c mice were distributed into four groups: control (non-infected animals); Trypanosoma cruzi infected; T. cruzi infected and submitted to benznidazole therapy; and only treated with benznidazole. The results obtained with the single cell gel (comet) assay showed that T. cruzi was able induce DNA damage in heart cells of both benznidazole treated or untreated infected mice. Similarly, T. cruzi infected animals showed an increase of DNA lesions in spleen cells. Regarding nitric oxide synthesis, statistically significant differences (p<0.05) were observed in all experimental groups compared to negative control, the strongest effect observed in the T. cruzi infected group. Taken together, these results indicate that T. cruzi may increase the level of DNA damage in mice heart and spleen cells. Probably, nitric oxide plays an important role in DNA damaging whereas benznidazole was able to minimize induced T. cruzi genotoxic effects in spleen cells.     

Biological, biochemical and molecular features of Trypanosoma cruzi strains isolated from patients infected through oral transmission during a 2005 outbreak in the state of Santa Catarina, Brazil: its correspondence with the new T. cruzi Taxonomy Consensus (2009)

We examined strains of Trypanosoma cruzi isolated from patients with acute Chagas disease that had been acquired by oral transmission in the state of Santa Catarina, Brazil (2005) and two isolates that had been obtained from a marsupial (Didelphis aurita) and a vector (Triatoma tibiamaculata). These strains were characterised through their biological behaviour and isoenzymic profiles and genotyped according to the new Taxonomy Consensus (2009) based on the discrete typing unities, that is, T. cruzi genotypes I-VI. All strains exhibited the biological behaviour of biodeme type II. In six isolates, late peaks of parasitaemia, beyond the 20th day, suggested a double infection with biodemes II + III. Isoenzymes revealed Z2 or mixed Z1 and Z2 profiles. Genotyping was performed using three polymorphic genes (cytochrome oxidase II, spliced leader intergenic region and 24Sα rRNA) and the restriction fragment length polymorphism of the kDNA minicircles. Based on these markers, all but four isolates were characterised as T. cruzi II genotypes. Four mixed populations were identified: SC90, SC93 and SC97 (T. cruzi I + T. cruzi II) and SC95 (T. cruzi I + T. cruzi VI). Comparison of the results obtained by different methods was essential for the correct identification of the mixed populations and major lineages involved indicating that characterisation by different methods can provide new insights into the relationship between phenotypic and genotypic aspects of parasite behaviour.     

Biological and molecular characterization of a raccoon isolate of Trypanosoma cruzi from South Carolina

Biological and molecular characteristics of a raccoon isolate of Trypanosoma cruzi (R36) were compared with those of a known virulent strain (Brazil). Included in the characterization were growth rate in liver infusion tryptose medium, infectivity for murine fibroblasts, intracellular amastigote replication and trypomastigote release rates, polymerase chain reaction (PCR) profiling of the mini-exon gene, isoenzyme and random amplified polymorphic DNA (RAPD) profiles, and in vivo virulence for C3H/HeJ mice. Similar growth curves were noted for both strains; however, infectivity and rates of intracellular amastigote replication and trypomastigote release were significantly lower for the R36 isolate than for the Brazil strain. To determine virulence, C3H/ HeJ mice were exposed intraperitoneally to the R36 isolate. No parasite was observed in blood by direct examination or in tissues by histology; however, T. cruzi was detected by PCR in tissues (quadriceps and spleen) at 21 days postinfection. Analyses of the mini-exon gene, isoenzyme, and RAPD profiles indicate that R36 is in the T. cruzi II group and the Brazil strain is in the T. cruzi I group. Although infectivity and virulence of the raccoon isolate were lower than those for the Brazil strain, autochthonous infections in the United States have been reported, which suggests the need for further study of local T. cruzi isolates.     

Integrate study of a Bolivian population infected by Trypanosoma cruzi,the agent of Chagas disease

A cross section of a human population (501 individuals) selected at random, and living in a Bolivian community, highly endemic for Chagas disease, was investigated combining together clinical, parasitological and molecular approaches. Conventional serology and polymerase chain reaction (PCR) indicated an active transmission of the infection, a high seroprevalence (43.3%) ranging from around 12% in < 5 years to 94.7% in > 45 years, and a high sensitivity (83.8%) and specificity of PCR. Abnormal ECG tracing was predominant in chagasic patients and was already present among individuals younger than 13 years. SAPA (shed acute phase antigen) recombinant protein and the synthetic peptide R-13 were used as antigens in ELISA tests. The reactivity of SAPA was strongly associated to Trypanosoma cruzi infection and independent of the age of the patients but was not suitable neither for universal serodiagnosis nor for discrimination of specific phases of Chagas infection. Anti-R-13 response was observed in 27.5% only in chagasic patients. Moreover, anti-R13 reactivity was associated with early infection and not to cardiac pathology. This result questioned previous studies, which considered the anti-R-13 response as a marker of chronic Chagas heart disease. The major clonets 20 and 39 (belonging to Trypanosoma cruzi I and T. cruzi II respectively) which circulate in equal proportions in vectors of the studied area, were identified in patients' blood by PCR. Clonet 39 was selected over clonet 20 in the circulation whatever the age of the patient. The only factor related to strain detected in patients' blood, was the anti-R-13 reactivity: 37% of the patients infected by clonet 39 (94 cases) had anti-R13 antibodies contrasting with only 6% of the patients without clonet 39 (16 cases).     

Colombian Trypanosoma cruzi major genotypes circulating in patients: Minicircle homologies by cross-hybridization analysis

Here we present compelling evidence of Trypanosoma cruzi genotypes infecting 77 human cases of Chagas disease in Santander Department of Colombia. The patients were clinically studied and classified according to the presence of cardiac symptoms. We describe the distribution of the major T. cruzi genotypes circulating in this area by means of direct PCR analysis of blood samples. PCR was directed to minicircles and amplified DNAs were hybridized using genotype-specific DNA probes. These samples were previously genotyped with miniexon, 24 α rRNA and cytochrome oxidase subunit II (COII) markers. Minicircle DNA analyses were more sensitive than miniexon, 24 α rRNA and CO II genes in detecting infective T. cruzi II (Tc II). Two Tc II genotypes were identified by hybridization using two complementary DNA probes in 27.3% of the patients, with 15.3% using all three markers. These corresponded to 10 cases genotyped only by hybridization. The lineage Tc I, determined by hybridization, was the most prevalent singly or combined with different genotypes (72.7%), and at least three different T. cruzi genotypes were identified. Attempts to find two T. cruzi genotypes Tc I and Tc II in other endemic areas of Colombia revealed that one similar to the most prevalent Tc I genotype was detected in distant geographical areas. A similar Tc II genotype was found in Bolivia and Chile, revealing the great distribution of some ancestral T. cruzi genotypes. We did not detect any association between infective Tc I and Tc II lineages and the severity of the patients’ cardiac symptoms.     

Trypanosoma cruzi and human ubiquitin are immunologically distinct proteins despite only three amino acid difference in their primary sequence

The high similarity between Trypanosoma cruzi and human ubiquitin prompted us to characterize the human humoral immunity to host and parasite ubiquitin in Chagas disease and its possible role in Chagas autoimmunity. We have used a simplified one step purification procedure to partially purify T. cruzi ubiquitin. Using this preparation we have performed ELISA and Western blots, to show that chagasic sera recognise T. cruzi but not human or Leishmania ubiquitin indicating a species-specific response. Our results show that despite the high degree of similarity in the primary structure of human and T. cruzi ubiquitins, the three amino acid difference is sufficient to distinguish parasite versus host proteins.