Trypanosoma cruzi: immunoglobulin isotype profiles during the acute phase of canine experimental infection with metacyclic or blood trypomastigotes.

A detailed investigation has been carried out about the serological profiles of groups of dogs experimentally infected with metacyclic (MT) or blood (BT) trypomastigotes of Berenice-78 Trypanosoma cruzi strain. Peripheral blood was collected from infected dogs and uninfected controls, weekly during 35 days following the acute phase of infection, and immunoglobulin profiles were determined by ELISA. Dogs infected with BT exhibited unaltered levels of IgG2, increases in IgM, IgE, IgA, IgG and IgG1. In contrast, dogs infected with MT presented unaltered levels of IgE and IgG1 and an increase in IgM, IgA, IgG and IgG2 levels. Compared with the MT group, animals infected with BT showed significant increases in IgM on days 7, 14 and 28, in IgA on days 7, 14 and 21, in IgE on days 7 and 14, in IgG on days 14 and 28, and in IgG1 on days 7, 14 and 21. Parasitemia levels of the infected animals were measured over the same time period. No correlations were found between the immunoglobulin profiles and the parasitemia levels. The results demonstrated that the inoculum source (BT or MT) influence the immunoglobulin isotype profile that may drive distinct outcome of acute canine Chagas disease.

Impact of Trypanosoma cruzi clonal evolution on its biological properties in mice.

Twenty Trypanosoma cruzi stocks attributed to the 19, 20, 39, and 32 clonal genotypes were comparatively studied in BALB/c mice during the acute and chronic phases of the infection to test the working hypothesis that T. cruzi clonal structure has a major impact on its biological properties. Fourteen parameters were assayed: (1) infectivity; (2) prepatent period; (3) patent period; (4) maximum of parasitemia; (5) day of maximum of parasitemia; (6) parasitemia; (7) mortality, (8) percentage of positive hemoculture, (9) tissue parasitism; (10) inflammatory process during the acute phase of the infection; (11) mortality, (12) percentage of positive hemoculture; (13) tissue parasitism; and (14) inflammatory process during the chronic phase of the infection. Statistical comparison showed that the results are overall consistent with the working hypothesis that biological differences are proportional to the evolutionary divergence among the genotypes. Thus, closely related genotypes (19 vs 20 and 32 vs 39) show in general fewer differences than distantly related groups (19 or 20 vs 32 or 39) except for the comparison between 19 and 32. The working hypothesis is even more strongly supported by the result of the nonparametric Mantel test, which showed a highly significant correlation (P = 2.3 x 10(-3)) between biological differences and genetic distances among all pairs of stocks. These data taken together emphasize that it is crucial to take into account the phylogenetic diversity of T. cruzi natural clones in all applied studies dealing with diagnosis, drug and vaccine design, epidemiological surveys, and clinical diversity of Chagas' disease. Index Descriptors and Abbreviations: Trypanosoma cruzi; phylogenetic distance; biological properties; clonal theory; multilocus enzyme electrophoresis (MLEE); randomly amplified polymorphic DNA (RAPD); acute phase (AP); chronic phase (CP); days after inoculation (d.a.i.); liver infusion tryptose (LIT); gastrointestinal tract (GIT); genitourinary tract (GUT); percentage of infectivity (%INF); percentage of mortality during the acute phase (%MORT AP); percentage of mortality during the chronic phase (%MORT CP); prepatent period (PPP); patent period (PP); maximum of parasitemia (MP); day of maximum of parasitemia (DMP); parasitemia (PAR); percentage of positive hemoculture during the acute phase (% + HC AP); percentage of positive hemoculture during the chronic acute phase (% + HC CP); tissue parasitism (TP); inflammatory process (IP); tissue parasitism during the acute phase (TP AP); tissue parasitism during chronic phase (TP CP); inflammatory process during acute phase (IP AP); inflammatory process chronic phase (IP CP); Mann-Whitney test (MW); Kruskal-Wallis (KW); Kolmogorow-Smirnov test (KS).

Trypanosoma cruzi: infectivity of clonal genotype infections in acute and chronic phases in mice.

Eight Trypanosoma cruzi stocks pertaining to the clonal genotypes 19/20, 32, and 39 have been characterized for three experimental parameters of infectivity in Balb/c mice: (i) percentage of mice with a patent parasitemia (% MPP), (ii) maximum parasitemia (MP), and (iii) percentage of mice with positive hemoculture (% MPH). By order of decreasing values, the values recorded for the clonal genotypes ranked as follows: 19/20, 32, and 39, except for the % MPP parameter, for which 19/20 and 32 were not statistically different. The rate of successful reisolation after infection in mice, analyzed by multilocus enzyme electrophoresis and random amplified polymorphic DNA typing, was statistically different according to the clonal genotype and was different for uniclonal infections and for mixed infections by two different clonal genotypes. These results confirm that T. cruzi clonal genotypes differ significantly in their infectivity in mice.

Experimental Trypanosoma cruzi biclonal infection in Triatoma infestans: detection of distinct clonal genotypes using kinetoplast DNA probes.

Monitored biclonal densities of parasites were offered to third-stage larvae of Triatoma infestans via an artificial feeding device and 30 days later, the gut contents of the insects were processed for microscopic examination and polymerase chain reaction (PCR) detection of Trypanosoma cruzi kinetoplast DNA [kDNA]). A total of 15 mixtures involving nine different stocks attributed to the 19/20, 32 and 39 major clonal genotypes of Trypanosoma cruzi were used. The presence of each T. cruzi clonal genotype after completion of the cycle through the insects was investigated by hybridising the PCR amplification products with genotype-specific minicircle kDNA probes. Sixty-five out of 90 examined insects (72.2%) were positive for parasites by microscopic examination and 85 (94.4%) were positive by PCR. The results show that almost half of the biclonal infections are not detectable after completion of the cycle, and that there are important differences in detection of such biclonal infections according to the clonal genotypes considered. Moreover, elimination of a clonal genotype by another is a frequent, but not constant, pattern in biclonal infections of T. infestans. The use of PCR and kDNA probes makes it possible to avoid the culture phase, which makes detection of mixed infections much easier in epidemiological surveys. Moreover, the fact that T. infestansdoes not transmit different T. cruzi clonal genotypes with the same efficiency has strong implications for the reliability of xenogiagnosis.

Heart autonomic innervation during the acute phase of experimental American trypanosomiasis in the dog.

Heart autonomic innervation was studied in dogs during the acute phase of the experimental infection with the Berenice-78 strain of Trypanosoma cruzi. A glyoxylic acid-induced fluorescence method for catecholamines and a thiocholine method for demonstrating acetylcholinesterase activity showed the sympathetic and the parasympathetic nerve fibers, respectively. At day 34 of infection, moderate-to-intense rarefaction of both cholinergic and noradrenergic nerve fibers occurred in the atria of all animals coincident with moderate to intense myocarditis. In the ventricles, sympathetic denervation was clearly present only when the inflammatory processes were moderate to intense. Preliminary results on the chronic phase indicate that normal autonomic innervation coexists with an incipient chronic fibrosing myocarditis.

Trypanosoma cruzi: compared vectorial transmissibility of three major clonal genotypes by Triatoma infestans.

Twenty Trypanosoma cruzi stocks attributed to the 19/20, 32, and 39 major clones (Tibayrenc et al. 1986) were used to infect experimentally third instar larvae of Triatoma infestans. Three variables were considered: (i) percentage of infected insects; (ii) number of flagellates per insect (NFI); and (iii) percentage of metacyclic trypomastigotes per insect. Differences between the genotypes under study for all parameters considered were detected. These differences were statistically significant (P < 10(-3)), except between the 39 and 32 clonal genotypes for the NFI parameter. The correlation coefficient between the genetic distance and the biological parameters determined by the nonparametric Mantel's test was strongly significant (P < 10(-4)). Data obtained suggest clearly that populations of parasites belonging to the 19/20 genotype are more efficiently transmitted (high transmissibility genotype) by the vector than the 32 genotype (low transmissibility genotype), while the 39 genotype presents intermediary characteristic. Results confirm the working hypothesis that the subdivision of T. cruzi into discrete clonal lineages has an impact on the vectorial competence of T. infestans, the most important vector of the chagasic infection in South America, and that different clonal lineages do not exhibit the same vectorial transmissibility. This fact is relevant both for Chagas' disease epidemiology and for the use of xenodiagnosis.

Compared vectorial transmissibility of pure and mixed clonal genotypes of Trypanosoma cruzi in Triatoma infestans.

A total of 15 mixtures involving 9 different stocks attributed to the 19/20, 32 and 39 major clonal genotypes of Trypanosoma cruzi were used to infect third-instar nymphs of Triatoma infestans via an artificial feeding device. Three biological parameters were considered: (1) the percentage of infected insects (%II), (2) the number of flagellates per insect (NFI), and (3) the percentage of trypomastigotes per insect (%DIF). Genetic characterization by both multilocus enzyme electrophoresis (MLEE) and random amplification of polymorphic DNA (RAPD) indicated that in almost all cases (87%), mixtures remained present after completion of the whole cycle in the insect vector. Two lines of comparison were performed: (1) pure clonal genotypes versus corresponding mixed clonal genotypes and (2) the actual behavior of mixed clonal genotypes versus the expected behavior of the theoretical mixture (i.e. the arithmetic mean of the results observed for each of the two clonal genotypes taken separately). Statistical analyses of the variables were made difficult because of the presence of large standard deviations. Nevertheless, in several cases, mixtures differed significantly from pure clonal genotypes, and in one case the actual mixture differed significantly from the theoretical mixture. In some cases, interaction (either potentialization or reciprocal inhibition) could be suspected.

Characterization of two isolates of Trypanosoma cruzi obtained from the patient Berenice, the first human case of Chagas' disease described by Carlos Chagas in 1909.

Two isolates of Trypanosoma cruzi were obtained from the patient Berenice, the first human case of Chagas' disease (Chagas 1909), when she was 55 and 71 years old, respectively. The isolates were characterized on the basis of their epimastigote-trypomastigote differentiation in liquid media and of the electrophoretic pattern of EcoR1 digestion products of kinetoplast DNA (k-DNA) minicircles (schizodeme) and isoenzyme patterns (zymodeme). Clear differences were found between the isolates, suggesting the occurrence of a heterogeneous population of T. cruzi in the infection of this patient.

[Quantitative and qualitative studies of the Auerbach and Meissner plexuses of the esophagus in dogs inoculated with Trypanosoma cruzi]. / Estudo quantitativo e qualitativo dos plexos de Auerbach e Meissner do esôfago de cães inoculados com o Trypanosoma cruzi.

A quantitative and qualitative study was conducted on the Auerbach and Meissner plexuses of the esophagus of four chagasic dogs sacrificed during the acute phase of infection. Ganglionitis and periganglionitis of the Auerbach plexus ranged from mild to moderate and induced significant neuronal lesions, especially in two animals. The ganglions of the Meissner plexus were observed in small number which did not permit any analysis. Mild or moderate myositis was observed mainly in the lower third of the esophagus and was rarely associated with amastigote nests. Ganglion and neuron counts did not demonstrate denervation. Although the formation of megaesophagus was not induced in any dog, lesions of the Auerbach plexus and myocells of the esophagus were observed during the acute phase of chagasic infection. To our knowledge, this is the first systematic quantitative and qualitative study of the Auerbach and Meissner plexuses of the esophagus in experimental trypanosomiasis cruzi.

Cardiac plexus of dogs experimentally infected with Trypanosoma cruzi: inflammatory lesions and quantitative studies.

Qualitative and quantitative aspects of the superficial and profound cardiac plexus of dogs experimentally infected with Be-62 and Be-78 strains of Trypanosoma cruzi were studied. Animals were autopsied in the acute phase of infection. The inflammatory process, lesions and number of parasites were more intense and frequent in animals infected with the Be-78 strain than in those infected with Be-62. Despite this, no statistically significant differences could be found between the number of neuron bodies in the ganglia of infected and control dogs.

Experimental Chagas' disease in dogs.

This paper describes the development of experimental Chagas' disease in 64 out-bred young dogs. Twenty-nine animals were inoculated with the Be-62 and 35 with Be-78 Trypanosoma cruzi strains. Twenty-six were infected with blood trypomastigotes by different inoculation routes and 38 with metacyclic trypomastigotes from the vector via the conjunctival route. Twenty of the 26 dogs infected with blood trypomastigotes were autopsied during the acute phase. Eleven died spontaneously and nine were sacrificed. Six remained alive until they died suddenly (two) or were autopsied. (four). Twelve of the 38 dogs infected with metacyclic trypomastigotes evolved naturally to the chronic phase and remained alive for 24-48 months. The parasitemia, clinical aspects and serology (IgM and IgG) as well as electrocardiogram, hemogram and heart anatomo-histopathologic patterns of acute and chronic cardiac forms of Chagas' disease as seen in human infections, were reproduced. The most important finding is the reproducibility of diffuse fibrosing chronic chagasic cardiopathy in all dogs infected with Be-78 T. cruzi strain autopsied between the 90th and 864th days of infection. Thus, the dog can be considered as a suitable experimental model to study Chagas' disease according to the requisites of the Word Health Organization (1984). Furthermore the animal is easily obtained and easy to handle and maintain in experimental laboratory conditions.

[Canine visceral leishmaniasis: evaluation of the serologic method used in epidemiologic studies]. / Leishmaniose visceral canina: avaliação da metodologia sorológica utilizada em inquéritos epidemiológicos.

A comparative study was made of eluates of the blood of dogs experimentally infected with different trypanosomatids. Using antigens prepared from promastigotes of Leishmania mexicana, L. braziliensis and L. chagasi, assessments were made by the indirect immunofluorescence test. The results showed a sensitivity of 87.5% in the diagnosis of canine visceral leishmaniasis, independent of antigen used. Cross-reactions occurred in 75% of cases of cutaneous leishmaniasis and 83.3% of dogs with chagas' disease. An epidemiological survey in an area of leishmaniasis confirmed that immunofluorescence tests on eluates of dogs' blood give cross-reactions between L. braziliensis and L. chagasi. The results suggest that such testing could be useful in public health campaigns but attention is drawn to the fact that the level of positive reactions cannot be used as an indicator of the prevalence of canine kala-azar.