Trypanosoma rangeli sialidase: kinetics of release and antigenic characterization.

The epimastigote stage of Trypanosoma rangeli release a sialidase with a high sialic acid hydrolysis capacity. We demonstrate that sialidase secretion is an active process that is reduced at low temperatures and in the presence of sodium azide. The enzyme is continuously released until certain maximally active concentrations are attained in the BHI culture medium when the parasite density reaches 2-3 x 10(6) cells. When introduced into culture medium already containing such enzyme levels, freshly harvested parasites do not secrete additional sialidase. These findings suggest a self-regulating mechanism and a biological role for the secreted T. rangeli sialidase. The secreted enzyme was purified to homogeneity by fractionation with ammonium sulphate and affinity chromatography. Antibodies raised against the purified molecule recognized antigens of similar molecular weights (73 kDa) in western immunoblotting analyses of T. rangeli and T. cruzi whole cell lysates. No antigenic recognition was recorded against T. cruzi active sialidase/trans-sialidase polypeptides or Clostridium perfringens and Vibrio cholerae commercial sialidases. These observations may indicate the expression of different antigenic domains in T. rangeli, T. cruzi and bacterial sialidases.

Trypanosoma rangeli: identification and purification of a 48-kDa-specific antigen.

Trypanosoma rangeli infects humans nonpathologically in some areas of Central and South America. Due to morphological and antigenic similarities with T. cruzi, the clear identification of this parasite is an important task. Here, we describe the identification and purification of a specific 48-kDa antigen from T. rangeli. By western blotting analysis, this molecule was not detected in T. cruzi epimastigotes and in Leishmania sp. promastigotes. Fluorescence-activated cell sorter analysis demonstrated that the protein is expressed uniformly by the T. rangeli cell population during axenic culture. Additionally, following immunostaining, a particular subcellular localization (present in organelles) is proposed for this antigen. These results suggest that this 48-kDa antigen may be a useful marker for the identification and characterization of T. rangeli isolates.

Experimental Trypanosoma rangeli infection in a murine model.

Trypanosoma rangeli experimental murine infections were performed in order to study parasitemias and anti-parasite antibody levels. Three groups of mice were used: a) mice infected with metatrypomastigotes derived from infected bugs; b) mice which received four reinoculations of metatrypomastigotes and c) mice immunosuppressed with cyclophosphamide. The results showed that bloodstream parasites can be found from the first day post inoculation reaching a peak at day 5 or 7 and then start to decline. Parasites disappeared completely from the circulation after 20-25 days. However in the immunosuppressed group, parasites were found in blood up to 45 days post infection. The humoral immune response was monitored using an ELISA test and low levels of specific IgG and IgM immunoglobulins were found. However the IgG titers were lower than the IgM. One could conclude that IgM was the predominant immunoglobulin isotype induced in a T. rangeli experimental infection because the highest titers were observed in the reinoculated group. IgM antibodies also showed the most prominent crossreactivities with T. cruzi antigens.

Serological diagnosis of Trypanosoma rangeli infected patients. A comparison of different methods and its implications for the diagnosis of Chagas' disease.

Venous blood from 65 Panamanian schoolchildren living in an area endemic for both Trypanosoma cruzi and T. rangeli were screened for the presence of these parasites. Trypanosoma rangeli were isolated and cultured from four individuals. Serological tests of all 65 sera were performed, including immunohaemagglutination (IHA), indirect immunofluorescence assay (IF) and ELISA using both T. rangeli and T. cruzi as antigens, as well as T. cruzi synthetic peptides in an ELISA assay. Results indicated a higher immunoreactivity to T. rangeli preparations than to T. cruzi within the studied population, which could be divided into four 'serological responder' groups. Interestingly, the panel of SAPA and other T. cruzi synthetic peptides were not useful in the discrimination of patients. Furthermore, patients from whom parasites had been isolated could not be distinguished from those of two other groups. Significant immunoreactivity to T. cruzi preparations was displayed in all responder sera, despite total lack of evidence of infection with this parasite. The immunobiological significance of T. rangeli infection is unclear, but these data indicate that it is a compounding problem in the accurate diagnosis of pathological T. cruzi infection by serological analysis. The relationship of these cohabiting species, in respect to infection outcome and immunological activation, is discussed.

Trypanosoma rangeli: epimastigote immunogenicity and cross-reaction with Trypanosoma cruzi.

Trypanosoma rangeli epimastigote components, able to elicit an immune response, were defined and compared with those found in Trypanosoma cruzi epimastigotes. Using polyclonal antibodies against these parasites and immunoblotting analysis, an antigenic similarity index of 0.48 was found when anti-T. cruzi antibodies were used and 0.60 with anti-T. rangeli antibodies. Additionally, immunoblotting analyses using specific antibodies against a T. rangeli polypeptide eluted from polyacrylamide gels confirm an antigen of 43 kDa as a specific marker for T. rangeli.

Karyotype variability in Trypanosoma rangeli.

The molecular karyotypes of several different protozoan parasites show high intra-species variation, including different kinetoplastids such as Trypanosoma brucei, Trypanosoma cruzi and Leishmania ssp. In this study, the molecular karyotype of Trypanosoma rangeli was examined. To evaluate potential intra-species molecular karyotype variations, 16 different samples were studied by pulsed field gel electrophoresis (PFGE) followed by ethidium bromide staining and hybridizations with 6 different probes. The result showed that different T. rangeli populations are highly polymorphic regarding the molecular karyotype, and thus suggests that PFGE analysis can be used for classification of different T. rangeli isolates. In addition, the molecular karyotype of T. rangeli was compared to molecular karyotypes of other kinetoplastids, and was shown to be distinctly different from that of T. cruzi, but shows some similarities with the karyotype described for T. brucei. Among the probes used one was identified as highly polymorphic, and thus informative for studies of different T. rangeli populations, and another was useful for differentiation between T. rangeli and T. cruzi.

Immunoparasitological studies of Trypanosoma cruzi low virulence clones from Panama: humoral immune responses and antigenic cross-reactions with Trypanosoma rangeli in experimentally infected mice.

The kinetics of humoral immune responses were investigated in mice experimentally infected with five clones of Trypanosoma cruzi isolated from different sources in Panama. Sera were collected at different timepoints post-infection. ELISA and IHA tests were used to detect antibodies against T. cruzi epimastigote antigens. The levels of T. cruzi specific antibodies increased during the course of infection; at day 90 post-infection the range was between 1:5120 and 1:10240. A high correlation was evident between ELISA and IHA results. Western blots revealed that these antibodies recognized polypeptides of 81, 76 and 71 KDa during the first weeks and 81, 76, 71, 50, 40, 28 and 12 KDa after 30-50 days. Only minor differences in antigen recognition patterns were demonstrated, suggesting that the major antigens may be represented in all clones. T. rangeli antigens were also recognized by T. cruzi seropositive sera. However, an ELISA test using antigens isolated from a genomic expression library of T. cruzi revealed that a hyperimmune rabbit serum against T. rangeli was unable to recognize the repeat sequence of SAPA (Shed Acute Phase Antigen) peptides but did recognize a number of other T. cruzi synthetic peptide antigens. The importance of these findings, in the context of Chagas' disease, is discussed.

Molecular techniques in the characterization of Leishmania isolates from Central America.

The public-health problems caused by leishmaniasis in most countries in Central America are becoming more severe. This is partly because of the increasing size of the human populations that are at risk and their migratory patterns. Annual incidence of the disease in Costa Rica, Honduras, Guatemala, Panama and Nicaragua is estimated to be as high as 20,000 cases. Regional changes in the epidemiology of the various Leishmania spp. present have emphasized the need for innovative, sensitive and accurate diagnostic tools. PCR and isoenzyme, monoclonal antibody, schizodeme, DNA-probe and random-amplified, polymorphic DNA analyses have been tested. Preliminary indications that Leishmania chagasi was present in Costa Rica and Honduras and that interspecific hybrids occurred in Nicaragua have been confirmed using these methods. The distribution of the mexicana complex was also found to be broader and more heterogeneous than initially expected. Overall, there was 87% concordance between the results produced using the different techniques.

[Evaluation of 4 immunobiochemical/molecular methods for the identification of Trypanosoma cruzi and Trypanosoma rangeli strains]. / Evaluación de cuatro métodos immunobioquímico/moleculares en la identificación de cepas de Trypanosoma cruzi y Trypanosoma rangeli.

In American man can be infected with two trypanosomes: Trypanosoma cruzi, the etiological agent of Chagas' disease and Trypanosoma rangeli, a suspected nonpathogenic parasite. In this communication are presented 4 methods in order to improve the current knowledge about the specific identification of these parasites. Using the SDS-PAGE technique it was possible to differentiate between T. rangeli. and T. cruzi based in at less 4 protein bands with a relative molecular weights of 93, 77-73, 63 and 54-52 KDa. These polypeptides were found only in T. rangeli electrophoretic profiles. An ELISA test showed that the antigenic composition found in the enzyme cisteine proteinase (cruzipain) is specific for T. cruzi epimastigotes. Antigenic analysis by Western blot assay, proved that T. rangeli and not T. cruzi present antigenic bands with a Mr of 142, 63, 54, 51, 49, 43, 39 and 24 KDa. Finally, using the Southern blot procedure, it was confirmed that SAPA, a DNA sequence originally identified in the T. cruzi, genome, is absent in T. rangeli nuclear DNA. These initial observations revealed that it is possible to identify both parasites using the described methods, however further works are required to clarify the biochemical, immunological and molecular relationship between T. rangeli and T. cruzi.

Evaluación de cuatro métodos immunobioquímico/moleculares en la identificación de cepas de Trypanosoma cruzi y Trypanosoma rangeli / Evaluation of 4 immunobiochemical/molecular methods for the identification of Trypanosoma cruzi and Trypanosoma rangeli strains

In American man can be infected with two trypanosomes: Trypanosoma cruzi, the etiological agent of Chagas' disease and Trypanosoma rangeli, a suspected nonpathogenic parasite. In this communication are presented 4 methods in order to improve the current knowledge about the specific identification of these parasites. Using the SDS-PAGE technique it was possible to differentiate between T. rangeli. and T. cruzi based in at less 4 protein bands with a relative molecular weights of 93, 77-73, 63 and 54-52 KDa. These polypeptides were found only in T. rangeli electrophoretic profiles. An ELISA test showed that the antigenic composition found in the enzyme cisteine proteinase (cruzipain) is specific for T. cruzi epimastigotes. Antigenic analysis by Western blot assay, proved that T. rangeli and not T. cruzi present antigenic bands with a Mr of 142, 63, 54, 51, 49, 43, 39 and 24 KDa. Finally, using the Southern blot procedure, it was confirmed that SAPA, a DNA sequence originally identified in the T. cruzi, genome, is absent in T. rangeli nuclear DNA. These initial observations revealed that it is possible to identify both parasites using the described methods, however further works are required to clarify the biochemical, immunological and molecular relationship between T. rangeli and T. cruzi

[Cloning of kDNA minicircles in different species of Leishmania and its use as probes for diagnosis]. / Clonaje de minicírculos de kADN en diferentes especies de Leishmania y su uso como sondas para diagnóstico.

The present study describes the cloning procedure for fragments of kinetoplast DNA minicircles from different Leishmania species and its use for detecting the presence of these parasites. Our methodology was as follow: the DNA of the kinetoplast from Leishmania mexicana amazonensis and Leishmania braziliensis panamensis was extracted, purified and digested with the enzyme Dra I. These fragments were cloned in the site for Hinc II in the plasmid pKS. E. coli was the bacterial strain used for transforming and amplifying the cloned fragments; the selection was carried out in LB medium supplemented with ampicillin. With the clones suspected to be positives we run a Southern blot and total kDNA, from each Leishmania species, was used as hybridization probe. Finally, the cloned purified fragments were tested as diagnostic probes against kDNA from eleven different species of Leishmania and one of Trypanosoma cruzi parasites. After cloning, transforming, amplifying and selecting, we obtained two probes of fragments of kDNA minicircles: one from L. m. amazonensis and the other from L. b. panamensis. Both probes showed high sensitivity for diagnosing cutaneous Leishmania complexes (Mexicana or Braziliensis); however, we observed a low grade crossreaction between some species belonging to the same complex. It is necessary to continue studies in order to obtain subfragments of these probes with a higher grade of specificity at the level of species and subspecies.

[Determination of soluble interleukin-2 receptor in patients with American Trypanosomiasis in Panama]. / Evaluación del receptor soluble de interleucina--2 en pacientes con Tripanosomiasis Americana en Panamá.

The quantitative determination of Inter leukin-2 Receptor (sIL-2R) in the serum of patients with acute or chronic Chagas disease is compared with values found in normal individuals. The mean value of soluble IL-2R in patients with acute Chagas' disease was found to be 3,282 +/- 171 U/ml. The mean value of sIL-2R in serum samples from chronic chagasic patients was 511 +/- 207 U/ml, while in the control or "normal" group of persons the mean value for sIL-2R was 366 +/- 108 U/ml. In patients with early or acute infections with T. cruzi, the serum levels of sIL-2R was usually above 1000 U/ml. However, the correlation with anti-T. cruzi antibodies and levels of sIL-2R was not always directly proportional. Specific antibodies anti-T. cruzi in serum from chronic chagasic patients, shown at low or high levels, did not reveal a proportional correlation with serum levels of sIL-2R which tend to be significantly lower than in early or acute infections. It is considered that high values for sIL-2R are related with the parasite activity and its pathologic interaction with the host. It is possible that high levels of sIL-2R could serve as an indicator of early or acute Chagas' disease and be useful in assessment of disease conditions and response to therapy.

[Hepatic polycystic hydatidosis. Clinical and histopathologic report of the second native case of echinococcosis in the Republic of Panama]. / Hidatidosis poliquística hepática. Informe clínico e histopatológico del segundo caso de equinococosis autoctono en la República de Panamá.

The second autochthonous case of hepatic hydatidosos by Echinococcus vogelli is reported for the Republic of Panama. The patient was a 8 year old female living in the Province of Colon (Maria Chiquita). She claimed never to have traveled outside panamanian territory. The multichambered cysts with fertile, numerous protoscolices were identified from biopsied material obtained after exploratory laparotomy at Manuel Amador Hospital in Colon (Republic of Panama). The cyst appeared multiloculated mostly affecting the left lobe of the liver, beside a cystic lesion on the right lobe. The large rostellar hooks, in a count of 100 protoscolices, had a total length of handles and blades which average 41 microns in diameter, which coincide with those of E. vogeli, the most prevalent hydatid disease in man in South and Central America.

[Evaluation of the development of immunologic response in patients with cutaneous leishmaniasis]. / Evaluación del desarrollo de la respuesta inmunológica en pacientes con Leishmaniasis cutánea.

In this study we present epidemiological and immunological data about cutaneous Leishmaniasis in Panama, in an area where recurrence occurs in 65% of the cases evaluated. The development of the cellular immune response, during clinical evolution of primary and recurrent cases, indicates that initial stimulation index (SI) less than 3 are observed in recurrent cases, while this level is higher than 3 in primary cases. The humoral immune response is related only with the time of clinical evolution. We point out the importance of looking at the population of T lymphocytes quantitatively, as well as looking at the levels of lymphokines, in order to explain the differences that we observed in the cellular immune response.

Host feeding profiles of Triatoma dimidiata in peridomestic habitats of western Panama.

Bloodmeal analysis of Triatoma dimidiata collected in peridomestic habitats of western Panama showed that avian feedings comprised 25% of this species' host selections; opossums, the principal reservoir of Chagas' disease in the republic, were not among mammalian feedings. These findings may account for the low infestation rates of Trypanosoma cruzi in the bugs and the hypoendemicity of Chagas' disease in western Panama.

Toxoplasmosis in Panama: a 10-year study.

We studied the prevalence of Toxoplasma antibody over a 10-year period in a rural population of 326 people in Chorrera Province of Panama using the dye test. Fifty-five seroconversions were found in 108 people at risk, and 48 (87%) in children between 2 and 13 years with a mean incidence rate of 8.6% per year. Antibody prevalence rose from 25% at 5 years to 50% at 10 years of age, and increased gradually, reaching 90% by 60 years. Mean antibody levels after seroconversion were 1:6,000 in the dye test; they fell to 1:1,000 after 1 year, 1:800 after 2 years, 1:200 after 3 years, and 1:333 after 7-9 years. About 10% of antibody titers ranged between 1:4 and 1:32. Toxoplasma antibody prevalence was also studied in the metropolitan Panama City population using 590 sera collected in the fall of 1981. Age-specific incidence rates were similar in the urban and rural setting (correlation coefficient 0.71). The number of cats observed in the rural area and in the city and the degree of soil contact appeared compatible with a hypothesis of transmission by oocysts.