Evaluation of 18S rDNA PCR assay using skin fragments as a diagnostic test for Trypanosoma caninum.

Trypanosoma caninum is a new species that has been recently identified in Brazil and infects domestic dogs. To date, no accurate diagnostic assays for this parasite have been established; thus, our aim was to evaluate more than one type of PCR for the diagnosis and molecular screening of T. caninum in 229 dogs living in Rio de Janeiro state. The tests were based on the amplification and sequencing of the 18S ribosomal DNA (rDNA) gene using healthy skin fragments. Additionally, PCR amplification of the kDNA minicircles region specific to the Leishmania genus was performed. The PCR results were compared with those of culture-based analysis performed with the same specimen. Using cultures, T. caninum and Leishmania chagasi were isolated from 11 and 12 dogs, respectively, whereas the 18S rDNA PCR assay detected parasitic infection in 35 dogs. Among these, 25 dogs showed an amplification pattern similar to T. caninum and 10 showed a pattern similar to L. chagasi; these results were confirmed by sequencing analysis. The kDNA PCR analysis showed that 14 dogs were positive for Leishmania infection. Of these, 2 dogs showed negative culture results and 12 were positive for L. chagasi, including 4 with negative 18S rDNA PCR results. Thus far, culture-based testing has been the only tool used successfully for T. caninum diagnosis. Our results demonstrate that 18S rDNA PCR-based test should be a useful diagnostic tool, particularly for distinguishing between T. caninum and L. chagasi infections in areas where these 2 parasites co-exist.

Isolation of Trypanosoma caninum in domestic dogs in Rio de Janeiro, Brazil.

SUMMARY: The domestic dog's involvement with different members of the Trypanosomatidae family has been the focus of several studies due to this animal's close proximity to man. Recently this animal has been infected by a new Trypanosoma species (T. caninum), described in Rio de Janeiro and 19 similar isolates were later obtained. The objective of this study was to identify these isolates. All samples were isolated from intact skin cultures and analysed morphologically, by biochemical isoenzyme electrophoresis assays and by several molecular PCR assays. Additionally, anti-Leishmania sp. antibodies were assessed using the indirect Immunofluorescence Antibody Test (IFAT) in all animals. The methodologies employed to identify the isolates, including partial nucleotide sequences of 18S rRNA gene, indicated patterns identical to T. caninum and patterns different from the other species, including T. cruzi and T. rangeli samples. A phylogenetic tree constructed with the partial 18S ribosomal sequence shows that T. caninum is clustered with T. pestanai. Ten (52.6%) animals presented anti-Leishmania sp. antibodies with titres varying from 1:40 to 1:320. Thus, the hypothesis that this protozoan has disseminated among the dogs in Rio de Janeiro must be considered. The importance of a correct diagnosis in those animals and the possible consequences in the areas where visceral leishmaniasis is found are discussed here.

Trypanosoma cruzi infection in Didelphis marsupialis in Santa Catarina and Arvoredo Islands, southern Brazil.

Between 1984 and 1993 the prevalence of the Trypanosoma cruzi infection in opossums (Didelphis marsupialis) was studied in Santa Catarina and Arvoredo Islands, State of Santa Catarina, Brazil. The association of the triatomine bug Panstrongylus megistus with opossums nests and the infection rate of these triatomines by T. cruzi was also studied. Thirteen different locations were studied in Santa Catarina Island (SCI), in which 137 D. marsupialis were collected. Sixty two opossums were collected at the Arvoredo Island (AI), located 12 miles north from SCI. All captured animals were submitted to parasitological examinations that revealed the presence of T. cruzi in 21.9% of the opossums captured in SCI and 45.2% among opossums captured in the AI. The presence of P. megistus was detected in most of the D. marsupialis nests collected in the SCI, however, in the non-inhabited AI only eight triatomines were collected during the whole study. The presence of T. cruzi-infected D. marsupialis associated with P. megistus in human dwellings in the SCI, and the high infection rate of D. marsupilais by T. cruzi in the absence of a high vector density are discussed.

Characterization of Leishmania sp. strains isolated from autochthonous cases of human cutaneous leishmaniasis in Santa Catarina State, southern Brazil.

Four Leishmania sp. samples were isolated from autochthonous human cases of American cutaneous leishmaniasis (ACL) in Santa Catarina State, southern Brazil. These strains were characterized using indirect immunofluorescence with a panel of Leishmania-specific monoclonal antibodies (MAbs), and by PCR amplification and hybridization assay of the mini-exon gene with group specific probes. The results obtained with the MAbs were in agreement with the genetic marker. Two isolates (MHOM/BR/89/JSC89-H1 and MHOM/BR/89/JSC89-H2) were identified as L. (Leishmania) amazonensis and two (MHOM/BR/96/LSC96-H3 and MHOM/BR/97/LSC97-H4) as L. (Viannia) braziliensis. The southernmost autochthonous cases of ACL in Brazil are due to two different Leishmania sp. species, confirming the spreading of ACL on the American continent.

Biological and isoenzymatic characterization of Trypanosoma cruzi strains isolated from sylvatic reservoirs and vectors from the state of Santa Catarina, Southern Brazil.

Sixty eight Trypanosoma cruzi strains were isolated in the state of Santa Catarina, Southern Brazil, from sylvatic reservoirs or naturally infected vectors and characterized by their biological behaviour in mice, morphology of bloodstream forms and isoenzyme profiles. Twenty eight strains were isolated from the triatomine bug (Panstrongylus megistus), 2 from rodents (Echimys dasythrix and Akodon sp) and 38 from opossums (Didelphis marsupialis). The infectivity in mice of 48 T. cruzi strains showed that 2 (4.2%) were of high virulence, 19 (39.6%) of medium virulence, 15 (31.2%) of low virulence and 12 (25.0%) produced subpatent parasitemia in mice. A morphological study of bloodstream trypomastigotes from 8 T. cruzi strains showed a predominance of stout forms. The isoenzyme pattern of 59 T. cruzi strains showed that 54 (91.5%) belonged to zymodeme Z1, 3 (5.1%) to zymodeme Z2 and 2 (3.4%) to mixed zymodemes, Z1 and Z2. All 34 T. cruzi strains analyzed from opossums were Z1. Three out of 5 strains isolated from adults of P. megistus collected inside houses, belonged to zymodeme Z2 and two strains exhibited mixed zymodemes, Z1 and Z2, in 5 out of 6 enzymes studied. Although the State of Santa Catarina is a non endemic region for human Chagas'disease, the presence of zymodeme Z2 parasites in the sylvatic vector, P. megistus, captured in domiciliary environments suggests the possibility of human and/or domestic mammal infection by T. cruzi.

[Colonization of artificial ecotopes by Panstrongylus megistus in Santa Catarina Island, Florianópolis, Santa Catarina, Brazil]. / Colonização de ecótopos artificiais pelo Panstrongylus megistus na Ilha de Santa Catarina, Florianópolis, Santa Catarina, Brasil.

The aim of this work was to verify the colonization of Panstrongylus megistus on artificial ecotopes in Florianópolis, in the Santa Catarina Island, South Brazil. For this purpose 443 houses of the Lagoa district and 779 house annexes (524 chicken-houses, 46 corrals and 209 storage-houses) in 9 different places were examined from 1985 to 1992. These ecotopes, which include ceilings and basements, were checked after application of dislodging liquid (Pirisa 5%). Colonization by P. megistus was verified in two houses, three chicken-houses and one storage-house of the Lagoa district, where eggs, nymphs and adults were collected. To verify local reports of P. megistus occurrence, another two houses and one school were investigated. The colonization at all of these places was confirmed. In the 9 artificial ecotopes examined, 559 eggs, 305 nymphs and 24 adults were collected. The infection rate of P. megistus by Trypanosoma cruzi was 55.3% (182/329). A similar infection rate of 56.5% (78/138) was obtained in adults of P. megistus from sylvatic ecotopes and in adults captured in the houses by the inhabitants between 1983 to 1991. Precipitin tests revealed blood from just one source in 94.0% of the insects (170/181). Human blood was found in 80.6% (25/31) of the adults and in 5.8% (1/17) of the nymphs captured in the houses. These results suggest the need to ally serious epidemiologic vigilance to the effort of the inhabitants in order to avoid the risk of domiciliation of P. megistus in the houses.

Trypanosoma rangeli (Tejera, 1920) isolated from a sylvatic rodent (Echimys dasythrix) in Santa Catarina island, Santa Catarina state: first report of this trypanosome in southern Brazil

A trypanosome strain isolated from a sylvatic rodent (Echimys dasythrix) from Santa Catarina Island (Santa Catarina State, Brazil) was characterized by the following methods: experimental transmission and development in invertebrate hosts, morphometry, cross protection, complement sensitivity, lectin agglutination and isoenzyme profiles. Comparasions were made with standard Trypanosoma cruzi and T. rangeli strains. All methods except isoenzyne analysis led to the identification of the isolate as T. rangeli. The isoenzyme differences found could be explained on the basis of polymorphism. Therefore this is the first report of T. rangeli in southern Brazil, increasing the geographical distribution of this parasite

Trypanosoma rangeli (Tejera, 1920) isolated from a sylvatic rodent (Echimys dasythrix) in Santa Catarina Island, Santa Catarina State: first report of this trypanosome in southern Brazil.

A trypanosome strain isolated from a sylvatic rodent (Echimys dasythrix) from Santa Catarina Island (Santa Catarina State, Brazil) was characterized by the following methods: experimental transmission and development in invertebrate and vertebrate hosts, morphometry, cross protection, complement sensitivity, lectin agglutination and isoenzyme profiles. Comparisons were made with standard Trypanosoma cruzi and T. rangeli strains. All methods except isoenzyme analysis led to the identification of the isolate as T. rangeli. The isoenzyme differences found could be explained on the basis of polymorphism. Therefore this is the first report of T. rangeli in southern Brazil, increasing the geographical distribution of this parasite.