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

Abstract

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.

Molecular study of Trypanosoma caninum isolates based on different genetic markers

“…Using this target, we identified three new T. caninum samples from the Mato Grosso state and confirmed the identity of the 18 isolates from Rio de Janeiro state, previously identified by other tools (Pinto et al 2010). Based on phylogenetic analysis using this molecular target, all T. caninum samples were grouped into the same cluster with sequences determined in our previous studies (Pinto et al 2010;Barros et al 2012). Indeed, there are many advantages in using this marker: it is a highly conserved region in the genome, it is present in thousands of copies, and its DNA sequence is widely available in public databases.…”

“…First, the species status of all samples were confirmed by sequencing of the 18S rDNA gene, since this marker is already been considered an acceptable molecular criterion for taxonomic identification (Hamilton and Stevens 2010;Pinto et al 2012). Using this target, we identified three new T. caninum samples from the Mato Grosso state and confirmed the identity of the 18 isolates from Rio de Janeiro state, previously identified by other tools (Pinto et al 2010). Based on phylogenetic analysis using this molecular target, all T. caninum samples were grouped into the same cluster with sequences determined in our previous studies (Pinto et al 2010;Barros et al 2012).…”

“…Amplification of the minicircle kDNA region yielded two products, one with 350 bp and other with 330 bp. Some samples were not analyzed by PCR assays and sequencing for the following reasons: (a) some stocks already been performed in other studies (Madeira et al 2009;Pinto et al 2010;Barros et al 2012), (b) due to lost by secondary contamination, or (c) reduced growth through the subcultures of the initial isolate (Table 2).…”

“…Flagellates of the Trypanosoma genus are parasites that belong to the Trypanosomatidae family, and this group includes species that infect a variety of vertebrate hosts (Hoare 1972). Trypanosoma caninum has been reported in dogs from different Brazilian states (Madeira et al 2009;Pinto et al 2010;Almeida et al 2011), and little is known about genetic data of this parasite. To date, partial 18S ribosomal DNA (rDNA) PCR following sequencing has been the only genetic approach used aiming to identify the various isolates.…”

Canine Macrophage DH82 Cell Line As a Model to Study Susceptibility to Trypanosoma cruzi Infection

“…Naturally infected dogs or seropositive dogs were detected in the United States (6), Mexico, Argentina, Venezuela, and Panama (7, 8). In Brazil, the presence of infected animals was also reported in different areas of the country (5, 9). The canine Chagas’ disease is becoming a major veterinarian concern in the Americas (8).…”