Molecular identification of Trypanosoma cruzi in domestic animals in municipalities of the State of Rio Grande do Norte, Brazil.

Trypanosoma cruzi, the etiologic agent of American trypanosomiasis, is a vector-borne zoonotic parasite which has been little studied regarding its infection in domestic animals. In this study, we evaluated the occurrence of natural infection by T. cruzi in farm animals using molecular markers and phylogenetic analysis in blood clot samples of 60 sheep (Ovis aires), 22 goats (Capra hircus), and 14 horses (Equus caballus) in eight municipalities located in an infection risk area in the state of Rio Grande do Norte (RN), Northeast Region of Brazil. Trypanosoma spp. infection was identified by amplifying the rRNA 18S SSU gene in 48.9% of the samples. The SH022 sample showed 99.8% similarity with the Y strain of T. cruzi in phylogeny, grouped in the DTU II clade. Blood clots of sheep, goats, and horses detected T. cruzi kDNA in 28.3% (17/60), 22.7% (5/22), and 15.4% (2/14) of the samples, respectively. These animals were distributed in the three studied mesoregions throughout the state of RN. The identification of natural infection in domestic animals contributes to expand the epidemiological transmission scenario in an area where T. brasiliensis is the main vector.

Trypanosomatids in Small Mammals of an Agroecosystem in Central Brazil: Another Piece in the Puzzle of Parasite Transmission in an Anthropogenic Landscape.

We surveyed infection by Trypanosoma spp. and Leishmania spp. in small wild mammals from Cumari, Goiás State aiming to investigate the diversity of trypanosomatid in a modified landscape of the Brazilian Cerrado (and possible infection overlapping with canids from the same area). Blood, skin, spleen, and liver samples were collected for parasitological, serological, and molecular assays. Gracilinanus agilis was the most abundant species (N = 70; 48.6%) and it was the only one with patent parasitemia. Characterization by mini-exon and 18SrDNA targets were achieved in 7/10 hemocultures with positive fresh blood examination, which confirmed the T. cruzi infection by Discrete Typing Units (DTU) TcI in single (N = 2) and mixed infections with other DTUs (N = 5). T. rangeli and T. dionisii were detected in skin fragments from Didelphis albiventris and Oecomys cleberi, respectively. G. agilis were found to be infected by L. braziliensis and L. guyanensis, while Leishmania sp. DNA was detected in the liver of Oligoryzomys nigripes and Calomys expulsus. Subpatent infection by T. cruzi and Leishmania sp. was serologically detected in 15% and 9% of the small mammal fauna, respectively. Small mammals from Cumari are included in T. cruzi and Leshmania spp. transmission cycles, showing a higher diversity of trypanosomatid species and/or genotypes than that observed in canids of the same agroecosystem.

Multiplex PCR as a tool for the diagnosis of Leishmania spp. kDNA and the gapdh housekeeping gene of mammal hosts.

BACKGROUND: The PCR assays usually employed for Leishmania diagnosis does not simultaneously detect a constitutive gene that would certify the viability of the DNA sample. We present a multiplex PCR approach for the simultaneous diagnosis of the Leishmania sp. kDNA fragment and a catalytic domain segment of a conserved region of the mammalian gapdh gene. METHODOLOGY: The proposed multiplex protocol was designed through in silico PCR. The annealing temperature, concentration of primer pairs, number of cycles, distinct polymerase enzymes and premix kit were defined to achieve an optimal reaction. The DNA detection sensitivity was tested with different concentrations of known L. tropica DNA, and the reproducibility of the assay was confirmed using samples from 106 wild mammals that were previously subject to Leishmania sp. kDNA analysis through singleplex reactions. PRINCIPAL FINDINGS: The following optimal conditions were established: 95°C for 1 min followed by 30 cycles of 95°C for 30 s, 61°C for 30 s, and 72°C for 30 s and a final extension at 72°C for 1 min. The multiplex PCR system was capable of detecting 0.1 ng of L. tropica diluted in 100 ng of mammalian DNA. Of 51 kDNA samples that were previously found to be positive, 45 (88.2%) were positive for both targets, two were positive only for kDNA and four were negative for both. Of 55 kDNA samples that were previously identified as negative, 38 (69.1%) were positive for gapdh whereas the other 17 were negative for both targets. CONCLUSIONS/SIGNIFICANCE: The proposed multiplex PCR system allows the simultaneous detection of the gapdh gene and Leishmania sp. kDNA in tissue samples derived from distinct wild mammal species. The amplification of the gapdh mammalian gene in the same reaction ensures the quality and viability of the DNA in the sample, eliminating the possibility of false-negative results that would impair an accurate description of the infection rates in a given population.

Distinct Leishmania species infecting wild caviomorph rodents (Rodentia: Hystricognathi) from Brazil.

BACKGROUND: Caviomorph rodents, some of the oldest Leishmania spp. hosts, are widely dispersed in Brazil. Despite both experimental and field studies having suggested that these rodents are potential reservoirs of Leishmania parasites, not more than 88 specimens were analyzed in the few studies of natural infection. Our hypothesis was that caviomorph rodents are inserted in the transmission cycles of Leishmania in different regions, more so than is currently recognized. METHODOLOGY: We investigated the Leishmania infection in spleen fragments of 373 caviomorph rodents from 20 different species collected in five Brazilian biomes in a period of 13 years. PCR reactions targeting kDNA of Leishmania sp. were used to diagnose infection, while Leishmania species identification was performed by DNA sequencing of the amplified products obtained in the HSP70 (234) targeting. Serology by IFAT was performed on the available serum of these rodents. PRINCIPAL FINDINGS: In 13 caviomorph rodents, DNA sequencing analyses allowed the identification of 4 species of the subgenus L. (Viannia): L. shawi, L. guyanensis, L. naiffi, and L. braziliensis; and 1 species of the subgenus L. (Leishmania): L. infantum. These include the description of parasite species in areas not previously included in their known distribution: L. shawi in Thrichomys inermis from Northeastern Brazil and L. naiffi in T. fosteri from Western Brazil. From the four other positive rodents, two were positive for HSP70 (234) targeting but did not generate sequences that enabled the species identification, and another two were positive only in kDNA targeting. CONCLUSIONS/SIGNIFICANCE: The infection rate demonstrated by the serology (51.3%) points out that the natural Leishmania infection in caviomorph rodents is much higher than that observed in the molecular diagnosis (4.6%), highlighting that, in terms of the host species responsible for maintaining Leishmania species in the wild, our current knowledge represents only the "tip of the iceberg."