Trypanosoma cruzi infection via oral route results in outbreaks or cases of acute Chagas disease (ACD) in different Brazilian regions and poses a novel epidemiological scenario. In the Espírito Santo state (southeastern Brazil), a fatal case of a patient with ACD led us to investigate the enzootic scenario to avoid the development of new cases. At the studied locality, Triatoma vitticeps exhibited high T. cruzi infection rates and frequently invaded residences. Sylvatic and domestic mammals in the Rio da Prata locality, where the ACD case occurred, and in four surrounding areas (Baia Nova, Buenos Aires, Santa Rita and Todos os Santos) were examined and underwent parasitological and serological tests. Triatomines were collected for a fecal material exam, culturing and mini-exon gene molecular characterization, followed by RFLP-PCR of H3/Alul. Paraffin-embedded cardiac tissue of a patient was washed with xylene to remove paraffin and DNA was extracted using the phenol-chloroform method. For genotype characterization, PCR was performed to amplify the 1f8, GPI and 18S rRNA genes. In the case of V7V8 SSU rRNA, the PCR products were molecularly cloned. PCR products were sequenced and compared to sequences in GenBank. Phylogenetic analysis using maximum likelihood method with 1000 bootstrap replicates was performed. None of the animals showed positive hemocultures. Three rodents and two dogs showed signs of infection, as inferred from borderline serological titers. T. vitticeps was the only triatomine species identified and showed T. cruzi infection by DTUs TcI and TcIV. The analysis of cardiac tissue DNA showed mixed infection by T. cruzi (DTUs I, II, III and IV) and Trypanosoma dionisii. Each case or outbreak of ACD should be analyzed as a particular epidemiological occurrence. The results indicated that mixed infections in humans may play a role in pathogenicity and may be more common than is currently recognized. Direct molecular characterization from biological samples is essential because this procedure avoids parasite selection. T. dionisii may under certain and unknown circumstances infect humans. The distribution of T. cruzi DTUS TcIII and TcIV in Brazilian biomes is broader than has been assumed to date.

Parasites are important components of the immense n-dimensional trophic network that connects all living beings because they, among others, forge biodiversity and deeply influence ecological evolution and host behavior. In this sense, the influence of Trypanosomatidae remains unknown. The aim of this study was to determine trypanosomatid infection and richness in rats, opossums, and dogs in the semiarid Caatinga biome. We submitted DNA samples from trypanosomatids obtained through axenic cultures of the blood of these mammals to mini exon multiplex-PCR, Sanger, and next-generation sequencing targeting the 18S rDNA gene. Phylogenetic analyses were performed to identify genetic diversity in the Trypanosomatidae family. Shannon, Simpson, equability, and beta-diversity indices were calculated per location and per mammalian host. Dogs were surveyed for trypanosomatid infection through hemocultures and serological assays. The examined mammal species of this area of the Caatinga biome exhibited an enormous trypanosomatid species/genotypes richness. Ten denoised Operational Taxonomic Units (ZOTUs), including three species (Trypanosoma cruzi,Trypanosoma rangeliandCrithidia mellificae) and oneTrypanosomasp. five genotypes/lineages (T. cruziDTU TcI, TcII, and TcIV;T. rangeliA and B) and four DTU TcI haplotypes (ZOTU1, ZOTU2, ZOTU5, and ZOTU10 merged), as well as 13 Amplicon Sequence Variants (ASVs), including five species (T. cruzi,T. rangeli,C. mellificae,Trypanosoma dionisii, andTrypanosoma lainsoni), five genotypes/lineages (same as the ZOTUs) and six DTU TcI haplotypes (ASV, ASV1, ASV2, ASV3, ASV5 and ASV13), were identified in single and mixed infections. We observed that trypanosomatids present a broad host spectrum given that species related to a single host are found in other mammals from different taxa. Concomitant infections between trypanosomatids and new host-parasite relationships have been reported, and this immense diversity in mammals raised questions, such as how this can influence the course of the infection in these animals and its transmissibility. Dogs demonstrated a high infection rate byT. cruzias observed by positive serological results (92% in 2005 and 76% in 2007). The absence of positive parasitological tests confirmed their poor infectivity potential but their importance as sentinel hosts ofT. cruzitransmission.

Trypanosoma rangeli is a generalist hemoflagellate that infects mammals and is transmitted by triatomines around Latin America. Due to its high genetic diversity, it can be classified into two to five lineages. In Brazil, its distribution outside the Amazon region is virtually unknown, and knowledge on the ecology of its lineages and on host species diversity requires further investigation. Here, we analyzed 57 T. rangeli samples obtained from hemocultures and blood clots of 1392 mammals captured in different Brazilian biomes. The samples were subjected to small subunit (SSU) rDNA amplification and sequencing to confirm T. rangeli infection. Phylogenetic inferences and haplotype networks were reconstructed to classify T. rangeli lineages and to infer the genetic diversity of the samples. The results obtained in our study highlighted both the mammalian host range and distribution of T. rangeli in Brazil: infection was observed in five new species (Procyon cancrivorous, Priodontes maximum, Alouatta belzebul, Sapajus libidinosus, and Trinomys dimidiatus), and transmission was observed in the Caatinga biome. The coati (Nasua nasua) and capuchin monkey (S. libidinosus) are the key hosts of T. rangeli. We identified all four T. rangeli lineages previously reported in Brazil (A, B, D, and E) and possibly two new genotypes.

Kinetoplastids include species economically important in agriculture, livestock, and human health. We evaluated the richness of kinetoplastids that infect small mammals in patches of unflooded forests in the Pantanal biome, an area where we hypothesize that its diversity is higher than currently recognized. Hemocultures (HC) and Next Generation Sequencing (NGS) targeting the 18S rDNA gene were employed for the detection of kinetoplastids. We grouped the positive samples into pools for each small mammal species (Monodelphis domestica, Thylamys macrurus, Oecomys mamorae, Thrichomys fosteri, Clyomys laticeps, and Holochilus chacarius). Eight parasite species were identified: Leishmania amazonensis, L. infantum; Trypanosoma cascavelli (HC + NGS), T. cruzi, T. lainsoni, T. rangeli (HC + NGS), Trypanosoma sp. DID, and Neobodo sp. The use of a tool as sensitive as NGS has increased our awareness of the diversity of kinetoplastids, as well as their host range, with emphasis on the species O. mamorae (seven kinetoplastid species, excepting T. cascavelli in a pool of nine individuals) and T. macrurus (four kinetoplastid species in a single individual). Furthermore, L. infantum and L. amazonensis infections were described in small mammals from this region for the first time. These findings make it mandatory to revisit the kinetoplastids/host associations proposed so far.

Trypanosomatids are hemoflagellate parasites that even though they have been increasingly studied, many aspects of their biology and taxonomy remain unknown. The aim of this study was to investigate the Trypanosoma sp. transmission cycle in nonflying small mammals in an area where a case of acute Chagas disease occurred in Mangaratiba municipality, Rio de Janeiro state. Three expeditions were conducted in the area: the first in 2012, soon after the human case, and two others in 2015. Sylvatic mammals were captured and submitted to blood collection for trypanosomatid parasitological and serological exams. Dogs from the surrounding areas where the sylvatic mammals were captured were also tested for T. cruzi infection. DNA samples were extracted from blood clots and positive hemocultures, submitted to polymerase chain reaction targeting SSU rDNA and gGAPDH genes, sequenced and phylogenetic analysed. Twenty-one wild mammals were captured in 2012, mainly rodents, and 17 mammals, mainly marsupials, were captured in the two expeditions conducted in 2015. Only four rodents demonstrated borderline serological T. cruzi test (IFAT), two in 2012 and two in 2015. Trypanosoma janseni was the main Trypanosoma species identified, and isolates were obtained solely from Didelphis aurita. In addition to biological differences, molecular differences are suggestive of genetic diversity in this flagellate species. Trypanosoma sp. DID was identified in blood clots from D. aurita in single and mixed infections with T. janseni. Concerning dogs, 12 presented mostly borderline serological titers for T. cruzi and no positive hemoculture. In blood clots from 11 dogs, T. cruzi DNA was detected and characterized as TcI (n = 9) or TcII (n = 2). Infections by Trypanosoma rangeli lineage E (n = 2) and, for the first time, Trypanosoma caninum, Trypanosoma dionisii, and Crithidia mellificae (n = 1 each) were also detected in dogs. We concluded that despite the low mammalian species richness and degraded environment, a high Trypanosoma species richness species was being transmitted with the predominance of T. janseni and not T. cruzi, as would be expected in a locality of an acute case of Chagas disease.

Background: In rural areas of Espírito Santo state, southeast Brazil, triatomine species attracted by light frequently invade residences. The aim of this study was to investigate the Trypanosoma cruzi discrete typing units (DTUs) harbored by these triatomines. Methods: Triatomine’s intestinal contents were examined, inoculated in mice, and the positive samples were cultivated. Flagellates obtained from infected mice hemoculture were submitted to DNA extraction using a salting-out method and to TcSC5D gene amplification. The amplified samples were sequenced, and polymorphism was analyzed for DTU identification. Results: Three hundred and ninety-four triatomines were identified: Triatoma vitticeps (90.03%), Panstrongylus geniculatus (8.89%), Panstrongylus megistus (0.54%), Panstrongylus diasi (0.27%), and Triatoma tibiamaculata (0.27%). Among the specimens, 251/394 (67.65%) presented flagellated forms similar to T. cruzi. After triatomine intestinal content inoculation into mice, 134 mice presented T. cruzi-like trypomastigotes from Tr. vitticeps and P. geniculatus and 89 samples were positive in hemoculture. Sixty-two samples were analyzed for the TcSC5D gene and TcI, TcII, TcIII, and TcIV DTUs were identified. Conclusions: We observed T. cruzi DTU diversity in Tr. vitticeps and P. geniculatus, which showed the predominance of TcII and occurrence of TcI, TcIII and TcIV. Triatomines presented high T. cruzi infection rates. Since little is known regarding the possible mammalian hosts that maintain the T. cruzi cycle, further studies are necessary to obtain a better understanding of the parasite transmission cycle in this region.