Corrigendum to "Trypanosoma spp. Neobats: Insights about those poorly known trypanosomatids" [Int. J. Parasitol. Parasites Wildl. 16 (2021) 145-152].

[This corrects the article DOI: 10.1016/j.ijppaw.2021.09.003.].

Trypanosomatid diversity in a bat community of an urban area in Campo Grande, Mato Grosso do Sul, Brazil.

Bats have a long evolutionary history with trypanosomatids, but the role of these flying mammals on parasite transmission cycles in urban areas, especially for Trypanosoma and Leishmania species, remains poorly known. The objective of this study was to evaluate the species richness of trypanosomatids parasitizing a bat community in Campo Grande (CG), a state capital within the Cerrado of the Brazilian Midwest. We evaluated 237 bats of 13 species by means of hemoculture and molecular detection in spleen samples. The bat community of CG appears to participate in the transmission cycles of various species of trypanosomatids. We report an overall trypanosomatid detection rate of 34.2% (n = 81), involving 11 out of 13 sampled bat species. We identified six species of trypanosomatids from 61 bats by analyzing SSU rRNA and/or kDNA: Trypanosoma cruzi DTU TcI, T. c. marinkellei, T. dionisii, Leishmania infantum, L. amazonensis, and T. janseni, with this latter being detected by hemoculture for the first time in a bat species. We also detected a Molecular Operational Taxonomic Unit, Trypanosoma sp. DID, in the phyllostomids Glossophaga soricina and Platyrrhinus lineatus. The highest trypanosomatid richness was observed for Sturnira lilium, which hosted three species: L. infantum, T. dionisii and T. janseni. Given that visceral leishmaniasis is endemic in CG, special focus should be placed on L. infantum. Moreover, L. amazonensis and T. cruzi warrant attention, since these are zoonotic parasites responsible for human cases of tegumentary leishmaniasis and Chagas disease, respectively. In this respect, we discuss how bat communities may influence the Leishmania spp. transmission in endemic areas.

Space-environment relationship in the identification of potential areas of expansion of Trypanosoma cruzi infection in Didelphis aurita in the Atlantic Rainforest.

Ecological Niche Modeling is widely used for animals, but rarely for understanding the parasite ecology. Trypanosoma cruzi is a heterogeneous and widely dispersed multi-host parasite. Didelphis aurita is a generalist species, both in terms of diet and environments. We modeled the D. aurita niche and T. cruzi infection in the Brazilian Atlantic Rainforest, using the models of two common vector species (Triatoma vitticeps and Panstrongylus megistus) as biotic variables, predicting their occurrence. Records of T. cruzi infected and non-infected D. aurita were analyzed through climate and landscape approaches by the Ecoland method. Models for each triatomine species and infected and noninfected D. aurita were produced considering climate and landscape: resolution of ~1km2 selected by Pearson's correlation [-0.7≤α≤0.7]. For modeling, seven algorithms available in ModleR package were used. True Skill Statistic was used to evaluate the models' performance (≥ 0.7). T. vitticeps indicates that there is a spatial dependence with warm areas in the southeastern region while P. megistus presented a distribution with high environmental suitability concentrated in the Southeast. High values of climatic suitability, landscape and potential presence of T. vitticeps and P. megistus were considered necessary, but not sufficient for the presence of D. aurita infected by T. cruzi. Climate models showed an ecological niche with suitability variations homogeneous, and landscape models showed a distribution of habitat conditions along the biome, with a fragmented profile and heterogeneous between locations. Ecoland demonstrated that D. aurita has different degrees of impact on its role in the enzootic cycle in different locations of the Atlantic Rainforest. Associating the models with the Ecoland method allowed the recognition of areas where D. aurita are important T. cruzi reservoirs. Areas of high suitability for the presence of marsupials are a necessary, but not sufficient for D. aurita to act as a reservoir for T. cruzi.

Corrigendum: Leishmania infantum infecting the carnivore Nasua nasua from urban forest fragments in an endemic area of visceral leishmaniasis in Brazilian Midwest.

[This corrects the article DOI: 10.3389/fcimb.2022.1050339.].

Kinetoplastid Species Maintained by a Small Mammal Community in the Pantanal Biome.

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.

Trypanosomatid Richness Among Rats, Opossums, and Dogs in the Caatinga Biome, Northeast Brazil, a Former Endemic Area of Chagas Disease.

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 rangeli and Crithidia mellificae) and one Trypanosoma sp. five genotypes/lineages (T. cruzi DTU TcI, TcII, and TcIV; T. rangeli A 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, and Trypanosoma 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 by T. cruzi as 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 of T. cruzi transmission.

Trypanosoma Species in Small Nonflying Mammals in an Area With a Single Previous Chagas Disease Case.

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.

Leishmania infantum infecting the carnivore Nasua nasua from urban forest fragments in an endemic area of visceral leishmaniasis in Brazilian Midwest.

Introduction: The aim of the present study was to investigate the occurrence of Leishmania infantum in South American coatis inhabiting two forest fragments in Campo Grande, Mato Grosso do Sul, Midwest region of Brazil, an endemic area of human and canine visceral leishmaniasis (VL). Material and methods: A total of 110 South American coatis were sampled in the conservation unit "Parque Estadual do Prosa" (PEP) and in the residential area "Vila da Base Aérea" (VBA) from March 2018 to April 2019. As a longitudinal study that include up to six recaptures of the same individual, a total of 190 capture events were obtained. Blood, bone marrow and skin samples were obtained for parasitological (axenic culture), serological (Enzyme Linked Immunosorbent Assay - ELISA and Dual-path Platform immunoassay - DPP® CVL) and molecular diagnostic assays (targeting kDNA for Leishmania spp. and L. infantum; and HSP70 followed by sequence analysis). Results: Seropositivity for L. infantum was found in 33 individuals, six in PEP and 27 in VBA. Furthermore, L. infantum was detected by molecular analysis in 16 individuals, seven from PEP and nine from VBA. We also isolated L. infantum from bone marrow of one individual and detected a single positive skin sample in molecular assay from other individual, both from VBA. Discussion: An overall infection rate of 36.4% (40/110) was observed, significantly higher in the VBA (49.1%) than in the PEP (21.6%), probably because VBA presents: (i) a large number of resident dogs and chickens that would be attracting sandflies; (ii) a denser population of this wild mammal species; and (iii) physical barriers and a lack of functional connectivity in the surroundings, preventing these animals to disperse out. We conclude that South American coati populations living in urban forest fragments of Campo Grande are affected by the epidemiological scenario of VL, known to involve dogs, vectors and humans. We highlight the importance of investigate the parasitism by L. infantum in this and other potential L. infantum reservoirs that inhabit urbanized regions endemic to VL.

Trypanosomatid Richness in Wild and Synanthropic Small Mammals from a Biological Station in Rio de Janeiro, Brazil.

Trypanosomatids are diverse and can infect several host species, including small mammals (rodents and marsupials). Between 2012 and 2014, 91 small mammals were surveyed for trypanosomatid infection in the Estação Biológica FIOCRUZ Mata Atlântica (EFMA), an Atlantic Forest area in Rio de Janeiro that presents different levels of conserved and degraded areas. Blood, skin, liver, and spleen samples were submitted to parasitological, serological, and molecular assays to detect the infection and determine the taxonomic status of their parasites. Sixty-eight individuals (74.7%; n = 91) were infected by trypanosomatids, including fourteen mixed infected by different trypanosomatid parasites. These hosts were infected by: T. cruzi DTU TcI (n = 12), T. cruzi DTU TcIV (n = 2), T. janseni (n = 15), T. dionisii (n = 1), and T. rangeli A (n = 1) detected in blood or tissue cultures, in addition to T. cruzi DTU TcI (n = 9) and Leishmania sp. (n = 1) only by the molecular diagnosis. Serological diagnosis was positive in 38 (71.6%) individuals for T. cruzi, the same amount for Leishmania spp., and 23 (43.3%) individuals were mixed infected. These data indicate a remarkable richness of trypanosomatid species/genotypes infecting small mammals, even in a disturbed area with low mammal species diversity-as is the case of the EFMA-reinforcing the generalist aspect of these parasites.

Trypanosoma spp. Neobats: Insights about those poorly known trypanosomatids.

Bats are infected with several trypanosomatid species; however, assessing the diversity of this interaction remains challenging since there are species apparently unable to grow in conventional culture media. Accordingly, the ecology and biology of the Molecular Operational Taxonomic Units (MOTUs) Trypanosoma spp. Neobats are unknown. Therefore, we performed the molecular characterization targeting the 18S small subunit rDNA from the blood clot of 280 bats of three Brazilian regions (Paraíba, Rio de Janeiro and Acre states), bypassing the selective pressure of hemoculture. From 68 (24%) positive blood clot samples, we obtained 49 satisfactory sequences. Of these successfully sequenced results, T. spp. Neobats (1, 3 and 4) represented 67%, with the most abundant T. sp. Neobat 4 (53%). Our results show: (1) high abundance and wide geographic range of T. sp. Neobat 4, restricted to Carollia bats; (2) high infection rate of T. sp. Neobat 4 in Carollia perspicillata populations (mean 26%); (3) infection with the monoxenous Crithidia mellificae; and (4) a new MOTU (T. sp. Neobat 5) in Artibeus cinereus, positioning in the Trypanosoma wauwau clade. These data corroborate the importance of bats as hosts of many Trypanosoma species and C. mellificae. They also show that the diversity of the T. wauwau clade is underestimated and warn about the high magnitude of trypanosomes we overpass with the hemoculture. Our findings combined with previous data show that T. spp. Neobats include host-specific and host-generalist species, probably playing different ecological roles: T. sp. Neobat 1 shows broad host range; T. spp. Neobat 3 and 4 are restricted to Artibeus and Carollia, respectively. Finally, T. Neobat 4 seems to be a well-succeeded parasite, especially within C. perspicillata metapopulations across a wide geographical distribution. This work is a step forward to understand the biology and life history of T. spp. Neobats.

Trypanosoma rangeli Genetic, Mammalian Hosts, and Geographical Diversity from Five Brazilian Biomes.

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.

Biological and Genetic Heterogeneity in Trypanosoma dionisii Isolates from Hematophagous and Insectivorous Bats.

This study describes the morphological, biochemical, and molecular differences among Trypanosoma dionisii isolates from hemocultures of hematophagous (Desmodus rotundus; n = 2) and insectivorous (Lonchorhina aurita; n = 1) bats from the Atlantic Rainforest of Rio de Janeiro, Brazil. Fusiform epimastigotes from the hematophagous isolates were elongated, whereas those of the insectivorous isolate were stumpy, reflected in statistically evident differences in the cell body and flagellum lengths. In the hemocultures, a higher percentage of trypomastigote forms (60%) was observed in the hematophagous bat isolates than that in the isolate from the insectivorous bat (4%), which demonstrated globular morphology. Three molecular DNA regions were analyzed: V7V8 (18S rDNA), glycosomal glyceraldehyde 3-phosphate dehydrogenase gene, and mitochondrial cytochrome b gene. The samples were also subjected to multilocus enzyme electrophoresis and random amplified polymorphic DNA analysis. All isolates were identified as T. dionisii by phylogenetic analysis. These sequences were clustered into two separate subgroups with high bootstrap values according to the feeding habits of the bats from which the parasites were isolated. However, other T. dionisii samples from bats with different feeding habits were found in the same branch. These results support the separation of the three isolates into two subgroups, demonstrating that different subpopulations of T. dionisii circulate among bats.

Serological evidence of arboviruses and coccidia infecting horses in the Amazonian region of Brazil.

BACKGROUND: Arboviruses and protozoans can cause neurologic disorders in horses. In Brazilian Amazon, several horses presenting signs compatible with disorders caused by these infectious agents have been observed. OBJECTIVE: To contribute to the knowledge of this epidemiological picture, we sought to construct a serological diagnostic panel for neurotrophic infectious agents in local horses. MATERIAL AND METHODS: A total of 213 blood samples from horses were collected from 29 farms in three municipalities. Samples were evaluated and considered positive when they met the following criteria: titers ≥ 1:80 with the indirect fluorescent antibody test (IFAT) for apicomplexan protozoans; positive recombinant enzyme-linked immunosorbent assay (ELISA) with subsequent titers ≥ 1:10 by the PRNt for viruses; and detection under direct microscopic examination for Trypanosoma evansi. RESULTS: No horses were found to be infected by T. evansi, and only two were infected Toxoplasma gondii and/or Neospora spp. The highest protozoan infection rate was observed for Sarcocystis neurona (40.3%; n = 86/213). Among the positive ELISA samples tested by the plaque reduction neutralization test (PRNT90), 92% (n = 76/83) were positive for St Louis Encephalitis virus, 43% (n = 6/14) were positive for West Nile virus and 33% (n = 16/48) were positive for Mayaro virus. Eighteen percent (n = 39/213) of horses were co-infected by S. neurona and at least one arbovirus, particularly SLEV and/or MAYV. CONCLUSION: Samples positive for SLEV associated with S. neurona, including samples from horses that had recovered from neurological signs were frequent, and must be considered when investigating the possible causes of neurological diseases in South Roraima horses.

Isolation and characterization of trypanosomatids, including Crithidia mellificae, in bats from the Atlantic Forest of Rio de Janeiro, Brazil.

We studied infection by Trypanosomatidae in bats captured in two areas with different degradation levels in the Atlantic Forest of Rio de Janeiro state: Reserva Ecológica de Guapiaçu (REGUA) and Estação Fiocruz Mata Atlântica (EFMA). Furthermore, we evaluated whether the diversity of trypanosomatids changes according to bat diversity and the different levels of preservation in the region. The results showed no influence of the level of preservation on bat species richness (15 and 14 species, respectively), with similar chiropterofauna and higher abundance of two common fruit-eating bat species in the tropics: Carollia perspicillata and Artibeus lituratus. Of the 181 bat specimens analyzed by LIT/Schneider hemoculture, we detected 24 infected individuals (13%), including one positive Sturnira lilium individual that was also positive by fresh blood examination. Molecular characterization using nested PCR targeting the 18 SSU rRNA-encoding gene fragment showed similar trypanosomatid infection rates in bats from the two areas: 15% in REGUA and 11% in EFMA (p = 0.46). Trypanosoma dionisii was the most frequently detected parasite (54%), followed by T. cruzi DTUs TcI and TcIV and Trypanosoma sp., in Neotropical phyllostomid bats (RNMO63 and RNMO56); mixed infections by T. dionisii/T. cruzi TcIII and T. dionisii/T. cruzi TcI were also observed. The T. cruzi DTUs TcI and TcIV are the genotypes currently involved in cases of acute Chagas disease in Brazil, and T. dionisii was recently found in the heart tissue of an infected child. Surprisingly, we also describe for the first time Crithidia mellificae, a putative monoxenous parasite from insects, infecting a vertebrate host in the Americas. Bats from the Atlantic Forest of Rio de Janeiro state harbor a great diversity of trypanosomatids, maintaining trypanosomatid diversity in this sylvatic environment.

Uncovering Trypanosoma spp. diversity of wild mammals by the use of DNA from blood clots.

Trypanosoma spp. infection in wild mammals is detected mainly through parasitological tests that usually display low sensitivity. We propose the use of DNA extracted directly from blood clots (BC), which are neglected sources of DNA for diagnosis and identification of Trypanosoma spp. This approach followed by nested PCR targeting the 18S SSU rDNA demonstrated to be sensitive and suitable to evaluate the diversity of trypanosomes infecting sylvatic mammals, including subpatent and mixed infections. Infection was detected in 95/120 (79.2%) samples from bats, carnivores and marsupials that included negative serological and hemoculture testing mammals. Thirteen Trypanosoma spp. or Molecular Operational Taxonomic Units (MOTUs) were identified, including two new MOTUs. The high diversity of trypanosomes species and MOTUs infecting bats and marsupials showed that these hosts can be considered as bio-accumulators of Trypanosoma spp., with specimens of Didelphis spp. displaying the highest trypanosome diversity. The use of blood clots allowed direct access to non-culturable parasites, mixed infections, besides bypassing the selective pressure on the parasites inherent to cultivation procedures. Trypanosoma cruzi was the species found infecting the highest number of individuals, followed by T. lainsoni. Positive PCR for T. cruzi was observed in 16 seronegative individuals and 30 individuals with negative hemocultures. Also, T. lainsoni, previously found only in rodents, showed to be capable of infecting bats and marsupials. This finding makes it clear that some species of Trypanosoma are more generalist than previously thought. Molecular diagnosis using nested PCR from DNA extracted from BC allowed the increase of the knowledge about host-spectrum and distribution of Trypanosoma spp. and allowed the identification of new MOTUs.

Trypanosoma cruzi transmission in the wild and its most important reservoir hosts in Brazil.

Trypanosoma cruzi (Kinetoplastea: Trypanosomatidae) infects all tissues of its hosts, which along with humans, include hundreds of mammalian species in the Americas. The epidemiology of T. cruzi has been changing in that currently the majority of the cases and/or outbreaks of Chagas disease occur by the ingestion of comestibles contaminated by T. cruzi metacyclic forms. These cases/outbreaks occur in distinct regional scenarios, mainly in the Amazon biome and are related to the local interaction mode of humans with their surroundings, as well as with the overall local ecological peculiarities. As trypanosomiasis caused by T. cruzi is primarily a zoonosis, understanding the variables that influences its transmission in the wild as well as the role played by the extant fauna in the maintenance of the parasite, is critical in establishing control measures. Here, we present the results of our studies of T. cruzi infection of free ranging wild mammalian fauna in the five biomes of Brazil, a country of continental dimensions. From 1992 up to 2017, we examined a total of 6587 free-ranging non-volant wild mammal specimens. Our studies found that 17% of mammals were seropositive and 8% of all animals displayed positive hemocultures indicative of high parasitemia and, consequently, of infectivity potential. We observed that opossums, mainly Philander spp. and Didelphis spp., the coati Nasua nasua, the capuchin monkey Sapajus libidinosus and the golden lion tamarin Leontopithecus rosalia, were mammal taxa that demonstrated higher rates of positive hemocultures. Additionally, Didelphis spp. demonstrated to be a competent bioaccumulator of TcI diversity. Chiroptera were distinguished for hosting the greatest diversity of species and genotypes of Trypanosoma spp. Additionally the observation of the higher host range of some Trypanosoma spp., shows the need to reassess the ecology of representatives of the taxon. Altogether, our results showed that each locality, may display distinct enzootiological and epidemiological scenarios that must be taken into account when it comes to establishing control and/or clarification campaigns of the local population.

High Trypanosoma spp. diversity is maintained by bats and triatomines in Espírito Santo state, Brazil.

The aim of this study was to reevaluate the ecology of an area in the Atlantic Forest, southeast Brazil, where Chagas disease (CD) has been found to occur. In a previous study, immediately after the occurrence of a CD case, we did not observe any sylvatic small mammals or dogs with Trypanosoma cruzi cruzi infections, but Triatoma vitticeps presented high T. c. cruzi infection rates. In this study, we investigated bats together with non-volant mammals, dogs, and triatomines to explore other possible T. c. cruzi reservoirs/hosts in the area. Seventy-three non-volant mammals and 186 bats were captured at three sites within the Guarapari municipality, Espírito Santo state. Rio da Prata and Amarelos sites exhibited greater richness in terms of non-volant mammals and bats species, respectively. The marsupial Metachirus nudicaudatus, the rodent Trinomys paratus, and the bats Artibeus lituratus and Carollia perspicillata were the most frequently captured species. As determined by positive hemocultures, only two non-volant mammals were found to be infected by Trypanosoma species: Monodelphis americana, which was infected by T. cascavelli, T. dionisii and Trypanosoma sp., and Callithrix geoffroyi, which was infected by T. minasense. Bats presented T. c. cruzi TcI and TcIII/V, T. c. marinkellei, T. dionisii, T. rangeli B and D, and Trypanosoma sp. infections. Seven dogs were infected with T. cruzi based only on serological exams. The triatomines T. vitticeps and Panstrongylus geniculatus were found to be infected by trypanosomes via microscopy. According to molecular characterization, T. vitticeps specimens were infected with T. c. cruzi TcI, TcII, TcIII/V, and TcIV, T. c. marinkellei and T. dionisii. We observed high trypanosome diversity in a small and fragmented region of the Atlantic Forest. This diversity was primarily maintained by bats and T. vitticeps. Our findings show that the host specificity of the Trypanosoma genus should be thoroughly reviewed. In addition, our data show that CD cases can occur without an enzootic cycle near residential areas.

Assessment of a quantitative 5' nuclease real-time polymerase chain reaction using groEL gene for Ehrlichia and Anaplasma species in rodents in Brazil.

New genotypes of Anaplasmataceae agents have been detected in wild carnivores, birds and deer in Brazil. The present work aimed to investigate the presence of Ehrlichia and Anaplasma species in rodents sampled in Brazil. Additionally, a newly designed quantitative 5' nuclease real-time multiplex PCR for Ehrlichia and Anaplasma spp. detection based on groEL gene amplification was designed, showing high specificity and sensitivity (10 groEL fragment copy/µL). Between 2000 and 2011, different rodent species [n=60] were trapped in 5 Brazilian biomes. Among 458 rodent spleen samples, 0.4% (2/458) and 2.4% (11/458) were positive for Ehrlichia and Anaplasma spp., respectively. Of 458 samples, 2.0% (9/458) and 1.1% (5/458) were positive for Anaplasma sp. and Ehrlichia sp., respectively, using conventional 16S rRNA PCR assays. Maximum Likelihood phylogenetic analyse based on a small region of 16S rRNA genes positioned the Anaplasma genotypes in rodents near Anaplasma phagocytophilum or Anaplasma marginale and Anaplasma odocoilei isolates. Ehrlichia genotypes were closely related to E. canis. There was a low occurrence of Anaplasma and Ehrlichia in wild and synanthropic rodents in Brazil, suggesting the circulation of new genotypes of these agents in rodents in the studied areas.

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.