Molecular detection of piroplasmids in mammals from the Superorder Xenarthra in Brazil.

Xenarthra mammals can be found from southern North America to southern South America, including all Brazilian biomes. Although it has been shown that Xenarthra mammals can play a role as reservoirs for several zoonotic agents, few studies investigate the diversity of piroplasmids (Apicomplexa: Piroplasmida) in this group of mammals. Taking into account that piroplasmids can cause disease in animals and humans, understanding the prevalence and diversity of piroplasmids in Xenarthra mammals would contribute to conservation efforts for this group of animals as well as to infer risk areas for transmission of emergent zoonosis. The present study aimed to investigate the occurrence and molecular identity of piroplasmids in free-living mammals of the Superorder Xenarthra from four Brazilian states (Mato Grosso do Sul, São Paulo, Rondônia, and Pará). For this, DNA was extracted from blood or spleen samples from 455 animals. A nested PCR based on the 18S rRNA gene was used as screening for piroplasmids. Of the 455 samples analyzed, 25 (5.5%) were positive. Additionally, PCR assays based on 18S rRNA near-complete, cox-1, cox-3, hsp70, cytB, ß-tubulin genes and the ITS-1 intergenic region were performed. Five out of 25 positive samples also tested positive for ITS-1-based PCR. The phylogenetic analysis positioned three 18S rRNA sequences detected in Priodontes maximus into the same clade of Babesia sp. detected in marsupials (Didelphis albiventris, Didelphis marsupialis, and Monodelphis domestica) and Amblyomma dubitatum collected from opossums and coatis in Brazil. On the other hand, the 18S rRNA sequence obtained from Dasypus novemcinctus was closely related to a Theileria sp. sequence previously detected in armadillos from Mato Grosso State, grouping in a subclade within the Theileria sensu stricto clade. In the phylogenetic analysis based on the ITS-1 region, the sequences obtained from Myrmecophaga tridactyla and Tamandua tetradactyla were placed into a single clade, apart from the other piroplasmid clades. The present study demonstrated the molecular occurrence of Piroplasmida in anteaters and Babesia sp. and Theileria sp. in armadillos from Brazil.

Genetic diversity of Trypanosoma cruzi strains isolated from chronic chagasic patients and non-human hosts in the state of São Paulo, Brazil.

BACKGROUND: Trypanosoma cruzi shows an exuberant genetic diversity. Currently, seven phylogenetic lineages, called discrete typing units (DTUs), are recognised: TcI-TcVI and Tcbat. Despite advances in studies on T. cruzi and its populations, there is no consensus regarding its heterogeneity. OBJECTIVES: This study aimed to perform molecular characterisation of T. cruzi strains, isolated in the state of São Paulo, to identify the DTUs involved and evaluate their genetic diversity. METHODS: T. cruzi strains were isolated from biological samples of chronic chagasic patients, marsupials and triatomines through culture techniques and subjected to molecular characterisation using the fluorescent fragment length barcoding (FFLB) technique. Subsequently, the results were correlated with complementary information to enable better discrimination between the identified DTUs. FINDINGS: It was possible to identify TcI in two humans and two triatomines; TcII/VI in 19 humans, two marsupials and one triatomine; and TcIII in one human host, an individual that also presented a result for TcI, which indicated the possibility of a mixed infection. Regarding the strains characterised by the TcII/VI profile, the correlation with complementary information allowed to suggest that, in general, these parasite populations indeed correspond to the TcII genotype. MAIN CONCLUSIONS: The TcII/VI profile, associated with domestic cycles and patients with chronic Chagas disease, was the most prevalent among the identified DTUs. Furthermore, the correlation of the study results with complementary information made it possible to suggest that TcII is the predominant lineage of this work.

Acute Chagas Disease Caused by Trypanosoma cruzi TcIV and Transmitted by Panstrongylus geniculatus: Molecular Epidemiological Insights Provided by the First Documented Autochthonous Case in Rondônia, Southwestern Amazonia, Brazil.

Recurrent outbreaks of oral infection and isolated cases characterize the new epidemiological scenario of Chagas disease (CD) in the Brazilian Amazon. Acute Chagas disease (ACD) is common in Pará and Amazonas, Northeastern and Northwestern Brazilian Amazonia. In the present study, we describe the first molecularly characterized autochthonous case of ACD in Rondônia, Southwestern Amazonia. The patient, a 39-year-old male resident in the small city of Cujubim, presented typical ACD symptoms: fever, asthenia, myalgia, progressive dyspnea, swelling of the legs, and tiredness at minimal efforts, all compatible with ACD and indicative of cardiac involvement. A thick blood drop test revealed trypomastigote forms of Trypanosoma cruzi genotyped as TcIV. An epidemiological investigation ruled out oral infection, and support for vectorial transmission included the finding of Panstrongylus geniculatus positive for T. cruzi (TcIII and TcIV) inside the tent used by the patient when harvesting forest timber, and a circular cutaneous lesion resembling a chagoma of inoculation. Treatment with benznidazole led to blood parasite clearance as confirmed by molecular tests. Altogether, our findings fitted well into the ecological scenario where deforestation and colonization of forested areas represent an important risk factor to the adaptation of P. geniculatus to human habitats, favoring vectorial transmission of CD in the Amazonian region.

Molecular investigation of haemotropic mycoplasmas and Coxiella burnetii in free-living Xenarthra mammals from Brazil, with evidence of new haemoplasma species.

Although mammals of the superorder Xenarthra are considered hosts of a wide range of zoonotic agents, works aiming at investigating the role of these animals as hosts for bacteria with zoonotic potential are rare. The present study aimed to investigate the occurrence and molecularly characterize Coxiella burnetii and haemoplasma (haemotropic mycoplasmas) DNA in blood and spleen samples from 397 free-living Xenarthra mammals (233 sloths, 107 anteaters and 57 armadillos) in five Brazilian states (Mato Grosso do Sul, São Paulo, Pará, Rondônia and Rio Grande do Sul). All biological samples from Xenarthra were negative in the qPCR for Coxiella burnetii based on the IS1111 gene. The absence of C. burnetii DNA in blood and spleen samples from Xenarthra suggests that these mammals may not act as possible hosts for this agent in the locations studied. When performed conventional PCR assays for the endogenous (gapdh) mammalian gene, 386 samples were positive. When screened by molecular assays based on the 16S rRNA gene of haemoplasmas, 81 samples were positive, of which 15.54% (60/386) were positive by conventional PCR and 5.44% (21/386) were positive by real-time PCR; three samples were positive in both assays. Of these, 39.74% (31/78) were also positive for the 23S rRNA gene and 7.69% (6/78) for the haemoplasma RNAse P gene. Among the samples positive for haemoplasmas, 25.64% (20/78) were obtained from anteaters (Tamandua tetradactyla and Myrmecophaga tridactyla), 39.74% (31/78) from sloths (Bradypus tridactylus, Bradypus sp. and Choloepus sp.) 34.61% (27/78) from armadillos (Priodontes maximus, Euphractus sexcinctus and Dasypus novemcinctus). A haemoplasma 16S rRNA sequence closely related and showing high identity (99.7%) to Mycoplasma wenyonii was detected, for the first time, in B. tridactylus. Based on the low identity and phylogenetic positioning of 16S rRNA and 23S rRNA sequences of haemoplasmas detected in anteaters and armadillos, the present study showed, for the first time, the occurrence of putative new Candidatus haemotropic Mycoplasma spp. ("Candidatus Mycoplasma haematotetradactyla" and "Candidatus Mycoplasma haematomaximus") in Xenarthra mammals from Brazil.

Leishmania diversity in bats from an endemic area for visceral and cutaneous leishmaniasis in Southeastern Brazil.

This study aimed to determine the occurrence of Leishmania infection in bats in urban and wild areas in an endemic municipality for visceral and cutaneous leishmaniasis in Minas Gerais, Brazil. Between April 2014 to April 2015, 247 bats were captured and classified into 26 species belonging to Phyllostomidae (90.7%), Vespertilionidae (8.1%) and Molossidae (1.2%) families. Blood samples from 247 bats were collected and submitted to nested-PCR, targeting the variable V7-V8 region of the SSU rRNA gene, followed by sequencing of the PCR product. The overall infection rate of Leishmania spp. in bats was 4.4%. Of the eleven bats infected, ten were frugivorous bats: Artibeus planirostris (8/11), Artibeus lituratus (1/11) and Artibeus cinereus (1/11) and one a nectarivorous bat (Glossophaga soricina). None of the individuals exhibited macroscopic alterations in the skin, spleen or liver. Phylogenetic analysis separated Leishmania species in clades corresponding to the subgenera Viannia, Leishmania, and Mundinia, and supported that the isolates characterized in the present study clustered closely with Leishmania (Viannia) sp., Leishmania (Leishmania) infantum and Leishmania (Leishmania) amazonensis. Here we report for the first time the bat Artibeus cinereus as a host of Leishmania (Leishmania) amazonensis. In the study we found that the mean abundance of bats did not differ in wild habitats and urban areas and that bat-parasite interactions were similarly distributed in the two environments. On the other hand, further studies should be conducted in more recent times to verify whether there have been changes in these parameters.

Genetic diversity of Trypanosoma cruzi strains isolated from chronic chagasic patients and non-human hosts in the state of São Paulo, Brazil

BACKGROUND Trypanosoma cruzi shows an exuberant genetic diversity. Currently, seven phylogenetic lineages, called discrete typing units (DTUs), are recognised: TcI-TcVI and Tcbat. Despite advances in studies on T. cruzi and its populations, there is no consensus regarding its heterogeneity. OBJECTIVES This study aimed to perform molecular characterisation of T. cruzi strains, isolated in the state of São Paulo, to identify the DTUs involved and evaluate their genetic diversity. METHODS T. cruzi strains were isolated from biological samples of chronic chagasic patients, marsupials and triatomines through culture techniques and subjected to molecular characterisation using the fluorescent fragment length barcoding (FFLB) technique. Subsequently, the results were correlated with complementary information to enable better discrimination between the identified DTUs. FINDINGS It was possible to identify TcI in two humans and two triatomines; TcII/VI in 19 humans, two marsupials and one triatomine; and TcIII in one human host, an individual that also presented a result for TcI, which indicated the possibility of a mixed infection. Regarding the strains characterised by the TcII/VI profile, the correlation with complementary information allowed to suggest that, in general, these parasite populations indeed correspond to the TcII genotype. MAIN CONCLUSIONS The TcII/VI profile, associated with domestic cycles and patients with chronic Chagas disease, was the most prevalent among the identified DTUs. Furthermore, the correlation of the study results with complementary information made it possible to suggest that TcII is the predominant lineage of this work.

Detection of Trypanosoma vivax in tissues of experimentally infected goats: what is the role of adipose tissue in the life cycle of this protozoon?

Trypanosomiasis, caused by Trypanosoma vivax, is responsible for great economic losses among livestock in Africa and South America. During the life cycle of these parasites, they may present different morphological, metabolic and physiological characteristics depending on the interactions that are encountered at each point of their life cycle. Although T. vivax is frequently reported in the circulation of its mammalian hosts, it has the ability to migrate to the tissues of these individuals. However, this characteristic is poorly understood. In this context, we aimed to investigate the presence of T. vivax and the changes caused in different tissues of experimentally infected goats. Despite the animals were not perfused before tissues collection, using different approaches, we demonstrated its presence in different samples, including in the adipose tissue and skin of infected animals. In addition, a mononuclear inflammatory reaction, mostly characterized by an infiltrate of lymphocytes, plasma cells and macrophages were observed. The results highlight the possibility that, like other trypanosomatids, T. vivax may use these tissues during its life cycle. Future studies aiming to elucidate the length of time for which T. vivax remains active in these sites, and whether it uses these sites as a refuge from trypanocidal drugs, and whether it is capable of recolonizing the blood circulation, are much needed.

Remarkable diversity, new records and Leishmania detection in the sand fly fauna of an area of high endemicity for cutaneous leishmaniasis in Acre state, Brazilian Amazonian Forest.

The species richness of Amazonian phlebotomines is considered to be one of the highest in the world. In the present study, we investigated the richness and diversity of phlebotomine fauna in Xapuri city, Acre state, Western Brazilian Amazonia, which is an area that is highly endemic for cutaneous leishmaniasis. Sand fly collections were performed monthly from August 2013 to July 2015 (288 h total of sampling effort) in intradomiciliary, peridomiciliary, and forested environments of two localities. Collected females were dissected, microscopically examined for flagellates in their guts, and preserved in ethanol. A total of 21,197 specimens comprising 14 genera and 57 species were collected, and the majority of these were Nyssomyia, Psychodopygus, and Trichophoromyia genera. Three new records of phlebotomine species for Acre are presented here, including Brumptomyia brumpti, Psathyromyia pradobarrientosi, and for the first time in Brazil, Th. omagua. In Xapuri, the phlebotomine fauna of different ecotopes was varied in regard to abundance, diversity, and frequency, and they included proven and permissive vectors of Leishmania spp. The fauna discovered in the forested areas (57 species) was richer and more diverse than was that (33 species) identified in the peri­ and intra-domiciles. The identification of Leishmania subgenera that were present in sand fly guts according to SSU rRNA sequences revealed ten and three species harboring Leishmania of subgenera Viannia and Leishmania (most likely Leishmania amazonensis), respectively. The presence of Leishmania (Leishmania) in sand flies are reported here for the first time in Acre. The presence of L. (Viannia) spp. in Brumptomyia sp. and Lutzomyia sherlocki. and the occurrence of mixed infections with Leishmania of both subgenera in Ps. lainsoni have been reported for the first time in Brazil. Taken together, data from previous studies and from the present study highlight the remarkable complexity of phlebotomine fauna that is possibly due to the well-preserved Xapuri forested areas sustaining vital economic activities of plant extraction and ecological tourism. Our findings also provide new insights into the ongoing adaptation of Trichophoromyia and Psychodopygus species to human habitats.

Genotypic Trypanosoma cruzi distribution and parasite load differ ecotypically and according to parasite genotypes in Triatoma brasiliensis from endemic and outbreak areas in Northeastern Brazil.

This study aimed to identify the Trypanosoma cruzi genotypes and their relationship with parasitic load in distinct geographic and ecotypic populations of Triatoma brasiliensis in two sites, including one where a Chagas disease (ChD) outbreak occurred in Rio Grande do Norte state, Brazil. Triatomine captures were performed in peridomestic and sylvatic ecotopes in two municipalities: Marcelino Vieira - affected by the outbreak; and Currais Novos - where high pressure of peridomestic triatomine infestation after insecticide spraying have been reported. The kDNA-PCR was used to select 124 T. cruzi positive triatomine samples, of which 117 were successfully genotyped by fluorescent fragment length barcoding (FFLB). Moreover, the T. cruzi load quantification was performed using a multiplex TaqMan qPCR. Our findings showed a clear ecotypic segregation between TcI and TcII harboured by T. brasiliensis (p<0.001). Although no genotypes were ecotypically exclusive, TcI was predominant in peridomestic ecotopes (86%). In general, T. brasiliensis from Rio Grande do Norte had a higher T. cruzi load varying from 3.94 to 7.66 x 106T. cruzi per insect. Additionally, TcII (median value=299,504 T. cruzi/intestine unit equivalents) had more than twice (p=0.1) the parasite load of TcI (median value=149,077 T. cruzi/intestine unit equivalents), which can be attributed to a more ancient co-evolution with T. brasiliensis. The higher prevalence of TcII in the sylvatic T. brasiliensis (70%) could be associated with a more diversified source of bloodmeals for wild insect populations. Either TcI or TcII may have been responsible for the ChD outbreak that occurred in the city of Marcelino Vieira. On the other hand, a smaller portion of T. brasiliensis was infected by TcIII (3%) in the peridomicile, in addition to T. rangeli genotype A (1%), often found in mixed infections. Our results highlight the need of understanding the patterns of T. cruzi genotype´s development and circulation in insect vectors and reservoirs as a mode of tracking situations of epidemiologic importance, as the ChD outbreak recently recorded for Northeastern Brazil.

Follow-up of dairy cattle naturally infected by Trypanosoma vivax after treatment with isometamidium chloride.

Trypanosoma vivax infections cause nonspecific clinical signs in cattle associated with aparasitemic intervals, making disease diagnosis a challenge. In Brazil, diminazene aceturate and isometamidium chloride (ISM) are available to treat bovine trypanosomosis. The objective of this study was to follow-up, by molecular and serological techniques, dairy cattle naturally infected by T. vivax after ISM treatment. Thirty cattle naturally infected with T. vivax received two applications of ISM, at a dosage of 1.0 mg/kg intramuscularly, on days 0 and 150. For T. vivax diagnosis, EDTA-blood and serum samples were evaluated on 0, 7, 15, 30, 60, 90, 120, 150, 180, 210, and 240 days after treatment PCR, Loop-mediated isothermal amplification (LAMP) and ELISA. Animals with persistent detection of T. vivax DNA by both PCR and LAMP were found and continuous detection of anti-T. vivax IgG antibodies by ELISA, suggesting the presence of T. vivax resistance to ISM. The combination of LAMP and ELISA tests can prevent misdiagnosis of the parasite clearance in treated cattle, contributing to better disease control. This is the first experiment that demonstrates the persistence infection of T. vivax under ISM treatment in a natural infected herd and evidence of ISM chemotherapy-resistant T. vivax in Brazil.

PhyloQuant approach provides insights into Trypanosoma cruzi evolution using a systems-wide mass spectrometry-based quantitative protein profile.

The etiological agent of Chagas disease, Trypanosoma cruzi, is a complex of seven genetic subdivisions termed discrete typing units (DTUs), TcI-TcVI and Tcbat. The relevance of T. cruzi genetic diversity to the variable clinical course of the disease, virulence, pathogenicity, drug resistance, transmission cycles and ecological distribution requires understanding the parasite origin and population structure. In this study, we introduce the PhyloQuant approach to infer the evolutionary relationships between organisms based on differential mass spectrometry-based quantitative features. In particular, large scale quantitative bottom-up proteomics features (MS1, iBAQ and LFQ) were analyzed using maximum parsimony, showing a correlation between T. cruzi DTUs and closely related trypanosomes' protein expression and sequence-based clustering. Character mapping enabled the identification of synapomorphies, herein the proteins and their respective expression profiles that differentiate T. cruzi DTUs and trypanosome species. The distance matrices based on phylogenetics and PhyloQuant clustering showed statistically significant correlation highlighting the complementarity between the two strategies. Moreover, PhyloQuant allows the identification of differentially regulated and strain/DTU/species-specific proteins, and has potential application in the identification of specific biomarkers and candidate therapeutic targets.

Total Ortholog Median Matrix as an alternative unsupervised approach for phylogenomics based on evolutionary distance between protein coding genes.

The increasing number of available genomic data allowed the development of phylogenomic analytical tools. Current methods compile information from single gene phylogenies, whether based on topologies or multiple sequence alignments. Generally, phylogenomic analyses elect gene families or genomic regions to construct phylogenomic trees. Here, we presented an alternative approach for Phylogenomics, named TOMM (Total Ortholog Median Matrix), to construct a representative phylogram composed by amino acid distance measures of all pairwise ortholog protein sequence pairs from desired species inside a group of organisms. The procedure is divided two main steps, (1) ortholog detection and (2) creation of a matrix with the median amino acid distance measures of all pairwise orthologous sequences. We tested this approach within three different group of organisms: Kinetoplastida protozoa, hematophagous Diptera vectors and Primates. Our approach was robust and efficacious to reconstruct the phylogenetic relationships for the three groups. Moreover, novel branch topologies could be achieved, providing insights about some phylogenetic relationships between some taxa.

Follow-up of dairy cattle naturally infected by Trypanosoma vivax after treatment with isometamidium chloride / Acompanhamento de bovinos leiteiros naturalmente infectados com Trypanosoma vivax após tratamento com cloreto de isometamidium

Abstract Trypanosoma vivax infections cause nonspecific clinical signs in cattle associated with aparasitemic intervals, making disease diagnosis a challenge. In Brazil, diminazene aceturate and isometamidium chloride (ISM) are available to treat bovine trypanosomosis. The objective of this study was to follow-up, by molecular and serological techniques, dairy cattle naturally infected by T. vivax after ISM treatment. Thirty cattle naturally infected with T. vivax received two applications of ISM, at a dosage of 1.0 mg/kg intramuscularly, on days 0 and 150. For T. vivax diagnosis, EDTA-blood and serum samples were evaluated on 0, 7, 15, 30, 60, 90, 120, 150, 180, 210, and 240 days after treatment PCR, Loop-mediated isothermal amplification (LAMP) and ELISA. Animals with persistent detection of T. vivax DNA by both PCR and LAMP were found and continuous detection of anti-T. vivax IgG antibodies by ELISA, suggesting the presence of T. vivax resistance to ISM. The combination of LAMP and ELISA tests can prevent misdiagnosis of the parasite clearance in treated cattle, contributing to better disease control. This is the first experiment that demonstrates the persistence infection of T. vivax under ISM treatment in a natural infected herd and evidence of ISM chemotherapy-resistant T. vivax in Brazil.

Resumo Em bovinos, infecções por Trypanosoma vivax geram sinais clínicos inespecíficos que, associados a intervalos aparasitêmicos, faz com que o diagnóstico da enfermidade seja desafiador. No Brasil, somente aceturato de diaminazeno e cloridrato de isometamidum (ISM) estão disponíveis para o tratamento da tripanossomose bovina. Este trabalho teve como objetivo acompanhar bovinos leiteiros naturalmente infectados por T. vivax, após o tratamento com ISM por meio de técnicas moleculares e sorológica. Foram utilizados 30 bovinos naturalmente infectados com T. vivax, sendo estes tratados com duas aplicações de ISM, na dosagem de 1,0 mg/kg por via intramuscular profunda, nos dias 0 e 150. Foram avaliadas, para diagnóstico de T. vivax, amostras de sangue acrescido de EDTA e soro, colhidas nos 0, 7, 15, 30, 60, 90, 120, 150, 180, 210 e 240 dias após os tratamentos pela reação em cadeia da polimerase (PCR), amplificação circular isotérmica do DNA (LAMP) e ensaio de imunoabsorção enzimático (ELISA). Verificou-se a presença de animais com persistência na detecção de DNA de T. vivax pela PCR e LAMP, bem como detecção contínua de anticorpos IgG anti-T. vivax pelo método de ELISA, sugerindo a presença de resistência de T. vivax ao ISM. A combinação dos testes LAMP e ELISA pode evitar falsos diagnósticos da eliminação do parasita nos bovinos tratados, contribuindo para um melhor controle da doença. Este é o primeiro experimento que demonstra infecção persistente do T. vivax em rebanho naturalmente infectado, tratado com ISM, e evidencia possível resistência ao quimioterápico no Brasil.

Detection of Trypanosoma vivax in tissues of experimentally infected goats: what is the role of adipose tissue in the life cycle of this protozoon? / Detecção de Trypanosoma vivax em tecidos de caprinos experimentalmente infectados: qual é o papel do tecido adiposo no ciclo de vida desse protozoário?

Abstract Trypanosomiasis, caused by Trypanosoma vivax, is responsible for great economic losses among livestock in Africa and South America. During the life cycle of these parasites, they may present different morphological, metabolic and physiological characteristics depending on the interactions that are encountered at each point of their life cycle. Although T. vivax is frequently reported in the circulation of its mammalian hosts, it has the ability to migrate to the tissues of these individuals. However, this characteristic is poorly understood. In this context, we aimed to investigate the presence of T. vivax and the changes caused in different tissues of experimentally infected goats. Despite the animals were not perfused before tissues collection, using different approaches, we demonstrated its presence in different samples, including in the adipose tissue and skin of infected animals. In addition, a mononuclear inflammatory reaction, mostly characterized by an infiltrate of lymphocytes, plasma cells and macrophages were observed. The results highlight the possibility that, like other trypanosomatids, T. vivax may use these tissues during its life cycle. Future studies aiming to elucidate the length of time for which T. vivax remains active in these sites, and whether it uses these sites as a refuge from trypanocidal drugs, and whether it is capable of recolonizing the blood circulation, are much needed.

Resumo A tripanossomíase, causada por Trypanosoma vivax, é responsável por grandes perdas econômicas na bovinocultura da África e da América do Sul. Durante seu ciclo de vida, o parasita pode apresentar diferentes características morfológicas, metabólicas e fisiológicas em função das interações que ele encontra em cada ponto do seu ciclo. Embora o T. vivax seja reportado, frequentemente, na circulação dos seus hospedeiros mamíferos, o protozoário tem a capacidade de migrar para os tecidos desses indivíduos. Entretanto, essa característica é pobremente conhecida. Neste contexto, o objetivo foi verificar a presença, assim como as alterações causadas pelo T. vivax nos diferentes tecidos de caprinos experimentalmente infectados. Apesar dos animais não terem sido perfundidos antes da coleta dos tecidos, utilizando-se diferentes abordagens, foi evidenciada a presença do T. vivax em diferentes amostras teciduais, incluindo no tecido adiposo e pele dos animais infectados. Além disso, foi observada reação inflamatória mononuclear, caracterizada majoritariamente por infiltrado de linfócitos, plasmócitos e macrófagos. Os resultados evidenciam a possibilidade de que, assim como outros tripanossomatídeos, T. vivax pode usar esses tecidos durante o seu ciclo de vida. São necessários futuros estudos, objetivando elucidar o período em que o T. vivax permanece ativo nesses sítios, se ele utiliza esses locais como refúgio das drogas tripanocidas, e se ele é capaz de recolonizar a circulação sanguínea.

Ehrlichia spp. and Anaplasma spp. in Xenarthra mammals from Brazil, with evidence of novel 'Candidatus Anaplasma spp.'

Anaplasmataceae agents are obligatory intracellular Gram-negative α-proteobacteria that are transmitted mostly by arthropod vectors. Although mammals of the Superorder Xenarthra (sloths, anteaters, and armadillos) have been implicated as reservoirs for several zoonotic agents, only few studies have sought to detect Anaplasmataceae agents in this group of mammals. This study aimed to investigate the occurrence and genetic diversity of Anaplasma spp. and Ehrlichia spp. in blood and spleen samples of free-living Xenarthra from four different states in Brazil (São Paulo, Mato Grosso do Sul, Rondônia, and Pará). Nested and conventional PCR screening assays were performed to detect the rrs and dsb genes of Anaplasma spp. and Ehrlichia spp., respectively. The assays were positive in 27.57% (91/330) of the Anaplasma spp. and 24.54% (81/330) of the Ehrlichia spp. Of the 91 positive Anaplasma spp. samples, 56.04% were positive in a conventional PCR assay targeting the 23S-5S intergenic region. Phylogenetic and distance analyses based on the rrs gene allocated Anaplasma sequences from sloths captured in Rondônia and Pará states in a single clade, which was closely related to the A. marginale, A. ovis, and A. capra clades. The sequences detected in southern anteaters from São Paulo were allocated in a clade closely related to sequences of Anaplasma spp. detected in Nasua nasua, Leopardus pardalis, and Cerdocyon thous in Brazil. These sequences were positioned close to A. odocoilei sequences. Genotype analysis corroborated previous findings and demonstrated the circulation of two distinct Anaplasma genotypes in animals from north and southeast Brazil. The first genotype was new. The second was previously detected in N. nasua in Mato Grosso do Sul state. The intergenic region analyses also demonstrated two distinct genotypes of Anaplasma. The sequences detected in Xenarthra from Pará and Rondônia states were closely related to those in A. marginale, A. ovis, and A. capra. Anaplasma spp. sequences detected in Xenarthra from São Paulo and were allocated close to those in A. phagocytophilum. The analyses based on the dsb gene grouped the Ehrlichia spp. sequences with sequences of E. canis (São Paulo, Mato Grosso do Sul, and Pará) and E. minasensis (Rondônia and Pará). The data indicate the occurrence of E. canis and E. minasensis and two possible new Candidatus species of Anaplasma spp. in free-living mammals of the Superorder Xenarthra in Brazil.

High Trypanosoma vivax infection rates in water buffalo and cattle in the Brazilian Lower Amazon.

Highly sensitive and accurate molecular diagnostic methods have not yet been employed for livestock trypanosomosis in the Brazilian Lower Amazon although the first reports of Trypanosoma vivax and Trypanosoma evansi in Brazil were in water buffalo (Bubalus bubalis) in this region. The present study assessed trypanosomosis in buffalo and cattle raised in communal and seasonally flooding pastures in the state of Pará using the fluorescent fragment length barcoding (FFLB) method. T. evansi was not detected, but high infection rates of T. vivax and T. theileri were revealed by a simplified FFLB standardized in the present study that discriminates all trypanosome species infective to livestock in South America. T. vivax infection rates detected by TviCATL-PCR were 24.6% for cattle (n = 61) and 28.1% for buffalo (n = 89). Using the FFLB method, overall T. vivax infection rates increased to 59.6% and 44.3% for buffalo and cattle, respectively. Furthermore, the predominance of a single microsatellite-based genotype of T. vivax was reinforced in the Lower Amazon. Relevant T. vivax infection rates detected in clinically healthy buffalo and cattle through the sampled years (2008-2017) highlight the need for systematic studies to demonstrate the endemic steady state of T. vivax in this region. Our findings provide baseline information for livestock management, including control of T. vivax dispersal, and the introduction of naïve animals. The growing international trade of live livestock from this very important livestock breeding region represents a serious risk for T. vivax spreading outside Amazonia and Brazil.

Multi-locus sequencing reveals a novel Bartonella in mammals from the Superorder Xenarthra.

The superorder Xenarthra consists of sloths, anteaters and armadillos, mammals originated from South America and currently distributed from the south of North America to the south of South America. The present study aimed to investigate the occurrence and genetic diversity of Bartonella spp. in blood and spleen samples from free-living Xenarthra mammals in the states of São Paulo (SP), Mato Grosso do Sul (MS), Rondônia (RO) and Pará (PA). Based on a quantitative real-time PCR (qPCR) assay, a Bartonella spp. nuoG gene fragment was detected in 1.51% (5/330) of the samples: 4 six-banded armadillos (Euphractus sexcinctus) sampled in the MS and 1 southern tamandua (Tamandua tetradactyla) sampled in the PA. Eight sequences (5 ftsZ, 2 gltA and 1 rpoB) were obtained in the conventional PCR assays. In both phylogenetic analyses based on Bayesian and distance (SplitsTree) methods, the obtained ftsZ, gltA and rpoB sequences were positioned in a distinct clade, but related to B. washoensis. The analysis of SplitsTree and genotype networks based on B. washoensis sequences from several hosts from various localities of the world showed that the sequences of the present study were allocated in a group separated from the other sequences, indicating that they probably originated from median vectors and large numbers of mutational events. Additionally, the analyses performed by BLAST showed low percentages of identities of the sequences obtained in the present study when compared to those previously deposited in GenBank. Therefore, we propose a new Candidatus to Bartonella occurring in Xenarthra in Brazil. The present study was the first to report the occurrence of Bartonella sp. in mammals of the superorder Xenarthra in the world, and it was the first to describe a new Candidatus related to B. washoensis in Brazil.

Molecular eco-epidemiology on the sympatric Chagas disease vectors Triatoma brasiliensis and Triatoma petrocchiae: Ecotopes, genetic variation, natural infection prevalence by trypanosomatids and parasite genotyping.

Triatoma petrocchiae is the newly member of the Triatoma brasiliensis species complex. This species overlaps with T. brasiliensis in geographic and ecotypic occupation in the sylvatic habitat because both inhabit rocky outcrops in the semi-arid portion of Brazilian northeast. In this region T. brasiliensis is the most important Chagas disease vector because it constantly colonizes domiciles. In contrast, T. petrocchiae is rarely found in peri or intradomiciliary habitats - reason why little is known about this species. Therefore, Here, we present information for the first time on. the T. petrocchiae ecotopes, genetic diversity, Trypanosoma cruzi prevalence/genotyping in comparison to T. brasiliensis. We found T. brasilensis (N = 223) and T. petrocchiae (N = 69) in co-habitation in rocky outcrops in three Districts of Paraíba and Rio Grande do Norte states. Forty-tree T. petrocchiae insects of eleven sampling spots (composing three geographic populations) were genotyped for the mitochondrial Cyt B gene and little geographic structure was observed. Tajima's D test suggested that species is evolving toward a mutation-drift equilibrium in our collection range. Sylvatic T. petrocchiae had 4% (3/68) of infected insects by T. cruzi, whereas T. brasiliensis had 26% (59/223). Fluorescent Fragment Length Barcoding demonstrated that all three T. petrocchiae harbored TcI whereas T. brasiliensis had TcI, but also TcIII, TcII/TcVI and T. rangeli genotype A, sometimes under mixed infections. None of infected T. petrocchiae were carrying mixed infections. However, this result should be confirmed using a larger pool of infected bugs. We here presented the first documentation of T. rangeli infecting T. brasiliensis. The finding of infected T. petrocchiae calls for constant vector monitoring because the epidemiologic scenario is dynamic and sylvatic vectors are progressively found in adaptation to anthropic environments.

Leishmania (Viannia) braziliensis type 2 as probable etiological agent of canine cutaneous leishmaniasis in Brazilian Amazon.

Canine cutaneous leishmaniasis (CCL) is a zoonosis of public health interest, and in the Americas, Leishmania (Viannia) braziliensis has been identified as the main etiological agent. The present study sought to investigate Leishmania spp. infection in domestic dogs from a rural area of the Xapuri municipality, Acre state, Brazilian Amazonia. For this purpose, visits were carried out to domiciles where the human cases of American cutaneous leishmaniasis (ACL) occurred, followed by the clinical evaluation of the animals in search of clinical signs suggestive of CCL. Blood samples were collected from 40 dogs, 13 of which had lesions suggestive of CCL, and biopsies of these lesions were performed. The methods used were Neal, Novy, and Nicolle's (NNN) medium cultures and direct parasitological examination. Further, to detect and characterize Leishmania DNA some molecular techniques were performed such as conventional polymerase chain reaction (PCR) and sequencing targeting SSU rDNA and ITS1, restriction fragment length polymorphism (RFLP) and high resolution melting (HRM) analysis targeting hsp70. The investigation revealed that the results obtained from the parasitological methods were negative. In PCR by ITS1 and network topology sequences, six strains from dogs, isolated from the Peruvian Andes, appeared identical to Leishmania (Viannia) braziliensis type 2 (99-100%). By other molecular methods these samples turned out to be positive to Leishmania (Viannia) sp.. The diagnosis of Leishmania in domestic dogs from Acre state showed a high proportion of infected animals, and the occurrence of L. braziliensis type 2 in Brazil for the first time. This new report suggests that L. braziliensis type 2 is both trans- and cis-Andean. However, more studies are needed regarding the clinical and diagnostic aspects of this species of Leishmania.

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.