Genomes of Endotrypanum monterogeii from Panama and Zelonia costaricensis from Brazil: Expansion of Multigene Families in Leishmaniinae Parasites That Are Close Relatives of Leishmania spp.

The Leishmaniinae subfamily of the Trypanosomatidae contains both genus Zelonia (monoxenous) and Endotrypanum (dixenous). They are amongst the nearest known relatives of Leishmania, which comprises many human pathogens widespread in the developing world. These closely related lineages are models for the genomic biology of monoxenous and dixenous parasites. Herein, we used comparative genomics to identify the orthologous groups (OGs) shared among 26 Leishmaniinae species to investigate gene family expansion/contraction and applied two phylogenomic approaches to confirm relationships within the subfamily. The Endotrypanum monterogeii and Zelonia costaricensis genomes were assembled, with sizes of 29.9 Mb and 38.0 Mb and 9.711 and 12.201 predicted protein-coding genes, respectively. The genome of E. monterogeii displayed a higher number of multicopy cell surface protein families, including glycoprotein 63 and glycoprotein 46, compared to Leishmania spp. The genome of Z. costaricensis presents expansions of BT1 and amino acid transporters and proteins containing leucine-rich repeat domains, as well as a loss of ABC-type transporters. In total, 415 and 85 lineage-specific OGs were identified in Z. costaricensis and E. monterogeii. The evolutionary relationships within the subfamily were confirmed using the supermatrix (3384 protein-coding genes) and supertree methods. Overall, this study showed new expansions of multigene families in monoxenous and dixenous parasites of the subfamily Leishmaniinae.

Molecular detection of blood-borne agents in vampire bats from Brazil, with the first molecular evidence of Neorickettsia sp. in Desmodus rotundus and Diphylla ecaudata.

Bats (Mammalia, Chiroptera) represent the second largest group of mammals. Due to their ability to fly and adapt and colonize different niches, bats act as reservoirs of several potentially zoonotic pathogens. In this context, the present work aimed to investigate, using molecular techniques, the occurrence of blood-borne agents (Anaplasmataceae, Coxiella burnetii, hemoplasmas, hemosporidians and piroplasmids) in 198 vampire bats sampled in different regions of Brazil and belonging to the species Desmodus rotundus (n = 159), Diphylla ecaudata (n = 31) and Diaemus youngii (n = 8). All vampire bats liver samples were negative in PCR assays for Ehrlichia spp., Anaplasma spp., piroplasmids, hemosporidians and Coxiella burnetii. However, Neorickettsia sp. was detected in liver samples of 1.51% (3/198) through nested PCR based on the 16S rRNA gene in D. rotundus and D. ecaudata. This is the first study to report Neorickettsia sp. in vampire bats. Hemoplasmas were detected in 6.06% (12/198) of the liver samples using a PCR based on the 16S rRNA gene. The two 16S rRNA sequences obtained from hemoplasmas were closely related to sequences previously identified in vampire and non-hematophagous bats from Belize, Peru and Brazil. The genotypic analysis identified a high diversity of bat-associated hemoplasma genotypes from different regions of the world, emphasizing the need for studies on this subject, in order to better understand the mechanisms of co-evolution between this group of bacteria and their vertebrate hosts. The role of neotropical bat-associated Neorickettsia sp. and bats from Brazil in the biological cycle of such agent warrant further investigation.

Vivaxin genes encode highly immunogenic, non-variant antigens on the Trypanosoma vivax cell-surface.

Trypanosoma vivax is a unicellular hemoparasite, and a principal cause of animal African trypanosomiasis (AAT), a vector-borne and potentially fatal livestock disease across sub-Saharan Africa. Previously, we identified diverse T. vivax-specific genes that were predicted to encode cell surface proteins. Here, we examine the immune responses of naturally and experimentally infected hosts to these unique parasite antigens, to identify immunogens that could become vaccine candidates. Immunoprofiling of host serum shows that one particular family (Fam34) elicits a consistent IgG antibody response. This gene family, which we now call Vivaxin, encodes at least 124 transmembrane glycoproteins that display quite distinct expression profiles and patterns of genetic variation. We focused on one gene (viv-ß8) that encodes one particularly immunogenic vivaxin protein and which is highly expressed during infections but displays minimal polymorphism across the parasite population. Vaccination of mice with VIVß8 adjuvanted with Quil-A elicits a strong, balanced immune response and delays parasite proliferation in some animals but, ultimately, it does not prevent disease. Although VIVß8 is localized across the cell body and flagellar membrane, live immunostaining indicates that VIVß8 is largely inaccessible to antibody in vivo. However, our phylogenetic analysis shows that vivaxin includes other antigens shown recently to induce immunity against T. vivax. Thus, the introduction of vivaxin represents an important advance in our understanding of the T. vivax cell surface. Besides being a source of proven and promising vaccine antigens, the gene family is clearly an important component of the parasite glycocalyx, with potential to influence host-parasite interactions.

Molecular epidemiological insights into Trypanosoma vivax in Argentina: From the endemic Gran Chaco to outbreaks in the Pampas.

Argentina is a home to millions of beef and dairy cattle and is one of the world's major exporters of meat. In the present study, Trypanosoma vivax was prevalent (2016-2018) in two major livestock farming regions, the Gran Chaco and the Pampas. In the Gran Chaco, 29% and 51% of animals (n = 72, taurine x zebuine crossbreed) were, respectively, positive by TviCATL-PCR and the more sensitive fluorescent fragment length barcoding (FFLB) method. While 18.4/38.8% of breeding cows (n = 49) tested positive by PCR/FFLB, infection increased to 52.2/78.3% in an outbreak of acute infection in steers (n = 23, taurine breed) brought from a non-endemic area. In the Pampas, overall infection rates in dairy cows (n = 54, taurine breed) were comparable (p > .01) between PCR (66.7%) and FFLB (62.9%) and showed a remarkable increase (PCR / FFLB) from 48.3/44.8% in 2017 to 88/84% in 2018. Infected dairy cattle exhibited anaemia, fever, anorexia, enlarged lymph nodes, emaciation and neurological signs. In contrast, beef cows (taurine x zebuine crossbreed) from the Pampas (n = 30) were asymptomatic despite exhibiting 16.7% (PCR) and 53.3% (FFLB) infection rates. Microsatellite genotyping revealed a remarkable microheterogeneity, seven genotypes in the Gran Chaco, nine in the Pampas and five shared between both regions, consistent with regular movement of T. vivax infected livestock. Data gathered in our study support the Gran Chaco being an endemic area for T. vivax, whereas the Pampas emerged as an outbreak area of acute infection in dairy cattle with critical negative impact in milk production. To the best of our knowledge, this is the first molecular study of T. vivax in Argentina, and results indicated the need for preventive measures to control T. vivax spread from the Gran Chaco to vast livestock farming areas across Argentina.

Remarkable kinetoplast, cytostome-cytopharynx complex, and storage-related structures as dissected by three-dimensional reconstruction of Trypanosoma sp. 858 isolated from a toad (Amphibia: Anura).

In Brazil, the Trypanosoma sp. 858 was isolated from a toad (Anura: Bufonidae: Rhinella ictericus) and successfully maintained in cultures. We previously demonstrated that this trypanosome is different but tightly clustered phylogenetically with other trypanosomes from anurans. In this study, we addressed the ultrastructural features of cultured epimastigotes of this new trypanosome. Our results showed very long and thin free motile forms exhibiting a long flagellum and remarkable large and loose K-DNA network. In addition, the anterior portion contained many acidocalcisomes and a well-developed spongiome tubules-contractile vacuole system. One of the main morphological features of this anuran trypanosome was the presence of a complex cytostome-cytopharynx with a specialized membrane coating at the entrance, which is often hidden by the flagellum. Other conspicuous features are the presence of lipid-like droplets, lamellar membrane limited inclusions, and one very large reservosome, all at the posterior portion of the cell body. This new trypanosome may constitute an excellent model for organelles studies related to endocytosis and lipid storage, as demonstrated herein using scanning and transmission electron microscopy and three-dimensional models obtained by either electron microscopy tomography or dual-beam slice and view series.

Endosymbiont Capture, a Repeated Process of Endosymbiont Transfer with Replacement in Trypanosomatids Angomonas spp.

Trypanosomatids of the subfamily Strigomonadinae bear permanent intracellular bacterial symbionts acquired by the common ancestor of these flagellates. However, the cospeciation pattern inherent to such relationships was revealed to be broken upon the description of Angomonas ambiguus, which is sister to A. desouzai, but bears an endosymbiont genetically close to that of A. deanei. Based on phylogenetic inferences, it was proposed that the bacterium from A. deanei had been horizontally transferred to A. ambiguus. Here, we sequenced the bacterial genomes from two A. ambiguus isolates, including a new one from Papua New Guinea, and compared them with the published genome of the A. deanei endosymbiont, revealing differences below the interspecific level. Our phylogenetic analyses confirmed that the endosymbionts of A. ambiguus were obtained from A. deanei and, in addition, demonstrated that this occurred more than once. We propose that coinfection of the same blowfly host and the phylogenetic relatedness of the trypanosomatids facilitate such transitions, whereas the drastic difference in the occurrence of the two trypanosomatid species determines the observed direction of this process. This phenomenon is analogous to organelle (mitochondrion/plastid) capture described in multicellular organisms and, thereafter, we name it endosymbiont capture.

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.

The Importance of Glycerophospholipid Production to the Mutualist Symbiosis of Trypanosomatids.

The symbiosis in trypanosomatids is a mutualistic relationship characterized by extensive metabolic exchanges between the bacterium and the protozoan. The symbiotic bacterium can complete host essential metabolic pathways, such as those for heme, amino acid, and vitamin production. Experimental assays indicate that the symbiont acquires phospholipids from the host trypanosomatid, especially phosphatidylcholine, which is often present in bacteria that have a close association with eukaryotic cells. In this work, an in-silico study was performed to find genes involved in the glycerophospholipid (GPL) production of Symbiont Harboring Trypanosomatids (SHTs) and their respective bacteria, also extending the search for trypanosomatids that naturally do not have symbionts. Results showed that most genes for GPL synthesis are only present in the SHT. The bacterium has an exclusive sequence related to phosphatidylglycerol production and contains genes for phosphatidic acid production, which may enhance SHT phosphatidic acid production. Phylogenetic data did not indicate gene transfers from the bacterium to the SHT nucleus, proposing that enzymes participating in GPL route have eukaryotic characteristics. Taken together, our data indicate that, differently from other metabolic pathways described so far, the symbiont contributes little to the production of GPLs and acquires most of these molecules from the SHT.

Genomic Organization and Generation of Genetic Variability in the RHS (Retrotransposon Hot Spot) Protein Multigene Family in Trypanosoma cruzi.

Retrotransposon Hot Spot (RHS) is the most abundant gene family in Trypanosoma cruzi, with unknown function in this parasite. The aim of this work was to shed light on the organization and expression of RHS in T. cruzi. The diversity of the RHS protein family in T. cruzi was demonstrated by phylogenetic and recombination analyses. Transcribed sequences carrying the RHS domain were classified into ten distinct groups of monophyletic origin. We identified numerous recombination events among the RHS and traced the origins of the donors and target sequences. The transcribed RHS genes have a mosaic structure that may contain fragments of different RHS inserted in the target sequence. About 30% of RHS sequences are located in the subtelomere, a region very susceptible to recombination. The evolution of the RHS family has been marked by many events, including gene duplication by unequal mitotic crossing-over, homologous, as well as ectopic recombination, and gene conversion. The expression of RHS was analyzed by immunofluorescence and immunoblotting using anti-RHS antibodies. RHS proteins are evenly distributed in the nuclear region of T. cruzi replicative forms (amastigote and epimastigote), suggesting that they could be involved in the control of the chromatin structure and gene expression, as has been proposed for T. brucei.

Pan-American Trypanosoma (Megatrypanum) trinaperronei n. sp. in the white-tailed deer Odocoileus virginianus Zimmermann and its deer ked Lipoptena mazamae Rondani, 1878: morphological, developmental and phylogeographical characterisation.

BACKGROUND: The subgenus Megatrypanum Hoare, 1964 of Trypanosoma Gruby, 1843 comprises trypanosomes of cervids and bovids from around the world. Here, the white-tailed deer Odocoileus virginianus (Zimmermann) and its ectoparasite, the deer ked Lipoptena mazamae Rondani, 1878 (hippoboscid fly), were surveyed for trypanosomes in Venezuela. RESULTS: Haemoculturing unveiled 20% infected WTD, while 47% (7/15) of blood samples and 38% (11/29) of ked guts tested positive for the Megatrypanum-specific TthCATL-PCR. CATL and SSU rRNA sequences uncovered a single species of trypanosome. Phylogeny based on SSU rRNA and gGAPDH sequences tightly cluster WTD trypanosomes from Venezuela and the USA, which were strongly supported as geographical variants of the herein described Trypanosoma (Megatrypanum) trinaperronei n. sp. In our analyses, the new species was closest to Trypanosoma sp. D30 from fallow deer (Germany), both nested into TthII alongside other trypanosomes from cervids (North American elk and European fallow, red and sika deer), and bovids (cattle, antelopes and sheep). Insights into the life-cycle of T. trinaperronei n. sp. were obtained from early haemocultures of deer blood and co-culture with mammalian and insect cells showing flagellates resembling Megatrypanum trypanosomes previously reported in deer blood, and deer ked guts. For the first time, a trypanosome from a cervid was cultured and phylogenetically and morphologically (light and electron microscopy) characterised. CONCLUSIONS: In the analyses based on SSU rRNA, gGAPDH, CATL and ITS rDNA sequences, neither cervids nor bovids trypanosomes were monophyletic but intertwined within TthI and TthII major phylogenetic lineages. One host species can harbour more than one species/genotype of trypanosome, but each trypanosome species/genotype was found in a single host species or in phylogenetically closely related hosts. Molecular evidence that L. mazamae may transmit T. trinaperronei n. sp. suggests important evolutionary constraints making tight the tripartite T. trinaperronei-WTD-deer ked association. In a plausible evolutionary scenario, T. trinaperronei n. sp. entered South America with North American white-tailed deer at the Pliocene-Pleistocene boundary following the closure of the Panama Isthmus.

Variant antigen diversity in Trypanosoma vivax is not driven by recombination.

African trypanosomes (Trypanosoma) are vector-borne haemoparasites that survive in the vertebrate bloodstream through antigenic variation of their Variant Surface Glycoprotein (VSG). Recombination, or rather segmented gene conversion, is fundamental in Trypanosoma brucei for both VSG gene switching and for generating antigenic diversity during infections. Trypanosoma vivax is a related, livestock pathogen whose VSG lack structures that facilitate gene conversion in T. brucei and mechanisms underlying its antigenic diversity are poorly understood. Here we show that species-wide VSG repertoire is broadly conserved across diverse T. vivax clinical strains and has limited antigenic repertoire. We use variant antigen profiling, coalescent approaches and experimental infections to show that recombination plays little role in diversifying T. vivax VSG sequences. These results have immediate consequences for both the current mechanistic model of antigenic variation in African trypanosomes and species differences in virulence and transmission, requiring reconsideration of the wider epidemiology of animal African trypanosomiasis.

Update on Chagas disease in Venezuela during the period 2003-2018. A review.

The present article reviews the status of Chagas disease in Venezuela during the period 2003-2018, based on the detection of Trypanosoma cruzi-infection in 3,343 blood samples of individuals from rural localities and 182 patients referred from health centers to confirm presumptive clinical diagnostic. The study involved samples from 81 rural localities of 17 states located at different regions and ecological life zones of the country. Analysis by parasitological (fresh microscopic observation, hemoculture and Giemsa stained blood smears), serological (DAT, IFAT-polyvalent, IgM, IgG tests) and molecular (PCR) tests, revealed 10.7% seroprevalence and 42.8% T. cruzi-infection, in individuals from rural localities and referred patients, respectively. In both groups T. cruzi-infection was detected at any age, revealing active transmission in children under 10-years-old. Clinical profile detected in referred patients, showed significantly major number of symptoms in orally infected patients than in infected by vectorial route (P<0.01). Genetic characterization of T. cruzi isolates obtained from orally and vectorial transmitted acute Chagas disease in western Venezuela, revealed the circulation of DTUI and DTUIII in the former, and DTUI, DTUII and DTUIII in patients infected by vectorial route. DTUI predominated in both cases, and haplotype Ib was the most frequently found in this genotype. Statistical analysis of clinical profile - T. cruzi DTUs - transmission route relationships did not show association among these variables and, consequently, chagasic patient's clinical condition did not depend of T. cruzi genotype or its route of transmission. In addition, differences in clinical severity may be associated with host susceptibility and/or parasite load received by the human receptor in spite of the T. cruzi genotype itself. The epidemiological implications of the present findings are discussed, and the need for developing efficient tools as well as implementation of urgent and radical changes in the public health policy to control Chagas disease transmission in the Venezuelan territory are suggested.

Expanding our knowledge on African trypanosomes of the subgenus Pycnomonas: A novel Trypanosoma suis-like in tsetse flies, livestock and wild ruminants sympatric with Trypanosoma suis in Mozambique.

Among the subgenera of African tsetse-transmitted trypanosomes pathogenic to livestock, the least known is the subgenus Pycnomonas, which contains a single species, Trypanosoma suis (TSU), a pathogen of domestic pigs first reported in 1905 and recently rediscovered in Tanzania and Mozambique. Analysis by Fluorescent Fragment Length Barcoding (FFLB) revealed an infection rate of 20.3% (108 out of 530 tsetse flies) in a recent study in the Gorongosa and Niassa wildlife reserves in Mozambique, and demonstrated two groups of Pycnomonas trypanosomes: one (14.1%, 75 flies) showing an FFLB profile identical to the reference TSU from Tanzania, and the other (6.2%, 33 flies) differing slightly from reference TSU and designated Trypanosoma suis-like (TSU-L). Phylogenetic analyses tightly clustered TSU and TSU-L from Mozambique with TSU from Tanzania forming the clade Pycnomonas positioned between the subgenera Trypanozoon and Nannomonas. Our preliminarily exploration of host ranges of Pycnomonas trypanosomes revealed TSU exclusively in warthogs while TSU-L was identified, for the first time for a member of the subgenus Pycnomonas, in ruminants (antelopes, Cape buffalo, and in domestic cattle and goats). The preferential blood meal sources of tsetse flies harbouring TSU and TSU-L were wild suids, and most of these flies concomitantly harboured the porcine trypanosomes T. simiae, T. simiae Tsavo, and T. godfreyi. Therefore, our findings support the link of TSU with suids while TSU-L remains to be comprehensively investigated in these hosts. Our results greatly expand our knowledge of the diversity, hosts, vectors, and epidemiology of Pycnomonas trypanosomes. Due to shortcomings of available molecular diagnostic methods, a relevant cohort of trypanosomes transmitted by tsetse flies to ungulates, especially suids, has been neglected or most likely misidentified. The method employed in the present study enables an accurate discrimination of trypanosome species and genotypes and, hence, a re-evaluation of the "lost" subgenus Pycnomonas and of porcine trypanosomes in general, the most neglected group of African trypanosomes pathogenic to ungulates.

Zoonotic Trypanosomes in Rats and Fleas of Venezuelan Slums.

Rattus spp. are reservoirs of many human zoonoses, but their role in domestic transmission cycles of human trypanosomiasis is underestimated. In this study, we report trypanosome-infected Rattus norvegicus and Rattus rattus in human dwellings in slums neighboring Maracay, a large city near Caracas, the capital of Venezuela. Blood samples of R. norvegicus and R. rattus examined by PCR and FFLB (fluorescent fragment length barcoding) revealed a prevalence of 6.3% / 31.1% for Trypanosoma lewisi (agent of rat- and flea-borne human emergent zoonosis), and 10.5% / 24.6% for Trypanosoma cruzi (agent of Chagas disease). Detection in flea guts of T. lewisi (76%) and, unexpectedly, T. cruzi (21.3%) highlighted the role of fleas as carriers and vectors of these trypanosomes. A high prevalence of rats infected with T. lewisi and T. cruzi and respective flea and triatomine vectors poses a serious risk of human trypanosomiasis in Venezuelan slums. Anthropogenic activities responsible for growing rat and triatomine populations within human dwellings drastically increased human exposure to trypanosomes. This scenario has allowed for the reemergence of Chagas disease as an urban zoonosis in Venezuela and can propitiate the emergence of atypical T. lewisi infection in humans.

Mapping benznidazole resistance in trypanosomatids and exploring evolutionary histories of nitroreductases and ABCG transporter protein sequences.

The nitro-heterocyclic compound benznidazole (BZ) is the first-line drug for the treatment of Chagas disease, caused by the protozoan Trypanosoma cruzi. However, therapeutic failures are common for reasons that include the influences of parasite and host genetics, the effects of toxicity on adherence to treatment, and difficulties in demonstrating parasitological cure. To obtain information on the origin of the resistance to BZ and eliminate from the scenery the participation of the host, initially we mapped the susceptibility to the drug in thirteen species of seven genera of the family Trypanosomatidae. We verified that all Trypanosoma species are sensitive to low concentrations of the drug (IC50 2.7 to 25 µM) while Non-Trypanosoma species are highly resistant to these concentrations. The two groups of parasites correspond to the major phylogenetic lineages of trypanosomatids. Next, we searched in the trypanosomatid genome databases homologs of two type-I nitroreductases (NTR-1 and OYE) and an ABC transporter (ABCG1) that have been associated with BZ resistance in T. cruzi. The predicted proteins were characterized regarding domains and used for phylogenetic analyses. Homologous NTR-1 genes were found in all trypanosomatids investigated and the structural characteristics of the enzyme suggest that it may be functional. OYE genes were absent in BZ-sensitive African trypanosomes, which excludes the participation of this enzyme in BZ bio-activation. Two copies of ABCG1 genes were observed in most BZ resistant species, while Trypanosoma species exhibit only one copy per haploid genome. Functional studies are required to verify the involvement of these genes in BZ resistance. In addition, since multiple mechanisms can contribute to BZ susceptibility, our study poses a range of organisms highly resistant to BZ in which these aspects can be investigated. Preliminary studies on BZ uptake indicate marked differences between BZ-sensitive and BZ-resistant species.

Genetic diversity of Bartonella spp. in vampire bats from Brazil.

Recently, an increasing number of Bartonella species have been emerged to cause human diseases. Among animal reservoirs for Bartonella spp., bats stand out due to their high mobility, wide distribution, social behaviour and long-life span. Although studies on the role of vampire bats in the epidemiology of rabies have been extensively investigated in Latin America, information on the circulation and genetic diversity of Bartonella species in these bat species is scarce. In the present work, 208 vampire bats, namely Desmodus rotundus (the common vampire bat; n = 167), Diphylla ecaudata (the hairy-legged vampire bat; n = 32) and Diaemus youngii (the white-winged vampire bat; n = 9) from 15 different states in Brazil were sampled. DNA was extracted from liver tissue samples and submitted to real-time PCR (qPCR) and conventional PCR (cPCR) assays for Bartonella spp. targeting five genetic loci, followed by phylogenetic and genotype network analyses. Fifty-one out of 208 liver samples (24.51%) were positive for Bartonella DNA in the ITS real-time PCR assay [40 (78.43%) of them were from D. rotundus from 11 states, and 11 (21.57%) samples from D. ecaudata from three states. Eleven genotypes were found for each gltA and rpoB genes. Several ITS sequences detected in the present study clustered within the lineage that includes B. bacilliformis and B. ancachensis. The Bayesian phylogenetic inference based on the gltA gene positioned the obtained sequences in six different clades, closely related to Bartonella genotypes previously detected in D. rotundus and associated ectoparasites sampled in Latin America. On the other hand, the Bartonella rpoB genotypes clustered together with the ruminant species, B. schoenbuchensis and B. chomelii. The present study describes for the first time the molecular detection of Bartonella spp. in D. ecaudata bats. It also indicates that Bartonella spp. of vampire bats are genetically diverse and geographically widespread in Brazil.

Shared species of crocodilian trypanosomes carried by tabanid flies in Africa and South America, including the description of a new species from caimans, Trypanosoma kaiowa n. sp.

BACKGROUND: The genus Trypanosoma Gruby, 1843 is constituted by terrestrial and aquatic phylogenetic lineages both harboring understudied trypanosomes from reptiles including an increasing diversity of crocodilian trypanosomes. Trypanosoma clandestinus Teixeira & Camargo, 2016 of the aquatic lineage is transmitted by leeches to caimans. Trypanosoma grayi Novy, 1906 of the terrestrial lineage is transmitted by tsetse flies to crocodiles in Africa, but the vectors of Neotropical caiman trypanosomes nested in this lineage remain unknown. RESULTS: Our phylogenetic analyses uncovered crocodilian trypanosomes in tabanids from South America and Africa, and trypanosomes other than T. grayi in tsetse flies. All trypanosomes found in tabanids clustered in the crocodilian clade (terrestrial lineage) forming six clades: Grayi (African trypanosomes from crocodiles and tsetse flies); Ralphi (trypanosomes from caimans, African and Brazilian tabanids and tsetse flies); Terena (caimans); Cay03 (caimans and Brazilian tabanids); and two new clades, Tab01 (Brazilian tabanid and tsetse flies) and Kaiowa. The clade Kaiowa comprises Trypanosoma kaiowa n. sp. and trypanosomes from African and Brazilian tabanids, caimans, tsetse flies and the African dwarf crocodile. Trypanosoma kaiowa n. sp. heavily colonises tabanid guts and differs remarkably in morphology from other caiman trypanosomes. This species multiplied predominantly as promastigotes on log-phase cultures showing scarce epimastigotes and exhibited very long flagellates in old cultures. Analyses of growth behavior revealed that insect cells allow the intracellular development of Trypanosoma kaiowa n. sp. CONCLUSIONS: Prior to this description of Trypanosoma kaiowa n. sp., no crocodilian trypanosome parasitic in tabanid flies had been cultured, morphologically examined by light, scanning and transmission microscopy, and phylogenetically compared with other crocodilian trypanosomes. Additionally, trypanosomes thought to be restricted to caimans were identified in Brazilian and African tabanids, tsetse flies and the dwarf crocodile. Similar repertoires of trypanosomes found in South American caimans, African crocodiles and tabanids from both continents support the recent diversification of these transcontinental trypanosomes. Our findings are consistent with trypanosome host-switching likely mediated by tabanid flies between caimans and transoceanic migrant crocodiles co-inhabiting South American wetlands at the Miocene.

Genetic diversity of trypanosomes pathogenic to livestock in tsetse flies from the Nech Sar National Park in Ethiopia: A concern for tsetse suppressed area in Southern Rift Valley?

In Ethiopia, home to the largest African herd of cattle, animal trypanosomiasis is a major constraint to the efforts made for food self-sufficiency. We searched for trypanosomes in tsetse flies caught in the Nech Sar National Park (NSNP), Southern Rifty Valley, Ethiopia, at the district of Arba Minch where intensive tsetse control is successfully improving cattle productivity. Despite narrow geographical and temporal scales of our survey, we found a remarkable diversity of trypanosomes using the sensitive and discriminative method of fluorescent fragment length barcoding. We also found a high density of Glossina pallidipes (47.8 flies/trap/day) showing relevant cytochrome oxidase I gene variability. The identification of blood meal sources through cytochrome b gene sequences revealed cattle and warthog as preferential ungulate hosts of tsetse flies in the study area. Our survey identified trypanosomes in 38% of the 287 flies examined (42% of proboscises and 32% of guts), and the following infection rates for each species: Trypanosoma vivax 23%, T. simiae 23%, T. congolense 22%, T. theileri 19.9%, T. (Trypanozoon) spp. 10.5%, T. godfreyi 9.4%, T. simiae Tsavo 6.3%, and mixed infections in proboscises (30%) and guts (61%). Phylogenetic analysis revealed T. vivax of the "West African-South American" genotype, T. congolense of Savannah (16.7%), Kilifi (3.5%) and Forest (2.1%) lineages, and new genotypes of T. simiae. To our knowledge, this is the first survey of trypanosomes in the NSNP, and the most comprehensive molecular characterisation of trypanosomes in tsetse flies of Ethiopia, including the comparison with samples from West and other East African countries. Our results support the diversification of T. vivax in East Africa, and the dispersion of the genotype herein identified in Ethiopia across West Africa and then in South America. Altogether, tsetse density and infection rate, repertoire of trypanosomes and feeding behavior indicate a high risk of transmission of trypanosomes pathogenic to ungulates by tsetse flies from the NSNP, a hotspot of tsetse infestation and trypanosome diversity. Our findings reinforce the need for constant surveillance, and the reliance on community efforts to prevent reinvasion of tsetse and animal trypanosomiasis in suppressed areas of Southern Rift Valley.

Trypanosoma madeirae sp. n.: A species of the clade T. cruzi associated with the neotropical common vampire bat Desmodus rotundus.

Molecular phylogenetic studies have revealed the growing diversity of bat trypanosomes. Here, 14 isolates from blood samples of the vampire bat Desmodus rotundus (Phyllostomidae) from Rio de Janeiro, Southeast Brazil, were cultivated, and morphologically and molecularly characterized. All isolates represent a novel species named Trypanosoma madeirae n. sp. positioned in the Neobat lineage of the clade T. cruzi. The Neobat lineage also comprises closely related trypanosomes of clades Neotropic 1, 2 and 3 from diverse phyllostomid species. Trypanosomes of Neotropic 1, found in Trachops cirrhosus and Artibeus jamaicensis (phyllostomids), likely represent a different species or genotype closely related to T. madeirae. Consistent with its phylogenetic positioning, T. madeirae differs from Trypanosoma cruzi in morphology of both epimastigote and trypomastigote culture forms and does not infect Triatoma infestans. Similar to its closest relatives of Neobat lineage, T. madeirae was unable to develop within mammalian cells. To date, PCR-surveys on archived blood/liver samples unveiled T. madeirae exclusively in D. rotundus from Southern to Northern Brazil. The description of a new species of bat trypanosome associated with vampire bats increases the repertoire of trypanosomes infecting D. rotundus, currently comprised of Trypanosoma cruzi, T. cruzi marinkellei, Trypanosoma dionisii, Trypanosoma rangeli, Trypanosoma pessoai, and Trypanosoma madeirae.

Shared species of crocodilian trypanosomes carried by tabanid flies in Africa and South America, including the description of a new species from caimans, Trypanosoma kaiowa n. sp.

Background The genus Trypanosoma Gruby, 1843 is constituted by terrestrial and aquatic phylogenetic lineages both harboring understudied trypanosomes from reptiles including an increasing diversity of crocodilian trypanosomes. Trypanosoma clandestinus Teixeira & Camargo, 2016 of the aquatic lineage is transmitted by leeches to caimans. Trypanosoma grayi Novy, 1906 of the terrestrial lineage is transmitted by tsetse flies to crocodiles in Africa, but the vectors of Neotropical caiman trypanosomes nested in this lineage remain unknown. Results Our phylogenetic analyses uncovered crocodilian trypanosomes in tabanids from South America and Africa, and trypanosomes other than T. grayi in tsetse flies. All trypanosomes found in tabanids clustered in the crocodilian clade (terrestrial lineage) forming six clades: Grayi (African trypanosomes from crocodiles and tsetse flies); Ralphi (trypanosomes from caimans, African and Brazilian tabanids and tsetse flies); Terena (caimans); Cay03 (caimans and Brazilian tabanids); and two new clades, Tab01 (Brazilian tabanid and tsetse flies) and Kaiowa. The clade Kaiowa comprises Trypanosoma kaiowa n. sp. and trypanosomes from African and Brazilian tabanids, caimans, tsetse flies and the African dwarf crocodile. Trypanosoma kaiowa n. sp. heavily colonises tabanid guts and differs remarkably in morphology from other caiman trypanosomes. This species multiplied predominantly as promastigotes on log-phase cultures showing scarce epimastigotes and exhibited very long flagellates in old cultures. Analyses of growth behavior revealed that insect cells allow the intracellular development of Trypanosoma kaiowa n. sp. Conclusions Prior to this description of Trypanosoma kaiowa n. sp., no crocodilian trypanosome parasitic in tabanid flies had been cultured, morphologically examined by light, scanning and transmission microscopy, and phylogenetically compared with other crocodilian trypanosomes. Additionally, trypanosomes thought to be restricted to caimans were identified in Brazilian and African tabanids, tsetse flies and the dwarf crocodile. Similar repertoires of trypanosomes found in South American caimans, African crocodiles and tabanids from both continents support the recent diversification of these transcontinental trypanosomes. Our findings are consistent with trypanosome host-switching likely mediated by tabanid flies between caimans and transoceanic migrant crocodiles co-inhabiting South American wetlands at the Miocene.