Andréa Pereira da Costa

Phylogenetic relationships of Leishmania species based on trypanosomatid barcode (SSU rDNA) and gGAPDH genes: taxonomic revision of Leishmania (L.) infantum chagasi in South America

Phylogenetic studies on trypanosomatid barcode using V7V8 SSU rRNA and gGAPDH gene sequences have provided support for redefining some trypanosomatid species and positioning new isolates. The genus Leishmania is a slow evolving monophyletic group and including important human pathogens. The phylogenetic relationships of this genus have been determined by the natural history of its vertebrate hosts, vector specificity, clinical manifestations, geographical distribution and molecular approaches using different markers. Thus, in an attempt to better understand the phylogenetic relationships of Leishmania species, we performed phylogenetic analysis on trypanosomatid barcode using V7V8 SSU rRNA and gGAPDH gene sequences among a large number of Leishmania species and also several Brazilian visceral Leishmania infantum chagasi isolates obtained from dogs and humans. Our phylogenetic analysis strongly suggested that Leishmania hertigi and Leishmania equatoriensis should be taxonomically revised so as to include them in the genus Endotrypanum; and supported ancient divergence of Leishmania enriettii. This, together with recent data in the literature, throws light on the discussion about the evolutionary southern supercontinent hypothesis for the origin of Leishmania ssp. and validates L. infantum chagasi from Brazil, thus clearly differentiating it from L. infantum, for the first time.

Isolation and Phylogenetic Relationships of Bat Trypanosomes from Different Biomes in Mato Grosso, Brazil

Abstract:  In the order Chiroptera, more than 30 trypanosome species belonging to the subgenera Herpetosoma, Schizotrypanum, Megatrypanum, and Trypanozoon have been described. The species Trypanosoma cruzi, Trypanosoma cruzi marinkellei, and Trypanosoma dionisii are the most common in bats and belong to the Schizotrypanum subgenus. Bats from 2 different biomes, Pantanal and Amazonia/Cerrado in the state of Mato Grosso, Brazil, were evaluated according to the presence of trypanosome parasites by means of hemoculture and PCR in primary samples (blood samples). A total of 211 bats from 20 different species were caught and the trypanosome prevalence, evaluated through hemoculture, was 9.0% (19), 15.5% (13), and 4.8% (6) in the municipalities of Confresa (Amazonia/Cerrado biome) and Poconé (Pantanal biome). Among the 123 primary samples obtained from the bats, only 3 (2.4%) were positive. Phylogenetic analysis using trypanosomatid barcoding (V7V8 region of SSU rDNA) identified all the isolates and primary samples as T. c. marinkellei. The sequences of the isolates were segregated according to the bat host genus or species and suggest that co-evolutionary patterns exist between hosts and parasites. Further studies in different Brazilian regions and biomes need to be conducted in order to gain real understanding of the diversity of trypanosomes in bats.

Humoral responses and immune protection in mice immunized with irradiated T. gondii tachyzoites and challenged with three genetically distinct strains of T. gondii

Toxoplasma gondii is an obligate intracellular parasite that infects a variety of mammals and birds. T. gondii also causes human toxoplasmosis; although toxoplasmosis is generally a benign disease, ocular, congenital or reactivated disease is associated with high numbers of disabled people. Infection occurs orally through the ingestion of meat containing cysts or by the intake of food or water contaminated with oocysts. Although the immune system responds to acute infection and mediates the clearance of tachyzoites, parasite cysts persist for the lifetime of the host in tissues such as the eye, muscle, and CNS. However, T. gondii RH strain tachyzoites irradiated with 255Gy do not cause residual infection and induce the same immunity as a natural infection. To assess the humoral response in BALB/c and C57BL/6J mice immunized with irradiated tachyzoites either by oral gavage (p.o.) or intraperitoneal (i.p.) injection, we analyzed total and high-affinity IgG and IgA antibodies in the serum. High levels of antigen-specific IgG were detected in the serum of parenterally immunized mice, with lower levels in mice immunized via the oral route. However, most serum antibodies exhibited low affinity for antigen in both mice strain. We also found antigen specific IgA antibodies in the stools of the mice, especially in orally immunized BALB/c mice. Examination of bone marrow and spleen cells demonstrated that both groups of immunized mice clearly produced specific IgG, at levels comparable to chronic infection, suggesting the generation of IgG specific memory. Next, we challenged i.p. or p.o. immunized mice with cysts from ME49, VEG or P strains of T. gondii. Oral immunization resulted in partial protection as compared to challenged naive mice; these findings were more evident in highly pathogenic ME49 strain challenge. Additionally, we found that while mucosal IgA was important for protection against infection, antigen-specific IgG antibodies were involved with protection against disease and disease pathogenesis. Most antigen responsive cells in culture produced specific high-affinity IgG after immunization, diverse of the findings in serum IgG or from cells after infection, which produced low proportion of high-avidity IgG.

A Survey of Ectoparasites Infesting Urban and Rural Dogs of Maranhão State, Brazil

ABSTRACT This study evaluated for the first time, ectoparasite infestations on dogs from urban and rural areas of the continental land of the state of Maranhâo, northeastern Brazil. In total, 622 dogs were examined for ectoparasite infestations. Overall, 392 (63.0%) were infested with ectoparasites, 154 (51.3%) of 300 urban dogs and 238 (73.9%) of 322 rural dogs. Five species of ectoparasites were found, three ticks [Rhipicephalus sanguineus (Latreille), Amblyomma ovale Koch, and Amblyommacajennense (F.) ], one flea [Ctenocephalides felis (Bouché) ], and one louse [ (Heterodoxus spininger (Enderlein) ]. The frequency of infestation by R. sanguineus tended to be higher in urban than in rural areas, whereas infestations by Amblyomma ticks and C. felis fleas tended to be higher among rural dogs. Louse (H. spininger) infestations were similarly low among all areas. Mixed infestations by at least two species of ectoparasites on the same dog were significantly more frequent on rural than on urban dogs. The most frequent mixed infestation was by R. sanguineus and C. felis, found on 11.4% of the dogs. Further studies are warranted to evaluate canine vector-borne agents in Maranhão, especially because most of the ectoparasites here reported are vectors of major vector-borne diseases, including zoonoses of continental importance.

Survey of Trypanosoma and Leishmania in Wild and Domestic Animals in an Atlantic Rainforest Fragment and Surroundings in the State of Espírito Santo, Brazil

ABSTRACT Trypanosoma and Leishmania infections affect wild and domestic animals and human populations. The growing process of deforestation and urbanization of Atlantic Rainforest areas has given rise to introduction of humans and domestic animals to the sylvatic cycles of Trypanosoma and Leishmania species. Serological, parasitological, and molecular surveys among wild and domestic animals in the Corrego do Veado Biological Reserve, which is an Atlantic Rainforest fragment in the state of Espírito Santo, southeastern Brazil, were evaluated. In total, 154 wild animals of 25 species and 67 domestic animals (47 dogs and 20 horses) were sampled. All the domestic animals were serologically negative for anti-Leishmania infantum chagasi antibodies and negative in parasitological approaches. Only the Order Chiroptera presented positive blood cultures and cryopreserved isolates. The phylogenetic trees based on SSU rDNA and gGAPDH genes confirmed the occurrence of Trypanosoma dionisii and provided the first record of Trypanosoma cruzi marinkellei in southeastern Brazil. The studies conducted in Atlantic Rainforest remaining trees provide the knowledge of parasite diversity or detect parasites that can accelerate the loss of hosts diversity.

A serological and molecular survey of Babesia vogeli, Ehrlichia canis and Rickettsia spp. among dogs in the state of Maranhão, northeastern Brazil

This study evaluated exposure and infection by tick-borne agents (Babesia vogeli, Ehrlichia canis and Rickettsia spp.) in 172 dogs in rural areas and 150 dogs in urban areas of the municipality of Chapadinha, state of Maranhão, northeastern Brazil, using molecular and serological methods. Overall, 16.1% of the sampled dogs (52/322) were seroreactive to B. vogeli, with endpoint titers ranging from 40 to 640. For E. canis, 14.6% of the dogs (47/322) were seroreactive, with endpoint titers from 80 to 163,840. Antibodies reactive to at least one of the five species of Rickettsia were detected in 18.9% of the dogs (61/322), with endpoint titers ranging from 64 to 4,096. High endpoint titers were observed for Rickettsia amblyommii. Three (0.9%) and nine (2.8%) canine blood samples were PCR-positive for Babesia spp. and E. canis. The ticks collected from urban dogs were all Rhipicephalus sanguineus sensu lato, whereas the rural dogs were infested by R. sanguineus s.l, Amblyomma cajennense sensu lato and Amblyomma ovale. One A. ovale tick was found to be infected by Rickettsia bellii. This study provides an epidemiological background for controlling and preventing canine tick-borne diseases in a neglected region of Brazil.

Diversity of bats trypanosomes in hydroeletric area of Belo Monte in Brazilian Amazonia

The Trypanosoma comprises flagellates able to infect many mammalian species and is transmitted by several groups of invertebrates. The order Chiroptera can be infected by the subgenera Herpetosoma, Schizotrypanum, Megatrypanum and Trypanozoon. In this study, we described the diversity of bats trypanosomes, inferring the phylogenetic relationships among the trypanosomes from bats caught Belo Monte Hydroeletric area (Brazilian Amazonia). Trypanosomes from bats were isolated by haemoculture, and the molecular phylogeny based on small subunit rDNA (SSU rDNA) and glycosomal-3-phosphate dehydrogenase (gGAPDH) gene sequences. Morphological characterization included light and scanning electron microscopy. A total of 157 bats were caught in the area belonging 6 Families (Emballonuridae, Furipteridae, Mormoopidae, Natalidae, Phyllostomidae and Vespertilionidae) and 34 species. The bat trypanosome prevalence, as evaluated through haemoculture, was 5,7%. Phylogenetic trees grouped the isolates in T. cruzi branch (TCI and TCbat lineage), T. cruzi marinkellei and Trypanosoma wauwau from Pteronotus parnellii. This is the first isolate from T. wauwau in Para state. The occurrence of T. cruzi in the ​​ Belo Monte Hydroeletric area (UHE Belo Monte) in Amazon/Brazil attentive to the risk of migration human population required for the works of the dam and new cities that grow in the vicinity of these businesses, but it is a zoonosis already known to the Amazon region, and the presence of unclassified Trypanosoma species, attend to the large parasitic biodiversity still unknown.

Morphological and molecular characterization and phylogenetic relationships of a new species of trypanosome in Tapirus terrestris (lowland tapir), Trypanosoma terrestris sp. nov., from Atlantic Rainforest of southeastern Brazi.

BackgroundThe Lowland tapir (Tapirus terrestris) is the largest Brazilian mammal and despite being distributed in various Brazilian biomes, it is seriously endangered in the Atlantic Rainforest. These hosts were never evaluated for the presence of Trypanosoma parasites.MethodsThe Lowland tapirs were captured in the Brazilian southeastern Atlantic Rainforest, Espírito Santo state. Trypanosomes were isolated by hemoculture, and the molecular phylogeny based on small subunit rDNA (SSU rDNA) and glycosomal-3-phosphate dehydrogenase (gGAPDH) gene sequences and the ultrastructural features seen via light microscopy and scanning and transmission electron microscopy are described.ResultsPhylogenetic trees using combined SSU rDNA and gGAPDH data sets clustered the trypanosomes of Lowland tapirs, which were highly divergent from other trypanosome species. The phylogenetic position and morphological discontinuities, mainly in epimastigote culture forms, made it possible to classify the trypanosomes from Lowland tapirs as a separate species.ConclusionsThe isolated trypanosomes from Tapirus terrestris are a new species, Trypanosoma terrestris sp. n., and were positioned in a new Trypanosoma clade, named T. terrestris clade.

Anuran trypanosomes: phylogenetic evidence for new clades in Brazil

Trypanosomes of anurans and fish are grouped into the Aquatic Clade which includes species isolated from fish, amphibians, turtles and platypus, usually transmitted by leeches and phlebotomine sand flies. Trypanosomes from Brazilian frogs are grouped within the Aquatic Clade with other anuran trypanosome species, where there seems to be coevolutionary patterns with vertebrate hosts and association to Brazilian biomes (Atlantic Forest, Pantanal and Amazonia Rainforest). We characterised the anuran trypanosomes from two different areas of the Cerrado biome and examined their phylogenetic relationships based on the SSU rRNA gene. A total of 112 anurans of six species was analysed and trypanosome prevalence evaluated through haemoculture was found to be 7% (8 positive frogs). However, only three isolates (2.7%) from two anuran species were recovered and cryopreserved. Analysis including SSU rDNA sequences from previous studies segregated the anuran trypanosomes into six groups, the previously reported An01 to An04, and An05 and An06 reported herein. Clade An05 comprises the isolates from Leptodactylus latrans (Steffen) and Pristimantis sp. captured in the Cerrado biome and Trypanosoma chattoni Mathis & Leger, 1911. The inclusion of new isolates in the phylogenetic analyses provided evidence for a new group (An06) of parasites from phlebotomine hosts. Our results indicate that the diversity of trypanosome species is underestimated since studies conducted in Brazil and other regions of the world are still few.

Rickettsia amblyommatis infecting ticks and exposure of domestic dogs to Rickettsia spp. in an Amazon-Cerrado transition region of northeastern Brazil

This study was performed in Maranhão state, a transition area two Brazilian biomes, Amazon and Cerrado. During 2011–2013, 1,560 domestic dogs were sampled for collection of serum blood samples and ticks in eight counties (3 within the Amazon and 5 within the Cerrado). A total of 959 ticks were collected on 150 dogs (9.6%). Rhipicephalus sanguineus sensu lato (s.l.) was the most abundant tick (68% of all collected specimens), followed by Amblyomma cajennense sensu lato (s.l.) (12.9%), Amblyomma parvum (9.2%), and Amblyomma ovale (5.2%). Other less abundant species (<1%) were Amblyomma oblongoguttatum, Rhipicephalus microplus, Haemaphysalis juxtakochi, and Amblyomma rotundatum. Females of A. cajennense s.l. ticks were morphologically identified as A. cajennense sensu stricto (s.s.) or A. sculptum. Molecular analyses of 779 canine ticks revealed three Rickettsia species: Rickettsia amblyommatis in 1% (1/100) A. cajennense s.l., ‘Candidatus Rickettsia andeanae’ in 20.7% (12/58) A. parvum, Rickettsia bellii in 6.8% (3/44) A. ovale and 100% (1/1) A. rotundatum ticks. An additional collection of A. sculptum from horses in a Cerrado area, and A. cajennense s.s. from pigs in an Amazon area revealed R. amblyommatis infecting only the A. cajennense s.s. ticks. Serological analysis of the 1,560 canine blood samples revealed 12.6% canine seroreactivity to Rickettsia spp., with the highest specific seroreactivity rate (10.2%) for R. amblyommatis. Endpoint titers to R. amblyommatis were significantly higher than those for the other Rickettsia antigens, suggesting that most of the seroreactive dogs were exposed to R. amblyommatis-infected ticks. Highest canine seroreactivity rates per locality (13.1–30.8%) were found in Amazon biome, where A. cajennense s.s. predominated. Lowest seroreactivity rates (1.9–6.5%) were found in Cerrado localities that were further from the Amazon, where A. sculptum predominated. Multivariate analyses revealed that canine seroreactivity to Rickettsia spp. or R. amblyommatis was statistically associated with rural dogs, exposed to Amblyomma ticks.