Oral and Intragastric: New Routes of Infection by Leishmania braziliensis and Leishmania infantum?

Although Leishmania transmission in nature is associated with the bite of an infected sandfly vector, other possible transmission routes are speculated to occur, such as the oral route. We evaluated the possibility of infection by this route in golden hamsters (Mesocricetus auratus) using Leishmania braziliensis (Lb) and Leishmania infantum (Li). Hamsters were exposed to experimental oral or intragastrical infection with axenic promastigotes, besides oral ingestion of a suspension of cultivated macrophages infected with amastigotes, lesion-fed Lutzomyia longipalpis, skin lesion or infective spleen fragment. The parasite's isolation, besides a positive PCR and IFAT, confirmed the intragastric infection by promastigote parasites. The oral ingestion of macrophages infected with L. braziliensis amastigotes was also infective. These results confirmed that Leishmania parasites could infect mammals by the intragastric route through the ingestion of promastigote forms (what can happen after a sandfly ingestion) and by the oral ingestion of infected macrophages (what can happen in nature in a predator-prey interaction). The better understanding of these alternative routes is essential to understand their transmission dynamics in nature. As far as we know, this is the first time that oral and intragastric Leishmania transmission has been experimentally demonstrated, constituting new infection routes, at least for L. infantum and L. braziliensis.

Landmarks of the Knowledge and Trypanosoma cruzi Biology in the Wild Environment.

Trypanosomatids are ancient parasitic eukaryotes that still maintain prokaryotic characteristics. Trypanosoma cruzi, a primarily wild mammal parasite, infected humans already long before European colonization of the Americas. T. cruzi heterogeneity remains an unsolved question, and until now, it has still not been possible to associate T. cruzi genotypes with any biological or epidemiological feature. One of the first biochemical attempts to cluster the T. cruzi subpopulations recognized three main subpopulations (zymodemes) that have been associated with the transmission cycles in the wild (Z1; Z3) and in the domestic environment (Z2). The description of wild mammal species harboring Z2 two decades later challenged this assemblage attempt. Currently, the genotypes of T. cruzi are assembled in seven discrete typing units (DTUs). The biology of T. cruzi still shows novelties such as the description of epimastigotes multiplying and differentiating to metacyclic trypomastigotes in the lumen of the scent glands of Didelphis spp. and the capacity of the true meiosis in parallel to clonal reproduction. The study of the transmission cycle among wild animals has broken paradigms and raised new questions: (i) the interaction of the T. cruzi DTUs with each of its mammalian host species displays peculiarities; (ii) the impact of mixed genotypes and species on the transmissibility of one or another species or on pathogenesis is still unknown; (iii) independent T. cruzi transmission cycles may occur in the same forest fragment; (iv) the capacity to act as a reservoir depends on the peculiarities of the host species and the parasite genotype; and (v) faunistic composition is a defining trait of the T. cruzi transmission cycle profile. The development of models of environmental variables that determine the spatial distribution of the elements that make up T. cruzi transmission by spatial analysis, followed by map algebra and networking, are the next steps toward interpreting and dealing with the new profile of Chagas disease with its many peculiarities. There is no way to solve this neglected disease once and for all if not through a multidisciplinary look that takes into account all kinds of human and animal activities in parallel to environmental variations.

Natural Trypanosoma (Trypanozoon) evansi (Steel, 1885) infection among mammals from Brazilian Amazon.

Trypanosoma evansi (Kinetoplastea Trypanosomatidae) is the Trypanosoma species that infects the greatest variety of mammals worldwide. In 2014, a dog from Rio Branco/AC, in the Brazilian Amazon region, presented flagellates without evident kinetoplasts in blood and symptoms of T. evansi infection. Our aim was to investigate the occurrence of T. evansi in dogs, bats and capybaras from Rio Branco. Blood was collected from 78 dogs from residential areas near the Zoobotanical Park (PZ). The serological diagnosis by IFAT detected the presence of anti-T. evansi antibodies in 21.9% of the evaluated dogs. T. evansi DNA was detected in one dog using a higly specific target of a repeated monomer of the satellite DNA of Trypanosoma (Trypanozoon) sp. Molecular diagnosis was also performed on 182 bat spleen samples collected inside PZ, and one Carollia perspicillata was positive. The DNA sequences obtained from these two samples showed similarities with T. brucei satellite DNA. Anti-T. evansi IFAT was carried out in 46 capybaras from rural and urban areas and the infection detected in 17.4% of them. We confirmed for the first time the presence of T. evansi in Acre State and describe three putative host species involved in the parasite transmission in that Amazon region. Moreover, this is the first study that confirms the infection by T. evansi through DNA sequence analysis in the Brazilian Amazon Region.

Trypanosoma janseni n. sp. (Trypanosomatida: Trypanosomatidae) isolated from Didelphis aurita (Mammalia: Didelphidae) in the Atlantic Rainforest of Rio de Janeiro, Brazil: integrative taxonomy and phylogeography within the Trypanosoma cruzi clade

BACKGROUND Didelphis spp. are a South American marsupial species that are among the most ancient hosts for the Trypanosoma spp. OBJECTIVES We characterise a new species (Trypanosoma janseni n. sp.) isolated from the spleen and liver tissues of Didelphis aurita in the Atlantic Rainforest of Rio de Janeiro, Brazil. METHODS The parasites were isolated and a growth curve was performed in NNN and Schneider's media containing 10% foetal bovine serum. Parasite morphology was evaluated via light microscopy on Giemsa-stained culture smears, as well as scanning and transmission electron microscopy. Molecular taxonomy was based on a partial region (737-bp) of the small subunit (18S) ribosomal RNA gene and 708 bp of the nuclear marker, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes. Maximum likelihood and Bayesian inference methods were used to perform a species coalescent analysis and to generate individual and concatenated gene trees. Divergence times among species that belong to the T. cruzi clade were also inferred. FINDINGS In vitro growth curves demonstrated a very short log phase, achieving a maximum growth rate at day 3 followed by a sharp decline. Only epimastigote forms were observed under light and scanning microscopy. Transmission electron microscopy analysis showed structures typical to Trypanosoma spp., except one structure that presented as single-membraned, usually grouped in stacks of three or four. Phylogeography analyses confirmed the distinct species status of T. janseni n. sp. within the T. cruzi clade. Trypanosoma janseni n. sp. clusters with T. wauwau in a well-supported clade, which is exclusive and monophyletic. The separation of the South American T. wauwau + T. janseni coincides with the separation of the Southern Super Continent. CONCLUSIONS This clade is a sister group of the trypanosomes found in Australian marsupials and its discovery sheds light on the initial diversification process based on what we currently know about the T. cruzi clade.

Trypanosoma janseni n. sp. (Trypanosomatida: Trypanosomatidae) isolated from Didelphis aurita (Mammalia: Didelphidae) in the Atlantic Rainforest of Rio de Janeiro, Brazil: integrative taxonomy and phylogeography within the Trypanosoma cruzi clade.

BACKGROUND: Didelphis spp. are a South American marsupial species that are among the most ancient hosts for the Trypanosoma spp. OBJECTIVES: We characterise a new species (Trypanosoma janseni n. sp.) isolated from the spleen and liver tissues of Didelphis aurita in the Atlantic Rainforest of Rio de Janeiro, Brazil. METHODS: The parasites were isolated and a growth curve was performed in NNN and Schneider's media containing 10% foetal bovine serum. Parasite morphology was evaluated via light microscopy on Giemsa-stained culture smears, as well as scanning and transmission electron microscopy. Molecular taxonomy was based on a partial region (737-bp) of the small subunit (18S) ribosomal RNA gene and 708 bp of the nuclear marker, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes. Maximum likelihood and Bayesian inference methods were used to perform a species coalescent analysis and to generate individual and concatenated gene trees. Divergence times among species that belong to the T. cruzi clade were also inferred. FINDINGS: In vitro growth curves demonstrated a very short log phase, achieving a maximum growth rate at day 3 followed by a sharp decline. Only epimastigote forms were observed under light and scanning microscopy. Transmission electron microscopy analysis showed structures typical to Trypanosoma spp., except one structure that presented as single-membraned, usually grouped in stacks of three or four. Phylogeography analyses confirmed the distinct species status of T. janseni n. sp. within the T. cruzi clade. Trypanosoma janseni n. sp. clusters with T. wauwau in a well-supported clade, which is exclusive and monophyletic. The separation of the South American T. wauwau + T. janseni coincides with the separation of the Southern Super Continent. CONCLUSIONS: This clade is a sister group of the trypanosomes found in Australian marsupials and its discovery sheds light on the initial diversification process based on what we currently know about the T. cruzi clade.

Trypanosoma sp. diversity in Amazonian bats (Chiroptera; Mammalia) from Acre State, Brazil.

Bats are ancient hosts of Trypanosoma species and their flying ability, longevity and adaptability to distinct environments indicate that they are efficient dispersers of parasites. Bats from Acre state (Amazon Biome) were collected in four expeditions conducted in an urban forest (Parque Zoobotânico) and one relatively more preserved area (Seringal Cahoeira) in Rio Branco and Xapuri municipalities. Trypanosoma sp. infection was detected by hemoculture and fresh blood examination. Isolated parasite species were identified by the similarity of the obtained DNA sequence from 18S rDNA polymerase chain reaction and reference strains. Overall, 367 bats from 23 genera and 32 species were examined. Chiropterofauna composition was specific to each municipality, although Artibeus sp. and Carollia sp. prevailed throughout. Trypanosoma sp. infection was detected in 85 bats (23·2%). The most widely distributed and prevalent genotypes were (in order) Trypanosoma cruzi TcI, T. cruzi marinkellei, Trypanosoma dionisii, T. cruzi TcIV and Trypanosoma rangeli. At least one still-undescribed Trypanosoma species was also detected in this study. The detection of T. cruzi TcI and TcIV (the ones associated with Chagas disease in Amazon biome) demonstrates the putative importance of these mammal hosts in the epidemiology of the disease in the Acre State.

The multiple and complex and changeable scenarios of the Trypanosoma cruzi transmission cycle in the sylvatic environment.

In this study, we report and discuss the results generated from over 20 years of studies of the Trypanosoma cruzi sylvatic transmission cycle. Our results have uncovered new aspects and reviewed old concepts on issues including reservoirs, true generalist species, association of mammalian species with distinct discrete typing units - DTUs, distribution of T. cruzi genotypes in the wild, mixed infections, and T. cruzi transmission ecology. Using parasitological and serological tests, we examined T. cruzi infection in 7,285 mammalian specimens from nine mammalian orders dispersed all over the Brazilian biomes. The obtained T. cruzi isolates were characterized by mini-exon gene sequence polymorphism and PCR RFLP to identify DTUs. Infection by T. cruzi was detected by serological methods in 20% of the examined animals and isolated from 41% of those infected, corresponding to 8% of all the examined mammals. Each mammal taxon responded uniquely to T. cruzi infection. Didelphis spp. are able to maintain high and long-lasting parasitemias (positive hemocultures) caused by TcI but maintain and rapidly control parasitemias caused by TcII to almost undetectable levels. In contrast, the tamarin species Leontopithecus rosalia and L. chrysomelas maintain long-lasting and high parasitemias caused by TcII similarly to Philander sp. The coati Nasua nasua maintains high parasitemias by both parental T. cruzi DTUs TcI or TcII and by TcII/TcIV (formerly Z3) at detectable levels. Wild and domestic canidae seem to display only a short period of reservoir competence. T. cruzi infection was demonstrated in the wild canid species Cerdocyon thous and Chrysocyon brachyurus, and positive hemoculture was obtained in one hyper carnivore species (Leopardus pardalis), demonstrating that T. cruzi transmission is deeply immersed in the trophic net. T. cruzi DTU distribution in nature did not exhibit any association with a particular biome or habitat. TcI predominates throughout (58% of the T. cruzi isolates); however, in spite of being significantly less frequent (17%), TcII is also widely distributed. Concomitant DTU infection occurred in 16% of infected mammals of all biomes and included arboreal and terrestrial species, as well as bats. TcI/TcII concomitant infection was the most common and widely dispersed, with mixed TcI/TcII infections especially common in coatis and in Didelphimorphia. The second most common pattern of concomitant infection was TcI/TcIV, observed in Chiroptera, Didelphimorphia and Primates. Taken together, our results demonstrate the complexity of T. cruzi reservoir system and its transmission strategies, indicating that there is considerably more to be learned regarding ecology of T. cruzi.

Autochthonous transmission of Chagas disease in Rio de Janeiro State, Brazil: a clinical and eco-epidemiological study.

BACKGROUND: After the control of the main modes of Chagas disease (CD) transmission in most endemic countries, it is important to identify the participation of native sylvatic vectors in CD transmission. Although CD is not considered endemic in Rio de Janeiro State (RJ), Brazil, we identified patients with CD born in RJ and investigated the possible autochthonous transmission in the state. METHODS: Patients born in RJ and followed in our institution between 1986 and 2011 were retrospectively analyzed. The cases identified as autochthonous transmission were submitted to epidemiological, clinical, serological, parasitological and molecular studies. Sectional field study with serological survey, research of sylvatic reservoirs and vectors was conducted in rural areas where patients were born. RESULTS: Among 1963 patients, 69 (3.5%) were born in RJ. From these, 15 (21.7%) were considered to have acquired the infection by autochthonous transmission. Cardiac form was the commonest form of presentation (60%). In rural areas in RJ northern region, sylvatic cycles of Trypanosoma cruzi and domestic invasion by Triatoma vitticeps were identified, and CD prevalence among inhabitants was 0.74%.TcI genotype was identified in sylvatic reservoirs and vectors. The genotype (mixed infection TcI/TcVI) could be identified in one of the autochthonous cases. CONCLUSIONS: The autochthonous vectorial transmission of CD occurs in RJ, probably due to wild cycles of T. cruzi and sylvatic vectors, such as T. vitticeps. Therefore, the health authorities should evaluate if RJ should be included in the original endemic area of CD and CD should be included in the diagnostic work out of cardiomyopathy of patients born in RJ. Moreover, control and educational measures should be put into place in the risk areas.

Distinct Leishmania species infecting wild caviomorph rodents (Rodentia: Hystricognathi) from Brazil.

BACKGROUND: Caviomorph rodents, some of the oldest Leishmania spp. hosts, are widely dispersed in Brazil. Despite both experimental and field studies having suggested that these rodents are potential reservoirs of Leishmania parasites, not more than 88 specimens were analyzed in the few studies of natural infection. Our hypothesis was that caviomorph rodents are inserted in the transmission cycles of Leishmania in different regions, more so than is currently recognized. METHODOLOGY: We investigated the Leishmania infection in spleen fragments of 373 caviomorph rodents from 20 different species collected in five Brazilian biomes in a period of 13 years. PCR reactions targeting kDNA of Leishmania sp. were used to diagnose infection, while Leishmania species identification was performed by DNA sequencing of the amplified products obtained in the HSP70 (234) targeting. Serology by IFAT was performed on the available serum of these rodents. PRINCIPAL FINDINGS: In 13 caviomorph rodents, DNA sequencing analyses allowed the identification of 4 species of the subgenus L. (Viannia): L. shawi, L. guyanensis, L. naiffi, and L. braziliensis; and 1 species of the subgenus L. (Leishmania): L. infantum. These include the description of parasite species in areas not previously included in their known distribution: L. shawi in Thrichomys inermis from Northeastern Brazil and L. naiffi in T. fosteri from Western Brazil. From the four other positive rodents, two were positive for HSP70 (234) targeting but did not generate sequences that enabled the species identification, and another two were positive only in kDNA targeting. CONCLUSIONS/SIGNIFICANCE: The infection rate demonstrated by the serology (51.3%) points out that the natural Leishmania infection in caviomorph rodents is much higher than that observed in the molecular diagnosis (4.6%), highlighting that, in terms of the host species responsible for maintaining Leishmania species in the wild, our current knowledge represents only the "tip of the iceberg."

Wild and synanthropic reservoirs of Leishmania species in the Americas.

The definition of a reservoir has changed significantly in the last century, making it necessary to study zoonosis from a broader perspective. One important example is that of Leishmania, zoonotic multi-host parasites maintained by several mammal species in nature. The magnitude of the health problem represented by leishmaniasis combined with the complexity of its epidemiology make it necessary to clarify all of the links in transmission net, including non-human mammalian hosts, to develop effective control strategies. Although some studies have described dozens of species infected with these parasites, only a minority have related their findings to the ecological scenario to indicate a possible role of that host in parasite maintenance and transmission. Currently, it is accepted that a reservoir may be one or a complex of species responsible for maintaining the parasite in nature. A reservoir system should be considered unique on a given spatiotemporal scale. In fact, the transmission of Leishmania species in the wild still represents an complex enzootic "puzzle", as several links have not been identified. This review presents the mammalian species known to be infected with Leishmania spp. in the Americas, highlighting those that are able to maintain and act as a source of the parasite in nature (and are thus considered potential reservoirs). These host/reservoirs are presented separately in each of seven mammal orders - Marsupialia, Cingulata, Pilosa, Rodentia, Primata, Carnivora, and Chiroptera - responsible for maintaining Leishmania species in the wild.

Trypanosoma cruzi among wild and domestic mammals in different areas of the Abaetetuba municipality (Pará State, Brazil), an endemic Chagas disease transmission area.

The presence of acute Chagas disease (ACD) due to oral transmission is growing and expanding in several South American countries. Within the Amazon basin, the Abaetetuba municipality has been a site of recurrent cases spanning across distinct landscapes. Because Chagas disease is primarily a zoonotic infection, we compared the enzootic Trypanosoma cruzi transmission cycles in three different environmental areas of Abaetetuba to better understand this new epidemiological situation. Philander opossum was the most abundant mammalian species collected (38% of the collected mammals) with a T. cruzi prevalence of 57%, as determined by hemocultures. Didelphis marsupialis was abundant only in the area with the higher level of environmental disturbance (approximately 42%) and did not yield detectable parasitemia. Despite similarities observed in the composition of the small mammalian fauna and the prevalence of T. cruzi infection among the studied areas, the potential of these hosts to infect vectors differed significantly according to the degree of land use (with prevalences of 5%, 41%, and 64% in areas A3, A1 and A2, respectively). Domestic mammals were also found to be infected, and one canine T. cruzi isolate was obtained. Our data demonstrated that the transmission of T. cruzi in the Amazon basin is far more complex than had been previously taught and showed that the probability of humans and domestic mammals coming into contact with infected bugs can vary dramatically, even within the same municipality. The exposure of dogs to T. cruzi infection (indicated by positive serology) was the common feature among the studied localities, stressing the importance of selecting domestic mammals as sentinels in the identification of T. cruzi transmission hotspots.

Mixed infection in the anteater Tamandua tetradactyla (Mammalia: Pilosa) from Pará State, Brazil: Trypanosoma cruzi, T. rangeli and Leishmania infantum.

Some Trypanosoma and Leishmania species are multi-host parasites whose distribution overlaps in several parts of the Brazilian Amazon basin. Despite being a common trait among wild mammals, mixed infections and their consequences for the host's health and parasite transmission are still a poorly known phenomenon. Here we describe a triple mixed infection - Trypanosoma cruzi, T. rangeli and Leishmania infantum - in a bone marrow sample from an anteater Tamandua tetradactyla captured in a house backyard from the endemic Abaetetuba municipality in the Amazon basin. T. cruzi was also isolated from blood samples. The mini-exon multiplex PCR characterization detected the infection by T. rangeli and T. cruzi (TcI genotype), while L. infantum infection was confirmed by an ITS-PCR followed by amplicon sequencing. This is the first description of T. rangeli isolation from bone marrow and the first report of L. infantum infection in xenarthrans. The implications of this finding are discussed considering the influence of mixed infections in the role of this mammal species as a putative reservoir host of these 3 trypanosomatid species.

Trypanosoma cruzi transmission cycle among wild and domestic mammals in three areas of orally transmitted Chagas disease outbreaks.

We report Trypanosoma cruzi infection in wild and domestic mammals from three orally acquired Chagas disease outbreak areas in Brazil. Cachoeiro do Arari (Pará) displayed a panzootic scenery (positive mammals in all ecologic strata), and human cases were probably the consequence of their exposure within the sylvatic T. cruzi transmission cycle. In Navegantes (Santa Catarina), Didelphis spp. was the main reservoir host, given that 93% were infected. In Redenção (Ceará), Monodelphis domestica and Thrichomys laurentius were also important for parasite maintenance. TCI was present in the three studied areas. Additionally, Z3 was detected in an armadillo from Pará and TCII in a triatomine from Navegantes. Domestic animals showed a high seroprevalence and should be considered sentinels in surveillance programs. The importance of a reduction in wild mammalian fauna diversity and selection of suitable T. cruzi reservoir hosts are discussed as risk factors for the re-emergence of Chagas disease.

Maintenance and breeding of Thrichomys (Trouessart, 1880) (Rodentia: Echimyidae) in captivity.

South American histricognath rodents Thrichomys apereoides laurentius and Thrichomys pachyurus are natural hosts of Trypanosoma cruzi, agent of Chagas disease. We established breeding colonies of these species to serve as experimental models in various parasitological studies. Both species of Thrichomys have all the requirements necessary to become excellent laboratory models: they can be easily maintained in the standard laboratory conditions and breed throughout the year and they do not have any special dietary demands and can be fed by standard food pellets designed for laboratory mice. Both species produce precocious offspring that have their eyes and ears open, teeth erupted, fur well developed, and can eat solid food in the first week of life. T. a. laurentius has larger litter sizes and lower body masses at birth and weaning than T. pachyurus. Moreover, females of T. a. laurentius reach puberty earlier and with lower body mass than T. pachyurus.

Maintenance and breeding of Thrichomys (Trouessart, 1880) (Rodentia: Echimyidae) in captivity

South American histricognath rodents Thrichomys apereoides laurentius and Thrichomys pachyurus are natural hosts of Trypanosoma cruzi, agent of Chagas disease. We established breeding colonies of these species to serve as experimental models in various parasitological studies. Both species of Thrichomys have all the requirements necessary to become excellent laboratory models: they can be easily maintained in the standard laboratory conditions and breed throughout the year and they do not have any special dietary demands and can be fed by standard food pellets designed for laboratory mice. Both species produce precocious offspring that have their eyes and ears open, teeth erupted, fur well developed, and can eat solid food in the first week of life. T. a. laurentius has larger litter sizes and lower body masses at birth and weaning than T. pachyurus. Moreover, females of T. a. laurentius reach puberty earlier and with lower body mass than T. pachyurus.