Leishmania 360°: Guidelines for Exosomal Research.

Leishmania parasites are a group of kinetoplastid pathogens that cause a variety of clinical disorders while maintaining cell communication by secreting extracellular vesicles. Emerging technologies have been adapted for the study of Leishmania-host cell interactions, to enable the broad-scale analysis of the extracellular vesicles of this parasite. Leishmania extracellular vesicles (LEVs) are spheroidal nanoparticles of polydispersed suspensions surrounded by a layer of lipid membrane. Although LEVs have attracted increasing attention from researchers, many aspects of their biology remain unclear, including their bioavailability and function in the complex molecular mechanisms of pathogenesis. Given the importance of LEVs in the parasite-host interaction, and in the parasite-parasite relationships that have emerged during the evolutionary history of these organisms, the present review provides an overview of the available data on Leishmania, and formulates guidelines for LEV research. We conclude by reporting direct methods for the isolation of specific LEVs from the culture supernatant of the promastigotes and amastigotes that are suitable for a range of different downstream applications, which increases the compatibility and reproducibility of the approach for the establishment of optimal and comparable isolation conditions and the complete characterization of the LEV, as well as the critical immunomodulatory events triggered by this important group of parasites.

African Trypanosomiasis: Extracellular Vesicles Shed by Trypanosoma brucei brucei Manipulate Host Mononuclear Cells.

African trypanosomiasis or sleeping sickness is a zoonotic disease caused by Trypanosoma brucei, a protozoan parasite transmitted by Glossina spp. (tsetse fly). Parasite introduction into mammal hosts triggers a succession of events, involving both innate and adaptive immunity. Macrophages (MΦ) have a key role in innate defence since they are antigen-presenting cells and have a microbicidal function essential for trypanosome clearance. Adaptive immune defence is carried out by lymphocytes, especially by T cells that promote an integrated immune response. Like mammal cells, T. b. brucei parasites release extracellular vesicles (TbEVs), which carry macromolecules that can be transferred to host cells, transmitting biological information able to manipulate cell immune response. However, the exact role of TbEVs in host immune response remains poorly understood. Thus, the current study examined the effect elicited by TbEVs on MΦ and T lymphocytes. A combined approach of microscopy, nanoparticle tracking analysis, multiparametric flow cytometry, colourimetric assays and detailed statistical analyses were used to evaluate the influence of TbEVs in mouse mononuclear cells. It was shown that TbEVs can establish direct communication with cells of innate and adaptative immunity. TbEVs induce the differentiation of both M1- and M2-MΦ and elicit the expansion of MHCI+, MHCII+ and MHCI+MHCII+ MΦ subpopulations. In T lymphocytes, TbEVs drive the overexpression of cell-surface CD3 and the nuclear factor FoxP3, which lead to the differentiation of regulatory CD4+ and CD8+ T cells. Moreover, this study indicates that T. b. brucei and TbEVs seem to display opposite but complementary effects in the host, establishing a balance between parasite growth and controlled immune response, at least during the early phase of infection.

Cutaneous Leishmaniasis: The Complexity of Host's Effective Immune Response against a Polymorphic Parasitic Disease.

This review is aimed at providing a comprehensive outline of the immune response displayed against cutaneous leishmaniasis (CL), the more common zoonotic infection caused by protozoan parasites of the genus Leishmania. Although of polymorphic clinical presentation, classically CL is characterized by leishmaniotic lesions on the face and extremities of the patients, which can be ulcerative, and even after healing can lead to permanent injuries and disfigurement, affecting significantly their psychological, social, and economic well-being. According a report released by the World Health Organization, the disability-adjusted life years (DALYs) lost due to leishmaniasis are close to 2.4 million, annually there are 1.0-1.5 million new cases of CL, and a numerous population is at risk in the endemic areas. Despite its increasing worldwide incidence, it is one of the so-called neglected tropical diseases. Furthermore, this review provides an overview of the existing knowledge of the host innate and acquired immune response to cutaneous species of Leishmania. The use of animal models and of in vitro studies has improved the understanding of parasite-host interplay and the complexity of immune mechanisms involved. The importance of diagnosis accuracy associated with effective patient management in CL reduction is highlighted. However, the multiple factors involved in CL epizoology associated with the unavailability of vaccines or drugs to prevent infection make difficult to formulate an effective strategy for CL control.

Intracellular and extracellular effector activity of mouse neutrophils in response to cutaneous and visceral Leishmania parasites.

Neutrophils are short-lived phagocytic cells equipped with several receptors for pathogen recognition and phagocytosis and have intracellular and extracellular effector mechanisms that can inactivate pathogens. Leishmaniases are diseases caused by different species of Leishmania that mainly afflicts poorer populations of tropical and subtropical regions and immunocompromised individuals. Thus, the present study aims to investigate the effector response of murine neutrophils to species of Leishmania causing American cutaneous leishmaniasis and zoonotic visceral leishmaniasis by evaluating pattern recognition receptors (PRR) and intracellular and extracellular effector microbicide activity. When exposed to Leishmania parasites, mouse neutrophils produced superoxide, released enzymes in the extracellular space and generated neutrophil extracellular traps, although PRR gene expression is negatively regulated. L. infantum, L. guyanensis, and L. shawi inhibited enzymatic activity, whereas L. amazonensis reduced the emission of extracellular structures. These findings indicate that although neutrophils trigger several microbicide mechanisms, Leishmania parasites can manipulate extracellular effector mechanisms. The present study also provides evidence that neutrophils can internalize parasites by coiling phagocytosis.

Occurrence of eggs and oocysts of gastrointestinal parasites in passerine birds kept in captivity in Para State, Brazil / Ocorrência de ovos e oocistos de parasitos gastrointestinais em aves Passeriformes mantidas em cativeiro no estado do Pará, Brasil

ABSTRACT: The objective of this study was to detect helminth eggs and protozoan oocysts in samples of feces from birds of the order Passeriformes in Para State, Brazil. Fecal samples were collected individually from 403 passerine birds seized and kept in captivity in Para State. Samples were processed by the double centrifugation technique in saturated sucrose solution and the coccidial oocyst-positive samples were submitted to sporulation in potassium dichromate 2.0%. Helminth eggs and/or protozoan oocysts were observed in 43.18% (174/403) of the fecal samples examined. Coccidial oocysts were detected in 93.68% (163/174) of the positive samples, whereas helminth eggs were observed in 10.34% (18/174) of the positive samples. Oocyst sporulation occurred in 43.56% (71/163) of the samples, and only Isospora spp. oocysts were detected. Nematode eggs of the superfamilies Trichostrongyloidea (4.60%; 8/174), Ascaridoidea (0.57%; 1/174), and Trichuroidea (0.57%; 1/174) were diagnosed in the positive samples. Cestoda eggs were diagnosed in 2.87% (5/174), whereas Trematoda eggs were detected in 2.30% (4/174) of positive samples. Passerine birds seized and kept in captivity in the visited local presented parasitism by intestinal helminths and protozoan, with a predominance of infection with coccidia of the gender Isospora.

RESUMO: O objetivo do presente estudo foi detectar ovos de helmintos e oocistos de protozoários em amostras de fezes de aves da ordem Passeriformes no estado do Pará, Brasil. Amostras de fezes foram coletadas individualmente de 403 aves Passeriformes oriundas de apreensão e mantidas em cativeiro no estado do Pará. As amostras foram processadas usando a técnica de dupla centrifugação em solução saturada de sacarose e as amostras positivas para oocistos de coccídios foram submetidas à esporulação em dicromato de potássio 2,0%. Ovos de helmintos e/ou oocistos de protozoários foram observados em 43,18% (174/403) das amostras fecais examinadas. Oocistos de coccídios foram detectados em 93,68% (163/174) das amostras positivas, enquanto que ovos de helmintos foram observados em 10,34% (18/174). A esporulação de oocistos ocorreu em 43,56% (71/163) das amostras, e somente oocistos de Isospora spp. foram detectados. Ovos de nematódeos das Superfamílias Trichostrongyloidea (4,60%; 8/174), Ascaridoidea (0,57%; 1/174) e Trichuroidea (0,57%; 1/174) foram diagnosticados nas amostras positivas. Ovos de Cestoda foram diagnosticados em 2,87% (5/174), enquanto que ovos de Trematoda foram detectados em 2,30% (4/174) das amostras positivas. Aves Passeriformes oriundas de apreensão e mantidas em cativeiro nas áreas visitadas estavam parasitadas por helmintos e protozoários, predominando a infecção por coccídios do gênero Isospora.

Molecular detection of Trypanosoma evansi (Kinetoplastida: Trypanosomatidae) in procyonids (Carnivora: Procyonidae) in Eastern Amazon, Brazil / Detecção molecular de Trypanosoma evansi (Kinetoplastida: Trypanosomatidae) em procionídeos (Carnivora: Procyonidae) na Amazônia Oriental, Brasil

ABSTRACT: The present study aimed to diagnose the natural infection of captive and free-living procyonids with Trypanosoma evansi in the states of Amapá and Pará, Brazil. From February 2012 to August 2013, whole blood samples and blood smears were obtained from 45 free-living procyonids and from nine procyonids kept in captivity in wild life refuges and zoobotanical parks in the states of Amapá and Pará. Whole blood samples were collected and kept at -20ºC for the detection of T. evansi DNA by PCR using the RoTat 1.2 forward and RoTat 1.2 reverse primers. In addition, the blood smears were processed and examined for the presence of trypomastigote forms of T. evansi. T. evansi DNA was detected in 18.52% (10/54) of the procyonids, namely, in captive crab-eating raccoons and captive and free-living coatis in Pará State. No trypomastigote forms were observed in the blood smears. DNA from T. evansi was detected in P. cancrivorus and N. nasua in Pará State, being this the first such report in P. cancrivorus.

RESUMO: O objetivo do presente trabalho foi realizar o diagnóstico da infecção natural por Trypanosoma evansi em procionídeos de vida livre e de cativeiro dos estados do Amapá e Pará, Brasil. Durante o período de fevereiro de 2012 a agosto de 2013, amostras de sangue total e esfregaços sanguíneos foram obtidos de 45 procionídeos de vida livre e de nove mantidos em cativeiro em mantenedores e Parques Zoobotânicos dos estados do Amapá e Pará. As amostras de sangue total foram coletadas e mantidas a -20ºC para pesquisa de DNA de T. evansi pela PCR utilizando-se os iniciadores RoTat 1.2 forward e RoTat 1.2 reverse. Os esfregaços sanguíneos também foram processados e examinados para a pesquisa de formas tripomastigotas do agente. DNA de T. evansi foi detectado em 18,52% (10/54) dos procionídeos, ocorrendo em mãos-peladas de cativeiro e quatis de vida livre e de cativeiro no estado do Pará. Não foram observadas formas tripomastigotas nos esfregaços sanguíneos. DNA de T. evansi foi detectado em P. cancrivorus e N. nasua no estado do Pará, sendo este o primeiro relato em P. cancrivorus.