New insights on severe clinical manifestations and deaths from visceral leishmaniasis in free-living crab-eating foxes (Cerdocyon thous) in Brazil

Visceral leishmaniasis (VL) is a zoonotic and severe neglected tropical disease, with worldwide distribution, that still cause many deaths among dogs and humans. Brazil is the country responsible for about 97% of the cases of leishmaniasis in the Americas and the disease is still considered a serious public health concern. Among wild mammalians, studies demonstrate the involvement of Cerdocyon thous in the biological cycle of Leishmania. Nevertheless, several authors consider the clinical manifestation of the disease to be rare or mild in free-living animals. Herein, we demonstrate the occurrence of severe clinical sings and deaths caused by VL in free-living crab-eating foxes. Three specimens of foxes collected from periurban areas were diagnosed with VL. The animals presented cutaneous, ophthalmological, gastrointestinal, locomotor and hematological alterations, and died after clinical progression. We identified the presence of anti-Leishmania antibodies by immunochromatographic test in all specimens. We also observed intra and extracellular amastigotes in skin cytology and lymph node aspirate. Furthermore, Leishmania infantum DNA was identified in all samples by the polymer chain reaction technique. Additionally, we performed blood count and stool parasitological tests and observed hematological disorders common to VL, such as anemia and lymphopenia. Taken together, our data demonstrate that VL can induce clinical complications and even cause death in C. thous and corroborate that this crab-eating fox is an adequate host for L. infantum.

The Potent Trypanocidal Effect of LQB303, a Novel Redox-Active Phenyl-Tert-Butyl-Nitrone Derivate That Causes Mitochondrial Collapse in Trypanosoma cruzi

Chagas disease, which is caused by Trypanosoma cruzi, establishes lifelong infections in humans and other mammals that lead to severe cardiac and gastrointestinal complications despite the competent immune response of the hosts. Furthermore, it is a neglected disease that affects 8 million people worldwide. The scenario is even more frustrating since the main chemotherapy is based on benznidazole, a drug that presents severe side effects and low efficacy in the chronic phase of the disease. Thus, the search for new therapeutic alternatives is urgent. In the present study, we investigated the activity of a novel phenyl-tert-butyl-nitrone (PBN) derivate, LQB303, against T. cruzi. LQB303 presented trypanocidal effect against intracellular [IC50/48 h = 2.6 μM] and extracellular amastigotes [IC50/24 h = 3.3 μM] in vitro, leading to parasite lysis; however, it does not present any toxicity to host cells. Despite emerging evidence that mitochondrial metabolism is essential for amastigotes to grow inside mammalian cells, the mechanism of redox-active molecules that target T. cruzi mitochondrion is still poorly explored. Therefore, we investigated if LQB303 trypanocidal activity was related to the impairment of the mitochondrial function of amastigotes. The investigation showed there was a significant decrease compared to the baseline oxygen consumption rate (OCR) of LQB303-treated extracellular amastigotes of T. cruzi, as well as reduction of “proton leak” (the depletion of proton motive force by the inhibition of F1Fo ATP synthase) and “ETS” (maximal oxygen consumption after uncoupling) oxygen consumption rates. Interestingly, the residual respiration (“ROX”) enhanced about three times in LQB303-treated amastigotes. The spare respiratory capacity ratio (SRC: cell ability to meet new energy demands) and the ATP-linked OCR were also impaired by LQB303 treatment, correlating the trypanocidal activity of LQB303 with the impairment of mitochondrial redox metabolism of amastigotes. Flow cytometric analysis demonstrated a significant reduction of the ΔΨm of treated amastigotes. LQB303 had no significant influence on the OCR of treated mammalian cells, evidencing its specificity against T. cruzi mitochondrial metabolism. Our results suggest a promising trypanocidal activity of LQB303, associated with parasite bioenergetic inefficiency, with no influence on the host energy metabolism, a fact that may point to an attractive alternative therapy for Chagas disease.