Enhanced apoptosis and inflammation allied with autophagic and apoptotic Leishmania amastigotes in the seemingly undamaged ear skin of clinically affected dogs with canine visceral Leishmaniasis

Programmed cell death plays a relevant role in the pathogenesis of visceral Leishmaniasis. Apoptosis selects suitable parasites, regulating parasite density, whereas autophagy eliminates pathogens. This study aimed to assess the inflammation and apoptosis in inflammatory cells and presents a unique description of the presence of autophagic and apoptotic Leishmania amastigotes in naturally Leishmania-infected dogs. Fragments from seemingly undamaged ear skin of sixteen Leishmania-infected dogs and seven uninfected dogs were evaluated through histomorphometry, ultrastructural, immunohistochemical and transmission electron microscopy (TEM) analyses. Leishmania amastigotes were present on seemingly undamaged ear skin only in clinically affected dogs. Parasite load, morphometrical parameters of inflammation and apoptotic index of inflammatory cells were higher in clinically affected animals and were related to clinical manifestations. Apoptotic index and morphometric parameters of the inflammatory infiltrate in undamaged ear skin were positively correlated with parasite load. Apoptotic and non-apoptotic Leishmania amastigotes were observed within neutrophils and macrophages. Leishmania amastigotes were positive for Bax, a marker for apoptosis, by immunohistochemistry. Morphological characteristics of apoptosis and autophagy in Leishmania amastigotes were observed only in phagocytes of clinically affected dogs. Positive correlations were found between histomorphometry and clinical manifestations. Our results showed that apoptosis and autophagy in Leishmania amastigotes may be related to both the increase in parasite load and apoptotic index in inflammatory cells, and with the intensity of the inflammatory response in clinically affected dogs. Thus, our study suggests that apoptotic and autophagy Leishmania within phagocytes may have facilitate the survival of the parasite and it appears to play an important role in the process of Leishmania infection.