Higher Expression of CCL2, CCL4, CCL5, CCL21, and CXCL8 Chemokines in the Skin Associated with Parasite Density in Canine Visceral Leishmaniasis

Several previous studies correlated immunopathological aspects of canine visceral leishmaniasis (CVL) with tissue parasite load and/or the clinical status of the disease. Recently, different aspects of the immune response in Leishmania-infected dogs have been studied, particularly the profile of cytokines in distinct compartments. However, the role of chemokines in disease progression or parasite burdens of the visceralising species represents an important approach for understanding immunopathology in CVL. We found an increase in inflammatory infiltrate, which was mainly composed of mononuclear cells, in the skin of animals presenting severe forms of CVL and high parasite density. Our data also demonstrated that enhanced parasite density is positively correlated with the expression of CCL2, CCL4, CCL5, CCL21, and CXCL8. In contrast, there was a negative correlation between parasite density and CCL24 expression. These findings represent an advance in the knowledge of the involvement of skin inflammatory infiltrates in CVL and the systemic consequences and may contribute to developing a rational strategy for the design of new and more efficient prophylactic tools and immunological therapies against CVL

Canine visceral leishmaniasis biomarkers and their employment in vaccines.

The natural history of canine visceral leishmaniasis (CVL) has been well described, particularly with respect to the parasite load in different tissues and immunopathological changes according to the progression of clinical forms. The biomarkers evaluated in these studies provide support for the improvement of the tools used in developing vaccines against CVL. Thus, we describe the major studies using the dog model that supplies the rationale for including different biomarkers (tissue parasitism, histopathology, hematological changes, leucocytes immunophenotyping, cytokines patterns, and in vitro co-culture systems using purified T-cells subsets and macrophages infected with L. infantum) for immunogenicity and protection evaluations in phases I and II applied to pre-clinical and clinical vaccine trials against CVL. The search for biomarkers related to resistance or susceptibility has revealed a mixed cytokine profile with a prominent proinflammatory immune response as relevant for Leishmania replication at low levels as observed in asymptomatic dogs (highlighted by high levels of IFN-? and TNF-? and decreased levels in IL-4, TGF-? and IL-10). Furthermore, increased levels in CD4+ and CD8+ T-cell subsets, presenting intracytoplasmic proinflammatory cytokine balance, have been associated with a resistance profile against CVL. In contrast, a polyclonal B-cell expansion towards plasma cell differentiation contributes to high antibody production, which is the hallmark of symptomatic dogs associated with high susceptibility in CVL. Finally, the different studies used to analyze biomarkers have been incorporated into vaccine immunogenicity and protection evaluations. Those biomarkers identified as resistance or susceptibility markers in CVL have been used to evaluate the vaccine performance against L. infantum in a kennel trial conducted before the field trial in an area known to be endemic for visceral leishmaniasis. This rationale has been a guiding force in the testing and selection of the best vaccine candidates against CVL and provides a way for the veterinary industry to register commercial immunobiological products

Parasite Burden in Hamsters Infected with Two Different Strains of Leishmania (Leishmania) infantum: “Leishman Donovan Units” versus Real-Time PCR

To develop and test new therapeutics and immune prophylaxis strategies for visceral leishmaniasis (VL), understanding tissue parasitism evolution after experimental infection with Leishmania infantum is important. Experimental infection in a hamster model (Mesocricetus auratus) reproduces several typical aspects of canine and human VL that are closely related to the inoculum’s route. We quantified the parasitism in the liver and spleen of hamsters experimentally infected by various routes (intradermal, intraperitoneal, and intracardiac [IC]) and different strains of L. infantum (MHOM/BR/74/PP75 and Wild) and compared two different methodologies to evaluate tissue parasitism (Leishman Donovan units [LDU] and real-time qPCR). In addition, the quantification of specific total-IgG in the serum of uninfected and infected hamsters was determined by ELISA. The animals were followed for 1, 3, 6 and 9 months post-infection for survival analysis. We found that infection with the Wild strain by the IC route resulted in higher mortality. Positive antibody (IgG) responses were detected with higher peaks at 6 and 9 months in the IC group inoculated with PP75 strain. However, in animals infected with the Wild strain the IgG levels were elevated in all infected groups during all the time evaluated. We also observed by LDU analysis that the IC route lead to higher parasitism in the liver and spleen with both strains. Furthermore, qPCR showed higher sensitivity for identifying animals with low parasitic burden. In conclusion, qPCR can be useful for assessing parasitism in the spleen and liver of a hamster model infected with L. infantum independent of the route of infection, and this technique may become an essential tool for assessing parasite density in the hamster model after experimental treatment or immunization with potential vaccine candidates