Development of real-time PCR assays for evaluation of immune response and parasite load in golden hamster (Mesocricetus auratus) infected by Leishmania (Viannia) braziliensis.
BACKGROUND
Cutaneous leishmaniasis (CL) is a neglected disease with a broad spectrum of clinical manifestations, ranging from small cutaneous nodules to severe mucosal tissue destruction. Leishmania (Viannia) braziliensis is the main species attributed to CL in the Americas. However, studies of experimental infection are limited in the murine model due to the self-resolutive pattern of the disease. Previously, our group demonstrated that the hamster model reproduces many of the clinical and histopathological features observed in humans. Herein, we standardized a RT-qPCR gene expression assay to evaluate a panel of immunological markers and a qPCR assay in order to quantify with high sensitivity and reproducibility the parasite load in skin lesions.


METHODS
Hamsters were intradermally infected in the footpad with 10(5) promastigotes of L. (V.) braziliensis and 110 days post-infection skin lesions and popliteal lymph nodes were removed for RNA and DNA extraction, both from the same tissue fragment. Gene expression of IFN-ɣ, IL-10, TGF-β TNF, IL-4, IL-6, iNOS and arginase were measured using non-infected animal tissue as a calibrator. Parasite load was quantified from DNA extracted from lesions by qPCR targeting Leishmania kDNA and normalized by hamster GAPDH, using a SYBR Green-based absolute quantification methodology.


RESULTS
A relative quantification RT-qPCR assay was standardized for the evaluation of mRNA levels from skin and lymph node samples of golden hamsters, with PCR efficiencies ranging from 92.3 to 116.4 %. In uninfected animals, higher basal mRNA levels in lymph nodes were observed for IFN-ɣ, TGF-β, TNF and IL-4 (111.4 ± 92.2; 5.6 ± 1.2; 5.3 ± 1.7; and 60.3 ± 26.8, respectively) in comparison to skin. In golden hamsters infected with L. (V.) braziliensis, an increase in the expression of all immunological markers evaluated was observed, ranging from 2.7 ± 0.2 for TGF-β to 1018.5 ± 809.0 for iNOS in skin lesions, and 2.4 ± 1.6 for TGF-β to 600.2 ± 666.4 for iNOS in popliteal lymph nodes. Interestingly, significantly higher levels of IFN-ɣ, TNF and IL-10 mRNA were observed in skin in comparison to lymph nodes, while a lower significant level of arginase mRNA was observed in skin. In parallel, parasite loads were quantified by qPCR from the skin lesions of infected animals, ranging from 27.0 to 6647.0, with a median of 553.4 (416.7-1504.0) parasites/mg skin equivalents, whereas lesion size varied from 0.3 to 3.1 mm. Despite the tendency of larger lesions to present higher parasite load, the correlation observed was not statistically significant.


CONCLUSIONS
In this study, we describe for the first time a sensitive, reproducible and cheaper molecular assay to quantify from the same tissue fragment the gene expression of immunological markers and the parasite load in skin lesions, observing a mixed profile of immune response in the hamster model infected by L. (V.) braziliensis.
['Ribeiro-Romão|Raquel Peralva|RP|', 'Saavedra|Andrea Franco|AF|', 'Da-Cruz|Alda Maria|AM|', 'Pinto|Eduardo Fonseca|EF|', 'Moreira|Otacilio C|OC|']
D000818:Animals D006224:Cricetinae D016207:Cytokines D016054:DNA, Protozoan D005260:Female D005786:Gene Expression Regulation D007892:Leishmania braziliensis D016773:Leishmaniasis, Cutaneous D008647:Mesocricetus D059208:Parasite Load D012333:RNA, Messenger D016053:RNA, Protozoan D060888:Real-Time Polymerase Chain Reaction D012680:Sensitivity and Specificity D012867:Skin
Parasite load Golden hamster Mesocricetus auratus Leishmania (Viannia) braziliensis RT-qPCR qPCR Cytokine gene expression Real time PCR