Development of real-time PCR assays for evaluation of immune response and parasite load in golden hamster (Mesocricetus auratus) infected by Leishmania (Viannia) braziliensis

Abstract

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.

Assessment of the intestinal immune response in Giardia duodenalis experimentally infected rats using quantitative real-time PCR

“…The results of the present study are consistent with those of Bayraktar et al [32] and Ribeiro-Romão et al [22] as we found that the recorded profile of cytokines revealed the presence of a mixed type of immune response (Th1 and Th2 cytokines) and the dominant production level related to Th1 cytokines (IFN-γ and TNF-α). A study conducted by Singer and Nash [7] disagreed with our results as they reported that neither a specific Th1 nor a specific Th2 response seem to be necessary for giardiasis.…”

“…In line with the evaluation of the immune response, the use of a reproducible, efficient and fast method to provide this precise assessment is extremely beneficial. Quantitative real time-PCR has been considered by many authors to be of great benefit for assessment of immune responses in giardiasis [18][19][20][21] and leishmaniasis [22] ; and is of greater sensitivity than many other procedures [23] . Despite all efforts made on this issue, the available knowledge about the mechanisms of the immune response to giardiasis is still unsatisfactory.…”

Efficacy of cryotherapy plus topical Juniperus excelsa M. Bieb cream versus cryotherapy plus placebo in the treatment of Old World cutaneous leishmaniasis: A triple-blind randomized controlled clinical trial

“…This finding may be due to technical errors in sampling of the tissue and/or processing of the PCR. On the other hand, it is worth mentioning that the sensitivity of PCR for detection of leishmaniasis is not perfect [ 96 ]. Four patients out of 33 in group A and one out of 29 patients in group B (a total of 5 patients with PCR-undetected strains of Leishmania) , had complete cure in this study.…”

Comparison of parasite load by qPCR and histopathological changes of inner and outer edge of ulcerated cutaneous lesions of cutaneous leishmaniasis

“…The DNA concentration obtained from the extraction was 9.1 ng/μL determined using the Qubit Fluorimeter ® . The Leishmania standard curve was built from 10-fold serial dilutions ranging from 1.0 x10 4 to 1.0 x10 -2 parasite DNA equivalents/reaction [ 12 ].…”