title: 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: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Laboratório Interdisciplinar de Pesquisas Médicas, Instituto Oswaldo Cruz (FIOCRUZ/RJ), Rio de Janeiro, Brazil.
authors.forename: Raquel Peralva
authors.initials: RP
authors.lastname: Ribeiro-Romão
authors.affiliation: Laboratório Interdisciplinar de Pesquisas Médicas, Instituto Oswaldo Cruz (FIOCRUZ/RJ), Rio de Janeiro, Brazil.
authors.forename: Andrea Franco
authors.initials: AF
authors.lastname: Saavedra
authors.affiliation: Laboratório Interdisciplinar de Pesquisas Médicas, Instituto Oswaldo Cruz (FIOCRUZ/RJ), Rio de Janeiro, Brazil.
authors.forename: Alda Maria
authors.initials: AM
authors.lastname: Da-Cruz
authors.affiliation: Laboratório Interdisciplinar de Pesquisas Médicas, Instituto Oswaldo Cruz (FIOCRUZ/RJ), Rio de Janeiro, Brazil.
authors.forename: Eduardo Fonseca
authors.initials: EF
authors.lastname: Pinto
authors.affiliation: Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz (FIOCRUZ/RJ), Rio de Janeiro, Brazil. otacilio@ioc.fiocruz.br.
authors.forename: Otacilio C
authors.initials: OC
authors.lastname: Moreira
mesh_terms: D000818:Animals; D006224:Cricetinae; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D016054:DNA, Protozoan / Q000032:analysis / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation; D007892:Leishmania braziliensis; D016773:Leishmaniasis, Cutaneous / Q000097:blood / Q000276:immunology* / Q000469:parasitology; D008647:Mesocricetus; D059208:Parasite Load; D012333:RNA, Messenger; D016053:RNA, Protozoan; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012867:Skin / Q000469:parasitology
chemical_list: D016207:Cytokines; D016054:DNA, Protozoan; D012333:RNA, Messenger; D016053:RNA, Protozoan
keywords: Cytokine gene expression; Golden hamster; Leishmania (Viannia) braziliensis; Mesocricetus auratus; Parasite load; RT-qPCR; Real time PCR; qPCR