Exploring the immune signalling pathway-related genes of the cattle tick Rhipicephalus microplus: From molecular characterization to transcriptional profile upon microbial challenge.

In dipteran insects, invading pathogens are selectively recognized by four major pathways, namely Toll, IMD, JNK, and JAK/STAT, and trigger the activation of several immune effectors. Although substantial advances have been made in understanding the immunity of model insects such as Drosophila melanogaster, knowledge on the activation of immune responses in other arthropods such as ticks remains limited. Herein, we have deepened our understanding of the intracellular signalling pathways likely to be involved in tick immunity by combining a large-scale in silico approach with high-throughput gene expression analysis. Data from in silico analysis revealed that although both the Toll and JAK/STAT signalling pathways are evolutionarily conserved across arthropods, ticks lack central components of the D. melanogaster IMD pathway. Moreover, we show that tick immune signalling-associated genes are constitutively transcribed in BME26 cells (a cell lineage derived from embryos of the cattle tick Rhipicephalus microplus) and exhibit different transcriptional patterns in response to microbial challenge. Interestingly, Anaplasma marginale, a pathogen that is naturally transmitted by R. microplus, causes downregulation of immune-related genes, suggesting that this pathogen may manipulate the tick immune system, favouring its survival and vector colonization.

Parasite burden in Leishmania (Leishmania) amazonensis-infected mice: validation of luciferase as a quantitative tool.

Given the lack of effective and safe alternatives to the drugs already in use, considerable efforts are being applied to the search of new therapeutic options to treat leishmaniasis. A necessary step in the discovery of antileishmanial drugs is the validation of drug candidates in mouse models. The standard methods to quantify the parasite burden in animal models, mainly culture-based, are time consuming and expensive. In recent years, in vivo imaging systems have been proposed as a tool to overcome these problems, allowing parasite detection in living organisms. Here we compared different treatment efficacy evaluation approaches. Recombinant Leishmania (L.) amazonensis lines expressing the luciferase gene (La-LUC) were obtained and characterized for biological properties as compared with the wild type (WT) parental line. Bioluminescence generated by La-LUC was shown to correlate with the number of promastigotes in vitro. La-LUC promastigotes and intracellular amastigotes were equally sensitive to amphotericin B (AmB) as the WT parasites. The clinical pattern of lesion development upon infection with the transgenic lines was similar to lesions observed after infection with the WT strain. The half maximal effective dose (ED50) of AmB was determined in La-LUC infected mice through quantification of bioluminescence in vivo and ex vivo, by limiting dilution and using clinical parameters. There was agreement in the ED50 determined by all methods. Quantification of bioluminescence in vivo and/or ex vivo was elected as the best tool for determining parasite burden to assess drug efficacy in infected mice. Furthermore, the detailed analysis of AmB effectiveness in this model generated useful data to be used in drug combination experiments.