Selection of Molecules with Immunological Potential from Excretory and Secretory Products from the Nematode Haemonchus placei by Cell Proliferation and Gene Expression Assays.

The nematodeHaemonchus placeiis a pathogenic parasite, the most seriously affecting ruminant's health and being responsible for enormous economic losses all over the world. The present protocol describes different in vitro techniques to select potential candidate antigens with immune-protective activity from excretory and secretory products (ESP) fromH. placeitransitory infective larvae (xL3). ESP from xL3were obtained from the in vitro infective larvae (L3) maintained in Hank's medium at 37 °C with 5% CO2for 48 h. Then, the presence of ESP proteins was confirmed by SDS-PAGE to be used in an in vitro proliferation assay with bovine peripheral blood mononuclear cells (PBMCs). The ESP were exposed to the PBMCs during two different periods (24 and 48 h). Genes associated with immune response against the nematode were analyzed using relative gene expression and bioinformatic tools. These are simple, economic, and helpful tools to identify potential immune-protective molecules under in vitro conditions for confirming the efficacy of future in vivo assays. Graphical overview.

Assembly and Analysis of Haemonchus contortus Transcriptome as a Tool for the Knowledge of Ivermectin Resistance Mechanisms.

Haemonchus contortus (Hc) is an important parasitic nematode of small ruminants. In this study we assembled the transcriptome of Hc as a model to contribute to the knowledge about the profile of the differential gene expression between two Mexican Hc strains under different anthelmintic resistance statuses, one susceptible and the other resistant to ivermectin (IVMs and IVMr, respectively), in order to improve and/or to have new strategies of control and diagnosis. The transcript sequence reads were assembled and annotated. Overall, ~127 Mbp were assembled and distributed into 77,422 transcript sequences, and 4394 transcripts of the de novo transcriptome were matched base on at least one of the following criteria: (1) Phylum Nemathelminthes and Platyhelminthes, important for animal health care, and (2) ≥55% of sequence identity with other organisms. The gene ontology (GO) enrichment analysis (GOEA) was performed to study the level of gene regulation to IVMr and IVMs strains using Log Fold Change (LFC) filtering values ≥ 1 and ≥ 2. The upregulated-displayed genes obtained via GOEA were: 1993 (for LFC ≥ 1) and 1241 (for LFC ≥ 2) in IVMr and 1929 (for LFC ≥ 1) and 835 (for LFC ≥ 2) in IVMs. The enriched GO terms upregulated per category identified the intracellular structure, intracellular membrane-bounded organelle and integral component of the cell membrane as some principal cellular components. Meanwhile, efflux transmembrane transporter activity, ABC-type xenobiotic transporter activity and ATPase-coupled transmembrane transporter activity were associated with molecular function. Responses to nematicide activity, pharyngeal pumping and positive regulation of synaptic assembly were classified as biological processes that might be involved in events related to the anthelmintic resistance (AR) and nematode biology. The filtering analysis of both LFC values showed similar genes related to AR. This study deepens our knowledge about the mechanisms behind the processes of H. contortus in order to help in tool production and to facilitate the reduction of AR and promote the development of other control strategies, such as anthelmintic drug targets and vaccines.

In silico analysis of two Haemonchus spp. serine protease peptides (S28) and their immunomodulatory activity in vitro.

The aim of this study was to evaluate the in vitro immune modulation of two de novo peptides with hypothetical identity to the serine protease family (S28) from Haemonchus spp. Expression of mRNAs encoding these peptides was confirmed by RTqPCR in L3 and adult stage parasites. Antibodies from serum samples collected from an H. contortus-infected lamb at 60 days post infection detected both peptides, as assessed by indirect ELISA. Lamb peripheral blood mononuclear cells (PBMCs) were exposed to each peptide, as well as to the peptide mixture, and cell proliferation assays were performed at 24, 48 and 72 h. The relative expression of the IL4, IL5, IL6, IL13, CXCL8 and FCεR1A genes was quantified by RTqPCR from lamb PBMCs exposed to the peptide mixture at 24 and 48 h. With respect to immune gene expression, 15- and 3-fold upregulation at 24 h was observed with IL5 and CXCL8, respectively, and 2-fold upregulation of CXCL8 at 48 h. In contrast, downregulation of IL5 was stimulated at 48 h. These data suggest that these peptides (pep-hsp and pep-pcx), which show high identity with intestinal and excretion/secretion serine proteases, can trigger immunogenic activity, and suggest that they may be useful as potential parasite vaccines.