Excreted/Secreted 15-kDa Proteins and Serine Protease Peptides from Haemonchus contortus Act as Immune-Response Enhancers in Lambs.

This study assessed the immunoprotective effect in lambs of a native excreted/secreted 15-kDa protein and two synthesised S28 peptides derived from the infective transitory larvae (xL3) and adult stages (AS) of Haemonchus contortus. Twenty-two Pelibuey lambs were divided into negative and positive control groups, as well as immunised lamb groups, with 100 µg of the 15-kDa native protein (15kDaNP) and S28 peptides (S28P). The eggs per gram (EPG) and haematocrit were measured, and AS were counted and morphologically measured. To assess the immunoprotection in lambs, indirect enzyme-linked immunosorbent assays and relative expression analyses of immune cytokines were performed using serum and abomasal samples. Our results showed a 72.28% reduction in adult worms (AW) in the 15kDaNP-immunised group, achieving a high clinical response with 41% haematocrit and low EPG values (436 ± 661). Conversely, the S28P group achieved the highest IgG levels (2.125 ± 0.880 OD), with AW exhibiting the greatest body length (p > 0.05) and upregulation of the IL5 and FCεR1A genes associated with nematode control. The 15kDaNP group showed increased expression of genes related to nematode control and anti-inflammatory responses, including IL4, IL5, IL6, and IL13 (p < 0.05). The S28P and 15kDaNP should be explored as potential vaccines against sheep haemonchosis.

Molecular study of the transcription factor SKN-1 and its putative relationship with genes that encode GST and antioxidant enzymes in Haemonchus contortus.

Haemonchus contortus is a parasitic nematode of ruminants. Once inside its host, it is exposed to reactive oxidative species and responds by synthesising antioxidant enzymes as a defence. In Caenorhabditis elegans, antioxidant genes are regulated by the transcription factor skinhead-1 (Cel-SKN-1). However, there is little information about the function of SKN-1 in H. contortus (Hco-SKN-1). Therefore, we performed a molecular investigation to characterise Hco-SKN-1 and its putative relationship with genes encode antioxidant enzymes, namely glutathione S-transferases (Hco-GSTs, n = 3), superoxide dismutase (Hco-SOD) and catalase (Hco-CAT), which are involved in haematophagy and defence against the host. We used in silico sequence analysis of Hco-SKN-1 and Hco-GSTs to design and perform relative expression assays involving H. contortus eggs, infective larvae (L3) and adults. Furthermore, we exposed H. contortus transitional infective larvae (xL3) to erythrocytes or hydrogen peroxide (H2O2) and evaluated the relative expression of antioxidant genes at 24 or 48 h. Gene Ontology (GO) analysis revealed 31 functions associated with Hco-SKN-1 and Hco-GSTs, including stress resistance, larval development and the active immune response. Hco-GST-5957 and Hco-SOD showed the highest expression in adults, indicating a relationship with specific functions at this mature stage. xL3 exposed to erythrocytes or H2O2 showed significant upregulation of Hco-SKN-1, but it occurred after upregulation of the antioxidant genes, indicating that these genes are not regulated by Hco-SKN-1 during the blood-feeding stage. Additional investigation is necessary to understand the putative regulation of antioxidant genes by Hco-SKN-1 during the blood-feeding stage.

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.

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.

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.

Study of a Mexican isolate of Arthrobotrys musiformis (Orbiliales): Predatory behavior and nematocidal activity of liquid culture filtrates against Haemonchus contortus (Trichostrongylidae), protein profile and myco-constituent groups.

A Mexican isolate of the nematophagous fungus Arthrobotrys musiformis was obtained from a soil sample from the Chapultepec ecological reserve zone, in Cuernavaca, Morelos, Mexico. This isolate demonstrated an important predatory activity (74.9%) against the parasitic nematode Haemonchus contortus (L3) and its fungal liquid culture filtrates (LCF) grown in two media showed the following highest nematocidal activities (NA): In Czapek-DoxBroth (CzDoxB) 80.66% and potato-dextrose broth (PDB) 49.84%. Additionally, two major compounds derived from carboxylic acids and two derivates from alkane group were identified by GC-MS. These compounds have been associated to many biological activities. On the other hand, the protein profile analysis by SDS-electrophoresis followed by a zymogram revealed a 10 kDa protein with protease activity. This study provides important information for future experiments focused to explore the potential use of this protein as well as the identified bioactive compounds presents in the LCF as potential candidates against sheep haemonchosis.

Cell death induction by mycelium extracts from Pleurotus spp. on cervical cancer cell lines.

Mushrooms have health benefits, including anti-tumoral properties. We evaluated the cytotoxic and cell death induction effects of water-soluble extracts of Pleurotus ostreatus and Pleurotus eryngii mycelium in the cervical cancer cell lines HeLa (HVP18+) and SiHa (HVP16+) as well as the non-tumoral cell line HaCaT. Both Pleurotus extracts presented similar protein patterns from 190 to 10 kDa and displayed protease activity on a gelatine substrate. The mycelium extracts of both Pleurotus strains induced a dose-dependent cytotoxic effect on HPV+ cells IC50 65 µg), whereas HaCaT cells were less susceptible (IC50 90 µg). The cytotoxic effect at the IC50 concentration was not associated with apoptosis, the activation of Caspases-3/7 was not significantive; only P. eryngii induced a moderate (1.2-fold) increase in SiHa cells. Pleurotus extracts induced autophagy, mainly in SiHa cells (4.3-fold). Neither extracts induced changes in p53 protein expression, suggesting that the cytotoxic effect could be due to p53-independent pathways.

Analysis of the immunomodulatory activity of excreted and secreted products from Haemonchus placei transition infective larvae (xL3).

The excretory/secretory (E/S) products released by infective transitory larvae (xL3) of Haemonchus placei have an important biological function in stimulating immune mechanisms during the invasive process. Our objective was to analyse the modulatory activity of 15 and 70 kDa E/S products from H. placei xL3. Both E/S products were collected from xL3in vitro cultures at 24 and 72 h. Proteins were confirmed by SDS-PAGE, and the corresponding spots were elicited by gel isoelectrofocusing (IEF) and characterised by mass spectrometry. Additionally, flow cytometry of CD4+/γδ+ T cells and immune gene expression were performed by proliferation assays using each E/S product to stimulate lymphocyte and peripheral blood mononuclear cells (PBMCs) from non-infected calves. The IEF results displayed two spots of 7.0 and 5.7 pI for the 15 and 70 kDa products, respectively. Additionally, 29 and 17 peptides from the 15 and 70 kDa E/S products, respectively, were identified with the hypothetical neurotransmitter and enzymatic functions necessary for larval development. The relative expression displayed upregulation of IL4, 5, 6, 8, 10, 13, IFNγ, and FCεR1A genes (from 2.0- to 17.6-fold, p < 0.05) stimulated by the 15 and 70 kDa proteins, indicating specific genes against haemonchosis. Although the percentage of median florescence intensity (MFI%) of CD4+/γδ+ T cells did not change for both E/S products compared to the negative control and concanavalin-A stimulated cells as the positive control (p > 0.05), the 15-kDa protein reduced the levels of both T cells, and the 70-kDa proteins increased the γδ+ cells slightly. Additionally, there was increased PBMCs proliferation by the 70 kDa proteins (p < 0.05), denoting the biological role of other immune cells. The 15 and 70 kDa protein E/S products from H. placei xL3 showed modulation of the immune response, and although more studies are required, they indicate important functions in the host/parasite interaction.

Immune response related to Pelibuey sheep naturally infected with gastrointestinal nematodes in a tropical region of Mexico.

We analysed the immune response involved in sheep naturally infected with gastrointestinal (GI) nematodes. Fifteen Pelibuey lambs were grazed in paddocks contaminated with GI nematodes for 13 weeks. To assess the infection, the number of eggs per gram (epg) and the percentage of packed cell volume (pcv) were evaluated. Blood and abomasal tissue samples were collected at week 8 post-infection to analyse the expression levels of IL-4, IL-5, IL-6, IL-8, IL-13, TGF-ß and FCεR1A genes. The nematode Haemonchus contortus was the main species identified. In addition, two groups of lambs were classified based on the x ± SE of epg and pcv values: G-1, with 151 ± 28 and 29 ± 0.33%, respectively, and G-2, with 475 ± 59.5 and 26 ± 0.38%, respectively. For G-1, upregulation of IL-4, IL-8, IL-13, TGF-ß and FCεR1A genes from 2.42- to 14.99-fold was observed in blood and abomasal tissue samples (p > .05), and IL-5, IL-8 and TGF-ß genes had significant gene expression levels in blood (p < .05). For G-2, moderate gene expression levels, ranging from 1.22- to 3.45-fold, were observed in abomasal tissue (p > .05), and the IL-5 gene presented significant gene expression in blood (p < .05). Strong positively correlated values (r) between pcv and IL-4, IL-8 and TGF-ß genes were observed in G-1. In contrast, significant negative correlations between epg and IL-4, IL-5 and FCεR1A genes indicate acute infection for G-2. Our results suggest that IL-4, IL-5, IL-13, TGF-ß and FCεR1A genes are important modulators of GI nematode infections of Pelibuey lambs.