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.

The aim of the study was to compare the relative gene expression of Haemonchus contortus P-glycoprotein genes (Hco-pgp) between fourth (L₄), infective (L₃), and transitory infective (xL₃) larval stages as laboratory models to study ivermectin (IVM) resistance. The H. contortus resistant to IVM (IVMr) and susceptible to IVM (IVMs) strains were used to develop xL₃in vitro culture and to infect Meriones unguiculatus (gerbils) to collect L₄ stages. Morphometric differences were evaluated from 25 individuals of H. contortus from each strain. Relative gene expression from xL₃ and L₄ was determined between comparison of IVMr stages and from IVMr vs IVMs stages. Seven Hco-pgp genes (1, 2, 3, 4, 9, 10, and 16) were analysed by RT-qPCR using L₃ stage as control group, per strain, and GAPDH and β-tubulin as constitutive genes. Morphological changes were confirmed between xL₃ and L₄ developing oral shape, oesophagus, and intestinal tube. In addition, the body length and width showed statistical differences (p < 0.05). The Hco-pgp1, 2, 3, and 4 genes (p < 0.05) were upregulated from 7.1- to 463.82-fold changes between IVMr stages, and Hco-pgp9 (13.12-fold) and Hco-pgp10 (13.56-fold) genes showed differences between L₄ and xL₃, respectively. The comparative study between IVMr vs IVMs strains associated to xL₃ and L₄ displayed significant upregulation for most of the Hco-pgp genes among 4.89–188.71 fold-change. In conclusion, these results suggest the use of H. contortus xL₃ and L₄ as suitable laboratory models to study IVMr associated with Hco-pgp genes to contribute to the understanding of anthelmintic resistance.

The upregulation of P-glycoprotein genes in Haemonchus contortus suspected of ivermectin resistance (IVM-Res) was studied in sheep farms. A faecal egg count reduction test (FECRT) was used to evaluate IVM-Res on four farms (from A to D). Faecal samples positive to gastrointestinal nematodes (GINs) were used to identify infective larval stages (L3) pre- and post-IVM treatment by Polymerase Chain Reaction (PCR) test. Ten P-glycoprotein (pgp) genes were analysed to estimate the over expression level in H. contortus L3. Genes were quantified using the Retro Transcription–real time PCR technique. FECRT indicated low IVM efficacy on the four farms from zero to 33% of eggs per gram and Haemonchus, Cooperia and Trichostrongylus were the GINs identified after IVM treatment. H. contortus isolates displayed different pgp gene upregulation levels (p < 0.05): pgp4 from 19.03 to 25.96-fold on four farms; 3 and 16 pgp from 32.19 to 134.21-fold on three farms; 1, 2 and 12 pgp from 7.14 to 56.43-fold on two farms; 10 and 11 pgp from 6.82 to 32.86-fold on one farm. In conclusion, the examination of FECRT showed low IVM efficacy against GINs. Additionally, pgp3, 4 and 16 were the most frequent genes involved in sheep farms with IVM-Res problems in relation to IVM resistant H. contortus.

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.

Parasitic infections, particularly those caused by Haemonchus contortus (H. contortus), severely impact livestock production, with growing resistance to commercial anthelmintics posing a major challenge. Green-synthesized metallic nanoparticles using Artemisia cina (A. cina), a plant with known anthelmintic and antioxidant properties, represent a promising sustainable alternative for parasite control. In this study, silver nanoparticles (AgNPs) were synthesized using an aqueous extract of A. cina to evaluate their anthelmintic activity against infective larvae (L3) of H. contortus and their effect on the expression of oxidative stress-related genes. The larval mortality bioassay was conducted in 96-well microtiter plates by incubating L3 larvae with increasing AgNP concentrations for 24 h. To investigate oxidative stress responses, larvae were exposed to sublethal concentrations of AgNPs, A. cina aqueous extract, AgNO₃, and H₂O₂. Expression levels of SOD, GPx, and CAT genes were then quantified by RT-qPCR at multiple post-exposure time intervals. The synthesis was optimized by varying parameters such as pH, temperature, and extract volume. The nanoparticles were characterized using UV-Vis spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), and Electrophoretic Light Scattering (ELS). Overall, synthesis at pH 8 yielded small, spherical, stable, and abundant AgNPs. In vitro assays on L3 larvae showed a mortality rate of 91.33% at the highest AgNP concentration (500 μg/mL), with lethal concentration (LC50 and LC90) values of 4.128 ppm (μg/mL) and 17.993 μg/mL, respectively. Relative expression analyses revealed that AgNPs induced the overexpression of the SOD gene, highlighting its role in the oxidative stress response. In contrast, the expression levels of GPx and CAT genes were markedly downregulated. These results suggest that SOD could serve as a potential biomarker of oxidative stress induced by AgNPs in combination with A. cina metabolites, influencing the infective stages of H. contortus.