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.

Coffee Pulp: A Natural Alternative for Control of Resistant Nematodes in Small Ruminants.

Goat production in Mexico is an important economic activity that is affected by different gastrointestinal nematode (GIN) species. GINs resistant to commercial anthelmintics have been reported. Plant extracts or agro-industrial by-products, such as coffee pulp, have been proposed as control alternatives, given their secondary metabolite content. The aim of the present study was to determine the anthelmintic activity of the hydroalcoholic extract of coffee pulp against benzimidazole-resistant GINs. Stool samples were collected from goats, from which GIN eggs were identified and quantified. Molecular techniques confirmed the genus of GINs and their benzimidazole resistance profile. The percentage of egg hatching inhibition (% EHI) and larval mortality (% LM) with the hydroalcoholic extract of coffee pulp was determined at concentrations from 200 to 0.39 mg/mL. The genera Haemonchus spp. and Trichostrongylus spp. were identified, and the presence of the ß-tubulin gene mutation, associated with benzimidazole (BZ) resistance, was determined. Hydroalcoholic extract of coffee pulp inhibited 100% of egg hatching at 200 and 100 mg/mL, with no larvicidal effect at the evaluated concentrations.

The immune gene expression induced by Cooperia punctata in naturally infected calves with resistance and susceptible phenotype traits.

The analysis of the immune gene expression was performed in Zebu × Holstein calves with resistant and susceptible phenotypes naturally infected with Cooperia punctata. Fourteen calves of 4 months old were grazed for 11 weeks under a tropical climate. The parasitic infection showed an average epg value of 1055 ± 1155 and an IgG optical density of 0.814 ± 0.0.037 with statistic differences among the different weeks (p < 0.05), and a pcv value of 24 ± 2.0 % (p > 0.05). High variation in epg value was observed, between 7 ± 7.14 and 4657 ± 1886, and, based on these differences; the infected hosts were classified as five resistant calves with epg ≤ 200 and nine susceptible calves with epg ≥ 300. Moreover, IgG levels displayed statistical differences between resistance and susceptible calves to C. punctata infection. The immune gene expression was analysed in three resistant and susceptible calves, respectively. Nine cytokine genes and the FCεR1A receptor were analysed at the 3rd and 11th weeks post-infection. In the first period upregulation was found, from 2.19- to 9.45-fold, (p < 0.05) for IL-2, -5, - 6, -10, TGF-ß and FCεR1A in the resistant group; the expression was decreased at the 11th week with low level of IgG. In contrast, downregulation for susceptible calves was found for nine immune genes and upregulation for INF-γ in both periods together with increased IgG levels. In conclusion, immune gene expression was regulated at the begging infection of C. punctata in resistant grazing calves. In contrast, suppression of important genes was involved in calves susceptible to C. punctata.

Detection of Gastrointestinal Nematode Populations Resistant to Albendazole and Ivermectin in Sheep.

Gastrointestinal parasite infections represent a major welfare problem in small ruminants reared in extensive systems, which may be exacerbated by anthelmintic resistance. Therefore, we aimed to study the efficacy of albendazole and ivermectin in sheep. Eighty-six animals were selected from commercial farms in the temperate area of the State of Mexico at the age of seven months. These animals were randomly distributed into three groups: Group A, treated with albendazole, Group I, treated with ivermectin and Group C, left untreated. Faecal samples were collected before the anthelmintic was administered and 15 days post-treatment. Both Group A and Group I displayed a significant decrease of faecal egg counts when pre- and post-treatment values were compared (p = 0.003 and p = 0.049, respectively), and a significantly lower faecal egg count when compared with Group C after the treatment (p < 0.05). However, the faecal egg count reduction test showed that gastrointestinal nematodes (GIN) developed anthelmintic resistance to both albendazole and ivermectin. The results of the polymerase chain reaction (PCR) allowed the identification of Cooperia spp., and Trichostrongylus colubriformis. The allele-specific PCR results confirmed that T. colubriformis was resistant to albendazole. In conclusion, this study showed the presence of resistant GIN to albendazole and ivermectin in sheep reared in Mexican temperate zones. Therefore, nematode infections should be systematically monitored in order to implement integrated management strategies to prevent the spread of anthelmintic resistance.