High rates of benzimidazole-resistance-associated alleles in Haemonchus contortus and detection of resistance against macrocyclic lactones in strongylids from German alpaca herds.

The population of South American camelids (SAC) has been steadily growing in Europe, where they are confronted with the regional endoparasite population of ruminants. As there are no anthelmintic drugs registered for use against nematode infections in SACs, anthelmintics (AH) available for ruminants or horses are usually applied. Reports indicating potential failures in administered AH are increasing. However, the generally low egg counts in SACs complicate the application of resistance tests in the field. The present study reports a follow-up study on SAC farms where anthelmintic resistance (AR) was suspected. The aims were (i) to repeat faecal egg count reduction tests (FECRTs) on potentially affected farms identified in a previous study with larger sample sizes, (ii) to verify suspected AR of Haemonchus contortus against benzimidazoles (BZ) by performing a single-nucleotide polymorphism (SNP) analysis using digital polymerase chain reaction (dPCR), and (iii) to apply the mini-FLOTAC technique for more reliable results at low egg counts in line with current recommendations. Seven farms (9-46 animals each) were examined by coproscopy, larval differentiation and SNP analysis. A FECRT was performed on six of these farms with moxidectin (three farms), monepantel (two farms) and ivermectin (one farm). The FEC was calculated according to the current World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines with the clinical protocol (a newly introduced variant of FECRT which can be used for smaller sample sizes and lower egg counts on the cost of sensitivity) and an expected efficacy of 99%. A high level (> 90%) of BZ-resistance-associated SNPs on codon 200 of H. contortus was observed on all farms. With the FECRT, resistance was demonstrated for ivermectin (74% FECR), while it remained inconclusive for one farm for moxidectin treatment. Sustained efficacy was demonstrated for the remaining treatments. This study showed an advanced level of BZ resistance in H. contortus of SACs and the development of AR against macrocyclic lactones on some farms. Thus, constant monitoring of AH treatment and sustainable worm control methods both need to be applied.

Regulatory effect of Balanites aegyptiaca ethanol extract on oxidant/antioxidant status, inflammatory cytokines, and cell apoptosis gene expression in goat abomasum experimentally infected with Haemonchus Contortus.

This experiment aimed to assess the regulatory effects of treatment with Balanites aegyptiaca fruit ethanol extract (BA-EE) on oxidant/antioxidant status, anti-inflammatory cytokines, and cell apoptosis gene expression in the abomasum of Haemonchus contortus-infected goats. Twenty goat kids were assigned randomly to four equal groups: (G1) infected-untreated, (G2) uninfected-BA-EE-treated, (G3) infected-albendazole-treated, (G4) infected-BA-EE-treated. Each goat in (G1), (G3), and (G4) was orally infected with 10,000 infective third-stage larvae. In the fifth week postinfection, single doses of albendazole (5 mg/kg.BW) and BA-EE (9 g/kg.BW) were given orally. In the ninth week postinfection, the animals were slaughtered to obtain abomasum specimens. The following oxidant/antioxidant markers were determined: malondialdehyde (MDA), glutathione (GSH), glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT). The mRNA gene expression of cytokines (IL-3, IL-6, IL-10, TNF-α) and cell apoptosis markers (Bax, Bcl-2) were estimated. (G1) showed significantly reduced GSH content and GST and SOD activities but a markedly increased MDA level. (G3) and (G4) revealed a markedly lower MDA level with pronouncedly elevated GSH, SOD, and GST levels. The antioxidant properties of BA-EE were superior to those of albendazole. The mRNA gene expressions of IL-3, IL-6, IL-10, TNF-α, and Bax-2 were upregulated in (G1) but downregulated in (G3) and (G4). Bcl-2 and Bcl-2/Bax ratio expression followed a reverse course in the infected and both treated groups. We conclude that BA-EE treatment has a protective role in the abomasum of H. contortus-infected goats. This could be attributed to its antioxidant properties and ability to reduce pro-inflammatory cytokines and cell apoptosis.

Comparison of P-glycoprotein gene expression of two Haemonchus contortus isolates from Yucatan, Mexico, with resistant or susceptible phenotype to ivermectin in relation to a susceptible reference strain.

The variability in the expression of different P-glycoprotein (P-gp) genes in parasitic nematodes of ruminants such as Haemonchus contortus (Hco-pgp) may be caused by different factors including nematode biology, geographical region and anthelmintic pressure. This study analysed the relative expression level of 10 P-gp genes in two H. contortus (Hco-pgp) field isolates from Yucatan, Mexico: 1) PARAISO (IVM-resistant) and 2) FMVZ-UADY (IVM-susceptible). These isolates were compared with a susceptible reference isolate from Puebla, Mexico, namely "CENID-SAI". In all cases H. contortus adult males were used. The Hco-pgp genes (1, 2, 3, 4, 9, 10, 11, 12, 14 and 16) were analysed for each isolate using the RT-qPCR technique. The Hco-pgp expressions were pairwise compared using the 2-ΔΔCt method and a t-test. The PARAISO isolate showed upregulation compared to the CENID-SAI isolate for Hco-pgp 1, 3, 9, 10 and 16 (P < 0.05), and the PARAISO isolate showed upregulation vs. FMVZ-UADY isolate for Hco-pgp 2 and 9 (P < 0.05), displaying 6.58- and 5.93-fold differences (P < 0.05), respectively. In contrast, similar Hco-pgp gene expression levels were recorded for FMVZ-UADY and CENID-SAI isolates except for Hco-pgp1 (P <0.1), which presented a significant upregulation (6.08-fold). The relative expression of Hco-pgp allowed confirming the IVM-resistant status of the PARAISO isolate and the IVM-susceptible status of the FMVZ-UADY isolate when compared to the CENID-SAI reference isolate. Therefore, understanding the association between the Hco-pgp genes expression of H. contortus and its IVM resistance status could help identifying the genes that could be used as molecular markers in the diagnosis of IVM resistance. However, it is important to consider the geographic origin of the nematode isolate and the deworming history at the farm of origin.

Variants of NLRP3 Protein in Haemonchus contortus Infected Sheep: Impact on Immune Cell Responsiveness to LPS In Vitro.

Pathogen recognition is an essential component to achieve the desired outcome of host protection. Nod-like receptor pyrin containing domain 3 (NLRP3) is a cytoplasmic pattern recognition receptor (PRR) with a wide array of agonists, such as PAMPs, DAMPs, ATP, bacterial product and viral products. Stimulation of the NLRP3 inflammasome results in proteolytic activation of IL-1ß and IL-18, cell pyroptosis and classically, the induction of proinflammatory responses. St. Croix (STC) sheep have resistance traits exhibiting the appropriate T-helper type 2 immune response ensuing protection during helminth parasitic infection whereas parasite-susceptible Suffolk (SUF) sheep have an impaired response resulting in parasite establishment and adverse symptoms. The objective of these experiments was to determine if NLRP3 protein in H. contortus-infected SUF sheep was defective using the classical activation pathway of NLRP3 inflammasome. Peripheral blood mononuclear cells (PBMCs) derived from H. contortus-infected STC and SUF sheep were isolated from whole blood and treated (MCC950 treatment for 2 h followed by LPS treatment for 3 h, 1400 W treatment for 2 h followed by LPS treatment for 3 h, LPS treatment for 3 h or culture media for 3 h). qPCR analysis of LPS-stimulated PBMC revealed an upregulation in inflammatory associated genes IL-1ß, TLR4, TNFα and NFκB (p < 0.0001) in STC PBMC and downregulation in IFNγ, IL-6 and iNOS for SUF PBMC. Pharmacological inhibition of iNOS in SUF PBMC resulted in an upregulation in the expression of IFNγ. These preliminary data begin to discover a relationship between NLRP3 activation and TLR4 signalling in PBMC of STC and SUF sheep.

In vitro and in vivo anthelmintic effect of essential oil obtained from Thymus capitatus flowers against Haemonchus contortus and Heligmosomoides polygyrus.

Sheep haemonchosis is a disease that causes serious losses in livestock production, particularly with the increase of cases of anthelmintic resistance around the world. This justifies the urgent need of alternative solutions. The aim of this study was to determine the chemical profile, in vitro, and, in vivo, anthelmintic properties of Thymus capitatus essential oil. To evaluate the, in vitro, anthelmintic activity of the T. capitatus EO on Haemonchus contortus, two tests were used: egg hatch assay (EHA) and adult worm motility (AWM) assay. The nematicidal effect of this oil was evaluated, in vivo, in mice infected artificially with Heligmosomoides polygyrus using faecal egg count reduction (FECR) and total worm count reduction (TWCR). Chromatographic characterization of T.capitatus composition using gas chromatography coupled to mass spectrometry (GC-MS) demonstrated the presence of carvacrol (81.16%), as the major constituents. The IC50 values obtained was 1.9 mg/mL in the EHT. In the AWM assay; T. capitatus essential oil achieved 70.8% inhibition at 1 mg/mL after 8 h incubation. The in vivo, evaluation on H. polygyrus revealed a significant nematicidal effect 7 days post-treatment by inducing 49.5% FECR and 64.5% TWCR, using the highest dose (1600 mg/kg). The results of present study, demonstrate that T.capitatus EO possess a significant anthelmintic properties. Furthermore, it could be an alternative source of anthelmintic agents against gastrointestinal infections caused by H. contortus.

In vitro ovicidal and larvicidal activity of a hydroalcoholic extract and its fractions from Cyrtocarpa procera fruits on Haemonchus contortus.

This study describes the in vitro anthelmintic effect of a hydroalcoholic extract (HA-E) and its fractions from Cyrtocarpa procera fruits against Haemonchus contortus eggs and infective larvae. The HA-E was subjected to bipartition using ethyl acetate, which resulted in an aqueous fraction (Aq-F) and an organic fraction (EtOAc-F). The HA-E and both fractions were tested using the egg hatching inhibition assay (EHIA) and the larval mortality test (LMT). Fractionation of the EtOAc-F was achieved using different chromatographic processes, i.e., open glass column and HPLC analysis. Fractionation of the EtOAc-F gave 18 subfractions (C1R1-C1R18), and those that showed the highest yields (C1R15, C1R16, C1R17 and C1R18) were subjected to anthelmintic assays. The HA-E and the EtOAc-F displayed 100% egg hatching inhibition at 3 and 1 mg/mL, respectively, whereas Aq-F exhibited 92.57% EHI at 3 mg/mL. All subfractions tested showed ovicidal effect. Regarding the larval mortality test, HA-E and EtOAc-F exhibited a larvicidal effect higher than 50% at 50 and 30 mg/mL, respectively. The subfractions that showed the highest larval mortality against H. contortus were C1R15 and C1R17, with larval mortalities of 53.57% and 60.23% at 10 mg/mL, respectively. Chemical analysis of these bioactive subfractions (C1R15 and C1R17) revealed the presence of gallic acid, protocatechuic acid, and ellagic acid. This study shows evidence about the ovicidal and larvicidal properties of C. procera fruits that could make these plant products to be considered as a natural potential anthelmintic agents for controlling haemonchosis in goats and sheep.

An alkaloidal fraction of Annona tomentosa possesses anthelmintic activity and induces surface modifications in Haemonchus contortus.

The nematode Haemonchus contortus is, as a parasite, responsible for most mortality of small ruminants, causing significant economic losses. Numerous plant-derived compounds have exhibited promising anthelmintic activities against this nematode. Notably, the Annona genus stands out for demonstrated anthelmintic effects by extracts from several of its species against different nematodes. This study aimed to assess the effect of an Annona tomentosa fraction, rich in alkaloids, on H. contortus. This fraction, named Alk.F, is derived from the methanolic extract of the plant's stem bark. Chemical characterization of Alk.F was performed by liquid chromatography coupled with mass spectrometry. Among the nine predominant peaks obtained, seven alkaloids were identified: reticuline, reticuline N-oxide, reticuline N-oxide isomer, cyclanoline, asimilobine, tetrahydropalmatine and anonaine. Alk.F inhibited the larval development of H. contortus with an IC50 of 0.026 mg/mL, inhibited larval exsheathment with an IC50 of 0.38 mg/mL, and displayed low hemolytic activity towards sheep erythrocytes. Furthermore, atomic force microscopy revealed that Alk.F altered adhesive forces and the height profile on the surface of H. contortus larvae. In conclusion, A. tomentosa alkaloids alter the cuticle structure of H. contortus, inhibiting larval development and exsheathment, thus offering possibilities for contributing to the development of new anthelmintic drugs.

Nemabiome metabarcoding shows a high prevalence of Haemonchus contortus and predominance of Camelostrongylus mentulatus in alpaca herds in the northern UK.

Gastrointestinal nematodes (GINs) are a common threat faced by pastoral livestock. Since their major introduction to the UK in the early 1990s, South American camelids have been cograzed with sheep, horses, and other livestock, allowing exposure to a range of GIN species. However, there have been no molecular-based studies to investigate the GIN populations present in these camelids. In the current study, we sampled nine alpaca herds from northern England and southern Scotland and used high-throughput metabarcoded sequencing to describe their GIN species composition. A total of 71 amplicon sequence variants (ASVs) were identified representing eight known GIN species. Haemonchus contortus was the most prevalent species found in almost all herds in significant proportions. The identification of H. contortus in other livestock species is unusual in the northern UK, implying that alpacas may be suitable hosts and potential reservoirs for infection in other hosts. In addition, the camelid-adapted GIN species Camelostrongylus mentulatus was identified predominantly in herds with higher faecal egg counts. These findings highlight the value of applying advanced molecular methods, such as nemabiome metabarcoding to describe the dynamics of gastrointestinal nematode infections in novel situations. The results provide a strong base for further studies involving cograzing animals to confirm the potential role of alpacas in transmitting GIN species between hosts.

Analysis of lncRNA-related studies of ivermectin-sensitive and -resistant strains of Haemonchus contortus.

In this study, 858 novel long non-coding RNAs (lncRNAs) were predicted as sensitive and resistant strains of Haemonchus contortus to ivermectin. These lncRNAs underwent bioinformatic analysis. In total, 205 lncRNAs significantly differed using log2 (difference multiplicity) > 1 or log2 (difference multiplicity) < - 1 and FDR < 0.05 as the threshold for significant difference analysis. We selected five lncRNAs based on significant differences in expression, cis-regulation, and their association with the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. These expressions of lncRNAs, namely MSTRG.12610.1, MSTRG.8169.1, MSTRG.6355.1, MSTRG.980.1, and MSTRG.9045.1, were significantly downregulated. These findings were consistent with the results of transcriptomic sequencing. We further investigated the relative expression of target gene mRNAs and the regulation of mRNA and miRNA, starting with lncRNA cis-regulation of mRNA, and constructed a lncRNA-mRNA-miRNA network regulation. After a series of statistical analyses, we finally screened out UGT8, Unc-116, Fer-related kinase-1, GGPP synthase 1, and sart3, which may be involved in developing drug resistance under the regulation of their corresponding lncRNAs. The findings of this study provide a novel direction for future studies on drug resistance targets.

Potential distribution modelling for Haemonchus contortus (Nematoda: Trichostrongylidae) in South America.

The species Haemonchus contortus occurs in many regions worldwide, mainly parasitising small ruminants and economically impacting animal production. Climate change is considered a driving force for the risk of diseases caused by helminths and can also affect relationships between parasites and their hosts, with the potential to cause losses in both animal production and biodiversity in general. The aim of this study was to model the potential distribution of H. contortus in South America. We used MaxEnt to perform the analyses and describe the contribution of important bioclimatic variables involved in the species distribution. Our results show that H. contortus colonised most of the areas with habitats that suit the species' environmental requirements and that this parasite presents habitat suitability in a future scenario. Understanding the effects of climate change on the occurrence and distribution of parasite species is essential for monitoring these pathogens, in addition to predicting the areas that tend to present future parasite outbreaks and identify opportunities to mitigate the impacts of the emergence of diseases caused by these organisms.

Single-Cell Transcriptomic Profiling Unveils Dynamic Immune Cell Responses during Haemonchus contortus Infection.

BACKGROUND: Haemonchus contortus is a parasite widely distributed in tropical, subtropical, and warm temperate regions, causing significant economic losses in the livestock industry worldwide. However, little is known about the genetics of H. contortus resistance in livestock. In this study, we monitor the dynamic immune cell responses in diverse peripheral blood mononuclear cells (PBMCs) during H. contortus infection in goats through single-cell RNA sequencing (scRNA-Seq) analysis. METHODS AND RESULTS: A total of four Boer goats, two goats with oral infection with the L3 larvae of H. contortus and two healthy goats as controls, were used in the animal test. The infection model in goats was established and validated by the fecal egg count (FEC) test and qPCR analysis of the gene expression of IL-5 and IL-6. Using scRNA-Seq, we identified seven cell types, including T cells, monocytes, natural killer cells, B cells, and dendritic cells with distinct gene expression signatures. After identifying cell subpopulations of differentially expressed genes (DEGs) in the case and control groups, we observed the upregulation of multiple inflammation-associated genes, including NFKBIA and NFKBID. Kyoto Encyclopedia of the Genome (KEGG) enrichment analysis revealed significant enrichment of NOD-like receptor pathways and Th1/Th2 cell differentiation signaling pathways in CD4 T cells DEGs. Furthermore, the analysis of ligand-receptor interaction networks showed a more active state of cellular communication in the PBMCs from the case group, and the inflammatory response associated MIF-(CD74 + CXCR4) ligand receptor complex was significantly more activated in the case group, suggesting a potential inflammatory response. CONCLUSIONS: Our study preliminarily revealed transcriptomic profiling characterizing the cell type specific mechanisms in host PBMCs at the single-cell level during H. contortus infection.

Synergistic Interaction Effect of Artemisia cina n-hexane Extract and Tagetes lucida Ethyl Acetate Extract on Haemonchus Contortus.

PURPOSE: We analysed the possible synergistic activity among active extracts from Artemisia cina and Tagetes lucida combinations on Haemonchus contortus, a nematode parasitising sheep. METHODS: The work was carried out in vitro on eggs and infective larvae (L3) of H. contortus. The results were analysed with SAS 9.1, applying the ANOVA and Tukey test, and the lethal concentration (LC) values LC50 and LC90 were determined with regression analysis, employing Proc Probit of SAS 9.1. Additionally, the lethal concentration (LC) was calculated with LC50 and LC90 to determine the synergistic effect. RESULTS: The results demonstrated a high efficacy of the two plants studied on both nematode eggs and L3 larvae as well as of their combinations. The highest egg hatching inhibition was obtained with a 50/50 combination, and the best larvae mortality was obtained with 25% A. cina and 75% T. lucida at 10 mg/mL. Additionally, this combination showed a synergistic effect. CONCLUSION: The two plant species studied here can be applied as natural anthelmintic alternatives due to their high bioactive effect and synergistic response.

The in vitro rumen exsheathment test for studying the effect of plant extracts on the exsheathment of Haemonchus contortus infective larvae.

This study applied the in vitro rumen exsheathment test (IVRET) to evaluate the exsheathment kinetics of Haemonchus contortus infective larvae (L3) incubated in ruminal liquor (RL) containing acetone:water extracts of Acacia pennatula (AP), Gymnopodium floribundum (GF), Havardia albicans (HA) or Lysiloma latisiliquum (LL). The role of polyphenols in the biological activity of the evaluated extracts was also determined. Larvae were incubated in RL either alone or added with a different plant extract (AP, GF, HA, or LL) at 1200 µg/mL. Polyethylene glycol (PEG) was added to block polyphenols in each treatment (RL+PEG, AP+PEG, GF+PEG, HA+PEG, and LL+PEG). After incubation times of 0, 1, 3, 6, 9, and 24 h, the exsheathment process was stopped to count the number of ensheathed and exsheathed L3. A Log-Logistic model was used to determine the L3 exsheathment kinetics in the different RL treatments. The inflection point of the respective kinetic curves, which indicates the time to reach 50 % exsheathed L3 (T50), was the only parameter that differed when comparing the exsheathment models (99 % probability of difference). The T50 values obtained for GF, HA, and LL treatments (T50 = 7.11 - 7.58 h) were higher in comparison to the T50 of RL (5.72 h) (≥ 70 % probability of difference). The L3 incubated in RL added with GF, HA, and LL extracts delayed their exsheathment at 3 and 6 h of incubation (28.71 - 48.06 % exsheathment reduction) compared to the RL treatment. The T50 value for AP, AP+PEG, GF+PEG, HA+PEG, and LL+PEG were similar to RL and RL+PEG (T50 = 5.34 - 6.97 h). In conclusion, the IVRET can be used to identify plants with the potential to delay the exsheathment of H. contortus L3 in the ruminal liquor. The acetone:water extracts of G. floribundum, H. albicans, and L. latisiliquum delayed the T50 of H. contortus exsheathment, which was evident at 3 and 6 h of incubation in ruminal liquor. The observed exsheathment delay was attributed to the polyphenol content of the extracts.

Anthelmintic efficacy of an organic fraction from Guazuma ulmifolia leaves and evaluation of reactive oxidative stress on Haemonchus contortus.

This study describes the anthelmintic efficacy of an organic fraction (EtOAc-F) from Guazuma ulmifolia leaves and the evaluation of its reactive oxidative stress on Haemonchus contortus. The first step was to assess the anthelmintic effect of EtOAc-F at 0.0, 3.5, 7.0 and 14 mg kg of body weight (BW) in gerbil's (Meriones unguiculatus) artificially infected with H. contortus infective larvae (L3). The second step was to evaluate the preliminary toxicity after oral administration of the EtOAc-F in gerbils. Finally, the third step was to determine the relative expression of biomarkers such as glutathione (GPx), catalase (CAT), and superoxide dismutase (SOD) against H. contortus L3 post-exposition to EtOAc-F. Additionally, the less-polar compounds of EtOAc-F were identified by gas mass spectrophotometry (GC-MS). The highest anthelmintic efficacy (97.34%) of the organic fraction was found in the gerbils treated with the 14 mg/kg of BW. Histopathological analysis did not reveal changes in tissues. The relative expression reflects overexpression of GPx (p<0.05, fold change: 14.35) and over expression of SOD (p≤0.05, fold change: 0.18) in H. contortus L3 exposed to 97.44 mg/mL of EtOAc-F compared with negative control. The GC-MS analysis revealed the presence of 4-hydroxybenzaldehyde (1), leucoanthocyanidin derivative (2), coniferyl alcohol (3), ferulic acid methyl ester acetate (4), 2,3,4-trimethoxycinnamic acid (5) and epiyangambin (6) as major compounds. According to these results, the EtOAc-F from G. ulmifolia leaves exhibit anthelmintic effect and increased the stress biomarkers on H. contortus.

Reducing fecal egg count through selective breeding alters dorper lamb response to Haemonchus contortus in an artificial challenge trial.

Infection by gastrointestinal nematodes (GIN), particularly Haemonchus contortus, can be detrimental to sheep health and performance. Genetic susceptibility to GIN varies between breeds, with those lacking high levels of natural resistance often requiring frequent anthelmintic treatment when facing parasitic challenge. Genetic technology can serve as a tool to decrease GIN susceptibility via selection for sheep with reduced fecal egg count (FEC) estimated breeding values (EBVs). However, the physiological changes that result from implementation of this strategy are not well described. Additionally, there is a need for comparison of animals from recent selective breeding against breeds with inherent GIN resistance. In this study we administered a challenge of H. contortus to Dorper x White Dorper (DWD; n = 92) lambs that have been genetically selected for either low (DWD-) or high (DWD+) FEC EBVs and Barbados Blackbelly x Mouflon (BBM; n = 19) lambs from a genetically resistant breed backgrounds. Lamb FEC, packed-cell volume (PCV) and serum IgG were measured at intermittent levels over 5 weeks. At day 21 and day 35, the selectively bred DWD- had a lower mean FEC compared to DWD+, but were higher than BBM. Reductions in both PCV and serum IgG from initial day 0 levels were observed in DWD lambs, but not in BBM. Furthermore, from a subset of lambs (n = 24) harvested at day 21, DWD- only tended (p = 0.056) to have lower mean worm counts than DWD+, with BBM having the lowest mean worm count. Differentially expressed genes (DEGs) identified via RNA-sequencing of abomasal tissue at day 21 indicate a more pronounced Th2 immune response and more rapid worm expulsion occurred in iBBM than iDWD- and iDWD+ lambs. However, gene expression in DWD- suggests an association between reduced FEC EBV and gastric acid secretion and the ability to limit worm fecundity. Ultimately, selection of Dorper sheep for low FEC EBV can reduce susceptibility to GIN, but it will likely require multiple generations with this trait as a breeding priority before presenting a similar resistance level to Caribbean breeds.

Multi-omics data elucidate parasite-host-microbiota interactions and resistance to Haemonchus contortus in sheep.

BACKGROUND: The integration of molecular data from hosts, parasites, and microbiota can enhance our understanding of the complex biological interactions underlying the resistance of hosts to parasites. Haemonchus contortus, the predominant sheep gastrointestinal parasite species in the tropics, causes significant production and economic losses, which are further compounded by the diminishing efficiency of chemical control owing to anthelmintic resistance. Knowledge of how the host responds to infection and how the parasite, in combination with microbiota, modulates host immunity can guide selection decisions to breed animals with improved parasite resistance. This understanding will help refine management practices and advance the development of new therapeutics for long-term helminth control. METHODS: Eggs per gram (EPG) of feces were obtained from Morada Nova sheep subjected to two artificial infections with H. contortus and used as a proxy to select animals with high resistance or susceptibility for transcriptome sequencing (RNA-seq) of the abomasum and 50 K single-nucleotide genotyping. Additionally, RNA-seq data for H. contortus were generated, and amplicon sequence variants (ASV) were obtained using polymerase chain reaction amplification and sequencing of bacterial and archaeal 16S ribosomal RNA genes from sheep feces and rumen content. RESULTS: The heritability estimate for EPG was 0.12. GAST, GNLY, IL13, MGRN1, FGF14, and RORC genes and transcripts were differentially expressed between resistant and susceptible animals. A genome-wide association study identified regions on chromosomes 2 and 11 that harbor candidate genes for resistance, immune response, body weight, and adaptation. Trans-expression quantitative trait loci were found between significant variants and differentially expressed transcripts. Functional co-expression modules based on sheep genes and ASVs correlated with resistance to H. contortus, showing enrichment in pathways of response to bacteria, immune and inflammatory responses, and hub features of the Christensenellaceae, Bacteroides, and Methanobrevibacter genera; Prevotellaceae family; and Verrucomicrobiota phylum. In H. contortus, some mitochondrial, collagen-, and cuticle-related genes were expressed only in parasites isolated from susceptible sheep. CONCLUSIONS: The present study identified chromosome regions, genes, transcripts, and pathways involved in the elaborate interactions between the sheep host, its gastrointestinal microbiota, and the H. contortus parasite. These findings will assist in the development of animal selection strategies for parasite resistance and interdisciplinary approaches to control H. contortus infection in sheep.

Growth hormone stimulates the in vitro development and establishment of Haemonchus contortus in sheep.

The physiologic increase in some sex hormones has been associated with an increase in the parasite load caused by Haemonchus contortus in ewes, especially prolactin. In lambs that are especially susceptible to hemonchosis, the levels of sex hormones are low; in contrast, the levels of another pituitary hormone, growth hormone (GH), which is structurally very similar to prolactin, are high. In this study, the in vitro and in vivo effects of GH on H. contortus larvae development and establishment were evaluated. The addition of 20 ng/mL GH for 5 and 10 days to cultures of H. contortus larvae induced an enlargement (p<0.01) and an L3/L4 molting rate (p<0.03) greater than that of untreated larvae or those treated with other concentrations of the hormone. Flow cytometry showed that 3.8% of the largest and most complex cells of newly obtained larvae of H. contortus were positive for the GH receptor, and by immunofluorescence with confocal microscopy, it was observed that these receptors are located in the intestinal region larvae. In the in vivo assay, the administration of recombinant GH to gonadectomized lambs produced an increase in FEC (p<0.03), the number of female adult worms in the abomasum (p<0.05) and the levels of specific antibodies (p<0.04) in relation to the control lambs; however, it did not affect the fertility of H. contortus females. Although many factors affect the development and implantation of H. contortus in the abomasum of sheep, the results of this study strongly suggest that GH participates in the development and establishment of the parasite in sheep, mainly in young sheep.

Association of ß-globin polymorphisms and tolerance to haemonchosis in ewes and lambs of different sheep breeds.

Gastrointestinal nematodes (GIN), especially Haemonchus contortus, represent a significant challenge for sheep production. Given the global concern about GIN anthelmintic resistance, alternative control methods able to reduce the dependence on these drugs are highly advisable. Since previous studies have shown that sheep carrying the Hb-A allele of ß-globin are more resistant to H. contortus, this study aimed to investigate the relationship between the different haplotypes (Hb-AA, Hb-AB and Hb-BB) and phenotypes in Santa Inês (SI), Texel (TX) and White Dorper (DO) breeds infected with H. contortus. Blood samples were collected from 180 ewes and 123 lambs of the three breeds for DNA extraction followed by qPCR using a hydrolysis probe to identify the ß-globin haplotypes. Phenotypic data, including fecal egg count (FEC), packed cell volume (PCV), FAMACHA score and body condition score for ewes and lambs, as well as weight gain for lambs, were collected. The genotypic frequencies of ß-globin for ewes and lambs were, respectively: 21.7% and 21.4% Hb-AA, 50% and 50% Hb-AB and 28.3% and 28.6% Hb-BB in SI; 0% and 0% Hb-AA, 18.6% and 9.4% Hb-AB and 81.4% and 90.6% Hb-BB in TX; and 0% and 0% Hb-AA, 13.1% and 0% Hb-AB and 86.9% and 100% Hb-BB in DO. In ewes, mean PCV differed (p<0.05) between the three haplotypes, with higher PCV in Hb-AA animals, followed by Hb-AB and Hb-BB. When considering each breed separately, SI Hb-AA ewes presented higher PCV (p<0.05), highlighting that even in a breed already considered resistant, animals with Hb-AA haplotype showed superior performance. Lambs with the Hb-AA haplotype exhibited a higher (p<0.05) mean PCV compared to those with Hb-AB and Hb-BB. The same pattern was found in SI when analyzing each breed separately. No significant association was found between ß-globin haplotypes and FEC, FAMACHA score, body condition score, or weight gain. Nevertheless, given that anemia is the major clinical sign of haemonchosis, our findings on PCV reinforce that sheep carrying the Hb-A allele of ß-globin are more tolerant to haemonchosis. This study may support the development of a valuable tool, targeting genetic selection for GIN control, reducing the dependence on anthelmintics and boosting sheep production worldwide.

Putative SNPs in Ovar-DRB1 and GALNTL6 Genes Conferring Susceptibility to Natural Infection of Haemonchus Contortus in Southern Indian Sheep.

AIM: To explore associations between phenotypic traits and polymorphisms in the DRB1 and GALNT6 gene in Nellore, Deccani and Kenguri sheep naturally infected with Haemonchus contortus. MATERIALS AND METHODS: Blood and faecal samples were collected to evaluate fecal worm egg counts (FEC), packed cell volume (PCV), hemoglobin (Hb), eosinophilia and for DNA isolation. RESULTS: Animals were grouped into susceptible and resistant groups based on EPG counts. FEC and circulating eosinophilia were higher in a susceptible group. Log FEC was negatively correlated (P < 0.01) with PCV, and Hb estimates. The second exon of DRB1 and intron variant of GALNTL6 genes were amplified from DNA samples of resistant and susceptible sheep. Characterization of Ovar-DRB1 amplicon by RFLP revealed two genotypes ('bb' and 'ab'). The genotype frequencies differed significantly between both groups (P < 0.05). The 'bb' genotypes had higher (P < 0.05) log FEC value than 'ab' genotypes and 'b' allele was linked with susceptibility to haemonchosis in sheep. The mean FEC of Nellore sheep was high indicating susceptibility of the breed and also in which the frequency of 'b' allele was more compared to the other two breeds. OVAR-DRB1 genotypes associated with FEC did not affect PCV and Hb. PCR-RFLP assay developed to determine the genotypes with respect to SNP rs424521894 of GALNTL6 revealed monomorphic nature at the locus in the breeds studied. CONCLUSION: MHC polymorphism could be used as a genetic marker for the selection of sheep resistant to H. contortus. However, a more intensive study, involving controlled infections and other GALNTL6 SNPs may be enforced to make any decisive assertion.

Rapid, automated quantification of Haemonchus contortus ova in sheep faecal samples.

Haemonchus contortus is one of the most pathogenic nematodes affecting small ruminants globally and is responsible for large economic losses in the sheep and goat industry. Anthelmintic resistance is rampant in this parasite and thus parasite control programs must account for drug efficacy on individual farms and, sometimes, whether H. contortus is the most prevalent trichostrongylid. Historically, coproculture has been the main way to determine the prevalence of H. contortus in faecal samples due to the inability to morphologically differentiate between trichostrongylid egg types, but this process requires a skilled technician and takes multiple days to complete. Fluoresceinated peanut agglutinin (PNA) has been shown to specifically bind H. contortus and thus differentiate eggs based on whether they fluoresce, but this method has not been widely adopted. The ParasightTM System (PS) fluorescently stains helminth eggs in order to identify and quantify them, and the H. contortus PNA staining method was therefore adapted to this platform using methodology requiring only 20 min to obtain results. In this study, 74 fecal samples were collected from sheep and analyzed for PNA-stained H. contortus, using both PS and manual fluorescence microscopy. The percentage of H. contortus was determined based on standard total strongylid counts with PS or brightfield microscopy. Additionally, 15 samples were processed for coproculture with larval identification, and analyzed with both manual and automated PNA methods. All methods were compared using the coefficient of determination (R2) and the Lin's concordance correlation coefficient (ρc). ParasightTM and manual PNA percent H. contortus results were highly correlated with R2 = 0.8436 and ρc = 0.9100 for all 74 fecal samples. Coproculture versus PS percent H. contortus were also highly correlated with R2 = 0.8245 and ρc = 0.8605. Overall, this system provides a rapid and convenient method for determining the percentage of H. contortus in sheep and goat fecal samples without requiring specialized training.