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The aim of the study was to purify and characterise recombinant proteins with the potential as an anti-parasite vaccine. Full-length cDNAs encoding seryl-tRNA synthetase (srs-2) were cloned from Haemonchus contortus (HcSRS-2) and Teladorsagia circumcincta (TcSRS-2). TcSRS-2 and HcSRS-2 cDNA (1458bp) encoded proteins of 486 amino acids, each of which was present as a single band of about 55 kDa on SDS-PAGE. Multiple alignments of the protein sequences showed homology of 94% between TcSRS-2 and HcSRS-2, 76-93% with SRS-2s of eight nematodes and 68% with Mus musculus SRS-2. The predicted three-dimensional structures revealed an overall structural homology of TcSRS-2 and HcSRS-2, highly conserved binding and catalytic sites, and minor differences in the tautomerase binding site residues in other nematode SRS-2 homologues. A phylogenetic tree was constructed using helminth and mammalian SRS-2 sequences. Soluble C-terminal SRS-2 proteins were expressed in Escherichia coli strain AY2.4 and purified. Recombinant HcSRS-2 assay shows that the recombinant enzyme was active and stable. The K m and V max for ATP were 3.9 ± 1.0 μM and 2.7 ± 0.1 μmol min -1 mg -1 protein, respectively. Antibodies in serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant HcSRS-2 and TcSRS-2 in enzyme-linked immunosorbent assays. Recognition of the recombinant proteins by antibodies generated by exposure of sheep to the native enzyme indicates similar antigenicity of the two proteins., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Millions of livestock animals worldwide are infected with the haematophagous barber's pole worm, Haemonchus contortus, the aetiological agent of haemonchosis. Despite the major significance of this parasite worldwide and its widespread resistance to current treatments, the lack of a high-quality genome for the well-defined strain of this parasite from Australia, called Haecon-5, has constrained research in a number of areas including host-parasite interactions, drug discovery and population genetics. To enable research in these areas, we report here a chromosome-contiguous genome (∼280 Mb) for Haecon-5 with high-quality models for 19,234 protein-coding genes. Comparative genomic analyses show significant genomic similarity (synteny) with a UK strain of H. contortus, called MHco3(ISE).N1 (abbreviated as "ISE"), but we also discover marked differences in genomic structure/gene arrangements, distribution of nucleotide variability (single nucleotide polymorphisms (SNPs) and indels) and orthology between Haecon-5 and ISE. We used the genome and extensive transcriptomic resources for Haecon-5 to predict a subset of essential single-copy genes employing a "cross-species" machine learning (ML) approach using a range of features from nucleotide/protein sequences, protein orthology, subcellular localisation, single-cell RNA-seq and/or histone methylation data available for the model organisms Caenorhabditis elegans and Drosophila melanogaster. From a set of 1,464 conserved single copy genes, transcribed in key life-cycle stages of H. contortus, we identified 232 genes whose homologs have critical functions in C. elegans and/or D. melanogaster, and prioritised 10 of them for further characterisation; nine of the 10 genes likely play roles in neurophysiological processes, germline, hypodermis and/or respiration, and one is an unknown (orphan) gene for which no detailed functional information exists. Future studies of these genes/gene products are warranted to elucidate their roles in parasite biology, host-parasite interplay and/or disease. Clearly, the present Haecon-5 reference genome and associated resources now underpin a broad range of fundamental investigations of H. contortus and could assist in accelerating the discovery of novel intervention targets and drug candidates to combat haemonchosis., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

This study examined Haemonchus contortus prevalence and benzimidazole resistance in eight districts of Marathwada, Maharashtra, India. A comprehensive investigation of 264 abomasa of goats collected from abattoirs and goats necropsied at the College of Veterinary Sciences and Animal Husbandry, Parbhani, revealed 21.21 % a prevalence of H. contortus. The incidence of H. contortus did not vary much across seasons and it was highest in summer (23.42 %), followed by monsoon (22.89 %), and lowest in winter (15.71 %). Statistically non-significant (p < 0.05) prevalence was observed in male and female animals. A detailed examination of 168 adult H. contortus worms from eight districts revealed the presence of all conceivable genotypes including homozygous resistant (rr), susceptible (SS), and heterozygous (Sr) BZ susceptible genotypes. The rr was the most frequent at 50 %, followed by SS at 27 % and Sr at 22 %. The presence of the SNP was observed in in all eight randomly selected and sequenced samples., Competing Interests: Declaration of competing interest The authors affirm that they do not possess any identifiable conflicting financial or personal interests that could have potentially influenced the findings presented in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 (L 3 ) and adults. Furthermore, we exposed H. contortus transitional infective larvae (xL 3 ) to erythrocytes or hydrogen peroxide (H 2 O 2 ) 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. xL 3 exposed to erythrocytes or H 2 O 2 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., Competing Interests: Declaration of Competing Interest We also state that there is no conflict of interest associated with this second manuscript version and there is not financial or personal relationship with other people or organization that could inappropriately influence our work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Haem is essential but toxic for metazoan organisms. Auxotrophic nematodes can acquire sufficient haem from the environment or their hosts in the meanwhile eliminate or detoxify excessive haem through tightly controlled machinery. In previous work, we reported a role of the unique transporter protein HRG-1 in the haem acquisition and homeostasis of parasitic nematodes. However, little is known about the haem efflux and detoxification via ABC transporters, particularly the multiple drug resistance proteins (MRPs)., Results: Here, we further elucidate that a member of the mrp family (mrp-3) is involved in haem efflux and detoxification in a blood-feeding model gastrointestinal parasite, Haemonchus contortus. This gene is haem-responsive and dominantly expressed in the intestine and inner membrane of the hypodermis of this parasite. RNA interference of mrp-3 resulted in a disturbance of genes (e.g. hrg-1, hrg-2 and gst-1) that are known to be involved in haem homeostasis and an increased formation of haemozoin in the treated larvae and lethality in vitro, particularly when exposed to exogenous haem. Notably, the nuclear hormone receptor NHR-14 appears to be associated the regulation of mrp-3 expression for haem homeostasis and detoxification. Gene knockdown of nhr-14 and/or mrp-3 increases the sensitivity of treated larvae to exogenous haem and consequently a high death rate (> 80%)., Conclusions: These findings demonstrate that MRP-3 and the associated molecules are essential for haematophagous nematodes, suggesting novel intervention targets for these pathogens in humans and animals., (© 2024. The Author(s).)

Xenobiotic biotransformation is an important modulator of anthelmintic drug potency and a potential mechanism of anthelmintic resistance. Both the free-living nematode Caenorhabditis elegans and the ruminant parasite Haemonchus contortus biotransform benzimidazole drugs by glucose conjugation, likely catalysed by UDP-glycosyltransferase (UGT) enzymes. To identify C. elegans genes involved in benzimidazole drug detoxification, we first used a comparative phylogenetic analysis of UGTs from humans, C. elegans and H. contortus, combined with available RNAseq datasets to identify which of the 63 C. elegans ugt genes are most likely to be involved in benzimidazole drug biotransformation. RNA interference knockdown of 15 prioritized C. elegans genes identified those that sensitized animals to the benzimidazole derivative albendazole (ABZ). Genetic mutations subsequently revealed that loss of ugt-9 and ugt-11 had the strongest effects. The "ugt-9 cluster" includes these genes, together with six other closely related ugts. A CRISPR-Cas-9 deletion that removed seven of the eight ugt-9 cluster genes had greater ABZ sensitivity than the single largest-effect mutation. Furthermore, a double mutant of ugt-22 (which is not a member of the ugt-9 cluster) with the ugt-9 cluster deletion further increased ABZ sensitivity. This additivity of mutant phenotypes suggest that ugt genes act in parallel, which could have several, not mutually exclusive, explanations. ugt mutations have different effects with different benzimidazole derivatives, suggesting that enzymes with different specificities could together more efficiently detoxify drugs. Expression patterns of ugt-9, ugt-11 and ugt-22 gfp reporters differ and so likely act in different tissues which may, at least in part, explain their additive effects on drug potency. Overexpression of ugt-9 alone was sufficient to confer partial ABZ resistance, indicating increasing total UGT activity protects animals. In summary, our results suggest that the multiple UGT enzymes have overlapping but not completely redundant functions in benzimidazole drug detoxification and may represent "druggable" targets to improve benzimidazole drug potency., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Among gastrointestinal nematodes, Haemonchus contortus (Rudolphi) Cobb (order Strongylidae; family Trichostrongylidae) is one of pathogenic and economic importance in domestic and wild ruminants, including the European bison, Bison bonasus Linnaeus (order Cetartiodactyla; family Bovidae); a species on the International Union for Conservation of Nature's Red List of Threatened Species. Carabus granulatus Linnaeus (order Coleoptera; family Carabidae) is one of the most prevalent species of ground beetle, inhabiting a wide range of terrestrial ecosystems in Poland. Twenty-six ground beetles of this species inhabiting the Białowieża Primeval Forest in eastern Poland were screened for the presence of DNA of pathogenic gastrointestinal nematodes of ruminants. Extracted DNA was sequenced and compared to reference sequences. In six insects, the presence of H. contortus DNA was detected. The obtained nucleotide sequences were homologous to each other and to the majority of the published DNA sequences of H. contortus isolates. The sequences were also identical to a sequence of H. contortus isolated from European bison in Poland. The study provides the first molecular evidence of the presence of H. contortus DNA in C. granulatus. The finding suggests that ground beetles may play a role in the transmission dynamics of this parasite., (© 2024 The Authors. Medical and Veterinary Entomology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

To understand the benzimidazole (BZ) resistance of Haemonchus contortus in Southern Xinjiang, three single nucleotide polymorphisms (SNPs) designated as F167Y, E198A, and F200Y, in the isotype-1 β-tubulin gene which are associated with BZ resistance, were investigated for H. contortus populations from sheep in Hejing and Minfeng counties of Southern Xinjiang. In brief, a total of 190 H. contortus adults were collected from 52 out of 70 slaughtered sheep in city abattoirs across two regions in Southern Xinjiang. The species identity of each adult worm was confirmed by PCR amplification of ITS-2 using H. contortus-specific primers targeting the ITS-2. The samples were then investigated for BZ-related SNPs at locus 167, 198, and 200, by PCR-sequencing of the isotype-1 β-tubulin gene. The results showed that only E198A and F200Y mutations were detected in the investigated H. contortus populations. The E198A mutation (homozygous and heterozygote resistant: found in 40% and 30% of sequenced samples from Minfeng and Hejing counties, respectively) was predominant compared with the F200Y mutation (homozygous and heterozygote resistant: found in 14% and 13.3% of sequenced samples from Minfeng and Hejing counties, respectively). The results indicate a high prevalence of BZ resistance in H. contortus populations from certain areas of Southern Xinjiang. Our findings provide valuable information for the prevention and control of H. contortus in areas with similar conditions., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Haemonchus contortus (the barber's pole worm)-a highly pathogenic gastric nematode of ruminants-causes significant economic losses in the livestock industry worldwide. H. contortus has become a valuable model organism for both fundamental and applied research (e.g., drug and vaccine discovery) because of the availability of well-defined laboratory strains (e.g., MHco3(ISE).N1 in the UK and Haecon-5 in Australia) and genomic, transcriptomic and proteomic data sets. Many recent investigations have relied heavily on the use of the chromosome-contiguous genome of MHco3(ISE).N1 in the absence of a genome for Haecon-5. However, there has been no genetic comparison of these and other strains to date. Here, we assembled and characterised the mitochondrial genome (14.1 kb) of Haecon-5 and compared it with that of MHco3(ISE).N1 and two other strains (i.e., McMaster and NZ&#95;Hco&#95;NP) from Australasia. We detected 276 synonymous and 25 non-synonymous single nucleotide polymorphisms (SNPs) within Haecon-5. Between the Haecon-5 and MHco3(ISE).N1 strains, we recorded 345 SNPs, 31 of which were non-synonymous and linked to fixed amino acid differences in seven protein-coding genes ( nad 5, nad 6, nad 1, atp 6, nad 2, cyt b and nad 4) between these strains. Pronounced variation (344 and 435 SNPs) was seen between Haecon-5 and each of the other two strains from Australasia. The question remains as to what impact these mitogenomic mutations might have on the biology and physiology of H. contortus , which warrants exploration. The high degree of mitogenomic variability recorded here among these strains suggests that further work should be undertaken to assess the nature and extent of the nuclear genomic variation within H. contortus .

Nuclear hormone receptors (NHRs) are emerging target candidates against nematode infection and resistance. However, there is a lack of comprehensive information on NHR-coding genes in parasitic nematodes. In this study, we curated the nhr gene family for 60 major parasitic nematodes from humans and animals. Compared with the free-living model organism Caenorhabditis elegans, a remarkable contraction of the nhr family was revealed in parasitic species, with genetic diversification and conservation unveiled among nematode Clades I (10-13), III (16-42), IV (33-35) and V (25-64). Using an in vitro biosystem, we demonstrated that 40 nhr genes in a blood-feeding nematode Haemonchus contortus (clade V; barber's pole worm) were responsive to host serum and one nhr gene (i.e., nhr-64) was consistently stimulated by anthelmintics (i.e., ivermectin, thiabendazole and levamisole); Using a high-throughput RNA interference platform, we knocked down 43 nhr genes of H. contortus and identified at least two genes that are required for the viability (i.e., nhr-105) and development (i.e., nhr-17) of the infective larvae of this parasitic nematode in vitro. Harnessing this preliminary functional atlas of nhr genes for H. contortus will prime the biological studies of this gene family in nematode genetics, infection, and anthelmintic metabolism within host animals, as well as the promising discovery of novel intervention targets., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Aldo-keto reductases (AKRs), a superfamily of NADP(H)-dependent oxidoreductases, catalyze the oxidoreduction of a wide variety of eobiotic and xenobiotic aldehydes and ketones. In mammals, AKRs play essential roles in hormone and xenobiotic metabolism, oxidative stress, and drug resistance, but little is known about these enzymes in the parasitic nematode Haemonchus contortus. In the present study, 22 AKR genes existing in the H. contortus genome were investigated and a phylogenetic analysis with comparison to AKRs in Caenorhabditis elegans, sheep and humans was conducted. The constitutive transcription levels of all AKRs were measured in eggs, larvae, and adults of H. contortus, and their expression was compared in a drug-sensitive strain (ISE) and a benzimidazole-resistant strain (IRE) previously derived from the sensitive strain by imposing benzimidazole selection pressure. In addition, the inducibility of AKRs by exposure of H. contortus adults to benzimidazole anthelmintic flubendazole in vitro was tested. Phylogenetic analysis demonstrated that the majority of AKR genes in H. contortus lack orthologues in the sheep genome, which is a favorable finding for considering AKRs as potential drug targets. Large differences in the expression levels of individual AKRs were observed, with AKR1, AKR3, AKR8, and AKR10 being the most highly expressed at most developmental stages. Significant changes in the expression of AKRs during the life cycle and pronounced sex differences were found. Comparing the IRE and ISE strains, three AKRs were upregulated, and seven AKRs were downregulated in adults. In addition, the expression of three AKRs was induced by flubendazole exposure in adults of the ISE strain. Based on these results, AKR1, AKR2, AKR3, AKR5, AKR10 and AKR19 in particular merit further investigation and functional characterization with respect to their potential involvement in drug biotransformation and anthelmintic resistance in H. contortus., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2024. The Author(s).)

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., Competing Interests: Declaration of competing interest Authors have no potential conflict of interest pertaining to this submission., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Over the years, comprehensive explorations of the model organisms Caenorhabditis elegans (elegant worm) and Drosophila melanogaster (vinegar fly) have contributed substantially to our understanding of complex biological processes and pathways in multicellular organisms generally. Extensive functional genomic-phenomic, genomic, transcriptomic, and proteomic data sets have enabled the discovery and characterisation of genes that are crucial for life, called 'essential genes'. Recently, we investigated the feasibility of inferring essential genes from such data sets using advanced bioinformatics and showed that a machine learning (ML)-based workflow could be used to extract or engineer features from DNA, RNA, protein, and/or cellular data/information to underpin the reliable prediction of essential genes both within and between C. elegans and D. melanogaster . As these are two distantly related species within the Ecdysozoa, we proposed that this ML approach would be particularly well suited for species that are within the same phylum or evolutionary clade. In the present study, we cross-predicted essential genes within the phylum Nematoda (evolutionary clade V)-between C. elegans and the pathogenic parasitic nematode H. contortus -and then ranked and prioritised H. contortus proteins encoded by these genes as intervention (e.g., drug) target candidates. Using strong, validated predictors, we inferred essential genes of H. contortus that are involved predominantly in crucial biological processes/pathways including ribosome biogenesis, translation, RNA binding/processing, and signalling and which are highly transcribed in the germline, somatic gonad precursors, sex myoblasts, vulva cell precursors, various nerve cells, glia, or hypodermis. The findings indicate that this in silico workflow provides a promising avenue to identify and prioritise panels/groups of drug target candidates in parasitic nematodes for experimental validation in vitro and/or in vivo.

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 β-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 β-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 β-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 β-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 β-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., Competing Interests: Declaration of Competing Interest The authors report no declarations of competing interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Complex cross-talk occurs between gastrointestinal nematodes and gut symbiotic microbiota, with consequences for animal metabolism. To investigate the connection between methane production and endoparasites, this study evaluated the effect of mixed infection with Haemonchus contortus and Trichostrongylus colubriformis on methanogenic and methanotrophic community in rumen microbiota of lambs using shotgun metagenomic and real-time quantitative PCR (qPCR). The rumen content was collected from six Santa Inês lambs, (7 months old) before and after 42 days infection by esophageal tube. The metagenomic analysis showed that the infection affected the microbial community structure leading to decreased abundance of methanotrophs bacteria, i.e. α-proteobacteria and β-proteobacteria, anaerobic methanotrophic archaea (ANME), protozoa, sulfate-reducing bacteria, syntrophic bacteria with methanogens, geobacter, and genes related to pyruvate, fatty acid, nitrogen, and sulfur metabolisms, ribulose monophosphate cycle, and Entner-Doudoroff Pathway. Additionally, the abundance of methanogenic archaea and the mcrA gene did not change. The co-occurrence networks enabled us to identify the interactions between each taxon in microbial communities and to determine the reshaping of rumen microbiome associations by gastrointestinal nematode infection. Besides, the correlation between ANMEs was lower in the animal's postinfection. Our findings suggest that gastrointestinal parasites potentially lead to decreased methanotrophic metabolism-related microorganisms and genes., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

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., (© 2024. The Author(s).)

Recently, a S168T variant in the acetylcholine receptor subunit ACR-8 was associated with levamisole resistance in the parasitic helminth Haemonchus contortus. Here, we used the Xenopus laevis oocyte expression system and two-electrode voltage-clamp electrophysiology to measure the functional impact of this S168T variant on the H. contortus levamisole-sensitive acetylcholine receptor, L-AChR-1.1. Expression of the ACR-8 S168T variant significantly reduced the current amplitude elicited by levamisole compared to acetylcholine, with levamisole changing from a full to partial agonist on the recombinant L-AChR. Functional validation of the S168T mutation on modulating levamisole activity at the receptor level highlights its critical importance as both a mechanism and a marker of levamisole resistance., Competing Interests: Declaration of competing interest The authors report no conflict of interest for this work., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Our understanding of anthelmintic resistance in the gastrointestinal nematodes of Australian cattle relies exclusively on small-scale phenotypic reports utilising traditional faecal egg count reduction tests. This approach is not readily scalable to establish the national prevalence of resistance, nor is it conducive of routine longitudinal surveillance for the emergence of resistance in its early stages. This study introduces the benefits of applying mixed amplicon metabarcoding longitudinally for timely and cost-efficient molecular surveillance of multiple anthelmintic resistance mutations, as they emerge on farms. Using opportunistically collected faecal samples from a cattle herd in central west New South Wales (2019-2023), we detected the early emergence of Haemonchus spp. levamisole-resistant S168T shortly after levamisole introduction, while benzimidazole-resistant allele frequencies remained constant. Additionally, we observed the possible spill-over of resistant Haemonchus contortus from sheep, along with variations in faecal burdens and species diversity influenced by climate stochasticity and host immunity. This study emphasises the power of molecular diagnostics for farm-level anthelmintic resistance management, providing essential evidence to support its integration into routine surveillance programmes., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Editorial Board of Veterinary Parasitology (JŠ)., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The faecal egg count reduction test (FECRT) is the most widely used method to assess treatment efficacy against gastrointestinal nematodes (GIN). Information on genera composition of the GIN community is not available with this test and it is commonly obtained by identifying cultured third-stage larvae (L3) or through molecular assays in the post-treatment survey, but results provided are usually only qualitative or semi-quantitative. The updated WAAVP guidelines now recommend assessing anthelmintic efficacy for each GIN genus/species separately (genus-specific FECRT), but this approach is poorly employed in Europe and in goats especially. For this reason, four FECRT trials were conducted using oxfendazole and eprinomectin in two Italian goat farms. Samples were processed individually using the McMaster technique and then pooled to create two samples from faeces of 5 animals each. Pooled samples were analysed using the McMaster and cultured for seven days at 26°C to obtain L3s. The genus-specific FECRT was based on larval identification, integrating coproculture and FEC results. Larvae were identified as Haemonchus, Trichostrongylus, Teladorsagia, Oesophagostomum / Chabertia and Bunostomum. Molecular assays (a multiplex real-time PCR and two end-point PCRs) were also implemented on pooled samples to support the morphological identification. The Spearmann Rho test confirmed a high correlation between the two approaches (Rho = 0.941 and Rho = 0.914 respectively for Haemonchus and Trichostrongylus, the two most common genera). Both oxfendazole and eprinomectin were effective in one farm, while none in the other farm (FECR = 75.9% and 73.3% respectively). In the second farm, the genus-specific FECRT highlighted a different response to treatment among genera: oxfendazole lacked efficacy against both Haemonchus and Trichostrongylus spp., eprinomectin only against Haemonchus, while all other genera were susceptible to both drugs. This study brings new attention on the importance of adopting a genus-specific approach to identify and quantify differences in susceptibility to anthelmintics among genera in goats, providing support for FECRT interpretation, anthelmintic resistance evaluation and evidence-based GIN control., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Haemonchus contortus (H. contortus) is the most common parasitic nematode in ruminants and is prevalent worldwide. H. contortus resistance to albendazole (ABZ) hinders the efficacy of anthelmintic drugs, but little is known about the molecular mechanisms that regulate this of drug resistance. Recent research has demonstrated that long noncoding RNAs (lncRNAs) can exert significant influence as pivotal regulators of the emergence of drug resistance., Results: In this study, transcriptome sequencing was conducted on both albendazole-sensitive (ABZ-sensitive) and albendazole-resistant (ABZ-resistant) H. contortus strains, with three biological replicates for each group. The analysis of lncRNA in the transcriptomic data revealed that there were 276 differentially expressed lncRNA (DElncRNA) between strains with ABZ-sensitive and ABZ-resistant according to the criteria of |log2Foldchange|≥ 1 and FDR < 0.05. Notably, MSTRG.12969.2 and MSTRG.9827.1 exhibited the most significant upregulation and downregulation, respectively, in the resistant strains. The potential roles of the DElncRNAs included catalytic activity, stimulus response, regulation of drug metabolism, and modulation of the immune response. Moreover, we investigated the interactions between DElncRNAs and other RNAs, specifically MSTRG.12741.1, MSTRG.11848.1, MSTRG.5895.1, and MSTRG.14070.1, involved in regulating drug stimulation through cis/trans/antisense/lncRNA‒miRNA-mRNA interaction networks. This regulation leads to a decrease (or increase) in the expression of relevant genes, consequently enhancing the resistance of H. contortus to albendazole. Furthermore, through comprehensive analysis of competitive endogenous RNAs (ceRNAs) involved in drug resistance-related pathways, such as the mTOR signalling pathway and ABC transporter signalling pathway, the relevance of the MSTRG.2499.1-novel-m0062-3p-HCON&#95;00099610 interaction was identified to mainly involve the regulation of catalytic activity, metabolism, ubiquitination and transcriptional regulation of gene promoters. Additionally, quantitative real-time polymerase chain reaction (qRT-PCR) validation indicated that the transcription profiles of six DElncRNAs and six DEmRNAs were consistent with those obtained by RNA-seq., Conclusions: The results of the present study allowed us to better understand the changes in the lncRNA expression profile of ABZ-resistant H. contortus. In total, these results suggest that the lncRNAs MSTRG.963.1, MSTRG.12741.1, MSTRG.11848.1 and MSTRG.2499.1 play important roles in the development of ABZ resistance and can serve as promising biomarkers for further study., (© 2024. The Author(s).)

The aim of the study was to compare the relative gene expression of Haemonchus contortus P-glycoprotein genes ( Hco-pgp ) between fourth (L 4 ), infective (L 3 ), and transitory infective (xL 3 ) 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 xL 3 in vitro culture and to infect Meriones unguiculatus (gerbils) to collect L 4 stages. Morphometric differences were evaluated from 25 individuals of H. contortus from each strain. Relative gene expression from xL 3 and L 4 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 L 3 stage as control group, per strain, and GAPDH and β-tubulin as constitutive genes. Morphological changes were confirmed between xL 3 and L 4 developing oral shape, oesophagus, and intestinal tube. In addition, the body length and width showed statistical differences ( p < 0.05). The Hco-pgp 1, 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 L 4 and xL 3 , respectively. The comparative study between IVMr vs IVMs strains associated to xL 3 and L 4 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 xL 3 and L 4 as suitable laboratory models to study IVMr associated with Hco-pgp genes to contribute to the understanding of anthelmintic resistance.

Haemonchus contortus is a parasitic haematophagous nematode that primarily affects small ruminants and causes significant economic loss to the global livestock industry. Treatment of haemonchosis typically relies on broad-spectrum anthelmintics, resistance to which is an important cause of treatment failure. Resistance to levamisole remains less widespread than to other major anthelmintic classes, prompting the need for more effective and accurate surveillance to maintain its efficacy. Loop-primer endonuclease cleavage loop-mediated isothermal amplification (LEC-LAMP) is a recently developed diagnostic method that facilitates multiplex target detection with single nucleotide polymorphism (SNP) specificity and portable onsite testing. In this study, we designed a new LEC-LAMP assay and applied it to detect the levamisole resistance marker S168T in H. contortus. We explored multiplexing probes for both the resistant S168T and the susceptible S168 alleles in a single-tube assay. We then included a generic probe to detect the acr-8 gene in the multiplex assay, which could facilitate the quantification of both resistance markers and overall genetic material from H. contortus in a single step. Our results showed promising application of these technologies, demonstrating a proof-of-concept assay which is amenable to detection of resistance alleles within the parasite population, with the potential for multiplex detection, and point-of-care application enabled by lateral flow end-point detection. However, further optimisation and validation is necessary., (Copyright © 2023. Published by Elsevier Ltd.)

The intestine of Haemonchus contortus is an essential tissue that has been indicated to be a major target for the prevention of haemonchosis caused by this parasitic nematode of small ruminants. Biological peculiarities of the intestine warrant in-depth exploitation, which can be leveraged for future disease control efforts. Here, we determined the intestinal ncRNA (lncRNA, circRNA and miRNA) atlas using whole-transcriptome sequencing and bioinformatics approaches. In total, 4846 novel lncRNA, 982 circRNA, 96 miRNA (65 known and 31 novel) and 8821 mRNA were identified from the H. contortus intestine. The features of lncRNA, circRNA and miRNA were fully characterized. Comparison of miRNA from the intestines and extracellular vesicles supported the speculation that the miRNA from the latter were of intestinal origin in H. contortus. Further function analysis suggests that the cis-lncRNA targeted genes were involved in protein binding, intracellular anatomical structure, organelle and cellular process, whereas the circRNA parental genes were mainly enriched in molecular function categories, such as ribonucleotide binding, nucleotide binding, ATP binding and carbohydrate derivative binding. The miRNA target genes were related to the cellular process, cellular response to stimulus, cellular protein modification process and signal transduction. Moreover, competing endogenous RNA network analysis revealed that the majority of lncRNA, circRNA and mRNA only have one or two binding sites with specific miRNA. Lastly, randomly selected circRNA, lncRNA and miRNA were verified successfully using RT-PCR. Collectively, these data provide the most comprehensive compilation of intestinal transcripts and their functions, and it will be helpful to decipher the biological and molecular complexity of the intestine and lay the foundation for further functional research., (© 2023. The Author(s).)

Anthelmintic-resistant parasitic nematodes present a significant threat to sustainable livestock production worldwide. The ability to detect the emergence of anthelmintic resistance at an early stage, and therefore determine which drugs remain most effective, is crucial for minimising production losses. Despite many years of research into the molecular basis of anthelmintic resistance, no molecular-based tools are commercially available for the diagnosis of resistance as it emerges in field settings. We describe a mixed deep amplicon sequencing approach to determine the frequency of the levamisole (LEV)-resistant single nucleotide polymorphism (SNP) within arc-8 exon 4 (S168T) in Haemonchus spp., coupled with benzimidazole (BZ)-resistant SNPs within β-tubulin isotype-1 and the internal transcribed spacer-2 (ITS-2) nemabiome. This constitutes the first known multi-drug and multi-species molecular diagnostic developed for helminths of veterinary importance. Of the ovine, bovine, caprine and camelid Australian field isolates we tested, S168T was detected in the majority of Haemonchus spp. populations from sheep and goats, but rarely at a frequency greater than 16%; an arbitrary threshold we set based on whole genome sequencing (WGS) of LEV-resistant Haemonchus contortus GWBII. Overall, BZ resistance was far more prevalent in Haemonchus spp. than LEV resistance, confirming that LEV is still an effective anthelmintic class for small ruminants in New South Wales, Australia. The mixed amplicon metabarcoding approach described herein paves the way towards the use of large scale sequencing as a surveillance technology in the field, the results of which can be translated into evidence-based recommendations for the livestock sector., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Haemonchus contortus is the most pathogenic and economically restrictive gastrointestinal nematode in the small ruminant industry globally. Morbidity, poor cross-bodily state, and mortality of sheep in Lesotho suggest the presence of H. contortus. The present study investigated the morphological, molecular, and population genetics of H. contortus third-stage larvae infecting sheep in four ecological zones (EZ) of Lesotho. Coprocultures were prepared for larval morphological identification and PCR determination. Larvae were identified morphologically as 100% H. contortus. The Second Internal Transcribed Spacer (ITS-2) gene of the ribosomal DNA of H. contortus isolates in the present study revealed nucleotide homology ranging from 97 to 100% when compared with selected GenBank reference sequences. Pairwise evolutionary divergence among H. contortus isolates was low, with 0.01318 recorded as the highest in the present study. Five haplotypes resulted from 14 Lesotho sequences. Haplotype diversity and nucleotide diversity were 0.76923 and 0.00590, respectively. Genetic differentiation among isolates was low but not statistically significant. An analysis of molecular variance revealed that most molecular variation was distributed within topographic populations at 94.79% (F ST  = 0.05206, p > 0.05) and 5.21% among populations. There was high gene flow and no definite population genetic structure among Lesotho isolates., Competing Interests: Declaration of Competing Interest The authors do not declare any competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Although ivermectin (IVM) has a wide spectrum and long half-life, its frequent use as an anthelmintic for the last 42 years led to its worldwide tolerance by Haemonchus contortus. We evaluated the combination of limonene (LIM), a P-glycoprotein (Pgp) modulator, with IVM in lambs infected with a multidrug-resistant H. contortus. Twenty-four male Dorper lambs were artificially infected with two doses (seven days apart) of 8000 infective larvae of a multidrug-resistant isolate of H. contortus. The infection was patent 25 days later. Fifteen days before treatment with IVM (DAY -15), animals were divided into 4 groups: Infected-untreated control (CTL), IVM, LIM, and LIM+IVM. From DAY -15 to DAY + 14, groups LIM and LIM+IVM received 200 mg/kg of body weight/day of LIM via oral. On DAY 0, a single dose of IVM at 200 µg/kg of body weight was administered orally to groups IVM and LIM+IVM. On DAY + 7 and DAY + 14, fecal egg counts (FEC) were performed and on DAY + 14 animals were euthanized for total worm count (TWC), worm length, fecundity of females, and Pgp-9 gene expression. On DAY + 7, group LIM+IVM had 96.29% efficacy based on Fecal Egg Count Reduction TEST (FECRT) and a highly significant reduction in FEC (P = 0.0005) when compared to CTL. On DAY + 14, the efficacy of LIM+IVM was 82.87% on FECRT, although no differences were found among groups for FEC, TWC, worm length, or Pgp-9 gene expression. Female worms from the CTL group had higher egg counts in their uterus when compared to LIM. No differences were found for hematological or biochemical parameters, body weight, or weight gain among groups. Thus, LIM given daily at 200 mg/kg was safe for animals and, when combined with IVM, decreased egg shedding and could reduce pasture contamination, although it was unable to kill multidrug-resistant H. contortus., Competing Interests: Declaration of Competing Interest The authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest or non-fi nancial interest in the subject matter or materials discussed in this manuscript., (Copyright © 2023. Published by Elsevier B.V.)

Haemonchus contortus is a blood-sucking gastrointestinal nematode that infects all ruminants and causes significant economic losses in production. Characterizing the genetic variability of H. contortus populations is crucial for understanding patterns of disease transmission and developing effective control strategies against haemonchosis. This study aimed to identify the genetic variability of H. contortus isolates in small ruminants from slaughterhouses in Bangladesh. During January to December 2015, 400 abomasa samples were collected and 186 were found to be positive for Haemonchus. A 321-bp fragment of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA and an 800-bp fragment of the mitochondrial nicotinamide dehydrogenase subunit-4 gene (nad4) were amplified using polymerase chain reaction (PCR) and directly sequenced. The results showed 10 genotypes (ITS-2) and 45 haplotypes (nad4) among the 186 worms. The sequences were 98.5 to 100% identical to reference sequences from the GenBank database. ITS-2 sequence analysis revealed four nucleotide substitutions at positions 30, 41, 42, and 216. There was one transition (C/T) at position 42 and three transversions (C/A at position 30, G/C at position 41, and T/A at position 216). The nad4 gene sequences showed 15 substitutions, all of which were transitions. The pairwise distance of ITS-2 between H. contortus populations ranged from 0.005 to 1.477. The nucleotide diversity (μ) among the populations was 0.009524 using ITS-2 and 0.00394 using nad4. This study indicated low genetic deviation among H. contortus populations in Bangladesh., (© 2023. The Author(s).)

Santa Ines (SI) and Ile de France (IF) sheep are known to be resistant and susceptible to Haemonchus contortus infection, respectively. Several studies have shown some genes as potential biological markers for sheep resistance against gastrointestinal nematodes using molecular tools, including transcriptomic analysis. In this study, we sequenced the polyadenylated RNA of the abomasal tissue of SI and IF suckling lambs to identify mucosa-specific transcript alterations between breeds artificially infected with H. contortus. Naïve SI (n = 4) and IF (n = 4) lambs were artificially infected every other day, over a period of 52 days, from 14 to 66 days old, with a total of 5,400 H. contortus infective larvae. Fundic abomasal tissue samples were collected at 68 days old, and submitted to high-throughput RNA sequencing (RNA-seq). Differential expression analysis (P value < 0.001 and False Discovery Rate (FDR) < 0.05) between SI and IF samples identified 292 genes, most of which showed greater expression in SI lambs. To help annotate and assign possible function to differentially expressed genes (DEGs), we used previously available single-cell RNA-seq (scRNA-seq) data from ovine abomasal mucosa to putatively identify cell types and possible mechanisms involved in resistance to H. contortus. In particular, genes associated with endothelial and tuft cells showed the greatest increases in expression in SI relative to IF lambs. SI lambs had higher percentages of tuft cells than IF lambs in the fundic abomasal mucosa. Although we found innate immunity (cell-mediated in mucosa) acting as a protagonist in impairing H. contortus infection, a stronger acquired immune response was being modulated at an earlier stage by SI lambs. We suggest that the complex connection between innate and adaptive immunity is via cellular antigen processing and presentation (APP). Based on comparison with scRNA-seq data, SI lambs showed a robust APP mechanism characterized mainly by greater T cell APP, macrophage differentiation, and cytokine signalling. We identified potential mechanisms and markers to advance knowledge for selection of H. contortus resistance at a very early age, in SI as well as in other commercial sheep breeds., (Copyright © 2023 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

The aim of this study was to investigate the occurrence of benzimidazole-resistant Haemonchus contortus in Norwegian sheep flocks. Screening was based on detection of one of the resistance-conferring mutations in the β tubulin isotype 1 gene (F200Y, TAC) in larvae (L3) cultivated from H. contortus eggs from naturally infected sheep. Faecal samples were collected in 2021/2022 from flocks in the northern (n = 34), central (n = 7), eastern (n = 40), southern (n = 1), and western (n = 87) areas of Norway. In total, samples were taken from 169 flocks (spring-ewes samples: 167, autumn-lambs samples: 134). Individual faecal samples were collected from 10 randomly selected ewes (spring) and 10 randomly selected lambs (autumn) in each flock. Faecal samples collected from each flock on each occasion were pooled (lamb and ewe samples pooled separately) and cultured for L3 development. After harvest of larvae (Baermann method), DNA was extracted and then analysed using droplet digital PCR with primer/probe sets targeting the BZ-associated F200Y (TAC) mutation. Haemonchus was found in 60% (80/134) of samples from lambs, and in 63% (106/167) from ewes. Among these, the F200Y mutation was detected in 73% (58/80) of larval samples from lambs and 69% (73/106) of larval samples from ewes, respectively. Although regional differences were evident, the mutation was detected in all areas indicating a widespread distribution and a strong potential for an increasing problem with treatment-resistant haemonchosis in Norwegian sheep flocks., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Protease inhibitors are major components of excretory/secretory products released by parasitic nematodes and have been proposed to play roles in host-parasite interactions. Haemonchus contortus (the barber's pole worm) encodes for several serine protease inhibitors, and in a previous study we identified a trypsin inhibitor-like serine protease inhibitor of this blood-feeding nematode, SPI-I8, as necessary for anticoagulation. Here, we demonstrated that a bovine pancreatic trypsin inhibitor/Kunitz-type serine protease inhibitor (BPTI/Kunitz) domain-containing protein highly expressed in parasitic stages, HCON&#95;00133150, is involved in suppressing proinflammatory cytokine production in mammalian cells. Fluorescent labelling of HCON&#95;00133150 revealed a punctate localisation at the inner hypodermal membrane of H. contortus, an organ closely related to the excretory column. Yeast two-hybrid screening and immunoprecipitation-mass spectrometry identified that the recombinant HCON&#95;00133150 physically interacted with a range of host proteins including the G protein subunit beta 1 of sheep (Ovis aries; OaGNB1), a negative regulator of NLRP3 inflammasome activation. Interestingly, heterologous expression of HCON&#95;00133150 enhanced the inhibitory effect of OaGNB1 on NLRP3 inflammasome and the maturation of proinflammatory cytokines IL-1β and IL-18 in transfected cells. 1-to-1 orthologues (n = 33) of BPTI/Kunitz inhibitor domain-containing proteins were predicted in clades III, IV and V (but not clade I) parasitic nematodes. Structural (tandem BPTI/Kunitz inhibitor domains inverted into the globular reticulation) and functional (a GNB1 enhancer) characterisation of HCON&#95;00133150 and its orthologues elucidated that these molecules might contribute to immune suppression by parasitic nematodes in animals and humans., Competing Interests: Declaration of Competing Interest The authors have declared that no competing interests exist., (Copyright © 2023 Elsevier B.V. All rights reserved.)

An associated microbiome of any host helps it in different metabolic processes ranging from the decomposition of food to the maturation of gametes. Organisms with a parasitic mode of life, though present at nutritious sites inside their host, maintain their own microbiome. Nevertheless, the comprehensive characterization and functionality of microbiome in parasitic organisms remain understudied. We selected two nematode parasites of Kashmir Merino sheep viz; Haemonchus contortus and Trichuris ovis based on their higher prevalence, difference in mode of nutrition, habitation site and effect on host. The objective of the study was to explore the bacteria associated with these parasitic nematodes of sheep. We adopted a 16S rRNA metagenomic sequencing approach to estimate and compare the bacterial communities present in these two nematode species. Nematode parasites from Kashmir Merino sheep were identified morphologically and confirmed with DNA characterization. H. contortus was dominated by phylum Proteobacteria (57%), Firmicutes (25%), Bacteroidota (15%) and Actinobacteriota (3%). Conversely, T. ovis showed Proteobacteria (78%) followed by Firmicutes (8%), Bacteroidota (8%), Actinobacteriota (1%), Fusobacteriota (1%) and other phyla (4%). This study provides a comprehensive account of the microbiome composition of H. contortus and T. ovis, both of which are highly prevalent among Kashmir Merino sheep. Additionally, T. ovis exhibited a greater bacterial diversity compared to H. contortus. Notably, these nematodes were found to harbor certain pathogenic bacteria. This study can further be carried forward in gaining insights into the complex relationship between the microbiota of a parasite and its pathogenicity, reproductive potential and host microbiome modification.

The Transforming Growth Factor-β mimic (TGM) multigene family was recently discovered in the murine intestinal parasite Heligmosomoides polygyrus. This family was shaped by an atypical set of organismal and molecular evolutionary mechanisms along its path through the adaptive landscape. The relevant mechanisms are mimicry, convergence, exon modularity, new gene origination, and gene family neofunctionalization. We begin this review with a description of the TGM family and then address two evolutionary questions: "Why were TGM proteins needed for parasite survival" and "when did the TGM family originate"? For the former, we provide a likely answer, and for the latter, we identify multiple TGM building blocks in the ruminant intestinal parasite Haemonchus contortus. We close by identifying avenues for future investigation: new biochemical data to assign functions to more family members as well as new sequenced genomes in the Trichostrongyloidea superfamily and the Heligmosomoides genus to clarify TGM origins and expansion. Continued study of TGM proteins will generate increased knowledge of Transforming Growth Factor-β signaling, host-parasite interactions, and metazoan evolutionary mechanisms., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Association between ovine β-globin polymorphisms and resistance against haemonchosis was described and related to the mechanism of high oxygen affinity β A ➔ β C switch during anaemia, but there are no studies regarding the involved local host responses. Phenotypic parameters and local responses were evaluated in sheep from two β-globin haplotypes naturally infected with Haemonchus contortus. Morada Nova lambs were monitored at 63, 84 and 105 days of age for faecal egg counts and packed cell volume (PCV) under natural infection with H. contortus. At 210 days of age, lambs of Hb-AA and Hb-BB β-globin haplotypes were euthanised, and the fundic region of abomasum was sampled for evaluation of microscopic lesions and relative expression of genes related to immune, mucin and lectin activities. Lambs harbouring the β A allele presented an improved resistance/resilience against clinical haemonchosis, showing higher PCV during infection. Hb-AA animals presented increased eosinophilia in the abomasum compared to Hb-BB animals, accompanied by higher Th2 profile, mucin and lectin activity transcripts, while the inflammatory response was increased in Hb-BB animals. This is the first report to demonstrate an enhanced local response in the primary site of H. contortus infection related to β A allele of β-globin haplotype., (© 2023 John Wiley & Sons Ltd.)

Background: The CAP superfamily proteins are distributed widely in eukaryotes and play crucial roles in various biological processes. However, very little is known about their functions in parasitic nematodes, including Haemonchus contortus, a socioeconomically important parasitic nematode. We have therefore studied a member of the CAP protein family of H. contortus, named Hc-CAP-15, with the aim to explore its roles in regulating the parasitic developmental process., Methods: The conservation and phylogenetic relationships, spatial expression and temporal transcription profiles of Hc-CAP/cap-15, as well its biological function during parasite development were investigated using bioinformatics, immunofluorescence, real-time PCR and RNA interference (RNAi)., Results: Hc-CAP-15 was found to be a single-domain CAP protein consisting of four conserved motifs that is localized in the cuticle, intestine and oocyte of adult worms. Hc-cap-15 was transcribed at all developmental stages of H. contortus, with the highest transcription level in parasitic fourth-stage larvae (L4s). Silencing of Hc-cap-15 resulted in a significant increase in the body length of L4s., Conclusions: The results suggested that Hc-CAP-15 is important for the development of H. contortus. Our findings provide a basis for further study of the functions of the CAP family proteins in H. contortus and related parasitic nematodes., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Haemonchus contortus is the most pathogenic nematode in small ruminants and anthelmintic resistance (AR) hampers its efficient control. Early detection of AR status is required to reduce selection for AR and cannot be achieved using phenotypic tests. For benzimidazoles (BZs), the detection of AR-associated alleles characterised by single nucleotide polymorphisms (SNPs) in the isotype 1 β-tubulin gene allows early AR detection in strongyles. The F200Y, F167Y, E198A and E198L polymorphisms have been described in BZ-resistant populations with a clear variation in frequencies between regions. A novel digital PCR (dPCR) enables the detection of all of the above-described polymorphisms in H. contortus. Assays were validated using synthetic DNA fragments containing these SNPs. Then, larvae obtained and pooled at farm level from 26 Austrian and 10 Italian sheep farms were analysed. For all assays a detection limit of 15 copies/μl of resistance alleles and a high level of accuracy were demonstrated, allowing to detect allele frequencies of 1% in most samples. In Austrian samples, elevated frequencies of F200Y resistance alleles were detected on all farms. Polymorphisms in codon 167 and codon 198 were identified in H. contortus from Austria for the first time. In Italian samples, the frequency of resistance alleles was still comparatively low, but F200Y resistance alleles were traceable. In conclusion we developed for the first time dPCR assays that target all SNPs of relevance associated with BZ-resistance in H. contortus. Future research on AR development could benefit from an early onset of SNP-based surveillance that would include the developed assays for all SNPs of relevance. Improved surveillance in the long term will include other important, though less pathogenic, nematode genera in the analyses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Biodiversity within the animal kingdom is associated with extensive molecular diversity. The expansion of genomic, transcriptomic and proteomic data sets for invertebrate groups and species with unique biological traits necessitates reliable in silico tools for the accurate identification and annotation of molecules and molecular groups. However, conventional tools are inadequate for lesser-known organismal groups, such as eukaryotic pathogens (parasites), so that improved approaches are urgently needed. Here, we established a combined sequence- and structure-based workflow system to harness well-curated publicly available data sets and resources to identify, classify and annotate proteases and protease inhibitors of a highly pathogenic parasitic roundworm (nematode) of global relevance, called Haemonchus contortus (barber's pole worm). This workflow performed markedly better than conventional, sequence-based classification and annotation alone and allowed the first genome-wide characterisation of protease and protease inhibitor genes and gene products in this worm. In total, we identified 790 genes encoding 860 proteases and protease inhibitors representing 83 gene families. The proteins inferred included 280 metallo-, 145 cysteine, 142 serine, 121 aspartic and 81 "mixed" proteases as well as 91 protease inhibitors, all of which had marked physicochemical diversity and inferred involvements in >400 biological processes or pathways. A detailed investigation revealed a remarkable expansion of some protease or inhibitor gene families, which are likely linked to parasitism (e.g., host-parasite interactions, immunomodulation and blood-feeding) and exhibit stage- or sex-specific transcription profiles. This investigation provides a solid foundation for detailed explorations of the structures and functions of proteases and protease inhibitors of H. contortus and related nematodes, and it could assist in the discovery of new drug or vaccine targets against infections or diseases.

Haemonchus contortus is a parasitic nematode of small ruminants responsible for significant economic losses and animal health concerns globally. Detection of gastrointestinal nematode (GIN) infection in veterinary practice typically relies on microscopy-based methods such as the faecal egg count and morphological identification of larval culture. However, mixed co-infections are common and species-specific identification is typically time-consuming and expertise-intensive. Compounded by increasing anthelmintic resistance, there is an urgent need to implement the molecular diagnosis of GIN in the livestock industry, preferably in field settings. Advances in isothermal amplification techniques including recombinase polymerase amplification (RPA) assays could improve this. Yet, constraints in RPA kit availability and amplicon detection systems limit the use of this technology in point of care settings. In this study, we present an early-stage, proof-of-concept demonstration of RPA targeting the internal transcribed spacer (ITS2) region of H. contortus. Having tested against eight closely related nematodes and also against five farm isolates in Eastern Hungary, preliminary results derived from a comparative analysis of 3 primer sets showed the assay detects H. contortus DNA and has a limit of detection of 10 -5 ng/μl. We also tested an end-result naked eye detection system using various DNA binding dyes, of which EvaGreen® dye was successful for a qualitative RPA detection that could be adaptable at farm sites., Competing Interests: Declaration of Competing Interest The authors declare that they have no direct conflict of interest with this work., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Nematodes have lost the ability to synthesise necessary lipids de novo and have complementally evolved the capacity to acquire fatty acids and their derivatives from a diet or host animal. Nematode-specific fatty acid- and retinol-binding protein (FAR) family is one approach that facilitates lipid acquisition, representing an Achilles heel and potential target against roundworms of socioeconomic significance. However, little is known about their detailed functional roles in either free-living or parasitic nematodes., Methods: A genome-wide identification and curation were performed to screen the FAR family members of Haemonchus contortus. Their transcription patterns in worms were also analysed to identify the targets. Ligand binding assay and molecular docking were conducted to verify the fatty acid binding activities of FAR proteins of interest. RNA interference (RNAi) and heterologous expression (rescuing) experiments were designed to explore the potential roles of the selected FAR protein in nematodes. Localisation of the protein was shown in sections of paraffin-embedded worms after an immunohistochemistry (IHC) assay., Results: Here, an orthologue of far-6 in the model organism Caenorhabditis elegans (Ce-far-6) was functionally characterised in a parasitic nematode, H. contortus (Hc-far-6). It is demonstrated that knockdown of Ce-far-6 gene did not affect worm fat content, reproduction, or lifespan, but decreased worm body length at an early life stage of C. elegans. In particular, the Ce-far-6 mutant associated phenotype was completely rescued by Hc-far-6, suggesting a conserved functional role. Surprisingly, there were distinct tissue expression patterns of FAR-6 in the free-living C. elegans and parasitic H. contortus. High transcriptional level of Hc-far-6 and dominant expression of FAR-6 in the intestine of the parasitic stage of H. contortus link this gene/protein to nematode parasitism., Conclusions: These findings substantially enhance our understanding of far genes and the associated lipid biology of this important parasitic nematode at a molecular level, and the approaches established are readily applicable to the studies of far genes in a broad range of parasites., (© 2023. The Author(s).)

Gastrointestinal parasites are among the most economically important pathogens of small ruminants causing serious economic losses and animal welfare problems for the livestock industry worldwide. The emergence of anthelmintic resistant H. contortus in small ruminants is a serious problem because it undermines effective helminth control and results in reduced productivity. Little is known about resistance to benzimidazoles (BZ) in Haemonchus in goats and sheep in Uganda. The objective of this study was to determine the prevalence of gastrointestinal parasites and to identify the presence of benzimidazole resistance associated mutations in the β-tubulin isotype 1 gene of Haemonchus contortus in goats from selected districts of Uganda. A total of 200 goats from 10 districts of Uganda slaughtered at Kalerwe abattoir in Kampala were sampled for H. contortus adult worms. Faecal samples were also collected to detect other intestinal parasites. Faecal microscopy and analysis were performed using flotation and sedimentation techniques. DNA was extracted from adult worms and PCR and sequencing of the ITS- 2 region and β-tubulin isotype 1 gene performed to identify H. contortus species and to determine the presence of mutations associated with anthelmintic resistance respectively. Faecal microscopy showed that the most prevalent intestinal parasites were coccidia (98%), strongyles (97.5%), Strongyloides (82%), Paramphistomum (74.5%), Moniezia (46%), Fasciola (1.5%) and Trichuris (1%). Most goats had a high intestinal burden of coccidia (≥ 5000 oocyst per gram) and strongyles (≥ 1000 egg per gram), 65% and 67.5%, respectively. The prevalence of H. contortus adult worms was 63% (126/200). Sequencing of the partial β-tubulin isotype 1 gene of 54 Haemonchus contortus adult male isolates revealed the presence of mutations associated with anthelmintic resistance. The F200Y mutation was the most common mutation (13% of samples with good beta-tubulin sequences) followed by the E198A and E198K mutations, both found in 9% of sequenced samples. Mutation F167Y was not identified in any of the samples and there were no heterozygous individuals for any of the SNPS associated with BZ resistance that were identified in this study. These findings highlight the need for controlled use of anthelmintics especially benzimidazoles, to enable sustainable control of H. contortus in Uganda, and a need for further investigation to understand the resistance of other parasites identified in this study., Competing Interests: Declaration of Competing Interest This study was supported by the Africa Livestock Productivity and Health Advancement (ALPHA) Initiative, co-funded by Zoetis and the Bill & Melinda Gates Foundation. Two of the co-authors (E.M., G.V.) are employed by Zoetis. E.M. and G.V. were involved in the writing of the manuscript. There are no potential areas of bias which may confer any advantage to Zoetis., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

In humans, nematode infections are prevalent in developing countries, causing long-term ill health, particularly in children. Worldwide, nematode infections are prevalent in livestock and pets, affecting productivity and health. Anthelmintic drugs are the primary means of controlling nematodes, but there is now high prevalence of anthelmintic resistance, requiring urgent identification of new molecular targets for anthelmintics with novel mechanisms of action. Here, we identified orthologous genes for phosphoethanolamine methyltransferases (PMTs) in nematodes within the families Trichostrongylidae , Dictyocaulidae , Chabertiidae , Ancylostomatoidea , and Ascarididae . We characterized these putative PMTs and found that they possess bona fide PMT catalytic activities. By complementing a mutant yeast strain lacking the ability to synthesize phosphatidylcholine, the PMTs were validated to catalyze the biosynthesis of phosphatidylcholine. Using an in vitro phosphoethanolamine methyltransferase assay with PMTs as enzymes, we identified compounds with cross-inhibitory effects against the PMTs. Corroboratively, treatment of PMT-complemented yeast with the PMT inhibitors blocked growth of the yeast, underscoring the essential role of the PMTs in phosphatidylcholine synthesis. Fifteen of the inhibitors with the highest activity against complemented yeast were tested against Haemonchus contortus using larval development and motility assays. Among them, four were found to possess potent anthelmintic activity against both multiple drug-resistant and susceptible isolates of H. contortus , with IC 50 values (95% confidence interval) of 4.30 μM (2.15-8.28), 4.46 μM (3.22-6.16), 28.7 μM (17.3-49.5), and 0.65 μM (0.21-1.88). Taken together, we have validated a molecular target conserved in a broad range of nematodes and identified its inhibitors that possess potent in vitro anthelmintic activity., Competing Interests: The authors declare no conflict of interest.

MHC gene is highly polymorphic and plays central molecule in immune system in recognition of pathogens and parasites. The present study examined the variability of MHC class II DRB exon 2 gene using microsatellite analysis, and its association with Haemonchus contortus infestation in Salem Black goat population. Animals were naturally exposed to mixed infestation of endoparasites, predominantly Haemonchus contortus . Pooled fecal coproculture and larval identification showed predominant presence of haemonchus (L3) larva. Fecal egg count (FEC) and packed cell volume (PCV) were used as indicator traits. All the three studied loci, OarCP73 , DYA , and ODRB1.2 , were polymorphic having three, four, and four alleles and four, eight, and nine genotypes, respectively. The loci showed low to moderate values of polymorphic information content. The mean fecal egg count estimates were 477.12 ± 34.14 eggs per gram of feces and the mean PCV values were within the normal range; however, they showed negative correlation with FEC values. All the three microsatellite loci and the effect of genotypes on indicator traits were found to be statistically significant ( p  ≤ 0.05). The present study will be helpful in selection and breeding of goats for Haemonchosis resistance.

Garole sheep exhibits within-breed difference in resistance to natural gastrointestinal nematode infection predominated by Haemonchus contortus. In the present study, interferon gamma gene (IFN-γ) was characterized in relation to parasitological, haematological, and immune response against H. contortus in resistant and susceptible Garole sheep. Resistant and susceptible Garole sheep were selected from the field based on consistent low faecal egg counts (FEC) for one year and single nucleotide polymorphisms (SNPs) in the IFN-γ gene. The partial amplification of IFN-γ gene (1282 bp) revealed 4 SNPs exclusively in resistant sheep and 3 SNPs were shared between resistant and susceptible Garole sheep. The selected resistant and susceptible Garole sheep were challenged with H. contortus infection. The parasitological, haematological, immunological responses, and expression of IFN-γ gene were compared between the resistant and susceptible Garole sheep. The FEC of resistant sheep was significantly (P < 0.05) lower than the susceptible sheep infected with H. contortus. There was spontaneous elimination of H. contortus from 28 to 33 days post infection (DPI) in resistant sheep. Haemoglobin and packed cell volume were significantly (P < 0.05) higher in resistant sheep than the susceptible sheep. The serum concentration of immunoglobulin (Ig)G 1 and IgA and cytokine IFN-γ activity and also the expression of IFN-γ gene were significantly (P < 0.05) higher in the infected resistant sheep from 14 to 28 DPI compared to the susceptible sheep. In resistant sheep, IgA and IgG 1 and cytokine IFN-γ positively correlated with expression of IFN-γ gene, and the SNPs recorded in the resistant sheep only might play an important role in conferring resistance against H. contortus. Further studies are required to elucidate the role of IFN-γ gene in H. contortus resistance in Garole sheep., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Haemonchus contortus is an important parasite that causes disease that seriously endangers ruminant animals cattle, sheep, goat, and camel. Here, we compared the proeomics analysis of three adult Haemonchus contortus isolates from mouflons ( Ovis ammon ). A total of 1,299 adult worm proteins were identified, and 461 proteins were quantified, of which 82 (108), 83 (97), and 97 (86) significantly upregulated (downregulated) differentially expressed proteins (DEPs) were detected among pairwise comparisons (1-vs.-3, 2-vs.-3, and 2-vs.-1). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatic analysis indicated that these DEPs are mainly concentrated in cellular composition, molecular function, biological function, and catabolism pathways. In addition, Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were carried out to screen the DEPs. The main biological processes involved were nucleotide, nucleotide phosphate, ribonucleotide, purine-containing compound, purine ribonucleotide, single-organism, oxoacid, organic, carboxylic, oxoacid metabolic processes and single-organism catabolic processes. The majority of KEGG pathways were found to be related to metabolic pathways, biosynthesis of secondary metabolites, biosynthesis of antibiotics, carbon metabolism, and microbial metabolism in diverse environments. Moreover, we also found differences in the expression of some important or novel regulatory proteases, such as serine hydroxymethyl transferase (SHMT), dihydrolipoyl dehydrogenase (DLD), and transket pyr domain-containing protein (TKPD). In summary, label-free proteomic analysis of adult H. contortus worms displayed significant differences in three different individual isolates, which helps to improve our understanding of the growth and metabolic mechanisms of H. contortus in different individuals and relative natural environments and provides novel drug targets for the treatment of parasitic diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Liu, Liu, Han, Wang and Li.)

Short-chain dehydrogenases/reductases (SDRs) regulate the activities of many hormones and other signaling molecules and participate in the deactivation of various carbonyl-bearing xenobiotics. Nevertheless, knowledge about these important enzymes in helminths remains limited. The aim of our study was to characterize the SDR superfamily in the parasitic nematode Haemonchus contortus. Genome localization of SDRs was explored, and phylogenetic analysis in comparison with SDRs from free-living nematode Caenorhabditis elegans and the domestic sheep (Ovis aries, a typical host of H. contortus) was constructed. The expression profile of selected SDRs during the life cycle along with differences between the drug-susceptible and drug-resistant strains, were also studied. Genome sequencing enabled the identification of 46 members of the SDR family in H. contortus. A number of genes have no orthologue in the sheep genome. In all developmental stages of H. contortus, SDR1, SDR3, SDR5, SDR6, SDR14, and SDR18 genes were the most expressed, although in individual stages, huge differences in expression levels were observed. A comparison of SDRs expression between the drug-susceptible and drug-resistant strains of H. contortus revealed several SDRs with changed expression in the resistant strain. Specifically, SDR1, SDR12, SDR13, SDR16 are SDR candidates related to drug-resistance, as the expression of these SDRs is consistently increased in most stages of the drug-resistant H. contortus. These findings revealing several SDR enzymes of H. contortus warrant further investigation., (© 2023. The Author(s).)

Haemonchus contortus is one of the most detrimental gastrointestinal nematode parasites for small ruminants, especially in tropics and subtropics. Gastrointestinal nematode and microbiota share the same microhabitat; thus they interact with each other and their host. Metagenomics tools provide a promising way to examine the alterations in the gastric microbial composition induces by gastrointestinal parasites. In this study, we used metagenomics tools to characterize the impact of H. contortus infection on the caprine abomasal microbiota at early and late stage of infection and compared it with non-infected control. Our results showed that H. contortus infection caused a significant increase in abomasal pH at early (7 days post-infection) and late stage of infection (56 days post-infection). The analysis of alpha and beta diversity showed that the microbiota diversity both in number and in proportion was significantly affected at early and late stage of infection. All microbiota classes are impacted by H. contortus infection but Clostridia and Bacteroidia are more concerned. In infected animals, the genera Prevotella decreased at 7 and 56 days post-infection. Here we showed that the abomasal microbiota was significantly affected early after H. contortus infection, and these changes persist at late stage of the infection., (© 2023. The Author(s).)

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 L 3 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., Competing Interests: Declaration of Competing Interest The authors of this manuscript have no financial or personal relationship with other people or organization that could be inappropriately influence the content of this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Parasitic roundworms (nematodes) have lost genes involved in the de novo biosynthesis of haem, but have evolved the capacity to acquire and utilise exogenous haem from host animals. However, very little is known about the processes or mechanisms underlying haem acquisition and utilisation in parasites. Here, we reveal that HRG-1 is a conserved and unique haem transporter in a broad range of parasitic nematodes of socioeconomic importance, which enables haem uptake via intestinal cells, facilitates cellular haem utilisation through the endo-lysosomal system, and exhibits a conspicuous distribution at the basal laminae covering the alimentary tract, muscles and gonads. The broader tissue expression pattern of HRG-1 in Haemonchus contortus (barber's pole worm) compared with its orthologues in the free-living nematode Caenorhabditis elegans indicates critical involvement of this unique haem transporter in haem homeostasis in tissues and organs of the parasitic nematode. RNAi-mediated gene knockdown of hrg-1 resulted in sick and lethal phenotypes of infective larvae of H. contortus, which could only be rescued by supplementation of exogenous haem in the early developmental stage. Notably, the RNAi-treated infective larvae could not establish infection or survive in the mammalian host, suggesting an indispensable role of this haem transporter in the survival of this parasite. This study provides new insights into the haem biology of a parasitic nematode, demonstrates that haem acquisition by HRG-1 is essential for H. contortus survival and infection, and suggests that HRG-1 could be an intervention target candidate in a range of parasitic nematodes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)