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

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

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

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

Functional validation of novel levamisole resistance marker S168T in Haemonchus contortus.

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.

Analysis of P-gp genes relative expression associated to ivermectin resistance in Haemonchus contortus larval stages from in vitro cultures (L3 and xL3) and from gerbils (Meriones unguiculatus) (L4) as models of study.

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

Transcriptome reveals the roles and potential mechanisms of lncRNAs in the regulation of albendazole resistance in Haemonchus contortus.

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_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.

Faecal egg count reduction test in goats: Zooming in on the genus level.

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.

The proof is in the poo-ding: Benefits of the longitudinal molecular surveillance of drug resistance demonstrated in a New South Wales cattle herd.

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.

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

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

Non-coding RNA in the gut of the blood-feeding parasitic worm, Haemonchus contortus.

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.

A mixed amplicon metabarcoding and sequencing approach for surveillance of drug resistance to levamisole and benzimidazole in Haemonchus spp.

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 ß-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.

Real-time single-base specific detection of the Haemonchus contortus S168T variant associated with levamisole resistance using loop-primer endonuclease cleavage loop-mediated isothermal amplification.

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.

Combined effects of Limonene and Ivermectin on P-glycoprotein-9 gene expression of lambs Infected with Haemonchus contortus.

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.

Benzimidazole-resistance associated mutation in Haemonchus contortus in Norwegian sheep, as detected by droplet digital PCR.

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 ß 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.

Genetic variability of Haemonchus contortus isolates in small ruminants from slaughterhouses in Bangladesh.

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.

Characterization and population genetics of Haemonchus contortus in Merino sheep in Lesotho.

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% (FST = 0.05206, p > 0.05) and 5.21% among populations. There was high gene flow and no definite population genetic structure among Lesotho isolates.

Bacteria associated with ovine gut parasites Trichuris ovis and Haemonchus contortus.

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.

Abomasal RNA-seq reveals a strong local cellular response in suckling lambs with resistance against Haemonchus contortus.

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.

Convergent Evolution in a Murine Intestinal Parasite Rapidly Created the TGM Family of Molecular Mimics to Suppress the Host Immune Response.

The Transforming Growth Factor-ß 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-ß signaling, host-parasite interactions, and metazoan evolutionary mechanisms.

A member of the CAP protein superfamily, Hc-CAP-15, is important for the parasitic-stage development of Haemonchus contortus.

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.

Genome-Wide Analysis of Haemonchus contortus Proteases and Protease Inhibitors Using Advanced Informatics Provides Insights into Parasite Biology and Host-Parasite Interactions.

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.