A type 2 immune circuit and arachidonic acid metabolism role in anti-nematode infection: evidence from transcriptome and targeted metabolome data in goat.

The gastrointestinal nematode infection poses a covert threat to both humans and domestic animals worldwide, eliciting a type 2 immune response within the small intestine. Intestinal tuft cells detect the nematode and activated group 2 innate lymphoid cells. Tuft cell-derived leukotrienes (one of the metabolites of arachidonic acid) were found to drive rapid anti-helminth immunity, but it is still poorly understood whether the tuft cell-mediated type 2 immune circuit and arachidonic acid metabolism modulate anti-parasitic immunity in the gastric epithelium. This study was designed to evaluate the immunological responses of goats inoculated with or without H. contortus. Results showed that H. contortus infection induced a systemic type 2 immune response, characterised by lymphocyte proliferation and greater eosinophils both in peripheral blood and abomasal mucosa, as well as increased type 2 cytokines IL-4, IL-5, and IL-13. Infection of H. contortus altered the transcriptome of the abomasum epithelium, especially tuft cell-mediated circuit-key genes. The infection also influenced the abomasal microbiota, arachidonic acid metabolism and related lipid metabolites, accompanying with great increases in the secretion of leukotrienes and prostaglandins. These findings demonstrate the role of tuft cells mediated circuit in sensing H. contortus infection and immune activation, reveal the candidate function of arachidonic acid involved in anti-helminth immunity, and suggest novel strategies for the control of parasitic diseases in livestock and humans.

Characterisation of seryl tRNA synthetase (srs-2) in Haemonchus contortus and Teladorsagia circumcincta.

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 Km and Vmax 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.

Exploring alternative anthelmintic compounds: Impact of peruvin, hentriacontane/1-nonacosanol and their synergistic effect on the health of Meriones unguiculatus infected with Haemonchus contortus.

Haemonchus contortus is a highly pathogenic blood-feeding parasite affecting sheep and goats, leading to significant economic losses. With increasing resistance to conventional anthelmintics, exploring plant-based alternatives is crucial. In vitro, studies suggest that peruvin and hentriacontane/1-nonacosanol, isolated from Artemisia cina (Asteraceae), may synergistically control Haemonchus contortus. However, their in vivo efficacy and safety are unestablished. This study evaluated these compounds' anthelmintic activity and health effects and their synergistic mixture in Meriones unguiculatus (gerbils). The compounds were isolated using open-column chromatography and identified by spectroscopic techniques. Gerbils were artificially infected with H. contortus following dexamethasone treatment to enhance infection. Anthelmintic activity was assessed by larval reduction in the stomach, blood biochemical parameters using a blood chemistry analyzer, and the anatomopathological changes in kidney and liver tissues. Peruvin (0.4 mg/kg) and hentriacontane/1-nonacosanol (2.60 mg/kg) achieved larvicidal reductions of 84.86 % and 74.05 %, respectively, while their synergistic mixture (0.08/0.0017 mg/kg) resulted in a 100 % reduction. Histopathological analysis revealed minor inflammation and albuminous degeneration, primarily affecting the liver. The peruvin-treated group showed notable kidney damage, while hepatic alterations were similar across both compounds. Although effective, further research is needed to optimize dosing and ensure safety.

Prevalence status and detection of benzimidazole resistance using AS-PCR in Haemonchus contortus of goats from Marathwada region, Maharashtra, India.

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.

Chromosome-contiguous genome for the Haecon-5 strain of Haemonchus contortus reveals marked genetic variability and enables the discovery of essential gene candidates.

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.

Molecular signatures of Haemonchus contortus infection in sheep: A comparative serum proteomic study on susceptible and resistant sheep breeds.

Due to the negative impact of Haemonchus contortus in the tropics and subtropics, the detection of serum protein profiles that occur in infected sheep is of high relevance for targeted selective treatment strategies (TST). Herein, we integrated proteomics with phenotypic traits to elucidate physiological mechanisms associated to H. contortus infection in susceptible (Dorper - D) and resistant (Santa Inês - S) sheep breeds. Naïve female lambs were infected with H. contortus third-stage larvae on day zero (D0), and samples were collected weekly, for 28 days. Feces were used for individual fecal egg counts (FEC) blood for packed cell volume (PCV) and serum for specific antibody quantification through ELISA. Sera was collected on D0 (-) and D21 (+), and analyzed using a LC-MS/MS based proteomics approach. FEC, PCV, and anti-H. contortus antibody levels confirmed the absence of infection on D0. On D28 there was a significant difference between the two breeds for logFEC means (D = 3774 and S = 3141, p=0.033) and PCV means (D = 16.3 % and S = 24.3 %, p=0.038). From a total of 754 proteins identified, 68 differentially abundant proteins (DAPs) were noted. Phosphopyruvate hydratase (ENO3) was a DAP in all comparisons, while S+ vs D+ and S- vs D- shared the highest number of DAPs (8). Each of the four experimental groups clustered separately in a principal component analysis (PCA) of protein profile. Among the DAPs, proteins associated with the innate and adaptive immune system were detected when comparing S- vs D- and S+ vs D+. In D-, some proteins were linked to stress response to handling, sampling and heat. Focusing on the consequences of infection in each breed, in the D+ vs D- comparison, upregulated proteins were associated with inflammation control and immune response, where downregulated proteins pointed to a negative impact of infection on tissue anabolism, compromising muscle growth and fat deposition. In the S+ vs S- comparison, upregulated proteins were related to immune response, while the downregulated proteins were possibly linked to muscular development and growth, impaired by infection. Collectively, it can be concluded that ENO3 regulation emerges as a potential factor underlying the differential immune response observed between Santa Inês and Dorper sheep infected with H. contortus. In turn, detected acute phase proteins (APPs) reinforce their relation with infection, inflammation and stress conditions, whereas THEMIS-like may contribute to the immune system in Dorper. GSDMD, Guanylate-binding protein and ACAN warrant further investigation as possible biomarkers for TST strategy development.

In vivo anthelmintic activity of Combretum mucronatum schumach & Thonn leaf extract against Haemonchus contortus in goats.

Parasitic infections with gastrointestinal nematodes are a serious problem for the health and welfare of domestic animals and negatively affect the economics of animal production. Haemonchus contortus is a haematophagous nematode of small ruminants responsible for significant mortality and morbidity. In addition, the widespread resistance to synthetic anthelmintic drugs emphasizes the urgent need of alternative treatment options against haemonchosis. This work aims to investigate the anthelmintic activity of an hydroethanolic Combretum mucronatum leaf extract (CMLE) against Haemonchus contortus in goats. Goats were artificially infected with 3500 third-stage larvae of H. contortus, and 21 days later, treated with CMLE (1000, 500, 250 mg/kg) for 4 consecutive days. Different parameters such as faecal egg count reduction, weight and haematocrit were monitored during the experimental period. The number of eggs per gram of faeces (EPG) was concentration-depended lower and significantly reduced compared to the untreated control (p < 0.0001). The effect of the highest CMLE dose (4 ×1000 mg/kg body weight) was similar to the effect of albendazole (1 ×5 mg/kg of body weight). The ED50 and ED90 values calculated were 189.17 and 392.33 mg/kg body weight respectively. ED50 and ED90 values were time-dependent. Moreover, CMLE improved haematocrit and weight of goats in dose-dependent and time-dependent manner. These results showed that CMLE could be used for haemonchosis treatment in small ruminants.

The first molecular detection of benzimidazole resistance in Haemonchus contortus from sheep in Hejing and Minfeng counties of Southern Xinjiang.

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 ß-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 ß-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.

The Mitogenome of the Haecon-5 Strain of Haemonchus contortus and a Comparative Analysis of Its Nucleotide Variation with Other Laboratory Strains.

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_Hco_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 (nad5, nad6, nad1, atp6, nad2, cytb and nad4) 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.

Target screening using RNA interference in the sheep abomasal nematode parasite Haemonchus contortus.

RNA interference (RNAi) on parasitic nematodes has been described as a valuable tool for screening putative targets that could be used as novel drug and/or vaccine candidates. This study aimed to set up a pipeline to identify potential targets using RNAi for vaccine/anti-parasite therapy development against Haemonchus contortus, a blood-feeding abomasal nematode parasite. The available H. contortus sequence data was mined for targets, which were tested for essentiality using RNAi electroporation assays. A total of 56 genes were identified and tested for knockdown using electroporation of first-stage larvae (L1) H. contortus with the target double-stranded RNA. Electroporation of L1 proved to be effective overall; 17 targets had a strong phenotype and significant reduction in alive H. contortus, and another 24 had a moderate phenotype with a significant reduction in larvae development. A total of 28 targets showed a significant reduction in the development of H. contortus larvae to the infective stage (L3) following the RNAi assay. Down-regulation of target transcript levels was evaluated in some targets by semi-quantitative PCR. Four out of five genes tested showed complete knockdown of mRNA levels via semi-quantitative PCR, whereas the knockdown was partial for one. In conclusion, the results indicate that the RNAi pathway is confirmed in H. contortus and that several target genes have the potential to be investigated further as possible vaccine candidates.

Australian Marine and Terrestrial Streptomyces-Derived Surugamides, and Synthetic Analogs, and Their Ability to Inhibit Dirofilaria immitis (Heartworm) Motility.

A bioassay-guided chemical investigation of a bacterium, Streptomyces sp. CMB-MRB032, isolated from sheep feces collected near Bathurst, Victoria, Australia, yielded the known polyketide antimycins A4a (1) and A2a (2) as potent inhibitors of Dirofilaria immitis (heartworm) microfilaria (mf) motility (EC50 0.0013-0.0021 µg/mL), along with the octapeptide surugamide A (3) and the new N-methylated analog surugamide K (4). With biological data suggesting surugamides may also exhibit activity against D. immitis, a GNPS molecular network analysis of a library of microbes sourced from geographically diverse Australian ecosystems identified a further five taxonomically and chemically distinct surugamide producers. Scaled-up cultivation of one such producer, Streptomyces sp. CMB-M0112 isolated from a marine sediment collected at Shorncliff, Qld, Australia, yielded 3 along with the new acyl-surugamides A1-A4 (5-8). Solid-phase peptide synthesis provided additional synthetic analogs, surugamides S1-S3 (9-11), while derivatization of 3 returned the semi-synthetic surugamide S4 (12) and acyl-surugamides AS1-AS3 (13-15). The natural acyl-surugamide A3 (7) and semi-synthetic acyl-surugamide AS3 (15) were shown to selectively inhibit D. immitis mf motility (EC50 3.3-3.4 µg/mL), however, unlike antimycins 1 and 2, were inactive against the gastrointestinal nematode Haemonchus contortus L1-L3 larvae (EC50 > 25 µg/mL) and were not cytotoxic to mammalian cells (human colorectal carcinoma SW620, IC50 > 30 µg/mL). A structure-activity relationship (SAR) study on the surugamides 3-15 revealed that selective acylation of the Lys3-ε-NH2 correlates with anthelmintic activity.

Excreted/Secreted 15-kDa Proteins and Serine Protease Peptides from Haemonchus contortus Act as Immune-Response Enhancers in Lambs.

This study assessed the immunoprotective effect in lambs of a native excreted/secreted 15-kDa protein and two synthesised S28 peptides derived from the infective transitory larvae (xL3) and adult stages (AS) of Haemonchus contortus. Twenty-two Pelibuey lambs were divided into negative and positive control groups, as well as immunised lamb groups, with 100 µg of the 15-kDa native protein (15kDaNP) and S28 peptides (S28P). The eggs per gram (EPG) and haematocrit were measured, and AS were counted and morphologically measured. To assess the immunoprotection in lambs, indirect enzyme-linked immunosorbent assays and relative expression analyses of immune cytokines were performed using serum and abomasal samples. Our results showed a 72.28% reduction in adult worms (AW) in the 15kDaNP-immunised group, achieving a high clinical response with 41% haematocrit and low EPG values (436 ± 661). Conversely, the S28P group achieved the highest IgG levels (2.125 ± 0.880 OD), with AW exhibiting the greatest body length (p > 0.05) and upregulation of the IL5 and FCεR1A genes associated with nematode control. The 15kDaNP group showed increased expression of genes related to nematode control and anti-inflammatory responses, including IL4, IL5, IL6, and IL13 (p < 0.05). The S28P and 15kDaNP should be explored as potential vaccines against sheep haemonchosis.

Genomic Regions Associated with Resistance to Gastrointestinal Parasites in Australian Merino Sheep.

The objective of this study was to identify genomic regions and genes associated with resistance to gastrointestinal nematodes in Australian Merino sheep in Uruguay, using the single-step GWAS methodology (ssGWAS), which is based on genomic estimated breeding values (GEBVs) obtained from a combination of pedigree, genomic, and phenotypic data. This methodology converts GEBVs into SNP effects. The analysis included 26,638 animals with fecal egg count (FEC) records obtained in two independent parasitic cycles (FEC1 and FEC2) and 1700 50K SNP genotypes. The comparison of genomic regions was based on genetic variances (gVar(%)) explained by non-overlapping regions of 20 SNPs. For FEC1 and FEC2, 18 and 22 genomic windows exceeded the significance threshold (gVar(%) ≥ 0.22%), respectively. The genomic regions with strong associations with FEC1 were located on chromosomes OAR 2, 6, 11, 21, and 25, and for FEC2 on OAR 5, 6, and 11. The proportion of genetic variance attributed to the top windows was 0.83% and 1.9% for FEC1 and FEC2, respectively. The 33 candidate genes shared between the two traits were subjected to enrichment analysis, revealing a marked enrichment in biological processes related to immune system functions. These results contribute to the understanding of the genetics underlying gastrointestinal parasite resistance and its implications for other productive and welfare traits in animal breeding programs.

Effect of feeding a blackseed meal-sericea lespedeza leaf meal pellet on gastrointestinal nematode and coccidia infection and animal performance in young goats.

Gastrointestinal nematode (GIN) infection poses the most significant obstacle to the sustainable development of small ruminant (sheep and goat) farming globally. Resistance of GINs to synthetic anthelmintic drugs has led to rising interest in exploring alternative methods for parasite control, such as the utilization of bioactive plants with anti-parasitic properties. In this investigation, black seed (Nigella sativa), a shrub high in secondary antioxidant compounds, and sericea lespedeza (Lespedeza cuneata), a perennial legume high in tannins with anti-parasitic properties were combined to determine if two bioactive plants containing different types of secondary compounds can provide a stronger anti-parasitic effect than sericea lespedeza alone. In a 49-day trial, naturally parasitized 6-7-month-old intact male Spanish goats (n = 15/treatment) were fed pelletized feeds encompassing sericea lespedeza leaf meal (SL), a combination of black seed meal (BS) and sericea lespedeza leaf meal (BS-SL - 75 % SL, 25 % BS), or alfalfa (Medicago sativa, control parasitized; CONP), with an additional group of dewormed kids given the alfalfa pellets (Control treated; CONT). Weekly measurements of animal weights and samples of blood and feces were collected to determine the packed cell volume (PCV), GIN fecal egg counts (FEC), and coccidia fecal oocyte counts (FOC), respectively. All animals were processed at the end of the trial (60 total), with adult Haemonchus contortus worms recovered from the abomasum of each goat for counting and sex determination. Carcass weights were recorded after processing. Goats given the SL and BS-SL treatments had lower FEC (P<0.05) than the parasitized alfalfa (CONP) goats. At the end of the study, the SL and BS-SL groups' FOC values were lower (P < 0.05) than the CONT and CONP groups. A rise in PCV values was seen over time for all groups; SL, BS-SL, and CONT animals exhibited higher PCV values (P < 0.05) in comparison to the CONP goats. The parasitized goats fed SL-only pellets showed greater feed intake and animal body weights (P < 0.05) compared to goats fed BS-SL or alfalfa pellets. However, the treatments had no effect on the weight of the goats' carcasses. Although the H. contortus adult worm counts in the CONT goats (alfalfa-dewormed) were lower (P < 0.05) than in the CONP goats (alfalfa-parasitized), they did not differ from the SL or BS-SL animals. This study indicates that sericea lespedeza leaf meal pellet diet, either by itself or in combination with black seed meal, showed promising results as an anthelmintic and may prove to be an effective alternative to exclusive use of conventional deworming drugs. The addition of black seed did not appear to enhance the effectiveness of sericea lespedeza in this study.

Molecular study of the transcription factor SKN-1 and its putative relationship with genes that encode GST and antioxidant enzymes in Haemonchus contortus.

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 (L3) and adults. Furthermore, we exposed H. contortus transitional infective larvae (xL3) to erythrocytes or hydrogen peroxide (H2O2) 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. xL3 exposed to erythrocytes or H2O2 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.

Anthelmintic effects of Podang mango (Mangifera indica) fruit peel waste extract through in vivo application on Indonesian Etawa goat production and health.

Background and Aim: The continuous use of anthelmintic drugs has led to global issues of resistance. One breakthrough to address this problem is the utilization of bio-anthelmintics derived from active compounds in agro-industrial waste. This in vivo study investigated the effectiveness of Podang mango (Mangifera indica L.) fruit peel waste extract for anthelmintic purposes, using concentrations up to 5%. Materials and Methods: This study included 28 Etawa crossbred goats aged 17 months. Goats were randomly assigned to four groups: A negative control, an aqueous fruit peel extract (AFPE) group at 2.5%, another AFPE group at 5%, and a positive control receiving ivermectin. Goats chosen had egg per gram (EPG) counts surpassing 1000 before exposure to Haemonchus contortus. For 7 days within a 9-week study, AFPE from Podang mangoes was given. On the 7th day, the positive control group was administered ivermectin. AFPE dosage relied on the average abomasum fluid per kilogram of animal weight. The feeding regimen consisted of concentrate and Pennisetum purpureum cv. Mott is customized for the nutritional needs of livestock. Data on feed consumption, digestibility, average daily gain, percentage reduction in fecal egg count, body condition score, and clinical parameters were collected throughout the study. Results: At higher treatment levels (AFPE), there was a greater reduction in both EPG and fecal egg counts. The expected and normal ranges were maintained for consumption and digestibility. While body weight increased, FAMACHA parameters showed a decrease. Compared to the negative control group, substantial disparities (p < 0.05) existed for hemoglobin, red blood cells, and hematocrit in both the positive control and the treatment groups. Blood urea nitrogen and creatinine, indicative of liver and kidney health, were within normal ranges. Conclusion: At a concentration of up to 5%, Podang mango waste extract (AFPE) can function as a substitute for traditional helminth medicines or bio-anthelmintics in goats, enhancing their production.

Phylogenetic and transcriptomic study of aldo-keto reductases in Haemonchus contortus and their inducibility by flubendazole.

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.

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

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

Inference of Essential Genes of the Parasite Haemonchus contortus via Machine Learning.

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.

Efficacy of a Haemonchus contortus vaccine under field conditions in young alpacas.

Anthelmintic resistance to Haemonchus contortus creates increasing management challenges with small ruminants and camelids. The commercial vaccine, Barbervax®, contains H11 and H-gal-GP antigens, derived from gut mucosal membrane enzymes of H. contortus involved in digesting blood. Antibody neutralization of these antigens causes failure of H. contortus to digest blood, resulting in parasite death. H11 and H-gal-GP are considered "hidden" antigens, meaning the host immune system does not encounter these proteins under natural infection. Therefore, repeat immunization is required to maintain protective humoral responses. One previous study evaluated the safety of Barbervax® in camelids but the efficacy could not be assessed due to lack of successful infection in the controls. The objective of the current study was to evaluate clinical parameters of anemia, fecal egg counts (FECs), and humoral immune responses of healthy alpacas after immunizing with Barbervax® compared to non-vaccinated controls, all under natural environmental exposure on parasite-laden pastures. A crossover-like study was performed where twenty alpacas (298 ± 66 days of age) were assigned to be initially vaccinated with Barbervax® (n=10) or receive no treatment (n=10). Three doses of Barbervax® were administered at three-week intervals. Feces and blood were collected on Day -10, 0, 21, 43, 64, 85, 106, and 135 to evaluate FECs, packed cell volume (PCV), and antibody titers. Each group was kept on separate adjacent pastures. Tracer sheep (n=2 per study group) were introduced on Day 43 for a three-week period to ensure parasite acquisition. For the crossover-like component on Day 85, the initial non-vaccinated group was administered Barbervax® with dosing repeated on Day 106 and 135. Results indicated all initially vaccinated alpacas produced antibody titers to vaccine antigen that corresponded to lower mean FECs compared to the initially non-vaccinated group. A reduced mean FEC in the vaccinate group was observed 21 days after peak antibody titers. Similarly, when pooled vaccinate antibody titers were noted to wane on Day 106, an increase in FEC was observed at the following time point (Day 135). Conclusions from our study support the use of Barbervax® to reduce H. contortus burdens in alpacas. Furthermore, a less than 30-day lag time between antibody titer and resultant effect in FECs was observed. Additional studies assessing the ability of Barbervax® to reduce H. contortus burdens during subsequent grazing seasons would provide even greater information regarding the use of Barbervax® within alpaca herds to modulate H. contortus infections, refugia, and anthelmintic use.