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.

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.

UNASSIGNED: 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%.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

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.

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.

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.

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.

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.

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.

Gastrointestinal helminth infection, particularly by Haemonchus contortus, poses significant challenges to sheep farming worldwide. While anthelmintic drugs have been traditional control measures, the emergence of resistance calls for alternative strategies. Understanding the interaction between parasites, host, and their microbiome is crucial for management of helminth infection. This study intricately explores the interactions between microbial communities in Kashmir Merino sheep infected with H. contortus, to understand the complex interplay between host, parasite, and their microbiome. Sheep abomasal contents and H. contortus were collected from infected and control groups, processed for DNA extraction, and subjected to metagenomic sequencing of the 16 S rRNA gene. Downstream analysis unveils distinct microbial patterns, where Proteobacteria were dominant in H. contortus, while Bacteroidota and Firmicutes prevailed in the sheep abomasum. The revelation of unique genera and shifts in diversity indices underscored helminth-induced disruptions in the host. Beta diversity analysis further showed significant variations in bacterial profiles, providing insights into the intricate host, parasite, and microbiome dynamics. Additionally, this study elucidated the presence of pathogenic bacteria within H. contortus, accentuating their potential role in exacerbating sheep health issues. This finding underscores the complexity of the host-parasite-microbiome interaction showing helminth-induced microbiome alterations of the host.