Haemonchus contortus (H. contortus) is a gastrointestinal parasite affecting small ruminants, leading to a significant decline in animal productivity. In this study, we developed a nanovaccine by encapsulating the recombinant protein rHcES-15, derived from the excretory/secretory products of H. contortus, within biodegradable poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs). To construct the nanovaccine, PLGA NPs were prepared using a modified double emulsion solvent evaporation technique. Scanning electron microscopy (SEM) illustrated successful encapsulation of rHcES-15 within PLGA NPs, with a size ranging between 350-400 nm. The encapsulation efficiency (EE) of the antigen in the nanovaccine was determined to be 72%. A total of forty experimental mice were divided into five groups, receiving the nanovaccine on day 0 and being humanely sacrificed at the end of the 14-day trial. The stimulation index (SI) from mice vaccinated with the nanovaccine indicated an amplified lymphocyte proliferation and a significant increase in anti-inflammatory cytokines (IL-4, IL-10, and IL-17). Furthermore, the percentages of T-cells (CD4+, CD8+) and dendritic cell phenotypes (CD83+, CD86+) were substantially upregulated in mice immunized with the nanovaccine compared to control groups and the rHcES-15 group. Similarly, higher levels of antigen-specific serum immunoglobulins (IgG1, IgG2a, IgM) were observed in response to the nanovaccine compared to both the antigenic (rHcES-15) and control groups. In conclusion, the data strongly supports the notion that encapsulation of rHcES-15 within PLGA NPs effectively stimulates immune cells in vivo, ultimately augmenting antigen-specific adaptive immune responses against H. contortus. This discovery highlights the promising potential of the nanovaccine, justifying additional investigations to ascertain its efficacy finally.

Abstract: Assessing infection by a parasite, such as Haemonchus contortus, requires time and experienced personnel. Field-based diagnostics using metagenomic species-specific targeting methods would be beneficial. Here, we examine two helminth control methods, zoledronic acid (ZA), an activator of γ δ T cells, and a neutralizing antibody (AB) against γ δ T cells. We conducted a randomized complete block designed investigation for the effects of Haemonchus contortus (Hc) infection on Alpine wethers (young castrated male goats) to identify if metagenomic methods will provide strong evidence for diagnostics. Different tissues were collected from wethers and the microbiome compositions were identified. The relative proportion of microbial community members in the environments; the richness of the microbial community; and the evenness of distribution in microbial community members were determined. It was hypothesized that numerous parameters that include many data points will provide a more precise means to identify infection or health based on similarity to uninfected control data parameters. We found that samples did not sequence well, when multiplexed with high concentration libraries, giving taxonomic numbers that had residual and unmeasured confounding bias related to analysis or selection that define significance of OTUs in the tissue samples at the phylum or species level.

The emergence of drug-resistant parasitic nematodes of both humans and livestock calls for development of alternative and cost-effective control strategies. For the economically important ruminant strongylid Haemonchus contortus, Barbervax® remains the only registered vaccine available. Here we compared the microbiome, genome-wide diversity and transcriptome of H. contortus adult male populations that survived vaccination with an experimental vaccine after inoculation in sheep. Our genome-wide SNP analysis revealed 16 putative candidate vaccine evasion genes. However, we did not identify any evidence for changes in microbial community profiling based of 16S rRNA gene sequencing results of vaccine surviving parasite populations. A total of 58 genes were identified as significantly differentially expressed with six being long non-coding (lnc) RNAs and no putative candidate SNP associated genes. The genes highly upregulated in surviving parasites from vaccinated animals were associated with GO terms belonging to predominantly molecular function and a few biological processes that may have facilitated evasion or potentially lessened the effect of the vaccine. These included five targets: astacin (ASTL), carbonate dehydratase (CA2), phospholipase A2 (PLA2), glutamine synthetase (GLUL) and fatty acid-binding protein (FABP3). We searched all five DEG targets against the proteomes of selected Nematoda (Clades III, V, IV, C, I) and Platyhelminthes (Clades Monogenea, Trematoda, Cestoda, Rhabditophora) to determine homologs within the H. contortus NZ_HCO_NP v1.0 genome and identified single-copy orthologous groups (OGs) in selected proteomes. All but one (FABP3) demonstrated high levels of duplication and wide-spread occurrence in closely related Caenorhabditis elegans and Pristionchus pacificus, with complete absence of all five gene targets among other Clade III (Toxocara canis) and V (Ascaris suum, Ascaris lumbricoides and Parascaris univalens) nematodes, further supporting their vital biological functions in nematodes. Phylogenetic analyses inferred the presence of only ASTL and CA2 in almost all Nematoda, platyhelminthes and metazoans examined, with loss of GLULs observed among all outgroup vertebrate species and the presence of FABP3 in only three other species (Schmidtea mediterranea, Fasciola gigantica and F. hepatica). Our tertiary structure predictions and modelling analyses were used to perform in silico searches of all published and commercially available inhibitor molecules or substrate analogues with potential broad-spectrum efficacy against nematodes of human and veterinary importance.

The individual and combined effects of extracellular products (ECP) from Arthrobotrys oligospora (Ao) and A. musiformis (Am) growth in liquid media against Haemonchus contortus L3 (HcL3) were assessed. The following methodological steps were established: isolation, morphological (MI) and molecular identification (Mol-I), assessment of nematocidal activity (NA) of fungal liq-uid culture filtrates (LCF) produced in two liquid media alone and in combination and finally, the myco-compound profile identification (MCP). The MI of both isolates suggested that the fungi corresponded to the species Ao and Am. This result was confirmed by PCR analysis followed by sequencing, alignment and obtaining coverage and similarity with respect to isolates reported in the NCBI database. Likewise, the highest Hc mortalities were 91.4% with individual LCF of Am and 86.2% with those of Ao at the highest concentration (100 mg/mL) in Czapek-Dox Broth. The combination of both LCF resulted in a similarly high larval mortality with no statistical differ-ences in relation to individual activity (P>0.05). The MCP showed the presence of alkaloids in both fungi. Coumarins, triterpenes and saponins were found only in Ao. Both fungi produced ECP with a high NA that could be identified and assessed in future studies as potential natural an-thelmintic compounds.

We conducted an experiment on lambs to determine the effect of infection with the gastrointestinal nematode (GIN) Haemonchus contortus on growth performance, nutrient composition, microbiota, and ruminal fermentation in vivo and in vitro. Twelve female lambs were equally divided into two groups: uninfected animals (CON) and animals infected with approximately 5000 third-stage ISE larvae derived from the MHCo1 strain of H. contortus (INF). Two lambs per group were killed on days 48, 49, and 50 after infection and ruminal content was collected separately from each lamb. Batch cultures of ruminal fluid from CON and INF were incubated in vitro with 0.25 g meadow hay, a plant mixture and chicory used as substrates. The ruminal populations of protozoa, bacteria, total Archaea, and the archaean orders Methanobacteriales and Methanomicrobiales were significantly higher in INF than CON. In vivo concentrations of n-butyrate, iso-butyrate, and iso-valerate were significantly lower in INF than CON. In vitro values after 24 h of ruminal fermentation varied within the substrate. GIN infection by H. contortus was associated with increased methanogenesis. In conclusion, chicory was a promising substrate for mitigating methane production and may modulate the ruminal microbiome of GIN-infected lambs due to its chemical and phytochemical composition.