In vitro evaluation of the potential of mites of the family Macrochelidae (Acari: Mesostigmata) as macrobiological agents against the nematode Haemonchus contortus (Strongylida: Trichostrongylidae).

Small ruminants (sheep and goats) constantly suffer from endoparasitoses caused by gastrointestinal nematodes. Among these, the species Haemonchus contortus (Rudolphi, 1803) is considered to be the one of greatest importance within sheep farming. This nematode is difficult to control due to its resistance to most commercial anthelmintics. The aim of the present study was to assess the potential of macrochelid mites as macrobiological agents for controlling endoparasitoses of sheep caused by the nematode, H. contortus. For this, novel in vitro methodology was used, in which assessments were made not only of the predatory ability but also the population growth of mite species (Macrocheles merdarius, Macrocheles robustulus and Holostaspella bifoliata) when offered larvae of the nematode, H. contortus. The predatory ability of the mites, M. merdarius and H. bifoliata were efficient regarding their predatory ability against H. contortus nematode larvae. The mite, M. merdarius exhibited the highest predation rate with mean distribution values for the treated group of 18656 ± 10091 and for the control group of 1178 ± 712 (P < 0.0001). The species, H. bifoliata presented the highest population growth rate, with a percentage acarid recovery rate of 263% in relation to the number added initially. The data from this in vitro predation experiment suggest that, M. merdarius and H. bifoliata showed promise as macrobiological agents for controlling gastrointestinal endoparasitoses of sheep caused by the nematode, H. contortus given that both species reduced the population of this helminth by more 70% and the number of mites recovered was three times greater than the number added.

Single-Cell Transcriptomic Profiling Unveils Dynamic Immune Cell Responses during Haemonchus contortus Infection.

BACKGROUND: Haemonchus contortus is a parasite widely distributed in tropical, subtropical, and warm temperate regions, causing significant economic losses in the livestock industry worldwide. However, little is known about the genetics of H. contortus resistance in livestock. In this study, we monitor the dynamic immune cell responses in diverse peripheral blood mononuclear cells (PBMCs) during H. contortus infection in goats through single-cell RNA sequencing (scRNA-Seq) analysis. METHODS AND RESULTS: A total of four Boer goats, two goats with oral infection with the L3 larvae of H. contortus and two healthy goats as controls, were used in the animal test. The infection model in goats was established and validated by the fecal egg count (FEC) test and qPCR analysis of the gene expression of IL-5 and IL-6. Using scRNA-Seq, we identified seven cell types, including T cells, monocytes, natural killer cells, B cells, and dendritic cells with distinct gene expression signatures. After identifying cell subpopulations of differentially expressed genes (DEGs) in the case and control groups, we observed the upregulation of multiple inflammation-associated genes, including NFKBIA and NFKBID. Kyoto Encyclopedia of the Genome (KEGG) enrichment analysis revealed significant enrichment of NOD-like receptor pathways and Th1/Th2 cell differentiation signaling pathways in CD4 T cells DEGs. Furthermore, the analysis of ligand-receptor interaction networks showed a more active state of cellular communication in the PBMCs from the case group, and the inflammatory response associated MIF-(CD74 + CXCR4) ligand receptor complex was significantly more activated in the case group, suggesting a potential inflammatory response. CONCLUSIONS: Our study preliminarily revealed transcriptomic profiling characterizing the cell type specific mechanisms in host PBMCs at the single-cell level during H. contortus infection.

Analysis of lncRNA-related studies of ivermectin-sensitive and -resistant strains of Haemonchus contortus.

In this study, 858 novel long non-coding RNAs (lncRNAs) were predicted as sensitive and resistant strains of Haemonchus contortus to ivermectin. These lncRNAs underwent bioinformatic analysis. In total, 205 lncRNAs significantly differed using log2 (difference multiplicity) > 1 or log2 (difference multiplicity) < - 1 and FDR < 0.05 as the threshold for significant difference analysis. We selected five lncRNAs based on significant differences in expression, cis-regulation, and their association with the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. These expressions of lncRNAs, namely MSTRG.12610.1, MSTRG.8169.1, MSTRG.6355.1, MSTRG.980.1, and MSTRG.9045.1, were significantly downregulated. These findings were consistent with the results of transcriptomic sequencing. We further investigated the relative expression of target gene mRNAs and the regulation of mRNA and miRNA, starting with lncRNA cis-regulation of mRNA, and constructed a lncRNA-mRNA-miRNA network regulation. After a series of statistical analyses, we finally screened out UGT8, Unc-116, Fer-related kinase-1, GGPP synthase 1, and sart3, which may be involved in developing drug resistance under the regulation of their corresponding lncRNAs. The findings of this study provide a novel direction for future studies on drug resistance targets.

Potential distribution modelling for Haemonchus contortus (Nematoda: Trichostrongylidae) in South America.

The species Haemonchus contortus occurs in many regions worldwide, mainly parasitising small ruminants and economically impacting animal production. Climate change is considered a driving force for the risk of diseases caused by helminths and can also affect relationships between parasites and their hosts, with the potential to cause losses in both animal production and biodiversity in general. The aim of this study was to model the potential distribution of H. contortus in South America. We used MaxEnt to perform the analyses and describe the contribution of important bioclimatic variables involved in the species distribution. Our results show that H. contortus colonised most of the areas with habitats that suit the species' environmental requirements and that this parasite presents habitat suitability in a future scenario. Understanding the effects of climate change on the occurrence and distribution of parasite species is essential for monitoring these pathogens, in addition to predicting the areas that tend to present future parasite outbreaks and identify opportunities to mitigate the impacts of the emergence of diseases caused by these organisms.

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.

Anthelmintic screening of Bangladeshi medicinal plants and related phytochemicals using in vitro and in silico methods: An ethnobotanical perspective.

ETHNOPHARMACOLOGICAL RELEVANCE: Infections caused by parasitic worms or helminth continue to pose a great burden on human and animal health, particularly in underdeveloped tropical and subtropical countries where they are endemic. Current anthelmintic drugs present serious limitations and the emergence of drug resistance has made it increasingly challenging to combat such infections (helminthiases). In Bangladesh, medicinal plants are often used by indigenous communities for the treatment of helminthiases. Knowledge on such plants along with screening for their anthelmintic activity has the potential to lead to the discovery of phytochemicals that could serve as novel molecular scaffolds for the development of new anthelminthic drugs. AIM OF THE STUDY: The purpose of this study was i) to conduct an ethnobotanical survey to gather data on Bangladeshi medicinal plants used in the treatment of helminthiases, ii) to test plants with the highest use values for their in vitro anthelmintic activity, and iii) to carry out in silico screening on phytochemicals present in the most active plant extract to investigate their ability to disrupt ß-tubulin function in helminths. METHODS: The ethnobotanical survey was conducted across three sub-districts of Bangladesh, namely Mathbaria, Phultala and Khan Jahan Ali. The in vitro screening for anthelmintic activity was performed in a motility test using adult Haemonchus contortus worms. Virtual screening using PyRx was performed on the phytochemicals reported from the most active plant, exploring their interactions with the colchicine binding site of the ß-tubulin protein target (PDB ID: 1SA0). RESULTS: The survey respondents reported a total of 32 plants for treating helminthiases. Based on their use values, the most popular choices were Ananas comosus (L.) Merr., Azadirachta indica A.Juss., Carica papaya L., Citrus maxima (Burm.) Merr., Curcuma longa L., Momordica charantia L., Nigella sativa L. and Syzygium cumini (L.) Skeels. In vitro anthelmintic testing revealed that A. indica leaves and bark had the highest activity with LC50 values of 16 mg/mL in both cases. Other plant extracts also exhibited good anthelmintic activity with LC50 values ranging from 16 to 52 mg/mL, while the value for albendazole (positive control) was 8.39 mg/mL. The limonoids nimbolide and 28-deoxonimbolide showed a binding affinity of -8.9 kcal/mol, and satisfied all drug-likeness parameters. The control ligand N-deacetyl-N-(2-mercaptoacetyl)colchicine had a binding affinity of -6.9 kcal/mol. CONCLUSION: Further in silico and in vitro studies are warranted on the identified limonoids to confirm the potential of these derivatives as novel drug templates for helminthiases. The current study supports the need for an ethnobotanical survey-based approach to discover novel drug templates for helminthiases.

Reducing fecal egg count through selective breeding alters dorper lamb response to Haemonchus contortus in an artificial challenge trial.

Infection by gastrointestinal nematodes (GIN), particularly Haemonchus contortus, can be detrimental to sheep health and performance. Genetic susceptibility to GIN varies between breeds, with those lacking high levels of natural resistance often requiring frequent anthelmintic treatment when facing parasitic challenge. Genetic technology can serve as a tool to decrease GIN susceptibility via selection for sheep with reduced fecal egg count (FEC) estimated breeding values (EBVs). However, the physiological changes that result from implementation of this strategy are not well described. Additionally, there is a need for comparison of animals from recent selective breeding against breeds with inherent GIN resistance. In this study we administered a challenge of H. contortus to Dorper x White Dorper (DWD; n = 92) lambs that have been genetically selected for either low (DWD-) or high (DWD+) FEC EBVs and Barbados Blackbelly x Mouflon (BBM; n = 19) lambs from a genetically resistant breed backgrounds. Lamb FEC, packed-cell volume (PCV) and serum IgG were measured at intermittent levels over 5 weeks. At day 21 and day 35, the selectively bred DWD- had a lower mean FEC compared to DWD+, but were higher than BBM. Reductions in both PCV and serum IgG from initial day 0 levels were observed in DWD lambs, but not in BBM. Furthermore, from a subset of lambs (n = 24) harvested at day 21, DWD- only tended (p = 0.056) to have lower mean worm counts than DWD+, with BBM having the lowest mean worm count. Differentially expressed genes (DEGs) identified via RNA-sequencing of abomasal tissue at day 21 indicate a more pronounced Th2 immune response and more rapid worm expulsion occurred in iBBM than iDWD- and iDWD+ lambs. However, gene expression in DWD- suggests an association between reduced FEC EBV and gastric acid secretion and the ability to limit worm fecundity. Ultimately, selection of Dorper sheep for low FEC EBV can reduce susceptibility to GIN, but it will likely require multiple generations with this trait as a breeding priority before presenting a similar resistance level to Caribbean breeds.

The in vitro rumen exsheathment test for studying the effect of plant extracts on the exsheathment of Haemonchus contortus infective larvae.

This study applied the in vitro rumen exsheathment test (IVRET) to evaluate the exsheathment kinetics of Haemonchus contortus infective larvae (L3) incubated in ruminal liquor (RL) containing acetone:water extracts of Acacia pennatula (AP), Gymnopodium floribundum (GF), Havardia albicans (HA) or Lysiloma latisiliquum (LL). The role of polyphenols in the biological activity of the evaluated extracts was also determined. Larvae were incubated in RL either alone or added with a different plant extract (AP, GF, HA, or LL) at 1200 µg/mL. Polyethylene glycol (PEG) was added to block polyphenols in each treatment (RL+PEG, AP+PEG, GF+PEG, HA+PEG, and LL+PEG). After incubation times of 0, 1, 3, 6, 9, and 24 h, the exsheathment process was stopped to count the number of ensheathed and exsheathed L3. A Log-Logistic model was used to determine the L3 exsheathment kinetics in the different RL treatments. The inflection point of the respective kinetic curves, which indicates the time to reach 50 % exsheathed L3 (T50), was the only parameter that differed when comparing the exsheathment models (99 % probability of difference). The T50 values obtained for GF, HA, and LL treatments (T50 = 7.11 - 7.58 h) were higher in comparison to the T50 of RL (5.72 h) (≥ 70 % probability of difference). The L3 incubated in RL added with GF, HA, and LL extracts delayed their exsheathment at 3 and 6 h of incubation (28.71 - 48.06 % exsheathment reduction) compared to the RL treatment. The T50 value for AP, AP+PEG, GF+PEG, HA+PEG, and LL+PEG were similar to RL and RL+PEG (T50 = 5.34 - 6.97 h). In conclusion, the IVRET can be used to identify plants with the potential to delay the exsheathment of H. contortus L3 in the ruminal liquor. The acetone:water extracts of G. floribundum, H. albicans, and L. latisiliquum delayed the T50 of H. contortus exsheathment, which was evident at 3 and 6 h of incubation in ruminal liquor. The observed exsheathment delay was attributed to the polyphenol content of the extracts.

Anthelmintic efficacy of an organic fraction from Guazuma ulmifolia leaves and evaluation of reactive oxidative stress on Haemonchus contortus.

This study describes the anthelmintic efficacy of an organic fraction (EtOAc-F) from Guazuma ulmifolia leaves and the evaluation of its reactive oxidative stress on Haemonchus contortus. The first step was to assess the anthelmintic effect of EtOAc-F at 0.0, 3.5, 7.0 and 14 mg kg of body weight (BW) in gerbil's (Meriones unguiculatus) artificially infected with H. contortus infective larvae (L3). The second step was to evaluate the preliminary toxicity after oral administration of the EtOAc-F in gerbils. Finally, the third step was to determine the relative expression of biomarkers such as glutathione (GPx), catalase (CAT), and superoxide dismutase (SOD) against H. contortus L3 post-exposition to EtOAc-F. Additionally, the less-polar compounds of EtOAc-F were identified by gas mass spectrophotometry (GC-MS). The highest anthelmintic efficacy (97.34%) of the organic fraction was found in the gerbils treated with the 14 mg/kg of BW. Histopathological analysis did not reveal changes in tissues. The relative expression reflects overexpression of GPx (p<0.05, fold change: 14.35) and over expression of SOD (p≤0.05, fold change: 0.18) in H. contortus L3 exposed to 97.44 mg/mL of EtOAc-F compared with negative control. The GC-MS analysis revealed the presence of 4-hydroxybenzaldehyde (1), leucoanthocyanidin derivative (2), coniferyl alcohol (3), ferulic acid methyl ester acetate (4), 2,3,4-trimethoxycinnamic acid (5) and epiyangambin (6) as major compounds. According to these results, the EtOAc-F from G. ulmifolia leaves exhibit anthelmintic effect and increased the stress biomarkers on H. contortus.

Machine learning prediction of multiple anthelmintic resistance and gastrointestinal nematode control in sheep flocks.

The high prevalence of Haemonchus contortus and its anthelmintic resistance have affected sheep production worldwide. Machine learning approaches are able to investigate the complex relationships among the factors involved in resistance. Classification trees were built to predict multidrug resistance from 36 management practices in 27 sheep flocks. Resistance to five anthelmintics was assessed using a fecal egg count reduction test (FECRT), and 20 flocks with FECRT < 80% for four or five anthelmintics were considered resistant. The data were randomly split into training (75%) and test (25%) sets, resampled 1,000 times, and the classification trees were generated for the training data. Of the 1,000 trees, 24 (2.4%) showed 100% accuracy, sensitivity, and specificity in predicting a flock as resistant or susceptible for the test data. Forage species was a split common to all 24 trees, and the most frequent trees (12/24) were split by forage species, grazing pasture area, and fecal examination. The farming system, Suffolk sheep breed, and anthelmintic choice criteria were practices highlighted in the other trees. These management practices can be used to predict the anthelmintic resistance status and guide measures for gastrointestinal nematode control in sheep flocks.

Epidemiology and field efficacy of anthelmintic drugs associated with gastrointestinal nematodes of sheep in Nejo district, Oromia, Ethiopia.

Gastrointestinal nematodes (GINs) are major constraints to health and productivity of small ruminants. Methods of their control relies mainly on anthelmintic drugs; however, the indiscriminate use of these drugs could lead to the development of anthelmintic resistance (AR). This study aimed to investigate the epidemiology of GINs infection, and field evaluation of anthelmintic efficacy in sheep. The epidemiological data were collected using a cross-sectional study design while a farm-based field study design was employed for the evaluation of anthelminthic efficacy. Furthermore, standard parasitological techniques were employed for qualitative and quantitative worm identification. The overall prevalence indicated 50.3%. Six genera of GINs (Haemonchus, Trichostrongylus, Oesophagostomum/Chabertia, Trichuris, Teladosargia/Ostertagia and Nematodirus) were identified. Among the identified genera, Haemonchus (25.4%) and Trichostrongylus (24.8%) were the dominant genera followed by mixed infection (21.8%), Oesophagostomum/Chabertia (10.4%), Trichuris (7.8%), Teladosargia (Ostertagia) (5.7%) and Nematodirus (4.1%). Mixed infections consisted either of double infections with Haemonchus and Trichostrongylus, or triple infections with Haemonchus, Trichostrongylus and Trichuris. The McMaster egg counting results showed that the mean EPG of infected sheep was 845.6. The results also showed 66 (34.2%), 101 (52.3%) and 26 (13.5%) sheep had low, moderate and heavy worm burden, respectively. Albendazole and Ivermectin showed low efficacy (percentage reductions = 90% and 92%; 95% lower confidence limit = 82.1% and 83.6% respectively) whereas Tetramisole was effective (FECR% = 96.8%; 95% LCL = 93.4%). Factors such as age, body condition, management system and past deworming history of sheep were found to have a statistically significant (p < 0.05) influence on the occurrence and burden of the worms. This is further explained as the highest prevalence and worm burden was detected in sheep of young age (p = 0.008; OR = 0.58; 95% CI = 0.39-0.87), poor body condition (p = 0.001; OR = 0.08; 95% CI = 0.04-0.16) and sheep kept under semi-intensive (p = 0.04; OR = 1.53; 95% CI = 1.02-2.29) with no deworming history for the last two months (p = 0.001; OR = 2.97; 95% CI = 1.94-4.56). The study results revealed that nematode infections were among sheep health constraints that could hurt their productivity while low efficacy of Albendazole and Ivermectin were detected. Therefore, the appropriate management techniques of GIN infections should be designed and implemented. Moreover, a further study involving more sensitive techniques (e.g. Mini-FLOTAC, molecular, and serological techniques) should be conducted by considering different host and environmental risk factors such as production level and seasons.

Revisiting anthelmintic resistance in sheep flocks from São Paulo State, Brazil.

Haemonchus contortus and Trichostrongylus colubriformis are the most important gastrointestinal nematodes causing serious losses in sheep production of tropical and subtropical regions. Prophylaxis of gastrointestinal nematode infections is based on anthelmintics use, but their frequent administration selects multiple-resistant parasites. To evaluate how the situation has changed over the last decades, the anthelmintic resistance status of gastrointestinal nematodes in sheep flocks was assessed in the current study and compared to previous surveys. In each one of the 15 flocks evaluated, animals (n ≥ 7) were allocated into at least five groups and treated as follows: 1) untreated control; 2) albendazole; 3) levamisole; 4) ivermectin; and 5) monepantel. If more animals were available, two additional groups were included: 6) closantel, and 7) moxidectin. The faecal egg count reduction test (FECRT) was carried out to evaluate the pre- and post-treatment using the SHINY tool. Haemonchus spp. was the most prevalent nematode from faecal cultures. The mean efficacy of albendazole was 40%. Only in two farms, levamisole presented a relatively high percentage of reduction in the FECRT about 90%, while ivermectin and moxidectin presented the worst mean efficacy of 34% and 21% among all farms, respectively. Like other anthelmintics, closantel demonstrated low efficacy (63%) across all farms evaluated. Monepantel presented an overall mean efficacy of 79%, but it was the only anthelmintic that presented efficacy ≥95%, in five farms. The results revealed that gastrointestinal nematodes with multiple anthelmintic resistance were prevalent in all 15 sheep herds. The research suggests that nematodes are becoming more and more resistant to various anthelmintic compounds, which has made the problem worse. This circumstance highlights the necessity to put into practice sustainable and long-lasting methods to prevent gastrointestinal nematode infections in sheep husbandry.

Crude Extracts of Pleurotus Spp. and the Presence of Their Proteins in the Ovicidal and Larvicidal Activity of Haemonchus contortus.

Mushrooms of the genus Pleurotus have shown nematophagous activity as it produces many chemical compounds and enzymes affecting parasitic nematodes. This study aimed to extract the inhibitory activity of the five strains of the fungus Pleurotus spp. It was evaluated against eggs and larvae of Haemonchus contortus. The extract of P. ostreatus obtained the highest level of inhibition of eggs at 97.6% (1341 µg/mL) followed by P. pulmonarius (EPP) at 81.2% (774 µg/mL). The extract selected for evaluation against larvae was P. pulmonarius, showing no effect for L3 larvae, but for L4 larvae an immobility effect of 56.93% was observed at 900 µg/mL. The protein profile showed the presence of 23 protein bands in the extract. The crude extract of P. pulmonarius showed degradation of tissues both inside the eggs and larvae L1. Metabolites produced by Pleurotus mushrooms can consider using in agriculture sustainable by utilizing in producing of ovicidal and larvicidal against H. contortus instead of chemical compounds.

Multi-omics data elucidate parasite-host-microbiota interactions and resistance to Haemonchus contortus in sheep.

BACKGROUND: The integration of molecular data from hosts, parasites, and microbiota can enhance our understanding of the complex biological interactions underlying the resistance of hosts to parasites. Haemonchus contortus, the predominant sheep gastrointestinal parasite species in the tropics, causes significant production and economic losses, which are further compounded by the diminishing efficiency of chemical control owing to anthelmintic resistance. Knowledge of how the host responds to infection and how the parasite, in combination with microbiota, modulates host immunity can guide selection decisions to breed animals with improved parasite resistance. This understanding will help refine management practices and advance the development of new therapeutics for long-term helminth control. METHODS: Eggs per gram (EPG) of feces were obtained from Morada Nova sheep subjected to two artificial infections with H. contortus and used as a proxy to select animals with high resistance or susceptibility for transcriptome sequencing (RNA-seq) of the abomasum and 50 K single-nucleotide genotyping. Additionally, RNA-seq data for H. contortus were generated, and amplicon sequence variants (ASV) were obtained using polymerase chain reaction amplification and sequencing of bacterial and archaeal 16S ribosomal RNA genes from sheep feces and rumen content. RESULTS: The heritability estimate for EPG was 0.12. GAST, GNLY, IL13, MGRN1, FGF14, and RORC genes and transcripts were differentially expressed between resistant and susceptible animals. A genome-wide association study identified regions on chromosomes 2 and 11 that harbor candidate genes for resistance, immune response, body weight, and adaptation. Trans-expression quantitative trait loci were found between significant variants and differentially expressed transcripts. Functional co-expression modules based on sheep genes and ASVs correlated with resistance to H. contortus, showing enrichment in pathways of response to bacteria, immune and inflammatory responses, and hub features of the Christensenellaceae, Bacteroides, and Methanobrevibacter genera; Prevotellaceae family; and Verrucomicrobiota phylum. In H. contortus, some mitochondrial, collagen-, and cuticle-related genes were expressed only in parasites isolated from susceptible sheep. CONCLUSIONS: The present study identified chromosome regions, genes, transcripts, and pathways involved in the elaborate interactions between the sheep host, its gastrointestinal microbiota, and the H. contortus parasite. These findings will assist in the development of animal selection strategies for parasite resistance and interdisciplinary approaches to control H. contortus infection in sheep.

Growth hormone stimulates the in vitro development and establishment of Haemonchus contortus in sheep.

The physiologic increase in some sex hormones has been associated with an increase in the parasite load caused by Haemonchus contortus in ewes, especially prolactin. In lambs that are especially susceptible to hemonchosis, the levels of sex hormones are low; in contrast, the levels of another pituitary hormone, growth hormone (GH), which is structurally very similar to prolactin, are high. In this study, the in vitro and in vivo effects of GH on H. contortus larvae development and establishment were evaluated. The addition of 20 ng/mL GH for 5 and 10 days to cultures of H. contortus larvae induced an enlargement (p<0.01) and an L3/L4 molting rate (p<0.03) greater than that of untreated larvae or those treated with other concentrations of the hormone. Flow cytometry showed that 3.8% of the largest and most complex cells of newly obtained larvae of H. contortus were positive for the GH receptor, and by immunofluorescence with confocal microscopy, it was observed that these receptors are located in the intestinal region larvae. In the in vivo assay, the administration of recombinant GH to gonadectomized lambs produced an increase in FEC (p<0.03), the number of female adult worms in the abomasum (p<0.05) and the levels of specific antibodies (p<0.04) in relation to the control lambs; however, it did not affect the fertility of H. contortus females. Although many factors affect the development and implantation of H. contortus in the abomasum of sheep, the results of this study strongly suggest that GH participates in the development and establishment of the parasite in sheep, mainly in young sheep.

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.

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.

Application of the SeDeM system for the preparation of antiparasitic tablets from mesquite flour for use in sheep.

The use of herbal medicine to treat various diseases is becoming increasingly important as an alternative therapy. Numerous plants have been traditionally used for different purposes, including antiparasitic in humans and animals. Diseases caused by gastrointestinal parasites in ruminants, especially by the nematode Haemonchus contortus, cause large economic losses to the producers, whether by complications of the diseases or the cost of treatment. The main way of handling nematodiasis is by administering anthelmintic drugs, but their excessive use has the disadvantage of causing drug resistance; therefore, an alternative is the use of herbal medicine for this purpose. Mesquite (Prosopis spp.) has been used in Mexico to treat gastrointestinal diseases attributed to helminths. The present study aimed to characterize the rheological properties of mesquite flour using the SeDeM Expert System to determine its suitability for tablet production by direct compression. Direct compression technology facilitates the tableting process by reducing manufacturing costs. The results of the present study indicate that mesquite flour can be processed by direct compression. The latter could allow the manufacturing of economic tablets to treat infections by H. contortus in ruminants.

Ecology of the free-living stages of cattle nematodes in the dry season in the Lerma Valley, Salta province, Argentina.

The objective of this work was to describe the dynamics of development and survival of the free-living stages of cattle gastrointestinal nematodes (GIN) in fecal matter (FM) and pasture during the dry season in the Lerma Valley, Salta province, northwestern Argentina (NWA) to contribute to GIN management. The climate in the region is characterized by a rainy summer followed by a dry season from middle autumn to early spring. Fecal matter from calves naturally infected with GIN was deposited on three experimental field plots in April, July and October 2019, corresponding to the beginning, middle and end of the dry season, respectively. Each experimental unit consisted of 7 stools of about 800 g and had four repetitions. To determine the development from egg to infective larvae (L3), the first sampling (5 g fecal matter) was performed from the 10th day post-contamination and continued every 3 days until L3 were found. Subsequently, a monthly sampling was made until two consecutive negative results were obtained. Sampling of pasture began three days after the L3 recovery from FM, and continued monthly until two negative results were obtained. The following parameters were evaluated: development time and development rate from egg to L3; permanence time of L3 in feces; time of appearance on pasture; migration rate; and permanence time of L3 on pasture. The main genera of parasites present were Cooperia and Haemonchus. Significant differences were observed in the development time among contamination months (p < 0.001); development time was highest in the July contamination (28 days), with October and April contamination averaging 9 and 10 days, respectively. Development time also showed significant differences (p < 0.01) among contamination months, being highest in October (31.48%). The highest permanence time in fecal matter values were recorded in the July contamination (183 days) and migration rate was highest in the October contamination (42.49%). The highest time of appearance on pasture value was recorded in the July contamination (117 days). Finally, the highest permanence time of L3 in feces values were detected in the October contamination (148 days). The results of this work show that fecal contamination in the NWA region in the dry season would play an epidemiological role in the GIN cycle as a source of infection for the next productive cycle in the rainy season.

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.