Variants of NLRP3 Protein in Haemonchus contortus Infected Sheep: Impact on Immune Cell Responsiveness to LPS In Vitro.

Pathogen recognition is an essential component to achieve the desired outcome of host protection. Nod-like receptor pyrin containing domain 3 (NLRP3) is a cytoplasmic pattern recognition receptor (PRR) with a wide array of agonists, such as PAMPs, DAMPs, ATP, bacterial product and viral products. Stimulation of the NLRP3 inflammasome results in proteolytic activation of IL-1ß and IL-18, cell pyroptosis and classically, the induction of proinflammatory responses. St. Croix (STC) sheep have resistance traits exhibiting the appropriate T-helper type 2 immune response ensuing protection during helminth parasitic infection whereas parasite-susceptible Suffolk (SUF) sheep have an impaired response resulting in parasite establishment and adverse symptoms. The objective of these experiments was to determine if NLRP3 protein in H. contortus-infected SUF sheep was defective using the classical activation pathway of NLRP3 inflammasome. Peripheral blood mononuclear cells (PBMCs) derived from H. contortus-infected STC and SUF sheep were isolated from whole blood and treated (MCC950 treatment for 2 h followed by LPS treatment for 3 h, 1400 W treatment for 2 h followed by LPS treatment for 3 h, LPS treatment for 3 h or culture media for 3 h). qPCR analysis of LPS-stimulated PBMC revealed an upregulation in inflammatory associated genes IL-1ß, TLR4, TNFα and NFκB (p < 0.0001) in STC PBMC and downregulation in IFNγ, IL-6 and iNOS for SUF PBMC. Pharmacological inhibition of iNOS in SUF PBMC resulted in an upregulation in the expression of IFNγ. These preliminary data begin to discover a relationship between NLRP3 activation and TLR4 signalling in PBMC of STC and SUF sheep.

Curcumin-PVP improves the in vitro efficacy of ivermectin against resistant and susceptible Haemonchus contortus.

Ivermectin (IVM) resistance in parasitic nematodes such as Haemonchus contortus has spurred a search for substances that help to recover its efficacy. One potential agent is the natural product curcumin (CUR). In this study, CUR was combined with polyvinylpyrrolidone (PVP) (CUR/PVP) to improve its solubility and biological applicability. This study determined the effect of CUR preincubation on the effective concentration 50% (EC50) of IVM in three H. contortus isolates with different susceptibilities to IVM. The IVM EC50 was determined for three H. contortus isolates with different IVM susceptibilities using the larval migration inhibition (LMI) test. The three isolates were (i) PARAISO (IVM resistant), (ii) FMVZ-UADY (IVM susceptible), and (iii) CENID-SAI INIFAP (reference IVM susceptible). The L3 of each isolate were preincubated for 3 h with one of three concentrations of CUR (µg curcumin/mL): CONC-1 (3.67), CONC-2 (5.67), or CONC-3 (8.48). Corresponding controls were performed without CUR. The EC50 of IVM was determined for each isolate after they were exposed to the different CUR concentrations. The EC50 of IVM differed between the isolates PARAISO > FMVZ-UADY > CENID-SAI INIFAP (P < 0.05). The CUR preincubation at CONC-1 did not decrease the EC50 of IVM for any of the three isolates, suggesting a hormetic effect. By contrast, CUR preincubation at CONC-2 or CONC-3 decreased the IVM EC50 for the PARAISO isolate (P < 0.05) compared with the reference isolate and reduced the EC50 of IVM for the FMVZ-UADY and CENID-SAI INIFAP isolates below the EC50 for the CENID-SAI INIFAP isolate without CUR preincubation. In conclusion, preincubation of H. contortus L3 with CUR reduced the EC50 of IVM for field isolates classified as resistant and susceptible to IVM. The CUR preincubation reduced the IVM resistance factor in the different isolates tested.

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.

In vitro ovicidal and larvicidal activity of a hydroalcoholic extract and its fractions from Cyrtocarpa procera fruits on Haemonchus contortus.

This study describes the in vitro anthelmintic effect of a hydroalcoholic extract (HA-E) and its fractions from Cyrtocarpa procera fruits against Haemonchus contortus eggs and infective larvae. The HA-E was subjected to bipartition using ethyl acetate, which resulted in an aqueous fraction (Aq-F) and an organic fraction (EtOAc-F). The HA-E and both fractions were tested using the egg hatching inhibition assay (EHIA) and the larval mortality test (LMT). Fractionation of the EtOAc-F was achieved using different chromatographic processes, i.e., open glass column and HPLC analysis. Fractionation of the EtOAc-F gave 18 subfractions (C1R1-C1R18), and those that showed the highest yields (C1R15, C1R16, C1R17 and C1R18) were subjected to anthelmintic assays. The HA-E and the EtOAc-F displayed 100% egg hatching inhibition at 3 and 1 mg/mL, respectively, whereas Aq-F exhibited 92.57% EHI at 3 mg/mL. All subfractions tested showed ovicidal effect. Regarding the larval mortality test, HA-E and EtOAc-F exhibited a larvicidal effect higher than 50% at 50 and 30 mg/mL, respectively. The subfractions that showed the highest larval mortality against H. contortus were C1R15 and C1R17, with larval mortalities of 53.57% and 60.23% at 10 mg/mL, respectively. Chemical analysis of these bioactive subfractions (C1R15 and C1R17) revealed the presence of gallic acid, protocatechuic acid, and ellagic acid. This study shows evidence about the ovicidal and larvicidal properties of C. procera fruits that could make these plant products to be considered as a natural potential anthelmintic agents for controlling haemonchosis in goats and sheep.

In vitro and in vivo anthelmintic effect of essential oil obtained from Thymus capitatus flowers against Haemonchus contortus and Heligmosomoides polygyrus.

Sheep haemonchosis is a disease that causes serious losses in livestock production, particularly with the increase of cases of anthelmintic resistance around the world. This justifies the urgent need of alternative solutions. The aim of this study was to determine the chemical profile, in vitro, and, in vivo, anthelmintic properties of Thymus capitatus essential oil. To evaluate the, in vitro, anthelmintic activity of the T. capitatus EO on Haemonchus contortus, two tests were used: egg hatch assay (EHA) and adult worm motility (AWM) assay. The nematicidal effect of this oil was evaluated, in vivo, in mice infected artificially with Heligmosomoides polygyrus using faecal egg count reduction (FECR) and total worm count reduction (TWCR). Chromatographic characterization of T.capitatus composition using gas chromatography coupled to mass spectrometry (GC-MS) demonstrated the presence of carvacrol (81.16%), as the major constituents. The IC50 values obtained was 1.9 mg/mL in the EHT. In the AWM assay; T. capitatus essential oil achieved 70.8% inhibition at 1 mg/mL after 8 h incubation. The in vivo, evaluation on H. polygyrus revealed a significant nematicidal effect 7 days post-treatment by inducing 49.5% FECR and 64.5% TWCR, using the highest dose (1600 mg/kg). The results of present study, demonstrate that T.capitatus EO possess a significant anthelmintic properties. Furthermore, it could be an alternative source of anthelmintic agents against gastrointestinal infections caused by H. contortus.

Natural resistance to worms exacerbates bovine tuberculosis severity independently of worm coinfection.

Pathogen interactions arising during coinfection can exacerbate disease severity, for example when the immune response mounted against one pathogen negatively affects defense of another. It is also possible that host immune responses to a pathogen, shaped by historical evolutionary interactions between host and pathogen, may modify host immune defenses in ways that have repercussions for other pathogens. In this case, negative interactions between two pathogens could emerge even in the absence of concurrent infection. Parasitic worms and tuberculosis (TB) are involved in one of the most geographically extensive of pathogen interactions, and during coinfection worms can exacerbate TB disease outcomes. Here, we show that in a wild mammal natural resistance to worms affects bovine tuberculosis (BTB) severity independently of active worm infection. We found that worm-resistant individuals were more likely to die of BTB than were nonresistant individuals, and their disease progressed more quickly. Anthelmintic treatment moderated, but did not eliminate, the resistance effect, and the effects of resistance and treatment were opposite and additive, with untreated, resistant individuals experiencing the highest mortality. Furthermore, resistance and anthelmintic treatment had nonoverlapping effects on BTB pathology. The effects of resistance manifested in the lungs (the primary site of BTB infection), while the effects of treatment manifested almost entirely in the lymph nodes (the site of disseminated disease), suggesting that resistance and active worm infection affect BTB progression via distinct mechanisms. Our findings reveal that interactions between pathogens can occur as a consequence of processes arising on very different timescales.

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.

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.

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.

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

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

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.

Acyl-CoA oxidase ACOX-1 interacts with a peroxin PEX-5 to play roles in larval development of Haemonchus contortus.

Hypobiosis (facultative developmental arrest) is the most important life-cycle adaptation ensuring survival of parasitic nematodes under adverse conditions. Little is known about such survival mechanisms, although ascarosides (ascarylose with fatty acid-derived side chains) have been reported to mediate the formation of dauer larvae in the free-living nematode Caenorhabditis elegans. Here, we investigated the role of a key gene acox-1, in the larval development of Haemonchus contortus, one of the most important parasitic nematodes that employ hypobiosis as a routine survival mechanism. In this parasite, acox-1 encodes three proteins (ACOXs) that all show a fatty acid oxidation activity in vitro and in vivo, and interact with a peroxin PEX-5 in peroxisomes. In particular, a peroxisomal targeting signal type1 (PTS1) sequence is required for ACOX-1 to be recognised by PEX-5. Analyses on developmental transcription and tissue expression show that acox-1 is predominantly expressed in the intestine and hypodermis of H. contortus, particularly in the early larval stages in the environment and the arrested fourth larval stage within host animals. Knockdown of acox-1 and pex-5 in parasitic H. contortus shows that these genes play essential roles in the post-embryonic larval development and likely in the facultative arrest of this species. A comprehensive understanding of these genes and the associated ß-oxidation cycle of fatty acids should provide novel insights into the developmental regulation of parasitic nematodes, and into the discovery of novel interventions for species of socioeconomic importance.

Enhanced mucosal response in sheep harbouring ßA beta-globin against haemonchosis.

Association between ovine ß-globin polymorphisms and resistance against haemonchosis was described and related to the mechanism of high oxygen affinity ßA ➔ ßC switch during anaemia, but there are no studies regarding the involved local host responses. Phenotypic parameters and local responses were evaluated in sheep from two ß-globin haplotypes naturally infected with Haemonchus contortus. Morada Nova lambs were monitored at 63, 84 and 105 days of age for faecal egg counts and packed cell volume (PCV) under natural infection with H. contortus. At 210 days of age, lambs of Hb-AA and Hb-BB ß-globin haplotypes were euthanised, and the fundic region of abomasum was sampled for evaluation of microscopic lesions and relative expression of genes related to immune, mucin and lectin activities. Lambs harbouring the ßA allele presented an improved resistance/resilience against clinical haemonchosis, showing higher PCV during infection. Hb-AA animals presented increased eosinophilia in the abomasum compared to Hb-BB animals, accompanied by higher Th2 profile, mucin and lectin activity transcripts, while the inflammatory response was increased in Hb-BB animals. This is the first report to demonstrate an enhanced local response in the primary site of H. contortus infection related to ßA allele of ß-globin haplotype.

Immunization with thiol-binding proteins from Haemonchus contortus adult worms partially protects goats against infection during prepatency.

To contribute of the knowledge of the immune mechanisms underlying the response to the immunization of goats with thiol-binding proteins fractions (PBS-TSBP) from Haemonchus contortus (H. contortus) adult worms, this study analyzed the degree of protection and the immune responses developed against the parasite after vaccination with this antigenic complex during the time-elapsing between challenge with L3 of the parasite and the development of adult worms, evidenced by the appearance of first faecal eggs (prepatent period or prepatency). Goat kids immunized with PBS-TBSP generated an immune response during the prepatency which translates into a reduction in the number of worms, as well as a lower reduction on packed cell volume and plasma protein levels in relation to the non-vaccinated animals. As previously described in other studies carried out after the prepatent period, this protection was associated with a systemic humoral response. At the local level, a specific humoral response was also observed, together with an immune-inflammatory infiltrate in the gastric mucosa of MCH-II + cells and CD4+ lymphocytes, whose number was associated with a reduction in the number of worms and an increase in plasma proteins. A high peripheral eosinophilia was detected, but no corresponding increased infiltration of the gastric mucosa by eosinophils or globular leukocytes was observed. In agreement with previous data on the immunolocalization of the antigens used here, the results obtained contribute to the idea that these may be excretion/secretion (E/S) products necessary for parasite survival, whose inactivation during the larval and/or pre-adult stages may have contributed to immunoprotection.

The anthelmintic potential of Bacillus thuringiensis to counter the anthelmintic resistance against Haemonchus contortus.

Haemonchus contortus (H. contortus) has developed resistance to nearly all available anthelmintic medications. Hence, alternative strategies are required to counter anthelmintic resistance. The present study investigated the anthelmintic potential of Bacillus thuringiensis (B. thuringiensis) against H. contortus. Bacterial spp were identified by conventional methods and confirmed by PCR; In addition, PCR amplification of the bacterial 16S rRNA gene detected B. thuringiensis at 750 base pairs (bps). The amplified products were sequenced, and the sequence data were confirmed using the Basic Local Alignment Tool (BLAST), which showed a significant alignment (97.98%) with B. thuringiensis and B. cereus. B. thuringiensis were selected to isolate purified crystal proteins (toxins), The protein profile confirmed by SDS-PAGE showed three prominent bands at 70, 36, and 15 kDa. In addition, the larval development of H. contortus was examined in vitro using two different treatments. Purified crystal protein diluted in 10 mM NaCl at a concentration of 2 mg/ml significantly reduced (P < 0.001) larval development by 75.10% compared to 1 × 108 CFU/ml spore-crystal suspension reduced (43.97%). The findings of in vitro experiments indicated that purified crystal protein was more toxic to the H. contortus larva than the spore-crystal suspension and control group. Moreover, To test the antinematodal effects of B. thuringiensis toxins in vivo, we chose 12 male goats (6 months old) and reared these animals in parasite-free conditions. We performed Fecal egg count reduction tests (FECRT) on samples collected before and after treatment at various times denotes 48 h post-treatment with Purified crystal proteins was significantly decreased (842 ± 19.07) EPG compared to 24 (2560 ± 233.66) and 12 h (4020 ± 165.22). Similarly, after 48 h of treatment, the FECRT of the Spores-crystal mix was reduced (2920 ± 177.20) EPG followed by 24- and 12-h denotes (4500 ± 137.84) and (4760 ± 112.24), respectively. Results of the above experiment suggested that purified crystal proteins have more anthelmintic potential in vivo. Current findings determine that B. thuringiensis toxin against H. contortus could be used in small ruminants to counter anthelmintic resistance. This study also suggested that future research structured on these proteins' pharmacokinetics and mode of action.

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

Haemonchus contortus is a blood-sucking gastrointestinal nematode that infects all ruminants and causes significant economic losses in production. Characterizing the genetic variability of H. contortus populations is crucial for understanding patterns of disease transmission and developing effective control strategies against haemonchosis. This study aimed to identify the genetic variability of H. contortus isolates in small ruminants from slaughterhouses in Bangladesh. During January to December 2015, 400 abomasa samples were collected and 186 were found to be positive for Haemonchus. A 321-bp fragment of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA and an 800-bp fragment of the mitochondrial nicotinamide dehydrogenase subunit-4 gene (nad4) were amplified using polymerase chain reaction (PCR) and directly sequenced. The results showed 10 genotypes (ITS-2) and 45 haplotypes (nad4) among the 186 worms. The sequences were 98.5 to 100% identical to reference sequences from the GenBank database. ITS-2 sequence analysis revealed four nucleotide substitutions at positions 30, 41, 42, and 216. There was one transition (C/T) at position 42 and three transversions (C/A at position 30, G/C at position 41, and T/A at position 216). The nad4 gene sequences showed 15 substitutions, all of which were transitions. The pairwise distance of ITS-2 between H. contortus populations ranged from 0.005 to 1.477. The nucleotide diversity (µ) among the populations was 0.009524 using ITS-2 and 0.00394 using nad4. This study indicated low genetic deviation among H. contortus populations in Bangladesh.

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

An associated microbiome of any host helps it in different metabolic processes ranging from the decomposition of food to the maturation of gametes. Organisms with a parasitic mode of life, though present at nutritious sites inside their host, maintain their own microbiome. Nevertheless, the comprehensive characterization and functionality of microbiome in parasitic organisms remain understudied. We selected two nematode parasites of Kashmir Merino sheep viz;Haemonchus contortus and Trichuris ovis based on their higher prevalence, difference in mode of nutrition, habitation site and effect on host. The objective of the study was to explore the bacteria associated with these parasitic nematodes of sheep. We adopted a 16S rRNA metagenomic sequencing approach to estimate and compare the bacterial communities present in these two nematode species. Nematode parasites from Kashmir Merino sheep were identified morphologically and confirmed with DNA characterization. H. contortus was dominated by phylum Proteobacteria (57%), Firmicutes (25%), Bacteroidota (15%) and Actinobacteriota (3%). Conversely, T. ovis showed Proteobacteria (78%) followed by Firmicutes (8%), Bacteroidota (8%), Actinobacteriota (1%), Fusobacteriota (1%) and other phyla (4%). This study provides a comprehensive account of the microbiome composition of H. contortus and T. ovis, both of which are highly prevalent among Kashmir Merino sheep. Additionally, T. ovis exhibited a greater bacterial diversity compared to H. contortus. Notably, these nematodes were found to harbor certain pathogenic bacteria. This study can further be carried forward in gaining insights into the complex relationship between the microbiota of a parasite and its pathogenicity, reproductive potential and host microbiome modification.

Relationship between faecal egg count and health status in Nguni goats reared on semi-arid rangelands.

Gastrointestinal parasitism is a major constraint to goat productivity, particularly in resource-limited production systems. The objective of the study was to determine the relationship between faecal egg count and the health status of different classes of Nguni goats. Body condition score (BCS), packed cell volume (PCV), FAMACHA score, and faecal egg count (FEC) were measured in 120 goats of different classes (weaners, does and bucks) across seasons. The identified gastrointestinal nematodes (GIN) were Strongyloides (30 %), Haemonchus contortus (28 %), Trichostrongylus sp. (23 %), Oesophagostomum sp. (17 %) and Ostertagia (2 %), which showed higher prevalence at the hot-wet season compared to other seasons. An interaction (P<0.05) between class and season on BCS was observed. Lower PCV were observed in weaners (24.6 ± 0.79) in the post-rainy season, whereas does 27.4 ± 0.86 and bucks (29.3±1.03) had the highest PCV in the same season. Higher FAMACHA scores were observed in the hot seasons for all goat classes, while lower in the cool-dry season. Linear relationships between FAMACHA scores and FEC were observed in all seasons. The rate of change in FAMACHA score was higher in the post-rainy season (P<0.01) than in other seasons as FEC increased in weaners and does. Bucks had a higher rate of change in FAMACHA in the hot-wet season (P<0.0001) as FEC increased. The rate of BCS decline was higher in the post-rainy season in weaners and does (P<0.01) and bucks (P<0.05) than in other seasons. The decline in PCV was faster during the wet than in the dry seasons. It can be concluded that class and season affected BCS, FAMACHA, and PCV. A linear relationship between FEC and FAMACHA score suggests that FAMACHA could be a good indicator of GIN burden.

Hepatocellular carcinoma-associated antigen 59 and ADP-ribosylation factor 1 with poly (lactic-co-glycolic acid): A promising candidate as nanovaccine against haemonchosis.

Haemonchus contortus (H. contortus) ADP-ribosylation factor 1 (Hc-ARF1) and Hepatocellular carcinoma-associated antigen 59 (Hc-HCA59) are recognized to largely regulate the immune responses of host cells. However, studies about the protective efficacy of the two molecules are poorly unknown. In this research, combinations of recombinant Hc-HCA59 (rHc-HCA59) and Hc-ARF1 (rHc-ARF1) proteins were amalgamated with poly (lactic-co-glycolic acid) (PLGA) nanoparticles adjuvant in order to investigate their protection potential against H. contortus in goats. The results demonstrated that the levels of IgG, IgA, IgE, and IL-4 were noticeably enhanced in the rHc-HCA59 and rHc-ARF1 (rHc-HCA59+rHc-ARF1) group before H. contortus third-stage larvae (L3) challenge. After the L3 challenge, the levels of IL-17, IL-9, and TGF-ß were considerably upregulated in the rHc-HCA59+rHc-ARF1 group. In the meantime, the abomasal worm burdens and the fecal eggs were reduced by 63.2% and 69.4% respectively in the rHc-HCA59+rHc-ARF1 group. According to the studies, PLGA nanoparticles immobilized with rHc-HCA59 and rHc-ARF1 proteins conferred partial protection and were expected to be a potential candidate for developing nano vaccines to combat goat haemonchosis.