BACKGROUND:Benin remains highly burdened by malaria, and data on antimalarial resistance surveillance are limited. This study assesses the evolution of molecular markers associated with antimalarial resistance in southern Benin between 2017 and 2024. METHODS:We analysed 358 dried blood spot samples collected from children in southern Benin (Kétonou, Kpomè and Klouékanmè) in 2017 and 2024. Molecular markers in the PfKelch13, Pfmdr1, Pfcrt, Pfdhfr, and Pfdhps genes associated with antimalarial drug resistance were investigated. Pfcrt haplotypes were characterised using quantitative PCR, while the remaining markers were analysed by nested PCR followed by Sanger sequencing. RESULTS:None of the detected non-synonymous PfKelch13 mutations (7/281) was a WHO-validated, candidate or potential marker of artemisinin partial resistance. The Pfcrt CVIET haplotype declined significantly from 88.1% (59/67) in 2017 to 35.4% (101/285) in 2024 (p < 0.001). The Pfmdr1 N86Y decreased from 11.6% (8/69) to 6.0% (17/285), while the NFD haplotype increased slightly from 56.9% (37/65) to 61.9% (169/273). No mutations were observed at Pfdhfr I164L or Pfdhps K540E. The Pfdhfr triple-mutant IRNI haplotype remained nearly fixed over time at 97.0% (64/66) in 2017 vs 96.4% (268/278) in 2024. A similar trend was observed for Pfdhps A437G at 98% in 2017 and 2024. The Pfdhps I431V rose from 8.3% (5/60) in 2017 to 10.5% (29/275) in 2024, whereas Pfdhps A581G remained stable at 8%. The combined Pfdhfr/PfdhpsIRN_G haplotype remained highly prevalent (96.5% vs 94.6%). Aside from the significant decline in CVIET, none of the other changes was statistically significant. CONCLUSION:Our findings show no mutations compromising ACT efficacy, nor any immediate threat to the continued use of SP. In addition, the observed re-emergence of chloroquine-sensitive genotypes suggests a shift in parasite population dynamics over time. Nevertheless, continuous molecular surveillance remains essential for detecting emerging resistance and informing timely policy decisions.
Anthelmintic resistance constitutes a global threat to the control of parasitic nematodes. Current research has primarily focused on parasite-intrinsic genetic mechanisms, while the contribution of the symbiotic microbial community remains a key knowledge gap. Here, we report that ivermectin (IVM) resistance in the gastrointestinal nematode Haemonchus contortus is associated with the abundance of the bacterium Stenotrophomonas maltophilia. A representative strain, designated SM1, was isolated from resistant populations, and its abundance was associated with the resistant phenotype. Depletion of SM1 increased larval susceptibility to IVM, whereas reintroduction of the bacterium partially enhanced IVM tolerance. Metabolic analysis indicated that SM1 converts IVM into demethylated and oxo-derivatives (M1, M4, and M7). Using in silico analysis, the putative cytochrome P450 monooxygenase (Cmp08160) with a possible participation in IVM biotransformation was identified. Collectively, these findings suggest that symbiotic bacteria can influence IVM susceptibility in H. contortus and highlight the relevance of considering host-microbiota interactions in studies of anthelmintic resistance.
There are restricted treatment options for Chagas disease, a global health risk causing 12,000 fatalities annually. This situation is exacerbated by environmental changes and migration, which is altering patterns of disease transmission, and by expansion of the vector range. The disease is caused by the protozoan parasite Trypanosoma cruzi, which possesses a divergent translation apparatus, characterized by enlarged ribosomes and a unique mRNA cap appended to the 5' end of a 39-nucleotide spliced leader sequence at the 5' end of all its mRNAs. Given that many antimicrobial agents are protein synthesis inhibitors and that there are significant differences between the T. cruzi protein synthesis machinery and that of its mammalian host, it is likely that T. cruzi-specific protein synthesis inhibitors could be employed for the treatment of Chagas disease. Such inhibitors can be identified by screening chemical libraries in an in vitro translation assay. However, lysates of T. cruzi cannot reinitiate translation of exogenous mRNAs. Here, we demonstrate that T. cruzi extracts derived from a strain deficient for hemin accumulation efficiently translated a capped and polyadenylated reporter mRNA. We optimized assay conditions and adapted them to a 384-well format. We further miniaturized this assay to a 5 μL volume, demonstrating its suitability for ultra-high throughput screening. Further, in a proof-of-principle pilot study, we identified quinazoline compounds that inhibit translation of reporter mRNAs by T. cruzi extracts. These compounds inhibited protein synthesis and proliferation of insect-derived forms and wild-type parasites growing in mammalian cells at a low micromolar range and a selectivity index higher than 200. This first-of-its-kind T. cruzi in vitro translation system will enable high-throughput screening of very large chemical libraries, while first-in-class hit compounds identified in the pilot study can be developed into lead compounds by design and synthesis of focused libraries to develop therapeutics for Chagas disease.
Gastrointestinal nematodes, including Haemonchus contortus, represent a major constraint to livestock production globally, and the increasing prevalence of anthelmintic resistance necessitates the discovery of new chemotypes. Natural products provide a rich and underexplored source of bioactive scaffolds for anthelmintic development. However, there remains a need to systematically optimise such scaffolds to define structure-activity relationships in whole-organism systems. Here, we investigated neolignan compounds from the fruits of Styrax suberifolius and assessed the impact of halogenation on their anthelmintic activity. Two known neolignans, suberifolioside A and equiselignan B, were isolated and used to generate a series of 11 halogenated derivatives using N-halosuccinimide chemistry. In addition, a new glycoside, suberifolioside B, together with two known metabolites, 7R,8S-dihydrodehydrodiconiferyl alcohol and 3',4-O-dimethylcedrusin, were characterised by spectroscopic and spectrometric methods, and the absolute configuration of equiselignan B was confirmed by X-ray crystallography. All compounds were evaluated for activity against larval stages of H. contortus using an established in vitro phenotypic assay. While no compounds significantly affected exsheathed third-stage larvae, multiple derivatives displayed activity against fourth-stage larvae, indicating pronounced stage-specific susceptibility. Notably, halogenation of the neolignan scaffold modulated biological activity, with several derivatives inducing substantial reductions in larval motility and distinct abnormal phenotypes. This modulation was non-linear, with both mono- and poly-halogenated derivatives displaying activity. A monobrominated ether analogue exhibited maximal motility inhibition approaching that observed for moxidectin under the assay conditions, highlighting the potential of this scaffold for optimisation. These findings demonstrate that halogenated neolignans represent a tractable chemical class for anthelmintic discovery and reveal stage-specific vulnerabilities in H. contortus. This work provides a foundation for further optimisation and prioritisation of neolignan derivatives for downstream studies of mechanism of action and translational potential.
Trichomoniasis, caused by Trichomonas vaginalis, is the most prevalent nonviral sexually transmitted infection worldwide. The occurrence of treatment failure with metronidazole (MTZ) and the adverse effects of currently available nitroimidazoles underscore the necessity for novel therapeutic candidates. From a library of synthetic thienopyrimidine derivatives, G-18 was identified as a lead compound. G-18 exhibited potent anti-T. vaginalis activity against the tested T. vaginalis isolate, with a minimum inhibitory concentration (MIC) of 4 μg/mL and a half-maximal inhibitory concentration (IC50) of 1 μg/mL at 24 h, and it significantly reduced trophozoite viability within 2 h. Flow cytometry confirmed a higher proportion of propidium iodide (PI)-positive parasites following G-18 treatment compared to MTZ treatment (51.2% vs. 10.4%). G-18 demonstrated limited cytotoxicity towards mammalian cells under the tested conditions. Scanning and transmission electron microscopy revealed pronounced ultrastructural damage, including surface roughening, membrane disruption, and organelle disorganization. These findings support G-18 as a promising anti-trichomonal lead compound with rapid in vitro activity and a preliminary selectivity profile under the tested conditions.
The Plasmodium falciparum multidrug resistance-1 (pfmdr1) gene, which encodes the ABC P-glycoprotein homolog PgH1, accumulates mutations that result in amino acid changes, namely single-nucleotide polymorphisms at codons N86Y, Y184F, and D1246Y, associated with altered parasite susceptibility to multiple antimalarial drugs. Among these antimalarials are compounds used in artemisinin-based combination (ACT) therapies, which combine a fast-acting but rapidly clearing artemisinin with a longer-lasting partner drug. The in vitro susceptibility of each of these drugs varies with the pfmdr1 genotype of the treated parasites. This study characterized sequence diversity within the pfmdr1 hinge domain and the association with pfmdr1 point mutations at codons 86, 184 and 1246 in population samples from Cameroon, the Democratic Republic of Congo, and Benin, two decades after the introduction of ACTs. The data were evaluated and compared with similar data reported before or during the early implementation of ACT-based treatment strategies. Among 780 successfully sequenced isolates, a high diversity of Arginine-rich repeats in the hinge domain was observed. A significant correlation between the (Asn-Asp-Asn) 7-2-9 variant and both the 86Y and the 1246Y mutation was observed (p < 0.005, OR = 12.6, respectively). We also uncovered, when examining haplotypes across the N86Y, Y184F, and D1246Y codons, that an accumulation of mutations was inversely correlated with diversity in the hinge region. The data suggest patterns consistent with selective pressure and/or linkage with reduced diversity as a potential result of ACT implementation.
Naegleria fowleri is a free-living amoeba that causes a rare but almost always fatal brain infection known as primary amoebic meningoencephalitis (PAM). Although amphotericin B (AmB) is considered the primary treatment for PAM, it is often recommended in combination with several other drugs due to side effects associated with high concentrations. In this study, we investigated whether histone deacetylase inhibitors (HDACis) enhance the amoebicidal activity of AmB against N. fowleri. Anti-proliferative and cytotoxic activities were assessed using the cell counting kit and lactate dehydrogenase assays, respectively, and apoptosis induction was evaluated by flow cytometry. Class-specific HDACis, including class I inhibitors MS275 and MGCD0103 and a class II inhibitor MC1568, showed minimal effects on trophozoite viability. In contrast, the pan-HDACis SAHA and JNJ significantly reduced trophozoite viability in a dose- and time-dependent manner. Furthermore, 10 μM SAHA and JNJ significantly reduced the encystation ratio of N. fowleri. Combined treatment with low doses of HDACi (1 μM) and AmB (0.5 μM) produced stronger amoebicidal and pro-apoptotic effects than either agent alone. These combination treatments also exhibited low cytopathic effects on human brain tumor cells. These findings suggest that SAHA and JNJ, particularly in combination with AmB, hold promise as therapeutic candidates against N. fowleri infection while potentially mitigating the severe side effects associated with AmB.
Reduced anthelmintic efficacy against strongyles in equids is a widespread concern, but up-to-date field data are scarce. The aim of this study was to evaluate, over a three-year period, the efficacy of the anthelmintic (AH) molecules available in France (fenbendazole, pyrantel, ivermectin and moxidectin) against strongyles in equids using faecal egg count reduction tests (FECRTs). A total of 104 complete FECRTs (i.e., required sample size reached) were conducted in 98 groups, involving 749 equids kept in 70 facilities. Fenbendazole-resistant populations were detected in 8/9 groups (89%) from 7/8 facilities (88%; upper 90% CI range: 30.5%-83%) and pyrantel-resistant populations in 15/22 groups (68%) from 12/17 facilities (71%; upper 90% CI range: 56%-97%). Regarding macrocyclic lactone efficacy, ivermectin-resistant populations were identified in 14/51 groups (27%) kept in 11/41 facilities (27%; upper 90% CI range: 85.6%-99.9%) and moxidectin-resistant populations in 5/22 groups (23%) kept in 4/18 facilities (22%; upper 90% CI range: 85.3%-99.7%). Resistance was mainly detected in the Normandy region and in breeding farms (25/70), but also in riding schools offering boarding services (3/70), experimental platforms (2/70), and a zoological park (1/70). In conclusion, this study provides the first evidence in France of resistance to all four available AH molecules and documents the spread of these resistances across the facilities. It also highlights the urgent need for regulatory changes requiring the use of AH only after a diagnostic assessment (e.g., targeted selective treatment based on faecal egg count results) in order to reduce the selective pressure exerted on parasite populations.
Metronidazole (MTZ) remains the first-line therapy for trichomoniasis; however, increasing resistance in Trichomonas vaginalis threatens treatment efficacy. Because iron availability enhances MTZ sensitivity, we investigated whether MTZ-induced parasite death involves ferroptosis-related mechanisms and explored alternative pathways contributing to drug resistance. Exposure of metronidazole-sensitive and -resistant isolates to ferroptosis inducers reduced parasite viability, yet neither ferroptosis inhibition nor iron chelation restored survival, and lipid peroxidation was not detected. These findings indicate that MTZ-associated cytotoxicity does not proceed via canonical ferroptotic pathways in T. vaginalis. Instead, resistant parasites exhibited elevated expression of thioredoxin-associated transcripts, consistent with enhanced redox defense capacity. Functional suppression of thioredoxin reductase (TrxR) activity by mitomycin C (MMC) significantly enhanced MTZ-mediated killing and produced a synergistic antiparasitic effect in resistant isolates. Although selective TrxR inhibition alone was insufficient to fully reproduce this phenotype, these findings implicate thioredoxin-dependent redox regulation as a contributor to MTZ susceptibility. Combination treatment was accompanied by selective metabolic reprogramming, including upregulation of pentose phosphate pathway genes linked to NADPH generation. Collectively, our results demonstrate that MTZ induces a non-ferroptotic form of cell death and identify thioredoxin-dependent redox buffering as a resistance-associated vulnerability. Targeting redox homeostasis through rational combination therapy may represent a mechanistically guided strategy to overcome metronidazole resistance in trichomoniasis.
OBJECTIVES:Anthelmintic resistance (AR) in gastrointestinal nematode (GIN) parasites of ruminant livestock has been widely reported in Africa but analysis of variation between regions and over time is lacking. The objective of this paper was to collate data on AR and, through meta-analyses, identify patterns in its occurrence in order to guide future research, practice and policy. RESEARCH DESIGN AND METHODS:A systematic literature review following PRISMA guidelines retrieved publications on AR, from which comparable data on treatment effectiveness using the faecal egg count reduction (FECR) test were extracted for meta-analysis using logistic and linear regression. RESULTS:Data were skewed towards East Africa and small ruminants and spanned 1995-2025. Of 646 articles identified, 60 were retained for analysis. There was evidence of failure across all drug groups: percent FECR (FECR%) was below 95% in sheep and goats in 48% of tests using benzimidazoles (BZ), and 57% for imidazothiazoles (IMTZ) and macrocyclic lactones (ML); in cattle, similar levels of treatment failure were reported but sample size was very low. FECR<95% was more commonly reported in sheep in southern and North Africa than in other regions, and in goats failure rates were higher for BZ and IMTZ than other drug groups and increased over time. FECR% declined in small ruminants over the reporting period for all drug groups and frequently involved survival of Haemonchus after treatment. CONCLUSIONS:Treatment failure due to AR appears to be widespread in ruminant livestock in Africa. This first meta-analysis shows increasing levels of AR over time for the three major anthelmintic drug groups in small ruminants, and similar levels of AR in GIN in cattle. These findings reinforce the need to adapt parasite control strategies to preserve anthelmintic efficacy. They also provide a baseline for future studies, which would benefit from clear and comparable methods for measuring AR and new in vitro technologies.
Therapeutic failure in Acanthamoeba Keratitis (AK) is largely driven by the high resistance of cysts to antimicrobial agents. However, the development of effective cysticidal drugs is hindered by the lack of standardized in vitro encystation protocols. Inconsistencies in induction methods can produce immature cells that lack the structural defenses of mature clinical walls, potentially leading to false-positive results in drug screening. This study aims to evaluate the impact of cyst maturity on drug resistance assays by systematically comparing two common liquid media-glucose-supplemented PBS (PBSEB) and alkaline encystation buffer (EB2)-within Acanthamoeba Genotype T4 backgrounds. We assessed morphological transitions, transcriptional profiles (EMSP, ATG8, and CS-I), ultrastructure (TEM), and functional resistance to sodium dodecyl sulfate (SDS) and chlorhexidine gluconate (CHG) using environmental reference and clinical isolates, including the stress-tolerant NCKU_D strain. Although both media induced morphological rounding and upregulated the autophagy marker ATG8, functional and molecular assays revealed distinct developmental outcomes. PBSEB-treated cells failed to significantly upregulate the critical early serine proteinase EMSP and exhibited insufficient CS-I expression, remaining highly susceptible to SDS lysis and lacking intact walls under TEM. Conversely, EB2 triggered a robust 11.8-fold increase in EMSP, yielding structurally mature, SDS-resistant cysts. Importantly, functional assays demonstrated that EB2-induced cysts exhibited significantly higher chemical resistance to CHG than the PBSEB group at 48 h, with this resistance advantage further widening by 72 h of induction. Based on these distinct profiles, we suggest that while PBSEB serves as a useful model for investigating cell detachment kinetics and early autophagic pathways, the EB2 protocol is more suitable for evaluating downstream therapeutic efficacy and product safety where functional, double-walled resistance is required to avoid false-positive outcomes in Genotype T4 drug screening.
The emergence and spread of artemisinin-resistant Plasmodium falciparum threaten recent progress in malaria control and highlight the urgent need for antimalarial agents with new mechanisms of action. Here, we performed a high-throughput phenotypic screening of the Nagoya Chemical Library, comprising 36,160 compounds. This yielded 1391 (3.8%) primary hits, further reduced by cytotoxicity profiling. Differential screening against wild-type 3D7 and transgenic 3D7-yDHODH parasites expressing yeast dihydroorotate dehydrogenase (DHODH) was done to identify mitochondrial electron transport chain inhibitors. This approach identified a compound with modest activity against parasite DHODH, confirmed by enzymatic assays with recombinant PfDHODH. A subset of 441 potent compounds with half-maximal concentration (EC50) < 6.5 μM was selected and profiled against the Dd2-derived multidrug-resistant panel. This analysis identified compounds likely targeting key parasite pathways, including phosphatidylinositol 4-kinase (PfPI4K) and P-type ATPase 4 (PfATP4). Chemical clustering of a selected subset of 350 compounds (<10 μM) revealed novel scaffolds with antiplasmodial potency that bear no structural resemblance to known antimalarial compounds. Collectively, these findings highlight novel chemotypes associated with validated antiplasmodial targets (PfDHODH, PfPI4K, and PfATP4) and uncover previously uncharacterized scaffolds with unknown targets. These compounds provide a foundation for further investigation of their mechanisms of action and optimization as potential antimalarial agents.
Theileria annulata, the causative agent of tropical theileriosis, poses a significant threat to cattle industries, a challenge further intensified by increasing drug resistance. In this study, the food-grade phenolic compound octyl gallate (OG) was evaluated for its antitheilerial activity and identified as a potent and selective inhibitor of T. annulata. Comparative screening of gallic acid (GA) derivatives revealed that OG exhibited the strongest activity against T. annulata-transformed cell lines, with IC50 values of 374.0 nM in TaNM cells and 448.7 nM in the buparvaquone-resistant TaXJS cells, while maintaining more than 90% viability in bovine peripheral blood mononuclear cells (PBMCs) at concentrations up to 50 μM, indicating a favorable selectivity profile. At the molecular and cellular levels, OG treatment led to significant downregulation of the parasite genes TaSP and Tap104 and led to the disintegration of the annulate lamellae (AL), a parasite-induced, host-derived structure implicated in host cell manipulation, together with a concentration-dependent induction of apoptosis in TaNM and TaXJS cells. Collectively, these results identify OG as a promising lead compound with activity against T. annulata, combining direct effects on the parasite with apoptosis-associated effects in infected host cells. This study provides experimental support for further investigation of OG as a potential therapeutic option for the control of tropical theileriosis, particularly in the context of emerging drug resistance.
Brain infection by the pathogenic free-living amoebae Naegleria fowleri is a life-threatening illness, with a mortality rate >95% in the US. One of the challenges faced in treating these infections is the lack of efficacious drugs. Here, we further characterize two leads. The first, a N. fowleri enolase inhibitor (HEX ((1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid)), emerged from a target-based approach while the other, BDGR-20191 (rac-(4aR,13bS)-10,11-dichloro-4-(oxetan-3-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one), was identified in a phenotypic screen for anti-amoebic agents. Chronic low dose exposure to these agents led to cell lines that were tolerant to each. However, the tolerance phenotypes were reversed in the absence of selection with the agent. Differential gene expression analyses suggest that HEX tolerant cells upregulate cellular components that could impact levels of accumulated glycolytic intermediates, while BDGR-20191 tolerant cells could be targeting membrane biosynthesis. Unbiased metabolomics of the lines supported these possibilities. While HEX and BDGR-20191 are not synergistic with one another or with other standard-of-care drugs, their additive nature suggests they could be a useful addition to current therapeutic cocktails.
Trichomonas gallinae is a protozoan parasite of major concern in avian medicine, particularly in domestic pigeons (Columba livia). This study investigated the nitroimidazole susceptibility and T. gallinae occurrence in domesticated pigeons from Eastern Spain, kept for different competitions. A total of 220 pigeons from 11 lofts were sampled and examined by microscopy and culture, revealing 63.6% of infected birds. Genotyping identified genotype C as predominant, with occasional detection of genotype A, mixed A/C infections, and one isolate of Lineage III. In vitro susceptibility testing of 42 isolates showed a high occurrence (45.2%) of elevated metronidazole minimum lethal concentrations (MLC) values (MLC ≥40 μg/mL) ranging from 5 to >100 μg/mL in 9/11 pigeon lofts examined. MLC values were not associated with sex, age, or reproductive status. In contrast, higher IC50 values were observed in isolates obtained from pigeons bred for Pica competitions, and metronidazole exposure was associated with higher MLC values compared with ronidazole. No significant associations were found between in vitro nitroimidazole susceptibility values and environmental or management parameters, although suboptimal hygiene and high bird density were common in lofts with strains with reduced susceptibility to metronidazole. The findings of this study underscore the urgent need to establish evidence-based therapeutic protocols, strengthen biosecurity measures, and promote the development of novel therapeutic alternatives for the control of avian trichomonosis, in order to mitigate the spread of T. gallinae strains resistant to currently available drugs in pigeon populations.
Anthelmintic resistance in gastrointestinal nematodes (GIN) of cattle is of increasing concern, but the number of studies monitoring anthelmintic efficacy is insufficient to provide a representative picture. In this study, resistance to macrocyclic lactones (ML) and benzimidazoles (BZ) in GINs from 14 Scottish dairy farms (four non-organic, 10 organic) was evaluated using multiple approaches. The faecal egg count reduction test (FECRT) remains the primary tool for evaluating anthelmintic resistance in the field and was used on the four non-organic farms. Differing methodologies and recent guideline updates complicate the interpretation of FECRT results across studies, as alternative statistical frameworks produce varying confidence intervals that can alter conclusions. Nonetheless, resistance to BZs and MLs in Ostertagia ostertagiwas consistently detected with all analytical approaches on 3/3 and 4/4 non-organic farms, respectively. The egg hatch test, combined with nematode differentiation, was used to assess BZ resistance across all 14 farms. The 95% effective concentration values observed at both the population and species levels, especially for O. ostertagi (EC95: 0.045-1.294 μg/ml), were consistent with BZ resistance on 13/14 farms. Mixed amplicon sequencing was applied to 10 populations from seven farms, including both pre- and post-treatment FECRT samples. Resistance-associated polymorphisms in the β-tubulin isotype-1 gene were detected on 5/7 farms and were present at over 25% frequency in O. ostertagi, Cooperia oncophora, and Trichostrongylus spp. populations.The presence of resistance to both BZs and MLs on 3/4 non-organic farms and higher than expected EC values for BZ on 13/14 farms, showing reduced efficacy of BZ across nearly all farms, including organic, underscoring that anthelmintic resistance is not confined to a single management system. This finding emphasizes the need for sustainable parasite control across all production systems to prevent further development and spread of resistance. Although these data originate from Scotland, the high level of animal movement across the UK suggests that the findings are likely relevant to grazing systems throughout much of the country.
Cryptosporidium parvum is a zoonotic intestinal protozoan that causes severe diarrhea in children and immunocompromised individuals. The lack of effective therapies underscores the urgent need for novel drug targets. As C. parvum primarily relies on glycolysis for energy, fructose-1,6-bisphosphate aldolase (FBA) represents a promising target. Here, we characterized CpFBA and evaluated its potential as a therapeutic target. Sequence analysis revealed that CpFBA contains a signature domain associated with the type I fructose-bisphosphate aldolase superfamily and adopts a tetrameric structure. CpFBA protein localizes to both the cytoplasm and membrane of C. parvum sporozoites and presents in C. parvum excretory-secretory products (ESPs). Recombinant CpFBA protein (rCpFBA) exhibits typical aldolase activity, with a higher affinity for fructose-1,6-bisphosphate (FBP) (Km = 81.23 μM). Subsequently, molecular docking computationally predicted that morin may potentially bind to CpFBA. Anti-CpFBA serum or morin treatment significantly reduces glucose uptake and ATP levels of C. parvum sporozoites, and decreases parasite number in vitro. In C. parvum-infected suckling mice, morin treatment (60 mg/kg/day) significantly reduces oocyst shedding by 61.8%, alleviates C. parvum-induced weight loss and intestinal damage, preserves the expression of tight junction proteins (ZO-1, occludin), and suppresses the production of pro-inflammatory cytokines. These findings suggest that CpFBA is a promising drug target against C. parvum, and the natural compound morin resists C. parvum infection.
Benzimidazole resistance in Haemonchus contortus represents a widespread challenge in small ruminant production worldwide. Benzimidazoles remain one of the most commonly used drug classes, due to their broad spectrum of activity, affordability, and ease of administration. However, extensive and often inappropriate use of these compounds has led to the rapid emergence and global spread of resistance, particularly in H. contortus, a highly pathogenic and genetically diverse gastrointestinal nematode. Resistance to benzimidazoles is primarily associated with point mutations in the isotype-1 β-tubulin gene, most notably at codons 167 (F167Y), 198 (E198A), and 200 (F200Y), which result in modifications to the β-tubulin that prevent drug binding and compromise treatment efficacy. This review summarises the molecular mechanisms underlying benzimidazole resistance and provides an overview of currently available diagnostic approaches, including in vivo and in vitro assays, as well as molecular techniques such as real-time PCR, digital PCR, pyrosequencing, deep amplicon sequencing, and isothermal amplification methods. Despite regional variation in the distribution of resistance-associated polymorphisms, there is a consistent global predominance of the F200Y mutation. Finally, this review highlights the importance of integrating molecular surveillance with sustainable parasite control strategies to slow the further spread of benzimidazole resistance and preserve the long-term efficacy of available anthelmintics.
Trichinellosis is a food-borne zoonosis caused by Trichinella spiralis, for which effective treatment against the muscle larvae stage remains limited. To identify new anti-T. spiralis agents from medicinal plants, 30 extracts with reported or presumed antiparasitic relevance were screened in vitro. Among them, the 80% ethanol extract of Anaphalis margaritacea (AMEE) showed the strongest larvicidal activity, causing complete muscle larvae mortality within 72 h at 100 μg/mL and exhibiting a clear dose-dependent effect. Preliminary chemical characterization by UPLC-Q-TOF-MS/MS led to the tentative identification of eight major constituents, mainly organic acids and flavonoids. In a murine model of T. spiralis infection, oral administration of AMEE reduced muscle larvae burden by up to 59.7% in a dose-dependent manner. AMEE treatment also alleviated muscle fibre damage and inflammatory infiltration in infected muscle, accompanied by reduced levels of IL-6, IL-13, and TGF-β and decreased mRNA expression of Nqo1 and Sod2. To explore the basis of its antiparasitic activity, integrated proteomics, mitochondrial enzyme activity assays, and transmission electron microscopy were performed. AMEE markedly suppressed the activities of mitochondrial respiratory complexes I, II, and IV, reduced complex V activity, and induced mitochondrial swelling and cristae disruption in muscle larvae. These findings show that AMEE exerts significant anti-T. spiralis activity in vitro and in vivo, with evidence consistent with mitochondrial dysfunction in muscle larvae and attenuation of infection-associated muscle pathology.
