Localized cutaneous leishmaniasis (LCL) caused by Leishmania mexicana is a neglected tropical disease with limited therapeutic options due to the toxicity, high cost, and prolonged administration associated with meglumine antimoniate (Glucantime®), highlighting the need for safer and more accessible treatments. In the present study, topical cream formulations containing aqueous extracts of Allium sativum (1%, 5%, and 10%) were evaluated in an experimental BALB/c mouse model of LCL. The 1% formulation showed the highest efficacy, significantly reducing lesion size and parasite burden compared to both the excipient and Glucantime® control groups. Histopathological analysis revealed re-epithelialization and improved tissue repair, while no significant differences in IFN-γ expression were observed among groups. Chemical characterization by GC-MS and UHPLC-MS/MS identified organosulfur and phenolic compounds, supporting a mechanistic basis for the observed leishmanicidal and wound-healing effects. Overall, the 1% A. sativum formulation demonstrated superior therapeutic performance under the evaluated conditions, combining antiparasitic activity, tissue repair, and good tolerability. These findings highlight the potential of A. sativum-based topical formulations as a safe, effective, and less invasive alternative for the treatment of localized cutaneous leishmaniasis.

Entomopathogenic nematodes (EPNs) are useful as biological agents for the control of insect pests and disease vectors. They are also important for understanding nematode pathogenicity and host anti-nematode response. The pathogenic properties of EPNs towards insects are attributed to effector molecules, which are produced by both the nematodes and their symbiotic bacteria during infection. Here, we hypothesized that the contribution of the mutualistic bacterium Photorhabdus luminescens to the virulence of Heterorhabditis bacteriophora is host-dependent. To test this hypothesis, we compared the survival responses of two natural lepidopteran hosts, the greater wax moth Galleria mellonella and the Indianmeal moth Plodia interpunctella, following infection with either axenic H. bacteriophora (lacking P. luminescens) or symbiotic nematodes carrying their bacterial partner. We find that the presence of P. luminescens enhances the pathogenicity of H. bacteriophora towards G. mellonella larvae, whereas P. interpunctella larvae show broadly similar survival trajectories following infection with symbiotic or axenic nematodes, despite a shorter median survival time after symbiotic infection. These findings imply that EPNs may exert distinct infection strategies against different but related natural insect hosts, which may in turn activate differential immune mechanisms to deal with the combined threat of the nematodes and their symbiotic bacterial partners.

Coproparasitological examinations are essential non-invasive tools for detecting endoparasites in wild animals; however, the lack of methodological standardization and the variable performance of available techniques remain important challenges. This study evaluated the agreement and comparative performance of the Simplified Parasite Detection Method (SPDM) relative to three routinely used coproparasitological comparator methods: spontaneous sedimentation, Willis-Mollay flotation, and Mini-FLOTAC®. A total of 143 pooled fecal samples from mammals, birds, and reptiles were analyzed. Overall, SPDM showed moderate to substantial agreement with the comparator methods (Cohen's κ = 0.509 [C.I 95% 0.362-0.656]; 0.624 [CI 95% 0.491-0.757]; p < 0.001), together with high relative specificity, negative predictive values, and overall agreement. In addition to its comparative performance, SPDM required approximately 5 min of processing time per sample, involved minimal laboratory infrastructure, and had a low implementation cost, making it a practical alternative for routine coproparasitological examinations in both laboratory and field settings. Comparative performance varied among animal groups, with the highest agreement observed in birds and lower agreement in reptiles, probably reflecting the limited sample size and biological characteristics of reptile feces. SPDM also detected parasitic structures in some samples classified as negative by the comparator methods, highlighting its potential as an additional coproparasitological technique. Because no independent gold standard was available, the diagnostic indices reported should be interpreted as measures of comparative performance relative to the evaluated comparator methods rather than estimates of absolute diagnostic accuracy.

Pentatrichomonas hominis and Blastocystis sp. are enteric protists with the capacity to infect both non-human primates (NHPs), humans and other animal hosts, yet their epidemiology and coinfection patterns in NHPs remain poorly understood. Given the phylogenetic proximity of NHPs to humans, and the role of the Crab-eating macaque as a key animal model for human diseases, this species is considered a potential reservoir for zoonotic protists. Here, we investigate the occurrence and genetic profiles of P. hominis and Blastocystis sp. in NHPs from China. In this study, fresh fecal samples were collected from Crab-eating macaques in Beijing (n = 212) and Suzhou (n = 292). All samples were analyzed using species-specific PCR and SSU rRNA-based phylogenetic analysis. The resulting sequences were subjected to molecular analysis for species confirmation of P. hominis and subtype identification of Blastocystis sp. The overall prevalence of P. hominis was 55.16% (60.85% in Beijing, 51.03% in Suzhou), with juveniles showing significantly higher infection rates than adults at both sites (p < 0.0001). The overall prevalence of Blastocystis sp. was 50.6% (40.57% in Beijing, 57.88% in Suzhou), with adults exhibiting higher rates than juveniles. Two Blastocystis sp. genotypes were identified: ST1 (216 samples) and ST2 (39 samples). Coinfection of the two protists occurred at an overall rate of 18.65%. Phylogenetic analysis revealed that our P. hominis isolates are closely related to reference sequences AY758392 and JX565035, while Blastocystis sp. isolates formed two major clades (ST1 and ST2), with ST1 being the dominant subtype. Together, these results detail the epidemiology of P. hominis and Blastocystis sp. in Crab-eating macaques in China, including prevalence, age-dependent infection patterns, ST1 dominance, and coinfection rate. These extend global knowledge of these protists, particularly regarding subtype distribution and host age associations.

Malaria, caused by Plasmodium falciparum is a deadly disease and remains a global health concern. With the emergence of resistance to artemisinin; it is pivotal to identify potential therapeutic drug targets against malaria. This study employs the use of machine learning and computational approaches to predict the druggable protein among the ∼1000 essential hypothetical genes of P. falciparum. While traditional target prioritization strategies rely on either manual curation or single-parameter thresholds, neither approach is conducive to working with literally thousands of essential proteins nor can they capture non-linear relationships between several features influencing druggability. In contrast, machine learning can integrate heterogeneous descriptors such as sequence, structure, interaction, and annotation features and learn complex patterns associated with known druggable proteins, enabling scalable and more objective prediction of druggable essential hypothetical proteins. The screening criteria involved five point classification as EC number, molecular weight, PPI, orthology, and GO annotation, for which several web tools were employed. Machine learning algorithms like Decision Tree, Random Forest, SVM, Perceptron, and Logistic Regression were imputed to train the system and predict the druggable proteins using training and test data set accordingly, in the ratio of 70:30. Along with accuracy scores, ROC-AUC curve was generated for better visual representation. The study reported nine proteins as druggable. Furthermore, assignment of a normalized weighted score for each criterion to prepare a ranked list of putative drug targets identified PfCERLI1 as prioritized drug target. Additionally, the target protein structure obtained from AlphaFold was refined and underwent quality checking using Galaxy WEB and SAVES meta-server respectively. Schrödinger Glide was employed for molecular docking of the target protein with phytochemical ligand dataset, reported AD34 (Anthraquinone) as the best inhibitor with a binding affinity score of -13.05 kcal/mol. Molecular Dynamics(MD) simulation study of the protein-ligand complex for 100ns revealed the presence of hydrogen bond between the inhibitor and the active site residues Leu147 and Thr224 throughput the simulation time-frame. The above study proposed, PfCERLI1, as the novel essential hypothetical protein of P. falciparum for therapeutic intervention study in future.

The increasing resistance of Aedes aegypti L. to conventional larvicides has intensified the search for novel bioactive compounds through molecular design. This study explores the larvicidal efficacy and toxicological profiles of piperine and its saturated analog, tetrahydropiperine, by integrating in silico molecular docking and experimental bioassays. Both compounds were subjected to docking studies using as target enzymes human and insect acetylcholinesterases (PDB: 4EY6 and 6XYU) and the juvenile hormone-binding protein (PDB: 5V13). Tetrahydropiperine exhibited higher binding affinities, with binding energies of -9.36 kcal mol[-1] (4EY6), -9.32 kcal mol[-1] (6XYU), and -10.60 kcal mol[-1] (5V13), compared to piperine, which showed energies of -9.56, -10.83, and -10.49 kcal mol[-1], respectively. In larvicidal bioassays, tetrahydropiperine demonstrated greater activity, with an LC50 of 54.5 μΜ, whereas piperine showed an LC50 of 78.8 μΜ after 24 h of exposure. Toxicological evaluation in Swiss mice revealed that tetrahydropiperine, at 300 mg kg[-1], caused no significant adverse effects, while piperine induced mortality and behavioral alterations at doses above 50 mg kg[-1]. These results suggest that the unsaturation in piperine side chain may act as a toxicophoric moiety, and that its saturation enhances larvicidal selectivity while reducing systemic toxicity. Tetrahydropiperine thus emerges as a promising scaffold for the development of selective, safe, and effective larvicidal agents.

Given the need to develop sustainable strategies for the management of ectoparasites, the inclusion of eco-friendly mycoacaricide and/or mycoinsecticide products has become increasingly imperative. Rhipicephalus microplus is an ectoparasitic arthropod of major economic and sanitary importance in livestock production, acting as a vector of pathogens of veterinary relevance and causing substantial production losses. Control of this tick species has become progressively more challenging due to the widespread development of resistance to synthetic chemical acaricides commonly used in cattle production systems, highlighting the need for alternative approaches within integrated tick management programs. In this context, the present study evaluated the in vitro efficacy of the entomopathogenic fungi Beauveria bassiana IBCB66 and Metarhizium anisopliae IBCB425 against different developmental stages of R. microplus, including engorged females, eggs, and unfed larvae. Bioassays were conducted using conidial suspensions at concentrations ranging from 10[6] to 10[9] conidia/mL. Both fungal isolates exhibited high conidial viability (>90%) after 24 h of incubation. In the engorged-female immersion assay, B. bassiana IBCB66 at 10[9] conidia/mL significantly reduced egg mass and key reproductive parameters, whereas M. anisopliae IBCB425 affected hatchability of eggs laid by treated females. In the egg bioassay, direct exposure of eggs to conidial suspensions reduced hatching at the highest concentrations. In the unfed larval bioassay, both isolates caused significant larval mortality across the tested concentrations. These results demonstrate that B. bassiana IBCB66 and M. anisopliae IBCB425 exert biological effects at multiple stages of the R. microplus life cycle and support their potential use as environmentally sustainable components of integrated tick control strategies.

Leishmaniasis is a parasitic disease caused by several Leishmania species, including Leishmania (Leishmania) amazonensis, and is associated with oxidative stress and tissue damage. The objective was to evaluate 11 β-carboline derivatives for their predicted toxicity and drug-likeness properties, antileishmanial activity, hemolytic potential, antioxidant capacity. In silico analyses indicated favorable physicochemical parameters, as well as good permeability and solubility profiles. In vitro assays demonstrated antileishmanial activity against promastigotes after 72 h, with IC50 values ranging from 5.15 to 41.19 μM (8) and (5) were the most active compounds. None of the derivatives showed hemolytic activity in human erythrocytes. Antioxidant assays revealed significant free radical scavenging, with (11) showing the highest activity, however all compounds outperformed the control. These findings identify β-carboline derivatives as promising candidates with combined antileishmanial, antioxidant, and low-toxicity profiles. Further studies are warranted to investigate their mechanisms of action and in vivo efficacy.

In this research, we investigated the genetic diversity of Cryptosporidium spp. in 65 free-living white-eyed parakeets (Psittacara leucophthalmus) from the central-eastern region of São Paulo state, Brazil. Using real-time nested PCR of the 18S rRNA gene and conventional nested PCR of the actin and heat shock protein (HSP-70) genes, two samples were positive for Cryptosporidium spp., representing an prevalence of 3.08% (95% CI: 0.85-10.54%). However, oocysts of this protozoan were not observed through coproparasitological examination, nor were clinical signs characteristic of cryptosporidiosis noted in any bird. The amplicons obtained in this study were subjected to bidirectional Sanger sequencing. Among the positive samples, one presented sequences of sufficient quality for analysis of the 18S rRNA and HSP70 genes, exhibiting 100% and 99.64% genetic similarity, respectively, with Cryptosporidium meleagridis sequences deposited in GenBank. Phylogenetic analysis using HSP-70 gene sequences from Cryptosporidium spp. confirmed that the amplified Cryptosporidium sequence is closely related to C. meleagridis sequences, grouping within the same clade. Nested PCR targeting the parasite surface glycoprotein gene (GP60) was performed; however, a sufficient number of amplicons were not obtained, making subgenotyping impossible. We believe this to be the first report of C. meleagridis, a protozoan with zoonotic potential in the synanthropic white-eyed parakeets (P. leucophthalmus).

This study investigated the diversity of culturable bacteria in the gut of Bithynia siamensis goniomphalos, the first intermediate host of trematode parasites. It examined the relationship between bacterial communities and trematode infection. Snails were collected from freshwater habitats in Udon Thani and Khon Kaen provinces, Thailand, and classified as infected or uninfected using the cercarial shedding method. A total of 132 bacterial isolates were obtained and identified based on 16S rRNA gene sequencing. Bacterial abundance differed significantly between the two groups, with higher counts observed in uninfected snails (2.06-8.60 × 103 CFU/ml) than in infected snails (1.06-1.60 × 103 CFU/ml) (p < 0.05). Bacterial composition also varied according to infection status. Bacterial isolates from infected snails were predominantly associated with Acinetobacter, Vogesella, and Stenotrophomonas, whereas Exiguobacterium spp. were mainly found in uninfected snails. Several taxa, including Bacillus spp., Chryseobacterium spp., and Micrococcus spp., were detected in both groups. These findings suggest that parasite infection is associated with shifts in both the abundance and composition of gut bacterial communities in B. siamensis goniomphalos. The results provide new insights into host-microbe-parasite interactions and may contribute to a better understanding of parasite transmission dynamics in freshwater ecosystems.

The development of new drugs against afflictions that disproportionately impact poorly resourced areas around the globe is an expensive endeavor. As cost-effective alternatives, strategic combinations of approved drugs can be used to enhance the efficacy against Plasmodium falciparum. Understanding the metabolic consequences of such combinations is essential for optimizing treatment strategies and delaying drug resistance. An integrated metabolomic and pharmacological analysis was performed on P. falciparum parasites exposed to chloroquine (CQ), pyrimethamine (PY), sulfadoxine (SD), and their combinations (SDPY and SDCQ). Dose‒response assays were used to quantify drug potency, whereas untargeted metabolomic profiling was used to assess pathway-level perturbations associated with individual and combined treatments. Dose‒response assays confirmed the nanomolar potency of PY (IC50 = 12.5 nM) and CQ (IC50 = 11 nM) compared with the micromolar efficacy of SD (IC50 = 9.1 μM), which is consistent with the role of PY as an antifolate partner drug capable of enhancing combinatorial, pathway-level metabolic perturbations. Metabolomic profiling revealed that PY strongly disrupted folate-dependent pyrimidine biosynthesis, leading to deoxyuridine and dUMP accumulation, whereas SD caused milder perturbations, which was consistent with DHPS inhibition. CQ produced modest metabolic effects alone but markedly amplified antifolate-induced stress when combined with PY. Drug combinations generated metabolic responses that are distinct from those resulting from individual treatments. Across antifolate combinations, consistent trends included reduced amino acid pools, suppression of thiamine and glutathione metabolism, and enhanced PPP inhibition, leading to broad disruption of nucleotide, redox, and carbon metabolism. Pyrimidine suppression has emerged as the central hallmark of antifolate-based therapy in P. falciparum. The distinct metabolic perturbations observed with drug combinations suggest enhanced pathway-level responses that may contribute to improved antiplasmodial activity and inform future combination strategies. These findings provide a mechanistic foundation for guiding antimalarial combination policies, optimizing therapeutic regimens, and strengthening rational drug-design strategies.

Tick paralysis in companion animal pets is primarily caused by holocyclotoxins secreted by the salivary glands of the Australian eastern paralysis tick, Ixodes holocyclus. These toxins have been traditionally identified and quantified using in vivo neonate assays that are relatively reproducible but constrained by animal ethics. Thus improved analytical methods are needed to support the standardisation of antiserum products, toxin characterisation, and the development of next-generation therapeutics to treat companion animal paralysis. Specific anti-holocyclotoxin (anti-HT) monoclonal antibodies were produced against predicted B cell epitopes from HT-1 and HT-5. The monoclonal antibodies were characterised using surface plasmon resonance kinetics and used to develop ELISAs. The sensitivity, reproducibility, and specificity of the monoclonal antibody-based ELISAs for the detection of HT-1 and HT-5 are high. The results from the Surface Plasmon Resonance (SPR) kinetics showed that the selected antibodies have high affinities for the these HTs. The optimised ELISAs enabled high-affinity HT-1 detection across a 1- 60 ng/mL range and distinguished toxin-neutralising capacity among commercial anti-tick sera. Finally, this immunoassay, which utilises monoclonal antibodies against HT-1 and HT-5, represents a major step forward for the quantification of HTs and provides a much more ethical alternative to in vivo testing. Its application in the assessment of tick antisera makes this technique highly relevant and provides potential for the development of recombinant anti-toxin treatments. Future research should focus on developing a panel of mAbs to detect all 19 HT types.

We previously reported that Plasmodium yoelii 17XNL (Py) protected mice from collagen-induced arthritis (CIA). However, the host factors required for this protection are not fully understood. As interferon-gamma (IFN-γ) is often induced by protozoan infections and is also known as an anti-arthritic cytokine in CIA, we here investigated the requirement of IFN-γ for the anti-arthritic effects of Py infection. CIA was induced by immunization with collagen type II (CII) emulsified with Freund's complete adjuvant in IFN-γ knockout (IFN-γKO) and wild-type (WT) mice. At 2-week post-immunization, mice were inoculated with Py. In addition to evaluation of arthritis severity, serum levels of anti-CII IgG antibodies and splenic cytokine production were determined. In IFN-γKO mice, Py infection suppressed CIA development and downregulated pro-arthritic cytokines (IL-17A and TNF-α) during the period of parasitemia. However, a marked rebound of arthritis occurred after parasite clearance, associated with a surge in serum anti-CII IgG2a levels. Our findings demonstrate that host IFN-γ is not required for the initial suppression of CIA by Py infection during parasitemia. The subsequent rebound of arthritic symptoms after parasite clearance in the IFN-γKO mice was associated with a surge in anti-CII IgG, particularly the IgG2a subclass.

Psoroptic mange represents a significant challenge in livestock production due to economic losses and animal welfare concerns. The present study aimed, for the first time, to evaluate the compatibility of the entomopathogenic fungus Metarhizium pingshaense, Mentha piperita essential oil (EO) and its major constituents (menthol and menthone), as well as their acaricidal potential, individually and in combination, against adult Psoroptes ovis var. cuniculi mites collected from rabbits. Mites were also evaluated using scanning electron microscopy (SEM) 24h and 48h after the treatment. The results indicated that M. piperita EO did not compromise M. pingshaense conidial germination. M. piperita EO, menthol, menthone (at 0.01 mg/mL), and the fungus, individually or combined, exhibited significant acaricidal potential, reducing mite survival. Two days post-treatment, mortality rates ranged from 21.67% to 100%. By day 4, groups treated with the lowest fungal concentrations exhibited mortality rates above 75%, differing significantly from the control (40%). From day 6 onward, mortality rates exceeded 95% in all groups, including the control, highlighting the limitations of in vitro assays and the need for improved experimental models. SEM analysis revealed progressive mycelial development on treated mites with structural cuticular alterations consistent with fungal penetration including formations suggestive of appressoria. These findings reinforce the potential of integrating entomopathogenic fungi with plant-derived bioactive compounds as part of sustainable control strategies for mites of veterinary importance.

This study evaluated the insecticidal properties of polyporphyrin, a natural compound isolated from Carica papaya seed oil, against Anopheles gambiae, a major vector of malaria. Polyporphyrin was purified through column chromatography of the methanol extract. Larvicidal activity was assessed using WHO standard protocols, and the compound showed strong effects, recording 100% mortality at 0.032 mg/mL with LD50 and LD90 values of 0.0068 and 0.0144 mg/mL, respectively. Adulticidal activity was evaluated using the CDC bottle assay, where polyporphyrin caused up to 100% mortality at 0.032 mg/mL, with LD50 and LD90 values of 0.0058 and 0.0116 mg/mL. Ovicidal testing revealed 80.20% inhibition of egg hatching at the same concentration. Molecular docking indicated effective interaction with the quadruple mutant PfDHFR-TS enzyme, suggesting potential against insecticide resistance. Molecular electrostatic potential analysis showed distinct electron-rich and electron-poor regions, supporting its specificity and reactivity as an insecticidal agent. Overall, polyporphyrin demonstrates strong multi-stage activity against An. gambiae. Further studies should assess environmental safety and field performance to support its development as an eco-friendly malaria vector control agent.