Visceral leishmaniasis (VL) remains a major public health concern in Latin America, particularly in Brazil, where Leishmania infantum is primarily transmitted by the sand fly Lutzomyia longipalpis. Understanding parasite development within the vector is essential for elucidating transmission dynamics, yet classical microscopic assessments often lack sensitivity for detecting early or low intensity infections. In this study, Lu. longipalpis females were experimentally infected with either promastigote or amastigote forms of Le. infantum and the infection dynamics were evaluated by both optical microscopy and digital PCR (dPCR). Microscopic examination revealed successful midgut colonization in both groups, with higher parasite counts in amastigote-infected sand fly females during the early phase of infection. Quantification by dPCR confirmed this pattern, detecting significantly higher parasite loads at the initial time points in amastigote-fed sand flies. However, parasite loads converged between groups at later stages, indicating comparable infection dynamics regardless of the parasite infective form. Both methods showed consistent patterns of parasite dynamics, whereas dPCR provided greater sensitivity for detecting and quantifying low parasite burdens, particularly during periods of reduced infection intensity. Overall, our findings provide new insights into parasite-vector interactions and validate dPCR as a precise and sensitive tool for assessing Leishmania infections in sand fly vectors, with potential applications for entomological surveillance and transmission studies.

Acanthamoeba castellanii is a free-living amoeba that is found in trophozoite and cyst forms in the environment. While trophozoites rely primarily on mitochondrial oxidative phosphorylation for ATP production, cysts exhibit metabolic adaptations that enable survival under adverse conditions, including anaerobic ATP synthesis. Inorganic phosphate (Pi) plays a central role in cellular bioenergetics, as it is required for ATP synthesis. Pi uptake is mediated by specific membrane transporters that are functionally coupled to ion gradients. Emerging data indicate that inorganic phosphate transport is tightly integrated with both aerobic and anaerobic energy metabolism in A. castellanii, influencing ATP production, substrate utilization, and cellular viability. In this review, we summarize current knowledge on inorganic phosphate transport systems in A. castellanii and discuss their relationship with energy metabolism in both trophozoites and cysts.

Primary Amoebic Meningoencephalitis (PAM), also known as Naegleriasis, has historically been rare but is re-emerging as a major public health concern. This acute, often fatal infection is caused by a free-living freshwater amoebic protozoan Naegleria fowleri, which invades the host central nervous system (CNS), inducing severe neuroinflammation. The immunopathogenesis of PAM remains poorly understood. Herein, the entire N. fowleri proteome was computationally analyzed, revealing Cathepsin B as a putative virulence factor with a potential to form a stable complex with human TLR1/2. This finding was further supported by molecular dynamics simulation analyses of the complex. Interestingly, Cathepsin B has been reported to mediate amoebic invasion of the host nasal mucosa, which reinforces our prediction. Based on these findings, a novel vaccine construct was designed using immunoreactive T and B cell epitopes from Cathepsin B and coupled with different TLR adjuvants. Computational analyses demonstrated the strong binding affinity of TLR4 adjuvant-coupled multi-epitope vaccine with TLR4, forming the most stable interactions. Theoretical immune simulation studies revealed that this vaccine elicits robust IgM and IgG responses upon triple administration, maintains B cell memory and triggers the expression of IFN-γ. Our data thus provide insight into the pathogenesis of PAM and offer a promising lead toward the development of an effective vaccine to combat the disease, which currently lacks reliable and effective treatments.

This research investigated the acaricidal activity of essential oils (EOs) derived from Ocimum gratissimum (OO), Monarda citriodora (MO), and Cymbopogon nardus (RO), both individually and in binary and ternary mixtures, against the brown dog tick, Rhipicephalus sanguineus using the larval packet test. Seven concentrations (0.1, 0.2, 0.4, 0.8, 1.2, 1.6, and 2.0%) were evaluated for each oil and their combinations. Lethal concentration values (LC50, LC95, and LC99) with 95% confidence limits (CL) were determined, and interaction effects were assessed through the Combination Index (CI) and Dose Reduction Index (DRI) to identify additive, synergistic, or antagonistic responses. Among the tested individual oils, OO demonstrated the greatest acaricidal effectiveness, exhibiting the lowest LC50 value (0.19%). The binary mixture of OO + MO showed enhanced efficacy (LC50 = 0.12%) with a CI of 0.464, indicating the combination achieves the same biological effect with less drug exposure. Furthermore, the ternary blend (OO + MO + RO) produced the strongest synergistic interaction (CI = 0.359) and achieved substantial dose reductions, with DRI values of 10.66, 7.85, and 7.21 for OO, RO and MO, respectively. Low CI value with higher DRI values in ternary mixture indicated enhanced efficacy with reduced required doses. These results demonstrate the potential of EOs, both alone and in combination, against the larvae of R. sanguineus, and support their integration into pest management strategies.

The life cycle of Schistosoma mansoni includes a short-lived miracidial stage, during which miracidia must locate and penetrate suitable Biomphalaria snail hosts. Although several snail-derived attractants have been reported, the physicochemical properties underlying miracidial chemotaxis remain poorly defined. To examine the contribution of acidity and carboxyl groups to miracidial attraction, we first evaluated chemotactic responses to a previously reported snail-derived peptide (P12) and its amino acid-substituted derivatives under acidic and neutral conditions. Under acidic conditions, miracidia accumulated toward all peptide solutions. Neutralization did not significantly affect miracidial attraction to P12, whereas it significantly reduced attraction to P12-D/N at all concentrations tested and to P12-P/A at intermediate concentrations. Circular dichroism analysis revealed that all peptides adopted random conformations irrespective of pH, indicating that peptide secondary structure did not account for the observed differences in attraction. Based on these findings, we examined miracidial chemotaxis toward simple acidic solutions. S. mansoni miracidia were attracted to organic acids containing carboxyl groups, including acetic, succinic, and citric acids, at specific concentrations, whereas hydrochloric acid induced attraction only within a limited concentration range and not at higher acidity. These results indicate that miracidial attraction is influenced by both acidity and the presence of carboxyl groups, rather than by acidity alone. As several organic acids tested here can be produced by snail hosts, the present findings provide insight into the chemical basis of host-finding behavior in S. mansoni miracidia in vitro.

Cryptosporidiosis remains a significant health concern, particularly in immunocompromised hosts, for whom available treatments may have limited efficacy. This study evaluated the therapeutic effects of geraniol (GR), alone and in combination with nitazoxanide (NZ), in immunosuppressed mice experimentally infected with Cryptosporidium parvum. The mice were treated in groups with varying doses of GR (25 and 50 mg/kg), NZ (100 mg/kg), and their combination for 7 days. Treatment effects were assessed based on fecal oocyst shedding, intestinal antioxidant enzyme activities, Th1-associated cytokine gene expression, and selected hepatic and renal biochemical markers. GR significantly reduced oocyst shedding and improved antioxidant and immune-related parameters compared with untreated infected/immunosuppressed mice (p < 0.01). The combination of GR with NZ produced a greater reduction in oocyst shedding than either treatment alone, with the GR 50 mg/kg + NZ 100 mg/kg regimen producing the greatest overall effect (p < 0.001). The combined treatment was also associated with restoration of CAT and SOD activities and increased expression of IL-12 and IFN-γ (p < 0.001). No evident treatment-related hepatic or renal toxicity was observed based on the evaluated biochemical markers. Overall, these findings provide preliminary experimental evidence that GR, particularly in combination with NZ, may warrant further investigation as an adjunctive approach for cryptosporidiosis. However, validation in additional animal models and ultimately well-designed clinical studies is required.

Toxoplasmosis is a zoonosis widespread worldwide, although it primarily affects populations in underdeveloped countries. In humans, infection can lead to serious complications, particularly in immunocompromised individuals and pregnant women. Despite advances in the diagnosis of this disease, therapeutic regimens for treating toxoplasmosis are not fully effective, and new drugs are highly desirable. In this context, we evaluated 4'-(4-methylphenyl)-2,2':6',2''-terpyridine (L) and its previously reported Pt(II) complex (1) against Toxoplasma gondii. The free ligand L exhibited greater antiparasitic activity (IC50 < 0.78 μM) than 1 (IC50 = 2.4 μM), significantly reducing intracellular parasite proliferation in BeWo cells. Both compounds showed high selectivity toward host cells, with selectivity indices (SIs) exceeding 128 and 41 for L and complex 1, respectively. Molecular docking studies against TgCDPK1 and TgDHFR-TS were performed to investigate the potential binding modes of the compounds. Simulations suggested that both molecules can accommodate the ATP binding site of TgCDPK1 and the cofactor binding pockets of TgDHFR-TS. The free ligand L showed higher docking scores compared to the complex 1. This observation may be attributed to the greater conformational flexibility and lower steric hindrance of the free ligand, which potentially allows for a more optimized fit in the catalytic cavity under the simulated conditions. These findings indicate that terpyridine derivatives represent promising candidates for the development of new anti-T. gondii agents.

Cutaneous leishmaniasis (CL) is a neglected tropical disease. The treatment is restricted to a handful number of drugs that exhibit toxic effects and limited efficacy. The development of new alternative therapies, including the identification of effective drugs for topical treatment of CL, is of utmost interest. The aim of this study was to prepare, characterize and evaluate the efficacy of topical therapy with lapachol-micelle in the experimental treatment of CL. The lapachol-micelle was prepared using polaxamer 407. A preliminary stability study was conducted for 15 days at room temperature. The antileishmanial efficacy was performed in BALB/c mice infected with Leishmania (Leishmania) major, the treatment groups were: topical lapachol-micelle, intraperitoneal Glucantime, and control (untreated). Therapeutic efficacy was assessed by parasite load, and lesion size. To evaluate toxicity, the animals were weighed and blood was collected for biochemical analysis. Lapachol-micelle presented adequate characteristics: size 15 nm, zeta potential -3mV, and polydispersity index 0.3; remaining stable in preliminary stability study. Treatment with lapachol-micelle resulted in significant improvement, leading to stabilization of lesion size, while in the other groups, lesions increased in size. No significant changes were observed in weight or biochemical parameters. Topical treatment with lapachol-micelle may be a promising alternative for the treatment of CL.

The aim of the present study was to evaluate the protection against tissue cyst formation from T. gondii in pigs immunized with a DNA vector plasmid encoding partial rop18 and sag1 genes. Ten pigs were divided into three groups (G): G1, and G2 received 150 μg of pcDNA3.1+rop18, and 150 μg of pcDNA3.1+sag1 by intradermal (ID) and intramuscular (IM) routes, respectively. G3 received 300 μg of pcDNA 3.1 alone by IM. All groups received 2% (v/v) of levamisole as an adjuvant. Four immunizations were carried out on days 0, 14, 35, and 56; the challenge was performed with ∼ 4 × 10[3] oocysts of the ME49 strain by oral route at day 70, and animals were culled on day 100. Humoral immune response of IgM and IgG antibodies was assessed using indirect ELISA, and tissue cysts were evaluable by mouse bioassay. The results showed that the animals immunized with the DNA vaccine (sag1+rop18) developed partial protection against the formation of tissue cysts, however, more studies with higher number of pigs should be conducted.

Plants are a sustainable source of bioactive components for medical and agricultural fields. This study evaluated the chemical composition, antioxidant capacity, antibacterial activity, and larvicidal potential of Rosmarinus officinalis (rosemary) and Jasminum officinale (jasmine) essential oils against Culex pipiens and Aedes aegypti larvae. GC/MS analysis revealed that rosemary oil was dominated by oxygenated monoterpenes (eucalyptol 29.51% and camphor 21.44%), while jasmine oil primarily contained aromatic esters (benzyl acetate 41.0%) and monoterpene alcohols (β-linalool 8.38%). Rosemary oil exhibited higher total phenolic and flavonoid contents, correlating with superior antioxidant (DPPH, FRAP, ABTS) and antibacterial activities against Escherichia coli, Staphylococcus aureus, Klebsiella spp., and Proteus spp. Larvicidal bioassays demonstrated that rosemary oil was more potent, with lower LC50 and LC90 values against both mosquito species. Biochemical analyses of treated larvae revealed significant inhibition of acetylcholinesterase, esterases, and metabolic enzymes (GABA-T, amylase, lipase), alongside elevated oxidative stress markers (LPO, TPC) and reduced antioxidant defenses (SOD, CAT, GST). These findings highlight the potential of rosemary and jasmine essential oils, particularly rosemary, as natural, eco-friendly agents with antioxidant, antibacterial, and insecticidal properties, supporting their application in vector control and as functional bioactive agents in food and health sectors.

Artificial feeding systems have become essential tools for studying tick physiology, pathogen acquisition, and transmission without relying on vertebrate hosts. While in vitro feeding has been successfully established for several ixodid species, including Ixodes ricinus, Dermacentor spp., and Rhipicephalus spp., no system has yet supported complete engorgement and reproduction of Ixodes hexagonus, a widespread European tick and vector of Borrelia spp. and the tick-borne encephalitis virus. In this study, we report the first successful in vitro feeding of adult I. hexagonus using a silicone-membrane system. Two trials were conducted with ticks of different ages and starvation period. Ticks were fed on silicone membrane at 27°C and appr. 75% relative humidity. Out of 38 and 20 females, 29 and 18 successfully fed to repletion (76 and 90%, respectively) with a mean weight of 111.1 mg for the first and 132.6 mg for the second trial. The mean feeding durations were 7.2 and 8.8 days and the oviposition rates were 90 and 100% in trial 1 and 2, respectively. The mean number of eggs produced was 402 ± 171 for trial 1 and 847 ± 378 for trial 2, whereby all females produced fertile eggs with a mean hatching rate of 92.1 ± 14.5% and 99.4 ± 0.61% for the first and the second trial, respectively. These results demonstrate that I. hexagonus might be reliably maintained and reproduced under artificial conditions, establishing this species as a promising experimental model for controlled studies on pathogen acquisition, transmission dynamics, and vector competence. The development of this feeding system expands the methodological toolkit available for European tick research and provides a foundation for future work on the epidemiology of hedgehog-associated tick-borne pathogens.

Leishmaniasis is a parasitic disease caused by Leishmania species and constitutes a significant global public health concern. Pentavalent antimonials, the current first-line therapy for cutaneous leishmaniasis, are limited by toxicity, high cost, and emerging drug resistance. This study aims to evaluate the antipromastigote and intracellular antiamastigote activity of the polyphenolic compounds cynarin (CYN), a hydroxycinnamic acid derivative, and the flavonoids apigenin (API) and luteolin (LUT) against Leishmania tropica. Cytotoxicity was assessed in THP-1-derived macrophages using the CellTiter-Glo® assay. Antileishmanial activity against promastigotes and intracellular amastigotes was assessed using microscopic enumeration and viability assays, with amphotericin B serving as the reference drug. Selectivity index values (SI) were calculated as CC50/IC50, with SI ≥ 10 regarded as therapeutically relevant. CYN, API, and LUT exhibited CC50 values of 561 ± 18.1 μM, 247.3 ± 30.8 μM, and 67.5 ± 7.9 μM, respectively. Among the tested compounds, CYN (IC50: 130.1 ± 9.3 μM) showed the most pronounced antipromastigote activity, whereas API (IC50: 674.6 ± 16.3 μM) and LUT (IC50: 863.8 ± 16 μM) were active only at high concentrations. Both CYN (SI: 21.3) and LUT (SI: 11.6) displayed selective activity against intracellular amastigotes, while API exhibited comparatively modest activity (SI: 8.0), though its effect remained noteworthy. Microscopic analysis confirmed pronounced morphological alterations in both parasite forms at concentrations ≥ IC50. CYN demonstrated the highest selectivity, whereas LUT exhibited enhanced activity against intracellular amastigotes, indicating distinct pharmacological profiles. Nonetheless, comprehensive pharmacokinetic and pharmacodynamic studies in advanced in vivo models are required to validate their translational potential.

Bacillus thuringiensis Berliner 1911 has high effectiveness against important mosquito vectors of human pathogens; however, there are few studies on the selection of Brazilian isolates of B. thuringiensis with activity against Anopheles darlingi Root, 1926, the main transmitter of human malaria parasites in the Amazon region. The present study sought to select native isolates of B. thuringiensis acquired from soil samples from three Brazilian biomes, with pathogenicity to An. darlingi larvae under laboratory conditions. Fifteen isolates of B. thuringiensis from the Maranhão Entomopathogenic Bacilli Bank, Brazil, were used, selected for their high larvicidal activity against Aedes aegypti Linnaeus, 1962, in a previous trial. A selective bioassay was performed with 15 isolates, of which thirteen (86.6%) were effective against An. darlingi and were subsequently used in quantitative bioassays. Considering the observations at 24, 48, and 72 h, the most toxic isolates were BtMA-689 and BtMA-690, for which the lowest values of LC50 and LC90 were achieved across the three reading intervals. However, six isolates (BtMA-681, BtMA-1114, BtMA-687, BtMA-690, BtMA-703, and BtMA-750) caused mortality rates similar to the standard strain Bti T14 001, for which no statistically significant differences were detected. The study confirms new Brazilian isolates of B. thuringiensis with larvicidal activity against An. darlingi, which can be used in the manufacture of specific by-products containing these insects.

Psoroptic mange caused by Psoroptes cuniculi is an important parasitic dermatopathy in rabbits and is commonly associated with pruritus, accumulation of ceruminous exudate, crust formation, and inflammatory auricular lesions. Although several isoxazolines have demonstrated efficacy against mite infestations in companion animals, information regarding their use in rabbits remains limited. The present study evaluated the efficacy and safety of single-dose oral administration of afoxolaner, fluralaner, and sarolaner in rabbits naturally infested with P. cuniculi. Thirty-two New Zealand White rabbits were randomly allocated into four experimental groups (n = 8/group): untreated control, afoxolaner (2.5 mg/kg), fluralaner (25 mg/kg), or sarolaner (2 mg/kg). Parasitological and clinical evaluations were performed on Days 0, +3, +7, +14, +21, +28, and +35. Rabbits treated with afoxolaner and fluralaner achieved complete parasitological cure from Day +3 onwards, while those treated with sarolaner reached complete parasitological cure on Day +7. No significant differences in parasitological cure rates were observed among the treated groups after treatment (P > 0.05). All treated groups showed progressive clinical improvement throughout the study period. No adverse effects were observed following treatment administration. In contrast, rabbits from the untreated control group remained positive and showed persistence of clinical lesions throughout the experimental period. Under the conditions of this study, single-dose oral administration of afoxolaner, fluralaner, and sarolaner demonstrated therapeutic efficacy against natural P. cuniculi infestation in rabbits, supporting the potential use of isoxazolines for the treatment of psoroptic mange in this species.