Parasites & Vectors最新文献

Background: The life cycle of Leishmania infantum is maintained mainly in dogs in anthropogenic environments and in many other wild animals in the sylvatic cycle. The ecological plasticity of some wild canids facilitates their role as hosts for Leishmania spp. in different endemic regions. Although red foxes (Vulpes vulpes) frequently test positive for L. infantum in Europe, little is known about their clinical presentation, immune response, or treatment outcomes. This study investigated the prevalence, clinical, and immunological features of L. infantum infection in foxes from southern Italy, complemented by an in vitro evaluation of cytokine responses in fox macrophages.

Methods: Wild foxes from a wildlife rehabilitation center in southern Italy were molecularly and serologically screened for L. infantum. One sick fox underwent a complete diagnostic confirmation, treatment, and follow-up through hematological, biochemical, cytological, and molecular evaluations. In addition, peripheral blood mononuclear cells (PBMCs) isolated from a healthy fox were differentiated into macrophages and experimentally infected with L. infantum to assess early host-parasite interactions and cytokine gene expression profiles at two time points (4 h and 48 h).

Results: Overall, 13 out of 54 foxes (24.1%) were molecularly positive for L. infantum, with a prevalence of 25% (5/20) in necropsied animals and 22% (9/41) in live animals. One individual tested seropositive for L. infantum and Ehrlichia sp. using the SNAP Leish 4Dx^® test (2.4%, 1/41). The sick fox treated with meglumine antimoniate and allopurinol showed marked clinical and laboratory improvement. In the in vitro evaluation, the percentage of infected macrophages decreased from 32.8% at 4 h (2.25 parasites/cell) to 21.5% at 48 h (2 parasites/cell). Cytokine gene expression at 4 h and 48 h showed an increase in interleukin-6 (IL-6) (0.164-0.552) and IL-10 (0.828-4.245), stable IL-4 (0.505-0.708), a decrease in IL-12 (1.793-1.223) and IFN-γ (1.507-0.613), and consistently low TNF-α (0.377-0.411).

Conclusions: The present study confirmed a high L. infantum prevalence of infection in red foxes from southern Italy. Serological findings herein and in the literature, together with in vitro cytokine gene expression, suggested that most foxes may remain subclinically infected. The high molecular positivity and the synanthropic nature of red foxes reinforce their role as reservoirs of L. infantum in endemic areas.

Background: Tick-borne encephalitis virus (TBEV, Orthoflavivirus encephalitidis) is an arbovirus of the family Flaviviridae. It is the etiological agent of tick-borne encephalitis (TBE), a severe disease affecting the central nervous system. Among arboviral infections, TBE represents the greatest burden in northern Eurasia, both in terms of emerging infection risk and mortality. Globalization and climate change increase the risk of TBEV introduction into nonendemic countries. They may also lead to the emergence of new viral variants featuring increased virulence for humans or altered antigenic characteristics. Hence, sensitive and specific TBEV detection methods are needed not only for diagnostics but also for One Health approach goals (surveillance and identification of viral sources in the environment).

Methods: Here, we describe a newly developed reverse transcription PCR (RT‒PCR) assay for TBEV detection. The assay was developed and evaluated using armored RNA positive control particles (ARCs). The assay was evaluated using several sample types: (1) a panel of heterologous viral and bacterial RNA/DNA; (2) RNA from TBEV strains isolated in different years in various Russian regions; and (3) RNA from TBEV-positive and TBEV-negative ticks (collected in northwest Russia).

Results: The limit of detection (LOD) of the assay is 10³ copies/mL (20 copies/reaction) of TBEV RNA. The developed demonstrated 100% analytical specificity. The assay was compared with the two most commonly used Russian commercial kits for TBEV diagnostics.

Conclusions: The results indicate that the developed RT‒PCR assay is a reliable and competitive method for the detection of TBEV RNA, which establishes its value as a tool for diagnosing and monitoring the virus.

Background: In East Africa, bovine schistosomiasis, although common, is poorly appreciated and managed, detrimentally impacting upon livestock health. In certain settings, bovine schistosomiasis may be involved in zoonotic transmission of human schistosomiasis. Better disease management and more effective control of bovine schistosomiasis require the development of sensitive and specific serological screening and rapid diagnostic tools.

Methods: We developed an enzyme-linked immunosorbent assay (ELISA) for Schistosoma bovis detection in cattle, utilizing nine shortlisted potential diagnostic protein targets. These shortlisted candidates, STI, IPP, OP, PGK1, COG, PDZ, and Sbp80 (as three fragments), were identified from Schistosoma japonicum homologs already reported with the highest diagnostic potential. In S. bovis, these proteins participate in various biological processes, including metabolic pathways, transcriptional regulation, glycolysis, phosphorylation, and cell signalling, although their real diagnostic potential has not been explored until now.

Results: The ELISA was optimized using bovine blood serum samples from regions in Tanzania and validated for sensitivity and specificity. Two targets of specific focus, Conserved Oligomerix Golgi complex subunit 4 (COG) and a domain of the cysteine protease calpain (Sbp80), achieved the highest specificity and sensitivity among the recombinant proteins, with 92% and 88% sensitivity and 100% and 80% specificity, respectively. We further evaluated the COG-based and calpain-based ELISA on further "real-world" bovine serum samples from abattoir sites in Zanzibar, detecting S. bovis in 59.1% of tested animals.

Conclusions: Both COG and calpain are promising candidates for serological screening and later inclusion in portable diagnostic tests for S. bovis infection in cattle. Such future diagnostic assays will enable better point-of-detection monitoring, and once scalable, aid in the control of disease in cattle.

Background: Olfactory detection of host odors is fundamental to mosquito host-seeking behavior. Although the olfactory pathways of model species such as Anopheles gambiae have been well characterized, the molecular basis of human odor detection in Anopheles sinensis, an important malaria vector in Asia with opportunistic feeding habits, remains poorly understood. This study systematically investigates the functional role of the odorant-binding protein AsOBP21f in this process.

Methods: AsOBP21f was cloned and characterized using bioinformatics, quantitative reverse transcription PCR (RT-qPCR), and phylogenetic analyses. Recombinant AsOBP21f protein was expressed and purified for fluorescence competitive binding assays with 35 human odorants. Molecular docking was performed to elucidate ligand-binding interactions. Electroantennogram (EAG) recordings and behavioral assays were conducted to evaluate mosquito responses to high-affinity ligands. RNA interference (RNAi) knockdown was used to assess the functional role of AsOBP21f in host-seeking and blood-feeding behavior.

Results: AsOBP21f was predominantly expressed in olfactory tissues of mosquito females, including the antennae and proboscis. Its protein exhibited notable selectivity for hydrophobic odor molecules with C10-C15 carbon chains, and had strong binding affinities for methyl tridecanoate, dodecanal, decanal, and pentadecanoic acid. Behavioral experiments further demonstrated dose-dependent effects of these ligands; methyl tridecanoate showed significant attraction, while dodecanal exhibited clear repellency. RNAi-mediated silencing of AsOBP21f significantly reduced the antennal electrophysiological response of mosquito females to host odors and markedly decreased blood-feeding success.

Conclusions: These results highlight the role of AsOBP21f in host-seeking behavior through the detection of human odors, such as methyl tridecanoate, in An. sinensis, providing a potential target for malaria control.

Background: Drug-induced liver injury (DILI) remains a significant barrier to the safe and efficient use of antimalarial medicines. Many promising compounds fail in late-stage development or post-marketing owing to unforeseen toxicity, particularly DILI. Incorporating a predictive hepatotoxicity assessment is therefore critical to reduce clinical risk and development costs. The Medicines for Malaria Venture (MMV) Global Health Priority Box (GHPB) provides a library of compounds with demonstrated or potential antimalarial activity, yet their hepatotoxicity risk remains poorly defined. Our early work developed and validated a nanoscaffold-based three-dimensional (3D) liver spheroid platform. Here, we apply this validated model for the first time to assess the hepatotoxicity of clinically used and candidate antimalarial GHPB compounds.

Methods: Our validated nanoscaffold-based 3D liver spheroid platform was used to evaluate the hepatotoxicity of approved antimalarial drugs of known liver toxicity (quinine, primaquine, amodiaquine, sulfadoxine/pyrimethamine, and artemisinin) and six GHPB candidate compounds. Half maximal inhibitory concentration (IC₅₀)-derived data from the approved antimalarial drugs were used to generate a reference framework on the basis of established DILI classifications, which was then applied to categorize the GHPB candidate compounds relative to hepatotoxic risk.

Results: Our nanoscaffold-based 3D liver spheroid platform accurately reproduced the known DILI rankings of the approved antimalarials, confirming its predictive validity. Using these referenced IC₅₀-derived profiles, candidate compounds from the GHPB were classified into distinct hepatotoxicity categories, ranging from low: MMV1167451 (compound 01) and MMV020192 (compound 02), moderate: MMV1797658 (compound 03) and MMV1435700 (compound 04), to high: MMV006344 (compound 05) and MMV006931 (compound 06) risk, demonstrating the model's capacity to support early-stage animal-free antimalarial hepatotoxicity screening.

Conclusions: This study demonstrates the translational application of a validated nanoscaffold-based 3D human liver spheroid model for antimalarial drug in vitro hepatotoxicity assessment. By establishing a reference framework from clinically approved antimalarials and applying it to candidate compounds from the MMV GHPB, our platform enabled early classification of hepatotoxicity risk using a human-relevant, non-animal method. The findings support the integration of advanced 3D in vitro systems into antimalarial drug discovery pipelines to improve safety prediction, reduce reliance on animal testing, and accelerate the development of safer, more effective antimalarial therapies.

Background: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) protein profiling has emerged over the last decade as a method of choice for species identification of many medically important arthropods. However, the influence of intraspecific variability on the performance of this popular technique has seldom been tested. This study provides the first standardized comparison of different geographical populations of Phlebotomus perniciosus, a vector of Leishmania infantum and Toscana virus in the western Mediterranean, by MALDI-TOF MS protein profiling.

Methods: Phlebotomus perniciosus males were collected in five countries (Portugal, Spain, France, Italy, Croatia) that represent most of its distribution in Europe. All samples were trapped, stored and processed according to a highly standardized protocol to avoid effects other than geographical origin on their protein spectra acquired by MALDI-TOF MS protein profiling. The obtained protein spectra were compared with laboratory-reared specimens of Ph. perniciosus.

Results: Twenty-two analysed specimens from five geographical populations provided protein spectra that were highly similar, species-specific and clustering according to their quality. No grouping according to geographical origin was observed, and the protein spectra of field-collected specimens showed similar composition and complexity to spectra from Ph. perniciosus laboratory colony-reared in captivity for several decades.

Conclusions: Our findings demonstrate that in samples of a same sex, with the same collection method and storage time, MALDI-TOF MS protein profiling does not reflect the geographical origin of analysed specimens, confirming the value of this technique for high-fidelity and reproducible species identification of sand flies regardless of their geographical origin.

Background: The Three-River-Source Region of the Qinghai-Tibet Plateau is a hyperendemic focus for echinococcosis, with Echinococcus granulosus, E. multilocularis, and E. shiquicus circulating between definitive canid hosts (dogs and foxes) and intermediate hosts (livestock and rodents). However, the extent of environmental contamination by Echinococcus eggs remains understudied and poses significant risks to human and animal health.

Methods: From 2019 to 2021, we collected 631 canid fecal samples (296 from dogs and 335 from foxes) and 398 adjacent soil samples across endemic counties in the Three-River-Source Region of the Qinghai-Tibet Plateau. Multiplex real-time PCR was employed to detect Echinococcus species DNA in feces and soil samples.

Results: The overall Echinococcus prevalence in canid feces was 7.13% (45/631), with 3.01% in E. multilocularis (19/631), 2.06% in E. granulosus (13/631), and 2.06% in E. shiquicus (13/631). Foxes presented increased E. multilocularis (3.88%, 13/335) and E. shiquicus (2.69%, 9/335) infections, whereas dogs presented increased E. granulosus prevalence (2.70%, 8/296). Soil contamination with Echinococcus species was detected in 2.51% (10/398) of the samples. The primary contaminants were E. multilocularis and E. shiquicus (1.01% each, 4/398), whereas E. granulosus was less frequent (0.50%, 2/398). Moreover, the soil near fox feces was contaminated with both E. multilocularis and E. shiquicus, whereas the dog-associated soil was contaminated with all three species.

Conclusions: This study suggests widespread environmental deposition of Echinococcus eggs on the Qinghai-Tibet Plateau, driven by canid defecation. If these eggs remain viable, their persistence in soil would indicate a potential zoonotic transmission risk, highlighting the need for integrated control strategies targeting both domestic and wild canids.

Background: The ecdysone receptor (EcR) is a central regulator of mosquito physiology, best known for its role in vitellogenesis. However, its contribution to antiviral defense and dengue virus (DENV) replication in Aedes aegypti remains poorly understood.

Methods: RNA interference was used to silence Aedes aegypti EcR (AaEcR). Effects on DENV replication, immune gene expression, ovarian development, vitellogenin (Vg) synthesis, and target of rapamycin (TOR) pathway activity were assessed using molecular, cellular, and phenotypic analyses.

Results: Silencing AaEcR markedly suppressed DENV replication, viral protein expression, and virion production. These antiviral effects coincided with increased expression of antimicrobial peptides and activation of innate immune pathways, indicating that AaEcR facilitates viral replication by dampening host defenses. In addition, AaEcR proved essential for reproductive output. Knockdown impaired ovarian development, reduced follicle size and number, and lowered egg production by ~30%, although egg viability was unaffected. At the molecular level, AaEcR depletion strongly reduced Vg transcription and protein abundance, along with decreased phosphorylation of S6 kinase, suggesting that AaEcR promotes fecundity through both transcriptional activation and TOR-Vg signaling.

Conclusions: AaEcR functions as a dual regulator of mosquito biology, suppressing antiviral immunity while enhancing reproductive output. This tradeoff between immunity and fecundity highlights AaEcR as a promising molecular target for vector control. Disrupting EcR signaling could simultaneously reduce mosquito population size and limit arboviral transmission, offering a potential strategy for integrated management of mosquito-borne diseases.

The irradiation of female mosquitoes has been shown to influence blood feeding behavior, depending on dose, life stage at irradiation, and mating status. In sterile male releases during the sterile insect technique (SIT) programs, a small number of sterile females can be released accidentally, which may pose a manageable but nonnegligible risk regarding disease transmission. In this study, female Aedes aegypti, Aedes albopictus, and Anopheles arabiensis were exposed to gamma irradiation at male-sterilizing doses and the blood feeding rates and longevity of mated versus unmated sterile females were assessed. Although some differences were observed between the three species, generally females irradiated at the pupal stage showed a marked decrease in blood feeding rates, while irradiated adults showed slightly higher feeding rates, more pronounced in the second week (for Aedes spp.). Overall, blood feeding females irradiated as pupae that were mated died soonest, while in the non-blood fed groups, irradiation only correlated with negative effects on survival in Ae. aegypti, and mating status correlated with diminished survival in Ae. albopictus. Although the overall risk of a small number of mated, irradiated females participating in disease transmission in SIT programs is expected to be very low, rigorous sex separation and quality control will minimize this risk, and developing genetic sexing strains to ensure safety should be prioritized.

Background: Bacillus thuringiensis is widely employed for biological control. It can effectively suppress populations of various mosquito species, including Aedes aegypti. However, the precise mechanism underlying the action of Cry protein produced by Bacillus thuringiensis on Ae. aegypti remains elusive. On the basis of our previous research findings, five Aedes aminopeptidase N proteins (AeAPNs) were identified from the brush border membrane vesicles (BBMV) of Ae. aegypti that could bind to Cry4Ba or Cry11Aa. Further analysis confirmed that AeAPN1 and AeAPN2 are not functional receptors for these proteins. In this study, we investigated an additional aminopeptidase N (AeAPN3, AAEL012774) as a potential binding receptor for Cry proteins.

Methods: Comprehensive bioinformatics analysis involving whole-genome screening, genetic mapping, structural characterization, phylogenetic analysis, and spatiotemporal expression profiling were used to identify Ae. aegypti aminopeptidase N homologs. Ligand blotting and enzyme-linked immunosorbent assay (ELISA) were used to measure binding affinity to Cry4Ba. To elucidate its functional role as a potential receptor mediating Cry4Ba activity in Ae. aegypti midgut cells, AeAPN3 was knocked out with CRISPR/Cas9 technology.

Results: A total of 29 homologs of Ae. aegypti aminopeptidase N were identified in this study. Then, we expressed GST-APN3 fusion protein in E. coli and found that it had high-affinity binding to Cry4Ba protein (K_d = 20.53 nM). Mosquito larvae had approximately threefold higher resistance against Cry4Ba after AeAPN3 knockout, indicating its significant involvement as an active receptor mediating Cry4Ba activity.

Conclusions: Overall, this study provides a foundation for elucidating the specific larvicidal mechanisms of Bacillus thuringiensis (Bt) against mosquito populations.