Parasites & Vectors最新文献

Background: Golden apple snails (Gastropoda: Ampullariidae: Pomacea) were introduced into China in the 1980s for aquaculture and have since become widespread agricultural pests across East Asia. In addition to their invasive impact, they are a key intermediate host of the rat lungworm Angiostrongylus cantonensis (Secernentea: Angiostrongylidae) in China, the causative agent of eosinophilic meningitis in humans.

Methods: We conducted a malacological survey of 55 freshwater sites across Shanghai and neighboring East China provinces to assess Pomacea distribution, genetic diversity, and A. cantonensis infection status. A total of 700 Pomacea snails were examined for A. cantonensis using traditional lung microscopy and molecular xenomonitoring (PCR and LAMP). Mitochondrial COI barcoding was performed on 200 individuals from 20 high-density sites to assess species composition and genetic diversity.

Results: Pomacea snails were found at 81.8% (45/55) of sites surveyed. No A. cantonensis infections were detected by microscopy or molecular assays. Genetic analyzes revealed three Pomacea species (P. canaliculata, P. maculata, and P. occulta) and nine distinct COI haplotypes. Pomacea canaliculata was the most common and genetically diverse species, with four unique haplotypes (H5-H8) occurring only in Shanghai, indicative of recent introductions. Overall, populations showed moderate haplotype diversity (Hd = 0.73) and population structure (F_ST = 0.24).

Conclusions: Although no A. cantonensis infections were detected in the snails examined in this survey, these negative findings do not preclude the possibility of low-prevalence or newly emerging infections. The wide distribution and high genetic diversity of Pomacea populations across Shanghai and East China highlight that suitable hosts are already well-established, emphasizing the ongoing risk of parasite introduction and spread into currently nonendemic regions. Continued molecular surveillance, public awareness, and strengthened biosecurity measures remain essential to effectively manage invasive snail populations and mitigate future public health threats.

Background: The parasite Leishmania is the causative agent of leishmaniasis and relies on a single "mitochondrion" as its "powerhouse". It also has a compromised antioxidant defense system. Consequently, potential therapeutic strategies include triggering mitochondrial dysfunction along with subversion of host metabolic bioenergetics; however, such information remains poorly defined. The focus of this study was to delineate the impact of antileishmanials amphotericin B (Ampho B) and miltefosine (hexadecylphosphocholine [HePC]) on the metabolic bioenergetics of Leishmania parasites vis-à-vis mammalian macrophages.

Methods: In promastigotes, the redox status was evaluated by flow cytometry; levels of ATP were measured by chemiluminescence; and oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and substrate utilization were assessed by XFp Analyzer. In Leishmania donovani (L. donovani)-infected macrophages, expression of metabolic bioenergetics related regulatory molecules was assessed by droplet digital polymerase chain reaction (ddPCR) and immunoblotting.

Results: In promastigotes, at their respective 50% and 90% inhibitory concentrations (IC₅₀ and IC₉₀), Ampho B and HePC disrupted redox homeostasis, enhanced generation of mitochondrial superoxide, depleted OCR and ATP, and caused a greater degree of mitochondrial inhibition with HePC than with Ampho B. The L. donovani promastigotes sourced acetyl CoA primarily from the fatty acid oxidation pathway for mitochondrial tricarboxylic acid cycle, and inhibition of the mitochondrial fatty acid oxidation was higher with Ampho B. In L. donovani-infected macrophages, there was significantly increased expression of the Ampk axis (Ampk-Lkb1-Sirt1), mitochondrial biogenesis marker Pgc1α, and markers of oxidative phosphorylation (Cox IV, Atp synthase). The rate-limiting enzymes of glycolysis, namely  HkII, Pfk, Pkm2, and glucose transporter (Glut1), were enhanced, but expression of Mtor was decreased. All markers of the Ampk axis and oxidative phosphorylation were significantly curtailed by both antileishmanials in favor of parasite clearance, whereas glycolytic markers remained unchanged; overall, the dampening of metabolic bioenergetics by HePC was greater than that caused by Ampho B.

Conclusions: Targeting the "mitochondrion" and metabolic reprogramming are effective leishmanicidal strategies adopted by HePC and Ampho B, with the degree of inhibition by HePC exceeding that of Ampho B. Accordingly, screening for compounds capable of mediating metabolic reprogramming could augment the limited armamentarium of antileishmanials.

Background: Leishmania parasites are transmitted through the bite of infected female sand flies. The sand fly inoculum contains both the parasite and the salivary proteins, which can modulate the immune system's function. Some of these salivary proteins have the potential to be used as a vaccine candidate. Since there have been fewer studies investigating the salivary proteins of Phlebotomus (Ph.) sergenti, this prompted us to select among the three protein members of Ph. sergenti apyrase family (PsSP40, PsSP41, and PsSP42) and measure its effectiveness as vaccine candidate against Leishmania (L.) tropica.

Methods: To select among the three family members as the candidate for immunization, different parameters including the physicochemical characters, three-dimensional structure, virtual immune stimulatory potential, and human leukocyte antigen (HLA) class II-binding epitope content were considered. To investigate the effect of immunization with the selected antigen through immunoinformatics analysis (PsSP42) against L. tropica infection, we immunized BALB/c mice with two distinct recombinant plasmids (conventional VR1020 and new-generation NTC9385R) two times at 3-week intervals followed by immediate electroporation. Eight weeks post-challenge, the parasite load in draining lymph nodes was measured by quantitative real-time polymerase chain reaction (PCR). The interferon (IFN)-γ and interleukin (IL)-4 cytokines before (against recombinant Leishmania tarentolae expressing PsSP42) and after (against parasite frozen/thawed antigens) L. tropica infection (2 × 10⁷ parasite per footpad plus Ph. sergenti salivary gland homogenate (SGH)) were measured by enzyme-linked immunosorbent assay (ELISA).

Results: On the basis of immunoinformatics analysis of three apyrase salivary proteins from Ph. sergenti, PsSP42 demonstrated superior HLA class II-binding peptides compared with the other two proteins (PsSP40 and PsSP41) and was selected for immunization studies. Our findings indicated that NTC-PsSP42 and not VR1020-PsSP42 plasmid immunization relatively reduced the parasite load in the draining lymph nodes. This was assigned to a significant higher IFN-γ to IL-4 ratio induced by NTC-PsSP42 immunization in comparison with pertinent controls.

Conclusions: In our study, although the expected protective response was not achieved by any of the recombinant plasmids, the NTC-PsSP42 platform induced a weak Th1-polarized immune response, which partially influenced the parasite load. Since the new generation of plasmids are worth evaluating owing to the lack of antibiotic resistance genes on the backbone, we recommend further assessment of NTC-PsSP42 potential adjuvnated with immunostimulatory sequences such as as CpG motifs or even in heterologous prime-boost regimens.

Canine vector-borne diseases pose a continuous threat to dogs worldwide. Various vector-borne agents, including bacteria, protozoa, helminths, and, to a lesser extent, viruses, cause these diseases. These pathogens are transmitted primarily by arthropod vectors, including ticks, mosquitoes, phlebotomine sand flies, fleas, lice, tabanid flies and triatomine bugs. The diagnosis and treatment of these diseases can be challenging, and co-infections may further complicate management. Besides being potentially fatal to dogs, some of these pathogens are zoonotic. Many biotic and abiotic factors (e.g. climate change, increased mobility of people and animals, urban expansion and land-use changes) are affecting the distribution and activity of vectors and the pathogens they transmit worldwide. The evolving relationship between dogs and humans may also increase owners' exposure to ectoparasites and the diseases they transmit. In this article, we review key aspects of canine vector-borne diseases and discuss the importance of year-round prevention, as recommended by established international guidelines from leading veterinary parasitology organizations.

Background: Plasmodium falciparum merozoite invasion of erythrocytes is an essential step in the asexual blood-stage cycle and a major target for antimalarial intervention. Rhoptry neck proteins play key roles in the formation and function of the tight junction, yet many remain poorly characterized. RALP1, a conserved rhoptry neck-associated leucine zipper-like protein, has been proposed to participate in erythrocyte binding and invasion. Conventional gene disruption attempts have been unsuccessful, suggesting that RALP1 may be essential for parasite survival. Nevertheless, its precise role and broader molecular impact during intraerythrocytic development remain to be fully elucidated.

Methods: We generated a 3 × HA-tagged conditional knockdown line (ralp1-ha-glmS) using CRISPR-Cas9-mediated homologous recombination. RALP1 abundance and subcellular localization were evaluated by Western blotting and immunofluorescence assays. Effects on parasite growth, schizont maturation, merozoite invasion, and merozoite numbers were assessed using tightly synchronized cultures and established invasion and cytological assays. Transcriptomic changes following GlcN-induced RALP1 knockdown were analyzed by RNA-seq at early ring and schizont stages. Sequence-based structural and epitope features were examined using IUPred2A, ANCHOR2, AlphaFold3, NetMHCpan, and NetMHCIIpan.

Results: Precise integration of the ha-glmS cassette enabled GlcN-inducible reduction of RALP1 protein levels, most prominently in schizonts. RALP1 knockdown reduced parasite proliferation, impaired schizont maturation, decreased merozoite numbers, and lowered erythrocyte invasion efficiency. RNA-seq showed limited effects in early rings but widespread downregulation of invasion- and host-parasite interaction-related genes in schizonts after correction for glucosamine-responsive transcripts, with GO enrichment highlighting processes related to host cell interaction, biological adhesion, and membrane-associated components. Sequence-based analyses indicated that RALP1 contains extensive intrinsically disordered regions with multiple predicted interaction motifs, while predicted B- and T-cell epitope hotspots concentrated within the C-terminal RBC-binding domain. AlphaFold3 modeling yielded low global confidence (pTM = 0.23), consistent with a primarily disordered architecture.

Conclusions: RALP1 is required for normal schizont maturation and efficient erythrocyte invasion in P. falciparum. Its partial knockdown perturbs transcription of key invasion ligands and apical components, indicating a broader role in preparing merozoites for host-cell entry. The extensive disorder, epitope-rich C-terminal region, and essential function of RALP1 highlight its potential as a candidate for therapeutic or vaccine targeting.

Background: Sri Lanka is experiencing a re-emergence of brugian filariasis 4 decades after its elimination in 1969. A comprehensive understanding of the mosquito species that can facilitate the development of the brugian parasite is essential for implementing targeted surveillance and control measures. This study evaluated the vector potentiality of field-caught mosquitoes for brugian parasites across endemic districts within the filarial transmission belt in Sri Lanka.

Methods: Mosquito surveillance was conducted at six sites across five districts with the highest reported brugian cases during 2021-2022. Mosquitoes were collected at the site of the most recently reported human brugian case in each district using dog-baited, window and gravid traps to maximize species diversity and abundance in the sample. Mosquitoes were identified morphologically, and randomly selected mosquitoes were molecularly confirmed via a PCR targeting the COΙ region. Vector potentiality was evaluated by observing nematode parasites upon dissection, molecular confirmation via PCR and sequencing the Brugia sp.-specific HhaΙ region. Mosquitoes harboring the infective L3 stage brugian parasites were tested for the presence of human DNA to investigate their involvement in human brugian filariasis transmission. Statistical analyses were performed using generalized linear mixed models.

Results: A total of 766 mosquitoes of 15 species were dissected to obtain L3 larvae of the brugian parasite. Of these, 10.05% (n = 77) from nine species across four genera were identified to support the development of Brugia spp. to the infective L3 larval stage within the head and thoraces of field-caught mosquitoes: Mansonia annulifera, Ma. indiana, Ma. uniformis, Culex lophoceraomyia, Cx. tritaeniorhynchus, Cx. quinquefasciatus, Cx. vishnui, Armigeres subalbatus and Coquillettidia crassipes. Notably, Ma. indiana, which has not previously been identified as a potential vector for brugian filariasis in Sri Lanka, showed the highest weighted infectivity at the S₁ site. Site-based risk assessment identified the S₁ site as having the highest risk of brugian filariasis followed by S₆.

Conclusions: Many mosquito genera supporting the development of Brugia spp. to the infective L3 larval stage in field-caught mosquitoes were identified expanding beyond the previously known Mansonia vectors. The diversity of potentially infective species indicates complex transmission dynamics requiring integrated surveillance approaches.

Background: While Cystoisospora suis is well established as a primary pathogen in suckling piglets, it can also infect weaned piglets. In this context, we investigated its co-infection with enterotoxigenic Escherichia coli (ETEC), a major cause of post-weaning diarrhea.

Methods: Weaned piglets were randomly divided into four groups: a negative control group (NC), an ETEC single-infection group (EC), a C. suis single-infection group (CS), and a co-infection group (EC-CS). Following infection, clinical symptoms were recorded, and samples were collected to evaluate intestinal histopathological damage, expression of tight junction protein genes, inflammatory cytokine levels, and gut microbiota changes.

Results: Compared to single-infection groups, piglets in the co-infection group exhibited more severe diarrhea, growth retardation, and intestinal damage, characterized by near-total loss of villus and crypt structures. Co-infection significantly impaired intestinal barrier function, as evidenced by a marked downregulation of claudin-1 messenger RNA (mRNA) expression compared to both single-infection groups, and triggered more intense local and systemic inflammatory responses. 16S ribosomal RNA (rRNA) sequencing revealed that co-infection exacerbated gut microbiota dysbiosis and promoted the proliferation of pathogenic bacteria.

Conclusions: Co-infection with C. suis and ETEC exerts a synergistic pathogenic effect in weaned piglets. The mechanism involves a vicious cycle of intestinal barrier disruption, microbiota dysbiosis, and amplified inflammatory responses. These findings provide a novel theoretical basis for the clinical prevention and control of complex intestinal co-infections.

Background: Aedes aegypti mosquitoes infected with the endosymbiotic bacterium Wolbachia pipientis have been released as a sustainable strategy to mitigate arbovirus transmission. Among the strains successfully deployed, wMel and wAlbB have shown promising blocking effects against dengue virus (DENV). However, the strength of viral inhibition depends on Wolbachia density within mosquito tissues, the genetic backgrounds of both host and virus, and the viral dose. In this study, we aimed to investigate the vector competence for DENV-1 of Ae. aegypti with Brazilian genetic background infected with wMel or wAlbB.

Methods: A total of 493 wild and wMel- and wAlbB-infected mosquitoes were orally challenged with low (5 × 10⁴ FFU/mL) and high (5 × 10⁵) titers of DENV-1. Relative Wolbachia density was measured by quantitative polymerase chain reaction (qPCR), and viral infection in mosquito bodies and saliva was assessed by qPCR with reverse transcription (RT-qPCR). Transmission potential was tested through saliva microinjection into susceptible mosquitoes. The infection prevalence and viral loads in mosquito bodies were analyzed at 7, 14, and 21 days post infection (dpi).

Results: Both Ae. aegypti groups infected with wMel and wAlbB had reduced (albeit distinct) DENV-1 infection and transmission relative to wild type mosquitoes. wMel-infected mosquitoes exhibited less abundant bacteria in their bodies but a greater degree of DENV-1 inhibition compared with those carrying wAlbB, indicating that DENV-1 blocking is strain specific rather than Wolbachia density-driven. Moreover, we observed that Wolbachia had a protective effect on mosquitoes by decreasing the DENV-1 loads in their bodies, but with a constant presence of virus. Viral transmission rates in the saliva were similar among wild and wMel- and wAlbB-infected mosquitoes at 7 and 14 dpi but lower in wMel and wAlbB mosquitoes at 21 dpi.

Conclusions: The similar DENV-1 loads in mosquito bodies over time (7, 14, and 21 dpi) infected with either the wMel or wAlbB strain, regardless of the viral titer of the infectious blood meal, suggest that Wolbachia may have a maximum pathogen-blocking capacity beyond which additional virus suppression cannot be achieved. The viral suppression only after 21 dpi in the saliva raises concerns and warrants further investigation, as females may transmit before Wolbachia blockage becomes effective. While wAlbB may exhibit comparable DENV-1 blocking to wMel, its enhanced thermal tolerance makes it epidemiologically relevant in tropical regions. Continuous monitoring of Wolbachia dynamics and DENV genomic variation in the field remains essential to evaluate long-term effectiveness and detect potential adaptive viral responses.

Background: The start of eukaryotic translation requires recruitment of messenger RNA (mRNA) through the action of the eukaryotic initiation factor 4F (eIF4F) complex. eIF4F is formed by joining of the eIF4G and eIF4E subunits and generally also requires the eIF4A helicase. In Leishmania infantum, five eIF4Gs form multiple eIF4F-like complexes, with those based on the related EIF4G3 and EIF4G4 being active during translation, but with likely nonredundant roles that need to be better defined.

Methods: To further investigate the roles of EIF4G3 and EIF4G4 in Leishmania infantum, we generated transgenic cell lines expressing each protein tagged with a C-terminal hemagglutinin (HA) epitope. Expression analyses were then carried out during different phases of promastigote growth, followed by gene knockout and complementation assays investigating the essentiality of the targeted eIF4Gs. The HA-tagged proteins were then used as baits in a large-scale investigation of potential protein partners, from different growth phases: early exponential, late exponential, and stationary.

Results: EIF4G3 and EIF4G4 were expressed as multiple isoforms during promastigote growth, with EIF4G4 isoforms changing according to the growth phase. The two HA-tagged proteins were capable of replacing the corresponding native proteins after deletion of the endogenous genes. EIF4G3-HA and EIF4G4-HA were always found with their known eIF4E partners, respectively EIF4E4 and EIF4E3. EIF4G3-HA also more consistently coprecipitated with poly(A)-binding protein 1 (PABP1), RNA-binding protein 23 (RBP23), and EIF4AI, with EIF4G4-HA having greater association with PABP3 and the HEL67 helicase. A variable number of translation factors and ribosomal proteins were found with both baits, reflecting roles in translation. Our extensive analyses, investigating also proteins with possible moonlighting roles and uncharacterized polypeptides, not only revealed new proteins bound to both baits but also identified new specific partners for EIF4G3, and possibly EIF4G4, some of those being restricted to selected growth phases.

Conclusions: Overall, new and more defined binding partners were observed for EIF4G3, with EIF4G4 having an increased coprecipitation with other translation initiation factors. Newly identified partners, for both eIF4Gs, might facilitate specific mRNA recognition or function regulating translation during growth. Further studies on some of those might reveal unique and conserved aspects of the Leishmania translation and might help define targets for novel translation inhibitors.