The Anopheles maculipennis complex comprises closely related mosquito species of epidemiological importance across the Palearctic region. Anopheles messeae and Anopheles daciae are among its most widely distributed members but are morphologically indistinguishable, hindering species-specific vector surveillance and ecological and epidemiological studies. This limitation highlights the need for rapid, sensitive, specific, and high-throughput molecular methods for their identification. We report the development and validation of a TaqMan SNP genotyping assay targeting the Internal Transcribed Spacer 2 (ITS2) locus to discriminate between two closely related members of the Anopheles maculipennis complex, An. messeae and An. daciae. The assay targets a diagnostic single-nucleotide polymorphism (SNP) within the ITS2 region of ribosomal DNA. The optimized TaqMan SNP genotyping assay was applied to 293 field-collected mosquitoes. The assay showed high analytical sensitivity, with a detection limit of 0.1 ng of genomic DNA, and high specificity, with no false-positive results among the non-target species tested. The TaqMan assay showed 98
Myocardial ischemia–reperfusion (IR) injury is a critical complication in patients with myocardial infarction following the restoration of coronary blood flow. Echinococcus granulosus cyst fluid protein antigen B (EgAgB) is known to modulate host immune response. This study investigates whether the recombinant antigen B subunit 2 (rEgAgB8/2) can mitigate IR injury by suppressing inflammation and promoting myocardial tissue repair. An IR injury model was induced by ligating the left anterior descending (LAD) coronary artery for 1 h, followed by 24 h reperfusion. Recombinant EgAgB8/2 protein expressed in E. coli was administered intraperitoneally at the onset of reperfusion to evaluate its therapeutic potential. After 24 h of reperfusion, cardiac function and injury were assessed using echocardiography, TTC/Evans Blue staining, and histopathological analysis. Protein and mRNA levels of cytokines (TNF-α, IL-6, IL-10, and TGF-β) in sera and cardiac tissue were quantified using ELISA and RT-qPCR, respectively. In vitro studies, cytokine secretion in bone marrow-derived macrophages (BMDMs) culture supernatants was measured by ELISA. Macrophage polarization was evaluated via flow cytometry by identifying surface markers for M1 (CD86⁺) and M2 (CD206⁺) phenotypes. Expression levels of proteins in the cGAS/STING/p-TBK1/p-IRF3 signaling pathway in both myocardial tissues and BMDMs were detected by Western blotting. Treatment with rEgAgB8/2 significantly improved cardiac function and reduced myocardial injury markers (LDH, CK-MB) and infarct size in mice with IR injury. Compared to untreated IR controls, rEgAgB8/2 treatment significantly reduced the levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in both serum and cardiac tissue, and downregulated the M1 macrophage marker iNOS in cardiac tissue. Concurrently, it increased the expression of anti-inflammatory cytokines (IL-10 and TGF-β) in serum and cardiac tissue, along with upregulating the M2 macrophage marker Arg-1 in cardiac tissue. This reduction in the inflammatory response in IR-injured cardiac tissue was associated with enhanced polarization toward the M2 macrophage phenotype. In vitro studies further showed that rEgAgB8/2 promotes M2 macrophage polarization associated with reduced activation of the cGAS/STING/TBK1/IRF3 signaling pathway. rEgAgB8/2 promotes macrophage polarization toward M2 phenotype in a manner associated with suppression of the cGAS/STING/TBK1/IRF3 signaling pathway to attenuate myocardial IR injury and associated inflammation. It provides a novel therapeutic strategy for ischemia–reperfusion injury and related inflammatory diseases.
Lipid droplets (LDs) are dynamic organelles involved in lipid storage, energy metabolism, cellular signaling, and host–pathogen interactions. In trypanosomatids, the lipid droplet kinase (LDK) has been associated with LD biogenesis; however, its function in Trypanosoma cruzi, the causative agent of Chagas disease, remains unknown. To investigate the role of LDK, we generated LDK-null mutants in the T. cruzi Dm28c strain using CRISPR/Cas9-mediated gene disruption. Gene deletion was confirmed by PCR and quantitative real-time PCR. The effects of LDK ablation on parasite growth, lipid droplet accumulation, susceptibility to benznidazole and hydrogen peroxide, and infectivity in L929 fibroblasts were evaluated. Phenotypic characterization revealed that LDK is not essential for T. cruzi survival under the conditions evaluated, as LDK-null parasites remained viable and displayed growth kinetics comparable to those of the parental line. Likewise, LDK deletion did not affect benznidazole susceptibility. In contrast, knockout parasites exhibited significantly reduced LD accumulation after 96 h of growth, with reductions of 42.1–45.5
Coyotes (Canis latrans) are highly adaptable mesopredators whose increasing overlap with domestic dogs and humans may facilitate parasite transmission at the wildlife–domestic interface. However, parasite diversity and transmission dynamics between wild and domestic canids remain poorly characterized in Central America. This study investigated gastrointestinal helminths infecting coyotes and domestic dogs in Costa Rica using an integrative molecular, morphological and ecological approach. Fecal samples were collected from coyotes and domestic dogs across protected, fragmented, and urban landscapes in Costa Rica. Host species identity was confirmed using cytb RFLP and 12S metabarcoding, and individual coyotes were identified through SNP genotyping. Parasite communities were characterized using microscopy and deep amplicon sequencing of the nematode cytochrome oxidase 1 (cox1) gene. Phylogenetic and haplotype analyses were conducted for dominant parasites, and vertebrate 12S metabarcoding was used to evaluate coyote diet. Moreover, a reservoir capacity model was applied to estimate the potential contribution of coyotes to parasite transmission. Thirty-one individual coyotes and 149 domestic dogs were analyzed. Seven helminth groups were detected in metabarcoding, whereas microscopy identified four parasite species or groups. Ancylostoma caninum was the most prevalent nematode in both hosts, with 38.7
House flies are suspected mechanical transmitters of faecal-borne helminth diseases. To better understand their role in disease transmission, controlled experimental studies are required. This study investigates the short-term carriage of helminth eggs on the exoskeleton and inside the digestive tract of house flies (Musca domestica L.) following experimental exposure to faeces containing eggs of Ascaris suum and Taenia saginata. Forty-eight house flies, allocated to eight cages, were exposed to non-human primate faeces spiked with 10,000 A. suum and 5000 T. saginata eggs per gram of faeces under controlled environmental conditions (30 °C, 60
