Enteric protozoan infections—cryptosporidiosis, giardiasis, microsporidiosis, and coccidiosis—constitute a significant global burden on human and animal health. Yet comprehensive parasitological surveys of wildlife in the eastern Tibetan Plateau, where anthropogenic, livestock, and wildlife activities extensively intersect, remain deficient. We collected 359 fecal specimens from wild carnivores across 98 random quadrats and employed molecular techniques for host identification and protozoan screening. Seven carnivore species were identified, with Tibetan fox (Vulpes ferrilata) predominating. Intestinal protozoa were exclusively recovered from Vulpes species, yielding a prevalence of 19.37% (68/351; 95% CI, 15.24%–23.51%). Eimeria falciformis (7.41%, 26/351) and Eimeria banffensis (7.12%, 25/351) were the most frequently detected, followed by Enterocytozoon bieneusi (3.70%, 13/351). Cryptosporidium was confirmed in one V. ferrilata, and Giardia intestinalis in 2 samples (V. ferrilata and Vulpes vulpes). This investigation furnishes the first molecular evidence that wild carnivores, especially V. ferrilata, harbor zoonotic protozoa including G. intestinalis assemblage AII and E. bieneusi genotypes PigEBITS5 and a D-like variant. The concurrent detection of Cryptosporidium yak genotype and E. bieneusi subgroup 1i in foxes, pikas, yaks, and sheep implies potential interspecies transmission at the wildlife–livestock interface. These findings fill knowledge gaps in regional parasite diversity and emphasize the need for enhanced surveillance of wildlife-borne protozoa under a One Health framework, thereby providing scientific evidence for zoonotic risk assessment and public health planning.

The study of Plasmodium falciparum intraerythrocytic developmental cycle relies on synchronized parasite cultures. This study aimed to quantitatively evaluate 4 common synchronization methods for the culture of P. falciparum 3D7, incorporating a novel nanomaterial-based tool to
objective
ly assess late-stage parasite enrichment. The 4 methods including 5% D-sorbitol, isosmotic Percoll-Sorbitol (60% PS), discontinuous Percoll-Sorbitol gradients (70%–40% PS), and the Plasmion method. Synchronization efficiency was assessed through microscopic examination and flow cytometry. In addition to SYBR Green I as the stain for flow cytometry, we innovatively employed 20 nm polystyrene fluorescent microspheres (PFMs20), which bind to parasites following the production of new permeability pathways, to provide an
objective
validating the enrichment of mature stages. The binding rate was defined as the ratio of PFMs20 positivity to SYBR Green I positivity. Microscopic examination of Giemsa-stained blood smears served as the gold standard for assessing the stage-specific profiles achieved by each synchronization method. The 4 methods yielded distinct efficiency profiles: 5% D-sorbitol achieved 88.37% ring-stage purity; 60% PS demonstrated high late-stage enrichment with PFMs20 binding rates reaching 87.47%; 70%–40% PS uniquely enabled visible hemozoin-rich layer separation and showed 88.12% binding efficiency in its schizont-enriched fraction; while Plasmion yielded a binding rate of 73.37% in its late-stage fraction. Our results formulate a clear decision matrix for synchronization protocol selection. The sorbitol method is ideal for basic ring-stage studies, Percoll-based gradients support rigorous stage-specific investigations, and the Plasmion method is well-suited for resource-limited settings. This comparative analysis provides preliminary guidelines for synchronization method selection that require validation in diverse laboratory and field contexts.