The protein in question is NcDYRK2, a member of the dual-specificity tyrosine phosphorylation-regulated kinase family. DYRKs are ancient and highly conserved serine/threonine kinases found across eukaryotes, where they orchestrate processes as varied as cell cycle progression, cellular differentiation and signal transduction. While their functions in model organisms such as yeast, flies and mammals have been mapped in considerable detail, the roles of DYRK family members in apicomplexan parasites have remained largely unexplored. Given that neosporosis lacks effective chemotherapy, the researchers reasoned that identifying parasite kinases governing virulence and development could expose promising drug targets that would be absent, or at least very different, in the host animal.

To interrogate NcDYRK2’s function, the team turned to CRISPR-Cas9 gene editing. They generated a knockout strain lacking the dyrk2 gene, and in parallel constructed complemented parasites in which the gene was restored, allowing any observed defects to be attributed specifically to the deleted kinase rather than to off-target effects of editing. They also engineered parasites carrying an epitope-tagged version of the native protein, which enabled them to pinpoint where NcDYRK2 resides inside the cell by immunofluorescence microscopy. The answer was striking: the kinase localizes predominantly to the endoplasmic reticulum, the membrane network that in most eukaryotes serves as a major intracellular calcium store.

That ER localization proved to be a meaningful clue rather than a curiosity. When the researchers compared the knockout parasites with wild-type controls across a battery of standard fitness assays, a paradoxical picture emerged. Loss of NcDYRK2 significantly enhanced gliding motility, the distinctive substrate-crawling movement that apicomplexans use to traverse tissues and invade new cells, and it also boosted plaque formation, a composite readout of a parasite population’s ability to spread and destroy a host cell monolayer. Yet the more fundamental steps of the parasite’s life cycle inside the host cell were untouched: host cell invasion, intracellular replication and egress all proceeded normally in the knockout strain.

Because gliding motility in apicomplexans is tightly governed by intracellular calcium, the team next examined calcium dynamics directly using real-time fluorescence imaging. The Δdyrk2 parasites showed elevated basal concentrations of intracellular Ca²⁺, suggesting that in the absence of the kinase the parasite’s calcium homeostasis is chronically disturbed. Even more telling was the response to ionomycin, an ionophore that floods the cytosol with calcium: knockout parasites mounted a diminished Ca²⁺ response compared with wild type. Together these observations indicate that NcDYRK2 helps regulate both the resting calcium set point and the parasite’s capacity to mobilize calcium from internal stores, consistent with its position on the endoplasmic reticulum.

To understand how a single kinase could produce such broad phenotypic effects, the researchers performed RNA sequencing on knockout and wild-type parasites. The transcriptomic comparison revealed extensive gene expression alterations: 554 differentially expressed genes in total, of which 393 were downregulated and 161 upregulated. Principal component analysis cleanly separated the knockout group from the parental controls along the first principal component, which accounted for over 70 percent of the total variance, and biological replicates clustered tightly, with intra-group correlation coefficients above 0.986, underscoring the robustness of the dataset.

Among the downregulated genes were several with well-known roles in stage differentiation, a process of central importance to the parasite’s biology and transmission. Bradyzoite-associated genes including BPK1, CST1, MCP3, MCP4 and MAG2 were all transcriptionally reduced in the knockout strain, as was MIC17A, a gene associated with the merozoite stage. Bradyzoites are the slowly dividing, cyst-forming stage that persists chronically in infected animals, and the cyst wall proteins they produce are what allow the parasite to hide from the immune system and from drugs. The downregulation of this entire developmental program implicates NcDYRK2 as a regulator of stage differentiation, not merely a housekeeping enzyme.

The calcium signaling machinery itself also bore the fingerprint of NcDYRK2 loss. Calmodulin genes CAM1 and CAM3 were transcriptionally upregulated in the knockout parasites, a pattern that could reflect compensatory responses to the disrupted calcium balance, while PIPLCβ, encoding phosphoinositide phospholipase C beta, an enzyme that generates calcium-mobilizing second messengers, was downregulated. Quantitative reverse transcription PCR confirmed these transcriptional changes independently. The coordinated alteration of calmodulins and phospholipase signaling provides a mechanistic bridge between the kinase’s ER location, the observed calcium dysregulation and the hypermotile phenotype of the knockout parasites.

The most consequential result came from experiments in living animals. When mice were infected with the knockout parasites, virulence was markedly attenuated compared with wild-type infection, and quantitative PCR of brain tissue at 40 days post-infection revealed significantly reduced brain cyst burdens. This finding ties the in vitro molecular phenotypes directly to pathogenicity: a parasite that cannot properly regulate its calcium signaling and cannot fully express its bradyzoite developmental program is also a parasite that establishes fewer chronic infections in the brain. All animal work was conducted under the approval of the Institutional Animal Care and Use Committee of China Agricultural University, with measures taken to minimize animal suffering.

Taken together, the study establishes NcDYRK2 as a critical coordinator of calcium-dependent motility, stage-specific gene expression and virulence in N. caninum. What makes the kinase especially interesting from a drug discovery standpoint is its unusual profile of dispensability and necessity: the parasite can invade, replicate and exit host cells without it, yet it loses much of its disease-causing capacity in its absence. Inhibiting such a target would not need to kill the parasite outright in culture but could instead defang it in the host, blunting tissue migration and chronic cyst formation, the hallmarks of neosporosis. The work also adds to a growing appreciation that apicomplexan parasites have co-opted conserved eukaryotic kinase families for parasite-specific purposes, and that the endoplasmic reticulum, long studied as a calcium store, functions in these organisms as a signaling platform integrated with motility and differentiation. The research was supported by the National Natural Science Foundation of China, and the authors suggest that NcDYRK2 now merits further evaluation as a candidate target for future anti-neosporosis strategies, a prospect that could not come soon enough for an industry losing calves to this silent abortifacient.

Subject of Research: Role of the endoplasmic reticulum-associated kinase NcDYRK2 in regulating calcium dynamics, motility, development and virulence in Neospora caninum

Article Title: Endoplasmic reticulum-associated NcDYRK2 regulates Ca2⁺ dynamics, motility, and development gene expression in Neospora caninum

Article References: Yang, S., Wang, X., Yu, S., Fu, Y., Liu, Q., & Liu, J. (2026). Endoplasmic reticulum-associated NcDYRK2 regulates Ca2⁺ dynamics, motility, and development gene expression in Neospora caninum. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07681-x

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07681-x

Keywords: Neospora caninum, NcDYRK2, calcium signaling, endoplasmic reticulum, gliding motility, transcriptomics, virulence, bradyzoite differentiation, CRISPR-Cas9, apicomplexan parasites, kinases, neosporosis

The study, led by Taner Gürel of the Samsun Veterinary Control Institute together with Şinasi Umur of Ondokuz Mayıs University, set out to fill a gap in molecular epidemiological data for Turkish wild boar populations. Wild boars occupy a pivotal position in the sylvatic cycles of Sarcocystis, Toxoplasma gondii, and Neospora caninum, three cyst-forming parasites of the phylum Apicomplexa that infect both animals and, in some cases, humans. Because wild boar populations have expanded dramatically across Europe and Anatolia, and because their meat is increasingly consumed as game, understanding what parasites these animals carry has direct implications for food safety, veterinary medicine, and public health.

To detect the parasites, the team combined classical parasitological techniques with a battery of molecular assays. Muscle tissue samples were first subjected to artificial digestion, a method that liberates cysts from meat fibers, and to histopathological examination to look for the characteristic sarcocysts embedded in muscle. The researchers then deployed a multiplex polymerase chain reaction targeting the mitochondrial cytochrome c oxidase subunit I gene, known as cox1, alongside a conventional PCR targeting the 18S ribosomal RNA gene to identify and differentiate Sarcocystis species. For the other two parasites, they used species-specific PCRs: one amplifying the 529 base pair repetitive element of Toxoplasma gondii, a target prized for its sensitivity because it is repeated hundreds of times in the parasite genome, and another targeting the NC5 region of Neospora caninum.

The results were unambiguous for Sarcocystis. Sarcocystis miescheriana was detected in 92 percent of the sampled animals, making it by far the dominant species in the population. Notably, the zoonotic species Sarcocystis suihominis, which can infect humans who eat undercooked pork or wild boar meat, was not found in any of the samples. That absence is reassuring from a food safety standpoint, though the authors caution that continued surveillance is warranted. Phylogenetic analysis of the cox1 sequences obtained from the Turkish isolates placed them close to European strains of S. miescheriana, consistent with the picture of a parasite lineage circulating widely among wild suids across the continent. The 18S rRNA sequence generated in the study has been deposited in GenBank under accession number PV104359.

Co-infections emerged as a defining feature of the population. Fifty-six percent of the wild boars harbored at least two of the three parasite species, and 28 percent carried all three simultaneously. Such high rates of mixed infection reflect the overlapping transmission routes of these parasites: Sarcocystis miescheriana cycles between pigs and canids, with dogs and other carnivores shedding sporocysts that contaminate the environment and are ingested by intermediate hosts; Toxoplasma gondii is transmitted both through oocysts shed by cats and through tissue cysts in meat; and Neospora caninum, whose definitive hosts are canids, is a major cause of abortion in cattle. A wild boar foraging in a contaminated landscape can readily pick up all three pathogens over its lifetime.

To identify risk factors for infection, the researchers applied multivariable logistic regression adjusted for age and sex. Adult age emerged as a significant predictor of Toxoplasma gondii infection, with adult animals showing roughly 3.77 times the odds of infection compared with younger boars, a statistically significant association. A similar but non-significant trend was observed for Neospora caninum, with an adjusted odds ratio of 3.21. Sex was not a significant predictor for either parasite. Triple infections were numerically more common in older animals, a pattern consistent with cumulative environmental exposure over time, although this trend did not reach statistical significance. The age effect fits the biology of these parasites: animals that live longer encounter more contaminated soil, water, and prey, and once infected with tissue cyst-forming protozoa they typically remain infected for life.

The detection of Toxoplasma gondii DNA in half of the sampled boars carries particular public health weight. Toxoplasmosis is one of the most common zoonotic infections worldwide, and while healthy adults usually experience mild symptoms, the parasite can cause severe disease in immunocompromised individuals and in fetuses when a woman acquires a primary infection during pregnancy. Game meat is an increasingly recognized route of exposure, and cases of ocular toxoplasmosis following wild game consumption have been documented in the medical literature. The 529 base pair repetitive element sequences generated in this study have been deposited in GenBank under accession numbers PV133829 through PV133831, adding to the genetic record of the parasite in the region.

Neospora caninum, detected in 38 percent of the animals, is less often a direct human health concern but is economically significant in livestock. The parasite is a leading cause of abortion and neonatal mortality in cattle worldwide, and wildlife reservoirs are thought to contribute to its persistence and spread. Previous surveys in other countries have found antibodies against N. caninum in feral swine and non-carnivorous wildlife, and the Turkish study adds molecular confirmation of the parasite’s presence in wild boar tissues from the region. The finding reinforces the idea that wild boars, which share habitats with livestock and are scavenged upon or hunted by canids, participate in the epidemiology of a parasite that costs the dairy industry substantially each year.

The Turkish results align with a growing body of European literature. Molecular studies in Italy, Greece, Latvia, Iran, and China have repeatedly identified Sarcocystis miescheriana as the predominant Sarcocystis species in wild boars, and surveys in Switzerland have documented both Sarcocystis and Toxoplasma in muscles of wild boars destined for human consumption. What distinguishes the new study is its combined assessment of all three parasites in the same animals, its use of both multiplex and conventional PCR platforms, and its formal statistical modeling of age and sex as risk factors. By quantifying co-infection patterns rather than treating each parasite in isolation, the study offers a more realistic picture of the parasite ecology within a single wild population.

The authors conclude that their results update the molecular epidemiology of tissue cyst-forming apicomplexans in Turkish wild boars and reinforce the importance of sustained One Health surveillance at the wildlife-livestock-human interface. As wild boar numbers continue to grow and the popularity of game meat rises, the study serves as a reminder that hunters, processors, and consumers all have a stake in safe handling and thorough cooking of wild game. It also highlights the value of molecular tools, which can distinguish morphologically similar parasite species and reveal co-infections that microscopy alone would miss. For now, the wild boars of Türkiye’s Black Sea coast stand as a vivid example of how much invisible parasite traffic moves through a seemingly healthy wildlife population.

Subject of Research: Molecular prevalence, co-infections, and genetic characterization of Sarcocystis, Toxoplasma, and Neospora parasites in wild boars from Türkiye

Article Title: Molecular Occurrence, Co-infections and Genetic Characterization of Sarcocystis miescheriana, Toxoplasma gondii and Neospora caninum in Wild Boars (Sus scrofa) from Türkiye

Article References: Molecular Occurrence, Co-infections and Genetic Characterization of Sarcocystis miescheriana, Toxoplasma gondii and Neospora caninum in Wild Boars (Sus scrofa) from Türkiye. (n.d.). https://doi.org/10.1007/s11686-026-01397-9

Image Credits: AI Generated

DOI: 10.1007/s11686-026-01397-9

Keywords: Sarcocystis miescheriana, Toxoplasma gondii, Neospora caninum, wild boar, Türkiye, co-infection, molecular epidemiology, PCR, game meat, food safety, One Health, parasitology