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More than 60 zoonoses are linked to small mammals, including some of the most devastating pathogens in human history. Millions of museum-archived tissues are available to understand natural history of those pathogens. Our goal was to maximize the value of museum collections for pathogen-based research by using targeted sequence capture. We generated a probe panel that includes 39,916 80-bp RNA probes targeting 32 pathogen groups, including bacteria, helminths, fungi, and protozoans. Laboratory-generated, mock-control samples showed that we are capable of enriching targeted loci from pathogen DNA 2,882‒6,746-fold. We identified bacterial species in museum-archived samples, including Bartonella, a known human zoonosis. These results showed that probe-based enrichment of pathogens is a highly customizable and efficient method for identifying pathogens from museum-archived tissues.

Toxoplasmosis is a disease of worldwide distribution, causing high morbidity and mortality in humans, as well as heavily impacting animal health and the economy. Toxoplasma gondii, the causative agent, is an intracellular parasite with a complex life cycle whose completion entails asexual, pre-sexual, and sexual stage conversions. Pre-sexual and sexual differentiation take place only within the intestinal epithelium of felines. Recently, several transcriptional factors and epigenetic components crucial to trigger parasite stage transitions within the cat have been identified, allowing, through precise genetic manipulation, obtaining pre-sexual stages known as merozoites in vitro. Through conditional depletion of two pre-sexual stage-specific gene silencing transcription factors, AP2XII-1 and AP2XII-2, we have characterized the interplay between cell division and the sequence of events leading up to differentiation of tachyzoites into merozoites. We explored genome duplication, assembly of daughter cells, karyokinesis, and cytokinesis, characterizing the differential cell division modes and kinetics undergone by critical structures along the differentiation axis. Building onto the pre-existing body of knowledge, primarily describing the underpinnings of these forms of division by transmission electron microscopy, our work contributes previously unexplored temporal and spatial resolution to the transitions between endodyogeny and endopolygeny, providing a conceptual framework for understanding and exploring T. gondii’s route of sexual differentiation.IMPORTANCESexual development in Toxoplasma gondii is essential for transmission, but remains poorly understood, largely because pre-sexual stages are restricted to the feline intestine and have only recently become experimentally accessible. Here, we leverage an in vitro differentiation system to resolve how parasites transition toward merozoite formation at the cellular level. By combining expansion microscopy, stage-specific markers, and quantitative analyses, we define the temporal sequence of nuclear division and daughter cell assembly during merogony, addressing longstanding ambiguity regarding division modes in these stages. Our findings reveal that parasites can adopt alternative division strategies emerging from a polyploid intermediate, highlighting an unexpected degree of flexibility in how cell division is executed during differentiation. Beyond refining this developmental framework, this work establishes a foundation for future mechanistic studies of pre-sexual biology and provides broader insight into the diversity of eukaryotic cell division strategies.

The lytic cycle of Toxoplasma gondii is critical for parasite dissemination and disease progression in the host. Calcium signaling plays a crucial role in driving these processes; however, the molecules that control calcium storage and release remain poorly understood. The endoplasmic reticulum, likely the largest calcium reservoir in T. gondii , has been understudied in the context of calcium signaling. Here, we uncover a direct link between ER redox regulation and calcium homeostasis, showing that ER redox activity can influence calcium signaling events that govern microneme protein maturation and secretion, parasite invasion, and replication. Our findings indicate that redox-dependent calcium regulation in the ER contributes to control of the parasite lytic cycle and reveals a previously unrecognized mechanism that may influence parasite virulence.

RNS are necessary for cell-autonomous immunity to T. gondii infection; however, the molecular mechanisms by which RNS regulate parasite control remain poorly understood. Our findings support a model in which post-translational modification of ROP5 by RNS is a conserved mechanism of inhibiting the functions of divergent ROP5 paralogs. These data provide a specific example of how host RNS are used to counter T. gondii immune evasion effectors that can be applied to understand how nitrosylation regulates the function of other parasite effectors and the role of RNS in the control of other intracellular pathogens.

Toxoplasma gondii is an obligate parasite whose infection can be detrimental when combined with pregnancy or immunodeficiency. Studies on T. gondii virulence have revealed various secretory proteins that inhibit the host interferon-gamma (IFN-γ) immune response. However, much of the broader virulence landscape remains unclear. To explore the unknown molecular pathways of T. gondii virulence in mice, we searched for immunosuppressive functions in genes encoding non-secretory proteins, associated with fundamental cellular processes of the virulent type I strain. Here, we found that TgJosephin, a highly conserved deubiquitinase, was important for virulence in wild-type mice but not mice lacking the IFN-γ receptor (IFNγR). In addition, TgJosephin expression was dependent on TgRad23, and loss of TgJosephin led to increased ubiquitination of a microtubule protein SPM1. Our results suggest a novel anti-IFN-γ pathway of T. gondii mediated by TgJosephin and SPM1 deubiquitination.

Intracellular bacteria and protists rely on the host cell to supply many metabolites, but the mechanisms through which pathogens manipulate host metabolism to their benefit are not understood. Here, we demonstrate that when the obligate intracellular parasite secretes its rhoptry organelle contents into the host cytoplasm before invasion-a process called \"kiss and spit\"-host cell metabolite abundance is altered in nucleotide synthesis, the pentose phosphate pathway, glycolysis, and amino acid synthesis. U-13C6-labeling metabolomics confirmed that kiss and spit increased the flow of carbon through the pentose phosphate pathway and nucleotide synthesis. An increase in 2,3-bisphosphoglycerate abundance led us to investigate the activation of host cytosolic nucleosidase II (cN-II) to provide purines for the parasite. We found that manipulates the host cN-II enzyme to dephosphorylate GMP and IMP that it needs for replication. Furthermore, we found that the approved anti-cancer drug fludarabine, which inhibits cN-II, also inhibits replication. These results reveal host cell manipulation and highlight potential therapies for toxoplasmosis.IMPORTANCEA fundamental challenge in parasitology is understanding how intracellular parasites rapidly reprogram host metabolism to support replication. This study reveals that initiates profound metabolic reprogramming through a \"kiss-and-spit\" mechanism, secreting effector molecules without invasion. We demonstrate that specifically hijacks host cytosolic 5'-nucleotidase II (cN-II) by elevating 2,3-bisphosphoglycerate levels, which allosterically activates this enzyme to generate purines essential for parasite survival. Genetic deletion of host cN-II significantly impairs parasite replication, establishing cN-II as a critical host dependency factor. These findings have important implications for antiparasitic drug development while advancing our understanding of purine metabolism in apicomplexan parasites. More broadly, elucidating the molecular mechanism linking parasite effector secretion to specific host enzyme activation provides a framework for understanding metabolic manipulation across other intracellular pathogens.

Onchocerca lupi , a zoonotic parasite, causes ocular onchocerciasis in both domestic and wild carnivores, as well as humans. Despite recent scientific advances, gaps remain in both the biology and genetic structure of this parasite. To date, two genotypes have been described (genotype 1 distributed in Europe, Asia, and the United States, and genotype 2 circulating in the Iberian Peninsula) based on mitochondrial gene analysis. This study provided three distinct genomes (nuclear, mitochondrial, and Wolbachia endosymbiont) of O. lupi isolated from a dog living in Portugal. Overall, the data presented here corroborate the divergence between the two genotypes and provide new insights into the identification of genes that could serve as novel therapeutic targets for this filarial disease.

To provide data that can be used to inform treatment and prevention strategies for zoonotic pathogens in animal and human populations, we assessed the occurrence of zoonotic pathogens and their vectors on 2,381 client-owned dogs and cats living in metropolitan areas of 8 countries in eastern and Southeast Asia during 2017-2018. Overall exposure to ectoparasites was 42.4% in dogs and 31.3% in cats. Our data cover a wide geographic distribution of several pathogens, including Leishmania infantum and zoonotic species of filariae, and of animals infested with arthropods known to be vectors of zoonotic pathogens. Because dogs and cats share a common environment with humans, they are likely to be key reservoirs of pathogens that infect persons in the same environment. These results will help epidemiologists and policy makers provide tailored recommendations for future surveillance and prevention strategies.

The role of invasive alien species in the transmission dynamics of zoonotic pathogens is often overlooked, despite the rapid escalation in biological invasions globally. Here we synthesise available information on the influence of invasive alien species on zoonotic pathogen dynamics in invaded ranges, focussing on Europe, and identify key associated knowledge gaps. We identified 272 documented interactions between alien species and zoonotic pathogens within invaded ranges. The majority of these involved invasive alien mammals followed by birds with only a few occurrences of other taxa documented. A wide range of potential interactions between invasive alien species and zoonotic pathogens were identified but few studies considered transmission to humans and so there was limited evidence of actual impacts on human health. However, there is an urgent need to raise awareness of the potential risks posed to human health by the transmission of zoonotic diseases by invasive alien species; the role of invasive alien species in zoonotic disease transmission may exceed that of native wildlife and occur in a relatively short period following the arrival of an invasive alien species within a new region. Ecological and social mechanisms govern the dynamics of zoonotic disease transmission but wildlife diseases are not consistently included within animal, plant and human policies. Rapid advances in the development of systems frameworks that integrate the ecological, economic and social processes promoting spillover in rapidly changing environments will increase understanding to inform decision-making.

Migratory birds can transport infected ticks across continents. We evaluated pathogens in ticks collected from migratory birds in Italy. We found DNA from Rickettsia aeschlimannii, R. africae, and R. raoultii bacteria, all of which can cause disease in humans. Bird migrations might facilitate the spread of these pathogens into new areas.