The eco-epidemiology of zoonoses is often oversimplified to host-pathogen interactions while findings derived from global datasets are rarely directly transferable to smaller-scale contexts. Through a systematic literature search, we compiled a dataset of naturally occurring zoonotic interactions in Austria, spanning 1975-2022. We introduce the concept of zoonotic web to describe the complex relationships between zoonotic agents, their hosts, vectors, food, and environmental sources. The zoonotic web was explored through network analysis. After controlling for research effort, we demonstrate that, within the projected unipartite source-source network of zoonotic agent sharing, the most influential zoonotic sources are human, cattle, chicken, and some meat products. Analysis of the One Health 3-cliques (triangular sets of nodes representing human, animal, and environment) confirms the increased probability of zoonotic spillover at human-cattle and human-food interfaces. We characterise six communities of zoonotic agent sharing, which assembly patterns are likely driven by highly connected infectious agents in the zoonotic web, proximity to human, and anthropogenic activities. Additionally, we report a frequency of emerging zoonotic diseases in Austria of one every six years. Here, we present a flexible network-based approach that offers insights into zoonotic transmission chains, facilitating the development of locally-relevant One Health strategies against zoonoses.

Phytophthora cinnamomi Rands is a highly prevalent phytopathogen worldwide, ranking among the top ten in terms of distribution. It inflicts crown rot, canker, and root rot on numerous plant species, significantly impacting the biodiversity of both flora and fauna within affected environments. With a host range spanning over 5,000 species, including important plants like Quercus suber, Quercus ilex, Castanea sativa, and commercially significant crops such as avocado (Persea americana), maize (Zea mays), and tomato (Solanum lycopersicum), Phytophthora cinnamomi poses a substantial threat to agriculture and ecosystems. The efficient dissemination of the oomycete relies on its short-lived asexually motile zoospores, which depend on water currents to infect host roots. However, managing these zoospores in the laboratory has long been challenging due to the complexity of the life cycle. Current protocols involve intricate procedures, including alternating cycles of growth, drought, and flooding. Unfortunately, these artificial conditions often result in a rapid decline in virulence, necessitating additional steps to maintain infectivity during cultivation. In our research, we sought to address this challenge by investigating zoospore survival under various conditions. Our goal was to develop a stable stock of zoospores that is both easily deployable and highly infective. Through direct freezing in liquid nitrogen, we have successfully preserved their virulence. This breakthrough eliminates the need for repeated culture transfers, simplifying the process of plant inoculation. Moreover, it enables more comprehensive studies of Phytophthora cinnamomi and its interactions with host plants.

The study of virus-host interactions is essential to achieve a comprehensive understanding of the viral replication process. The commonly used methods are yeast two-hybrid approach and transient expression of a single tagged viral protein in host cells followed by affinity purification of interacting cellular proteins and mass spectrometry analysis (AP-MS). However, by these approaches, virus-host protein-protein interactions are detected in the absence of a real infection, not always correctly compartmentalized, and for the yeast two-hybrid approach performed in a heterologous system. Thus, some of the detected protein-protein interactions may be artificial. Here we describe a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect protein partners during the infection (AP-MS in viral context). This way, virus-host protein-protein interacting co-complexes can be purified directly from infected cells for further characterization.

Intestinal epithelial cells (IECs) play pivotal roles in nutrient uptake and in the protection against gut microorganisms. However, certain enteric pathogens, such as Salmonella enterica serovar Typhimurium (S. Tm), can invade IECs by employing flagella and type III secretion systems (T3SSs) with cognate effector proteins and exploit IECs as a replicative niche. Detection of flagella or T3SS proteins by IECs results in rapid host cell responses, i.e., the activation of inflammasomes. Here, we introduce a single-cell manipulation technology based on fluidic force microscopy (FluidFM) that enables direct bacteria delivery into the cytosol of single IECs within a murine enteroid monolayer. This approach allows to specifically study pathogen-host cell interactions in the cytosol uncoupled from preceding events such as docking, initiation of uptake, or vacuole escape. Consistent with current understanding, we show using a live-cell inflammasome reporter that exposure of the IEC cytosol to S. Tm induces NAIP/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected S. Tm mutants devoid of these invasion-relevant ligands were able to grow in the cytosol of IECs despite the absence of T3SS functions, suggesting that, in the absence of NAIP/NLRC4 inflammasome activation and the ensuing cell death, no effector-mediated host cell manipulation is required to render the epithelial cytosol growth-permissive for S. Tm. Overall, the experimental system to introduce S. Tm into single enteroid cells enables investigations into the molecular basis governing host-pathogen interactions in the cytosol with high spatiotemporal resolution.

The transmission of pathogens from reservoir to recipient host species, termed pathogen spillover, can profoundly impact plant, animal, and public health. However, why some pathogens lead to disease emergence in a novel species while others fail to establish or do not elicit disease is often poorly understood. There is strong evidence that deformed wing virus (DWV), an (+)ssRNA virus, spills over from its reservoir host, the honeybee Apis mellifera, into the bumblebee Bombus terrestris. However, the low impact of DWV on B. terrestris in laboratory experiments suggests host barriers to virus spread in this recipient host. To investigate potential host barriers, we followed the spread of DWV genotype B (DWV-B) through a host\'s body using RT-PCR after experimental transmission to bumblebees in comparison to honeybees. Inoculation was per os, mimicking food-borne transmission, or by injection into the bee\'s haemocoel, mimicking vector-based transmission. In honeybees, DWV-B was present in both honeybee faeces and haemolymph within 3 days of inoculation per os or by injection. In contrast, DWV-B was not detected in B. terrestris haemolymph after inoculation per os, suggesting a gut barrier that hinders DWV-B\'s spread through the body of a B. terrestris. DWV-B was, however, detected in B. terrestris faeces after injection and feeding, albeit at a lower abundance than that observed for A. mellifera, suggesting that B. terrestris sheds less DWV-B than A. mellifera in faeces when infected. Barriers to viral spread in B. terrestris following oral infection may limit DWV\'s impact on this spillover host and reduce its contribution to the community epidemiology of DWV.

After mosquitoes, ticks are among the most important vector of pathogens of concern for animal and public health, but unless mosquitoes ticks remain attached to their hosts for long time periods providing an opportunity to analyse their role in the dispersal and dynamics of different zoonotic pathogens. Given their interest in public health it is important to understand which factors affect their incidence in different hosts and to stablish effective surveillance programs to determine the risk of transmission and spill-over of zoonotic pathogens. Taking benefit of a large network of volunteer ornithologists, we analysed the life-history traits associated to the presence of ticks using information of 620,609 individuals of 231 avian species. Bird phylogeny, locality and year explained a large amount of variance in tick prevalence. Non-colonial species non breeding in grasslands and non-spending the non-breeding season as gregarious groups or isolated individuals (e.g. thrushes, quails and finches) had the higher prevalence of ticks and appear as good candidates for zoonosis surveillance programs based on the analyses of ticks collected from wild birds. Ringers underestimated tick prevalence but can be considered as an important source of information of ticks for public and animal health surveillance programs if properly trained for the detection and collection of the different tick development phases.

Bombyx mori nucleopolyhedrovirus (BmNPV) is the most important virus that threatens sericulture industry. At present, there is no effective treatment for BmNPV infection in silkworms, and lncRNA plays an important role in biological immune response and host-virus interaction, but there are relatively few studies in silkworms. In this study, the four midgut tissue samples of the resistance strain NB (NB) and susceptible strain 306 (306) and the NB and 306 continuously infected with BmNPV for 96 h are used for whole transcriptome sequencing to analyze the differences in the genetic background of NB and 306 and the differences after inoculation of BmNPV, and the significantly different mRNA, miRNA and lnRNA between NB and 306 after BmNPV inoculation were screened. By comparing NB and 306, 2651 significantly different mRNAs, 57 significantly different miRNAs and 198 significantly different lncRNAs were screened. By comparing NB and 306 after BmNPV inoculation, 2684 significantly different mRNAs, 39 significantly different miRNAs and 125 significantly different lncRNAs were screened. According to the significantly different mRNA, miRNA and lncRNA screened from NB and 306 and NB and 306 after virus inoculation, the mRNA-miRNA-lncRNA regulatory network was constructed before and after virus inoculation, and the BmBCAT-Bomo_chr7_8305-MSTRG.3236.2 regulatory axis was screened from them, and it was found that BmBCAT was not Bomo_chr7_8305 regulated in the genetic background, after viral infection, MSTRG.3236.2 competes for binding Bomo_chr7_8305 regulates BmBCAT. The whole transcriptome sequencing results were verified by qPCR and the time-series expression analysis was performed to prove the reliability of the regulatory network. The BmBCAT-Bomo_chr7_8305-MSTRG.3236.2 regulatory axis may play a potential role in the interaction between silkworms and BmNPV. These results provide new insights into the interaction mechanism between silkworms and BmNPV.

The ability of a pathogen to survive and cause an infection is often determined by specific interactions between the host and pathogen proteins. Such interactions can be both intra- and extracellular and may define the outcome of an infection. There are a range of innovative biochemical, biophysical and bioinformatic techniques currently available to identify protein-protein interactions (PPI) between the host and the pathogen. However, the complexity and the diversity of host-pathogen PPIs has led to the development of several high throughput (HT) techniques that enable the study of multiple interactions at once and/or screen multiple samples at the same time, in an unbiased manner. We review here the major HT laboratory-based technologies employed for host-bacterial interaction studies.

The epithelium of the gastrointestinal tract has been extensively characterized using advanced histological and RNA sequencing techniques, which has revealed great cellular diversity. Pathogens, such as viruses and bacteria, are highly adapted to their host and often exhibit not only species-specificity, but also a preference or tropism for specific gastrointestinal segments or even cell types - some of these preferences are so specific, that these pathogens still cannot be cultured in the lab. Organoid technology now provides a tool to generate human cell types, which enables the study of host cell tropism. Focusing on the gastrointestinal tract, we provide an overview about cellular differentiation in vivo and in organoids and how differentiation in organoids and their derived models is used to advance our understanding of viral, bacterial, and parasitic infection. We emphasize that it is central to understand the composition of the model, as the alteration of culture conditions yields different cell types which affects infection. We examine future directions for wider application of cellular heterogeneity and potential advanced model systems for gastrointestinal tract infection studies.

This protocol is focused on using the recently established planarian infection model system to study host-pathogen interactions during fungal infection. Here, we describe in detail the infection of the planarian Schmidtea mediterranea with the human fungal pathogen Candida albicans. This simple and reproducible model system allows for rapid visualization of tissue damage throughout different infection timepoints. We note that this model system has been optimized for use with C. albicans, but should also be applicable for use with other pathogens of interest.