The Development of Ovine Gastric and Intestinal Organoids for Studying Ruminant Host-Pathogen Interactions

Gastrointestinal (GI) infections in sheep have significant implications for animal health, welfare and productivity, as well as being a source of zoonotic pathogens. Interactions between pathogens and epithelial cells at the mucosal surface play a key role in determining the outcome of GI infections; however, the inaccessibility of the GI tract in vivo significantly limits the ability to study such interactions in detail. We therefore developed ovine epithelial organoids representing physiologically important gastric and intestinal sites of infection, specifically the abomasum (analogous to the stomach in monogastrics) and ileum. We show that both abomasal and ileal organoids form self-organising three-dimensional structures with a single epithelial layer and a central lumen that are stable in culture over serial passage. We performed RNA-seq analysis on abomasal and ileal tissue from multiple animals and on organoids across multiple passages and show the transcript profile of both abomasal and ileal organoids cultured under identical conditions are reflective of the tissue from which they were derived and that the transcript profile in organoids is stable over at least five serial passages. In addition, we demonstrate that the organoids can be successfully cryopreserved and resuscitated, allowing long-term storage of organoid lines, thereby reducing the number of animals required as a source of tissue. We also report the first published observations of a helminth infecting gastric and intestinal organoids by challenge with the sheep parasitic nematode Teladorsagia circumcincta, demonstrating the utility of these organoids for pathogen co-culture experiments. Finally, the polarity in the abomasal and ileal organoids can be inverted to make the apical surface directly accessible to pathogens or their products, here shown by infection of apical-out organoids with the zoonotic enteric bacterial pathogen Salmonella enterica serovar Typhimurium. In summary, we report a simple and reliable in vitro culture system for generation and maintenance of small ruminant intestinal and gastric organoids. In line with 3Rs principals, use of such organoids will reduce and replace animals in host-pathogen research

Differences in immune responses to Haemonchus contortus infection in the susceptible Ile de France and the resistant Santa Ines sheep under different anthelmintic treatments regimens

Understanding the immunological basis of resistance to gastrointestinal nematode infections in livestock is important in order to develop novel methods of parasite control such as vaccination or genetic selection for parasite resistance. The present study aimed to investigate differences in immune response between parasite resistant Santa Ines and susceptible Ile de France sheep breeds to natural Haemonchus contortus infection. Parasitological parameters, humoral immunity, local and circulating cellular immune responses were evaluated in 19 Santa Ines and 19 Ile de France lambs undergoing different anthelmintic treatments regimens: suppressive treatments (SUP) or targeted selective treatments (TST) over a 5-month grazing period. Santa Ines lambs had significantly lower Haemonchus faecal egg count and worm burden compared to Ile de France regardless of treatment regime. In addition, circulating blood eosinophils count and parasite-specific IgG levels were significantly higher and more rapidly induced in Santa Ines lambs. Abomasal immune responses were generally greater in the resistant breed, which had significantly higher levels of parasite-specific IgA in mucus, and elevated number of globule leukocytes and CD3+ T cells within the abomasal mucosal. Furthermore, numbers of POU2F3+ epithelial cells, a tuft-cell specific transcription factor, were also elevated in the Santa Ines breed, suggesting that this breed is better able to initiate T-helper type 2 immune responses within the abomasum. In conclusion, the differential immunological responses detailed here are relevant to understanding resistance to gastrointestinal nematodes in other host breeds, as well as to resistance breeding as a sustainable control approach for parasitic infections

Development of bovine abomasal organoids as a novel in-vitro model to study host-parasite interactions in gastrointestinal nematode infections

Gastro-intestinal nematode (GIN) parasites are a major cause of production losses in grazing cattle, primarily through reduced growth rates in young animals. Control of these parasites relies heavily on anthelmintic drugs; however, with growing reports of resistance to currently available anthelmintics, alternative methods of control are required. A major hurdle in this work has been the lack of physiologically relevant in vitro infection models that has made studying precise interactions between the host and the GINs difficult. Such mechanistic insights into the infection process will be valuable for the development of novel targets for drugs, vaccines, or other interventions. Here we created bovine gastric epithelial organoids from abomasal gastric tissue and studied their application as in vitro models for understanding host invasion by GIN parasites. Transcriptomic analysis of gastric organoids across multiple passages and the corresponding abomasal tissue showed conserved expression of tissue-specific genes across samples, demonstrating that the organoids are representative of bovine gastric tissue from which they were derived. We also show that self-renewing and self-organising three-dimensional organoids can also be serially passaged, cryopreserved, and resuscitated. Using Ostertagia ostertagi, the most pathogenic gastric parasite in cattle in temperate regions, we show that cattle gastric organoids are biologically relevant models for studying GIN invasion in the bovine abomasum. Within 24 h of exposure, exsheathed larvae rapidly and repeatedly infiltrated the lumen of the organoids. Prior to invasion by the parasites, the abomasal organoids rapidly expanded, developing a ‘ballooning’ phenotype. Ballooning of the organoids could also be induced in response to exposure to parasite excretory/secretory products. In summary, we demonstrate the power of using abomasal organoids as a physiologically relevant in vitro model system to study interactions of O. ostertagi and other GIN with bovine gastrointestinal epithelium

New technologies to study helminth development and host-parasite interactions

How parasites develop and survive, and how they stimulate or modulate host immune responses are important in understanding disease pathology and for the design of new control strategies. Microarray analysis and bulk RNA sequencing have provided a wealth of data on gene expression as parasites develop through different life-cycle stages and on host cell responses to infection. These techniques have enabled gene expression in the whole organism or host tissue to be detailed, but do not take account of the heterogeneity between cells of different types or developmental stages, nor the spatial organisation of these cells. Single-cell RNA-seq (scRNA-seq) adds a new dimension to studying parasite biology and host immunity by enabling gene profiling at the individual cell level. Here we review the application of scRNA-seq to establish gene expression cell atlases for multicellular helminths and to explore the expansion and molecular profile of individual host cell types involved in parasite immunity and tissue repair. Studying host-parasite interactions in vivo is challenging and we conclude this review by briefly discussing the applications of organoids (stem-cell derived mini-tissues) to examine host-parasite interactions at the local level, and as a potential system to study parasite development in vitro. Organoid technology and its applications have developed rapidly, and the elegant studies performed to date support the use of organoids as an alternative in vitro system for research on helminth parasites

Tuft cells increase following ovine intestinal parasite infections and define evolutionarily conserved and divergent responses

Helminth parasite infections of humans and livestock are a global health and economic problem. Resistance of helminths to current drug treatment is an increasing problem and alternative control approaches, including vaccines, are needed. Effective vaccine design requires knowledge of host immune mechanisms and how these are stimulated. Mouse models of helminth infection indicate that tuft cells, an unusual type of epithelial cell, may ‘sense’ infection in the small intestine and trigger a type 2 immune response. Currently nothing is known of tuft cells in immunity in other host species and in other compartments of the gastrointestinal (GI) tract. Here we address this gap and use immunohistochemistry and single cell RNA-sequencing to detail the presence and gene expression profile of tuft cells in sheep following nematode infections. We identify and characterize tuft cells in the ovine abomasum (true stomach of ruminants) and show that they increase significantly in number following infection with the globally important nematodes Teladorsagia circumcincta and Haemonchus contortus. Ovine abomasal tuft cells show enriched expression of tuft cell markers POU2F3, GFI1B, TRPM5 and genes involved in signaling and inflammatory pathways. However succinate receptor SUCNR1 and free fatty acid receptor FFAR3, proposed as ‘sensing’ receptors in murine tuft cells, are not expressed, and instead ovine tuft cells are enriched for taste receptor TAS2R16 and mechanosensory receptor ADGRG6. We also identify tuft cell sub-clusters at potentially different stages of maturation, suggesting a dynamic process not apparent from mouse models of infection. Our findings reveal a tuft cell response to economically important parasite infections and show that while tuft cell effector functions have been retained during mammalian evolution, receptor specificity has diverged. Our data advance knowledge of host-parasite interactions in the GI mucosa and identify receptors that may potentiate type 2 immunity for optimized control of parasitic nematodes