No abstract

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.

Livestock abortion is an important cause of productivity losses worldwide and many infectious causes of abortion are zoonotic pathogens that impact on human health. Little is known about the relative importance of infectious causes of livestock abortion in Africa, including in subsistence farming communities that are critically dependent on livestock for food, income, and wellbeing. We conducted a prospective cohort study of livestock abortion, supported by cross-sectional serosurveillance, to determine aetiologies of livestock abortions in livestock in Tanzania. This approach generated several important findings including detection of a Rift Valley fever virus outbreak in cattle; high prevalence of C. burnetii infection in livestock; and the first report of Neospora caninum, Toxoplasma gondii, and pestiviruses associated with livestock abortion in Tanzania. Our approach provides a model for abortion surveillance in resource-limited settings. Our findings add substantially to current knowledge in sub-Saharan Africa, providing important evidence from which to prioritise disease interventions.

S U M M A R YCryptosporidium is a well-known cause of neonatal enteritis in cattle worldwide. Cattle are commonly infected with four different species of Cryptosporidium but only one of these, Cryptosporidium parvum, is associated with clinical disease. Identification of species in cases of calf scour can give an indication if Cryptosporidium is the causative agent or not. In addition, C. parvum is a zoonotic species and so has implications for human health, for this reason it is important to identify the species of Cryptosporidium infecting cattle particularly where a farm is implicated in an outbreak of cryptosporidiosis in humans. Here a multiplex PCR test, which can identify the four common cattle-adapted Cryptosporidium species, including C. parvum, has been developed. This test allows quick and accurate detection of Cryptosporidium species in cattle fecal samples including mixed infections, which could be missed by the more common method of sequencing the same gene.

Toxoplasma gondii is an intracellular protozoan parasite that can infect many warm-blooded animal species and humans. Despite substantial knowledge of the biology, epidemiology, and host-pathogen interactions of T. gondii, there are still very few effective control strategies to prevent oocyst shedding in cats, tissue cysts in livestock for consumption, and infection and disease in humans. This article reviews current progress and targets for vaccination against T. gondii.