The Development of 3D Bovine Intestinal Organoid Derived Models to Investigate Mycobacterium Avium ssp Paratuberculosis Pathogenesis

Abstract

Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of Johne's Disease, a chronic enteritis of ruminants prevalent across the world. It is estimated that approximately 50% of UK dairy herds are infected with MAP, but this is likely an underestimate of the true prevalence. Infection can result in reduced milk yield, infertility and premature culling of the animal, leading to significant losses to the farming economy and negatively affecting animal welfare. Understanding the initial interaction between MAP and the host is critical to develop improved diagnostic tools and novel vaccines. Here we describe the characterisation of three different multicellular in vitro models derived from bovine intestinal tissue, and their use for the study of cellular interactions with MAP. In addition to the previously described basal-out 3D bovine enteroids, we have established viable 2D monolayers and 3D apical-out organoids. The apical-out enteroids differ from previously described bovine enteroids as the apical surface is exposed on the exterior surface of the 3D structure, enabling study of host-pathogen interactions at the epithelial surface without the need for microinjection. We have characterised the cell types present in each model system using RT-qPCR to detect predicted cell type-specific gene expression, and confocal microscopy for cell type-specific protein expression. Each model contained the cells present in the original bovine intestinal tissue, confirming they were representative of the bovine gut. Exposure of the three model systems to the K10 reference strain of MAP K10, and a recent Scottish isolate referred to as C49, led to the observation of intracellular bacteria by confocal microscopy. Enumeration of the bacteria by quantification of genome copy number, indicated that K10 was less invasive than C49 at early time points in infection in all model systems. This study shows that bovine enteroid-based models are permissive to infection with MAP and that these models may be useful in investigating early stages of MAP pathogenesis in a physiologically relevant in vitro system, whilst reducing the use of animals in scientific research.Bos taurus: urn:lsid:zoobank.org:act:4C90C4FA-6296-4972-BE6A-5EF578677D64

Using Species a Rotavirus Reverse Genetics to Engineer Chimeric Viruses Expressing SARS-CoV-2 Spike Epitopes

“…3D bovine enteroids were prepared as described in Hamilton et al ( 112 ). Infection of organoids was carried out as described by Derricott et al ( 70 ) and Blake et al ( 113 ) with modifications. 3D organoids were mechanically disrupted into multicellular fragments by pipetting to expose the apical surface of the cells in 80% of the enteroids.…”

“…The sheared enteroids were counted using a bright-field microscope and diluted to the final concentration of 2,500 enteroids/well. Each enteroid was estimated to contain approximately 40 cells as described by Blake et al ( 113 ). The sheared enteroids were then diluted to the appropriate concentration with IntestiCult Organoid Growth Medium (STEMCELL Technologies) supplemented with 10 μM ROCK pathway inhibitor (Cayman chemicals), 10 μM Galunisertib (Cayman Chemicals), and 10 μM p38 inhibitor (Enzo) and aliquoted into 15 mL falcon tubes for each condition.…”

Bovine Enteroids as an In Vitro Model for Infection with Bovine Coronavirus

“…Human intestinal organoids have proven successful for culturing human rotavirus (HuRoV), norovirus, and enteroviruses [ 22 , 25 , 26 ], and porcine enteroids have been used to study infection caused by porcine enteric coronaviruses and the host innate response [ 27 ]. Although studies on bovine enteroids infected with enteric pathogens are limited to date, there are some studies on the characterization of cell types and culture of bovine rotavirus (BRoV) group A, Salmonella typhimurium, Toxoplasma gondii, and Mycobacterium avium subspecies paratuberculosis [ 17 , 28 , 29 ].…”

Polarity reversal of canine intestinal organoids reduces proliferation and increases cell death

“…Apical‐out organoids were first published by Co et al, who have demonstrated that human intestinal organoids that usually grow embedded in a three‐dimensional extracellular matrix (ECM) can reverse their polarity when they are cultured in a floating manner in medium without ECM 12 . This method has now been adopted by many other labs and expanded to different animal species, for example, pigs, 13 chickens, 14 cows 15 and dogs, 16 and also organoids derived from human iPSCs and embryonic stem cells 17,18 . Remarkably, regardless of the species from which the organoids were derived, the majority of these studies utilised their apical‐out organoids within the initial 7 days following the induction of polarity reversal 13,16,17,19–21 .…”