Next generation sequencing (NGS) can be applied to identify and characterize the entire set of microbes within a sample. However, this platform does not provide a morphological context or specific association between the viral or bacterial sequences detected and the histological lesions. This limitation has generated uncertainty whether the sequences identified by NGS are actually contributing or not for the clinical outcome. Although in situ hybridization (ISH) and immunohistochemistry (IHC) can be used to detect pathogens in tissue samples, only ISH has the advantage of being rapidly developed in a context of an emerging disease, especially because it does not require development of specific primary antibodies against the target pathogen. Based on the sequence information provided by NGS, ISH is able to check the presence of a certain pathogen within histological lesions, by targeting its specific messenger RNA, helping to build the relationship between the pathogen and the clinical outcome. In this mini review we have compiled results of the application of NGS-ISH to the investigation of challenging diagnostic cases or emerging pathogens in pigs, that resulted in the detection of porcine circovirus type 3, porcine parvovirus type 2, Senecavirus A, and Mycoplasma hyorhinis.
This thesis is dedicated to my parents Kathy and Greg who have blessed me with their guidance to this point over great distance with love and patience. Everything you have taught me has brought me to a place where I have found purpose, passion and endless opportunities. I will never forget how fortunate I am. This thesis is also dedicated to those whose greatest challenge in life is themselves.
Lawsonia intracellularis (LI)is an obligate intracellular bacterium that causes proliferative enteropathy (PE) in various species including pigs, horses, rabbits, dogs and hamsters. Infection by LI results in the expansion of proliferative SOX9 positive cells and reduction of enterocytes and goblet cells. In a previous study, RNAseq analysis of laser micro‐dissected ilea of infected pigs showed up‐regulation of ADAMTS1 metallopeptidase which codes for surface proteins with adhesion and protease activity. The immune related marker unc‐93 homolog (UNC93B1, necessary for toll‐like receptors response) and the tetraspanin CD63, that participates in cell differentiation, were downregulated. We hypothesize that LI regulates cell proliferation and differentiation through ADAMTS1 and CD63 genes and avoids immune response through UNC93B1. In the current study, swine enteroids were used to model LI infection. We analyzed gene expression of MUC2 (marker for goblet cells), SOX9, ADAMTS1, CD63 and UNC93B1 in swine enteroids infected with LI for 3, 5, 7, 14 and 21 days. Published studies showed that expression of MUC2 and SOX9 in infected pigs decreased on day 7 and was followed by a decreased SOX9 expression at days 14 and 21. We observed no changes in the expression of MUC2 in infected enteroids. SOX9 expression in infected compared with uninfected enteroids at the same time points reached a maximum of 44% increase on day 7 followed by a decrease in expression by 68% and 33% on days 14 and 21 respectively. Both infected enteroids (fold change: 6.63) and tissue samples (fold change: 1.4) from infected pigs showed increased expression of ADAMTS1 on day 5. Expression of UNC93B1 in infected tissues increased from days 5 (19%) to 15 (43%) while no change was observed on day 5 and increased expression was observed on day 7 (fold change: 2.98) in enteroids. CD63 expression in the tissues increased from day 5 (45%) to 15 (22%) while increase in expression on day 5 (fold change: 1.23) and day 7 (fold change: 3.28) were also observed in enteroids. Overall, swine enteroids replicated most of the changes observed in pigs for MUC2, SOX9, ADAMTS1 and UNC93B1, suggesting infected swine enteroids are a viable model to study PE‐associated changes. A probable explanation for the differences in response of enteroids to LI infection in MUC2 and SOX9 expression can be the permissiveness of the culture system for infection since there is no bacterial competition, passage through the gastric environment, or anti‐microbial products present in the in vitro system.
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