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Johne’s Disease in Dairy Cattle: Validation of Genetic Markers and Assessment of Salivary Gland Transcriptome

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Title: Johne’s Disease in Dairy Cattle: Validation of Genetic Markers and Assessment of Salivary Gland Transcriptome
Author: Mallikarjunappa, Sanjay
Department: Department of Animal Biosciences
Program: Animal and Poultry Science
Advisor: Karrow, Niel
Abstract: Johne’s disease (JD) is a severe production limiting disease in cattle caused by Mycobacterium avium susbp. paratuberculosis (MAP) infection. Issues associated with currently available JD diagnostic methods have prompted evaluation of new diagnostic techniques. While the genetic basis of MAP infection status is supported via identification of various candidate genes and single nucleotide polymorphisms (SNPs), their validation prior to inclusion in a selective breeding program for JD resistance is essential. The purposes of this thesis was to evaluate salivary mRNA biomarkers as a potential MAP exposure diagnostic tool, identify new SNPs and also validate previously reported JD SNPs, and validate the functional relevance of the candidate gene IL10RA using a gene edited IL10RA knockout (KO) mammary epithelial cell line (MAC-T cells). RNA-seq transcriptome profile analysis of mandibular and parotid salivary glands revealed differences in expression of some immunoregulatory and anti-microbial genes between MAP-exposed and control unchallenged cattle. Subject to their validation, the identified expressed genes could serve as potential salivary biomarkers of MAP exposure. Fine mapping of previously identified JD quantitative trait loci by our lab, by imputation to high density (HD) SNP genotypes, enabled identification of new SNPs associated with MAP infection status. Follow-up validation of previously reported JD SNPs was also undertaken by testing their association with sire estimated breeding value (EBV) for milk ELISA test scores. A total of 498 sires grouped as high or low EBV group were genotyped using a custom SNP panel comprised of 155 JD SNPs, and 5 of these SNPs were confirmed for their association with JD. Functional relevance of a JD candidate gene was investigated by creation of IL10RA KO MAC-T cells using CRISPR/cas9 gene editing approach. Stimulation of IL10RA KO cells with immunostimulants like MAP lysate+/- IL10, revealed the regulatory role of IL10RA through its ligand IL-10 during MAP lysate stimulation. The findings of this thesis have addressed some of the knowledge-gaps specific to JD diagnosis and control. They provide a future framework for validation of salivary biomarkers, validation of JD SNPs using different JD phenotypes, and characterizing the functional importance of other JD candidate genes by gene editing approach.
URI: http://hdl.handle.net/10214/17650
Date: 2019-12
Rights: Attribution-NonCommercial-NoDerivatives 4.0 International
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Attribution-NonCommercial-NoDerivatives 4.0 International Except where otherwise noted, this item's license is described as Attribution-NonCommercial-NoDerivatives 4.0 International