Title page for ETD etd-03202012-143927

Type of Document Master's Thesis
Author Tyler, Jr., Ronald Dale
Author's Email Address rontyler@vt.edu
URN etd-03202012-143927
Title Evaluation of a Paratuberculosis Quantitative Polymerase Chain Reaction Assay with Microscopic Correlation
Degree Master of Science
Department Veterinary Medical Sciences
Advisory Committee
Advisor Name Title
Sriranganathan, Nammalwar Committee Chair
LeRoith, Tanya Committee Co-Chair
Pickrell, Gary R. Committee Member
Prater, Mary Renee Committee Member
Sponenberg, Daniel Phillip Committee Member
  • Johne’s disease
  • paratuberculosis
  • histopathology
  • qPCR
Date of Defense 2012-05-01
Availability restricted
Paratuberculosis is an intestinal condition in ruminants infected with Mycobacterium avium subspecies paratuberculosis (MAP) and precedes Johne’s disease, a chronic enteric disorder in ruminants caused by MAP infection. Necropsy with histopathology provides definitive diagnosis of Johne’s disease and positive culture of MAP from tissues provides definitive diagnosis of paratuberculosis. To determine assay sensitivity, 85 formalin-fixed paraffin-embedded (FFPE) tissues from ruminants diagnosed with Johne’s disease were tested with a commercial paratuberculosis quantitative polymerase chain reaction (qPCR) assay and had a sensitivity of 92%. To determine assay specificity, 21 FFPE tissues from animals without gastrointestinal disease combined with 13 FFPE tissues from non-ruminant animals (frog, dove, turtle, dog, and 2 cats) with non-paratuberculosis mycobacterial diseases were tested with the commercial qPCR assay and had a specificity of 100%.

Slides prepared from the FFPE tissue blocks were stained with hematoxylin & eosin (H&E) and Ziehl-Neelsen’s (acid fast stain), then examined for granulomatous inflammation and scored on a scale from 0-4 based on the quantity of acid fast bacteria (AFB). Digital microscopy and morphometric software were used to compute an acid fast bacteria area index (AFBAI) to evaluate a more precise correlation with the qPCR results. The quantity of AFB in tissue slides showed medium to strong correlation with the appropriate qPCR results.

The results indicate that the commercial qPCR assay can be used on FFPE tissues with good results and the qPCR results have medium-strong correlation with quantitative acid fast histopathology.

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