Detection of Mycobacterium avium spp. paratuberculosis (Map) in samples of sheep paratuberculosis (Johne’s disease or JD) and human Crohn’s disease (CD) using liquid phase RT-PCR, in situ RT-PCR and immunohistochemistry

The association between the human Crohn’s disease (CD) and Mycobacterium avium ssp. paratuberculosis (Map), the etiological agent of the sheep paratuberculosis (Johne’s disease or JD), has been controversial because of technical limits to detection of the microorganism. Intestinal samples from 10 sheep naturally affected with JD (5 paucibacillary and 5 multibacillary infections), 8 humans with CD and 1 sheep experimentally infected with a reference strain (ATCC 43015) of Map isolated from a patient with CD were collected. A procedure for the extraction of RNA from formalin-fixed, paraffin embedded tissues was optimized. Archived tissue samples from cases of JD and CD were examined by light microscopy using Haematoxyline and Eosin and Ziehl–Neelsen stains. Liquid phase RT-PCR, in situ RT-PCR and immunohistochemistry were also performed on the same samples. In situ RT-PCR targets were the IS900 sequence and the gene locus F57. The effectiveness of the primer–probes was demonstrated using Dot-Blot testing. A diffuse granulomatous enteritis was present in samples from all sheep with JD; lesions were categorized as subtypes 3b and 3c (Perez classification). Human CD samples appeared very similar to the lymphocytic paucimicrobial form of JD (subtype 3c) and the experimentally infected sheep had an enteritis with lesions compatible with Perez type 2. Liquid phase RT-PCR and Dot-Blot test were positive for Map in all sheep with JD and negative in all samples from CD patients as well as the experimentally infected sheep. In situ RT-PCR was positive for the presence of Map both in JD and CD infected samples. Immunohistochemistry confirmed the in situ RT-PCR results in all JD and CD samples, with the exception of the experimentally infected sheep, which resulted negative. This study demonstrated the effectiveness of the in situ RT-PCR technique in the contribution to establish Map as the etiological agent of CD.