Paratuberculosis (Johne's disease) is a chronic intestinal disease of
ruminants caused by Mycobacterium avium subspecies paratuberculosis
(MAP). Three forms have been described in sheep — multibacillary,
paucibacillary and asymptomatic. Real-time RT-PCR (qPCR) and
microarray analyses were used to compare gene expression in ileal tissue
from sheep with the three forms of the disease in order to understand the
immune responses underpinning these three defined pathologies. All
animals from the infected flocks were IS900 positive by qPCR and
therefore infected with MAP. Asymptomatic sheep had no clinical signs of
disease, showed no evidence of acid-fast bacteria (ZN-), exhibited normal
histology of the terminal ileum and were seronegative. Paucibacillary
sheep were ZN- and showed lymphocyte/eosinophil infiltrate into the
lamina propria. 2/6 of the paucibacillary animals were seropositive.
Multibacillary sheep had high numbers of ZN+ bacteria associated with
infiltrating sheets of epithelioid macrophages and were seropositive.
Control sheep were IS900 negative and thus uninfected with MAP.qPCR experiments compared the expression of the following genes
- IL-la, IL-1B, IL-3, IL-6, IL-8, IL-10, IL-12p40, IL-18, CD34, CXCR4,
GM-CSF, IFNy, IGFBP-2, IGFBP-6, TGF6, TIRAP, TNFa, TRAF-land
TRAM. The results confirmed that pauci- and multibacillary forms are
linked to the differential expression of IFNy and IL-10 respectively, but
imply that polarisation is incomplete, with upregulation of both
proinflammatory IL-18 and anti-inflammatory TGFB in both disease
forms. Increased levels of the proinflammatory cytokines IL-ip, IL-8,
TNFa and TRAF-1, indicative of persistent inflammatory lesions, were
observed in clinical tissues. IL-3 was detected at low levels in all infected
animals but never in uninfected control samples. IGFBP-6 was upregulated
and CXCR4 down-regulated in paucibacillary samples compared to multibacillary samples. SNP analysis was carried out on
these genes, identifying three novel SNPs in each gene, but none were
linked to disease pathology.Microarray experiments discovered 63 differentially expressed
genes. Four genes were found to be differentially expressed in infected
tissue compared to uninfected controls, and a further eight in clinical
tissues compared to uninfected controls. Eight genes were differentially
expressed in clinical tissue compared to asymptomatic tissue. Seven
genes were quantified by qPCR and validated the microarray data well.
Pathway analysis of the microarray data identified several immune
pathways that are involved in pathogenesis. Infected tissues displayed
up-regulation of the genes involved in complement activation, and downregulation
of TCR signalling and MHC class II genes. In addition,
clinical tissues displayed up-regulation of genes involved in the JAKSTAT
and TLR2 signalling pathways, NK cell cytotoxicity and antibody
production. Multibacillary tissues also displayed up-regulation of genes
involved in leukocyte migration.Overall, these data confirm that multibacillary pathology is linked
to type 2 and paucibacillary pathology is linked to type 1 immune
responses, and identify novel genes and gene pathways for future
analyses
