Proceedings of the 10th International Colloquium on Paratuberculosis

#153 SNP discovery in bovine Sp110 gene and its genetic association with infetion by MAP
Otsanda Ruiz, Mikel Iriondo, Carmen Manzano, Iratxe Montes, Elena Molina, Joseba M. Garrido, Ramon A.
Juste, Patricia Vazquez, Ad Koets, Andone Estonba
University <strong>of</strong> <strong>the</strong> Basque Country, Spain; NEIKER-Tecnalia, Spain; Utrecht University, The Ne<strong>the</strong>rlands ;
Three polymorphisms <strong>of</strong> <strong>the</strong> Sp110 gene have been associated with tuberculosis susceptibility in humans
(Tosh et al. 2006). Moreover, Ipr1 gene in mice, a Sp110 homolog, has been reported to confer genetic resistance
to Mycobacterium tuberculosis and Listeria monocytogenes through apoptotic pathway activation in
infected macrophages (Pan et al. 2005). Therefore, we hypo<strong>the</strong>size that <strong>the</strong> bovine Sp110 gene could be involved
in <strong>the</strong> innate immunity response against Mycobacterium avium subsp. paratuberculosis (MAP) infection
in cattle. To test <strong>the</strong> hypo<strong>the</strong>sis, a SNP discovery study on <strong>the</strong> Sp110 gene for use on a case-control association
study has been undertaken comparing 500 healthy and 350 infected Holstein-Friesian cattle.
As nucleotide variation <strong>of</strong> bovine Sp110 gene is poorly described, we focused on SNP (single nucleotide
polymorphisms) discovery by comparative sequencing. Looking for functional variation, 9980 bp out <strong>of</strong>
51532 bp <strong>of</strong> <strong>the</strong> gene were sequenced in 30 animals (15 healthy / 15 infected). This included <strong>the</strong> 300 bp <strong>of</strong>
<strong>the</strong> promoter, 12 exons, and both 3’- and 5’- UTR regions <strong>of</strong> <strong>the</strong> gene. In all, 20 SNPs were detected for <strong>the</strong>
first time in Holstein-Friesian cattle. The application <strong>of</strong> <strong>the</strong> selected SNP polymorphisms to <strong>the</strong> case-control
samples has not yet revealed any association.
#156 Different cytokine responses after interaction between human macrophages and
Mycobacterium avium subsp. paratuberculosis type II and type III strains
Erika Borrmann, Petra Moebius, Heike Köhler
Institute <strong>of</strong> Molecular Pathogenesis, Friedrich-Loeffler-Institute, Jena, Germany
Objective: The crucial process in <strong>the</strong> pathogenesis <strong>of</strong> paratuberculosis is <strong>the</strong> internalization <strong>of</strong> Mycobacterium
avium subsp. paratuberculosis (MAP) by intestinal host macrophages, its survival and intracellular multiplication.
The knowledge about <strong>the</strong>se pathogenic mechanisms is still limited. Fur<strong>the</strong>rmore, despite similarities <strong>of</strong>
paratuberculosis to Crohn`s disease (CD) concerning <strong>the</strong>ir histopathology and clinical symptoms, <strong>the</strong> putative
pathogenic role <strong>of</strong> MAP in CD is unknown. The objective <strong>of</strong> <strong>the</strong> current study was to investigate differences in
<strong>the</strong> cytokine responses <strong>of</strong> human monocytes after interaction with three MAP strains <strong>of</strong> different genotypes.
Materials and Methods: The human monocyte cell line THP-1 was incubated with two MAP strains <strong>of</strong>
type II (reference strain ATCC19698, one bovine field isolate) and one MAP strain <strong>of</strong> type III (intermediate type)
characterized by IS900-RFLP and MIRU-VNTR genotyping before. An in vitro model was established and <strong>the</strong>
test conditions were standardized. As parameters for <strong>the</strong> determination <strong>of</strong> <strong>the</strong> impact <strong>of</strong> <strong>the</strong>se strains on <strong>the</strong>
macrophage responses, <strong>the</strong> production <strong>of</strong> mRNA <strong>of</strong> <strong>the</strong> cytokines TNF-alpha, IL-1, and IL-10 was determined
by quantitative real-time PCR using GAPDH as housekeeping gene. The biological active proteins IL-1 and IL-
10 were measured using ELISA, TNF-alpha by a cytotoxicity test.
Results: Differences in <strong>the</strong> expression <strong>of</strong> mRNA and <strong>the</strong> production <strong>of</strong> <strong>the</strong> proteins IL-1, IL-10 and TNFalpha
were detected after interaction <strong>of</strong> human monocytes with <strong>the</strong> three MAP-strains. These differences were
determined between MAP type III and MAP type II as well as between <strong>the</strong> MAP type II reference and field
strains.
Conclusion: It seems that MAP strains <strong>of</strong> diverse genotypes induce different responses <strong>of</strong> macrophages.
Fur<strong>the</strong>r investigations will show if <strong>the</strong>re is a correlation <strong>of</strong> <strong>the</strong> cytokine responses <strong>of</strong> macrophages and <strong>the</strong> virulence
properties <strong>of</strong> <strong>the</strong>se specific MAP strains.
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