Proceedings of the 10th International Colloquium on Paratuberculosis

#215 Development <strong>of</strong> an antigen delivery system for elicitation <strong>of</strong> protective immune response
against Mycobacterium avium subsp. paratuberculosis using attenuated Salmonella
enterica serovar Typhimurium
Yung Fu Chang, Subhash Chandra, Jenn-Wei Chen, Sen Liu, Tzu-Yi Ma, Maria A. P. Moreira,
College <strong>of</strong> Veterinary Medicine, Cornell University, USA
Background: Mycobacterium avium subspecies paratuberculosis (MAP), <strong>the</strong> causing agent <strong>of</strong> Johne’s disease
in cattle and o<strong>the</strong>r ruminants has adversely affected <strong>the</strong> production and economy <strong>of</strong> dairy industry. As being intracellular
pathogens similar to M. tuberculosis, MAP-infected host requires essentially T-cell immunity against
MAP to eliminate its infection. Therefore to elicit T-cell immunity by a vaccine, <strong>the</strong> cytosolic antigen delivery is
considered a prerequisite.
Methods: Attenuated Salmonella engineered to produce heterologous proteins present a versatile tool
to deliver <strong>the</strong> targeted antigen(s) into cytosol <strong>of</strong> macrophage and into o<strong>the</strong>r antigen presenting cells using its
type III secretion system. Hence we, by using homologues recombination method: λ phage mediated, have
made deletion mutant <strong>of</strong> aroA, and yeJ, <strong>of</strong> serovar Typhimurium, Newport and Dublin. Fur<strong>the</strong>r we have tested
<strong>the</strong>se strain to deliver MAP antigens Ag85A, Ag85B, SOD and 74F into culture medium via its type III secretion
system. To evaluate delivery efficacy <strong>of</strong> MAP antigens by attenuated Salmonella, we have made several
truncated constructs <strong>of</strong> Ag85A, Ag85B, SOD and 74F and fusion Ag85A -SOD C 1-72-Ag85B gene with sopE104 a
C
N-terminal fragment sopE <strong>of</strong> Salmonella and sopE promoter and compared <strong>the</strong> delivery efficacy by attenuated
Salmonella strains.
Results: Our primary data show that truncated fragments and fusion <strong>of</strong> two or <strong>the</strong>re protective antigens;
Ag85A, Ag85B, SOD and 74F were expressed well by Salmonella (delta aroA+yeJ) and its promoter, and secreted
into culture medium via type III secretion system.
Conclusion: Attenuated Salmonella (delta aroA+yeJ) strains are efficient tool for targeted antigen delivery
<strong>of</strong> MAP antigen to elicit T cell immunity. By using type III secretion system <strong>of</strong> Salmonella a T cell epitope or
whole optimized antigen or many fused T-cell antigens can be delivered into cytosol to induce protective immunity.
Thus Salmonella delivery system could be used to develop an effective vaccine against MAP .
#221 Comparative analysis <strong>of</strong> transcriptome changes in bovine macrophages infected with
SuperShedding strains <strong>of</strong> Mycobacterium avium subspecies paratuberculosis
Edward Alan Kabara, Paul Coussens, Michigan State University, USA
Mycobacterium avium subspecies paratuberculosis is <strong>of</strong>ten spread between animals through ingestion <strong>of</strong>
feces-contaminated food. This mode <strong>of</strong> contamination is <strong>of</strong> substantial concern when considering a subgroup
<strong>of</strong> MAP called SuperShedders. Infection with <strong>the</strong>se MAP strains can increase <strong>the</strong> fecal colony load <strong>of</strong> <strong>the</strong>
animal over 1000 fold <strong>the</strong> levels observed in infection with o<strong>the</strong>r strains <strong>of</strong> MAP. Previous research in this area
has shown that SuperShedders share many common transcriptome changes in infected macrophages when
compared to <strong>the</strong> transcriptome <strong>of</strong> macrophages infected with o<strong>the</strong>r MAP strains. The objective <strong>of</strong> this research
is to better understand what changes occur in macrophages infected with SuperShedders that leads to higher
fecal colony loads. Through careful reexamination <strong>of</strong> previously collected microarray data on experimentally
infected monocyte-derived macrophages, we found 78 bovine genes that were significantly differentially
regulated in SuperShedder infected macrophages when compared to o<strong>the</strong>r MAP strains. DAVID s<strong>of</strong>tware, a
program designed to examine functional groupings, was used to study enrichment in several annotation categories.
We found significant differences in lymphotoxin and glucose transport regulation. Several genes were
selected from <strong>the</strong> DAVID results. These genes were tested for alternative expression via RT-qPCR validation <strong>of</strong>
RNA extracted from monocyte-derived macrophages infected with 10 strains <strong>of</strong> MAP. Two strains used in this
study are SuperShedders. Based on <strong>the</strong>se results, we hypo<strong>the</strong>size that regulation <strong>of</strong> glucose transportation
and lymphotoxin regulation play import roles in MAP proliferation and fecal colony load.
142