Proceedings of the 10th International Colloquium on Paratuberculosis

#67 The γδ cells as marker <strong>of</strong> non-seroconverted cattle naturally infected with Mycobacterium
avium subspecies paratuberculosis
Fateh A Badi, Amal I Al Haroon, Ahmed Mohammed Alluwaimi
College <strong>of</strong> Veterinary Medicine, King Faisal University, Saudi Arabia; Immunology Unit, Regional diagnostic
laboratory, Ministry <strong>of</strong> Health, Saudi Arabia
Early diagnosis <strong>of</strong> MAP infection is a pressing need that enables efficient intervention with <strong>the</strong> spread <strong>of</strong> MAP
infection in herds. Hence, study <strong>of</strong> lymphocyte subsets and <strong>the</strong>ir expressed adhesion molecules could contribute
in defining a distinct diagnostic marker (or markers) at <strong>the</strong> subclinical period <strong>of</strong> <strong>the</strong> infection that could
in turn facilitate <strong>the</strong> development <strong>of</strong> effective diagnostic approach. In accordance with this objective, milk and
blood samples were collected from two groups <strong>of</strong> cattle naturally infected with MAP and <strong>the</strong>ir corresponding
negative controls. Group(C) comprised 3-4 year-old ELISA negative/PCR positive-cattle that were considered
as subclinical seronegative low shedder group (early stage). Group (A) included 6-8 year-old ELISA positive
cattle, which were considered as a clinical seropositive group (late stage). Flow cytometry <strong>of</strong> B cells, CD8 + ,
CD4 + and gd cells and <strong>the</strong> adhesion molecules CD44 + , CD62L, LFA-1 and LPAM-1 indicated increase ingroup
A. CD4 + and B cells levels were higher in blood than milk <strong>of</strong> group A, and significant expression <strong>of</strong> CD44 + in
blood and milk and LPAM-1 in blood only. The CD8 + cells count in milk was higher than blood in <strong>the</strong> late stage.
The peculiar feature <strong>of</strong> <strong>the</strong> early stage (group C) was <strong>the</strong> high level <strong>of</strong> gd cells in <strong>the</strong> blood and milk, with tendency
to express high level <strong>of</strong> CD62L. Compelling evidence could support <strong>the</strong> assumption that <strong>the</strong> dominant
gd cells at early stage <strong>of</strong> MAP infection could be <strong>of</strong> CD8CD2 - WC+1 + phenotype. gd cells appear as promising
markers in defining early changes <strong>of</strong> MAP infection due to <strong>the</strong>ir important role in priming innate and cell mediated
immunity. Possible utilization <strong>of</strong> this peculiar changes in <strong>the</strong> gd cells level in <strong>the</strong> early diagnosis <strong>of</strong> MAP
infection should be <strong>the</strong> subject <strong>of</strong> fur<strong>the</strong>r research.
#70 Pr<strong>of</strong>iling immune responses to <strong>the</strong> PE and PPE protein families in naturally MAP infected
cattle
Nick Alexander Mackenzie, Jeroen De Buck
University <strong>of</strong> Calgary, Canada
The PE and PPE proteins make up two families <strong>of</strong> polymorphic proteins specific to mycobacteria. These proteins
are composed <strong>of</strong> conserved N-terminal domains and highly variable C-termini. While <strong>the</strong>se proteins make
up a significant portion <strong>of</strong> <strong>the</strong> coding capacity <strong>of</strong> pathogenic mycobacteria, <strong>the</strong>ir functional roles are largely
unknown. Of <strong>the</strong> current hypo<strong>the</strong>ses, <strong>the</strong> most widespread is that <strong>the</strong>y play a role in antigenic variation. Their
polymorphic nature, as well as <strong>the</strong>ir observed immunogenicity in mycobacterial infections makes <strong>the</strong>m interesting
diagnostic and vaccine candidates. Indeed, if <strong>the</strong>se proteins were involved in antigenic variation, we would
expect to see patterns <strong>of</strong> immune responses emerge to <strong>the</strong>se proteins following disease progression. To this
end, we are developing and testing a novel protein array to study bovine immune responses to <strong>the</strong> 10 MAP
PE proteins, <strong>the</strong> 36 MAP PPE proteins, and a panel <strong>of</strong> previously identified antigens, including: Map0857c,
Map0862, Map1087, Map1204, Map1272c, Map1637c and Map1730c. This represents <strong>the</strong> first time a protein
array approach has been used to study immune responses against polymorphic protein families in <strong>the</strong>ir entirety.
Representatives <strong>of</strong> conserved regions <strong>of</strong> each PE and PPE subfamily were produced recombinantly in
Eschericia coli and purified by nickel affinity purification alongside all variable regions <strong>of</strong> <strong>the</strong> MAP PE/PPE repertoire,
allowing demonstration <strong>of</strong> specific immune responses. Animals in different stages <strong>of</strong> disease with varying
ELISA, fecal and tissue culture results (n=16) were tested in Western and dot blot assays as well as negative
samples (n=8) from non-infected controls. Using this approach we identified variable immune responses
between animals and categories <strong>of</strong> infection. This information will be invaluable in <strong>the</strong> identification <strong>of</strong> early
disease markers and provides clues as to <strong>the</strong> role <strong>of</strong> <strong>the</strong>se proteins in MAP pathogenesis and host responses.
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