Proceedings of the 10th International Colloquium on Paratuberculosis

#167 Analysis <strong>of</strong> Mycobacterium avium subsp. paratuberculosis antigens used in an in-house
enzyme-linked immunosorbent assay for Johne’s disease
Shigetoshi Eda1 , M. Cathy Scott1 , John P. Bannantine2 1 University <strong>of</strong> Tennessee Knoxville, USA; 2 National Animal Disease Center, USDA-ARS, Ames, Iowa, USA
We developed a novel enzyme-linked immunosorbent assay (ELISA), called EVELISA, for <strong>the</strong> detection <strong>of</strong>
Mycobacterium avium subsp. paratuberculosis (MAP) infection in cattle which showed a higher sensitivity
than current ELISA test. We previously reported that <strong>the</strong> use <strong>of</strong> heat-killed M. flavascens for pre-absorption <strong>of</strong>
cross-reactive antibodies improved specificity <strong>of</strong> <strong>the</strong> EVELISA. Fur<strong>the</strong>r, we showed that EVELISA antigens can
also be used for detection antibodies against MAP in a micr<strong>of</strong>luidic system (<strong>the</strong> 9th <strong>International</strong> <strong>Colloquium</strong> on
Paratuberculosis). The EVELISA uses surface antigens removed from <strong>the</strong> bacteria with 80% ethanol followed
by gentle vortex agitation. To test <strong>the</strong> hypo<strong>the</strong>sis that <strong>the</strong> high sensitivity <strong>of</strong> EVELISA was due to MAP-specific
cell surface antigens, we used thin layer chromatography (TLC) to compare <strong>the</strong> extract <strong>of</strong> MAP against that <strong>of</strong>
a series <strong>of</strong> bacteria that are ei<strong>the</strong>r closely related to MAP or known to cause false positive antibody reaction
in commercially available ELISAs. Surface antigen extracts were fractionated using <strong>the</strong> Folch wash method
followed by cold-acetone precipitation and loaded onto aluminum-backed silica-gel-60 plates, <strong>the</strong>n developed
with a series <strong>of</strong> solvents, including ceric sulphate/ammonium molybdate, ninhydrin and α-naphthol solution.
MAP-specific molecules were detected in <strong>the</strong> chlor<strong>of</strong>orm fraction <strong>of</strong> <strong>the</strong> Folch wash and were not precipitated
by <strong>the</strong> cold acetone treatment. Fractions obtained after <strong>the</strong> Folch wash and acetone precipitation were tested
for anti-MAP antibody detection in an ELISA format. We found that none <strong>of</strong> <strong>the</strong> fractions could achieve <strong>the</strong>
level <strong>of</strong> EVELISA sensitivity, indicating that <strong>the</strong> cocktail <strong>of</strong> antigens in EVELISA was <strong>the</strong> key for EVELISA’s high
sensitivity. The protein components contained in <strong>the</strong> aqueous fraction are being analyzed by using polyclonal
antibodies produced against <strong>the</strong> MAP extract.
#186 Identification <strong>of</strong> Mycobacterium avium subsp. paratuberculosis in fecal specimens by
culture, real-time PCR, and nested PCR
Ching Ching Wu, J. Elliot Williams, Tsang Long Lin, Gilles R. G. Monif
Purdue University, USA; University <strong>of</strong> Florida, USA; Infectious Diseases Incorporated, USA
Direct fecal Mycobacterium avium subsp. paratuberculosis (Map) real-time PCR based on heat shock protein
gene (hsp) combining DNA extraction procedures with PCR has been commercially available (Tetracore
VetAlert TM Johne’s Real-Time PCR, Rockville, MD). Direct fecal Map nested PCR based on insertion sequence
(IS)1311 has been recently developed (FecaMap ® , Infectious Diseases Incorporated, Bellevue, Nebraska).
These two PCR tests were compared to culture for direct detection <strong>of</strong> Map in fecal specimens obtained from
two dairy herds in Florida. All three tests were applied to 327 fecal specimens and <strong>the</strong> results were analyzed
and compared. Direct fecal real-time PCR was performed at Purdue University and direct fecal nested PCR
was carried out at University <strong>of</strong> Florida. Two hundred and sixty-eight fecal specimens were negative for Map in
all three tests. Of <strong>the</strong>se negative fecal cultures, 14 were positive in both <strong>the</strong> real-time and nested PCR tests.
Twelve real-time PCR tests positive for Map occurred in <strong>the</strong> absence <strong>of</strong> confirmation by <strong>the</strong> fecal culture or
nested PCR. Seven positive nested PCR tests were recorded without correspondence in <strong>the</strong> fecal culture or
real-time PCR. Fifty-nine fecal specimens were culture positive for Map. The results <strong>of</strong> direct fecal real-time
and nested Map PCR agreed with those <strong>of</strong> culture in 13 fecal samples. The overall correlation <strong>of</strong> culture with
direct fecal real-time PCR and direct fecal nested PCR was 24/59 (40.8%) and 20/59 (34%), respectively.
When <strong>the</strong> data were corrected for <strong>the</strong> 14 presumed false negative cultures, direct fecal real-time and nested
Map PCR identified 38/73 (52%) and 34/73 (47%) cultures positive for Map, respectively. The results <strong>of</strong> direct
fecal real-time and nested Map PCR are comparable and reflect a difference in <strong>the</strong> amount <strong>of</strong> sample used in
<strong>the</strong> test, as <strong>the</strong> nested PCR used 1/<strong>10th</strong> <strong>the</strong> amount <strong>of</strong> feces processed for <strong>the</strong> real-time PCR.
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