Proceedings of the 10th International Colloquium on Paratuberculosis

Missouri). Serum from a cow with documented Johne’s disease constituted <strong>the</strong> positive
control. Final analytical readings were done at 24 and 48 hours. The appearance <strong>of</strong> one or
more clearly definable precipitation lines before or at 48 hours constituted a positive result.
The AGID tests were done with both nonabsorded and Mycobacterium phlei absorbed sera.
Pre-absorbed ELISA test: Test sera were pre-absorbed with M. phlei. The ELISA results
were calculated from wavelength readings at optical density (OD) 405 nm. All readings less
than 1.6 OD were considered negative; readings between 1.5 and 1.9 OD were deemed
suspicious/inconclusive; and readings <strong>of</strong> 2.0 to 2.5 OD were called low positive. A high
positive was any reading 2.6 OD or above.
RESULTS
Frequency <strong>of</strong> double precipitation bands within <strong>the</strong> AGID test: Of <strong>the</strong> 71 individual animals
identified having a positive AGID test, 13 (18%) exhibited, at one point or ano<strong>the</strong>r, a double
immunoprecipitation band.
Relationship <strong>of</strong> double precipitation bands to corresponding ELISA optical density readings:
The mean ELISA optical density reading (OD) for <strong>the</strong> 13 cows whose AGID test
demonstrated a double precipitation band was 3.88 with a range from 2.3 to 5.8. Only one
serum had an OD reading <strong>of</strong> less than 2.6.
Single, primary precipitation bands were not tightly correlated with <strong>the</strong> presence <strong>of</strong> a
diagnostic Map ELISA test. Nine <strong>of</strong> <strong>the</strong> 71 (12.7%) cases in which a double band occurred
had a negative UF ELISA test. Ano<strong>the</strong>r 10 (14%) sera were deemed to be, at best,
suspicious (Table 1). The greatest number <strong>of</strong> cases correlated with strongly positive Map
ELISA tests 39/71 (54.9%). Serial observation in three cows demonstrated <strong>the</strong> unmasking <strong>of</strong>
a second immunoprecipitation band when <strong>the</strong> corresponding ELISA titer became markedly
elevated above that value at which <strong>the</strong> primary immunoprecipitation band occurred (Table 2)
When AGID positive sera were absorbed against varying concentration <strong>of</strong> M. plea, <strong>the</strong>
results for <strong>the</strong> immunoprecipitation bands remained unchanged.
Table 1. Distribution <strong>of</strong> serum Map ELISA titers among Holstein cows with a positive AGID
test
ELISA Titer Number <strong>of</strong> Cows (Percentage)
0 – 1.5 – negative 9 (12.7%)
1.6 – 1.9 – suspicious 10 (14%)
2.0 – 2.5 - positive 13 (18.3%)
Greater than 2.5 – strong positive 39 (54.9%)
DISCUSSION
The common absence <strong>of</strong> an immunoprecipitation band in <strong>the</strong> face <strong>of</strong> a high Map ELISA test
result has been largely ascribed to <strong>the</strong> amount <strong>of</strong> specific antibody required for an
immunoprecipitation band in contrast to <strong>the</strong> significantly smaller amount <strong>of</strong> specific antibody
that is required for a diagnostic Map ELISA reading.
Unlike <strong>the</strong> primary immunoprecipitation band, <strong>the</strong> second immunoprecipitation band appears
to have a positive correlation with markedly elevated Map ELISA test readings. This
observation implies that <strong>the</strong> second immunoprecipitation band identifies predominantly <strong>the</strong>
same Map antigen complexes that are used in <strong>the</strong> Map ELISA test.
The resultant interpretation <strong>of</strong> this data was that a better Map ELISA test could be developed
using selected antigenic subsets embedded in <strong>the</strong> primary immunoprecipitation band.
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