Significance of Single and Double Map
Agar Gel Immunodiffusion Precipitation
Bands
Gilles R. G. Monif1, 2J. Elliot Williams2
Infectious Diseases Incorporated, Bellevue, NE

1

University of Florida College of Veterinary Medicine,
Department of Infectious Diseases and Pathobiology, Gainesville, FL

2

KEY WORDS: Cows, MAP ELISA, IPBand, necropsy
ABSTRACTA retrospective analysis was performed todetermine the significance of single anddouble MAP AGID immunoprecipitation(IP) bands. Of the 58 sera that exhibited asingle MAP IP band, 15.5% lacked corresponding anti-MAP antibodies. Of the 71
sera that tested positive in a MAP AGID
test, 13 (18%) exhibited a second IP band.
The second MAP IP band occurred only
when the OD test reading exhibited a furtherincrease. The test data infers that the first
IP band is caused by a dominant cell-wall
mycobacterium antigen. The second IP bandis specific for MAP isolates. The presence ofa positive MAP AGID IP band in an animal
with necropsy documented Johne’s disease
and no anti-MAP antibodies argue for theexistence of polyphonic genomic variant of
MAP as being a causes of Johne’s disease.
INTRODUCTION
Agar gel immunodiffusion test (AGID)
was initially developed as a screening test
for the detection of Johne’s disease in flock
ruminants. While AGID positivity has anapparent 100% specificity with the presenceof chronic granulomatous enteritis (Johne’sdisease), it has only 5% sensitivity in detect-190

ing infected cows1-3. The development ofabsorbed enzyme-linked immunoabsorbent
assay (ELISA) tests and documentation of
their great sensitivity have led to the relative
abandonment of AGID in herd management
schema of dairy herds1-2.
Cows with documented Johne’s disease
can have a diagnostic MAP ELISA reading
and not have a positive AGID test. Conversely, cows with necropsy documented
Johne’s disease may have a positive immunoprecipitation band and not have a diagnostic MAP ELISA titer. Occasionally, a secondimmunoprecipitation band is identified in
AGID tests.
The purposes of this report are to describe the relationship between the single
and the second immunoprecipitation bands
as they relate to their corresponding MAPELISA titers.
MATERIALS and METHODS
Study Populations: The AGID test data,
derived from a five year herd management
program which was implemented betweenJanuary 2001 and January 2006, were reviewed to identify the number of cows witha positive AGID test. Immunoprecipitation
bands were identified in sera obtained from71 cows.
Study Material: The corresponding MAP
Vol. 13, No.3, 2015 • Intern J Appl Res Vet Med.

ELISA titers were identified and tabulated
for the cows with a single and double immunoprecipitations bands. Multiple observations on three cows were available inwhich the presence or absence of the second
precipitation band varied.
Nineteen single IP band sera camefrom cows that had been necropsied at theUniversity of Florida College of Veterinary
Medicine. In each case, the histopathology
confirmed Johne’s disease due to acid-fastbacilli.
AGID: Petri dishes were poured with sterile
saline 1% agarose prepared in 0.1 M TrisHCL buffer at pH 10. Well distances were
8 mm. Well sizes were 4 mm for the six peripheral wells and 3 mm for the central well.
The peripheral well received 45 ul of the test
serum. The central well was inoculated with
35 ul of a crude protoplasmic antigen (Allied Monitor, Fayette Missouri). Serum froma cow with documented Johne’s disease constituted the positive control. Final analyticalreadings were done at 24 and 48 hours. The
appearance of one or more clearly definable precipitation lines before or at 48 hours
constituted a positive result. The AGID
tests were done with both non-absorbed andMycobacterium phlei absorbed sera.
Pre-absorbed ELISA Test: Test sera werepre-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 considerednegative; readings between 1.5 and 1.9 OD
were deemed suspicious/inconclusive; andreadings of 2.0 to 2.5 OD were called low
positive. A high positive was deemed to be
any reading of 2.6 OD or above.
RESULTS
Pre-absorbed MAP ELISA Test: The MAP
ELISA test had USDA certification for diagnostic use. Protoplastic protein derivativefrom IS 900 MAP isolate (Allied Monitor)
constituted the test’s antigen core array. Test
sera were pre-absorbed with M. phlei. TheELISA results were calculated from wavelength readings at optical density (OD) 405
nm. All readings less than 1.5 OD were conIntern J Appl Res Vet Med • Vol. 13, No. 3, 2015.

sidered negative; readings between 1.6 and1.9 were deemed suspicious/inconclusive;and readings between 2.0 to 2.5 OD werecalled low positives. A high positive reading
was a value 2.6 OD or above.
Overall Relationship of a Single IP
Band to MAP ELISA Test Reading: Single
IP bands were not tightly correlated to thecorresponding MAP ELISA. Of the 54
sera identified as being AGID test positive
with a single IP band, 9 (15.5%) lacked thedemonstrable presence of anti-MAP antibodies. Another 10 (17.2%) had inconclusive
readings. Thirty-eight of the 54 exhibited astrong positive OD reading.
Frequency of a Double IP Band within
AGID Positive Tests: Of the 71 sera identified as having a positive MAP AGID test 13(18%) had a double band.
Relationship of a Double IP Band to its
Corresponding MAP ELISA Optical Density
Reading: The mean MAP ELISA opticaldensity reading for the 13 sera with a double
IP band was 3.88 with a range from 2.3 to5.8. Only one serum had an OD reading ofless than 2.6.
Serial OD Readings on Sera from a Cow
Exhibiting a Double IP Band: Serial MAP
ELISA OD readings from the same cow
were available in three instances.
Cow #4151’s initial sera exhibited asingle IP band at an OD reading of 2.32.
When she developed clinical disease andher OD reading increased to 4.1, a second IPband was present.
Cow #3882’s sera exhibited a single IPband at an OD reading of 3.09 When her OD
reading increased to 4.96, a second IP band
was present. The second band disappeared
when the OD reading dropped below 4.0.
Six AGID and MAP ELISA test results
were available for Cow #3917. Three sera
obtained over a seven day period exhibiteda double IP band. Her OD readings were5.5, 5.0 and 4.1. When her OD readings fell
below 4.0, the next three consecutive AGID
tests collected over a three day period demonstrated the presence of a single IP band.

191DISCUSSION
It had long been presumed that both theMAP AGID and MAP ELISA tests identified
similar or corresponding antigens. The absence of an immunoprecipitation band in theface of a high MAP ELISA test reading had
been largely ascribed to the amount of MAP
antibody required for immunoprecipitationin contrast with that required for a positive
ELISA test. Analysis of the double band
phenomena challenges this line of reasoning. What is inferred is that the primary/first
immunoprecipitation band is due to antigenantibody complex elicited by an antigenicarray common to mycobacteria that are derived from Mycobacterium avium (MAA) or
Mycobacterium hominissuis5. It should havebeen anticipated that in the MAP’s evolution
from MAA, disease, the procss would haveproduced pathogenic polymorphic genomic
variants. IS900 MAP isolates are a causeof Johne’s disease, but the sole cause of theclinical/pathological presentation termed
Johne’s disease. Fifteen point five percentof sera with a single IP band lacked antibodies that identified IS 900 MAP.

192

REFERENCES
1.

2.

3.

4.

5.

Sockett D. C., Conrad T. A., Thomas C. B., Collins
M. T.: 1992. Evaluation of four serological tests
for bovine paratuberculosis. J. Clin. Microbiol.
30:1134-1139
McNab W.B., Meek A. H., Duncan J. R., Brooks B.
W., Van Dreumel A. A., Martin S. W., Nielsen K.
H., Sugden E. A., Turcotte C.: 1991. An evaluationof selected screening tests for bovine paratuberculosis. Can. J. Vet. Med. 55:252-259
Sergeant E. S. G., Marshall D. J., Eamens G. J.Kearns C. Whittington R. J..: 2003. Evaluation ofan absorbed ELISA and an agar-gel immunodiffusion tests in ovine paratuberculosis in sheep inAustralia. Prevent. Vet. Med. 61:235-2484
Williams E., Monif G. R. G., Buergelt C. D.: 2008.
Comparative analysis of different MAP ELISA
tests. The Paratuberculosis Newsletter- March:
page 7Frothingham C. R.: Evolutionary bottlenecks in theagents of tubercu¬losis, leprosy and paratuberculosis. Med. Hypothesis 1999; 52: 95-99

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