An alternative serological test for the
diagnosis of Johne’s disease
Student: Leslie Nekkers
Studentnumber: 3347710
Supervisors Calgary, Canada: Rienske Mortier & Jeroen De Buck
Supervisor Utrecht, the Netherlands: Susanne Eisenberg
Date: 8th of June, 2012
An alternative serological test for the diagnosis of Johne’s disease
Index
Summary
Introduction
Materials and methods
Results
Discussion
Acknowledgements
Appendices:
Appendix I
Appendix II
Appendix III
Appendix IV
References
p. 2
p. 3-4
p. 5-8
p. 9-17
p. 18-19
p. 20
FCM protocol
ELISA protocol
Table for test characteristics
Statistics
p. 21
p. 22
p. 23-24
p. 25-26
p. 27
Page | 1
An alternative serological test for the diagnosis of Johne’s disease
Summary
Johne’s disease (JD), also known as paratuberculosis, causes a chronic intestinal infection
in ruminants and is caused by Mycobacterium avium subsp. paratuberculosis (MAP).
Due to Johne’s disease, there is a decrease in milk yield, which causes significant
production losses.
Since there is no cure available for animals infected with MAP, the main focus of
management programs for Johne’s disease is prevention of infection and detecting
infected animals with diagnostic tests. The issue with the current serological tests is that
they have a low sensitivity.
Lately, a paper has been published by Eda et al. (2005) in which a new test has been
suggested: the Flow Cytometry Method (FCM).
The objective of this study was to make a comparison between the described FCM and the
ELISA (IDEXX) to determine the sensitivity and specificity of the FCM as an alternative
diagnostic test for Johne’s disease.
The hypothesis was that the FCM would have a sensitivity and specificity similar to the
ELISA (IDEXX).
FCM was optimized in our lab using samples from an infection trial as well as from herds
with a known JD status. The results measured by flow cytometry are displayed in graphs.
When a sample is negative, a peak in the histogram can be seen above the zero on the x axis, and thus the histogram will be on the left side of the graph. When a sample is
completely positive, the histogram will shift towards the right side of the graph.
A wide variety of positivity can be seen in different samples.
It was decided to use a cut-off of 70% for the FCM, since this cut-off produced the highest
sensitivity (93%) and the highest specificity (84%) for the FCM. When the FCM was
compared to the ELISA (IDEXX), the kappa value had an outcome of 0,7760. The
positive predictive value is 85% and the negative predictive value is 93%.
The FCM is a good candidate to be developed into a sensitive diagnostic test for detection
of MAP infection, but further research still needs to be conducted.
Page | 2
An alternative serological test for the diagnosis of Johne’s disease
Introduction
Johne’s disease, also known as paratuberculosis, causes chronic enteritis in ruminants and
is caused by Mycobacterium avium subsp paratuberculosis (MAP).¹ Johne’s disease is a
chronic intestinal infection, causing lesions and thickening of the mucous membrane and
submucosa of the intestinal wall. 2
Annually the Canadian dairy industry loses about 15 million CND$ due to Johne’s
disease. The major economic losses are the result of the symptoms that this chronic
disease causes; the loss of weight and the reduced milk production, owing to the decreased
protein uptake in the gut. 3 Also, the animals have a decreased slaughter value, increased
calving intervals and suffer from infertility. 4
The progress of Johne’s disease can be divided in four phases. The first phase is a long
latent phase, called ‘Silent infection’. 2 In this phase it is suspected that the animals are
infected while they are still calves, but that there are no clinical symptoms visible yet and
there is an indication that they are shedding very low quantities of MAP. Due to the low
quantities it is hard to detect to shedding in this phase. ⁵ In this phase there is also no
detectable humoral response present. 2
In the second phase, the ‘Carrier stage’, there are still no clinical symptoms, but
intermittent shedding of MAP through the feces is possible. It should be possible to detect
a humoral response with serum ELISA as well, in this phase. 2
In the third phase, the ‘Clinical stage’, the clinical symptoms become very clear: diarrhea,
weight loss, detectable shedding of MAP and positive results from the serological tests in
most cases.
In the fourth phase, the ‘Advanced clinical stage’, the animals are lethargic, weak and
have intermandibular edema due to the hypoproteinemia through the decreased protein
uptake from the intestinal lumen. ⁵ This stage always results in death because no treatment
is available for Johne’s disease.
Since there is no cure for animals infected with MAP, the main focus of management
programs for Johne’s disease is prevention of infection of susceptible animals and
detecting infected animals.
For the latter part of the management programs, there should be diagnostic tests available
which have a high sensitivity and specificity for an infection with MAP.
Until now it was believed that calves up to six months were susceptible to MAP infection,
so management protocols are all based on this assumption. There is actually little research
conducted on this subject to support this assumption. To test this theory, an infection trial
is being conducted at the UofC. In this trial calves, collected at the moment of birth, are
infected with MAP at different ages and with different doses to learn more about age
dependent susceptibility and the influence of different doses on establishing MAP
infection (Age and dose dependent susceptibility to MAP infection in dairy calves,
Rienske Mortier, Herman Barkema, Jeroen De Buck). The research on an alternative
serological test for the diagnosis of Johne’s disease will be conducted as a part of this
infection trial.
Diagnosing Johne’s disease is often proven difficult because of a low sensitivity of the
available serological tests, due to the chronic nature of the disease in the gut, which makes
it difficult to detect the disease systemically. The other available diagnostic tests, which
do have a higher sensitivity and specificity, are harder to execute or ask more experience.
Page | 3
An alternative serological test for the diagnosis of Johne’s disease
The diagnostic tests that are available are fecal culture, IFN-γ ELISA (this one is noncommercial) and serum and milk ELISA. After euthanasia there also are tissue culture
(which is the gold standard) and histology available to form the diagnosis.
Fecal culture is one of the most commonly applied tests for detecting MAP because it has
a high specificity, which is considered to be 100%, if it is confirmed with PCR. The
sensitivity of fecal culture for detection of infected animals is in the range 65-70%,
depending on the stage of infection. 6 The disadvantage of fecal culture is that it only gives
a positive result when there are MAP-bacteria present in the feces. But even when there
are bacteria present in the feces, it is not a guarantee that the animal is actually infected,
since infected material can pass the digestive tract without infecting the animal itself . This
would lead to falsely assuming that the animal has Johne’s disease.
The animal can also show intermittent shedding. Thus a fecal culture can show up
negative, when an animal is actually infected but not shedding at that point of time.
Another disadvantage is that fecal culture takes 5 to 16 weeks incubation time. 7 The
following three tests are based on detecting a MAP specific immune response.
The IFN-γ ELISA’s sensitivity for detection of infectious animals varies from 0.13 to
0.85.6
It proves to be helpful to detect subclinical infected cows, because the only cellular
immune response that can be measured in a subclinical infected cow is the IFN- γ
production of mononuclear cells. The problem with the IFN- γ test is that it is not often
applied in field research due to the circumstances in which it should be executed. This
because the test has to be performed on fresh blood samples in the lab, within 6 hours of
sampling and because the interpretation of results is challenging. 4
The last test is the ELISA-test (IDEXX) for antibodies in serum with a specificity of 97%
and a sensitivity of 60% according to the manufacturers. 8
The great range in specificity and sensitivity for the ELISA’s comes from the fact that
there are so many different (commercial and in-house) ELISA’s available. The main
reason though, is that the ELISA is usually compared to different gold standards.
Sometimes the fecal culture is used, sometimes tissue culture. Sensitivity of a test always
depends on the test it is compared with. There is also a difference in how sensitive and
specific the tests are, depending on the age of the affected animal and the antigenpreparations.6
Recently a new experimental method for detecting Johne’s disease has been developed by
Eda et al. (2005). This method is called the flow cytometry method (FCM).
According to Eda et al. all serological tests up to now only use whole cell lysates or
portions of MAP and never the whole bacilli to detect a MAP infection. Based on this
fact, Eda et al. came up with the hypotheses that intact MAP bacilli with a complete
repertoire of surface antigens would be a more suitable method for detecting a MAP
infection.
Not only could be concluded from this study that FCM has a higher sensitivity (95,2%)
and specificity (96,7%) than the already existing serological tests and fecal culture, it also
appeared to be capable to detect an infection of MAP far before any other test could (6-44
months earlier for fecal culture). 7
The objective of this research paper is to make a comparison between the described FCM
and the ELISA-test for antibodies in serum of blood samples.
The hypothesis is that the FCM will have a specificity and sensitivity that are similar to
those of the ELISA.
Page | 4
An alternative serological test for the diagnosis of Johne’s disease
Materials and methods
To test the FCM and eventually compare it with the ELISA, serum samples were used
from two different sources:
One part came from the barn that is part of the ‘Age and dose dependent susceptibility to
MAP infection in dairy calves’ trial. These serum samples came from the first run of the
trial and were already tested with the ELISA.
The other part of the serum samples came from farms around Calgary, which were tested
for the ‘Age and dose dependent susceptibility to MAP infection in dairy calves’ trial.
Materials and methods for the barn:
Animals
The animals used for the trial ‘Age and dose-susceptibility to experimental MAP infection
in dairy calves’ are male Holstein-Friesian calves, who all were collected right after birth
from heifers or second year calvers only and avoiding contact with the dam or feces at all
times.
The total sample size of the study is 33 animals, but only a part of the serum samples of
this group were tested with the FCM.
The calves were assigned into different groups according to age and dose. Because
housing facilities were limited, the study was divided into two repeated runs in ord er to
achieve maximal potential.
Housing
The animals were individually housed in custom made housing units, which included the
pen which contained the calf, and a marked zone in front of the pen, where bootdip, boots,
coveralls and gloves for that particular calf were provided. These items in the marked
zone were not switched between calves, but stayed with the same calf during his stay in
the housing unit, to prevent cross contamination at all times.
The animals will stay here until they are 17 months of age and necropsy will be performed
on the calves.
Experimental infection
The inoculum contained a virulent MAP strain, isolated from a clinical case (Cow 69),
which showed an identical IS900 – RFLP profile as the strain K10, which is the reference
strain. The MAP strain was grown in BD Difco Middelbrook 7H9 broth (BD).
The culture was then tested on viability with the Live/Dead stain, dose and contamination
with real-time PCR. The inoculum was given to the calves with a syringe at the back of
their tongue.
Sampling
Feces, whole blood and serum, and urine samples were taken on a regular basis. The
samples were routinely collected every 12 weeks before infection. In the first month after
infection, samples were taken every week. After the first month, the samples were
collected monthly, up to necropsy.
Serum samples were tested with the IDEXX ELISA and results were available upon
arrival for analysis.
Page | 5
An alternative serological test for the diagnosis of Johne’s disease
Materials and methods for the farms:
Sampling
The farms that were tested around Calgary were sampled twice; once in 2009 and once in
2011.
Samples were taken from 24 farms located north and south of Calgary to identify their
Johne’s disease status. Samples taken were serum, fecal and milk samples and these were
processed in the Johne’s disease Bacteriology lab at the University of Calgary. Serum and
milk samples were analyzed with Pourquier and IDEXX ELISA™ (Institut Pourquier,
Montpellier, France and IDEXX Laboratories, Inc., Westbrook, ME), fecal samples were
cultured in liquid medium and presence of MAP bacteria was confirmed with IS900 PCR.
Based on the outcome of these samples, twenty low prevalence farms were selected to be
the source of the calves for this study.
From this group 44 positive serum samples were taken and matched with 44 negative
samples according to age and days in milk. These 88 samples were used to calculate the
test characteristics of the FCM. All 88 samples had been tested previously with the ELISA
and results were available for analysis upon arrival.
Serum ELISA
The serum ELISA test used for analyzing the samples is the IDEXX (IDEXX
Laboratories, Inc., Westbrook, ME), which was performed according to the instructions of
the manufacturer (see appendix II for the complete protocol). The positivity of the serum
sample, tested with ELISA, will be calculated through the s/p ratio (sample to positive
ratio).
S/p ratio = (Sample - Negative average)/(Positive average – Negative average) 8.
If the outcome is above 60%, which was the used cut-off for the ELISA, the sample is
considered positive. The outcome is always in comparison to the standard positive control
of the ELISA, so if a sample has a calculated outcome of 200%, it means that the sample
is 2 times as positive as the ELISA’s positive control.
Materials and methods for the FCM:
Study design of the Flow Cytometry Method(FCM) 9
FITC-antibody
MAP cells
Serum antibody
Fig 1. On the bottom of the
eppendorf cup there is a layer of
MAP cells (from reference strain
K10, Cow 69 or from a cultured
biofilm from the strain of cow 69)
on which the antibodies from the
serum sample will attach. Then a
FITC – labeled anti bovine IgG
antibody can be added, so that the
FCM can detect whether
fluorescence is present.
In short, MAP was cultured in a middlebrook medium, then pelleted and washed with
buffer A in a microcentrifuge tube. After these steps, the serum sample was added and
incubated and then pelleted and washed again and resuspended in a buffer containing
FITC – labeled anti bovine IgG antibody and incubated again.
The mixture for the FCM would then be analyzed by the Flow cytometer, by detecting the
fluorescence of the FITC-antibodies who did bind to the serum antibodies. (See appendix
I for the complete protocol.)
Page | 6
An alternative serological test for the diagnosis of Johne’s disease
The Flow cytometer (BD LSRII, BD Biosciences, Canada) analyzes the sample solution
through a stream of single cells, generated by the fluidics system (Fig 2.). The stream of
single cells is achieved by letting the sample solution pass through an inner sheath, while
the outer sheath contains faster flowing fluid in the same direction. This causes a massive
drag effect on the sample solution, leading it through the narrowing inner sheath. This
effect is called hydrodynamic focusing and causes the cell solution to pass the detector
cell by cell. When cells are too big or clump, the nozzle of the instrument would become
blocked, which would make it impossible to analyze the samples.
While cells are passing by the detector, information is gathered on the cells properties. In
this case, the size and the fluorescence of the cells.
A laser beam is shot at the passing cells which will scatter the light beam. The light that is
scattered forward is collected by a detector, also known as the forward scatter channel
(FSC). The FSC can be used to determine the size of the particle and to distinguish
between cellular debris and living cells.
Light measured at 90º to the excitation line is called side scatter and collected by the side
scatter channel (SSC). The SSC can provide information on the granular content of a
particle.
For detecting fluorescence, Flow cytometers use separate fluorescence (FL-) channels.
The ability of fluorochromes to accept light energy (excitation) at any given wavelength
and re-emit it at a longer wavelength (emission) makes it possible to use fluorescence
dyes in the Flow cytometer. In this experiment, by using the fluorescence dye FITC, we
were able to detect whether antibodies against MAP-cells were present.
After the light is re-emitted, it will hit a photodetector which will produce a small current.
The associated voltage is converted into electrical signals large enough to be plotted
graphically.
Visualizing the cells of interest in graphs and eliminating all other particles is an
important principle of the flow. This procedure is called gating.
The two graphs used in this particular experiment were the density plot and the singleparameter histogram.
The density plot was used to determine whether the cells that were analyzed, were
approximately all similar in size and granularity. On the axes forward vs side scatter are
projected. So in this graph it is important to see just one group of dots, to confirm that we
were only seeing MAP-cells with or without antibodies.
The single-parameter histogram was used to analyze whether a sample was positive or
negative for MAP-antibodies in the serum. On the y-axis is forward scatter on the x-axis
the fluorescence is projected. The histogram also shows the percentage of fluorescent
(positive) cells.
Page | 7
An alternative serological test for the diagnosis of Johne’s disease
Fig 2. Schematic overview of a Flow cytometer setup
Before analyzing the serum of the calves, definite negative controls would first go through
the procedure, to establish whether the machine was working correctly and to optimize the
reference values and to set the gating. The negative controls were samples with only cells
and PBS, so no serum and FITC-antibodies was added. The positive control was a definite
positive serum sample, He12.
In this experiment several conditions were tested, to optimize the standards under which
the FCM for detecting antibodies for MAP-cells can be performed. To optimize the
protocol for the FCM we tried different concentrations of TW80, cells and serum. The
concentrations that led to the best results were processed in the protocol and used as the
standard during the rest of the experiment.
Page | 8
An alternative serological test for the diagnosis of Johne’s disease
Results
Optimizing protocol FCM
For the first run with the FCM, there were four different serum samples used, of which the
following dilution series were produced:
1:1, 1:2, 1:4, 1:16 and 1:32.
The serum samples that were analyzed, were:
- C20 (Calf 20, which was part of the first run of the ‘Age and dose dependent
susceptibility to MAP infection in dairy calve’ trial. He had an s/p ratio of 249 for
ELISA, was shedding MAP in the last seven months and was positive on tissue
culture.)
- C25 (Calf 25, which stayed negative for all the tests during the whole trial and was
not infected in the trial with MAP.)
- Serum used as the positive control in the ELISA-test
- Serum used as the negative control from the ELISA-test
The negative controls used for the FCM, were:
- Only MAP-cells
- MAP-cells and serum
- MAP-cells and the FITC-labeled antibody
- PBS, serum and the FITC-labeled antibody
The cell culture used was the K10 strain.
The negative controls showed all up negative in the graphs.
The most positive reaction was seen for C20 with dilution 1:2, in which 63% of the cell
population showed fluorescence.
Fig 3. Graph of serum sample C20, dilution 1:2
As seen in the graph, the sample still only reached a positivity of 65,2%.
The same outcome, neither decisively positive nor negative, was the case for C25. C25
only reached a positivity of 7,9% at the highest dilution, the 1:32 dilution.
The positive control for ELISA serum sample showed up negative for the whole dilution
series, as did the negative ELISA serum sample.
Based on these results, it was decided to use a biofilm for the cell culture, to intensify the
washing steps and to add M. smegmatis to the serum.
The decision of using a biofilm for half of the samples and using C69 for the other half,
was based on that Eda et al. used a biofilm in their trial and seemed to get better results
with it. The C69 strain was chosen to see whether the antibodies in the serum would react
stronger to this strain than to the K10 strain, since the calves where infected with the C69
strain.
Page | 9
An alternative serological test for the diagnosis of Johne’s disease
Intensifying the washing steps was done to make sure the pellet of cells was totally clean,
so only MAP would bind and that there would be no background noise on the graphs. The
Tween80 (TW 80) concentration was increased from 0,05% to 0,1% for several samples.
M. smegmatis was added to the serum sample to get rid of all the non-specific antibodies.
These non-specific antibodies might get in the way of the specific antibodies, which
would make it harder for the specific antibodies to bind. Thus in the second run M.
smegmatis was added to the pellet and in the following runs the M. smegmatis was added
to the serum samples before adding the serum sample to the pellet of cells.
Using different MAP-strains
So in half of the samples the biofilm cells were used and in the other half of the samples
the cells from the C69 strain were used. Those two groups were split in half as well, one
half was washed with the 0,05%TW 80 and the other half with the 0,1%TW 80. Also M.
smegmatis was added to the pellets. (Fig 4.)
0,05%TW 80
0,1%TW 80
C69 strain
Biofilm
C20 1:1
C20 1:2
C25
Positive control
ELISA
Negative control
ELISA
Only MAP cells
Only MAP cells with
serum
Only M. smegmatis
with serum and FITC
Only MAP with
FITC
C20 1:1
C20 1:2
C25
Positive control
ELISA
Negative control
ELISA
Only MAP cells
Only MAP cells with
serum
Only M. smegmatis
with serum and FITC
Only MAP with
FITC
C20 1:1
C20 1:2
C25
Positive control ELISA
Fig 4. Table with
the set-up of the
second run
Negative control ELISA
Only MAP cells
Only MAP cells with
serum
Only M. smegmatis with
serum and FITC
Only MAP with FITC
Negative
controls
C20 1:1
C20 1:2
C25
Positive control ELISA
Negative control ELISA
Only MAP cells
Only MAP cells with
serum
Only M. smegmatis with
serum and FITC
Only MAP with FITC
Negative
controls
Page | 10
An alternative serological test for the diagnosis of Johne’s disease
The serum samples that were used in this run were the same as in the first run, with the
same dilution series.
The results from the group with the 0,1%TW 80 gave definite stronger positive reactions,
compared to the group with 0,05% TW 80:
Fig 5. Serum sample C20, washed with
0,05%TW 80
Fig 6. Serum sample C20, washed with
0,1%TW 80
Also the positive ELISA serum sample showed a positive shift with 0,1%TW 80:
Fig 7. Serum sample positive control ELISA,
washed with 0,1%TW 80
The biofilm did not lead to any improvement in the results, the samples stayed negative
because there was an overload of cells. This due to the way the cells were obtained, by
scraping the surface of the biofilm and using that for one sample. There was also no
noticeable difference between using the K10 strain and the C69 strain.
Determination amount of cells
Based on the information that there might be an overload of cells and not enough serum
with antibodies to bind, so that in comparison there will be few bindings detected in
respect to the size of the sample, it was decided to do a serial dilution of cells:
1:1, 1:2, 1:10, 1:100 for the samples with the C69 strain and 1:1 and 1:10 for K10.
The same serum samples as in the previous runs were used.
Page | 11
An alternative serological test for the diagnosis of Johne’s disease
The graphs showed that there was a definite shift for the positive serum samples from the
negative to the positive side when the cells were more diluted. The positive serum control
from the ELISA reached a positivity of 88,8% at a dilution of 1:100 (Fig 9.), compared to
a positivity of 16,7% at a dilution of 1:1 (Fig 8.):
Fig 8. Serum sample positive control
ELISA with cells 1:1
Fig 9. Serum sample positive control ELISA
with cells diluted 1:100
This was also the case for serum sample C20. The negative controls and the negative
serum sample from the ELISA stayed all negative.
Based on these results the amount of cells was decreased for the following runs.
Determination amount of antibodies
In this run a lower dilution of serum was tried, to see whether the same results with a
dilution of 1:100 of cells would be achieved at a lower dilution of serum. The dilution set
was: 1:1 (pure serum), 1:2, 1:10 and 1:20. The serum sample used for this dilution set was
C20, one set without M. smegmatis and one set with M. smegmatis.
The 1:100 dilution of cells was used in this run.
There were also a number of known negative samples tested. The choice for testing more
negative samples in this run was made by observing that although the FCM was able to
test samples positive, it tested also most of the negative serum samples positive in
previous runs. To make sure that these were not false positives, a list of serum samples
tested negative by ELISA, was included.
Together with these two sets, another set of serum samples was tested, to determine how
accurate the FCM would be with samples that were already tested with the ELISA. These
samples came from the farms tested around Calgary.
The negative samples were:
The positive samples were:
Hu163
He95
Vdsluijs 120
He178
NC607
Ge456
By400
He12
Brc557
He37
NC508
Another set, consisting of six samples from six calves in the first run of the ‘Age and dose
dependent susceptibility to MAP infection in dairy calves’ trial, was also tested. These
were pre-infection samples taken in the first two weeks of the trial, when no Johne’s
disease could have infected the calves yet through other calves or dust. These were serum
samples taken on March 15, 2010 from Cow 16, 17, 19, 20, 21 and 22.
The last samples used in this run were two negative controls with only MAP cells, no
antibodies added, and one serum sample C25 with M. smegmatis.
Page | 12
An alternative serological test for the diagnosis of Johne’s disease
The results showed that diluting the serum even further, decreased the binding and the
positivity of the sample became lower. Due to these results it was decided to keep the
undiluted serum with the 1:100 cell ratio as the standard for the FCM.
The results from the pre-infection group, which were thought to be negative, were not
consistently negative. Sample C16 actually reached a positivity of 68%.
Some of the graphs from the serum samples of the farms tested around Calgary were
clearly positive or negative as was expected, but there were also a few samples which
were not definitely positive or negative. These samples remained more in the middle of
the graph. These were serum samples He37 (Fig 10.), He95, Ge456 and Brc557.
Fig 10. Serum sample He37, with a positivity of 58,2%
In the following runs these serum samples were taken and tested under several different
circumstances.
Determination of cross-reactivity with M. smegmatis
Two of the serum samples that were neither positive nor negative were tested in this run
under different circumstances. The used serum samples were Ge456 and Brc557. The
main objective of this run was to evaluate the capacity of M. smegmatis to get rid of the
observed cross reactivity.
The first set was a dilution set of the serum itself:
1:1, 1:2, 1:10 and 1:100.
The next set was a dilution set of the serum sample with M. smegmatis added only to the
first dilution (1:1) and then taking the rest of the dilution set from that first dilution.
The last set was also the same dilution set, but in this set M. smegmatis was added to all
the serum dilutions separately.
In addition to these samples, two negative controls with only MAP cells were also used.
The results showed no improvement when M. smegmatis was added to the dilutions, the
outcome remained almost the same.
Page | 13
An alternative serological test for the diagnosis of Johne’s disease
A different approach was applied in the following four runs, using a very clear positive
serum sample, a sample that remained more or less in the middle and a very clear negative
serum sample:
+ serum sample He178
+ serum sample He178 with M. smegmatis
Cells
5µl
10µl
10µl
10µl
10µl
Cells
5µl
10µl
10µl
10µl
10µl
Serum
8µl
8µl
4µl
4µl 1:2
4µl 1:10
Serum
8µl
8µl
4µl
4µl 1:2
4µl 1:10
+/- serum sample He37
+/- serum sample He37 with M. smegmatis
Cells
5µl
10µl
10µl
10µl
10µl
Cells
5µl
10µl
10µl
10µl
10µl
Serum
8µl
8µl
4µl
4µl 1:2
4µl 1:10
Serum
8µl
8µl
4µl
4µl 1:2
4µl 1:10
- serum sample NC607
- serum sample NC607 with M. smegmatis
Cells
5µl
10µl
10µl
10µl
10µl
Cells
5µl
10µl
10µl
10µl
10µl
Serum
8µl
8µl
4µl
4µl 1:2
4µl 1:10
Serum
8µl
8µl
4µl
4µl 1:2
4µl 1:10
Page | 14
An alternative serological test for the diagnosis of Johne’s disease
The graphs showed for all the serum samples, including serum sample NC607 which was
supposed to be negative as showed in the previous run, a high percentage of positivity.
(Fig 11. and 12.)
Fig 11. Positive serum sample He178,
5μl cells with 8μl serum
Fig 12. Negative serum sample NC607,
5μl cells with 8μl serum
The graphs for the serum dilutions became more negative, as expected due to the
decreased saturation.
Because of these results, three more runs based on the same principle but with different
serum samples were implemented.
The serum samples used in following run were He12 (the definite positive serum sample),
He95 (the in the middle serum sample) and NC508 (the definite negative serum sample).
This time, the negative sample remained negative and serum sample He95 with M.
smegmatis was also negative. The rest of the samples were positive.
Fig 13. Positive serum sample He12,
10μl cells with 4μl serum
Fig 14. Negative serum sample NC508,
10μl cells with 4μl serum
Due to these results, it was decided to test the repeatability of the FCM. In the next run,
the same serum samples were tested and the same results were obtained. It was concluded
that the FCM was repeatable.
Since M. smegmatis did not give the desired results, the same was attempted with M.
phlei.
Since the M. phlei did not arrive until later on, the ninth run was done without the M.
smegmatis or M. phlei. In this run, serum samples from calves in the first run from the
‘Age and dose dependent susceptibility to MAP infection in dairy calves’ trial were taken.
These serum samples came from four calves, two who remained negative through the
whole trial on fecal culture, serum ELISA, IFN-ɣ ELISA and tissue culture and two who
became very positive during the trial and remained positive for the rest of the trial on the
same tests.
Page | 15
An alternative serological test for the diagnosis of Johne’s disease
These were samples from C1 and C20, who were repeatedly positive, and C25 (non infected) and C13 (infected), who were negative on ELISA during the whole infection
trial. The samples that were tested, were a pre-infection sample for the positive calves and
from every two months after infection. Results showed that the pre-infection samples all
tested negative. The positive samples showed an increasing percentage of positivity and
the negative cows started out negative but showed positive peaks as well over time. This
will be reviewed in the discussion.
Determination of cross-reactivity with M. phlei
In this run, the set-up as described in the runs using M. smegmatis, was tried one last time,
this time with M. phlei. The serum samples used were: He12, He37 and NC508.
It was decided, after comparing the results with M. smegmatis results, that M. phlei would
be a better option. It decreased the background noise, as did M. smegmatis, but the serum
samples remained more consistent with M. phlei.
Using two gates
Since there was still some difficulty on how to interpret the samples that had a positivity
of 50-60%, it was decided to set two gates. The first gate was set on the negative control
and the second gate was set on the positive control, which were the first two samples
tested in every run. By doing so, the positivity of the other samples was determined by the
first and the second gate, which gave every sample two outcomes.
Determination of the test characteristics
As most of the protocol was settled after this run, 88 serum samples from the farms
around Calgary were tested with the FCM to determine the test characteristics.
Every positive serum sample was matched to a negative serum sample from a different
cow according to age and days in milk. As shown in the table (see appendix III for
contents), there are two numbers for each sample in the FCM, one percentage of positivity
based on the first gate and one based on the second gate. By doing so, there would be
more options to determine the best cut-off for the FCM.
FCM
ELISA
1st gate
2nd gate
Pos. (44)
90,26±13,02
47,52±28,31
Neg. (44)
39,98±25,47
6,27±5,35
Fig 15. Table with mean and standard deviation
Page | 16
An alternative serological test for the diagnosis of Johne’s disease
Calculating different cut-offs
Based on the results of these 88 samples, several cut-offs were taken and used to calculate
the sensitivity, specificity, positive predictive value, negative predictive value and kappa
value. These cut-offs were decided upon by the gates set on the negative control and on
the positive control. The first three cut-offs are based on the gate set on the negative
control and the last three cut-offs are based upon the gate set on the positive control. It
was taken into account that the prevalence was 50%, since samples were chosen manually.
The calculations for sensitivity, specificity, positive predictive value, negative predictiv e
value of the FCM and the kappa value9:
ELISA
FCM
Pos.
Neg.
Pos.
A
C
A+C
Neg.
B
D
B+D
A+B
C+D
Total
Sensitivity (Se): A/A+C
Specificity (Sp): D/B+D
Positive predictive value (PPV): A/A+B
Negative predictive value (NPV): D/C+D
Kappa value: (2(A*D-B*C))/((A+B)*(C+D)+(A+C)*(B+D))
Interpretations of the kappa value10:
<0,2
Slight agreement between the two compared tests
0,2-0,4
Fair agreement
0,4-0,6
Moderate agreement
0,6-0,8
Substantial agreement
>0,8
Almost perfect agreement
Outcomes for the cut-offs set on the first gate:
50%
60%
70%
Se
100%
95%
93%
Sp
59%
68%
84%
PPV
71%
75%
85%
NPV
100%
94%
93%
Kappa value
0,6449
0,6609
0,7760
NPV
94%
83%
65%
Kappa value
0,7011
0,7857
0,5340
Outcomes for the cut-offs set on the second gate:
10%
20%
50%
Se
93%
80%
45%
Sp
75%
97,7%
100%
PPV
77%
97%
100%
Based on these outcomes, the cut-off of 70% for the first gate was chosen as the best
option.
Page | 17
An alternative serological test for the diagnosis of Johne’s disease
Discussion
The FCM is a good candidate to be developed into a sensitive diagnostic test for detection
of MAP infection, but further research still needs to be conducted.
Further research would include evaluating the FCM in the infection trial that is being
executed at UofC, since there was not enough time to run all the samples from that trial
during this project.
Taking these results into account when calculating the sensitivity and specificity of the
test, together with the already known 88 samples, a more accurate outcome could be
given.
Not only should the research on the FCM for Johne's disease have a bigger sample size, it
should also be compared to other diagnostic tests for Johne’s disease. An example could
be comparing the FCM with tissue culture or fecal culture, as other gold standards. The
reason for comparing the FCM with the ELISA in this paper was that comparing it with
another serological test available for Johne’s disease seemed a good first step.
Comparing the FCM with other gold standards will give a better understanding in how
high the tests’ specificity and sensitivity really are and how early the FCM can detect a
MAP infection. Especially since the ELISA has a low sensitivity (60%, IDEXX)8 and
specificity, it is advised that the FCM should be compared with a diagnostic test that gives
more accurate results and thus has a higher sensitivity and specificity.
Furthermore, it was noted in this project that several serum samples, which were negative
according to the ELISA, tested negative the first time with the FCM but became positive
in the several runs after that. It has been observed before that when sera go through
several freeze and thaw cycles, the sera change their classification and can become more
positive.11
Due to these results, several aliquots were made of each serum sample and kept in the
freezer at a temperature of -20°C.
Also, it was suggested that the incubation method of an hour of the serum sample with M.
phlei in the incubator at a temperature of 37°C was perhaps less effective than an
incubation overnight at room temperature, which is done with other serological tests.12
There were also no dilution series made for the M. phlei, to observe at which dilution it
will give the best results.
These last two conditions were not executed and might give better results on excluding
background noise in the graphs.
Another issue that was observed, was that several samples remained more in the middle of
the graph and were not decisively negative nor positive (50-60%), even after adding M.
phlei to the serum samples. This could be a result of the fact that Johne’s disease is a
chronic disease with several stages, which differ in the reaction that the immune system
produces. Thus, if an infected cow is still in phase one or two, it will be harder to even
almost impossible to detect an immune response. While phase three could give a higher
response and phase four a definite positive result.
It was also noted that every immune system reacts differently to a pathogen and might
produce more specific antibodies than another individual immune system. 13
Page | 18
An alternative serological test for the diagnosis of Johne’s disease
The cows could also be infected with different MAP strains and might react less strongly
to the C69 strain.
The serum samples that came from the two negative calves (9 th run, C25 and C13) in the
first run of the ‘Age and dose dependent susceptibility to MAP infection in dairy calves’
trial and which showed several positive peaks, could be explained by taking into account
the set-up of the barn and the fact that it could be possible that the calves at a certain
period actually did ingested MAP (perhaps through dust particles etc.), but did not get
infected, only showed a low response. C13 was a special case, since it got experimentally
infected, but did not show up positive on any of the other tests. Yet it may explain why
the FCM did show some positive serum samples.
The other inconsistent results came from the pre-infection group. These samples were
thought to be negative, but were not consistently so.
Due to these results, it was concluded that some (maternal) antibodies are likely to show
cross reactivity that may be causing a partly positive reaction or that positive peaks might
also be due to non-specific or cross-reactive antibodies in any of the animals.
It was decided to use only one cut-off for the FCM, but the next step could be to evaluate
samples based on two cut-offs.
The FCM will produce results within a day, which give it an advantage to diagnostic tests
like the fecal culture, which take longer. Disadvantage of the FCM is that it needs to be
performed in a lab and that a flow cytometer needs to be available to retrieve the result s.
Furthermore, the FCM is labour intensive and the costs for appropriate equipment are
high.
Page | 19
An alternative serological test for the diagnosis of Johne’s disease
Acknowledgements
I would like to thank my supervisors in Calgary and Utrecht for their support in this
project.
More specifically, I would like to thank Rienske Mortier for all the time that she invested
in this project and for all the great advice. I am also very grateful for all the opportunities
that she offered me and for all the fun that we had.
I would like to thank Dr. Jeroen De Buck, for listening and offering his advice when the
project got stuck.
I would like to thank Susanne Eisenberg, for all the advice and helping me with the
preparations for the project before going to Canada.
I would also like to thank Karen Poon, who helped me and Rienske with analyzing the
serum samples in the Flow cytometer and for making us laugh when things were not going
too well.
And I would like to thank Dr. Karin Orsel, for giving us the opportunity to come to
Canada and arranging so much, which made us feel really welcome.
Page | 20
An alternative serological test for the diagnosis of Johne’s disease
Appendix I
Flow cytometry protocol
Add 37.5 µL of M. phlei to 75 µL of serum sample
Incubate for 1 hour at 37 °C
Spin down serum samples at 6000 g for 10 min
Resuspend 50 µL of C69 glycerol stock in 450 µL PBS
Take 10 µL and resuspend in 400 µL PBS
Spin down at 16 000 g for 10 min
(highest speed on small centrifuge)
7) Discard supernatant
Wash
8) Resuspend pellet in 100 µl Buffer A
a. Buffer A: PBS (pH 7.0) + 10% Superblock (PIERCE Technology) + 0.1%
TW 80 (ACROS Organics)
9) Spin down at 16000 g for 10 min
10) Discard supernatant
11) Resuspend pellet in 100 µl Buffer A + 4 µl serum sample
12) Incubate at 37°C for 1h
13) Spin down at 6000 g for 10 min
14) Discard supernatant
Wash 1
15) Resuspend pellet in 100 µl Buffer A
16) Spin down at 6000 g for 10 min
17) Discard supernatant
Wash 2
18) Resuspend pellet in 100 µl Buffer A
19) Spin down at 6000 g for 10 min
20) Discard supernatant
21) Resuspend pellet in 50 µl Buffer A + 1 µl FITC – labeled anti bovine IgG antibody
22) Incubate at 37°C for 1h
23) Spin down at 6000 g for 10 min
24) Discard supernatant
Wash 1
25) Resuspend pellet in 100 µl Buffer A
26) Spin down at 6000 g for 10 min
27) Discard supernatant
Wash 2
28) Resuspend pellet in 100 µl Buffer A
29) Spin down at 6000 g for 10 min
30) Discard supernatant
31) Resuspend pellet in 500 µl PBS (pH 7.0) (Only 300 µl needed for FCM analysis)
32) FCM analysis
a. (Forward scatter 691 eV
b. Side scatter 307 eV, threshold 200
c. FL1 channel 590 eV)
1)
2)
3)
4)
5)
6)
Page | 21
An alternative serological test for the diagnosis of Johne’s disease
Appendix II
Serum ELISA (IDEXX) protocol
1) Obtain the antigen-coated plates and record the sample position
2) Dilute the samples in sample diluents at the recommended dilution factor and
allow them to incubate for 15 minutes to two hours at room temperature, 18° 25°C
3) Dispense 100 µl of diluted negative control into two wells of the antigencoated plates
4) Dispense 100 µl of diluted positive control into two wells of the antigencoated plates
5) Dispense 100 µl of each diluted sample (serum, plasma or milk) into the
applicable well
6) Cover the plate and incubate for 45 minutes ±3 minutes at room temperature,
18°-25°C
7) Aspirate the liquid contents of all the wells into an appropriate waste reservoir
8) Wash each well three times with approximately 300 µl of wash solution
Aspirate the liquid contents of all the wells after each wash
Avoid plate drying between plate washings and prior to the addition of the
conjugate. Following the final wash fluid aspiration, gently, but firmly, tap
the residual wash fluid from each plate onto an absorbent material
9) Dispense 100 µl of diluted HRPO conjugate into each well
10) Cover the plate and incubate for 30 minutes ±3 minutes at room temperature,
18°-25°C
11) Aspirate the liquid contents of all the wells into an appropriate waste reservoir
12) Wash each well three times with approximately 300 µl of wash solution
Aspirate the liquid contents of all the wells after each wash
Avoid plate drying between plate washings and prior to the addition of the
substrate. Following the final wash fluid aspiration, gently, but firmly, tap the
residual wash fluid from each plate onto an absorbent material
13) Dispense 100 µl of TMB substrate solution into each well
NOTE: All wells should be observed for color development during the first 1 3 minutes of incubation with the TMB substrate. Abnormal color development
includes variation within areas or ‘spots’ in the well. If this occurs, the results
for those samples should be considered invalid and the samples should be
retested in duplicate using a fresh dilution. However, the other results on the
plate can be considered valid
14) Incubate for 10 minutes at room temperature, 18°-25°C
15) Dispense 100 µl of stop solution into each well of the test plate to stop the
reaction
16) Measure and record the absorbance ate A(450)nm
17) Calculate the results
Page | 22
An alternative serological test for the diagnosis of Johne’s disease
Appendix III
Table with samples for calculating test characteristics (88 samples in total, of which 44
positive for ELISA):
Positive serum samples for ELISA:
Negative serum samples for ELISA:
Fecal (0): Negative outcome for fecal culture
Fecal (1): Positive outcome for fecal culture
Sample ‘10/’11
WC70
VDS49 (’09)
VDV488
VDS186
IV1000
HUM54
HU219
HUM1600
HE114
VDS221
HE196
VDS210
HE76
VDS209
HE135
VDS201
HE28
VDS167
HE37
VDS184
HE75
VDS169
HE80
VDS182
HE193
VDS161
HE12
VDS140
HE118
VDS94
HE5
VDS79
HE9
VDS47
HE6
VDS58
HE219
VDS85
FC961
HUM395
CH227
VDS223
Flow(%)
96; 51
53; 8
99; 66
36; 4
100; 78
11; 1
95; 40
29; 3
77; 10
20; 2
100; 72
9; 1
94; 35
4,4;0,4
100; 90
22; 2,5
84; 28
70; 13,1
99; 88
14; 1,5
78; 20
45; 7
99; 72
63; 14
99; 64
50; 7
100; 100
19; 2
83; 28
76; 16
99; 41
60; 9
97; 41
40; 4
99; 54
88; 9
78; 14
41; 5
50; 7
78; 13
76; 14
62; 13
ELISA
70,5
-0,33
192,38
-0,06
85,97
7,05
61,39
-3,91
85,22
-0,19
207,65
5,25
141,23
-0,06
210,06
3,95
125,47
10,3
234,76
1,23
87,65
0,97
151,92
20,93
229,66
1,49
256,96
4,33
103,89
23,16
131,1
4,66
86,62
1,67
175,4
-0,33
62,28
-0,95
82,58
4,5
91,49
5,9
Fecal
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
1
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
Page | 23
An alternative serological test for the diagnosis of Johne’s disease
BY399
VDS105
Sample ’09
HE53
NC577
HE178
VDS120
GE409
NC471
HE72
BY105
HE150
VDS68
RI162
BY348
GE282
BY262
HE65
VDS49 (’10)
FC12
HUM363
GE136
HUM341
GE301
BY390
RI113
NC586
VDD1840
NC570
HE58
HUM178
DIJ422
BY431
DIJ407
VDS139
HE145
BRI544
VDP539
BRI517
HE189
VDS106
GE316
BY93
HE219
HUM396
WO375
BRI472
72; 15
79; 21
Flow
100;97
73; 15
99,8; 89,9
63; 10
70; 22
39; 5
99; 70
10; 1
100; 98
8; 0,7
99; 68
56; 12
99; 60
67; 10
99; 39
42; 4
99; 52
16; 2
99; 81
15; 1,4
99; 38
9; 0,9
71; 13
13,5; 1,1
93; 45
10; 1,3
99; 73
50; 8,4
69; 15
5,4; 0,6
75; 14
8,7; 0,8
97; 37
12;1
58; 9
45; 5
98; 37
69; 14
99,6; 64
72; 12
87; 20
46; 5
88; 21
60; 8
68,9
19,98
ELISA
256,429
23,84
211,54
17,30
76,29
7,1
220,54
4,98
260,7
3,35
291,3
6,09
159,25
1,33
174,1
4,56
268,12
18,94
218,97
1,59
89,63
2,55
304,84
5,89
73,07
1,66
141,48
6,15
100,56
2,10
67,04
-0,14
217,06
2,84
90,28
1,96
62,22
33,67
69,51
7,53
116,11
0,22
79,15
2,67
0
0
Fecal
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
Page | 24
An alternative serological test for the diagnosis of Johne’s disease
Appendix IV
With a cut-off of 50% for the first gate:
ELISA
FCM
Pos.
Neg.
Pos.
44
0
44
Neg.
18
26
44
62
26
88
Neg.
14
30
44
56
32
88
Neg.
7
37
44
48
40
88
Sensitivity: 100%
Specificity: 59%
PPV: 71%
NPV: 100%
Kappa value= 0,6449
With a cut-off of 60% for the first gate:
ELISA
FCM
Pos.
Neg.
Pos.
42
2
44
Sensitivity: 95%
Specificity: 68%
Positive predictive value (PPV): 75%
Negative predictive value (NPV): 94%
Kappa value: 0,6609
With a cut-off of 70% for the first gate:
ELISA
FCM
Pos.
Neg.
Pos.
41
3
44
Sensitivity: 93%
Specificity: 84%
PPV: 85%
NPV: 93%
Kappa value= 0,7760
Page | 25
An alternative serological test for the diagnosis of Johne’s disease
With a cut-off of 10% for the second gate:
ELISA
FCM
Pos.
Neg.
Pos.
41
2
44
Neg.
12
33
44
53
35
88
Sensitivity: 93%
Specificity: 75%
PPV: 77%
NPV: 94%
Kappa value= 0,7011
With a cut-off of 20% for the second gate:
ELISA
FCM
Pos.
Neg.
Pos.
35
9
44
Neg.
1
43
44
36
52
88
Sensitivity: 80%
Specificity: 97,7%
PPV: 97%
NPV: 83%
Kappa value= 0,7857
With a cut-off of 50% for the second gate:
ELISA
FCM
Pos.
Neg.
Pos.
20
24
44
Neg.
0
44
44
20
68
88
Sensitivity: 45%
Specificity: 100%
PPV: 100%
NPV: 65%
Kappa value=0,5340
Page | 26
An alternative serological test for the diagnosis of Johne’s disease
References
1. Nielsen SS, Toft N. A review of prevalences of paratuberculosis in farmed animals
in europe. Prev Vet Med. 2009 88:1-14.
2. Eisenberg S. Within-farm dispersion of Mycobacterium avium subspecies
paratuberculosis by bioaerosols. Proefschrift 2011 Dec 6:2
3. Chi J, Van Leeuwen JA, Weersink A, Keefe GP. Direct production losses and
treatment costs from bovine viral diarrhoea virus, bovine leukosis virus,
mycobacterium avium subspecies paratuberculosis, and neospora caninum. Prev
Vet Med. 2002 Sep 30;55(2):137-53.
4. Johnson-Ifearulundu Y, Kaneene JB, Lloyd JW. Herd-level economic analysis of
the impact of paratuberculosis on dairy herds. Jour of the Am Vet Med Assoc.
1999 214:822-825.
5. Whitlock RH, Buergelt C. Preclinical and clinical manifestations of
paratuberculosis (including pathology). Vet Clin North Am Food Anim Pract. 1996
Jul 12(2):345-56.
6. Nielsen SS, Toft N. Ante mortem diagnosis of paratuberculosis: A review of
accuracies of ELISA, interferon-gamma assay and faecal culture techniques. Vet
Microbiol. 2008 Jun 22 129(3-4):217-35.
7. Eda S, Elliott B, Scott MC, Waters WR, Bannantine JP, Whitlock RH, et al. New
method of serological testing for mycobacterium avium subsp. paratuberculosis
(johne's disease) by flow cytometry. Foodborne Pathog Dis. 2005 Fall 2(3):250-62.
8. Rossiter C, Hansen D. Johne’s Disease Diagnostic Tests - the ELISA, Part 2 of 4
on the topic of Johne’s disease testing. New York State Cattle Health Assurance
Program
9. Rahman, M. Introduction to Flow Cytometry 2006:4-8, 12, 16-19, 24-25.
10. Veterinary epidemiologic research, Dohoo et al., 2003:91-92, 95-100.
11. Boadella, M and Gortázar, C. Effect of haemolysis and repeated freeze-thawing
cycles on wild boar serum antibody testing by ELISA. BMC Veterinary Research
Volume 4, 2011:498.
12. Marassi, CD et al. Interference of anti-M. bovis antibodies in serological tests for
paratuberculosis. Proceedings of 8ICP 2005, Theme 5: Diagnosis.
13. Bakker D, Willemsen PTJ & van Zijderveld FG. 2000: Paratuberculosis
recognized as a problem at last: A review, Vet Quart, 22(4):200-204.
Page | 27