Application and Field Validation of a PCR Assay for the Detection of Mycoplasma Hyopneumoniae from Swine Lung Tissue Samples

Abstract

A PCR assay was validated for the detection of Mycoplasma hyopneumoniae in porcine lung tissue. The detection limit of the assay was 0.18 colony-forming units/g of lung sample spiked with M. hyopneumoniae. In field validation, 426 pigs from 220 cases were examined for M. hyopneumoniae infection by M. hyopneumoniae PCR and a fluorescent antibody (FA) test. In total, 103 pig lungs (24.2%) were positive in the PCR test, and 69 pig lungs (16.2%) were positive in the FA test, among which, 62 pigs were positive for both PCR and FA test. Most of the PCR-positive but FA test-negative cases had lesions compatible with M. hyopneumoniae infection. With Bayesian modeling, the diagnostic sensitivity and specificity of the PCR were determined to be 97.3% and 93.0%, respectively.

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Journal of Veterinary Diagnostic
http://vdi.sagepub.com/content/19/1/91
The online version of this article can be found at:
DOI: 10.1177/104063870701900115
2007 19: 91J VET Diagn Invest
Grant Maxie
Hugh Y. Cai, Tony van Dreumel, Beverly McEwen, Geoff Hornby, Patricia Bell-Rogers, Pat McRaild, Gaylan Josephson and
Swine Lung Tissue Samples
fromMycoplasma HyopneumoniaeApplication and Field Validation of a PCR Assay for the Detection of
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Application and field validation of a PCR assay for the detection of Mycoplasma
hyopneumoniae from swine lung tissue samples
Hugh Y. Cai,
1
Tony van Dreumel, Beverly McEwen, Geoff Hornby, Patricia Bell-Rogers,
Pat McRaild, Gaylan Josephson, Grant Maxie
Abstract. A PCR assay was validated for the detection of Mycoplasma hyopneumoniae in porcine lung
tissue. The detection limit of the assay was 0.18 colony-forming units/g of lung sample spiked with M.
hyopneumoniae. In field validation, 426 pigs from 220 cases were examined for M. hyopneumoniae infection by
M. hyopneumoniae PCR and a fluorescent antibody (FA) test. In total, 103 pig lungs (24.2%) were positive in
the PCR test, and 69 pig lungs (16.2%) were positive in the FA test, among which, 62 pigs were positive for
both PCR and FA test. Most of the PCR-positive but FA test–negative cases had lesions compatible with M.
hyopneumoniae infection. With Bayesian modeling, the diagnostic sensitivity and specificity of the PCR were
determined to be 97.3% and 93.0%, respectively.
Key words: Bayesian modeling; enzootic pneumonia; FA test; Mycoplasma hyopneumoniae; PCR.
<!?show "fnote_aff1"$^!"content-markup(./author-grp[1]/aff|./author-grp[1]/dept-list)>
Mycoplasma hyopneumoniae causes enzootic pneumonia,
which has been reported worldwide and is the most
common and economically important swine disease.
14
Because of the slow growth of M. hyopneumoniae and the
overgrowth of other mycoplasmas (e.g., Mycoplasma
hyorhinis), isolation of this organism by culture is difficult.
Other antigen or DNA detection tests include the
fluorescent antibody (FA) test, immunohistochemistry
(IHC), in situ hybridization, and PCR. The advantages
and disadvantages of these tests have been reviewed
recently.
19
The FA test has been the major antigen-
identification assay; however, similar to the IHC, it may
generate false-negative results if airway is not included in
the tissue section.
19
In addition, false-negative results may
occur if the samples are not fresh or not stored (frozen)
properly. Since the early 1990s, many M. hyopneumoniae
PCR assays, including single PCR or nested PCR, have
been described
19
and have been found to be similar or more
sensitive than the FA test.
3,18,20
Recently, 2 real-time PCR
assays have been described to be highly sensitive for the
detection of M. hyopneumoniae from bronchial or nasal
swabs at the herd level, using both assays in combination,
although the sensitivity was low when using only 1 of the
assays or at the individual animal level.
7,22
In the past several years, many studies have been
conducted to determine the diagnostic value of different
sample types for PCR testing, and the conclusion remains
indefinite. Bronchoalveolar lavage fluid samples were found
to be suitable and superior to tonsil and tracheobronchial
swabs, lung tissue samples, and tracheobronchial brush
samples.
2,10
Nasal swabs were found to be good indicators
of the presence of M. hyopneumoniae in the bronchi
17
and
were more suitable than tonsil tissue samples.
17
In another
study, lung tissue and nasal swabs were found not to be
reliable indicators of experimentally induced M. hyopneu-
moniae infection,
10
and it was recommended that nasal
swabs should only be used for monitoring disease status at
the herd level.
13
Another report described the detection rate
as higher in lung homogenates than in nasal swabs in pigs
naturally infected with M. hyopneumoniae.
13
Nested PCR
has been described to be a sensitive tool for the detection of
M. hyopneumoniae.
6,10,13,20
However, nested PCR may be
prone to cross-contamination
1
and may be unnecessary if
the proper samples are used.
10
Although many M.
hyopneumoniae PCRs have been validated using clinical
samples, very few large-scale field validations have been
reported; therefore, the information on diagnostic specific-
ity/sensitivity is limited.
A PCR test targeting the 16S rRNA gene has been
developed in nested and non-nested PCR formats and
applied for detection of M. hyopneumoniae in nasal
swabs.
5,6,11
This study was conducted to optimize, validate,
and apply the non-nested PCR assay for the detection of
M. hyopneumoniae in swine lung tissue. Since there is no
perfect test (gold standard) for the detection of M.
hyopneumoniae, the diagnostic specificity and sensitivity
were analyzed using Bayesian rather than frequentist
statistics so that the uncertainty about all parameters was
modeled with probability that reflected the scientific
uncertainty of the unknown quantities.
All bacteria used in this study were obtained from the
American Type Culture Collection (ATCC) or were
isolated and characterized at the Animal Health Labora-
tory (AHL), University of Guelph, Guelph, Ontario. The
Mycoplasma strains were stored at 270uC in Friis broth
a,
15
and other bacteria in brain heart infusion
b
broth containing
From the Animal Health Laboratory, Laboratory Services
Division, University of Guelph, Box 3612, Guelph, Ontario, N1H
6R8, Canada.
1
Corresponding Author: Dr. Hugh Cai, Animal Health
Laboratory, Laboratory Services Division, University of Guelph,
PO Box 3612, Guelph, Ontario, N1H 6R8, Canada. hcai@
lsd.uoguelph.ca
J Vet Diagn Invest 19:91–95 (2007)
Brief Communications 91

30% glycerol. Before use, the Mycoplasma strains were
propagated in Friis broth, and other bacterial strains were
cultured in trypticase soy broth.
a
From the year 2000 to 2004, lung tissue samples were
selected randomly from cranioventral (CV) and caudal
lobes with or without pneumonic lesions from routine
submissions to the AHL. The same portions of the lung
samples were tested by PCR, FA test, and histopathology
examination. Lung tissue DNA was extracted using a DNA
extraction kit
c
following the manufacturer’s protocol.
Briefly, 3 or more pieces of tissue (total 20–25 mg) were
cut from each lung and placed into a sterile safe-lock
microcentrifuge (manufacturer: Eppendorf, Missisauga,
Ont, Canada) 1.5-ml tube with 180 ml of lysis buffer and
digested with 20 ml of proteinase K (.600 mAU/ml) in
a shaking dry bath (55uC) till the tissues were completely
lysed (2–4 hours or overnight). The complete homogenate
was used for DNA extraction following the manufacturer’s
instruction exactly, except that 50 ml of elution buffer was
used to elute DNA. DNA was extracted from pure cultures
of Mycoplasma organisms and other bacteria using
a commercial DNA extraction kit.
d
The PCR assay was
done in a 25-ml reaction volume containing 2 mM MgCl
2
,
e
0.2 millimoles each of deoxynucleotide triphosphate,
e
0.75 U of heat-activating polymerase,
f
0.2 mm of each
primer,
g
and 2 ml of DNA extract. Primers MH649F (59-
GAG CCT TCA AGC TTC ACC AGG A-39)and
MH649R (59-TGT GTT AGT GAC TTT TGC CAC C-
39) have been described previously.
11
Reactions were carried out in a thermocycler.
h
The
reaction started with an initial polymerase-activating
temperature of 95uC for 12 minutes, followed by 35 cycles
of denaturing at 94uC for 20 seconds, annealing at 60uC for
30 seconds, elongating at 72uC for 40 seconds, and a final
elongation step at 72uC for 7 minutes. After the PCR was
completed, 18 ml of PCR products were electrophoresed in
a 1.5% agarose gel, stained with ethidium bromide, and
visualized using an ultraviolet camera.
i
In each PCR test
run, known M. hyopneumoniae positive and negative lung
tissue samples were included in the whole procedures, from
sample preparation through DNA extraction and PCR
amplification. In addition, M. hyopneumoniae DNA
samples and pure water were also included as template
and nontemplate controls.
M. hyopneumoniae on the surface of the bronchial and
bronchiolar epithelium were visualized using the FA test.
21
Briefly, the lung tissue was cut into 12-mm slices and
mounted on a glass slide. A drop (10 ml) of 1:80 diluted M.
hyopneumoniae rabbit antiserum
j,
15
was applied to the 1
section of the cut tissue, and 10 ml of 1:80 diluted
heterologous hyperimmune rabbit serum
k,
15
on the adjoin-
ing section as control. The slide was incubated at room
temperature for 30 minutes then rinsed with phosphate
buffered saline (PBS). After a drop of goat antirabbit
fluorescein-labeled antiserum conjugate was applied to the
tissue sections, the slide was incubated at room temperature
for 30 minutes, then washed again in PBS before being
examined under an epifluorescence microscope. The test
was considered positive if micro-organisms within and/or
on the surface of the airways fluoresced with M.
hyopneumoniae antisera and did not fluoresce with the
heterologous hyperimmune rabbit serum.
For histologic examination, sections of lung were taken
from CV and caudal lobes, routinely processed and stained
HE, and examined by the same pathologist. The pig was
classified as having lesions compatible with M. hyopneu-
moniae infection if it met the following criteria: hyperplasia
of bronchiolar epithelial cells, peribronchial lymphocytic
cuffing, presence of moderate to large numbers of macro-
phages in alveoli, and perivascular lymphocytic cuffing.
Mixed infection was diagnosed when more than 1 pathogen
was identified by microbiology testing and/or histology
lesions compatible with mixed infection were found.
Diagnostic sensitivity and specificity were calculated by
Bayesian modeling,
4,9
using the freeware program Win-
BUGS (http://www.mrc-bsu.cam.ac.uk/bugs). Prior distri-
bution of model parameters was determined by obtaining
expert opinion.
8,9
Pairwise and overall agreement among
current diagnostic tests (FA test, histopathology, and PCR)
were determined using a kappa statistic.
16
Lung tissue samples spiked with M. hyopneumoniae
culture were tested to determine the analytic sensitivity and
specificity of the PCR assay. A pure culture of M.
hyopneumoniae strain SuiJ was propagated in Friis broth
for 48 hours at 37uC. The colony-forming unit (cfu) was
determined by plating 10 ml of each dilution onto Friis agar
plates and counting the colonies after incubating the plates
at 37uC for 48 hours. Serial dilutions of the culture were
spiked in lung tissues (25 mg each) to a final concentration
of 0.018 to 180,000 cfu of M. hyopneumoniae per gram of
lung tissue. Among these, the samples of 0.18–180,000 cfu/g
were PCR positive.
Of the 5 mycoplasma species, including Mycoplasma
arginini 108, Mycoplasma flocculare MS42, Mycoplasma
hyopneumoniae SuiJ, Mycoplasma hyorhinis MH31, Myco-
plasma hyosynoviae S16, and 14 other bacterial species,
including Actinobacillus suis AHL110, Arcanobacterium
pyogenes AHL104, Escherichia coli ATCC 25922, Erysipe-
lothrix rhusiopathiae AHL111, Klebsiella pneumoniae
AHL82, Pasteurella multocida AHL83, Pseudomonas aer-
uginosa ATCC27852, Proteus mirabilis AHL107, Serratia
marcescens AHL86, Staphylococcus aureus ATCC25923,
Staphylococcus hyicus AHL112, Streptococcus equisimilis
AHL105, Streptococcus intermedius AHL106, and Strepto-
coccus suis AHL109, only the M. hyopneumoniae strain was
PCR positive; all other bacteria were PCR negative.
Total of 426 pig lung samples from 220 cases was
examined for M. hyopneumoniae infection by M. hyopneu-
moniae PCR in parallel with an FA test. An example of the
PCR results is shown in Fig. 1. In total, there were 103 pig
lung samples (24.2%) positive in the PCR test and 69 pig
lungs (16.2%) positive in the FA test. There were 378
samples with correlated PCR and FA test results and 48
samples with differing PCR and FA test results (Table 1).
Agreement between FA test and PCR was moderate with
a kappa value of 0.654. The Bayesian statistics showed that
the diagnostic sensitivity and the diagnostic specificity were
97.3% and 93.0%, respectively, for the M. hyopneumoniae
PCR; and the prevalence of M. hyopneumoniae was 20.2%
with 95% probability interval of 16.0–24.9 (Table 2). The
92 Brief Communications

following prior information was used in determining the
beta distributions of each parameter: M. hyopneumoniae
FA test, sensitivity prior 5 0.3, beta 5 (13.34, 29.81);
specificity prior 5 0.97, beta 5 (167.2, 42.5); for M.
hyopneumoniae PCR, sensitivity prior 5 0.95, beta 5
(329.7, 11.2); specificity prior 5 0.8, beta 5 (19, 1);
prevalence prior 5 0.3, beta 5 (4, 10).
It has been noted that there is no true gold standard
available for M. hyopneumoniae tests in a diagnostic
setting.
7
Although the M. hyopneumoniae FA test is
a traditional test used to diagnose infected animals, it is
not a gold standard (i.e., perfect test). Therefore comparing
a new, possibly more accurate test such as the M.
hyopneumoniae PCR would lead to misclassification of
data and test sensitivity and specificity. With Bayesian
rather than frequentist statistics, the uncertainty about all
parameters is modeled with probability that reflects
scientific uncertainty of the unknown quantities.
9
The
reason for uncorrelated results between PCR and FA tests
may be that the FA test has a lower sensitivity. Among 426
samples tested, 48 samples did not have correlated PCR
and FA test results, with 41 samples being PCR positive
and FA test negative and 7 samples being PCR negative but
FA test positive. Except for 6 samples with no histology
results, most of the PCR-positive but FA test–negative
samples (16/19) were found to have lesions compatible with
M. hyopneumoniae infection. There were 2 samples with
a positive or suspicious FA test but negative PCR. The
sample with a suspicious FA test but negative PCR had no
obvious mycoplasma lesion (histologic examination was
not done on the case positive in the FA test but negative in
PCR) (Table 3). Therefore, as displayed by the Bayesian
statistical analysis, the PCR assay appeared to be more
sensitive than the FA test.
PCR is an amplification method; it may detect lower
numbers of M. hyopneumoniae cells compared with the
antibody–antigen reaction-based FA test. In addition, the
FA test requires bronchiolar epithelial cells to be intact in
the samples, since M. hyopneumoniae infects the luminal
surface of the bronchiolar epithelial cells. In mail-in frozen
samples, if the lungs thawed during transport, the
bronchiolar epithelial cells would slough and the antigen
would not be detected by FA test. We tested both mail-in
samples and fresh tissue from the AHL postmortem room.
Among the cases with uncorrelated FA and PCR tests, 69%
(18/26) were mail-in samples (Table 3). Destruction of
bronchiolar epithelial cells in sick pigs may be another
reason for low sensitivity of the FA test. The majority of
pneumonias in pigs with uncorrelated PCR and FA test
results had multiple infections with porcine respiratory and
reproductive syndrome virus, swine influenza viruses,
porcine circovirus type 2, M. hyopneumoniae, and other
bacteria (Table 3). The co-infections may have destroyed
the bronchiolar epithelial cells to the point where the
number of epithelial cells were insufficient for observation
in the FA test. This does not appear to have a significant
impact on the PCR test. The Bayesian statistical analysis
demonstrated that the PCR had lower specificity than did
the FA test (Table 2). The lower specificity could be due to
more false-positives than the FA test.
Choice of prior information is also important in
Bayesian analysis, and the input for PCR specificity was
lower than that for the FA test. Additionally, diagnostic
sensitivity and specificity are often inversely related.
Figure 1. Example of M. hyopneumoniae PCR on clinical samples. Lanes 1 and 20: DNA molecular-weight marker. Lanes 2–15:
clinical lung samples. Lane 16: negative control lung. Lane 17: positive control lung. Lane 18: no template, negative control. Lane 19:
positive DNA control. Clinical samples in lanes 4 and 7–10 were PCR positive, and the sample in lane 15 was PCR weak positive. The
positive-PCR products were 645 base pairs in size.
Table 1. Comparison of PCR and fluorescent antibody (FA)
tests for the detection of M. hyopneumoniae in lung.
FA test + FA test 2 Total
PCR + 62 41 103
PCR – 7 316 323
Total 69 357 426
Table 2. Diagnostic sensitivity and specificity for M.
hyopneumoniae fluorescent antibody (FA) test and PCR, using
Bayesian statistics (n 5 426).
FA test (95% PI)* PCR (95% PI)*
Sensitivity 79.0% (73.2, 84.3) 97.3% (91.3, 99.9)
Specificity 97.3% (96.1, 98.6) 93.0% (88.9, 97.1)
Positive predictive
value 88.5% (82.1, 93.8) 77.9% (65.4, 90.9)
*PI5 probability interval.
Brief Communications 93

Biologic factors such as disease attributes and sampling
techniques will also affect the sensitivity and specificity of
tests. The positive predictive value (PPV) is the probability
that given a positive test, the animal actually has the
disease. It is dependent upon prevalence; it actually
increases if the test has a high specificity (no false positives),
which accounts for the higher PPV of the FA test compared
with the PCR. If cost is not an issue, we would recommend
the use of PCR and FA tests in combination to increase
their sensitivity or specificity.
The PCR assay was originally reported for testing swab
samples. The detection limit of the PCR was described to
be 5 cfu, determined by testing the lysed cells directly
without other DNA extraction treatment.
11
This study
adapted and optimized the PCR for testing of lung tissue
samples. The PCR detection limit was improved in this
study by using a commercial DNA extraction kit, which
has been shown to be superior to some other DNA
extraction methods.
12
In addition, the use of hot-start PCR
in this study reduced the competition from nonspecific
amplification, which may be another reason for increased
PCR sensitivity. We found that carry-over contamination
could be prevented by using the proteinase K digestion
method to replace the mechanical homogenization (data
not shown). Although the problem of mechanical homog-
enizer causing cross-contamination has not been described
previously, we found from our experience that it was very
difficult to clean or degrade the DNA in the mechanical
tissue homogenizer, which can cause PCR cross-contami-
nation. In addition, we compared nested and non-nested
format M. hyopneumoniae PCRs in our preliminary
experiments, and we experienced cross-contamination
frequently in the nested PCR. Therefore, we decided not
to use the nested PCR.
In conclusion, the PCR optimized and validated in this
study is sensitive and specific and is a better alternative to
the FA test for the detection of M. hyopneumoniae in lung
tissue samples.
Acknowledgements. This project was financially sup-
ported in part by Ontario Pork and the Ontario Ministry of
Agriculture, Food and Rural Affairs.
Sources and manufacturers
a. Difco, Detroit, MI.
b. Becton Dickinson, Cockeysville, MD.
c. Qiagen DNeasy Tissue kit, Qiagen, Mississauga, Ontario,
Canada.
d. Instagen Matrix, BioRad, Mississauga, Ontario, Canada.
e. PE Applied Biosystems, Mississauga, Ontario, Canada.
f. AmpliTaq Cold polymerase, PE Applied Biosystems, Mis-
sissauga, Ontario, Canada.
g. Molecular Supercenter, University of Guelph, Guelph, On-
tario, Canada.
Table 3. Cases with uncorrelated Mycoplasma hyopneumoniae PCR and fluorescent antibody (FA) test results.
PCR FA test
Lesions compatible with
M. hyopneumoniae infection Co-Infection
Sample
freshness
03-30562 + 2 + Pasteurella multocida, Porcine circovirus type 2 (PCV2) Mail-in
03-41953 + 2 autolysed P. multocida, Arcanobacterium pyogenes Mail-in
03-44031-2 + 2 + P. multocida, PCV2, Swine influenza virus (SIV) Mail-in
03-44365 + 2 + P. multocida Mail-in
03-44768-2 + 2 + Not detected Fresh
03-44981 + 2 + P. multocida, PCV2 Mail-in
03-46710-1 + 2 Not done Porcine respiratory and reproductive syndrome virus (PRRSV) Mail-in
03-46710-2
03-47242 + 2 + P. multocida, Streptococcus suis, PRRSV Fresh
03-49875-1 2 +/2* 2 SIV Fresh
03-50247 + 2 + PCV2, PRRSV Mail-in
03-53148 + 2 Not done PRRSV Mail-in
03-53518 + 2 + P. multocida, SIV Fresh
03-53979 + 2 Not done Not detected Mail-in
03-56953-1 + 2 + P. multocida, S. suis, PRRSV Fresh
03-59571-2 + 2 + S. suis, PCV2, PRRSV Mail-in
04-11591 + 2 Not done Haemophilus parasuis, P. multocida Mail-in
04-11470-3 + 2 + PRRSV Fresh
04-13669-3 + 2 + Bordetella bronchiseptica, S. suis, PRRSV Fresh
04-13743 + 22PRRSV Mail-in
04-14204 + 2 + P. multocida, S. suis, PCV2, PRRSV Mail-in
04-18729-1 + 22TGE{ Fresh
04-18729-2 + 22TGE{
04-26362 2 + Not done No done Mail-in
04-27961 + 2 + Actinobacillus pleuropneumoniae Mail-in
04-31118 + 22Not detected Mail-in
04-36262 + 2 + P. multocida, A. pyogenes Mail-in
04-37722 + 2 + P. multocida, A. pyogenes Mail-in
* Suspicious.
{ TGE 5 Transmissible gastroenteritis of swine.
94 Brief Communications

h. PCR System 9600, PE Applied Biosystems, Mississauga,
Ontario, Canada.
i. Gel Documentation System, BioRad, Mississauga, Ontario,
Canada.
j. Rabbit antiserum against M. hyopneumoniae strain SuiJ
(Aarhus University, Denmark), Animal Health Laboratory,
University of Guelph, Guelph, Canada.
k. Rabbit antiserum against Mycoplasma hyorhinis strain 10118
(National Collection of Type Cultures, London, England),
Animal Health Laboratory, University of Guelph, Guelph,
Canada.
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