Detection of Corynebacterium pseudotuberculosis from sheep

60
ILHAN (Z.)
Detection of Corynebacterium
pseudotuberculosis from sheep lymph
nodes by PCR
Z. ILHAN1*
Department of Microbiology, Faculty of Veterinary Medicine, Yuzuncu Yıl University, 65080 Campus, Van, TURKEY.
1
Corresponding author: [email protected]
*
SUMMARY
RÉSUMÉ
Corynebacterium pseudotuberculosis is a facultative intracellular
bacterium that causes caseous lymphadenitis in sheep and goats. This
study was designed to evaluate the validity of PCR assay protocol for the
direct detection of C. pseudotuberculosis in 147 samples of lymph nodes
(prescapular and mediastinal) from carcasses of naturally infected sheep
and to compare its performance with the traditional bacteriological culture
technique. C. pseudotuberculosis was isolated in 81 samples mainly from
prescapular nodes and a specific 203 bp PCR amplified pld gene DNA
fragment was detected in 85 samples. The total agreement score between the
2 methods was 95.92%, the relative PCR sensitivity and specificity to culture
being 98.76% and 92.42%, respectively. The positive and negative predictive
probabilities given by PCR were 98.4% and 94.1%, respectively. The C.
pseudotuberculosis detection limit given by PCR was 2.1x104-2.1x103 CFU/
mL in sheep lymph tissue samples. In conclusion, the PCR assay proved to
be a sensitive and rapid method for the detection of C. pseudotuberculosis in
lymph node samples from naturally infected sheep.
Détection par PCR de Corynebacterium pseudotuberculosis dans les
nœuds lymphatiques de moutons
Keywords:
Corynebacterium
pseudotuberculosis,
caseous lymphadenitis, sheep, PCR, bacteriological
culture.
Corynebacterium
pseudotuberculosis est une bactérie intracellulaire
facultative responsable de la lymphadénite caséeuse des moutons et des
chèvres. Cette étude a été réalisée afin d’évaluer la validité d’une méthode
de détection directe de C. pseudotuberculosis par PCR dans 147 nœuds
lymphatiques (préscapulaires et médiastinaux) prélevés sur des carcasses
de moutons naturellement infectés et de comparer les performances de
ce test avec celles de la culture bactériologique classiquement utilisée. C.
pseudotuberculosis a été isolé à partir de 81 prélèvements, principalement de
nœuds lymphatiques préscapulaires et un fragment d’ADN amplifié par PCR
de 203 bp correspondant au gène pld de la bactérie a été mis en évidence dans
85 échantillons. L’agrément total entre les 2 méthodes a été de 95.92%, la
sensibilité et la spécificité de la PCR par rapport à la culture bactériologique
ont été respectivement de 98.76% et de 92.42% et les valeurs prédictives
positive et négative ont été de 98.4% et 94.1%. La limite de détection de C.
pseudotuberculosis dans les échantillons de nœuds lymphatiques donnée par
la PCR a été de 2.1x104-2.1x103 CFU/mL. En conclusion, la PCR apparait être
une méthode sensible et rapide pour la détection de C. pseudotuberculosis
dans des échantillons de nœuds lymphatiques provenant de moutons
naturellement infectés.
Mots-clés : Corynebacterium pseudotuberculosis,
lymphadénite caséeuse, mouton, PCR, culture
bactériologique.
Introduction
Corynebacterium pseudotuberculosis is a Gram-positive,
rod-shaped, non-spore-forming, facultative intracellular
bacterium that causes caseous lymphadenitis (CLA) in
sheep and goats [8, 20, 24]. It is also the causative agent of
ulcerative lymphangitis in horses, and sporadic suppurative
disease in other animal species, including humans [2, 3, 23].
A protein exotoxin, commonly known as phospholipase
D (PLD) and lipids of the bacterial cell wall (mycolic and
meso-diaminopimelic acids) have been identified as possible
virulence factors of C. pseudotuberculosis [2, 3, 8, 23, 24].
CLA is a chronic disease in adult small ruminants and is
characterized in the external form by fibrous encapsulated
abscesses in the peripheral lymph nodes; in the internal
form, abscesses sometimes form in the lungs and other
visceral organs. These encapsulated abscesses contain
concentric layers of yellow-green granular pus [3, 8, 29].
Enlargement of lymph nodes and the development of
abscesses can rupture and contaminate the milk, lambs, kids,
other animals and environment [2, 8, 26]. CLA is a highly
prevalent disease in sheep and goat population’s worldwide
[2, 6, 8, 26]. The prevalence rate is high in most countries;
for example, 42.4% of 4,089 culled sheep originating in five
regions of the western United States were positive [26] and
53.7% of 4,574 adult ewes slaughtered at a western Australian
abattoir exhibited the disease [6]. Major economic losses are
incurred due to decreased milk production, condemnation
of portions of or entire carcasses, reproductive inefficiency,
and devaluation of hides; and to a lesser extent deaths [2, 8,
9]. C. pseudotuberculosis has also public health significance,
causing human lymphadenitis. Moreover, it is likely that
sheep are the source of infection in humans [19] and the
possible public health risk also contributes to economic
losses [2, 19].
Revue Méd. Vét., 2013, 164, 2, 60-66
PCR DETECTION OF CORYNEBACTERIUM PSEUDOTUBERCULOSIS FROM SHEEP LYMPH NODES
The diagnosis of CLA poses many problems.
Palpation of affected lymph nodes is unreliable; it is not
specific and does not detect early cases or cases in which only
deep-seated lymph nodes and organs are involved [25]. The
most commonly used diagnostic methods for the detection
of C. pseudotuberculosis in animals are bacteriological culture
techniques and serological assays. C. pseudotuberculosis
grows relatively slowly on bacteriological culture media, so
conventional bacteriological methods for the recovery and
identification of this agent from different clinical samples can
take three to five days and can only detect living organisms
[10, 15, 21, 23]. Several serological tests have been used with
variable results. Serum antibodies to C. pseudotuberculosis
can be detected by haemolysis inhibition [9], anti-haemolysin
inhibition [25], indirect haemagglutination [25], complement
fixation [21], gel-diffusion precipitation [25], slide-and-tube
agglutination tests [25], and several ELISA techniques [13,
27]. Although serological tests are faster, these tests are not
enough specific or sensitive; furthermore, some tests are
difficult to standardize, especially those based on haemolysis
[13, 25, 27].
The lack of clinical features of CLA combined with the
drawbacks of bacteriological and serological detection
methods emphasize the need for reliable alternative
diagnostic methods to detect C. pseudotuberculosis [10,
27] in clinical materials such as lymph nodes. Polymerase
chain reaction (PCR)-based diagnostic techniques have the
potential to meet the need for better diagnostic tools for
infectious diseases caused by fastidious or slow-growing
bacteria. There are a few studies on the detection of C.
pseudotuberculosis DNA from pure culture [10, 12, 18] or pus
samples [18]. However, according to the available data, direct
PCR detection of C. pseudotuberculosis in lymph nodes has
not been reported. In this study, it was aimed to validate and
standardize a PCR protocol to detect C. pseudotuberculosis
DNA in lymph node samples from naturally infected sheep
and to compare its performance with the conventional
bacteriological culture method.
Materials and methods
ANIMALS AND SAMPLES
The carcasses of 3-6 years old sheep, not previously
immunized against CLA, slaughtered for human
consumption as food were examined for the presence of
CLA lesions in the Van Branch Slaughterhouse, Meat and
Fish Institute, Eastern Turkey; a total of 1,913 and 2,382
sheep carcasses were examined in the winter and summer
seasons, respectively. Abscessed lymph nodes (prescapular
and mediastinal) were taken from 147 carcasses under strict
aseptic precautions, with a sterile scalpel and placed in
individual sterile containers. The samples were immediately
transported to the laboratory and processed for culture, and
stored at -70°C before PCR processing.
Revue Méd. Vét., 2013, 164, 2, 60-66
61
BACTERIOLOGICAL ANALYSES
Swabs from the prescapular and mediastinal lymph
nodes were inoculated onto blood-agar base (Merck,
Darmstad, Germany) containing 5% (v/v) defibrinated sheep
blood. The plates were incubated at 37°C for five days and
were periodically checked for growth. Dry, yellowish-white,
opaque, and crumbly colonies were selected and transferred
onto brain-heart-infusion agar (BHI agar) (Oxoid,
Basingstoke, England) for pure culture. Identification of C.
pseudotuberculosis strains was performed using standard
classification tests: colony morphology; haemolytic activity
on blood agar; Gram stain; catalase and urease activity;
fermentation of glucose, maltose, galactose, mannose,
trehalose, lactose, arabinose, esculin, salicin, inositol and
xylose; methyl red; motility; and nitrate reduction. CAMP
(Christie, Atkins, Munch-Peterson) tests with Staphylococcus
aureus and Rhodococcus equi were also performed [8, 15, 17,
21].
POLYMERASE CHAIN REACTION (PCR) ASSAY
To avoid possible contamination, the extraction,
amplification and electrophoresis stages of the PCR were
performed in a separate cabinet. All plastic ware used
was DNase and RNase free, disposable and not re-used
throughout the experiment. Different sets of micropipettes
were used at each step of sample processing, DNA extraction,
PCR-mix preparation and electrophoresis.
DNA extraction from lymph nodes:
Immediately prior to DNA extraction, samples were
thawed at room temperature and any fat, pus or caseous mass
present was removed using a sterile scalpel. A 4 g portion
of lymph node sample (pooled prescapular and mediastinal
lymph nodes) was placed in a stomacherTM (Seward Ltd.,
West Sussex, UK) with 8 mL of sterile phosphate-buffered
saline (PBS, pH 7.2) and homogenized for 10 minutes.
An aliquot of the homogenate (300 µL) was placed in a
microtube for DNA extraction. Next, the same volume of
lysis solution (10 mM Tris-HCl, 1% SDS, 100 mM NaCl,
2% Triton-X100, pH 8.0) and 15 mL of proteinase K (20 mg/
mL) (Qiagen, Hilden, Germany) were added to the samples,
and the contents were mixed thoroughly. Following 1 hour
of boiling, saturated phenol (liquid phenol containing 0.1%
8-hydroxyquinoline, stabilized with 100 mM Tris-HCl, pH
8.0, and 0.2% 2-mercaptoethanol, 300 mL) was added; the
contents were mixed vigorously for 5 minutes and then
centrifuged at 11,600 g for 10 minutes at room temperature.
An equal volume of chloroform-isoamyl alcohol (24:1)
(Applichem, Darmstadt, Germany) was added to the aqueous
layer and after mixing thoroughly for 5 minutes, tubes were
centrifuged as before. Then, 0.1 volume of 3 M sodium
acetate (pH 5.2) and 2 volumes of 95% ethanol were added
to the upper layer and mixed thoroughly. After incubation
overnight at -20°C to precipitate the DNA and centrifugation
62
ILHAN (Z.)
Primer name
PLD-F
PLD-R2
Primer sequence (5’-3’)
PCR product
ATA AGC GTA AGC AGG GAG CA
ATC AGC GGT GAT TGT CTT CCA GG
203 bp
Table I: Primers used for PCR analyses of Corynebacterium pseudotuberculosis from lymph nodes in sheep [18].
at 13,000 g for 10 minutes at 0°C, the supernatants were
discarded and the pellets were washed sequentially with
95% and 70% ethanol. The samples were then vortexed and
centrifuged at 11,600 g for 5 minutes at 4°C. The pellets
were dried and suspended again in 50 µL of TE (TrisEDTA) buffer (Applichem). In addition, for standardization
of the extraction method, a commercial DNA-extraction
kit (DNeasy Blood&Tissue Kit®) (Qiagen) was also used
according to the manufacturer’s recommendations. For this
purpose, 25 mg of the pooled lymph tissue samples were
used for the initial extraction material. DNA concentrations
were determined spectrophotometrically (GBC, Dadenong,
Australia) by reading absorbance at 260 and 280 nm. Samples
were stored at -20°C until used as templates for amplification.
Primers, amplification conditions and agarose gel
electrophoresis:
The oligonucleotide primers used for PCR were from
the pld gene of C. pseudotuberculosis. Their sequences were
described by PACHECO et al. [18] and were presented in
Table I.
Amplification-reaction mixtures were prepared in
volumes of 50 µL containing 5 µL of 10X PCR master mix
(Fermentas, Vilnius, Lithuania), 5 µl of 25 mM MgCl2, 0.2
µL of 10 mM dNTP mixture (Fermentas), 2 U of Taq DNA
polymerase (Fermentas), 1 µmol of 25 mM each primer,
0.1% Triton-X100 (Applichem) and 5 µL of template. Bovine
serum albumin (BSA, 10 g/L, 1 mL) (Sigma-Aldrich Corp., St.
Louis, MO, USA) in 0.85% NaCl was added as an enhancer
to each PCR reaction mixture. PCR was performed in a
DNA thermocycler (Thermo Electron Corp., Waltham, MA,
USA) and amplifications were performed using the following
protocol: after an initial denaturation at 94°C for 5 minutes,
the next 35 cycles were 94°C for 1 minute (denaturation),
56°C for 1 minute (annealing), and 72°C for 2 minutes
(extension). The final extension was at 75°C for 5 minutes.
The negative control contained sterile, DNase/RNase free,
DEPC (diethylpyrocarbonate)-treated water (Applichem)
instead of DNA template. As a positive control, DNA isolated
from C. pseudotuberculosis Pl 18 strain (isolated strain
from a sheep with CLA and identified by biochemical tests,
inhibition of β-haemolysis by S. aureus (ATCC 25923) and
the CAMP test with R. equi (ATCC 33701) [16]) was supplied
by the Department of Microbiology, Faculty of Veterinary
Medicine, University of Ankara, and was included in all tests.
To determine the reliability of the results and to detect any
external contaminations, all PCR samples were processed in
duplicate.
The amplified products were analyzed by electrophoresis
on a 2% (w/v) agarose gel run at 80-90 volts for 1.5-2
hours and then stained with ethidium bromide (0.3 mg/L).
Amplified products were visualized using a computerized
image-analysis system (Spectronics Co., Westburg, NY,
USA). PCR products with a molecular size of 203 bp were
considered positive for C. pseudotuberculosis.
PCR limit of detection in lymph tissue samples:
C. pseudotuberculosis Pl 18 strain was grown on BHI agar
at 37°C for 48 hours. A single colony was removed from BHI
agar, placed in 25 mL of BHI broth (Oxoid) containing 0.05%
Tween 80 and incubated at 37°C for 48 hours. The culture
was then held at 4°C for 12 hours and centrifuged at 4,500
g for 20 minutes. The supernatant was discarded and pellet
was washed three times with sterile PBS (pH 7.2). Next, the
pellet was suspended again in 10 mL of sterile PBS and 10fold dilutions (from 10-1 to 10-10) were made. From each of
these dilutions, 0.1 mL was inoculated onto duplicate BHI
agar plates and incubated at 37°C for 48 hours; following
incubation, the colonies present were enumerated. Sterile
distilled water (Applichem) served as the negative control
and no growth was detected after incubation [16]. The
number of organisms in the dilutions was estimated
spectrophotometrically at 630 nm, and the concentration of
the original C. pseudotuberculosis suspension was estimated
at 2.1 x 108 colony-forming units (CFU)/mL.
To assess the PCR detection limit, prescapular and
mediastinal lymph node samples were collected from
14 slaughtered sheep that were considered to be free of
CLA (i.e., no history of CLA and no positive results from
bacteriological culture samples). These lymph nodes (pooled
prescapular and mediastinal lymph nodes) were then
artificially contaminated with known concentrations of C.
pseudotuberculosis Pl 18 strain ranging from 2.1 x 107 to 2.1
x 101 CFU/mL at final concentrations. C. pseudotuberculosis
DNA was extracted from all dilutions of lymph tissue samples
and processed by PCR as described earlier. All experiments
were done in triplicate.
STATISTICAL ANALYSIS
Eighty-one sheep tested positive according to the
bacteriological culture and were accepted as true positives
(gold standard) in the statistical analyses for the calculation
of sensitivity and specificity. The chi-squared test was used
to evaluate statistical significance and p < 0.05 was accepted
as significant [28]. The level of agreement between the
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PCR DETECTION OF CORYNEBACTERIUM PSEUDOTUBERCULOSIS FROM SHEEP LYMPH NODES
63
C. pseudotuberculosis positive samples (n = 81)
Season
Winter
Summer
Location
Prescapular lymph node
Mediastinal lymph node
Both locations
32
49
50
12
19
Table II: Distribution of the samples positive for C. pseudotuberculosis by microbiological analysis among the abscessed lymph nodes (n = 147) collected in
slaughterhouse according to the season and to the anatomical location
Bacteriological results
Positive (n = 81)
Negative (n = 66)
80
1
5
61
PCR positive
PCR negative
Positive agreement score
Negative agreement score
Total agreement score
93.02% (80/86)
91.04% (61/66)
95.92% (141/147)
Relative PCR sensitivity
Relative PCR specificity
Positive predictive probability
Negative predictive probability
98.76% (80/81)
92.42% (61/66)
98.4%
94.1%
Agreement score: number of identical (positive, negative or both) results with the 2 methods among the number of tested samples (positive, negative or both);
Sensitivity: frequency of true positive results among positive cultures; Specificity: frequency of true negative results among negative cultures; Positive and negative
predictive probabilities: probability of giving a positive and negative result, respectively by PCR.
Table III: Comparison between bacteriological and PCR results obtained from 147 sheep abscessed lymph node samples.
methods (PCR assay and bacteriological culture method)
was analyzed (with a 95% confidence interval) using the one
proportion method in Minitab (http://www.minitab.com,
released version 16; accessed 20 August 2011).
nodes (Table II). C. pseudotuberculosis was not isolated from
the remaining 44.8% (66/147) samples, but 50.0% (33/66) of
them were positive for Staphylococcus spp., 25.8% (17/66) for
Streptococcus spp., and 21.2% (14/66) for Escherichia coli.
Results
MOLECULAR DETECTION OF C. PSEUDOTUBERCULOSIS
MICROBIOLOGICAL FINDINGS
A PCR amplified DNA fragment of 203 bp specific
for the pld gene of C. pseudotuberculosis was evidenced in
85 lymph node samples (57.8%) (figure 1). Positive PCR
results were detected in different aliquots containing a C.
pseudotuberculosis density of at least 2.1 x 104 - 2.1 x 103 CFU/
mL in lymph tissue samples (figure 2).
Smears
of
colonies
revealed
Gram-positive
coryneform microorganisms. Isolates were identified as C.
pseudotuberculosis with positive catalase, urease, glucose,
galactose, maltose, mannose and methyl red tests and negative
trehalose, arabinose, lactose, esculin, xylose, inositol, salicin,
nitrate-reduction and motility tests. Isolates also inhibited
the activity of S. aureus β-haemolysin and enhanced the
haemolysis of R. equi.
C. pseudotuberculosis was isolated from 55.1% (81/147) of
abscessed lymph nodes collected in 147 animals, with 39.5%
(32/81) isolated in winter and 60.4% (49/81) in the summer.
In addition, 61.7% (50/81) isolates were cultured from
prescapular lymph nodes, 14.8% (12/81) from mediastinal
lymph nodes, and 23.4% (19/81) were present in both lymph
Revue Méd. Vét., 2013, 164, 2, 60-66
When the culture and PCR results were compared, 80
samples were positive (positive agreement score: 93.02%
(80/86)) and 61 samples were negative (negative agreement
score: 91.04% (61/66)) with the 2 methods, leading to a high
total agreement score of 95.92% (141/147). However, 3.40%
(5/147) samples tested negative by culture were positive by
PCR and 0.68% (1/147) sample was positive by bacteriological
culture but was negative by PCR (Table III). Consequently,
the sensitivity (frequency of true positive samples among
positive cultures) and specificity (frequency of true negative
64
Figure 1: Corynebacterium pseudotuberculosis polymerase chain reaction
(PCR) assay products obtained from sheep lymph tissue samples separated on a 2% (w/v) agarose gel.
M: Molecular marker; Lanes 1-4: culture negative, PCR positive
samples; Lanes 5-7: culture positive, PCR positive samples; Lane 8:
control negative (DNase / RNase free sterile water); Lane 9: control
positive (DNA from C. pseudotuberculosis Pl 18 strain).
samples among negative cultures) of PCR related to the
bacteriological culture were 98.76% and 92.42%, respectively,
leading to 98.4% as positive predictive probability and 94.1%
as negative predictive probability (with a 95% confidence
interval). In addition, differences in positive rates by the PCR
and culture methods were not statistically significant.
Discussion
The results obtained in previous studies showed that the
distribution of CLA lesions varies according to the anatomical
localization [10, 15, 26]. Prescapular lymph nodes were the
most common site of superficial lesion development in the
external form of the disease; the mediastinal and bronchial
lymph nodes were the most frequently affected thoracic lymph
nodes in the internal form [24]. Accordingly, prescapular
and mediastinal lymph node samples were analysed in this
study as representatives of the external and internal forms
of CLA, respectively. C. pseudotuberculosis was isolated from
85.19% of the prescapular lymph node samples and 38.27%
of the mediastinal lymph nodes. This result indicates that
prescapular lymph nodes are more suitable for culture than
mediastinal lymph nodes.
In a previous study, 41 abscessed lymph nodes from
sheep were examined by the bacteriological culture method
[15]: 85.4% of them were microbiologically positive,
and C. pseudotuberculosis was isolated in 46.3% of cases.
Additionally, Micrococcus spp. were cultured from 19.5%
of the samples, S. aureus and Pseudomonas aeruginosa
from 7.3%, and S. epidermidis from 4.8% [15]. In another
study, C. pseudotuberculosis was isolated from 78.8% of
118 abscessed lymph nodes (89 from sheep and 29 from
goats) [10]. Moreover, C. pseudotuberculosis was isolated
from all pus samples collected from small ruminants [18].
In the current study, C. pseudotuberculosis was isolated from
55.1% of the lymph node samples, 39.5% of positive samples
were found in winter and 60.4% in summer. In addition,
Staphylococcus spp. was prominently identified in the other
ILHAN (Z.)
Figure 2: Detection limit of C. pseudotuberculosis Pl 18 strain in inoculated sheep lymph tissue samples by PCR assay and visualized on a 2%
(w/v) agarose gel.
M: Molecular marker; Lanes 1-6: decreasing C. pseudotuberculosis
concentrations (CFU/mL) inoculated in lymph tissues (2.1 x 102; 2.1
x 103; 2.1 x 104; 2.1 x 105, 2.1 x 106 and 2.1 x 107, respectively); Lane 7:
control negative; Lane 8: control positive (DNA from C. pseudotuberculosis Pl 18 strain).
tissue samples negative for C. pseudotuberculosis following
by Streptococcus spp. and E. coli. These results indicate that
several bacterial organisms are present in CLA lesions;
however, C. pseudotuberculosis has been implicated as the
major pathogen in CLA [24]. In this study, the isolation rate
of C. pseudotuberculosis was higher in the summer season
than in winter. This could be because infections in sheep
are often introduced through superficial skin wounds; these
wounds can commonly occur during shearing, castration,
tagging and docking, procedures that all take place in the
summer. The fact that the abattoir materials in the region
of Van mostly represent unproductive and unhealthy sheep
might have also contributed to this finding. The breeders
are eager to slaughter the animals in a short period of time,
meaning the animals may be slaughtered during the summer
rather than at the beginning of winter.
Researches performed on naturally infected sheep
materials to determine the diagnostic sensitivity and
specificity of the PCR assay in comparison to bacteriological
culture methods are scarce. PACHECO et al. [18] analysing
pus samples collected from abscessed lymph nodes of 12
naturally infected sheep and 44 goats by bacteriological
culture and multiplex PCR (mPCR) reported that the relative
mPCR sensitivity was 91.7% in sheep, 95.4% in goats and
94.6% in both species. In the present study, 81 (55.1%) and
85 (57.8%) of the lymph-node samples from sheep were
positive by culture and PCR, respectively. Comparing these
techniques, 5 (3.40%) of the samples were PCR positive but
culture negative and would be considered as false positive
results, and only one sample (0.68%) was PCR negative
but culture positive and would be considered as false
negative result. Consequently, the relative PCR sensitivity
and specificity compared to cultures in the present study
were 98.76% and 92.42% respectively, leading to a positive
predictive probability of 98.4% and to a negative predictive
probability of 94.1%. The diagnostic gain obtained here was
higher than previously reported by PACHECO et al. [18].
Several factors may influence the sensitivity, specificity and
efficiency of PCR assays, including the nucleic acid extraction
Revue Méd. Vét., 2013, 164, 2, 60-66
PCR DETECTION OF CORYNEBACTERIUM PSEUDOTUBERCULOSIS FROM SHEEP LYMPH NODES
method, physic and chemical conditions of the reaction, total
reaction volume and the concentration of target DNA in the
samples [5]. Indeed, different samples and different input
volumes of the extraction materials were used and different
extraction protocols were applied in the two studies. Another
plausible explanation for these differences could be the
presence of various inhibitory substances such as haemoglobin
or blood in the samples. PACHECO et al. [18] used pus samples
that may have included more inhibitory substances than the
lymph-tissue samples used in this study. Several methods have
been attempted to decrease the effect of inhibitors, including
sample dilution, the addition of cationic surfactants, or other
sample facilitators such as BSA or polyethylene glycol [11, 14].
Differing from PACHECO et al. [18], BSA was added to the
PCR reaction mixtures in this study, which may enhance the
sensitivity of PCR assays. These findings may indicate that the
analysis of lymph tissue samples by PCR is a better option for
the detection of C. pseudotuberculosis DNA than the analysis of
pus samples from post-mortem sheep.
Studies concerning the limits of detection for genomic
DNA from C. pseudotuberculosis by PCR assays are also
limited. Blood samples from healthy goats were seeded with
101-1010 CFU/mL of C. pseudotuberculosis and then tested by
mPCR. The amplification products were detected in reactions
containing 106-103 CFU/mL of C. pseudotuberculosis [18].
In the present study, when the lymph-node samples from
healthy sheep were artificially contaminated, the detection
limit of the PCR assay was 2.1x104-2.1x103 CFU /mL of C.
pseudotuberculosis. This detection limit is lower than those
previously reported [18]. This may be due to several reasons.
First, the spiked samples used were different in the two studies.
Second, other inhibitory substances, such as such as collagen,
lactoferrin and PCR conditions may affect the detection limit
of the PCR assay [1, 22]. This detection limit does not pose a
problem in testing samples for CLA as very large numbers of
the microorganisms are present in CLA-infected sheep [7, 15,
27].
The cell-wall content of Corynebacteria is somewhat unique
and is very high in lipid content, the most notable being
mycolic and meso-diaminopimelic acids. On the basis of this
lipid content, the Corynebacteria genus is often grouped with
Mycobacteria, Nocardia and Rhodococci to form the “CMNR”
group [4, 8]. Pathogen members of the CMNR group cause
chronic and suppurative infections in animals [8, 21, 26]. This
PCR protocol might be applied for the diagnosis of CMNRgroup infections, but further studies in a variety of animal
species will be required.
Bacteriological culture and serological assays have
traditionally been used for the diagnosis of CLA, but these
techniques have a number of drawbacks. Thus, an alternative,
reliable method is needed to detect C. pseudotuberculosis in
clinical materials. This study presents a standardized PCR
protocol using lymph node samples from sheep; the positive and
negative predictive probabilities of this assay were determined
to be 98.4% and 94.1%, respectively. As a conclusion, data
Revue Méd. Vét., 2013, 164, 2, 60-66
65
from the current study suggests that, due to its high sensitivity,
relatively high specificity and rapidity, the PCR protocol could
be utilized as an alternative to traditional bacteriological
culture methods for the detection of C. pseudotuberculosis in
lymph node samples from naturally infected sheep. The results
of the present study are particularly important in aiding the
understanding of the epidemiology of CLA and control of the
disease.
Acknowledgments
The author thanks to Dr. E. EYDURAN, Biometry and
Genetics Unit, Department of Animal Science, Faculty of
Agriculture, Igdır University, Igdır, TURKEY, for statistical
analysis.
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