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Journal of Clinical Microbiology, April 1998, p. 1050-1055, Vol. 36, No. 4
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Detection of Mycobacterium tuberculosis
Complex in Cattle by PCR Using Milk, Lymph Node Aspirates, and
Nasal Swabs
Fabrizio
Vitale,*
Giuseppina
Capra,
Letizia
Maxia,
Stefano
Reale,
Gesualdo
Vesco, and
Santo
Caracappa
Istituto Zooprofilattico Sperimentale Della
Sicilia, Palermo, Italy
Received 28 April 1997/Returned for modification 12 November
1997/Accepted 14 January 1998
 |
ABSTRACT |
The PCR technique was applied to the diagnosis of tuberculosis in
live cattle, and both skin-test-negative and skin-test-positive animals
were studied. DNA was taken from various sources including specimens of
lymph node aspirates, milk, and nasal swabs. After slaughter and visual
inspection, tissues such as lymph nodes, lungs, and udders from
tuberculin reactors were tested by the same technique. Specific
oligonucleotide primers internal to the IS6110 insertion
element were used to amplify a 580-bp fragment. A 182-bp fragment was
obtained by designating a nested PCR from the first amplification
product. This fragment was cloned and sequenced, and after being
labeled it was employed in dot blot hybridization. A total of 100 cattle were tested, and PCR analysis was performed using nasal swab,
milk, and lymph node aspirate. Sixty skin-test-positive cows were also
tested to detect mycobacterial DNA in tissue samples from lymph nodes,
lungs, and udders, and the infection was confirmed in all of the
animals. Using PCR analysis of tissue samples from slaughtered animals
as a "gold standard" we calculated 100% values for sensitivity,
specificity, and positive and negative predictive values for milk and
lymph node aspirate samples. The respective values for nasal swab
samples were 58, 100, 100, and 28%. The respective values for all of
the samples were 74, 100, 100, and 35%, while for visual inspection
the values were 81, 100, 100, and 58%, respectively. PCR analysis of
specimens of lymph node aspirates, milk, and nasal swabs from
skin-test-negative animals showed that 52% of these skin test results
were false negatives. These animals, not being removed from the farms,
represent a potential source of further infection.
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INTRODUCTION |
Mycobacterium bovis, the
cause of tuberculosis in cattle, is also a pathogen for a large number
of other animals, and its transmission to humans constitutes a public
health problem (10). The diagnosis of bovine tuberculosis in
live animals mainly depends on clinical manifestations of the disease,
skin testing, and subsequent identification of the pathogen by
biochemical testing. It is known that the skin test lacks sufficient
sensitivity and specificity in many cases (8, 9, 16, 21).
Neill and coworkers (15) have reported that M. bovis may be isolated from the secretions of skin-test-negative
cattle and, furthermore, that these animals were not anergic, as is
sometimes the case in the later stages of the disease.
Identification of the mycobacterium is based on the traditional method
with the Ziehl-Neelsen acid-fast stain and on the pigmentation, growth
rate, and gross and microscopic colony morphologies of cultures of the
isolated causative organism. Biochemical methods such as tests for
niacin, catalase, nitrate reduction, and urease are used to identify
different species.
The Ziehl-Neelsen stain is very rapid but lacks specificity and cannot
be used to distinguish between the various members of the family
Mycobacteriaceae, while the other procedures usually require
4 to 8 weeks to obtain good growth. In order to be certain of the
diagnosis of tuberculosis postmortem histopathological examination of
organ lesions is carried out.
In the past few years molecular approaches to diagnosis have been
transforming the investigation of tuberculosis, especially in human
medicine. The introduction of PCR and nucleic acid hybridization has
greatly reduced identification time (3), and the use of PCR
has improved the level of detection in clinical specimens. It has been
previously reported (14) that by amplifying species-specific DNA sequences, and hybridizing the amplified sequence with a labeled probe, 5 fg of mycobacterial DNA (corresponding to one mycobacterium) can be detected in clinical samples. Rapid diagnosis by PCR with a
number of different targets (11, 17), including the
IS6110 insertion sequence, has been previously described
(2, 6, 7, 12, 23). IS6110 has only been detected
in species belonging to the M. tuberculosis complex
(M. tuberculosis, M. bovis, M. africanum, and M. microti) which present this sequence
in multiple copies. In the classical human M. tuberculosis
variant, the IS6110 element is usually present in 8 to 20 copies. In M. bovis strains the IS6110 element is
present in two to six copies (4, 5, 24-26). Only M. bovis BCG has a single copy of IS6110, as has been demonstrated in many studies using restriction fragment length polymorphism patterns (12, 13, 22).
The aim of this work was to evaluate the possible application of the
PCR technique to the diagnosis of tuberculosis in live cattle. PCR
analyses of biological samples such as milk, nasal swabs, and lymph
node aspirates taken from animals with known skin test reactions are
described. PCR analysis was also performed using tissue specimens from
slaughtered skin-test-positive animals to confirm our results. The
sensitivity and specificity of PCR and nucleic acid hybridization
methods were compared with those of the skin test. The results indicate
that these methods could become useful diagnostic tools especially for
the large-scale screening of cattle.
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MATERIALS AND METHODS |
Mycobacterial cultures.
The following mycobacterial
reference strains were obtained from the collections of the Pasteur
Institute (Paris, France): M. bovis (B7292), M. tuberculosis (140010002IP), M. avium (140310001IP), M. chelonae (140420003IP), M. phlei
(141300001IP), and M. fortuitum (140410001IP). M. paratuberculosis (ATCC 19698) was obtained from the American Type
Culture Collection. All the mycobacterial cultures were maintained on
Lowenstein-Jensen agar slopes and were grown in 100 ml of Dubos medium
enriched with 10% Dubos medium albumin and 5% equine serum
(Microbiological Diagnostici). The cultures were incubated for 25 to 28 days at 37°C. M. bovis was isolated routinely from
sacrificed bovines submitted to the Istituto Zooprofilattico Sperimentale della Sicilia in Palermo, Italy, where it was identified by conventional testing which included growth rate, gross and microscopic colony morphologies, and pigmentation of cultures and tests
for niacin, catalase, nitrate reduction, and urease.
Preparation of mycobacterial DNA.
Large-scale DNA
extractions were performed as described by B. C. Ross et al.
(18) with some modifications. Mycobacterial cultures in 100 ml of Dubos medium were centrifuged for 15 min (3,500 × g, 4°C) in a GPR Beckman centrifuge. The pellet was washed twice with STE buffer (100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA [pH
8.0]) and suspended in 4 ml of lysis buffer (50 mM Tris-HCl, 50 mM
EDTA [pH 8.5] 15% [wt/vol], 4% sodium dodecyl sulfate [SDS]). Lysozyme (Boehringer Mannheim) was added to a final concentration of 2 mg/ml. The mixture was incubated in a thermostatic bath at 37°C for
3 h. Proteinase K (Sigma Chemical Co.) was added to a final
concentration of 100 µg/ml, and incubation was continued at 37°C
for 1 h. After two rounds of phenol-chloroform extraction, the DNA
was precipitated with ammonium acetate (final concentration, 0.3 M),
overlaid with 2.5 volumes of ice-cold ethanol, and mixed by inversion
(19). Genomic DNA was recovered by centrifugation at
13,800 × g for 30 min, washed with 70% (vol/vol)
ethanol, and suspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH
8.0]). The DNA was incubated with RNase A (Boehringer Mannheim) (100 µg/ml) at 37°C for 1 h and further purified with another
phenol-chloroform extraction and precipitation step. The concentration
and purity of extracted DNA were calculated by readings of
A260 and A280 with a
Hitachi U-1100 spectrophotometer. The reagents were supplied by Sigma
Chemical Co.
DNA purification from bovine samples.
DNA was extracted from
samples of lymph node aspirates, milk, and nasal swabs from live
animals and from lymph node, lung, and udder tissues taken from
slaughtered animals. The extraction from the live animal samples was
performed with a QIAamp Blood and Tissue Kit (QIAGEN). Nasal swabs were
washed in 0.5 ml of phosphate-buffered saline solution. A total of 200 µl of each sample was incubated with Proteinase K in 1 volume of a
suitable lysis buffer, and then the enzyme was inactivated by heating
to 70°C for 10 min. Ethanol (0.525 volumes) was added, and the
mixture was applied onto a QIAamp spin column. After two rounds of
washing, the DNA was eluted with 200 µl of the supplied buffer
preheated to 70°C.
After slaughter, DNA extraction from tissue samples was performed by
lysis with chaotropic reagent in guanidinium thiocyanate (GuSCN)
(Eastman Kodak Company), as described by Boom et al. (1). A
small piece of tissue, about 25 mg, was lysed in 900 µl of
GuSCN-containing lysis buffer (120 g of GuSCN in 100 ml of 100 mM
Tris-HCl [pH 6.4] to which 20 ml of 36 mM EDTA [pH 8]-2%
[wt/vol] Triton X-100 was added) with 40 µl of diatom suspension
(Sigma Chemical Co.). The diatom-DNA complexes were rapidly collected
by centrifugation at 12,500 × g for 30 s in a
Beckman microcentrifuge. The pellet was washed twice with 1 ml of
GuSCN-containing washing buffer (120 g of GuSCN in 100 ml of 100 mM
Tris-HCl [pH 6.4]), twice with 1 ml of 70% (vol/vol) ethanol, and
once with 1 ml of acetone. After draining at 56°C for 10 min, the DNA
was eluted with 100 µl of TE buffer preheated to 56°C.
DNA amplification by PCR.
The target DNA for amplification
was a 580-bp fragment of IS6110, an insertion sequence-like
element currently used to identify members of the M. tuberculosis complex.
The primers used were the oligonucleotides 295 up
(5'-dGGACAACGCCGAATTGCGAAGGGC-3') and 851 down
(5'-dTAGGCGTCGGTGACAAAGGCCACG-3'),
which correspond to base
pairs 295 to 318 and 851 to 874 of the
IS
6110 insertion
element, respectively. The oligonucleotide sequence
was chosen because
of its GC content by using the Mac-Vector 5.0
program sequence analysis
software (Oxford Molecular Group).
To generate a sequence-specific probe, we designated an amplicon-nested
PCR product of 182 bp from the amplified 580-bp fragment.
It was
obtained by using primer 505 up (5'-dACGACCACATCAACCGGG-3')
and primer 669 down (5'-dGAGTTTGGTCATCAGCCG-3'), which
correspond
to base pairs 505 to 523 and 669 to 686, respectively. All
the
oligonucleotides were supplied by Cruachem Ltd. (Glasgow, United
Kingdom). PCR amplification was carried out in 100-µl reaction
mixtures containing (final concentrations) 2.0 mM MgCl
2, 50 mM
KCl, 10 mM Tris-HCl [pH 9.3], 0.01% Triton X-100, 200 µM (each)
deoxynucleoside triphosphate, 200 nM each primer, 72 µl of template
DNA solution, and 2.5 U of DNA
Taq polymerase (Promega). The
reactions
were performed in an automated thermal cycler (Mini Cycler;
MJ
Research, Inc.). The conditions were set as follows: denaturation
at
95°C for 1 min, annealing at 65°C for 1 min, and extension
at
72°C for 1 min. A 7-min extension period at 72°C was added
after 30 cycles. A positive control containing 10 ng of
M. bovis (B/29292) DNA and a negative control, without template DNA, were
included.
Electrophoresis.
Purified DNA and PCR products were analyzed
by electrophoresis through 0.8 and 1.5% neutral agarose gels,
respectively, containing 0.1 µg of ethidium bromide (Bio-Rad
Laboratories)/ml in TBE buffer (0.089 M Tris-HCl, 0.089 M boric acid,
0.002 M EDTA). The gels were visualized under UV light with a
transilluminator (UV-GENTM; Bio-Rad Laboratories) and photographed with
Polaroid 667 film. The DNA markers used were 
-HindIII
or ladder 100 (Pharmacia).
Cloning and sequencing of the specific DNA probe.
The DNA
probe, originated by PCR, was cloned by using a TA cloning kit
(Invitrogen). The recombinant plasmid was obtained by ligation of 50 ng
of pCR 2.1 vector with 10 ng of fresh PCR product. This plasmid was
employed to transform INV
F' One Shot competent cells. Briefly, 2 µl of 0.5 M 
-mercaptoethanol and 2 µl of the ligase reaction
product were added to 50-µl vials of frozen cells. After 30 min on
ice, the cells were heat shocked for 30 s in a 42°C water bath
and incubated in 250 µl of purchased SOC medium at 37°C for 1 h with rotary shaking at 225 rpm. Aliquots of 50 and 200 µl from each
transformation were spread on Luria-Bertani agar plates containing 50 µg of ampicillin/ml and X-Gal
(5-bromo-4-chloro-3-indolyl--D-galactopyranoside). The
plates were incubated for 18 h at 37°C. The picked colonies were
grown overnight in 20 ml of Terrific Broth. The nucleotide sequence of
the cloned fragment was determined by using pCR 2.1 primer with the
Sequenase kit from USB (20).
DNA labeling of the 182-bp DNA probe.
The 182-bp amplified
fragment was labeled with digoxigenin-dUTP by using a DIG DNA Labeling
Kit (Boehringer Mannheim) according to the instructions of the
manufacturer. For each reaction 60 ng of DNA was labeled at 37°C for
2 h and employed in hybridization.
Dot blot assay.
The amplified DNA was denatured for 5 min at
100°C and kept on ice. Samples of 5 µl were spotted on a positively
charged nylon membrane (Boehringer Mannheim) and fixed by UV exposure.
The blots were treated with 0.4 M NaOH for 3 min and neutralized with 1 M Tris-HCl (pH 8.0) for 3 min.
Hybridization and detection.
The filter was hybridized at
65°C overnight in an incubation bag. The prehybridization mix
consisted of 4× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium
citrate) 5× Denhardt solution (2% Ficoll, 2% albumin, bovine
fraction V, 2% polyvinylpyrrolidone), and 1% SDS. About 4 ml of
hybridization solution was used for the 100-cm2 membrane.
The hybridization solution contained 15 ng of the freshly denatured (10 min, 100°C) digoxigenin-dUTP-labeled 182-bp probe/ml. After
incubation the membrane was washed twice in 1× SSC-1% SDS (100 ml/100-cm2 membrane) for 15 min and twice in 1× SSC-0.1%
SDS at 65°C for 15 min. The presence of a digoxigenin-labeled probe
was detected by using an alkaline phosphatase-conjugated antibody and
CSPD substrate according to the instructions for the DIG-dUTP-DNA
detection kit (Boehringer Mannheim). The chemiluminescent signal was
revealed on X-ray film (X AR Omat; Kodak) after exposure for 20 min at room temperature.
| |
RESULTS |
Specificity and sensitivity of PCR.
The primers 295 up and 851 down were used for PCR analysis of purified DNAs of the eight
mycobacterial species listed in Materials and Methods. A 580-bp product
was found only in mycobacteria belonging to the M. tuberculosis complex, as was confirmed by dot blot analysis (data
not shown).
The sensitivity of the PCR was determined by adding mixtures containing
decreasing amounts of
M. bovis DNA in a range between
20 ng
and 1 fg to the reaction vials. One femtogram of DNA could
be amplified
and, in dot blot analysis, gave a detectable hybridization
signal with
the 182-bp probe (data not shown).
Detection of M. bovis DNA in different biological
samples.
During a 12-month period, 100 cattle were tested by PCR
using DNA extracted from lymph node aspirates, milk, and nasal swabs. All 60 of these animals which were skin test positive were
slaughtered, and DNA was purified from samples of lymph node, lung, and
udder tissues. PCR was carried out using these DNA samples to confirm the diagnosis of M. bovis infection.
Gel electrophoresis analysis of representative examples of PCR products
is shown in Fig.
1. The 580-bp fragment
of IS
6110 was amplified in all purified DNA from nasal swabs
(Fig.
1A),
milk (Fig.
1B) and lymph node aspirates (Fig.
1C) as
demonstrated
by comparison with the positive control containing 10 ng
of
M. bovis DNA (Fig.
1, lane 2). Successful amplification
of IS
6110 fragments was also obtained with DNA extracted
from tissue specimens.
Figure
2 shows
results for four samples each of lymph node, lung,
and udder tissues.
These tissue samples were taken postmortem
from four cows that were
also used for the analysis shown in Fig.
1 (lanes 3 to 6).
Hybridization of the 182-bp probe with amplified
DNA from specimens of
nasal swabs, milk, and lymph node aspirates
is shown in Fig.
3. The dot blot of amplified DNA from
lymph node,
lung, and udder tissues is shown in Fig.
4, and only the last
spot, corresponding
to the sample in lane 14 of Fig.
2, gave no
hybridization signal.
Amplification of the 580-bp fragment which
was not detectable by gel
analysis (Fig.
2, lane 13) was proved
by dot blot hybridization. These
results show the greater sensitivity
of dot blot hybridization compared
to gel electrophoresis.
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FIG. 1.
Analysis of PCR-amplified 580-bp fragment by 1.5%
agarose gel electrophoresis. DNA was extracted from nasal swab (A),
milk (B), and lymph node aspirate (C) samples. Procedures for DNA
preparation and PCR amplification, and sequences of primers used are
given in the text. A total of 70 µl of PCR products was analyzed.
Lane 1, phage digested with HindIII (panels A and B)
or ladder 100 (panel C) as a DNA molecular size marker; lane 2, positive control amplified from 10 ng of M. bovis DNA. (A)
Lanes 3 to 20, 580-bp amplified fragments from nasal swab samples from
skin-test-positive cows; lane 21, control without mycobacterial DNA.
(B) Lanes 3 to 14, 580-bp amplified fragments from milk samples from
skin-test-positive cows; lane 15, control without mycobacterial DNA.
(C) Lanes 3 to 12, 580-bp amplified fragments from lymph node aspirate
samples from skin-test-positive cows; lane 13, control without
mycobacterial DNA.
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FIG. 2.
Analysis of PCR-amplified 580-bp fragment by 1.5%
agarose gel electrophoresis. Procedures for DNA preparation and PCR
amplification and sequences of primers used are given in the text. DNA
was extracted from tissues of four skin-test-positive cows that were
also used for the experiment described in Fig. 1 (lanes 3 to 6). Lane
1, ladder 100 as a DNA molecular size marker; lane 2, target DNA
amplified from 10 ng of M. bovis DNA; lanes 3 to 6, DNA from
lymph node tissue; lanes 7 to 10, DNA from lung tissue; lanes 11 to 14, DNA from udder tissue; lane 15, control without mycobacterial DNA.
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FIG. 3.
Analysis of PCR products by dot blot hybridization with
11-dUTP-labeled 182-bp probe. Procedures for PCR amplification,
sequences of primers used, and description of cloning and sequencing of
probe are given in the text. Samples are the same as those used in the
experiment described in the legend to Fig. 1. Spot 1a, target DNA
amplified from 10 ng of M. bovis DNA; spots 2a to 6c, PCR
products from nasal swab samples; spot 7c, control without
mycobacterial DNA; spot 1d, target DNA amplified from 10 ng of M. bovis DNA; spots 2d to 6e, PCR products from milk samples; spot
7e, control without mycobacterial DNA; spot 1g, target DNA amplified
from 10 ng of M. bovis DNA; spots 2g to 5g, PCR products
from lymph node aspirate samples; spot 6g, control without
mycobacterial DNA.
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FIG. 4.
Analysis of PCR products by dot blot hybridization with
11-dUTP-labeled 182-bp probe. Procedures for PCR amplification,
sequences of primers used, and description of cloning and sequencing of
probe are given in the text. Samples are the same as those used for the
experiment described in the legend to Fig. 2. Spot 1, target DNA
amplified from 10 ng of M. bovis DNA; spots 2 to 5, PCR
products from lymph node tissue; spots 6 to 9, PCR products from lung
tissue; spots 10 to 13, PCR products from udder tissue; spot 14, control without mycobacterial DNA.
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Samples were deemed positive when the 580-bp fragment hybridized with
the 182-bp probe in the dot blot test. Samples were
considered negative
when no detectable signal was found in the
dot blot hybridization with
the labeled probe.
We calculated the sensitivity, specificity, and positive and negative
predictive values of the combined PCR-dot blot test
performed for the
60 animals that were skin test positive. For
these calculations we used
the results obtained by PCR using both
samples from live cattle and
tissue samples as a "gold standard."
PCR analysis using 54 milk
samples and 49 lymph node aspirates
gave the 100% values for
sensitivity, specificity, and positive
and negative predictive
values. PCR using 50 nasal swabs shown
high specificity (100%) but a
low sensitivity (58%); the positive
predictive value was also 100%
while the negative predictive value
was only 28% (Table
1). The lower values for nasal swabs
account
for the total sample values of 74, 100, 100, and 35% for
sensitivity,
specificity, and positive and negative predictive values,
respectively
(Table
2).
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TABLE 1.
Detection of M. tuberculosis complex by PCR
using nasal swabs, milk, and lymph node aspirates taken from
live skin-test-positive cattle versus PCR detection using
tissue samples from slaughtered cattle
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TABLE 2.
Detection of M. tuberculosis complex by PCR
using samples taken from live skin-test-positive cattle versus PCR
detection using tissue samples from slaughtered cattle
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As shown in Table
3, we compared the
results of visual inspection with those of PCR detection using tissue
samples. We observed
43 tissue specimens with typical lesions and 10 showing no visible
lesions during the veterinary inspection; all of
these samples
were PCR positive. The remaining seven skin-test-positive
cows
were negative by PCR-dot blot hybridization for all samples
examined.
We calculated the sensitivity, specificity, and positive and
negative
predictive values for visual inspection after slaughtering as
being 81, 100, 100, and 58%, respectively.
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TABLE 3.
Detection by visual inspection of typical M. tuberculosis complex lesions in tissue samples taken from
slaughtered skin-test-positive cattle versus PCR detection using the
same tissue samples
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The main goal of this study was to check
M. bovis infection
in cattle with high precision and compare the results with those
from
skin testing. We obtained interesting results especially
in the cases
of dubious reactivity to the skin test and when a
nonspecific reaction
occurred in animals in the absence of typical
clinical manifestations.
This happened in seven skin-test-positive
animals that, submitted to
veterinary inspection, had not shown
the typical
M. bovis
lesions. Two of these cows were tested by
PCR using milk, lymph node
aspirates, lymph node tissue, and lung
tissue. One of these cows was
positive to the skin test, while
the other showed a low reaction;
however, being officially positive,
both were slaughtered. The
skin-test-positive cow was PCR and
dot blot negative, while the results
for the cow with a dubious
reaction were negative for all of the
samples tested (data not
shown).
To establish if our test could detect false skin-test-negative
subjects, we examined 40 animals considered officially
M. bovis free. Of these a group of seven bulls and a group of seven
cows
on two different farms gave interesting results. All the animals
had been negative to the skin test performed 1 year before our
investigation. The dot blot hybridization of amplified DNA samples
from
the bull lymph node aspirates and cow milk showed that all
of these
animals were positive, except one bull (data not shown).
One week later
the animals were retested by the skin test and
were examined
clinically, and all results were negative. However,
on the basis of our
results, the skin test was repeated 6 months
after our test, and again
all the results were negative. These
animals are considered officially
M. bovis free.
In Table
4 the results from the 100 animals tested by PCR during the period of our investigation are
summarized. Of the 40
skin-test-negative animals, 19 were confirmed by
PCR, while 21
resulted positive in at least two of the tests performed
using
milk, lymph node aspirates, and nasal swabs.
| |
DISCUSSION |
The data presented here represent a successful attempt to satisfy
the need for a more sensitive, specific, and rapid test for the
diagnosis of tuberculosis in cattle. In particular, the utility of PCR
as a tool to test M. bovis infection in biological samples
taken from live animals was studied. The farms tested in this study
were randomly selected in Sicily, and the specimens examined were taken
and kept in sterile conditions to avoid contamination between different
cows from the same farm. We performed PCR using samples of milk, lymph
node aspirates, and nasal swabs from 100 cows and on tissue samples,
taken after slaughter, 60 skin-test-positive animals. IS6110
was chosen as the target sequence for the PCR test because it is
specific for mycobacteria belonging to the M. tuberculosis
complex, and the results obtained were in accordance with those of
other authors (12, 14, 23). Amplification of the 580-bp
fragment, revealed by detection of the 182-bp hybridized probe, can
reveal as little as 1 fg of DNA, corresponding to one mycobacterial
genome (14). Excellent sensitivity, specificity, and
positive and negative predictive values were found for the PCR-dot blot
test performed using milk samples and lymph node aspirates. So this
kind of sample could be considered useful in the screening for
tuberculosis in cattle, having a higher accuracy than the skin test.
Sampling milk did not present difficulties, and we obtained samples
from 54 of the 60 skin-test-positive cows (the remaining 6 were
pregnant). A total of 87% of these milk samples were positive by PCR,
and the results for the "no visible lesion reactors" were negative.
This latter result was also confirmed by nasal swab and lymph node
aspirate examination, revealing 12% false positives to the skin test.
It was hypothesized that this highly sensitive technique could be
employed to diagnose early tuberculosis in cattle and to prevent the
spread of infection. The nasal swab analysis only permitted
identification of 58% of infected animals and 28% of noninfected
cattle. Our results agree with known clinical data that report few
cases of open tuberculosis in cattle. Nevertheless PCR using nasal
swabs has high specificity and positive predictive value and could be
used together with PCR using other samples from the same subjects,
giving a clear sign of airborne contamination within herds. Taking
nasal swabs is easier and quicker than other more-invasive sampling
methods. It is therefore very useful particularly in the case of
generalized tuberculosis in which it is known that some animals may
fail to respond to skin testing while M. bovis may be
present in nasal fluid.
Another significant fact was the 81% sensitivity for the visual
inspection test versus PCR using tissue samples. The absence of
macroscopic lesions does not exclude the presence of early infection.
On the other hand the 58% negative predictive value indicates that a
significant number of nonlesion reactors are slaughtered erroneously.
The easy recognition of typical tuberculosis lesions in slaughtered
cattle gives 100% specificity and positive predictive value.
Only 28 milk samples, 30 lymph node aspirates, and 26 nasal swabs from
33 cows and 7 bulls that were negative to the skin test were examined,
because we could take only one or two kinds of sample from each animal.
In fact on many farms, officially free from infection, complete
sampling was not possible. A total of 11 milk, 10 lymph node aspirate,
and 13 nasal swab samples were positive by PCR, and the subjects for
which at least two samples were positive by PCR were deemed positive.
The data suggest that 52% of the skin-test-negative animals tested in
our study may be false negatives. The low sensitivity and specificity
of the bovine skin test (8, 9, 16, 21) is the cause of decreased efficacy in eradication campaigns and leads to a greater risk
in public health programs and also to economic losses in the cattle
industry.
The specific PCR analysis reported here can be performed on biological
samples easily taken from animals on the farm. The simplicity of its
application is typified by the fact that milk samples and lymph nodes
aspirates, which give precise results, provide excellent material for
diagnosis. The method is rapid, requiring only 48 to 72 h from
sampling for the detection of amplified DNA in dot blot hybridization.
It may therefore be very useful to do PCR in parallel with the
officially approved skin test, especially in the case of dubious
reactions, anergy, or when in the presence of cross-reactivity with
correlated antigenic determinants. Moreover, it may be possible to use
this test in epidemiological studies aimed at determining the
prevalence of bovine tuberculosis in areas in which the disease has not
been eradicated.
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ACKNOWLEDGMENT |
This study was supported by grants from the Italian Ministry of
Health.
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FOOTNOTES |
*
Corresponding author. Mailing address: Istituto
Zooprofilattico Sperimentale Della Sicilia, Via Rocco Dicillo No. 4, 90129 Palermo, Italy. Phone: (39) 91-6565111. Fax: (39) 91-6565233. E-mail: izspa{at}interbusiness.it.
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Journal of Clinical Microbiology, April 1998, p. 1050-1055, Vol. 36, No. 4
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
