© CAB International 2003 Animal Health Research Reviews 4(2); 73–93

ISSN 1466-2523 DOI: 10.1079/AHRR200359

Molecular genetic methods in the veterinary

clinical bacteriology laboratory: current
usage and future applications
Hugh Y. Cai1,2, Marie Archambault1, Carlton L. Gyles2 and
John F. Prescott2*
1Laboratory Services Division, Animal Health Laboratory, and 2Department of Pathobiology,
University of Guelph, Guelph, Ontario N1G 2W1, Canada

Received 22 September, 2003; Accepted 27 October, 2003

Abstract

In the last 5 years, numerous molecular methods have been published for the detection and
characterization of bacteria in the field of veterinary medicine. PCR has been the most com-
monly used technology. Although not currently used for clinical veterinary diagnosis, new
technologies such as liquid-phase hybridization, real-time PCR, pathogen load determination
and DNA/protein microarray have been described and have many possible applications in
the clinical bacteriology laboratory because of their sensitivity and efficiency. This review
describes the basic principles and application of recently published DNA-based molecular
techniques for the purpose of veterinary clinical bacteriological diagnosis. It covers advances
in probe hybridization technology, DNA/RNA amplification techniques and other molecular
detection methods, including 16S rRNA analysis for bacterial characterization and DNA
microarrays for bacterial detection. The review briefly summarizes the application of molecu-
lar methods for the diagnosis of specific important bacterial infections of animals, and for
other animal pathogens that are slow or difficult to isolate in the clinical bacteriology labora-
tory. In addition, the molecular detection of antimicrobial resistance genes and of bovine
mastitis pathogens is briefly described and current commercially available tests are listed.

Keywords: molecular genetics, methods, veterinary, clinical bacteriology, molecular beacons,

displacement hybridization, polymerase chain reaction (PCR), real-time PCR

Introduction The overall purpose is to provide an overview that

Since the late 1980s, many molecular genetic methods
have been developed for the detection and genotyping
of bacteria. The purpose of this review is to describe
various DNA-based molecular techniques for use in vet-
erinary clinical bacteriology for the purpose of animal
health diagnostics. The principles on which these tech-
niques are based, and their use, will be outlined and
more recently published molecular methods with poten-
tial for bacterial pathogen diagnosis will be described.
*Corresponding author: Department of Pathobiology, University of
Guelph, Guelph, Ontario N1G 2W1, Canada
E-mail: prescott@uoguelph.ca
speeds the adoption of these powerful and important
molecular techniques in animal health diagnosis.
DNA based molecular tests
Advances in probe hybridization technology
Hybridization assays using DNA probes were developed
in the early 1960s. Originally, the assays were performed
in solution and involved complicated procedures,
requiring either ultracentrifugation or chromatography
and filtration to separate hybridized from unhybridized
probes. In the mid-1960s a solid-based hybridization
74
assay, Southern blotting, overcame the complexity of
working with solutions. In this well-known procedure,
DNA is transferred onto a solid membrane or filter, and
the hybridization is performed on the membrane. The
technique revolutionized DNA detection and dominated
the field for over 20 years (Demidov, 2003).
Hybridization on a solid system has the limitation of
low sensitivity because of the lack of amplification of
targets and adsorption of the probe. In addition, it is
impossible to perform real-time monitoring because it
requires washing to remove unhybridized probes. To
overcome these drawbacks, a new generation of solu-
tion-based hybridization approaches has been
developed since the mid-1990s. These methods elimi-
nate the need to remove unhybridized probes. As a
result, in the near future the solution-based hybridiza-
tion approach may become more popular than PCR in
microbial detection because of its high sensitivity, sim-
plicity and minimal requirement for specialized
equipment.
Traditional nucleic acid probes
Nucleic acid probes bind to a complementary target
DNA or RNA with high affinity, and can be used for the
identification of microorganisms. A specific sequence is
selected and a probe is generated by synthesis, by
cloning or by PCR amplification of a genome carrying
the sequence. Nucleic acids extracted from bacteria are
transferred to a nitrocellulose membrane, fixed by UV
cross-linking or baking, then hybridized with the spe-
cific probe. Unbound probe is washed away, and the
bound probe is detected by radioactive or enzymatically
active moieties incorporated into the probe.
Various techniques are used for hybridization. In the
dot blot, target DNA is spotted directly on the mem-
brane filter. In the Southern blot a specific DNA
fragment is separated electrophoretically on a gel before
transfer onto the membrane. Its advantage is that the
size of the target DNA fragment is known. The northern
blot is similar to the Southern blot except that the target
is RNA.
Molecular beacons
Molecular beacons, first developed by Tyagi et al. in
1996, are single-stranded DNA molecules, with a fluo-
rophore attached at the 5 end and a quencher at the 3
end. The loop portion of the probe is designed to be
specific to the target sequence. The stem contains five to
seven complementary nucleotides. Therefore, in the
absence of a specific target, the probe forms a hairpin-
like stem–loop structure, keeping the fluorophore and
the quencher together. The energy from the fluorophore
passes to the quencher and is converted into heat.
H. Y. Cai et al.
When the loop hybridizes with a specific target, the
annealed stem is forced to separate and the energy in
the fluorophore is converted into fluorescence (Fig. 1).
Molecular beacons can be used to identify specific
nucleic acids in homogeneous solutions, e.g. PCR prod-
ucts. Amplicons can be detected by adding a PCR
product in a micrometer-sized well containing the previ-
ously immobilized probe, and reading the fluorescence
generated. Further transfers or washing steps are not
required. This method has been applied to detect PCR
products of a number of bacteria, including E. coli
O157:H7 (McKillip and Drake, 2000). Besides detecting
the PCR end products, molecular beacons can be used
to monitor the synthesis of specific nucleic acids in real
time (real-time PCR), which will be discussed later.
The presence of the hairpin stem in the molecular
beacon significantly enhances its specificity because the
hairpin stem competes with the non-perfect matched
targets, which enables a molecular beacon to discrimi-
nate between DNA sequences differing by only a single
nucleotide (Tyagi et al., 1998). Therefore, the molecular
beacon approach is valuable for detection of single base
mutations in DNA, for example in antimicrobial resist-
ance genes of Mycobacterium tuberculosis (Piatek et al.,
2000). For this, the isoniazid or rifampin resistance-
related genes and intergenic regions were PCR-amplified
then probed by nine molecular beacon probes, which
covered the complete amplicons. An isolate was deter-
mined to be drug resistant if one of the nine beacon
probes did not generate fluorescent signals.
Tripartite molecular beacon and displacement
hybridization
Compared with conventional DNA probes, molecular
beacon probes are more expensive and difficult to syn-
thesize. In addition, since both ends of molecular
beacon probes are covalently modified with fluorophore
and quencher, they do not offer the flexibility for fluo-
rophore change and the capability for surface
immobilization (e.g. microarray spotting) through free
DNA ends. To overcome these problems, alternative
molecular beacons designated tripartite molecular bea-
cons (TMBs) have been developed (Nutiu and Li, 2002).
A tripartite molecular beacon contains an unmodified
oligodeoxyribonucleotide (ODN) that forms a molecular
beacon-like structure with two universal single-stranded
arms. The arms are made with the sequence to anneal a
universal pair of ODNs modified separately with a fluo-
rophore and a quencher (Fig. 1). A TMB is simply a
probe with two standard arms that are interchangeable,
so different probes can be synthesized in a conventional
way, and then combined with universal fluorophore and
quencher labeled arms. TMBs are as effective as stan-
dard molecular beacons in detecting matching or
single-base-mutated nucleic acid targets.
Molecular genetic methods in the veterinary clinical bacteriology laboratory 75

A
Target

Standard molecular beacon Target

Target
B

Target

Tripartite molecular beacon

Fig. 1. Molecular beacon. (A) The standard molecular beacon is a stem-and-loop structure. The sequences at the ends of the
probe match and bind, creating the stem, while the rest of the probe is unmatched and unbound, creating the loop that is
specific to the target sequence. While folded this way, the fluorophore at one end of the probe is next to the quencher at the
other end. When the probe binds to a single-stranded DNA template, the structure unfolds, separating the quencher from the
dye and allowing fluorescence. (B) In a tripartite molecular beacon (TMB), the fluorophore and quencher are covalently
linked to two separate short oligodeoxyribonucleotides, which hybridize strongly with corresponding single-stranded arms
extended beyond the short hairpin stem. The fluorophore and quencher are close to each other, and the fluorescence is
quenched. In the presence of the nucleic acid target, the closed-state TMB is transformed into the open state, emitting a
strong fluorescence signal.

Displacement hybridization Peptide nucleic acid probes and locked nucleic acid

Displacement hybridization is another approach used to
overcome the shortcomings of conventional molecular
beacons (Li et al., 2002). Instead of a hairpin structure,
two complementary ODNs of different length are
labeled with a fluorophore and a quencher. The probes
normally form a double-stranded structure and the fluo-
rescence is quenched, but, in the presence of
complementary target DNA, the probes are displaced
and the longer probe hybridizes with the target, so that
the probes become fluorescent. The presence of the
complementary DNA strand was also found to signifi-
cantly enhance the specificity of the probes. Although
we could find no reports of the application of TMBs and
displacement hybridization for the detection of bacteria,
it appears that these two techniques will work as well as
or better than conventional molecular beacons, and flex-
ibility and ease of synthesis should make them more
popular.
Peptide nucleic acid (PNA) molecules are artificially syn-
thesized nucleic acids. In contrast to natural DNA, the
negatively charged sugar–phosphate backbone is
replaced by a neutral polyamide backbone formed by
repetitive units of N-(2-aminoethyl) glycine (Fig. 2;
Stender et al., 2002). Similar to DNA, PNA consists of
individual nucleotide bases, which enable PNA to
hybridize to complementary nucleic acid targets accord-
ing to the Watson and Crick base-pairing rules. Because
of the lack of electrostatic repulsion, a PNA probe has
stronger binding ability for complementary targets, and
hybridizes independently of the salt concentration. With
an unnatural backbone, a PNA probe resists digestion by
nucleases and other enzymes. For these reasons, PNA
probes have a better shelf life, are more suitable for
antisense therapy, and are more stable as self-reporting
probes for real-time PCR methods. In contrast also to
DNA probes, PNA probes are not degraded during PCR
76 H. Y. Cai et al.

OH OH NH
O O Base
Base Base

N
O
O O O O
O O
P P P
O–
O– O n
n n
LNA DNA PNA

Fig. 2. Chemical structures of DNA, locked nucleic acid (LNA) and peptide nucleic acid (PNA). In PNA, the sugar–phosphate
backbone of DNA is replaced by a polyamide backbone, keeping the spacing between the nucleotide bases the same, and
nucleotide bases are attached to the uncharged protein-like (polyamide) backbone. An LNA comprises a lightly modified
RNA backbone, in which the ribose moiety in the sugar–phosphate backbone is constrained by a methylene bridge between
the 2-oxygen and the 4-carbon atoms.

by the endonuclease activity of Taq DNA polymerase
(Wilhelm et al., 2001). One very useful feature for micro-
bial identification is that PNA probes penetrate the
hydrophobic cell wall of bacteria following preparation
of a standard bacteriological smear on a glass slide,
because of the relatively hydrophobic character of PNA
compared with that of DNA probes. PNA probes have
been used to identify bacteria such as M. tuberculosis
and Staphylococcus aureus directly from slide smears, a
process that could be completed within 3 hours (Stender
et al., 1999; Oliveira et al., 2002). This method may thus
revolutionize bacterial direct examination approaches in
clinical microbiology because it is rapid and specific, and
works for mixed cultures directly on samples without the
need for DNA extraction or amplification. A PCR–PNA-
based enzyme-linked immunosorbent assay (ELISA)
method was described as able to recognize point muta-
tions in genes associated with isoniazid and rifampin
resistance in M. tuberculosis (Bockstahler et al., 2002).
Similar to PNA, the locked nucleic acid (LNA) is
another type of nucleic acid mimic. LNA comprises a
lightly modified RNA backbone, in which the ribose
moiety in the sugar–phosphate backbone is constrained
by a methylene bridge between the 2-oxygen and 4-
carbon atoms (Fig. 2). The link fixes the sugar ring in
the 3 endo conformation, favoring the formation of
hybrids with complementary DNA or RNA sequences, so
that LNA probes have high binding affinity. Like PNA,
LNA is not cleavable by nucleases. Compared with PNA,
however, LNA has less ability to invade duplex DNA
because it does not contain an electrically neutral
pseudopeptide backbone (Demidov, 2003).
ized molecular biology. The PCR method was devised
and named by Mullis and Faloona (1987). The PCR uses
the principle of primer extension reaction, which hap-
pens in vivo and is catalysed by DNA polymerase. The
enzyme synthesizes a complementary strand of DNA in
the 5 to 3 direction. Instead of using one primer, the
PCR uses two primers, each complementary to opposite
strands of the region of DNA. Consequently, each primer
extension reaction directs the synthesis of DNA towards
the other and the amount of DNA that is synthesized
increases exponentially. A few copies of the target DNA
can be amplified to millions of copies after 30–40 cycles
of PCR. Since PCR became practical in the late 1980s,
many techniques have been developed to improve the
efficiency and accuracy of PCR reactions.
In ‘nested’ PCR, two pairs of PCR primers are used for
a single target DNA sequence. The first pair amplifies the
target in the usual PCR reaction, and the second pair
(nested primers) binds within the first PCR product and
produces a second PCR product that is shorter than the
first. The major purpose is to increase the sensitivity of
PCR reactions by a second amplification. However,
nested PCR is so sensitive that DNA contamination within
the PCR laboratory is a major concern. The nested PCR
also serves to confirm the specificity of the first PCR.
‘Multiplex’ PCR is a PCR reaction using more than one
pair of primers to amplify more than one target
sequence in the same PCR reaction. This technique is
useful for the detection of multiple pathogens or multi-
ple genes at the same time.
Real-time PCR

Polymerase chain reaction Conventionally, the size and purity of PCR products are
detected in a separate gel electrophoresis step after the
The PCR is a widely used technique for the amplification PCR is completed. Real-time PCR is an important and
of specific DNA sequences in vitro that has revolution- relatively recently introduced technology that can
Molecular genetic methods in the veterinary clinical bacteriology laboratory 77

detect PCR products during amplification. In a real-time
PCR instrument, an optical device is connected to the
reaction tubes to read the reaction signal during each
cycle of PCR reaction. Compared with conventional
PCR, real-time PCR is faster and the risk of contamina-
tion is reduced with a closed tube system. In regular
PCR (sometimes called ‘black PCR’), the amplification
of different concentrations of templates reaches log
phase sooner or later so that it is not possible to quan-
titate accurately the initial templates by measuring the
end PCR products. By monitoring the PCR reaction in
real time, the time (or cycle) in which the log phase
appears can be determined. By comparing the time to
log phase of the initial template with that of standard
controls, the initial template concentration can be
determined.
The current approaches used for monitoring PCR in
real time include double-stranded DNA (dsDNA) fluores-
cence dye, fluorescence-labeled probes or primers (Fig.
3). SYBR Green I is a commonly used dsDNA-binding
dye, which shows over a 100-fold enhancement of fluo-
rescence once bound to dsDNA. This dye binds to any
dsDNA so that no design or optimization of probes is
required, but unfortunately it also binds to both specific
and non-specific DNA, and primer-dimers. Therefore it
works well only for highly specific PCRs.
Real-time PCR probes are labeled with fluorescence
that is quenched before hybridization. For example,
exonuclease probes (hydrolysis probe or TaqMan probe)
produce fluorescence after the probe is hydrolysed by
Taq polymerase, which has 5–3exonuclease activity.
The hydrolysis separates fluorescein from a quenching
dye, which allows emission of fluorescence.
Hybridization probes are another example. These are
double probes designed to hybridize side by side on the
PCR product. The 3 end of the upstream probe is
labeled with fluorescein, which is the donor of fluores-
cence resonance energy transfer (FRET). The 5 end of
the downstream probes is labeled with an acceptor dye.
It is only when the two probes hybridize closely that
FRET happens and that fluorescence can be detected.
Molecular beacons can also be used to perform real-
time PCR monitoring. Compared with conventional
probes, some real-time PCR probes are more discrimina-
tory between perfect and imperfect matches, because
double probes are used, or the second structure, e.g. the
hairpin of the molecular beacon, competes against an
imperfectly matched target.

Fluorescent dye that SYBR Green I

binds to double-
stranded DNA Target
single-stranded DNA PCR products

Hybridization probe
(FRET probe)
Target

primer Probe
Hydrolysis probe
(TaqMan probe)
polymerase Target PCR products

Light upon extension Extended primer

(LUX) fluorogenic Hairpin primer
Target
primers

Target

Molecular beacon
Probe

Before amplification After amplification

Fig. 3. Different fluorescence monitoring schemes for real-time PCR. SYBR Green I is a double-stranded DNA-binding dye,
which shows more than 100-fold enhancement of fluorescence once bound to PCR products (double-stranded DNA).
Exonuclease probes (hydrolysis probe or TaqMan probe) produce fluorescence after the probe is hydrolysed by Taq poly-
merase. Hybridization probes are double probes that hybridize side by side on the specific PCR product and introduce
fluorescence resonance energy transfer (FRET), which generates strong fluorescence. LUX (light upon extension) fluorogenic
primers emit light strongly when extended in PCR products. Molecular beacons generate fluorescence when they bind to a
specific DNA fragment.
78 H. Y. Cai et al.

Invitrogen (Carlsbad, CA, USA) recently developed a
new type of real-time probe called LUXTM (light upon
extension) fluorogenic primers. LUX primers have
stem–loop structures that do not contain a quencher
moiety. Rather, the fluorescent oligonucleotides are
designed to self-quench based on sequence context.
LUX primers quench when free in solution, fluoresce
weakly when denatured, and only emit light strongly
when extended in PCR products. LUX fluorogenic
primers are less expensive than dual-labeled probes.
Similar to the SYBR Green methods, if the primers non-
specifically anneal to the target template, false-positive
results may occur. The advantage over SYBR Green is
that LUX primers can be labeled with different fluores-
ceins, so that multiplex real-time PCR is possible.
After the real-time PCR is completed, the melting tem-
perature (Tm) of the double-stranded PCR products can
be determined by measuring the fluorescence intensity
under different temperatures. Since the Tm of a DNA
duplex is determined by its nucleotide composition, Tm
profiles can be used for bacterial strain typing.
Many real-time PCR assays have been described for
the detection of pathogenic microorganisms (Table 1).
Although no application has been reported using quanti-
tative real-time PCR to solve veterinary clinical
diagnostic problems, this certainly will be useful when
the techniques are mature and field-validated. For exam-
ple, healthy carrier pigs are estimate to shed 102 to 103
Brachyspira hyodysenteriae cells per gram of feces,
whereas pigs with acute swine dysentery can contain 8
× 107 B. hyodysenteriae cells per gram of mucosa
(Kinyon et al., 1977). A quantitative PCR would help
accurate diagnosis of the disease, which currently can-
not be made simply based on positive/negative PCR
results.
Nucleic acid sequence-based amplification (NASBA)
NASBA is an amplification technology that targets RNA
based on the simultaneous activity of reverse transcrip-
tase, RNase and T7 RNA polymerase with two
oligonucleotide primers. In NASBA reactions, RNA is
first reverse-transcribed into cDNA that is transcribed
into antisense RNA by T7 RNA polymerase. The anti-
sense RNA is further reverse-transcribed to cDNA. By
repeating this cycle, NASBA can amplify a desired frag-
ment more than 1012-fold in 90–120 minutes (Fig. 4).
The amplified RNA product can be detected through the
use of a target-specific capture probe bound to magnetic
particles in conjunction with a ruthenium-labeled detec-
tor probe and an instrument (NucliSens Reader;
bioMérieux) capable of measuring electrochemilumines-
cence. Alternatively, RNA amplified by NASBA can be

Sense RNA 5
Reverse transcriptase Anti-sense primer with T7 RNA
polymerase promotor overhang (P1)
Sense RNA 5

Rnase H 5 DNA

Sense primer (P2)

Reverse transcriptase 5 DNA

5
DNA
T7 RNA polymerase 5 DNA

5 T7 RNA polymerase
Anti-sense RNA 5
5
5

5
DNA
P2 5 Anti-sense RNA 5 DNA
Reverse transcriptase
Reverse transcriptase

DNA 5
DNA 5 5 Anti-sense RNA
RNase H P1

Fig. 4. Principle of nucleic acid sequence-based amplification (NASBA). RNA is first reverse-transcribed into cDNA that is
transcribed into antisense RNA by T7 RNA polymerase. The antisense RNA is further reverse-transcribed to cDNA. By repeat-
ing this cycle, NASBA can amplify a desired fragment more than 1012-fold in 90–120 minutes. NASBA is a continuous,
isothermal process, which can be carried at a constant temperature, e.g. 41°C.
Table 1. Examples of applications of molecular methods for identification of animal pathogens that are difficult to isolate or identify using conventional phenotypic approaches

Organism Molecular assays Highlights and Advantages Disadvantages References

Anaplasma PCR (16S rRNA gene) and
(and Ehrlichia) reverse line blot assay (probe
hybridization with PCR
products)
A. marginale MSP1a gene
(msp1α ) PCR
A. marginale MSP5 gene
nested PCR and
hybridization with
PCR products
Bartonella B. henselae, B. quintana,
and R. prowazekii
gltA PCR and PCR–RFLP
Bartonella gene sequence-
based species identification
Borrelia B. burgdorferi flagellin gene
Simultaneously detects and differentiates Ehrlichia
ruminantium, Anaplasma ovis, A. centrale, A. marginale,
A. bovis, E. ovina, Ehrlichia sp. Strain Omatjenne
and A. phagocytophila from ruminant blood or
Amblyomma variegatum ticks
Identifies and genotypes A. marginale based on number
of repeat units of the gene. Simple to use compared
with other typing methods
MSP5 gene is the major target for PCR assays. Detect 30
A. marginale infected erythrocytes per ml of blood
Amplifies citrate synthase gene (gltA) of B. henselae,
B. quintana and R. prowazekii, then uses PCR–RFLP
to differentiate the three species
Identifies and differentiates different Bartonella
species based on sequences of house keeping gene,
e.g. rpoB and gltA
Isolation and detection of B. burgdorferi DNA from
Sensitivity was not determined; only Bekker et al., 2002
positive results are meaningful. May
miss some recently described strains
of A. centrale
Size of the specific PCR product Lew et al., 2002
varies;difficult to identify non-specific
amplification
Nested PCR is subject to contamination. Torioni de Echaide
Hybridization is tedious et al., 1998
Validated on pure cultures only Norman et al., 1995
Limited database of these house- La Scola et al., 2003
keeping genes is available currently.
No universal primers available;
may miss some undescribed strains
Analytical validation only. Use Exner and Lewinski, 2003
Molecular genetic methods in the veterinary clinical bacteriology laboratory

(fla) real-time PCR
Brucella B. abortus species-specific
multiplex PCR
B. abortus IS711 and alkB
gene and real-time PCR
B. melitensis IS711 PCR
B. ovis IS6501 PCR
clinical sample. Different DNA extraction systems of robotic DNA extraction is not as
including a robot system were compared, which had effective when dealing with
sensitivity between 25 and 50 cells/ml of fluid samples small number of samples
Identification and discrimination of B. abortus field PCR may be further optimized Bricker et al., 2003
strains (wild-type biovars 1, 2 and 4) from B. abortus to sharpen the PCR product
vaccine strains, other Brucella species and non-Brucella bands. Some strains of B. suis,
bacteria B. abortus and B. melitensis
share the same PCR profile
Syb Green I, TaqMan probe and hybridization probes real SYBR Green assay false positive on Newby et al., 2003
time PCR had same sensitivity: detected as low as two non-abortus Brucella and non-
genocopies of B. abortus Brucella strain; TaqMan probe
and hybridization probes
reacted with some B. canis strains
When tested with 126 aborted sheep fetuses, diagnostic High threshold of detection Leyla et al., 2003
sensitivity was 97.4% and specificity was 100% using 1.4 × 108 c.f.u./ml (stomach
culture as gold standard content)
Threshold of detection equivalent to culture in detecting The PCR also amplified other Manterola et al., 2003
B. ovis infection in 192 samples of ram semen. For B. abortus and B. melitensis,
experimentally infected rams, culture had a lower the other Brucella that were tested
threshold in the first 5 weeks and PCR had a lower
79

threshold 6–14 weeks after infection continued


Table 1. continued
Organism Molecular assays
B. abortus, B. melitensis
and B. suis IS711 real-time
PCR
Campylobacter C. jejuni ORF C TaqMan
real-time PCR
Campylobacter spp.
LightCycler real-time PCR
Chlamydia and C. suis ompA gene nested
Chlamydophila PCR
23S rRNA gene and
Omp1 FRET real-time PCR
Ehrlichia HE1/HE3 direct PCR,
chaffeensis VLPT nested PCR,
16S rRNA gene TagMan
real-time PCR and
Syb Green real-time PCR
Helicobacter 16S rRNA gene sequencing
Helicobacter pylori 23S
rRNA gene hybridization
probe real-time PCR
Pathogenic Leptospira
multiplex PCR (DNA
fragments of unknown
function)
80
Highlights and Advantages Disadvantages References
Three separate LightCycler FRET assays specific for Validated on pure cultures only. Redkar et al., 2001
B. abortus, B. melitensis and B. suis with B. abortus assay had relatively
detection limit of 25–50 cells low CP value on positive samples
and was sometimes positive
with B. suis. B. suis assay negative
on B. suis biovars 2, 3, 4 and 5.
E. coli was the only non-Brucella
used for specificity test
C. jejuni specific and as sensitive as culture when Ability to quantify was difficult Sails et al., 2003
validated with food samples to evaluate because some cells
may be dead or injured and the
culture method may have
underestimated the total
number of cells
Genus-specific PCR, followed by three separate Need to develop a multiplex Logan et al., 2001
real-time PCR assays to identify C. jejuni, real-time PCR (would reduce
C. coli, C. lari, C. upsaliensis, C. hyointestinalis, the procedures) and software
and C. fetus using probe and melting peak analysis to interpret the complicated
results
Sensitivity and specificity of PCR vs culture as standard No highly acceptable gold Sachse et al., 2003
were 94.4 and 81.0% respectively in testing of standard available (culture was
109 clinical samples from pigs experimentally 300 times less sensitive)
infected with C. suis
Quantitative detection of C. psittaci and C. pecorum DeGraves et al., 2003
in cattle virginal swabs. Highly sensitive in detecting
sub-clinical virginities in virgin heifers (53%
prevalence, most with one genome per specimen)
Four assays were compared. TaqMan real-time Syb Green assay detected E. canis, Loftis et al., 2003
PCR was more specific than Syb Green PCR and more E. ewingii, E. muris and
sensitive (detects 10 gene copies) than conventional PCR A. phagocytophila, as well
as E. chaffeensis. Validated in
very limited number of biological
samples
Characterization of mixed infection of spiral-shaped Cannot discriminate Helicobacter Misawa et al., 2002
organism from a puppy with bloody diarrhea and Flexispira at species/genus level
Sensitivity 97.0% (64 of 66 biopsy samples); Gold standard: culture and histology Lascols et al., 2003
specificity 94.6% (123 of 130 samples)
Identifies pathogenic Leptospira. Better than other Needs more validation Gravekamp et al., 1993;
PCR assays in detecting L. hardjo from bovine urine on other species of animals Wagenaar et al., 2000
H. Y. Cai et al.
Leptospira Leptospira 23S rRNA Validated with 145 dog urine samples. Diagnostic Gold standard: compatible Harkin et al., 2003
gene PCR sensitivity 100%, specificity 88.3%. PCR positive clinical signs and seroconversion
before seroconversion titer ≥1:400

Bovine semen 16S genes PCR Detects 100 bacteria/ml. Differentiates L. interrogans,
and PCR–RFLP L. borgpetersenii, L. noguchii, L. santarosai and L. biflexa to identify. Polyacrylaminde gels
Mycobacterium Multiplex PCR for M. bovis
bovis (specific fragment of
unknown function) and
M. tuberculosis (pncA)
Multiplex PCR for M. bovis
(hsp65) and Brucella
abortus (BCSP31K gene)
DR region of M. tuberculosis
complex conventional
(double run) and real-time
PCR with magnetic
sequence capture method
Distinguishes M. bovis from M. tuberculosis with one
PCR reaction. Highly specific. Threshold of detection:
4,000 genomes
Identifies milk M. bovis (10 c.f.u./ml) and
B. abortus (2,000 c.f.u./ml)
Both methods had a detection limit of 36 cells.
Conventional PCR detected 26 of 28 culture-positive
cattlelymph node specimens (93%), and the
LightCycler PCR detected 20 of 28 culture-positive
cattle lymph node specimens (71%)
Restriction patterns were difficult Heinemann et al., 2000
and silver stain are tedious. An
alternative can be sequencing of the
PCR products
Validated only on pure cultures Shah et al., 2002
Relatively low sensitivity for B. abortus Sreevatsan et al., 2000;
The reduced sensitivity of the Taylor et al., 2001
LightCycler PCR was considered
as the perturbation of fluorescent
signals by the red iron oxide
present in the sequence capture
magnetic beads, which may be
alleviated by heat treatment and
centrifugation to remove the
magnetic beads
Molecular genetic methods in the veterinary clinical bacteriology laboratory

Mycobacterium IS900 PCR IS900 is widely used as a PCR target
paratuberculosis
IS900 SYBR Green LightCycler
Real-time PCR
IS900 molecular beacon
real-time PCR
IS900 TaqMan real-time
PCR
ISMav2 PCR
Mycoplasma DGGE of a 16S rRNA
gene PCR
Syb Green LightCycler
Real-time PCR
Multiplex TagMan
IS900-like sequences exist in other Englund et al., 2001,
Mycobacterium spp. 2002; Garrido et al., 2000
O’Mahony and Hill, 2002
Fang et al., 2002
Kim et al., 2002
ISMav2 is unique to M. paratuberculosis. Uses False positive with high cell number Stratmann et al., 2002
peptide-mediated magnetic separation method to of M. marinum and M. scrofulaceum
increase sensitivity to 10 cells/ml milk ISMav2 has not been widely used or
validated as a PCR target
Differentiates all 10 avian and 7/10 small ruminant DGGE is tedious McAuliffe et al., 2003
Mycoplasma spp. tested
Validated for detection of M. gallisepticum using 98 Gold standard was serology Carli and Eyigor, 2003
chicken trachea swabs with sensitivity and specificity test only. No non-Mycoplasma
of 64.2% (18/28 chickens) and 100% respectively strains were tested to evaluate
specificity. Syb Green real-time
has been found to have
false-positive result in general
Validated using 50 feline blood samples for detection Quantitation was based on DNA Tasker et al., 2003
81

real-time PCR and quantitation of M. haemofelis and ‘Candidatus copy number. True correlation continued
82 H. Y. Cai et al.

detected specifically in real time through the use of

molecular beacon probes included in the amplification
reaction. One advantage of NASBA over PCR is that it is

Pusterla et al., 2000

a continuous, isothermal process and does not require

Mott et al., 1997

expensive thermocycling equipment. In addition, since
the target of NASBA is RNA instead of DNA, a positive
References

result indicates the presence of viable organisms (which

produce mRNA). The NASBA technique has already
been successfully applied for the detection of human
and animal pathogens, such as West Nile virus (Lanciotti
Slightly less sensitive than culture and Kerst, 2001), Chlamydia trachomatis (Morré et al.,
1996), Campylobacter spp. (Uyttendaele et al., 1994) and
number of organisms/µl was not
between copy number and the

Mycoplasma pneumoniae (Loens et al., 2002), and the

possible as the organisms are

No comparison with culture

identification of Mycobacterium spp. (Van der Vliet et

al., 1993).
c.f.u., colony-forming unit; DGGE, denaturing gradient gel electrophoresis; RFLP, restriction fragment length polymorphism.
Disadvantages

Strand displacement amplification (SDA)

unculturable

SDA is an isothermal process that uses primers, DNA

polymerase and a restriction enzyme to exponentially
amplify a unique nucleic acid sequence (Walker et al.,
Sensitivity and specificity similar to those of conventional

1992). SDA includes two phases, a target generation

phase and an exponential amplification phase. In target
nested PCR; detected 10 copies of the 16S rRNA gene

generation, dsDNA is heat-denatured (65°C, 10 minutes)

Detects 0.02 pg of N. risticii DNA, field-validated
simultaneously with a detection limit of less than

before primers with an overhang of a restriction enzyme

site anneal to the target. The exponential amplification
M. haemominutum’ in feline blood samples

phase is carried out in an isothermal environment

Validated with 153 horse blood samples.

(37°C). DNA polymerase, lacking 5–3 exonuclease

activity, extends the primers to generate products with a
restriction digestion site in one strand. Because a special
deoxyadenosine, 5-[α-thio]triphosphate (dATP[αS]), is
Highlights and Advantages

used to replace dATP, the restriction site of the newly

synthesized strand is inactivated. In the amplification
4 copies of templates

phase, the restriction enzyme nicks one strand of the

dsDNA products and allows polymerase to extend the
of the bacteria

DNA from the nicked site to displace the nicked strand.

After new dsDNAs are synthesized, the restriction
enzyme nicks them and the DNA extension starts again
(Fig. 5). Commercial SDA kits for detection of M. tuber-
culosis and Neisseria gonorrhoeae are available (BD
Diagnostic Systems, Sparks, MD, USA), and have been
shown to be robust, specific and sensitive (Akduman et
N. risticii 16S rRNA gene

N. risticii 16S rRNA gene

al., 2002; Barrett et al., 2002). SDA methods are simple,

TaqMan real-time PCR

take as little as 20 minutes to complete, and do not

require a thermocycler.
Molecular assays

nested PCR

16S rRNA analysis

Sequencing of the 16S ribosomal RNA (rRNA) gene has

Table 1. continued

been used as an important tool for phylogenetic analysis

and classification of bacteria for about two decades
Neorickettsia

(Lane et al., 1985). The 16S rRNA gene is well conserved

Organism

in all organisms and contains species-specific variable

regions allowing specific species identification. The 16S
rRNA is about 1500 base pairs long and consists of 10
Molecular genetic methods in the veterinary clinical bacteriology laboratory 83

Primer Polymerase
G-T-T-G-A-C
Target
dCTP, dGTP, dTTP, dATPaS

G-T-T-G-A-C
C-AsAsC-T-G
Nicked by restriction enzyme (Hinc II)

G-T-T G-A-C
C-AsAsC-T-G

Polymerization and strand displacement

G-
A-
G-T-T C
C-AsAsC-T-G

G-
A-
C

G-T-T G-A-C
G-T-T-G-A-C
C-AsAsC-T-G
C-AsAsC-T-G

Fig. 5. Principle of strand displacement amplification (SDA). SDA primers that include a HincII site overhang anneal to DNA
targets. DNA replication using dCTP, dGTP, dTTP and dATP[αS] produces a double-stranded DNA with a HincII site in one
strand. HincII nicks the unprotected strand. DNA polymerase extends the 3 end at the nick and displaces the downstream
fragment. The polymerase and displacement regenerate a double-stranded DNA with a HincII site in one strand. Nicking,
polymerization and displacement steps cycle continuously and amplify the DNA target exponentially in an isothermal
environment.

widely conserved regions and 10 divergent regions. The
divergent regions accumulate mutations with a slow but
constant rate of about 1% per 50 million years and thus
can be used for bacterial identification by sequence
comparison (Trotha et al., 2001). It has been proposed
that strains with rRNA similarity less then 97% belong to
different species.
Sequencing of 16S rRNA has been used to identify
slow-growing, unusual and fastidious bacteria, and bac-
teria that are poorly identified by conventional methods.
For example, Bacillus angiomatosis and Tropheryma
whippelii are non-culturable bacteria that were first iden-
tified by a 16S rRNA gene sequencing method (Relman
et al., 1990, 1992). 16S rRNA sequencing kits are avail-
able (PE Applied Biosystems, Forster City, CA, USA) and
are accurate and rapid for the identification of
Mycobacterium spp. (Patel et al., 2000) and clinical
coryneform bacterial isolates (Tang et al., 2000). This
method has also been used to identify a novel mycobac-
terial species as an agent of canine leproid granuloma
(Hughes et al., 2000). Cai et al. (2003) reported that 16S
rRNA sequencing was readily applicable to difficult-to-
identify bacteria in a veterinary clinical bacteriology
laboratory, and provided details of the costs and choice
of primers.
There are some problems with the use of 16S rRNA
gene sequencing for bacterial identification. Some closely
related species of bacteria, such as Shigella spp. and E.
coli, are difficult to classify by 16S rRNA analysis
(Christensen et al., 1998). For these bacteria, the gyrB
sequence analysis has been suggested (Fukushima et al.,
2002). Other housekeeping genes, such as the RNA poly-
merase -subunit gene (rpoB), have also been
investigated as alternatives for speciation of some bacte-
ria, such as Anaplasma spp., Bartonella spp., Ehrlichia
spp., Mycoplasma spp. and Neorickettsia spp. (Kim et al.,
2003; La Scola et al., 2003; Taillardat-Bisch et al., 2003). In
addition, some foreign DNA sequences, called intervening
sequences (IVS), of ~140 base pairs, may exist in 16S
rRNA genes (Linton et al., 1994; Etoh et al., 1998).
Therefore, possible IVS should be identified if a 16S rRNA
gene sequence is larger than the usual size (~1.5 kp).
DNA microarrays
A DNA microarray is essentially a miniaturized form of
multiple dot blots. It is composed of DNA probes bound
to a solid substrate, such as glass. During hybridization,
a target DNA sample covers the complete array area and
84
hybridizes with the probes that have a complementary
sequence. Hybridized targets are detected using a
reporter system. Either PCR products or synthesized
oligonucleotides can be used as probes and spotted on
the supporting matrix. PCR product probes are less
expensive and simpler to generate when limited number
of primers are needed to generate the probes. However,
their lengths make it difficult to discriminate between
minor sequence mismatches. An oligonucleotide probe
array is more expensive to construct but can be more
specific because of the short length of the probe. To
detect a target DNA or RNA, it usually requires PCR or
reverse transcription–PCR to amplify the targets so as to
achieve adequate sensitivity. Single or multiple reporter
molecules (e.g. biotin and Cy-3) can be incorporated
directly via 5 primer modifications, nick translation or
chemical incorporation. After hybridization of targets
with the microarray, a fluorescence scanner can be used
to scan a solid (e.g. glass) array, whereas a phosphoim-
ager can be used for isotopic reporters and
membrane-based arrays.
Microarrays have been widely used to study bacteria,
but their application in clinical microbiology is hindered
by low sensitivity when used directly on cultures or
samples. PCR is required to amplify the target DNA
before it can be detected by a DNA array. Therefore a
DNA microarray does not have an advantage over a reg-
ular PCR in detecting individual bacteria or individual
virulence genes. In order to detect multiple bacteria or
multiple virulence genes, it requires multiplex PCR to
amplify multiple targets, but multiplex PCR is difficult to
optimize and may include a maximum of about 10
sets of primers. To circumvent this obstacle, the 16S
rRNA gene has been used as a target, which can be
amplified using a set of universal primers and
hybridized with a DNA microarray consisting of species-
specific 16S rRNA gene probes (Zimmermann et al.,
2003). It is likely that microarrays will become common
in clinical microbiology in the future. In particular, they
could be used to identify organisms with complex dis-
ease-causing abilities, for example by detecting
virulence genes to identify the pathotypes of E. coli
(Bekal et al., 2003); or they could be used to screen for
multiple antibiotic resistance genes.
Published molecular genetic methods for bacterial
pathogens
Table 1 lists the most recent applications of molecular
methods for the diagnosis of specific important bacterial
infections of animals, and for other animal pathogens
that are slow or difficult to isolate in the clinical bacteri-
ology laboratory. As an example, the molecular
identification of Escherichia coli will be discussed in
detail. There is clearly tremendous scope for the
application of molecular methods to hard-to-isolate
H. Y. Cai et al.
bacteria, including newly reclassified members of the
order Rickettsiaceae (genera Anaplasma, Ehrlichia,
Neorickettsia and Rickettsia) (Dumler et al., 2001). This
order contains a series of emerging or newly recognized
pathogens, whose epidemiology is being elucidated
largely through molecular diagnostic approaches.
Similarly, as many other bacteria, such as Bartonella,
Borrelia and Mycoplasma, are reclassified based on
molecular findings (e.g. 16S rRNA gene sequences)
(Brenner et al., 1993; Birtles et al., 1995; Neimark et al.,
2001), there will be increased reliance on molecular
methods for diagnostic and identification purposes.
Escherichia coli
Escherichia coli is part of the normal intestinal
microflora, but small subsets (pathotypes) of the species
are capable of causing enteric and extra-intestinal dis-
eases. Table 2 lists the common types of pathogenic E.
coli that cause disease in animals. The implication of
extraintestinal E. coli in disease is straightforward as it is
typically based on recovery of the organisms from nor-
mally sterile sites. Pathogenic enteric isolates, however,
must be differentiated from normal flora isolates. Gene-
based methods are particularly valuable as they
circumvent the problem of expression of virulence fac-
tors in vitro and they may rapidly provide information
on the complement of virulence genes present in the
isolate.
Enterotoxigenic E. coli (ETEC) are among the most
common of the pathogenic E. coli implicated in enteric
diseases of animals and the vast majority of samples
submitted for diagnosis of E. coli enteric disease are
feces or intestine from calves or pigs suspected to be
suffering from ETEC-induced diarrhea. Identification of
ETEC is based on detection of the colonization fimbriae
and enterotoxin(s) or the genes that encode them (Table
2). Colonization fimbriae may be detected by immuno-
logical tests, such as agglutination, latex particle
agglutination or ELISA. However, certain fimbriae (such
as F18) are not readily expressed in vitro, and agglutina-
tion tests are prone to yield false-positive reactions.
There are no practical tests for the routine detection of
the enterotoxins in the diagnostic laboratory and sole
reliance on detection of the colonization fimbriae will
falsely identify enterotoxin-negative, fimbriae-positive
isolates as ETEC.
In the last decade, DNA probes and PCR methods
have been developed for the detection of the genes
coding for ETEC fimbriae and enterotoxins. The DNA
probes are highly suited to investigations in which large
numbers of isolates are examined simultaneously. The
PCR is well suited to routine application in the diagnos-
tic laboratory. A common approach is to use a multiplex
PCR that targets the genes for enterotoxins and fimbriae.
We have accumulated some experience with a multiplex
Molecular genetic methods in the veterinary clinical bacteriology laboratory
Table 2. Genes for toxins and adhesins that are targeted for identification of E. coli that cause enteric diseases in animals
Animal Pathotype of E. coli
Pigs
Neonatal Enterotoxigenic E. coli (ETEC)
Weaned ETEC
Shiga toxin-producing E. coli (STEC)
Enteropathogenic E. coli (EPEC)
Calves and lambs ETEC
EPEC
STEC
Dogs STEC O157:H7
Rabbits EPEC
LT, heat-labile enterotoxin; STa and STb, heat-stable enterotoxins a and b respectively; HUS, hemolytic uremic syndrome.
PCR which targets nine genes, namely genes that
encode the LT, STa and STb enterotoxins, and the F4
(K88), F5 (K99), F6 (987P), F18 and F41 fimbriae, as
well as the Stx2e Shiga toxin, which is implicated in
edema disease of pigs (Bosworth and Casey, 1997). We
have found that results are considerably improved with
the use of the Qiagen Multiplex Master Mix, which leads
to few or no non-specific bands. It is not necessary to
target all nine genes in order to satisfactorily identify
ETEC. For example, the PCR may be modified to target
only three genes (genes for STa, F5 and F41) for bovine
isolates (Franck et al., 1998).
Edema disease of pigs is usually so characteristic in its
clinical appearance that laboratory tests that identify the
isolates as Shiga toxin-producing E. coli (STEC) are usu-
ally not necessary. However, the multiplex PCR
described above can identify edema disease isolates by
its detection of genes for Stx2e and F18. Many of the
edema disease isolates will also have genes for entero-
toxins, and are called ‘STEC/ETEC’. Bosworth et al.
(1998) described a single-strand confirmation polymor-
phism assay that differentiates F18ab from F18ac. This
differentiation is not necessary for the identification of
edema disease strains of E. coli and the test is not suit-
able for application in a clinical diagnostic laboratory.
No molecular methods have been developed for the
specific identification of enteropathogenic E. coli (EPEC)
in pigs, cattle or rabbits. Considerable dependence is
placed on the detection of attaching and effacing lesions
in the intestine of affected animals. The PCR may be
used to identify EPEC isolates from these animals by the
presence of the eae (E. coli attaching and effacing) gene
and the absence of stx genes. STEC, especially those of
serogroups 5, 26, 111 and 118, have been implicated in
diarrhea and/or dysentery in calves. A multiplex PCR
that detects eae, stx1, stx2 and ehly (enterohemorrhagic
E. coli hemolysin) may be used for this type of STEC.
Typically, these isolates are eae+ and stx1+.
E. coli O157:H7 is implicated in disease in dogs (albeit
rarely) and its presence in cattle has public health signif-
85
Disease Target virulence genes
Diarrhea eltA (LT), estA (STa), estB (STb), faeG (F4),
fanA (F5), fim41a (F41), fasA (987P), (AIDA-1)
Diarrhea eltA, estA, estB, faeG, fedA (F18)
Edema disease stx2e (Shiga toxin 2e), fedA
Diarrhea eae (intimin)
Diarrhea estA, fanA, fim41a
Diarrhea eae
Diarrhea/dysentery eae, stx1, stx2
Bloody diarrhea, rfbE (O157), fliC (H7), eae
skin lesions, HUS stx1, stx2, ehly (EHEC hemolysin)
Diarrhea eae
icance. Preliminary identification is based on the aggluti-
nation of sorbitol-negative suspect colonies in O157
antiserum, but false positives occur. Confirmation and
initial characterization are based on the detection of rfbE
and fliC genes for the O and H antigens respectively,
the eae gene, and the stx1 and stx2 genes. The plasmid-
encoded ehly gene is sometimes also detected. Several
multiplex PCR protocols have been reported that target
these genes (Paton and Paton, 1998; Hu et al., 1999;
Botteldoorn et al., 2003).
Recently, real-time PCR assays have been developed to
detect pathogenic E. coli, mostly using molecular beacon
probes on TaqMan or LightCycler platforms. Some assays
have been applied to animal samples. TaqMan real-time
assays were described for the detection of STEC in milk
by detecting stx1 and stx2 genes separately (Burk et al.,
2002), by detecting the stx2 gene (McKillip and Drake,
2000), or by detecting the rfbE gene specific for O157
(Fortin et al., 2001). Sharma (2002) reported two sets of
TaqMan real-time PCR assays for the simultaneous detec-
tion and quantitation of EHEC in beef and bovine feces
by detection of a portion of the eae gene specific to the
EHEC O26, O111 and O157 serotypes, and by detection
of the stx1 and stx2 genes. Amplification and specific
detection of one or two genes of E. coli can be com-
pleted within 60 minutes (Reischl et al., 2002).
Ge and colleagues (2002) identified enterohemor-
rhagic E. coli (EHEC) by targeting the EHEC-hlyA gene
and amplifying probe–target hybrids by SDA. The assay
identified EHEC in 35 minutes with a detection limit of
4.3 bacteria and specificity of 100%. Although SDA is rel-
atively costly and complex to prepare and is susceptible
to minor variations in experimental condition, once opti-
mized and commercialized it will be simpler than PCR to
perform, because it uses an isothermal reaction and the
readout conditions require simple instruments. Most
recently, DNA microarrays have been described for
detecting and genotyping E. coli (Call et al., 2001;
Chandler et al., 2001; Bekal et al., 2003).
86
Detection of antimicrobial resistance genes
The molecular detection of antimicrobial resistance in
human medicine and its impact on clinical practice has
been reviewed in detail (Bergeron, 2000; Fluit et al.,
2001). The simultaneous rapid genotypic identification of
bacteria and their antibiotic resistance genes may have a
major impact on the treatment of infectious disease while
contributing to better control of antimicrobial resistance.
In addition, molecular detection of resistance also offers
the potential identification of resistance genes that are
expressed in vivo but not in vitro (Fluit et al., 2001).
However, phenotypic assays are still the method of
choice for most resistance determinations because of the
simplicity of the assays and the large numbers of antibi-
otic resistance genes that may exist for a single antibiotic.
Nucleic acid-based assays have the potential to
increase the rapidity and accuracy of resistance testing.
For example, molecular detection of the methicillin
resistance-encoding mecA gene in staphylococci and
rifampin resistance in M. tuberculosis has been widely
used in human medicine (Fluit et al., 2001). Because of
the emergence of multidrug-resistant microorganisms in
veterinary medicine, such as multidrug-resistant
Salmonella Newport, rapid testing by molecular meth-
ods that monitor specific resistance genes may become a
useful tool for veterinary teaching hospitals and veteri-
nary clinics. This approach could also be used at the
farm level for the surveillance of antimicrobial resistance
genes in food-producing animals. Molecular methods
may also be useful in the rapid detection of resistance
from normally sterile clinical specimens, such as blood,
cerebrospinal fluid and joint fluid (Bergeron, 2000).
There are limitations in the use of molecular assays
for the detection of antibiotic resistance. For example,
several dozen plasmid-mediated ß-lactamases cause
resistance by inactivating ß-lactam antibiotics, and a
large number of aminoglycoside resistance genes exist
in Gram-negative microorganisms (Fluit et al., 2001).
Problems associated with having many genes that
encode resistance to a single antibiotic are illustrated in
studies by Yang et al. (2001), who described a rapid
assay for the detection of multiple antibiotic-resistant
Salmonella Enteritidis and Salmonella Typhimurium iso-
lates from animals. The assay is a multiplex PCR that
amplifies antibiotic resistance genes and the Salmonella
spp.-specific gene SipB/C. When this multiplex PCR was
later compared with phenotypic assays, the drug resist-
ance patterns did not match well with PCR amplification
types (Yang et al., 2002). For example, two ampicillin-
resistant Salmonella isolates were negative for both
blaPSE-1 and blaTEM. The ampicillin resistance may have
been conferred by other ß-lactamase genes, such as
blaSHV and oxa-2, which were not investigated in the
experiments. The large number of different resistance
genes for some antibiotics makes full coverage by
probes or PCR very difficult.
H. Y. Cai et al.
New technology, including real-time PCR and
microarrays, has been applied to the detection of some
bacterial resistance genes (Killgore et al., 2000; Torres et
al., 2000; Westin et al., 2001). DNA arrays and chips are
technologies which allow the mass screening of genes.
If all known resistance genes could be represented on a
single chip or array, this would provide a complete test.
However, these techniques are still very expensive and
new resistance genes will continually emerge, so that
current genetic tests will miss the new mechanisms.
Furthermore, new antimicrobial drugs will also be regu-
larly developed, as illustrated by the continuing
appearance of newer quinolones with increased
potency. Frequent investigation will be needed to
update genetic tests so as to detect resistance associated
with these new drugs and with genetic changes.
Another problem with the detection of resistance genes
is that their presence does not lead automatically to treat-
ment failure since their expression may be low in vivo.
This is particularly true for ß-lactamase production by
enterobacteria, in which the resistance depends on the
degree of expression (Fluit et al., 2001). The extreme sen-
sitivity of molecular-based detection tests for resistance
genes also brings a major problem in detecting resistance
genes for non-sterile sites, polymicrobial infections, com-
plex microflora and contaminated clinical specimens.
In summary, the DNA-based detection of antibiotic
resistant bacteria can potentially be a rapid and sensitive
alternative to the current phenotypic detection method.
However, extensive comparison between the two meth-
ods is required to determine the sensitivity and
specificity before the DNA-based methods can be used
for clinical diagnosis. It is likely that phenotypic meas-
urement of antimicrobial susceptibility and resistance
will continue to be an important function of clinical bac-
teriology laboratories in the future and an area where
molecular methods may find less application.
PCR for detection of bovine mastitis pathogens
Early identification of the agents that cause bovine mas-
titis is important as it facilitates appropriate treatment,
minimizes the development of chronic disease, and con-
tributes to the control of infection in the herd. Bovine
mastitis can be classified into contagious and environ-
mental mastitis. Contagious mastitis is caused by a small
number of pathogens that include S. aureus,
Streptococcus agalactiae, Mycoplasma bovis and
Corynebacterium bovis, whereas environmental mastitis
is caused by a wide range of pathogens that exist in the
environment and contaminate the tips of the teats
(Quinn et al., 2002).
Routine bacteriological identification of mastitis
pathogens is usually performed by traditional culture
followed by biochemical tests on bacterial isolates. The
advantages of the culture method are that viable
Molecular genetic methods in the veterinary clinical bacteriology laboratory
causative bacteria can be identified and antimicrobial
susceptibility can be performed, providing guidance on
the selection of antimicrobials for treatment. However,
there are disadvantages. The sensitivity of culture may
not be sufficient to detect intermittent shedders with low
and high shedding cycles during lactation, subclinically
infected glands with low numbers of bacteria, or bacte-
ria inhibited by residual therapeutic antimicrobials or
leukocytes. Environmental contaminants and intracister-
nal microorganisms can also represent a major problem
in the interpretation of culture results. Another problem
is that certain bacteria, such as Streptococcus uberis and
S. parauberis (S. uberis type II) cannot be distinguished
by biochemical tests (Jayarao et al., 1991). Moreover,
bacterial culture and identification is time consuming
and requires more than 48 hours to complete.
Because of the limitations of conventional culture, sim-
plex and multiplex PCR protocols have been developed
for identification of the various mastitis pathogens
(Jayarao et al., 1991; Forsman et al., 1997; Ghadersohi et
al., 1997; Khan et al., 1998; Hirose et al., 2001; Kim et al.,
2001; Pinnow et al., 2001; Riffon et al., 2001; Daly et al.,
2002; Meiri-Bendek et al., 2002; Phuektes et al., 2001,
2003). These PCR methods offer the option of identifica-
tion of bacteria within hours. PCR can also improve the
level of detection because of its ability to detect low
numbers of organisms. Mastitis pathogens could proba-
bly be detected in carrier animals or at earlier stages of
infection in clinical cases. PCR also has the potential to
be extremely specific and to discriminate between
closely related microorganisms, such as S. parauberis and
S. uberis. The disadvantages of PCR methods include
their very low threshold of detection, since minor con-
taminants and non-viable bacteria in milk samples could
lead to misdiagnosis. In addition, PCR does not provide
information on antimicrobial sensitivities.
To our knowledge, real-time PCR has not yet been
described for the identification of mastitis pathogens.
These multiplex PCR protocols could be adapted to a real-
time platform PCR in order to increase the ability to detect
low numbers of organisms as well as turn-around time. In
addition, the real-time PCR offers quantitative data, which
might help in addressing the issue of minor contaminants
found in milk samples. While quantitative PCR has an
obvious place in distinguishing contaminants from gen-
uine infection, further work is needed to define what
would be regarded as genuinely positive PCR findings.
A species-specific multiplex PCR based on detection of
the 16S to 23S rRNA spacer region was developed and
compared with traditional culture by Phuektes et al.
(2001) for four mastitis pathogens, Staphylococcus aureus,
Streptococcus agalactiae, Streptococcus dysgalactiae and
Streptococcus uberis. This multiplex PCR was significantly
more effective than culture for the detection of low num-
bers of Staphylococcus aureus and Streptococcus uberis.
However, there were no significant differences in detec-
tion of low numbers of bacteria between PCR and culture
87
for Streptococcus agalactiae and S. dysgalactiae. More
recently, Phuektes et al. (2003) have used a multiplex
PCR to detect the same microorganisms in bulk tank milk
samples, in which correlations with bulk milk somatic cell
counts (BMCC), total bacterial counts and thermoduric
bacterial counts were evaluated. There was no relation-
ship in their study between the presence of S. aureus, S.
agalactiae or S. uberis and BMCC, total bacterial counts
or thermoduric bacteria counts, but the presence of S.
agalactiae was associated with high BMCC and total bac-
terial counts. Their results have shown that regular
analysis of bulk milk using this multiplex PCR assay may
be a useful tool for monitoring herd status for S. agalac-
tiae, but it is of less value for monitoring of S. aureus, S.
dysgalactiae and S. uberis.
Riffon et al. (2001) have developed molecular probes
reacting in PCR with bacterial DNA from bovine milk,
providing direct and rapid detection of E. coli,
Staphylococcus aureus, Streptococcus agalactiae,
Streptococcus dysgalactiae, Streptococcus parauberis and
Streptococcus uberis. The two sets of primers appeared
to discriminate the close phylogenetic species
Streptococcus agalactiae and Streptococcus dysgalactiae,
and Streptococcus uberis and Streptococcus parauberis.
The primers for E. coli, Staphylococcus aureus,
Streptococcus parauberis and Streptococcus uberis were
designed on the basis of 23S rRNA, and those for
Streptococcus agalactiae and Streptococcus dysgalactiae
were based on 16S rRNA sequence.
Several publications have described PCR methods for
the direct detection of Mycoplasma including M. alka-
lescens, M. bovigenitalium, M. bovirhinis and M. bovis
(Forsman et al., 1997; Hirose et al., 2001; Kim et al.,
2001; Pinnow et al., 2001). The threshold of detection
for these PCR tests ranged from 1 to 1500 colony-form-
ing units/ml of milk.
In summary, the area of molecular genetic tests for
the identification of mastitis pathogens is underdevel-
oped. Because of the enormous economic importance of
mastitis to the dairy industry, this is an area in which
rapid, specific and inexpensive tests have the potential
to be applied extensively in the veterinary clinical
microbiology laboratory.
Commercially available tests
Many laboratories use in-house molecular methods but
some tests are available commercially as kits, which
have been evaluated analytically or diagnostically to
varying degrees. Compared with in-house methods, the
commercial kits usually are easier to use because the
reagents are premixed, and results may be more consis-
tent between laboratories as the same reagents are used
in a standardized way. However the price is usually
higher. Current commercially available molecular test
kits are listed in Table 3.

Table 3. Commercially available kits and systems for molecular tests
Pathogen
Actinobacillus pleuropneumoniae
Bacillus anthracis
Borrelia burgdorferi and B. afzelli
Brachyspira hyodysentariae and
B. pilosicoli
Escherichia coli O157:H7
Helicobacter pylori
Lawsonia intracellularis
Mycobacterium paratuberculosis
Mycoplasma
Salmonella
Bacteria genotyping system
Bacterial 16S and 23S rRNA
identification
88
Kits Test format Company
Adiavet® APP PCR tests PCR Adiagène, France
Bacillus Anthracis Detection kit LightCycler real-time PCR Roche Diagnostics, Basel, Switzerland
RealArtTMBorrelia PCR kit PCR Artus Biotech USA, USA
BOR B. burgdorferi and B. afzelli detection kit Nested PCR AB Analitica, Italy
Adiavet® Brachy PCR test (differentiating PCR Adiagène, France
B. hyodysenteriae, B. pilosicoli, B. innocens
and B. intermedia)
BAX® System PCR assay for screening Automated PCR system DuPont Qualicon, Wilmington, DE, USA
E. coli O157:H7
TaqMan® pathogenic E. coli (0157:H7) TaqMan real-time PCR Applied Biosystem, Foster City, CA, USA
detection kits
HP H. pylori detection kit Nested PCR AB Analitica, Italy
Adiavet® law PCR test kit PCR Adiagène, France
paratub test kit
Adiavet® PCR Adiagène, France
IDEXX M.pt DNA test kit PCR and dot or Southern blot Idexx, Westbrook, ME, USA
Adiavet® myco av PCR test kit (for PCR Adiagène, France
M. gallisepticum, M. synoviae and
M. meleagridis)
Adiavet® M.HYOP PCR test (for PCR Adiagène, France
M. hyopneumonaie)
RealArtTM Salmonella PCR kit (for all LightCycler real-time PCR or PCR Artus Biotech USA,USA
Salmonella spp.)
BAX® System PCR assay for screening PCR automatic system DuPont Qualicon, Wilmington, DE, USA
Salmonella
TaqMan® Salmonella gold detection kit TaqMan real-time PCR Applied Biosystem, Foster City, CA, USA
RiboPrinter® microbial characterization Automatic ribotyping system DuPont Qualicon, Wilmington, DE, USA
system (for fingerprinting of any bacteria)
MicroSeq® microbial identification PCR and DNA sequencing Applied Biosystem, Foster City, CA, USA
system (kit)
H. Y. Cai et al.
Molecular genetic methods in the veterinary clinical bacteriology laboratory
Concluding remarks
In the last 5 years, numerous molecular methods have
been published for the detection and characterization of
bacteria in the field of veterinary medicine. The PCR has
been the most commonly used technology. Although
not currently used routinely for clinical veterinary diag-
nosis, new technologies such as liquid-phase
hybridization, real-time PCR, pathogen load determina-
tion and DNA/protein microarray have been described
and have many possible applications in the clinical bac-
teriology laboratory because of their sensitivity and
efficiency.
Veterinary molecular diagnosis has not yet been
applied in the way that it could be in clinical bacteriol-
ogy laboratories. Only a limited number of detection
assays have been developed into commercial diagnostic
kits, and only a few molecular tests are offered by vet-
erinary diagnostic laboratories. Most available kits or
laboratory services are for the detection of pathogens or
toxins, and few are for molecular typing. The molecular
detection of antimicrobial resistance genes is still a
largely unexplored area in veterinary clinical diagnosis.
There are several obstacles to the application of molecu-
lar diagnosis in veterinary bacteriology laboratories.
First, most current accredited tests are based on tradi-
tional methods. Without updating of these accreditation
standards, the molecular tests are not acceptable to
accreditation authorities. Secondly, little information is
available on method validation. Although a lot of effort
has been devoted to the development of molecular tests,
most published research stops at analytical validation
and does not take the additional steps that are required
for field validation. The fact that these steps are time-
consuming and costly is probably responsible for this.
An additional problem in the adoption of molecular
methods is that of the determination of gold standards
against which the sensitivity and specificity of molecular
tests can be measured. For example, it is highly likely
that these may turn out in some cases to be far more
sensitive than isolation or immunological approaches,
leading to a tendency to discount the reliability of the
newer assays. If these tests are in fact more reliable than
conventional tests, they may lead to changed under-
standing of the epidemiology of some bacterial
infections of animals, but gaining such acceptance will
require careful documentation. However, more robust
and reliable methods need to be developed and vali-
dated in order to reduce the cost of testing and to
facilitate application in a diagnostic laboratory.
Proficiency testing is also urgently needed in molecular
veterinary clinical diagnostic laboratories, as it remains
largely unregulated. Most available molecular test labo-
ratory services use procedures developed in-house. It
would be useful for an organization such as the
American Association of Veterinary Laboratory
Diagnosticians or the Office International des Epizooties
89
to accredit and standardize all molecular methods used
in veterinary bacteriology diagnostic laboratories and
provide standard controls, especially for quantitation
purposes.
In summary, the door to molecular diagnosis has just
opened. Many molecular methods are waiting to be put
into practical use for more efficient, rapid and sensitive
laboratory diagnosis of bacterial infections in animals.
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