Veterinary Microbiology 90 (2002) 435±446

PCR as a diagnostic tool for brucellosis

Betsy J. Bricker*
United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center,
2300 Dayton Road, Ames, IA 50010, USA

Abstract

Numerous PCR-based assays have been developed for the identi®cation of Brucella to improve
diagnostic capabilities. Collectively, the repertoire of assays addresses several aspects of the diagnostic
process. For some purposes, the simple identi®cation of Brucella is adequate (e.g. diagnosis of human
brucellosis or contamination of food products). In these cases, a genus-speci®c PCR assay is suf®cient.
Genus-speci®c assays tend to be simple, robust, and somewhat permissive of environmental in¯uences.
The main genetic targets utilized for these applications are the Brucella BCSP31 gene and the 16S±23S
rRNA operon.
Other instances require identi®cation of the Brucella species involved. For example, most govern-
ment-sponsored brucellosis eradication programs include regulations that stipulate a species-speci®c
response. For epidemiological trace back, strain-speci®c identi®cation is helpful. Typically, differential
PCR-based assays tend to be more complex and consequently more dif®cult to perform. Several
strategies have been explored to differentiate among Brucella species and strains, including locus
speci®c multiplexing (e.g. AMOS-PCR based on IS711), PCR-RFLP (e.g. the omp2 locus), arbitrary-
primed PCR, and ERIC-PCR to name a few. This paper reviews some of the major advancements in
molecular diagnostics for Brucella including the development of procedures designed for the direct
analysis of a variety of clinical samples. While the progress to date is impressive, there is still room for
improvement.
# 2002 Elsevier Science B.V. All rights reserved.

Keywords: Brucella; Brucellosis; PCR; PCR-RFLP; Molecular diagnostics; DNA-typing

1. Introduction

Brucellosis, caused by species of the Gram-negative bacterium Brucella, continues to be

a problem for humans and animals throughout the world. Even in the countries where all
forms of the disease have been eradicated, continued vigilance is essential for prevention of

*
Tel.: ‡1-515-663-7310; fax: ‡1-515-663-7458.
E-mail address: bbricker@nadc.ars.usda.gov (B.J. Bricker).

0378-1135/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 1 3 5 ( 0 2 ) 0 0 2 2 8 - 6
436 B.J. Bricker / Veterinary Microbiology 90 (2002) 435±446

the reintroduction of the disease. Critical to success is the availability of good diagnostic
tools.
The ``gold standard'' continues to be based on isolation of ``suspicious'' bacterial
colonies from host tissues, milk or vaginal exudates, followed by bacteriological char-
acterization (Alton et al., 1975). This process has withstood the test of time because it is
reliable and usually de®nitive. Depending on the number of phenotypic traits analyzed, the
Brucella organisms can be characterized to genus, to species, to sub-species (biotypes), and
sometimes to strain, particularly in the case of vaccine strains.
But this process also has drawbacks. First, the length of the process from clinical
specimen to de®nitive identi®cation takes time, typically 2 weeks. During this period, the
livestock producer, food processor or ill patient may be subjected to unnecessary hardship
if the suspicion is unsubstantiated. Second, the tests are complex and must be performed in
an authorized laboratory by highly skilled personnel. Some of the characteristics are
subjective, such as colony morphology, and require the trained eye and experience of an
expert. Third, the zoonotic nature of most Brucella species is a potential hazard for
laboratory personnel who must manipulate the infectious agent during testing. Finally, the
results are not always de®nitive. Because of the large number of traits examined and the
small number of differences among some species and biovars, a minor mutation may result
in con¯icting data and complicate the interpretation.
As with any disease, control of brucellosis would bene®t from improvements in
diagnostic methods that could address some of these issues. Furthermore, as we enter
a new era of biological warfare and agroterrorism, rapid tests that are highly sensitive and
highly speci®c are needed more than ever. PCR has the potential to meet the need for better
diagnostic tools. It is highly sensitive, very speci®c, inexpensive, and easily adapted to high
volume demands. The process is rapid, simple, and requires little manual labor. As long as
careful attention is given to avoid contamination, the method is very reliable and usually
highly reproducible at any properly equipped laboratory. Since PCR was ®rst introduced in
1987 (Mullis and Faloona, 1987) researchers have made excellent progress in developing
quality PCR-based tests for Brucella.

2. Overview

Since 1987, numerous PCR-based assays for Brucella have been developed and
published. The earliest assays were designed to exploit a single unique genetic locus that
was highly conserved in Brucella (e.g. the BCSP31 or the 16S rRNA genes). The advantage
of these types of assays is that they tend to have a simple design and are very robust. Such
tests are useful for screening or for identi®cation when species or biovar designations are
not critical.
The Brucella genus is composed of six classical species based mainly on host
preference: B. abortus, B. canis, B. melitensis, B. neotomae, B. ovis and B. suis. Based
on phenotypic traits, some species of Brucella have been subtyped into biovars: B. abortus
(7 biovars), B. melitensis (3 biovars), and B. suis (5 biovars). The number of species will
probably increase with the recent discovery of Brucella in a diversity of marine mammals.
PCR assays have been developed to differentiate among Brucella species and/or biovars.
 B.J. Bricker / Veterinary Microbiology 90 (2002) 435±446 437

These assays are directed toward genetic loci that are variable among the species/biovars.
Such targets are uncommon in Brucella since the genus is remarkably homogeneous and
has been proposed to be a single species (Verger et al., 1985, 1987). While some large
deletions or rearrangements have been reported within a species or biovar (e.g. Ficht et al.,
1996; Jumas-Bilak et al., 1998; Cloeckaert et al., 2000a; Bricker et al., 2000), most genetic
differences consist of single nucleotide polymorphisms. Particularly rare are regions of
hypervariability among species and biovars. Differential PCR-based assays are particularly
useful for epidemiological trace back, or for species-speci®c eradication programs.
However, the ability to discriminate usually involves a more complicated assay mix,
additional processing steps or problems with reproducibility.
While most of the data so far have been acquired by testing cultured bacteria, several
laboratories have been productive developing methods for the direct analysis of clinical
samples: tissues, semen, milk, and blood. These assays have the advantage that the results
can be determined immediately. The disadvantage is that they usually require more
extensive sample preparation to remove PCR inhibiting components. This paper reviews
the major PCR-based assays, their strengths and their weaknesses, in the context of today's
diagnostic requirements.

3. Genus-specific identification of Brucella

The earliest PCR-based assays developed for Brucella concentrated on genus-speci®c

loci for identi®cation. At this point in time, very few Brucella genes had been cloned
and sequenced so the number of available known targets was small. The ®rst published
PCR-based diagnostic assay was reported by Fekete et al. (1990). This assay was based
on the ampli®cation of a 635-bp sequence from a gene encoding a 43-kDa outer
membrane protein of B. abortus strain S19. They were able to demonstrate the assay
was speci®c for Brucella, applicable to all species and biovars, and very sensitive (less
than 100 bacteria). However, the primers and target sequence were never published for
proprietary reasons and so this initial assay was never adopted by other laboratories.
Nevertheless, their success encouraged further investigation into the application of PCR
technology.
The next Brucella gene target to be explored was the 16S rRNA gene (Herman and De
Ridder, 1992). The B. abortus 16S sequence had been published by Dorsch et al., 1989. The
authors selected unique primers based on alignment of the B. abortus 16S sequence with
those of several other bacteria including that of closely related Agrobacterium tumefaciens.
Successful ampli®cation of a predicted 800-bp amplicon from B. abortus and other species
of Brucella demonstrated that the sequences were highly conserved and that the test could
be extended to the entire genus. To assess speci®city, the assay was applied to a panel of
17 other bacteria. No products were ampli®ed from any non-Brucella species except
Ochrobactrum anthropi, the closest known relative to Brucella.
The rRNA operon was subsequently used by other laboratories to develop Brucella
speci®c tests. Romero et al. (1995a) analyzed nine different primer combinations to derive
their assay. Their results were similar to those of Herman and de Ridder. All strains of
Brucella examined were recognized by the assay, and all other bacterial species were
438 B.J. Bricker / Veterinary Microbiology 90 (2002) 435±446

negative except Ochrobactrum anthropi. Interestingly, Romero's group examined three

strains of O. anthropi including one ®eld isolate and found that only one strain cross-
reacted in the test. The following year Rijpens et al. developed an assay based on the
16S±23S intergenic spacer region, a region found to be highly variable among bacteria. In
this assay 9 strains of O. anthropi were examined along with 47 other bacterial strains.
Ampli®cations performed with a 50 8C annealing step permitted some cross-reactions with
certain O. anthropi strains, however increasing the annealing temperature by 5 8C
eliminated the cross-reactivity. In all three studies, the authors point out that infections
by Ochrobactrum species are rare and usually opportunistic in immuno-compromised
hosts, a situation not likely to be confused with brucellosis.
In 1992, a new PCR assay was published by Baily et al. (1992) based on the gene
encoding BCSP31. The BCSP31 gene was the ®rst published Brucella loci to be cloned
and sequenced (May®eld et al., 1988). This gene encodes an antigenic, periplasmic
protein of unknown function. It is conserved in all species and biovars of Brucella
examined; however, it is not expressed in Brucella ovis (Bricker et al., 1988). The PCR
assay developed by Baily et al. contained a single pair of oligonucleotide primers
designed to amplify a 223-bp product. The authors demonstrated that the assay was
robust, sensitive and speci®c for B. abortus and B. melitensis. No other Brucella species
were included in the panel of bacteria used to evaluate the procedure, nor was the close
relative Ochrobactrum anthropi. However, a more thorough investigation was reported by
Da Costa et al. (1996) in which all the Brucella species and biovars were tested and
recognized. Da Costa's group also surveyed 98 non-Brucella bacteria and found that
all organisms except one strain of Ochrobactrum were negative for ampli®cation. The
assay has subsequently been adopted by other laboratories and continues to be robust and
highly speci®c.

4. Differentiation of Brucella species and/or biovars by PCR

For some purposes, identi®cation of Brucella at the species level is not immediately
necessary to initiate action (e.g. diagnosis of human brucellosis or contamination of food
products). In these cases, a genus-speci®c PCR assay is suf®cient. However, there are many
instances where the Brucella species involved is relevant to the initiation of an appropriate
action. For example, brucellosis eradication programs are typically species-speci®c and the
associated regulatory actions are species dependent. Furthermore, differential assays are
particularly useful for epidemiological trace back to its source of infection. For this
purpose, even the classical procedures have limited value since single biovars tend to
predominate over large geographic areas.
For nearly a decade, there has been steady progress toward more sophisticated differential
assays despite the high level of conservation among Brucella species and strains.
Strategically, attempts to develop more speci®c PCR assays have taken three
approaches: (1) assays designed with highly speci®c primers and stringent assay condi-
tions; (2) assays designed with semi-speci®c primers and mildly permissive assay
conditions; and (3) assays based on ampli®cation with random primers under very
permissive conditions.
 B.J. Bricker / Veterinary Microbiology 90 (2002) 435±446 439

4.1. Assays designed with highly specific primers and stringent assay conditions

This strategy employs primer pairs that are either multiplexed to amplify strain-speci®c
targets, or that enclose regions of DNA sequence hypervariability. The advantage to this
strategy is that the use of speci®c, long (20±25 bp) primers permits the use of stringent
assay conditions which reduces the risk of false-positive reactions due to mis-priming. The
major drawback is that substantial sequence information is needed and in the case of
Brucella, divergence is dif®cult to ®nd.
This approach was ®rst used in our laboratory (Bricker and Halling, 1994) exploiting the
multi-copy element IS711 (Halling et al., 1993), also known as IS6501 (Ouahrani et al.,
1993). Although the number and placement of the IS elements in Brucella species are
typically conserved (Bricker and Halling, 1994), most species contain at least one copy of
the IS element at a unique chromosomal location. The assay was named the AMOS-PCR
assay for the Brucella species it can identify and differentiate (B. abortus biovars 1, 2 and 4;
B. melitensis, B. ovis and B. suis biovar 1). The multiplex design consists of one common
primer anchored in the IS element and a species-speci®c primer that binds to the unique
sequence ¯anking that insertion site. The assay primers were chosen so that species
discrimination was determined by the size of the amplicon. As more Brucella gene
sequences were published (Sangari et al., 1994), the assay was modi®ed to include strain-
speci®c primers for the two commonly used vaccine strains: S19 and RB51 (Bricker and
Halling, 1995). This was a signi®cant improvement since discrimination between vacci-
nated and ®eld infected animals is critical to any eradication program.
The assay has since been adopted by the National Veterinary Services Laboratories
(Animal and Plant Health Inspection Service, United States Department of Agriculture),
where most of the suspected veterinary samples from the United States are submitted for
identi®cation. Over a period of several years, the PCR assay performed at 100% accuracy
for 231 samples that were also tested by conventional biochemical tests. Samples were
correctly identi®ed as either B. abortus ®eld strain, B. abortus vaccine strain RB51 or B.
abortus vaccine strain S19 (Ewalt and Bricker, 2000).
Other laboratories reported success with the published protocol (Adone et al., 2001) or a
modi®ed version (Redkar et al., 2001). It should be noted that at least one laboratory had
dif®culties reproducing the results for the AMOS-PCR assay, remarking that the test would
only perform well if the published protocol was followed precisely (Leal-Klevezas et al.,
1995b). This has been our experience also. Due to the multiplex nature of the assay, the
assay components and the cycling parameters must be carefully controlled to allow
maximum performance of each primer pair. Recently, the assay was redesigned to improve
performance (Ewalt and Bricker, 2002). Some primers were changed to allow a more even
distribution of amplicon sizes for easier analysis, and an internal control was included to
detect PCR inhibitors. Based on the work of Vemulapalli et al. (1999), the vaccine strain
RB51 primer was relocated to exclude the parental strain, S2308, from which it was
derived. The new formulation still has the drawback that assay conditions must be carefully
reproduced, but it also has the advantage of being one of the few PCR-based tests that can
differentiate vaccine strains from ®eld strains.
Several laboratories have designed differential PCR assays targeting the omp2 locus.
This locus, originally characterized by Ficht et al. (1989), consists of two genes, omp2A
440 B.J. Bricker / Veterinary Microbiology 90 (2002) 435±446

and omp2B, arranged head to head on opposite strands, each encoding a 36-kDa outer
membrane protein. The two genes are approximately 85% homologous in sequence, although
it appears that only the omp2B gene is actually expressed. While the gene sequences are
highly conserved across Brucella species, signi®cant sequence polymorphisms were dis-
covered. For example, Ficht noted that B. abortus biovars 1, 2 and 4 contained a 115-bp
deletion in the omp2A gene. This polymorphism was exploited by Leal-Klevezas et al.
(1995a) to develop a PCR assay that differentiates B. abortus from other Brucella species.
The same year, Cloeckaert et al. also developed a PCR assay based on the omp2A and
omp2B gene sequences. He greatly enhanced the discriminatory power of the PCR assay by
incorporating restriction endonuclease cleavage of the amplicons after ampli®cation.
Certain strain-speci®c sequence variations modi®ed restriction sites, resulting in differ-
ential RFLP patterns. As a result, most Brucella species and some biovars could be
identi®ed and differentiated with the correct combination of restriction enzymes.
Following Cloeckaert's success, a number of Brucella genes have been examined by
PCR-RFLP with a wide range of restriction endonucleases, but success has been limited.
Cloeckaert et al. studied the omp25 gene (1995), and the dnaK gene (Cloeckaert et al.,
1996), with at least nine different restriction enzymes, but was only able to differentiate B.
melitensis and B. ovis from the other Brucella species. In 1996, Da Costa et al. examined
six Brucella genes by PCR-RFLP: BCSP31, 16S rRNA, groEL, dnaK, dnaJ and htrA. For
each locus he tested all six classical Brucella species, reference strains and vaccine strains,
but was unable to ®nd detectable polymorphisms in any of the genes. Cloeckaert et al.
(2000b) examined PCR-RFLP analysis on 7 genes associated with LPS-O antigen
synthesis but was unable to ®nd any signi®cant differences even among rough strains.
Vizcaino et al. (1997) did have success in differentiating most Brucella species by PCR-
RFLP of the omp31 locus. Although the method could not differentiate B. suis from
B. neotomae, it was able to differentiate B. suis from B. canis.
PCR-RFLP of the omp2 locus as described by Cloeckaert or as modi®ed by other labo-
ratories (Sifuentes-Rincon et al., 1997) has become a preferred method in many laboratories
for differential identi®cation of Brucella species or for characterizing new species or strains
(Miller et al., 1999; Clavareau et al., 1998; Brew et al., 1999; Cloeckaert et al., 2001).
Another application of differential PCR entails the multiplexing of genus- or species-
speci®c primer pairs targeting several different bacteria that might be identi®ed from the
sample environment. Two laboratories have applied this technology to Brucella identi®ca-
tion. Sreevatsan et al. (2000) developed a multiplex assay for B. abortus and Mycobacter-
ium bovis, targeting the BCSP31 gene of Brucella and the hsp65 locus of M. bovis. In 2001,
McDonald et al. described a multiplex assay for the identi®cation of potential biowarfare
agents. Their assay was designed to differentiate Coxiella burnetti, Brucella melitensis,
Bacillus anthracis and Yersinia pestis. This report also describes the dif®culties of doing
research under high security conditions.

4.2. Differentiation of Brucella strains by PCR with semi-specific primers and

moderately permissive assay conditions

This strategy exploits repeated sequences that are very similar but not identical.
Mismatched or degenerate primers must be used to anneal to a family of targets. Because
 B.J. Bricker / Veterinary Microbiology 90 (2002) 435±446 441

the primers are not perfect homologues, moderately permissive conditions must be used to
maximize hybridization to the target. The permissive environment increases the incidence
of mis-priming or partial priming resulting in variable numbers and yields of amplicons.
Because the annealing conditions are marginal, primer annealing and amplicon production
are readily in¯uenced by even small variations in the assay conditions. At the same time,
the primers are homologous over most of their sequence providing the necessary
speci®city. If the target sequence is large enough, lengthening the primer can offset some
of the mismatch effect. A key factor is positioning the largest stretch of homology at the 30
end of the primer. ERIC-PCR and REP-PCR are examples of PCR with semi-speci®c
primers. ERIC (enterobacterial repetitive intergenic consensus) sequences and REP
(repetitive extragenic palindromic) sequences are two classes of small DNA repeats
(approximately 35±125 bp) found dispersed throughout the chromosome(s) of most
bacteria. The sequences are homologous but polymorphic. Since these repeats are short
and widely dispersed, ERIC-PCR and REP-PCR function by hybridizing consensus or
degenerate primers to a family of target sites and amplifying the sequence between the
repeats. Depending on the number of elements clustered together, their orientation and
distance apart, a collection of different amplicons will be produced.
Both Mercier et al. (1996), and Tcherneva et al. (1996) investigated ERIC- and REP-
PCR for differential typing of Brucella. They both used identical primer pairs for
ampli®cation but with different cycling parameters and annealing temperatures. Interest-
ingly, they had different results. Mercier found ERIC-PCR to have greater discriminating
power among isolates while Tcherneva's group found more diversity with REP-PCR.
Furthermore, the published ®ngerprints from both laboratories look very different. This
illustrates that degenerate and mismatched primers are hypersensitive to nuances in assay
conditions with consequential affects on PCR performance.
In a different approach, Ouahrani-Bettache et al. (1996) introduced IS-anchored
arbitrary PCR. In this strategy, a speci®c primer homologous to a portion of IS6501
(IS711) was paired with a random primer. During ampli®cation, the speci®c IS primer was
designed to anchor the ampli®cation to the terminal sequence from each IS copy, extending
synthesis into the ¯anking DNA. An arbitrary primer would then anneal randomly to
regions of partial homology. Similar to REP-PCR and ERIC-PCR, reproducibility is
in¯uenced by assay conditions due to the tenuous binding of the arbitrary primer to semi-
homologous sequences.

4.3. PCR assays based on amplification with random primers under

very permissive conditions

One drawback to PCR typing with highly speci®c primers is the need to identify
polymorphous loci and obtain the corresponding DNA sequence(s). Arbitrary Primed PCR
(AP-PCR) or Random Ampli®ed Polymophic DNA PCR (RAPD-PCR), was developed to
bypass the need for speci®c target sequences. Short, arbitrarily designed primers anneal to
genomic DNA sequences under permissive conditions that permit hybridization to random,
partially homologous sequences. At a low frequency, another arbitrary primer will
fortuitously anneal to the synthetic sequence initiating the chain reaction. To promote
annealing, these primers are typically short and the hybridization is weak. Consequently,
442 B.J. Bricker / Veterinary Microbiology 90 (2002) 435±446

minute changes in the assay environment can easily in¯uence the annealing ef®ciency and
signi®cantly alter the results. Fekete et al. (1992b) was the ®rst group to apply this
technology to Brucella. Tcherneva et al. followed in 2000. In both laboratories, the
technique was found to be highly discriminating. In response to concerns about reprodu-
cibility, Tchernava's group methodically examined assay parameters to develop an
optimized protocol. However, the likely impact of uncontrollable factors such as equip-
ment performance and sample milieu on reproducibility makes it doubtful that this
technology will be widely adopted.

5. Identification of Brucella by PCR of field samples

In the initial phase of assay development, the test samples generally consist of puri®ed
DNA obtained from the cultured organism. However, in practice it would be prudent to
identify the organism directly from clinical specimens without having to grow the
organism. With respect to biowarfare or other emergency conditions, it may be critical
to perform analyses directly in the ®eld. Therefore, efforts have been made to develop
methods for analyzing infected or contaminated materials directly. Sample preparation is
key to success since most of these sources are rich in PCR inhibitors. Furthermore,
concentration of the initial sample is also usually necessary for achieving acceptable
detection limits since only minute quantities (1±10 ml) can be assayed.
As a veterinary diagnostic tool, PCR has been applied to tissues [mainly aborted fetuses
and associated maternal tissues (Cortez et al., 2001; Cetinkaya et al., 1999; Gallien
et al., 1998; Fekete et al., 1992a), blood (Guarino et al., 2000); milk (Tantillo et al., 2001;
Leal-Klevezas et al., 2000, 1995b; Romero and Lopez-Goni, 1999; Romero et al., 1995b;
Rijpens et al., 1996); nasal secretions (Sreevatsan et al., 2000) and semen (Amin et al.,
2001). Each type of clinical sample has inherent and unique dif®culties for adequate
sample preparation. Common themes of many procedures involve cell lysis to release
bacterial DNA (and often host DNA as well); DNA clean-up, usually by phenol
extraction and concentration by alcohol precipitation of the DNA (e.g. Leal-Klevezas
et al., 1995b) or with commercial kits (e.g. Sreevatsan et al., 2000); followed by
resuspension or elution in a minimal volume. The most common dif®culties arise from
co-puri®cation of PCR inhibitors with the DNA and from interference by excessive host
DNA (Morata et al., 1998; Queipo-Ortuno et al., 1999; Amin et al., 2001; Leal-Klevezas
et al., 1995b). Once these problems are overcome, the various assays perform very
well and may exceed the sensitivity achieved by direct culture (Queipo-Ortuno et al.,
1997).
PCR technology has also been applied to the detection of Brucella contamination in food
products, namely milk and soft cheeses. There has been greater emphasis on sample
preparation from milk (Tantillo et al., 2001; Leal-Klevezas et al., 2000, 1995b; Romero and
Lopez-Goni, 1999; Romero et al., 1995b; Rijpens et al., 1996), probably since this is
currently used as a diagnostic sample as well as being a food product. In addition, the liquid
matrix is easier to work with. Nevertheless, the consumption of tainted, unpastuerized soft
cheeses is a major source of human brucellosis (Chomel et al., 1994; Corbel, 1997). So far,
few laboratories have been developing PCR-based protocols for cheese and other milk
 B.J. Bricker / Veterinary Microbiology 90 (2002) 435±446 443

products. Since the presence of any Brucella in food products is unacceptable, the two
protocols developed for cheese samples are genus-speci®c, utilizing the BCSP31 gene
target (Serpe et al., 1999; Tantillo et al., 2001). More attention to detection of Brucella in
food products is greatly needed.
In endemic areas, human brucellosis, particularly when caused by B. melitensis, is a
serious health concern. The requirements for a diagnostic medical test are different from
the requirements for testing livestock. To date, nearly all PCR-based diagnostics for human
brucellosis target peripheral blood as the sample source. Beginning with Matar et al., 1996,
the preferred assay for human use is the BCSP31-based, genus-speci®c assay developed by
Baily and colleagues (Queipo-Ortuno et al., 1997, 1999; Morata et al., 1998, 1999; Casanas
et al., 2001; Zerva et al., 2001).
Advancements in sample processing have progressively lowered the limits for detection
by improved removal of polymerase inhibitors, particularly heme compounds, from whole
blood. Recently, Queipo-Ortuno et al. (1999) reported that treatment of lymphocyte pellets
with hydrogen peroxide prior to lysis would inactivate residual heme without permanent
damage to the DNA. However, complete removal of the hydrogen peroxide was necessary
prior to PCR. Zerva et al. (2001) found suf®cient DNA in the serum fraction to detect
infection. In their studies, processed serum was a better sample medium than processed
whole blood, presumably because sera contains fewer PCR inhibitors. In a survey of 31
naturally infected patients, serum-PCR had a 94% sensitivity level compared to 61% for
whole blood.
Other PCR assays have been utilized for human diagnostics. The ®rst report of human
brucellosis from a marine isolate (Brew et al., 1999) was con®rmed by PCR using the
PCR/RFLP method of Cloeckaert et al. (1995). Leal-Klevezas et al. (1995b) successfully
detected Brucella from human and animal blood using the omp2 locus. Morata et al. (2001)
recently demonstrated that brucellosis could also be diagnosed by PCR using clinical
samples taken from patients with focalized brucellosis, even though culture isolation from
the same samples was uncommon.

6. Conclusions

Over the past decade, there have been major advancements in all aspects of molecular
diagnostics with regard to human and animal brucellosis. PCR-based tests are proving to be
faster and more sensitive than traditional methods and the assays will only improve further
in the future. So far acceptance of molecular diagnostics has been slow. This is reasonable
considering the consequences at stake. However, threats of biological warfare and agro-
terrorism may accelerate the process. Already, assays speci®c to this circumstance are
being considered (McDonald et al., 2001) and real-time detection is being explored
(Redkar et al., 2001).
More research is needed in the areas of molecular subtyping and direct analysis of
clinical samples. Additional data are needed about the best choice of specimens and how
long there is detectable DNA in those specimens over the course of infection. Finally, we
need to develop a coherent, long-term strategy for implementing and utilizing molecular
diagnostics for human and animal diseases.
444 B.J. Bricker / Veterinary Microbiology 90 (2002) 435±446

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