Download A new Brucella canis species-specific PCR assay for the diagnosis

Comparative Immunology, Microbiology and Infectious Diseases 37 (2014) 237–241
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Comparative Immunology, Microbiology
and Infectious Diseases
journal homepage: www.elsevier.com/locate/cimid
A new Brucella canis species-specific PCR assay for the
diagnosis of canine brucellosis
Sung-Il Kang a , Sang-Eun Lee b , Ji-Yeon Kim a , Kichan Lee a ,
Jong-Wan Kim a , Hyang-Keun Lee a , So-Ra Sung a , Young-Ran Heo c ,
Suk Chan Jung a , Moon Her a,∗
a
The OIE Reference Laboratory for Brucellosis, Bacterial Disease Division, Animal and Plant Quarantine Agency, 480 Anyang,
Gyeonggi-do 430-757, South Korea
b
Division of Malaria & Parasitic Diseases, Korea National Institute of Health, Korea Centers for Disease Control & Prevention,
Chengwon-gun, South Korea
c
Department of Food and Nutrition, Chonnam National University, Yongbongdong, Gwanju, South Korea
a r t i c l e
i n f o
Article history:
Received 25 February 2014
Received in revised form 30 June 2014
Accepted 13 July 2014
Keywords:
Brucella canis
Buffy coat
Diagnostics
Species-specific PCR assay
Zoonosis
a b s t r a c t
Brucellosis is a zoonotic disease that is transmitted from animals to humans, and the development of a rapid, accurate, and widely available identification method is essential for
diagnosing this disease. In this study, we developed a new Brucella canis species-specific
(BcSS) PCR assay and evaluated its specificity and sensitivity. A specific PCR primer set
was designed based on the BCAN B0548-0549 region in chromosome II of B. canis. The PCR
detection for B. canis included amplification of a 300-bp product that is, not found on other
Brucella species or, genetically or serologically related bacteria. The detection limit of BcSSPCR assay was 6 pg/␮l by DNA dilution, or 3 × 103 colony-forming units (CFU) in the buffy
coats separated from whole blood experimentally inoculated with B. canis. Using the buffy
coat in this PCR assay resulted in approximately 100-times higher sensitivity for B. canis as
compared to detect directly from whole blood. This is the first report of a species-specific
PCR assay to detect B. canis, and the new assay will provide a valuable tool for the diagnosis
of B. canis infection.
© 2014 Elsevier Ltd. All rights reserved.
1. Introduction
The genus Brucella is gram-negative, facultative intracellular pathogens. Since the first isolation of a Brucella
strain, this genus has been found to be prevalent in
South America, Africa, Southeast Asia, and some European
countries. This disease has also occurred sporadically in
South Korea [1–3]. Currently, the genus Brucella consists of
10 species preference of animal hosts: B. abortus, B. canis, B.
suis, B. ovis, B. neotomae, B. melitensis, B. ceti, B. pinnipedialis,
∗ Corresponding author. Tel.: +82 31 467 1776; fax: +82 31 467 1778.
E-mail address: [email protected] (M. Her).
http://dx.doi.org/10.1016/j.cimid.2014.07.003
0147-9571/© 2014 Elsevier Ltd. All rights reserved.
B. microti, and B. inopinata. Of these, B. melitensis, B.
abortus, B. suis, and B. canis are transmitted to humans
[4]. B. ceti and B. pinnipedialis originated from marine
mammals are also known to have zoonotic potential
[5].
With the rapid growth of the pet and companion animal
industries, Brucella infection could become a serious public
health issue. Currently, canine brucellosis resulting from
B. canis infection is thought to be underestimated worldwide [6]. In dogs, B. canis can cause contagious abortion,
testicular atrophy, infertility and lymphadenitis. The infectious route is mainly through direct contact with the foetus,
placenta, foetal fluids, and/or vaginal discharge after abortions [7]. Humans generally become infected through close
238
S.-I. Kang et al. / Comparative Immunology, Microbiology and Infectious Diseases 37 (2014) 237–241
contact with infected dogs or abortion-related materials
[8].
Generally, diagnosis of canine brucellosis is based on
classical biotyping methods, serological tests or molecular techniques [4,9–11]. Whole blood is a useful sample
for isolation of B. canis because of the prolonged bacteraemia, which characterizes the disease [6,12]. However,
bacterial isolation has its disadvantages, because B. canis
is a slow-growing organism that requires a long incubation period and blind subcultures. This can also pose risks to
researchers working with the organism [6,13]. The serological tests routinely used for diagnosis of canine brucellosis
have the disadvantages of low sensitivity and varying
specificity [11]. Owing to these shortcomings, a variety of
molecular techniques using Brucella DNA have been developed to identify B. canis, such as a real-time PCR using
single nucleotide polymorphisms (SNPs) and a multiplex
differential PCR [14,15]. These molecular techniques have
their own shortcomings, including high expenditure and
expensive equipment. Currently using multiplex PCR it is
only possible to distinguish Brucella species by using a high
quality and concentration genomic DNA from the bacteria.
Therefore, this technique limits the choice of clinical specimens because of low sensitivity. On the hand, a high degree
of genetic similarity with other Brucella species, especially
B. suis, has interrupted the development of a simple PCR
assay to detect or identify B. canis until now [16,17].
Therefore, the aim of the current study was to develop
a specific PCR assay and evaluate its potential for detecting
B. canis from blood and abortion-related materials of dogs.
2. Materials and methods
2.1. Bacterial strains
Table 1
Bacteria strains used in this study and comparison of the results of two
PCR assays.
Species
Strains
PCR results
16S
rRNAa
B. canis
PCRb
Brucella species
B. abortus bv. 1 544
B. abortus bv. 2 86/8/59
B. abortus bv. 3 Tulya
B. abortus bv. 4 292
B. abortus bv. 5 B3196
B. abortus bv. 6 870
B. abortus bv. 9 C68
B. canis RM6/66
B. suis bv. 1 1330
B. suis bv. 2 Thomsen
B. suis bv. 3 686
B. suis bv. 4 40
B. suis bv. 5 513
B. ovis 63/290
B. neotomae 5K33
B. melitensis bv. 1 16M
B. melitensis bv. 2 63/9
B. melitensis bv. 3 Ether
B. ceti B1/94
B. pinnipedialis B2/94
B. microti CCM4915
B. inopinata B01
B. canis
ATCC 23448
ATCC 23449
ATCC 23450
ATCC 23451
ATCC 23452
ATCC 23453
ATCC 23455
ATCC 23365
ATCC 23444
ATCC 23445
ATCC 23446
ATCC 23447
NCTC 11996
ATCC 25840
ATCC 23459
ATCC 23456
ATCC 23457
ATCC 23458
NCTC 12891
NCTC 12890
BCCN 07-01
BCCN 09-01
94 isolates
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+
Non-Brucella organisms
Ochrobactrum anthropic
Escherichia coli O157:H7c
Pasteurella multocida
Salmonella Typhimurium
Campylobacter jejuni
Yersinia enterocolitica O: 9
Staphylococcus aureusc
Clostridium perfringens type A
Field strain
Field strain
ATCC 43017
ATCC 14028
ATCC 33560
NCTC 11174
Field strain
ATCC 13124
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a
The strains used in this study included 22 Brucella reference strains; 94 B. canis field isolates; and 8 non-Brucella
strains, including serologically cross-reacting strains or
phylogenetically related strains (Table 1). All of the B. canis
field isolates were obtained from dog-breeding farms during 2002–2011 and were identified by classical biotyping
assays and molecular methods [4,14]. Other non-Brucella
strains were identified by 16S rRNA sequence analysis and
VITEK 2 Compact (Biomerieux, Seoul, South Korea).
The Brucella strains were cultured on tryptic soy agar
(TSA) (BD, Franklin Lakes, NJ) supplemented with 5% foetal
bovine serum (FBS) (GIBCO, Grand Island, NY, USA) for 3
days at 37 ◦ C under 5% CO2 or aerobic conditions. Other
non-Brucella organisms were grown on 5% sheep blood agar
or MacConkey agar for 18–24 h at 37 ◦ C.
Brucella genus specific PCR assay that targets the 16S rRNA [17].
B. canis species-specific PCR assay developed in this study.
c
The field strains were identified as analysis of 16S ribosomal RNA gene
sequence.
b
were performed using a 20-␮l reaction mixture containing 2 ␮l of template DNA and 2 ␮l of each of the primers
(10 pmol). The PCR conditions consisted of an initial denaturation at 94 ◦ C for 7 min; 35 cycles of denaturation at
94 ◦ C for 35 s, annealing at 59 ◦ C for 40 s, and extension
at 72 ◦ C for 35 s, followed by a final extension at 72 ◦ C
for 10 min. All amplicons were analyzed by electrophoresis using 1.5% agarose gel with a 100-bp ladder (Bioneer
Co., Taejon, South Korea) as a molecular size marker and
ethidium bromide (0.02 ml/ml) for staining. Stained gels
were visualized and photographed under UV light with a
UV transilluminator (Bio-Rad Laboratories, Milan, Italy).
2.2. PCR primer design and amplification
2.3. Specificity and sensitivity of the BcSS-PCR assay
The PCR primer sets were designed at the BCAN B0548
region of B. canis chromosome II. This site was carefully analyzed with CLC Main Workbench software version
6.0 (Insilicogen Inc., South Korea). The primer pairs were
used as follows: forward 5 -CCAGATAGACCTCTCTGGA-3
and reverse 5 -TGGCCTTTTCTGATCTGTTCTT-3 . Amplification of specific DNA was performed using the amfiEco PCR
premix kit (GenDEPOT Inc., Barker, USA). The PCR reactions
The BcSS-PCR assay was investigated using the Brucella and non-Brucella strains listed in Table 1. The results
were compared to those of a 16S rRNA (F4/R2 primers)
PCR assay, which was used to identify Brucella species as
described previously [17]. The sensitivity of the BcSS-PCR
assay was calculated to 10-fold serial diluted DNA samples.
The concentration of B. canis DNA was determined by using
S.-I. Kang et al. / Comparative Immunology, Microbiology and Infectious Diseases 37 (2014) 237–241
an ND-1000 UV/VIS spectrophotometer (Nanodrop Tech.,
USA).
2.4. DNA extraction
239
Table 2
Direct detection from buffy coat of infected dogs by the B. canis speciesspecific PCR assay.
Specimen no.
Isolationa
Serological test
b
Genomic DNAs from all the strains and the buffy coat
was extracted using a commercial blood and tissue kit
(Qiagen Ltd., South Korea) according to the manufacturer’s
instructions.
2.5. Buffy coat preparation
The buffy coat was prepared using the Histopaque® 1083 solution (Sigma, South Korea). One millilitre of
whole-blood was layered onto 1 ml of the Histopaque® 1083 solution in a sterile 2-ml centrifuge tube. The tube was
centrifuged at 1500 × g for 30 min at room temperature.
The agglomerative white band of leukocytes (buffy coat)
was collected with a pipette and transferred to a sterile
1.5 ml eppendorf tube.
2.6. Comparison of the detection limit between buffy
coat and whole-blood
To determine the detection limit of the assay, fresh B.
canis reference strains (RM6/66) were diluted with saline
by 10-fold serial dilution. One-hundred microliters of each
serially diluted solution was inoculated in triplicate onto
TSA supplemented with 5% foetal bovine serum. After incubation for 3 days at 37 ◦ C, the colonies were counted and the
number of inoculated bacteria was calculated. One hundred
microliters of the serially diluted B. canis reference strains
were inoculated into 900 ␮l of fresh canine whole blood.
The whole blood then settled for 2 h at 37 ◦ C to permit
invasion of B. canis into peripheral blood mononuclear cells
such as macrophages. The DNA from 200 ␮l of the inoculated whole blood or from the buffy coat was extracted and
compared to the sensitivity of the BcSS-PCR assay.
2.7. Clinical specimen
Specimens were collected from 2 aborted foetuses and
13 whole blood samples from individual dogs in a breeding farm (Table 2). The foetal tissues were ground in 1 ml
of PBS buffer, and 0.1 ml was plated onto TSA medium
supplemented with 5% foetal bovine serum (FBS) (GIBCO,
Grand Island, NY, USA). Into the same medium as above
1
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7
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9
10
11
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13
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BcSS-PCR
c
2-ME RSAT
ICA
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a
Isolation: there were identified using the classical biotyping assay and
differential multiplex-PCR assay [14].
b
2-ME RSAT, 2-mercaptoethanol rapid slide agglutination test.
c
ICA, immuno-chromatographic assay [18].
was directly inoculated 0.1 ml of whole blood. The DNA
extraction from tissues and buffy coat was performed
using the DNeasy blood and tissue kit (Qiagen Ltd., South
Korea). Distilled water was used as a negative control. An
in house 2-mercaptoethanol rapid slide agglutination test
kit (2-ME RSAT) with the B. canis M-strain and an immunochromatographic kit (Bionote Inc., South Korea) were used
for serological tests [18].
3. Results
Specific primer sets for detecting B. canis were designed
carefully via alignments of a 12-bp deleted genetic site
with those from other Brucella species. The forward and
reverse primers were designed from BCAN B0548 encoding a hypothetical protein and from BCAN B0549 encoding
a helix-turn-helix domain – containing protein. Amplification with these primers provided a 300-bp fragment.
The BcSS-PCR assay was only positive for B. canis field
strains and yielded a negative reaction for other Brucella
species and non-Brucella bacterial species (Table 1). O.
anthropi and Staphylococcus aureus were amplified by the
16S rRNA gene PCR assay as reported previously [19]. In
our BcSS-PCR, these strains were not amplified. The optimal conditions of this species-specific PCR assay were
Fig. 1. The detection limit of the new Brucella canis specific polymerase chain reaction assay determined by the use of 10-fold serially diluted DNA. Lane
M: 100-bp DNA ladder, lane 1: 60 ng/␮l, lane 2: 6 ng/␮l, lane 3: 0.6 ng/␮l, lane 4: 60 pg/␮l, lane 5: 6 pg/␮l, lane 6: 0.6 pg/␮l, lane 7: 60 fg/␮l, lane 8: negative
control.
240
S.-I. Kang et al. / Comparative Immunology, Microbiology and Infectious Diseases 37 (2014) 237–241
Fig. 2. Comparison of the detection limit of the new Brucella canis specific polymerase chain reaction assay using whole blood inoculated with a B. canis
strain or buffy coat. DNA extracted from whole blood directly (A) or from buffy coat concentrated by a commercial solution, Histopaque® -1083 (B). Lane
M: 100-bp DNA ladder, lane 1: 3 × 108 , lane 2: 3 × 107 , lane 3: 3 × 106 , lane 4: 3 × 105 , lane 5: 3 × 104 , lane 6: 3 × 103 , lane 7: 3 × 102 , lane 8: 3 × 101 , lane
9: 3 × 100 CFU/ml; lane 10, negative control.
established, and the sensitivity was determined using 10fold serially diluted genomic DNA.
The sensitivity of the BcSS-PCR assay was 6 pg/␮l of
DNA (Fig. 1). The detection limit for bacterial cells was
3 × 105 CFU/ml when whole blood samples spiked with B.
canis were used (Fig. 2a). However, using the buffy coat
extracted by Histopaque from whole blood, the detection
limit was 3 × 103 CFU/ml (Fig. 2b). The buffy coat was 5
times more concentrated than the whole blood, but the
sensitivity of the PCR assay using the buffy coat was approximately 100 times greater than that of whole blood (Fig. 2).
The B. canis strains were isolated from 2 aborted foetuses and 13 whole blood samples. These isolated strains
have been characterized using the classical Brucella biotyping assay previously described, i.e., CO2 requirement, urea
hydrolysis, oxidase, catalase test, dye medium test (basic
fuchsin and thionin), and phage typing (Tbilisi [Tb], 104 Tb,
Weybridge [Wb], and R/C) [4].
Serologically, 11 out of 13 specimens were positive by
RSAT with 2-mercaptoethanol (2-ME RSAT). However, 3
specimens from the 2-ME RSAT positive dogs were negative
by immuno-chromatographic assay (ICA). In contrast, two
2-ME RSAT negative dogs were positive by ICA (Table 2).
The BcSS-PCR assay was also applied to these specimens to
test its clinical utility. Abortion materials gave strong positive band of 300-bp for B. canis (data not shown). Compared
with the isolation, 8 of 13 blood samples tested to be positive by the BcSS-PCR assay. Abortion materials were also
positive for B. canis (data not shown).
4. Discussion
Canine brucellosis is a zoonotic disease that poses a risk
for public health, and it is also an important disease in pet
dogs [6]. Canine brucellosis causes reproductive problems
in dogs and economic losses to dog breeders. As the number
of pet dogs increases, the risk of disease also increases due
to direct or indirect transfer from dogs to people. Quick
and accurate diagnostic tools are required to prevent the
transfer of this disease between pets and humans and to
minimize public health hazards and economic loss. A PCR
assay is known to be the most effective diagnostic methods
for the detection of Brucella strains. However, a one-step
PCR assay for the detection of the B. canis strain has not
been developed so far.
Our BcSS-PCR assay provided specific amplification for
all B. canis isolates. This result was compared to the performance of a previously reported genus specific Brucella
PCR assay based on the 16S rRNA gene. The field strains
of Ochrobactrum anthropi and Staphylococcus aureus were
falsely positive using PCR assay with the 16S rRNA gene
(F4/R2 primer sets) [20,21].
The sensitivity of the PCR assay using buffy coat was
approximately 100 times higher than using DNA extracted
from whole blood. This finding can be attributed to a concentration of peripheral blood mononuclear cells and the
absence of the inhibitory materials found in whole blood
[22,23]. The inhibitory materials of DNA amplification may
include anticoagulants and haemoglobin resulting from
the concentration of peripheral blood mononuclear cells in
whole blood [13]. According to previous reports, the detection limit of the BcSS-PCR described in the current study is
equal to the sensitivity of the BCSP31 PCR with primer pairs
of B4/B5, and less than that of the 16S rRNA PCR. The greater
sensitivity of this PCR could be because it amplifies a region
of the 16S rRNA gene, present in several copies in the bacterial genome [19]. In addition, the BcSS-PCR showed higher
sensitivity than the PCR assay using JPF/JPR primer pairs of
a gene encoding an outer membrane protein (omp-2) that,
is used to detect Brucella species [19].
Of the 13 dogs studied, 2 were found negative for B. canis
infection by results by 2-ME RSAT and 3 by ICA. In a serodiagnosis report by Keid et al. [24], among the blood-culture
positive dogs, only 82.81% by RBT (rapid slide agglutination) and 39.06% by 2-ME RSAT had positive reaction. In
addition, as reported by Abernethy et al. [25], the sensitivity of the RSAT was 76.6%. Kim et al. [18] reported that
the kappa value between 2-ME RSAT and ICA was 0.89.
Any single serological method cannot definitively diagnose
canine brucellosis owing to low specificity and sensitivity,
so substitutive or complementary diagnostic methods are
required.
The BcSS-PCR assay was evaluated with specimens
including a buffy coat from whole blood and aborted foetal
S.-I. Kang et al. / Comparative Immunology, Microbiology and Infectious Diseases 37 (2014) 237–241
materials. Our PCR assay was not able to detect B. canis
in some samples (number 6–10) irrespective of serological
results. This may have been due to the low number of B.
canis bacteria in blood or due to a loss of buffy coat during separation, which is dependent on the condition of the
whole blood.
In conclusion, the BcSS-PCR assay can specifically detect
B. canis strains by a simple and easy method that could
be available in a diagnostic centre, inspection agency or
animal hospital. This BcSS-PCR assay has a comparatively
high sensitivity when using a buffy coat sample from live
animals. Therefore, our PCR assay could be used as an
alternative diagnostic method to culture and serology. This
BcSS-PCR assay could also be used as a routine screening
tool for the diagnosis of B. canis in infected animals or
humans, particularly in underdeveloped and developing
countries.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
This study was supported by a grant (Project No: CAD13-2010-12-01) from the Animal and Plant Quarantine
Agency (QIA) of the Ministry of Agriculture, Food and Rural
Affairs (MAFRA) of the South Korea.
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