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First Identification of Taylorella equigenitalis From Genital Tracts of Thoroughbred Horses From the Inland Area of South Korea by Multilocus Sequence Typing
Accepted Manuscript First identification of Taylorella equigenitalis from genital tracts of Thoroughbred horses from the inland area of South Korea by multi-locus sequence typing (MLST) Ji Yong Hwang, Gil Jae Cho PII:
S0737-0806(17)30035-7
DOI:
10.1016/j.jevs.2017.09.011
Reference:
YJEVS 2390
To appear in:
Journal of Equine Veterinary Science
Received Date: 8 February 2017 Revised Date:
29 August 2017
Accepted Date: 22 September 2017
Please cite this article as: Hwang JY, Cho GJ, First identification of Taylorella equigenitalis from genital tracts of Thoroughbred horses from the inland area of South Korea by multi-locus sequence typing (MLST), Journal of Equine Veterinary Science (2017), doi: 10.1016/j.jevs.2017.09.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title page
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Manuscript title
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First identification of Taylorella equigenitalis from genital tracts of Thoroughbred horses
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from the inland area of South Korea by multi-locus sequence typing (MLST)
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Author’s names and affiliations
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First author: Ji Yong Hwang1,
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Corresponding author : Gil Jae Cho*1
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Author’s affilations
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1
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University, 1370, Sangyeok-dong, Buk-gu, Daegu, 702-701, South Korea.
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PHONE: +82-53-950-5978
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FAX: +82-53-950-5955
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Laboratory of Equine Science, College of Veterinary Medicine, Kyungpook National
Corresponding author’s e-mail address: [email protected]
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Author’s declaration of interests
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There are no conflicts of interests to declare.
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Sources of funding
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This research did not receive any specific grant from funding agencies in the public,
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commercial, or not-for-profit sectors.
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Author contributions and authorship 1
ACCEPTED MANUSCRIPT Ji Yong Hwang designed the study, conducted experiments, contributed to the data
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acquisition and interpretation and prepare the manuscript. Gil Jae Cho executed the study. All
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authors approved the final manuscript is true.
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Abstract
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The bacterium, Taylorella equigenitalis (T. equigenitalis) is reponsible for the disease
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Contagious Equine Metritis (CEM), a highly contagious venereal disease of horses. There
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have been substantial economic losses reported in various equine industries across the world
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as a result of CEM. So far, there had been no reported cases of T. equigenitalis in the inland
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area of South Korea.
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This study was performed to determine the prevalence and the genotype of T. equigenitalis in
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the inland area of South Korea. In this study, one of 38 Thoroughbred horses were found
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positive for T. equigenitalis using bacterial culture. Multilocus sequence typing (MLST) and
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construction of a neighbor-joining tree based on the T. equigenitalis MLST database
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(http://pubmlst.org/taylorella) indicated that the inland South Korean T. equigenitalis strain in
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this study showed a distinct genotype and no epidemiological relationship with other regional
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strains suggesting that the inland South Korean T. equigenitalis strain is a unique strain. In
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order to prevent serious repercussions to the South Korean equine industry, a full
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epidemiological investigation and comprehensive treatment regimen are needed.
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Contagious equine metritis; Taylorella equigenitalis; South Korea; MLST; Phylogenetic tree
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Keywords
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1. Introduction
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Contagious Equine Metritis (CEM) is a highly contagious venereal disease of horses caused
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by the bacterium, Taylorella (T.) equigenitalis. T. equigenitalis is a gram-negative, non-
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motile, facultative anaerobic coccobacillus. The first outbreak of CEM was reported in 1977
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ACCEPTED MANUSCRIPT from the United Kingdom (UK) and Ireland [7,39]. The causal agent was initially proposed to
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be Haemophillus equigenitalis in 1978 [35], and was finally transferred to the Genus
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Taylorella gen. nov. as Taylorella equigenitalis comb. nov. [33]. Since this first reported
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outbreak, CEM has been found worldwide, including in a number of European countries,
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South and North America, Australia, South Africa and Japan [2,3,23,24]. CEM causes
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substantial economic losses due to temporary mare infertility, international movement
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restrictions and the cost of epidemiological investigations, quarantine and treatment regimens
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in affected countries [12,31].
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The OIE "gold standard" for diagnosis of T. equigenitalis is bacterial culture of samples taken
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from the external genitalia of affected horses [17], however, due to its fastidious nature, very
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low growth rate and suppression of growth by other commensals in genital tract, isolation of
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T. equigenitalis by bacterial culture is difficult and time-consuming [26]. In addition,
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Taylorella asinigenitalis (T. asinigenitalis), the other Taylorella spp. isolated from donkeys
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representing very similar cultural and biochemical natures, may be misidentified as T.
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equigenitalis [4,16,19,27,29,40]. In order to overcome such drawbacks, various polymerase
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chain reaction (PCR) assays, ranging from ordinary PCR [2,3,6,9,41] to real-time PCR,
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which have very high sensitivity, specificity and rapidity have been developed [27,29,30,40].
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Recently, there are various epidemiological studies such as pulsed-field gel electrophoresis
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(PFGE) which is currently the main tool used to genotype CEM isolates [1], and several other
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molecular typing tools including field inversion gel electrophoresis (FIGE) [5], crossed-field
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gel electrophoresis (CFGE) [25] and chromosomal DNA fingerprinting [36]. However, these
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molecular epidemiological tools are poorly portable because of their index of variation and it
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is difficult to compare results among laboratories [21]. Thus, these molecular tools are not
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well suited for the global epidemiological studies of CEM outbreaks due to the difficulty in
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investigating the genetic relationship between isolates from different outbreaks. In light of
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ACCEPTED MANUSCRIPT overcoming these difficulties, multilocus sequence typing (MLST) seems to be more
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appropriate for large-scale global epidemiological studies [8]. MLST is based on sequence
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comparison of internal fragments of housekeeping genes which play vital function and are
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present in all isolates of a given species. Because mutations of housekeeping genes are
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assumed to be neutral [21,32] and nucleotide changes of them are relatively slow, MLST is
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ideal molecular tool for global epidemiology [11].
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The aim of this study is to isolate T. equigenitalis in inland South Korea by traditional
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bacteriological method and to genotype inland South Korea T. equigenitalis strain through
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MLST with previously genotyped strains from worldwide CEM outbreaks for the
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epidemiological analysis of CEM outbreak in South Korea.
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2. Materials and methods
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2.1. Clinical samples
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A total of 38 genital swabs were obtained from 38 Thoroughbred horses (3 stallions and 35
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mares) during the 2016 breeding season from Korea racing authority (KRA) studfarm (Jangsu,
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Jeollabuk-do, South Korea) and nearby Thoroughbred raising farms at Jeollabuk-do, South
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Korea. The external genital swabs were obtained according to OIE recommendations [26]
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using CultureSwab Plus Amies Gel with Charcoal (Becton Dickinson, Franklin Lakes, New
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Jersey, USA). Genital swabs were transported to the laboratory on ice within 24 hours.
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2.2. Reference strain
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Japanese strain CEMO-37 was imported from the Microbiology division, Equine Research
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Institute, Japan Racing Association (JRA). CEMO-37 was isolated from the clitoris of
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Thoroughbred mare in 2000. CEMO-37 didn’t represent any clinical signs.
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2.3. Bacterial culture
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Genital swabs were inoculated onto chocolate (heated blood) agar and two selective agar
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plates produced using a blood agar base and 5% defibrinated sheep whole blood mixture.
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Using heating block, the mixture was heated at 80
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to make the two different selective agar plates, antibiotics were added at 45
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trimethoprim (1
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amphotericin B (5
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selective agar plates, the plates were incubated in 5% (v/v) CO2 in air at 37
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as follows: ( )
), clindamycin (5
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) [17,40]. After inoculation onto the chocolate agar and two for 7 days.
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for 15 min and cooled to 45
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2.4. Preparations of genomic DNAs
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2.4.1. Culture plates
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Genomic DNAs from culture plates were prepared by modifications of previous boiling
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method [2]. Briefly, a loopful of bacterial colony was suspended in 1
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buffered saline (PBS) (pH 7.4) in 1.5
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at 13,000 rpm for 1.5 min. After centrifugation, the supernatant was discarded and the pellet
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was resuspended in 50
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block. After heating, the tubes were centrifuged at 13,000 rpm for 4 min. 50
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supernatant was harvested and used as the template for PCR amplification.
of 0.1 M Phosphate tube was centrifuged
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Eppendorf tube. The 1.5
of nuclease free water and lysed for 15 min at 97
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with heating of
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2.4.2. Genital swabs
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Genomic DNAs from genital swabs were prepared by modifications of previous boiling
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methods [27, 40]. Briefly, genital swabs were agitated for 5 s in 200 4
of 0.1 M Phosphate
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buffered saline (PBS) (pH 7.4) in 1.5
Eppendorf tube. The 1.5
tube was centrifuged
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at 13,000 rpm for 30 s. After centrifugation, the supernatant was discarded and the pellet was
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resuspended in 100
of nuclease free water and lysed for 15 min at 97
with heating
block. After heating, the tubes were centrifuged at 13,000 rpm for 1 min in order to eliminate
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insoluble pellet materials. 100
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PCR amplification.
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of supernatant was harvested and used as the template for
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2.5. Multilocus sequence typing (MLST)
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A total of 7 MLST loci (gltA, gyrB, fh, shmt, tyrB, adk and txn) and all primer pairs were
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chosen based on the previous study [8]. Direct sequencing of 7 MLST loci was performed by
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a commercial service (Bioneer, Daejeon metropolitan city, South Korea) according to the
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previous study's protocol [8]. Allelic profiles and Sequence types (STs) were assigned using
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the T. equigenitalis MLST database (http://pubmlst.org/taylorella) [18]. The MLST profiles
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were analyzed by eBURST v3 program at the default setting [13] and a population snapshot
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was depicted to show the relationship between the STs in this study and other T. equigenitalis
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STs in the MLST database samples. In order to extrapolate our results globally, we compared
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South Korea strain with other strains from different countries (USA, UK, UAE, Australia,
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Japan, France, Germany, Belgium, Switzerland, Poland and South Africa). Full details of all
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of the strains in this study are available on the T. equigenitalis MLST database
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(http://pubmlst.org/taylorella) [18]. STs were classified as single-locus variants (SLVs),
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double-locus variants (DLVs) or individual unlinked STs.
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2.6. Statistical analysis
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The phylogenetic tree was constructed based on concatenated nucleotide sequences from the
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7 MLST loci in order to show genetic relationship between all of the T. equigenitalis STs
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using neighbor-joining method with the bootstrap values at 1,000 replicates by MEGA 6 [34].
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3. Results
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3.1. Bacterial culture and isolation
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Of the 38 genital swabs, T. equigenitalis colonies were confirmed on chocolate and two
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selective agar plates from 1 stallion taken in 2016 breeding season. The appearance of T.
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equigenitalis colony was small in diameter (1.0-1.5 mm), convex, greyish-white, opaque,
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shiny and smooth. Field isolate from 1 stallion was named KITE-1.
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3.2. Multilocus sequence typing (MLST)
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A total of 36 STs were assigned to the 243 T. equigenitalis strains. When only SLVs were
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considered, six clonal complexes (CCs) were identified (CC1, CC2, CC3, CC4, CC8 and
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CC9) and 10 STs (ST3, ST4, ST5, ST9, ST30, ST31, ST42, ST44, ST50 and ST55) were
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assigned to the singletons which were not included in any clonal complexes (CCs) (Table 1
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and Fig. 1). Among 10 STs included in singletons, one strain from South Korea inland
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(KITE-1) was ST55. Of the 243 strains, 72 strains (29.6%) were singletons and each clonal
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complex (CC) accounted for 94 strains (38.7%), 59 strains (24.3%), 2 strains (0.82%), 6
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strains (2.47%), 5 strains (2.06%) and 5 strains (2.06%) for CC1, CC2, CC3, CC4, CC8 and
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CC9, respectively. Among 6 clonal complexes (CCs), only CC1 represented the predicted
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founder (ST1) and two subgroup founders (ST2 and ST33) (Fig. 1).
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When DLVs were considered, an additional links were represented; 1) CC1 and CC3 are
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linked since ST10 has two DLVs with ST1 and ST13. 2) CC2 and CC4 are linked since ST15
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ST45 and ST46. In addition to prior DLVs, ST15 of CC2 has one DLV with unlinked ST5. 3)
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CC4 and unlinked ST42 are linked since ST42 has two DLVs with ST17 and ST18. 4) CC8
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and unlinked ST5 are linked since ST5 has two DLVs with ST45 and ST46. 5) Unlinked ST4
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and ST5 has one DLV, unlinked ST55 has two DLVs with unlinked ST5 and ST44, and
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unlinked ST42 has one DLV with inlinked ST30 and are linked with CC1 since ST42 has one
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DLV with ST43 (Fig. 2).
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The neighbor-joining tree based on the 7 concatenated housekeeping gene sequences
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(3,521bp) represented the phylogenetic relationship between 36 STs in this study (Fig. 3).
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4. Discussion
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Since the first CEM outbreak reported during the 1977 breeding season in the UK and Ireland
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[7,40], CEM has resulted in significant economic losses worldwide [3,7,26,28,33,35,37,38].
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Due to the highly contagious nature of T. equigenitalis and its capacity to spread rapidly
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within a natural breeding system, rapid diagnosis and immediate treatment of T.
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equigenitalis-positive horses are essential [26,28,35,37,38]. However, due to the insidious
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nature of T. equigenitalis and the fact that, particularly stallions, can become inapparent
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carrier animals, the disease can easily spread and cause serious repercussions in the equine
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breeding industry [26,28,35,37,38]. In particular, recent CEM outbreak reports reflect a novel
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epidemiologic manifestation with a markedly different risk association for transmission via
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artificial insemination (AI). Artificial breeding has a high association with horizontal or
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fomite-associated transmission which appears to be an underestimated feature of this
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infection [24,31]. These suspected fomites included a contaminated breeding phantom at the
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AI facility, equipment or personnel during semen collection and handling, tack and grooming
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equipment [23].
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Due to the shortcomings of bacterial culture, various rapid, sensitive and specific PCR assays
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have
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[2,3,6,9,27,29,30,40,41]. PCR also has the added benefit of being able to distinguish T.
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equigenitalis from the closely related, donkey-associated T. asinigenitalis [40] which could
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be naturally infected to mares and stallions without any clinical signs and recovered from
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infected horses, causing misidentification as T. equigenitalis [4,16,19].
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MLST is a well-established molecular typing method ideal for the determination of the
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population structure of many bacterial species [8,20,21]. Based on the previous MLST study
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of T. equigenitalis, strains that have different geographic origin were assigned to STs and
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CCs [8]. A total of 36 STs and 6 CCs were identified in this study including one strain from
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South Korea belonging to ST55. Of the 6 CCs, the CC1, still in circulation in France,
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contains isolates from the first CEM outbreaks that simultaneously emerged in several
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countries in the late 1970s [8]. The CC1 had one founder (ST1) and two subgroup founder
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(ST2 and ST33) and a number of DLVs. Aside from the CC1, there have been STs without
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any genetic relationship to CC1 in different countries (e.g. France, Japan and United Arab
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Emirates), suggesting the existence of a natural worldwide reservoir unidentified so far [8].
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South Korea inland T. equigenitalis isolate (KITE-1) was genotyped by MLST for the first
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time in this study. When considered only SLVs, KITE-1 showed the distinct genetic
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relationship with the other previous strains, representing no links with any other STs (Fig. 1)
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in the default group definition (6/7) [13] However, when the group definition became relaxed
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(5/7) and considered DLVs additionally, KITE-1 showed two links with two STs (ST5 and
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ST44) (Fig. 2). Although KITE-1 showed very little genetic relationship with French strains
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(ST5 and ST44) according to the relaxed definition (5/7) (Fig. 2) [13] and the neighbor-
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relationship with other regional strains.
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MLST, based on the nucleotide sequence, is highly discriminatory and provides unambiguous
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results that can be directly analyzed among laboratories worldwide via the Internet [10].
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Furthermore, the recent characterization of whole genome sequences (WGS) of Taylorella
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spp. [14,15] allows the application of MLST, a new method to study the population biology
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and epidemiology of these bacteria. Therefore, MLST is a powerful tool for molecular
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epidemiological studies and population biology studies of the T. equigenitalis.
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5. Conclusions
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In conclusion, bacterial culture, MLST and phylogenetic analysis of T. equgenitalis isolate
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taken from South Korea inland Thoroughbred resulted in the identification of a unique
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epidemiological genotype of South Korea strain.
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Due to the small size of South Korea T. equigenitalis isolate, future genotyping of the
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additional South Korea T. equigenitalis field isolate using molecular epidemiological
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methods such as PFGE or MLST will further our understanding of T. equigenitalis
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epidemiology and will assist in the control of CEM in the South Korea equine industry.
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6. Acknowledgements We are grateful to Dr. Yuta Kinoshita from Microbiology division, Equine Research Institute, Japan Racing Association (JRA) who supplied us the reference strain CEMO-37.
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[30] Pusterla N, Madigan JE, Leutenegger CM. Real-time polymerase chain reaction : A
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novel molecular diagnostic tool for equine infectious diseases. Journal of Veterinary Internal
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Medicine 2006;20:3-12.
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[31] Schulman ML, May CE, Keys B, Guthrie AJ. Contagious equine metiritis: Artificial
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reproduction changes the epidemiologic paradigm. Veterinary Microbiology 2013;167: 2-8.
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[32] Selander RK, Caugant DA, Ochman H, Musser JM, Gilmour MN, Whittam TS. Methods
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of multilocus enzyme electrophoresis for bacterial population genetics and systemetics.
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Applied and Environmental Microbiology 1986;51: 873-84.
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[33] Sugimoto C, Isayama Y, Sakazaki R, Kuramochi S. (1983) Transfer of Haemophilus
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equigenitalis Taylor et al. 1978 to the Genus Taylorella gen. nov. as Taylorella equigenitalis
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comb. nov. Current microbiology 1983;9: 155-62.
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[34] Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular
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Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 2013;30:
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2725-9.
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organism of contagious equine metritis 1977: Proposal for a new species to be known as
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Haemophilus equigenitalis. Equine veterinary Journal 1978;10:136-44.
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[36] Thoresen SI, Jenkins A, Ask E. Genetic homogeneity of Taylorella equigenitalis from
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Norwegian horses revealed by chromosomal DNA fingerprinting. Journal of Clinical
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Microbiology. 1995;33: 233-4.
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[37] Timoney PJ. Contagious equine metritis. Comparative Immunology, Microbiology and
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Infectious Disease 1996;19:199-204.
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[38] Timoney PJ, Powell DG. Contagious equine metritis-epidemiology and control. Equine
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veterinary science 1988;8:42-6.
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[39] Timoney PJ, Ward J, Kelly P. A contagious genital infection of mares. The Veterinary
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Record 1977;101:103.
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[40] Wakeley PR, Errington J, Hannon S, Roest HIJ, Carson T. Hunt B, et al. Development
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of a real time PCR for the detection of Taylorella equigenitalis directly from genital swabs
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and discrimination from Taylorella asinigenitalis. Veterinary Microbiology 2006;118:247-54.
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[41] Zdovc I, Ocepek M, Gruntar I, Pate M, Klobucar I, Krt B Prevalance of Taylorella
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equigenitalis infection in stallions in Slovenia : Bacteriology compared with PCR
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examination. Equine Veterianry Journal 2005;37:217-21.
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1 (38)a
1-1-1-1-1-1-1
1
2 (7) 3 (6)
1-1-1-1-2-1-1 1-1-3-2-2-2-3
1 -c
4 (41)
1-2-5-3-2-2-3
-
5 (2) 6 (1) 7 (1) 8 (1) 9 (1) 10 (1) 11 (1) 12 (1) 13 (1) 14 (1) 15 (4)
1-2-1-1-2-2-3 1-1-1-1-2-4-1 1-1-1-1-3-1-4 1-1-1-1-4-1-1 1-1-2-1-1-2-2 1-1-5-3-1-1-1 1-1-5-3-5-1-1 1-1-6-1-2-1-1 1-1-7-1-1-1-1 1-1-8-1-1-1-4 1-2-4-1-2-2-4
1 1 1 3 3 1 1 1 2
16 (55)
1-2-4-1-2-3-4
17 (5)
1-3-6-1-2-2-4
18 (1) 19 (1) 20 (1) 30 (3) 31 (1) 33 (6) 34 (31) 35 (1) 39 (1) 42 (1) 43 (1) 44 (4) 45 (1)
1-3-9-1-2-2-4 1-4-1-1-1-1-1 2-1-1-1-1-1-1 1-3-1-1-2-3-22 1-11-2-1-14-3-3 1-1-1-1-1-1-4 1-1-1-1-1-1-5 1-1-1-1-1-1-6 11-1-1-1-1-1-5 1-3-1-1-2-16-4 1-1-1-1-2-1-4 1-2-1-2-16-2-4 1-2-20-1-2-3-3
4 1 1 1 1 1 1 1 8
46 (4)
1-2-3-1-2-3-3
8
47 (1)
1-2-4-1-17-1-1
9
48 (4)
1-2-21-1-17-1-1
9
50 (12)
1-14-23-1-2-3-1
-
339 340 341 342
Country UK (1)d USA (1) Australia (5) France (30) Unknown (1) France (7) Japan (6) France (7) South Africa (34) France (2) France (1) France (1) France (1) Japan (1) France (1) France (1) France (1) France (1) France (1) France (4) Belgium (10) France (27) Poland (4) Switzerland (14) Belgium (1) France (1) UAE (3) France (1) USA (1) Australia (1) Poland (3) UAE (1) France (6) France (31) France (1) France (1) UAE (1) France (1) France (4) Switzerland (1) Belgium (1) Germany (2) Switzerland (1) Switzerland (1) Belgium (2) Switzerland (2) Belgium (2) Poland (10) France (1) South Korea (1)
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Allelic profileb
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52 (1) 1-16-1-1-1-1-1 1 55 (1) 1-2-1-2-2-2-1 a The numbers in parentheses are the number of strains belonging to each ST. b Allelic profile of gltA-gyrB-fh-shmt-tyrB-adk-txn loci, respectively. c ‘-‘ means the singletons not assigned to any clonal complexes (CCs) d The numbers in parentheses are the number of respective national strains belonging to each ST.
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Figure 1. Population snapshot of the Taylorella equigenitalis. Clonal complexes (CCs) and
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individual unlinked STs are displayed as a single eBURST diagram by setting the group
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definition to zero of seven shared alleles. Circles indicate STs of T. equigenitalis strains; the
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size of a circle is proportional to the number of strains within each ST. Primary founders and
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subgroup founders are represented as blue and yellow colors, respectively. Black lines
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indicate single-locus variant (SLV) links and the length of lines are arbitrary to the distances
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between STs. Note the South Korea inland T. equigenitalis strain is represented as ST55.
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Figure 2. Population snapshot of the Taylorella equigenitalis. Single locus variants (SLVs)
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are represented as black and purple lines and double locus variants (DLVs) are represented as
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blue lines. DLVs were obtained with the relaxed definition (5/7) of eBURST program. This
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option helps to explore alternative equally plausible patterns of descent.
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Figure 3. Neighbor-joining tree of concatenated nucleotide sequences (3,521bp) of 7
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housekeeping gene loci. This tree shows genetic relationship between the 36 STs of T.
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equigenitalis. The number at each branch show bootstrap value (1,000 replications). The
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number of strains with a given ST is shown in parentheses. Bar, nucleotide substitution. 18
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epidemiological investigation is essential.
Therefore, we conducted multilocus sequence typing (MLST) for the inland strain of T. equigenitalis with T. equigenitalis MLST database.
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(T. equigenitalis) was isolated from Thoroughbred stallion in the inland area of South Korea.
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South Korea inland area has been free of CEM so far, but recently Taylorella equigenitalis
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Highlights
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S0737-0806(17)30035-7
DOI:
10.1016/j.jevs.2017.09.011
Reference:
YJEVS 2390
To appear in:
Journal of Equine Veterinary Science
Received Date: 8 February 2017 Revised Date:
29 August 2017
Accepted Date: 22 September 2017
Please cite this article as: Hwang JY, Cho GJ, First identification of Taylorella equigenitalis from genital tracts of Thoroughbred horses from the inland area of South Korea by multi-locus sequence typing (MLST), Journal of Equine Veterinary Science (2017), doi: 10.1016/j.jevs.2017.09.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title page
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Manuscript title
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First identification of Taylorella equigenitalis from genital tracts of Thoroughbred horses
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from the inland area of South Korea by multi-locus sequence typing (MLST)
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Author’s names and affiliations
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First author: Ji Yong Hwang1,
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Corresponding author : Gil Jae Cho*1
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Author’s affilations
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1
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University, 1370, Sangyeok-dong, Buk-gu, Daegu, 702-701, South Korea.
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PHONE: +82-53-950-5978
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FAX: +82-53-950-5955
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*
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Laboratory of Equine Science, College of Veterinary Medicine, Kyungpook National
Corresponding author’s e-mail address: [email protected]
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Author’s declaration of interests
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There are no conflicts of interests to declare.
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Sources of funding
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This research did not receive any specific grant from funding agencies in the public,
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commercial, or not-for-profit sectors.
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Author contributions and authorship 1
ACCEPTED MANUSCRIPT Ji Yong Hwang designed the study, conducted experiments, contributed to the data
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acquisition and interpretation and prepare the manuscript. Gil Jae Cho executed the study. All
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authors approved the final manuscript is true.
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Abstract
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The bacterium, Taylorella equigenitalis (T. equigenitalis) is reponsible for the disease
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Contagious Equine Metritis (CEM), a highly contagious venereal disease of horses. There
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have been substantial economic losses reported in various equine industries across the world
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as a result of CEM. So far, there had been no reported cases of T. equigenitalis in the inland
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area of South Korea.
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This study was performed to determine the prevalence and the genotype of T. equigenitalis in
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the inland area of South Korea. In this study, one of 38 Thoroughbred horses were found
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positive for T. equigenitalis using bacterial culture. Multilocus sequence typing (MLST) and
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construction of a neighbor-joining tree based on the T. equigenitalis MLST database
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(http://pubmlst.org/taylorella) indicated that the inland South Korean T. equigenitalis strain in
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this study showed a distinct genotype and no epidemiological relationship with other regional
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strains suggesting that the inland South Korean T. equigenitalis strain is a unique strain. In
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order to prevent serious repercussions to the South Korean equine industry, a full
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epidemiological investigation and comprehensive treatment regimen are needed.
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Contagious equine metritis; Taylorella equigenitalis; South Korea; MLST; Phylogenetic tree
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Keywords
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1. Introduction
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Contagious Equine Metritis (CEM) is a highly contagious venereal disease of horses caused
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by the bacterium, Taylorella (T.) equigenitalis. T. equigenitalis is a gram-negative, non-
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motile, facultative anaerobic coccobacillus. The first outbreak of CEM was reported in 1977
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ACCEPTED MANUSCRIPT from the United Kingdom (UK) and Ireland [7,39]. The causal agent was initially proposed to
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be Haemophillus equigenitalis in 1978 [35], and was finally transferred to the Genus
27
Taylorella gen. nov. as Taylorella equigenitalis comb. nov. [33]. Since this first reported
28
outbreak, CEM has been found worldwide, including in a number of European countries,
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South and North America, Australia, South Africa and Japan [2,3,23,24]. CEM causes
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substantial economic losses due to temporary mare infertility, international movement
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restrictions and the cost of epidemiological investigations, quarantine and treatment regimens
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in affected countries [12,31].
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The OIE "gold standard" for diagnosis of T. equigenitalis is bacterial culture of samples taken
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from the external genitalia of affected horses [17], however, due to its fastidious nature, very
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low growth rate and suppression of growth by other commensals in genital tract, isolation of
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T. equigenitalis by bacterial culture is difficult and time-consuming [26]. In addition,
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Taylorella asinigenitalis (T. asinigenitalis), the other Taylorella spp. isolated from donkeys
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representing very similar cultural and biochemical natures, may be misidentified as T.
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equigenitalis [4,16,19,27,29,40]. In order to overcome such drawbacks, various polymerase
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chain reaction (PCR) assays, ranging from ordinary PCR [2,3,6,9,41] to real-time PCR,
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which have very high sensitivity, specificity and rapidity have been developed [27,29,30,40].
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Recently, there are various epidemiological studies such as pulsed-field gel electrophoresis
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(PFGE) which is currently the main tool used to genotype CEM isolates [1], and several other
44
molecular typing tools including field inversion gel electrophoresis (FIGE) [5], crossed-field
45
gel electrophoresis (CFGE) [25] and chromosomal DNA fingerprinting [36]. However, these
46
molecular epidemiological tools are poorly portable because of their index of variation and it
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is difficult to compare results among laboratories [21]. Thus, these molecular tools are not
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well suited for the global epidemiological studies of CEM outbreaks due to the difficulty in
49
investigating the genetic relationship between isolates from different outbreaks. In light of
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appropriate for large-scale global epidemiological studies [8]. MLST is based on sequence
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comparison of internal fragments of housekeeping genes which play vital function and are
53
present in all isolates of a given species. Because mutations of housekeeping genes are
54
assumed to be neutral [21,32] and nucleotide changes of them are relatively slow, MLST is
55
ideal molecular tool for global epidemiology [11].
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The aim of this study is to isolate T. equigenitalis in inland South Korea by traditional
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bacteriological method and to genotype inland South Korea T. equigenitalis strain through
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MLST with previously genotyped strains from worldwide CEM outbreaks for the
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epidemiological analysis of CEM outbreak in South Korea.
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2. Materials and methods
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2.1. Clinical samples
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A total of 38 genital swabs were obtained from 38 Thoroughbred horses (3 stallions and 35
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mares) during the 2016 breeding season from Korea racing authority (KRA) studfarm (Jangsu,
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Jeollabuk-do, South Korea) and nearby Thoroughbred raising farms at Jeollabuk-do, South
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Korea. The external genital swabs were obtained according to OIE recommendations [26]
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using CultureSwab Plus Amies Gel with Charcoal (Becton Dickinson, Franklin Lakes, New
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Jersey, USA). Genital swabs were transported to the laboratory on ice within 24 hours.
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2.2. Reference strain
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Japanese strain CEMO-37 was imported from the Microbiology division, Equine Research
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Institute, Japan Racing Association (JRA). CEMO-37 was isolated from the clitoris of
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Thoroughbred mare in 2000. CEMO-37 didn’t represent any clinical signs.
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2.3. Bacterial culture
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Genital swabs were inoculated onto chocolate (heated blood) agar and two selective agar
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plates produced using a blood agar base and 5% defibrinated sheep whole blood mixture.
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Using heating block, the mixture was heated at 80
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to make the two different selective agar plates, antibiotics were added at 45
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trimethoprim (1
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amphotericin B (5
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selective agar plates, the plates were incubated in 5% (v/v) CO2 in air at 37
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) [17,40]. After inoculation onto the chocolate agar and two for 7 days.
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2.4. Preparations of genomic DNAs
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2.4.1. Culture plates
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Genomic DNAs from culture plates were prepared by modifications of previous boiling
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method [2]. Briefly, a loopful of bacterial colony was suspended in 1
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buffered saline (PBS) (pH 7.4) in 1.5
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at 13,000 rpm for 1.5 min. After centrifugation, the supernatant was discarded and the pellet
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was resuspended in 50
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block. After heating, the tubes were centrifuged at 13,000 rpm for 4 min. 50
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supernatant was harvested and used as the template for PCR amplification.
of 0.1 M Phosphate tube was centrifuged
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of nuclease free water and lysed for 15 min at 97
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with heating of
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2.4.2. Genital swabs
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Genomic DNAs from genital swabs were prepared by modifications of previous boiling
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methods [27, 40]. Briefly, genital swabs were agitated for 5 s in 200 4
of 0.1 M Phosphate
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buffered saline (PBS) (pH 7.4) in 1.5
Eppendorf tube. The 1.5
tube was centrifuged
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at 13,000 rpm for 30 s. After centrifugation, the supernatant was discarded and the pellet was
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resuspended in 100
of nuclease free water and lysed for 15 min at 97
with heating
block. After heating, the tubes were centrifuged at 13,000 rpm for 1 min in order to eliminate
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insoluble pellet materials. 100
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PCR amplification.
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of supernatant was harvested and used as the template for
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2.5. Multilocus sequence typing (MLST)
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A total of 7 MLST loci (gltA, gyrB, fh, shmt, tyrB, adk and txn) and all primer pairs were
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chosen based on the previous study [8]. Direct sequencing of 7 MLST loci was performed by
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a commercial service (Bioneer, Daejeon metropolitan city, South Korea) according to the
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previous study's protocol [8]. Allelic profiles and Sequence types (STs) were assigned using
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the T. equigenitalis MLST database (http://pubmlst.org/taylorella) [18]. The MLST profiles
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were analyzed by eBURST v3 program at the default setting [13] and a population snapshot
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was depicted to show the relationship between the STs in this study and other T. equigenitalis
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STs in the MLST database samples. In order to extrapolate our results globally, we compared
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South Korea strain with other strains from different countries (USA, UK, UAE, Australia,
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Japan, France, Germany, Belgium, Switzerland, Poland and South Africa). Full details of all
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of the strains in this study are available on the T. equigenitalis MLST database
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(http://pubmlst.org/taylorella) [18]. STs were classified as single-locus variants (SLVs),
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double-locus variants (DLVs) or individual unlinked STs.
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2.6. Statistical analysis
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The phylogenetic tree was constructed based on concatenated nucleotide sequences from the
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7 MLST loci in order to show genetic relationship between all of the T. equigenitalis STs
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using neighbor-joining method with the bootstrap values at 1,000 replicates by MEGA 6 [34].
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3. Results
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3.1. Bacterial culture and isolation
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Of the 38 genital swabs, T. equigenitalis colonies were confirmed on chocolate and two
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selective agar plates from 1 stallion taken in 2016 breeding season. The appearance of T.
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equigenitalis colony was small in diameter (1.0-1.5 mm), convex, greyish-white, opaque,
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shiny and smooth. Field isolate from 1 stallion was named KITE-1.
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3.2. Multilocus sequence typing (MLST)
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A total of 36 STs were assigned to the 243 T. equigenitalis strains. When only SLVs were
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considered, six clonal complexes (CCs) were identified (CC1, CC2, CC3, CC4, CC8 and
134
CC9) and 10 STs (ST3, ST4, ST5, ST9, ST30, ST31, ST42, ST44, ST50 and ST55) were
135
assigned to the singletons which were not included in any clonal complexes (CCs) (Table 1
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and Fig. 1). Among 10 STs included in singletons, one strain from South Korea inland
137
(KITE-1) was ST55. Of the 243 strains, 72 strains (29.6%) were singletons and each clonal
138
complex (CC) accounted for 94 strains (38.7%), 59 strains (24.3%), 2 strains (0.82%), 6
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strains (2.47%), 5 strains (2.06%) and 5 strains (2.06%) for CC1, CC2, CC3, CC4, CC8 and
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CC9, respectively. Among 6 clonal complexes (CCs), only CC1 represented the predicted
141
founder (ST1) and two subgroup founders (ST2 and ST33) (Fig. 1).
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When DLVs were considered, an additional links were represented; 1) CC1 and CC3 are
143
linked since ST10 has two DLVs with ST1 and ST13. 2) CC2 and CC4 are linked since ST15
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145
ST45 and ST46. In addition to prior DLVs, ST15 of CC2 has one DLV with unlinked ST5. 3)
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CC4 and unlinked ST42 are linked since ST42 has two DLVs with ST17 and ST18. 4) CC8
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and unlinked ST5 are linked since ST5 has two DLVs with ST45 and ST46. 5) Unlinked ST4
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and ST5 has one DLV, unlinked ST55 has two DLVs with unlinked ST5 and ST44, and
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unlinked ST42 has one DLV with inlinked ST30 and are linked with CC1 since ST42 has one
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DLV with ST43 (Fig. 2).
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The neighbor-joining tree based on the 7 concatenated housekeeping gene sequences
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(3,521bp) represented the phylogenetic relationship between 36 STs in this study (Fig. 3).
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4. Discussion
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Since the first CEM outbreak reported during the 1977 breeding season in the UK and Ireland
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[7,40], CEM has resulted in significant economic losses worldwide [3,7,26,28,33,35,37,38].
157
Due to the highly contagious nature of T. equigenitalis and its capacity to spread rapidly
158
within a natural breeding system, rapid diagnosis and immediate treatment of T.
159
equigenitalis-positive horses are essential [26,28,35,37,38]. However, due to the insidious
160
nature of T. equigenitalis and the fact that, particularly stallions, can become inapparent
161
carrier animals, the disease can easily spread and cause serious repercussions in the equine
162
breeding industry [26,28,35,37,38]. In particular, recent CEM outbreak reports reflect a novel
163
epidemiologic manifestation with a markedly different risk association for transmission via
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artificial insemination (AI). Artificial breeding has a high association with horizontal or
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fomite-associated transmission which appears to be an underestimated feature of this
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infection [24,31]. These suspected fomites included a contaminated breeding phantom at the
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AI facility, equipment or personnel during semen collection and handling, tack and grooming
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equipment [23].
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Due to the shortcomings of bacterial culture, various rapid, sensitive and specific PCR assays
170
have
171
[2,3,6,9,27,29,30,40,41]. PCR also has the added benefit of being able to distinguish T.
172
equigenitalis from the closely related, donkey-associated T. asinigenitalis [40] which could
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be naturally infected to mares and stallions without any clinical signs and recovered from
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infected horses, causing misidentification as T. equigenitalis [4,16,19].
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MLST is a well-established molecular typing method ideal for the determination of the
176
population structure of many bacterial species [8,20,21]. Based on the previous MLST study
177
of T. equigenitalis, strains that have different geographic origin were assigned to STs and
178
CCs [8]. A total of 36 STs and 6 CCs were identified in this study including one strain from
179
South Korea belonging to ST55. Of the 6 CCs, the CC1, still in circulation in France,
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contains isolates from the first CEM outbreaks that simultaneously emerged in several
181
countries in the late 1970s [8]. The CC1 had one founder (ST1) and two subgroup founder
182
(ST2 and ST33) and a number of DLVs. Aside from the CC1, there have been STs without
183
any genetic relationship to CC1 in different countries (e.g. France, Japan and United Arab
184
Emirates), suggesting the existence of a natural worldwide reservoir unidentified so far [8].
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South Korea inland T. equigenitalis isolate (KITE-1) was genotyped by MLST for the first
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time in this study. When considered only SLVs, KITE-1 showed the distinct genetic
187
relationship with the other previous strains, representing no links with any other STs (Fig. 1)
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in the default group definition (6/7) [13] However, when the group definition became relaxed
189
(5/7) and considered DLVs additionally, KITE-1 showed two links with two STs (ST5 and
190
ST44) (Fig. 2). Although KITE-1 showed very little genetic relationship with French strains
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(ST5 and ST44) according to the relaxed definition (5/7) (Fig. 2) [13] and the neighbor-
developed
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relationship with other regional strains.
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MLST, based on the nucleotide sequence, is highly discriminatory and provides unambiguous
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results that can be directly analyzed among laboratories worldwide via the Internet [10].
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Furthermore, the recent characterization of whole genome sequences (WGS) of Taylorella
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spp. [14,15] allows the application of MLST, a new method to study the population biology
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and epidemiology of these bacteria. Therefore, MLST is a powerful tool for molecular
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epidemiological studies and population biology studies of the T. equigenitalis.
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5. Conclusions
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In conclusion, bacterial culture, MLST and phylogenetic analysis of T. equgenitalis isolate
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taken from South Korea inland Thoroughbred resulted in the identification of a unique
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epidemiological genotype of South Korea strain.
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Due to the small size of South Korea T. equigenitalis isolate, future genotyping of the
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additional South Korea T. equigenitalis field isolate using molecular epidemiological
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methods such as PFGE or MLST will further our understanding of T. equigenitalis
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epidemiology and will assist in the control of CEM in the South Korea equine industry.
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6. Acknowledgements We are grateful to Dr. Yuta Kinoshita from Microbiology division, Equine Research Institute, Japan Racing Association (JRA) who supplied us the reference strain CEMO-37.
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7. References
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ACCEPTED MANUSCRIPT Table 1. Taylorella equigenitalis MLST data
1 (38)a
1-1-1-1-1-1-1
1
2 (7) 3 (6)
1-1-1-1-2-1-1 1-1-3-2-2-2-3
1 -c
4 (41)
1-2-5-3-2-2-3
-
5 (2) 6 (1) 7 (1) 8 (1) 9 (1) 10 (1) 11 (1) 12 (1) 13 (1) 14 (1) 15 (4)
1-2-1-1-2-2-3 1-1-1-1-2-4-1 1-1-1-1-3-1-4 1-1-1-1-4-1-1 1-1-2-1-1-2-2 1-1-5-3-1-1-1 1-1-5-3-5-1-1 1-1-6-1-2-1-1 1-1-7-1-1-1-1 1-1-8-1-1-1-4 1-2-4-1-2-2-4
1 1 1 3 3 1 1 1 2
16 (55)
1-2-4-1-2-3-4
17 (5)
1-3-6-1-2-2-4
18 (1) 19 (1) 20 (1) 30 (3) 31 (1) 33 (6) 34 (31) 35 (1) 39 (1) 42 (1) 43 (1) 44 (4) 45 (1)
1-3-9-1-2-2-4 1-4-1-1-1-1-1 2-1-1-1-1-1-1 1-3-1-1-2-3-22 1-11-2-1-14-3-3 1-1-1-1-1-1-4 1-1-1-1-1-1-5 1-1-1-1-1-1-6 11-1-1-1-1-1-5 1-3-1-1-2-16-4 1-1-1-1-2-1-4 1-2-1-2-16-2-4 1-2-20-1-2-3-3
4 1 1 1 1 1 1 1 8
46 (4)
1-2-3-1-2-3-3
8
47 (1)
1-2-4-1-17-1-1
9
48 (4)
1-2-21-1-17-1-1
9
50 (12)
1-14-23-1-2-3-1
-
339 340 341 342
Country UK (1)d USA (1) Australia (5) France (30) Unknown (1) France (7) Japan (6) France (7) South Africa (34) France (2) France (1) France (1) France (1) Japan (1) France (1) France (1) France (1) France (1) France (1) France (4) Belgium (10) France (27) Poland (4) Switzerland (14) Belgium (1) France (1) UAE (3) France (1) USA (1) Australia (1) Poland (3) UAE (1) France (6) France (31) France (1) France (1) UAE (1) France (1) France (4) Switzerland (1) Belgium (1) Germany (2) Switzerland (1) Switzerland (1) Belgium (2) Switzerland (2) Belgium (2) Poland (10) France (1) South Korea (1)
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52 (1) 1-16-1-1-1-1-1 1 55 (1) 1-2-1-2-2-2-1 a The numbers in parentheses are the number of strains belonging to each ST. b Allelic profile of gltA-gyrB-fh-shmt-tyrB-adk-txn loci, respectively. c ‘-‘ means the singletons not assigned to any clonal complexes (CCs) d The numbers in parentheses are the number of respective national strains belonging to each ST.
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Figure 1. Population snapshot of the Taylorella equigenitalis. Clonal complexes (CCs) and
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individual unlinked STs are displayed as a single eBURST diagram by setting the group
347
definition to zero of seven shared alleles. Circles indicate STs of T. equigenitalis strains; the
348
size of a circle is proportional to the number of strains within each ST. Primary founders and
349
subgroup founders are represented as blue and yellow colors, respectively. Black lines
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indicate single-locus variant (SLV) links and the length of lines are arbitrary to the distances
351
between STs. Note the South Korea inland T. equigenitalis strain is represented as ST55.
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Figure 2. Population snapshot of the Taylorella equigenitalis. Single locus variants (SLVs)
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are represented as black and purple lines and double locus variants (DLVs) are represented as
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blue lines. DLVs were obtained with the relaxed definition (5/7) of eBURST program. This
363
option helps to explore alternative equally plausible patterns of descent.
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Figure 3. Neighbor-joining tree of concatenated nucleotide sequences (3,521bp) of 7
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housekeeping gene loci. This tree shows genetic relationship between the 36 STs of T.
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equigenitalis. The number at each branch show bootstrap value (1,000 replications). The
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number of strains with a given ST is shown in parentheses. Bar, nucleotide substitution. 18
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epidemiological investigation is essential.
Therefore, we conducted multilocus sequence typing (MLST) for the inland strain of T. equigenitalis with T. equigenitalis MLST database.
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(T. equigenitalis) was isolated from Thoroughbred stallion in the inland area of South Korea.
According to the MLST results, South Korea inland T. equigenitalis strain is a unique strain epidemiologically.
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South Korea inland area has been free of CEM so far, but recently Taylorella equigenitalis
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Highlights
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