Isolation, antibiotic resistance, virulence traits and phylogenetic analysis of Corynebacterium pseudotuberculosis from goats in southwestern China

Accepted Manuscript Title: Isolation, antibiotic resistance, virulence traits and phylogenetic analysis of Corynebacterium pseudotuberculosis from goats in southwestern China Authors: Hexian Li, Haoyue Yang, Zuoyong Zhou, Xiaoxia Li, Wenyi Yi, Yangyang Xu, Zhiying Wang, Shijun Hu PII: DOI: Reference:

S0921-4488(18)30432-2 https://doi.org/10.1016/j.smallrumres.2018.09.015 RUMIN 5761

To appear in:

Small Ruminant Research

Received date: Revised date: Accepted date:

23-5-2018 24-9-2018 25-9-2018

Please cite this article as: Li H, Yang H, Zhou Z, Li X, Yi W, Xu Y, Wang Z, Hu S, Isolation, antibiotic resistance, virulence traits and phylogenetic analysis of Corynebacterium pseudotuberculosis from goats in southwestern China, Small Ruminant Research (2018), https://doi.org/10.1016/j.smallrumres.2018.09.015 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.

Isolation, antibiotic resistance, virulence traits and phylogenetic analysis of Corynebacterium pseudotuberculosis from goats in

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southwestern China

Hexian Lia, Haoyue Yanga, Zuoyong Zhoua, b*, Xiaoxia Lia, Wenyi Yia, Yangyang Xua,

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Zhiying Wanga, b, Shijun Hua, b

College of Animal Science, Rongchang Campus of Southwest University, No. 160

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a

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Institutional addresses:

Veterinary Science Engineering Research Center of Chongqing, No. 160 Xueyuan

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b

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Xueyuan Road, Rongchang District, Chongqing, 402460, China

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Road, Rongchang District, Chongqing, 402460, China

*Corresponding

author.

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Zuoyong Zhou:

E-mail address: [email protected] ; Tel.: +86-23-46751588; Fax: +86-23-46751588

Highlights:

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The C. pseudotuberculosis isolation rate was 39.22% in abscess of goats in Southwestern China. The C. pseudotuberculosis isolates were sensitive to many kinds of antibiotics.

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All C. pseudotuberculosis strains were harbored multiple virulence genes.

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C. pseudotuberculosis biovar ovis and biovar equi can be differentiated by

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

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fusA.

Corynebacterium

pseudotuberculosis,

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gram-positive

facultative

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The

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Abstract:

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intracellular bacteria, usually resulted in covert but huge economic losses in goats and

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sheep industry. Limited information is available rewardingly in southwestern China,

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one of the most important regions for goats production. This study reported for the first time the isolation and molecular characteristics of C. pseudotuberculosis from

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external abscess of goats in southwestern China, and found that (1) the isolation rate

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of C. pseudotuberculosis in abscess of goats was 39.22% (40/102), (2) all of isolated strains were sensitive to vancomycin, norfloxacin, cefradine, clarithromycin and cefepime, but resistant to nitrofurantoin and furazolidone, (3) the virulence or putative

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virulence genes including PLD, FagA, FagB, FagC, FagD, SigE, SpaC, SodC, PknG, NanH, OppA, OppB, OppC, OppD, OppF, CopC, NrdH and CpaE were presented in all C. pseudotuberculosis strains, (4) the housekeeping gene fusA can effectively differentiate biovar ovis from biovar equi in phylogenic analysis and can be employed

for further epidemiological study of C. pseudotuberculosis. Briefly, this study provided the basic information for infection of C. pseudotuberculosis in goats and will help in controlling of this pathogen in southwestern China. Keywords: Corynebacterium pseudotuberculosis; Goats; Antimicrobial resistance;

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Virulence genes; Phylogenetic analysis

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Introduction

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Corynebacterium pseudotuberculosis, a gram-positive facultative intracellular

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pathogen, is responsible for several chronic diseases including Caseous lymphadenitis

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(CLA) of small ruminants (Windsor and Bush, 2016), Oedematous skin disease (OSD) of buffalo (Viana et al., 2017), ulcerative lymphangitis of horse (Barauna et al., 2017),

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bovine mastitis (Silva et al., 2011), and necrotizing lymphadenitis of humans (Trost et

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al., 2010). C. pseudotuberculosis are divided into two distinct biovars according to their nitrate-reducing ability, biovar ovis (usually nitrate reductase negative) which infect sheep and goats, and biovar equi (usually nitrate reductase positive) that infect

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horses, cattle, and buffalo (Oliveira et al., 2016). Clinically, goats and sheep are the most commonly animals infected with C. pseudotuberculosis, and the characteristics of infection are the formation of abscesses in the lymph nodes and visceral organs, which seriously restricted the economic benefits of the goat farming. While the

infection of C. pseudotuberculosis in goats was reported recently in many different countries (Algaabary et al., 2009; Bush et al., 2012; Jung et al., 2015; Guerrero et al., 2018), limited information is available from China. According to official statistics, at the end of 2016, the number of goats in China

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was 139.77 million, of which was 21.18 million among Chongqing municipality, Sichuan province, and Guizhou province in southwestern China (China Statistical

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Yearbook, 2017). Although more than 30 reports have been recorded on C. pseudotuberculosis infection in goats, sheep, and camels in China (Zhou et al., 2016),

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most of them were just case report. In recent years, the skin abscess of goats were

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found at numerous farms in southwestern China, and the morbility is up to 60% in

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some farms, suggesting that C. pseudotuberculosis infection would be emergency in

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goats in the south of China. The aim of this study were (1) to investigate

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epidemiology of C. pseudotuberculosis in goats, (2) to test drug sensitivity and virulence-related genes by using C. pseudotuberculosis strains, and (3) to analysis the

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phylogenetic relationship of C. pseudotuberculosis.

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2. Materials and Methods 2.1 Sample collection and C. pseudotuberculosis isolation From May 2015 to September 2017, one hundred and two external abscess

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samples were collected from 12 goat ranchses Chongqing, Sichuan and Guizhou province in southwestern. Except for marasmus, the goats with abscess were seems to be healthy with the normal mental state, temperature and appetite. The surface of abscess were disinfected with alcohol, and cutted with a surgical blade, the pus or

cheese-like substance were extruded and collected in sterilized tubes and sent to lab in ice box. The collected samples were inoculated in agar plate with 5% rabbit blood and cultivated at 37℃ for 48 h. As the previous reports (de Sá Guimar Es Et Al, 2011; Guerrero et al., 2018; Sá et al., 2013), the colonies suspected to be C.

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pseudotuberculosis were primarily identified by colony morphology and Gram staining, and then sub-cultured in broth containing 10% fetal bovine serum

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(Biological Industries, Kibbutz Beit-Haemek, Israel), and confirmed by PCR

amplification of phospholipase D (PLD) gene (Table 1) with the following conditions:

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denaturation for 5 min at 94 °C, followed by 30 cycles for 30s at 94 °C, 30 s at 54 °C

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2.2 Antimicrobial resistance profile

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were identified by nitrate reductase test.

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and 90 s at 72 °C, and a final extension at 72 °C for 10 min. The biotype of isolates

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Antimicrobial susceptibility test were performed using disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2013).

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Following antibiotics with stated concentrations (μg/disc) were used: amoxicillin (10),

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cefepime (30), cefotaxime (30), cefoxitin (30), ceftriaxone (30), chloramphenicol (30), clarithromycin (15), furazolidone (300), gentamicin (10), kanamycin (30), levofloxacin (5), lincomycin (2), macrodantin (300), minocyline (30), norfloxacin

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(10), streptomycin (10), roxithromycin (15), tetracycline (30), trimethoprim (5) and vancomycin (30). 2.3 Detection of virulence genes DNA of C. pseudotuberculosis was extracted with a commercial DNA extraction

kit (Dalian TaKaRa Biotechnology Co., Ltd.) following the manufacturer’s instructions. Eighteen C. pseudotuberculosis virulence or putative virulence genes including phospholipase D (PLD), integral membrane protein (FagA), iron enterobactin transporter (FagB), ATP binding cytoplasmic membrane protein (FagC),

zinc-dependent

superoxide

dismutase

(SodC),

protein

kinase

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iron siderophore binding protein (FagD), sigma factors E (SigE), tip protein C (SpaC), G

(PknG),

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neuraminidase NanH, oligopeptide permease A (OppA), OppB, OppC, OppD, OppF, copper resistance protein (CopC), the glutaredoxin-like protein (NrdH) and Pilus

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assembly protein(CpaE) (Trost et al., 2010; Sá et al., 2013; Santana-Jorge et al., 2016;

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Silva et al., 2017), were detected by PCR with the primers (Table 1) synthesized by

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Bioligo Biotechnology Co., Ltd (Shanghai, China) .The PCR amplification conditions

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were: initial denaturation for 5 min at 94 °C, followed by 30 cycles of 94 °C for 40s,

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61 °C for 40s (58 °C for FagA, 55 °C for FagB, SigE, SpaC, SodC, PknG, NanH, OppA, OppB, OppC, OppD, OppF, CopC, NrdH and CpaE, 60 °C for FagC), and

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72 °C for 40 s, with a final elongation of 72 °C for 10 min.

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2.4 16S rRNA, fusA gene amplification and sequencing The 16S rRNA gene and elongation factor P (fusA) were amplified with the

following program: 5 min of initial denaturation at 94°C and then 30 cycles at 94°C

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for 30 s, at 54°C for 30s, and at 72°C for 90 s and 10 min of the final elongation at 72 °C. PCR products were sequenced both in forward and reverse directions by Shanghai Invitrogen Biotechnology Co., Ltd. The obtained 16S rRNA and fusA sequences were performed the online alignment with BLAST program, and the

confirmed sequences were deposited in GenBank. 2.5 Phylogenetic analysis 16S rRNA and fusA sequence of Corynebacteria from different species including human, goats, sheep, bovine, llama, antelope, horse, bubalus bubalis, cow and

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obtained in this research were used for phylogenetic analyses (Table 2). The sequences were aligned using the CLUSTAL X program with default parameters

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followed by manual inspection. MEGA 4.0 was employed to construct

neighbor-joining trees (Tamura et al., 2007). Bootstrapping was performed with 1000

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replicates.

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3. Results

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3.1 Isolation and identification of C. pseudotuberculosis

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Most of the goats selected for abscess collection in this study are already in the

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middle and late stages of the disease, because the abscess is relatively large and soft, the cheesy pus in the abscess were milky white or yellow-green (Fig. 1). Of the 102

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abscess samples from goats in southwestern China, 40 (39.22%) were identified as C.

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pseudotuberculosis infection. The C. pseudotuberculosis isolates grew well on agar medium with 5% rabbit blood, and the diameter of colonies were about 1.0 mm after inoculation for 48 h, with the characteristics of porcelain white, opaque, dry, low

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adhesion to the medium plate, and a narrow β-hemolysis can be seen from the back of the plate. Gram staining and microscopic examination resulting in club-like and pleomorphic Gram positive bacilli (Fig. 2), and all the isolates were tested nitrate reductase negative.

3.2 Antimicrobial resistance profile The antimicrobial susceptibility test showed that the most of the C. pseudotuberculosis isolates were susceptible to a majority of antibiotics and the resistance rates were below 15%, with the exceptions of following: nitrofurantoin

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(100% resistant), furazolidone (100% resistant) and streptomycin (18.42% resistant). And the most sensitive antibiotics to C. pseudotuberculosis were vancomycin,

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norfloxacin, cefradine, clarithromycin and cefepime, followed by amoxicillin, cefoxitin, levofloxacin, minocycline (Table 2).

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3.3 Distribution of virulence genes among C. pseudotuberculosis isolates

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All the virulence or putative virulence genes including PLD, FagA, FagB, FagC,

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FagD, SigE, SpaC, SodC, PknG, NanH, OppA, OppB, OppC, OppD, OppF, CopC,

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NrdH and CpaE were found in 40 isolates.

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3.4 Phylogenetic analysis of C. pseudotuberculosis Twenty sequences of 16S rRNA (Genbank accession number: KX580967.1、

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MF772499.1-MF772519.1) and 21 sequences of fusA gene (Genbank accession

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number: MG252234-MG252254) were obtained from C. pseudotuberculosis after PCR amplification

and sequenced. Phylogenetic analysis of 16S rRNA can easily

separated C. pseudotuberculosis from other Corynebacterium spp., but it is powerless

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to differentiate biovar ovis from biovar equi (Fig. 3). While the phylogenetic analysis of fusA can clearly differentiate biovar ovis from biovar equi (Fig. 4).

4. Discussion C. pseudotuberculosis infection in goats was first recorded in 1962 in China (Jia

and Liu, 1962). In recent years, the infection of this pathogen in goats were reported in Sichuan (Zhang et al., 2015), Shaanxi (Wu et al., 2018; Zhu et al., 2017), Fujian (Li et al., 2014), Jiangsu (Li et al., 2018) and Guizhou (Liao et al., 2018), and there is a tendency of expanding. However, these reports are mainly case descriptions.

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This study reported for the first time the isolation and molecular characteristics of C. pseudotuberculosis from external abscess of goats in southwestern China. The

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isolation rate of C. pseudotuberculosis in this study was higher than the results (25.66%) recorded from goats with CLA in Egypt (Algaabary et al., 2009) , whereas

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lower than the results reported by Tripathi et al. (2016) and Kumar et al.(2012) who

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showed an isolation rate of 48.18% and 51.9% , respectively (Kumar et al., 2012;

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Tripathi et al., 2016). Although C. pseudotuberculosis isolation is a confirmatory

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standard method but isolation failure is a problem (Oreiby et al., 2015). The reasons

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including contaminants over-growth, sterile abscesses or old calcified lesions contain little pus and few viable organisms, may result in C. pseudotuberculosis isolation

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failure (Baird and Fontaine, 2007) . Anyway, this study confirmed a relatively high

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infection rate of C. pseudotuberculosis which responsible for external abscess of goats in southwestern China. There may be several reasons contribute to the infection and transmission of C. pseudotuberculosis in some goat farms: (1)

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quarantine failure resulted in introduction of goats with the pathogen, (2) strong resistance to the external environment and multiple infection routes of C. pseudotuberculosis causing this pathogen to be widespread (Umer et al., 2017), and most of the goats in Chongqing are mainly grazing, which easily cause skin wounds

and provide conditions for the infection of this pathogen, (3) some farmers did not pay much attention to the disease, allowing the abscess to rupture on its own (personal communications with farmers), resulted in a large spread of the pathogen. The antimicrobial susceptibility test demonstrated that the majority of C.

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pseudotuberculosis isolates were sensitive to many kinds of antibiotics including vancomycin, norfloxacin, cefradine, clarithromycin, cefepime, amoxicillin, cefoxitin,

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levofloxacin, minocycline, trimethoprim, lincomycin, gentamicin, tetracycline and

kanamycin, similar results have been reported in previous studies (Chirino-Zárraga

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et al., 2006; Abebe and Sisay, 2015). While all the isolates were found resistant to

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nitrofurantoin and furazolidone which is consistent with the other report (Mittal et al.,

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2010). The relatively high sensitivity spectrum of C. pseudotuberculosis from

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southwestern China is likely due to a limited use of antimicrobials for goats in this

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region.

To characterize the carry level of virulence genes in C. pseudotuberculosis, 18

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virulence or putative virulence genes including PLD, FagA, FagB, FagC, FagD, SigE,

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SpaC, SodC, PknG, NanH, OppA, OppB, OppC, OppD, OppF, CopC, NrdH and CpaE (Trost et al., 2010; Sá et al., 2013; Santana-Jorge et al., 2016; Silva et al., 2017) were detected and found in all the isolated strains. The results of this study are

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different from the previous reports that the presence of FagD gene in 95.23% isolates (Sá et al., 2013), one of the reasons might be related to the relatively small number of C. pseudotuberculosis isolates we detected. Although all the strains have the same virulence genes profile, the expression level of virulence genes and the virulence of C.

pseudotuberculosis strains to goats deserved further research. The classification and relationship of C. pseudotuberculosis between biovar ovis and biovar equi has attracted a large number of researchers. Some molecular methods including restriction fragment length polymorphism (RFLP) of chromosomal DNA,

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ribotyping, and whole genome sequence analysis were used for the diferentiation of C. pseudotuberculosis biotypes (Sutherland et al., 1996; Ruiz et al., 2011; Soares et al.,

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2013) , and recently, two biotypes of C. pseudotuberculosis were differentiated by concatenated partial sequence of four housekeeping genes (dnaK, groEL1, inf B, and

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leuA) (Sellyei et al., 2017). In this study, we found that fusA can effectively

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differentiate biovar ovis from biovar equi of C. pseudotuberculosis, and it can be

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employed for further epidemiological study of this pathogen.

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In conclusion, the present study revealed a relatively high infection C.

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pseudotuberculosis responsible for the external abscess of goats, a high antibiotics sensitivity spectrum and virulence genes presentation in the isolated strains for the

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first time in southwestern China. In addition, this study provided a housekeeping

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gene candidate used for C. pseudotuberculosis biotype differentiation. These findings not only enrich information of C. pseudotuberculosis infection, but also provide the reference materials for the prevention of external abscess in goats in

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southwestern China.

Acknowledgements This work was supported by Fundamental Research Funds for the Central Universities

(XDJK2018C054) and in part by the Social Undertakings and Livelihood Security Technology Innovation Projects of Chongqing (CSTC2018JSCX-MSYBX0216) and the Plan of Entrepreneurial and Innovative Support for Overseas Students in Chongqing (CX2017103). We really appreciate Dr. Jianjun Wen in University of

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Texas Medical Branch (UTMB) for reviewing of this manuscript. Declaration

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The experiments comply with the current laws of the countries in which the experiments were performed.

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1596-1599. Tripathi, B.N., Kumar, J., Sonawane, G.G., Kumar, R., Dixit, S.K., 2016. Microbiological and Molecular Investigation of Clinically Suspected Caseous Lymphadenitis Cases in Goats. Agricultural Research. 5, 1-7. Trost, E., Ott, L., Schneider, J., Schroder, J., Jaenicke, S., Goesmann, A., Husemann,

IP T

P., Stoye, J., Dorella, F.A., Rocha, F.S., Soares, S.C., D'Afonseca, V., Miyoshi,

A., Ruiz, J., Silva, A., Azevedo, V., Burkovski, A., Guiso, N., Join-Lambert, O.F.,

SC R

Kayal, S., Tauch, A., 2010. The complete genome sequence of Corynebacterium

pseudotuberculosis FRC41 isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-regulatory networks contributing to 11, 728.

U

virulence. BMC Genomics.

N

Umer, M., Abba, Y., Abdullah, F., Saleh, W., Haron, A., Saharee, A., Ariff, A., Baiee,

A

F., Hambali, I., Sharif, A., 2017. Caseous lymphadenitis in small ruminants: An overview on reproductive implications. International Journal of Veterinary

M

Sciences and Animal Husbandry. 2, 23-31.

ED

Viana, M., Figueiredo, H., Ramos, R., Guimaraes, L.C., Pereira, F.L., Dorella, F.A., Selim, S., Salaheldean, M., Silva, A., Wattam, A.R., Azevedo, V., 2017. Comparative genomic analysis between Corynebacterium pseudotuberculosis

PT

strains isolated from buffalo. PLoS One. 12, e176347. Windsor, P.A., Bush, R.D., 2016. Caseous lymphadenitis: Present and near forgotten

CC E

from persistent vaccination? Small Ruminant Res. 142, 6-10.

Wu, H., Xu, X., Ren, J., Zhang, S., Fu, M., 2018. Isolation and identification of

A

Corynebacterium pseudotuberculosis in goats. Animal Husbandry ＆ Veterinary Medicine. 50, 74-77. (in Chinese).

Zhou, Z., Li, H., Zhang, M., Wang, Z., Zhou, R., Hu, S., Li, X., Song, X., Zhu, Z., 2016. Genome Sequence of Corynebacterium pseudotuberculosis Strain XH02 Isolated from a Boer Goat in Xuanhan, China. Genome Announc. 4, e01329-16. Zhu, W., Hou, Y., Huo, N., Sang, F., Zhang, H., Gao, Y., Li, Y., Wang, Y., Chen, D., 2017. Isolation and identification of Corynebacterium pseudotuberculosis from

goats. Progress in Veterinary Medicine. 38, 127-131. (in Chinese).

IP T

Figure legends: Fig. 1. The abscess of goats infected with C. pseudotuberculosis. The abscess

) were closed to parotid lymph node (A, B) , prescapular lymph node (C) , and

SC R

(

abdominal lymph node (C) of goats.

U

Fig. 2. Cultural and staining characteristics of C. pseudotuberculosis isolates from

N

goats. The C. pseudotuberculosis isolates grew well on the blood agar medium, and

A

the diameter of colonies were about 1 mm after inoculation for 48 h, with the

M

characteristics of porcelain white, opaque, dry, low adhesion to the medium plate (A)

ED

and β-hemolysis (B). The C. pseudotuberculosis isolates were Gram-stained positive (C) .

PT

Fig. 3. Phylogenetic tree of C. pseudotuberculosis isolates based on the sequence of

CC E

16S rRNA. The phylogenetic tree was reconstructed by MEGA4.0 using Neighbor-Joining methods. Supports of 1000 bootstraps are indicated at the nodes. Fig. 4. Phylogenetic tree of C. pseudotuberculosis isolates based on the sequence of

A

fusA. The phylogenetic tree was reconstructed by MEGA4.0 using Neighbor-Joining methods. Supports of 1000 bootstraps are indicated at the nodes.

A ED

PT

CC E C

A B

IP T

SC R

U

N

A

M

Fig. 1

D

A ED

PT

CC E A B C

IP T

SC R

U

N

A

M

Fig. 2

A ED

PT

CC E

IP T

SC R

U

N

A

M

Fig. 3

CP001829.1 Biotype ovis Sheep Australia KX580967.1 Biotype ovis Goat China CP013697.1 Biotype ovis Sheep Mexico CP013260.1 Biotype equi Horse USA CP013699.1 Biotype ovis Sheep Egypt MF772507.1 Biotype ovis Goat China MF772517.1 Biotype ovis Goat China

MF772516.1 Biotype ovis Goat China

IP T

CP003077.1 Biotype equi Horse USA

CP002924.1 Biotype ovis Sheep Argentina MF772518.1 Biotype ovis Goat China MF772508.1 Biotype ovis Goat China

SC R

MF772513.1 Biotype ovis Goat China CP003385.1 Biotype ovis Wildebeest ... CP012136.1 Biotype equi Horse Chile

CP003421.3 Biotype equi Buffalo Egypt CP003082.1 Biotype equi Horse USA

MF772503.1 Biotype ovis Goat China MF772505.1 Biotype ovis Goat China

N

64

MF772511.1 Biotype ovis Goat China

A

MF772500.1 Biotype ovis Goat China

CP002097.1 Biotype ovis Human France

ED

M

MF772504.1 Biotype ovis Goat China

67

PT

72

MF772509.1 Biotype ovis Goat China CP013146.1 Biotype ovis Sheep Equatorial Guinea CP012837.1 Biotype ovis Goat Brazil CP009927.1 Biotype ovis Goat Brazil CP008924.1 Biotype ovis Goat Norway CP008923.1 Biotype ovis Goat Norway CP008922.1 Biotype ovis Human Norway CP003062.1 Biotype ovis Sheep Australia MF772519.1 Biotype ovis Goat China

99

MF772502.1 Biotype ovis Goat China

CC E

CP002251.1 Biotype ovis Cow Israel CP013327.1 Biotype ovis Sheep Brazil CP003152.1 Biotype ovis Sheep Scotland MF772510.1 Biotype ovis Goat China MF772512.1 Biotype ovis Goat China

A

CP003407.1 Biotype ovis Llama USA MF772514.1 Biotype ovis Goat China CP013698.1 Biotype ovis Goat Portugal CP011474.1 Biotype ovis Sheep Brazil MF772515.1 Biotype ovis Goat China C. ulcerans KF564646.1

65

C. ulcerans NR 029292.1 C. diphtheriae GQ118344.1

99

C. diphtheriae GQ118341.1 M. tuberculosis MG995565.1

C. pseudotuberculosis

U

MF772499.1 Biotype ovis Goat China

Fig. 4 CP012837.1 Goat Brazil CP003062.1 Sheep Australia CP009927.1 Goat Brazil MG252235.1 Goat China CP013698.1 Goat Portugal MG252234.1 Goat China CP008924.1 Goat Norway

MG252249.1 Goat China CP013699.1 Sheep Egypt MG252242.1 Goat China MG252239.1 Goat China

SC R

MG252248.1 Goat China

IP T

CP012695.1 Goat Portugal

CP011474.1 Sheep Brazil

MG252246.1 Goat China MG252240.1 Goat China

U

MG252237.1 Goat China

MG252254.1 Goat China

62

N

MG252236.1 Goat China MG252252.1 Goat China MG252243.1 Goat China

A

CP013327.1 Sheep Brazil

M

CP013146.1 Sheep Equatorial Guinea

C. pseudotuberculosis Biotype ovis

MG252251.1 Goat China

CP002097.1 Human France

MG252241.1 Goat China MG252253.1 Goat China MG252244.1 Goat China

ED

CP002251.1 Cow Israel

PT

64

CP003407.1 Llama USA CP003152.1 Sheep Scotland MG252250.1 Goat China MG252247.1 Goat China CP003385.1 Wildebeest South Africa CP013697.1 Sheep Mexico

CC E

MG252238.1 Goat China CP008923.1 Goat Norway

MG252245.1 Goat China CP001829.1 Sheep Australia CP003077.1 Horse USA CP003082.1 Horse USA CP013260.1 Horse USA

70

CP012136.1 Horse Chile CP003421.3 Buffalo Egypt C.ulcerans KM268781.1

84

C.ulcerans KM268780.1 C.diphtheriae BX248355.1

100

C.diphtheriae NZ_CP020410.2 M.tuberculosis AP018033.1

C. pseudotuberculosis Biotype equi

CP008922.1 Human Norway

91

A

CP002924.1 Sheep Argentina

Table 1. Primers used for C. pseudotuberculosis 16Sr RNA, fusA, and virulence genes amplification Genes

Primer sequence (5′to 3′)

Amplification size (bp)

Reference

16 S rRNA

SF:AGAGTTTGATCCTGGCTCAG

1500

this study

895

this study

235

this study

252

this study

SR:CGGCTACCTTGTTACGACTT fusA

SF:ACCGCTTACCGTAACAAGGG

sigE

SF:GGAGCAGGCCTATAACATCG

IP T

SR:CATCCTGGATACCTGCGTCC

SR:TTGGGCGTCTTCATTAGTCG SpaC

SF:ATATATCCGCAGCTTGCACC

NanH

SF:TGCAGTGGGGGAGGGTA

SC R

SR:CTGCAATCACCAAAACGGTC 217

this study

SR:TTTCGTGTTCTCGTAAGGCG PknG

SF:TGGTGGTATGGGTTGGATCT

198

this study

SF:AACTACCTCAAGATGCGCTG

OppA

SF:CGGCTAAGGACTTTTCCTCC

237

this study

237

this study

231

this study

244

this study

223

this study

242

this study

160

this study

214

this study

SR:GTTTACCCGACGCAGAGATT

SF:TGTTGGCGTTAAATGGGGAA

M

OppB

this study

A

SR:CTCAAGAATCAATCCGGCGA

270

N

SodC

U

SR:TCAACGCGGTGATCATCAAT

SR:ACGGGAATCAACGAGTTACG OppC

SF:ACGGACATCCTTTTGGCTTT

OppD

ED

SR:TAACAACGATCGCCGCTAAA SF:GCTTTGATCGTCAAGGTTGC SR:ACTGTGCGTTCCTGCTTAAA SF:CCTCGTCAAATCTCTGGTGG

PT

OppF

SR:ACATGCGATCGGAAACGTAA

CopC

SF:CCAAGGTCGCTTTCTCTGGT

CC E

SR:CCCGCCACCGTAAAAGTAGT

NrdH

SF:AAGCCAGCTTGCGTTCAATG SR:AGTGCTCGCCATTCACTTCA

CpaE

SR:CCCGGTGATTCCTCTATGCC

A

SF:ATGAGGGAGAAAGTTGTTTTA

PLD

Fag-A

SF:ATGAGGGAGAAAGTTGTTTTA

924

(Aquino et al., 2013)

245

(Aquino et al., 2013)

291

(Aquino et al., 2013)

173

(Aquino et al., 2013)

SR:TCACCACGGGTTATCCGC SF:AGCAAGACCAAGAGACATGC SR:AGTCTCAGCCCAACGTACAG

Fag-B

SF:GTGAGAAGAACCCCGGTATAAG SR:TACCGCACTTATTCTGACACTG

Fag-C

SF:GTTTGGCTATCTCCTTGGTATG

SR:CGACCTTAGTGTTGACATACCC Fag-D

SF:GAGACTATCGACCAGGCAGA

226

(Aquino et al., 2013)

SR:ACTTCTTGGGGAGCAGTTCT

Table 2. Results of drug sensitivity test for C. pseudotuberculosis (n=40). Antibiotics

No. of C. pseudotuberculosis R I S

Percentage of resistance or sensitivity Resistance rate (%)

Sensitive rate (%)

1

6

31

2.63%

81.58%

ceftriaxone

1

8

29

2.63%

76.32%

streptomycin

7

8

23

18.42%

kanamycin

4

5

29

10.53%

gentamicin

3

4

31

7.89%

tetracycline

6

2

32

15.00%

80.00%

chloramphenicol

1

1

38

2.50%

95.00%

lincomycin

6

1

33

15.00%

82.50%

nitrofurantoin

40

0

0

furazolidone

40

0

0

vancomycin

0

0

40

norfloxacin

0

0

40

cefradine

0

0

minocycline

1

1

amoxicillin

1

cefoxitin

0

clarithromycin

0

IP T

cefotaxime

60.53% 76.32%

U

100.00%

0.00%

0.00%

100.00%

0.00%

100.00%

40

0.00%

100.00%

38

2.50%

95.00%

0

39

2.50%

97.50%

1

39

0.00%

97.50%

0

40

0.00%

100.00%

1

1

38

2.50%

95.00%

1

2

37

2.50%

92.50%

trimethoprim

2

3

35

5.00%

87.50%

cefepime

0

0

40

0.00%

100.00%

levofloxacin

CC E

roxithromycin

PT

0.00%

ED

SC R

81.58%

M

A

N

100.00%

A

Note: “R” represents resistance; “I” represents intermediate; “S” represents susceptible.

I N U SC R

Table 3. 16S rRNA and fusA sequences of C. pseudotuberculosis used for phylogenetic analysis. 16S rRNA

fusA

accession number

accession number

Australia

CP001829.1

CP001829.1

Genbank

Human

Frence

CP002097.1

CP002097.1

Genbank

Bovine

Israel

CP002251.1

CP002251.1

Genbank

PAT10

Sheep

Argentina

CP002924.1

CP002924.1

Genbank

42/02-A

Sheep

Australia

CP003062.1

CP003062.1

Genbank

1/06-A

Horse

California

CP003082.1

CP003082.1

Genbank

ovis

3/99-5

Sheep

Scotland

CP003152.1

CP003152.1

Genbank

equi

316

Horse

California

CP003077.1

CP003077.1

Genbank

ovis

P54B96

Wildebeest

South Africa

CP003385.1

CP003385.1

Genbank

ovis

267

Llama

California

CP003407.1

CP003407.1

Genbank

equi

31

Buffalo

Egypt

CP003421.3

CP003421.3

Genbank

ovis

48252

Human

Norwegian

CP008922.1

CP008922.1

Genbank

ovis

CS_10

Goat

Norwegian

CP008923.1

CP008923.1

Genbank

ovis

Ft_2193/67

Goat

Norwegian

CP008924.1

CP008924.1

Genbank

ovis

VD57

Goat

Brazil

CP009927.1

CP009927.1

Genbank

ovis

12C

Sheep

Brazil

CP011474.1

CP011474.1

Genbank

equi

E19

Horse

Chile

CP012136.1

CP012136.1

Genbank

ovis

1002B

Goat

Brazil

CP012837.1

CP012837.1

Genbank

ovis

N1

Sheep

CP013146.1

CP013146.1

Genbank

ovis

PA01

Sheep

Brazil

CP013327.1

CP013327.1

Genbank

ovis

MEX25

Sheep

Mexico

CP013697.1

CP013697.1

Genbank

ovis

PO222/4-1

Goat

Portugal

CP013698.1

CP013698.1

Genbank

ovis

E56

Sheep

Egypt

CP013699.1

CP013699.1

Genbank

Strain

ovis

C231

ovis

FRC41

ovis

I19

ovis ovis

PT

ED

equi

A

Biovar

M

Bacteria

A

CC E

C. pseudotuberculosis

Host

County

Sheep

Equatorial Guinea

Reference

I MB11

ovis

TG

ovis

RC-1

ovis

GS-5

CP013260.1

CP013260.1

Genbank

Goat

China

MF772519.1

MG252254.1

This study

Goat

China

MF772517.1

MG252253.1

This study

Goat

China

MF772517.1

MG252252.1

This study

Goat

China

MF772516.1

MG252251.1

This study

Goat

China

MF772515.1

MG252250.1

This study

ovis

Yunyang-8

Goat

China

MF772514.1

MG252249.1

This study

ovis

Yunyang-2

Goat

China

MF772513.1

MG252248.1

This study

JJ-5

Goat

China

MF772512.1

MG252247.1

This study

JJ-3

Goat

China

MF772511.1

MG252246.1

This study

ovis

SN-6

Goat

China

MF772510.1

MG252245.1

This study

ovis

SN-4

Goat

China

MF772509.1

MG252244.1

This study

ovis

GZ-3

Goat

China

MF772508.1

MG252243.1

This study

ovis

GZ-2

Goat

China

MF772507.1

MG252242.1

This study

ovis

DJ-3

Goat

China

MF772505.1

MG252240.1

This study

ovis

DJ-1

Goat

China

MF772504.1

MG252239.1

This study

ovis

ZX-6

Goat

China

MF772503.1

MG252238.1

This study

ovis

ZX-5

Goat

China

MF772502.1

MG252237.1

This study

ovis

DZ-9

Goat

China

MF772500.1

MG252235.1

This study

ovis

DZ-6

Goat

China

MF772499.1

MG252234.1

This study

ovis

XH02

Goat

China

KX580967.1

MG252236.1

This study

ovis

WZ

Goat

China

-

MG252241.1

This study

-

UKR100

Homo sapiens

Ukraine

MG995565.1

-

Genbank

-

HN-024

Homo sapiens

Viet Nam

-

AP018033.1

Genbank

-

KL489

Hydromys chrysogaster

Germany

-

KM268780.1

Genbank

-

KL490

Hydromys chrysogaster

Germany

-

KM268781.1

Genbank

-

NCTC 7910

-

UK

NR_029292.1

-

Genbank

-

MIT07-3331

Ferret

USA

KF564646.1

-

Genbank

-

ATCC 700971

-

USA

GQ118344.1

-

Genbank

M

A

FL-3 Yunyang-11

ED PT A

CC E C. diphtheriae

California

ovis

ovis

C. ulcerans

Horse

ovis

ovis

M. tuberculosis

N U SC R

equi

I N U SC R

-

ATCC 27010

-

NCTC13129

-

FDAARGOS_197

A

CC E

PT

ED

M

A

Note: “-” means not available.

-

USA

GQ118341.1

-

Genbank

-

UK

-

BX248355.1

Genbank

-

UK

-

NZ_CP020410.2

Genbank