多位點序列分型(MLST)在艾伯特埃希菌鑒定中的應(yīng)用

劉 祥，許彥梅，王 斌，鄧建平，肖 波，孫松松，周 陽，熊衍文，王 紅

多位點序列分型(MLST)在艾伯特埃希菌鑒定中的應(yīng)用

劉 祥1，許彥梅2，王 斌1，鄧建平1，肖 波1，孫松松1，周 陽1，熊衍文2，王 紅1

目的 探討多位點序列分型技術(shù)在新病原艾伯特埃希菌發(fā)現(xiàn)與鑒定中的應(yīng)用。方法 采用MLST技術(shù)對生鮮肉中分離的30株疑似艾伯特埃希菌的7個管家基因進行PCR擴增、測序、DNAstar軟件分析，核酸序列上傳至大腸桿菌MLST數(shù)據(jù)庫進行比對，確定其等位基因編號及基因序列型(ST型)，并利用MEGA6.0軟件Neighbor-joining法構(gòu)建遺傳進化樹，與大腸埃希菌、志賀氏菌、艾伯特埃希菌和傷寒沙門氏菌參考菌株進行親緣性分析。結(jié)果 30株菌可分為7種新序列型(16株)和4種已知序列型(14株)，N-J分析顯示與艾伯特埃希菌參考菌株具有高度親緣性，而與大腸埃希菌、志賀氏菌、弗格森埃希菌和鼠傷寒沙門氏菌存在較大差異。結(jié)論 MLST在管家基因水平上準確地確定艾伯特埃希菌的種屬關(guān)系，首次在國內(nèi)發(fā)現(xiàn)艾伯特埃希菌的存在。

多位點序列分型技術(shù)(MLST)；艾伯特埃希菌；新病原

艾伯特埃希菌是一種新發(fā)現(xiàn)的致瀉性病原菌，它可引起人類的散發(fā)感染和食物中毒[1-7]。該菌最初從孟加拉腹瀉兒童的糞便中分離，被鑒定為蜂房哈夫尼菌[1]，通過生化表型特征、16S rDNA序列和DNA雜交分析，Huys等將此類蜂房哈夫尼菌命名為埃希氏菌屬種的一個新種——艾伯特埃希菌(Escherichiaalbertii)[8]。由于缺乏艾伯特埃希菌的生化特性，迄今仍無該菌種的商品化鑒定系統(tǒng)。常規(guī)的細菌鑒定系統(tǒng)經(jīng)常將艾伯特埃希菌鑒定為哈夫尼菌、沙門氏菌、魯氏耶爾森菌，最常見的確定為大腸埃希菌。缺乏動力、不發(fā)酵木糖、不發(fā)酵乳糖和產(chǎn)生β-D葡萄糖醛酸酶被認為是艾伯特埃希菌常見生化特性，并且攜帶有編碼緊密粘附素(Intimin)基因eae，此外菌株還通常攜帶編碼細胞腫脹壞死毒素(cytolethal distending toxin，CDT)基因cdtABC[3,13]。

多位點序列分型(MLST)通過對7個管家基因核酸序列的分析，將每個基因的序列與MLST數(shù)據(jù)庫比對，得到相應(yīng)的等位基因編號，按照指定排列順序形成ST型。ST型可用于國際實驗室間比對、溯源以及分子流行病學(xué)、遺傳進化等研究；同時根據(jù)等位基因圖譜使用配對差異矩陣方法構(gòu)建系統(tǒng)進化樹，與參考菌株的相應(yīng)序列進行親緣性分析，從而準確地研究菌株遺傳進化關(guān)系，以確定種屬[9-11]。通過MLST分析，艾伯特埃希菌明顯不同于腸桿菌科及埃希菌屬中的其它種[3-8]。

本研究對四川省自貢市農(nóng)貿(mào)市場生鮮肉食品中分離出的30株疑似艾伯特埃希菌進行多位點序列分型，通過與NCBI數(shù)據(jù)庫收錄的大腸埃希菌、志賀氏菌、艾伯特埃希菌和傷寒沙門氏菌的相應(yīng)序列進行親緣性分析，為確定艾伯特埃希菌提供依據(jù)。

1 材料與方法

1.1 菌株來源與鑒定 從2013年3月-2014年7月，在中國四川省自貢市的超市和農(nóng)貿(mào)市場共收集446份生肉，包含92份牛肉、41份豬肉、22份羊肉、53份雞肉、189份雞腸、21份鴨肉、28份鴨腸。根據(jù)艾伯特埃希菌相關(guān)研究資料，取生肉樣品25 g，接種225 mL EC，20 ℃增菌24～36 h，取增菌液進行eae基因PCR檢測[3]。eae陽性增菌液接種麥康凱瓊脂平板，36 ℃培養(yǎng)過夜，取不發(fā)酵乳糖的白色菌落進行eae基因檢測。最后分離eae陽性且不發(fā)酵乳糖、不發(fā)酵木糖、動力陰性、cdtB基因陽性的疑似艾伯特埃希菌30株。

1.2 儀器與試劑

(1)主要儀器：PCR儀(SensoQuest Labcycler)、Bio-Rad凝膠成像系統(tǒng)(GEL DocXR+)。

(2)主要試劑：2×Taq MasterMix(含染料)、DNA Marker(DM1000)、去離子水：購自康為世紀公司；核酸染料(GeneGreen)：購自天根生化科技(北京)有限公司；PCR引物合成及測序：由生工生物工程(上海)有限公司完成。

1.3 DNA模板制備及反應(yīng)體系 挑取少許疑似艾伯特埃希菌純培養(yǎng)物接種至EC營養(yǎng)肉湯，36 ℃培養(yǎng)18～24 h。吸取1 mL菌懸液于1.5 mL無菌離心管，8 000 r/min，離心10 min，用500 μL無菌去離子水重懸并煮沸10 min，將重懸液10 000 r/min離心5 min，取上清液作為PCR模板。PCR體積為50 μL，2×Taq MasterMix 25 μL，ddH2O 20μL，上、下游引物(10 μmol/L)各2 μL，模板DNA1 μL。

1.4 MLST引物及反應(yīng)程序 管家基因位點選擇參照大腸桿菌MLST數(shù)據(jù)庫(http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/documents/primersColi_html)提供的分型方案，PCR引物序列、退火溫度及產(chǎn)物大小詳見參考文獻[12]。反應(yīng)程序為94℃ 5min預(yù)變性，按照94 ℃ 30 s、退火30 s、72 ℃ 45 s進行30個循環(huán)，最后一個循環(huán)結(jié)束后72 ℃延伸7 min。每次擴增均以無DNA模板的體系作為空白對照。

1.5 結(jié)果觀察 取PCR產(chǎn)物5 μL以1.5%瓊脂糖凝膠(含1/10 000核酸染料)進行電泳，通過凝膠成像系統(tǒng)觀察是否擴增出目的大小片段，將PCR產(chǎn)物送至上海生工生物工程公司雙向測序。

1.6 數(shù)據(jù)分析 利用SeqMan軟件對PCR產(chǎn)物序列與數(shù)據(jù)庫中的管家基因標準序列進行拼接和校正，校正后的7個管家基因序列同時上傳至E.coliMLST數(shù)據(jù)庫，確定菌株的等位基因型和序列類型。并根據(jù)Ooka等人[3]描述，基于7個管家基因的串聯(lián)序列，利用MEGA6.0軟件Neighbor-joining法構(gòu)建系統(tǒng)進化樹，與國際數(shù)據(jù)庫中已收錄的大腸桿菌、志賀氏菌、艾伯特埃希菌和傷寒沙門氏菌進行親緣性比對，探究該30株菌的種屬關(guān)系。

2 結(jié) 果

2.1 MLST分型結(jié)果 通過不斷優(yōu)化擴增條件、選取引物，所有菌株的7個管家基因序列均得到雙向峰圖完好的序列，與MLST數(shù)據(jù)庫中的等位基因剪切比對，結(jié)果顯示14株菌分別為4種已知ST型，而其余16株菌的各等位基因雙向序列通過互聯(lián)網(wǎng)上傳至數(shù)據(jù)庫，被接受并確認為7種新ST型，見表1。

2.2 MLST聚類圖譜分析 根據(jù)7個管家基因首尾串聯(lián)整合成的3 423 bp核苷酸序列圖譜分析，表明研究中的30株菌與大腸埃希菌、志賀氏菌、弗格森大腸埃希菌、鼠傷寒沙門氏菌存在較大差異。

表1 30株菌MLST型別Tab.1 MLST STs of 30 strains

30株艾伯特埃希菌中鑒別出11種序列型，均與艾伯特埃希菌LMG20976、KF1具有高度親緣關(guān)系，其中SP140602和SP140701兩株菌的序列型與艾伯特埃希菌KF1一致，見圖1。結(jié)果表明30株菌全部鑒定為艾伯特埃希菌。

圖1 30株菌與大腸埃希菌、弗格森埃希菌、艾伯特埃希菌、志賀氏菌、傷寒沙門氏菌系統(tǒng)發(fā)育關(guān)系分析結(jié)果

Fig.1 Phylogenetic relationships of 30 strains withE.coli,E.fergusonii,E.albertii,Shigellaspp. andSalmonellaentericaserovar Typhi

2.3 GenBank核酸序列索取號 本研究中30株艾伯特埃希菌的7個管家基因已全部上傳至GenBank數(shù)據(jù)庫，登陸號為：KP015856-KP016011、KP064411-KP064472。

3 討 論

艾伯特埃希菌是一種新發(fā)現(xiàn)的腸道致病菌。起初通過常規(guī)檢測方法被鑒定為致病性大腸桿菌(EPEC)或者出血性大腸桿菌(EHEC)，對該菌的誤診漏診容易造成公眾健康潛在的危險，也會影響臨床治療效果。由于目前對其研究較少，仍沒有標準化檢測方法和商品化鑒定系統(tǒng)，利用分子生物學(xué)技術(shù)在基因水平上研究遺傳進化關(guān)系，則成為菌種鑒定的關(guān)鍵。

利用MLST對30株疑似艾伯特埃希菌分型過程中，存在個別菌株的管家基因序列在大腸桿菌MLST數(shù)據(jù)庫中未找到完全匹配的等位基因型，可能屬于新型別。采用雙向測序，將其完好的序列和峰圖文件上傳至MLST數(shù)據(jù)庫以待確認，上傳前需核查PCR產(chǎn)物序列和峰圖文件，避免誤判等位基因型及新型別；此外，在本實驗樣品7個管家基因PCR擴增時，除purA外的其余6個管家基因產(chǎn)物測序峰圖基本為單峰，個別出現(xiàn)重峰、雜峰的通過多次擴增和降低模板濃度得到改善；而全部樣品purA基因測序產(chǎn)物重峰、雜峰情況嚴重，存在非特異產(chǎn)物干擾，結(jié)果無法用于比對分析。利用提高退火溫度，降低模板濃度，多次擴增后測序效果并不理想，最終改用數(shù)據(jù)庫中purA其它引物進行擴增后，產(chǎn)物特異性明顯提高，正反向測序峰圖完好。這也提示我們發(fā)現(xiàn)測序峰圖不完好時，可通過多次PCR擴增測序、優(yōu)化擴增條件和選取不同引物進行解決。

傳統(tǒng)檢測方法在新病原菌鑒定中存在局限，而MLST可通過研究管家基因圖譜與參考菌株相應(yīng)序列構(gòu)建系統(tǒng)進化樹，根據(jù)聚類分析結(jié)果判斷菌種歸屬，在新病原菌的鑒定和常見細菌新的變異方面發(fā)揮積極的作用。本研究MLST聚類分析結(jié)果顯示30株菌為艾伯特埃希菌，屬國內(nèi)首次發(fā)現(xiàn)并證實艾伯特埃希菌的存在。然后MLST針對的7個管家基因是大腸埃希菌的保守基因，如若標本中混有大腸桿菌，PCR擴增為混合產(chǎn)物無法區(qū)分，故不能直接作為該細菌PCR快速診斷。目前擬針對艾伯特埃希菌的特異基因建立PCR快速診斷方法。

綜上所述，MLST為最終確認艾伯特埃希菌提供依據(jù)。同時也適用于其他新病原菌的診斷以及后續(xù)的分子流行病學(xué)研究、菌群遺傳多態(tài)性、溯源等，為制定有效的監(jiān)測體系和預(yù)防疾病的流行與暴發(fā)提供有力的技術(shù)支持。

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DOI:10.3969/j.issn.1002-2694.2015.11.010

Abstract：MicroRNAs (miRNAs) are key regulators of gene expression. The miR-36 family has only been authentically found in helminth includingAscarisspp.,Caenorhabditiselegans,Brugiamalayi, andSchistosomamansoni. And the family member named miR-36f was identified to exist only in the egg and larval stage ofAscarissuum(A.suum) in our previous study.We herein preliminarily evaluated the function of miR-36f via its mimics and inhibitors by usingA.suumas model organism. The miR-36f mimics, inhibitors, mimics negative controls and inhibitor negative controls were absorbed by the different groups larvae via soaking method. Three days later, the mice were infected with the larvae above. And the larvae were recovered from the liver and lung of the mice respectively. The larvae of each group were counted and their length and width were measured, and the two target genes of miRNA were analyzed by qRT-PCR.The results showed that development and infective numbers of theA.suuminfective larvae were significantly influenced by miR-36f inhibitors (P<0.05), when compared with the mimics (P>0.05).The expression of the two key target genes of miR-36f named OST48 and cytochrome b were similar to that of inhibitor and mimics. When them iR-36f was influenced by its inhibitor, the development and infective ability ofA.suumlarvae were reduced, which indicated that the miR-36f might be associated with development and infectivity of theA.suumlarvae. Furthermore, miR-36f inhibitors seemed more effective than its mimics. The results would be helpful for better understanding and novel control strategy for parasitic helminths.

Keywords:Ascarissuum; asu-miR-36f; infectivity; development

Supported by the Science Fundfor Creative Research Groups of Gansu Province (Grant No.1210RJIA006)

Corresponding author: Xu Min-jun, Email:xuminjun@caas.cn

MicroRNAs (miRNAs) are small non-coding RNAs with increasingly recognized regulating roles played in gene expression in animals, plants and parasites by binding to mRNAs in 3′-untranslated regions (3′-UTR), and also in coding domain sequences or 5′-UTR[1-4]. And up to now, members of the miR-36 family have been only authenticated in helminths, includingAscarisspp.[5],Caenorhabditiselegans[6],Brugiamalayi[7],Schmidteamediterranea[8], andSchistosomamansoni[9]. The miR-36 family members were highly conserved, stayed in cluster in different organisms, and adult- and larva-specifically expressed; knocking down the miR-36 cluster will lead to embryonic and larval deadliness[10-11]. These reports indicated that the miR-36 members play vital roles in worms. Besides, the main control measure for the epidemic of worms at present is anthelmintic drugs, which easily results in the drug resistance of the parasite and drug residues in animal products[12-14]. Therefore, alternative controlling measure is urgently needed for the replacement of anthelmintic drugs for animal welfare and food safety, and changing miRNA expression abundance with convenient way is likely to be a new way to prevent and control the parasites.

A.suumis a globally parasitic nematode which causes infection in pigs and human with high prevalence rates, especially in developing countries[15-17], and the identification of its genome, proteome, and transcriptome made the parasite be a fine model[18-20]. We herein usedA.suumas a model to evaluate the roles of one miR-36 member named miR-36f, which is specifically expressed in the egg and larval stage ofA.suum. Furthermore, two key target genes of miR-36f, named OST48 (gi|171279896) and cytochrome b (gi|76250725), were detected with qRT-PCR after the over- and down-regulation of miR-36f with the corresponding mimics and inhibitors.

Materials and methods

Larvae preparation

A.suumembryos were collected from the uteri of live adult females, which were obtained from slaughtered pigs in Lanzhou, Gansu Province of China. The embryos were incubated in a culture dish at 28 ℃ for 30 days to allow embryonic development. After that, eggshells were removed with 7.5% v/v sodium hypochlorite overnight at 37 ℃ and shaking with glass beads[21]. A total of 1.0×105larvae were obtained and cultured in DMEM (HyClone) containing 50% calf serum, penicillin G potassium (1 000 units/mL), amphotericin B (10 μg/mL), and streptomycin sulfate (1 mg/mL). The mixture was then evenly spread into a dish of 5 holes, and the miRNA mimics, inhibitors, mimics negative controls and inhibitor negative controls were added to the 4 holes respectively, with the last one left as blank control. The experimental nematodes were cultured at 37 ℃ with 5% CO2for 72 h following gently hand shook 2-3 times each day.

Animals

Fifty 8-week-old BALB/c mice of specific-pathogen-free (SPF) grade were purchased from Lanzhou Veterinary Research Institute Animal Center. The mice were equally divided into five groups, housed in sterile cages, and fed with pelleted food and wateradlibitum. And the mice were acclimatized to these conditions for one week before the experiment. Animal experiments were performed strictly according to the institutional guidelines for animal ethics of China.

Preparation of miRNA inhibitors, mimics and negative controls

The miRNA inhibitors, mimics, and their negative controls (Table 1) were obtained from Invitrogen (Shanghai) and prepared a final concentration of 20 μM by following the instructions respectively. Nucleotides of miRNA inhibitor and its control were modified with 2’-O-methyl.

Challenge infection and measurement of larvae

After soaking for 72 h, larvae of the 5 groups were collected separately. And the 5 groups BALB/c

Tab.1 Sequences of miRNA inhibitor, mimic and their negative controls

Note:aN.C indicates negative control.

mice were subjected to be orally infected with about 5 000 larvae for each. Baermann method was adopted to recover the larvae that migrated to the livers and lungs 4 days later[22]. In a nutshell, livers and lungs of the experimental and the control mice were collected and carefully ground with a mortar, and transferred to a small gauze bag containing 30 mL sterile physiological saline with kanamycin (100 mg/L) and ampicillin (100 mg/L) added, and incubated at 37 ℃ for 24 hours. The larvae recovered were counted and measured with a microscope having micrometer (Olympus) with 20 worms detected for each.

qRT-PCR of target genes after miRNA interventions

Two target genes named OST48 and cytochrome b were then quantified by qRT-PCR following the over-and/or down-regulation of miR-36f. Briefly, total RNA from the transfectedA.suumlarvae were extracted with Trizol reagent (Invitrogen) in accordance with the manufacture’s protocol. The purity of total RNA was confirmed by BioPhotometer plus (Eppendorf). Reverse transcription to obtain cDNA by using EasyScript First-Strand cDNA Synthesis SuperMix (Transgene, Beijing). Real-time quantitative PCR was conducted using an ABI PRISM?7500 Sequence Detection System. All of the primers (Table 2) were synthesized by Shenggong Co, Ltd., China. The actin gene (EU109284) was used as the endogenous control. The amplification cycle conditions were performed as follows: 95 ℃ for 5 min, followed by 40 cycles of 95 ℃ for 15 s, 65 ℃ for 15 s, and 72 ℃ for 32 s. The relative quantification of each miRNA and its target gene was calculated using the equation: N = 2-ΔCt, ΔCt = CtmiRNA-Ctactin[23]. All reactions were conductedin triplicate.

Tab.2 Sequences of primers used in the amplification of target and endogenous control genes

PrimerSequence(5′to3′)cytochromeb?FAGTTTTGGGGGTTGTGTCTTTGcytochromeb?RACACTGCCCAAGTCAACTCAAAOST48?FAAAAGTTGGAGTTTGAGGAGCAOST48?RATCCACAAGCACAAGAATACGAβ?actin?FCTCGAAACAAGAATACGATGβ?actin?RACATGTGCCGTTGTATGATG

Results and discussion

There was a significant difference between the inhibitor treated group and the control, including the larvae recovered number, body length and body width (P<0.05). For larvae recovered number, there was 42.5±23.72 larvae recovered from the inhibitor treated groups (P<0.05), while it was 69.44±34.86, 55.77±27.30, 67.86±27.82, 75.00±33.07 recovered from the mimics groups and other 3 control groups, respectively (Figure 1). For body length, it measures 367±133.29 μm in average in the inhibitor group, while that of controls is 460.82±165.12 μm in average (P<0.05) (Figure 2A). For the body width, it is 17.04±6.37 μm and 23.44±6.90 μm respectively for the inhibitor treated groups and controls (P<0.05)(Figure 2B). For the immune protection of the

host, most of the invasion larvae were killed with only those worms strongly enough to survive. Therefore, although 5 000 larvae were orally infected, only a small number of larvae were successfully recovered, similar to the report by Islam et al[24]. No significant differences were found (P>0.05) in mimics, which was similar to the study reported by Puthiyakunnon[25].This phenomenon might suggest that, for its specific and crucial regulating functions for the development and infectivity of larvae, miR-36f was also critically controlled by organism, and any changes over the saturation level will not obtain functional enhancement.

Data are expressed as the mean values±standard deviations in each group. The symbol"*" indicatesP<0.05.

Fig.1 Number of recoveredA.suumlarvae from the five experimental groups

Data are expressed as the mean values±standard deviations in each group. The symbol"*" indicates P<0.05.Fig.2 Mean body length (A) and width (B) of recovered larvae from the five experimental groups

The expression of OST48 genes was significantly up-regulated after inhibitor treatment(P<0.05), and only slightly decreased after mimics treated (Figure 3A). In contrast to cytochrome b, OST48 was significantly reduced with the mimics (P<0.01), and only slightly increased after inhibitors treatment (P>0.05) (Figure 3B). The two target genes were obtained via enrichment analysis from a total of 216 target genes of miR-36f in our previous study, indicating essential roles of the two genes in the regulating network of miR-36f for the organisms.OST48 is an important part of the oligosaccharyl transferase which plays an important role in N-linked glycosylation[26-27]. And cytochrome b is involved in the oxidative phosphorylation, and is important for cellular metabolism[28-30]. When treated with mimics or inhibitors,miR-36f of the organism was influenced, leading to a changed regulation and metabolism balance, and the expression changes of targets. The phenomenon indicated that miR-36f did have functions of gene regulation in the worms. Besides, it was found that the two targets were not changed as similar trends, which confirmed the previous conclusion that cellular pathways might be regulated with one or more miRNAs[31].

A: Quantification of OST48. B: Quantification of cytochrome b. The symbol"*" indicates P<0.05; "**" indicates P<0.01.

Fig.3 Target gene relative expression analysis of groups interfered with mimics, inhibitors, negative control, inhibitor negative control and normal saline by qRT-PCR

Conclusions

By usingA.suumas model organism, we identified that the egg and larva stage-specific miRNA, miR-36f, involves in the infectivity and development of the parasite, which would help for better understanding and novel control strategy development of parasitic helminths.

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Received:2015-09-03;Revision accepted:2015-10-26

Application of multi-locus sequence typing (MLST)in the identification ofEscherichiaalbertii

LIU Xiang1,XU Yan-mei2,WANG Bin1,DENG Jian-ping1,XIAO Bo1,SUN Song-song1,ZHOU Yang1,XIONG Yan-wen2,WANG Hong1

(1.ZigongCenterforDiseaseControlandPrevention,Zigong643000,China;2.StateKeyLaboratoryforInfectiousDiseasePreventionandControl,NationalInstituteforCommunicableDiseaseControlandPrevention,ChineseCenterforDiseaseControlandPrevention,Beijing102206,China)

We studied the application of multilocus sequence typing (MLST) in the discovery and identification of a new pathogenEscherichiaalbertii. MLST was performed according to theE.coliMLST database using seven housekeeping genes. The 7 housekeeping genes of 30 suspectedEscherichiaalbertiistrains isolated from retail raw meats were amplified, sequenced and analysed by DNA star software. The nucleotide sequences were uploaded toE.coliMLST database to confirm the allele number and the sequence type (ST type). To analyse the phylogenetic relationships between the 30 strains and the reference strains ofE.coli,E.fergusonii,E.albertii,Shigellaspp. andSalmonellaentericaserovar Typhi, the phylogenetic tree was constructed with the neighbor-joining method of MEGA 6. Results showed that 30 strains were classified into 7 new STs (16 strains) and 4 known STs (14 strains), the analysis of N-J showed high homology withE.albertiistrain, while were highly divergent fromE.coli,Shigellasp.,E.fergusoniiandSalmonellaentericaserotype Typhi stains. MLST accurately determine the relationship between species ofE.albertiiat the level of housekeeping genes. And it is the first time of discovering and confirming the existence ofE.albertiiin China.

multilocus sequence typing (MLST);E.albertii; new pathogens

s: Wang Hong, Email: 460973389@qq.com; Xiong Yan-wen, Email: xiongyanwen@icdc.cn

Effects of miR-36f on larval infection and development ofAscarissuum

FENG Sheng-yong1,FU Jing-hua1,2,SHAO Chang-chun1,3,ZHU Xing-quan1,XU Min-jun1,2

(1.StateKeyLaboratoryofVeterinaryEtiologicalBiology,KeyLaboratoryofVeterinaryParasitologyofGansuProvince,LanzhouVeterinaryResearchInstitute,ChineseAcademyofAgriculturalSciences,Lanzhou730046,China;2.CollegeofAnimalScience,SouthChinaAgriculturalUniversity,Guangzhou510642,China;3.CollegeofVeterinaryMedicine,YangzhouUniversity,Yangzhou225009,China)

自貢市重點科技計劃項目(No.2013S06)和四川省衛(wèi)生和計劃生育委員會科研課題(No.150259)

王 紅，Email: 460973389@qq.com； 熊衍文，Email: xiongyanwen@icdc.cn

1.四川省自貢市疾病預(yù)防控制中心，自貢 643000; 2.中國疾病預(yù)防控制中心傳染病預(yù)防控制所，傳染病預(yù)防控制國家重點實驗室，北京 102206

10.3969/j.issn.1002-2694.2015.11.009

R378

A

1002-2694(2015)11-1033-04

2015-02-05；

2015-07-26

Supported by the Zigong Key Science and Technology Program (No. 2013S06) and Grant from the Health and Family Planning Commission of Sichuan Province (No. 150259)