1株朱鹮源红斑丹毒丝菌的分离鉴定及生物学特征分析.pdf
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1、第45卷第12 期2023年12 月doi:10.3969/j.issn.1008-0589.2023070041株朱源红斑丹毒丝菌的分离鉴定及生物学特征分析中国预防兽医学报Chinese Journal of Preventive Veterinary MedicineVol.45,No.12Dec.2023邵乐乐t,杨永春 t,马武林,白洪青,李颖4,徐浩瑜,吴燕芬,宋厚辉,邱国强2*(1.浙江农林大学动物科技学院动物医学院/浙江省畜禽绿色生态健康养殖应用技术研究重点实验室,浙江杭州31130 0;2.德清县生态林业综合服务中心,浙江德清3132 0 0;3.德清县自然资源和规划局,浙江德
2、清3132 0 0;4.湖州市生态林业保护研究中心,浙江德清3130 0 0)摘要:为确定浙江省德清县朱抢救保护基地一只死亡朱鹦的发病原因及病原特征,本研究采集其肝脏、心脏等组织样品,通过TSA平板划线培养,观察菌落、革兰氏染色、生化鉴定和16 SrRNA基因的PCR扩增与序列分析。结果显示,从朱鹦体内分离的细菌为红斑丹毒丝菌(E.rhusiopathiae),并命名为E.rhusiopathiae-716株。采用纸片扩散(K-B)法检测该菌对8 类10 种药物的敏感性;采用微量结晶紫染色法鉴定分离菌的生物被膜(BF)形成能力,结果显示该菌对青霉素类的氨苄西林和青霉素、喹诺酮类的诺氟沙星等5类
3、6 种抗生素敏感,对四环素类的四环素、大环内酯类的红霉素、氨基糖苷类的庆大霉素和阿米卡星等3类4种抗生素耐药,且其BF形成能力适中。采用Ilumina二代测序平台对分离菌的全基因组测序,并采用SPAdes拼接序列;利用Prokka和PATRIC3.6.9分析该菌基因组的分子特征。利用CARD数据库预测分离菌的耐药基因;采用VFDB数据库预测分离菌的毒力基因。采用ParSNP软件绘制该分离菌与GenBank中不同国家不同宿主来源的红斑丹毒丝菌全基因组的系统发育树。全基因组的分子特征分析结果显示,该分离菌基因组全长1.7 8 x103kb,G C含量36.5%,含16 94个蛋白编码基因、55个t
4、RNA、6 个rRNA、1个tmRNA,无质粒、无前噬菌体。经软件预测结果显示,该分离菌携带3个耐药基因,分别是耐四环素类药物的tetM、耐大环内酯类药物的mefA及兼具大环内酯类、林可酰胺类以及原霉素B类的msrD基因,这与其对四环素、红霉素的耐药表型相符,但未携带庆大霉素和阿米卡星耐药相关基因;其携带7 6 个与菌毛、英膜、铁摄取、溶胞素、分泌、转运等毒力相关基因;系统发育树结果显示分离株E.rhusiopathiae-716与韩国海豚源红斑丹毒丝菌分离株KC-Sb-R1处于同一分支,进一步表明分离菌为红斑丹毒丝菌。将不同剂量的E.rhusiopathiae-716株分别经腹腔感染维鸭及小
5、鼠后在7 d观察期内评估其致病性。结果显示,中高剂量组(1x105cfu/mL1x 10 c f u/mL)雏鸭和小鼠均出现厌食,被毛杂乱现象,部分雏鸭久卧不起,低剂量(1x104cfu/mL)组及阴性对照组雏鸭和小鼠均无明显异常。经计算,该分离菌对雏鸭和小鼠的LDso分别为110 4.94cfu/mL和110 5.8 6 cfu/mL;病死鸭及小鼠剖检后观察到二者消化道有出血点,雏鸭气囊浑浊等剖检病变,并再次从这两种动物的肝脏中分离到该菌。上述结果表明该菌株对禽类和哺乳动物均具有潜在致病性。本研究首次分离鉴定到一株朱源致病性红斑丹毒丝菌,并证实该菌为朱鹞的致病菌,该结果为进一步研究红斑丹毒丝
6、菌的致病机制及朱相关疾病的防治提供了用药参考。关键词:红斑丹毒丝菌;朱;全基因组测序;生物学特性中图分类号:S852.61文献标识码:A文章编号:10 0 8-0 58 9(2 0 2 3)12-12 45-0 8Isolation,identification and biological characteristics ofErysipelothrix rhusiopathiae from crested ibis*Corresponding author收稿日期:2 0 2 3-0 7-0 8基金项目:国家级大学生创新创业训练计划项目(2 0 2 2 10 3410 47);国家自然科学
7、基金(32 10 2 6 7 1);湖州市科技计划项目(2 0 2 2 GZ40)十共同第一作者:邵乐乐(1995-),男,河南周口人,硕士研究生,主要从事细菌分子流行病学研究;杨永春(197 6-),女,黑龙江海林人,副教授,主要从事人兽共患病分子流行病学研究.*通信作者:E-mail:q i u g u o q i a n g 7 7 7 7 16 3.c o m1246中国预防兽医学报2023年SHAO Le-let,YANG Yong-chun,MA Wu-lin,BAI Hong-qing,LI Ying*,XU Hao-yu,WU Yan-fen,SONG Hou-huil,QIU
8、 Guo-qiang*(1.Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province,College of Animal Science andTechnology&College of Veterinary Medicine,Zhejiang A&F University,Hangzhou 311300,China;2.Deqing County Ecological ForestryComprehensive Service Center,Deqing 31
9、3200,China;3.Deqing Natural Resources and Planning Bureau of Zhejiang,Deqing 313200,China;4.Huzhou Ecological Forestry Protection Research Center,Deqing 313000,China)Abstract:To determine the etiology and pathogenic characteristics of a dead crested ibis(nipponia nippon)in the rescue andprotection b
10、ase of crested ibis in Deqing County,Zhejiang Province,the liver,heart and other tissue were sampled aseptically.Bacterial colonies were observed through streak culture on TSA plates,followed by Gram staining,biochemical identification,andPCR amplification sequencing and analysis of the 16S rRNA gen
11、e.The results showed that one Erysipelothrix rhusiopathiae(E.rhusiopathiae)isolate were obtained from the crested ibis,which were named as E.rhusiopathiae-716.The sensitivity of theisolate to 10 antibiotics in 8 categories was detected by the universal Kirby Bauer(K-B)test,and its biofilm formation(
12、BF)abilitywas detected by the microplate crystal violet staining method.The results showed that the strain was sensitive to 5 categories and 6antibiotics such as ampicillin and penicillin of penicillins and norfloxacin of quinolones,and resistant to 3 categories and 4antibiotics such as tetracycline
13、 of tetracyclines,erythromycin of macrolides,gentamicin of aminoglycosides and amikacin,and ithad moderate BF ability.The whole genome of the isolate was sequenced using Ilumina second-generation sequencing platform,and the sequence was assembled using SPAdes.Prokka and PATRIC 3.6.9 were used to ana
14、lyze the functional characteristics ofthe genome,and the CARD database and VFDB database were used to predict the antibiotics resistance genes and the virulencegenes of the isolate,respectively.A phylogenetic tree of the whole genome of the isolate and E.rhusiopathiae from dfferentcountries and host
15、 sources in GenBank was constructed using ParsNP.The functional annotation results of the whole genomeshowed that the genome of the isolate was 1.78x10kb in length,with a GC content of 36.5%,containing 1694 protein-coding genesequences,55 tRNAs,6 rRNAs,1 tmRNA,and no plasmid,no prophagosome.The resu
16、lts of software prediction showed that theisolate carried three antibiotics resistance genes,namely tetM resistant to tetracycline,mefA resistant to macrolide,and msrD generesistant to macrolide,lincomonamide,and protomycin B.This was consistent with its resistance phenotypes to tetracycline anderyt
17、hromycin,but did not carry gentamicin and amikacin resistance-related genes;It carried 76 virulence-related genes such as pili,capsule,iron uptake,cytolysin,secretion,and transport.The phylogenetic tree showed that the isolate E.rhusiopathiae-716 was inthe same branch as the strain KC-Sb-R1 from dol
18、phin in South Korea,which further indicated that the isolate was E.rhusiopathiae.Ducklings and mice were infected intraperitoneally with different doses of E.rhusiopathiae-716,and the pathogenicity of the strainwas evaluated during a 7-day observation period.The results showed that the ducklings and
19、 mice in the medium and high dosegroups(1x10cfu/mL-1x10cfu/mL)exhibited anorexia,messy fur,and some ducklings were unable to lie up for a long time.Nosignificant abnormalities were observed in ducklings and mice of the low-dose group(1x10cfu/mL)and negative control group.After calculation,The LDso o
20、f the isolate to ducklings and mice were 1 x104.94cfu/mL and 1x105.8cfu/mL,respectively.Afterautopsy,bleeding spots were observed in the digestive tract of dead ducklings and mice,as well as turbidity was observed in the airsacs of the ducklings.The bacteria were recovered from the livers of these t
21、wo animals.This indicated that the strain had potentialpathogenicity to both poultry and mammals.In this study,a strain of pathogenic E.rhusiopathiae from crested ibis was isolated andidentified for the first time,and it was confirmed that this strain was the pathogen to crested ibis,providing a bas
22、is for furtherresearch on the pathogenesis of E.rhusiopathiae and the prevention and treatment of crested ibis related diseases.Key words:E.rhusiopathiae;crested ibis;whole genome sequence;biological characteristics朱(Crested ibis,Ni p p o n i a n i p p o n),被誉为“东方宝石”和“鸟中大熊猫”,为世界珍稀濒危鸟类,曾被世界自然保护联盟(IUC
23、N)列为极危(CR)物种,国家一级保护动物。疫病一直是威胁朱健康的重要阻碍,目前已报道的朱感染的细菌有拟肺炎克雷伯菌、大肠杆菌和产气荚膜梭菌等1-3,但未见红斑丹毒丝菌感染朱的报道。红斑丹毒丝菌(Erysipelothrix rhusiopathiae)是一第12 期邵乐乐,等.1株朱源红斑丹毒丝菌的分离鉴定及生物学特征分析1247种纤细或微弯曲的兼性厌氧革兰氏阳性杆菌,可感染多种动物引发丹毒4。其中,最易感动物为猪,还可感染羊、马等,人也可以感染该菌引发类丹毒,严重可导致死亡5。本研究通过细菌分离培养、16SrRNA基因和全基因组测序与序列分析,从2 0 2 2年6 月德清县下渚湖1只死亡朱
24、(40 日龄左右)体内分离鉴定到1株红斑丹毒丝菌,采用药敏试验检测其耐药表型,利用全基因组测序并分析其基因组特征,预测其携带的耐药基因、毒力基因,采用雏鸭和小鼠感染试验鉴定其致病性。为朱红斑丹毒丝菌感染的防治提供了参考依据。1材料与方法1.1主要实验材料病死朱来自浙江省德清县朱抢救保护基地。1日龄麻鸭购自杭州萧山汉潮家禽有限公司;5周龄ICR小鼠购自杭州医学院。金黄色葡萄球菌ATCC25923株由本实验室保存;TSA、TSB培养基购自上海儒安生物科技有限公司;KODone DNA聚合酶购自东洋纺(上海)生物科技有限公司;DL2000DNAMarker购自南京诺唯赞生物科技股份有限公司;细菌基因
25、组DNA提取试剂盒(离心柱型)购自天根生化科技(北京)有限公司;8 类10 种药敏纸片购自杭州微生物试剂有限公司;细菌微量生化鉴定管购自青岛海博生物技术有限公司。1.2细菌的分离鉴定和生长曲线的测定无菌剖检病死朱,采集肝脏、心脏等样品,采用TSA平板分离培养,37 培养2 4h,挑取单菌落纯化。采用革兰氏染色镜检,观察细菌的形态特征。纯化后菌液过夜培养,取2 L滴加于0.3%半固体培养基中心,37 培养2 4h后测量菌落直径,观察其运动性。将纯化后的细菌进行葡萄糖、乳糖、山梨醇、蔗糖、过氧化氢酶与氧化酶试验等生化鉴定。用PBS将过夜培养的菌液离心洗涤3遍,将其ODo0mm值调整为0.2 后采用
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