流产伊犁马驹主要致病菌的分离鉴定及其致病性研究

doi:10.16431/j.cnki.1671-7236.2025.07.034

摘要/Abstract

摘要： 【目的】了解造成伊犁马流产的主要致病细菌，为其临床指导用药提供科学依据。【方法】本试验采集5匹自然流产马驹的心脏、肝脏、脾脏、肺脏、肾脏、脐带、羊膜等35份组织样品进行增菌培养、纯化、PCR鉴定及测序，并对分离菌株进行耐药性及致病性试验。【结果】通过细菌分离培养、形态学观察从流产马驹组织中分离出6株菌，其中1株在琼脂平板上为较大的灰白色黏液菌落，挑取易拉丝，革兰阴性菌，短粗的杆状，单独或成双排列的杆菌，疑似为克雷伯菌；1株表面具有光泽，边缘不整齐半透明或不透明，革兰阴性，两端钝圆的短杆状菌，疑似为短杆菌；1株灰色或白色边缘光滑且明亮的圆形菌落，革兰阳性菌，堆聚成葡萄串状的球菌，疑似为葡萄球菌；1株表面光滑的圆形白色或乳白色菌落，革兰阴性，两端钝圆的长杆菌，疑似为大肠杆菌；1株在绵羊血琼脂平板菌落呈灰白色圆形，凸起较小、有β溶血环的菌落，革兰阳性球菌,成双或呈长链状排列的球菌，疑似为链球菌；1株在沙门菌显色培养基呈表面光滑呈紫色，边缘整齐的圆形菌落，革兰阴性，两端钝圆的短杆菌，疑似为沙门菌。6株分离菌株16S rRNA 基因PCR扩增结果显示，菌株均在1 540 bp处出现单一条带；1株分离株与GenBank上公布的克雷伯菌相似性＞95%，5株分离株分别与弗氏柠檬酸杆菌、缓慢葡萄球菌、大肠杆菌、马链球菌兽疫亚种和沙门菌的相似性＞97%，各菌株毒力基因检测结果与预期结果一致，最终确定分离的6种致病菌分别为克雷伯菌、弗氏柠檬酸杆菌、缓慢葡萄球菌、大肠杆菌、马链球菌兽疫亚种和沙门菌，分别命名为23-KLB-16S、23-NM-16S、23-HM-16S、23-DC-16S、23-ML-16S、23-SM-16S。相似性及遗传进化结果显示，分离株23-KLB-16S与克雷伯菌、23-NM-16S与弗氏柠檬酸杆菌、23-HM-16S与缓慢葡萄球菌、23-DC-16S与大肠杆菌、23-ML-16S与马链球菌兽疫亚种、23-SM-16S与沙门菌参考菌株相似性和亲缘关系较近。药敏试验结果显示，6株分离株均具有多重耐药，对四环素类及氟喹诺酮类药物较敏感，对林可酰胺类药物耐药。小鼠攻毒试验结果显示，上述6种致病菌对小鼠的半数致死量分别为1.54×10²、2.59×10⁷、3.62×10⁷、7.02×10⁷、6.08×10⁶和8.15×10⁵ CFU/只，其中克雷伯菌、马链球菌兽疫亚种及沙门菌致病性较强。小鼠剖检结果显示，肝脏、脾脏均出现不同程度的淤血与肿大，肺脏出血。【结论】从流产马驹的组织样品中分离出克雷伯菌、弗氏柠檬酸杆菌等6种主要致病菌株，并发现存在混合感染的情况，且6株菌均具有多重耐药，对四环素类及氟喹诺酮类药物较敏感，对林可酰胺类药物耐药，其中克雷伯菌、马链球菌兽疫亚种及沙门菌具有较强的致病性。

关键词: 流产; 马驹; 组织病理变化; 分离鉴定; 致病性

Abstract: 【Objective】 This study was conducted to understand the main pathogenic bacteria causing abortion in Yili horses,and provide scientific basis for their clinical guidance of medication. 【Method】 In this experiment,35 tissue samples of heart,liver,spleen,lung,kidney,umbilical cord and amniotic membrane from 5 spontaneously aborted foals were collected for bacterial enrichment and culture,purification,PCR identification and sequencing,and the isolated strains were tested for drug resistance and pathogenicity. 【Result】 Through bacterial isolation and culture,morphological observation,6 strains were isolated from the tissue of the aborted foal.One strain was a large grayish-white mucus colony on the agar plate,easy to stretch when picked,Gram-negative,short,thick,rod-shaped,individually or in pairs arranged bacilli,suspected to be Klebsiella sp.One strain was glossy surface,with an untidy edge of semi-transparent or opaque,Gram-negative,short,bluntly rounded at the ends,suspected to be Bacillus sp. One stain was stered into clusters of cocci,suspected to be Staphylococcus spp. One stain was round,white or creamy-white colony with smooth surfaces,Gram-negative,and long rods with bluntly rounded ends,suspected to be Escherichia coli.One strain was greyish-white round colony with a small bulge and β-hemolysis ring on a sheep blood agar plate,Gram-positive cocci,in pairs or in long chains,suspected to be Streptococcus spp.One strain was a round colony with a smooth surface and purple color,with neat edges,in the Salmonella chromogenic medium,and was Gram-negative,and short bacilli with bluntly rounded ends,suspected to be Salmonella.The results of PCR amplification of 16S rRNA gene of six isolates showed that the strains all showed a single band at 1 540 bp.BLAST alignment of 16S rRNA gene sequencing results showed that one isolate had more than 95% similarity to Klebsiella sp. published in GenBank, and five isolates had more than 97% similarity to Citrobacter freundii, Staphylococcus lentus, Escherichia coli, Streptococcus equi subsp. zooepidemicus and Salmonella published in GenBank, respectively.The virulence gene detection results of each strain were consistent with the expected results.The six strains of isolated pathogenic bacteria were finally identified as Klebsiella sp., Citrobacter freundii,Staphylococcus lentus,Escherichia coli, Streptococcus equi subsp.zooepidemicus and Salmonella,and named 23-KLB-16S,23-NM-16S,23-HM-16S,23-DC-16S,23-ML-16S and 23-SM-16S，respectively.The similarity and genetic evolution results showed that the isolates 23-KLB-16S and Klebsiella sp.,23-NM-16S and Citrobacter freundii,23-HM-16S and Staphylococcus lentus,23-DC-16S and Escherichia coli,23-ML-16S and Streptococcus equi subsp.zooepidemicus,23-SM-16S and Salmonella reference strains were similar and closely related.The results of drug sensitivity showed that the isolates were multi-drug resistant,generally more sensitive to tetracyclines and fluoroquinolones,and resistant to lincosamide.The results of the mouse attack test showed that the LD₅₀ of the above six pathogenic bacteria were 1.54×10²,2.59×10⁷,3.62×10⁷,7.02×10⁷,6.08×10⁶ and 8.15×10⁵ CFU/mouse,respectively,with Klebsiella sp.,Streptococcus equi subsp.zooepidemicus,and Salmonella being more pathogenic.The autopsy results of mice showed varying degrees of bruising and enlargement of liver and spleen,and hemorrhage in lung. 【Conclusion】 Six main pathogenic strains,including Klebsiella sp.and Citrobacter freundii,were isolated from tissue samples of aborted foals,and mixed infections were found.All six strains were multidrug-resistant and generally sensitive to tetracyclines and fluoroquinolones antibiotics,but resistant to lincosamides.Among them,Klebsiella sp.,Streptococcus equi subsp. zooepidemicus and Salmonella had strong pathogenicity.

Key words: abortion; foal; histopathological changes; isolation and identification; pathogenicity

中图分类号:

卢冲, 王语嫣, 付涵, 李桐阳, 缪荣浩, 卢亚宾, 李建龙, 刘建华, 郭庆勇, 买占海, 况玲. 流产伊犁马驹主要致病菌的分离鉴定及其致病性研究[J]. 中国畜牧兽医, 2025, 52(7): 3344-3358.

LU Chong, WANG Yuyan, FU Han, LI Tongyang, MIAO Ronghao, LU Yabin, LI Jianlong, LIU Jianhua, GUO Qingyong, MAI Zhanhai, KUANG Ling. Isolation and Identification of the Main Pathogenic Bacteria of Aborted Yili Foals and Their Pathogenicity[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(7): 3344-3358.

参考文献

[1] MAVADIYA S,RAVAL S,MEHTA S,et al.Economic aspects of some horse diseases[J].The Indian Journal of Field Veterinarians,2010,6(1):48-50.
[2] SEBASTIAN M M,BERNARD W V,RIDDLE T W,et al.Mare reproductive loss syndrome[J].Veterinary Pathology,2008,45(5):710-722.
[3] LEON A,PILLON C,TEBOURSKI I,et al.Overview of the causes of abortion in horses,their follow-up and management[J].Reproduction in Domestic Animals,2023,58:93-101.
[4] 吴海青,吴学良,布仁巴特,等.马匹繁殖率的主要影响因素[J].畜牧与饲料科学，2017,38(6):21-25.WU H Q,WU X L,BURENBAT,et al.The main factors influencing the reproduction rate of horses[J].Animal Husbandry and Feed Science,2017,38(6):21-25.(in Chinese)
[5] JUFFO G D,ANTONIASSI N A,BASSUINO D M,et al.Causes of abortion,stillbirth,and perinatal mortality in horses[J].Pesquisa Veterinária Brasileira,2022,42:e06808.
[6] MAI Z H,FU H,MIAO R H,et al.Serological investigation and isolation of Salmonella abortus equi in horses in Xinjiang[J].BMC Veterinary Research,2024,20(1):103.
[7] HUMPHRIES R,BOBENCHIK A M,HINDLER J A,et al.Overview of changes to the clinical and laboratory standards institute performance standards for antimicrobial susceptibility testing,M100[J].Journal of Clinical Microbiology,2021,59(12):e0021321.
[8] AI-KARBOLII M M,ALWAN M J.Bacterial isolation and identification from aborted placenta of women and ewes and goat in Al-Fallujah city[J].Plant Archives,2020,20(1):125-131.
[9] JIAN L W,YUAN Y S,SHOU Y G,et al.Serotype and virulence genes of Klebsiella pneumoniae isolated from mink and its pathogenesis in mice and mink[J].Scientific Reports,2017,7(1):17291.
[10] JOOB B,WIWANITKIT V.Klebsiella pneumoniae invasive liver abscess syndrome and endophthalmitis[J].Journal of Emergency Medicine,2017,53(6):917.
[11] MARTAK D,GUTHER J,VERSCHUUREN T D,et al.Populations of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae are different in human-polluted environment and food items:A multicentre European study[J].Clinical Microbiology and Infection,2022,28(3):447
[12] 王孝友,杨睿,沈克飞,等.兔肺炎克雷伯氏菌的分离与鉴定[J].中国兽医杂志,2012,48(2):43-44.WANG X Y,YANG R,SHEN K F,et al.Isolation and identification of Klebsiella pneumoniae in rabbits[J].Chinese Journal of Veterinary Medicine,2012,48(2):43-44.(in Chinese)
[13] 叶勇刚,廖党金,张明国,等.流产奶牛胎儿肺炎克雷伯氏菌的分离与鉴定[J].中国动物传染病学报,2018,26(6):68-72.YE Y G,LIAO D J,ZHANG M G,et al.Isolation and identification of Klebsiella pneumoniae in aborted cow fetus[J].Chinese Journal of Animal Infectious Diseases,2018,26(6):68-72.(in Chinese)
[14] 王佳宁,张自强,孔德婧,等.家兔肺炎克雷伯菌的分离鉴定[J].畜牧兽医学报,2023,54(12):5198-5206.WANG J N,ZHANG Z Q,KONG D J,et al.Isolation and identification of Klebsiella pneumoniae in rabbits[J].Acta Veterinaria et Zootechnica Sinica,2023,54(12):5198-5206.(in Chinese)
[15] 薛巧,赵战勤,刘会胜,等.弗氏柠檬酸杆菌对动物和人致病性研究进展[J].动物医学进展,2015,36(7):81-85.XUE Q,ZHAO Z Q,LIU H S,et al.Research progress of the pathogenicity of Citrobacter fredii in animals and humans[J].Progress in Veterinary Medicine,2015,36(7):81-85.(in Chinese)
[16] 李雨琪,许静茹,朱智豪,等.五株华南虎源弗氏柠檬酸杆菌的分离鉴定及其生物学特性研究[J].中国兽医科学,2024,54(1):70-78.LI Y Q,XU J R,ZHU Z H,et al.Isolation,identification and biological characteristics of five strains of Citrobacter fredii from South China[J].Chinese Veterinary Science,2024,54(1):70-78.(in Chinese)
[17] 黄晓东,周慧华,安健,等.中华绒螯蟹致病性弗氏柠檬酸杆菌的分离鉴定及其药敏特性[J].南方农业学报,2019,50(7):1613-1619.HUANG X D,ZHOU H H,AN J,et al.Isolation,identification and drug susceptibility of pathogenic Citrobacter fredii from Eriocheir sinensis[J].Journal of Southern Agriculture,2019,50(7):1613-1619.(in Chinese)
[18] 刘会胜,赵战勤,薛云,等.羊源致病性弗氏柠檬酸杆菌的分离与鉴定[J].畜牧兽医学报,2015,46(9):1593-1599.LIU H S,ZHAO Z Q,XUE Y,et al.Isolation and identification of the pathogenic Citrobacter fredii from sheep[J].Acta Veterinaria et Zootechnica Sinica,2015,46(9):1593-1599.(in Chinese)
[19] 白杰,周美红,胡秀彩,等.龟源弗氏柠檬酸杆菌的分离鉴定及药敏特性研究[J].大连海洋大学学报,2019,34(6):798-803.BAI J,ZHOU M H,HU X C,et al.Isolation,identification and drug susceptibility of Citrobacter fredii[J].Journal of Dalian Ocean University,2019,34(6):798-803.(in Chinese)
[20] 肖双燕,韦冬冬,刘明珠,等.广西地区美国牛蛙源弗氏柠檬酸杆菌的分离鉴定、耐药性及组织病理[J].水产学报,2024,48(5):118-130.XIAO S Y,WEI D D,LIU M Z,et al.Isolation,identification,drug resistance and histopathology of Citrobacter fredii from American bullfrog in Guangxi[J].Journal of Fisheries of China,2024,48(5):118-130.(in Chinese)
[21] IRLINGER F.Safety assessment of dairy microorganisms:Coagulase-negative staphylococci[J].International Journal of Food Microbiology,2008,126(3):302-310.
[22] PIETTE A,VERSCHRAEGEN G.Role of coagulase-negative staphylococci in human disease[J].Veterinary Microbiology,2009,134(1-2):45-54.
[23] PYORALA S,TAPONEN S.Coagulase-negative staphylococci—Emerging mastitis pathogens[J].Veterinary Microbiology,2009,134(1-2):3-8.
[24] 廖智香,古力米热.新疆伊犁地区485株葡萄球菌分布及耐药分析[J].医药世界,2006,11:35-36.LIAO Z X,GULIMI R.Distribution and drug resistance analysis of 485 strains of Staphylococcus in Yili,Xinjiang[J].Chronic Pathematology Journal,2006,11:35-36.(in Chinese)
[25] 殷小敏,管怀进,邵义祥.B6-Co小鼠角膜混浊的细菌学研究[J].交通医学,2008,22(6):600-602.YIN X M,GUAN H J,SHAO Y X.Bacteriological study of corneal opacity in B6-Co mice[J].Medical Journal of Communications,2008,22(6):600-602.(in Chinese)
[26] 朱思明,丁庆华,闫阗,等.鹅源缓慢葡萄球菌的分离鉴定及生物学特性研究[J].中国家禽,2021,43(7):106-111.ZHU S M,DING Q H,YAN T,et al.Isolation,identification and biological characteristics of Staphylococcus slowus from geese[J].China Poultry,2021,43(7):106-111.(in Chinese)
[27] 吴杰,伍茜,程建国,等.林麝缓慢葡萄球菌和大肠杆菌混合感染的病原分离鉴定及致病性[J].微生物学通报,2024,51(10):4195-4211.WU J,WU X,CHENG J G,et al.Pathogen isolation,identification and pathogenicity of mixed infection of Staphylococcus and Escherichia coli in musk deer[J].Microbiology China,2024,51(10):4195-4211.(in Chinese)
[28] 盛相鹏,杜崇涛,许会会,等.仓鼠源缓慢葡萄球菌的分离鉴定及生物学特性研究[J].动物医学进展,2010,31(7):30-35.SHENG X P,DU C T,XU H H,et al.Identification and biological characteristics of Staphylococcus lentus isolated from hamster[J].Progress in Veterinary Medicine,2010,31(7):30-35.(in Chinese)
[29] 刘富来,冯翠兰.猪源缓慢葡萄球菌的分离鉴定及致病性研究[J].中国动物检疫，2011,28(4):56-58.LIU F L,FENG C L.Identification and pathogenicity study of Staphylococcus lentus isolated from swine[J].China Animal Health Inspection,2011,28(4):56-58.(in Chinese)
[30] HILLIER S L,NUGENT R P,ESCHENBACH D A,et al.Association between bacterial vaginosis and preterm delivery of a low-birth-weight infant[J].New England Journal of Medicine,1995,333(26):1737-1742.
[31] BEUTLER B,GREENWALD D,HULMES J,et al.Identity of tumour necrosis factor and the macrophage-secreted factor cachectin[J].Nature,1985,316(6028):552-554.
[32] CHAOUAT G,MENU E,CLARK D,et al.Control of fetal survival in CBA×DBA/2 mice by lymphokine therapy[J].Reproduction,1990,89(2):447-458.
[33] DEB K,CHATURVEDI M M,JAISWAL Y,et al.Gram-negative bacterial LPS induced poor uterine receptivity and implantation failure in mouse:Alterations in IL-1β expression in the preimplantation embryo and uterine horns[J].Infectious Diseases in Obstetrics and Gynecology,2005,13(3):125-133.
[34] ALVARADO T D,BERASAIN M,SALEM A Z,et al.Antimicrobial and antioxidant activities of two medicinal plants Cuphea aequipetala var.hispida (Cav.) Koehne and Eryngium comosum Delaroche F against bacteria related to equine infections[J].Journal of Equine Veterinary Science,2020,94:103269.
[35] TANG Y W,HINDIYEH M,LIU D Y,et al.Molecular Medical Microbiology[M].3rd ed.Amsterdam:Elsevier,2023.
[36] DENAMUR E,CLERMONT O,BONACORSI S,et al.The population genetics of pathogenic Escherichia coli[J].Nature Reviews Microbiology,2021,19(1):37-54.
[37] 张召兴,贾青辉,张艳英,等.致貉流产死胎肠外致病性大肠杆菌的分离鉴定及生物学特性[J].中国兽医学报,2022,42(8):1626-1631.ZHANG Z X,JIA Q H,ZHANG Y Y,et al.Isolation and biological characterization of extraintestinal pathogenic Escherichia coli causing stillbirths in raccoon abortion[J].Chinese Journal of Veterinary Science,2022,42(8):1626-1631.(in Chinese)
[38] 刘勃兴,赵安奇,刘畅,等.貉流产死胎分离出致病大肠杆菌[J].野生动物学报,2021,42(1):181-191.LIU B X,ZHAO A Q,LIU C,et al.Disease-causing Escherichia coli isolated from stillborn raccoon abortions[J].Chinese Journal of Wildlife,2021,42(1):181-191.(in Chinese)
[39] 黄雨茅,李志舒,项文娜,等.孕晚期孕妇阴道定植大肠杆菌毒力基因分析及对妊娠结局的影响[J].中国计划生育学杂志,2022,30(12):2739-2743.HUANG Y M,LI Z S,XIANG W N,et al.Genetic analysis of the virulence of vaginally colonized Escherichia coli in pregnant women in late pregnancy and its effect on pregnancy outcome[J].Chinese Journal of Family Planning,2022,30(12):2739-2743.(in Chinese)
[40] 胡哲,郭奎,王金慧,等.我国马、驴流产沙门菌病的研究进展[J].中国畜牧业,2022,12:49-50.HU Z,GUO K,WANG J H,et al.Research progress of aborted salmonellosis in horses and donkeys in China[J].China Animal Industry,2022,12:49-50.(in Chinese)
[41] 伊恒博,王小雨,何伟,等.伊犁州马流产沙门氏菌病流行病学调查[J].新疆畜牧业,2024,40(1):33-36.YI H B,WANG X Y,HE W,et al.Epidemiological investigation of aborted salmonellosis in horses in Yili prefecture[J].Xinjiang Animal Husbandry,2024,40(1):33-36.(in Chinese)