冀北地区养殖场奶/肉犊牛腹泻中志贺菌的分离鉴定及特征分析

Isolation,identification and characteristic analysis of Shigella from diarrhea of milk/meat calves in breeding farms in Northern Hebei Province

为鉴定引起冀北地区养殖场中奶/肉犊牛腹泻的病原菌并分析其生物学特征,本研究于2022-2023年采集冀北地区养殖场患腹泻病奶/肉犊牛腹泻的肛拭子、粪便及病料组织273份,采用细菌分离培养、生化鉴定及PCR方法对志贺菌(Shigella)进行分离鉴定,采用人工感染小鼠试验、玻板凝集试验、PCR法和K-B药敏纸片法分别检测志贺菌的致病性、血清型、毒力基因,用人工感染小鼠试验和改良寇氏法测定其优势血清型代表株的半数致死量(LD50);采用RT-PCR对牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)、牛轮状病毒(bovine rotavirus,BRV)、牛冠状病毒(bovine coronavirus,BCoV)、牛诺瓦病毒(bovine norovirus,BNoV)和牛纽布病毒(bovine nebovirus,BNeV)5种病原检测。结果表明,5种病毒性病原检测为阴性;从采集的病料组织中分离得到94株志贺菌,其中52株志贺菌引起小白鼠发病与致死,为致病性志贺菌,对小鼠致死率在40%以上;52株致病性志贺菌中以福氏志贺菌F2a型(21.2%)、F1b型(25.0%),宋内志贺菌Ⅰ相(19.2%)和痢疾志贺菌(13.5%)为优势流行血清型;52株致病性志贺菌毒力基因ipaH、ipa BCD、ial、Set1A、Set1B、sen、Sat、sigA的检出率在51.9%~100.0%之间;52株致病性志贺菌对磺胺间甲氧嘧啶、土霉素、多西环素等12种药物的耐药率在51.9%以上,其他药物的耐药率在5.8%~34.6%之间,分离的菌株呈现MDR且至少耐4类药物,4株优势血清型代表株HS-1(福氏志贺菌F2a型)、HS-2(福氏志贺菌F1b型)、HS-3(宋内志贺菌Ⅰ相)、HS-4(痢疾志贺菌)的LD50分别为2.51×10^(6)、5.01×10^(6)、6.68×10^(6)和2.11×10^(7)CFU/mL。本研究为冀北地区养殖场中奶/肉犊牛源志贺菌病的流行病调查与防控提供参考依据。

To identify the pathogenic bacteria causing diarrhea in milk/meat calves in livestock farms in the Northern Hebei region and analyze their biological characteristics,this study collected273 anal swabs,feces,and diseased tissue samples from livestock farms in the Northern Hebei region from 2022 to 2023.Bacterial isolation and culture,biochemical identification and PCR methods were used to isolate and identify Shigella.The pathogenicity,serotype and virulence genes of Shigella were detected by artificial infection test in mice,glass plate agglutination test,PCR method and K-B drug sensitivity paper method,respectively.The median lethal dose of the representative strains of its dominant serotypes was determined by artificial infection animal test and modified Koch's method.RT-PCR was used to detect five pathogens:BVDV,BRV,BCoV,BNoV and BNeV.The results showed that the detection of five viral pathogens was negative;94 strains of Shigella were isolated from collected diseased tissues,of which 52 strains were pathogenic Shigella causing disease and death in mice,with a mortality rate of over 40%in mice;52 pathogenic Shigella strains were dominated by Shigella flexneri F2a(21.2%)and F1b(25.0%),Shigella sonnei has PhaseⅠ(19.2%)and Shigella dysentery(13.5%)as the dominant serotypes;the detection rates of 52 pathogenic Shigella virulence genes ipaH,ipa BCD,ial,Set 1A,Set 1B,sen,Sat and sigA were between 51.9%and 100%.The resistance rate of 52 pathogenic Shigella strains to 12 drugs such as sulfamethoxine,oxytetracycline,and doxycycline was more than 51.9%,and the resistance rate of other drugs was 5.8%-34.6%.The isolated strains showed MDR and were resistant to at least four kinds of drugs.The LD_(50)of the four dominant serotype representative strains HS-1(F2a),HS-2(F1b),HS-3(Shigella sonneiⅠ),and HS-4(Shigella dysenteriae)were 2.51×10^(6),5.01×10^(6),6.68×10^(6)and 2.11×10^(7) CFU/mL,respectively.This study provides a reference basis for the epidemiological investigation and prevention and control of shigellosis originating from milk and meat calves in livestock farms in the Northern Hebei region.