副猪嗜血杆菌与猪丹毒丝菌混合感染病原的分离鉴定及生物学特性分析

从疑似副猪嗜血杆菌和猪丹毒丝菌混合感染病猪无菌采集心脏、肝脏、脾脏、肺及心包积液,采用常规分离培养法分离病原菌,通过染色镜检、生化鉴定、卫星试验和核酸测序同源性比对,确诊为副猪嗜血杆菌与猪丹毒丝菌混合感染。为进一步了解2株菌的生物学特性,测定了2株菌以及混合菌液的半数致死量(LD50)以及对常用抗生素的药物敏感性。结果显示:(1)将2株菌的序列与GenBank数据库中已发表序列进行同源性比对,副猪嗜血杆菌与猪丹毒丝菌的同源性各达到96%和99%;(2)致病性试验中,SPF鼠均在接种后24h左右开始死亡,从其心脏、肝脏、脾脏及肺均回收到攻毒菌,表明2株菌均为此次疫病的主要致病菌;(3)通过测定副猪嗜血杆菌和猪丹毒丝菌的LD50分别为5.28×109、3.58×104 CFU/mL,进一步证明二者具有较强毒性。根据2株菌的LD50等体积混合2种菌液注射SPF鼠,副猪嗜血杆菌与猪丹毒丝菌LD50均出现下调,分别为3.70×109、2.51×104 CFU/mL,表明混合感染增强了二者的致病力;(4)药敏试验结果显示副猪嗜血杆菌与猪丹毒丝菌对常用抗生素的耐药性与临床耐药谱相一致,未出现新的耐受药物;同时筛选出了2种菌的共敏感药物氧氟沙星和氟苯尼考,有利于对疾病的快速治疗与防控。     

鱼源鸡源猪丹毒杆菌对抗菌药物的敏感性试验

关于猪丹毒杆菌对抗菌药物的敏感性研究,以往的资料仅涉及猪源菌株,未见有鱼源和鸡源菌株的有关报道。我们以16种常用抗菌药物对分离自鱼和鸡的17株猪丹毒杆菌进行了本试验。现将结果报告如下。     

1株猪丹毒杆菌的分离鉴定及药敏试验

无菌操作采集某规模养猪场疑似感染猪丹毒杆菌的病死猪肺脏等组织进行细菌分离,从中分离出一株病原菌,经细菌分离培养、形体特性及生化鉴定,确定为猪丹毒杆菌。通过药敏试验表明,该菌对阿莫西林、青霉素及头孢类药物高度敏感,为临床用药防治猪丹毒杆菌病提供科学根据。     

四川攀西地区奶牛乳房炎病原菌调查与敏感药物筛选试验

为了解四川攀西地区奶牛乳房炎病原菌和主要病原菌的耐药性情况,采用常规方法对3个奶牛场92个牛奶样品中的病原菌进行了分离鉴定,结果分离到17种菌,共计86株,分离到的病原菌数量最多的依次是大肠杆菌23株、绿脓杆菌17株、葡萄球菌15株、链球菌7株、沙雷菌属4株、弗劳氏枸橼酸杆菌3株、沙门氏菌3株、阴沟肠杆菌复合菌3株、粪肠球菌2株、猪丹毒丝菌2株以及坐皮肤球菌、产酸克雷伯氏菌、屎肠球菌、睾丸酮丛毛单胞菌、枯草芽孢杆菌、施氏假单胞杆菌、奇异变形杆菌各1株。采样量为12头、16头、18头奶牛的3个场,分别分离出14种、13种、16种乳房炎病原菌。同时,采用常规药敏试验方法对分离病原菌株数量最多的8种主要病原菌即大肠杆菌、绿脓杆菌、葡萄球菌、链球菌、沙雷菌、弗劳氏枸橼酸杆菌、沙门氏菌、阴沟肠杆菌复合菌进行了常用抗生素敏感试验,结果表明他们均存在不同程度的耐药性。     

仔猪副猪嗜血杆菌的分离鉴定及耐药性检测

为了了解海南藏族自治州共和县规模化猪场中仔猪副猪嗜血杆菌分离株的流行情况及对常用抗菌药的耐药情况。本试验于2022—2023年采集海南藏族自治州共和县规模化猪场中患疑似副猪嗜血杆菌病的仔猪鼻拭子、关节液、胸腔积液、肺脏等病料,共计283份。采用细菌分离鉴定、PCR方法对采集的病料进行副猪嗜血杆菌分离鉴定,采用人工感染小鼠试验验证分离株的致病性,采用纸片扩散法(Kirby-Bauer,简称K-B法)检测致病性副猪嗜血杆菌分离株对临床中常用抗菌药的耐药性。结果显示：从采集的283份病料中分离到128株副猪嗜血杆菌,通过致病试验显示,91株副猪嗜血杆菌能引起小鼠不同程度发病与死亡,为致病性菌株;分离的91株致病性副猪嗜血杆菌对磺胺间甲氧嘧啶、阿莫西林、氨苄西林等6种药物的耐药率为85.7%以上,对其他药物的耐药率为3.3%～25.3%,本研究为该地区仔猪副猪嗜血杆菌合理用药及防控提供参考。     

白鹭大肠埃希菌的分离鉴定与药敏试验

对3份白鹭新鲜粪便进行细菌分离纯化、染色镜检和生化鉴定,并用Kirby-Bauer法对分离细菌用30种抗菌药进行了耐药性试验。结果表明,分离到3株革兰阴性小杆菌为大肠埃希菌,分别编号为E1、E2和E3。3株白鹭大肠埃希菌都对青霉素G、阿莫西林、万古霉素和洁霉素耐药,对常用广谱抗菌药物有2重以上耐药性,但对试验用的氨基苷类抗生素、喹诺酮类药物和复方新诺明高度敏感。     

一株猪丹毒杆菌的分离及鉴定

猪丹毒为我国三大猪传染病之一,给养猪业造成严重的经济损失。为了诊断某猪场猪只发病情况,试验从送检病料中分离到一株分离菌,对分离菌株进行镜检、生化鉴定和16S rDNA基因序列分析,结果判定分离菌株为猪丹毒杆菌,从而将此病例确诊为猪丹毒,为养殖场防治该病提供理论依据。     

一株猪丹毒杆菌分离株的致病性与耐药性分析

从某猪场病死猪心血、肝、脾等组织分离到1株革兰阳性杆菌,经细菌形态、16S rRNA基因扩增,确定分离菌为猪丹毒杆菌。对分离菌进一步开展了药物敏感性试验、耐药基因的检测、细菌致病性试验及免疫保护性试验。结果发现:分离株对β-内酰胺类、大环内酯类、氟苯尼考高敏,但对氨基糖苷类、四环素、林可霉素、诺氟沙星等耐药;分离株可扩增出氨基糖苷类耐药基因aac C2和四环素耐药基因tetB,但菌株中没有扩增到四环素耐药基因tetA。细菌致病性试验表明该毒株对小鼠的LD50为1.77×10~5cfu/mL。小鼠免疫保护试验结果表明,疫苗G4T10免疫后28 d,以分离株攻毒,对照组全部死亡(5/5),免疫组小鼠攻毒后没有死亡,仅表现为体重减轻,表明现用猪丹毒疫苗能保护小鼠抵抗猪丹毒的致死性攻击。     

一株猪丹毒杆菌的分离及鉴定

猪丹毒为我国三大猪传染病之一,给养猪业造成严重的经济损失。为了诊断某猪场猪只发病情况,试验从送检病料中分离到一株分离菌,对分离菌株进行镜检、生化鉴定和16S rDNA基因序列分析,结果判定分离菌株为猪丹毒杆菌,从而将此病例确诊为猪丹毒,为养殖场防治该病提供理论依据。     

猪丹毒杆菌分离鉴定及体外抑菌试验

为了确诊临床疑似猪丹毒病例,采集病死猪的内脏,用常规生化法和PCR法进行细菌分离鉴定,用牛津杯法做体外抑菌试验。结果:分离菌H_2S、靛基质、枸橼酸盐等试验为阴性;分解葡萄糖、麦芽糖等,不能分解蔗糖;对青霉素、头孢唑啉等高度敏感,对氯霉素、链霉素等中度敏感;枯草芽孢杆菌、鸡唾液乳酸杆菌和猪小肠乳酸杆菌对其均有较强的抑制作用。结论:分离菌为猪丹毒杆菌,该病是由猪丹毒杆菌引起的急性热性传染病,防治首选药物为青霉素,枯草芽孢杆菌、鸡唾液乳酸杆菌和猪小肠乳酸杆菌可减少或部分取代抗生素对该病的防治。     

猪链球菌7型的分离鉴定及药敏试验

某猪群暴发类似猪链球菌病,为确诊和防制该病,通过将病料接种到血液营养琼脂培养基和TSA培养基分离细菌,对分离菌株进行生化鉴定和PCR鉴定,并接种小白鼠进行动物试验和药敏试验。结果表明,分离的细菌为猪链球菌7型,对小白鼠具有致病性,且对恩诺沙星和头孢唑啉高度敏感。该结果对临床防制猪链球菌病具有参考意义。     

猪源益生菌的分离筛选及部分生物学特性研究

经形态、染色特性观察,从健康仔猪肠道分离出5株杆菌、1株链球菌及3株芽孢杆菌,初次筛选出产酸及抑菌效果好的杆菌、芽孢杆菌各2株,链球菌1株,经生化鉴定为乳酸杆菌2株,乳酸链球菌1株,地衣芽孢杆菌、蜡样芽孢杆菌各1株,对其进行了耐酸、耐胆汁及动物安全性试验。结果表明,筛选菌株有较强的产酸、耐酸及耐胆汁特性,对常见肠道致病菌抑制作用明显,动物使用安全,可作为猪用益生素制剂的候选菌种。     

Comparison of methods for antimicrobial susceptibility testing and MIC values for pleuromutilin drugs for Brachyspira hyodysenteriae isolated in Germany

In Germany treatment of swine dysentery is hampered by Brachyspira hyodysenteriae strains showing elevated MIC values to the few antibiotics licensed. Therefore, susceptibility testing of clinical isolates is an important service to the swine practitioner. This study compares the established agar dilution procedure for antimicrobial susceptibility testing of this fastidious anaerobe to the broth microdilution test newly developed [Anim. Health Res. 2 (2001) 59; Vet. Microbiol. 84 (2002) 123; J. Clin. Microbiol. 41 (2003) 2596]. A total of 221 isolates were examined twice with either test procedure using tiamulin and valnemulin as antibiotics. Both methods gave reproducible results, and the MIC values for the reference strains B. hyodysenteriae B204 and Staphylococcus aureus ATCC 29213 corresponded to previously published data. However, the results for individual strains differed significantly for both tests (P < 0.001) with MIC values being on average one dilution step lower in the broth dilution method. The 221 strains used for comparing test procedures were isolated between 1989 and 2001. An additional 102 strains isolated in 2002 were tested only with the broth dilution procedure. A significant rise in the average MIC value for both pleuromutilins could be demonstrated when comparing earlier isolates to those from 2000 to 2001 (P < 0.05), while in 2002 the average MIC significantly decreased when compared to the value in 2000 (P < 0.05). However, strains with MIC values for tiamulin as high as 8 microg/ml (broth dilution) could still be isolated.     

Characterization of Erysipelothrix rhusiopathiae strains isolated from acute swine erysipelas outbreaks in Eastern China

Recently, a series of acute swine erysipelas outbreaks occurred in Eastern China. Eight strains isolated from cases of septicemia were determined as serotype 1a, and 4 of the isolates were resistant to acriflavine. One isolate strain named {"type":"entrez-nucleotide","attrs":{"text":"HX130709","term_id":"383559396","term_text":"HX130709"}}HX130709 was attenuated on agar media containing acriflavine dye. The 432-bp hypervariable region in spaA gene of the field and attenuated strains were amplified and sequenced. It was further compared with the vaccine strain G₄T₁₀, and thus, the eight field strains can be divided into four spaA-types. The partial spaA gene analysis also showed that no point mutations occurred among different archived passages of {"type":"entrez-nucleotide","attrs":{"text":"HX130709","term_id":"383559396","term_text":"HX130709"}}HX130709 during the attenuation. Results of pulsed-field gel electrophoresis showed that eight distinct patterns with 22 to 30 DNA fragment bands were produced from field strains, and twelve distinct patterns with 23 to 27 DNA fragment bands were produced from different passages of the attenuated strains. Mouse pathogenicity test showed that the mortality of the mice infected with 10⁴ CFU field strains was 100% and the attenuation of strain {"type":"entrez-nucleotide","attrs":{"text":"HX130709","term_id":"383559396","term_text":"HX130709"}}HX130709 occurred between 46 and 50 passages. All the field and attenuated strains were highly sensitive to β-lactam antibiotics, tetracyclines and macrolides. So, we can make conclusions that the acute swine erysipelas outbreaks in Eastern China were caused by serotype 1a E. rhusiopathiae strains with different biochemical characteristics, and the virulence of serotype 1a E. rhusiopathiae strains is unrelated with some point mutations in 432-bp hypervariable region of the spaA gene.     

Assessment of Antimicrobial Susceptibility of Virulent Strains of Rhodococcus equi Isolated From Foals and Soil of Horse Breeding Farms With and Without Endemic Infections

Rhodococcus equi is an opportunistic, intracellular saprophyte that causes severe pyogranulomatous pneumonia in foals. The bacterium displays in vitro susceptibility to many antibiotics. The highest efficacy against R. equi in vitro and in vivo is achieved by using a combination of rifampicin and macrolide antibiotics. Recent years have seen an upward trend in the minimum inhibitory concentration (MIC) of rifampicin and erythromycin, suggesting increasing resistance of R. equi to these antibiotics. The aim of the study was to determine the antimicrobial activity of 24 selected antibiotics against R. equi strains isolated from dead foals and from the environment of horse breeding farms with and without endemic R. equi infections. Minimum inhibitory concentration gradient strips were used to determine the lowest concentration of the antibiotic that inhibited the growth of R. equi. Based on normal MIC distribution, an epidemiologic cutoff values (ECOFF) were assessed for particular antibiotics and R. equi strains. The results were compared with ECOFFs for S. aureus, according to the European Committee on Antimicrobial Susceptibility Testing data. The data indicate that the lowest MIC values were obtained for clarithromycin, rifampicin, imipenem, and vancomycin. The majority of R. equi strains can be classified as wild type in relation to the majority of antibiotics. A small percentage of strains presented non-WT (NWT) with the exception of SXT, for which 35% of strains were classified as NWT. The lack of interpretative criteria for R. equi creates a real problem in the assessment of antibiotic sensitivity both for clinical and scientific purposes.     

广东地区动物源性大肠杆菌的分离及药敏分析

由广东多个地区猪及禽类养殖场采集疑似大肠杆菌病病料,分离出猪源及禽源大肠杆菌共71株。血清型鉴定实验确定52株的血清型,其中猪源大肠杆菌28株,优势血清型为O101及O157,这两种血清型分别占25％与17．9％;禽源大肠杆菌24株,优势血清型为O119,占62．4％。选用10种抗生素试纸进行药敏试验（庆大霉素、复方新诺明、氟哌酸、红霉素、羧苄青霉素、头孢氨苄、青霉素G、氯霉素、氨苄青霉素、丁胺卡那霉素）,结果表明实验菌株耐药性程度较高。     

新疆地区2株驴源马流产沙门菌的分离鉴定及致病性分析

[目的] 研究确定导致新疆地区2个规模化驴场出现流产的疑似病原沙门菌,并探究其致病能力和耐药性情况。[方法] 通过细菌分离培养、形态学观察、生化试验对分离菌进行了鉴定,并对分离菌的鞭毛基因FliC进行了PCR扩增及序列分析,通过致病性测定、荷菌量检测及病理组织学观察,鉴定和分析了分离菌的致病性,并通过药敏试验分析其耐药性。[结果] 通过细菌分离培养、形态学观察、生化试验,确定分离到的2株细菌均为马流产沙门菌,分别命名为G1-1和XD1-2。对这2个分离株的鞭毛基因FliC的遗传进化分析结果显示,2株分离菌FliC氨基酸序列之间的相似性为99.0%,2株分离菌FliC氨基酸序列与爱尔兰马源马流产沙门菌分离株Ireland-HE801373、Ireland-HE801378株相似性均最高,且均为99.3%;分离株G1-1与爱尔兰马源马流产沙门菌分离株Ireland-HE801373和Ireland-HE801378及国内马源马流产沙门菌分离株China-KJ486797.1和China-KJ486769.1亲缘关系较近,分离株XD1-2则与美国肠炎沙门菌分离株USA-EBQ1214032.1亲缘关系较近,相似性分析与进化树结果一致。小鼠致病性试验显示,2株分离菌对小鼠的毒性均较强且致病性存在差异,XD1-2对小鼠的致病性强于G1-1。分离菌G1-1对9种抗菌药物耐药,对11种抗菌药物敏感。分离菌XD1-2对11种抗菌药物耐药,对5种抗菌药物敏感。[结论] 本试验成功分离到2株驴源沙门菌,不同养殖场来源的驴源沙门菌的致病能力和耐药性有一定差异,对鞭毛基因FliC的进化分析也显示出差异,本研究结果为驴源沙门菌的防治提供了一定的参考依据。     

奶牛流产胎儿及阴道分泌物病原菌的检测和药物敏感性分析

调查北京地区7个奶牛场奶牛流产的发病原因,采集流产胎儿及恶露不尽母牛的阴道分泌物进行细菌分离鉴定、致病性及中西药的药敏试验,并测定中药的MIC和MBC。共分离出9种42株细菌,其中金黄色葡萄球菌11株、腐生葡萄球菌6株、表皮葡萄球菌2株、变形菌属5株、克雷伯氏菌属3株、埃希氏菌属2株、蜡样菌属3株、黏液奈瑟氏菌属4株、浅黄奈瑟氏菌属2株、其他未鉴定出种属的菌4株;其中,葡萄球菌占45.2%、杆菌占23.8%、其他革兰氏阴性菌占31.0%。通过致病性试验,有28株为致病菌。对其中几种分离菌进行了25种常用抗生素、7种中药及一个复方的体外抑菌试验,结果表明,这些菌对多数抗生素有耐药性,如青霉素和链霉素,大多数革兰氏阴性菌对喹诺酮类药物比较敏感,头孢菌素类药物则对革兰氏阳性菌有较好的抑制效果,中药金银花和瓜蒌对这些菌的体外抑菌作用较好。     

扭角羚肺炎克雷伯氏菌的分离鉴定

本试验旨在报告四川省野生扭角羚自然生存过程中致病性细菌的检测及生物学特征。试验对发病死亡扭角羚病变组织进行细菌学检查及分离,对分别从肝脏、脾脏和肺脏分离得到的4株优势菌进行形态特征、生化特性和16SrDNA分子鉴定,判定为肺炎克雷伯氏菌。这4株菌对供试小白鼠均有致病性。测其LD50,分别为2.9×107、2.9×107、4.5×107和1.1×108 CFU/只。4株分离菌对供试的先锋霉素等敏感,对阿米卡星等中度敏感,对红霉素等耐药。推测这4株肺炎克雷伯氏菌是导致扭角羚死亡的主要病原菌。     

四种兽用抗生素的体外抑菌效果评价

本实验用试管药物稀释了四种兽用抗生素对猪常见病原菌和正常菌的最小抑菌浓度 （minimal inhibitory concentration, MIC） 和最小杀菌浓度 （minimal bactericidal concentration, MBC）。通过实验发现：各种菌对四种药物的敏感性存在很大的差异,大多数细菌对药物C和D较敏感而对药物A和B存在较严重的耐药。