两株不同血清型禽源多杀性巴氏杆菌的理化特性研究

禽源多杀性巴氏杆菌Ｐ１０５９（８：Ａ型）和Ｃ４８．１（５：Ａ）对营养的需要较高,在一般培养基上生长不良,相比之下,Ｃ４８－１比Ｐ１０５９生长更差,但在胰蛋白胨肉汤和加５ｍＬ／Ｌ血清的普通培养基中两株菌均生长丰盛;两株菌的生长曲线显示,Ｃ４８－１在对数生长期的繁殖速度较快,最高菌数略低,但衰落期细胞死亡速度较慢;对培养基中ＮａＣｌ含量的要求,Ｃ４８－１为０～２．７％,Ｐ１０５９为０．９％～２．１％;     

18株不同源多杀性巴氏杆菌耐药性分析

通过微量稀释法药敏试验及四环素类耐药基因的扩增对18株不同源多杀性巴氏杆菌进行耐药性分析。并采用PCR及其产物测序检测上述菌株中一类整合酶携带率。结果表明,18株不同源的多杀性巴氏杆菌对氯霉素的耐药率为83.33%,对四环素、强力霉素与庆大霉素的耐药率为50%~61.11%,对氧氟沙星、卡那霉素、恩诺沙星与氟苯尼考的耐药率为27.78%~38.89%。18株菌株均未检测到tet A耐药基因,22.22%的多杀性巴氏杆菌携带tet B耐药基因,5.56%的多杀性巴氏杆菌携带tet O耐药基因,tet K与tet Q两种耐药基因的携带率均为27.28%,而对于tet G的携带率为100%,Ⅰ型整合酶携带率为22.22%。本研究为进一步研究多杀性巴氏杆菌耐药机制及跨物种传播奠定了基础。     

天津地区禽多杀性巴氏杆菌分离株血清型鉴定

通过Ｃａｒｔｅｒ氏荚膜型鉴定方法和琼脂扩散试验,对天津地区禽多杀性巴氏杆菌血清型进行鉴定。结果１６株分离菌中１１株为Ａ：１（４）,４株为Ａ：１（３,４）,１株为－：１（４）表明引起天津地区禽霍乱的致病菌与国内报道的以Ａ：１型为主有一定的差异。     

猪源多杀性巴氏杆菌的生物学特性与ToxA基因鉴定

对17株分离自猪萎缩性鼻炎临床症状猪群的多杀性巴氏杆菌(Pasteurella multocida,Pm),进行了生物学特性试验与ToxA基因鉴定。基于Pm对甘露醇、卫茅醇、山梨醇、海藻糖的发酵能力和产生鸟氨酸脱羧酶的特性,鉴定出10株Pm多杀亚种(P.multocidasubsp.multocida);7株Pm败血亚种(P.multocidasubsp.septica)。根据PCR荚膜分型结果,有8株A型,9株D型。针对ToxA基因中的1 230 bp片段,采用T1/T4引物,4株Pm分离菌株被确定为产毒多杀性巴氏杆菌(ToxingenicP.multocida,T+Pm),占23.53%(4/17)。同时对17株Pm分离株进行药物抗性测定和分析得知,17株Pm对青霉素、先锋霉素Ⅴ、卡那霉素、庆大霉素、丙氟哌酸、氟哌酸、多黏菌素B和利福平的敏感率均在52.9%~100%,而对链霉素和氯洁霉素均不太敏感。     

貉源多杀性巴氏杆菌分离、鉴定及特征分析

为了鉴定引起貉呼吸道疾病的病菌并分析其生物学特征,2018—2020年采集河北地区养殖场中患呼吸道疾病貉的咽拭子、病死貉肺脏心血等病料组织291份,采用分离培养、形态学观察、生化鉴定及PCR等方法对多杀性巴氏杆菌(Pasteurella multocida,PM)进行鉴定,采用人工感染小鼠试验、PCR方法和K-B药敏纸片法分别检测PM的致病性、荚膜血清型、毒力基因及耐药性,根据致病性、荚膜血清型和毒力基因携带情况选择代表株,并检测其半数致死量(LD₅₀)。结果显示,分离得到了163株PM,其中94株能引起小鼠的死亡,其死亡率在40.0%～100.0%之间;94株致病性PM属于4种血清型,以荚膜血清A型(31.9%),荚膜B型(25.5%),荚膜D型(37.2%)为优势流行血清型;94株PM毒力基因psl、exbD、exbB、fimA、oma87、tonB、sodA、fur、sodC、plpB、hgbA、hgbB、ompA、ompH、oxA、pfhA检出率在50.0%～100.0%之间,其他毒力检出率在34.0%～48.9%之间,致病性菌株与携带的毒力基因具有一定相...     

福建及邻近省份禽源多杀性巴氏杆菌的分离鉴定

本研究收集2007~2013年福建省及邻近省份的疑似禽霍乱病死亡鸡、鸭、鹅,进行细菌分离,PCR进行种和荚膜群的鉴定,琼脂扩散试验鉴定其Heddleston氏耐热菌体抗原型。结果共分离鉴定多杀性巴氏杆菌95株,其中鸭源74株,鸡源17株,鹅源4株。其荚膜群全为A型,1型耐热菌体抗原型占67.4%。与20世纪80年代以来我国已有的报道相同,禽源(不含火鸡)多杀性巴氏杆菌血清型仍然以A:1为主。     

多杀性巴氏杆菌耐药机制研究进展

多杀性巴氏杆菌属于革兰氏阴性杆菌,能感染多种宿主,可引起禽霍乱、猪萎缩性鼻炎、猪肺疫、牛羊及兔的出血性败血症。随着抗生素的广泛使用,该病原菌的耐药性也在逐年增加。近几年的研究中更是经常发现部分病原菌会对β-内酰胺类、大环内酯类、喹诺酮类、四环素类、氯霉素类、磺胺类抗生素产生较强的耐药性。本文就多杀性巴氏杆菌对6类抗生素的药物敏感性及耐药机制进行了总结,为新药的研究以及新型疗法的建立提供理论依据。     

一株鸡源荚膜A型多杀性巴氏杆菌分离鉴定及耐药性分析

从疑似禽霍乱病死鸡中分离一株细菌,采用16SrRNA方法鉴定为多杀性巴氏杆菌,PCR方法分析其血清型为A型,对其进行耐药性分析,结果显示分离株对氨苄青霉素、头孢噻肟、恩诺沙星、多粘菌素敏感,对青霉素、链霉素、四环素、氟苯尼考高度耐受.本研究结果为鸡源多杀性巴氏杆菌的防治提供参考.     

禽源多杀性巴氏杆菌保菌方法研究

通过对禽源多杀性巴氏杆菌Ｐ１０５０株和Ｃ４８－１株及６株田间分离细菌的保存观察试验，表明用脱纤羊血和感染小鼠肝脾组织在－２０℃条件下，禽源多杀性巴氏杆菌可存活２年，用鲜血斜面封盖石蜡油保存法，禽源多杀性巴氏杆菌在４℃条件下可存活３个月，菌株保存前生生物学特性及毒力不发生变化，试验提供的方法对禽霍乱流行病学调查和血清分型研究具有实际意义。     

我国禽源多杀性巴氏杆菌血清型研究

本试验用Carter的荚膜群鉴定法和Heddlesten的热稳定抗原鉴定法对我国116株禽源多杀性巴氏杆菌进行了血清学分型研究。研究表明,A∶1型是我国最主要的血清型,占69.8%,其次为A∶1(14)型和A∶3(4)型,分别占6.8%和4.3%。并对我国目前使用和试用的8株禽霍乱弱毒菌苗株进行了定型,其中3株为A∶1型,与主要血清型相同,5株与主要血清型不一致。在A∶1型菌株中,大部分菌株的耐热抗原与1型标准血清反应,形成1条沉淀线,部分菌株(9株)形成2条沉淀线,表明在A∶1型菌株中还存在有抗原差异。     

8株禽多杀性巴氏杆菌外膜蛋白H基因序列分析

为了解国内禽多杀性巴氏杆菌（Pasteurella multocida,Pm）流行株外膜蛋白 H（OmpH）基因的变异情况,参考GenBank中已发表的多杀性巴氏杆菌序列设计1对特异性引物,采用PCR方法对不同来源的8株禽Pm菌株和3个荚膜型参考株（A、B、D）的OmpH基因进行扩增、测序。结果显示,11个菌株的OmpH基因开放阅读框在1002~1071 bp 之间;SignalIP 4.0预测结果表明,信号肽均为N端20个氨基酸残基,成熟蛋白氨基酸残基数量在314~337 aa之间,推测的分子质量在33.76~37.04 ku之间。与GenBank中15个菌株OmpH基因序列比对结果发现,核苷酸同源性在84.9%~100.0%之间;氨基酸同源性在81.5%~100.0%之间;其中C48-1、1010、9003、890920、921012、XJ-e 6个国内禽Pm分离株OmpH序列同源性为100.0%。试验结果表明,国内禽Pm菌株OmpH基因非常保守。     

猪多杀性巴氏杆菌的分离鉴定及生物学特性研究

用PCR方法配合生化鉴定，从有肺炎症状猪的肺脏及进行性萎缩性鼻炎(Progressive atrophic rhinitis，PAR)症状猪的鼻拭子中分离出66株多杀性巴氏杆菌(Pasteurella multocida，Pm)。然后做了药敏试验，并用PCR方法对这66株Pm进行分型及毒素基因的检测，用豚鼠皮肤坏死试验及小鼠致死试验对产毒素多杀性巴氏杆菌(Toxigenie Pasteurella multocida，T^ Pm)进一步鉴定。结果显示PCR鉴定与生化鉴定Pm结果完全一致；PCR分型表明有46株为D型Pm，18株为A型：Pm，1株为B型Pm，1株无法定型；有8株用PcR检测为T^ Pm；豚鼠皮肤坏死试验及小鼠致死试验对这8株T^ Pm的进一步鉴定也表明均为产毒素菌株。所鉴定的8株T^ Pm都为D型，都分离于有严重PAR症状的猪。     

Characteristics of a haemolytic extract from avian Pasteurella multocida

In experimental fowl cholera, the intramuscular inoculation of Pasteurella multocida induces tissue damage that implies proteolytic or cytolytic activity of the bacteria. Such activity could not be demonstrated by conventional in vitro tests. The treatment of P. multocida strain VP21 with Tween-80 yielded an extract that lysed washed chicken red cells. Extracts were active to a maximum titre of 64. Haemolytic activity of the extract was neither affected by boiling nor by extremes of pH, indicating the active component was not a simple protein. Treatment with trypsin had no effect, but it was inactivated by Proteinase K. Yields were highest from bacteria grown in dextrose starch- or casein sucrose-yeast broths; were similar if cultured in air or anaerobically, but were reduced if the bacteria were grown in 5% CO(2). Haemolytic activity was eliminated on exposure to serum or serum albumen. The extract from strain VP21 haemolysed red cells from the chicken, rabbit, sheep, horse, bovine and human, with the highest titres observed on chicken cells. Six other avian strains and seven out of 10 strains of P. multocida from other species yielded an extract which haemolysed chicken red cells. The elaboration of this cytotoxic substance in vivo and its role in pathogenesis remains to be determined.     

体内繁殖禽巴氏杆菌的提取及其交叉保护特性

通过不同攻毒途径、不同攻毒剂量、不同采血时间及不同采血途径的试验表明，以翅静脉途径攻击多杀巴氏杆菌，剂量10亿／只，在鸡濒死挣扎时的即刻颈静脉无菌放血，可以自鸡体采集到最大量的含菌血，每只鸡37～39mL，再将血液进行差速和蔗糖密度梯度离心，可最大限度地提取到血液中的巴氏杆菌，每只鸡300～400亿的总菌量。该结果与报道的提取方法相比，效率提高了1000倍以上。提取的细菌灭活后免疫鸡，经异型菌交叉攻毒试验表明，体内繁殖的巴氏杆菌具有交叉保护特性，而体外培养菌则没有。用该方法提取的巴氏杆菌，其总蛋白和总糖含量均高于培养基培养菌，提示二者之间的差异可能与交叉保护特性存在着一定的相关性。     

恩诺沙星在健康及巴氏杆菌感染鸡体内的药物代谢动力学研究

健康及巴氏杆菌感染鸡 (10CFUC4 8- 1多杀性巴氏杆菌 /只鸡 ,im)按 5mg/kg恩诺沙星单次im后 ,采用反相高效液相色谱法检测用药后不同时间点的血浆药物浓度。结果表明 ,恩诺沙星在健康及巴氏杆菌感染鸡体内的消除均符合二室开放模型。巴氏杆菌感染可显著加速鸡体内恩诺沙星的消除 ,推测可能与感染鸡体内巴氏杆菌裂解后产生的内毒素有关。内毒素可刺激骨髓产生大量白细胞 ,而恩诺沙星又恰恰能在吞噬细胞中富集 ,且在富集后可迅速向周围炎性组织中释放 ,因此导致血中药物浓度下降。提示在应用恩诺沙星预防、治疗细菌性疾病时 ,利用其在健康鸡体内的药物动力学参数指导临床用药 ,有时是不适宜的。     

Antibiotic Sensitivity Patterns among Indian Strains of Avian Pasteurella multocida

An investigation was carried out to study the antibiotic sensitivity of avian strains of Pasteurella multocida and to select an effective antimicrobial agent for control of avian pasteurellosis in India. A total of 123 strains of P. multocida recently isolated from different avian species (chicken, duck, turkey, quail, and goose), from different regions of India were subjected to antibiotic sensitivity tests using 20 different antibiotics. Absolute resistance was observed against sulfadiazine. The studies indicated that the strains were most sensitive to chloramphenicol (73.98%), followed by enrofloxacin (71.54%), lincomycin (64.23%), norfloxacin (61.79%) and doxycycline-HCl (56.91%). The majority of the strains were found to exhibit intermediate sensitivity. Chloramphenicol was selected and suggested for treatment. Antibiogram studies also revealed the emergence of multidrug-resistant strains of P. multocida among Indian poultry.     

我国猪群中多杀性巴氏杆菌的基因型分析

本研究旨在了解我国猪群中流行的多杀性巴氏杆菌(Pm)的主要基因型。利用荚膜基因分型、脂多糖(LPS)基因分型、多位点序列分型(MLST)对2013年12月—2017年12月4年间来源于我国各地区规模化猪场患有疑似呼吸系统疾病的病死猪肺、鼻拭子、气管、肝等样品中分离鉴定的Pm进行基因型分析,并对23种主要毒力基因进行检测。结果表明,当前在我国猪群中流行的Pm的荚膜基因型为A(48.85%)、D(42.75%)、F(2.67%),优势基因型为A和D;LPS基因型为L3(25.00%)和L6(75.00%)型,优势基因型为L6;MLST基因型为ST3(21.25%)、ST10(27.50%)、ST11(42.50%)、ST12(2.50%)、ST16(2.50%)、ST74(1.25%)及ST75(2.50%),优势基因型为ST3、ST10和ST11。如果将荚膜基因型、LPS基因型和MLST基因型组合起来看,当前在我国猪群中流行的Pm的主要基因型为荚膜:脂多糖:MLST基因型A:L3:ST3(20.00%)、A:L6:ST10(26.25%)及D:L6:ST11(42.50%)。毒力基因检测的结果发现部分毒力基因的分布表现出一定的"基因型偏好性"。结果提示,D:L6:ST11是我国猪群中流行的多杀性巴氏杆菌的主要基因型,可能与我国猪群中由多杀性巴氏杆菌导致的呼吸道疾病密切相关。     

Deoxyribonucleic acids from Pasteurella multocida and Pasteurella haemolytica]

Pasteurella multocida and P. haemolytica strains contain between 1.5 and three per cent phosphorus, between nine and 14 per cent nitrogen, between two and four per cent DNA, and between five and 18 per cent RNA, the precise figures depending on culturing conditions. High-molecular DNA may be isolated by means of bacteriolysis, using deoxycholate or dodecylsulphate and the usual steps of purification, with yield and purity differing by strains. DNA with sufficient purity can be obtained from Sepharose 2 B by gel chromatography. The isolated DNA yields were characterised, base values being between 37 and 38 per cent GC for P. haemolytica and between 41 and 48 per cent GC for P. multocida. Highly suitable precursors to DNA synthesis for tritium labelling are 3H-thymidine, which is incorporated in excess of 3H-thymine by a factor of 255, as well as 3H-uracil, with its activity being recovered also from the pyrimidine bases of DNA via pyrimidine biosynthesis.     

Adaptive Acquisition of Novobiocin Resistance in Pasteurella multocida Strains of Avian Origin

Naturally occurring strains of Pasteurella multocida are atypically susceptible to hydrophobic antibiotics such as novobiocin, despite their Gram-negative cell envelope ultrastructure. Four strains adaptively resistant to 1000 g/ml of novobiocin were obtained by sequentially subculturing cell surface hydrophobic variants of avian origin in the presence of increasing antibiotic concentrations. Adaptive novobiocin resistance was accompanied in all cases by the concomitant acquisition of resistance to coumermycin, a hydrophobic antibiotic possessing the same mechanism of action, but not to the functionally disparate hydrophobic antibiotic rifamycin. The acquisition of resistance was not accompanied by alterations in the lipid composition of the cell envelope. Subsequent growth of adaptively resistant strains in the absence of novobiocin did not result in the restoration of susceptibility to either novobiocin or coumermycin. Acquisition of adaptive resistance in encapsulated parental strains resulted in an inability to synthesize capsular material and enhanced cell surface hydrophobicity; however, parental encapsulation and decreased cell surface hydrophobicity were restored upon removal of novobiocin. These data suggest that acquisition of adaptive resistance to novobiocin conferred in this manner is the result of a stable genetic event affecting the mechanistic target of both novobiocin and coumermycin rather than a physiological adaptation involving outer membrane impermeability.