Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS)

Comprehensive identification of conditionally essential genes requires efficient tools for generating high-density transposon libraries that, ideally, can be analysed using next-generation sequencing methods such as Transposon Directed Insertion-site Sequencing (TraDIS). The Himar1 (mariner) transposon is ideal for generating near-saturating mutant libraries, especially in AT-rich chromosomes, as the requirement for integration is a TA dinucleotide, and this transposon has been used for mutagenesis of a wide variety of bacteria. However, plasmids for mariner delivery do not necessarily work well in all bacteria. In particular, there are limited tools for functional genomic analysis of Pasteurellaceae species of major veterinary importance, such as swine and cattle pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, respectively. Here, we developed plasmids, pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the transposase gene), that allow delivery of mariner into both these pathogens, but which should also be applicable to a wider range of bacteria. Using the pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant libraries in both A. pleuropneumoniae and P. multocida that showed a near random distribution of insertions around the respective chromosomes as detected by TraDIS. A preliminary screen of 5000 mutants each identified 8 and 14 genes, respectively, that are required for growth under anaerobic conditions. Future high-throughput screening of the generated libraries will facilitate identification of mutants required for growth under different conditions, including in vivo, highlighting key virulence factors and pathways that can be exploited for development of novel therapeutics and vaccines.

养殖石鲽鱼病原菌鸭瘟巴斯德氏菌(Pasteurella anatipestifer)初步研究

1998年7月从寻山水产集团公司养鱼场养殖的石鲽鱼病鱼肠道中分离到多株细菌,经人工感染试验证明其中1株为致病菌。该菌株为革兰氏阴性短杆菌,无动力, 32℃以上不生长。氧化酶阳性。发酵葡萄糖产酸不产气。经BIOLOG MICROSTATION SYSTEM鉴定为鸭瘟巴斯德氏菌(Pasteurella anatipestifer)。药敏试验表明,该菌株对呋喃唑酮、呋喃妥因、氟哌酸、丁胺卡那和环丙沙星敏感。

Construction and Virulence of Filamentous Hemagglutinin Protein B1 Mutant of Pasteurella multocida in Chickens

Pasteurella multocida, a Gram-negative nonmotile coccobacillus, is the causative agent of fowl cholera, bovine hemorrhagic septicemia, enzoonotic pneumonia and swine atropic rhinitis. Two filamentous hemagglutinin genes, fhaB1 and JhaB2, are the potential virulence factors. In this study, an inactivationfhaB1 mutant ofP. multocida in avian strain C48-102 was constructed by a kanamycin-resistance cassette. The virulence of thefhaB1 mutant and the wild type strain was assessed in chickens by intranasal and intramuscular challenge. The inactivation offhaB1 resulted in a high degree of attenuation when the chickens were challenged intranasally and a lesser degree when challenged intramuscularly. ThefhaB1 mutant and the wild type strain were investigated their sensitivity to the antibody-dependent classical complement-mediated killing pathway in 90% convalescent chicken serum. ThefhaB1 mutant was serum sensitive as the viability has reduced between untreated serum and heat inactivated chicken serum （P〈0.007）. These results confirmed that FhaB1 played the critical roles in the bacterial pathogenesis and further studies were needed to investigate the mechanism which caused reduced virulence of the fhaB1 mutant.

Development of Multiplex-PCR for Identification of Pasteurella multocida,Haemophilus parasuis and Actinbacillus pleuropneumoniae

[ Objective] To develop multiplex-PCR for diagnosis of mixed infection caused by Pasteurella multocida （ PM）, Haomophilus parasuis （HPS） and Actinbaci/lus pleuropneumoniae （App）. [ Method ] PCR method was developed to detect single infection caused by PM, HPS or App. The conditions of amplification and primers were optimized, and the multiple-PCR was developed to detect mixed infection of PM, HPS and App. [ Result] A 457-bp band, a 821-bp band and a 342-bp band were simultaneously amplified in the one PCR reaction system. The method had high sensitivity and specificity. [ Conduslon] The multiple-PCR is successfully developed and can be used for differential diagnosis of PM, HPS and App.

Presence of <i>Actinobacillus pleuropneumoniae, Streptococcus suis, Pasteurella multocida, Bordetella bronchiseptica, Haemophilus parasuis and Mycoplasma hyopneumoniae</i>in upper respiratory tract of swine in farms from Aguascalientes, Mexico

Respiratory diseases are one of the most important health problems in pig herds. The porcine respiratory disease complex (PRDC) is the term used to describe pneumonic diseases caused by multiple infectious agents that provoke weight loss in animals or death. In the PRDC multiple pathogens (bacteria and/or viruses) work in combination to induce this respiratory disease. Within this complex, Actinobacillus pleuropneumoniae, Streptococcus suis, Pasteurella multocida, Bordetella bronchiseptica, Haemophilus parasuis and Mycoplasma hyopneumoniae are the main bacterial pathogens involved in great economic losses to the swine industry. The aim of this work was to estimate the presence of A. pleuropneumoniae, S. suis, P. multocida, B. bronchiseptica, H. parasuis and M. hyopneumoniae in the upper respiratory tract of pigs in representative swine farms inAguascalientes,Mexico, using PCR technique. The study was performed in 14 swine farms. We obtained a total of 212 nasal swabs. Near 20% of samples were positive for A. pleuropneumoniae (located in the 79% of farms);17% were positive for S. suis (in 86% of farms), of these, 3% were S. suis serovar 2;30% were positive for H. parasuis (93% of farms);23% of the samples to P. multocida (in 79% of farms);and 19% to M. hyopneumoniae (in 64% of farms). B. bronchiseptica was not detected in this study. The results obtained show that bacterial pathogens of PRDC were present in the upper respiratory tract of pigs in all farms studied;therefore, these pathogens are widely disseminated in pig farms of Aguascalientes, Mexico.

Isolation and Identification of Mannheimia haemolytica and Pasteurella multocida Species from Ruminants in Six Different Regions in Morocco

Pasteurella species is considered the principal pathogen of the respiratory tract.Mannheimia haemolytica and Pasteurella multocida were investigated and typed from nasal swabs and tissues taken from sheep,goat and cattle.Indeed,41 lung and 121 nasal swabs samples were collected from animals with respiratory diseases during 2015 to 2017 in six different regions in Morocco.At first,a screening of Pasteurella species using the real time PCR(RT-PCR)was carried out,then all isolated strains on agar blood were confirmed by PCR gel based assay specific for M.haemolytica and P.multocida.Pathogenicity was evaluated in mice and histopathological examination was done on some of lung tissue.The results revealed that 34 samples of which 28(55%)from nasal swabs and six(38%)from lungs were positive for M.haemolytica and nine samples of which seven(14%)from nasal swabs and two(13%)from lungs were positive on P.multocida serogroup A.Seventy-two percent(72%)isolates were highly pathogenic to mice,which is in accordance with the results obtained by histopathology examination.This is the first report for widespread infections of Pasteurella(M.haemolytica&P.multocida)in ruminants in Morocco.Therefore,measures including development of vaccines are highly required to mitigate the impact of the bacteria in animals.

Molecular analysis of Pasteurella multocida strains isolated from fowl cholera infection in backyard chickens

Objective:To characterize Pasteurella isolated from backyard chickens using whole cell protein lysate profiles and random amplified polymorphic DNA(RAPD)techniques to show their genetic relationship because Pasteurclla multocida(P.multocida)is an important cause of fatal infections in backyard chickens.Methods:Twenty one P.multocida isolates were recovered previously from clinical cases of fowl cholera belonging to individual owners and phenotypically analysed using biochemical tests and serotyping were used far the genetic characterization.Results:Phylogenetic study based on both methods revealed that the recovered population of P.multocida isolated from backyard chickens differs markedly,constituting a well-separated cluster and appearance of 3 distinguishing lineages with greater discrimination shown by RAPDPCR that resulted in two suclusters in cluster A and three subclusters in cluster B and were related greatly with capsular serogroups for the examined strains.The whole cell protein revealed the presence of dominant protein bands at approximately 41 and 61 kDa in all of the examined isolates that may be a virulent proteins share in the increasing of its pathogenicity.Clear distinctive bands ranged from 123 to 1534 bp.Conclusions:Based on the previous findings,there are three spreading clusters that may indicate the association of a small number of P.multocida variants with the majority of cases suggesting that certain clones of P.multocida are able to colonue the examined backyard chickens.Also,the ease and rapidity of RAPD-PCR support the use of this technique as alternative to the more labour-intensive SDS-PACE system for strain differentiation and epidemiological studies of avian P.multocida.Further application of RAPD technology to the examination of avian cholera outbreaks in commercially available flocks may facilitate more effective management of this disease by providing the potential to investigate correlations of P.multocida genotypes,to identify affiliations between bird types and bacterial genotypes,and to elucidate the role of specific bird species in disease transmission.

Adjuvant activity of Pasteurella multocida A strain,Pasteurella multocida B strain and Salmonella typhimurium bacterial DNA on cellular and humoral immunity responses against Pasteurella multocida specific strain infections in Balb/c mice

Objective: To evaluate the effects of Pasteurella multocida(P. multocida) vaccines on the expression and release of antibodies, interleukin(IL)-6 and IL-12 by serum. Methods: Balb/c mice were immunized with two formalin and iron inactivated vaccine doses within 2 weeks. The vaccines were adjuvant with P. multocida A strain, P. multocida B strain and Salmonella typhimurium bacterial DNA(AbDNA, BbDNA and SbDNA for short, respectively). The animals were challenged 4 weeks after immunization. Blood of mice was collected to detect the change of specific antibody, IL-6, and IL-12 using ELISA. Results: The specific antibody and interleukins in the immunized group increased significantly compared to the control mice after vaccination and challenge(P<0.05). The highest release of these cytokines was obtained by P.multocida inactivated with iron and adjuvant with AbDNA at a concentration of 25 μg/mL. The antibody titer peak was 0.447 in mice vaccinated with iron-killed whole-cell antigen adjunct with AbDNA. The time-courses of release showed that bacterial DNA was able to stimulate IL-6 and IL-12 production more than alum(P<0.05). Conclusions: Our findings introduce that bacterial DNA is capable of releasing an immunological response with several cytokines.These indicate that bacterial DNA entrapped with killed P. multocida antigen is a new and effective adjuvant to enhance specific immunity and resistance of animal against the infectious pathogen, which could simplify the development of highly promising strong adjuvant.

Pasteurella multocida capsular:lipopolysaccharide types D:L6 and A:L3 remain to be the main epidemic genotypes of pigs in China

Pasteurella multocida is a leading cause of respiratory disorders in pigs.This study was designed to understand the genotypical and antimicrobial resistant characteristics of P.multocida from pigs in China.To achieve this,we briefly investigated 158 P.multocida isolates from pigs with respiratory disorders in China between 2019 and 2020.Genotyping through multiplex PCR assays assigned these 158 isolates into capsular genotypes A(60.13%,95/158),D(35.44%,56/158),F(4.43%,7/158),and/or lipopolysaccharide(LPS)genotypes L3(28.48%,45/158)and L6(66.46%,105/158).In addition,eight isolates(5.06%,8/158)were found to be nontypable using the LPS genotyping method.When combining the capsular genotypes and the LPS genotypes,D:L6(34.81%,55/158)and A:L6(31.65%,50/158)were the predominant genotypes,followed by A:L3(24.05%,38/158).PCR detection of virulence factor-encoding genes showed that over 80%of the isolates were positive for exbB,tonB,exbD,ompH,ptfA,fimA,sodA,sodC,fur,ompA,oma87,plpB,hsf-2,nanH and hgbB,suggesting the presence of these genes were broad characteristics of P.multocida.We also found approximately 63.92%(101/158),51.27%(81/158),8.86%(14/158),7.59%(12/158),3.16%(5/158),0.63%(1/158),and 0.63%(1/158)of the isolates grew well in media with the presence of colistin(4μg/mL),tetracycline(16μg/mL),tigecycline(1μg/mL),ampicillin(32μg/mL),chloramphenicol(32μg/mL),cefepime(16μg/mL),and ciprofloxacin(1μg/mL),respectively.This study contributes to the understanding of genotypes and antimicrobial resistance profile of P.multocida currently circulation in pigs of China.

Enzyme-Linked Immunosorbent Assay for Pasteurella Multocida in Rabbits and Serotypes of the Isolates

The control and prevention of pasteurellosis in rabbits which makes the hosts unsuitable for experimental use raised the needs to improve and simplify the procedures of Enzyme-Linked Immunosorbent Assay (ELISA) for detect of antibody against P.multocida. A comparison on the sensitivity and specificity of bacterial culture of antemortem and postmortem samples, complement fixation test and enzyme-linked immunosorbent assay of 11 apparently healthy adult rabbits was conducted. The incidence rates showed 45.45%,54.54% and 72.73% respectively. The sensitivity for the three methods were 0.63, 0.67,and 1.00,and specificity for them were 1.00, 0.67 and 1.00 respectively. Somatic serotypes of isolates of P.multocida from rabbits of three groups (rabbits of group 2 were with clinic signs, those of groups 1 and 3 were apparently healthy) revealed no remarkable differences,and the predominant types were type 3 and type 3, 4. This was somewhat different from the reports derived from other states. As the antigen of different serotype used in ELISA may have different sensitivity and specificity, which is affected also by different preparation method, a type non-specific antigen should be selected to meet such request. The trial of accomplishment of ELISA without positive and negative controls was presented for discussion.

Structural and functional evidence for two separate oligosaccharide binding sites of Pasteurella multocida hyaluronan synthase

Pasteurella multocida hyaluronan synthase (PmHAS) is a bi-functional glycosyltransferase, containing a β1,3-glucuronyltransferase and β1,4-N-acetylglucosaminetransferase domain. PmHAS catalyzes the elongation of hyaluronan (HA) through the sequential addition of single monosaccharides to the non-reducing end of the hyaluronan chain. Research is focused on the relation between the length of the HA oligosaccharide and the single-step elongation kinetics from HA4 up to HA9. It was found that the turnover number kcat increased with length to maximum values of 11 and 14 s-1 for NAc- and UA-transfer, respectively. Interestingly, the specificity constant kcat/KM increased with polymer length from HA5 to HA7 to a value of 44 mM-1s-1, indicating an oligosaccharide binding site with increasing specificity towards a heptasaccharide at the UA domain. The value of kcat/KM remained moderately constant around 8 mM-1s-1 for HA4, HA6, and HA8, indicating a binding site with significantly lower binding specificity at the NAc domain than at the UA domain. These findings are further corroborated by a structural homology model of PmHAS, revealing two distinct sites for binding of oligosaccharides of different sizes, one in each transferase domain. Structural alignment studies between PmHAS and glycosyltransferases of the GT-A fold showed significant similarity in the binding of the UDP-sugars and the orientation of the acceptor substrate. These similarities in substrate orientation in the active site and in essential amino acid residues involved in substrate binding were utilized to localize the two HA oligosaccharide binding sites.

Biological Characterisation and Pathogenicity of a Pasteurella multocida Isolate from Sheep in Morocco

In this study,55 suspected pasteurellosis clinical cases from different provinces of Morocco were investigated.Molecular analysis revealed that 47%of samples were positive for Pasteurella multocida,all typed as serogroup A,and 11%positive for Mannheimia heamolytica.Eight isolates were recovered from 26 P.multocida positive samples,and characterized by biochemical and molecular typing methods.Among these isolates,two strains(S13 and S14)were selected for genes(RNA16S and rpoB)sequence analysis and virulence study in mice,guinea pigs and sheep.Phylogeny study showed similarities of both S14 and S13 isolates with strains from other species.In laboratory animals,the strain S14 was more virulent than S13 and induced severe illness in sheep.The high mortality of infected mice suggests that this model may represent an alternative for testing pathogenicity and vaccine efficacy.

Evaluation Preliminary of a Dry Emulsion System as a <i>Pasteurella multocida</i>Oral Carrier for Pigs

Background: This work evaluated the capacity of a dry emulsion as a carrier of viable microorganisms with potential use as prophylaxis of infectious diseases. Methods: The aqueous phase containing P. multocida not viable in PBS was emulsified in mineral oil to obtain a w/o emulsion. The microorganisms remained stable and only in two cases (n = 6) did the bacterial concentration decrease. Scanning Electron Microscopy (SEM) revealed a structure of a system with the organized association of particles with cubic symmetry. Using two ex vivo bioadhesion systems, it was demonstrated that the disperse-adsorbed system is capable of adhering to the intestinal mucosa and remains adhered for long periods of time. Results: The no viability of the bacteria in the dry emulsion and the possibility of controlled release were confirmed. In vivo trial was conducted in pigs. It was possible to locate the emulsion and the bacteria attached to the gut of the living animal. An ELISA kit was used to monitor the mean antibody titer of treated pigs over a 2-week period, and a classic primary response curve occurred when the titer was plotted against time. Conclusion: We propose the disperse-adsorbed system as an alternative to commonly used vehicles for immunogens in the oral vaccines.

Deadly case of Pasteurella multocida aortitis and mycotic aneurysm following a cat bite

Animal bites are frequently encountered in the emergency department(ED). Aortitis leading to mycotic abdominal aortic aneurysm is a rare and potentially deadly complication of Pasteurella multocida(P. multocida) following an animal bite. We present the case of a 68-year-old male who presented to the ED after falling at home. He complained of weakness and abdominal pain. He was in septic shock and was treated empirically with broad-spectrum antibiotics and intravenous fluids. He reported previous antibiotic treatment of a cellulitis secondary to a cat bite injury to his right thumb four weeks prior. Abdominal ultrasound and subsequent computed tomography scan revealed a leaking mycotic abdominal aneurysm that was surgically repaired. Blood cultures and aortic wall tissue cultures grew P. multocida. Given how common animal bite presentations are in the ED, this case highlights the need to consider aortitis and mycotic abdominal aortic aneurysm in an unwell patient with an animal bite.

黄芩苷对多杀性巴氏杆菌感染猪巨噬细胞后炎症因子表达及信号通路影响的研究

为研究不同浓度黄芩苷对多杀性巴氏杆菌(Pm)HN-13株感染的猪巨噬细胞(HD11)内炎症因子表达的影响及与炎症信号通路的相互作用关系,本研究采用western blot检测HN-13株感染(0、30 min、60 min、90 min)HD11细胞中促分裂原活化蛋白激酶(MAPK)信号通路3个关键蛋白(p-ERK/ERK、p-p38/p38、p-JNK/JNK)的表达水平;采用ELISA和荧光定量RT-PCR(RT-qPCR)分析检测低浓度剂量黄芩苷(L组,20μmol/L)、中浓度剂量黄芩苷(M组,40μmol/L)和高浓度剂量黄芩苷(H组,60μmol/L)对Pm感染的猪巨噬细胞内肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和IL-1β分泌水平及其m RNA转录水平的影响,同时设置对照组(Control)和模型组(HN-13);利用抑制剂SCH772984阻断ERK通路,采用ELISA和RT-qPCR检测黄岑苷对Pm感染的HD11细胞内炎症因子TNF-α、IL-6和IL-1β表达及m RNA转录水平的影响,同时设置对照组(Control)、模型组(HN-13)、抑制组(HN-13+抑制剂)。结果显示:与HN-13株处理0 min组相比,随着感染时间的延长,HN-13株感染HD11细胞中p-ERK/ERK的蛋白表达量逐渐升高(P<0.01),而p-p38/p38和p-JNK/JNK蛋白表达量均未发生显著变化(P>0.05),表明,HN-13株可使HD11细胞炎症相关ERK通路蛋白表达增强,且与作用时间呈正相关。与模型组(HN-13)相比,3个浓度剂量的黄芩苷均显著抑制TNF-α、IL-6和IL-1β的分泌水平及m RNA转录水平(P<0.05),且抑制作用随着黄芩苷浓度升高而增强。与抑制组相比,黄芩苷与抑制剂共处理组可以显著抑制HN-13感染的HD11细胞中TNF-α、IL-1β和IL-6的分泌及m RNA转录水平(P<0.05)。综上所述,黄芩苷可以抑制Pm感染的HD11细胞中ERK通路相关蛋白的表达和分泌,进而发挥抗炎作用。本研究为研制防治Pm引起的猪肺疫的新药提供了思路。

江苏省肉种鸡场禽多杀性巴氏杆菌流行病学调查

为了解江苏省禽多杀性巴氏杆菌流行情况,促进肉种鸡场的禽多杀性巴氏杆菌病防控,研究通过细菌鉴定方法、PCR检测、荚膜血清学分型、致病性分析以及药物敏感性试验,对分离自江苏省17个肉种鸡场的禽多杀性巴氏杆菌进行菌落形态、培养特性、生化发酵、荚膜型、菌落荧光型、药敏性等生物学特性研究,并对部分种鸡场进行血清学检测。结果显示:共鉴定13株禽多杀性巴氏杆菌,所有分离株荚膜分型均为A型,菌落荧光观察均属于强毒株;分离株对氨基糖苷类药物和氯霉素类药物具有一定的耐药性,而对β-内酰胺类药物高度敏感;确诊种鸡场阳性率显著高于未确诊种鸡场(P<0.01)。研究表明,江苏省部分地区肉种鸡场存在荚膜A型多杀性巴氏杆菌流行,强毒株比例较高,但耐药性较弱。

鸭源多杀性巴氏杆菌的分离鉴定及药敏试验

目的:从疑似禽霍乱的4只病死鸭的病料中分离并鉴定多杀性巴氏杆菌(Pasteurella multocida,Pm),并通过药敏试验筛选敏感药物用于治疗。方法:无菌采集肝脏组织病料,接种至普通营养琼脂、麦康凯及血琼脂培养基上进行病原菌的培养、分离纯化;对纯化后的分离菌进行革兰染色、镜检,提取其核酸作为模板,进行16S rRNA基因的PCR鉴定;圆纸片扩散试验测定分离菌株对18种抗菌药物的敏感程度。结果:在血琼脂培养基上分离菌株表现为稍微隆起、表面光滑、湿润、灰白色、露珠样、半透明的圆形菌落,但在普通营养琼脂和麦康凯培养基上不生长。革兰染色镜检显示分离菌为一种革兰染色阴性、菌体两端着色深、中间着色较浅的球状短小杆菌。PCR扩增16S rRNA出现1 500 bp的目的条带,其序列分析与Pm菌株Q的16S rRNA基因的相似性为99.86%;药敏试验表明,分离菌株对米诺环素敏感性最高,抑菌圈直径达19 mm,对四环素、强力霉素、庆大霉素、氨苄西林、头孢哌酮表现中度敏感,其他药物均为耐药。结论:本研究从一例疑似禽霍乱的病死鸭中分离鉴定出1株多杀性巴氏杆菌,分离菌株对米诺环素较为敏感,建议用于临床治疗。

两株兔源多杀性巴氏杆菌分离鉴定及荚膜血清分型

为探明浙江省规模兔场主要传染病流行现状,明确其致病菌及血清型,开展了家兔流行病学调查与病原分离鉴定。调研发现,不少兔场存在以打喷嚏和鼻腔流出浆液性、黏液性或脓性分泌物为主要临床特征的呼吸道传染病,进而对浙江省家兔主产区发病严重兔场进行采样,从嵊州、宁波三家患病兔鼻腔中分离到两株病原菌,进行了病原分离鉴定、荚膜血清分型及药敏试验。根据分离菌的菌落形态、革兰氏染色特征、特异性PCR以及荚膜血清分型PCR鉴定结果,确定两株致病菌均为荚膜血清A型多杀性巴氏杆菌,分别命名为ZJNB0321和ZJSZ0322。构建系统发育树,结果表明,两分离株属于同一分支,且与多杀性巴氏杆菌处于同一分支,并拥有独立的分支。药敏试验结果表明,不同地区分离株药敏特性不尽相同。研究结果为兔场合理用药及有效防控提供了科学依据。

1株牛源D:L3:L5型多杀性巴氏杆菌的分离鉴定及病原特性评价

【目的】通过探讨青海省西宁市某规模化奶牛场病死牛源多杀性巴氏杆菌的荚膜血清型、耐药性和毒力基因分布情况,为多杀性巴氏杆菌引起的牛呼吸道疾病的治疗和预防提供参考。【方法】对病死牛肺脏样本进行细菌分离,通过菌落观察和染色镜检、生化鉴定、特异性引物扩增、16S rRNA测序分析及系统发育树构建、荚膜血清及脂多糖分型、毒力基因检测、致病性试验和药敏试验对分离菌进行鉴定和病原特性评价。【结果】肺脏样本中分离得到的菌落呈现灰白色中间微凹陷的露珠样,未出现溶血环;革兰染色可见粉红色球杆菌;瑞氏染色可见两端浓染的蓝色球杆菌。生化鉴定结果显示,分离菌株D-葡萄糖、D-甘露醇、D-甘露糖、酪氨酸芳胺酶、磷酸酶、COURMARATE和ELLMAN反应结果为阳性;16S rRNA测序结果显示,分离菌株与多种多杀性巴氏杆菌相似性>95%,鉴定分离得到的菌株为多杀性巴氏杆菌,命名为XN222。荚膜血清分型及脂多糖分型结果显示,分离菌株XN222的荚膜血清分型为D型,其脂多糖分型为L3和L5型。毒力基因扩增结果显示,分离菌株XN222携带ptfA、fimA、hsf-2、sodA、tbpA、sodC等共计19种毒力基因。小鼠致病性试验结果显示,注射0.2 mL 1.5×10^(8)CFU/mL菌液,可导致100%(10/10)小鼠死亡,说明分离菌株XN222具有较强的致病能力。药敏试验结果显示,分离菌株XN222对头孢噻呋、左氧氟沙星、恩诺沙星、马波杀星、四环素和青霉素等15种抗菌药均敏感,对泰乐菌素中度敏感。【结论】本研究分离获得1株致病力较强的D型多杀性巴氏杆菌,该菌株的发现丰富了国内牛源多杀性巴氏杆菌亚型,可为多杀性巴氏杆菌引起的牛呼吸道疾病的防治、病原学调查和巴氏杆菌多价苗研制等提供生物素材。

多杀性巴氏杆菌的分离鉴定和全基因组测序分析

多杀性巴氏杆菌(Pm)可感染多种动物,引起肺炎或全身性败血症,造成严重的经济损失。为鉴定河北昌黎县某肉牛养殖场导致牛大批病死的病原,本研究从病牛肺脏中分离到疑似Pm的菌株,对其进行革兰氏染色观察,16S rDNA基因的PCR扩增与序列分析,并对其荚膜和脂多糖进行PCR分型,PCR扩增其7个管家基因后分析分离菌的多位点序列分型(MLST)。结果显示,分离株为革兰氏阴性菌,与Pm的16S rDNA基因序列同源性达99.87%,表明分离到一株Pm。PCR分型鉴定结果显示其为血清B型和脂多糖L2型,MLST分型为ST44型,将该Pm分离株命名为PmB-1。将PmB-1以4.88 cfu/只经腹腔注射感染小鼠,进行致病性试验,结果显示PmB-1能引起小鼠内脏出血病变。采用IlluminaHiseq二代结合PacBio三代测序平台对PmB-1进行全基因组测序及生物信息学分析。结果显示,PmB-1基因组含1条染色质,长2 331 836 bp,编码2 141个基因,并含19个rRNA,58个tRNA和42个sRNA;含有3个前噬菌体、5个基因组岛,5个获得性免疫(CRISPR-Cas)系统、3个插入序列以及1个转座子元件。致病性预测分析结果显示,PmB-1含244个毒力相关基因,224个耐药基因,495个基因参与病原与宿主的互作,14个基因编码分泌系统蛋白,535个基因编码转运蛋白,190个基因编码分泌蛋白,499个基因编码跨膜蛋白,17个基因编码双组分调控系统。本研究分离到一株Pm,并对其进行小鼠致病性试验,全基因组测序和生物信息学分析,为预防Pm的感染、流行和研究其致病机制提供了参考依据。