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.

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.

A large-scale fatal outbreak of Pasteurella multocida among wild rodents in southwestern China

Background:Pasteurella multocida is an important and old zoonotic pathogen worldwide which has an impressive host spectrum including numerous domestic and wild animals as well as birds,causing specific diseases or outbreak with great economic impact.It has never been reported that P.multocida can cause an epidemic in wild rodents.In June 5–17,2016,more than 1000 rodent deaths of an unknown cause quickly spread in the PuEr City,Yunnan province,southwestern China.Methods:The rodents in affected areas and outside of the epidemic areas were collected and screened for possible known pathogens including Yersinia pestis,rabies virus and hantavirus as well as other bacteria.The possible bacterial pathogens were isolated both by culture medium and by mouse inoculation in parallel.The isolates were identified by the Vitek GNI card and PCR assays for 16S rRNA genes.The pathogen strains were selected for whole genome sequencing analysis.Results:A total of 123 rodents were collected from 25 sample sites at affected area,among of which,all 119 dead rodents were negative for the pathogen under consideration except P.multocida,and all four live rodents were negative for P.multocida.In addition,480 rodents collected from other 23 counties outside of the epidemic area in Yunnan were negative for with P.multocida.A total of 14 strains of P.multocida(six directly isolated from the field rodents and eight from the experimental mice that were injected with the organ substrates from the dead rodents)belonged to serogroup A and serogroup F represented by 9 N and 20 N were identified in these epidemic areas.Whole genome sequencing revealed that the serogroup F strain shared 99%similarity to P.multocida Pm70 from chicken,but contained a 50 k bp insertion sequence.The serogroup A strain shared 95%similarity to P.multocida FDAARGOS_385 from a human patient,but contained four large structural differences.Histological abnormalities were identified in the livers,lungs,hearts and brains of the inoculated mice.Conclusions:The simultaneous occurrence of both serotypes of P.multocida may have caused this sudden onset of mortality across the local rodent population in Yunnan Province,China.Further attention should be paid to this old bacterium in the world.

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.

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.

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.

多杀性巴氏杆菌诊断技术及防控策略研究进展

多杀性巴氏杆菌是动物呼吸道常见的致病细菌,在世界范围内严重威胁牲畜健康。该病原广泛存在于各种健康动物的鼻腔、咽及肺等部位,当机体受到外界不良刺激造成免疫水平下降时,该菌大量繁殖并引发疾病。从多杀性巴氏杆菌培养特性、发病机制、诊断技术和防控策略等方面进行综述,为多杀性巴氏杆菌疾病的防治奠定基础。

Subcutaneous abscess caused by Pasteurella multocida in a patient due to a cat bite

P asteurella multocida (P. multocida), a Gram-negative pathogen, is mainly associated with animal infections. Human P. multocida infections are occasionally encountered and most human cases are due to the animal exposure or contact, especially due to cat bites. 1 In China, P. multocida infections are rarely described. CASE REPORT A 50-year old woman was sent to the Emergency Department of West China Hospital on June 25, 2004, due to the complain of an onset of pain, erythema, bleeding, and swelling at a wound on the right leg, caused by a pet cat bite 11 hours ago. She was diagnosed as having a wound infection and received intravenous penicillin G 4 million U/day. One day later, though the bleeding was ceased, she felt worse. Then, she was admitted to the Department of Infectious Diseases. She has no fever nor other systemic symptoms when she was admitted. On examination, three close, small and deep holes were found on the right leg near the ankle. Around the holes, the skin and soft tissue were red swelling and tenderness with a high skin temperature. Little pus was presented when the wound was crushed. Regional lymphadenopathy in right groin was found. No dyskinesia and tenderness of the ankle joint was found. Examination of her respiratory, cardiovascular, and gastrointestinal systems was unremarkable. Initial blood routine examination for her revealed: haemoglobin 12.5 g/dl; white blood cell count 7×10 9/L (neutrophils 4.48, lymphocytes 2.45); platelet, 184×10 9/L. But the white cell count increased to 11.2×10 9/L (neutrophils 8.52, lymphocytes 2.58) on the next day. Routine biochemical examination for her revealed normality.

一例山羊链球菌与多杀性巴氏杆菌混合感染的调查分析

对2020年8月广西某养羊场送检的1份羊病料采用细菌学和分子生物学方法进行诊断,对分离菌株进行形态学特性、生理生化、16S rDNA测序,结果鉴定为链球菌,应用PCR方法检测到多杀性巴氏杆菌阳性。对分离菌进行药敏试验,结果显示该菌对青霉素、恩诺沙星、氧氟沙星、土霉素、头孢噻呋钠高度敏感。对分离菌进行致病性试验,结果显示该菌有较强的致病性。检测结果表明该病为链球菌与多杀性巴氏杆菌混合感染引起的。