Development of a typing system for epidemiological studies of porcine toxin-producing Pasteurella multocida ssp. multocida in Denmark

The aim of the present study was to evaluate capsular-typing, plasmid-profiling, phage-typing and ribotyping for epidemiological studies of toxin-producing Pasteurella multocida ssp. multocida in Denmark. The evaluation of methods was based on 68 strains from nasal swabs and 14 strains from pneumonic lungs. Strains from lungs were all of capsular Type A, whereas strains from nasal swabs were of both capsular Types A and D. Only 9% of the strains contained plasmids, which could not be associated with antibiotic resistance. Phage-typing divided 61% of strains into 10 groups, while 39% were non-typable. CfoI ribotyping divided strains into four groups of which one type contained 94% of isolates. HindIII ribotyping divided strains into 18 types. A total of 18 strains from The Netherlands, UK and USA were subjected to HindIII ribotyping, resulting in 13 types of which six were identical to ribotypes of Danish strains. Phage-typing of isolates from an outbreak of atrophic rhinitis involving six herds in 1985 showed the existence of an epidemic strain. This type was recognised in the herd suspected of being the source of the infections and in four of the five infected herds. These findings were supported by HindIII ribotyping, as 85% of isolates from all herds were assigned to one ribotype. In conclusion, HindIII ribotyping seems to represent a useful tool for epidemiological studies of toxigenic P. multocida ssp. multocida.     

Bactericidal activity in the sera of mice vaccinated with Pasteurella multocida type A

The susceptibility of Pasteurella multocida to killing by serum and the ability of protective vaccines to stimulate this mechanism of immunity in mice were investigated. P. multocida type of bovine origin was used to prepare a vaccine incorporating heat killed organisms and for homologous infection of mice. Bactericidal capacity and ELISA antibody titres were determined for individual mouse sera. Protection was clearly associated with bactericidal antibodies raised by vaccination. The bactericidal assay may be useful as a rapid, simple screening test of vaccinated mice for functional protective antibody levels.     

禽多杀性巴氏杆菌ompa DNA疫苗在小鼠体内免疫效果试验

应用PCR技术扩增获得禽多杀性巴氏杆菌的ompa基因片段,克隆到pUCm-T载体,再亚克隆到真核表达质粒载体pCDNA3.1(+)上,构建重组质粒pcA,体外转染Vero细胞,RT-PCR和间接免疫荧光试验检测其转录表达情况。动物免疫分为3组:pCDNA3.1(+)组、PBS对照组和pcA组,每组16只BALB/c小鼠,pCDNA3.1(+)组和pcA组以100μg/只的剂量肌注免疫,PBS组每只小鼠肌注100μL 1×PBS,各组均免疫3次,每次间隔2周。间接ELISA检测免疫后小鼠血清特异性抗体水平,MTT法检测免疫小鼠脾淋巴细胞增殖情况,三免2周后检测脾淋巴细胞IFN-γ分泌情况。强毒攻击,计算小鼠存活数目及保护率。结果显示,间接免疫荧光试验和RT-PCR检测结果均表明pcA可在体外培养的Vero细胞中表达目的蛋白。动物免疫后,pcA组免疫小鼠血清抗体水平持续上升,与pCDNA3.1(+)组和PBS组相比差异极为显著(P<0.01)。经提取的禽多杀性巴氏杆菌总外膜蛋白(Omps)刺激后,pcA组的刺激值(SI值)与pCDNA3.1(+)组及PBS免疫组相比均差异显著(P<0.05)。脾细胞产生的IFN-...     

产毒素多杀性巴氏杆菌的鉴定

采用菌落多重PCR方法对分离保存的28株多杀性巴氏杆菌进行种型和毒素基因的检测,结果表明,菌株C51-6、M-4和P-2237为产毒素多杀性巴氏杆菌,菌株C51-6和P-2237为荚膜血清D型,菌株M-4为荚膜血清A型。同时用金黄色葡萄球菌抑制试验、中性吖啶黄沉淀试验和豚鼠皮肤坏死试验对PCR方法进行了验证。基于对甘露醇、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,3株菌株鉴定为多杀性巴氏杆菌多杀亚种。     

Atypical Pasteurella strains producing a toxin similar to the dermonecrotic toxin of Pasteurella multocida subspecies multocida

This paper is the first report of the production of a dermonecrotic toxin by pasteurella strains that do not belong to the species Pasteurella multocida subspecies multocida. Four strains, isolated from cattle with atrophic rhinitis, were characterised phenotypically. The strains were related to pasteurellaceae, but their taxonomic position remained unclear. The strains produced a toxin that caused a haemorrhagic dermonecrosis in guinea pigs and was lethal to mice. Both effects were neutralised by an antiserum against the purified dermonecrotic toxin of P multocida subspecies multocida. Western blot analysis of culture filtrates of the bovine strains revealed a protein, with the same molecular weight as dermonecrotic toxin, which reacted with both polyclonal and monoclonal antibodies against the toxin. In an immunodiffusion test, anti-dermonecrotic toxin serum did not discriminate between the toxin of the bovine strains and the toxin of P multocida subspecies multocida. It is concluded that these atypical pasteurella strains produce a toxin that is closely related to the dermonecrotic toxin of P multocida subspecies multocida.     

Pasteurella multocida infection in heterophil-depleted chickens

The present study was aimed at elucidating the role of heterophil granulocytes during the initial infection with Pasteurella multocida subsp. multocida in chickens. Chickens (17 and 19 wk old) were depleted of their heterophil granulocytes by 5-fluorouracil treatment. When the heterophil blood counts were significantly reduced, the birds were inoculated intratracheally with 1.8-4.3 x 10(4) colony-forming units of P. multocida. Twelve, 24, or 48 hr postinoculation, the birds were euthanatized and examined for macroscopic and histologic lesions in the lungs. Bacterial invasion was determined by culture of P. multocida from the spleen. Recruitment of heterophils into the respiratory tract during infection was found to contribute considerably to the lung lesions in chickens and was found to mediate tissue damage, possibly allowing a more rapid systemic spread of P. multocida. However, during progression of the infection, the heterophil-mediated necrosis in chickens seemed to stimulate giant cell demarcation of infected lung tissue, which coincided with the clearance of P. multocida from the spleen, thus hampering further invasion. Consequently, heterophil activation plays a dual role for the outcome of a P. multocida infection in chickens, where it initially seems to promote invasion and systemic spread but subsequently helps limit the infection by giant cell formation and bacterial clearance.     

牛多杀性巴氏杆菌znuA基因的原核表达及其免疫保护作用的研究

为研究牛多杀性巴氏杆菌(P.multocida)高亲和力锌吸收蛋白znuA的免疫学活性及其免疫保护作用,本研究利用PCR方法扩增了P.multocida znuA基因,构建表达载体pET-28a-znuA,将其转化E.coli BL21后经诱导表达。表达产物经SDS-PAGE和western blot分析显示,重组蛋白约40 ku;以重组znuA蛋白免疫小鼠后用P.multocida菌株Y-1攻毒,结果显示重组znuA蛋白对免疫组小鼠保护率为60%,表明其具有免疫保护作用。本研究首次在原核系统中表达了P.multocida znuA蛋白,且验证了其免疫保护力,为深入探究znuA基因在P.multocida致病过程中的作用及其亚单位疫苗的开发奠定了基础。     

Experimental Pasteurella multocida pneumonia in calves

Pasteurella multocida was isolated from the lungs of calves that died on a farm in the south of England. This organism was inoculated experimentally into 13 calves by the intratracheal route: in all but two of the calves mild clinical disease resulted and at necropsy, three or four days later, pneumonic consolidation involving up to 22 per cent of the lung was observed. P multocida was isolated from all but two of the lungs. Of two calves inoculated intravenously with P multocida, one showed mild clinical disease and slight pneumonic consolidation at necropsy and the other remained normal. Control calves inoculated intratracheally and intravenously with sterile broth showed no signs of illness and no pneumonic consolidation. Histologically the lung lesions comprised a fibrinous bronchopneumonia with variable sized areas of coagulative necrosis, extensive deposition of fibrin and massive dilatation and oedema of the interlobular and pleural lymphatics. It is concluded that P multocida should receive more recognition as a primary pathogen.     

Long-chain fatty acids of Pasteurella multocida nad Pasteurella haemolytica

The relative contents of long-chain fatty acids in P. multocida and P. haemolytica were investigated. A dependence on the composition of the broth was established. Accordingly, comparative quantitative studies on fatty acid contents have to be conducted using bacteria grown with the same lot of broth medium. As for P. multocida, there were significant differences between the serovars (C14 in TDHM and C16, delta 2C18 in BPL). These differences are, however, not significant to replace serotyping. Highly significant differences were also detected between P. multocida isolates from nasal swabs and pneumonic lungs (interims of C14, delta C16 on BPL and BRU). The largest differences were measured for strains grown on BRU, which is interpreted as an expression of virulence. Significant differences were found between biotypes A and T of P. haemolytica, namely for C14, C16 in TDHM, and C14, delta C16, C16, C18 in BPL medium.     

Protective effect of Pasteurella multocida cell-free antigen and toxoid against challenge with toxigenic strains of pasteurella multocida in mice

The cell-free antigen (CFA) obtained from the culture supernatant of Pasteurella multocida (P. multocida) and the toxin (PMT) purified from CFA were inactivated and mixed with oil adjuvant to prepare a trial vaccine. Both of the mice immunized with CFA and PMT toxoid vaccine were noticeably protected against intratracheal challenge with toxigenic strains of P. multocida. Nevertheless, the protective indices of the mice immunized with CFA vaccine indicate that it is more protective and clears away the bacteria more promptly than in the mice immunized with PMT vaccine. The results suggested that CFA would possibly be good as an effective antigen to toxigenic strains of P. multocida infection.     

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.     

多杀性巴氏杆菌毒素的研究进展

多杀性巴氏杆菌毒素(Pasteurella multocida toxin,PMT)是一种由D型多杀性巴氏杆菌分泌的毒素蛋白,作为猪萎缩性鼻炎的主要致病因子而备受关注。PMT作为一种有丝分裂原,通过细胞内多条信号通路影响细胞的生长和分化。PMT可分为N端和C端两大结构域,N端被认为与受体结合以及跨膜转运有关,C端具有催化活性。近年来,也有人认为PMT与致癌因素有关。随着PMT研究的深入,PMT疫苗也正成为研究的一个热点。本研究就PMT胞内活性、结构与功能等最新研究进展进行概述。