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。     

Factors influencing the adherence of strains of Streptococcus bovis and Escherichia coli isolated from ruminal epithelium

Two strains of Streptococcus bovis (A1 and A5) and one strain of Escherichia coli (0141: H28) isolated from the surface of bovine ruminal mucous epithelium were examined for adherence to isolated and cultured ruminal epithelial cells. The E. coli adhered to the target cell by means of fimbriae, which had several common properties with type 1 common fimbriae and caused mannose-sensitive haemagglutination. The A1 strain of S. bovis was devoid of fimbriae and its adherence to the epithelial surface was not inhibited by treatment with sugars or phenol-treated bacterial membrane from the same organism. It was therefore postulated that the bacterial glycocalyx of the S. bovis organisms acted as ligand. The extent of bacterial adherence depended on the state of differentiation of the target cell in both the isolated and the cultured ruminal cell systems. The receptors for both adherent bacterial species were in all probability associated with the glycocalyx of the target cells.     

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.     

Molecular Variability among Strains of Pasteurella multocida Isolated from an Outbreak of Haemorrhagic Septicaemia in India

The applicability of conventional and molecular methods for rapid detection and differentiation of Pasteurella multocida serogroup B isolates involved in an outbreak of haemorrhagic septicaemia affecting Indian buffaloes, was studied. Five isolates were obtained and were subjected to phenotypic and genotypic characterization. None of the five isolates could be differentiated on the basis of cultural, biochemical, pathogenicity and antimicrobial sensitivity patterns. Polymerase chain reaction (PCR)-based techniques were found to be specific and sensitive for rapid detection and differentiation of isolates. Repetitive extragenic palindromic (REP-) PCR, enterobacterial repetitive intergenic consensus (ERIC-) PCR and single-primer PCR differentiated all the five isolates into different profiles. All the isolates involved in the outbreak were found to have a genetic profile different from standard P. multocida strain (P52). However, three isolates had similar profiles, whereas each of the remaining two had a different profile. The study indicates the involvement of multiple strains of P. multocida in a single outbreak of haemorrhagic septicaemia in buffaloes. The results also indicate that molecular methods of detection and typing are superior to conventional methods for rapid epidemiological investigations of haemorrhagic septicaemia.     

A Single-chain Fragment Variable Recombinant Antibody against F5 Fimbria of Enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> Inhibits Agglutination of Horse Red Blood Cells Induced by F5 Protein

Bovine colibacillosis caused by enterotoxigenic Escherichia coli (ETEC) is a worldwide problem. Adhesion of ETEC to intestinal cell receptors mediated by the surface protein F5 fimbriae is the initial step in the establishment of colibacillosis. Prevention of ETEC F5⁺ adhesion to enterocytes protects newborn calves against collibacillosis. On the enterocytes, the F5 fimbriae bind to a ganglioside that is also found on horse red blood cells. Thus, the presence of F5 fimbriae induces haemagglutination, which is useful as an indicator in a functional assay system. In this study, recombinant anti-F5 scFv antibody fragment produced in E. coli HB2151 reacted with F5 fimbriae in ELISA and Western immunoblot, and prevented haemagglutination induced by the binding of the F5 fimbriae to its natural host receptors on horse red blood cells. Given the ease with which recombinant antibodies can be mass-produced, the presently described scFv may hold promise as a prophylactic agent for colibacillosis.     

The expression of plasmid mediated afimbrial adhesin genes in an avian septicemic Escherichia coli strain

An Escherichia coli strain (SEPT13) isolated from the liver of a hen presenting clinical signs of septicaemia had a LD₅₀ of 4.0 × 10⁵ CFU/ml in one-day-old chickens, expressed Ia, Ib, E1, E3, K and B colicins and aerobactin. The strain was ampicillin and streptomycin resistant, and found to have fimA, csgA and tsh DNA related sequences; it could adhere to and invade HEp-2 and tracheal epithelial cells, expressed fimbriae (observed by electron microscopy), and had five plasmids of 2.7, 4.7, 43, 56, and 88 MDa. Transposon mutagenesis of strain SEPT13, with transposon TnphoA, resulted in a mutant strain named ST16 that had a LD₅₀ of 1.2 × 10¹² CFU/ml. All other biological characteristics of strain ST16 were the same as those detected for strain SEPT13 except for the migration of an 88 MDa plasmid to the 93 MDa position indicating the insertion of the transposon into the 88 MDa plasmid. The 93 MDa plasmid of strain ST16 was transferred, by electroporation assay, to non-pathogenic receptor strains (E. coli strains K12 MS101 and HB101), resulting in transformant strains A and B, respectively. These strains exhibited adhesion properties to in vitro cultivated HEp-2 cells but did not have the capacity for invasion. The adherence occurred despite the absence of fimbriae; this finding suggests that the 88 MDa plasmid has afimbrial adhesin genes.     

Study of the toxin-producing ability of Pasteurella multocida in mice

Cell-free sonicated extracts and broth cultures of Pasteurella multocida strains of pig origin were examined for their lienotoxicity in mice. P. multocida strains represented capsular types A and D with or without dermonecrotoxic (DNT) activity in the guinea pig skin test. Mouse lienotoxicity test was suitable for determining the toxigenicity of P. multocida strains only when bacterium-free extracts were tested. In that case both toxigenic type A and D strains were lethal to intravenously inoculated mice and caused a remarkable reduction in spleen mass when sublethal doses were used. The extracts of atoxic strains were not lethal and induced splenic hyperplasia. By testing viable cells no correlation was demonstrable between toxin production and virulence of P. multocida to mice. In one experiment the concentrated sterile culture fluids of a toxigenic type D P. multocida and a toxigenic B. bronchiseptica strain were compared. The former caused deaths and splenic atrophy among mice, while the latter was nontoxic and induced slight hyperplasia of the spleen. This fact indicates that P. multocida secretes its toxin into the culture fluid.     

Antigenic relationship between the dermonecrotic toxins produced by Pasteurella multocida type D and type A

Crude dermonecrotic toxins (DNT) were prepared from Pasteurella multocida (P.m.) type D and type A strains isolated from pigs with atrophic rhinitis. Rabbits were immunized with the DNT of P.m. type D. This serum neutralized the DNT of P.m. type A to the same degree as the homologous one both in vitro (cytopathogenicity for tissue culture cells) and in vivo (mouse lethality and dermonecrotic activity in guinea pig).     

Comparative sequence analysis of 16S rRNA gene of Pasteurella multocida serogroup B isolates from different animal species

The phylogenetic relationships of five isolates of Pasteurella multocida serotype B:2 belonging to buffalo, cattle, pig, sheep and goat were investigated by comparative sequence analysis of 16S rRNA gene. The 1468bp fragment of 16S rRNA gene sequence comparison showed that the isolates of cattle (PM75), pig (PM49) and sheep (PM82) shared 99.9% homology with the buffalo isolate (vaccine strain P52) whereas, the goat isolate (PM86) shared 99.8% homology with the vaccine strain. The 16S rRNA gene sequences of these isolates were also found monophyletic with type B reference strain NCTC 10323 of P. multocida subsp. multocida. The present study indicated the close relationships of haemorrhagic septicaemia causing P. multocida serotype B:2 isolates of buffalo and cattle with other uncommon hosts (pig, sheep and goat).     

Induction of nasal turbinate atrophy in germ-free pigs, using Pasteurella multocida as well as bacterium-free crude and purified dermonecrotic toxin of P multocida

To establish the role of the dermonecrotic toxin (DNT) of Pasteurella multocida in the cause and pathogenesis of atrophic rhinitis, germ-free pigs were inoculated with several strains of P multocida, crude DNT, or purified DNT. In some experiments, the aforementioned inocula were combined with Bordetella bronchiseptica. All DNT-producing P multocida strains induced severe turbinate atrophy. Histologic examination of the remnants of the nasal turbinates revealed intact, but undulated, ciliated epithelium and numerous osteoclasts. Inflammation was minimal or absent. A DNT-producing B bronchiseptica strain induced only mild turbinate atrophy. The lesions were characterized histologically by loss of cilia and ciliated cells and by an infiltration of predominantly mononuclear cells. Bone formation seemed impaired. Turbinate lesions were most severe in pigs infected with a combination of B bronchiseptica and a DNT-producing P multocida strain. Intranasal administration of sterile DNT-containing culture filtrate of P multocida or purified DNT of P multocida did not result in turbinate atrophy. In contrast, turbinate atrophy developed when these preparations were injected IM or when intranasal administration of DNT was preceded by inoculation of B bronchiseptica.     

An abattoir survey of pneumonia and pleuritis in slaughter weight swine from 9 selected herds. IV. Bacteriological findings in chronic pneumonic lesions.

A total of 855 pig lungs were collected at slaughter and evaluated macroscopically. Bacteriological examinations were carried out on tissue samples from chronic pleuropneumonic lesions (n = 196) and from chronic bronchopneumonic lesions with suppuration (n = 14). Samples from normal lung tissue (n = 22) were also included. Pasteurella multocida was isolated from 54%, Actinobacillus (Haemophilus) pleuropneumoniae from 11%, and Streptococcus spp. from 14% of the pneumonic lesions, respectively. From normal lung tissue P. multocida was isolated from 3 (14%) of the samples, A. pleuropneumoniae was not recovered and streptococci were isolated from only 1 (5%) of these samples. The above mentioned bacterial species were recovered either in pure cultures or mixed with various other microbes. A total of 109 P. multocida strains were further characterized by capsular serotyping and testing for production of dermonecrotic toxin. Ninety-nine (91%) of the strains were capsular type A 10 (9%) were type D. Out of the type A and the type D strains 94% and 90% were toxigenic, respectively. Most of the A. pleuropneumoniae strains were serotype 2. Strains of serotypes 1 and 7 were also identified. The majority of the streptococci were identified as either Streptococcus suis or Streptococcus dysgalactiae. Actinomyces pyogenes was isolated from 14% of the lesions and anaerobic bacteria from 18%, respectively. The significance of the various bacterial species in relation to the development of chronic pneumonic lesions is discussed. Special attention is paid to P. multocida, and it is concluded that this bacterial species is probably of importance for the development of both types of chronic pneumonias.     

致猪水肿病大肠埃希菌多重PCR检测方法的建立及应用

本研究旨在建立一种快速鉴定致猪水肿病大肠埃希菌的多重PCR检测方法.分别针对大肠埃希菌16S rDNA、志贺毒素Stx2e A亚基和菌毛F18ab A亚基保守序列设计合成3对特异性引物,优化多重PCR反应条件,并进行特异性和敏感性检测.结果显示,阳性对照菌株扩增产物大小分别为1 062、733和313 bp.特异性和灵敏性检测结果表明,与肠炎沙门菌、多杀性巴氏杆菌、胸膜肺炎放线杆菌、副猪嗜血杆菌、支气管败血波氏杆菌和猪链球菌等猪常见致病菌均无交叉反应;菌体直接扩增法最低检出量为1 875 CFU.利用建立的多重PCR检测方法对分离收集的128株大肠埃希菌进行鉴定,得到36株致猪水肿病大肠埃希菌,其中30株既有菌毛F18ab又产志贺毒素Stx2e,另外6株仅产志贺毒素Stx2e.结果表明,本试验所建立的多重PCR检测方法对致猪水肿病大肠埃希菌的快速诊断和流行病学调查具有一定的应用价值.     

Expression of the dermonecrotic toxin by Bordetella bronchiseptica is not necessary for predisposing to infection with toxigenic Pasteurella multocida

This experiment was designed to determine whether a Bordetella bronchiseptica mutant that does not produce dermonecrotic toxin (DNT) is still capable of predisposing pigs to infection with toxigenic Pasteurella multocida. Three groups of pigs were initially inoculated intranasally with a wild type B. bronchiseptica that produces DNT, an isogenic mutant of B. bronchiseptica that does not produce DNT, or PBS. All pigs were then challenged intranasally with a toxigenic strain of P. multocida 4 days later. P. multocida was recovered infrequently and in low numbers from pigs initially inoculated with PBS, and no turbinate atrophy was present in these pigs. P. multocida was isolated in similar numbers from the pigs initially inoculated with either the wild type or the DNT mutant of B. bronchiseptica, and turbinate atrophy of a similar magnitude was also seen in pigs from both of these groups. Thus, although the DNT has been shown to be responsible for much of the pathology seen during infection with B. bronchiseptica by itself, infection with non-DNT-producing strains can still predispose to secondary respiratory infections with P. multocida.     

Purification of three fragments of the dermonecrotic toxin from Pasteurella multocida

Dermonecrotic toxin purified from sonicates of Pasteurella multocida was mildly trypsinized. The trypsinized preparations were reversibly dissociated into three polypeptides, with molecular weights of about 23,000 (fragment a), about 64,000 (fragment b), and about 74,000 (fragment c) by treatment with 100 mM dithiothreitol and 6 M urea. Upon removal of dithiothreitol and urea from the dissociated toxin by dialysis, the fragments reassociated and formed dermonecrotic toxin indistinguishable from the native toxin. The three fragments were separated from the dissociated toxin by gel filtration on a Sephadex G-200 column equilibrated with buffer containing 4 M urea and 1 mM dithiothreitol. The purified fragments a, b, and c did not show dermonecrotic activity for guinea pigs. Immunodiffusion and immunoelectrophoretic analysis with rabbit anti-dermonecrotic antiserum showed that the three purified fragments were antigenically distinct but had partial identity with the native toxin.     

Pasteurella multocida antigen-induced in vitro lymphocyte immunostimulation, using whole blood from cattle and turkeys.

A whole blood lymphocyte stimulation assay to study cell-mediated immune responses in bovine pasteurellosis was developed. Peripheral blood lymphocytes from cattle artificially immunised with three Asiatic haemorrhagic septicaemia strains of Pasteurella multocida exhibited higher stimulation indices when incubated with antigen preparations from homologous strains than with the heterologous shipping fever strain. Lymphocytes from cattle immunised with the shipping fever strain of P multocida exhibited a higher stimulation index when incubated with an antigen preparation from the homolgous strain than with antigen preparations from heterologous haemorrhagic septicaemia strains. These results suggest that immunogenic differences exist between the haemorrhagic septicaemia strains and the shipping fever strain of P multocida. An assay using turkey whole blood lymphocytes was also developed. The use of small amounts of whole blood, microtitre plates, either 125I iododeoxyuridine or 3H-thymidine as the labelling agent, and a multiple cell-culture harvester makes the method simple, rapid and suitable for the study of immune competence and cell-mediated immune responses in turkeys on a flock basis.     

Identification of toxigenic Pasteurella multocida in atrophic rhinitis of pigs by in vitro characterisation

Toxigenic strains of Pasteurella multocida were readily differentiated from non-toxigenic strains by an agarose overlay method using bovine turbinate cells or bovine lung cells. Cells which were young and densely confluent were best suited to this assay. The incubation period required to distinguish toxigenic strains was dependent on the confluence of the monolayers, which was affected by the seeding rate, cell passage level and growth time prior to overlay. The agarose overlay method correctly identified 11 of 11 reference strains of Pasteurella multocida, and visible cytotoxic changes were present in the monolayers after 48 to 65 h. Outbreaks of the enzootic form of atrophic rhinitis in 2 New South Wales piggeries were associated with the isolation of toxigenic type D strains of P. multocida.     

The type 4 fimbrial subunit gene of pasteurella multocida

Colonisation of host tissue by Gram- negative bacteria is facilitated by various adhesins, one of which is type 4 fimbriae (pili). These structures have been associated with pathogenesis in several bacterial species, and have been shown to mediate colonisation of epithelial surfaces. Recently, type 4 fimbriae were identified and characterised from P. multocida strains A, B and D. The type 4 fimbrial subunit protein (PtfA) was identified as an 18-kDa protein which was isolated from whole membrane fractions.We report here the isolation and characterisation of the gene (ptfA) encoding the PtfA protein from P. multocida VP161 (serotype A:1). Part of the gene was cloned on a 2-kb genomic DNA fragment. The complete ptfA gene was obtained using inverse PCR. The gene and its flanking regions were characterised, and the deduced PtfA amino acid sequence was compared to type 4 subunit protein sequences from other bacterial species. The ptfA gene was amplified and sequenced from several P. multocida strains. Comparison of these sequences revealed variation within the type 4 subunit gene of P. multocida.     

Purification of a heat labile dermonecrotic toxin from culture fluid of Pasteurella multocida

A highly pure heat-labile dermonecrotic toxin (DNT) of Pasteurella multocida was isolated from bacterium-free broth culture fluid. The protocol for the isolation included the following steps: ammonium sulphate precipitation, gel filtration, ion exchange chromatography and preparative polyacrylamide gel electrophoresis (PAGE). About 1 mg of purified DNT was recovered from 3 l of broth culture fluid. The final product was toxic for embryonic bovine lung (EBL) cells, lethal for mice, dermonecrotic in the guinea pig skin test and inactivated by heating at 56 degrees c. The recovery of biological activity was about 5% that of the original culture fluid and the specific activity had increased about 4000 times. After sodium dodecyl sulphate (SDS)-PAGE and silver staining a single band appeared, indicating that the purified DNT was free from contaminating proteins. The molecular weight of the toxin was approximately 125,000 daltons. The minimal toxic dose of DNT protein for embryonic bovine lung cells was about 2 ng, the minimal dermonecrotic dose in the guinea pig skin test was about 80 ng and the 50% lethal dose for mice about 300 ng.     

Haemolytic Escherichia coli isolated from dogs with diarrhea have characteristics of both uropathogenic and necrotoxigenic strains

Twenty-four haemolytic Escherichia coli strains were isolated from dogs with diarrhea. The strains were serotyped and analysed by polymerase chain reaction (PCR) for genes encoding virulence factors associated with E. coli that cause diarrhea in animals. Adhesion antigen production was deduced from haemagglutination experiments. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of heat extracts was also used as an indication for the production of adhesive structures. The majority of the strains was shown to produce this type of virulence factor. Adhesion and invasion tests of the strains and Caco-2 cells showed that all strains adhered and that two were invasive. The two invasive strains were positive in the intimin PCR and one of them also contained genes encoding CS31A. The PCR for heat stable toxin (ST) was positive in only four strains, as was the presence of F17 fimbrial genes. Surprisingly, 19 strains had intact P fimbrial operons, coding for an adhesin involved in urinary tract infection (UTI). The cytotoxic necrotising factor 1 (CNF1) gene, also mainly found in UTI was likewise detected in these 19 strains. Cytolethal distending toxin (Cdt) genes were found in five strains. The high number of strains positive for CNF1 and P fimbriae prompted us to test the strains in a multiplex PCR used to test E. coli isolated from UTI in various species for 30 virulence associated genes. The data showed that the majority of the diarrhea isolates have virulence factor profiles highly similar to UTI E. coli isolates from dogs. This raises the question whether these isolates are real intestinal pathogens or "innocent bystanders". However, since CNF1 producing necrotoxic E. coli (NTEC) strains isolated from humans, pigs and calves with diarrhea appear to be highly related to our strains, it might be that in dogs this type of isolate is capable of causing not only UTI, but also diarrhea. If this is the case and this type of isolate is "bifunctional", domestic animals likely constitute a reservoir of NTEC strains which can be also pathogenic for humans.     

Further development of P. multocida vaccines for swine

149 strains with antigen fractions of both A and D type could be found out of 446 P. multocida field strains of porcine origin. Most of them are producing the dermonecrotizing toxin. These A/D strains proved to be virulent in mice and piglets as well. In mice, the vaccination with one of the most virulent and immunogenic A/D strains, inactivated and A1(OH)3 adsorbed caused an immunity against challenge infections with P. multocida of types A, D and A/D. This effect could be confirmed on SPF piglets.