副猪嗜血杆菌外膜蛋白表型分析

为揭示副猪嗜血杆菌外膜蛋白与毒力的关系,采用SDS-PAGE测定了82个副猪嗜血杆菌分离株细胞外膜蛋白(OMP),比较了不同临床背景分离株的OMP表型差异,根据外膜蛋白的电泳迁移率Rf值和蛋白含量对OMP与毒力菌株的相关性和PAGE分型进行了聚类分析。图谱表型分析结果表明,82个分离株分成以相对分子质量36～40ku为特征的Ⅰ型和以42～45ku为特征的Ⅱ型2种类型,约34%患病猪分离株属于PAGEⅠ型,只有8.7%健康猪分离株属于PAGEⅠ型。Rf值聚类分析结果显示外膜蛋白分为3种类型,PAGEⅠ、PAGEⅡ和PAGEⅢ型。图谱蛋白含量聚类分析显示5个PAGE型。结果提示,36～40ku蛋白与菌株的毒力相关。     

Characters of Escherichia coli 078 isolated from septicaemic animals

Twenty-one Escherichia coli isolates of serogroup 078 from animal septicaemia were obtained from laboratories in France, England and Canada. The bacteria were compared for outer membrane protein (OMP) patterns, lipopolysaccharide patterns, surface proteins of fimbrial types, biotypes, antibiotypes, colicin production, hydroxamate production and virulence in mice. Sixteen isolates from bovine, ovine, porcine and avian species in France and England had a similar OMP pattern. This characteristic associated with minor properties like surface proteins type, colicin V production and virulence in mice made these 16,078 E. coli isolates from 4 animal species, good candidates for the same clonal grouping. The five other bovine isolates with "Vir" or "31a" phenotypes were heterogeneous for most of the characteristics studied.     

Comparative Analysis of the Outer Membrane Protein Profiles of Isolates of the Pasteurella multocida (B:2) Associated with Haemorrhagic Septicaemia

Outer membrane proteins (OMP) of P. multocida (serotype B:2) field isolates (n = 6) and a vaccine strain (P-52) were extracted by a sarkosyl method and characterized using SDS-PAGE and immunoblotting. About 20 polypeptide bands were observed in the profile of the vaccine strain with MW ranging from 16 to 90 kDa and, based on band thickness and intensity of staining, three polypeptides of MW 31, 33 and 37 kDa were considered to be the major OMPs. The profiles of the field isolates showed minor differences when compared with that of the vaccine strain. The OMP of 33 kDa was only expressed by the vaccine strain. Four field isolates expressed an OMP of 39 kDa, which did not appear in the profiles of the remaining two field isolates and the P-52 strain. Similarly, an OMP of 25 kDa was exclusively seen in the profile of a single isolate. By immunoblotting studies, using anti-P. multocida (P-52) whole-cell hyperimmune serum raised in rabbits as well as buffalo immune sera, it became evident that the polypeptide of 37 kDa was the most antigenic OMP in the profiles of all the isolates, including the P-52 strain. Other polypeptides were either weakly antigenic or visible in the profile of only a few of the isolates. The study thus identified the major OMP of P. multocida (B:2) and suggested that this highly antigenic 37 kDa OMP has potential for further protective and immunodiagnostic studies.     

陕西省部分禽源性大肠杆菌的外膜蛋白型

从陕西省部分优势血清型的禽源性大肠杆菌中提取外膜蛋白 (outer membrane protein,OMP) ,用 SDS- PAGE进行 OMP分型。 18株菌共产生了 3种 OMP型 ,其中 4株 O1菌株 ,4株 O2菌株 ,4株 O78菌株和 3株 O89菌株各属2个 OMP型 (OMP- 1,2型 ) ;3株 O75菌株属 3个 OMP型 (OMP- 1,2 ,3型 )。结果表明 ,陕西省分离的优势血清型禽源大肠杆菌具有多样性的 OMP型 ,且多种血清型间具有共同的 OMP型。     

禽源大肠杆菌O2,O78分离株外膜蛋白型的研究

从１７个禽大肠杆菌病病例的Ｏ２、Ｏ７８分离株提取的主要外膜蛋白（ＯＭＦ），在ＳＤＳ－ｐＡＧＥ中出现了２个ＯＭＰ型。其中，９个Ｏ２分离株属２个ＯＭＰ型，８个Ｏ７８分离株均属其中的１个ＯＭＰ型。结果表明，分离到的Ｏ２、Ｏ７８大肠杆菌具有多样性的ＯＭＰ型，而且两者存在着共同的ＯＭＰ型。     

Effect of Incubation Temperature and Salinity on Expression of the Outer Membrane Protein Profile of Edwardsiella tarda

Abstract

Outer membrane proteins (OMP) of 10 isolates of Edwardsiella tarda were compared by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The OMP profile of the type strain E. tarda ATCC 15947 cultured at 25°C had five major protein bands of 40, 36.5, 34, 28.5, and 25 kDa and a large number of minor proteins ranging in size from approximately 10 to 120 kDa. Differences between the OMP profiles of the isolates of E. tarda included the inconsistent presence of the 34- or 36.5-kDa proteins in five isolates of E. tarda and two major bands of 47 and 44 kDa that were present in only two isolates of E. tarda. There were no differences in the outer membrane protein profiles of 9 out of 10 isolates of E. tarda incubated at a temperature of 25°C compared with those at 35°C. To evaluate the effect of salinity, 10 isolates of E. tarda were cultured in brain heart infusion broth containing 0.5, 1.5, and 3.0% sodium chloride. Reactions of isolates of E. tarda to the different salinity levels were placed into three groups. The first group expressed more or fewer protein bands at 1.5% sodium chloride. The second group lost major bands at 3% salinity, whereas the third group had no change in the OMP profile with salinity. The OMP profile differences and the different reactions to salinity levels suggest that the isolates are heterogeneous.     

Identification of virulence associated markers in the cell wall of pigeon Streptococcus gallolyticus strains

The cell wall protein profiles of 56 isolates of Streptococcus gallolyticus of differing virulence for pigeons were compared by SDS-PAGE. Additionally, Western blot analysis was performed on the cell wall proteins of 14 strains using sera of pigeons, experimentally infected with A(+)T1 or A(-)T2 strains of S. gallolyticus. The profile of silver stained gels exhibited a complex array of 20-50 bands ranging from less than 6.5-210kDa. A band with molecular mass of 114kDa was only observed in isolates that belonged to the highly virulent A(+)T1, A(+)T2, A(+)T3 and A(-)T1 culture supernatant groups. A band with a slightly higher molecular mass (115kDa) as well as a 207kDa band were only detected in isolates that belonged to the moderately A(-)T3 or low A(-)T2 virulent culture supernatant groups. The 114 and 115kDa band were recognised by all homologous and heterologous pigeon sera used whereas the 207kDa band was only recognised by sera of pigeons infected with a A(-)T2 strain. These findings may indicate that the 114, 115 and 207kDa bands are useful as additional virulence associated markers for pigeon S. gallolyticus strains.     

Characterization and comparison of antimicrobial susceptibilities and outer membrane protein and plasmid DNA profiles of Pasteurella haemolytica and certain other members of the genus Pasteurella.

The outer membrane protein (OMP), plasmid, and antimicrobial resistance profiles of Pasteurella haemolytica serotypes 1 through 12, a bovine isolate of P multocida, a chicken isolate of P multocida, and an unidentified Pasteurella species of bovine origin were examined. Isolates of P haemolytica serotypes belonging to the same biotype possessed similar OMP profiles. Biotype A isolates contained 2 prominent OMP of 43 kilodaltons (kD) and 29 kD, whereas biotype-T serotypes contained 3 major OMP of 43, 36, and 25 kD. The major OMP profiles of the 2 P multocida isolates and the unidentified Pasteurella species were different from each other and from P haemolytica isolates. Plasmid DNA screening indicated both plasmid-containing and plasmid-free P haemolytica and P multocida isolates. Multiple drug resistance was found in pasteurellae isolates with and without plasmids. However, a relationship between drug resistance and plasmid isolation was found in 3 of 4 haemolytica serotype 1 field isolates, all of which contained a 2.51-megadalton plasmid and had multiple drug resistance for benzylpenicillin, ampicillin, streptomycin, and tetracycline.     

Immunogenicity of iron-regulated outer membrane proteins of Pasteurella multocida A:3 in cattle: molecular characterization of the immunodominant heme acquisition system receptor (HasR) protein

The iron-regulated outer membrane proteins (IROMPs) of Pasteurella multocida A:3 strain 232 (Pm232), a bovine isolate, were investigated as potential immunogens in cattle. We addressed the ability of P. multocida IROMP-enriched fractions to induce antibody responses in cattle by different vaccination strategies and the protective efficacy of these antibodies using a P. multocida-induced pneumonia challenge model. Vaccination of cattle with outer membrane-enriched fractions derived from Pm232 grown on either iron-depleted (IROMPs) or iron-sufficient (OMPs) conditions induced significant antibody responses; however, the correlation with lung lesion scores was not significant (P = 0.01 and P < 0.07, respectively). SDS-PAGE, Western blots and densitometric analyses of Pm232 grown under iron-deficient conditions revealed five major IROMPs including an immunodominant 96 kDa protein band. Mass spectrometry analysis of the 96kDa protein band suggested homology with the heme acquisition system receptor (HasR) of avian P. multocida (strain Pm70) and was confirmed by DNA sequence analysis of the cloned Pm232 hasR gene. Further analyses indicated that Pm232 HasR is a surface-exposed OMP and conserved among most P. multocida isolates investigated. In addition, cattle vaccinated with live Pm232 or IROMPs had significantly higher antibody responses to the 96 kDa protein band and the correlation with lung lesion scores approached significance (P = 0.056). These results indicate that antibody responses in cattle are induced by P. multocida IROMPs, and that the 96 kDa HasR protein is an immunodominant IROMP.     

Protein variability among Mycoplasma hyopneumoniae isolates

Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to study the protein variability of Mycoplasma hyopneumoniae isolates. Fifty-six M. hyopneumoniae isolates from 6 different countries and 37 different herds were used. From eight herds, more than one isolate was available. All SDS-PAGE patterns of isolates originating from different herds were clearly divergent. Intra-species protein variability was quantified using the reference strain J and seven field strains all obtained from different herds and classified according to virulence. Between the field strains, a variability of 25% was found, while the culture-adapted strain J was clearly divergent and showed 30% variability with the field strains. No clustering according to virulence was obtained, but a protein band of about 181 kDa was present in the two highly virulent isolates whereas this protein band was absent in the moderately and low virulent isolates. Protein patterns of isolates derived from different animals from the same herd, were identical or differed in only a few protein bands. This study clearly indicates that, in agreement with previous studies on genomic diversity of M. hyopneumoniae isolates, proteomic variability within the species is high. Our study did not find clear evidence that more than one M. hyopneumoniae isolate circulates within a herd at a specific time point. The minor differences found between M. hyopneumoniae isolates from the same herd might reflect the organism's ability to alter its proteomic expression profile under field conditions.     

Plasmid profile analysis, phage typing, and antibiotic sensitivity of Salmonella dublin from clinical isolates in the United States.

One hundred clinical isolates of Salmonella choleraesuis subsp. choleraesuis serovar dublin (Salmonella dublin) were examined for phage sensitivity, antibiotic resistance patterns, and plasmid content. Computer analysis of the lysis patterns observed by using 27 typing phages divided the S. dublin isolates into 26 groups. One lytic pattern (Designated pattern 16) contained 52% of the isolates examined whereas 16 isolates had unique patterns, and nine patterns had fewer than ten members. Although 14 antibiotic resistance patterns were observed among the 100 isolates, 79% of the isolates grouped in three major patterns. Seven plasmid groups were identified and designated A-G based on the large plasmids found in the isolates. Of the 100 isolates, 28 contained the plasmid profile of Group A, 28 were Group B, 7 were Group C, 34 were Group D, and 1 isolate each was observed in Groups E, F, and G. The strong association between antibiotic resistance pattern and plasmid type suggest that the drug resistance genes are plasmid borne.     

Outer membrane protein profiles of Yersinia ruckeri

The outer membrane protein (OMP) profiles of 135 isolates of Yersinia ruckeri, obtained from nine European countries (100 isolates), North America (23 isolates), Australia (six isolates) and South Africa (two isolates), and including four reference strains, were examined by SDS-PAGE. Outer membranes were isolated by selective solubilisation of the cytoplasmic membrane with 0.5% (w/v) sodium N-lauroyl sarcosinate (Sarkosyl). Outer membrane proteins were stable after in vitro passage and there was no variation in OMP profiles due to colony selection. With the exception of a 39.5 kDa peptidoglycan-associated protein there was also no variation at different stages of the growth cycle. The 39.5 kDa protein was not produced during logarithmic growth phase but increased in abundance as the stationary phase progressed. Interstrain variation occurred in the possession of a 36.5 or 38 kDa heat-modifiable protein and in the possession of peptidoglycan-associated proteins in the molecular weight range 36.5 to 40.5 kDa. Based on variation of these proteins five OMP-types, designated OMP-types 1-5, were identified among the 135 isolates examined. Outer membrane protein analysis was demonstrated to be useful in epidemiological studies of Y. ruckeri.     

Pasteurella multocida and bovine respiratory disease

Pasteurella multocida is a pathogenic Gram-negative bacterium that has been classified into three subspecies, five capsular serogroups and 16 serotypes. P. multocida serogroup A isolates are bovine nasopharyngeal commensals, bovine pathogens and common isolates from bovine respiratory disease (BRD), both enzootic calf pneumonia of young dairy calves and shipping fever of weaned, stressed beef cattle. P. multocida A:3 is the most common serotype isolated from BRD, and these isolates have limited heterogeneity based on outer membrane protein (OMP) profiles and ribotyping. Development of P. multocida-induced pneumonia is associated with environmental and stress factors such as shipping, co-mingling, and overcrowding as well as concurrent or predisposing viral or bacterial infections. Lung lesions consist of an acute to subacute bronchopneumonia that may or may not have an associated pleuritis. Numerous virulence or potential virulence factors have been described for bovine respiratory isolates including adherence and colonization factors, iron-regulated and acquisition proteins, extracellular enzymes such as neuraminidase, lipopolysaccharide, polysaccharide capsule and a variety of OMPs. Immunity of cattle against respiratory pasteurellosis is poorly understood; however, high serum antibodies to OMPs appear to be important for enhancing resistance to the bacterium. Currently available P. multocida vaccines for use in cattle are predominately traditional bacterins and a live streptomycin-dependent mutant. The field efficacy of these vaccines is not well documented in the literature.     

Analyses of lipopolysaccharides, outer membrane proteins and DNA fingerprints reveal intraspecies diversity in Moraxella bovis isolated in Argentina

Intra-specific diversity within Moraxella bovis was investigated analysing DNA fingerprints, outer membrane proteins (OMP) and lipopolysaccharides (LPS) profiles. Three collection strains and 57 isolates of M. bovis, collected during 3 years from cattle with infectious bovine keratoconjunctivitis (IBK) symptoms, from diverse geographical locations of Argentina, were examined. The LPS and OMP profiles were studied through SDS–PAGE analysis and genotype was determined by PCR-DNA fingerprinting. Genotyping identified five DNA types while analysis of LPS and OMP profiles identified three rough LPS types and three OMP types among the 60 isolates of M. bovis including the three collection strains. None of the three methods employed to assess diversity was discriminating when used alone because the degree of heterogeneity in each group of surface structures was limited, but when data of each typing method were combined, 15 distinct subgroups were determined. This subgrouping was clearly able to differentiate isolates of the same genotype. These typing methods appear to be useful to assess different aspects of the disease such as the diversity within a population of M. bovis associated to epidemic conditions, track the causal agent in an outbreak of the disease, monitoring vaccination programs and studies on virulence.     

Outer membrane protein profiles of Edwardsiella ictaluri from fish.

Outer membrane proteins (OMP) prepared with sodium N-lauroyl sarcocinate (SLS) from 33 Edwardsiella ictaluri isolates from fish were examined by electrophoresis. Twenty-eight isolates from channel catfish (Ictalurus punctatus) had similar OMP profiles. Ten bands (71 kilodaltons [kD] to 19.5 kD) were identified in all isolates from channel catfish. One major 35-kD protein comprised most of the protein content of the outer membrane of isolates from channel catfish. Differences existed among isolates in the amount of protein within minor OMP bands. Edwardsiella ictaluri ATCC 33202 contained larger quantities of the 38.5- and 37-kD proteins than did the other isolates. Outer membrane protein profiles of E ictaluri derived from Bengal danio (Danio devario) and walking catfish (Clarias batrachus) were identical to OMP profiles of isolates from channel catfish. In contrast, OMP profiles from single isolates from green knife fish (Eigemannia virescens) and white catfish (Ictalurus catus) were different. Variations in incubation time, SLS extraction time, SLS extraction number, and in vivo and in vitro passage had no effect on the OMP profile of E ictaluri ATCC 33202. An increase in duration of sample solubilization did affect the OMP profile of E ictaluri ATCC 33202 by decreasing the amount of protein in 52-, 46-, and 43.5-kD bands. Accompanying the decrease were increased staining intensity in the 31.5- and 28.5-kD bands and the appearance of 4 new bands (34, 33, 25.5, and 22.5 kD). Edwardsiella ictaluri, a gram-negative bacterium in the family Enterobacteriaceae, is the cause of enteric septicemia of catfish.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diversity of avian Pasteurella multocida strains based on capsular PCR typing and variation of the OmpA and OmpH outer membrane proteins

One hundred avian Pasteurella multocida isolates recovered from cases of fowl cholera and related infections in England and Wales over a 13-year period were characterised by capsular PCR typing and analysis of outer membrane protein (OMP) profiles. Sixty-eight percent of the strains were of capsular type A, 14% were type F, 5% were type D, 4% were type B and 9% were untypable. Nineteen distinct OMP profiles (OMP-types) were identified based mainly on molecular mass heterogeneity of the heat-modifiable (OmpA) and porin (OmpH) proteins. Fifty-six percent of the isolates were represented by 15 OMP-types, whereas 44% of the isolates were associated with four OMP-types. The extensive molecular mass heterogeneity of the OmpA and OmpH proteins supports previous findings that avian P. multocida strains are very diverse. Furthermore, the isolates studied were associated with different clinical symptoms and were recovered from a wide range of lesions and tissues. The high degree of strain diversity together with the wide variety of clinical symptoms suggest that certain avian strains of P. multocida are opportunistic pathogens of relatively low virulence. Strains of capsular types B, D and F, as well as the untypable isolates, were associated exclusively with specific OMP-types and represent distinct and widely disseminated clonal groups. These observations support the view that avian strains of P. multocida have a clonal population structure. Based on previous studies, the molecular mass heterogeneity of the OmpA and OmpH proteins might provide a selective advantage to P. multocida by generating antigenic variation.     

Strain- and growth condition-dependent variability in outer membrane protein expression by Bordetella bronchiseptica isolates from dogs.

OBJECTIVE: To characterize strain-dependent and growth condition-dependent variability in outer membrane protein (OMP) expression of Bordetella bronchiseptica isolates from dogs and evaluate the systemic immune response to OMP of B bronchiseptica among infected dogs. SAMPLE POPULATION: 8 strains of B bronchiseptica isolated from dogs, including a historic reference strain, 2 commercially available vaccine strains, and 5 field strains, and serum samples collected from 3 specific-pathogen-free (SPF) dogs before and 1 month after infection with B bronchiseptica. PROCEDURE: OMP were isolated from cultures in the late exponential phase of growth and compared among strains and, within strains, among growth conditions by means of polyacrylamide gel electrophoresis and immunoblotting. Serum samples were probed with OMP from 1 of the field strains. RESULTS: Strain-dependent variability in OMP profiles and growth condition-dependent and strain-dependent variability in expression of filamentous hemagglutinin (FHA) and pertactin was found, along with heterogeneity of the pertactin proteins produced by these B bronchiseptica strains. All 3 SPF dogs seroconverted to proteins with estimated molecular masses of 200 and 66 kDa, suggesting that FHA and pertactin were involved in the immunologic response of these dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that there is growth condition and strain variability in expression of OMP, FHA, and pertactin proteins produced by B bronchiseptica. This information could be useful in the improvement of vaccines for prevention of bordetellosis in dogs.     

Comparison of seroreactivity of rams with brucellosis in a complement fixation test, whole cell ELISA and by immunoblotting

In rams with ovine brucellosis, a high degree of serological correlation exists between the complement fixation (CF) test which utilises antigen extracted from bacteria with hot saline, and the ELISA reactivity using methanol-fixed Brucella ovis as the assay reagent. Since the whole cell ELISA (CELISA) detects mainly antibodies against surface antigens of B. ovis, it was concluded that the similar findings of the two serological tests is due in part to the presence of membrane antigens in the CF test antigen following hot saline extraction of intact bacteria. Immunoblots with pooled sera representing different CF titres confirmed that the major immunoreactive antigens of B. ovis were located in four zones: alpha, beta, gamma 1 and 2 with corresponding apparent molecular masses of 55 and 60 kDa; 27 and 29 kDa; 18.5-20 kDa and 17-18 kDa, respectively. These zones of reactivity were consistently present in immunoblots when assayed against different B. ovis isolates even though Coomassie brilliant blue staining of SDS-PAGE gels revealed some differences in polypeptide banding patterns. However, these intensely-stained CBB bands located at 38 and 40 kDa which distinguished three of the seven B. ovis isolates were considerably less reactive in immunoblots compared to polypeptides that were located at positions equivalent to alpha, beta or gamma reactivities. Intensity of immunoblot reactivity against polypeptides located in the alpha, beta and gamma zones intensified with increasing CF titre. Sera with CF titres greater than 32 also tended to react against bands of higher apparent molecular masses located at 65, 70, 73, 78, 80 and 86 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of monoclonal antibodies to bovine herpesvirus type I, Los Angeles strain.

We produced monoclonal antibodies (mAbs) against bovine herpesvirus type 1 (BHV-1), Los Angeles strain, and then evaluated them as potential candidates for preparing diagnostic reagents. Of the 318 cell lines expressing antibodies to the virus, 60% (192) secreted IgG and 40% (126) secreted IgM. Twenty-six mAbs were selected based on enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) titers and characterized by immunoprecipitation, immunofluorescence and immunoblots. The selected mAbs were assigned to one of four groups based on their immunoprecipitation patterns. Group A (4 mAbs) precipitated a complex of three glycoproteins with molecular weight (MW) 130 kDa, 72 kDa and 55 kDa, which presumably represented gI of BHV-1. Monoclonal antibodies of this group were highly reactive in ELISA but had low VN titers. Group B (4 mAbs) precipitated a glycoprotein with MW of 71 kDa (gIV). This group of mAbs had high VN titers. Group C (16 mAbs) precipitated a 97 kDa glycoprotein (gIII). Monoclonal antibodies of this group had high ELISA but low VN titers. Group D (2 mAbs) precipitated a double band of non-glycosylated proteins with MW of 37/32 kDa; these proteins could not be assigned to any of the antigens of BHV-1 previously described. ELISA and VN titers of this group of mAbs were low. To test the antigenic variability of the antigenic determinants which were recognized by these 4 groups of mAbs, we adapted Madin Darby bovine kidney cell-propagated BHV-1 Los Angeles strain to Crandell's feline kidney cell line. After the tenth passage in feline kidney cells, the epitopes on the 37/32 kDa peptide recognized by the mAbs group D were no longer detectable. Additional changes were noted in the electrophoretic mobility of the 130 kDa and 71 kDa glycoproteins (gI) identified by mAb of group A shifted downward. The 71 kDa glycoprotein (gIV) reactive with mAb group B and the 97 kDa (gIII) reactive with mAb group C remained stable. Since clone No. 191 of group B mAb was potent in ELISA, VN, immunoblots and immunofluorescence, and recognized an epitope which did not change under selective pressure, we feel that the mAb produced by this clone are a good candidate for the production of diagnostic reagents.