金黄色葡萄球菌凝集因子A的免疫原性

为研究金黄色葡萄球菌(Staphylococcus aureus)凝集因子A(ClfA)免疫原性及免疫保护作用,应用PCR方法扩增出金黄色葡萄球菌Newman、Wood46和HLJ23-1株的clfa基因并进行序列分析,再将Newman株的clfa基因插入到pQE-30载体上,导入宿主菌Escherichia coli M15(pREP4)并诱导表达和纯化ClfA重组蛋白。用纯化的ClfA免疫小鼠,检测血清中抗体和细胞因子水平,首次免疫后35 d时用金黄色葡萄球菌Wood46、HLJ23-1、Newman株对小鼠攻毒。结果发现:clfa基因序列高度保守;ClfA重组蛋白在E.coli M15中获得表达;在首次免疫后35 d时血清抗体效价和细胞因子浓度与对照组相比,均显著升高(P<0.05);攻毒结果为蛋白免疫组小鼠获得一定的免疫保护。由此表明,ClfA重组蛋白有较好的免疫原性和免疫保护力。     

牛源金黄色葡萄球菌粘附因子Efb与ClfA的免疫生物学特性比较

为评价与比较金黄色葡萄球菌的粘附因子Efb(Extracellular fibrinogen-binding protein)和Clf A(Clumping factor A)的抗原性、粘附特性及其抗血清对金黄色葡萄球菌菌体的识别能力、抗粘附能力及免疫保护力等免疫生物学特性,分别构建Efb和Clf A的重组表达载体,并用纯化的重组蛋白免疫实验动物,分别检测家兔抗血清对菌体的识别能力、抗粘附能力以及小鼠抗血清的抗体效价和免疫保护作用。结果显示,Efb和Clf A均有与纤维蛋白原(Fibrinogen,Fg)结合的能力,且Efb蛋白对纤连蛋白(Fibronectin,Fn)的结合能力优于Clf A(P0.01),Clf A免疫组抗血清对全细菌的识别能力优于Efb免疫组(P0.01)。与对照组相比Efb和Clf A免疫组的抗血清均能显著抑制金黄色葡萄球菌对Fg和Fn的粘附(P0.01),Efb免疫组对金黄色葡萄球菌的粘附抑制优于Clf A,两种粘附因子免疫小鼠后产生的血清抗体效价与对照组比较有显著的升高,抗体滴度可达1∶40 500,攻毒结果显示Efb与Clf A免疫组均对小鼠具有良好的保护效果。该研究结果对Efb在金黄色葡萄球菌的亚单位疫苗的应用奠定了基础。     

Opsonic and Protective Properties of Antibodies Raised to Conjugate Vaccines Targeting Six Staphylococcus aureus Antigens

Staphylococcus aureus is a major cause of nosocomial and community-acquired infections for which a vaccine is greatly desired. Antigens found on the S. aureus outer surface include the capsular polysaccharides (CP) of serotype 5 (CP5) or 8 (CP8) and/or a second antigen, a β-(1→6)-polymer of N-acetyl-D-glucosamine (PNAG). Antibodies specific for either CP or PNAG antigens have excellent in vitro opsonic killing activity (OPKA), but when mixed together have potent interference in OPKA and murine protection. To ascertain if this interference could be abrogated by using a synthetic non-acetylated oligosaccharide fragment of PNAG, 9GlcNH₂, in place of chemically partially deacetylated PNAG, three conjugate vaccines consisting of 9GlcNH₂ conjugated to a non-toxic mutant of alpha-hemolysin (Hla H35L), CP5 conjugated to clumping factor B (ClfB), or CP8 conjugated to iron-surface determinant B (IsdB) were used separately to immunize rabbits. Opsonic antibodies mediating killing of multiple S. aureus strains were elicited for all three vaccines and showed carbohydrate antigen-specific reductions in the tissue bacterial burdens in animal models of S. aureus skin abscesses, pneumonia, and nasal colonization. Carrier-protein specific immunity was also shown to be effective in reducing bacterial levels in infected lungs and in nasal colonization. However, use of synthetic 9GlcNH₂ to induce antibody to PNAG did not overcome the interference in OPKA engendered when these were combined with antibody to either CP5 or CP8. Whereas each individual vaccine showed efficacy, combining antisera to CP antigens and PNAG still abrogated individual OPKA activities, indicating difficulty in achieving a multi-valent vaccine targeting both the CP and PNAG antigens.     

Fibrinogen binding sites P336 and Y338 of clumping factor A are crucial for Staphylococcus aureus virulence

We have earlier shown that clumping factor A (ClfA), a fibrinogen binding surface protein of Staphylococcus aureus, is an important virulence factor in septic arthritis. When two amino acids in the ClfA molecule, P(336) and Y(338), were changed to serine and alanine, respectively, the fibrinogen binding property was lost. ClfAP(336)Y(338) mutants have been constructed in two virulent S. aureus strains Newman and LS-1. The aim of this study was to analyze if these two amino acids which are vital for the fibrinogen binding of ClfA are of importance for the ability of S. aureus to generate disease. Septic arthritis or sepsis were induced in mice by intravenous inoculation of bacteria. The clfAP(336)Y(338) mutant induced significantly less arthritis than the wild type strain, both with respect to severity and frequency. The mutant infected mice developed also a much milder systemic inflammation, measured as lower mortality, weight loss, bacterial growth in kidneys and lower IL-6 levels. The data were verified with a second mutant where clfAP(336) and Y(338) were changed to alanine and serine respectively. When sepsis was induced by a larger bacterial inoculum, the clfAP(336)Y(338) mutants induced significantly less septic death. Importantly, immunization with the recombinant A domain of ClfAP(336)SY(338)A mutant but not with recombinant ClfA, protected against septic death. Our data strongly suggest that the fibrinogen binding activity of ClfA is crucial for the ability of S. aureus to provoke disease manifestations, and that the vaccine potential of recombinant ClfA is improved by removing its ability to bind fibrinogen.     

Clumping factor A-mediated virulence during Staphylococcus aureus infection is retained despite fibrinogen depletion

Clumping factor A (ClfA), a fibrinogen-binding protein expressed on the Staphylococcus aureus cell surface, has previously been shown to act as a virulence factor in experimental septic arthritis. Although the interaction between ClfA and fibrinogen is assumed to be of importance for the virulence of S. aureus, this has not been demonstrated in any in vivo model of infection. Therefore, the objective of this study was to investigate the contribution of this interaction to ClfA-mediated virulence in murine S. aureus-induced arthritis. Ancrod, a serine protease with thrombin-like activity, was used to induce in vivo depletion of fibrinogen in mice. Ancrod treatment significantly aggravated septic arthritis following inoculation with a ClfA-expressing strain (Newman) compared to control treatment. Also, ancrod treatment tended to enhance the arthritis induced by a clfA mutant strain (DU5876), indicating that fibrinogen depletion exacerbates septic arthritis in a ClfA-independent manner. Most importantly, the ClfA-expressing strain was much more arthritogenic than the isogenic clfA mutant, following inoculation of fibrinogen-depleted mice. This finding indicates that the interaction between ClfA and free fibrinogen is not required for ClfA-mediated functions contributing to S. aureus virulence. It is conceivable that ClfA contributes to the virulence of S. aureus through interactions with other host ligands than fibrinogen.     

Solid matrix-antibody-antigen complexes induce antigen-specific CD8+ cells that clear a persistent paramyxovirus infection

We have previously shown that the adoptive transfer of splenocytes, isolated from mice immunized by infection with the paramyxovirus simian virus 5 (SV5), enhance the speed of clearance of SV5 from the lungs of immunodeficient mice; clearance is mediated primarily through CD8+ effector cells and not by serum neutralizing antibody (D.F. Young, R.E. Randall, J.A. Hoyle, and B.E. Souberbielle, J. Virol. 64:5403-5411, 1990). In this article we demonstrate that immunization of mice with solid matrix-antibody-antigen (SMAA) complexes also induces CD8+ effector cells that are responsible for clearing persistent SV5 infections in immunodeficient mice. The demonstration that immunization with SMAA complexes (an exogenous antigen) can induce class I-restricted cytotoxic T lymphocytes (CTLs) suggests that that these cells may be responsible for virus clearance in vivo. This premise is supported indirectly by the observation that immunization with SMAA complexes was less efficient in inducing class I-restricted CTLs (as measured in vitro) than was infectious virus and that splenocytes isolated from mice immunized with SMAA complexes were also less efficient in clearing virus from lungs of immunodeficient mice than were splenocytes isolated from mice immunized by infection with virus. This was not because the SMAA complexes were generally less immunogenic than infectious virus, since mice immunized with SMAA complexes (which contained the HN protein of SV5) produced higher levels of neutralizing antibody than mice immunized with infectious virus. In the majority of experiments, fixed and killed suspensions of Staphylococcus aureus Cowan strain A were used as the solid matrix in the construction of SMAA complexes. However, in this article we present evidence that alum-antibody-antigen complexes are as immunogenic as S. aureus A-antibody-antigen complexes. These results suggest that the immunological reactivity of the solid matrix itself does not influence the intensity of the immune response to the antigens of interest in the SMAA complexes. The significance of these results for vaccine design are discussed.     

Fasciola hepatica: host responders and nonresponders to parasite glutathione S-transferase.

Fasciola hepatica glutathione S-transferase (FhGST) was isolated from adult worms by glutathione agarose affinity chromatography. SDS-PAGE shows three proteins of M(r) ranging from 29-27.8 kDa. Western immunoblot analyses using SDS-PAGE separated adult worm extracts and probed with a rabbit anti-FhGST antiserum reveal two bands in the same M(r) range. Mice and rabbits immunized with purified FhGST develop copious amounts of anti-FhGST antibodies. Moreover, antisera to F. hepatica adult worms and excretion-secretion products also react with FhGST. Cross-reactivity with schistosomes is evidenced in the reactivity with FhGST of anti-Schistosoma mansoni adult worm antisera and, to a lesser extent, antisera to S. mansoni-soluble egg antigens. The time of appearance of anti-FhGST antibodies in different species of animals infected with F. hepatica was determined. Sheep and a New Zealand white rabbit developed anti-FhGST antibodies detectable by ELISA as early as 2 weeks postexposure with F. hepatica. However, neither mice nor calves infected with F. hepatica developed antibodies to FhGST through the 5-10 weeks of infection tested. But mice infected with S. mansoni developed anti-FhGST cross-reacting antibodies by 6 weeks of infection. Calves immunized with a Fasciola/Schistosoma cross-reactive, cross-protective antigen complex in which a 12,000-kDa protein (Fh12) has been shown to contain immunoprophylactic activity, also developed antibodies to FhGST. Since FhGST is a novel potential vaccine, its protection-inducing capability in a multivalent vaccine combined with Fh12 clearly warrants study. In summary, it appears that hosts with fascioliasis are either responders to FhGST (rabbits, sheep) or nonresponders (mice, cattle), offering interesting models for studying the immune response.     

14型肺炎球菌荚膜多糖-破伤风类毒素结合疫苗的研制

将14型肺炎球菌的荚膜多糖(PS)与破伤风类毒素(TT)通过化学方法结合,制备成多糖-蛋白结合疫苗(PS14-TT)。用该结合疫苗免疫小鼠,在小鼠体内产生了高滴度的PS-IgG抗体和TT-IgG抗体,且再次注射后有加强应答效应,表明制备的结合疫苗保留了完好的抗原性,具有胸腺依赖性抗原的特性。     

Induction of protective immunity against Schistosoma mansoni by a non living vaccine. I. Partial characterization of antigens recognized by antibodies from mice immunized with soluble schistosome extracts

A single intradermal injection of frozen and thawed schistosomula in conjunction with the bacterial adjuvant Mycobacterium bovis strain Bacille Calmette Guerin, Phipps substrain (BCG) induced significant levels of resistance to challenge Schistosoma mansoni infection in C57BL/6 mice. Immunization with the aqueous fraction remaining after 100,000 X G centrifugation of the larval lysate was also protective under these conditions, suggesting that some immunogenic determinants may not be membrane associated. Frozen-thawed cercariae and soluble components of adult worms also protected against challenge infection in these experiments. These observations indicate that soluble immunogens are present in both early and late developmental stages of the parasite, and therefore may be good candidate antigens for an immunochemically defined vaccine against schistosomiasis. Induction of humoral reactivity against soluble or membrane antigens was examined in mice protected against cercarial challenge by prior exposure to frozen-thawed larvae, soluble larval, or soluble adult antigens plus BCG. Animals that were immunized with frozen-thawed larvae produced low but significant levels of antibodies against larval surface antigens when examined by indirect immunofluorescence or by immunoprecipitation of surface-labeled schistosomula. Mice immunized with soluble antigens, however, showed negligible antibody reactivity against surface membrane antigens. Because mice immunized with soluble antigens were resistant to challenge infection, these results strongly suggest that anti-surface membrane reactivity is not required in the mechanism of protective immunity in this model. Sera from mice immunized with either total freeze-thaw larval lysate or soluble schistosome extracts all showed strong reactivity against soluble antigens, as detected by ELISA. Western blot analysis showed these antisera to react with a restricted number of high m.w. antigens that were present both in schistosomula and in adult worms. These antigens are therefore likely to play a major role in the development of resistance in this model as immunogens and/or as targets of protective immune response.     

Immunoaffinity-isolated antigens induce protective immunity against larval Strongyloides stercoralis in mice

The objective of this study was to identify soluble protein antigens that would induce protective immunity against infective-stage larvae (L-3) of Strongyloides stercoralis in mice. Deoxycholate (DOC)-soluble proteins derived from L-3, adsorbed to aluminum hydroxide, induced protective immunity in BALB/c mice. The immunized mice generated parasite-specific IgG that could transfer passive immunity to na?ve animals. The protective antibodies bound to parasite antigens found in the muscles and nerve cords of the L-3. An IgG affinity chromatography column generated with IgG from the sera of DOC-immunized mice was used to purify specific larval antigens. Proteins were eluted from the affinity column with sizes of 80, 75, 61, 57, 43, and 32 kDa. This antigen pool stimulated both proliferation and IL-5 production by splenocytes recovered from mice immunized with live L-3. Vaccination of mice with the immunoaffinity-isolated antigens led to significant protective immunity, with 83% of challenge larvae killed. This study demonstrates that IgG-isolated proteins are candidate antigens for a vaccine against larval S. stercoralis.     

The immunogenicity of mono- and associated vaccines made from strains of opportunistic bacteria]

In experiments on white mice the immunogenic potency of two variants of combined vaccines prepared from strains of opportunistic bacteria was compared with that of monovaccines used as the components of the combined vaccines. All the vaccines under study were found highly immunogenic. Decrease of monovaccine share in the combined vaccine did not reduce the immunogenic potency to any of the strains. An 8- to 16-fold increase in the level of specific antibodies in the colostric lactosera of cows immunized with combined vaccines was observed, and the survival rate of newborn calves increased 3- to 4-fold.     

Staphylococcus aureus strains isolated from bovine mastitis: virulence,antibody production and protection from challenge in a mouse model

Septic arthritis in mice was used as a model to evaluate the virulence of Staphylococcus aureus and coagulase-negative staphylococci (CNS) isolated from cases of bovine mastitis. In addition, the model was used to evaluate the cross protection elicited by heterologous antibodies. Mice were intramuscularly inoculated with serial bacterial doses of different strains of S. aureus or CNS, for virulence determination; they were monitored for arthritis, gangrene or death up to 20 days. Antibody response, cross reactivity and resistance to challenge were tested by subcutaneous inoculation with a low dose of one of the S. aureus or CNS strains followed by challenge with two S. aureus strains. S. aureus alpha-hemolysin isolate was the most virulent, followed by alpha+beta-hemolysin and beta-hemolysin isolates. The least virulent isolates were the non-hemolytic S. aureus strains but even they were more virulent than the CNS strains tested. Antibodies against three different S. aureus antigens were detected by the ELISA in all mice that were inoculated with the S. aureus strains but not in any of those with the CNS strains. Immunoblot test against various S. aureus strains as antigens showed high cross-reactivity among the S. aureus strains but only a slight similarity, restricted to the bands above 36 kDa, with the CNS sera. Low-dose inoculation of alpha or alpha+beta strains before challenge with homologous and heterologous strains protected the mice, whereas the two beta strains provided only partial protection. The inoculations of non-hemolytic S. aureus or the CNS strains did not elicit any protection. Our findings demonstrate that pre-exposure of mice to a low dose of certain S. aureus strains could provide protection and that the antibodies produced could have an important protective role.     

Expression of staphylococcal clumping factor A impedes macrophage phagocytosis

Clumping factor A (ClfA), a fibrinogen-binding protein linked to the Staphylococcus aureus cell wall, is an important virulence factor in infection models, e.g., of septic arthritis. However, the mechanism(s) by which ClfA contributes to the virulence of the bacterium is unknown. In the present study, the impact of ClfA expression on the phagocytosis of S. aureus by macrophages was investigated using clfA-positive and clfA-negative isogenic strains. Furthermore, the possible contribution of ClfA to the proinflammatory and immunostimulatory activity of S. aureus was studied. Our results indicate that ClfA expression significantly protects S. aureus against macrophage phagocytosis. This protection does not require the presence of intact fibrinogen, a ligand for ClfA. ClfA expression by S. aureus enhanced the proliferative response of spleen cells. On the other hand, a clfA mutant strain caused more release of proinflammatory mediators by macrophages than its clfA-positive parental strain. Both the protection against phagocytosis and the enhanced immunostimulatory activity provided by ClfA expression are likely to contribute to the in vivo virulence of S. aureus.     

Immunogenic and Invasive Properties of Brucella melitensis 16M Outer Membrane Protein Vaccine Candidates Identified via a Reverse Vaccinology Approach

Brucella is the etiologic agent of brucellosis, one of the most common and widely distributed zoonotic diseases. Its highly infectious nature, the insidious, systemic, chronic, debilitating aspects of the disease and the lack of an approved vaccine for human use in the United States are features that make Brucella a viable threat to public health. One of the main impediments to vaccine development is identification of suitable antigens. In order to identify antigens that could potentially be used in a vaccine formulation, we describe a multi-step antigen selection approach. We initially used an algorithm (Vaxign) to predict ORF encoding outer membrane proteins with antigenic determinants. Differential gene expression during acute infection and published evidence for a role in virulence were used as criteria for down-selection of the candidate antigens that resulted from in silico prediction. This approach resulted in the identification of nine Brucella melitensis outer membrane proteins, 5 of which were recombinantly expressed and used for validation. Omp22 and Hia had the highest in silico scores for adhesin probability and also conferred invasive capacity to E. coli overexpressing recombinant proteins. With the exception of FlgK in the goat, all proteins reacted to pooled sera from exposed goats, mice, and humans. BtuB, Hia and FlgK stimulated a mixed Th1–Th2 response in splenocytes from immunized mice while BtuB and Hia elicited NO release from splenocytes of S19 immunized mice. The results support the applicability of the current approach to the identification of antigens with immunogenic and invasive properties. Studies to assess immunogenicity and protective efficacy of individual proteins in the mouse are currently underway.     

IgY antibodies of chicken do not bind staphylococcal binder of immunoglobulin (Sbi) from Staphylococcus aureus

The immunoglobulin (Ig) binding proteins of Staphylococcus aureus namely staphylococcal protein A (SpA) and staphylococcal binder of immunoglobulin (Sbi) are responsible for false positives during immunoassays. Avian IgY antibodies were reported to have no affinity to SpA and thus are safe for use in immunoassays. However, the behaviour of Sbi with IgY was not reported. The purpose of the present study is to evaluate the interactions between IgY antibodies and Sbi protein from different S. aureus strains. Initially, heterologous cloning and expression of complete sbi gene in Escherichia coli was undertaken. Recombinant Sbi protein was utilized to generate polyclonal anti-Sbi IgY and anti-Sbi antibodies in chicken and BALB/c mice respectively. Indirect ELISA and Western blotting were performed to evaluate the reactivity of anti-Sbi antibodies. Non-reducing PAGE followed by Western blotting and double-antibody sandwich dot-ELISA were performed to analyze the reactivity of IgY antibodies with recombinant Sbi and native Sbi from S. aureus strains. To avoid the possible interference of enzyme-conjugated secondary antibodies from mammalian sources, mouse anti-Sbi revealing antibodies were labeled with biotin so that streptavidin-HRP was used as developing reagent for chromogenic reaction. Sbi was highly immunogenic in chicken and mouse with antibody titers of 1:128,000 and 1:64,000 dilutions respectively. We observed that unimmunized IgY antibodies showed no affinity to either recombinant Sbi or native Sbi from S. aureus strains in Western blotting and double antibody sandwich ELISA. In view of these observations, we recommend that IgY antibodies are safe and free from false positives due to SpA and Sbi in immunoassays involving detection of S. aureus antigens/exotoxins.     

The characterization of antibacterial antibodies in bovine immune sera to Staphylococcus aureus

Humoral antibody responses to the encapsulated Smith diffuse strain of Staphylococcus aureus were examined in cows immunized with the killed vaccine via different systemic routes. The sequential appearance of the antibody within different immunoglobulin classes in the sera during the course of immunization was followed by passive hemagglutination (PHA) and precipitation (PC) reactions and the mouse passive protection test. Repeated intravenous injections with the killed vaccine suspended in buffered saline stimulated production of IgM antibody exclusively during the whole period of immunization. On the contrary, following intramuscular administration with the vaccine incorporated in Freund's incomplete adjuvant, the antibodies appeared predominantly in IgG fractions of the sera. Specific antibody to the homologous strain used for vaccination was prepared from bovine immune sera by an absorption and elution process. The mouse passive protective activity of the antibody preparation was removed by absorption with the capsular polysaccharide antigen as well as by the whole cell adsorbent of the Smith diffuse strain, but not by the Smith compact and Cowan I strains of S. aureus. IgM, IgG1 and IgG2 proteins were isolated from the purified antibody and were compared, on a weight basis, with respect to their biological activities. Slightly higher activity of the IgG over the IgM antibody was demonstrated both in the mouse passive protection test and PC reaction, whereas in the PHA reaction, IgM antibody was shown to possess a significantly higher activity than IgG antibody. These studies suggest that IgG as well as IgM antibody might play an important role in protection against infection with encapsulated strains of S. aureus in cows.     

Expression of an Escherichia coli antigenic fusion protein comprising the heat labile toxin B subunit and the heat stable toxin, and its assembly as a functional oligomer in transplastomic tobacco plants

Enterotoxigenic Escherichia coli (ETEC) strains are important pathogens in developing countries. Some vaccine formulations containing the heat labile toxin B subunit (LTB) have been used in clinical trials; however, the induction of neutralizing antibodies against the heat-stable toxin (ST), a poor immunogenic peptide, is necessary, as most ETEC strains can produce both toxins. In this study, a plant optimized synthetic gene encoding for the LTB-ST fusion protein has been introduced into plastids of tobacco leaf tissues, using biolistic microprojectile bombardment, in an effort to develop a single plant-based candidate vaccine against both toxins. Transplastomic tobacco plants carrying the LTB-ST transgene have been recovered. Transgene insertion into the plastid was confirmed by both PCR and Southern blot analysis. GM1-ELISA revealed that the LTB-ST fusion protein retained its oligomeric structure, and displayed antigenic determinants for both LTB and ST. Western blot analysis, using LTB antisera, confirmed the presence of a 17-KDa protein in transplastomic lines, with the correct antigenicity of the fusion protein. Expression levels of this fusion protein in different lines reached up to 2.3% total soluble protein. Oral immunization of mice with freeze-dried transplastomic tobacco leaves led to the induction of both serum and mucosal LTB-ST specific antibodies. Following cholera toxin challenge, a decrease of intestinal fluid accumulation was observed in mice immunized with LTB-ST-containing tobacco. These findings suggest that tobacco plants expressing LTB-ST could serve as a plant-based candidate vaccine model providing broad-spectrum protection against ETEC-induced diarrhoeal disease.     

Antibody Response to Staphylococcus aureus in Rabbits: Sequence of Immunoglobulin Synthesis and Its Correlation with Passive Protection in Mice

Antibody in hyperimmune rabbit antisera specific for Staphylococcus aureus teichoic acid was shown to be associated with the IgM fraction. Treatment of such sera with mercaptoethanol destroyed its activity in passive mouse protection tests, whereas absorption with antirabbit IgG had no effect. Antibody response in normal rabbits immunized by a single or by three daily injections of a killed vaccine of S. aureus was followed by a sensitive passive hemagglutination test. Antibody detected during the "primary" response was completely susceptible to reduction with mercaptoethanol. Most of the antibody detected after a secondary antigenic stimulation at 10 weeks was also susceptible to mercaptoethanol. The antibody titers correlated well with mouse protective activity, and this activity of the serum was also shown to be associated with the IgM fraction.     

Corneal virulence of Staphylococcus aureus in an experimental model of keratitis

The aim of this study was to determine the pathogenic role of alpha-, beta-, and gamma-toxins in a rabbit model of Staphylococcus aureus keratitis. S. aureus strains 8325-4, Newman, and their isogenic mutants were intrastromally injected into rabbit corneas. Eyes were scored for pathology by slit lamp examination (SLE), histologic examination, and bacterial colony-forming units (CFU) per cornea were determined. Rabbits were immunized against alpha-toxin and subsequently challenged with S. aureus strain 8325-4 or Newman. All strains grew equivalently to approximately 7 log CFU/cornea at 25 h postinfection. SLE scores at 15, 20, and 25 h postinfection revealed that alpha-toxin - producing strains caused greater corneal pathology than strains deficient in alpha-toxin. A beta-toxin - deficient mutant produced significantly less ocular edema than its parent or rescued strains. The gamma-toxin-deficient mutant, relative to its parent strain or genetically rescued strain, had reduced virulence. These results demonstrate that the virulence of S. aureus involves mainly alpha-toxin and to a lesser extent gamma-toxin, with beta-toxin mediating minimal corneal pathology.     

Antigenic and immunogenic activity of flagella and fimbriae preparations from uropathogenic Proteus mirabilis.

The antigenic and immunogenic activities of fimbriae and flagella from three uropathogenic strains of Proteus mirabilis were compared. Flagella were obtained by mechanical treatment and fimbriae were isolated from cells by heat shock, ammonium sulfate precipitation, sodium deoxycholate and urea treatment, and gel filtration. Both preparations inoculated to mice demonstrated high antigenicity. Titers up to 1:80,000 were determined by an enzyme-linked immunosorbent assay either against the homologous or heterologous strains. When immunized mice were challenged with homologous or heterologous hematogenous infecting doses, a good cross protection was achieved only when fimbriae were used as antigens. Cross-reactivity found between the three fimbriae antisera, and the presence of common proteins in sodium dodecyl sulfate polyacrylamide gel electrophoresis patterns of fimbriae, should validate the study of these proteins to determine the existence of a shared adhesin.