Construction and immunogencity of a DeltaapxIC/DeltaapxIIC double mutant of Actinobacillus pleuropneumoniae serovar 1

The apxIC and apxIIC genes of the Actinobacillus pleuropneumoniae serovar 1 strain SLW01, encoding the ApxI- and ApxII-activating proteins, respectively, were deleted successively by a method involving sucrose counterselection. The resulting strain, SLW03, contained no foreign DNA and could secrete unactivated ApxIA and ApxIIA RTX toxins with complete antigenicity. Strain SLW03 was attenuated at least 1000-fold in Balb/C mice and caused no adverse effects in pigs at doses of up to 1 x 10(9) CFU mL(-1). SLW03 was able to induce a significant immune response and provide complete protection from clinical signs upon homologous (serovar 1) and heterologous (serovar 9) challenge of A. pleuropneumoniae. Pigs vaccinated via the intranasal (i.n.) route had significantly higher serum titers and fewer pulmonary lesions than pigs vaccinated via the intramuscular route postchallenge. These results suggest that the mutant strain SLW03 could be used as a candidate live vaccine that can induce reliable cross-serovar protection following i.n. immunization.     

胸膜肺炎放线杆菌apxIC基因插入失活突变株构建及免疫原性分析

胸膜肺炎放线杆菌是引起猪传染性胸膜肺炎(APP)的呼吸道病原菌,其分泌的Apx毒素是最重要的毒力因子之一。为构建APP突变弱毒菌株,在apxIC基因下游XhoI酶切位点处插入氯霉素抗性基因(Chlr)制备转移载体,通过电转化导入APP血清10型参考菌株(D13039)进行同源重组,筛选获得apxIC基因插入突变菌株D13039C-Chlr。该突变菌株特性鉴定结果表明其溶血活性完全丧失,可正常增殖和分泌ApxI毒素,连续10次传代后基因组中插入的Chlr基因可稳定遗传,利用5个剂量(2×108CFU~2×106CFU)对每组3只小鼠腹腔攻毒结果显示突变菌株毒力较母源菌株降低至少100倍以上,将突变菌株作为弱毒活疫苗经滴鼻途径免疫仔猪后利用APP血清1型(4074)和血清10型(D13039)菌株攻毒进行免疫原性鉴定,结果显示血清1型攻毒后非免疫组4头仔猪全部死亡而免疫组4头中死亡2头,非免疫组肺损伤指数(34.4)显著高于免疫组(17.5),血清10型攻毒后非免疫组肺损伤指数(17.5)也高于免疫组(10.5),同时鼻拭子和肺组织样品的细菌重分离数及PCR检测阳性数非免疫组也明显高于免疫组,表明突变菌株作为弱毒活疫苗对仔猪具有一定的交叉免疫保护力。该突变菌株的构建为鉴定ApxI毒素活性及研制具有交叉保护活性的APP弱毒活疫苗奠定了基础。     

Induction of antigen-specific immune responses by oral vaccination with Saccharomyces cerevisiae expressing Actinobacillus pleuropneumoniae ApxIIA

An effective way of inducing both mucosal and systemic immune responses to protect against Actinobacillus pleuropneumoniae serotype 2 Korean isolate was examined in mice by oral immunization using Saccharomyces cerevisiae expressing the ApxIIA protein. The immunogenicity of the yeast-derived ApxIIA antigen was confirmed by the challenge test and ApxIIA-specific IgG antibody response assay. The group subcutaneously immunized with the protein extracted from the yeast expressing ApxIIA showed a higher survival rate after challenging with A. pleuropneumoniae serotype 2 isolate and IgG antibody level in serum than the group injected with that prepared from the yeast harboring vector only. Feeding the yeast expressing ApxIIA to mice induced both systemic and mucosal immune responses against the antigen. ApxIIA-specific IgA antibody titers and the number of IgA-secreting cells of mice vaccinated with S. cerevisiae expressing ApxIIA dose-dependently increased from the third immunization in both intestine and lung (P<0.01). A similar tendency of ApxIIA-specific IgG antibody responses was observed in the sera. The protective efficacy of the oral immunization was then evaluated by a challenge with a minimal lethal dose (MLD, 4.5 x 10(7) CFU/ml) of the A. pleuropneumoniae serotype 2 isolate. Fifty percent of the 30 mg administered group and 30% of the 15 mg administered group survived while none of the mice in the control groups survived after 36 h. These results suggest that feeding animals the yeast expressing the antigen can be an effective strategy to induce protective immune responses against A. pleuropneumoniae infection.     

Truncation of the lipopolysaccharide outer core affects susceptibility to antimicrobial peptides and virulence of Actinobacillus pleuropneumoniae serotype 1

We reported previously that the core oligosaccharide region of the lipopolysaccharide (LPS) is essential for optimal adhesion of Actinobacillus pleuropneumoniae, an important swine pathogen, to respiratory tract cells. Rough LPS and core LPS mutants of A. pleuropneumoniae serotype 1 were generated by using a mini-Tn10 transposon mutagenesis system. Here we performed a structural analysis of the oligosaccharide region of three core LPS mutants that still produce the same O-antigen by using methylation analyses and mass spectrometry. We also performed a kinetic study of proinflammatory cytokines production such as interleukin (IL)-6, tumor necrosis factor-alpha, IL1-beta, MCP-1, and IL8 by LPS-stimulated porcine alveolar macrophages, which showed that purified LPS of the parent strain, the rough LPS and core LPS mutants, had the same ability to stimulate the production of cytokines. Most interestingly, an in vitro susceptibility test of these LPS mutants to antimicrobial peptides showed that the three core LPS mutants were more susceptible to cationic peptides than both the rough LPS mutant and the wild type parent strain. Furthermore, experimental pig infections with these mutants revealed that the galactose (Gal I) and d,d-heptose (Hep IV) residues present in the outer core of A. pleuropneumoniae serotype 1 LPS are important for adhesion and overall virulence in the natural host, whereas deletion of the terminal GalNAc-Gal II disaccharide had no effect. Our data suggest that an intact core-lipid A region is required for optimal protection of A. pleuropneumoniae against cationic peptides and that deletion of specific residues in the outer LPS core results in the attenuation of the virulence of A. pleuropneumoniae serotype 1.     

Lactococcus lactis AbiD1 abortive infection efficiency is drastically increased by a phage protein

Three genetically defined Actinobacillus pleuropneumoniae serotype 7 mutants with deletions in the small (tbpB), the large (tbpA), and both transferrin binding protein genes were constructed and examined in an aerosol infection model. Neither mutant caused clinical disease or could be reisolated, and no immune response could be detected 21 days after infection. This result clearly implies that each transferrin binding protein on its own is a virulence factor of A. pleuropneumoniae serotype 7.     

构建胸膜肺炎放线杆菌apxIC基因插入突变株

目的：构建胸膜肺炎放线杆菌（APP）apxIC基因插入突变菌株,以鉴定ApxⅠ毒素的生物学特性。方法：根据apxⅠ核酸序列（U05042）设计1对引物,用于自APP血清10型参考菌株（D13039）基因组DNA中扩增apxIC基因及其上下游约2．8kb的基因片段,经克隆测序后在apxIC基因下游xbI酶切位点处插入约0．9kb的氯霉素（Chl）抗性基因表达盒,构建用于转化的转移载体pUIC-Chl^r,将转移载体DNA经电转化导入APP血清10型参考菌株中进行同源重组,以获得突变菌株。结果：在含有氯霉素的培养基中经筛选获得2株丧失溶血活性的突变菌株（D13039C-Chl^r）;利用PCR和Southern blot对突变菌株鉴定,显示氯霉素抗性基因已被插入细菌基因组中。结论：利用电转化和同源重组技术构建成功APP apxIC基因插入突变菌株,为分析ApxⅠ毒素的生物学特性,进而研制APP基因工程减毒活疫苗奠定了基础。     

Demonstration of the third antigenically distinct outer membrane lipoprotein (OmlA) in Actinobacillus pleuropneumoniae serotype 7

The gene encoding an outer membrane lipoprotein (OmlA) of Actinobacillus pleuropneumoniae strain WF83 (serotype 7 reference strain), designated omlA₇, was sequenced. The amino acid sequence of OmlA₇ showed 64.5 and 71.6% identity to that of OmlA from serotypes 1 (OmlA₁) and 5 (OmlA₅), respectively. The first 134 amino acids of OmlA₇ were identical to those of OmlA₅. A Southern blot analysis revealed the presence of a gene highly homologous to the omlA₇ in the reference strains of serotypes 3, 4, 6, and 7. A Western blot analysis using a specific antiserum against a recombinant OmlA₇ detected expression of the homologous proteins in the serotypes 4, 6, and 7 reference strains and a serotype 3 field strain, but not in a serotype 3 reference strain. The data demonstrate the third antigenically distinct OmlA is expressed in A. pleuropneumoniae.     

苏北地区规模化猪场产肠毒素大肠杆菌的分离鉴定及灭活疫苗效果评估

[目的]产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)是引起仔猪腹泻的重要病原菌,本研究通过调查苏北地区规模化猪场ETEC的流行情况,分析其生物学特性,研制具有免疫保护效果的优势血清型菌株的灭活疫苗,以期对苏北地区ETEC的防控提供参考。[方法]从苏北地区规模化猪场采集3-30日龄的仔猪新鲜粪样、肛拭子及小肠组织样,分离出ETEC,对分离菌株进行血清型鉴定、耐药性测定、小鼠致病力测定;最后通过动物免疫试验研究优势血清型菌株灭活疫苗对小鼠的免疫保护效果。[结果]从21个规模化猪场采集病料562份,通过PCR鉴定及测序得到141株ETEC;血清凝集试验鉴定出85株菌的O抗原血清型,其中08、0101和0128为优势血清型,占定型菌株的61.2%(52/85),其他血清型包括09、03、020、0148、0149等;分析141株ETEC对14种常见抗生素的耐药情况,得出分离株对新霉素、红霉素、四环素、庆大霉素、强力霉素、阿莫西林、甲氧苄啶/磺胺甲恶唑高度耐药,耐药率均高达80%以上;对恩诺沙星敏感性较高,敏感率达50.4%(71/141);对多粘菌素B和头孢噻肟中介耐药,占比分别为66%(93/141)和51.8%(73/141);多重耐药现象严重,其中10重耐药的菌株占比最大,为19%(27/141);小鼠攻毒试验测得08血清型强毒株YC-6的半数致死量(median lethal dose,LD50)为1.4×10^7 CFU/只,最低致死量(minimum lethal dose,MLD)为3×10^7 CFU/只;08血清型强毒株YC-6和0101血清型强毒株LYG-3制备的单价灭活疫苗对小鼠的保护率均达到100%,因此利用08血清型强毒株YC-6和0101血清型强毒株LYG-3研制二价灭活疫苗,结果显示该二价疫苗对感染不同血清型ETEC小鼠的保护率在83%以上。[结论]本研究通过对苏北地区ETEC的流行病学调查,得出其优势血清型,并研制出针对对优势血清型免疫保护效果较好的二价灭活疫苗,给临床ETEC的监测和防控提供参考。     

First Chromosomal Restriction Map of Actinobacillus pleuropneumoniae and Localization of Putative Virulence-Associated Genes

Combined physical and genetic maps of the genomes of Actinobacillus pleuropneumoniae AP76 (serotype 7 clinical isolate) and of A. pleuropneumoniae ATCC 27088 (serotype 1 reference strain) were constructed by using the restriction endonucleases ApaI, AscI, NotI, and SalI. The chromosome sizes as determined by the addition of estimated fragment sizes were 2.4 Mbp, and both maps had a resolution of approximately 100 kbp. The linkages between the ApaI, AscI, NotI, and SalI fragments and their relative positions were determined by (i) fragment excision and redigestion and (ii) partial digests of defined fragments and Southern blot using end-standing probes. The single SalI site within the chromosome of strain A. pleuropneumoniae AP76 was defined as position 1 of the map; for the map of A. pleuropneumoniae ATCC 27088, the corresponding SalI site was chosen. Putative virulence-associated genes (apx, omlA, sodA, tbpBA, ureC, and a repeat element) and housekeeping genes (glyA, metJ, recA, and rhoAP) were positioned on the physical maps and located on the ApaI and NotI fragments of A. pleuropneumoniae serotype reference strains.     

Role of rpoS in acid resistance and fecal shedding of Escherichia coli O157:H7

Acid resistance (AR) is important to survival of Escherichia coli O157:H7 in acidic foods and may play a role during passage through the bovine host. In this study, we examined the role in AR of the rpoS-encoded global stress response regulator sigma(S) and its effect on shedding of E. coli O157:H7 in mice and calves. When assayed for each of the three AR systems identified in E. coli, an rpoS mutant (rpoS::pRR10) of E. coli O157:H7 lacked the glucose-repressed system and possessed reduced levels of both the arginine- and glutamate-dependent AR systems. After administration of the rpoS mutant and the wild-type strain (ATCC 43895) to ICR mice at doses ranging from 10(1) to 10(4) CFU, we found the wild-type strain in feces of mice given lower doses (10(2) versus 10(3) CFU) and at a greater frequency (80% versus 13%) than the mutant strain. The reduction in passage of the rpoS mutant was due to decreased AR, as administration of the mutant in 0.05 M phosphate buffer facilitated passage and increased the frequency of recovery in feces from 27 to 67% at a dose of 10(4) CFU. Enumeration of E. coli O157:H7 in feces from calves inoculated with an equal mixture of the wild-type strain and the rpoS mutant demonstrated shedding of the mutant to be 10- to 100-fold lower than wild-type numbers. This difference in shedding between the wild-type strain and the rpoS mutant was statistically significant (P 相似文献   

Actinobacillus pleuropneumoniae serotype 7 siderophore receptor FhuA is not required for virulence

A ferrichrome receptor, FhuA, was identified in Actinobacillus pleuropneumoniae serotype 7. An isogenic mutant with a deletion in the ferrichrome uptake receptor gene (fhuA) was constructed and examined in an aerosol infection model. The disease caused by the mutant was indistinguishable from disease induced by A. pleuropneumoniae serotype 7 wild-type; an isogenic mutant lacking expression of the exbB gene that is required for the uptake of transferrin-bound iron retained the ability to utilize ferrichrome, thereby indicating that an energy-coupling mechanism involved in ferrichrome transport remains to be identified.     

血清7型胸膜肺炎放线杆菌apxⅡC-/apxⅠA+基因缺失重组菌株的构建与鉴定

通过接合转移和SacB负向筛选方法,成功构建了一株apxⅡC缺失的血清7型胸膜肺炎放线杆菌重组菌株。首先构建重组转移质粒pEHA1。将pEHA1转化供体菌大肠杆菌(E.coliβ2155),并将其与野生型APP血清7型亲本菌混合培养约5h,然后涂到含氯霉素抗性的培养基培养,挑取阳性克隆,接种到无抗性液体培养基,培养后涂于含有蔗糖的的固体培养基,培养一定时间后挑取蔗糖抗性的克隆,即可得到目的突变株。通过PCR、遗传稳定性、外毒素分泌、重组位点序列分析证明重组菌构建成功。通过对重组菌生物学特性进行初步研究,表明突变株生长能力未受影响,对小鼠毒力显著降低。该突变株构建体系的建立为猪传染性胸膜肺炎减毒活疫苗的开发及对胸膜肺炎放线杆菌新基因的功能研究奠定了良好基础。     

Fhua and HgbA, outer membrane proteins of Actinobacillus pleuropneumoniae: their role as virulence determinants

For the recently described serotype 15 of biotype I and serotypes 13 and 14 of biotype II of Actinobacillus pleuropneumoniae, fhuA and hgbA were detected by polymerase chain reaction and DNA sequencing. To determine the substrate specificity of the iron receptors FhuA and HgbA and to study their role in the virulence of A. pleuropneumoniae, we used two isogenic A. pleuropneumoniae serotype 1 deletion mutants of fhuA and hgbA. Different sources of iron and siderophores were tested in growth promotion assays. FhuA and HgbA are specific for their ligands ferrichrome and hemoglobin, respectively. The virulence of the two deletion mutant strains was evaluated in experimental infections using specific pathogen-free piglets. While the fhuA mutant (DG02) was as highly virulent as the parental strain S4074, the virulence of the hgbA mutant (DeltahgbA) was reduced. Our data indicate that both FhuA and HgbA are conserved among all serotypes and biotypes of A. pleuropneumoniae and that HgbA, the receptor for porcine hemoglobin, may play a role in virulence.     

Evaluation of the hemoglobin-binding activity of Actinobacillus pleuropneumoniae using fluorescein-labeled pig hemoglobin and flow cytometry

In the present study, the hemoglobin (Hb)-binding activity of Actinobacillus pleuropneumoniae was examined using fluorescein-labeled pig Hb and flow cytometry. Comparison of the Hb-binding activity of A. pleuropneumoniae serotype 1 strain 4074 grown under iron-restricted conditions with cells grown under iron-sufficient conditions indicated that iron-restriction in A. pleuropneumoniae promotes the expression of Hb receptors, and that Hb-binding activity is, at least in part, iron-repressible. Hb-binding activity was also observed in representative strains of A. pleuropneumoniae belonging to serotypes 1 and 2. In addition, A. pleuropneumoniae serotype 1 LPS or capsule isogenic mutants were tested in flow cytometry in order to understand the influence of surface polysaccharides on Hb-binding activity. Experiments with an acapsulated mutant indicated that surface molecules with Hb-binding activity are more exposed at the cell surface in the absence of capsular polysaccharides. However, the Hb-binding activity of LPS mutants analyzed in this study was unchanged compared to the parent strain. The outer membrane proteins profile of A. pleuropneumoniae serotype 1 grown under iron-restricted or iron-sufficient conditions was also evaluated by polyacrylamide gel electrophoresis. Iron-regulated outer membrane proteins were observed under iron-restricted growth conditions which suggests that one or more of these outer membrane proteins may play a role in the Hb-binding activity detected by flow cytometry.     

胸膜肺炎放线杆菌血清10型apxIC-/p36+弱毒株的构建与鉴定

摘要：【目的】构建血清10型胸膜肺炎放线杆菌弱毒菌株,为胸膜肺炎放线杆菌减毒活疫苗研究奠定基础。【方法】通过细菌接合转移和SacB负向筛选标记完成突变株的构建与筛选,用PCR、Western blot、重组位点序列对突变株进行鉴定分析。首先构建含肺炎支原体p36基因的pEICALDH重组转移质粒,并转化供体大肠杆菌( E. coliX7213),将转化的阳性克隆子与野生型APP血清10型亲本菌混合培养6 h;然后涂至含氯霉素抗性和烟酰胺腺嘌呤二核苷酸（NAD）的TSA培养基培养,挑取阳性克隆,接种至无抗性的含NAD的TSB液体培养基,培养6～8 h后涂至含10%的蔗糖及NAD的TSA培养基,培养24 h后挑取蔗糖抗性的克隆,即得到目的突变株。【结果】小鼠毒力试验结果表明突变株比亲本株的毒力显著降低;生长特性分析结果显示突变株与亲本株的增殖能力无显著差异;同时免疫试验结果表明突变株与安全剂量的亲本株均可诱导小鼠产生较好的免疫反应,证明apxIC基因缺失并不影响APP的免疫活性。【结论】成功构建了含猪肺炎支原体p36基因的胸膜肺炎放线杆菌血清10型突变株,所获得的突变株有望成为猪传染性胸膜肺炎弱毒疫苗株。     

Pathogenic potential of a collagenase gene from Aeromonas veronii

The role of collagenase as a mechanism of bacterial pathogenicity in some pathogenic bacteria has been reported. The information on the role of collagenase in Aeromonas spp. pathogenesis is scant. In the present study, a mutant Aeromonas veronii RY001 that is deficient in the putative collagenase gene acg was constructed and compared with the wild-type strain for virulence factors. Bacterial cells and cell-free extracellular products of the mutant had significantly less collagenolytic activity, but there were not significant differences in caseinolytic, gelatinolytic, and elastolytic activities. Adhesion and invasion abilities of the mutant strain on epithelioma papillosum of carp cells was only 56% of that of the wild-type strain, and the cytotoxicity of the mutant strain to epithelioma papillosum of carp cells was only 42% of that of the wild-type strain. The LD50 values of the wild-type strain were determined as 1.6 x 10(6) and 3.5 x 10(5) cfu in goldfish and mice, respectively, whereas the mutant RY001 strain showed slightly higher values (i.e., 2.8 x 10(6) and 1.4 x 10(6) cfu in goldfish and mice, respectively). These results indicated the involvement of the collagenase gene in the pathogenesis of A. veronii.     

A Suitable Streptomycin-Resistant Mutant for Constructing Unmarked In-Frame Gene Deletions Using rpsL as a Counter-Selection Marker

The streptomycin counter-selection system is a useful tool for constructing unmarked in-frame gene deletions, which is a fundamental approach to study bacteria and their pathogenicity at the molecular level. A prerequisite for this system is acquiring a streptomycin-resistant strain due to rpsL mutations, which encodes the ribosomal protein S12. However, in this study no streptomycin resistance was found to be caused by rpsL mutations in all 127 clinical strains of Klebsiella pneumoniae isolated from liver abscess patients. By screening 107 spontaneous mutants of streptomycin resistance from a clinical strain of K. pneumoniae, nucleotide substitution or insertion located within the rpsL was detected in each of these strains. Thirteen different mutants with varied S12 proteins were obtained, including nine streptomycin-dependent mutants. The virulence of all four streptomycin-resistant mutants was further evaluated. Compared with the parental strain, the K42N, K42T and K87R mutants showed a reduction in growth rate, and the K42N and K42T mutants became susceptible to normal human serum. In the mice LD₅₀ (the bacterial dose that caused 50% death) assay, the K42N and K42T mutants were ∼1,000-fold less lethal (∼2×10⁵ CFU) and the K87R mutant was ∼50-fold less lethal (∼1×10⁴ CFU) than the parental strain (∼2×10² CFU). A K42R mutant showed non-observable effects on the above assays, while this mutant exhibited a small cost (P<0.01) in an in vitro growth competition experiment. In summary, most of the K. pneumoniae strains with streptomycin resistance caused by rpsL mutations are less virulent than their parental strain in the absence of streptomycin. The K42R mutant showed similar pathogenicity to its parental strain and should be one of the best choices when using rpsL as a counter-selection marker.     

Monitoring and survival of Lactobacillus paracasei LTH 2579 in food and the human intestinal tract

A PCR-based detection system specific for Lactobacillus paracasei LTH 2579 was developed and applied to follow the fate of the strain in complex ecosystems. This strain was isolated from fruit mash and was characterised as being highly resistant to low pH and bile at concentrations as they occur in the human digestive tract. The application of the subtraction hybridisation technique permitted to identify a 235 bp chromosomal DNA fragment of strain LTH 2579. Based on this target sequence a specific PCR system was developed and combined with the species-specific PCR system for L. paracasei. This combination of PCR based detection systems was successfully applied to monitor L. paracasei LTH 2579 in fermented sausages which were inoculated with this strain (2.0 x 10(7) CFU/g) together with the strongly competitive L. sakei LTH 681 (1.0 x 10(6) CFU/g). At the time of consumption of the sausages the respective counts were 1.8 x 10(7) and 1.4 x 10(8) CFU/g. After consumption of the sausages by three volunteers L. paracasei LTH 2579 was recovered from fecal samples. The counts determined for the strain ranged between 1.2 x 10(7) and 1.5 x 10(8) CFU/g of feces. The fortuitous lactobacilli constituted a share of 5-12% of the lactobacilli in the fecal flora.     

Isolation and Characterization of LPS Mutants of Actinobacillus pleuropneumoniae Serotype 1

The major adhesin of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, has been previously identified as lipopolysaccharide (LPS). The purpose of the present study was to isolate and characterize A. pleuropneumoniae LPS mutants. Screening of LPS mutants was performed with colony dot and sensitivity to novobiocin. One mutant obtained by colony dot (F19) and one mutant selected for its increased sensitivity to novobiocin (33.1) did not react with a monoclonal antibody against A. pleuropneumoniae serotype 1 O-antigen compared with the parent strain. Mutants F19 and 33.1 did not express high-molecular-mass LPS bands as determined in silver-stained SDS-PAGE gels. The core-lipid A region of mutant 33.1 and of the parent strain had similar relative mobilities and reacted with serum from a pig experimentally infected with the serotype 1 reference strain of A. pleuropneumoniae, while the same region in mutant F19 showed faster migration and did not react with this serum. Use of piglet tracheal frozen sections indicated that mutant F19 was able to adhere to piglet trachea as well as the parent strain, while mutant 33.1 adhered [half as much as] the parent strain. Finally, both LPS mutants were markedly less virulent in mice than the parent strain. Taken together, our observations support the idea that LPS is an important virulence factor of A. pleuropneumoniae. Received: 23 December 1996 / Accepted: 19 February 1997     

重组猪胸膜肺炎放线杆菌毒素ApxI对小鼠的急性毒性和免疫保护性研究

研究了猪胸膜肺炎放线杆菌毒素Ⅰ重组表达蛋白(包括粗提包涵体和经复性处理的重组蛋白)对小鼠的急性毒性以及免疫保护性,并和天然毒素Ⅰ(由APP血清10型菌的培养物上清提取)做了对比。在急性毒性试验中,3种蛋白均以200μg只的剂量腹腔注射小鼠,并分别于24h、7d和14d后眼眶放血致死,检测血常规和血液生化相关指标。结果表明,3种蛋白均不引起小鼠死亡,且重组表达蛋白对小鼠的生长、血常规和血液生化指标没有显著影响。在免疫保护性试验中,用3种蛋白乳化后免疫小鼠,2周后加强免疫1次,并在每次免疫后采血检测其效价,二免2周后用致死剂量的APP血清10型菌(1.09×108cfu)腹腔攻毒。结果表明,天然毒素和复性的重组表达蛋白均具有良好的免疫原性,对小鼠具有较好的免疫保护效力。