副猪嗜血杆菌aroA基因鉴定及遗传进化分析

[目的]细菌aroA基因参与芳香族氨基酸的生物合成,被成功应用于细菌分类和基因失活致弱突变菌株的构建.副猪嗜血杆菌(Hps)是感染猪出现多发性浆膜炎和关节炎的一种病原细菌,鉴定该菌aroA全基因序列将有助于鉴定遗传进化关系和突变分析.[方法]利用PCR和细菌基因组步移技术鉴定Hps的aroA基因序列,进而对不同血清型菌株该基因序列进行鉴定,并与其它革兰氏阴性细菌进行比对和遗传进化分析.[结果]自Hps血清5型基因组DNA中获得包含完整aroA基因的3.7 kb基因片段,其中aroA基因全长1314 bp,编码产物长度437 aa,分子量大小47.9 kDa,该基因上游为磷酸烯醇式丙酮酸羧化酶基因.自本试验选择的Hps不同血清型菌株中均可扩增出包含完整aroA基因的1476 bp片段,且这些不同血清型菌株间核酸序列同源性在97.7%以上.Hps血清5型aroA基因序列与巴氏杆菌科其它成员核酸序列同源性为70.6%-78.9%,与E.coli和S.typhi-murium的同源性分别为66.4%和67.2%.[结论]本试验首次对Hps的15个血清型国际参考菌株及地方分离株aroA全基因序列进行了鉴定,序列比较结果显示aroA基因在革兰氏阴性细菌中具有较高的同源性.aroA基因鉴定对构建基因失活突变菌株以研究Hps生物学特性奠定了基础.     

副猪嗜血杆菌蜂胶苗的制备及免疫效果试验

目的：以原场副猪嗜血杆菌为制苗菌株,制成自家细菌苗,预防本场猪格拉泽氏病。方法：用从河南济源一自繁自养大型养猪场发病猪分离到的副猪嗜血杆菌进行培养,经甲醛灭活,以蜂胶为佐剂,制成原场副猪嗜血杆菌灭活苗,含菌量为200亿/ml。母猪分别于产前30d和15d各注射4ml/头。仔猪出生后,分别在15、30日龄肌肉注射,1.5ml/头。结果：应用试验证明,该灭活苗保护率达98%。结论：该蜂胶苗安全、可靠,对猪格拉泽氏病有较好的预防作用。     

副猪嗜血杆菌毒力因子研究进展

副猪嗜血杆菌(Haemophilus parasuis,HPS)是猪上呼吸道的一种常在菌,但却因其被证明是以多发性浆膜炎、关节炎、呼吸困难、高热及高死亡率为特征的Glasser病的病原体而广受关注。介绍了巴氏杆菌科一些常见的毒力相关蛋白、神经氨酸酶、血清型以及毒力相关的三聚自身转运载体与HPS毒力之间的关系,另外就近年来对HPS毒力因子方面的一些研究报道和成果作一综述。     

副猪嗜血杆菌安徽株血清型、基因型及耐药性

【背景】副猪嗜血杆菌(Haemophilusparasuis,HPS)是猪革拉瑟氏病(Gl?sser’sdisease)的病原体,由于长期使用抗生素产生的耐药性以及灭活疫苗缺乏交互免疫保护力,目前对该病无法形成有效防控。【目的】了解安徽地区副猪嗜血杆菌血清型、基因型及耐药性特征,为猪革拉瑟氏病的有效防控提供科学依据和技术支撑。【方法】针对2008–2017年安徽地区分离的44株HPS,利用PCR反应鉴定血清型,应用多位点序列分型(multilocus sequence typing,MLST)进行基因分型,通过微量稀释法测定最小抑菌浓度(minimalinhibitoryconcentration,MIC)。【结果】44株HPS共检测出6种血清型(1、4、5、7、13、14),血清型4型和13型为优势血清型,各占40.91%;9种ST型(ST241、ST267、ST268、ST269、ST270、ST271、ST272、ST273和ST274)中ST267和ST268为优势基因型,各占40.91%;对庆大霉素、青霉素的敏感率分别为100%和93.2%,86.4%的分离株呈现多重耐药,耐药谱型有33种,其中以甲氧苄啶/磺胺甲噁唑-四环素构成比最大,为42.4%。【结论】安徽地区HPS流行血清型和基因型呈现多元化,具有较高的遗传异质性,多重耐药现象严重,血清型、ST型与耐药性之间无明显相关性。     

A rapid pulsed-field gel electrophoresis method of genotyping Haemophilus parasuis isolates

Aims: To develop a modified pulsed‐field gel electrophoresis (PFGE) method for characterizing Haemophilus parasuis isolates. Methods and Results: A modified PFGE procedure was designed using CpoI to generate restriction maps of H. parasuis genomic DNA. This approach was used to characterize 47 H. parasuis clinical isolates and 15 reference strains. All strains could be typed by this method, and the procedure was completed in 36 h. A total of 39 different PFGE patterns were identified among 47 epidemiologically unrelated clinical isolates. Conclusions: The modified PGFE described in this report efficiently characterized H. parasuis isolates. This method can be adopted for studying the epidemiology of Glässer’s disease outbreaks in addition to differentiating and classifying previously untypeable H. parasuis isolates. Significance and Impact of the Study: The modified PFGE method described is a novel means of characterizing H. parasuis isolates. It is also a highly discriminatory molecular typing method (discriminatory index of 0·98) that can overcome the limitations of serotyping.     

副猪嗜血杆菌血清4、13和14型TbpA对豚鼠的交互免疫保护性

【背景】副猪嗜血杆菌(Haemophilusparasuis,HPS)是猪革拉瑟氏病(Gl?sser?sdisease)的病原体,目前在对该病的防控中,由于长期使用抗生素产生的耐药性以及灭活疫苗缺乏交互免疫保护力,亟待寻求新的方法来解决这一问题。【目的】探究不同血清型HPS转铁结合蛋白A (TbpA)对豚鼠的交互免疫保护性,为进一步用仔猪开展相关研究奠定基础。【方法】采用HPS血清型4、13和14型重组TbpA以0.1mg/只、经2次间隔期为20d的免疫后,检测豚鼠血清IgG抗体和细胞因子(IL-2、IL-5、IL-8、IFN-γ、MCP-1、TNF-α)水平;以HPS血清型4、13和14型菌株5 LD50剂量腹腔攻毒,检查病理组织学变化及其免疫保护率。【结果】HPS血清型4、13和14型重组TbpA均能诱导豚鼠产生较高水平的特异性抗体IgG,并使细胞因子显著升高。HPS血清型4、13和14型重组TbpA在免疫后均能对豚鼠产生交互免疫保护,其中HPS血清型13型TbpA免疫组的交互免疫保护率最高,对血清型4、14型HPS均为50.0%,对相同血清型HPS的免疫保护率也最高,达到83.3%,病理组织学检查显示与HPS血清型4、14型TbpA免疫组相比,13型TbpA免疫组的组织切片病理变化与攻毒对照组之间差异更明显。【结论】HPS血清型4、13和14型TbpA均能诱导豚鼠的体液免疫和相关细胞因子的分泌,产生交互免疫保护力,其中HPS血清型13型Tbp A的交互免疫保护力最强,可作为一种新的疫苗候选抗原。     

Identification of sulI allele of dihydropteroate synthase by representational difference analysis in Haemophilus parasuis serovar 2

AIMS: Identification of genes differentially present in Haemophilus parasuis serovar 2 by representational difference analysis (RDA). METHODS AND RESULTS: Bacterial genomic DNA was extracted, cleaved with Sau3AI and ligated to oligonucleotide adapter pair. The optimal tester (H. parasuis serovar 2)/driver ratio (H. parasuis serovars 1, 3 and 5) for the hybridization was established and the mixture was hybridized, and amplified by PCR. The products were cloned and transformed into Escherichia coli TOP10 cells and checked for specificity by Southern blotting analysis. The RDA subtractive technique yielded six bands ranging from 1500 to 200 bp, which were cloned into pCR II-TOPO vector and 40 clones were analysed. A fragment of 369 bp was specific for H. parasuis serovar 2, and showed 99% homology to sulI gene encoding for dihydropteroate synthase (dhps). The dhps gene conferring sulfonamide resistance was detected in H. parasuis serovar 2 but was absent in serovars 1, 3, 5 and in most of the Actinobacillus pleuropneumoniae serotypes (except serotype 7). CONCLUSION: sulI allele of dihydropteroate synthase has been identified in H. parasuis serovar 2 by RDA technique. SIGNIFICANCE AND IMPACT OF THE STUDY: The RDA technique seems to be an useful method for the identification of genes that are differentially present in H. parasuis, a respiratory pathogen of veterinary interest.     

安徽PRSS发病猪副猪嗜血杆菌的分离鉴定及其耐药性分析

目的了解猪繁殖与呼吸综合征（PRRS）发病猪继发感染副猪嗜血杆菌（HPS）的情况及分离菌株的药物感受性。方法应用细菌分离培养、形态学检查、生化试验和PCR技术,对2007年1月至2008年12月从安徽不同地区采集的146头份PRRS发病猪的病料进行HPS检测,并采用标准K-B纸片法对分离菌株进行14种抗菌药物敏感试验。结果分离鉴定出12株HPS,检出率为8.22%（12/146）;12株HPS对氯霉素100%敏感,环丙沙星为91.7%,阿莫西林和新霉素为83.3%,对罗红霉素100%耐药,阿米卡星为83.3%。结论安徽省不同地区PRRS感染猪群中均存在程度不同的HPS感染,各地区HPS分离株表现出形态上的变化特征和一致的生化特性,且有对临床常用抗菌药物耐药性增强的趋势。     

Haemophilus parasuis infection in 3D4/21 cells induces autophagy through the AMPK pathway

Haemophilus parasuis (H. parasuis) is a common commensal in the upper respiratory tract of pigs, but causes Glässer's disease in stress conditions. To date, many studies focused on the immune evasion and virulence of H. parasuis; very few have focused on the role autophagy played in H. parasuis infection, particularly in porcine alveolar macrophages (PAMs). In this study, a PAM cell line, 3D4/21 cells were used to study the role of autophagy in H. parasuis infection. 3D4/21 cells tandemly expressing GFP, mCherry, and LC3 were infected with H. parasuis serovar 5 (Hps5). Western blot analysis and confocal and transmission electron microscopy showed that H. parasuis infection effectively induces autophagy. Using Hps strains of varying virulence (Hps4, Hps5, and Hps7) and UV‐inactivated Hps5, we demonstrated that autophagy is associated with the internalisation of living virulent strains into cells. In 3D4/21 cells pretreated with rapamycin and 3‐MA then infected by Hps4, Hps5, and Hps7, we demonstrated that autophagy affects invasion of H. parasuis in cells. AMPK signal results showed that Hps5 infection can upregulate the phosphorylation level of AMPK, which is consistent with the autophagy development. 3D4/21 cells pretreated with AICAR or Compound C then infected by Hps5 revealed that the autophagy induced by Hps5 infection is associated with the AMPK pathway. Our study contributes to the theoretical basis for the study of H. parasuis pathogenesis and development of novel drugs target for prevention Glässer's disease.     

A novel nicotinamide adenine dinucleotide control strategy for increasing the cell density of Haemophilus parasuis

Haemophilus parasuis is the causative agent of Glässer's disease and is a major source of economic losses in the swine industry each year. To enhance the production of an inactivated vaccine against H. parasuis, the availability of nicotinamide adenine dinucleotide (NAD) must be carefully controlled to ensure a sufficiently high cell density of H. parasuis. In the present study, the real-time viable cell density of H. parasuis was calculated based on the capacitance of the culture. By assessing the relationship between capacitance and viable cell density/NAD concentration, the NAD supply rate could be adjusted in real time to maintain the NAD concentration at a set value based on the linear relationship between capacitance and NAD consumption. The linear relationship between cell density and addition of NAD indicated that 7.138 × 10⁹ NAD molecules were required to satisfy per cell growth. Five types of NAD supply strategy were used to maintain different NAD concentration for H. parasuis cultivation, and the results revealed that the highest viable cell density (8.57, OD₆₀₀) and cell count (1.57 × 10¹⁰ CFU/mL) were obtained with strategy III (NAD concentration maintained at 30 mg/L), which were 1.46- and 1.45- times more, respectively, than cultures with using NAD supply strategy I (NAD concentration maintained at 10 mg/L). An extremely high cell density of H. parasuis was achieved using this NAD supply strategy, and the results demonstrated a convenient and reliable method for determining the real-time viable cell density relative to NAD concentration. Moreover, this method provides a theoretical foundation and an efficient approach for high cell density cultivation of other auxotroph bacteria.     

Elemental iron does repress transferrin, haemopexin and haemoglobin receptor expression in Haemophilusinfluenzae

The iron repressible nature of Haemophilus influenzae transferrin binding proteins suggests a regulatory role for elemental iron in their expression. The existence of a Haemophilus ferric uptake repressor (Fur) binding motif identified in the promoter region of both tbpA and tbpB further supports this hypothesis. However, a recent study using brain heart infusion growth medium suggested that transferrin binding protein synthesis in H. influenzae was haem- rather than iron-regulated. The present study re-investigates this observation and using a chemically defined medium, we demonstrate that elemental iron haem or protoporphyrin IX can each regulate Haemophilus influenzae transferrin, haemopexin and haemoglobin receptor expression.     

Siderophore-mediated iron acquisition in the staphylococci

Iron is frequently a growth-limiting nutrient due to its propensity to interact with oxygen to form insoluble precipitates and, therefore, biological systems have evolved specialized uptake mechanisms to obtain this essential nutrient. Many pathogenic bacteria are capable of obtaining stringently sequestered iron from animal hosts by one or both of the following mechanisms: extraction of heme from host erythrocyte and serum hemoproteins, or through the use of high affinity, iron-scavenging molecules termed siderophores. This review summarizes our current knowledge of siderophore-mediated iron acquisition systems in the genus Staphylococcus.     

Comparison of real-time PCR and culture isolation in colostrum-deprived pigs immunized and challenged with Haemophilus parasuis

Aims: A real‐time PCR (RT‐PCR) based on the detection of the infB gene of Haemophilus parasuis is compared with culture isolation (Frandoloso et al., (2011) Clin Vaccine Immunol 18 , 50–58.), evaluating different subunit or commercial vaccines. Methods and Results: Samples from different tissues of 24 experimentally infected and challenged colostrum‐deprived piglets were tested. The RT‐PCR gave globally a 23·3% more of positive results than culture, and all samples being positive by culture were positive by RT‐PCR also. H. parasuis could not be cultured from any of the samples of the piglets included in the three vaccinated groups resulting in a strong protection, but it could be detected by RT‐PCR in six samples in the group immunized with the commercial vaccine, in three in that vaccinated with native proteins with affinity to porcine transferrin (NPAPT) administered intramuscularly and in only two in that immunized with NPAPT intratracheally. Conclusions: The RT‐PCR was more sensitive than culture for H. parasuis detection in the organs compared. Significance and Impact of the Study: The RT‐PCR evidenced that NPAPT vaccines were those yielding the best protection results in terms of H. parasuis clearance.     

Effect of iron limitation on protein composition and ultrastructure of Haemophilus ducreyi

Protein profiles of whole cells of Haemophilus ducreyi grown in the presence or absence of the iron chelator desferal, were compared by polyacrylamide gel electrophoresis. Each of four strains produced novel proteins in the range 43-160 kDa when cultured under conditions of reduced iron availability. At some sub-inhibitory concentrations, desferal produced enhanced growth, possibly due to it functioning as an exogenous siderophore. Organisms grown under conditions of reduced iron availability ultrastructurally showed also large periplasmic spaces between cytoplasm and outer membrane.     

Identification of surface-exposed proteins Haemophilus ducreyi

The proteins exposed at the surface of intact whole cells of 6 strains of Haemophilus ducreyi were identified by radioactive labelling using ¹²⁵I and Iodogen, followed by autoradiography of SDS-polyacrylamide gels of cell lysates. The most highly labelled proteins were a 39-kDa protein found in all 6 strains, and a group of proteins which exhibited inter-strain variation ranging between 27 and 30.5 kDa.     

The solution structure of the C-terminal domain of TonB and interaction studies with TonB box peptides

The TonB protein transduces energy from the proton gradient across the cytoplasmic membrane of Gram-negative bacteria to TonB-dependent outer membrane receptors. It is a critically important protein in iron uptake, and deletion of this protein is known to decrease virulence of bacteria in animal models. This system has been used for Trojan horse antibiotic delivery. Here, we describe the high-resolution solution structure of Escherichia coli TonB residues 103-239 (TonB-CTD). TonB-CTD is monomeric with an unstructured N terminus (103-151) and a well structured C terminus (152-239). The structure contains a four-stranded antiparallel beta-sheet packed against two alpha-helices and an extended strand in a configuration homologous to the C-terminal domain of the TolA protein. Chemical shift perturbations to the TonB-CTD (1)H-(15)N HSCQ spectrum titrated with TonB box peptides modeled from the E.coli FhuA, FepA and BtuB proteins were all equivalent, indicating that all three peptides bind to the same region of TonB. Isothermal titration calorimetry measurements demonstrate that TonB-CTD interacts with the FhuA-derived peptide with a K(D)=36(+/-7) microM. On the basis of chemical shift data, the position of Gln160, and comparison to the TolA gp3 N1 complex crystal structure, we propose that the TonB box binds to TonB-CTD along the beta3-strand.     

Utilization of transferrin-bound iron by Haemophilus species of human and porcine origins

Haemophilus influenzae and H. haemolyticus acquired iron bound to human transferrin but not to human lactoferrin, ovo- or porcine transferrins. Conversely the swine pathogens H. pleuropneumoniae and H. parasuis used iron bound only to porcine transferrin. Growth under conditions of iron deprivation induced the production of siderophores and iron-repressible outer membrane proteins in H. parainfluenzae, H. paraphrophilus and H. parasuis but not in H. influenzae, H. haemolyticus or H. pleuropneumoniae. The latter 3 Haemophilus species appear to sequester transferrin bound iron via a siderophore-independent mechanism. However, the ability to produce iron chelating compounds did not enable H. parainfluenzae or H. paraphrophilus to utilize transferrin bound iron.