副猪嗜血杆菌检测技术研究进展

副猪嗜血杆菌是存在于健康猪上呼吸道的一种致病菌,也是格拉泽氏病的病原,近年来其发生与流行给养猪业造成巨大的经济损失,现已成为危害养猪业较严重的细菌性疾病之一.及时、准确地检测出该病以便采取相应的措施,是成功防制该病的关键.本文就从血清学、病原学、分子生物学等方面阐明了副猪嗜血杆菌的检测技术,从而为兽医临床诊断与治疗提供参考.     

一种新的人兽共患传染病——狐狸阴道加德纳氏菌病的研究 Ⅵ.阴道加德纳氏菌病灭活疫苗的研制

选用免疫原性优良的我国狐狸阴道加德纳氏菌主要流行型血清Ⅰ型GVF44号菌株,先后试验研制了氢氧化铝胶、蜂胶和油佐剂灭活苗。试验结果表明,铝胶苗的安全性优,免疫剂量为40亿菌/1ml,3倍免疫剂量的铝胶灭活苗接种狐狸后无任何不良反应,免疫后21d经100个ID_(50)强毒的攻击,对Ⅰ型的保护率达92％。对Ⅱ、Ⅲ型的保护率达80％以上。免疫持续期为6个月,4～10℃条件下可保存10个月。通过田间试验应用证实该疫苗安全、有效。     

一种新的人兽共患传染病——狐狸阴道加德纳氏菌病的研究 Ⅵ.阴道加德纳氏菌病灭活疫苗的研制

选用免疫原性优良的我国狐狸阴道加德纳氏菌主要流行型血清Ⅰ型GVF44号菌株,先后试验研制了氢氧化铝胶、蜂胶和油佐剂灭活苗。试验结果表明,铝胶苗的安全性优,免疫剂量为40亿菌/1ml,3倍免疫剂量的铝胶灭活苗接种狐狸后无任何不良反应,免疫后21d经100个ID_(50)强毒的攻击,对Ⅰ型的保护率达92％。对Ⅱ、Ⅲ型的保护率达80％以上。免疫持续期为6个月,4～10℃条件下可保存10个月。通过田间试验应用证实该疫苗安全、有效。     

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。     

西藏小型猪10种猪病血清学调查

目的对西藏小型猪进行10种猪病（猪瘟、伪狂犬病、篮耳病、猪流感、圆环病毒感染、细小病毒病、乙型脑炎、弓形体病、衣原体病和布氏杆菌病）的血清学调查。方法采用ELISA或凝集试验方法对10头已注射猪瘟疫苗的原代（F0代）母猪和40头20日龄乳猪、50头第一代（F1代）3～5月龄猪进行10种猪病的血清学检测。结果猪瘟病毒、细小病毒和乙型脑炎病毒抗体阳性率分别是母猪为80.0%、80.0%、70.0%;20日龄乳猪为45.0%、25.0%、45.0%;3～5月龄猪为4.0%、76.0%、80.0%。圆环病毒抗体阳性率母猪为60.0%,20日龄乳猪为5.0%。3～5月龄猪为0%;篮耳病病毒、伪狂犬病病毒、猪流感病毒、弓形体、衣原体、布氏杆菌在不同年龄西藏小型猪中抗体阳性率均为0%。结论不同年龄西藏小型猪未受篮耳病病毒、伪狂犬病病毒、猪流感病毒、弓形体病、衣原体病、布氏杆菌病感染。     

新型奶牛乳房炎多联苗的研制及免疫力试验

目的：筛选出毒力较强的金黄色葡萄球菌、大肠杆菌、无乳链球菌作为制苗菌株,制成多联菌苗,预防及治疗奶牛乳房炎。方法：通过菌株的分离、纯化、鉴定试验及毒力试验获得毒力较强的菌株,制成金黄色葡萄球菌类毒素及菌体蛋白、大肠杆菌和无乳链球菌灭活菌苗,合理配伍制成多联菌苗,进行安全、异常毒性及免疫力试验。结果：所选金黄色葡萄球菌、大肠杆菌、无乳链球菌的最小致死量分别是3．0×10^8／ml、1．5×10^8／ml、9．0×10^8／ml,多联苗安全、异常毒性试验合格,对免疫组小白鼠保护率为83．3％。结论：该多联菌苗具有安全、无毒、高效的特点,可以用来预防及治疗奶牛乳房炎。     

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。     

猪气喘病实验猪模型的建立

目的 研究建立猪气喘病人工发病模型。方法 将分离得到的一株猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)Js株进行各种试验鉴定,证实其为Mhp强毒株。每头猪肺内接种Js株培养物2ml,15～25d后观察临床症状和病理变化,采集病变组织,经冻干制成攻毒用组织毒。安检合格,批号为20000324。给15头小梅山二元杂交猪分别气管内注射以KM₂培养基作10^-2、10^-3、10^-4和10^-5稀释的强毒,每头猪5ml,对照组注射培养基。攻毒后25d观察试验猪临诊症状,X线透视,记录病理变化。结果 10^-2、10^-3、10^-4稀释的强毒试验组猪均出现了典型的猪气喘病临床症状和病理变化。结论 人工发病试验测得Mhp Js株组织强毒接种气管内注射最小发病剂量为10^-4稀释5ml,正式试验人工发病可用100个最小发病剂量即强毒冻干物1:100稀释气管内注射5ml,可确保攻毒成功。     

猪乳铁蛋白在4种重组乳杆菌中的表达及抑菌活性比较

为了获得优化的猪乳铁蛋白乳杆菌表达系统,并比较重组猪乳铁蛋白的抑菌活性,根据乳杆菌使用密码子的偏嗜性优化合成猪乳铁蛋白成熟肽编码序列,将其克隆到乳杆菌表达载体pPG612.1的XhoⅠ/BamHⅠ位点,获得了plf乳杆菌表达载体质粒pPG612.1-plf。将获得的重组质粒分别电转化入干酪乳杆菌ATCC393、戊糖乳杆菌KLDS1.0413、植物乳杆菌KLDS1.0344和副干酪乳杆菌KLDS1.0652细胞内,获得4种表达猪乳铁蛋白的重组乳杆菌。经木糖诱导,通过Western blotting和激光共聚焦检测重组猪乳铁蛋白的表达,用ELISA方法检测和比较4种重组菌上清中表达猪乳铁蛋白的量,并用琼脂孔穴扩散抑菌法检测4种重组乳杆菌表达乳铁蛋白的抑菌活性。结果表明,乳铁蛋白在4种重组乳杆菌中均得到正确表达,其产物分子量约73 kDa,重组干酪乳杆菌、重组戊糖乳杆菌、重组植物乳杆菌和重组副干酪乳杆菌的重组猪乳铁蛋白表达量分别为9.6μg/mL、10.8μg/mL、12.5μg/mL、9.9μg/mL。重组猪乳铁蛋白对大肠杆菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、巴氏杆菌和李氏杆菌均有一定的抑菌作用,对金黄色葡萄球菌的抑菌作用最强,且4种重组乳杆菌中重组植物乳杆菌表达产物的抑菌效果优于其他重组菌的表达产物。结果表明在4种乳杆菌中重组猪乳铁蛋白的最佳表达系统为植物乳杆菌,该结果为猪乳铁蛋白的乳杆菌表达系统进一步开发与应用奠定了基础。     

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。     

Comparison of Haemophilus parasuis reference strains and field isolates by using random amplified polymorphic DNA and protein profiles

ABSTRACT: BACKGROUND: Haemophilus parasuis is the causative agent of Glasser's disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for distinguishing H. parasuis reference strains and field isolates. RESULTS: The DNA and WCP lysate profiles of 15 reference strains and 31 field isolates of H. parasuis were analyzed using the Dice and neighbor joining algorithms. The results revealed unique and reproducible DNA and protein profiles among the reference strains and field isolates studied. Simpson's index of diversity showed significant discrimination between isolates when three 10mer primers were combined for the RAPD method and also when both the RAPD and WCP lysate typing methods were combined. CONCLUSIONS: The RAPD profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of DNA mutations in the genes of the samples. The recent field isolates had different WCP lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression.     

Soluble expression of OmpA from Haemophilus parasuis in Escherichia coli and its protective effects in the mouse model of infection

Haemophilus parasuis causes contagious porcine Gl?sser's disease leading to severe losses in the swine industry. In this study, we established an efficient Escherichia colibased system for the expression of H. parasuis major outer-membrane protein (MOMP) that has been known as a good vaccine candidate against Gl?sser's disease. Use of an E. coli-derived pelB leader sequence made it possible to produce recombinant MOMP (rMOMP) as the soluble forms without an additional refolding process. Using two different animal models, it was evaluated that the rMOMP was capable of inducing a significant immune response and providing protection against H. parasuis infection.     

猪流行性腹泻mRNA疫苗的制备与免疫原性评价

猪流行性腹泻(porcine epidemic diarrhea,PED)是一种可引起仔猪高致死率的传染性疾病,尽管已有灭活疫苗和减毒活疫苗上市,但由于病毒变异频繁导致保护效力欠佳,发病率依然居高不下。本研究首次制备了PED mRNA候选疫苗,并在小鼠和妊娠母猪上评价了其免疫原性,证明了基于病毒受体结合区异源二聚体的mRNA候选疫苗具有良好的免疫原性,可在小鼠上高效诱导体液和细胞免疫应答,单次免疫血清中和抗体滴度达到1:300以上,并在妊娠母猪上诱导了与灭活疫苗相近的中和抗体水平,实现了100%抗体转阳。本研究为PED mRNA疫苗的进一步产业化奠定了基础。     

Genotypic diversity of Haemophilus parasuis field strains

Haemophilus parasuis is the cause of Gl?sser's disease and other clinical disorders in pigs. It can also be isolated from the upper respiratory tracts of healthy pigs, and isolates can have significant differences in virulence. In this work, a partial sequence from the 60-kDa heat shock protein (Hsp60) gene was assessed as an epidemiological marker. We analyzed partial sequences of hsp60 and 16S rRNA genes from 103 strains of H. parasuis and other related species to obtain a better classification of the strains and examine the correlation with virulence. The results were compared with those obtained by enterobacterial repetitive intergenic consensus PCR. Our results showed that hsp60 is a reliable marker for epidemiological studies of H. parasuis and that the analysis of its sequence is a better approach than fingerprinting methods. Furthermore, the analysis of the hsp60 and 16S rRNA gene sequences revealed the presence of a separate lineage of virulent strains and indicated the occurrence of lateral gene transfer among H. parasuis and Actinobacillus strains.     

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.     

Isolation and characterization of the P5 adhesin protein of Haemophilus parasuis serotype 5

Haemophilus parasuis is a Gram-negative respiratory pathogen of young pigs that colonizes the upper respiratory tract and produces a number of symptoms collectiviely described as Gl?sser's disease. Recently, an H. parasuis P5-like outer membrane adhesin protein homologous to H. influenzae P5 was identified. The P5 adhesin was partially purified by anion exchange and size-exclusion chromatography. Final purification for functional studies was performed by elution of the protein from a polyacrylamide gel. Identification of the protein as a P5 adhesin homolog of H. influenzae was confirmed by N-terminal sequencing. The P5 protein had a molecular mass of 32,000 and a pI of 5.5. Unlike the H. influenzae P5 adhesin, the H. parasuis P5 protein did not bind carcinoembryonic antigen.     

Identification and characterization of the TonB region and its role in transferrin-mediated iron acquisition in Haemophilus parasuis

Haemophilus parasuis is the causative agent of Gl?sser's disease, which is responsible for considerable economic losses in the pig-rearing industry. The aim of the study reported here was the identification, sequencing and molecular characterization of the TonB region that includes tonB, exbBD, and tbpBA genes in H. parasuis. In addition, two fusion proteins were generated. One of them (pGEX-6P-1-GST-TbpB) contained the first 501 amino acids of H. parasuis TbpB protein, while the second (pBAD-Thio-TbpB-V5-His) included the first 102 amino acids of H. parasuis TbpB N-terminus domain. A panel of 14 hybridomas secreting monoclonal antibodies was raised against the two recombinant TbpB fusion proteins. Furthermore, to assess whether the expression of the H. parasuis ExbB, TbpB, and TbpA proteins was upregulated under conditions of restricted availability of iron, a rabbit polyclonal antibody against H. parasuis TbpB-His fusion protein was produced. A rabbit polyclonal antibody against serotype 7 of Actinobacillus pleuropneumoniae ExbB and TbpA proteins was also used for the detection of the homologous proteins in H. parasuis. Overall, the data indicate that H. parasuis, like other members of the Pasteurellaceae family, possesses the genetic elements of the TonB region for iron acquisition and the transferrin-binding proteins encoded under this region are upregulated under restricted iron availability.     

Genomic characterization of Haemophilus parasuis SH0165, a highly virulent strain of serovar 5 prevalent in China

Haemophilus parasuis can be either a commensal bacterium of the porcine respiratory tract or an opportunistic pathogen causing Gl?sser's disease, a severe systemic disease that has led to significant economical losses in the pig industry worldwide. We determined the complete genomic sequence of H. parasuis SH0165, a highly virulent strain of serovar 5, which was isolated from a hog pen in North China. The single circular chromosome was 2,269,156 base pairs in length and contained 2,031 protein-coding genes. Together with the full spectrum of genes detected by the analysis of metabolic pathways, we confirmed that H. parasuis generates ATP via both fermentation and respiration, and possesses an intact TCA cycle for anabolism. In addition to possessing the complete pathway essential for the biosynthesis of heme, this pathogen was also found to be well-equipped with different iron acquisition systems, such as the TonB system and ABC-type transport complexes, to overcome iron limitation during infection and persistence. We identified a number of genes encoding potential virulence factors, such as type IV fimbriae and surface polysaccharides. Analysis of the genome confirmed that H. parasuis is naturally competent, as genes related to DNA uptake are present. A nine-mer DNA uptake signal sequence (ACAAGCGGT), identical to that found in Actinobacillus pleuropneumoniae and Mannheimia haemolytica, followed by similar downstream motifs, was identified in the SH0165 genome. Genomic and phylogenetic comparisons with other Pasteurellaceae species further indicated that H. parasuis was closely related to another swine pathogenic bacteria A. pleuropneumoniae. The comprehensive genetic analysis presented here provides a foundation for future research on the metabolism, natural competence and virulence of H. parasuis.