猪肺炎支原体脂蛋白LppB的克隆表达及其编码氨基酸的生物学分析

本研究旨在获得猪肺炎支原体脂蛋白LppB的表达产物,分析其编码氨基酸的生物学特性,为进一步研究脂蛋白LppB的功能奠定基础。试验构建重组表达质粒pET30a-LppB,通过快速定点突变试剂盒将LppB基因中1 068,1 353,1 578nt处的A突变为G,使改造后的LppB基因能在大肠杆菌系统中正确表达,IPTG诱导重组蛋白的表达并进行蛋白纯化。SDS-PAGE分析结果显示,IPTG诱导表达后获得一条特异性蛋白条带,相对分子质量约为70 000,与预期蛋白大小一致。可溶性分析结果表明目的蛋白主要以可溶的形式表达于上清中;Western blot鉴定结果表明纯化的蛋白能与抗6×His标签的单抗发生特异性反应。采用DNAStar Protean模块对脂蛋白LppB进行了二级结构、蛋白质骨架柔性区域、抗原指数的预测,并确定了B细胞抗原优势表位的可能分布。     

Mycoplasma hyopneumoniae infection in pigs: Duration of the disease and evaluation of four diagnostic assays

200 SPF pigs were infected by aerosol with Mycoplasma hyopneumoniae and the development of clinical signs, serological and pathological reactions were studied. Mean time to onset of coughing was 13 days. A mean delay of 9 days was observed from onset of coughing until seroconversion against M. hyopneumoniae as measured by ELISA. At an individual level, the sensitivity for this ELISA was estimated to 98–100% and the specificity to 93–100%. Pasteurella multocida was isolated from the majority of the lungs 4 weeks post inoculation with M. hyopneumoniae and the lung lesions in pigs were significantly larger when P. multocida was present as compared to pigs with M. hyopneumoniae alone. An evaluation of cultivation, immunofluorescence, ELISA and polymerase chain reaction for demonstration of M. hyopneumoniae in lungs showed that all four methods have a high sensitivity in the acute stages of pneumonia. In the later stages the sensitivity of cultivation was superior to the other methods. No differences in specificity were observed between the methods. The antigen-ELISA OD values and the immunofluorescence scores revealed a strong positive correlation. Nasal swabs were additionally used for demonstration of M. hyopneumoniae and the polymerase chain reaction was found superior to the other methods.     

猪肺炎支原体及其生物学研究进展

猪肺炎支原体是引起猪支气管肺炎的主要病原 ,严重危害着养猪事业的发展。虽然猪肺炎支原体的结构简单 ,但由于它对营养要求较高 ,故培养难度大 ,也是人们不能对其深入研究的一个主要原因。近年来利用克隆技术 ,对猪肺炎支原体从基因水平上进行了多种研究 ,已经取得了一些可喜成果。文章从它的生物学特性、荚膜结构、膜蛋白研究以及基因水平的研究等方面进行了综合性论述 ,并简明地指出了目前研究中存在的问题以及未来展望     

Experimental evaluation of Mycoplasma hyopneumoniae bacterin against a Korean M. hyopneumoniae challenge

The objective of this study was to evaluate the efficacy of a new Mycoplasma hyopneumoniae bacterin against a Korean M. hyopneumoniae challenge under experimental conditions. Fifteen pigs were allocated randomly into 3 groups (5 pigs per group) that were designated in 1 of 3 ways: vaccinated-challenged, unvaccinated-challenged, or unvaccinated-unchallenged. The pigs in the vaccinated-challenged group were immunized with an M. hyopneumoniae whole-cell bacterin at a 1.0 mL dose-level at 21 d old. At 42 d old (0 d post-challenge), the pigs in the vaccinated-challenged and unvaccinated-challenged groups were inoculated intranasally with a strain of Korean M. hyopneumoniae. Vaccinated-challenged pigs elicited a strong cell-mediated immunity as measured by M. hyopneumoniae-specific interferon-γ secreting cells when compared with unvaccinated-challenged pigs. Vaccination of pigs with this new M. hyopneumoniae bacterin reduced nasal shedding and lung lesions. The evaluated vaccine was therefore considered effective in controlling M. hyopneumoniae infection.     

猪肺炎支原体特异性蛋白P36基因的克隆与表达

根据Genbank中猪肺炎支原体P36基因序列设计一对特异性引物,通过聚合酶链式反应(PCR)扩增出猪肺炎支原体ZCF23株特异性蛋白P36基因序列,经测序确认后,克隆人原核表达载体pGEX 6p-1的EcoR I和Xho I位点之间,转化宿主菌BL21,将筛选出的阳性克隆用IPTC诱导,通过SDS-PAGE电泳进行鉴定,结果表明,P36蛋白基因获得了表达,这为猪肺炎支原体免疫检测与诊断提供了重要条件.     

猪肺炎支原体P102基因C末端序列的克隆及原核表达

应用DNA重组技术,将猪肺炎支原体P102的C末端基因克隆到pET-28a表达载体上,并构建了重组表达载体;将鉴定正确的重组质粒转化大肠杆菌BL21,用终浓度为1．0mmol／L的IPTG诱导5h,然后用Bug BusterTM Ni—NTAHis·Bind纯化试剂盒在自然条件下对目的蛋白进行纯化。ELASE检测结果表明：该蛋白具有良好的生物学活性。利用纯化蛋白制备高免血清为检测P102蛋白在真核系统中的表达奠定基础。     

PCV2和猪肺炎支原体:协同感染和联合免疫接种

PCV2是养猪行业中最有经济学意义的重要病原体.现在,这种微小又无处不在的病毒被认为是很多疾病的病原体,这些疾病被共同命名为猪圆环病毒病(Porcine Circovirus Disease,PCVDs).在这些PCVDs中,PCV2全身性疾病[PCV2-SystemicDisease,PCV-SD,以前称之为断奶后多系统衰竭综合征(PMMWS)]是最重要的一种.虽然PCV2感染必然会引起PCV-SD的各种临床症状和损伤(图1),但在与其它病原体进行混合感染时,这种疾病能得到有效的复制.协同感染源之一是猪肺炎支原体(Mycoplasma hyponeumoniae,M.hyo).M.hyo是引起动物肺炎(Enzootic Pneumonia,EP)的主要病原体,EP是一种慢性呼吸道疾病,主要影响生长和育肥猪.     

A field evaluation of two vaccines against Mycoplasma hyopneumoniae infection in pigs

Background

A field trial was carried out with two Mycoplasma hyopneumoniae vaccines in order to investigate the benefit of vaccination under field conditions in modern Danish pig production facilities with pigs being positive for M. hyopneumoniae. The M. hyopneumoniae infection of the herd was confirmed through blood samples that were positive for antibodies against M. hyopneumoniae combined with gross lesions of the lungs related to M. hyopneumoniae at slaughter and detection of M. hyopneumoniae by polymerace chain reaction in these lesions.

Results

A total of 2,256 pigs from two herds were randomly divided into three groups. Group 1 received 2 mL ThoroVAX^®VET, Group 2 received 1 mL Ingelvac^®MycoFLEX, and Group 3 was a non-vaccinated control group. The vaccination was performed by a person who was not involved in the rest of the trial and vaccination status thereby blinded to the evaluators.The prevalence of lung lesions related to M. hyopneumoniae were significantly lower for pigs vaccinated with ThoroVAX^®VET but not for pigs vaccinated with Ingelvac^®MycoFLEX^®, when compared to non-vaccinated pigs. There was no significant effect of vaccination on growth rate, antibiotic consumption or mortality.

Conclusion

This trial demonstrated that vaccination with Thoro^®VAX VET was effective in reducing the prevalence of lung lesion in pig units infected with M. hyopneumoniae.     

Clinical evaluation of intradermal vaccination against porcine enzootic pneumonia (Mycoplasma hyopneumoniae)

The aim of the present study was to investigate the efficacy of single-dose intradermal vaccination against Mycoplasma hyopneumoniae on a commercial swine unit. A total of 1051 healthy suckling piglets of 28±3 days of age were randomly assigned to one of three experimental groups: (a) intradermal: 346 piglets vaccinated intradermally (Porcilis M Hyo ID Once, Intervet SPAH), (b) intramuscular : 351 piglets vaccinated intramuscularly (Porcilis M1 Intervet SPAH) and (c) controls: 354 piglets injected with a placebo (adjuvant only). Performance parameters such as average daily weight gain (ADG), as well as health parameters and lung lesion scores were monitored from four weeks of age until slaughter. The improvement in ADG over the controls, during the finishing phase, was 27 g/day for the intradermal group and 17 g/day for the intramuscular group. Both intradermal and intramuscular vaccinations were effective in reducing clinical signs and lung lesions caused by M hyopneumoniae. Compared with the controls, approximately 10.4 per cent fewer clinical cases were diagnosed in the intradermal group, and 6 per cent fewer in the intramuscular group, during the finishing period. In conclusion, performance results were better in the vaccinated groups than in the control group, while intradermal vaccination afforded greater protection than intramuscular vaccination, especially with regard to morbidity, lung lesion and pleuritis scores.     

Morphological and ultrastructural studies of Mycoplasma flocculare and Mycoplasma hyopneumoniae in vitro.

Cells of Mycoplasma flocculare were found to vary in size and shape, especially in the later phases of growth, whereas those of Mycoplasma hyopneumoniae were fairly uniform irrespective of growth phases. Filamentous cells were present in cultures of M flocculare in the stationary and declining phases, but were never found in cultures of M hyopneumoniae. The filamentous and bizarre forms observed when mycoplasmas were suspended in phosphotungstic acid probably result from the action of the hypotonic solution. The surface of all cells was covered by a fuzzy coat consisting of fine hairs or bristles. An electron-lucent region was usually seen in cells negatively stained after centrifugation, but was only occasionally seen in cells negatively stained directly from the medium. Intracytoplasmic membranes were present in sectioned cells. No attachment organelle was found in cells of either species.     

猪肺炎支原体中国分离株热休克蛋白Hsp70(Dnak)的全基因克隆及C末端基因的表达与免疫活性分析

猪肺炎支原体（Mycoplasma hyopneumoniae,MHP）是猪气喘病的病原。DnaK又称为热休克蛋白Hsp70,具有分子伴侣和免疫的作用。以MHP中国分离株Yin-1为模板,扩增了DnaK基因的全序列,将该序列克隆到pMD18-T载体上并测序。测序结果Yin-1株DnaK基因即Hsp70基因全长1803bp,编码600aa,第631～633位TGA在支原体编码色氨酸而不是作为终止密码子。将该序列与多种致病支原体DnaK基因进行分析比较,发现Yin-1株与232株、J株、7448株等MHP菌株的DNA同源性为99.3％～99．4％,氨基酸同源性为99．2％～99.3％,而与非同种支原体的DNA和氨基酸同源性仅为64．5％～74．4％和59.3％～77％。这表明DnaK基因在种内高度保守,种间差异较大。以pET28a为载体构建重组质粒,原核表达DnaKC末端大小为1kb左右的基因,对表达的蛋白进行纯化后,通过Westernblot检测它的免疫活性。原核表达得到大小为41ku的目的蛋白．经Westernblot证实．纯化蛋白可与MHP阳性猪血清反应,具有免疫活性。     

The growth response of Mycoplasma hyopneumoniae and Mycoplasma flocculare based upon ATP-dependent luminometry

Cultures of Mycoplasma hyopneumoniae and M. flocculare in Friis' broth grew faster and to higher titers in air than in 8% CO2; cultures in air grew better when shaken than when stationary. Under the optimal conditions, both species have generation times of about 10 h and achieve maximum titers of at least 10(9) organisms per ml. Maximum growth was reached near pH 7.0, before the phenol red indicator had noticeably changed colour. Changes in growth were readily detected by an ATP assay based upon the luciferin-luciferase reaction. Concentrations of ATP fell rapidly after peak growth. Although the addition of 27 mM glucose to the medium did not change the pattern of growth and gas chromatography gave no evidence of the production of volatile or non-volatile end-products, washed harvested cells of both species metabolized [14C]-glucose. The addition of 29 mM arginine to the medium inhibited growth.     

猪肺炎支原体DJ-166株的分离鉴定

从山西某猪场采集疑似猪支原体肺炎肺组织病料,接种CH培养基培养,克隆纯化获得一株菌株。该菌株经PCR、培养特性、生化特性和血清学特性试验鉴定为猪肺炎支原体,命名为DJ-166株。该分离菌株在人工合成培养基上传8代稳定后,活菌计数达到108CCU/m L;菌液培养物气管注射猪后,致病性较弱;免疫原性试验证明该分离菌株具有良好的免疫原性,此结论为猪支原体肺炎疫苗的研究提供了必要条件。     

猪肺炎支原体YN200901株的分离鉴定

采集疑似猪肺炎支原体病理变化的肺脏,接种到改良牛心培养基、猪胃消化液培养基进行培养,经瑞特染色、镜检,菌落狄氏染色、PCR鉴定及序列比对、生化试验鉴定,抗猪肺炎支原体血清生长抑制试验,分离获得一株猪肺炎支原体云南地方流行菌株,命名为YN200901。试验结果为云南本地株猪肺炎支原体的后续研究提供了物质基础。     

猪肺炎支原体S株的分离与鉴定

从典型猪肺炎支原体病变肺组织传代分离到一株支原体,经培养特性、血清学鉴定、生化鉴定、PCR检测及测序分析证明其为猪肺炎支原体,纯化后命名为S株。将S株P46基因和P97基因R1区的氨基酸序列与其他菌种的相应序列进行同源性分析,该菌株P46基因氨基酸序列与其他菌种同源高达99%以上,P97基因R1区的氨基酸重复数为11个,不同于其他菌株;免疫原性试验结果表明该菌株具有良好的免疫原性。     

猪肺炎支原体流行病学及防控研究进展

猪肺炎支原体是引起猪地方性肺炎的主要病原,流行于世界各地,给养猪业造成重大经济损失。本文综述了猪肺炎支原体的主要传播途径以及引起的临床症状和肺部病变,总结了其主要致病机理,分析了综合控制该疫病的方法。猪肺炎支原体的主要传播途径是与感染猪密切接触,也可通过空气远距离传播,但无垂直传播相关报道;猪肺炎支原体感染的主要临床特征是间歇性、强度可变的干咳,但在与其他病原继发或混合感染后,临床症状和肺部病变可能会加重;猪肺炎支原体主要通过释放黏附素引起纤毛发生病变、死亡,导致上皮细胞受损或脱落从而影响纤毛功能,也能通过产生剧毒的H2O2等代谢产物引发毒性效应;防治该病首先要通过生物安全措施控制猪肺炎支原体的传入,其次是采用抗菌药物进行治疗,最后是通过接种疫苗降低其传播率。本文为猪养殖场做好猪肺炎支原体病防控提供了参考。     

猪肺炎支原体检测芯片的研制及初步应用

本研究探索建立一种新的基因芯片方法检测和鉴定猪肺炎支原体。在猪肺炎支原体的16SrRNA、P36和P463个基因内设计3个靶基因片段,用PCR标记Cy3探针,建立了用于检测和鉴定猪肺炎支原体的检测芯片。试验结果显示,猪肺炎支原体与检测芯片的3个靶基因（Mhp-16S、Mhp-P46和Mhp-P36）杂交呈阳性,猪鼻支原体只与Mhp-16S靶基因杂交呈阳性,其它所测细菌和病毒不与检测芯片的靶基因杂交。检测芯片的检测灵敏度和PCR相同,能达到10pg基因组DNA／50μL标记体系或反应体系。用检测芯片鉴定了3株疑似猪肺炎支原体临床分离株,结果有2株为猪肺炎支原体,1株为其它猪支原体。用检测芯片、P36-PCR和分离鉴定分别对45头病猪临床样品选择混合培养物中的猪肺炎支原体进行了检测,结果它们的检出率分别为20％（9／45）、22．2％（10／45）和8．9％（4／45）。检测芯片还检出有26．7％（12／45）的病猪感染了其它猪支原体。试验结果表明,所建立的检测芯片是一种快速检测和鉴定猪肺炎支原体的敏感特异性方法。     

Dynamics and persistence of Mycoplasma hyopneumoniae infection in pigs

The purpose of this study was to describe the dynamics (shedding and transmission) of Mycoplasma hyopneumoniae infection within a population of swine and to determine the duration of the infection (persistence) through the identification of the agent in bronchial samples. Sixty-three 2-month-old pigs were used in this study. The pigs (n = 28) were experimentally infected by the intratracheal route with M. hyopneumoniae and considered as seeder pigs. The remaining pigs (n = 32) were not inoculated and randomly allocated to 2 different groups: direct contact exposure pigs (n = 12) and indirect contact exposure pigs (n = 20). Blood samples and nasal swabs were collected throughout the study on days 0, 28, 35, 42, 49, 63, 91, and 119 postinfection. To assess the duration of M. hyopneumoniae infection, 9 seeder and 6 contact exposure pigs were slaughtered at days 155 (group 1), 170 (group 2), and 185 (group 3) postinfection. Direct contact pigs showed evidence of infection on day 28 by polymerase chain reaction (PCR) and on day 35 by serology. The indirect contact exposure pigs presented a very delayed and slow seroconversion pattern; they did not present evidence of transmission until 42 d after the infection of seeder pigs. Identification of M. hyopneumoniae in bronchial swabs was confirmed by nested-PCR from days 155 to 185 postinfection. At the last slaughter date, 77.7% and 100% of the seeders and contact exposure pigs, respectively, tested positive. The results of this study reconfirmed direct infection of M. hyopneumoniae and suggest that indirect transmission can occur in a population. Finally, duration of the infection in this study was longer than previously described.     

Fluorescence serological and culture detection of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis in swine lungs

The present paper compares the fluorescence-serological detection of M. hyopneumoniae and M. hyorhinis in frozen sections with cultural procedures. In 161 lungs of swine the agent of enzootic pneumonia, M. hyopneumoniae, was found almost exclusively in frozen sections (22 times). Only one strain was cultivable. M. hyorhinis was detected 59 times by culture but only 18 times in the sections. For the detection of M. hyopneumoniae therefore the fluorescence-serological investigation of lung sections is the preferable method.     

猪肺炎支原体乳酸脱氢酶基因的克隆、表达、纯化及其间接ELISA检测方法的建立

根据GenBank中的猪肺炎支原体乳酸脱氢酶（LDH）基因序列设计1对引物，PCR扩增LDH基因，首先将扩增片段连接到克隆载体pMD18-T上，然后连接到表达载体pGEX—KG上，经测序正确后诱导表达。重组质粒在大肠杆菌中表达的目的蛋白以可溶性蛋白和包涵体2种形式存在。将超声波破碎的诱导菌液高速离心，其上清用Glutathione Sepharose4Bbead亲和层析纯化。用猪肺炎支原体的标准阳性血清对纯化蛋白作Western—blot检测，出现目的条带；以纯化蛋白为抗原建立了检测猪肺炎支原体抗体的间接ELISA方法，该方法具有较好的稳定性和重复性，较高的特异性与敏感性。用建立的ELISA方法与中国兽医药品监察所研制的IHA试荆盒同时检测120份临床血清，二者总符合率为92．5％。用建立的ELISA方法检测了671份临床送检不同年龄阶段的猪血清，结果显示断奶前仔猪猪肺炎支原体（Mycoplasma hyopnenmoniae，MHP）感染率为44．3％，保育猪为3．0％，育肥猪为17．44％，种猪为73．41％，这初步反映了猪喘气病在各个年龄阶段的感染率。