绵羊肺炎支原体Y98和丝状支原体丝状亚种PG3抗原的分析

用SDS-PAGE和双向电泳方法,对绵羊肺炎支原体标准株Y98和丝状支原体丝状亚种标准株PG3的全菌可溶性抗原进行分析.结果表明,用SDS-PAGE分析Y98有9条蛋白带,PG3有11条蛋白带,其中有2条相同蛋白带;用双向电泳分析Y98全菌可溶性抗原多肽斑点有288±9,主多肽斑点有47个,相对分子质量范围在27 000120 000;pI范围为4.436-7.164,PG3全菌可溶性抗原多肤斑点有243±11个,主多肽斑点有36个,相对分子质量范围在27 000～120 000,而pI范围为4.213-7.987,二者有21个相同多肽点,但其多肽含量略有差异.     

绵羊肺炎支原体与猪肺炎支原体交叉抗原研究

为了研究绵羊肺炎支原体标准株Y98与猪肺炎支原体标准株232全菌蛋白免疫原性的异同,试验采用SDS-PAGE和免疫印迹分析技术对其进行了研究,结果表明:绵羊肺炎支原体标准株Y98同猪肺炎支原体标准株232免疫印迹结果基本一致,在70,50,40,25 ku蛋白带处存在共同抗原。     

18种抗菌药物对绵羊肺炎支原体和丝状支原体分离株的抗菌活性

应用微量法测定了18种常用抗菌药物对绵羊肺炎支原体分离株HD1-goat和丝状支原体分离株XT1-goat的抗菌活性.结果表明,喹诺酮类药物及盐酸多西环素、酒石酸泰乐菌素对两个分离株均有很高的抗菌活性;硫氰酸红霉素对分离株HD1-goat无抑制生长作用,但对分离株XT1-goat有很高的抑制和杀灭活性;氨基糖苷类药物和...     

绵羊肺炎支原体的分离与鉴定

绵羊肺炎支原体是一种引起羊传染性胸膜肺炎的病原微生物,它不仅能感染绵羊同时也可感染山羊,发病率和死亡率较高,易给养羊业造成较大损失。近年来,内蒙古境内羊传染性胸膜肺炎也多有发生,为了查明病原,分别在内蒙古一些发病地区采取病料,经分离得到了疑似绵羊支原体菌株7株。对该病原微生物的分离培养、形态学特征、理化特性等进行了阐述,以期为该病的快速诊断及防治提供参考。     

绵羊肺炎支原体Y98 P30基因的克隆、表达及免疫试验

根据猪肺炎支原体（Mycoplasma hyopneumoniae,Mhp）跨膜蛋白P30基因序列设计引物,通过PCR克隆出绵羊肺炎支原体（Mycoplasma ovipneumoniae,MO）膜蛋白P30基因片段。通过对该序列的同源性分析表明MOP30基因与Mhp P30基因核苷酸序列同源性为79%。抗原表位及跨膜结构预测的结果表明,MO P30基因与Mhp P30基因的抗原表位及跨膜结构均具有高度的一致性。该片段中含1个TGA编码Trp,而不是终止密码子。将P30基因与原核表达载体pET-28a连接构建pET-28a-P30表达质粒,转化受体菌Rossta得到重组菌株Rossta（pET-28a-P30）。经诱导后SDS-PAGE表明有30 000左右的目的条带出现;Western blot表明Rossta（pET-28a-P30）表达的30 000蛋白主要以包涵体的形式存在;小鼠免疫试验表明Rossta（pET-28a-P30）原核表达的蛋白对小鼠具有一定的保护作用。     

绵羊肺炎支原体Y98株未知基因Hsp70（DnaK）的克隆

绵羊肺炎支原体（Mycoplasma ovipneumoniae,MO）是绵羊传染性胸膜肺炎（Contagious ovine pleuropneumo-nia）的主要致病菌。热休克蛋白Hsp70也叫（DnaK）,具有分子伴侣和免疫的作用。本试验以MO Y98基因组为模板,通过比对15种支原体Hsp70基因的序列并设计简并引物,分别利用同源克隆和染色体步移—Tail-PCR技术克隆到了四段Hsp70（DnaK）基因片断并进行了核苷酸序列测定。利用序列拼接软件对克隆的四段序列进行序列拼接,通过ORF Finder软件分析出了MO Y98的Hsp70的开放式阅读框架,预测分析该基因是由1 815bp组成,编码604个氨基酸。本试验于国内外首次克隆到了MO的Hsp70基因序列,为MO的抗原研究以及Hsp70的功能研究等奠定了基础。     

绵羊肺炎支原体Y98标准株P30外膜蛋白与IFN-γ的融合表达及其免疫原性

以绵羊肺炎支原体(Mycoplasma oumvipneonia,MO)标准株Y98的P30外膜蛋白为抗原,同时以细胞因子IFN-γ为佐剂,将两者串联融合表达.小鼠免疫保护试验结果显示,P30重组蛋白的免疫保护率为60％,IFN-γ重组蛋白的免疫保护率为20％,P30-IFN-γ重组蛋白的免疫保护率达到80％.间接ELISA试验证实,经100 μL与200 μL P30-IFN-γ重组蛋白免疫小鼠的血清中,P30抗体效价分别为1:32 000与1:64 000.以上结果证实,所制备的重组蛋白对小鼠实验个体基本安全,且P30-IFN-γ重组蛋白中的细胞因子IFN γ组分可以调节机体免疫机能,增强P30蛋白的免疫保护效果.     

绵羊肺炎支原体分离株交叉反应性的测定

绵羊肺炎支原体分离株致敏经过鞣酸和戊二醛处理的绵羊红细胞,制备成试验用抗原,与通过攻毒取得的几种血清进行间接血凝试验来检测其交叉反应性。通过检测,绵羊肺炎支原体分离株同无乳支原体、巴氏杆菌等几种可以引起肺炎症状的菌种无交叉反应性。     

绵羊肺炎支原体快速诊断抗原的制备及应用

绵羊支原体肺炎已成为规模化羊场中羊只发病率最高,死亡率最高的疾病之一.本试验制备出了绵羊支原体肺炎间接平板凝集诊断抗原,试验效果良好;通过实际应用,同支原体培养方法结果达100%.结果表明该诊断原可在临床上进行简单快速有效的疾病诊断.     

The identification of an organism isolated from maned sheep as Mycoplasma mycoides subsp. mycoides

The natural host-range of M. mycoides subsp. mycoides is generally believed to be restricted to cattle, although the production in experimental conditions of a generalized infection of sheep and goats has been reported on some occasions.     

The in vitro effect of six antimicrobials against Mycoplasma putrefaciens, Mycoplasma mycoides subsp. mycoides LC and Mycoplasma capricolum subsp. capricolum isolated from sheep and goats in jordan

Respiratory disease in sheep and goats is a major problem in Jordan and is often associated with Mycoplasma species. Without effective vaccines, control is mainly by chemotherapy, but the uncontrolled use of antimicrobials has led to concerns about the potential development of antimicrobial resistance. The in vitro effect of chloramphenicol, florfenicol, enrofloxacin, tylosin, erythromycin and oxytetracycline was determined against 32 isolates of Mycoplasma species-M. mycoides subsp. mycoides LC (6), M. capricolum subsp. capricolum (8) and M. putrefaciens (18), all isolated from either nasal swabs or milk, from sheep and goats in different regions of Jordan. The antimicrobial susceptibility showed some Mycoplasma species-specific differences, with M. capricolum subsp. capricolum being more susceptible to tylosin and erythromycin. Chloramphenicol and florfenicol were the least effective for all three Mycoplasma species. No trends or significant differences in antimicrobial susceptibilities were observed between sheep and goat isolates, between milk or nasal swab isolates, or between isolates from different regions of Jordan. Some isolates of M. capricolum subsp. capricolum and M. putrefaciens showed higher MIC levels with oxytetracycline, as did two isolates of M. mycoides subsp. mycoides LC with tylosin, possibly indicating signs of development of antimicrobial resistance.     

感染绵羊肺炎支原体后绵羊SPLUNC1 mRNA表达水平监测

为检测绵羊感染绵羊肺炎支原体(MO)前后的短腭、肺及鼻咽上皮克隆基因1(SPLUNC1)表达水平变化,对6只巴什拜羊和6只盘羊杂交羊分别人工感染MO,在感染前(第0天)和感染后第5、14、21天,采集口腔喉咽部的上腭组织,用Real-time PCR方法检测SPLUNC1mRNA的表达水平。结果:感染后两组羊SPLUNC1 mRNA的相对表达水平均升高,在感染后第14~21天,巴什拜羊SPLUNC1 mRNA水平均极显著高于盘羊杂交羊(P001)。该结果说明在临床上表现为抗MO感染的巴什拜羊,体内可表达高水平的SPLUNC1 mRNA,这为巴什拜羊抗MO感染的机理研究提供一定的参考依据。     

从湖羊肺脏中分离绵羊肺炎支原体的鉴定

从新疆湖羊肺脏病料中分离出一株支原体XJ-3f，用其培养物人工感染80日龄健康绵羊，28d后剖杀，剖检可见肺表面肉粉色实变，显微病理变化为大灶性融合性肺炎。参考国际已知支原体16SrRNA序列，设计一对扩增700bp片段的通用引物，直接提取肺脏组织DNA进行PCR扩增并克隆、测序。将该序列与GenBank中33种支原体序列比较，结果证明该序列与绵羊肺炎支原体（M．ovipneumonia）标准株Y．98同源性为99．9％，而与山羊支原体山羊亚种（M．capricolum subsp．capricolum，Mcc）、丝状支原体山羊亚种（M．mycoides subsp．Capri，Mmc）、丝状支原体丝状亚种LC型（M．mycoides subsp．mycoides LC，M．mmLC）等同源率为81％。以感染羊血清和Y．98与从发病羊肺脏中分离出的三株支原体茵体蛋白进行Western blot，证明均与感染羊血清有特异性反应，且与Y．98相比无明显差异，故确定分离株为绵羊肺炎支原体（M．ovipneumonia）。     

Characterisation of protein and antigen variability among Mycoplasma mycoides subsp. mycoides (LC) and Mycoplasma agalactiae field strains by SDS-PAGE and immunoblotting

Mycoplasma mycoides subsp. mycoides (LC) (Mmm LC) and Mycoplasma agalactiae are the most important mycoplasma species involved in the contagious agalactia syndrome. A total of 25 field strains from Spain and the two type strains were analysed by SDS-PAGE and immunoblotting. Two polyclonal antisera (PAbs) raised against a pool of strains of each mycoplasma species were used. The results revealed a high degree of protein variability among the field strains. The type strain of Mmm LC appeared to be representative of the field strains of this species, whereas this was not the case with the M. agalactiae type strain. Whereas M. agalactiae is known to possess a gene family regulating surface antigen diversity, there is a need to study the mechanisms used byMmm LC to generate antigenic variability in more detail.     

Molecular analysis of field strains of Mycoplasma capricolum subspecies capripneumoniae and Mycoplasma mycoides subspecies mycoides, small colony type isolated from goats in Tanzania.

A molecular analysis of strains of Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae) and Mycoplasma mycoides subsp. mycoides, small colony type (M. mycoides SC) isolated from goats was performed using the amplified fragment length polymorphism (AFLP) and pulsed-field gel electrophoresis (PFGE) fingerprinting techniques. Among the 11 field strains of M. capripneumoniae from Tanzanian goats, two AFLP patterns were demonstrated, with 10 of the strains showing indistinguishable patterns. Five Kenyan strains of M. capripneumoniae produced three AFLP patterns, with two of them being indistinguishable from the 10 identical Tanzanian and one Ugandan strain (M74/93) isolated from sheep. The AFLP pattern of the type strain (F38(T)) was identical to two Kenyan strains (Baringo and G183/82). On PFGE analysis, all the examined M. capripneumoniae strains exhibited identical PFGE profiles.Five field strains of M. mycoides SC isolated from goats displayed identical AFLP patterns except for one strain which differed from others at only one position. The AFLP pattern of the type strain of M. mycoides SC (PG1(T)) was different from the field strains. The five field strains of M. mycoides SC produced identical PFGE profiles, which were, however, different from the type strain. The AFLP and PFGE profiles of M. mycoides SC strains from goats were identical to those of six strains isolated from cattle affected with contagious bovine pleuropneumonia (CBPP) in the same areas. The results of this study suggest a close epidemiological linkage between strains of M. capripneumoniae and between M. mycoides SC type, respectively, isolated from goats in Tanzania.     

Effect of the caprine variant of Mycoplasma mycoides subsp mycoides on endothelium, monocytes, and complement of guinea pig, calf, sheep, and goat serum

The caprine variant of Mycoplasma mycoides subsp mycoides causes septicemia with coagulopathy in goats. Pathogenetic mechanisms that might explain the coagulopathy, the ability of the Mycoplasma to persist in the blood, and its specificity for goats were studied. Severe endothelial damage was seen by electron microscopy of goat aorta tissue exposed in vitro to 10(7) colony-forming units of mycoplasmas. The Mycoplasma did not damage 51Cr-labeled adherent cells from peripheral blood of goats. The hemolytic complement titer was reduced by 94%, 50%, 50%, and 25% in guinea pig, calf, sheep, and goat serum, respectively, 30 minutes after treatment with 8 X 10(9) colony-forming units of the Mycoplasma. Freshly prepared serum from these animal species killed the Mycoplasma. Heat-inactivated serum was not mycoplasmacidal. Complement from these 4 animal species was activated by the Mycoplasma through the classical pathway, because ethyleneglycoltetraacetic acid precipitation of serum Ca2+ inhibited activation. Proof that the classical pathway was functional in goats was not conclusive because Ca2+ supplementation of ethyleneglycoltetraacetic acid-treated serum did not restore complement activity. Endothelial damage and complement activation may explain the coagulopathy. The function that complement activation may have in the inflammatory response of this disease is not known. Difference in susceptibility of calves, sheep, and goats to M mycoides septicemia cannot be explained by species variation in complement mycoplasmacidal activity.     

In vitro antimicrobial susceptibility of Mycoplasma mycoides mycoides large colony and Arcanobacterium pyogenes isolated from clinical cases of ulcerative balanitis and vulvitis in Dorper sheep in South Africa

The in vitro activities of enrofloxacin, florfenicol, oxytetracycline and spiramycin were determined against field isolates of Mycoplasma mycoides mycoides large colony (MmmLC) by means of the broth microdilution technique. The minimum inhibitory concentrations (MICs) of these antimicrobial drugs were determined for a representative number of 10 isolates and 1 type strain. The susceptibility of Arcanobacterium pyogenes to enrofloxacin, oxytetracycline and tilmicosin was determined by means of an agar disk diffusion test. The MICs of enrofloxacin, florfenicol, oxytetracycline and spiramycin were within the ranges of 0.125-0.5, 1.0-2.0, 2.0-4.0 and 4.0-8.0 microg/ml, respectively. This study has shown that resistance of MmmLC against enrofloxacin, florfenicol, oxytetracycline and spiramycin was negligible. All the field strains of A. pyogenes that were tested were susceptible to enrofloxacin, oxytetracycline and tilmicosin with mean inhibition zones of 30.6, 42.3 and 35.8 mm, respectively. Although there is lack of data on in vivo efficacy and in vitro MIC or inhibition zone diameter breakpoints of these antimicrobial drugs for MmmLC, the MIC results indicate that these 4 classes of antimicrobial drugs should be effective in the treatment of ulcerative balanitis and vulvitis in sheep in South Africa.     

Mycoplasma mycoides subsp. capri and Mycoplasma mycoides subsp. mycoides LC can be grouped into a single subspecies

Mycoplasma mycoides subsp. capri and Mycoplasma mycoides subsp. mycoides LC can be combined into one taxon on the basis of several contributions on both DNA sequence and protein analyses reported in the literature. Moreover, for the differentiation and identification of mycoplasmas of the "mycoides cluster", we investigated the rpoB gene, encoding the beta-subunit of the RNA polymerase. A segment of 527 bp of the rpoB gene was amplified from 31 strains of ruminant mycoplasmas by PCR. The nucleotide sequences were determined and aligned, and accurate genetic relationships were calculated. Cluster analysis of rpoB DNA allowed species differentiation within the "mycoides cluster" and confirmed that M. mycoides subsp. capri and M. mycoides subsp. mycoides LC cannot be distinguished from each other. "Mycoplasma mycoides subsp. capri" is proposed as a common name for both subspecies.