斑点免疫结合试验在霉形体研究中的应用

本文报道了将斑点免疫结合试验(DIBA)用于霉形体种的鉴定,细胞培养中霉形体感染的检测和鸡胚实验感染霉形体的检测,都取得了满意的结果。应用DIBA对11种霉形体培养物和36个自感染细胞和污染疫苗中分离的霉形体的培养物进行了鉴定,结果与直接分离培养物用生长抑制试验,间接免疫荧光检测的结果完全一致。应用口腔霉形体、猪鼻霉形体、精氨酸霉形体和莱氏无胆甾体等4种霉形体的抗血清以DIBA对36株细胞培养物进行检测的结果,证明其中15株感染口腔霉形体,1株感染莱氏无胆甾体,1株为口腔霉形体和莱氏无胆甾体的混合感染,与对照用的分离培养方法结果一致,还有1株用分离培养方法和DNA荧光染色证明有霉形体感染,经鉴定此株霉形体不属于此4种霉形体,故用DIBA未检出。以鸡毒霉形体和滑液霉形体实验感染鸡胚各18枚,以DIBA从这些鸡胚的卵黄液及尿囊液中可检出各自感染的霉形体种。结果说明,在霉形体研究中DIBA为一种快速、简便和特异的血清学方法。     

从山羊分离出绵羊肺炎霉形体的报告

本文报道从辽宁、河北、甘肃等地区送检的13份疑似山羊传染性胸膜肺炎病料(肺、淋巴结、胸水)中,分离到13株霉形体,经鉴定12株为绵羊肺炎霉形体(M.ovipneumoniae)、1株为精氨酸霉形体(M.arginini)。从12株绵羊肺炎霉形体中选用辽宁源LG株、河北源HG株和甘肃源GG株进行了动物回归试验,结果人工感染的6只健康山羊肺部产生的病变与自然发病送检的病羊肺的病理变化一致,从病肺中均可重新分离到绵羊肺炎霉形体。证明上述地区山羊群或山羊和绵羊混合群中流行的肺炎是由绵羊肺炎霉形体所致。     

鸽源霉形体的分离与鉴定

对照株及标准抗血清 MMP1、MMP4、鸽霉形体694株及3种相应的抗血清。培养基 修改的F14-6培养基(含葡萄糖)、精氨酸培养基。试验鸽 武汉市郊某养鸽专业户及市场上购买的食用鸽共70只。     

用药物纸片进行霉形体药敏试验

以常用的14种国产药物的纸片,对上海奶牛场分离到的5株牛霉形体、5株牛鼻霉形体和从组织培养及小牛血清中分离到的3株精氨酸霉形体,以及定型菌种牛鼻霉形体、猪肺炎霉形体和猪鼻霉形体进行了药敏试验,试验结果表示出影响菌体蛋白合成的呋喃类、氯霉素类、四环素类、氨基苷类药物对霉形体有抑菌作用,一般在同一霉形体种中,有相似的药物敏感范围,但这些药物对不同种霉形体的抑制作用有所差别.     

从患肺炎的绵羊肺脏所分离霉形体的特性

绵羊肺炎在苏丹是一种重要的羊疾病,但这方面的工作做的很少。从患肺炎的绵羊肺脏中分离到霉形体在苏丹已有报道(Khan, 1960; Elmahi等人,1978;Harbi等人,1981),其中E Imahi等人(1978)共分离到11株霉形体,有3株被鉴定为精氨酸霉形体。本文将报告这些霉形体的特性。材料与方法 1978从患肺炎的绵羊肺脏分离的3株霉形体冻干物被送到设在丹麦阿尔胡斯大学的联合国粮农组织,动物霉形体合作中     

精氨酸霉形体的分离与鉴定

引言细胞培养已被广泛地应用于各种生物学研究,但一旦为霉形体污染就会造成细胞染色体的畸变,并导致细胞功能和特性的改变,并因此而影响研究结果,特别是传代细胞,被霉形体污染后就无法保持其原有的细胞特性,甚至无法继续传代。我们曾在生长于含双抗生长液中的IB-RS-2细胞培养物中和市售商品犊牛血清中分离到精氨酸霉形体,其鉴定的结果报道如下。     

污染的霉形体在传代细胞内增殖的观察

对霉形体进入DK传代细胞内,并进行增殖的情况,作了详细的电镜观察.结果如下:(1)霉形体靠近传代细胞膜表面呈串珠样排列;霉形体与传代细胞膜表面接触部位融合增厚,电子密度增高,向内凹陷,将霉形体裹入细胞质,形成由膜结构包着的包涵体.(2)在传代细胞质中,除可见由膜结构包裹着的霉形体性包涵体外,还可见没有任何膜包着的散在的霉形体,并且均可见到霉形体的裂殖增殖相和出芽增殖相.(3)在传代细胞核的核周池和核质中,均见到典型的霉形体及其裂殖增殖相.(4)霉形体在传代细胞中大量的增殖,致使细胞崩解,霉形体即被释放出来.(5)霉形体使传代细胞出现严重的超微病变.本文还讨论了传代细胞培养污染霉形体后难以救治的根本原因是,霉形体在传代细胞内增殖,故仅仅着眼于消除培养液中的霉形体是不能达到预期结果的.     

猪肺炎霉形体冻干保存期的测定

猪肺炎霉形体的保存,是进行猪肺炎霉形体研究必不可少的基础工作。国内外曾对真空冻干保存的猪肺炎霉形体作过测定,一般保存期为4～6年。我们冻干的猪肺炎霉形体保存于-20℃9年零260天仍存活力,现报导如下。一、供试菌株:共6个菌株(见附表),于-20℃低温冰箱保存。二、冻干方法:将经KM_2液体培养基复壮生木茂盛的猪肺炎霉形体培养物与等量灭菌脱脂奶混合。加1000u/ml青霉素,混匀后进行冻干。     

上海地区分离的牛霉形体及牛鼻霉形体的生理生化鉴定

本文报道从上海奶牛场分离到的5株牛霉形体和14株牛鼻霉形体,以定型菌种作为对照,进行了洋地黄敏感试验、葡萄糖发酵试验、精氨酸降解试验、四唑还原试验、薄膜斑点形成试验、产生过氧化氢活性及使绵羊红血球溶血等七项生理、生化特性鉴定。结果证明我们分离的这两种霉形体的生理生化特性与定型菌种基本一致。     

精氨酸霉形体和莱氏无（需）胆甾醇霉形体在鸡胚细胞内增殖的比较…

将精氨酸霉形体和莱氏无（需）胆甾醇霉形体，分别接种于鸡胚细胞，电镜下观察结果如下：（１）此２种霉形体在形态、细胞内分布、增殖形式和吞噬功能以及对培养细胞的选择性等方面是相同的；（２）此２种霉形体引起培养细胞的病理学过程不同。接种ＭＡ的鸡胚细胞，早期即出现髓模样结构和脂质体等退行性变化，晚期在细胞质内才能见到空泡出现；而接种ＡＬ的鸡胚细胞，早期即可出现细胞质内的空泡化现象，晚期细菌质内才可见到髓膜样     

鸡滑液囊支原体的分离鉴定及病理组织学观察

为了解宁夏及其周边不同地区蛋鸡场中的鸡滑液囊支原体的种类及其致病力,采用改良Frey氏鸡滑液囊支原体培养基,从疑似感染鸡滑液囊支原体的不同蛋鸡群中分离出病原株,设计鸡滑液囊支原体vlhA基因特异性引物,对分离到的病原进行基因序列扩增并测序,使用MEGA6.0软件中的邻接法(Neighbor-joining,NJ)构建分离株系统发育树并进行遗传进化分析,采用从临床症状最明显的鸡体分离的菌株进行动物感染试验,制作石蜡切片,HE染色,病理组织学观察。结果表明,所分离到的病原菌均为鸡滑液囊支原体,部分菌株之间极其相似;鸡滑液囊支原体不仅可以使鸡只关节肿胀、生长缓慢,也可引起鸡只的肝脏轻微肿大,肝细胞部分坏死、间质增生;脾脏结缔组织增生,出血。     

山羊支原体山羊肺炎亚种M1601生长曲线与pH值的测定

为了研究山羊支原体山羊肺炎亚种分离株M1601株在Thiaucourt肉汤培养基中的生长及繁殖特性。利用活菌计数方法对其在不同培养阶段的生长滴度及pH值进行了测定,并绘制了生长曲线。结果表明,传代稳定的M1601在Thiaucourt肉汤培养基中培养6～60h为对数生长期．pH值逐渐下降：培养60～72h进入稳定期．pH值降为6．27．培养72h进入衰亡期。这为山羊支原体山羊肺炎亚种培养特性研究和疫苗生产工艺研究提供了参考数据,以便在生产上更合理地应用其生长规律。     

Comparison of a modified Edward-type medium and a modified SP4-type medium for primary isolation of Mycoplasma gallisepticum (MG) from chickens vaccinated with the F strain of MG

The efficacy of two media, an Edward-type medium (EPJ) and a modified SP4-type medium (SP4-PS), were compared for primary isolation of Mycoplasma gallisepticum (MG) from commercial layer chickens (n = 58) vaccinated with the live F strain of MG. Three groups of chickens that differed in the interval after vaccinal exposure to the F strain (32, 41, and 102 weeks) were studied at necropsy. Mycoplasma isolation was attempted from the trachea, sinus, and cloaca using lavage and swab techniques but was successful only from the trachea and sinus. MG was isolated from 39 (8.4%) of 463 culture attempts from 58 tracheal inocula and 58 sinus inocula. Isolation of MG was successful more frequently using EPJ medium than SP4-PS medium, and isolation occurred more often from the sinus than from the trachea. Of the 58 chickens studied, 19 (33%) were shown by culture to be infected with MG. Isolation was successful only from 32- and 41-week post-vaccination exposure groups. However, all chickens studied were serologically positive for MG antibody by rapid-plate agglutination and hemagglutination-inhibition assays.     

内蒙古地区绵羊肺炎支原体的分离鉴定及序列分析

本试验无菌采取内蒙古地区某发病羊场的绵羊病变肺脏组织,接种于支原体液体培养基进行分离培养后获得1株支原体,根据分离株的培养特性、形态学观察及生化试验等,初步鉴定为绵羊肺炎支原体。然后提取分离株的基因组,用通用引物体外扩增出分离株16S rRNA序列,将该序列与GenBank中已知33种支原体序列进行比较,结果表明该序列与绵羊肺炎支原体标准株Y-98的16S rRNA序列的同源性为99%,鉴定该分离株为绵羊肺炎支原体。     

一株猪肺炎支原体的分离鉴定

从全国部分猪场采集到疑似猪支原体肺炎肺组织病料12份,提取DNA进行猪肺炎支原体PCR和多重PCR检测,将病料研磨后分离猪肺炎支原体,最终分离到1株疑似猪肺炎支原体;通过测序分析、形态观察、生化试验、血清学试验证实其为猪肺炎支原体。该菌株能适应人工培养基的培养,且传代生长良好,液体培养基中培养活菌滴度达109CCU/m L;菌株有一定的致病性,免疫原性好,可作为疫苗备用菌株,该菌株的分离鉴定为研制猪支原体肺炎疫苗奠定了基础。     

致犊牛肺炎牛支原体南疆株的分离鉴定及oppF基因序列分析

本研究旨在调查新疆喀什某规模化奶牛场的犊牛死亡原因,并确定病原体.无菌采集3份因肺炎死亡的犊牛肺脏病料样品.采用牛支原体专用液体培养基和1.0%牛支原体琼脂固体筛选培养基从3份病死犊牛肺脏病料中分离得到2株牛支原体(Mycoplasma bovis,M.bovis),分别命名为M.bovis-NJ-1和M.bovis-NJ-2.通过菌落形态学观察、特异性PCR和oppF测序比对对分离株进行鉴定.结果显示,2个分离株在固体培养基上的菌落呈现典型的"煎蛋状",且Dienes染色特点符合牛支原体菌落着色特征,中心呈深蓝色;PCR能扩增出牛支原体特异的448 bp目的片段;2个分离株的oppF基因序列与牛支原体国际标准株PG45的同源性分别为96.7%和95.3%.结果表明,引起犊牛发病死亡的病原是牛支原体,本研究为犊牛支原体肺炎的快速诊断和防制提供依据.     

Microflora of pneumonic lungs in a pig herd established by hysterectomy.

Mycoplasmas and other micro-organisms are recorded from a pig herd originally derived by hysterectomy. Complement fixation tests were positive for Mycoplasma hyopneumoniae 20 months after the first hysterectomy. Following a marked increase in pneumonic lesions found at slaughter, and after modifying the culture medium M hyopneumoniae was isolated. The principal micro-organism associated with this mycoplasma was Pasteurella multocida.     

牛支原体新疆株的分离鉴定

为调查新疆规模化奶牛场病牛死亡原因并确定病原,本研究无菌采集7份肺炎病死牛病变肺组织样,通过牛支原体液体培养基和固体培养基分离到1株支原体,采用形态学观察和生化试验鉴定该分离株,采用支原体特异性引物和牛支原体16S rRNA通用引物扩增基因序列并测序,使用DNAStar软件将分离菌株测序结果与GenBank中的标准株序列进行同源性比对,采用Mega 6.0软件中的邻接法(Neighbor-Joining,NJ)依据16S rRNA序列构建分离株系统进化树。结果显示,分离株菌落呈典型的"煎蛋样",菌落中心凹陷深入培养基,周边菲薄而透明,经Dienes染液染色后,菌落中心呈深蓝色。该分离株不分解葡萄糖、尿素、不水解精氨酸,血细胞吸附试验和溶血试验均呈阴性,氯化三苯基四氮唑还原反应呈阳性,产生膜和斑。PCR反应扩增出大小为1 911 bp的牛支原体特异性目的片段;分离株16S rRNA基因序列与牛支原体标准株PG45的序列同源性为99.8%,与牛支原体地方株(Mb NM2012、Mb HB0801、Mb Hubei-1、Mb Ningxia-1、Mb CQ-W70和Mb 08M)的同源性为99.3%～99.7%。系统进化树显示,分离株16S rRNA基因与Mb Ningxia-1株和Mb 08M株亲缘关系较近,处于同一分支。本研究结果证实了引起病牛死亡的病原为牛支原体,为新疆牛支原体病的防治提供了科学依据。     

Mycoplasma mycoides subspecies mycoides as the cause of a subauricular abscess and mastitis in a goat.

A 6-year-old female goat was admitted with right-sided subauricular swelling and facial nerve paralysis. Mastitis developed subsequently. The subauricular swelling localized to a mass and was excised. Mycoplasma mycoides subsp mycoides was cultured from samples obtained from the mass and mammary gland. After clinical improvement, the goat was discharged to the owner with instructions to isolate the goat and to submit milk samples from the rest of the herd for microbial culturing. Mycoplasma spp can spread to other tissues from an initial site of infection. In goats with clinical signs similar to those of the goat of this report, samples should be obtained for microbial culture on Mycoplasma medium. Goats infected with Mycoplasma spp should be isolated or culled because of the risk of transmission to uninfected animals.     

ISOLATION OF MYCOPLASMA HYOPNEUMONIAE AND ITS ASSOCIATION WITH PNEUMONIA OF PIGS IN AUSTRALIA

Nine strains of mycoplasmas were isolated from the lungs of 5 pigs with clinical signs of naturally acquired enzootic pneumonia. Mycoplasma hyopneumoniae was isolated from the lungs of 1 pig and M. hyorhinis from the lungs of 4. An unidentified mycoplasma, which utilized arginine, grew rapidly in broth culture and produced centred colonies on solid medium, was isolated from the lungs of 4 pigs. The pathogenicity of the isolated strain of M. hyopneumoniae was determined by inoculation of pigs from an enzootic pneumonia-free herd. Enzootic pneumonia was produced in the lungs of all 5 pigs inoculated intranasally and intratracheally with broth cultures of the organism isolatied by limit dilution techniques. Enzootic pneumonia was produced in 3 of 6 pigs inoculated intranasally and intratracheally with M. hyopneumoniae purified by the passage of colonies on agar blocks. M. hyopneumoniae was isolated in pure culture from the lungs of all pigs with induced pneumonic lesions.