Detection of Eperythrozoon suis DNA from swine blood by whole organism DNA hybridizations

A procedure is described for isolating Eperythrozoon suis DNA of sufficient quantity and purity to serve as a probe in whole-organism DNA hybridizations for detecting parasitized swine. The E. suis organisms were isolated from the blood of infected swine; the DNA was recovered and digested with restriction endonucleases and resolved on agarose gels. In DNA hybridizations using recovered E. suis DNA, blood samples from parasitized swine could be differentiated from uninfected, control samples. A high salt lysate recovery technique was used in sampling swine whole blood for E. suis DNA and found to offer many advantages in the collection and recovery process.     

附红细胞体自然感染猪血液生化指标的检测

附红细胞体 ( Eperythrozoon)是寄生于人畜血液、红细胞及骨髓中的一群微生物。附红细胞体病( Eperythrozoonosis)简称附红体病 ,本病危害的严重性主要表现在感染畜种基本齐全 ,病原分布逐年扩大 ,临床病例逐步增多 ,从 1 92 8年至今的 70多年中 ,在世界范围内已有 30多个国家发生此病。由于该病在多数情况下呈潜伏状态 ,临床发病的不显现性 ,并没有引起人们的足够重视。近年来 ,随着附红细胞体病临床病例的增多 ,家畜附红细胞体感染的暴发流行 ,影响畜牧业发展 ,对人类健康造成威胁 ,才逐渐被国内外学者关注。本试验在前人研究的基础上 ,…     

应用间接血凝试验诊断猪附红细胞体病

附红细胞体病 (Eperythrozoonosis)是由附红细胞体 (Eperythrozoon)寄生于红细胞表面、血浆及骨髓内而引起的一种以红细胞压积降低、血红蛋白下降、白细胞增多、贫血、黄疸、发热为主要临床特征的人兽共患病 [1,2 ]。近年来随着人附红细胞体病例的增多 ,家畜附红细胞体病的暴发流行 ,已引起国内外学者的广泛关注 [3 ,4]。本试验探索了将用 IHA于猪附红细胞体病的诊断 ,以便为该病的临床诊断提供一种较为敏感、特异、实用和快速的血清学诊断方法。1　材料与方法1.1　血清样品　猪附红细胞体病阳性血清和阴性血清均由本教研室提供 ,被检血清…     

附红细胞体自然感染猪血液流变学

选择临床健康的2月龄附红细胞体自然感染仔猪15头,每周采血1次,连续4次,检测血样的RBC、PCV、ηb10s-1、ηb150 s-1、ηp、ηre10 s、ηre150 s-1、AI、RI、TK.检测结果如下随着猪附红细胞体感染强度的增强,ηb10 s-1、ηre10s-1、ηre150 s-1、AI、RI、TK明显增高,而RBC、PCV、ηb150 s-1、ηp无明显变化,表明猪感染附红细胞体后,血液粘度增高,红细胞聚集性增高,红细胞变形能力降低,进而影响脏器组织的血液灌注和微循环功能.这些血液流变学改变,构成了血栓、炎症、变性、水肿、坏死等的病理学基础.     

附红细胞体自然感染对猪红细胞的影响

选择临床健康的 2月龄附红细胞体自然感染仔猪 1 5头 ,每周采血一次 ,连续 4次 ,观察红细胞的形态 ,并测定RBC、红细胞脆性 (最小脆性和最大脆性 )、红细胞ATPase和SOD活性、红细胞C3b受体 (E_C3bRR)率和红细胞免疫复合物花环 (E_ICR)率。检测结果 :随着猪附红细胞体感染强度的增强 ,所测定指标的变化无明显规律性或无明显差异 (P >0 .0 5 ) ,但红细胞脆性增高、红细胞ATPase和SOD活性及E_C3bRR率和E_ICR率均有降低趋势。表明猪感染附红细胞体后 ,红细胞易破裂而溶血、红细胞内外阳离子平衡失调、红细胞抗氧化能力降低、红细胞免疫功能下降。     

Indirect hemagglutination for Eperythrozoon suis detection in experimentally and spontaneously infected swine

Ten splenectomized and non-splenectomized pigs were experimentally infected with E. suis bearing red blood cells in order to determine the antibody response. All animals were monitored for antibody titer by indirect hemagglutination over a period of 80-290 days postinfection. Latent E. suis infection only yielded a detectable antibody titer in one pig. Acutely infected pigs had a titer ranging up to 1:640. Maximum antibody response lasted only 2 months and dropped below the level of detection of our assay within 2 to 3 months. At this time, the clinical symptoms could reappear and antibodies were again detectable. However, no booster effect was observed with this second outbreak. We also determined the antibody frequency in 138 pigs from 16 herds in Southern Germany. Pigs from only 4 out of 6 clinically positive herds had antibody titer against E. suis. 20 out of 78 pigs of the clinical positive herds demonstrated a detectable E. suis antibody titer. In 10 herds that were asymptomatic and presumed uninfected all 80 pigs were serologically negative for E. suis.     

应用间接ELISA方法检测猪附红细胞体抗体

近年来,国内外对附红细胞体病的研究已取得一定进展,针对此病的血清学诊断先后建立了CFT、IHA、ELISA、IFAT等检测方法,这些血清学诊断方法所用抗原均为附红细胞体的粗提抗原。而本试验对猪附红细胞体抗原进行了提纯,并进行了免疫性分析,用具有最好免疫性的提纯抗原建立了检测猪附红细胞体抗体间接ELISA方法,并与IHA进行了比较,旨在为猪附红细胞体病的流行病学调查及疫病监测提供简便、快速、特异、敏感的检测方法。1材料与方法1.1主要材料96孔酶标板为MaxiSorp公司产品;羊抗猪IgG-HRP为美国KPL公司产品;猪肺炎支原体、猪瘟、…     

猪败血性链球菌与附红细胞体混合感染病例

猪败血性链球菌病是猪链球菌感染引起的一种急性败血性传染病,猪附红细胞体病是由猪附红细胞体寄生于猪红细胞而引起的一种散在的热性、溶血性传染病.本文以某猪场这2种病混合感染病例为题材,从发病基本情况、临床症状、剖检变化、实验室检验、防治措施等6个方面对该混合感染病例做了详尽的阐述.     

An indirect hemagglutination test for the diagnosis of Eperythrozoon suis infection in swine.

An indirect hemagglutination test was developed to detect naturally occurring eperythrozoonosis in swine and to follow experimentally induced infections serologically. The antigen used was prepared from the plasma of acutely infected, splenectomized pigs. The test was specific and reliable in detecting swine latently infected with the disease.     

Evaluation of an enzyme-linked immunosorbent assay for detection of Eperythrozoon suis antibodies in swine.

An ELISA was developed and tested to detect antibodies to Eperythrozoon suis in swine. Results were compared with those of the indirect hemagglutination (IHA) test. Antigen isolated from swine heavily infected with E suis was used for both tests. Comparison of the ELISA with the IHA test revealed a significant (P less than 0.001) correlation between results. Of 114 samples obtained from 9 swine infected with E suis, 87.7% were seropositive (titer greater than or equal to 200) via the ELISA, and 80.7% were seropositive (titer greater than or equal to 20) via the IHA test. The sensitivity of the ELISA was greater than that of the IHA test. All blood samples obtained from specific-pathogen-free swine tested negative for E suis antibody. Cross-reactions were not observed between E suis antigen and antisera against various swine and cattle disease agents using ELISA. We concluded that the ELISA may be used for rapid and effective diagnosis of infection with E suis in swine.     

Porcine eperythrozoonosis: from Eperythrozoon suis to Mycoplasma suis

Mycoplasma suis (formerly known as Eperythrozoon suis ) is the most prevalent agent causing haemolytic anaemia in swine. The disease is also known as porcine eperythrozoonosis. M.suis is a small, pleomorphic bacteria parasitizing porcine erythrocytes. To date, no in vitro cultivation system for M.suis has been established and, therefore, our knowledge about the characteristics of M.suis and the pathogenesis of porcine eperythrozoonosis is rather limited. M.suis can cause acute disease, but the major significance of M.suis infections lies in the fact that M.suis can establish chronic and persistent infections leading to a higher susceptibility to other infections, especially of the respiratory and digestive tracts. The present article summarizes the current knowledge of the pathogen, the clinical signs and pathogenesis, diagnostic as well as therapy and prophylaxis.     

Development of a diagnostic PCR assay based on novel DNA sequences for the detection of Mycoplasma suis (Eperythrozoon suis) in porcine blood

An efficient method of control of porcine eperythrozoonosis (PE) caused by Mycoplasma suis is eradication of infection by detection and removal of infected carrier animals. At present, only a few tests are available for the diagnosis of these latent M. suis infections in pigs. The objective of this study was to develop a PCR assay based on novel DNA sequences for the identification of M. suis-infected pigs. A 1.8 kb EcoRI DNA fragment of the M. suis genome was isolated from the blood of pigs experimentally infected with M. suis. Specificity of the DNA fragment was confirmed by DNA sequence analysis and PCR using primers directed against sequences contained in the 1.8 kb fragment. PCR products of 782 bp in size were amplified only from M. suis particles prepared from the blood of experimentally infected pigs but not from any controls, comprising blood from gnotobiotic piglets and a panel of bacteria including other porcine mycoplasmas. PCR results were confirmed by dot blot hybridisation. The applicability of the PCR assay to diagnose M. suis infections in pigs was evaluated by investigating blood samples from 10 symptomatic pigs with clinical signs typical of porcine eperythrozoonosis and blood samples from 10 healthy pigs. The M. suis-specific PCR product was amplified from all samples taken at episodes of acute disease as well as from samples taken during the latent stage of infection, thus demonstrating the suitability of the PCR assay for detecting latent infected carrier animals.     

猪附红细胞体感染途径研究

为了探讨猪附红细胞体对动物的感染途径,本试验采用口服、伤口、皮下、皮肤以及呼吸道等5种途径对小鼠进行猪附红细胞体的感染试验.结果显示,5种方式均能使小鼠感染猪附红细胞体,其中伤口组感染率最高,在第9天感染率达到96％,口服组次之,在第12天感染率达到89％,呼吸道组也在第12天感染率达到80％,皮肤组和皮下组的感染率较低,分别为19％和20％.从第18天到第30天各试验组先后出现猪附红细胞体消亡的现象.说明口服、伤口、皮下、皮肤以及呼吸道等5种途径均能使小鼠感染猪附红细胞体.     

猪附红细胞体的形态学观察

应用光学显微镜和电子显微镜对猪附红细胞体显微结构和超微结构进行观察。结果显示,附红细胞体属典型的原核生物,多数为大小不等的球形、卵圆形、杆状等多形态生物体,直径为0.2μm~2.5μm,常在红细胞表面或血浆中单个或成团寄生。该病原无细胞壁,由单层界膜包裹着,无明显细胞器和细胞核。附红细胞体通过一些细丝连接到红细胞膜上,使红细胞膜表面出现皱褶、突起、凹陷,导致红细胞结构和功能改变。     

抗猪附红细胞体单克隆抗体的制备

以纯化的猪附红细胞体免疫BALB/c小鼠,运用淋巴细胞杂交瘤技术进行细胞融合,并用间接ELISA方法进行筛选,经过间接ELISA方法、免疫印迹和间接免疫荧光试验进行鉴定,共获得5株分泌抗猪附红细胞体单克隆抗体的杂交瘤细胞,分别命名为1H1、1H2、3A5、5B1和7E11,其单抗亚类鉴定分别属于IgG2b、IgG1、IgG2b、IgG2b和IgG2b。这5株McAb均能与猪附红细胞体全菌蛋白发生特异性反应,而不与猪肺炎支原体、猪链球菌、大肠杆菌和猪繁殖与呼吸综合征病毒发生反应,并且1H1、3A5和5B1能识别同一抗原位点,1H2和7E11识别另一抗原位点。     

抗附红细胞体有效药物的筛选

以PCR检测附红细胞体呈现阳性，且镜检染虫率达909／5以上的猪血样为研究对象，选择几种附红细胞体敏感的药物(血虫净，附红净，庆大霉素，博士914，土霉素，三毒清，红弓链914，附红120，红弓链克，乌金土霉素)和对支原体敏感的恩诺沙星，对立克次氏体敏感的红霉素，采用两种方法进行体外药效试验。附红净对附红细胞体的作用效果最明显，而庆大霉素对附红细胞体基本无效，其他药物对附红细胞体也有一定杀灭效果。     

猪附红细胞体检测方法研究概况

猪附红细胞体病是由猪附红细胞体引起的一种人兽共患传染病.其主要的临床特征是发热、黄疸和贫血.文章综述了猪附红细胞体的检测方法,包括光学显微镜检查、电子显微镜检查、补体结合试验、间接血凝试验、酶联免疫吸附试验、荧光抗体试验、聚合酶链式反应、DNA重组探针技术、DNA杂交技术、原位杂交技术等,为快速、准确地诊断附红细胞体病提供了参考.     

猪附红细胞体实验动物模型的建立

为建立一种合适的猪附红细胞体病动物模型，利用摘除脾和／或注射地塞米松的昆明小白鼠，以腹腔注射方式人工感染猪附红细胞体，通过血液涂片镜检、PCR检测、临床症状观察及病理剖检对感染情况进行了鉴定。结果显示，猪附红细胞体能够经腹腔注射感染昆明小白鼠，且感染鼠表现出与猪附红细胞体病相似的临床症状。结果表明，猪附红细胞体实验动物模型已成功建立，并证实啮齿类动物在附红细胞体的传播过程中发挥着重耍作用。     

猪附红细胞体几种染色方法的比较

为了筛选出比较理想的染色方法,使猪附红细胞体病的诊断和研究得到可靠的技术保证,本试验对附红细胞体病猪的同一份血液样本,分别采用了12种染色方法进行染色,并比较其染色效果。结果显示,丫啶橙染色和姬-瑞氏混合液染色效果最好,其次为亚伯特氏、瑞氏和姬姆萨氏等染色方法。