温氏附红细胞体PCR检测方法的建立

本研究旨在建立1种快速准确检测温氏附红细胞体感染的分子生物学诊断方法。根据温氏附红细胞体的16S rRNA基因参考序列的保守区,利用软件Primer Premier 5.0设计合成了1对特异性引物,对温氏附红细胞体的基因组DNA进行PCR检测。结果表明,扩增出1段985bp的DNA序列,EcoRⅠ酶切鉴定得到2条500bp左右的条带,与预期结果一致,说明该方法扩增出了温氏附红细胞体的特异性条带。通过敏感性、特异性、重复性和临床样品检测试验证明,该方法具有特异、灵敏、快速的优点。结果提示,所建立的方法具有较高的特异性和灵敏性,可用于牛附红细胞体病诊断和流行情况监测。     

奶牛附红细胞体感染PCR诊断方法的建立

为建立特异、敏感、快速的奶牛附红细胞体感染诊断方法，该研究根据本实验室已测得的奶牛附红细胞体16S rRNA基因序列，设计1对种特异性引物，建立了奶牛附红细胞体的PCR诊断方法。特异性试验和敏感性试验结果表明，该诊断方法与猪肺炎支原体、鸡毒支原体、大肠杆菌、肠道沙门氏菌、葡萄球菌、鸡艾美耳球虫、牛双芽巴贝斯虫无交叉反应，能检测的奶牛附红细胞体最低DNA量为0．154fg，同时能检测出在4℃存放长达3个月的血样。通过临床血样检测，证明该方法可用于本病的早期诊断。     

羊附红细胞体解离方法的比较研究

本试验为建立一种高效的羊附红细胞体解离方法,采集红细胞感染率大于90%的阳性抗凝血,分别应用水浴法和药物体外驱虫法对羊附红细胞体进行解离,观察分离前后附红细胞体感染率、红细胞感染强度、附红细胞体数、杂质含量和附红细胞体的运动性5项指标;提取附红细胞体抗原,制备全蛋白悬液,测定蛋白质含量,比较解离效果。将两种方法制备的羊附红细胞体抗原全蛋白进行SDS-PAGE试验,观察条带是否一致。结果显示,200 mL血液中加入1 mL双向红莲灭,4℃作用36 h,即可达到较好分离效果。与水浴法相比,解离后,红细胞感染率和感染强度明显下降,蛋白含量增加,收率高,纯度好。SDS-PAGE结果显示,体外驱虫法所制的羊附红细胞体抗原全蛋白悬液,其抗原蛋白带与水浴法相同,因此可用于粗制羊附红细胞体抗原。     

小尾寒羊附红细胞体的诊治

今年自入夏以来,华北和中原许多地区流行附红细胞体病,本病来势之凶猛,病状之严重,损失之重大,在养猪业、养羊业上触目惊心。关于猪附红细胞体的报道比较多见,但是小尾寒羊感染附红细胞体还未见报道。现就定州市某小尾寒羊饲养场发生附红细胞体的诊治情况报告如下。1发病情况 该羊场2002年8月,有部分羊出现结膜红肿、流泪、分泌异物,随后发病羊增加到60余只。有个别羊只出现精神萎顿,不     

羊附红细胞体解离方法的比较研究

羊附红细胞体病对羊群有一定的危害性,本文将展开对羊附红细胞体解离方法的研究。为了能够得到准确的实验结果,首先要获取红细胞感染率较高的阳性抗凝血作为实验样品,使用多种方法对红细胞体进行解离,然后比较这几种方法对羊附红细胞体的解离效果。主要采用SDS-PAGE方法检测。结果显示,在针对羊附红细胞体解离方法当中,在200m L的抗凝血液当中加入1m L的双向红莲灭,保持在4℃左右的情况下,36h后会有比较好的分离效果。传统的水溶法,其解离的效果会比较差,但可明显降低羊附红细胞的感染率及感染的强度,效率也比较高。希望能够通过本文的研究,为业界提供更多的参考。     

羊附红细胞体双抗体夹心ELISA诊断方法的建立

为快速准确诊断和检测羊附红细胞体病,及时采取防治措施,建立了检测羊附红细胞体抗原的双抗夹心ELISA诊断方法。选取规模化养殖场羊,镜检附红细胞体红细胞感染率> 90%,无菌采取血液,分离羊附红细胞体抗原,制备纯化兔抗羊附红细胞体抗体,应用辣根过氧化物酶标记抗体,进行双抗体夹心ELISA试验。试验结果表明,双抗体夹心ELISA方法的最佳工作条件为:抗体最佳包被量为82.91 μg/mL,酶标抗体最适工作浓度为1∶400,抗原最低检出量为7.81 μg/mL;而且与支原体、大肠杆菌、葡萄球菌以及牛、猪、兔附红细胞体均不出现交叉反应,表明该方法具有良好的特异性,可用于羊附红细胞体病的诊断和群体检测。     

猪附红细胞体50S核糖体基因PCR检测方法的建立及应用

根据GenBank上最新发布的猪附红细胞体基因组序列(NC-015155)设计一对引物,并以吉林省延边地区猪附红细胞体基因组DNA为模板,建立猪附红细胞体50 S核糖体基因PCR诊断方法,通过特异性、敏感性及临床应用试验验证,快速准确的检测出猪附红细胞体.试验结果显示,建立的猪附红细胞体PCR诊断方法扩增片段大小为10...     

羊附红细胞体巢式PCR检测方法的建立及临床应用

根据GenBank上发表的羊附红细胞体16SrRNA基因序列（登录号AF338268）设计2对引物,用巢式PCR方法扩增16SrRNA的部分序列,将目的片段克隆并测序。测序结果与AF338268相似性达99%以上,只有3bp的差异。该方法与猪附红细胞体、羊肺炎支原体、链球菌、葡萄球菌和大肠杆菌均无交叉反应,能检测到的最低DNA量为25fg。用于检测的28份临床样本中,23份为阳性。所建立的巢式PCR检测方法具有较高的敏感性和特异性,可用于羊附红细胞体病急性感染和隐性感染的早期诊断,为该病的临床检测、流行病学调查、进出口检疫和实验室研究提供了新的技术手段。     

羊泰勒虫可视化LAMP检测方法的建立

为了建立1种快速简便的羊泰勒虫检测方法，试验根据羊泰勒虫MPSP基因设计2组引物，建立羊泰勒虫环介导等温扩增(LAMP)检测方法，利用该方法分别检测羊泰勒虫、瑟氏泰勒虫、卵形巴贝斯虫、附红细胞体的DNA。结果：LAMP方法检测羊泰勒虫的结果为阳性，其他虫体均为阴性，具有较好的特异性；对羊泰勒虫DNA最低检测量为1.9×10^-9 ng/μL,是PCR方法的100倍。分别利用LAMP、PCR检测30份疑似羊泰勒虫感染病例，LAMP方法阳性检出率为20%(6/30),PCR方法阳性检出率为16.67%(5/30),LAMP方法准确率高于PCR方法。结论：建立的羊泰勒虫可视化LAMP检测方法特异性好、准确率高，可作为羊泰勒虫病诊断的检测方法。     

重庆地区牛附红细胞体16S rRNA基因部分序列的测定和分析

目的对牛附红细胞体的16SrRNA基因序列进行测定和系统进化分析。方法无菌采取感染附红细胞体的黄牛、奶牛、水牛血液,提取附红细胞体的基因组DNA,根据GenBank公布的奶牛附红细胞体16SrRNA序列设计的特异性引物进行PCR扩增,并对PCR产物进行序列测定和系统进化分析。结果PCR扩增出的DNA片段均为415bp左右。序列测定和系统进化分析显示,三者间的相似度分别为（黄牛：水牛=99．52％;黄牛：奶牛：99．28％;奶牛：水牛=99．76％）。与GenBank上公布的Mycoplasma wenyonii（武汉株）序列比较存在有4个突变位点,相似度均〉98％。结论表明本次在重庆地区从牛体分离的附红细胞体为温氏附红细胞体。     

绵羊附红细胞体人工感染小鼠模型的建立

分别用绵羊附红细胞体自然感染病羊的全血及分离纯化的绵羊附红细胞体对小白鼠进行攻毒,以建立绵羊附红细胞体人工感染小鼠模型。通过攻毒后症状观察、血液涂片镜检和绵羊附红细胞体特异性PCR检测法,对建立的模型进行评价。结果显示,各试验组小鼠人工感染后3~6 d,血液中均可检测到绵羊附红细胞体,而对照组小鼠未出现异常症状,且血液附红细胞体检查结果为阴性。该研究成功地构建了绵羊附红细胞体小白鼠感染模型,创建的模型可用于附红细胞体的生物学特性、致病机制、药物筛选等方面的研究。     

The prevalence of Eperythrozoon ovis infection in weaner and adult sheep in north eastern Victoria

A survey of weaner (6 to 12 months) and adult sheep for the presence of Eperythrozoon ovis antibodies using an immunofluorescent antibody assay was carried out. In 22 shires in north eastern Victoria over 2 years infection was demonstrated in 10% and 51% of weaner and adult sheep respectively.     

Humoral immune response of sheep to infection with Eperythrozoon ovis

Circulating antibody was detected by an indirect fluorescent antibody test (IFAT) in the serum of sheep infected experimentally with Eperythrozoon ovis. Antibodies were first detected 15 to 32 days after infection with E ovis and titres peaked at 41 days. This antibody may be associated, at least in part, with protection against infection with E ovis since the initial increase in antibody titre coincided with a fall in the primary parasitaemia. A role for antibody is suggested further by the fact that the prepatent period of infection was prolonged by one day and the parasitaemia initially remained at low levels in infected sheep protected by passively transferred hyperimmune serum. Moreover, following primary infection, acquired immunity was manifest by a lack of parasitaemia following challenge infections while increased IFA titres were observed. No evidence of opsonic activity was observed in an in vitro erythrophagocytosis test in that neither mouse macrophages nor sheep monocytes phagocytosed E ovis infected or uninfected erythrocytes sensitised with hyperimmune serum.     

A modified indirect immunofluorescent assay for the detection of antibody to Eperythrozoon ovis in sheep

Experimental ovine eperythrozoonosis was studied using Giemsa staining of blood films and a modified indirect immunofluorescent antibody assay (IFAA). The serums of 21 Border Leicester Merino cross lambs between 12 weeks and 7 months-of-age were analysed before and after infection with Eperythrozoon ovis (E. ovis) using the IFAA test. No rise in the IFAA titre was seen until day 7 and this coincided with the first detection of E. ovis organisms in blood smears stained with Giemsa. The percentage of E. ovis infected red blood cells peaked on day 14, but the IFAA titre did not peak until day 35. Titres to E. ovis, on average, had begun to drop by day 63. There was considerable individual variation in response to E. ovis infection as measured by the IFAA. Titres as high as 6,400 were observed in individual sheep at the peak of E. ovis parasitaemia of red cells. One sheep had a titre of 51,200 nineteen days after infection, and titres of 3,000 were maintained for several months in a few sheep. The assay proved reliable, and up to 100 samples per day could be tested. The antigenicity of the slide preparations was found to be satisfactory after storage for 6 months at -20 degrees C and 4 degrees C and for 28 months at -70 degrees C. Temperature fluctuations during storage rendered slides unsuitable for the IFAA after these times. A method of storing E. ovis infected blood in liquid nitrogen is described.     

THE EFFECT OF EPERYTHROZOON OVIS INFECTION ON THE GLUCOSE LEVEL AND SOME ACID-BASE FACTORS IN THE VENOUS BLOOD OF SHEEP

Eperythrozoon ovis infected sheep have low venous blood glucose levels and correspondingly increased blood lactic acid levels as compared with control sheep. Acid-base studies showed that these changes were accompanied by significant falls in venous pH, and standard bicarbonate as well as a negative base excess. All these changes were considered to result from the increased alvcolytic activity of infected erythrocytes. The acidosis and hypoglycaemia associated with E. ovis infection, while not having any apparent effect on young, well-fed sheep, could be potentially serious in pregnant ewes and in sheep on a low plane of nutrition.     

Role of the spleen and rosette-formation response in experimental Eperythrozoon ovis infection

The role of the spleen and rosette-formation responses was investigated in sheep experimentally infected with Eperythrozoon ovis. Phagocytic activity was observed in the spleen 19 days after primary infection. Phagocytosis of E. ovis-parasitised and non-parasitised erythrocytes by cordal reticular cells occurred. E. ovis organisms seemed to be detached from the erythrocytes by pseudopodia extending from macrophages and cordal reticular cells without causing damage to the plasmalemma of the erythrocyte. No phagocytic activity was observed in spleens removed 74 and 146 days after infection. Antigen-specific lymphoid cell responsiveness, assessed by rosette formation, indicated that 2.8, 15.4, 8.0 and 6.0% of lymphoid cells in the spleens of the four E. ovis-infected sheep, respectively, formed antigen-specific rosettes. Rosette formation did not occur when splenic lymphocytes from E. ovis-infected sheep were mixed with non-infected erythrocytes or when splenic lymphocytes from an uninfected sheep were used.     

Changing morphology of Eperythrozoon ovis

Light microscopy studies of Eperythrozoon ovis in sheep revealed that Giemsa stain was only less reliable than acridine orange as a means of parasite identification when low parasitaemias were present. The morphology of E ovis altered as the degree of parasitaemia increased.     

A cross-sectional study to show Eperythrozoon ovis infection is prevalent in Western Australian sheep farms

A serological survey and risk factor study was conducted to estimate the prevalence of Eperythrozoon ovis infection in Western Australian weaner sheep, the prevalence of farms with infected sheep, and to identify factors affecting initiation and maintenance of infection on the farm. The study was conducted on 91 farms, purposively chosen from 41 randomly selected regional shires stratified by sheep number and rainfall zones. Twenty sheep were selected systematically from a mixed-sex flock on each farm and tested for serum antibody to E ovis using an enzyme-linked immunosorbent assay. Information on putative risk factors was collected using an interview questionnaire. Antibody to E ovis was detected in 4.5% of sheep on 47% of the farms sampled. The prevalence of E ovis infection in sheep was estimated at the 95% confidence level to be between 3.6 and 5.5%, and the prevalence of farms with infected sheep was estimated to be between 37.5 and 56.5%. Most farms with serological evidence of infection occurred in the Great Southern agricultural region (79.5%), south-east of Perth through to Albany (latitude 32 to 34 degrees S, longitude 116 to 120 degrees E), and in the Northern region (12.8%) surrounding Geraldton (latitude 29 degrees S, longitude 114 degrees E). There were significantly more farms (P less than 0.05) with evidence of infection in the Great Southern region compared to the Central region between Geraldton and Perth, and on farms in the region south compared to north of latitude 32 degrees S. None of the putative risk factors examined in the questionnaire were associated with serological evidence of infection on the farm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Eperythrozoon ovis on the lysis of sheep erythrocytes in the complement fixation test

When erythrocytes from sheep experimentally infected with Eperythrozoon ovis were used in the titration of reagents for a standardised complement fixation test, increased amounts of both haemolysin and complement were required for erythrocyte lysis compared with preinfection titrations. The haemolysin requirement increased by up to 125% at 55 days post-infection and complement requirement increased by up to 40% at 40 days post-infection. These changes appeared to correlate with the development of a macrocytic anaemia in affected sheep rather than E. ovis parasitaemia. The results emphasise the need to carefully monitor the haematological parameters of sheep used as sources of erythrocytes for the complement fixation test.