Culture Experiment in vitro of Eperythrozoon of Mustela lutreola

[Objective] The research aimed to make the culture experiment in vitro of eperythrozoon of Mustela lutreola. [Method] 30% newborn bovine serum or Mustela lutreola was added in RPMI-1640, DMEM, M-199 and HL culture solutions. Epery-throzoon of M. lutreola was cultured in vitro under the conditions of 37 ℃, 5% CO2. The culture solutions were replaced every 24 hours. Appropriate amount of healthy erythrocytes were supplemented to make subculture. [Result] The infection rate and infection intensity of eperythrozoon in RPMI-1640 and HL culture solutions were higher than that in DMEM and M-199 culture solutions. The infection rate and infec-tion intensity of eperythrozoon in RPMI-1640 and HL culture solutions with adding 30% serum of M. lutreola were higher than that with adding 30% newborn bovine serum. And the infection rate and infection intensity of eperythrozoon in RPMI-1640 culture solution were slightly higher than that in HL culture solution. The infection rate was over 80% after culturing 12 hours. 23 generations of subculture in vitro was made. [Conclusion] The research provided an effective approach for screening out the drugs that could effectively control eperythrozoon of M. lutreola, preparing diagnostic antigen and producing the relevant vaccine.

In vitro Drug Sensitivity of Eperythrozoon from Mink

[Objective] This study aimed to test the sensitivity of Eperythrozoon from mink to various drugs in vitro. [Method] The red blood cells isolated from Eperythro- zoon positive mink was cultured in complete medium （70% RPMI-1640 medium and 30% calf serum）, supplemented with bernier, oxytetracycline, trichlorfon, tylosin, imi- docarb, florfenicol, Fuhongjuesha or primaquine phosphate at the working concentra- tions of 12, 24, 36, 48 and 96 μL/ml, incubated at 37.3 ℃, 5% CO2. [Result] Fuhongjuesha was most efficient for killing Eperythrozoon, followed by trichiorfon and primaquine phosphate, but trichlorfon is toxic. Bernier, imidocarb and florfenicol are efficient. [Conclusion] The study provides a scientific reference for clinical treatment of eperythrozoonosis.

Drug Screening Experiment in vitro of Fox Eperythrozoon

[Objective] The research aimed to make the drug screening experiment in vitro of eperythrozoon of fox. [Method] RPMI-1640 was used as the basic culture medium and 30% calf serum was added. Using Berenil, oxytetracycline, al icin, doxy-cycline,imidocarb,florfenicol,Fuhongjuesha,primaquine phosphate and other drug powder,the drug screening experiment in vitro of fox eperythrozoon was made under the conditions of 37.3 ℃, 5% CO2. [Result] The effects of Fuhongjuesha was the best,and that of primaquine phosphate and Berenil was the next. And imidocarb,al-licin and florfenicol were effective. [Conclusion] The research provided scientific and theoretical basis for the clinical treatment of eperythrozoonosis.

Investigation on Eperythrozoon Infection in Sheep in Xinjiang Region of China

[ Objective] To understand the epidemic status of Eperythrozoon ovis in sheep in Xinjiang region of China. [ Method ] A total of 850 blood samples were collected from seven sheep breeding areas in Xinjiang region. The Eperythrozoon ovis infection was detected by blood tablet examination, blood smear stained with Wright＇s stain and polymerase chain reaction （PCR）. [ Result] The infection rates were 97%, 97% and 75% when the samples were detected by blood tablet examination, blood smear stained with Wright＇s stain and PCR, respectively. Sheep eperythrozooo nosis occurred in the four seasons without seasonal epidemics. No obvious difference in infection rates was observed between the sheep breeding areas. The Eperythrozoon ovis infection was slight or mild in most cases, but the infection was severe in two periods, that is, from May to June and from November to December. [ Conclusion] The Eperythrozoon ovis infection is popular in sheep in Xinjiang region of China, and the main inducements may be changes in seasons and forage formula.

Study on Biological Characteristics of Livestock Eperythrozoon

Based on the study of infection and morphological characteristics of 12 kinds of livestock Eperythrozoon,disinfection test by drugs in vitro,disinfection test by ultraviolet light,Eperythrozoon survival test at different times and temperatures,drug sensitivity test and clinical treatment of infected swines were carried out on Eperythrozoon suis and Eperythrozoon wenyonii to further study the biological characteristics. Test results showed that infection level of livestock Eperythrozoon was high,while morbidity was low. Eperythrozoon suis and Eperythrozoon wenyonii could survive for 1 year at 3- 5 ℃,180 d at 16- 26 ℃ and 30 min at- 20 ℃,while which died instantly at 65 ℃. Eperythrozoon was sensitive to general chemical disinfection drugs,while not sensitive to ultraviolet light disinfection. Transmission of Eperythrozoon mainly included contagious transmission and vertical transmission. Results of drug sensitivity test showed that Eperythrozoon was sensitive to Tetracycline and antigen insect drugs. Sizes and shapes of different livestock Eperythrozoon were different. The study provided a scientific basis for the effective prevention and treatment of livestock Eperythrozoonosis.

一起东佛里生奶绵羊附红细胞体病流行病学调查、诊疗报告

为探究附红细胞体在内蒙古呼和浩特市托克托县地区的流行情况,接诊呼和浩特托县地区某东佛里生奶绵羊养殖场病例,该场近一周多圈舍出现产奶羊只体温升高、精神沉郁、瘫软、食欲减退或废绝等症状,有出现症状1~2 d迅速死亡病例,也有使用抗生素硫酸庆大霉素-青霉素联用结合补液治疗1周无好转或死亡病例,死亡总数超8只,仍有10余只相似症状正在接受治疗。经现场剖检、圈舍环境调查、样品实验室检测,结合临床诊疗,确诊此次疫情为奶绵羊附红细胞体病,且周边羊养殖场(户)羊只也存在附红细胞体感染。说明附红细胞体在托克托县呈地方性流行。

附红细胞体感染诱发补体异常活化导致溶血的研究

目的附红细胞体(Eperythrozoon,EP)病是人兽共患性血液传染病,可以引发溶血、出血、血栓形成、流产等,其主要的致病机制不清,本研究应用源于猪的EP感染研究其致病机制。方法收集EP感染养殖生猪27头和13例汉族饲养人员,对照组为无EP感染的同种猪26头和健康汉族人末梢血20份。ELISA和流式细胞检测猪和人感染EP后血清补体C3及其降解产物、补体调节蛋白CD55、CD59、补体膜攻击复合物(C5b-9)沉积及细胞因子在猪冠状动脉内皮中的表达。结果EP感染后猪和人血清C3降解产物C3a升高有统计学意义(P<0.05),猪红细胞CD55和CD59阴性比例显著增加具有显著的统计学意义(P<0.01),红细胞膜和猪血管内皮沉积大量C5b-9,同时血管内膜中细胞因子产生增加具有统计学意义(P<0.05)。结论EP感染诱发补体异常活化导致红细胞及血管内皮损伤,引发溶血、出血、血栓形成等。

猪附红细胞体16S rRNA基因的序列测定和系统进化分析

从确诊为猪附红细胞体感染的猪场,无菌采集血样,抽提猪附红细胞体基因组DNA,采用真细菌的通用引物进行16S rRNA基因扩增,对扩增产物进行克隆和测序。从3 个地理位置不同的猪场均成功地扩增出长度为1 469 bp的核苷酸序列。系统进化分析表明,3个猪场样品所测序列一致性达99 52%以上,具有相同的基因型,但与国外报道的猪附红细胞体Illinois株同源性为95%,属于同一基因群,但基因型不同;所有种类的附红细胞体和血巴尔通氏体组成同一进化分支,这类血营养菌与支原体科,支原体属的病原最靠近(75%),而与立克次氏体目的病原较远(70%)。上述研究证实,广东所流行的猪附红细胞体是一种新基因型的猪附红细胞体,建议命名为猪附红细胞体广东株型;为反映进化关系,猪附红细胞体和其它血营养菌应划归于支原体科的支原体属。

猪附红细胞体PCR检测方法的建立

根据已发表的部分猪附红细胞体序列设计了一对特异性引物,通过聚合酶链式反应(PCR)扩增出了一条约936bp的基因片段,并成功地将该基因克隆于克隆载体PMD18-T,经酶切分析和PCR进一步鉴定后进行序列测定,结果表明,所克隆的目的基因与GenBank中发表的序列一致。并利用特异性引物初步建立了猪附红细胞体病PCR检测技术。

猪附红细胞体PCR检测方法的建立和初步应用

基于猪附红细胞体广东株16S rRNA基因的序列特点,设计合成种特异性引物,建立了猪附红细胞体PCR检测方法。该方法能特异性扩增523 bp的猪附红细胞体16S rRNA基因片段,而对猪丹毒杆菌G 4T 10株、猪链球菌ST 171株、多杀性巴氏杆菌EO 630株、猪胸膜肺炎放线杆菌、猪肺炎支原体、鸡毒支原体和猫血巴尔通氏体CA株的基因组DNA没有扩增带出现。对猪附红细胞体基因组DNA的最小检测量为160 pg。通过对38份临床样品的检测,8份为猪附红细胞体感染阳性,其余为阴性。结果表明,建立的PCR检测方法具有极高的敏感性和特异性,可用于急性猪附红细胞体病和临床健康带菌猪的诊断。

舍饲小尾寒羊附红细胞体病的流行病学调查

20 0 2年 1至 12月对廊坊某羊场的舍饲小尾寒羊进行了流行病学调查 ,结果显示 ,3— 9月份附红细胞体呈上升趋势 ,10— 2月份附红细胞体呈下降趋势。证实了附红细胞体的感染情况与吸血昆虫出没密切相关。

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要作用。

猪附红细胞体电镜特点及药物治疗效果的电镜观察

近年来,国内外很多关于猪附红细胞体病诊断与防治的文章,但对猪附红细胞体本身形态学报道的文章却很少。本实验对猪附镜下的各形态、大小以及感染强度作了较详细的描述。球形、环形、圆盘形为E.suis的主要形态,体形较大的E.suis以芽苞生殖进行繁殖,感染强度随着病情的发展而呈现波浪形态变化。对致病机制作了进一步的阐述,在急性阶段,附红细胞体病会引起由一种经冷凝素IgM来介导的或与之相关的自身免疫溶血性贫血。最终结合电镜观察临床药物治疗效果,长效土霉素、贝尼尔在防治猪附红体病有一定的疗效。

猪附红细胞体的体外连续培养

临床疑似猪附红细胞体病猪红细胞,经Giemsa染色镜检及PCR扩增,证实感染猪附红细胞体(M.suis)。PCR扩增片段(781 bp)与M.suis已知序列(AJ504999)同源性达99.4%。经预试验对红细胞接种量、血清添加量、pH等条件优化后,选用添加新生牛血清、酵母浸出液、葡萄糖等成分的PPLO肉汤,将染虫红细胞接种到细胞培养板上,置于37℃、含5%CO2的培养箱培养,定期更换培养液,约72 h后红细胞溶血变为带虫血影(Ghost)。除首次接种外不添加任何红细胞,连续培养达280 d以上,染虫率维持近100%。对培养的带虫血影进行了光镜及扫描电镜观察、PCR鉴定、感染阴性红细胞试验、抗生素敏感性试验,证实与接种物为同一病原,并保持有感染阴性红细胞的能力。

附红细胞体病病原学研究概况

附红细胞体病是近几年来才引起医学和畜牧兽医工作者注意的一种人畜共患病。文章从附红细胞体的分类、种类、形态、染色特性、结构、在机体的分布及增殖方式、对理化因素的抵抗力、致病性。

猪和兔附红细胞体的形态学及生殖方式观察

应用光镜和电镜对猪和兔附红细胞体的显微和超微结构进行了观察。结果表明,猪和兔附红细胞体在光镜和电镜下的形态结构、大小以及在红细胞上的附着方式等均无显著差别,其形态多为球形、卵圆形,直径0.2～2.5μm,在红细胞表面以一根或数根纤丝连接成串珠状或单个寄生。在透射电镜下,首次观察到附红细胞体边缘有一段20～30nm厚的光亮区域,并且在扫描和透射电镜下同时观察到附红细胞体的出芽生殖现象。由此推测,附红细胞体的发育方式为成熟的附红细胞体以出芽的方式产生1个未成熟形态的附红细胞体,随着未成熟形态附红细胞体的发育,逐渐从成熟附红细胞体体内脱离出来,最后以纤丝与成熟附红细胞体相连接;脱离出来的未成熟形态的附红细胞体黏附到同一红细胞或临近红细胞的膜上,最终发育成1个成熟的附红细胞体。

附红细胞体病研究综述

对附红细胞体病(Eperythrozoonosis)的病原形态特征、病原分类、发病机制、流行病学、体外培养、治疗及预防等研究成果进行了综述,以期对该病的病原学及防治研究提供参考。

温氏附红细胞体16S rRNA基因的序列测定和分析

从自然感染附红细胞体的湖北黄牛无菌采集血液,分离附红细胞体并提取病原基因组,用血营养菌的16SrRNA基因的通用引物进行PCR扩增,得到长约1.5 kb的扩增片段,将其克隆到pMD18-T载体后进行测序和分析。结果表明该片段全长为1 471 bp(GenBank收录号为AY946266),同源性分析表明该序列与Neimark公布的温氏附红细胞体(AF016546)的16S rRNA基因序列同源性达到98.7%,证实该病原为温氏附红细胞体,从分子生物学水平证实了温氏附红细胞体在湖北省的存在。将该序列与5种支原体、14种血营养菌及边缘无浆体等的相应序列进行比较,建立系统发育树,结果表明温氏附红细胞体同边缘无浆体的关系较远,而与肺炎支原体组的亲缘关系较近。

羊附红细胞体病PCR检测方法的建立

根据羊附红细胞体的16SrRNA基因参考序列,设计1对特异性引物,建立了检测羊附红细胞体的PCR技术。用本方法从感染血样中特异扩增出1条预期大小为1169bp的片段。该方法灵敏、快速、特异性高,可用于羊附红细胞体病的早期快速诊断和流行病学调查。

反刍兽源附红细胞体感染小鼠的研究

目的 采用反刍兽源附红细胞体感染小鼠 ,研究附红细胞体的感染途径及在小鼠体内的消长规律 ,试图建立附红细胞体感染动物模型。方法 经小鼠腹腔、皮下接种含有附红细胞体的奶牛、山羊血液 ,脏器悬液和接触感染。结果 四种接种方法均获成功 ,附红细胞体在小鼠体内呈现一定的规律性。感染后第 8天附红细胞体在小鼠的血液可以查见。感染后第 15～ 18天附红细胞体的感染率达到高峰。感染后第 2 0d附红细胞体逐渐消失。在整个实验过程中未见感染小鼠明显的临床症状。结论 可以用小鼠建立附红细胞体感染动物模型。附红细胞体的感染方式有以下几种 :腹腔、皮下接种、接触感染。