流产衣原体检测技术与分型方法研究进展

流产衣原体(Chlamydia abortus)广泛分布于世界各地,能感染猪、牛、羊等多种家畜,引起孕畜流产,是危害畜牧业最严重的衣原体病原。针对流产衣原体抗体检测的血清学方法如间接血凝试验(IHA)、酶联免疫吸附试验(ELISA)等在兽医临床检测中广泛使用。以聚合酶链反应(PCR)为代表的病原分子生物学检测技术敏感性和特异性高,有效地弥补了血清学方法的不足。在流行病学研究中,进一步对流行菌株的分型分析在阐释流产衣原体遗传进化、毒力演变和跨物种传播以及疫情预警和疫苗研制等方面具有重要意义。论文就当前国内外广泛应用的流产衣原体检测技术和分型方法进行综述,可为我国动物衣原体病的诊断和防控提供参考。     

猪的衣原体感染

衣原体(Chlamydiaceae)是一类专性细胞内寄生的革兰氏阴性菌,可引起人和动物的多种疾病。在猪上已经分离到的衣原体包括猪衣原体(Chlamydia suis)、羊流产衣原体(Chlamydia abortus)、兽类衣原体(Chlamydia pecorum)和鹦鹉热衣原体(Chlamydia psittaci)。衣原体感染猪,可引起多种不同的病症,包括结膜炎、肺炎、心包炎、多发性关节炎、多发性浆膜炎、伪膜性或坏死性小肠炎;对母猪可致产后无乳征(PPDS)、阴道分泌物增加、返情、流产、木乃伊胎、弱仔、围产期和新生仔猪死亡增加;对公猪则引起精液品质下降、睾丸炎、附睾炎和尿道炎。不过衣原体病被看作是不太重要的猪病,因为关于猪衣原体病的报道微乎其微;此外,在兽医实验室的常规诊断中并不包括衣原体检测,因而衣原体常常不会引起重视;另外一方面,衣原体常常和其他病原混在一起,而这些病原更加容易被检查到。但是,近期的研究证实,衣原体对种公母猪和仔猪的感染比预想的要更为普遍,对经济效益的影响也很重要。本文对猪衣原体病作一概述,内容包括:猪衣原体病的分类学、诊断注意事项、流行病学和病理学、公共卫生意义和如何防治本病。     

天峻县土种藏系羊衣原体病的血清学调查

<正> 羊衣原体病是由鹦鹉热衣原体(Chlamydia psittaci)感染引起的一种接触性传染病。衣原体广泛寄生于人类、鸟类、哺乳动物,可引起不同种动物的多征候群疾病。藏羊感染后临床上以发热、流产和其他繁殖障碍为主要特征,也可引起肺炎、多发性关节炎、脑脊髓炎和腹泻等病症,该病又叫母羊地方性流产、母羊衣原体流产。为查清天峻县土种藏羊中该病的感染情况,本文应用IHA进行衣原体病血清学调     

藏系绵羊衣原体病血清学调查

<正>鹦鹉热衣原体(Chlamydia psittaci)是一种可在多种动物间广泛传播的人畜共患病病原,绵羊感染后可引起流产(妊娠后期)、早产、死胎及发热、肺炎、肠炎、多发性关节炎和脑脊髓炎等广泛性症候群。为摸清兴海县藏系羊衣原体感染情况,笔者应用衣原体抗体间接血凝试验(IHA)进行了藏系羊衣原体血清学调查,现将结果报告如下。     

青海天峻土种藏系羊衣原体病的血清学调查

土种藏系羊衣原体病是由鹦鹉热衣原体(Chlamydia Psittaci)感染引起的一种接触性传染病,衣原体广泛寄生于人类、鸟类、哺乳动物,它可引起不同种动物的多症候群疾病,藏羊感染后临床上以发热、流产和其他繁殖障碍为主要特征,也可引起肺炎、多发性关节炎、脑脊髓炎和腹泻等病症,该病又叫母羊地方性流产、母羊衣原体流产.为查清天峻县土种藏羊中该病的感染情况,根据本省动物疫病预防控制中心的安排,我们于2009年4～7月应用衣原体病IHA进行了血清学调查,结果报告如下.     

羊流产衣原体灭活苗田间免疫试验效果观察

羊衣原体性流产(羊地方性流产)流行于近50个国家,成为引起母羊流产的主要病原之一。对本病的预防,主要采用疫苗的免疫接种,并应用一些实用技术检测疫苗效力。本文就我们研制的羊流产衣原体卵囊黄甲醛灭活油佐剂苗进行较大规模田间免疫试验,并通过数理统计来评价该苗在不同地区的预防效果,用以衡量疫苗的免疫效果。     

共和县绵羊衣原体病流行情况的调查

正所谓的衣原体病是由鹦鹉热衣原体(Chlamydia Psittaci)感染引起的羊、牛、人等多种动物共患的传染病,鹦鹉热衣原体属于衣原体科、衣原体属。广泛寄生于人类、鸟类、哺乳动物体内,它可引起不同种动物的多征后群疾病,临诊病理特征为流产、肺炎、肠炎、结膜炎、多发性关节炎和脑炎。该病又叫地方性流产病、衣原体性流产。近几年的研究结果表明,随着布氏杆菌病的基本控制,衣原体病和弓形虫病已成为造成牛羊流产的主要     

羊流产衣原体的分离鉴定及间接免疫荧光法检测

对收集的疑似衣原体感染的羊流产胎儿样本接种鸡胚卵黄囊进行分离培养,通过PCR-RFLP(限制性片段长度多态性分析)方法鉴定为流产衣原体。将收集到的流产衣原体阳性样本接种Hela229细胞,应用基于流产衣原体MOMP单克隆抗体的间接免疫荧光法,在Hela229细胞浆内可见呈绿色荧光的衣原体包涵体,进一步在病原学水平上确定发生在甘肃省肃南县羊流产的病原为流产衣原体。     

鹦鹉热衣原体基因工程疫苗研究进展

鹦鹉热衣原体(Chlamydia psittaci, Cps)是专性细胞内寄生、革兰氏阴性病原体,能在鸟类、人类和其它哺乳动物中广泛传播。Cps能够导致禽类的呼吸道和消化道疾病,引起家禽高热、腹泻、异常分泌物以及产蛋下降。常规衣原体疾病防控主要依赖于抗生素,但随着对食品安全的重视、养殖端减抗替抗的推行,需要开展生物安全和疫苗免疫等防控技术研究以预防衣原体感染。本文综述了Cps亚单位疫苗、DNA疫苗和活载体疫苗等基因工程疫苗的研究进展。。     

L929细胞无血清悬浮驯化机器在流产衣原体灭活疫苗中的应用研究

为了给规模化生产流产衣原体提供悬浮细胞培养方法,本研究通过逐步降血清悬浮驯化法使贴壁L929细胞逐步适应悬浮培养环境,最终获得了一株可无血清悬浮培养的L929细胞株;将流产衣原体接种于L929悬浮细胞上,研究不同的促感染试剂以提高收菌量,并选择出最佳的培养方法;制备流产衣原体灭活疫苗,以25 μL 、50 μL、100 μL的剂量免疫Balb/c小鼠,攻毒后检测其免疫保护效果。结果显示,当该L929悬浮细胞初始接种密度为5×105个/mL 时,培养72 h后细胞浓度可达到约7×106个/mL,细胞活力在95%以上;在细胞密度为6×106个/mL,接菌量为5.5×104 IFU/mL,加入脂质体2000的量为1.7 μL/mL、放线菌酮浓度为0.4 μg/mL时,菌量增殖倍数最大,48 h内约扩增了52倍。经流产衣原体悬浮细胞灭活疫苗免疫后,小鼠产生的抗体水平随免疫剂量的增加而提升;接种50 μL、100 μL疫苗组小鼠的脾淋巴细胞增殖水平、IFN-γ、IL-2的含量远高于接种25 μL疫苗和注射PBS组的小鼠;对免疫后的小鼠进行攻毒,接种疫苗组小鼠的脾脏、十二指肠、子宫中的流产衣原体基因组含量远低于未接种疫苗组小鼠。注射PBS组及接种25 μL疫苗组的小鼠子宫出现了不同程度的坏死及炎症,而其余两组小鼠的子宫无异常变化。结果表明,本试验成功驯化出１株L929悬浮培养细胞株,并且通过悬浮培养工艺制备的流产衣原体灭活疫苗免疫效果良好,接种50 μL、100 μL该灭活疫苗可以有效抑制流产衣原体的感染,该L929细胞全悬浮培养株的成功驯化为疫苗规模化生产提供了科学依据。     

New challenges for vaccination to prevent chlamydial abortion in sheep

Ovine enzootic abortion (OEA) is caused by the obligate intracellular Gram-negative bacterium Chlamydia abortus. OEA remains a common cause of infectious abortion in many sheep-rearing countries despite the existence of commercially available vaccines that protect against the disease. There are a number of confounding factors that influence the uptake and use of these vaccines, which includes an inability to discriminate between infected and vaccinated animals (DIVA) using conventional serological diagnostic techniques. This suggests that the immunity elicited by current vaccines is similar to that observed in convalescent, immune sheep that have experienced OEA. The existence of these vaccines provides an opportunity to understand how protection against OEA is elicited and also to understand why vaccines can occasionally appear to fail, as has been reported recently for OEA. Interferon-gamma (IFN-γ), the cytokine that classically defines Th1-type adaptive immunity, is a strong correlate of protection against OEA in sheep and has been shown to inhibit the growth of C. abortus in vitro. Humoral immunity to C. abortus is observed in both vaccinated and naturally infected sheep, but antibody responses tend to be used more as diagnostic markers than targets for strategic vaccine design. A future successful DIVA vaccine against OEA should aim to elicit the immunological correlate of protection (IFN-γ) concomitantly with an antibody profile that is distinct from that of the natural infection. Such an approach requires careful selection of protective components of C. abortus combined with an effective delivery system that elicits IFN-γ-producing CD4+ve memory T cells.     

Protective adaptive immunity to Chlamydophila abortus infection and control of ovine enzootic abortion (OEA)

Ovine enzootic abortion (OEA) remains a major problem in sheep-rearing countries despite the availability of protective vaccines. The causative agent, Chlamydophila abortus, is a Gram-negative bacterium that can induce a persistent, subclinical infection in non-pregnant sheep. The development of a new safe, effective and practical vaccine requires a detailed understanding of host-pathogen interactions and the identification of clear correlates of protection. Since disease (abortion) is only observed during pregnancy, the nature of host immunity to C. abortus and the specialised immunological features that permit maternal acceptance of the semi-allogeneic fetus are central to the pathogenesis of OEA. We review the current literature on persistence of C. abortus, host immunity to infection and mechanisms of abortion. We identify the key outstanding questions surrounding OEA and discuss the current knowledge gaps with a view to developing improved control strategies.     

Chlamydial infections in small ruminants.

Chlamydophila abortus (formerly Chlamydia psittaci) is one of the most important causes of reproductive failure in sheep and goats, especially in intensively managed flocks. The disease is usually manifested as abortion in the last 2 to 3 weeks of gestation, regardless of when the animal was infected. Ewes that abort are resistant to future reproductive failure due to C. abortus, but they become inapparent carriers and persistently shed the organism from their reproductive tracts during estrus. Chlamydophila pecorum is the other member of the genus that affects small ruminants, and it is recognized as a primary cause of keratoconjunctivitis in sheep and goats and of polyarthritis in sheep.     

Abortion in small ruminants in Switzerland: investigations during two lambing seasons (1996-1998) with special regard to chlamydial abortions

Abortion cases of 144 goats und 86 sheep were investigated etiologically during 2 lambing seasons (1996/1997, 1997/1998). Macroscopic inspection of fetus and placenta was completed by histopathology and bacteriological isolation of agents. In addition, immunohistologically the following antigens were labeled in formalin-fixed and paraffin-embedded tissue sections: Toxoplasma gondii, Neospora caninum, Chlamydophila abortus (formerly Chlamydia psittaci serovar 1) and Border Disease Virus. From farms with abortions caused by Chlamydophila abortus specific data were recorded. In 75% of abortion cases in sheep and in 59% of cases in goats an etiologic diagnosis could be substantiated. Chlamydophila abortus is the most commonly involved agent in the etiology of caprine and ovine abortion (sheep 39%, goats 23%), followed by Toxoplasma gondii (sheep 19%, goats 15%) and Coxiella burnetti (sheep 1%, goats 10%). All other agents are of minor importance. An infectious cause of abortion based on histopathologic findings without isolation of agents was observed in sheep (10%) and goats (21%). Malformation occurred in sheep (2%) and goats (3%) and lesions suggestive for Vitamin E/Selenium deficiency were seen in goats only (2%).     

Immunopathology of Chlamydophila abortus infection in sheep and mice

Chlamydophila abortus targets the placenta, causing tissue damage, inflammation and abortion (enzootic abortion of ewes). It is one of the main infectious causes of abortion in ewes, resulting in major economic losses to agricultural industries worldwide. Although ruminants and pigs are the principal hosts, humans are also susceptible to infection. Control of disease requires a host inflammatory response, which is likely to contribute to pathology and abortion. Mouse models have been widely used to provide insight into the role of specific immune cells in controlling infection and disease. The use of such model systems for investigating the mechanisms of abortion, latency, persistence, and immunity to reinfection will result in the identification of novel vaccine control strategies for sheep.     

Abortion in small ruminants in the Netherlands between 2006 and 2011

During five successive lambing seasons between 2006 and 2011, 453 submissions of abortion material, 282 of ovine and 171 of caprine origin, were examined at the Animal Health Service in the Netherlands. Infectious agents as the most plausible cause of the abortion were found in 48 percent of the ovine submissions and in 34 percent of the caprine submissions. Submission of both aborted fetus and placental membranes increased the diagnostic yield of laboratory investigations (17 percent and 21 percent for ovine and caprine submissions, respectively). The main infectious causes of abortion in sheep were Chlamydia abortus, Campylobacter spp., Toxoplasma gondii, Listeria spp., and Yersinia pseudotuberculosis. The main infectious causes of abortion in goats were Coxiella burnetii, Chlamydia abortus, Listeria spp., Toxoplasma gondii, and Campylobacter spp. In 42 percent of the ovine and in 56 percent of the caprine submissions a causal agent was not identified. Furthermore, in 12 percent of the ovine and 10 percent of the caprine submissions evidence of placentitis, indicative of an infectious cause of the abortion, was found, but no infectious agent was identified. Most infectious causes of ovine and caprine abortion have zoonotic potential. Humans, especially pregnant women, who are in close contact with lambing sheep or goats should be aware of the importance of precautionary hygiene measures.     

Investigations concerning the prevalence of Coxiella burnetii and Chlamydia abortus in sheep in correlation with management systems and abortion rate in Lower Saxony in 2004

The intracellular bacteria Coxiella (C) burnetii and Chlamydia (Chl) abortus induce abortion in sheep and also affect humans. While Chl. abortus only infrequently infects humans, C burnetii is the aetiological agent of numerous Q fever outbreaks during the last decades. There is only limited knowledge about the prevalence of both pathogens in sheep, although sheep are involved in almost all Q fever outbreaks in Germany. The aim of our study was to investigate the prevalence of both pathogens in flocks located in Lower Saxony, Germany, in correlation to the management form and abortion rate. Serum samples of 1714 sheep from 95 flocks located in Lower Saxony were investigated by ELISA. 2.7% of these samples were positive, 1.3% showed inconclusive results in the C. burnetii-ELISA. Elevated intra-flock seroprevalences were only detected in three migrating flocks. Chlamydia-specific antibodies could be detected in 15.1% serum samples of mainly shepherded and migrating flocks. In one of these flocks with a high intra-flock seroprevalence for C burnetii (27%) and Chlamydia (44.9%), C burnetii was detected in 21.6% of the placenta samples of normal births and in 12.5% of the colostrum samples by PCR. Aborted fetuses and the corresponding placentas were negative in C burnetii-PCR, but in most of them and also in many other placenta samples Chl. abortus could be detected by PCR and DNA microarray. This survey shows a low overall prevalence of C. burnetii in sheep in Lower Saxony in the year 2004. However, three migrating flocks with a high intra-flock prevalence are localized in the southern parts of Lower Saxony. Spreading of C burnetii could occur, because of the large radius of grazing of all three flocks.     

Diagnosis of ovine chlamydial abortions by PCR-RFLP performed on vaginal swabs

Ovine enzootic abortion is an infectious and contagious disease clinically characterized by abortion and weak neonates, affecting sheep and goats. The etiological agent is Chlamydophila (C.) abortus, which is considered one of the most common animal pathogens of small ruminants; it has important economic implications and represents a significant zoonotic risk. Clinical diagnosis is often difficult because the clinical signs and the pathological lesions are not specific for C. abortus infection, in fact they can also be observed as a result of infections with other abortifacient agents. Moreover, the involvement of the laboratory is necessary to perform the definitive diagnosis. One hundred and seventeen vaginal swabs from sheep with clinical signs related to chlamydial infection were examined by a PCR-RFLP assay that demonstrated high specifity and sensitivity. Six samples were positive for C. abortus. Vaginal swabs are easy to handle and allow to deal with biohazardous material in safety conditions.