三株柔嫩艾美耳球虫(Eimeria tenella)的繁殖力和免疫原性的比较研究

分别用柔嫩艾美耳球虫早熟弱毒株、鸡胚适应株和强毒株孢子化卵囊１０００个／只，给１０日龄无球虫雏鸡免疫接种，逐日检查粪便并进行克粪便卵囊（Ｏ．Ｐ．Ｇ．）计数，结果发现早熟弱毒株和鸡胚适应株的潜隐期均有不同程度缩短，繁殖力亦有不同程度的下降。同时进行了柔嫩艾美耳球虫不同株球虫的免疫原性比较：分别用三株卵囊免疫雏鸡，免疫剂量为１０００个／只，免疫后１４ｄ各用１０００个／只强毒株卵囊攻虫，逐日进行Ｏ．Ｐ．Ｇ．计数，发现各组免疫鸡攻虫后的卵囊产量均大大下降，其中以强毒株免疫攻虫组的排出卵囊量最低，仅为未免疫组的１．２３％，早熟株和鸡胚株免疫鸡攻虫的排出卵囊量相近，分别为未免疫组的７．３５％和７．２３％。由此可见，三株柔嫩艾美尔球虫均表现出良好的免疫原性。     

宿主日龄对艾美耳球虫 (Eimeria)早熟虫株感染力及雏鸡球虫免疫力的影响(英文)

用 10 0 0个柔嫩艾美耳球虫 (E .tenella)早熟株孢子化卵囊 ,10 0 0个毒害艾美耳球虫 (E .neca trix)早熟株孢子化卵囊和 5 0 0个巨型艾美耳球虫 (E .maxima)早熟株卵囊分别接种 1,7和 2 5日龄雏鸡 ,接种后 14天分别用相应的 2 0 0 ,2 0 0和 10 0个亲本毒株孢子化卵囊进行攻虫。初次接种和攻虫后分别检查卵囊总产量。结果表明 ,3种早熟虫株的卵囊都能够成功的感染 1日龄和 7日龄雏鸡。初次接种后雏鸡都产生了不同程度的球虫免疫力。本研究证实 ,艾美耳球虫早熟株卵囊能够诱导初生和幼龄雏鸡产生球虫免疫力     

和缓艾美耳球虫激发宿主免疫应答的初步研究

和缓艾美耳球虫Eimeriamitis是7种鸡球虫中致病力弱的虫种,对于其免疫原性的认识至今仍有争论。本研究以和缓艾美耳球虫涿州株为研究对象,以初次免疫及二次免疫后鸡的卵囊排出量为标准评估了其免疫原性。同时利用ELISA检测了免疫后鸡群血清中球虫特异性IgY抗体的水平,并通过ELISPOT技术分析了二免后鸡群外周血单核淋巴细胞（PBMC）中分泌IFN-γ的特异性淋巴细胞的数量。结果显示,二次免疫后,鸡的卵囊排出量显著低于初次免疫,免疫组的血清特异性IgY抗体水平较低,而分泌IFN-γ的特异性淋巴细胞的数量较高。这些结果表明和缓艾美耳球虫涿州株具备良好免疫原性,且其激发宿主产生的保护性免疫以细胞免疫应答为主,有作为疫苗株的潜力。     

宿主日龄对艾美耳球虫（Eimeria）早熟虫株感染力及雏鸡球虫免疫力的影响

用1000个柔嫩艾美耳球虫(E.tenella)早熟株孢子化卵囊,1000个毒害艾美耳球虫(E.necatrix)早熟株孢子化卵囊和500个巨型艾美耳球虫(E.maxima)早熟株卵囊分别接种1,7和25日龄雏鸡,接种后14天分别用相应的200,200和100个亲本毒株孢子化卵囊进行攻虫.初次接种和攻虫后分别检查卵囊总产量.结果表明,3种早熟虫株的卵囊都能够成功的感染1日龄和7日龄雏鸡.初次接种后雏鸡都产生了不同程度的球虫免疫力.本研究证实,艾美耳球虫早熟株卵囊能够诱导初生和幼龄雏鸡产生球虫免疫力.     

三株柔嫩艾美耳球虫（Eimeria tenella）的繁殖力和免疫原？…

分别用柔蒋美耳球虫早熟弱毒株、鸡胚适应株和强毒株孢子化卵囊１０００个／只，给１０日龄无早雏鸡免疫接种，逐日粪便并进行克粪便卵囊计数，结果发现产毒株和鸡胚适应株的潜隐期均有不同程度缩短，繁殖力亦有不同程度的下降，同时进行了柔嫩艾美耳球虫不同株球虫的免疫原性比较，分别用三株卵囊免疫雏鸡，免疫剂理为１０００个／只，免疫后１４ｄ各用１０００个／只强毒株卵囊攻虫，逐日进行Ｏ．Ｐ．Ｇ．计数，发现各组免疫鸡攻虫     

福建三株柔嫩艾美耳球虫的分离、鉴定及致病性的初步研究

采用单卵囊分离技术,从福建省莆田市、福清市和连江县等三地鸡场的病鸡盲肠内容物样品中,分离获得3株柔嫩艾美耳球虫Eimeria tenella,代号分别为PT0705、FQ0709和LJ0711.为比较这3株柔嫩艾美耳球虫的致病性,分别用1×104、5×104和10×104个孢子化卵囊的剂量感染14日龄和21日龄健康无球虫雏鸡,通过临床表现、死亡率、增重、病变计分等指标进行统计和分析,确定PT0705为毒性最强虫株.     

柔嫩艾美耳球虫田间分离株对马杜霉素耐药性检测

以对马杜霉素完全敏感和柔嫩艾美耳球虫豪顿株为参照 ,检测柔嫩艾美耳球虫 2个河北分离株(HBZ、HBH)、 2个山东分离株 (SDZ、SDT)对马杜霉素的耐药程度。按每羽 5× 1 0 4个孢子化卵囊的剂量接种 7日龄试验鸡只 ,接种后第 7天剖杀所有试验鸡 ,观察盲肠病变 ,计算平均增重、盲肠内容物卵囊数、粪便中卵囊排出量和抗球虫指数 (ACI)。以ACI值作为判定耐药程度的标准 ,ACI≥ 1 80判为敏感 ,1 6 0≤ACI<1 80判为部分耐药 ,ACI<1 6 0判为耐药。结果是柔嫩艾美耳球虫豪顿株的ACI为 1 98 3,柔嫩艾美耳球虫河北分离株HBZ、HBH组的ACI分别是 1 4 7 5、 1 5 3 7,山东分离株SDZ、SDT组的ACI分别是1 2 7 2、 1 30 0。试验证明柔嫩艾美耳球虫 2个河北分离株 (HBZ、HBH)、 2个山东分离株 (SDZ、SDT)对马杜霉素具有耐药性     

兔球虫PCR检测方法的改进和应用

兔球虫病由艾美耳属球虫寄生于消化道而引起,对养兔业的危害极大。由于兔艾美耳球虫的11个虫种在致病性上有较大差异,因此进行准确的虫种鉴定对兔球虫病的诊断和防治有十分重要的意义。为提高和扩大PCR方法检测粪便样品中艾美耳球虫的灵敏度和应用范围,我们利用全基因组扩增技术对提取自粪便样品的球虫基因组进行预扩增,提高PCR反应初始模板含量,再利用兔艾美耳球虫ITS-1序列种特异性引物对WGA产物进行PCR扩增,以鉴定虫种。结果显示,经全基因组扩增后的样品的PCR检测能够在单个卵囊的水平上进行虫种鉴定。利用该方法,我们对10份卵囊含量较少的田间兔粪样品进行了PCR检测,对其中的虫种进行了的鉴定。检测结果显示,与卵囊形态鉴定相比,该方法不仅与能确定形态的形态学检测结果一致,还能够检测出形态学不易判断的虫种,体现了更高的准确性。该技术的应用可提高田间样品检测的敏感性和准确度,为兔球虫临床感染情况提供实践指导。     

鸡感染巨型艾美耳球虫后的体液、粘膜与细胞免疫应答

鸡的7种艾美耳球虫中,巨型艾美耳球虫免疫原性最强。为研究巨型艾美耳球虫激发宿主产生的免疫应答,我们检测了巨型艾美耳球虫3次感染后鸡只的抗体应答及细胞应答特征。ELISA检测显示,感染鸡只产生了显著高于未感染鸡只的特异性IgY和sIgA（肠道及胆汁）抗体;而酶联免疫斑点法（ELISPOT）检测显示,被感染鸡只脾脏中分泌IFN-γ的球虫特异性淋巴细胞的数量显著高于对照组。粪便中卵囊的检测则显示,第3次感染后的鸡只几乎无卵囊排出,证实巨型艾美耳球虫产生的免疫应答可对同源感染提供完全的免疫保护。     

柔嫩艾美耳球虫山西分离株的分离与致病性观察

运用单卵囊分离技术，从山西省某鸡场鸡球虫感染粪便样品中获得l株纯种球虫，鉴定为柔嫩艾美耳球虫并命名为Eimeria tenella SX010323，并对其致病性进行了研究。（1）该球虫卵囊寄生于盲肠、为宽卵圆形，卵囊壁光滑、褐色，无卵膜孔，有极粒，无内、外残体。孢子囊呈长卵圆形，有斯氏体。卵囊大小范围为（16．80～28．80）μm×（14．40～25．20）μm，平均大小为（21．54±0．24）μm×（17．85±0．24）μm，形状指数为1．21±0．05。子孢子，大小范围为（10．20～12．75）μm×（4．40—7．25）μm，平均大小为（11．54±0．24）μm×（6．27±0．43）μm。潜隐期为141h，最短孢子化时间为19h；（2）63只11日龄的海兰白公雏随机分为7组，分别口服感染此新鲜卵囊1×10^3、5×10^3、1×10^4、2×10^4、5×10^4、1．0×10^5个，并设立不感染对照组。结果表明：所有感染组增重均低于对照组（P〈O．05），各感染组间随着感染剂量的增加，增重和增重率显著降低，血便率和盲肠病变记分增加（P〈0．05）；（3）将4℃分别保存17、13、10、4、1个月和新鲜孢子化的虫株卵囊，以1．5×10^4个／只的剂量感染11日龄雏鸡，每组10只。结果表明：保存1个月和4个月活力较高，保存10个月活力开始下降，保存13、17个月活力下降较大。     

A low-virulence Eimeria intestinalis isolate from rabbit (Oryctolagus cuniculus) in China: molecular identification,pathogenicity, and immunogenicity

An Eimeria intestinalis isolated from a rabbit in China was first identified by amplifying the 18S small subunit (SSU) ribosomal RNA gene. The size of the amplified fragment was 1521 bp. The 18S SSU RNA gene of the E. intestinalis isolate shared 99 % sequence identity with E. intestinalis isolates from France and the Czech Republic, with 100 and 96 % coverage, respectively. Then, the pathogenicity and immunogenicity of the E. intestinalis isolate were evaluated in specific pathogen free (SPF) rabbits. In the pathogenicity assay, SPF rabbits in four groups were infected with 5?×?10³, 5?×?10⁴, 5?×?10⁵, and 0 sporulated oocysts, respectively. Clinical signs including diarrhoea, constipation, loss of appetite, and reduction of body weight gain were observed in rabbits inoculated with 5?×?10⁴ and 5?×?10⁵ oocysts. And one rabbit (25 %) inoculated with 5?×?10⁵ oocysts died 15 days after the inoculation. In the immunogenicity assay, SPF rabbits in five groups (named B1, B2, B3, B4, and B5) were immunised with 5?×?10¹, 5?×?10², 5?×?10³, 0, and 0 sporulated oocysts, respectively. All rabbits but the B5 group were challenged with 1?×?10⁶ oocysts. After the challenge, no or slight clinical signs were seen in rabbits of the B2 and B3 groups. Compared with the control, a 69.6 and 84.5 % reduction of oocyst output was observed in the B2 and B3 groups, respectively. The body weight gain of the two groups was obviously higher than that of the challenge control group. All the results show that the E. intestinalis isolate has low virulence but immunogenicity in rabbit.     

Selection and characterization of a precocious line of Eimeria media

Coccidiosis, caused by the infection of Eimeria parasites, is one of the most common diseases in domestic rabbits. Live anticoccidial vaccine formulated with attenuated precocious lines of pathogenic eimerian parasites is expected to be valuable for the control of rabbit coccidiosis as a similar strategy to produce anticoccidial vaccines against chicken coccidiosis has being used for several decades. Eimeria media, moderate pathogenic, is widespread in China. Therefore, attenuated anticoccidial vaccines against rabbit coccidiosis should contain vaccine strain(s) of E. media. In this study, a precocious line of E. media (Empre) was selected by collecting and propagating the early excreted oocysts with 16 successive generations. The prepatent period of Empre reduced from 108 h of its parental strain (Emwt) to 70 h. The fecundity of Empre was about 1/10 to 1/3 lower than that of Emwt. Each sporocyst of Empre sporulated oocyst contained only one large refractile body instead of two smaller ones seen in the parental strain. When vaccinated with 1 × 10³ or 1 × 10⁴ precocious line oocysts, the rabbits were completely protected against homologous challenge with the parental strain 14 days post challenge by terms of body weight gain and oocyst output counting, indicating the efficacy of Empre. Meanwhile, all immunized rabbits showed no clinical sign post immunization, indicating the safety of Empre. For co-immunization, 1 × 10³Empre oocysts and 5 × 10² oocysts of a precocious line of E. intestinalis (EIP8) were inoculated to each rabbit in a trial. No diarrhea or mortality was found after vaccination, and the weight gains of the vaccinated group were similar to that of unvaccinated-unchallenged control (UUC) group, while the weight gains of the vaccinated group were similar to that of unvaccinated-unchallenged control (UUC) group (P > 0.05), but significantly higher than that of UCC group (P < 0.01) after challenge, indicating it is safe and effective when using co-immunization. These results together show that Empre, as a precocious line, is a good candidate of precocious line of E. media for anticoccidial vaccine development.

     

Eimeria sp. from the rabbit (Oryctolagus cuniculus): Pathogenicity and immunogenicity ofEimeria intestinalis

The pathogenicity and immunogenicity ofEimeria intestinalis was evaluated in SPF rabbits. The antimals were given immunizing doses of 6, 6×10², 6×10³, and 6×10⁴ sporulated oocysts and were challenged with 3×10³ oocysts. The criteria analysed were the daily weight gain and the occyst output. This study showed thatE. intestinalis had strong immunogenicity, as the inoculation of 6 oocysts was sufficient to minimize the clinical expression of the disease following the challenge and to reduce the oocyst output by about 60%. The immunity towards the excretion of oocysts and the illness was absolute in animals inoculated with 600 or more oocysts. Moreover, this protection seemed to be efficient at least 8 weeks after the challenge. The present results also confirm the pathogenicity ofE. intestinalis, although the occurrence of diarrhoea may be irregular, and emphasize the fact that the capacity of thisEimeria for multiplication is not a criterion for clinical diagnosis of the disease.     

Oral inoculation of ultraviolet-irradiated Eimeria species oocysts protects chickens against coccidiosis

Prevention of coccidiosis is one of the best ways of controlling disease. Therefore, the present study was carried out to evaluate the protective effect of ultraviolet (UV)-irradiated sporulated oocysts of Eimeria species against coccidiosis in layer chickens. One hundred forty-four one-day-old layer chicks were randomly divided into 4 groups (n = 36), including non-immunized/non-challenged negative control group (NC group), non-immunized/challenged control group (NIC group), non-irradiated sporulated oocyst/challenged group (CA group), and UV-irradiated sporulated oocyst/challenged (UV group). At the age of 4 days, chickens in groups UV and CA were both orally inoculated with 1.0 × 10⁴ UV-irradiated and non-irradiated sporulated oocysts of Eimeria species, respectively. Chickens in groups NIC and NC were served as positive and negative controls, respectively. Chickens in all groups were orally challenged with 7.5 × 10⁴ sporulated oocysts of Eimeria species except the NC group at the age of 21 days. The results revealed that chicks receiving UV-irradiated sporulated oocysts had no signs of illness with minimal or no changes in the cecal integrity and a significantly lower oocyst shedding (OPG) than in the NIC group. Additionally, the cytokine gene expression profiles were evaluated. Expression levels of IL-2, IL-12, and IFN-γ were significantly higher in the spleen of chicks in the UV and CA groups than in the NC group post-challenge. As expected, treatment with irradiated oocysts resulted in a significant reduction in oocyst shedding and maintenance of cecal mucosal integrity. Furthermore, the body weight was higher in chickens inoculated with UV-irradiated oocysts than their non-irradiated counterparts. In conclusion, our results demonstrate that inoculation with UV-irradiated sporulated oocysts of Eimeria species can produce a substantial reduction in infection symptoms.

     

Selection and characterization of a precocious line ofEimeria intestinalis,an intestinal rabbit coccidium

A precocious line ofEimeria intestinalis was obtained by selection for early development of oocysts in rabbits and after six consecutive passages in animals. This line (EiP) was derived from a wild strain (EiO) isolated in 1975 from the caecal content of a rabbit with coccidiosis. The prepatent period of the EiP strain was reduced from 215 h to<144 h, the result being that the oocyst sporulation time was the same for both lines. The excreted and unsporulated oocysts had exactly the same shape, but microscopical examination of the sporulated oocysts showed a marked difference between EiP and EiO strains. A huge refractile globule was located in each of two sporocysts of the precocious line, whereas no refractile globule was seen in the other two. The EiP line had a reproductive potential much lower (1000 times) than that of its parent strain EiO and, as judged by the weight gain, mortality and lesions that also occurred in the jejunum and above all in the ileum, its pathogenicity was substantially reduced.     

Protective immunity against Eimeria acervulina following in ovo immunization with a recombinant subunit vaccine and cytokine genes

A purified recombinant protein from Eimeria acervulina (3-1E) was used to vaccinate chickens in ovo against coccidiosis both alone and in combination with expression plasmids encoding the interleukin 1 (IL-1), IL-2, IL-6, IL-8, IL-15, IL-16, IL-17, IL-18, or gamma interferon (IFN-gamma) gene. When used alone, vaccination with 100 or 500 mug of 3-1E resulted in significantly decreased oocyst shedding compared with that in nonvaccinated chickens. Simultaneous vaccination of the 3-1E protein with the IL-1, -15, -16, or -17 gene induced higher serum antibody responses than 3-1E alone. To evaluate protective intestinal immunity, vaccinated birds were challenged with live E. acervulina oocysts 14 days posthatch, and fecal-oocyst shedding and body weight gain were determined as parameters of coccidiosis. Chickens vaccinated with 3-1E protein showed significantly lower oocyst shedding and normal body weight gain than nonvaccinated and infected controls. Simultaneous immunization with 3-1E and the IL-2, -15, -17, or -18 or IFN-gamma gene further reduced oocyst shedding compared with that achieved with 3-1E alone. These results provide the first evidence that in ovo vaccination with the recombinant 3-1E Eimeria protein induces protective intestinal immunity against coccidiosis, and this effect was enhanced by coadministration of genes encoding immunity-related cytokines.     

Induction of protective immunity against Eimeria tenella, Eimeria maxima, and Eimeria acervulina infections using dendritic cell-derived exosomes

This study describes a novel immunization strategy against avian coccidiosis using exosomes derived from Eimeria parasite antigen (Ag)-loaded dendritic cells (DCs). Chicken intestinal DCs were isolated and pulsed in vitro with a mixture of sporozoite-extracted Ags from Eimeria tenella, E. maxima, and E. acervulina, and the cell-derived exosomes were isolated. Chickens were nonimmunized or immunized intramuscularly with exosomes and subsequently noninfected or coinfected with E. tenella, E. maxima, and E. acervulina oocysts. Immune parameters compared among the nonimmunized/noninfected, nonimmunized/infected, and immunized/infected groups were the numbers of cells secreting T(h)1 cytokines, T(h)2 cytokines, interleukin-16 (IL-16), and Ag-reactive antibodies in vitro and in vivo readouts of protective immunity against Eimeria infection. Cecal tonsils, Peyer's patches, and spleens of immunized and infected chickens had increased numbers of cells secreting the IL-16 and the T(h)1 cytokines IL-2 and gamma interferon, greater Ag-stimulated proliferative responses, and higher numbers of Ag-reactive IgG- and IgA-producing cells following in vitro stimulation with the sporozoite Ags compared with the nonimmunized/noninfected and nonimmunized/infected controls. In contrast, the numbers of cells secreting the T(h)2 cytokines IL-4 and IL-10 were diminished in immunized and infected chickens compared with the nonimmunized/noninfected and the nonimmunized/infected controls. Chickens immunized with Ag-loaded exosomes and infected in vivo with Eimeria oocysts had increased body weight gains, reduced feed conversion ratios, diminished fecal oocyst shedding, lessened intestinal lesion scores, and reduced mortality compared with the nonimmunized/infected controls. These results suggest that successful field vaccination against avian coccidiosis using exosomes derived from DCs incubated with Ags isolated from Eimeria species may be possible.