Potential of a recombinant antigen as a prophylactic vaccine for day-old broiler chickens against Eimeria acervulina and Eimeria tenella infections.

A genetically engineered Eimeria tenella antigen (GX3262), produced as a fusion protein with beta-galactosidase and identified with a monoclonal antibody, induced partial but significant protection in young broiler chickens against experimental E. tenella and Eimeria acervulina infections. The antigen appears to share a T-helper cell epitope with the parasite as evidenced by (a) booster inoculation with either the recombinant antigen or with a small number of live oocysts enhanced the protective immunity in GX3262 primed chickens, and (b) ability of the antigen to induce in vitro stimulation of T-cells from chickens immunized with antigen or parasite. These observations suggest the feasibility of a single vaccination of 1 or 2-day-old broilers with GX3262 to induce an acceptable degree of protective immunity. The implications of the observations reported here are far reaching in terms of a practical coccidiosis vaccine for poultry, and show for the first time that 1-day-old broiler chickens can be efficiently vaccinated with a recombinant antigen against one or more species of Eimeria.     

The effects of Eimeria acervulina infection on the metabolism of chick duodenal tissue

Broiler chicks, 2--3 weeks old were infected with eimeria acervulina, and the metabolism and ultrastructure of the infected duodenal tissue were studied during the period 3--14 days after inoculation (DAI). Between 4 and 5 DAI duodenal rings showed an increase in C-1/C-6 ratios of CO2 evolved from glucose as well as decreases in the rates of oxidation of glucose and octanoic acid. Between 4--7 DAI mitochondria from infected epithelial layers had reduced rates of octanoic acid and alpha-ketoglutaric acid oxidation as compared to controls. Electron microscopic observations confirmed the biochemical findings. At 5--6 DAI mitochondria in many uninfected cells were progressively swollen and then vacuolated as the cristae appeared to break down. Mitochondria in cells which contained parasites did not show these changes.     

鸡堆型艾美耳球虫早熟株的最小免疫剂量研究

为确定堆型艾美耳球虫(Eimeria acervulina)早熟株的最小免疫剂量,设立了6个早熟株1次免疫组、6个早熟株2次免疫组、2个空白对照组、2个攻毒组。SPF鸡4日龄时以200、300、400、500、600、1 000个/羽的剂量接种早熟株卵囊进行第1次免疫,于14日龄使用1次免疫2倍的剂量进行第2次免疫。一次/二次免疫组及各不免疫攻虫组分别于18或28日龄以2×105个/羽的同源强毒株进行攻毒,以相对增重率、病变记分、病变记分减少率、卵囊抑制率和抗球虫指数(ACI)等指标评价免疫效果,确定最小免疫剂量。结果显示,本次试验使用的早熟株具有良好的免疫原性,不同免疫剂量均能诱发雏鸡产生保护力,且随着免疫剂量的增加保护力逐渐提高。一次免疫剂量为1 000个/羽和二次免疫的首次免疫剂量大于等于400个/羽、第二次免疫800个/羽,可达到良好免疫效果,可分别定为E.acervulina早熟株一次免疫和二次免疫的最小免疫剂量。     

帝克珠利对鸡堆型艾美尔球虫的药效实验

球虫病是威胁养鸡业的重要疾病之一,目前对该病的防治仍主要依赖药物［１］。但耐药虫株的出现使现有许多抗球虫药物疗效降低甚至丧失。因此,除了采取轮换用药及穿梭用药等方法以减少或延缓耐药性的产生外,开发评价和应用新型抗型抗球虫药物极为重要和必要。本文报道我...     

Influence of coccidiosis on growth rate and feed conversion in broilers after experimental infections with Eimeria acervulina and Eimeria maxima

Under experimental conditions, the effects of subclinical Eimeria (E.) acervulina and E. maxima infections on growth and feed conversion in broilers of different ages were analysed. It was concluded that infection with E. acervulina and E. maxima led to a process which was independent of the age at which the birds were infected. The infection adversely affected growth and feed conversion for 2 to 3 weeks, followed by a recovery period of 2 to 3 weeks when compensatory growth took place. From this study it may be concluded that subclinical coccidiosis in the first weeks of life and in the last week of life of broilers does not lead to appreciable damage on growth and feed conversion. Since coccidiosis cannot be avoided in practice, systems in which broilers contact subclinical coccidiosis either in the first weeks of life or in the last week of life should be aimed for. It is suggested that in a coccidiostat programme an efficient anticoccidiosis agent is particularly desirable in the 3rd and 2nd week before slaughter. Examinations performed one week or less before slaughter can hardly be justified, on the grounds that there is a risk of a negative effect on growth an feed conversion due to subclinical coccidiosis.     

Oocyst output and transmission rates during successive infections with Eimeria acervulina in experimental broiler flocks

The infection dynamics of Eimeria species determine the clinical manifestation of the disease coccidiosis in poultry flocks, and a better understanding of the dynamics may contribute to improvement of control measures. Our aim was to study the course of infection and the transmission of Eimeria acervulina in groups of broilers by quantifying the transmission rate parameter and oocyst output. Three transmission experiments were carried out with groups of 20 male SPF broilers. At 2 days of age, one bird in each trial was orally inoculated with five sporulated E. acervulina oocysts (D0 post-inoculation, pi). One day after inoculation (D1 pi), the inoculated bird was housed with 19 non-inoculated contact birds. Individual faecal droppings were examined daily from D3-D32 pi to quantify the number of oocysts per gram faeces. The inoculated bird started shedding oocysts at D5 pi and contact birds between D10 and D17 pi. Contact birds that became infected due to oocyst excretion by the inoculated bird were characterized as first generation contact birds (C1). Contact birds excreting from D15 pi onwards (C2) became infected after the first C1 birds had started shedding and were considered to belong to a successive generation of the flock infection. Oocyst output was significantly lower for C1 compared to C2 birds, but the transmission rate parameter remained constant for both infection generations. These results suggest that although oocyst load increases, the transmission rate of E. acervulina remains constant between successive generations of infection in a flock.     

Immunohistochemical study of S100-like protein in Eimeria brunetti and Eimeria acervulina

We have investigated the expression of a calcium-binding protein, the S100 protein, in Eimeria brunetti and Eimeria acervulina stages. For this purpose, paraffin sections of distal ileum and bursa of Fabricius or duodenum from experimentally infected chickens were treated with anti-alpha-S100 (anti-alpha subunit of S100 protein) and anti-beta-S100 (anti-beta subunit of S100 protein) monoclonal antibodies and anti-S100 whole molecule polyclonal antibody. The avidin-biotin peroxidase method was used to demonstrate immunoreactivity. In the ileum, our results reveal a positive immunoreaction for the beta subunit and S100 whole molecule within the macrogametes of E. brunetti, whereas they were devoid of immunostaining after treatment of the paraffin sections with the anti-alpha-S100 antiserum. Schizonts and oocysts of E. brunetti and all the E. acervulina stages gave a negative reaction after treatment with any of the three antiserum used in the study. This result indicated that the S100 protein molecules within these stages were not recognized by the antibodies, suggesting that these molecules are different from those identified in macrogametes of E. brunetti. By contrast, in the epithelial cells, lining the lumen of the bursa of Fabricius, macrogametes of E. brunetti were stained by the three antibodies used. These results may indicate the existence of metabolic adaptations that enable the parasite to invade tissue sites different from those where the parasite usually develops.     

堆型艾美耳球虫(Eimeria acervulina)早熟株与毒株的繁殖力和致病性比较试验

对经15代选育的堆型艾美耳球虫(Eimeria acervulina)北京株的早熟株与其毒株的繁殖力和致病性进行了比较研究，证实早熟株的潜隐期比毒株缩短27h，繁殖力有所下降；对致病性的研究显示，早熟株感染后对鸡只增重的影响较小，而且肠道病变记分亦较毒株下降。由此认为，该早熟株符合球虫早熟株的基本生物学特性，适宜用于鸡球虫病早熟苗的制作。     

堆型艾美尔球虫的分离与病理学研究

采用单卵囊感染技术,从鸡球虫混合种中分离出一株堆型艾美尔球虫(Eimeria acenrulina).并对该株堆型艾美尔球虫的病理学做了进一步研究.利用堆型艾关尔球虫的寄生部位、肉眼病变、最小潜在期等生物学特性进行鉴定,并做回归试验,用此球虫卵囊1×105个感染雏鸡.取感染5d后有明显病理变化的十二指肠组织,采用石蜡切片技术,进行病理学观察.结果表明,虫体寄生于肠绒毛上皮细胞内,大量的肠绒毛遭到破坏、断裂和脱落.     

堆型艾美耳球虫早熟株免疫剂量研究

为确定堆型艾美耳球虫(Eimeria acervulina)早熟株合适的免疫剂量,设立7个早熟株免疫攻虫组、7个母株免疫攻虫组、1个不免疫攻虫组和1个不免疫不攻虫组,7个早熟株/母株免疫组的免疫剂量为孢子化卵囊100、200、400、600、800、1 000、2 000个/羽,经嗉囊感染,7日龄首次免疫,14日龄以同等剂量进行第二次免疫,21日龄以1.5×105个/羽的同源母株进行攻虫,28日龄结束试验,以增重、肠道病变记分和卵囊减少率为试验指标。并对早熟株中免疫保护效果较好的2个免疫剂量进行重复试验,免疫方法、试验周期、试验指标同第一批试验,攻虫剂量为2×105个/羽的同源母株。结果显示,免疫期间,各免疫组的增重与不免疫组差异不显著(P>0.05);攻虫期间,早熟株400、600、800、1 000个/羽和母株600、800、1 000个/羽免疫组的相对增重与不免疫不攻虫组差异不显著(P>0.05);肠道病变记分,除2 000免疫组明显低于不免疫攻虫组(P<0.05)外,其余各免疫组虽低于不免疫攻虫组,但与其差异不显著(P>0.05);卵囊减少率,早熟株和母株400～2 000个/羽免疫组均达90%以上,其中600、800个/羽免疫组达97%以上。用早熟株600、800个/羽进行免疫重复试验,两个免疫组攻虫期间的相对增重和肠道病变记分均与不免疫不攻虫组差异不显著(P>0.05),而与不免疫攻虫组差异显著(P<0.05),其卵囊减少率均在88%以上。研究结果表明,该堆型艾美耳球虫早熟株保持了良好的免疫原性,不同免疫剂量均能诱发鸡产生免疫保护力,其中以600、800个/羽的免疫效果更好,可考虑以600个/羽作为该早熟株在疫苗制备中的推荐免疫剂量。     

堆型艾美耳球虫ADF基因的原核表达与免疫原性

参考Gen Bank中收录的E.acervulina ADF基因序列设计1对特异性引物,以河北保定株堆型艾美耳球虫子孢子DNA为模板,用聚合酶链式反应(PCR)法扩增ADF基因。将扩增的ADF基因克隆至p MD18-T载体,转化感受态细胞DH5α,蓝白斑法筛选出阳性重组子,提取阳性质粒进行PCR、酶切鉴定及对序列确证。将扩增产物与原核表达载体p ET32a(+)连接,构建重组质粒p ET32a-ADF,转化大肠杆菌Rosetta(DE3)进行诱导表达,表达产物经SDS-PAGE鉴定。用E.acervulina重组质粒p ET32a-ADF免疫雏鸡,观察其对雏鸡细胞免疫和体液免疫功能的影响。结果显示,ADF基因扩增产物为729 bp,与预期结果相符。序列分析结果表明,该株的ADF基因序列与参考序列同源性达99.6%。表达出的ADF重组蛋白相对分子质量大小约为46 000。与健康对照组相比,重组质粒组淋巴细胞的增殖能力明显增强(P0.05),外周血中Ig G及IL-2含量均显著升高(P0.01),表明p ET32a-ADF质粒DNA能有效提高雏鸡的细胞免疫和体液免疫水平。     

Characterisation and histopathological observations of a selected Brazilian precocious line of Eimeria acervulina

Precocious lines of Eimeria acervulina "Cu" and "I" strains were obtained after 25 passages of oocysts in chickens that showed a shortening of the prepatent period for first oocyst output from 96 h to 81 and 82 h, respectively. Both precocious lines were evaluated for pathogenicity using as criteria weight gain, lesion score and total oocyst production. Infection of the "Cu" precocious line in chickens showed a high weight gain, low lesion score and low oocyst production, when compared to parent strain infected chickens. However, the results did not show a significant difference in relation to the criteria used above for the E. acervulina "I" precocious line when compared to its parent strain. This suggests a low degree of attenuation for the "I" strain but good attenuation for the precocious "Cu" line. The histopathological observations of chickens infected with the E. acervulina "Cu" parent strain and precocious line, comparing life cycle and intestinal lesions, showed: (1) parasite stages only in the border cells of infected chicken intestinal villi, for the precocious line; (2) parasite stages in the border cells of the intestinal villi and submucosa cells near the Lieberkühn glands of the intestine; and (3) high degree of inflammatory cells around the parasites in chickens infected with the parent strain. The "Cu" strain was also characterized for sensitivity against eight anticoccidial drugs. Sensitivity was observed for four anticoccidial drugs and partial resistance for four other drugs, although the strain had never had previous contact with anticoccidial drugs, suggesting the presence of a natural resistance factor. This Brazilian E. acervulina "Cu" precocious line showed attenuation for pathogenicity in chickens, suggesting that it could be a suitable strain for use as a live vaccine in Brazil.     

The effect of Eimeria acervulina infection on hearts of young broiler chicks

Hearts from 2-to-3-week-old broiler chicks that were inoculated with 2 X 10(6) sporulated oocysts/bird weighed significantly less than hearts from control chicks at 4 and 7 days postinoculation (PI). However, heart weights of infected chicks expressed as percentage of body weights were not significantly different from those of controls throughout the experiment. Infection also had no significant effect on heart protein content. Cardiac glycogen was significantly increased at 7 days PI but not at 4 days PI. Oxidation of both alpha-ketoglutaric and octanoic acids by mitochondrial preparations from hearts of infected chicks was significantly depressed at 4 days PI compared with controls; at 7 days PI, however, oxidation of octanoic acid was significantly increased, but that of alpha-ketoglutaric acid was not.