Coccidiosis, caused by various Eimeria species, is a major parasitic disease in chickens. However, our understanding on how chickens respond to coccidian infection is highly limited at both molecular and cellular levels. The present study employed the Affymetrix chicken genome array and performed transcriptome analysis on chicken cecal epithelia in response to infection for 4.5 days in vivo by the cecal-specific species E. tenella. By Significance Analysis of Microarrays (SAM), we have identified 7,099 probe sets with q-values at <0.05, in which 4,033 and 3,066 genes were found to be up- or down-regulated in response to parasite infection. The reliability of the microarray data were validated by real-time qRT-PCR of 20 genes with varied fold changes in expression (i.e., correlation coefficient between microarray and qRT-PCR datasets: R (2) = 0.8773, p<0.0001). Gene ontology analysis, KEGG pathway mapping and manual annotations of regulated genes indicated that up-regulated genes were mainly involved in immunity/defense, responses to various stimuli, apoptosis/cell death and differentiation, signal transduction and extracellular matrix (ECM), whereas down-regulated genes were mainly encoding general metabolic enzymes, membrane components, and some transporters. Chickens mustered complex cecal eipthelia molecular and immunological responses in response to E. tenella infection, which included pathways involved in cytokine production and interactions, natural killer cell mediated cytotoxicity, and intestinal IgA production. In response to the pathogenesis and damage caused by infection, chicken cecal epithelia reduced general metabolism, DNA replication and repair, protein degradation, and mitochondrial functions.

The security,stability and immunogenicity trials of recombinant Salmonella typhimurium X4550 carrying rabbit's Eimeria stiedai MIC-5 gene expression plasmid asd-pBMIC-5-IL-15(X4550/asd-pBMIC-5-IL-15) was assayed in this study.The results showed that the 109 CFU inoculation dose was safe in the experimental rabbits.The recombinant plasmid existed stably in X4550 after 30 passages,which displayed a good genetic stability.After two immunizations in rabbits,the recombinant Salmonella typhimurium induced the specific antibodies against rabbit Eimeria stiedai and significantly improved the level of lymphocyte proliferation.The anti-coccidia index reached 164.4.The results indicated that the regulation DNA vaccine mediated by Salmonella had a good security,stability and immunogenicity against rabbit Eimeria stiedai.

【Objective】 The aim of this study is to construct a recombinant co-expression vector expressing Eimeria stiedai microneme protein 5(MIC-5) and rabbit IL-15 in eukaryotic cells,which may lay a foundation for further study immune regulatory DNA vaccine against infection of Eimeria stiedai.【Method】 E.stiedai MIC-5 gene and rabbit's IL-15 gene were respectively cloned into plasmid pBudce4.1 to construct recombinant vector pB-MIC-5-IL-15 for co-expression,and then the recombinant plasmid pB-MIC-5-IL-15 was transfected into BHK-21 cell line for expression.The RT-PCR and indirect fluorescent immunoassay were used to detect the transcription and expression of interesting genes in BHK-21 cell line,respectively.【Result】 The results showed that E.stiedai MIC-5 gene and rabbit's IL-15 gene were successfully transcripted.And interesting genes of E.stiedai MIC-5 and rabbit's IL-15 were successfully expressed at 36,48 and 72 hour post-transfection,respectively.【Conclusion】 The recombinant co-expression vector was successfully constructed,which can express E.stiedai MIC-5 and rabbit IL-15 gene in eukaryotic cells.

鸡球虫病是由顶复器门(Apicom plexa)、孢子虫纲(Sporozoasida)、真球虫目(Eucoccidiorida)、艾美耳科(Eimeriidae)、艾美耳属(Eimeria)的球虫引起的一种呈全球性分布的原虫病,发病率高达50%～70%,死亡率为20%～30%,严重时高达80%[1-2].每年全世界因球虫病造成的经济损失超过30亿美元[3].

本文对采自山东的兔斯氏艾美耳球虫(E.stiedai)卵囊做毒性研究.主要研究了不同攻虫剂量兔斯氏艾美耳球虫对不同月龄兔的毒害作用.同时,研究了酶联免疫吸附试验(间接法)检测兔斯氏艾美耳球虫抗体的最适反应条件和在毒理学实验中的应用,并对攻虫后的各项检测指标结果做详细的比较.对兔斯氏艾美耳球虫致病性研究发现成年兔感染兔斯氏艾美耳球虫通常无明显的临床症状,而幼兔感染时则较为严重;人工感染试验结果发现,在一定的数量范围内,宿主吞食的卵囊数增加,随之疾病的严重性也增加;从特异性抗体的变化情况来看,Ⅰ,Ⅱ,Ⅲ.Ⅳ,Ⅴ组的抗体效价明显高于空白对照Ⅵ组,且随着感染时间的增长,抗体水平也明显增高.本试验结果为兔斯氏艾美耳球虫疫苗的研制提供理论依据.

Coccidiosis is a kind of sporozoan diseases in rabbits caused by coccidian of Eimeridae,which has been a great threat to rabbit breeding industry.In this paper,investigations on coccidian species and their epidemic in rabbits from 5 food markets of Lanzhou,Gansu province were performed.Analysis of rabbit coccidian epidemic will provide a basis for the separation of coccidian species and prevention.

The protective efficacy of a vaccine against coccidiosis was examined in Lingnanhuang female broilers kept in floor-pens.Three trail groups,including negative group(NC),positive group(PC) and vaccinated group(VC) was tested,and each group comprised 30 broilers.The results showed that the average relative ratio of weight gain of 3 groups were 100 %,84.77 % and 94.97 %,respectively.The OPG of PC group(2.32×106) was significantly higher(p0.01) than vaccinated group(2.69× 105) after challenge infection.The mortality rate of 3 groups were 0,10 % and 0,respectively.Furthermore,higher ACI were detected in NC(200) and VC birds(184.97),which were significantly higher than that of PC birds(154.77).

This experiment examined the virulence of 6 Eimeria spp.Lanzhou strain by infecting Jiahehuang female broilers at various dosages.Nine trial groups,including negative control group(NC) and 8 challenge infection groups(1-8) were tested.The average relative growth ratio(RGR) of 8 groups was 100 %,77.23 %,77.02 %,76.42 %,72.86 %,63.91 %,67.19 % and 42.06 %,respectively.The RGR of NC group was significantlg higher than other groups(p0.01).The RGRs of the 1st and the 2nd group were significant higher than the 5th and the 6th groups(p0.01) and the 7th group(p0.01).The feed conversion ratio(FCR) in the 7th group was significantlg higher than other groups.The OPG of the 7th group(150.11×104) was significantly higher than the 1st(3.05×104),the 2nd(2.35×104),the 3rd(32.20×104),the 4th(62.23×104) and the 5th group(60.45×104)(p0.01) after challenge.The mortality rate of 9 groups was 0,5.0 %,5.0 %,15 %,25 %,40.0 %,45.0 %,95 % and 100 %,respectively.These results indicated that the Eimeria spp.strains isolated from Lanzhou are highly virulent.Chickens infected with 6×104 oocysts suffered significant suppression in growth and 50 % mortality.The best challenge dosage was 1×104-1×105(each Eimeria spp.).

<正>鸡球虫病是一种呈世界性分布的原虫病,给养禽业造成严重的经济损失,据统计包括防治该病所花的药费在内全世界每年损失约80亿美元,仅美国一年损失就高达45

兔球虫病是家兔最常见且危害严重的一种原虫病,对养兔业危害极大,1～4月龄以内的幼兔感染率高达100%以上,死亡率可达60%～70%,耐过兔生长发育受阻.

To examine the immunoprotective effect of the recombinant antigen of 3-1E gene in chickens,recombinant surface antigen 3-1E gene of Eimeria tenella was administered to chicken at 7 days old and boosted at 14 days old.Chickens were chall-enge with E.tenella oocysts one week post immunization(21 days old).The results showed that the protection of 3-1E soluble protein were superior to that of inclusion body in relation to body-weight gain,oocysts number and cecal lesion scores.The 3-1E soluble protein could induce partial protection against against E.tenella infection.

Two experiments were designed to test the efficacy of LCV-2,a coccidiosis vaccine for chickens.In experiment 1,the efficacy of LCV-2 was evaluated on male broilers,Chinese Yellow strain,reared in wire cages on the basis of relative ratio of weight gain,OPG(oocysts per gram of stool),morbidity and score of intestinal tract lesions and anticoccidio index(ACI) after violent challenge.Results showed that in vaccinated challenged groups,the maximum percentage protection(survivors after challenge) against mixed species of Eimeria were 100%,and relative ratio of weight gain was observed in vaccinated non-challenged group(97.74%),which was significantly higher(P0.05)than vaccinated challenged group(89.72%) and unvaccinated challenge group(69.85%).Furthermore,the difference between vaccinated challenged group and unvaccinated challenged group was significantly higher(P0.05).After challenge,the average OPG of unvaccinated challenged group(1.26×105) was significantly higher(P0.05)than vaccinated challenged groups(5.813×104),the average score of intestinal lesions were also record in unvaccined challenged groups followed by vaccined challenged groups and vaccined non-challenged group(1).The higher ACI were calculated in vaccinated non-challenged birds(192.74)and vaccinated challenged birds(178.76),which significantly higher than unvaccinated challenged birds(62.18).In experiment 2,the efficacy of LCV2 were evaluated in vaccinated challenged groups,negative control group and unvaccinated non-challenge group(which reared in one floor-pen with vaccinated challenge group together) on the basis of relative ratio of average weight gain,average OPG and intestinal lesions,too.Results showed that chickens of vaccinated non-challenged groups(with the relative ratio of weight gain 99.39%) grown faster than vaccinated challenged(with the relative ratio of weight gain 91.67%),but no significant difference was found from these two groups.After challenge,the intestinal lesions of vaccinated challenged group got worst,which score of intestinal lesions was 4,and followed by unvaccinated no-challenged group(with intestinal lesions score:1),and no intestinal lesions was observed in negative groups(with intestinal lesions score 0).Furthermore,the OPG of unvaccinated non-challenged group(3.675×105) was significantly lower(P0.05)than vaccinated challenged group(6.025×105) after challenge,and significantly lower(P0.01)before challenged too(7.5×104,3.5×104).The highest ACI was calculated in unvaccinated non-challenged birds(193.39),which followed by vaccinated challenged birds(175.76).From these results,it can be concluded that the vaccinated chickens,no matter of which reared in cages or in floorpens were resistant to coccidiosis with the floor-reared chickens vaccinated by LCV-2 to be better.

【Objective】 The paper investigated bioactivity of Eimeria tenella 3-1E protein expressed in E.coli.【Method】 The recombinant plasmid pGEX-3-1E sequenced correctly was transformed into E.coli BL21,and was prokaryotic expressed in E.coli.After optimizing expression conditions,the purified proteins were Western-Blotted.【Result】 The expression fusion protein was a 44.7 ku,and was in agreement with deduction,the highest soluble expression level of recombinant protein was obtained after being induced at 28 ℃ for 6 h and the concentration of IPTG was 0.5 mmol/L.The purified proteins could react with the anti-sera derived from rabbits challenged with the E.tenella YL strain.【Conclusion】 The 3-1E protein possesses immunogenicity.

In order to study the pathogenicity and pathology of Eimeria stiedai,25 young rabbits were infected artificially by purified sporulated oocysts of Eimeria stiedai(E.stiedai).These rabbits were divided into 5 groups,one control group and four experimental groups.The sporulated oocysts were inoculated orally to four experiment groups with the dose of 2.0×103,1.0×104,5.0×104 and 2.5×105,respectively.The results showed that there were significantly differences in mean pathogenic scores,liver index,transaminase and pathologic histology between control and experimental groups(P0.05 or P0.01),indicating that E.stiedai may have a strong pathogenic effect in severe liver coccidiosos in young rabbits.

Microneme protein 5 gene of Eimeria tenalla LZ strain was amplified by RT-PCR and cloned into pMD18-T for sequencing,then subcloned into the prokaryotic expressing vector pET28a for expression.The amplified fragment length of Et MIC-5 gene was 918 bp,encoding 306 amino acids.Compared with the MIC-5 genes of Houghton strain,the identities of nucleotide sequence and deduced amino acid sequence were 99.2 % and 99.6 %,respectively.There were four Apple domains(15~89,90~160,173~242 and 245~306) in the deduced amino acid sequence,which implied that MIC-5 protein may play roles in invasion of Eimeria tenalla.Moreover,E.coli strain BL21(DE3) transformed with recombinant pETMIC-5 can express interesting protein,which amounts to 15.2 % in the total protein of the induced bacteria by the assaying of gel scanning.

Eimeria species were isolated successfully from chickens in Lanzhou,Gansu province,by single oocyst separating technique with plastic paper.Under experimental conditions,20 chickens were infected with single E.tenella oocyst.After 5~15 days,the oocysts in the feces were detected through saturated brine flotation.amoug which the oocysts of E.tenella were detected possitive in 15 chickens.The results show that the method used in this study is simple,convenient and effective with 75 percent of success rate and 100 percent of accuracy.Moreover,the material(plastic paper) is easily available and sterilizing.indicating that the technique with plastic paper charicterized with being practical,simplicity,convenience,higher success rate and accuracy.

Objective To isolate the pure specie of Eimeria stiedai by single oocyst separation technology and observe the sporulation process. Methods The pathogenicity of isolated specie was studied by a series of indexes such as latency period,mortality,liver index,transaminase,scores of pathological changes in artificial infected young rabbits.Results The shortest time of sporulation of oocyst was 39 hours under the temperature of 29 ℃.The latency period was 13 to 15 days and the infection rate was 20%. Conclusion The experiment results indicated that the pathogenicity of pure specie of E.stiedai was proportional to the inoculated dose of continuous proliferative oocysts in a range in the inoculatd groups.

To evaluate the mechanism of immune enhancement of β-lentinan(LTN) in chickens,both in vitro and in vivo trials were carried out to study the effect of LTN on NO productive capacity of the splenic and thymus lymphocytes and abdominal macrophage.One-hundred-twenty 21day-old chickens were randomly divided into 4 groups which were injected abdominally with 2 mL of 1 g/L LTN,10 g/L LTN,20 g/L sodium thioglycolate and normal saline solution,respectively.The NO production of the splenic and thymus lymphocytes and abdominal macrophage were measured on day 3 post-injection.And the macrophage activated by sodium thioglycolate were stimulated with LTN at level of 50 mg/L,100 mg/L,200 mg/L and 400 mg/L respectively and the NO production by macrophages were measured.In consequence,1 g/L and 10 g/L LTN stimulated the splenic and thymus lymphocytes and macrophages to produce NO.Different concentrations of LTN were able to stimulate macrophages to increase the production of NO.Moderate stimulation of immunocytes to produce NO may be one of the most important immunoactive mechanisms by LTN in chickens.