采用2对附红细胞体种特异性引物,对上海地区2个屠宰场采集的736份猪血液基因组DNA进行PCR检测,将小附红细胞体特异性引物扩增产物克隆到pMD18-T载体,进行序列测定和生物信息学分析.将小附红细胞体阳性血液分离物肌肉注射给5头切除脾的断奶广西巴马小型猪仔猪,感染后定期采血,进行显微镜检,并提取基因组DNA进行PCR检测.另设3头猪作为不感染对照组,进行相同的检测.结果显示,共有101份样品检测出附红细胞体阳性,阳性率为13.72％,其中54份为小附红细胞体单独感染,占7.34％.5头巴马小型猪在接种小附红细胞体后第13～16天起,至第34天试验结束,出现明显的临床症状;血液显微镜检和PCR检测均发现存在小附红细胞体感染;其序列测定显示,16S rRNA基因的部分序列与接种的小附红细胞体的相应序列完全一致.上述结果表明,本研究成功筛选出小附红细胞体阳性血液,并成功实现了小附红细胞体对巴马小型猪的人工感染试验.

雏鸡是指从刚孵出到50日龄阶段的小鸡,该阶段小鸡具有生长速度快、代谢旺盛、消化和体温调节能力差、对疾病抵抗力弱和环境适应能力差等方面的特点,因此饲养雏鸡要精心.雏鸡饲养的好坏直接关系到雏鸡的成活率和养鸡户的经济效益.本文阐述了育雏、饲养管理要点,供养殖户参考.

The oocyst wall protein CP41 gene of Cryptosporidium mouse genotype was amplified from total RNA by RT-PCR, sequenced and compared with that from C. parvum in GenBank. The sequences shared 90.5 % nucleotide and 87.57 % amino acid identities with the published sequences. The gene fragment was subcloned into pGEX-5x-3 vector and transformed into E.coli BL21 (DE3). Recombinant protein was expressed after IPTG induction and identified by SDS-PAGE and western blot. The expressed proteins were used to immunize rabbit, specific antibodies were detected by ELISA. The recombinant protein was about 46 ku and mainly existed in the form of inclusion body. The protein could be recognized the positive sera against Cryptosporidium rabbit genotype. High level antibodies against Cryptosporidium in rabbits could be produced after 3 times immunization with the protein, indicating that the fusion protein had high reactogenicity and immunogenicity.

In order to clone and analyze the sequence of internal transcribed spacer 1(ITS-1) of Eimeria tenella,E.necatrix,E.acervulina,E.maxima,E.praecox and E.mitis from chicken in Shanghai,a genus-specific primers was employed to amplify the ITS-1 of six Eimeria species.The amplified products were cloned into pMD18-T vector and their nucleotide sequences were determined.The phylogenetic relations were analyzed by compared together with sequences from GenBank.The results showed that there were two amplified fragments for E.maxima and E.mitis respectively,and only one for others species.The length of amplified fragments were 547 bp and 422 bp for E.maxima,627 bp and 493 bp for E.mitis,and 668 bp,691 bp,507 bp and 541 bp for E.tenella,E.necatrix,E.acervulina and E.praecox,respectively.The sequence similarities of six species ranged from 34% to 52%.Phylogenetic tree showed that each species was at the same cluster with corresponding species which sequence downloaded from GenBank.

Drug resistance is one of the main obstacles for prevention and control of the Eimeria species through prophylactic chemotherapy.At present,the mechanism of drug resistance for chicken Eimeria spp.is still poorly understood.This papers reviewed the progress on techniques of random amplified polymorphic DNA,mRNA differential display,suppression subtractive hybridization,cDNA array used in reseach of the genetic variation,ATPase activity detection,and polyacrylamide gel electrophoresis,two-dimensional gel electrophoresis combined with mass spectrometry protein analysis uesd in reseach of protein differential expression of chichen Eimeria spp.related with drug-resistance.The research direction of genetic variation and protein differential expression of Eimeria spp.related with drug-resistance were also proposed.

Total RNA was extracted from Cryptosporidium parvum oocysts isolated from mouse.The sporozoite surface antigen CP15/60 gene was amplified by RT-PCR and then cloned into pMD18-T vector.The verified DNA fragment was subcloned into pET-28b(+) vector,and the recombinant plasmids were transformed into Escherichia coli BL21(DE3) subsequently and induced by IPTG.The expressed recombinant protein was identified by SDS-PAGE and Western-blotting.Titers of serum antibody of SPF rabbits immunized with the expressed protein for 3 times were detected by ELISA.Sequence of the cloned gene shared 98.66% identity with that from GenBank.The gene was expressed efficiently in the form of soluble fusion protein.The recombinant protein made up approximately 57.5% of the total bacterial soluble proteins,and 95.2% of the purified bacterial proteins.The recombinant protein could be recognized specifically by the sera from rabbit infected with C.parvum.ELISA result showed that the titer of specific antibody against the expressed protein in sera from the immunized rabbits was significantly higher than that of the unimmunized rabbits.The results revealed that the recombinant protein had good reactogenicity and immunogenicity.