DNA was extracted from the tissue of the dead goose caused by the attack of Goose Parvovirus(GPV) as well as from allantoic liquid GPV,a pair of primers were designed according to the sequence of GPV published in GenBank,and an expected 440 bp segment was amplified by PCR.The sequencing result showed that the homology was 99% in comparison with the reference sequence of GPV strain B which was published in GenBank.This test showed that this PCR method could specially detect GPV and was more sensitive in detecting GPV than AGP assay method.The least amount of GPV that could be detected was 102.5GELD50/0.2mL.Using this PCR method to detect the distribution of GPV in goose challenged by GPV,the result indicated that GPV existed in the viscera,such as brain,liver,lung,kidney and intestine.All above results reveal that this PCR method for the detection of GPV has been established successfully,and it has strong speciality,high sensitivity and good repetition.

According to the conserved gene sequences of ALV in GenBank,a pair of the specific primers were designed.Through the optimization of reaction conditions,the PCR detection method was established.Then the ten tissue samples separated from different local chicken breeding bases in Jiangsu province were detected by PCR.The results showed that 90% of the suspected samples were ALV positive and the normal chicken liver tissue was ALV negative.The amplified target gene was cloned,sequenced and compared with the reference nucleotide sequence in GenBank,the results showed that the target gene fragment was 300 bp,and the homology of nucleotides was more than 97%.The study showed that J-subtype ALV existed in the local chickens in Jiangsu province.

According to the conserved gene sequences of Duck plague virus(DPV) in GenBank,one pair of specific primer was designed.A rapid diagnostic technique of PCR was established by optimizing the reaction condition.The DNA segments amplified were 1 293 bp.The experiments had proved that PCR possessed a high specificity.And the sensitivity test results indicated that PCR was more sensitive,which could detect DHV with only 15 pg·mL-1 DNA.Detection of field samples from viral diseased ducks also confirmed that the PCR method could identify the infection of DPV,which was specific,rapid and sensitive.

<正>正确使用兽药,重要的一条是要使用敏感的兽药,前提是在用药前要进行病原分离和兽药对病原的敏感性试验,根据试验结果,选择敏感的药物。1使用抗生素之前为什么要进行药敏试验细菌对抗生素产生耐药性是细菌在长期进化过程中所获得的生存本领,只要药物胁迫存在,细

The strain LH of goose parvovirus(GPV) was isolated from the infected young goose in Liuhe District of Nanjing.The virus were propagated in goose embryos of 12-day-old,and showed typical shape of parvovirus under electronic microscope.The goose of 8-day-old were artificially inoculated with the virus sample,infection rate and mortality was 100%,and the clinic symptom and pathological lesion were similar to the natural infected cases of GPV.The GPV antigen from allantoic fluid of infected embryos could react with positive serum of GPV by agar gel precipitation.The strain LH liquid was detected by PCR with the specific primers aim at GPV VP3,and the result showed a bound of 1.6 kb,coincide with expected bound.Sequence analysis showed 99%-100% nucleotide homology between the amplified product and the other published GPV strains,which indicated the isolated strain LH was goose parvovirus.

A pair of primers specific to ChIFN-γ gene were designed and synthesized according to the known cDNA sequences from GenBank,and ChIFN-γ gene was cloned and amplified by reverse transcripition-polymerase chain reaction(RT-PCR) from the total mRNA in the lymphocyte of chicken blood stimulated with ConA for 8 hours.RT-PCR product was cloned into pMD18-T vector.The recombinant plasmid was identifled by digestion with restriction endonucleases and the nucleotide sequence was determined.The ChIFN-γ gene was a 495 bp open reading frame encoding a polypeptide of 164 amino acids.The gene was inserted into the expression vector pPICZa-A,which had been cleaved by Kpn I and Not I.The recombinant vector was transfered into yeast Pichia pastoris strain X-33,and the ChIFN-γ gene was successfully expressed after induced by methanol.The activity of the recombinant ChIFN-γ was detected by the Western-blotting.The results showed that the recombinant yeast ChIFN-γ was a protein with re-activity.

A pair of primers specific to chicken γ-interferon gene were designed and synthesized according to the known sequences from GenBank,using the total mRNA in the lymphocyte in chicken blood as the template,the mature protein gene of γ-interferon was cloned and amplified through the reverse transcription polymerase chain reaction.The gene was then ligated to pET32a to establish the gene's non-fusion expressive vector.The best inducing time for the expression of this recombinant protein was tested through inducing the expression of selected positive plasmid with different time span

A pair of primers specific to ChIFN-γ gene were designed and synthesized according to the known sequences from the GenBank,and were used to amplify ChIFN-γ cDNA by RT-PCR from total RNA of chicken spleen cells induced by ConA.RT-PCR product was cloned into pMD18-T vector.The recombinant plasmid was identifled by digestion with restriction endonucleases and the nucleotide sequence was determined.The ChIFN-γ gene was 500 bp in length,with a 495 bp open reading frame encoding a polypeptide of 164 amino acids,the sequence homologous rate of ChIFN-γ and that from GenBank was 99.6%.

Based on the genome sequence of Newcastle disease virus(NDV) published in GenBank,ten pairs of gene-specific primers were designed to amplify cDNA of SHJ00 strain from diseased Sanhuang chicken by RT-PCR technique.Sequence analysis showed that the entire genome of SHJ00 consisted of 15 192 nucleotides.SHJ00 and NDV strains shared 6 common ORFs with the same order:3′-NP-P-M-F-HN-L-5′,but SHJ00 genome had extra six nucleotides(ACACTC)between NP and P genes.The F gene was 1 672 bp and contained a complete open reading frame encoding a protein of 553 amino acids.The amino acid sequence of cleavage site region was 112R-R-Q-K-R-F117 which was the same as virulent NDV strains.The SHJ00 belonged to NDV genotype Ⅶ,and had 80.9%-97.7%nucleotide homology with other published NDV strains.The nucleotide sequence of HN gene was 1 716 bp and encoded a protein of 571 amino acids.It shared 80.0%-98.1%nucleotide homology with other published NDV strains.

A wild strain of gosling new type viral enteritis virus(GNVEV)was isolated from the affected young goose in Nanjing.The viruses were propagated in 12-day-old goose embryos,the typical adenoviruses were observed by electronic microscope,they were 70～120 nm in diameter.The virus could infect goose embryos after neutralization test with gosling plague positive serum.The 4-day-old healthy and sensitive goslings were artificially inoculated with virus sample,the clinical symptoms and pathological lesion were similar to the GNVEV natural infected cases.The concentrated antigen from allantoic fluid of infected embryos could not react with positive serum of gosling plague by agar gel precipitation.

通过方阵法对间接ELISA抗原、抗体反应浓度进行筛选,再运用正交试验对不同的包被液、封闭液、底物进行比较和选择,最后对酶进行选择,最终确定运用ELISA法测定小鹅瘟抗体的最佳反应条件。即抗原最佳浓度为37.00μg/ml,血清最佳稀释倍数为1∶200,包被液为0.1 mol/L碳酸盐缓冲液,4℃包被12 h,封闭液为10%犊牛血清的PBS溶液,使用羊抗兔IgG酶标二抗,底物为联苯二胺(OPD)。

The biological characteristic of a strain H9N2 AIV that isolated from duck was investigated in this paper.The results indicated that IVPI was 0,ICPI was 0.26,NHAT was slow,HOT was 30 minutes.The assay of intravenous pathogenicity showed that the mortality of 26-day-old ducks was 40% and the reisolated rate of throat swabs of susceptible ducks was l00% on the 1~2 day after challenging with 108.0 ELD50 NJ01 virus per duck,respectively.In addition,the reisolated rate of cloaca swabs of 30-day-old special free chicks was l00% on the 4~6th day after challenging with the same dose.The Hemagglutinin(HA) gene was amplified by RT-PCR and the sequence analysis results indicated that the homology of HA nucleotide sequence was between 94.8% and 96.0% as compared with three strains isolated from Hongkong,the amino acid sequence of cleavage site region was-RSSRG-.It was concluded that the NJ0I strain was low pathogenic avian influenza virus and belong to Eurasian lineage.It can replicate in SPF chicken and non-immune ducks.

A strain of virus,which was suspected to be duck hepatitis virus(DHV),was isolated from the diseased and dead ducks in the duck farm in Anhui province in China.The virus strain was isolated by means of saline suspension and blastula inoculation.Animal test indicated that the death rate was 70%.Through observation of clinical performance and the results of dissection and passive conservancy blood serum test,the pathologic materials were diagnosed as DHV.Duck blastula neutralized test proved that the serotype of the virus was type Ⅰ.

从发病雏鸭体内分离到1株具血凝活性病毒,并鉴定为H9亚型禽流感病毒。取发病鸭肺作为病毒分离病料,接种10日龄SPF鸡胚,死亡鸡胚尿囊液具血凝活性,且不能被已知鸡新城疫(ND)阳性血清、产蛋下降综合症EDS-76单克隆抗体、禽流感H5亚型阳性血清抑制,但能被禽流感H9亚型阳性血清抑制,血凝抑制价为211。该毒株经中国农业科学院哈尔滨兽医研究所国家禽流感参考实验室鉴定为H9N2亚型。对分离株进行的研究结果表明,该毒株为低致病力毒株,其ICPI为0.26,IVPI为0。用第三代鸡胚尿囊液原液0.5 m l静脉注射8日龄樱桃谷鸭,雏鸭死亡率达50%。

以甲醛为灭活剂,研究在不同温度条件(37.4、25℃)下,不同甲醛浓度(0.1%、0.2%、0.3%、0.4%)对含鸭源H9亚型禽流感病毒NJ01株的尿囊液完全灭活所需时间的变化,为规模化生产疫苗提供可靠的灭活动力学试验依据。

为了建立一种简便的检测猪圆环病毒2型(PCV2)的方法,本实验将PCV2的ORF2基因片段整合到巴斯德毕赤酵母(Pichia pastoris)菌株X-33染色体上,构建了X-33(pPICZa-ORF2)重组工程菌.经甲醇诱导后,成功的表达出ORF2基因片段.经过Bradford 蛋白质总含量测定和凝胶薄层扫描结果表明,表达产物占重组工程菌培养上清总蛋白的58%,表达量可达47mg/ L.间接ELISA结果初步表明重组表达产物具有良好的抗原性,能够有效地区分PCV2型病毒标准阳性与阴性血清.

A strain of virus was isolated from 20 cloacal of ducks in Nanjing using SPF chicken embryos.The virus showed haemagglutination activity but not inhibited by positive sera against New castle disease virus(NDV) and egg-drop syndrome-76 virus(EDSV).HI test indicated that the isolate belonged to H9 subtype of influenza virus.The assay of intravenous pathogenicity showed that the isolate had low pathogenicity to chickens,but no pathogenicity to ducks.The study also indicated that the 5-week-mouses could be infected with morbidity and mortality.

根据已发表的马立克氏病病毒(MDV)血清I型与鸡白介素2(IL-2)DNA序列,设计两对引物,用PCR扩增出MDV gB基因和IL-2基因,并克隆入pEGFP-C1载体,构建了载体pC1-gB-IL.通过酶切分离外源基因表达盒,并将外源基因表达盒插入pTK2B质粒,成功构建了转移载体pT-C-gB-IL.将转移载体pT-C-gB-IL转染CEF细胞,培养36～48 h后,经荧光显微镜观察,有荧光出现,证明该载体得到表达,并通过Western-blotting鉴定证明MDV gB和IL-2的融合基因得到了有效的表达.

1 发病情况 南通市某肉鸭场于2003年7月份出孵雏鸭5 000余只,采用池边网床饲养.雏鸭16日龄开始发病,并出现死亡.随后2 d内死亡1 300多只.用链霉素、恩诺沙星给鸭饮水,但效果不佳,病情得不到控制.