毒害艾美耳球虫初次感染雏鸡免疫器官IL-2和淋巴细胞增殖功能变化

应用组织匀浆涂片和酸性α-醋酸萘酯酶（ANAE）染色及细胞培养技术和四甲基偶氮唑盐（MTT）测定法对毒害艾美耳球虫（eimeria necatrix，E．necatrix）初次感染雏鸡免疫器官的T细胞比例、白细胞介素-2（interleukin-2，IL-2）诱生活性、T细胞和B细胞对ConA或PMA的增殖功能的动态变化进行了较全面系统的研究。结果发现，E．necatrix初次感染雏鸡，其胸腺和脾脏T细胞比例分别于感染后7～21d和7～24d明显高于对照雏鸡；IL-2诱生活性分别于感染后16～18d和18～21d较对照雏鸡显著升高；T细胞对ConA的增殖反应分别在感染后14～16d和10～18d明显增加。法氏囊和脾脏B细胞对PMA的增殖反应分别于感染后14～24d和14～21d显著高于对照雏鸡。表明E．necatrix初次感染雏鸡免疫器官的IL-2调节及细胞免疫和体液免疫功能均明显提高。     

Validation and application of real-time polymerase chain reaction assays for representative rumen bacteria

Real‐time polymerase chain reaction (PCR) assays for 11 representative rumen bacterial species were validated. The sensitivity was tested by using the serially diluted target 16S rDNA from respective bacterial species. The recovery of the target DNA and the assay reproducibility were determined using DNA from rumen fluid spiked with different quantities of the target. Minimum detection levels for the target were 10–100 copies in pure culture. The recovery of the added target ranged from 82.4 to 116.6%. The intra‐ and inter‐assay variations of each assay were <9.4 and <12.6%, respectively. Therefore, the real‐time PCR assays evaluated in the present study are considered to be sufficiently reliable for monitoring all 11 bacterial species in the rumen. The assays were then applied to the monitoring of the bacterial species attached to ruminally incubated rice straw. Among the monitored fibrolytic species, Fibrobacter succinogenes was found to be the most dominant, accounting for 2.61% of total bacteria after 24 h incubation. Selenomonas ruminantium and Streptococcus bovis, non‐fibrolytics, were detected on the rice straw at 8.96% and 1.16% of total bacteria, respectively. Such high levels of non‐fibrolytics on the plant fiber suggest a synergistic relationship between fibrolytics and non‐fibrolytics.     

16S rRNA基因及外膜蛋白基因分析在鸭疫里默氏菌鉴定中的应用

本实验对鸭疫里默氏菌（包括6个血清型）、大肠杆菌、鼠伤寒沙门氏菌和多杀性巴氏杆菌进行了16S rRNA基因片段的扩增和测序，并将测序结果进行了同源性比较和酶切分析；同时根据标准血清2型鸭疫里默氏菌外膜蛋白的基因序列设计一对引物进行扩增。根据16S rRNA的测序结果及EcoRⅠ和HaeⅢ酶切片段分析，可将鸭疫里默氏菌与其它临床症状易混淆的细菌感染相区别，而外膜蛋白基因序列的特异性也为鸭疫里默氏菌的鉴别诊断提供了一种快速、准确的PCR鉴定方法。     

广西沼泽型水牛IL-18和TNF-α基因的克隆与序列分析

从健康沼泽型水牛静脉无菌采血,分离外周血单核细胞(PBMCs),用刀豆素A(ConA)诱导培养3,8和12 h后,提取细胞总RNA,以Oligo(dT)18为引物合成第一链cDNA,根据牛的白细胞介素18(IL-18)和绵羊的肿瘤坏死因子α(TNF-α)基因序列设计2对特异性引物,通过PCR方法扩增出水牛IL-18和TNF-α基因,将其克隆到pMD18-T载体,测序后进行序列分析。结果试验中所克隆到的IL-18和TNF-α基因序列的开放阅读框(ORF)分别为602 bp和715 bp,与GenBank所登录的水牛IL-18和TNF-α核苷酸序列同源性为99.1%和99.7%,其推导的氨基酸序列同源性分别是99.0%和98.7%。表明成功从水牛外周血中克隆到了水牛IL-18和TNF-α基因。     

Genetic, phenotypic and pathogenic diversity among xanthomonads isolated from pistachio (Pistacia vera) in Australia

Repetitive extragenic palindromic polymerase chain reaction (rep-PCR), sequencing of the 16S−23S rDNA internal transcribed spacer (ITS), biochemical and physiological tests, the Biolog microplate system, polyacrylamide gel electrophoresis (PAGE) of whole-cell proteins, and pathogenicity tests were used to characterize variability among xanthomonads isolated from pistachio trees suffering from bacterial dieback in four regions of Australia. ITS sequencing and rep-PCR revealed two distinct genotypes among the strains. The ITS sequencing suggested that the pistachio strains were closely related to Xanthomonas translucens pathovars, in particular X. translucens pv . poae . Results of physiological and biochemical tests, as well as Biolog microplate analysis and protein profiling, confirmed the existence of two groups. Furthermore, pathogenicity and host-range studies indicated that the two groups were biologically different. There was an association between the two groups and the geographical origin of the strains.     

16S rRNA基因在细菌菌种鉴定中的应用

本文综述了应用16S rRNA基因作为靶基因对细菌进行鉴定的各种分子生物学技术，并指出应用16S rRNA基因在乳酸菌鉴定方面的研究进展。     

猪IL-2真核表达质粒构建及对PRRSV-ORF5基因疫苗免疫佐剂作用的研究

本研究应用真核表达载体pcDNA3.1（＋）和猪白细胞介素2（IL-2）基因成功构建了IL-2的真核表达质粒（pcDNA-IL-2）,探讨了pcDNA-IL-2作为分子免疫佐剂对猪繁殖与呼吸综合征（PRRS）ORF5基因疫苗（pcDNA-PRRSV-SC2-ORF5）免疫猪的增强作用。经间接ELISA法、MTT比色法及流式细胞仪分别对pcDNA-IL-2与pcDNA-PRRSV-SC2-ORF5共同免疫猪、pcDNA-PRRSV-SC2-ORF5单独免疫猪、pcDNA3.1（＋）空载体和灭菌水免疫对照猪的血清抗PRRSV抗体IgG、外周血T淋巴细胞的增殖活性、CD4^＋和CD8^＋细胞比例进行检测,结果表明pcDNA-IL-2与pcDNA-PRRSV-SC2-ORF5共同免疫猪的IgG含量、外周血T淋巴细胞的增殖活性、CD4^＋和CD8^＋细胞比例与pcDNA-PRRSV-SC2-ORF5单独免疫猪相比有显著差异（P〈0.05）。说明pcDNA-IL-2能显著增强pcDNA-PRRSV-SC2-ORF5基因疫苗免疫猪的体液和细胞免疫应答,可作为PRRSV基因疫苗的良好佐剂。     

‘Candidatus Phytoplasma australiense’ is the phytoplasma associated with Australian grapevine yellows, papaya dieback and Phormium yellow leaf diseases

Sequence comparisons and phylogenetic analysis of the 16S rRNA genes and the 16S/23S spacer regions of the phytoplasmas associated with Australian grapevine yellows, papaya dieback and Phormium yellow leaf diseases revealed minimal nucleotide differences between them resulting in the formation of a monophyletic group. Therefore, along with Australian grapevine yellows, the phytoplasmas associated with Phormium yellow leaf and papaya dieback should also be considered as Candidatus Phytoplasma australiense.     

Genotypic and phenotypic characterization of lactic acid bacteria isolated from Italian ryegrass silage

Twenty-three lactic acid bacteria (LAB) isolated from three cultivars (Akiaoba, Nagahahikari and Tachiwase) of Italian ryegrass (Lolium multiflorum Lam.) silage were precisely characterized by a combination of phenotypic tests, genotypic 16S ribosomal DNA sequencing and rapid PCR-based analyses, focusing on their useful phenotypes for silage preparation as inoculants. We successfully identified both at the species and subspecies levels: phenotypically novel Lactococcus lactis subsp. lactis, Lactobacillus brevis, Lactobacillus coryniformis subsp. torquens, Lactobacillus curvatus, Lactobacillus plantarum subsp. plantarum, Lactobacillus sakei subsp. carnosus, Leuconostoc mesenteroides subsp. dextranicum and Pediococcus parvulus. This is the first report to elucidate the presence of Lactobacillus coryniformis ssp. torquens and Leuconostoc mesenteroides subsp. dextranicum in Italian ryegrass silages. Physiological and biochemical tests revealed that phenotypic characteristics are different among the different strains of the same species and subspecies, and that the isolates show unique and diverse phenotypes related to fermentation factors, such as available carbohydrates, optimal growth pH and temperature. These results suggest that, for various well-preserved silage preparations, the isolates may be useful in producing novel inoculants corresponding to their optimally climatic and ecological niches.     

circADAMTS16的表达谱分析及其对牛脂肪细胞分化的影响

旨在探究 circADAMTS16在牛不同组织和前体脂肪细胞中的表达水平及其对牛脂肪细胞分化的影响，为进一步研究circRNA在细胞分化和脂肪组织发育过程中的调控作用提供依据。采用组织块法分离培养牛原代前体脂肪细胞并诱导分化，模拟牛脂肪组织生长发育过程；通过qRT-PCR检测 circADAMTS16在牛不同组织（心、肝、脾、肺、肾、肌肉、脂肪）和脂肪细胞分化不同阶段（0 d、2 d、4 d、8 d、10 d）的表达水平；构建 circADAMTS16的过表达载体（pCD2.1- circADAMTS16），设计合成 circADAMTS16的小干扰RNA （si- circADAMTS16），采用（Lipofectamine3000）转染试剂将pCD2.1- circADAMTS16和si- circADAMTS16转染牛前体脂肪细胞，利用qRT-PCR法检测转染效果，同时检测脂肪分化标志基因PPARγ、C/EBPα和 C/EBPβ的表达量变化，并进行油红O染色，综合分析干扰和过表达 circADAMTS16对牛脂肪细胞分化的影响。结果如下：①qRT-PCR检测结果表明 circADAMTS16在心脏中表达量最高，肝脏次之，脾最低；②在牛原代前体脂肪细胞分化过程中， circADAMTS16在第2天显著高表达，随后逐渐降低，在第8天时达到最低。③在牛前体脂肪细胞中过表达circADAMTST16后，脂肪分化标志基因PPARγ、C/EBPα、C/EBPβ的表达量显著下降；油红O染色结果显示，过表达后脂滴形成数量明显少于对照组；而干扰 circADAMTS16表达后，脂肪分化标志基因PPARγ和C/EBPα表达量显著上升。综上所述， circADAMTS16对牛原代前体脂肪细胞的分化过程具有显著的调控作用，过表达 circADAMTST16可抑制牛原代前体脂肪细胞分化进程，而干扰 circADAMTS16表达可以促进牛原代前体脂肪细胞分化进程，探明 circADAMTS16对牛原代前体脂肪细胞分化的具体调控作用。