感染16型蓝舌病病毒后绵羊部分细胞因子消长特点研究

本试验旨在探明绵羊感染16型蓝舌病病毒（Bluetongue virus type 16,BTV16）后细胞因子IFN-γ、IL-2、IL-4和IL-10的消长特点。用实时荧光定量PCR检测方法对感染BTV16后3只绵羊上述4种因子mRNA进行检测,同时设立阴性对照绵羊,并以0 d mRNA为基准,计算mRNA的相对表达量,同时检测病毒抗体效价、测量绵羊体温。结果显示,接种BTV16的3只绵羊均不同程度产生抗体和体温症状,4种细胞因子的mRNA在接种病毒2~4 d内均出现显著上升,其中IFN-γ峰值在2.58~27.84倍之间,IL-2峰值在5.24~17.19倍之间,IL-4峰值在2.16~3.43倍之间,IL-10峰值在15.78~48.77倍之间,个体上升幅度存在显著差异,4种细胞因子均在高水平持续6 d左右后逐渐下降。对照绵羊上述参数在正常范围内波动。本研究阐明了接种BTV16后绵羊细胞因子IFN-γ、IL-2、IL-4、IL-10在转录水平上的消长特点,为进一步深入开展BTV感染特征、宿主机体免疫机制研究提供参考。     

伪狂犬病病毒在NF-κB家族p65基因敲除细胞系的复制规律研究

试验旨在研究伪狂犬病病毒(PRV)在NF-κB家族p65基因敲除细胞系中的复制规律。利用慢病毒介导的CRISPR/Cas9基因定点修饰技术构建猪肺泡巨噬细胞(3D4/21)p65基因稳定敲除细胞系。通过构建p65-sgRNA重组质粒,转染至HEK293T/17细胞,收取慢病毒,感染3D4/21细胞后利用嘌呤霉素筛选获得多克隆细胞系,T7核酸酶检测敲除效率,再通过有限稀释法获得3D4/21-p65^-/-的稳定细胞系。CCK-8试剂盒检测3D4/21细胞中敲除p65基因后对细胞增殖的影响;流式细胞术检测PRV-GFP感染3D4/21及3D4/21-p65^-/-细胞后病毒增殖的差异;实时定量PCR检测PRV感染3D4/21及3D4/21-p65^-/-细胞后PRV gB、TK基因mRNA表达水平及PRV感染细胞诱导的IL-1β和IL-6基因mRNA水平表达的变化;Western blotting检测PRV-QXX感染3D4/21及3D4/21-p65^-/-细胞后PRV gB、gE蛋白的表达;滴度测定检测PRV-QXX感染3D4/21及3D4/21-p65^-/-细胞后子代病毒滴度。结果表明,sgRNA2和sgRNA3的基因编辑效率较高,对其进行克隆化培养进而获得敲除p65基因的稳定表达细胞系;CCK-8试剂盒检测细胞活力表明,p65基因敲除对细胞活力无影响;流式细胞仪检测表明,同一时间点PRV-GFP在3D4/21-p65^-/-中的增殖显著高于对照细胞;实时荧光定量PCR表明在3D4/21细胞中敲除p65基因促进了PRV gB、TK基因的mRNA表达水平,而抑制了IL-1β、IL-6基因的mRNA表达;Western blotting结果表明,在3D4/21细胞中敲除p65基因促进了PRV gB、gE蛋白的表达;滴度测定结果表明,同一时间点PRV-QXX在3D4/21-p65^-/-细胞中子代病毒的复制显著高于对照细胞。以上结果均表明,p65基因敲除可促进PRV在3D4/21细胞中复制。     

伪狂犬病病毒在NF-κB家族p65基因敲除细胞系的复制规律研究

试验旨在研究伪狂犬病病毒（PRV）在NF-κB家族p65基因敲除细胞系中的复制规律。利用慢病毒介导的CRISPR/Cas9基因定点修饰技术构建猪肺泡巨噬细胞（3D4/21）p65基因稳定敲除细胞系。通过构建p65-sgRNA重组质粒,转染至HEK293T/17细胞,收取慢病毒,感染3D4/21细胞后利用嘌呤霉素筛选获得多克隆细胞系,T7核酸酶检测敲除效率,再通过有限稀释法获得3D4/21-p65^-/-的稳定细胞系。CCK-8试剂盒检测3D4/21细胞中敲除p65基因后对细胞增殖的影响;流式细胞术检测PRV-GFP感染3D4/21及3D4/21-p65^-/-细胞后病毒增殖的差异;实时定量PCR检测PRV感染3D4/21及3D4/21-p65^-/-细胞后PRV gB、TK基因mRNA表达水平及PRV感染细胞诱导的IL-1β和IL-6基因mRNA水平表达的变化;Western blotting检测PRV-QXX感染3D4/21及3D4/21-p65^-/-细胞后PRV gB、gE蛋白的表达;滴度测定检测PRV-QXX感染3D4/21及3D4/21-p65^-/-细胞后子代病毒滴度。结果表明,sgRNA2和sgRNA3的基因编辑效率较高,对其进行克隆化培养进而获得敲除p65基因的稳定表达细胞系;CCK-8试剂盒检测细胞活力表明,p65基因敲除对细胞活力无影响;流式细胞仪检测表明,同一时间点PRV-GFP在3D4/21-p65^-/-中的增殖显著高于对照细胞;实时荧光定量PCR表明在3D4/21细胞中敲除p65基因促进了PRV gB、TK基因的mRNA表达水平,而抑制了IL-1β、IL-6基因的mRNA表达;Western blotting结果表明,在3D4/21细胞中敲除p65基因促进了PRV gB、gE蛋白的表达;滴度测定结果表明,同一时间点PRV-QXX在3D4/21-p65^-/-细胞中子代病毒的复制显著高于对照细胞。以上结果均表明,p65基因敲除可促进PRV在3D4/21细胞中复制。     

Influence of four soil maintenance practices on Collembola communities in a Mediterranean vineyard

We studied the influence of four soil maintenance practices on Collembola communities in the soil of a Mediterranean vineyard: (a) postemergence herbicide with glyphosate; (b) postemergence and pre-emergence herbicides with glyphosate, terbuthylazine, diuron and oryzalin; (c) natural flora and (d) tillage to a depth of 10–15 cm. Total Collembola abundance, species diversity and species richness significantly varied between the four practices. Notably, the practice using postemergence and pre-emergence herbicides had significantly lower values. Identification of Collembola at species level allowed an interspecies comparison and revealed significant differences for the most common species between the four practices, with each practice being characterized by a different set of species. None of the species were found to be significantly more abundant in the plots treated with postemergence and pre-emergence herbicides.     

内蒙古红豆草黄萎病菌的分离鉴定及其生物学特性

依照柯赫氏法则,利用形态学结合分子生物学的方法首次对内蒙古自治区呼和浩特市中国农科院草原研究所沙尔沁基地红豆草疑似黄萎病的病样进行分离和鉴定,利用蘸根接种法对病原菌的致病性进行测定。同时,首次在室内条件下对病菌的生物学特性,如生长最适温度、酸碱度以及碳源、氮源对病原菌生长速率和产孢量的影响进行了研究。结果表明,引起沙尔沁基地红豆草黄萎病的病原菌为大丽轮枝孢菌,菌株具有较宽的温度和酸碱度适应范围,最适于菌株生长和产孢的温度均为25℃,最适于菌株生长和产孢的pH值范围分别为7~10和5~6。在碳源选择上,大丽轮枝孢菌在可溶性淀粉上的生长速度最快,而蔗糖最有利于其产孢。在氮源选择上,大丽轮枝孢菌在牛肉膏和L-脯氨酸上的生长速度较快,且L-脯氨酸最有利于其产孢。     

Production and Identification of Clone Sheep Using Bone Marrow Mesenchymal Stem Cells as Donor Cell

In order to obtain the cloned sheep using bone marrow mesenchymal stem cells（BMSCs）as the donor cells,the BMSCs of sheep were chosen and reconstructed embryos were built to transfer.10 published microsatellite markers were chosen,and the DNA samples from clone sheep,donor cells and surrogate ewes were amplified,and the relationship of father-son（RCP）was analyzed using the Quantity One for genotyping.The results showed that the reconstructed embryos were successfully built for electric fusion using sheep BMSCs as nuclear donor,and making nuclear transplantation into enucleated mature oocytes of which the fusion rate was 80.62%.20 surrogate ewe were chosen to be implanted with the reconstructed embryos at morula stage by implant surgery,and 5 lambs were born and only 3 were survived.The genotype of cloned sheep was in line with the dornor cell and the RCP were more than 99.999%.In conclusion,the first clone sheep were obtained successfully by using BMSCs as a nuclear donor in this experiment.     

Assessment of the socio-economic impacts associated with the arrival of apple snail (Pomacea canaliculata) in Mwea irrigation scheme,Kenya

Background

In Kenya, rice (Oryza sativa L.) is mainly produced under irrigation by small-scale farmers. Mwea irrigation scheme (MIS) in Kirinyaga County accounts for 80–88% of rice production. Here, rice is the main source of livelihood and a source of revenue generation for the county. However, a recently established invasive freshwater snail, Pomacea canaliculata (Lamarck) (family: Ampullariidae), a species of apple snail, presents a serious threat to rice production.

Results

Household surveys, focus group discussions and key informant interviews highlight apple snail as a serious problem in MIS. Households that observed at least a moderate level of infestation (>20% of cultivated area) experienced significant reductions in rice yield (~14%) and net rice income (~60%). Farmers reported increased use of chemical pesticides for management of apple snail. In addition, the cost of hired labor for physical removal of egg masses and snails is resulting in substantial negative effects on net income. Farmer age, area of land owned, responsibility for decision-making, receipt of extension advice, training, and membership of a farmer organization, were all statistically significant variables to explain farmers awareness of the need for area-wide apple snail management.

Conclusion

Strategies to limit the spread of apple snail are urgently needed. A Multi-Institutional Technical Team (MITT) has been established to spearhead management efforts and consolidate advice to farmers on how to manage apple snail. However, without action to mitigate spread, the consequences could be disastrous for rice production and food security in Kenya, and for other rice growing regions across Africa. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.