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The CRISPR/Cas9 induces large genomic fragment deletions of MSTN and phenotypic changes in sheep

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Journal of Integrative Agriculture 2020年第4期19卷 1065-1073页

作者： DING Yi ZHOU Shi-wei DING Qiang cai Bei ZHAO Xiao-e ZHONG Shu JIN Miao-han wang Xiao-long MA Bao-hua CHEN Yu-lin college of animal science and Technology Northwest A&F UniversityYangling 712100P.R.China college of veterinary medicine Northwest A&F UniversityYangling 712100P.R.China

The CRISPR/Cas9 system has been extensively used to engineer genetic loci for the generation of knockouts, insertions, and point mutations in animal models. However, many mutations that have been reported in animals are small insertions or deletions. This study used the CRISPR/Cas9 system to induce large DNA fragment deletions in MSTN via three guide RNAs in sheep. This successfully achieved the precise gene editing of the ovine MSTN gene by injecting both Cas9 m RNA and sg RNAs into embryos at the one-cell stage. Of 10 edited animals, 3 animals(30%) exhibited large genomic fragment deletions(~5 kb). Furthermore, the body weights of these 3 animals were significantly different(P01, P15=0.001, P30=0.005, P60=0.027) between lambs with large deletions and wildtype lambs. In addition, the edited lambs were also significantly different(P01, P151, P30=0.002, P60=0.011) compared with wildtype. These results suggest that the generated MSTN knockout sheep is a reliable and effective animal model for further study. Furthermore, this method is time-and labor-saving, and efficient for the creation of animal models for agriculture, biology, and medicine.

关键词： CRISPR/Cas9 sheep MSTN large deletion genetic engineering

Genomic analysis of indigenous goats in Southwest Asia reveals evidence of ancient adaptive introgression related to desert climate

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Zoological Research 2023年第1期44卷 20-29页

作者： Hojjat Asadollahpour Nanaei Yudong cai Akil Alshawi Jiayue Wen Tanveer Hussain Wei-Wei Fu Nai-Yi Xu Abdulameer Essa Johannes ALenstra Xihong wang Yu Jiang Key Laboratory of animal Genetics Breeding and Reproduction of Shaanxi ProvinceCollege of Animal Science and TechnologyNorthwest A&F UniversityYanglingShaanxi 712100China Department of Internal and Preventive medicine College of Veterinary MedicineUniversity of BaghdadIraqi Ministry of Higher Education and Scientific ResearchBagdad 10090Iraq Department of Molecular Biology Virtual University of PakistanLahore 54000Pakistan animal Genetics Resources Department Directorate of Animal Resourcesthe Ministry of Iraqi AgricultureBaghdad 10081Iraq Faculty of veterinary medicine Utrecht University3508 TD UtrechtThe Netherlands

Understanding how evolutionary pressures related to climate change have shaped the current genetic background of domestic animals is a fundamental pursuit of biology. Here, we generated wholegenome sequencing data from native goat populations in Iraq and Pakistan. Combined with previously published data on modern, ancient(Late Neolithic to Medieval periods), and wild Capra species worldwide, we explored the genetic population structure, ancestry components, and signatures of natural positive selection in native goat populations in Southwest Asia(SWA). Results revealed that the genetic structure of SWA goats was deeply influenced by gene flow from the eastern Mediterranean during the Chalcolithic period, which may reflect adaptation to gradual warming and aridity in the region. Furthermore, comparative genomic analysis revealed adaptive introgression of the KITLG locus from the Nubian ibex(C. nubiana) into African and SWA goats. The frequency of the selected allele at this locus was significantly higher among goat populations located near northeastern Africa. These results provide new insights into the genetic composition and history of goat populations in the SWA region.

关键词： Goat Adaptation KITLG Southwest Asia Nubian ibex

Emergence of a novel reassortant avian influenza virus(H10N3) in Eastern China with high pathogenicity and respiratory droplet transmissibility to mammals

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science China(Life sciences) 2022年第5期65卷 1024-1035页

作者： Kaituo Liu Pingyun Ding Yuru Pei Ruyi Gao Wenwen Han Huafen Zheng Zhuxing Ji Miao cai Jinyuan Gu Xiuli Li Min Gu Jiao Hu Xiaowen Liu Shunlin Hu Pinghu Zhang Xiaobo wang Xiaoquan wang Xiufan Liu college of veterinary medicine Yangzhou UniversityYangzhou 225009China Jiangsu Co-innovation Center for Prevention and Control of Important animal Infectious Diseases and Zoonosis Yangzhou UniversityYangzhou 225009China Jiangsu Key Laboratory of Zoonosis Yangzhou 225009China Institute of Translational medicine Key Laboratory of Geriatric Disease Prevention and Control of Jiangsu ProvinceMedical CollegeYangzhou UniversityYangzhou 225009China

Decades have passed since the first discovery of H10-subtype avian influenza virus(AIV) in chickens in 1949,and it has been detected in many species including mammals such as minks,pigs,seals and *** of human infections with H10N8viruses identified in China in 2013 have raised widespread *** novel reassortant H10N3 viruses were isolated from chickens in December 2019 in eastern China during routine surveillance for *** internal genes of these viruses were derived from genotype S(G57) H9N2 and were consistent with H5N6,H7N9 and H10N8,which cause fatal infections in *** viral pathogenicity and transmissibility were further studied in different animal *** two H10N3 isolates had low pathogenicity in chickens and were transmitted between chickens via direct *** viruses were highly pathogenic in mice and could be transmitted between guinea pigs via direct contact and respiratory *** importantly,these viruses can bind to both human-type SAα-2,6-Gal receptors and avian-type SAα-2,3-Gal *** shedding in chickens and good adaptability to mammals of these H10N3 isolates would make it easier to transmit to humans and pose a threat to public health.

关键词： H10N3 H9N2 receptor binding pathogenicity respiratory droplet transmissibility

甘肃某羊场免疫布鲁菌病疫苗后绵羊流产原因的调查及分析

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中国兽医科学 2024年第8期54卷 1103-1110页

作者：刘志杰刘萍吴锦艳尚佑军何继军王子坚蔡江聂帅帅李鹏飞杜国玉苟惠天曹小安甘肃农业大学动物医学院甘肃兰州川730070 中国农业科学院兰州兽医研究所动物疫病防控全国重点实验室甘肃兰州730046 甘肃省动物疫病预防控制中心甘肃兰州730046

应用疫苗免疫绵羊后,羊场依然出现大规模流产和不孕,为查明原因,收集该羊场31份流产羊和23份不孕羊的阴道分泌物拭子及其对应的羊血清进行检测。用竞争酶联免疫吸附试验(c ELISA)、布鲁菌抗体Ig G检测试验和试管凝集试验(SAT)检测血清;... 详细信息

应用疫苗免疫绵羊后,羊场依然出现大规模流产和不孕,为查明原因,收集该羊场31份流产羊和23份不孕羊的阴道分泌物拭子及其对应的羊血清进行检测。用竞争酶联免疫吸附试验(c ELISA)、布鲁菌抗体Ig G检测试验和试管凝集试验(SAT)检测血清;用荧光PCR做54份拭子流产相关的布鲁菌病、Q热和衣原体病的病原检测;对检测布鲁菌阳性的样本用AMOS-PCR进行布鲁菌种属鉴定,扩增利福平耐药基因(rpoB)并测序,将序列与其标准参考菌株比对后构建进化树。结果显示:用布鲁菌c ELISA检测54份血清,其中22份血清初筛阳性,阳性率为40.7%;SAT确诊其中16份样本为阳性,其中流产羊有6只阳性,阳性率为19.4%,不孕羊有10只阳性,阳性率为43.5%,总体阳性率为29.6%。用布鲁菌抗体Ig G检测试验检测感染的样本19份发现,流产羊有6只阳性,阳性率为19.4%,不孕羊有13只阳性,阳性率为56.5%,总体阳性率为35.2%。荧光PCR结果显示:拭子样本10份为布鲁菌阳性,阳性率为18.5%;12份为贝氏柯克斯体(Q热病原)阳性,阳性率为22.2%;衣原体阳性率为0。布鲁菌阳性样本AMOS-PCR扩增得到731 bp的条带,rpoB基因分析发现所有氨基酸序列都未发现利福平耐药突变,AMOS-PCR和rpoB基因系统进化树分析结果显示羊感染的为马耳他布鲁菌。结论:本场群发性流产的发生可能是由于布鲁菌病疫苗免疫失败后病原感染,并伴随着贝氏柯克斯体等病原的混合感染引起。

关键词：布鲁菌病 Q热流产 rpoB基因

Transmission of Carbapenem Resistance Between Human and animal NDM-Positive Escherichia coli Strains

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Engineering 2022年第8期8卷 24-33页

作者： Yingbo Shen Fupin Hu Yongqiang wang Dandan Yin Lu Yang Yiqiang Chen Chunyan Xu Jiyun Li Junyao Jiang Xueyang wang Yulin Fu Dongyan Shao Dejun Liu Tengfei Ma Chang cai Zhangqi Shen Shaolin wang Juan Li Rong Zhang Yuebin Ke Congming Wu Jianzhong Shen Timothy R.Walsh Yang wang Beijing Key Laboratory of Detection Technology for animal-Derived Food Safety College of Veterinary MedicineChina Agricultural UniversityBeijing 100193China CAS Key Laboratory of Pathogenic Microbiology and Immunology Institute of MicrobiologyChinese Academy of SciencesBeijing 100101China Institute of Antibiotics&Key Laboratory of Clinical Pharmacology of Antibiotics(MoH) Huashan HospitalFudan UniversityShanghai 200040China Department of Preventive veterinary medicine College of Veterinary MedicineChina Agricultural UniversityBeijing 100193China State Key Laboratory of animal Nutrition College of Animal Science and TechnologyChina Agricultural UniversityBeijing 100193China college of animal science and Technology College of Veterinary MedicineZhejiang A and F UniversityHangzhou 311300China State Key Laboratory for Infectious Disease Prevention and Control Collaborative Innovation Center for Diagnosis and Treatment of Infectious DiseaseNational Institute for Communicable Disease Control and PreventionChinese Center for Disease Control and Prevention(CDC)Beijing 102206China The Second Affiliated Hospital of Zhejiang University Zhejiang UniversityHangzhou 310009China Key Laboratory of Genetics&Molecular medicine of Shenzhen Shenzhen Center for Disease Control and PreventionShenzhen 518055China Department of Zoology University of OxfordOxford OX13SZUK

Although carbapenem use is prohibited in animals in China,carbapenem-resistant Escherichia coli(CREC),especially New Delhi metallo-β-lactamase(NDM)-producing strains,are widely prevalent in foodproducing *** present,the impact of livestock-associated CREC strains on human populations at the national level is ***,we conduct a retrospective cross-sectional study to investigate the prevalence of CREC from clinical settings across 22 Chinese provinces or municipalities and analyze anthropogenic factors associated with their *** also ascertain the blaNDMand blaKPCabundance among pig and chicken farms and present a detailed genomic framework for CREC of animal and human ***,631/29799(2.1%)clinical Escherichia coli(***)isolates were identified as *** analysis revealed that being male,an age below 1,an age between 13 and 18,provinces with greater chicken production,and provinces with higher pig production were associated with higher odds of CREC *** general,73.8%(n=45/61)of pig farms and 62.2%(n=28/45)of chicken farms had a blaNDMabundance of 1×10^(-5)to 1×10^(-3)and 1×10^(-3)to 1×10^(-2),*** all the Chinese NDM-positive ***(n=463)available at the National Center for Biotechnology Information(NCBI),the genomic analysis revealed that blaNDM-5and Inc X3 were the predominant carbapenemase gene-plasmid combination,while a highly homogeneous relationship between NDM-positive isolates from humans and animals was demonstrated at the plasmid and core genome *** the findings suggest frequent CREC transmission between humans and animals,indicating that further discussions on the use of antibiotics in animals and humans are needed,both in China and across the globe.

关键词： Carbapenem resistance Escherichia coli blaNDM-5 One Health Antimicrobial resistance transmission

Screening for Monoclonal Antibodies Against the Membrane Proteins of Chicken Embryo Fibroblast Blocking the Infection of IBDV

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Agricultural sciences in China 2010年第2期9卷 294-298页

作者： YANG Yan-yan ZHU Li-qian YANG Ji-fei TENG Man cai Shu-jun wang Li LUO Jun DENG Rui-guang ZHANG Gai-ping Henan Provincial Key Laboratory of animal Immunology Henan Academy of Agricultural Sciences Zhengzhou 450002 P.R.China college of veterinary medicine Yangzhou University Yangzhou 225009 P.R.China

This article aims to establish an efficient assay for screening monoclonal antibodies （McAbs） against the membrane proteins of chicken embryo fibroblast （CEF） for further studies of the cellular receptors of infectious bursal disease virus （IBDV）. McAbs against the membrane proteins of CEF were prepared by cell fusion. The monolayer CEF pre-incubated with the CEF-specific McAbs for 2 h were infected with IBDV and incubated with F22-EA6-biotin postinfection. Then, the cells were reacted with streptavidin-horseradish peroxidase （HRP） and finally stained by 3-amino-9-ethylcarbazole （AEC）. The inhibitive percentage of IBDV infection was calculated by counting the IBDV-infected cells to determine the inhibition efficiency of the CEF-specific McAbs. Compared with the control cells, the IBDV-infected cells pretreated with CEF-specific antibody significantly decreased; supernatant fluids of a total of 768 hybridomas were analyzed. The results of immunohistochemistry assays showed that six of them （1A5, 1H11, 2B 12, 3G1, 4D10, and 4B8） have the abilities to block the infection of IBDV to CEF, among which 4B8 can perfectly block the infection. This novel method is a sensitive and specific assay for the screening of CEF membrane protein-specific McAbs, which can block the infection of IBDV to CEF, and these McAbs can be used for the further investigations of the cellular receptors of IBDV.

关键词： CEF IBDV receptor biotin-streptavidin

Attenuation of Virulent Porcine Reproductive and Respiratory Syndrome Virus Strain CH-1a and Genetic Variation of ORF5 Gene

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Journal of Integrative Agriculture 2012年第12期11卷 2035-2042页

作者： cai Xue-hui WU Guo-jun LIU Yong-gang LIU Guang-qing SHI Wen-da wang Shu-jie MA Ping LI Cheng-jun HAN Wen-yu college of animal science and veterinary medicine Jilin University State Key Laboratory of veterinary Biotechnology Harbin Veterinary Research Institute Chinese Academy of Agricultural Sciences Division of Avian Infectious Diseases Shanghai Veterinary Research Institute Chinese Academy of Agricultural Sciences

To develop a modified live vaccine （MLV） against porcine reproductive and respiratory syndrome virus （PRRSV）, virulent CH-Ia strain was attenuated by serial passages up to 130 passage （P130） in Marc-145 cells. The virulence and immune efficacy of the attenuated CH-1 a were evaluated in pigs. The results showed that animals inoculated with P130 did not develop any clinical sign of the disease, but produced rapid and effective humoral immune responses against PRRSV challenge, indicating that attenuated CH-1 a P 130 is the candidate as the effective vaccine against PRRSV. To define the potential mutations in the attenuated CH-la genome, we sequenced and analyzed the ORF5 gene of CH-la strain of different passages （P39, P55, P65, P70, P85, P100, P115, P120, P125, and P130） and found that three mutations （C5Y, H38Q and L146Q） which may be related with the attenuation of CH-la. In addition, we also found a unique restriction enzyme site （TspEI） in the ORF5 gene of attenuated CH-la, which can be used as a genetic marker to distinguish original and attenuated CH- 1 a.

关键词： attenuation porcine reproductive and respiratory syndrome virus genetic variation ORF5 gene

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黑龙江畜牧兽医 2024年第18期 40-44页

作者：庞明月张太鹏蔡家宝王宏肖霖力宋玉清许晓玲白佳桦刘彦秦玉圣北京农学院动物科技学院北京102206 北京市农林科学院畜牧兽医研究所北京100097 河北工程大学生命科学与食品工程学院河北邯郸056038 河北省种猪繁育技术创新中心河北张家口075000

为了探讨Kisspeptin(KP)对颗粒细胞类固醇激素合成相关基因的mRNA表达水平及定时输精母猪繁殖性能的影响,试验先分离了母猪卵巢中的颗粒细胞,用KP-10处理颗粒细胞,以未处理颗粒细胞为对照,研究KP-10对颗粒细胞类固醇激素合成酶细胞色素P... 详细信息

为了探讨Kisspeptin(KP)对颗粒细胞类固醇激素合成相关基因的mRNA表达水平及定时输精母猪繁殖性能的影响,试验先分离了母猪卵巢中的颗粒细胞,用KP-10处理颗粒细胞,以未处理颗粒细胞为对照,研究KP-10对颗粒细胞类固醇激素合成酶细胞色素P450家族11亚家族A成员1(CYP11A1)、3β-羟基类固醇脱氢酶(3β-HSD)、促卵泡激素受体(FSHR)、促黄体生成素受体(LHCGR)基因的mRNA表达水平的影响;然后选择健康的大白×长白二元杂交初产断奶母猪202头,随机分为PMSG对照组(n=102)、KPL组(n=50)和KPH组(n=50),分别于断奶后24 h颈部肌肉注射孕马血清促性腺激素(PMSG)1000 IU/头、KP-100.5 mg/头和KP-101.0 mg/头,72 h后颈部肌肉注射100μg/头GnRH,16 h后进行第1次配种,24 h后进行第2次配种,统计各组母猪平均卵泡直径、断奶-发情间隔、发情率、妊娠率、分娩率、窝总产仔数和窝产活仔数,研究KP-10对定时输精母猪繁殖性能的影响。结果表明:KP-10处理的颗粒细胞中CYP11A1、3β-HSD、FSHR和LHCGR基因的mRNA表达水平较对照颗粒细胞显著或极显著提高(P1);PMSG对照组、KPL组、KPH组母猪断奶后第1,2,3,4,5天的平均卵泡直径均差异不显著(P>0.05);PMSG对照组、KPL组、KPH组断奶-发情间隔、发情率、分娩率、窝总产仔数和窝产活仔数均差异不显著(P>0.05),其中KPL组母猪发情率、分娩率高于PMSG对照组和KPH组(P>0.05);KPL组母猪妊娠率显著高于PMSG对照组和KPH组(P0.05)。说明KP-10可用于调控母猪卵泡同步发育,并且可提高定时输精母猪的妊娠率。

关键词： Kisspeptin 孕马血清促性腺激素卵泡发育定时输精繁殖性能

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Infection of Taenia asiatica in a Bai people in Dali

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Infection of Taenia asiatica in a Bai people in Dali