西安荷斯坦奶牛群5个基因座位遗传多态性的PCR-RFLP分析

应用PCR-RFLP方法对西安荷斯坦牛的κ-en、β-lg、β-lg5′侧翼区、CSN1S2、IGFBP-3共5个基因座位进行了多态性分析。结果表明，在西安荷斯坦牛群中，没有发现携带CSN1S2^P等位基因的个体，其多态信息含量为0。κ-en基因座位呈现低度多态(PIC=0．2366)，β-lg、β-lg5′侧翼区、IGFBP-3基因座位的多态信息含量分别为0．3168、0．3689、0．4439，均呈现中度多态。κ-en、β-lg、β-lg5′侧翼区、IGFBP-3、CSNIS2基因座位的杂合度和DNA多态度分别为0．2742、0．3947、0．4879、0．4891、0和0．0255、0．0116、0．0333、0．0112、0。而且，在西安荷斯坦牛群中，κ-en、β-lg、β-lg5′侧翼区、IGFBP-3共4个基因座位均处于Hardy-Weinberg平衡状态，CSN1S2基因座位处于纯合状态。     

亚洲璃眼蜱唾液腺一新功能基因的克隆与测序

HaB1是亚洲璃眼蜱雌成蜱唾液腺差异表达基因文库中的一个片段,根据其序列设计引物HaB1-GSP1和HaB1-GSP2,以唾液腺总RNA为模板,RACE法扩增获得HaB1的未知3′-末端。测定该末端序列,进行序列拼接,设计全长引物5-′CCAGTCCGAAGGAAGGGCG-3′和5-′TCTC-CGGGCACGTGAAGTGTC-3′。以cDNA第一链为模板,扩增获得基因全长。经核查表明,该基因为一新基因(登录号AY803896)。     

基因枪轰击不同剂量小鹅瘟病毒VP3基因疫苗在雏鹅体内的动态分布

本文开展了检测小鹅瘟病毒(GPV)VP3基因的实时荧光定量PCR(FQ-PCR)方法的建立和基因枪轰击不同剂量(1、3和6μg)GPV-VP3基因疫苗(pcDNA-GPV-VP3)在30日龄四川白鹅体内(心、肝、脾、肺、肾、法氏囊、胸腺、哈氏腺、十二指肠、空肠、回肠、直肠、盲肠、胰腺、血液、脑及注射部位皮肤)分布规律的研究。结果表明:①建立的FQ-PCR特异性强、灵敏度高、重复性好,核酸模板数与FQ-PCR测定的Ct值相关系数达到0.999,具有很好的直线相关性;②pcDNA-GPV-VP3各剂量免疫雏鹅1 h即可在各组织中检测到,其中注射部位含量最高,肝、肾、淋巴器官(脾、法氏囊、胸腺、哈氏腺)含量较高;③到免疫后217 d时,1μg组免疫雏鹅各个组织器官内仍检测到pcDNA-GPV-VP3的存在,但多数组织器官中的含量比1 h时约少了4个数量级,其中免疫部位减少了7个数量级;④血液中pcDNA-GPV-VP3的含量较少,且免疫后1 h~217 d各时间点的差异不显著(P≥0.05);⑤不同剂量pcDNA-GPV-VP3免疫雏鹅各组织中的含量呈现的总体规律为6μg组>3μg组>1μg组,但差异不显著(P≥0.05)。因此,FQ-PCR是定量检测pcDNA-GPV-VP3在免疫雏鹅体内含量的可靠方法,pcDNA-GPV-VP3免疫雏鹅后1 h时可分布至雏鹅体内各组织器官中并持续存在217 d以上。     

DNA methylation and targeted sequencing of methyltransferases family genes in canine acute myeloid leukaemia,modelling human myeloid leukaemia

Tumours shows aberrant DNA methylation patterns, being hypermethylated or hypomethylated compared with normal tissues. In human acute myeloid leukaemia (hAML) mutations in DNA methyltransferase (DNMT3A) are associated to a more aggressive tumour behaviour. As AML is lethal in dogs, we defined global DNA methylation content, and screened the C‐terminal domain of DNMT3 family of genes for sequence variants in 39 canine acute myeloid leukaemia (cAML) cases. A heterogeneous pattern of DNA methylation was found among cAML samples, with subsets of cases being hypermethylated or hypomethylated compared with healthy controls; four recurrent single nucleotide variations (SNVs) were found in DNMT3L gene. Although SNVs were not directly correlated to whole genome DNA methylation levels, all hypomethylated cAML cases were homozygous for the deleterious mutation at p.Arg222Trp. This study contributes to understand genetic modifications of cAML, leading up to studies that will elucidate the role of methylome alterations in the pathogenesis of AML in dogs.     

Establishment of Digital RT-PCR Assay for Detection of Swine Influenza Virus H3N2 Subtype

A new assay for the detection of swine influenza virus (SIV) was developed with a novel nucleic acid probe——Molecular beacon in this study. The specific primers and molecular beacon probes were designed according to the conserved region of H3 and N2 genes of SIV H3N2 subtype. A digital RT-PCR assay was developed for detection of SIV H3N2 subtype. The results showed that SIV H3N2 subtype could be identified simultaneously on this microarry with high sensitivity and reproducibility,which could reach to 10⁶ dilute viruse. The conclusion was that the digital RT-PCR method could analyze quantitatively the RNA templates.On the identification of H3N2 SIV,the digital RT-PCR method was much more scientific than Real-time quantitative PCR method.     

Molecular Characterization of Equine Rotavirus Group A Detected in Argentinean Foals During 2009–2014

Equine rotavirus group A (RVA) has been detected in several countries worldwide since its first detection in 1975. Currently, equine RVA is considered the major cause of dehydrating diarrhea in foals younger than 3 months, and the frequency of detection in clinical cases varies from 20% to 77%. The genotypes of epidemiologic relevance found in horses are G3P[12] and G14P[12]. In a survey conducted in Argentina from 1992 to 2008, equine RVA was detected in 21% and 39% of the fecal samples and outbreaks, respectively. Genotype distribution was 51% G3P[12] and 33% G14P[12]. In continuation with the surveillance, the aim of the present study was to characterize the equine RVA detected in Thoroughbred foals in Argentina from 2009 to 2014. A total of 436 stool samples (corresponding to 177 single diarrhea cases or outbreaks) were analyzed. Equine RVA was detected in 31% (135 of 436) of the samples, which corresponded to 42% (74 of 177) of outbreaks. From the positive cases, 42% (57 of 135) were genotyped. Of this, 63% were G3 (36 of 57) and 37% (21 of 57) were G14 genotype. Considering the whole data (1992–2014), equine RVA was detected in 25% (300 of 1,207) of the stool samples and 41% (119 of 293) of the diarrhea outbreaks. The results of this study also show a cyclic pattern of the G3 and G14 prevalence in the horse population with a change in G3:G14 frequencies from year to year. Furthermore, clustering in the phylogenetic tree suggests evolutionary and geographic relationships between the Argentinean strains compared with the strain circulating worldwide.     

3-甲基喹噁啉-2-羧酸的制备及其细胞毒性

通过贝鲁特反应和还原、脱羧反应在体外合成制备3-甲基喹噁啉-2-羧酸,并进行理化鉴定和抗菌活性测试;通过MTT方法研究3-甲基喹噁啉-2-羧酸对多种细胞的生长抑制作用,利用单细胞电泳研究其对细胞DNA的损伤作用,利用流式细胞术研究其对细胞周期的改变。结果显示,体外成功合成制备出3-甲基喹噁啉-2-羧酸,而3-甲基喹噁啉-2-羧酸几乎没有抗菌效果,对多种细胞的生长抑制作用较弱,在剂量检测范围内细胞抑制率不到30%,但在一定剂量下能导致细胞DNA损伤,主要表现为尾长和尾部DNA含量显著升高;并且3-甲基喹噁啉-2-羧酸也能改变Chang细胞的细胞周期,表现为S期阻滞。结果表明,3-甲基喹噁啉-2-羧酸具有一定的细胞毒性。     

Genetic background of CTX‐M‐15‐producing Enterobacter hormaechei ST114 and Citrobacter freundii ST265 co‐infecting a free‐living green turtle (Chelonia mydas)

CTX‐M‐type extended‐spectrum β‐lactamase (ESBL)‐producing Enterobacteriaceae have become identified in marine ecosystem constituting a serious ecological issue. In this respect, although contamination of coastal waters and seafood, and even colonization of seabirds and fishes have been increasingly reported, molecular data are lacking to elucidate the clinical impact of ESBL producers in infected marine animals. In this study, using a genomic approach, we have analysed the genetic background of CTX‐M‐15‐producing Enterobacter hormaechei (belonging to the international human clone ST114) and Citrobacter freundii (ST265) co‐infecting a free‐living green turtle (Chelonia mydas) suffering from septic arthritis, which progressed to generalized coelomitis and death. Wide resistome of these pathogens contributed to treatment failure and death of the animal.     

Follicular dynamics and changes in oestradiol‐17β, progesterone and LH profiles following PGF2α induced oestrus in early and late ovulating Beetal goats

This study compares the factors associated with variable interval to oestrus and ovulation between early versus late ovulating goats following PGF_2α administration. The time of ovulation in Beetal goats (n = 38) was monitored through transrectal ultrasound at every 6 hr following a single dose of PGF_2α (experiment 1). Variations in oestrus and ovulation times were further explored through the changes in follicular dynamics, endocrine profiles and behaviour in another set of goats (n = 13) following single PGF_2α given randomly during the luteal phase (experiment 2). The ovulation time varied between 60 and 96 hr, and 57% of ovulations occurred by 72 hr following PGF_2α (experiment 1). Accordingly, the goats (n = 13) in the second experiment were retrospectively divided either into early and/or late ovulating, that is, ≤72 and/or ≥84 hr following PGF_2α. The onset of oestrus, peak estradiol‐17β concentration and LH surge after PGF_2α was first observed in early than late ovulating goats (p < 0.05). The goats ovulating early had larger follicle and smaller CL in diameter at the time of PGF_2α administration than those ovulating late (5.4 ± 0.2 vs. 4.3 ± 0.2 mm and 10 ± 0.6 vs. 11.8 ± 0.3 mm, respectively; p < 0.05). Likewise, plasma progesterone concentration tended to be lower (p = 0.087) in early than late ovulating goats. In conclusion, the size of dominant follicle and CL at the time of PGF_2a determines the interval to ovulation following a single dose of PGF_2a during the luteal phase.