鸡白痢沙门菌和鸡伤寒沙门菌PCR-RFLP鉴别

根据鸡白痢沙门菌和鸡伤寒沙门菌flic基因可变区两端的保守序列设计1对引物,PCR扩增出约866 bp的产物,用HinplI对PCR产物进行酶切,经RFLP分析区分鸡白痢沙门菌和鸡伤寒沙门菌.利用该技术对1株鸡白痢沙门菌标准株及2株鸡伤寒沙门菌标准株进行分子鉴别,结果与预计的RFLP模式相符,证明该方法可行.在此基础上...     

肠炎沙门菌感染鸡lncRNA表达谱分析

研究旨在分析鸡感染肠炎沙门菌后长链非编码RNA(lncRNA)的表达谱,了解lncRNA在肠炎沙门菌感染过程中发挥的作用.通过提取脾脏组织总RNA进行高通量测序,利用生物信息学方法进行比较分析.根据差异显著的lncRNAs与mRNAs之间的位置关系预测lncRNAs顺式调控的邻近基因,并对邻近基因进行GO富集和KEGG...     

不同鸡种沙门菌感染率比较分析

试验采用血清平板凝集法,对江苏省某种鸡场中白耳鸡、仙居鸡、安卡鸡和清远麻鸡4个品种的沙门菌天然感染情况进行了评估。结果显示,公鸡中肉用型安卡鸡对2种沙门菌病的感染率最高,蛋用型品种居中;母鸡中蛋用型品种对2种沙门菌病的感染率最高,肉用型品种居中;不同品种、不同性别以肉蛋兼用型的清远麻鸡感染带菌率最低。     

鸡肠炎沙门菌病及其抗性相关基因的研究进展

肠炎沙门菌(Salmonella enteritidis,SE)是一种特殊的泛嗜性胞内病原菌,也是最为常见的食物源性病原菌之一,通过污染鸡蛋进行传播。肠炎沙门菌能导致成年鸡隐性感染且长期排菌,容易为生产所忽视;并且能在污染的鸡蛋中增殖,却不会引起蛋内结构明显的变化。肠炎沙门菌病不但对禽类生产造成的经济损失无法估量,而且对人类的身体健康也造成很严重的危害。宿主的遗传背景对肠炎沙门菌病的抗性有着深远的影响,探索抗性相关基因的遗传特性和差异表达规律是研究鸡对肠炎沙门菌病抗性的必要途径。本文对鸡肠炎沙门菌病的致病机理和抗性相关基因的研究进展进行了阐述,以期为鸡抗病育种研究提供借鉴。     

鸡白痢沙门菌感染产蛋期蛋鸡的致病性分析

为了解鸡白痢沙门菌感染产蛋期蛋鸡致病特点,采集2022年2-11月感染产蛋期蛋鸡的肝脏等组织开展研究。对病鸡的肝脏、脾脏进行细菌分离,对分离菌株进行多重PCR鉴定和血清型检测;取病变的肝脏等组织制备病理切片以观察其病理变化;通过qRT-PCR分析肝脏、脾脏组织中鸡白痢沙门菌毒力基因和细胞因子mRNA的表达水平。结果表明,从送检的蛋鸡中分离出18株细菌,在麦康凯培养基上生长无色小菌落,多重PCR鉴定和血清型为鸡白痢沙门菌;病理切片可见心肌、肝脏及肺脏炎性细胞群浸润和细胞坏死;qRT-PCR检测显示感染鸡白痢沙门菌的病鸡诱导生物被膜相关基因(CsgB、RpoS、bcsA和PagC)、SPI-1基因(HilA、InvA、PrgH和SipC)和SPI-2基因(SteE、RcsC、SseL和SpiC)等毒力基因的表达水平上调;感染产蛋鸡的肝脏中IL-1β、IL-6、IL-4和IL-13的表达水平上调,IL-18和IFN-γ表达水平降低;脾脏中IL-6高度表达,IL-1β和IL-18表达水平显著降低。本研究中感染病鸡组织中鸡白痢沙门菌使生物被膜、SPI-1、SPI-2相关毒力基因和TH2分泌的细胞...     

鸡白痢沙门菌研究进展

鸡白痢沙门菌是鸡白痢的病原,具有高度适应专一宿主的特点。它可引起雏鸡的急性全身性感染,发病率和病死率极高;成年鸡被感染后可引起体质量下降、产蛋率和孵化率降低、组织病变、白痢和生殖道畸形等,给很多国家的养鸡业带来巨大危害,并引起严重的经济损失。为了更好的预防和控制、甚至最终净化该病,需要对鸡白痢沙门菌进行深入的研究。本文从病原学及其基因组特征、流行病学、致病性及毒力基因、检测方法和防控措施等方面介绍了鸡白痢沙门菌的研究进展,并对其研究趋势进行了展望。     

鸡白痢沙门菌对四环素类药物耐药性和耐药基因调查分析

检测分析珠三角地区69株鸡白痢沙门菌对四环素的耐药性及5种四环素耐药基因[tet(A)、tet(B)、tet(C)、tet(M)和tet(X)]在分离株中的分布情况。结果显示,43株(62.7%)鸡白痢沙门菌分离株对四环素产生耐药性,耐药基因tet(A)的检出率为75.4%,未能检测到耐药基因tet(B)、tet(C)、tet(M)和tet(X),表明鸡白痢沙门菌对四环素的耐药机制以tet(A)基因介导的主动外排为主。     

不同鸡种对鸡白痢沙门菌定植与免疫反应差异分析

试验对鸡白痢沙门菌在鸡肠道内定植及其对鸡免疫性能的影响进行探讨。选取狼山鸡、罗斯鸡各40只,分为攻毒组、空白组,攻毒组口服灌注0.5mL鸡白痢沙门菌(107cfu/mL),观察攻毒鸡群的发病状况,并采集盲肠内容物和血液分别进行菌落数和免疫指标的测定。结果发现,狼山鸡和罗斯鸡在肠道中的载菌量分别为4.94×107cfu/mL和5.18×107cfu/mL。免疫指标方面,狼山鸡中除IgM外,其余指标与空白组差异显著(P0.05),罗斯鸡中IgY、白细胞介素-1β与空白组差异显著(P0.05)。表明这2个鸡种对鸡白痢沙门菌均易感,但狼山鸡免疫反应优于罗斯鸡。     

鸡白痢沙门菌基因组差异片段的筛选与分析

采用抑制性消减杂交技术（SSH）对鸡白痢沙门菌C79-13株与鸡伤寒沙门菌Sg9株进行了基因组差异片段分析.结果,从C79-13株中共检出13个特异性差异片段.经同源分析,这些序列可分为3类：噬菌体相关序列、质粒相关序列和已知功能序列.这些差异片段包含一些重要的沙门菌毒力相关基因,如编码大肠杆菌素、IpaJ蛋白、尾突蛋白、切除酶的基因.结果表明,鸡白痢沙门菌C79-13株与鸡伤寒沙门茵Sg9株基因组问存在较多差异基因,这些差异片段为确定鸡白痢沙门菌特异性遗传标志,建立分子鉴别新体系提供了基础.     

鸡白痢沙门菌的耐药性及磺胺类耐药基因的研究

本研究检测分析了珠三角地区鸡白痢沙门菌对磺胺药的耐药性及相关耐药基因,以进一步了解鸡白痢沙门菌对磺胺药耐药性的产生和传播机制。采用微量稀释法检测69株鸡白痢沙门菌分离株对磺胺药的耐药性,PCR检测了磺胺类耐药基因SulⅠ、SulⅡ和SulⅢ在分离株中的分布情况。结果显示,75.4%的受试菌株对磺胺异恶唑产生了耐药性;耐药基因SulⅡ检出率最高(79.7%),其次是耐药基因SulⅠ(33.3%),未能检出耐药基因SulⅢ。提示珠三角地区鸡白痢沙门菌对磺胺药的耐药情况相当严重,磺胺类耐药基因SulⅠ和SulⅡ在磺胺类耐药菌株的产生中起重要作用。     

Immunological and gene expression responses to a Salmonella infection in the chicken intestine

Besides infection in humans, Salmonella enteritidis can also cause serious illness in young chickens. However, the genetic and immunological parameters important for the disease in chickens are not well characterized. In this study, processes in the chicken intestine in response to a Salmonella infection were investigated in two different chicken lines. One-day-old chickens were orally infected with Salmonella. T-cell subpopulations, phagocytic properties of intestinal mononuclear cells and RNA expression levels of the jejunum were investigated. The two chicken lines differed in the amount of cfu in the liver and growth retardation after the infection. Differences in phagocytic activity of intestinal mononuclear cells were found between control and Salmonella infected chickens. The number of CD4+ T-cells of the intestine decreased after the Salmonella infection in one chicken line, while the number of CD8+ T-cells increased in both chicken lines, but the time post infection of this increase differed between the lines. In one chicken line the expression levels of the genes carboxypeptidase M and similar to ORF2 decreased after the Salmonella infection, which might be related to a decrease in the amount of macrophages. With the microarray, ten genes were found that were regulated in only one of the chicken lines, while we found six genes regulated in response to the infection in both chicken lines. So differences in genetic background of the chickens influence the intestinal host response of the Salmonella infection as observed by phagocytic activity, gene expression and changes in the number of T-cell subpopulations and macrophages.     

Differential expression of Wilms’ tumour 1 gene in porcine urogenital organs during development

Wilms’ tumour 1 gene (WT1) is essential for the development of mammalian urogenital system. However, the expression pattern of WT1 in the development of porcine urogenital organs is still unclear. Here, we examined the expression of WT1 mRNA and protein in porcine kidneys, ovaries and testes from embryonic days 35 and 60 (E35d, E60d, n = 3) to the newborn (0d, n = 4) and adult (210d, n = 3) stages, using real‐time PCR and immunofluorescent staining. Real‐time PCR analysis showed that porcine kidneys, ovaries and testes all expressed high level of WT1 mRNAs, especially in adult testes (p < 0.05 or 0.01 vs. kidney and ovary, respectively). Morphologically, characteristic microstructures of the kidneys, ovaries and testes were observed and discerned at all four stages. Immunofluorescently, WT1 expression was detected in a dynamic and context‐specific pattern during the development of these organs. Taken together, porcine urogenital organs express relatively high levels of WT1 mRNA. Dynamical and context‐specific expression profile of WT1 in these organs occurs during their development, implying its close association with the development and function of porcine kidney, ovary and testis.     

Orally administered attenuated Salmonella enteritidis reduces chicken cecal carriage of virulent Salmonella challenge organisms

Chickens were immunized orally with 10(9)cfu of the temperature-sensitive (T(s)) mutant E/1/3 of Salmonella enteritidis at 1, 2, 3 and 7 days of age. The animals were challenged with wild-type strains of Salmonella of different serotypes 7 or 14 days following immunization. Chickens receiving multiple oral doses of the vaccine strain showed no signs of disease. Immunized animals shed the vaccine strain for at least 2 weeks after the last inoculation; on the other hand, colonization by the attenuated mutant of internal organs such as spleen and liver was limited. Early exposure of the immunized animals to the virulent bacteria resulted in a reduced cecal colonization by the pathogen. Visceral invasion by the wild-type strain of S. enteritidis or S. gallinarum was drastically diminished in birds challenged 14 days after immunization. Significant differences in the number of these Salmonella were found in the cecal contents, spleen and liver of immunized birds compared with the control animals. In addition, cecal colonization by the virulent strain was reduced in birds challenged with S. typhimurium. These results demonstrate that immunization of newly hatched chickens with live attenuated T(s) mutant E/1/3 of S. enteritidis is safe and reduces Salmonella shedding.     

Cytokine gene expression in chicken cecal tonsils following treatment with probiotics and Salmonella infection

Probiotics are currently employed for control of pathogens and enhancement of immune response in chickens. In this study, we investigated the underlying immunological mechanisms of the action of probiotics against colonization of the chicken intestine by Salmonella enterica subsp. enterica serovar Typhimurium (Salmonella serovar Typhimurium). Birds received probiotics by oral gavage on day 1 of age and, subsequently, received Salmonella serovar Typhimurium on day 2 of age. Cecal tonsils were removed on days 1, 3 and 5 post-infection (p.i.), RNA was extracted and subjected to real-time quantitative RT-PCR for measurement of interleukin (IL)-6, IL-10, IL-12 and interferon (IFN)-gamma gene expression. There was no significant difference in IL-6 and IL-10 gene expression in cecal tonsils of chickens belonging to various treatment groups. Salmonella serovar Typhimurium infection resulted in a significant increase in IL-12 expression in cecal tonsils on days 1 and 5p.i. However, when chickens were treated with probiotics prior to experimental infection with Salmonella, the level of IL-12 expression was similar to that observed in uninfected control chickens. Treatment of birds with probiotics resulted in a significant decrease in IFN-gamma gene expression in cecal tonsils of chickens infected with Salmonella compared to the Salmonella-infected birds not treated with probiotics. These findings reveal that repression of IL-12 and IFN-gamma expression is associated with probiotic-mediated reduction in intestinal colonization with Salmonella serovar Typhimurium.     

Genetic variation of chicken MC1R gene in different plumage colour populations

1. Genetic variation in the chicken MC1R gene was analysed through bioinformatic methods after polymerase chain reaction (PCR) amplification and sequencing of the coding region of MC1R gene from 5 different populations with 4 plumage colours (black, grey plumage with black spots, yellow plumage with black spots, red). 2. A total of 11 novel variations were detected in Hebei chickens, of which 8 were non-synonymous. Allele distribution analysis showed that the wild-type e(+) (Brown Leghorn) allele was mainly found in Hy-Line Brown and Lohmann Brown, the dominant Extended black E(AY220304) allele was mainly found in Hebei chicken with black plumage, whereas the Buttercup (e(bc)) allele was rare. 3. Nucleotide diversity (π) within each colour strain of Hebei chickens (0·0047-0·0052) was significantly greater than that of Hy-Line Brown (0·0024) or Lohmann Brown (0·0043). 4. The results indicate that there is abundant polymorphism in the MC1R gene, especially in Hebei chicken, which was associated with its rich plumage colour diversity.     

鸡去泛素化相关因子1基因原核表达载体的构建及表达

构建鸡去泛素化相关因子1(Chicken ubiquitin-related factor 1,chUAF1)的原核表达载体,并对表达产物进行鉴定.将提取的鸡法氏囊cDNA根据哺乳动物基因组的特性5'-端GC含量高3'-端GC含量低的特点设计2对引物,PCR获取2段基因片段.将两者用搭桥部分的酶切位点进行酶切,胶回收,连接成chUAF1基因全长,再将基因克隆入pMD18-T载体,酶切鉴定后,将chUAF1基因亚克隆到pET28a原核表达载体,构建重组质粒pET28a-UAF1.转化BL21(DE3)大肠杆菌中,IPTG诱导表达并鉴定.结果显示,所构建的chUAF1基因表达载体pET28a-chUAF1经PCR及DNA测序结果表明构建正确,SDS-PAGE鉴定在预期位置出现阳性条带.结果表明,已成功构建了pET28a-chUAF1原核表达载体,为下一步试验提供了依据.     

Baculovirus-mediated gene expression in chicken primary cells

A recombinant baculovirus was constructed containing an expression cassette with a reporter gene, green fluorescent protein, directed by a constitutive mammalian promoter: a human cytomegalovirus immediate early promoter/enhancer (CMV-IE). High titer virus was prepared with ultracentrifugation. Efficient gene delivery and expression were observed in the virus-treated chicken primary culture, myoblast cells, and whole embryonic fibroblast cells. It was noticed that an addition of sodium butyrate (a selective histone deacetylase inhibitor) to viral transduction medium extremely enhanced the reporter-gene expression. However, there is no effect of presence of trichostatin A observed. To maximize the reporter-gene expression, the baculoviral infection condition was optimized with both cell types. Our approaches demonstrated that recombinant baculovirus could efficiently deliver its genome DNA into chicken primary cells and that CMV-IE, a mammalian-cell-active promoter, was functional in chicken primary cells and could direct a high level of gene expression. Clearly, the recombinant baculovirus provides an alternative means for foreign gene delivery into avian cells.     

山地乌骨鸡不同组织中CAPN 1基因表达的发育性变化

本试验采用SYBR GreenⅠ荧光定量法,首次测定了四川山地乌骨鸡1d、14d、28d、42d、56d、70d、84d的胸肌、腿肌、肝脏、心、脑组织中钙蛋白酶1CAPN1基因的相对表达量。结果表明：CAPN1基因在鸡体内普遍存在,CAPN1基因在肌肉组织中的表达量与鸡肌肉组织的生长发育呈相似的变化规律。