非洲猪瘟病毒生物学特性及在猪肉生产中的防控措施研究

非洲猪瘟是一种烈性外来疾病,不属于人畜共患病,但会造成大量猪死亡,自中国首次发现疫情以来,多个地区相继暴发疫情,扑杀大量生猪,给生猪养殖业带来巨大的损失。目前还没有针对非洲猪瘟的特效药和有效疫苗,还需要进一步的研究。该文从非洲猪瘟病毒的生物学特性、防控难点以及在猪肉食品生产中的防控方法等方面进行概述,提出食品企业应做好应对措施,在食品生产过程中全面防控,增强从业人员的防控意识,防止非洲猪瘟病毒通过猪肉产品在市场中传播,旨在为抑制非洲猪瘟的扩散和食品企业对非洲猪瘟病毒的防控提供参考。     

猪伪狂犬病毒(PRV)桂B株的分离和鉴定

从广西某市猪场采集发病仔猪大脑、肾、脾等混合病料中分离到一株病毒。病毒接种PK细胞和CER细胞均出现典型细胞病变,引起病变细胞形成台胞体,在PK细胞上毒价为TCID5010-5．3／0．1mL。病毒能被伪狂犬病病毒标准阳性血清中和,血清中和效价为1：316。病毒接种家兔后出现典型伪狂犬病症状。电镜观察到圆形、有囊膜、直径100～180um大小的病毒颗粒。PCR可扩增出特异性217bP的DNA片移。证实我们所分离到的病毒为伪狂犬病病毒,并命名为猪伪狂犬病病毒桂B株。     

猪瘟免疫失败的原因及对策

猪瘟是由猪瘟病毒引起的猪的一种高度传染性和致死性传染病,是目前危害我国养猪业最严重的疾病之一。生产实践证明,现时的猪场中,由于传染病引起的猪只死亡有40%～50%与猪瘟有关,而依靠购买仔猪进行肉猪生产的养殖户,因猪瘟造成的损失更为严重。     

应用免疫荧光技术研究水稻条纹病毒(RSV)侵染介体灰飞虱卵巢的过程

由水稻条纹病毒(Rice stripe virus,RSV)引起的水稻条纹叶枯病,在许多水稻种植区引起严重的减产,水稻条纹病毒由灰飞虱(Laodelphax striatellus Fallén)以持久增殖型传播,并可以经卵传播方式以较高的比例传给子代灰飞虱。为研究水稻条纹病毒的经卵传播特性及其侵染介体昆虫卵巢的过程,分别利用RT-PCR和免疫荧光技术检测虫体各个器官以及卵巢的带毒率,结果表明,饲毒后尽管灰飞虱的带毒率有28.6%,但是仅有5.3%的卵巢被病毒侵染。同时在利用免疫荧光技术对饲毒后不同时间灰飞虱卵巢中的RSV进行定位,发现RSV首先在卵巢管柄位置出现,随后侵染滤室细胞及生殖区,最后转移到胚盘细胞。结果认为,病毒可能是在滤泡细胞以及生殖区向正在发育的卵母细胞提供营养时,伴随这些营养物质进入卵母细胞。且生殖区的卵原细胞可发育成卵母细胞和滤泡细胞,我们推测,尽管RSV可伴随营养输送进入卵母细胞,但是最初带毒卵母细胞的病毒可能来源于位于卵巢生殖区的病毒。研究结果提示,RSV可能通过营养输送途径运送至卵母细胞,并最终侵染卵的胚盘细胞     

表达外源基因的猪繁殖与呼吸综合征病毒(PRRSV)感染性克隆的构建及病毒拯救

为建立表达标记基因的重组猪繁殖与呼吸综合征病毒(PRRSV)反向遗传操作平台,本研究在PRRSV HuN4-F112疫苗株感染性分子克隆的基础上,采用突变PCR技术将新城疫病毒(NDV)NP蛋白末端优势抗原区插入PRRSV NSP2蛋白的复制非必须区,经基因片段的克隆、拼接,构建了含有外源标记基因的感染性的PRRSV cDNA克隆psk-HuN4-F112-Δ52+NP49。用限制性内切酶SwaΙ将psk-HuN4-F112-Δ52+NP49线性化后通过细胞外转录获得病毒RNA,用脂质体法将病毒RNA转染叙利亚仓鼠肾细胞(BHK-21)包装出病毒粒子,再转接到猴胚胎肾上皮细胞(MACR-145)传代拯救病毒。对拯救的病毒进行RT-PCR扩增、酶切和序列分析鉴定。结果表明,拯救病毒含有不同于亲本病毒的分子标记(病毒基因组14667位人工产生的MluⅠ酶切位点)和插入的标记基因序列。间接免疫荧光试验表明,外源基因NP49在拯救的PRRSV中表达,且能够在PRRSV传代过程中稳定遗传。病毒生长特性比较显示,拯救病毒与亲本病毒在MARC-145细胞上具有相似的增殖特性。本研究构建了含有标记基因PRRSV的感染性克隆并获得了拯救病毒,为进一步开发新型PRRS疫苗提供了有效的反向遗传操作平台。     

两株广西伪狂犬病病毒的分离鉴定

从广西不同地方的发病仔猪脑内分离到两株病毒,分别命名为Ｂ株和Ｗ株。用这两株病毒分别接种家兔,产生的症状和病变与伪狂犬病病毒强毒ＭｉｎＡ株相似。将两株病毒在乳兔肾细胞和ＣＥＲ细胞上连续传代,均稳定地出现与ＭｉｎＡ株相同的细胞病变（ＣＰＥ）。在ＣＥＲ细胞上进行微量血清和试验,ＭｉｎＡ株的抗血清能完全中和Ｂ株和Ｗ株对细胞的致病变作用,表明两个毒株与ＰｒＶ强毒具有相似的抗原性。电镜下观察Ｂ株和Ｗ株病毒粒子     

利用侧流核酸试纸条快速检测非洲猪瘟病毒

为满足基层普通实验室或养殖场等资源有限的实验室对非洲猪瘟病毒(African swine fever virus,ASFV)的检测需求,该研究开发了一种简单、快速、低成本的检测技术,可以用肉眼观察检测结果。研究将传统聚合酶链式反应(polymerase chain reaction,PCR)与胶体金试纸条技术结合,开发一种低成本的侧流核酸测定试纸条(lateral flow nucleic acid assay,LFNAA)。该体系设计了独特的尾引物,避免了传统核酸试纸条的半抗原标记及抗体的使用。经过PCR扩增后产生一端带着单链寡核苷酸尾巴的双链DNA产物,能够与胶体金标记的寡核苷酸捕获探针结合,从而在试纸条上形成可以用肉眼观察的目标产物。该LFNAA试纸条能够在猪瘟病毒(Classical swine fever virus,CFSV)、猪瘟病毒猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)、猪圆环病毒1型(Porcine circovirus 1,PCV1)、猪圆环病毒2型(Porcine circovirus 2,PCV2)、猪伪狂犬病毒(Pseudorabies virus,PRV)、猪细小病毒(Porcine parvovirus virus,PPV)中特异的鉴定出ASFV的存在,其灵敏度与琼脂糖凝胶电泳的分析结果一致,均能达到103 copies/μL。因此,仅需要一台普通PCR仪,即可对ASFV进行快速灵敏的鉴定(<2 h),其低成本、操作简便的特点非常适合资源有限的实验室中非专业人员的操作。此外,该技术可进一步结合等温扩增技术,能够在食品安全和医学诊断中实现更快更简便的现场检测。     

鸡传染性喉气管炎的研究Ⅱ．鸡传染性喉气管炎病毒某些生物学特性的研究

对鸡传染性喉气管炎病毒（ＩＬＴＶ）的鸡胚、鸡胚肾细胞等培养系统进行研究；对病毒包涵体、病毒形态进行观察；对病毒毒价进行测定。结果，病毒在鸡胚绒毛尿囊膜产生痘斑和坏死灶，鸡胚发育不良并致死；鸡胚肾细胞稳定地产生合胞体等细胞病变；绒毛尿囊膜切片经Ｈ－Ｅ梁色，可观察到紫红色嗜酸性核内包涵体充满细胞核；病毒细胞培养物粗提后用负染法电镜观察，绒毛尿囊膜用超薄切片法电镜观察，均可见到病毒颗粒，病毒直径约２４０～３００ｎｍ，核衣壳直径约１００～１４０ｎｍ，外有圆形或椭圆形囊膜；经微量培养系统测定，病毒毒价为１０６．１２ＴＣＩＤ５０／０．０５ｍＬ。     

中国农科院哈尔滨兽医所发现调控猪瘟病毒复制宿主因子

正近日,中国农业科学院哈尔滨兽医研究所猪烈性传染病创新团队经过系统研究,发现了一个新的调控猪瘟病毒复制的宿主分子丝裂原活化的蛋白激酶激酶2(MEK2),并阐明了其调控病毒复制的分子机制。相关研究成果发表在国际著名病毒学专业期刊《Journal of Virology》(病毒学杂志)上,并被编委评为该杂志的亮点文章。据介绍,猪瘟是猪的一种烈性传染病,其传播快、死亡率高,给世界各国的养猪业造成重大经济损失,我国在     

多重连接探针扩增(MLPA)技术同时检测五种病毒的研究

现有的动物疫病诊断技术多是针对单一病原进行的,而动物疫病的流行却出现了多种病毒混合感染,现有诊断技术不能很好地满足国境口岸快速、高通量检疫的需求,动物疫病多重检测新技术的研究近来已经成为动物疫情监测、疫病控制领域关注的焦点。本研究利用多重连接探针扩增(MLPA)技术特异、敏感、高通量等技术优势,以猪流感病毒(SIV)、伪狂犬病病毒(PRV)、口蹄疫病毒(FMDV)、猪传染性胃肠炎(TGEV)和猪繁殖与呼吸综合症病毒(PRRSV)为研究对象,分别设计了这5种病毒的MLPA探针,将这5种探针混合,建立了可以同时检测这5种病毒的MLPA扩增方法。方法特异性试验表明,针对每种病毒的探针都只能扩增出其对应的病毒模板,其余病毒模板的扩增都是阴性结果;而且,5种探针混合物都只能从单个目的病毒中扩增出单一的特异性条带,而猪细小病毒(PPV)、猪瘟病毒(CSFV)和猪流行性腹泻病毒(PEDV)的扩增均为阴性。敏感性试验结果表明,5重MLPA扩增的最低检测限可以达到单个病毒核酸3000~6000个拷贝。本研究建立的5种病毒MLPA检测方法在国内外首次实现一次采样,一次分析,检测5种猪病的目的,该技术特异性强,敏感性高,加之其多重性检测优势,有望成为未来疫病检测的新方向。     

RT-PCR检测猪瘟病毒初探

本研究建立了反转录聚合酶链反应 ( RT PCR)技术检测猪瘟病毒 ( Hog Cholera Virus,HCV)方法。合成的二对引物 HCV 1 / HCV 2和 HCV A1 / HCV A2成功地扩增兔化弱毒株、法国株和石门株的核酸 ,并对采自博白县 3个、贵港市 1个和武宣县 1个猪场共 1 3份病料进行检测。结果从博白县和贵港市的 1 1份材料中检测到 HCV的核酸。     

Cinnamon polyphenol extract regulates tristetraprolin and related gene expression in mouse adipocytes

Cinnamon (Cinnamomum verum) has been widely used in spices, flavoring agents, and preservatives. Cinnamon polyphenol extract (CPE) may be important in the alleviation of chronic diseases, but the molecular evidence is not substantial. Tristetraprolin (TTP) family proteins have anti-inflammatory effects through the destabilization of pro-inflammatory mRNAs. TTP expression is reduced in fats of obese people with metabolic syndrome and brains of suicide victims. This study used quantitative real-time PCR to explore the effects of CPE on the regulation of TTP, VEGF, and related gene expression in mouse 3T3-L1 adipocytes. CPE (100 μg/mL) increased TTP mRNA levels by up to 10-fold, and this stimulation was sustained over 16 h. The levels of VEGF mRNA, a putative target of TTP, were decreased 40-50% by CPE. It also affected the expression of other genes coding for ZFP36L1 and ZFP36L3 (TTP homologues), GM-CSF, COX2, IL6, APP, G-CSF, and PAI1. This study demonstrated that CPE rapidly induces TTP mRNA and reduces VEGF mRNA and affects the expression of a number of other genes in the cultured adipocytes.     

Cacao polyphenol extract suppresses transformation of an aryl hydrocarbon receptor in C57BL/6 mice

Dioxins enter the body through the diet and cause various toxicological effects through transformation of an aryl hydrocarbon receptor (AhR). Plant extracts and phytochemicals including flavonoids are reported to suppress this transformation. This paper investigates the suppression by a cacao polyphenol extract (CPE) of AhR transformation in vivo. The CPE was administered orally to C57BL/6 mice at 100 mg/kg of body weight, followed 1 h later by 3-methylcholanthrene (MC), an AhR agonist, injected intraperitoneally at 10 mg/kg of body weight. CPE suppressed the MC-induced transformation to the control level by inhibiting the formation of a heterodimer between AhR and an aryl hydrocarbon receptor nuclear translocator in the liver at 3 h postadministration. It also suppressed MC-induced cytochrome P4501A1 expression and NAD(P)H:quinone-oxidoreductase activity, whereas it increased glutathione S-transferase activity at 25 h. CPE constituents and their metabolites might contribute, at least in part, to the suppression of AhR transformation. The results indicate that the intake of CPE suppressed the toxicological effects of dioxins in the body.     

Protection of human HepG2 cells against oxidative stress by cocoa phenolic extract

Cocoa is a rich source of flavanols and procyanidin oligomers with antioxidative properties, providing protection against oxidation and nitration. The present study investigated the potential protective effect of a polyphenolic extract from cocoa on cell viability and antioxidant defenses of cultured human HepG2 cells submitted to oxidative stress induced by tert-butylhydroperoxide (t-BOOH). Pretreatment of cells with 0.05-50 microg/mL of cocoa polyphenolic extract (CPE) for 2 or 20 h completely prevented cell damage and enhanced activity of antioxidant enzymes induced by a treatment with t-BOOH. Moreover, lower levels of GSH caused by t-BOOH in HepG2 cells were partly recovered by a pretreatment with CPE. Increased reactive oxygen species (ROS) induced by t-BOOH was dose-dependently prevented when cells were pretreated for 2 or 20 h with CPE. These results show that treatment of HepG2 in culture with CPE (within the physiological range of concentrations) confers a significant protection against oxidation to the cells.     

Enteric virus pollution of tyrrhenian areas

63 samples (53 seawater and 10 estuarine water samples) of 20 L were obtained during a bathing season from 47 seawater stations and from 1 estuarine station. To determine viral pollution, all samples were subjected to two different methods of viral concentration: tangential ultrafiltration and adsorption-elution with electropositive membranes. Detection of viruses was by cytopathic effect (CPE) in BGM and NA 104 cells. Isolates were identified by dot-blot hybridization and Polyacrylamide Gel Electrophoresis (PAGE). While estuarine water showed enterovirus and/or reovirus presence in 100% of samples, only 14 stations of 47 seawater station samples (30%) showed viral contamination: enteroviruses were isolated from 6 and reoviruses from 8 of the 14 stations. 28 unidentified viruses were detected from seawater in MA104 cells by CPE whereas these viruses were not detected in BGM cells. Enterovirus recovery seems to be better when water samples are concentrated by the tangential ultrafiltration than with absorption-elution with electropositive membranes. For reoviruses and the other viruses the two methods were almost equivalent. BGM cells seem to be more susceptible to enteroviruses, MA104 to reoviruses. Reoviruses failed to indicate enterovirus presence as most of enteroviruses were isolated in waters where reovirus was not observed. Isolated viral species distribution changed during bathing season.     

Effect of Drip Fertigation on Yield,Water Use Efficiency,and Nutrients Availability in Banana in West Bengal,India

Field studies were conducted during 2008–2009 and 2009–2010 at the Gangetic alluvial plains of West Bengal, India, to assess the different levels of drip fertigation at variable evaporation replenishment compared to surface irrigation and conventional soil fertilization on yield, water use efficiency, and nutrients availability in plant and ratoon crop of banana. The experiment was laid in an Augmented Factorial Complete Block Design with three replications having three drip irrigation schedules at 50%, 60%, and 70% of cumulative pan evaporation (CPE) and three drip fertigation schedules at 50%, 60%, and 80% of recommended nitrogen, phosphorus, and potassium (NPK) fertilizers with inclusion of conventional surface irrigation at 100% of IW/CPE. The results showed that fruit yield of plant and ratoon crop increased progressively with increasing levels of irrigation water (up to 60% CPE) and NPK fertigation through the drip system. However, maximum fruit yield and water use efficiency of crops was obtained with drip irrigation at 60% CPE with NPK fertigation at 80% of recommended dose. Drip irrigation, as a whole, registered higher fruit yields and water use efficiency with savings of 38.3–41.5% of water compared to surface irrigation. Availability of N, P, and K in soil at vegetative, shooting, and harvesting stages for plant and ratoon crop consistently increased with increasing rate of irrigation water and NPK fertigation through the drip system. Higher availability of macronutrients in soil was recorded with drip irrigation at 70% CPE with 80% of recommended drip NPK fertigation. Overall drip fertigation system improved the available plant nutrients in the soil as compared with traditional surface irrigation.     

Rapid and sensitive automated method for glucose monitoring in wine processing

A rapid and sensitive automated method for glucose monitoring that might be employed during wine fermentation and processing was developed. A flow injection (FI) system coupled with an automated dilutor and the "redox-versatile" modified electrode were used to directly measure glucose in wine. To avoid interferences during wine analysis, different formulations of enzymatically modified carbon paste electrodes (CPE) were used and evaluated in oxidation and reduction mode. The best selectivity and sensitivity for glucose monitoring in real samples was obtained in cathodic mode at a fixed potential of 0 V versus Ag/AgCl using a CPE modified with glucose oxidase, horseradish peroxidase, and ferrocene as redox mediator. A total linear range of 0.02-50 g/L glucose was covered using this automated system and allowed the measurement of glucose in dry, medium, and sweet white or red wines without any sample pretreatment. The results showed a good correlation with the standard method, and the proposed method is very rapid, simple, and reliable and does not need skilled operators.     

Performance of Turmeric and Soil Moisture Depletion Pattern Under Different Water Regimes and Nutrient Sources at New Alluvial Zone of Indo-Gangetic Plains,India

In India, the production of turmeric is not even half compared to its potential. To analyze the possibilities for higher production in turmeric, this study was conducted at the BCKV, Gayeshpur, West Bengal, during 2011 and 2012, to evaluate the effect of irrigation scheduling (0.6, 0.9, and 1.2 irrigation water (IW)/cumulative pan evaporation (CPE) and rainfed) in main plots and nutrient management [100% inorganic, 75% inorganic + 25% FYM (Farm yard manure), and 50% inorganic + 25% FYM + 25% vermicompost] in subplots on soil moisture depletion and productivity of turmeric. The highest value of fresh rhizome yield (23.90 kg ha^?1) and qualitative indices were obtained with irrigation schedule at 0.9 IW/CPE ratio water regimes and 50% inorganic + 25% FYM + 25% vermicompost. The correlation coefficients of water use and yield of turmeric were found at 0.943. The combination of 0.9 IW/CPE and 50% inorganic + 25% FYM + 25% vermicompost improved the overall performance of turmeric crop.     

Effects of different sources of organic matter on soil aggregate formation and stability: A laboratory study on a Lithic Rhodoxeralf from Turkey

In this study the influences of three different organic sources on aggregate formation and stability in soil were investigated.Our approach involves establishing plot experiment in the greenhouse with a Lithic Rhodoxeralf and a variety of carbon sources having specific chemical properties applied to the soil of individual plots. Treatments include K-Humate (KH, 25, 50 and 100 kg/ha), Concentrated Plant Extract (CPE, 50, 100, 200 kg/ha) and Molasses (M, 50, 100, 200 kg/ha). After a seven-month incubation period, aggregate formation and their stability were determined and their correlation to different C sources was developed.The effect of KH on aggregate formation was significant for 2–1 mm aggregates (P < 0.05) and 0.5–0.25 mm aggregates (P < 0.01). The effect of CPE on aggregate formation was significant for 0.5–0.25 aggregates (P < 0.05), while the M had no significant effect on aggregate formation in any aggregate size. The effect of KH on aggregate stability was significant for 8–4 mm and 1–0.5 mm aggregates (P < 0.01). The effect of CPE on aggregate stability was significant for 0.5–0.25 mm aggregates (P < 0.05), while the M was significant for 8–4 mm aggregates (P < 0.05).     

基于交流阻抗法的豆浆电特性检测

为实现豆浆品质快速检测和通电加热电源频率的优化,该文利用精密阻抗分析仪和DJS-10电导电极通过50 mV的激励电压在20 Hz～12 MHz频率范围内测量了不同温度（30～85℃）和不同固形物含量（1.01%～9.58%）的豆浆的交流阻抗。试验结果表明,豆浆阻抗模值与阻抗相位角随测量频率变化具有明显规律。豆浆的阻抗特性与温度以及固形物含量关系显著,且阻抗Nyquist 图存在明显差异。在全频段,豆浆阻抗模值随着温度和固形物含量的升高而减小;在低频段,豆浆阻抗相位角随温度以及固形物含量的升高而增大;在高频段,豆浆阻抗相位角随温度以及固形物含量的升高而减小。同时,研究提出了豆浆的电阻R、电容C、恒相位元件CPE（constant phase element）三元件等效电路模型,并用ZSimpWin软件进行拟合得到了试验条件下豆浆的等效电路元件参数。基于豆浆阻抗随频率变化规律及豆浆等效电路模型分析得出,豆浆通电加热电源的频率应在300 Hz～300 kHz范围内。豆浆的等效电阻R与温度以及固形物含量之间具有良好的负指数关系。CPE参数Q值随温度的升高而降低,当固形物含量超过3.5%后,Q值随固形物含量的升高迅速增加。CPE参数n值随温度的升高而升高,但在本研究中的固形物含量范围内n值并未随固形物含量的改变发生明显变化。本研究为豆浆通电加热电源的频率选择提供了参考依据,同时为基于豆浆电特性分析实现豆浆品质的快速检测奠定基础。