In vitro and in vivo modulation of the equine immune response by parapoxvirus ovis

REASON FOR PERFORMING STUDY: While immune modulators are used routinely in equine medicine, their mechanism of action is not always known. OBJECTIVES: To determine the effect of a commercial preparation of inactivated parapoxvirus ovis (Orf virus; PPVO) on cytokine gene expression by equine peripheral blood mononuclear cells (PBMC) both in vitro and in vivo. METHODS: PBMC were prepared from 6 mixed-breed yearlings and cultured in vitro with PPVO with or without Concanavalin A (Con A) for 24 h. Effects on the expression of IFNalpha, IFNbeta IFNgamma, TNFalpha and IL-18 were analysed by real time quantitative PCR (RT-PCR). In addition, 12 yearling horses were treated with PPVO and whole blood RNA samples were prepared at regular intervals to assess effects on in vivo cytokine gene expression. Six of those yearlings were later treated with saline and served as treatment controls. Nine additional yearlings were injected intradermally with a single dose and their injection sites biopsied at 24 and 48 h for cytokine expression. RESULTS: In vitro culture of PBMC with PPVO led to a significant increase in IFNalpha and IFNbeta gene expression compared to mock-stimulated cultures. In addition, expression of IFNgamma and TNFalpha was significantly higher in PBMC stimulated with PPVO and Con A, than those stimulated with Con A alone. No changes were observed in IL-18 gene expression in vitro. Treatment of horses with a 3-dose regimen of PPVO resulted in elevation of IFNgamma gene expression, which was detected 24 h after the first dose and declined thereafter. Intradermal inoculation led to increased expression of IFNgamma along with IFNbeta, IL-15 and IL-18. CONCLUSIONS: Together these results indicate that PPVO stimulated IFNgamma production both in vitro and in vivo. Increased cytokine expression could account for its immunomodulatory activity. POTENTIAL RELEVANCE: The absence of adverse reactions and clear indications of increased expression of cytokine gene expression supports previous clinical uses for this immune modulator in those situations when increased expression of IFNgamma is warranted.     

3种猪细胞因子mRNA实时荧光定量RT-PCR检测方法的建立与应用

为建立猪细胞因子SYBR Green Ⅰ实时荧光定量RT-PCR检测方法,根据GenBank中3种重要的猪细胞因子即猪白细胞介素-2(interleukin-2,IL-2)、α-干扰素(interferon α,IFN-α)和肿瘤坏死因子-α(tumor necrosis factor,TNF-α)的基因序列,设计特异引物扩增目的基因.将3种基因克隆至pMD18-T载体上,得到各自阳性克隆质粒,以3种阳性质粒为标准品建立标准曲线并进行熔解曲线分析以及灵敏性、特异性和重复性试验.结果表明,当标准品稀释度为1×10¹~1×10⁶ 拷贝/μL时,3种基因的Ct值与浓度间具有良好的线性关系,相关系数均≥0.992.熔解曲线分析表明,产物为特异性单峰且重复性较好.应用建立的方法对猪繁殖与呼吸综合征病毒(PRRSV)TJM-F92株免疫的30日龄猪外周血单核细胞(PBMC)中IL-12、IFN-α和TNF-α表达量进行检测,结果发现,免疫了PRRSV TJM-F92株的猪PBMC细胞内3种细胞因子表达量均极显著升高(P< 0.01).研究结果为IL-12、IFN-α和TNF-α的定量分析提供了技术平台.     

犬瘟热病毒强毒株HBF-1株的培育及致病性研究

为研究从北极狐病料样品中分离的一株强毒的致病性,本实验采用病例复制、RT-PCR检测、间接免疫荧光检测(IFA)和电镜观察等方法证实分离得到犬瘟热病毒(CDV),并命名为HBF-1。对该分离株H基因的核苷酸序列比对显示,HBF-1与疫苗株的同源性为91.0%～91.5%,与国内外分离株的同源性为93.5%～99.9%。病毒传代培育试验结果显示HBF-1已适应在北极狐、貉、水貂和犬体内繁殖,具有较广的感染范围。但各种动物的临床症状和剖检病理变化存在不同程度的差异,表明HBF-1分离株对北极狐、貉、水貂和犬的致病力不同;毒力测定结果显示其半数感染量分别为102.46 ID50/mL、102.95 ID50/mL、102.46 ID50/mL和102.58 ID50/mL,表明HBF-1为一株CDV强毒株,可以在不同的经济动物间进行水平传播。本研究结果为开发新的CDV疫苗提供了实验基础。     

Radiographic evaluation of cardiac size in flying fox species (Pteropus rodricensis, P. hypomelanus, and P. vampyrus).

Dilated cardiomyopathy is a relatively common pathology in captive flying foxes (Pteropus spp.). The goal of this study was to establish quantitative reference range measurements that could be used to support a diagnosis of cardiac disease in these animals. Lateral and ventrodorsal thoracic radiographs from apparently healthy flying foxes (n = 66) of three species (Rodriguez island flying fox, P. rodricensis, n = 18; small island flying fox, P. hypomelanus, n = 16; and Malaysian flying fox, P. vampyrus, n = 32) were evaluated objectively to describe the cardiac appearance. Absolute and relative cardiac dimensions also were measured. The same methods were used to evaluate radiographs from flying foxes (n = 9) with known dilated or acute cardiomyopathy. The following ratios were most appropriate for categorizing normal cardiac silhouette size. In the ventrodorsal projection, heart width to thoracic width and heart width to clavicle length were the preferred measurements. In the lateral projection, heart width compared with thoracic height was the preferred measurement. From radiographs of the bats with known dilated and acute cardiomyopathy, the apicobasilar heart length compared with thoracic height and heart width compared with thoracic height on lateral films were the most sensitive ratios for diagnosing cardiomegaly.     

Missense polymorphisms in the MC1R gene of the dog,red fox,arctic fox and Chinese raccoon dog

Coat colour variation is determined by many genes, one of which is the melanocortin receptor type 1 (MC1R) gene. In this study, we examined the whole coding sequence of this gene in four species belonging to the Canidae family (dog, red fox, arctic fox and Chinese raccoon dog). Although the comparative analysis of the obtained nucleotide sequences revealed a high conservation, which varied between 97.9 and 99.1%, we altogether identified 22 SNPs (10 in dogs, six in farmed red foxes, two in wild red foxes, three in arctic foxes and one in Chinese raccoon dog). Among them, seven appeared to be novel: one silent in the dog, three missense and one silent in the red fox, one in the 3′‐flanking region in the arctic fox and one silent in the Chinese raccoon dog. In dogs and red foxes, the SNPs segregated as 10 and four haplotypes, respectively. Taking into consideration the published reports and results of this study, the highest number of missense polymorphisms was until now found in the dog (9) and red fox (7).     

B亚型禽偏肺病毒SYBRGreenI荧光定量PCR检测方法的建立及应用

根据GenBank登录的B亚型禽偏肺病毒（JN224985．1）F基因序列设计1对特异性引物,经RT—PCR扩增出214bp的目的片段。回收目的片段并与pMD18-T载体连接后转化到基因工程菌DH5a中,提取重组质粒,经PCR、酶切及测序鉴定后,筛选出阳性质粒作为模板,建立SYBRGreenI荧光定量PCR标准曲线,并做敏感性试验、特异性试验和重复性试验。结果表明,标准曲线循环阈值与模板浓度呈良好的线性关系,产物T值在83．0～83．7℃之间,灵敏度为7．9×10^2拷贝／μL,特异性和重复性较好。本试验建立了检测B亚型禽偏肺病毒的sYBRGreenI荧光定量PCR方法,为该病的早期诊断以及感染程度的定量分析奠定了基础。     

狐狸脑炎病毒的分离鉴定

通过ＭＤＣＫ传细胞从黑龙江某狐场疑似狐狸脑炎病狐的肝脏中分离到对本动物具有较强致病能力的强毒株,定名为ＦＥＶ－Ｈ。经系统鉴定,并与已知国内分离毒株狐狸脑炎病毒ＦＥＶ－８８０１,狐喉气管炎病毒ＦＡＶ－２比较,证实为狐狸脑炎病毒,属犬１型腺病毒（ＣＡＶ－１）。     

猪传染性胃肠炎病毒S基因实时荧光定量RT-PCR检测方法的建立及初步应用简

据猪传染性胃肠炎病毒（transmissible gastroenteritis virus of swine,TGEV）S基因序列设计1对特异性引物,通过对实时荧光定量RT-PCR反应条件的优化,建立了SYBR GreenⅠ实时荧光定量RT-PCR检测TGEV的方法,同时对12份病料进行检测,并与常规RT-PCR进行比较。结果显示,该方法的敏感性达到43.07拷贝/μL,具有良好的特异性和重复性,而常规RT-PCR最低只检测到4.307×10³ 拷贝/μL,敏感性较低。本试验建立的检测TGEV S基因的SYBR GreenⅠ实时荧光定量RT-PCR方法为传染性胃肠炎的鉴别诊断及TGEV的分离鉴定奠定了技术基础。     

Development and validation of cytokine quantitative, real time RT-PCR assays for characterization of Asian elephant immune responses

Infectious disease is an important factor in Asian elephant health and long-term species survival. In studying disease pathogenesis, it is important to consider not only the pathogen, but also the effectiveness of the host immune response. Currently, there is a paucity of information available on elephant immune function. Measurement of cytokine levels within clinical samples can provide valuable information regarding immune function during health and disease that may elucidate disease susceptibility. To develop tools for assessment of elephant immune function, Asian elephant partial mRNA sequences for interleukin (IL)-2, IL-4, IL-10, IL-12, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and β-actin were determined. Sequence information was then utilized to design elephant-specific primers and probes for quantitative, real time, RT-PCR assays for the measurement of cytokine mRNA. Greater than 300 bps of Asian elephant mRNA sequence were determined for each cytokine of interest. Consistent and reproducible, real time, RT-PCR assays with efficiencies of greater than 93% were also developed. Assay sensitivities ranged from less than 1 to 5000 DNA copies with the exception of IL-12, which had a sensitivity of 42,200 copies. Employment of molecular techniques utilizing mRNA-based detection systems, such as real time, RT-PCR, facilitate sensitive and specific cytokine detection and measurement in samples from species for which commercial reagents are not available. Future studies utilizing these techniques to compare elephant immune function during health and in the face of infection will be useful for characterizing the contribution of the elephant immune system to disease.     

猪流行性腹泻病毒实时荧光定量RT-PCR检测方法的建立与应用

根据Gen Bank登录的猪流行性腹泻病毒（PEDV）毒株（登录号KJ960180）的M基因保守序列,设计1对扩增片段大小为299 bp的特异性引物,经PCR扩增、克隆、测序鉴定后,提取质粒作为阳性标准品,建立了PEDV的SYBR GreenⅠ荧光定量RT-PCR检测方法。该方法在1.21×10^3~1.21×10^8拷贝/μL范围内呈现良好的线性,相关系数（R2）为0.999,扩增效率为99%,扩增产物的熔解曲线为单个特异峰,产物Tm值为85.5~86℃,最低检测限为1.21×10^1拷贝/μL。本研究建立的SYBR GreenⅠ实时荧光定量RT-PCR检测方法特异性强、重复性好、成本低且操作简单,适用于PEDV的早期诊断、定量研究和流行病学监测。     

禽腺病毒血清4型感染鸡组织中NLRP3基因转录水平的实时荧光定量PCR分析

旨在分析禽腺病毒血清4型（FAdV-4）感染鸡组织中NLRP3基因的转录水平,本研究设计鸡NLRP3特异性引物,利用RT-PCR扩增NLRP3基因180 bp片段并克隆至pMD-18T载体,制备重组质粒pMD-18T-NLRP3。以pMD-18T-NLRP3质粒作为标准品进行荧光定量PCR并建立标准曲线。通过反应条件优化,成功建立了检测NLRP3基因的实时荧光定量PCR方法,并利用该方法对致病性FAdV-4感染鸡组织中NLRP3基因的转录水平进行了分析。结果显示,所设计的NLRP3引物可特异性扩增鸡NLRP3基因,建立的实时荧光定量PCR对鸡NLRP3标准质粒的扩增曲线良好,标准品的拷贝数与Cq值呈现良好的线性关系。与对照组相比,NLRP3分子在FAdV-4感染鸡肝和脾中的转录水平极显著高于对照组（P<0.001）,在盲肠扁桃体和法氏囊的表达显著高于对照组（P<0.01）。本研究所建立的鸡NLRP3基因SYBR Green Ⅰ实时荧光定量PCR可以检测FAdV-4感染鸡不同组织中NLRP3的转录水平;致病性FAdV-4感染所造成的组织炎症损伤与NLRP3分子密切相关。     

Comparative studies on in vitro mitogen-induced proliferation of peripheral blood lymphocytes in dog and breeding fox.

In vitro blastogenesis of dog and fox lymphocytes was compared by a microculture technique. The highest 3H-thymidine incorporation in cultures of dog lymphocytes was observed at day 3, while in those of fox at day 2, incubated either at 37 degrees C or at 39 degrees C. Lymphocytes cultured at 39 degrees C incorporated more tritiated thymidine than did cells cultured at 37 degrees C. The stimulation index (SI) of dog peripheral blood lymphocytes to both mitogens concanavalin A (Con A) and leucoagglutinin (LA) was in a similar range, while pokeweed mitogen (PWM) showed a weaker but significant stimulatory action. The blastogenesis of fox lymphocytes was the greatest in Con A stimulated cultures. The mitogenic potency of LA and PWM was about half of that of Con A, with no essential difference between them. Maximum lymphocyte proliferation of dog and fox was observed when culture media were supplemented with 10% fetal calf serum (FCS).     

Development of a microarray for studying porcine cytokine production in blood mononuclear cells and intestinal biopsies

A microarray for demonstration of a limited number of porcine cytokines was initiated. Polymerase chain reaction (PCR) products were synthesized for four house-keeping genes, cyclophilin, beta-actin, hypoxanthine phosphoribosyltransferase (HPRT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the following cytokines: interleukin (IL)-1beta, IL-4, IL-6, IL-8, IL-10, IL-12 p35, IL-12 p40, IL-18, interferon (IFN)-alpha, IFN-beta, IFN-gamma, tumour necrosis factor (TNF)-alpha, macrophage inhibition factor (MIF) and granulocyte/macrophage colony-stimulating factor (GM-CSF). Cytokine production was induced by incubation of porcine peripheral blood mononuclear cells (PBMC) with Concanavalin A (ConA) or oligodeoxynucleotide (ODN) 2216. RNA was isolated after 6 or 24 h from stimulated cells or unstimulated control cells and from intestinal biopsies. Cytokine expression was analysed using a 3-DNA Array 350(TM) labelling kit from Genisphere. Data were normalized using external control genes and analysed with the genepix pro 5.0 software. All the cytokines could be induced in PBMC and expressed on the array and the cytokines IL-6 and IFN-alpha were also analysed at protein level. All but one cytokine were expressed in samples from intestinal biopsies. Densitometric analyses of PCR products of the house-keeping genes were performed to validate the results from the microarray. Thus, this microarray will enable analyses of the cytokine profile during local and systemic infections in the pig.     

Development of a Real-time PCR Assay for Detection of Chicken IL-1β, IL-18 and Tumor Necrosis Factor α Genes

In order to develop a SYBR GreenⅠ Real-time PCR assay for detection of chicken IL-1β,IL-18 and tumor necrosis factor-α (TNF-α) genes, four specific primer pairs were designed according to the chicken's IL-1β,IL-18, TNF-α and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene sequences in GenBank. The four fragments were amplified by RT-PCR from chicken embryo fibroblasts, cloned and sequenced. The recombinant plasmids containing the target gene were constructed and used as the Real-time PCR standard templates. Real-time PCR assays based on SYBR GreenⅠfor detection of chicken IL-1β,IL-18, TNF-α and GAPDH were established. The results showed that each gene's melting curve also had a single peak,each gene's amplification efficiency was 101.2%, 95.6%, 100.1% and 98.2%, R² was 0.9996,0.9998,0.9957 and 0.9989. Moreover,the assays were highly sensitive,the detection limit of 100 copies in 35 Ct and each gene's coefficient of variation less than 1.4 percent for intra-assay. This reliable Real-time PCR assay might be used for decting chicken's IL-1β,IL-18 and TNF-α mRNA expressing and provided the basis for quantitative analysis of cytokine expression in host cell after virus infect which cause immunosuppressive diseases.