Expression of RANTES mRNA in skin lesions of feline eosinophilic plaque

Abstract One of the mechanisms of eosinophil infiltration is its induction by chemoattractants such as regulated upon activation, normal T-expressed and secreted (RANTES) which is a cysteine–cysteine chemokine that mediates chemotaxis and activation of eosinophils in humans and mice. Skin lesions of feline eosinophilic plaque are characterized by a predominant infiltration of eosinophils. The mechanism(s) of eosinophilic infiltration in the skin and/or mucosa of cats is unknown. It is possible that RANTES is involved. To investigate the presence of RANTES in the skin of cats with eosinophilic plaques and nonaffected skin, we cloned and sequenced the full-length feline RANTES cDNA gene, in order to determine whether it is present in the skin of cats with eosinophilic plaques and/or if it is present in normal adjacent skin. We were able to document the the expression of RANTES mRNAs in skin with feline eosinophilic plaque as well as in normal cat skin. The full-length cDNA sequence of the RANTES gene (742 bp) contained a single open reading frame of 276 bp encoding a protein of 92 amino acids. The amino acid sequence of feline RANTES shared 67 and 74% sequence identity with that of bovine and mouse RANTES genes, respectively. RT–PCR analysis on RANTES mRNA in the skin of cats with eosinophilic plaque revealed that its expression was higher in the eosinophilic plaque skin lesions than in the normal skin. The result suggested that RANTES might play a role to induce eosinophil infiltration in feline eosinophilic plaque lesions.     

Electron Microscopy and Molecular Characterization of Phytoplasmas Associated with Strawflower Yellows in the Czech Republic

Strawflower (Helichrysum bracteatum) with symptoms resembling those associated to phytoplasma infection were observed in several areas in the Czech Republic during the period 1994–2001. Plants with leaf bronzing, reddening and necrosis, proliferation of secondary shoots, flower abnormalities and dwarfing died in advanced stages of the disease. The disease incidence ranged from 2% to 70% and caused significant loss to the flower and seed production. Transmission electron microscopy showed phytoplasmas in sieve cells of affected plants, but not in healthy ones. Association of phytoplasmas with the disease was confirmed by polymerase chain reaction using phytoplasma universal ribosomal primers R16F2n/R16R2. An amplification product of the expected size (1.2 kb) was observed in all samples of the symptomatic strawflowers. The restriction profiles obtained following separate digestion with three endonucleases (AluI, HhaI, MseI) showed that phytoplasmas infecting strawflowers from different localities in the Czech Republic were uniform and undistinguishable from aster yellows (subgroup 16SrI-B). Sequence analysis of 1771 bp of the ribosomal operon amplified with primers P1/U3, R16F2n/R2 and 16R758/P7 indicated that the closest related phytoplasmas were those associated with 'Rehmannia glutinosa var. purpurea', both originating from Bohemia. This is the first report on the occurrence of a phytoplasma-associated disease of strawflower in the Czech Republic.     

Monitoring of atrazine treatment on soil bacterial,fungal and atrazine-degrading communities by quantitative competitive PCR

We report the development of quantitative competitive (QC) PCR assays for quantifying the 16S, 18S ribosomal and atzC genes in nucleic acids directly extracted from soil. QC-PCR assays were standardised, calibrated and evaluated with an experimental study aiming to evaluate the impact of atrazine application on soil microflora. Comparison of QC-PCR 16S and 18S results with those of soil microbial biomass showed that, following atrazine application, the microbial biomass was not affected and that the amount of 16S rDNA gene representing 'bacteria' increased transitorily, while the amount of 18S rDNA gene representing fungi decreased in soil. In addition, comparison of atzC QC-PCR results with those of atrazine mineralisation revealed that, in response to atrazine treatment, the amount of atzC gene increased transitorily in soil pre-treated with atrazine, suggesting that accelerated atrazine biodegradation in soil could be due to a transient increase in the size of the atrazine mineralising community.     

The Value of Tissue Imprint Hybridization for Rapid Detection of Infectious Bursal Disease Virus from Field Outbreaks

The use of a peroxidase labelled PCR generated probe followed by enhanced chemiluminescence hybridization assay detected infectious bursal disease virus directly from bursal imprints on a nylon membrane. Tissue imprint hybridization proved to be a simple, rapid and safe means of detecting IBD virus for screening large numbers of field samples. The PCR generated probe was highly specific for IBD virus and did not hybridize with cellular nucleic acids in control imprints. Tissue imprint hybridization was found to be a more sensitive method than conventional antigen detection assays.     

Detection of Brucella species in the milk of infected cattle,sheep, goats and camels by PCR

One hundred and three milk samples were collected from 52 cows, 21 ewes, 18 goats and 12 camels. The animals tested positive to at least one of the following: (1) standard tube agglutination test (SAT); (2) Rose Bengal plate test (RBPT); (3) milk ring test (MRT). All milk samples were examined by culture and single-step polymerase chain reaction (PCR) techniques for detection of Brucella species. The PCR assay amplified Brucella-DNA from 29 bovine milk samples, 10 from sheep, 13 from goats and one from a camel. The direct culture method detected Brucella organisms from 24 samples of cows' milk, 12 from sheep, 10 from goats and failed to detect any Brucella organisms from camels' milk. PCR detected up to 100 colony forming units (CFU) of B. abortus per millilitre of milk in 100% of diluted milk samples, and 1000 CFU of B. melitensis from 70% of milk samples. Although the overall sensitivity of the PCR was higher than the culture method, it should be possible to increase the sensitivity to detect lower numbers of Brucella organisms in field samples. The speed and sensitivity of the PCR assay suggest that this technique could be useful for detection of Brucella organisms in bovine milk, as well as in sheep, goat, and camels milk.     

猪伪狂犬病PCR诊断方法的建立

根据伪狂犬病病毒(PRV)gB基因的序列,设计并合成了一对引物,以闽A株细胞培养毒为模板,筛选最佳反应条件,建立了检测PRV的PCR方法,应用本方法对分离的病毒进行基因扩增,获得了217bp的特异性DNA片段,证明了对分离病毒检测的特异性和敏感性,为伪狂犬病的快速诊断提供了条件。     

猪链球菌PCR检测技术研究进展

猪链球菌是猪的一种重要病原菌,并且也会引起人的链球菌病。有35个荚膜血清型(1/21、~34),通常自发病或死亡猪体分离获得1,2,7,9型和14型菌株,其中2型是毒力最强的血清型。根据已知猪链球菌16 SrRNA及溶血素(sly)、谷氨酸脱氢酶(gdh)、荚膜多糖(cps)、胞壁蛋白或溶菌酶释放相关蛋白(mrp)、胞外因子(epf)编码基因序列设计特异性引物,建立猪链球菌群和1(14),2(1/2),7型和9型特异性PCR或多重PCR,建立2型致病性菌株和1型高致病性菌株毒力鉴定PCR或多重PCR,用于检测和鉴别临床病料和细菌分离物中的猪链球菌,具有高敏感性和高特异性,与其他致病菌及其他血清的猪链球菌型无交叉反应,为疫病诊断及流行病学的研究提供了快速、简便和有用的工具。     

实时荧光定量PCR技术研究进展及应用

实时荧光定量PCR(Real-time fluorescent quantitative polymerase chain reaction,Real-time PCR)技术是20世纪90年代中期发展起来的一种新型核酸定量技术.它利用荧光实时检测PCR反应,具有灵敏度高、特异性强、节约时间等优点.文章就常用的4种方法,即DNA结合染色、水解探针、分子信标和杂交探针的原理和特点进行介绍,同时综述了荧光定量PCR技术在实践中的应用.     

羊痘病毒实时荧光定量TaqManPCR检测方法的建立

参照羊痘病毒（CaPV）P32的基因序列，设计合成了2套引物和1条探针，建立了实时荧光定量PCR技术，对细胞培养物、皮肤丘疹、痂皮等组织病料中的GPV进行了特异性检测和敏感性试验。结果显示，用300nmol／L引物浓度和200nmol／L探针浓度，获得的CT值较小，而△Rn最大；可检测到相当于0．1TCID50的病毒DNA；制作的标准曲线中各浓度范围内有极好的线性关系且线性范围宽，相关系数为0．9995以上；组内和组间试验重复性的变异系数分别为2．3％和3．4％；与常规的PCR相比较，该方法具有快速、特异、敏感、可定量，可同时检测大量样品等优点。表明，荧光TaqMan PCR是一种检测CaPV的良好方法，可对组织病料中低含量的CaPV或持续带毒宿主进行准确检测。     

鸡白痢沙门菌等位基因特异性PCR检测方法的建立

根据鸡白痢沙门菌与鸡伤寒沙门菌的rfbS基因在第237和第598位碱基的不同，设计和合成了等位基因特异性PCR引物，建立了快速检测鸡白痢沙门菌的PCR方法，并应用该方法对鸡白痢沙门菌临床分离样品进行了PCR鉴定。结果显示，该PCR方法能够特异性地鉴定鸡白痢沙门菌，检测灵敏度达100PgDNA。对35个经常规方法鉴定的鸡白痢沙门菌分离株应用等位基因特异性PCR方法进行鉴定，鉴定出33株鸡白痢沙门菌，符合率为94．3％。表明，建立的等位基因特异性PCR方法能够准确而快速地鉴定鸡白痢沙门菌。