Mycoplasma iowae species-specific DNA probe

A Mycoplasma iowae (MI) species-specific DNA probe (designated pMI-2) of 6.0 kbp (kilobase pairs) was isolated from an MI strain I-695 genomic library prepared in plasmid pUC8 and Escherichia coli strain JM83. When labeled with [32]P by nick translation, the probe hybridized in dot blot assays with 6 reference strains and 8 field isolates of MI but not with 16 other known species of avian mycoplasmas. The pMI-2 probe detected a minimum of 1.5 ng of MI strain I-695 chromosomal DNA. Under identical conditions of hybridization, the probe did not hybridize with a high concentration (200 ng) of M. gallisepticum or M. synoviae chromosomal DNA.     

Fingerprinting of Mycoplasma gallisepticum strains isolated from multiple-age layers vaccinated with live F strain

Mycoplasma gallisepticum (MG) isolates were obtained from three multiple-age commercial layer farms on which live F strain vaccine had been administered to each replacement flock for at least 2 years. All such isolates had restriction endonuclease DNA and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein patterns characteristic of F strain. These cultures also hybridized in dot blot assays with both the MG strain-specific and species-specific DNA probes. In contrast, the original MG isolate that came from one of the farms before vaccination began clearly was not F strain. These results suggest that continuous use of live F strain vaccine in each replacement pullet flock on multiple-age commercial layer sites will result in displacement of the original field strain of MG with the vaccine strain.     

A Mycoplasma gallisepticum strain-specific DNA probe

Total DNA from the vaccine F strain (K810) and the reference S6-strain of Mycoplasma gallisepticum (MG) was cloned in Escherichia coli using the plasmid pUC8. A 6-kilobase fragment, specific for the vaccine strain, was identified by colony dot and Southern hybridization analyses. When labeled and used as a probe, this fragment hybridized with the homologous and one other vaccine F-strain (F2F10), but it did not hybridize with other MG strains (Fg38, S6, A5969, V503) or with three other species of avian mycoplasmas.     

滑液支原体WVU1853株热不稳定延伸因子的表达及黏附特性分析

旨在探究滑液支原体(Mycoplasma synoviae,MS)热不稳定延伸因子(elongation factor thermo unstable,EF-Tu)的黏附特性.参照GenBank中MS WVU1853株EF-Tu序列,设计引物对Tu-F/Tu-R,利用PCR扩增获得MS EF-Tu基因后,将其克隆入pE...     

滑液支原体GDPD基因的原核表达、酶活性分析及亚细胞定位

为了解滑液支原体（Mycoplasma synoviae，MS）甘油磷酸二酯磷酸二酯酶（glycerophosphodoester phosphodiesterase，GDPD）的生物学功能，本试验参照GenBank中MS WVU1853株序列设计特异性引物，应用PCR技术扩增获得MS WVU1853株GDPD基因，在测序及序列分析的基础上将其克隆至pET-28a（+）质粒构建原核表达载体pET-GDPD，转化大肠杆菌BL21（DE3）感受态细胞后经IPTG诱导表达，纯化表达产物并分析其酶促活性，进而制备其多克隆抗体，应用间接ELISA和Western blotting检测其免疫原性并分析其在MS内的分布。结果表明，MS WVU1853株GDPD基因CDS全长726 bp，编码242个氨基酸，重组蛋白分子质量约为28 ku。酶促活性检测表明，重组蛋白可催化对硝基苯磷酸二钠（pNPP）转化为对硝基苯酚，且其作用的最适pH为9.0，最佳温度为37℃，Pb²⁺具有较强的抑制作用，而Ca²⁺对其具有较强的促进作用。间接ELISA及Western blotting检测结果表明，MS GDPD具有良好的免疫原性，可刺激新西兰兔产生高水平的抗体，血清效价高达1：160000；亚细胞定位结果表明，MS GDPD在细胞膜和细胞浆内均有分布，但在细胞膜的含量略高于细胞浆。本研究结果为探究MS GDPD生物学功能提供了一定的参考依据。     

新城疫病毒F48E8株融合蛋白基因的克隆

以提纯的我国标准ＵＤＶＦ４８Ｅ８强毒株基因组ＲＮＡ为模板、化学合成的融合蛋白（Ｆ）基因特异寡核苷酸为引物，反转录合成Ｆ基因ｃＤＮＡ，并采用同聚物加尾的方法克隆到细菌质粒ｐＧＥＭ３Ｚｆ（－）。经ＡＩＸ平板筛选和酶切分析，并用Ｆ基因片段核酸探针作ｄｏｔ－ｂｌｏｔ检测，共获得插入片段长度在０．６￣２．８ｋｂ之间的阳性克隆３４个，其中插入片段大于１．５ｋｂ的阳性克隆８个。用多种限制性内切酶对阳性克隆ｐＦ７     

Cloning of Mycoplasma synoviae genes encoding specific antigens and their use as species-specific DNA probes.

A genomic library of Mycoplasma synoviae (MS) was generated by using bacteriophage lambda gt11 as a cloning and expression vector. Identification of recombinant clones highly specific to MS was achieved by screening the library for expression of MS proteins with polyclonal antiserum that had been preadsorbed with 6 heterologous avian mycoplasma species antigens. Expression of the recombinant clones in Escherichia coli followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the total cell lysates and immunoblot yielded a predominant reactive fusion protein of 165 kD. Two clones (MS2/28 and MS2/12) that yielded inserts of different size were selected. The 2 MS DNA inserts were subcloned in a plasmid vector, labeled with digoxigenin, and used as probes for the specific recognition of several MS strains. A high degree of conservation was demonstrated for the MS2/12 and MS2/28 genes in tested MS strains. In addition, neither DNA fragment recognized any other avian mycoplasma species (M. gallisepticum, M. meleagridis, M. gallinarum, M. iners, M. anatis, and M. iowae), thus indicating their high specificity to MS. The sensitivity of the slot blot hybridization method using digoxigenin-labeled MS2/12 and MS2/28 probes for direct detection of MS from broth cultures of field isolates was 10(5) colony-forming units/ml. These results demonstrate the effectiveness of adsorbed antisera for the isolation of species-specific mycoplasma DNA and the potential for its use as probes for the specific and direct detection of MS from broth cultures of field isolates.     

Ribosomal RNA gene probes to detect intraspecies heterogeneity in Mycoplasma gallisepticum and M. synoviae

Intraspecies genotypic heterogeneity among strains of Mycoplasma gallisepticum and M. synoviae was tested using genomic fingerprints with a ribosomal RNA (rRNA) gene probe. The organism's DNA was digested by a restriction endonuclease, electrophoresed, transferred to a nitrocellulose sheet, and hybridized with 32P-labeled pMC5 plasmid carrying the highly conserved rRNA genes of M. capricolum. The resulting hybridization patterns indicated a degree of genotypic heterogeneity among M. gallisepticum strains more pronounced than among the M. synoviae strains tested. Most importantly, the live vaccine F strain of M. gallisepticum could be distinguished from virulent field isolates of this species, enabling the detection and identification of the F strain in areas in which vaccination with this strain has taken place. Genomic fingerprints with an rRNA gene probe can thus be added to the battery of tools useful in taxonomy at the intraspecies level and in epidemiology of mycoplasmosis in poultry.     

Species-specific DNA probes for Campylobacter species isolated from pigs with proliferative enteritis

Cloned, chromosomal DNA probes from porcine isolates of Campylobacter hyointestinalis and C. mucosalis were developed for the detection and identification of these putative swine enteric pathogens. High molecular weight chromosomal DNA from each species was used to construct genomic libraries in plasmids. Recombinants were selected which hybridized strongly to the homologous organism, but not to any other species of Campylobacter. Species-specific recombinants were labeled with phosphorus-32 and tested for sensitivity by dot blot hybridization to various dilutions of DNA and bacteria from each swine species, including C. hyointestinalis, C. mucosalis, C. coli and C. jejuni. Specificity was tested by hybridizing these probes against various strains of C. hyointestinalis or C. mucosalis, and against reference strains of all other described Campylobacter species. A C. hyointestinalis-specific probe and a C. mucosalis-specific probe were identified which were capable of detecting 1 ng of DNA or 10(4) cfu by bacterial spot blotting on nylon membranes. These probes hybridized to intestinal mucosal scrapings containing C. hyointestinalis and C. mucosalis obtained from pigs with proliferative enteritis, but not to material from normal pigs. Thus, cloned, chromosomal DNA probes may be useful in the detection and identification of bacteria involved in swine proliferative enteritis.     

Development of genomic probes to Sarcocystis cruzi (Apicomplexa).

A genomic library of Sarcocystis cruzi sporozoite DNA was constructed in bacteriophage lambda gt10. Recombinant phages containing insert DNA were selected by growth on Escherichia coli strain C600 hflA150. Of 14 clones examined, 11 contained DNA inserts ranging in size from approximately 1.45 kilobase (kb) to 6.18 kb. Insert DNA from four of these clones specifically hybridized to 32P-labelled S. cruzi merozoite DNA. One of these insert DNA, clone SL41, was selected and labelled with 32P. This probe did not hybridize with the other ten DNA inserts nor with bovine cellular DNA, but it hybridized with sporozoite, merozoite and bradyzoite DNA preparations. The SL41 probe could detect merozoite DNA in as little as 17 ng total DNA. Genomic probes detecting developmental stages of Sarcocystis spp. could provide an improved means is diagnosis of acute bovine sarcocystosis.     

Characterization of a repetitive DNA probe for Babesia bigemina

A plasmid (p16) containing a Babesia bigemina DNA insert was selected and labeled with 32P. This probe was evaluated for specificity and sensitivity by dot blot hybridization. The probe was specific and hybridized with only Babesia bigemina DNA, and not DNA from Babesia bovis, bovine leukocyte, Trypanosoma brucei or Anaplasma marginale. The DNA probe detected as little as 10 pg of Babesia bigemina DNA. The probe hybridized with Babesia bigemina isolates from Mexico, the Caribbean region and Kenya. Genomic Babesia bigemina DNA of a Kenyan isolate was digested with restriction endonucleases, and the fragments were separated by gel electrophoresis and Southern blotted. The filter was hybridized with labeled p16 and each endonuclease digestion produced at least 16 resolvable DNA fragments. The inserted Babesia bigemina DNA was approximately 6.3 kb in size. A partial restriction map was constructed. A simple whole blood dot blot procedure was utilized to evaluate the sensitivity of the DNA probe. This probe would detect as few as 150 Babesia bigemina infected erythrocytes contained in a 1-microliter sample. The DNA probe has the potential to be a very sensitive and specific diagnostic tool.     

结核分枝杆菌分泌蛋白ESAT-6、CFP10基因的克隆、鉴定及其原核表达

以人型结核分枝杆菌 H37RV株基因组 DNA为模板 ,应用 PCR法对 ESAT- 6和 CFP10基因进行扩增 ,产物经纯化后与载体 PMD18- T连接、转化及酶切鉴定 ,亚克隆到原核表达载体 PGEX- 6 P- 1,构建原核重组表达质粒 ,转化入大肠杆菌 BL2 1中 ,以 1mmol/L IPTG诱导 ,进行 SDS- PAGE电泳。结果表明 ,ESAT- 6和 CFP10基因表达的融合蛋白相对分子质量分别为 32 0 0 0和 36 0 0 0 ,与实测相符。重组结核杆菌分泌蛋白 ESAT- 6和 CFP10的成功表达为结核病诊断及重组疫苗的构建打下了基础     

Virulence of Mycoplasma synoviae strains in experimentally infected broiler chickens

Seven field isolates of German origin and the type strain WVU 1853 of Mycoplasma synoviae (MS) were experimentally investigated for their virulence in mycoplasma-free broiler chickens. Two groups of birds were inoculated at 6 days of age with each isolate, one group into the thoracic air sac and the other group intravenously and all surviving birds were examined at necropsy 17 days post inoculation (pi). Groups of negative control birds received sterile Frey's broth medium by intravenous and intra-air sac inoculation, respectively. Variation in virulence was evaluated on the basis of significant differences in incidence, severity and extend of MS-induced airsacculitis and synovitis as well as isolation rates of MS especially from parenchymous organs. All the strains tested were pathogenic but varied in their virulence for broiler chickens. Based on differences of the virulence, the isolates were classified to the categories: (1.) highly virulent, (2.) virulent, (3.) moderately virulent and (4.) slightly virulent. (1) Strains WVU 1853 and 246-91 induced a systemic disease associated with multiple synovitis and bilateral airsacculitis (2) Strains 93-92 and 151-77 induced bilateral airsacculitis similar to WVU 1853 and 246-91 but rarely a systemic disease after exposure by intra-thoracic airsac inoculation. (3) In comparison, strains 27-79, 76-93 and 513-83 caused less frequently airsacculitis and even if, then only at the side of intra-airsac exposure. (4) Strain 91-93 has been found to differ significantly from all the other isolates in its capacity to produce disease independently from the inoculation route. After intravenous inoculation, findings gave no indications for strains with selective tropism to the epithelial membranes of the lower respiratory tract or to those of the joints, tendon sheaths and bursae. However, the presented data of the experiments suggest that the MS strains tested differ in their potential capacity to invade systemically and produce acute septicaemia.     

禽致病性大肠杆菌pilA基因的克隆及其在乳酸菌中的表达

根据GenBank中禽致病性大肠杆菌pilA基因序列设计合成1对引物,以本实验室分离的禽致病性大肠杆菌基因组DNA为模板,采用PCR技术扩增得到pilA基因片段,经测序鉴定准确后将其克隆到乳酸乳球菌表达载体pMG36e中,构建重组质粒并将其电转入乳酸乳球菌MG1363,得到重组乳酸乳球菌。SDS-PAGE分析显示,表达的蛋白约为19 ku,与预期相符。Western blot进一步证实了该蛋白的免疫反应性。     

Presence of pap-, sfa-, and afa-related sequences in necrotoxigenic Escherichia coli isolates from cattle: evidence for new variants of the AFA family.

Necrotoxigenic Escherichia coli (NTEC) are associated with intestinal and extraintestinal diseases in animals and human beings and produce Cytotoxic Necrotizing Factor 1 (CNF1) or 2 (CNF2). Fourty-three NTEC1, 42 NTEC2, and 32 CNF-negative isolates from cattle were tested by colony DNA hybridization, by plasmid DNA hybridization and by PCR assays for the presence of DNA sequences homologous to the operons coding for fimbrial (PAP/PRS, SFA/FIC, and F17) and afimbrial (AFA/Dr) adhesins of extraintestinal E. coli. Most NTEC1 isolates hybridized with the PAP probes and either the SFA probe (37%) or the AFA probes (49%). Most NTEC2 isolates, in contrast, hybridized with the F17 probe (45%), the AFA probes (19%), or the F17 and AFA probes (22%). A probe-positive plasmid was identified in each of the 19 NTEC2 isolates studied. They all hybridized with the CNF2 toxin probe (Vir plasmids) and most of them with the F17 (6 plasmids) or AFA (7 plasmids) probes. PCR amplification was obtained with 6 of the 11 NTEC isolates tested for the papGII/prsG genes; with all 5 NTEC isolates tested for the sfa and related operons; but with none of the 18 NTEC isolates tested for the afa and related operons. pap-, sfa-, and afa-related sequences are thus present in NTEC isolates from cattle in addition to f17-related operons and may code for adhesins corresponding to specific colonization factors. f17- and afa-related sequences can be located on the Vir plasmids along with the cnf2 gene. Existence of new variants of the AFA/Dr family is evident from the negative results of this family-specific PCR assay.     

蜜蜂抗菌肽Abaecin在枯草杆菌中的分泌表达

为了在枯草芽孢杆菌中分泌表达具有生物活性的Abaecin,采用重叠延伸PCR方法拼接合成含有编码Abaecin的基因及其相关调控元件的重组表达质粒pGplat.将构建的表达质粒pGplat电击转化至枯草杆菌,筛选正常生长的菌体,摇瓶发酵,取上清液冻干作为样品.通过Western blot试验检测Abaecin在枯草芽孢杆菌中的分泌表达情况.采用琼脂扩散法检测表达产物Abaecin的抑菌活性.Western blot试验结果证实在3.9 ku处有表达产物的目的条带.琼脂扩散法结果证明表达产物Abaecin对大肠杆菌、鸡沙门氏菌等革兰氏阴性菌有抑制活性.试验表明在枯草杆菌中能有效分泌表达具有生物活性的抗菌肽Abaecin.本研究结果为开发全新高效抗生素肽类药物和饲料添加剂提供了理论依据.     

Molecular cloning and characterization of Mycobacterium paratuberculosis promoters in Escherichia coli.

DNA fragments from Mycobacterium paratuberculosis were cloned in the promoter probe plasmid pKO1. Of 957 recombinant DNA clones, 24 induced synthesis of galactokinase (the reporter gene) when these plasmids were transformed into an Escherichia coli strain deficient for the enzyme. A DNA insert from one putative promoter-containing plasmid, designated pAG5, was sequenced and shown to contain, a characteristic RNA polymerase binding site, a probable ribosomal binding site and a putative open reading frame.     

新城疫病毒F48E8株cDNA文库的构建

以新城疫病毒（ＮＤＶ）中国标准强毒株为材料，分别用基因组ＲＮＡ及ｐｏｌｙ（Ａ）＋ｍＲＮＡ为模板反转录合成ｃＤＮＡ。将ｃＤＮＡ通过同聚物加尾ｐｏｌｙ（ｄＣ）后克隆于末端加有ｐｏｌｙ（ｄＧ）的线性化质粒ｐＧＥＭ－３Ｚｆ（－）上，转化大肠杆菌ＴＧ１，经ＩＰＴＧ和Ｘ－ｇａｌ筛选及电泳检查，其中有５６个克隆含有大小在０．２～２．７ｋｂ范围的外源片段。斑点杂交鉴定有５２个是ＮＤＶｃＤＮＡ克隆，平均长度为１．３１ｋｂ，从而构建了ＮＤＶｃＤＮＡ文库。文库的统计学质量检验表明，拥有５２个克隆的Ｆ４８Ｅ８株ｃＤＮＡ文库可能覆盖ＮＤＶ整个基因组     

Pathogenicity of Mycoplasma synoviae in chicken embryos.

Pathogenicity of Mycoplasma synoviae (MS) was examined in specific-pathogen-free (SPF) white leghorn chicken embryos. Six isolates of MS were inoculated into 7-day embryos via the yolk sac. Isolates were evaluated for gross and microscopic lesions through 19 days' incubation and for embryo lethality through 20 days' incubation. Isolates in decreasing order of lethality, from lowest to highest 50% embryo lethal dose, were WVU 1853, K1968, K1858, FMT, 92D8034, and F10-2AS. Embryo lethality was consistent with lesion incidence and severity. Embryo lethality did not correlate with previous results regarding pathogenicity of these same six isolates in SPF broiler chickens.     

Isolation of Mycoplasma synoviae from the brains of commercial meat turkeys with meningeal vasculitis

Mycoplasma synoviae (MS) was isolated from the brains of 22-week-old commercial meat turkeys displaying severe synovitis and infrequent central nervous system signs. Histological examination of the brains revealed mild-to-severe meningeal vasculitis. The vasculitis ranged from fibrinoid necrosis with little inflammation to a marked infiltration of lymphocytes and plasma cells disrupting the architecture of the vessel wall, accumulating as perivascular cuffs, and involving surrounding meninges. Occasional arteries were undergoing thrombosis. Similar lesions were occasionally seen in renal, synovial, and splenic vessels. MS isolates from the brain, trachea, and joint showed similar protein-banding patterns by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, the protein profile differed markedly from the standard MS reference strain, WVU 1853. This is the first known field case of MS isolation from the brains of turkeys.