Structural gene and strain specificity of a novel cysteine protease produced by Staphylococcus aureus isolated from a diseased chicken

Recently whole genome sequencing of Staphylococcus aureus has revealed the genes encoding cysteine proteases such as staphopain and SspB. In this study, we cloned and sequenced the structural gene (ScpA) encoding a cysteine (thiol) protease of S. aureus strain CH-91 from a chicken with dermatitis using polymerase chain reaction (PCR) and inverse PCR methods. The sequence information revealed a coding sequence (CDS) of 1200 nucleotides encoding the ScpA preproenzyme of 399 amino acids with a molecular mass of 45,071 Da. The deduced amino acid sequence of the ScpA differed at many positions from those of staphopain and SspB with identities of 64 and 42%, respectively. In the Southern blot analysis with a total DNA of S. aureus strain CH-91, the ScpA probe hybridized with a single 7.7 kb XbaI fragment or 2.8 and 0.8 kb EcoRI fragments, whereas the staphopain and SspB probes did not hybridize with these DNA fragments. These results suggest that this ScpA gene is a single-copy gene and is a novel gene, which is not found in the published whole genome sequences of S. aureus. In immunoblot, PCR, and Southern blot assays, the ScpA or its gene was detected in high protease-producing strains from chickens, but was not recognized in bovine and porcine strains or low protease-producing avian strains. These results indicate that the ScpA of CH-91 type may be specific to the high protease-producing strains of S. aureus from chickens, namely, there is a strain specificity of the ScpA.     

Expression of rabbit IgA heavy chain genes in E. coli and in murine myeloma cells

Rabbit IgA-heavy chain cDNA and germline genes were cloned into prokaryotic and eukaryotic expression vectors, respectively. The Fc alpha encoding portion of six C alpha cDNA clones were cloned into pUC8 and E. coli were transformed. Radioimmunoassay of the molecules synthesized by these clones showed that molecules with Fc alpha antigenic determinants were produced at the level of approximately 0.1 to 1.0 microgram per ml culture. Radiobinding analysis showed that each of the clones encoded heavy chains of the IgA-g subclass. Southern blot analysis of rabbit germline DNA revealed 10 germline C alpha genes. Five of these, isolated from recombinant cosmid libraries, were cloned into a eukaryotic expression vector containing a rearranged murine VDJ gene, the CH enhancer region and the Eco-gpt gene. Murine myeloma cells, J558L, were transfected with each of the heavy chain constructs and stable transfectants was selected with mycophenolic acid. The immunoglobulins produced by each transfectant were analyzed by radiobinding and by SDS-PAGE. Each transfectant were shown to synthesize IgA molecules and thus all five C alpha genes are expressible. The heavy chains from the transfectants ranged in size from 55,000 to 60,000 daltons. Radiobinding analyses indicated that four of the five genes encode molecules of the IgA-f subclass; the serological identity of the fifth gene is not yet established.     

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.     

Southern blot analysis of strain variation in Campylobacter mucosalis.

A panel of three DNA probes were derived at random from a genomic DNA library of Campylobacter mucosalis strain E8384-4. Each probe hybridized specifically to C. mucosalis DNA from bacteria fixed to nylon membranes. The probes did not hybridize to DNA from other Campylobacter species or to other bacteria even at 100-fold higher amounts. Each probe hybridized to all of 24 isolates of C. mucosalis which had been collected over time from different geographic locations. Southern blot analysis of selected C. mucosalis isolates was carried out to determine if the probes would be useful for differentiating among various isolates. It indicated that restriction fragment length polymorphisms (RFLPs) exist at the loci identified by our probes. These differences were used to characterize seven C. mucosalis isolates recovered from pigs in Minnesota. The results suggest that RFLP analysis may be a useful tool for epidemiological studies of C. mucosalis.     

Characterization of Listeria monocytogenes isolates by Southern blot hybridization

A synthetic deoxyribonucleotide probe for virulent Listeria monocytogenes, designated ADO7, was evaluated for its ability to identify restriction fragments of L. monocytogenes with nucleic acid sequences homologous with the beta-hemolysin gene by Southern blot hybridization of clinical and food isolates. The synthetic probe hybridized with three restriction fragments (approximately 1.1, 0.86, and 0.76 kb) of the serotype 1/2A isolates. Southern blot hybridization of the serogroup 4B isolates indicated that the nucleic acid sequences homologous with the beta-hemolysin gene probe were limited to a single restriction fragment of approximately 1 kb.     

我国鸡传染性支气管炎病毒地方性流行毒株S1基因的RT-PCR扩增及其克隆与鉴定

采用ＲＴ－ＰＣＲ方法,以ＩＢＶＳ１全基因特异性引物分别从我国华东（ＨＤ）、华北（ＨＢ）、华中（ＨＺ）、华南（ＨＮ）、西北（ＸＢ）及东北（ＤＢ）等地的ＩＢＶ流行株基因组中扩增出预期的１．７ｋｂ左右的ＤＮＡ片段。ＰＣＲ产物的ＨａｅＩＩ酶切分析及其与英国ＩＢＶＳ１全基因核酸探针的分子杂交证实所获６个ＩＢＶ流行株的ＰＣＲ产物为ＩＢＶＳ１基因。将此６个毒株的Ｓ１基因ＰＣＲ产物分别进行５’和３’端的ＢａｍＨＩ和ＨｉｎｄＩＩ酶切识别位点的分子修饰之后插入到克隆质粒ｐＵＣ１８的ＢａｍＨＩ／ＨｉｎｄＩＩ位点,在大肠杆菌中实现了目的基因的分子克隆。Ｓ１基因的ＲＦＬＰ分析表明我国ＩＢＶ已有分子水平的变异。     

Cloning and characterization of a genomic probe for malignant catarrhal fever virus

A genomic probe specific for malignant catarrhal fever (MCF) virus was cloned by using purified viral DNA from MCF-virus strain WCll. Restriction endonuclease analysis of the purified viral DNA was used to identify the cloned viral genomic fragment. Dot blot hybridization by use of the genomic probe (pRP-5) indicated that the probe hybridized specifically with WCll-MCF virus, as well as with one other isolate of MCF-associated herpesvirus. Hybridization also was observed to a non-MCF virus strain of bovine herpesvirus.     

Recombinant DNA probes for Mycoplasma synoviae

A genomic library was prepared from Mycoplasma synoviae (MS) strain WVU 1853 cloned in plasmid vector pUC8 and transformed in Escherichia coli host JM83. In dot blot assays, four transformed E. coli clones hybridized with 32P-labeled chromosomal DNA of MS but not with 32P-labeled chromosomal DNA of M. gallisepticum (MG) strain S6. In Southern hybridization, each of the CsCl-purified recombinant plasmid clones was shown to contain two MS DNA fragments between 1.0 to 2.3 kbp in length. 32P-Labeled probes prepared from each of the four recombinant plasmids hybridized in dot blot assays with MS strain WVU 1853 and nine MS field isolates but not with MG strains S6, K810, F2F10, four MG field isolates, and 15 other species of avian mycoplasmas.     

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.     

Detection of bovine viral diarrhea virus by spot hybridization with probes prepared from cloned cDNA sequences

A cytopathic strain of bovine viral diarrhea virus (BVDV) was purified from infected cell culture fluids by isopycnic density-gradient centrifugation. Genomic RNA was extracted and tailed with adenine residues at the 3' end with poly-A polymerase. Double-stranded complementary DNA (cDNA) was synthesized, using the poly-A-tailed RNA as a template and oligo-dT as a primer, and then cloned into the pUC9 plasmid. Virus-specific cDNA sequences, varying in length from 0.5 to 2.5 kilobases (kb), were obtained. One BVDV-specific sequence of cloned cDNA, 1.1 kb in length and with an internal Pst I restriction endonuclease cleavage site, was selected for use as a probe. The cloned cDNA insert was removed from the plasmid either with or without flanking plasmid sequences and labeled with 32P-nucleotides by nick translation for use as hybridization probes for BVDV. The performance of probes of smaller fragments of the insert was compared to that of the intact sequence in hybridization assays. In addition, 2 methods of specimen preparation were compared to establish optimum parameters for hybridization. The hybridization assay was 10-100 times more sensitive than infectivity assays for BVDV in infected cell cultures. Freezing of specimens reduced by 10-fold the sensitivity of hybridization for BVDV target sequences. The probes prepared from the cloned cDNA hybridized with all cytopathic and noncytopathic BVDV strains tested but not with uninfected cell cultures, cellular ribosomal RNA, bovine coronavirus, bluetongue virus, or bovine adenovirus 3. Probes prepared with native plasmid DNA did not hybridize with BVDV or uninfected cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

九种微孢子虫核糖体小亚单位RNA(SSUrRNA)基因拷贝数的研究

以已克隆测序的家蚕微孢子虫 (镇江株 ,Nosemabombycis)的核糖体小亚单位RNA(SSUrRNA)编码基因(12 0 5bp)为模板 ,用随机引物合成法标记的探针 ,在与 9种微孢子虫及家蚕基因组DNA的 6种限制性内切酶的酶切产物的Southern杂交图谱中 ,9种微孢子虫基因组DNA酶切产物的杂交图谱极为相似 ,而与家蚕基因组DNA的任何一种酶的酶切产物均无杂交信号 ,表明微孢子虫和家蚕的SSUrRNA基因同源性很低或没有同源性。同时还证实了已克隆的SSUrRNA基因来源于微孢子虫基因组DNA ,不是从家蚕基因组DNA扩增而来。在酶解较为完全的酶切产物的杂交图谱中 ,微孢子虫基因组DNA中SSUrRNA基因的拷贝数至少在 15以上     

DNA probes for the detection of toxin genes in Escherichia coli isolated from diarrhoeal disease in cattle and pigs

DNA gene probes specific for genes coding for heat labile toxin (LT), heat stable toxins (STpa, STpb) and Vero-cell toxins (VT1, VT2) were used to examine 1031 diarrhoeal disease isolates of E. coli (345 from cattle and 686 from pigs). Of the bovine strains, 60 hybridized with the STpa probe and most possessed the K99 (F5) or F41 adhesin. Five bovine strains possessed STpb genes and five either VT1 or VT2 genes. Of the porcine strains, 245 hybridized with one or more gene probes. Of 160 K88 (F4) positive strains, 133 possessed both LT and STpb genes, whilst 17 possessed LT or STpb or STpa alone or in combination. Ten K88 strains did not possess toxin genes. Isolates bearing the K99 (F5) adhesion possessed either STpa, STpb and VT2 genes alone or in combination; in one isolate only the LT gene was detected. Isolates belonging to serogroup 0138:K81 were more heterogeneous as to their toxin genes; of the 60 strains, fourteen carried only VT2 genes, thirty-two carried VT2, STpa and STpb genes, one carried LT, VT2, STpa and STpb genes, two carried STpb gene, four carried STpa and STpb genes, one carried LT and VT2 genes, two carried LT and STpa genes, whilst four carried none. Twenty-four percent of all toxigenic strains apparently did not possess adhesins.     

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.     

Development of a recombinant Anaplasma marginale DNA probe

An Anaplasma marginale DNA probe has been developed by using an improved method for the isolation of genomic DNA. Purified genomic A. marginale DNA from the St. Croix isolate was partially digested with Sau 3A1 into fragments (greater than or equal to 5.0 kb). The restriction fragments were cloned using standard techniques in the pBR322 vector and used to transform E. coli (DH5) host cells. The recombinant A. marginale DNA library was screened by the colony lifting procedure. Colonies containing plasmids with A. marginale DNA inserts were identified by hybridization with a genomic A. marginale DNA radiolabeled probe (32P). Seven recombinant A. marginale DNA probes were evaluated by dot-blot in vitro hybridization assays to identify candidates as diagnostic tools in bovine anaplasmosis studies. Specificity and sensitivity experiments were carried out by using heterologous and homologous DNAs. The heterologous panel contained bovine DNA (WBC) and blood parasites DNA from Babesia bovis (Bb), Babesia bigemina (Bbi), Eperythrozoon suis (Es) and Eperythrozoon wenyoni (Ew). The homologous DNA panel included A. marginale DNAs of 12 different isolates which were isolated in the Caribbean, Mexico, and the U.S.A. The selected diagnostic probe was identified as pSt. Croix A1, and labeled with 32P by using in vitro nick translation and random primer techniques. The pSt. Croix A1 probe demonstrated 100% specificity and high sensitivity by hybridization in dot blotting and Southern blotting. The probe can detect 500-1000 infected erythrocytes per microliters which corresponds to a parasitemia of less than 0.01%. The A. marginale DNA insert was approximately 6.4 kb in size and a partial restriction map has been constructed.     

Comparative prevalence of four enterotoxin genes among Escherichia coli isolated from swine

Presence of Escherichia coli enterotoxin genes LT (heat-labile enterotoxin), STaP (heat-stable enterotoxin a, porcine genotype), STaH (heat-stable enterotoxin a, human genotype), and STb (heat-stable enterotoxin b) among 874 swine isolates of E coli was determined, using DNA probes and the DNA colony hybridization technique. Of the 874 isolates evaluated, 45% hybridized with at least one of the enterotoxin gene probes and were designated as enterotoxigenic E coli (ETEC). Eighty-five percent of the ETEC were from pigs with enteric colibacillosis. The remaining 15% were from pigs with edema disease or various other diseases, and from healthy swine. Seventy-four percent of the ETEC hybridized with the STb probe, 52% with STaP, and 31% with LT; ETEC did not hybridize with the STaH probe. Most of the ETEC hybridized with more than one enterotoxin gene probe. Isolates that hybridized with the LT probe also hybridized with STb. The most prevalent gene combination was LT-STb. However, 35% of the ETEC from neonatal (less than or equal to 1 week old) swine with enteric colibacillosis were of the STaP-only genotype, and 33% of the ETEC from older swine with enteric colibacillosis were of the STb-only genotype.     

A specific DNA probe which identifies Babesia bovis in whole blood.

A genomic library of Babesia bovis DNA from the Mexican strain M was constructed in plasmid pUN121 and cloned in Escherichia coli. Several recombinants which hybridized strongly to radioactively labeled B. bovis genomic DNA in an in situ screening were selected and further analyzed for those which specifically hybridized to B. bovis DNA. It was found that pMU-B1 had the highest sensitivity, detecting 25 pg of purified B. bovis DNA, and 300 parasites in 10 microliters of whole infected blood, or 0.00025% parasitemia. pMU-B1 contained a 6.0 kb B. bovis DNA insert which did not cross-hybridize to Babesia bigemina, Trypanosoma evansi, Plasmodium falciparum, Anaplasma marginale, Boophilus microplus and cow DNA. In the Southern blot analysis of genomic DNA, pMU-B1 could differentiate between two B. bovis geographic isolates, Mexican strain M and Thai isolate TS4. Thus, the pMU-B1 probe will be useful in the diagnosis of Babesia infection in cattle and ticks, and in the differentiation of B. bovis strains.     

Genomic analysis of Theileria sergenti stocks in Japan with DNA probes.

Restriction fragment length polymorphisms of Theileria sergenti DNA from 18 different infections of cattle in 14 locations in Japan were analyzed by Southern blotting using T. sergenti genomic DNA fragments as probes. Probe pTs 2 hybridized with four fragments in BamHI digested piroplasm DNA, at 8.0, 7.3, 6.0 and 3.4 kb. Probe pTs 11-D1 hybridized with multiple fragments. With each probe, polymorphisms were observed among stocks from different locations. However, there was no correlation between the patterns of hybridization bands and the locations where parasites were collected. Analysis of the hybridization patterns of stocks obtained from individual cattle in the same grazing areas showed an almost identical pattern.