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.     

Rapid identification and differentiation of the vaccine strain Rac H from EHV 1 field isolates using a non-radioactive DNA probe.

A method for rapid differentiation between the EHV 1 live vaccine strain Rac H and field isolates is described. Total DNA was isolated from virus-infected small scale cell cultures. DNA fragments digested with restriction endonuclease BamHI were separated, transferred and immobilized on filter membranes. A Digoxigenin-labeled probe derived from EHV 1 was used for hybridization. This probe hybridized specifically to sequences of the inverted terminal repeat region which in case of Rac H include a deletion of 0.8 kb. By comparing the different migration patterns after blot hybridization it could be shown that in 65 isolates from cases of abortion the live vaccine strain Rac H was not involved.     

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.     

Isolation of genes encoding bovine IgM, IgG, IgA and IgE chains

Bovine C mu, C gamma, C alpha and C epsilon genes were cloned in an EMBL4 recombinant phage library using rabbit immunoglobulin switch mu (Su) and human C gamma as probes. Restriction mapping and Southern blot analyses of these clones identified one clone which hybridized with rabbit C mu and JH probes. The HG and C mu regions were separated by 6 kb of DNA. One C alpha and one C epsilon gene were found on overlapping clones and were separated by approximately 15 kb of DNA. Southern blot analysis of germline DNA with a bovine C alpha associated probe (S alpha) indicated that the germline contains a single C alpha gene. Similar analyses with a bovine C epsilon probe indicated that the germline contains either one C epsilon gene with allelic restriction polymorphism or two C epsilon genes. Three C gamma genes were cloned and did not overlap with one another. Southern blot analyses of germline DNA with a bovine C gamma probe indicated that the germline contains a total of four C gamma genes. The genes cloned correspond to three of the four genes identified by Southern blot analysis. The orientation of each CH gene was assigned by hybridization with S mu or S gamma probes. The S gamma probe hybridized to DNA immediately adjacent to all three C genes; the S probe hybridized to DNA immediately adjacent to the C mu, C alpha and C epsilon genes. Unexpectedly, the S mu probe also hybridized with a segment of DNA approximately 7 kb downstream of the C mu gene. This may represent a switch region for C gamma.     

Detection of infectious laryngotracheitis virus infected cells with cloned DNA probes.

A genomic library of infectious laryngotracheitis virus (ILTV) DNA BamH1 fragments was prepared and two cloned fragments were evaluated for their potential as probes for the detection of ILTV infected cells. The virus was purified by a modified sucrose density gradient procedure for the isolation of pure ILTV DNA. A genomic library was constructed using BamH1-digested ILTV DNA and pGEM7 as a vector. A 1.1 kb cloned BamH1 fragment of ILTV DNA was tested in a slot or dot blot assay for the detection of ILTV infected cells. The limit of detection for this probe was at least 0.12 ng of pure ILTV DNA. The probe was able to identify both chicken embryo liver (CELi) cells and choriallantoic membranes infected with ILTV. Chicken embryo liver cells infected with several field isolates and a vaccine strain of ILTV were positive by dot blot analysis using this probe. Some qualitative differences in the degree of hybridization to cells infected by different ILTV isolates were observed. Uninfected cells and cells infected with fowlpox virus, turkey herpesvirus, Marek's disease virus or Newcastle disease virus were negative by the same assay. Compared with the 1.1 kb fragment, a larger 6 kb cloned BamH1 fragment of ILTV DNA showed a stronger hybridization signal to DNA from ILTV infected cells.     

Practical application of nucleic acid techniques to avian disease problems

A workshop in which 17 practicing scientists participated was intended to address primarily people who use or could use biotechnology in their work and was confined to five techniques. Endonuclease fingerprinting and mapping involved cleaving nucleic acid with a specific restriction enzyme and separating the nucleic acid fragments by electrophoresis. Field and vaccine isolates of Pasteurella multocida could be distinguished; Salmonella enteritidis could be divided into three groups; chlamydia could be grouped into seven groups; and vaccinia, quail pox, and fowl pox could be clearly distinguished. Preparation of nucleic acid probes involved producing large amounts of labeled oligonucleotides, usually of unknown sequence. Successful probes had been made for infectious bursal disease virus, avian influenza virus, Newcastle disease virus, and infectious bronchitis virus. In Southern, Northern, and dot blotting, either DNA or RNA fragments were placed on or transferred to a solid substrate and probed. The procedure was able to detect infectious bursal disease virus, infectious bronchitis virus, Mycoplasma gallisepticum, and Marek's disease virus. In situ hybridization involved applying a labeled probe to frozen or fixed sections or to intact cells. In Polymerase chain reaction, two primers, some distance apart, were annealed to a denatured target DNA. Repeated cycles of DNA synthesis with a thermostable polymerase, denaturing, and reannealing resulted in great amplification of a rare sequence. After 30 cycles, a rare gene sequence could be amplified more than 10(6) times. It was used successfully to detect minute quantities of influenza virus and infectious bursal disease virus, and the process was used to facilitate DNA sequencing of coccidiosis gene segments.     

Restriction endonuclease analysis of alcelaphine herpesvirus 1 DNA and molecular cloning of virus genomic DNA for potential diagnostic use

Alcelaphine herpesvirus 1 (AHV-1) genomic DNA was analyzed using restriction enzymes having recognition sequences both low in guanine-cytosine content (BamHI, KpnI, HindIII) and high in guanine-cytosine content (SmaI, AvaI, ApaI). The results from the restriction enzyme analyses along with exonuclease treatment demonstrated that the termini of AHV-1 DNA are likely composed of polyrepetitive sequences high in guanine-cytosine content similar to those found in Herpesvirus saimiri DNA. Cleaving AHV-1 DNA with the restriction enzyme SmaI produced polyrepetitive sequences that appeared as low molecular weight supermolar bands less than 1 kilobase pairs (kb) in size. Analysis of AHV-1 DNA cleaved with HindIII indicated that the polyrepetitive sequences are approximately 29.5 kb in size. The total molecular weight for AHV-1 DNA was determined to be approximately 118 kb. Seventy-five percent of the AHV-1 genome was cloned into the plasmids pAT153 and pBR322. Cloned DNA fragments representing unique sequences of the AHV-1 genome hybridized with AHV-1 genomic DNA but showed no appreciable hybridization with AHV-2 DNA or bovine herpesvirus 1, 2, or 4 DNA.     

新城疫病毒特异性糖蛋白基因探针的制备及应用

应用ＲＴ－ＰＣＲ获取新城疫病毒Ｆ４８Ｅ８株的部分囊膜糖蛋白基因片段。扩增产物经琼脂糖凝胶电泳分析,长为９２６ｂｐ,与预期结果相符;将该基因片段定向克隆入质粒载体ＰＵＣ１９只,得到重组质粒Ｐ９２６,酶切分析结果与已发表的ＮＤＶ毒株酶切位点一致。     

Mapping of porcine parvovirus DNA and development of a diagnostic DNA probe

Dimeric and monomeric replicative forms of DNA of porcine parvovirus (PPV) strain NADL-2 were isolated and examined by restriction enzyme analysis and reciprocal Southern blot hybridization during development of a DNA probe for PPV. Genomic single stranded PPV DNA was 5.0 kb long, and results substantiated the rolling-hairpin model of parvovirus DNA replication with the primer sequence located in the 3' terminal hairpin loop. An additional finding was the generation of a 4.7 kb species of viral DNA which was considered to be a 0.3 kb deletion variant of genomic PPV DNA. A 3.0 kb DNA fragment obtained by Pst I/Hind III digestion of monomer replicative form DNA was cloned into a plasmid vector, pUC 19. The cloned fragment, recovered from transformed Escherichia coli strain TB1 and labelled with [32P] dCTP, was evaluated by dot hybridization as a probe for PPV in infected cell cultures. The probe was specific for PPV infected cells, and was 100 times more sensitive than the standard hemagglutination test.     

Use of a Salmonella typhimurium-derived virulence probe in the detection of Salmonella sp. and in the characterization of S. cholerae-suis virulence plasmids

A genetic probe encoding a virulence gene from Salmonella typhimurium was useful in the detection of Salmonella from feces during an outbreak of salmonellosis at a local dairy. A 3.2-kb BamHI restriction endonuclease fragment of the S. typhimurium virulence plasmid, pStSR100, has been useful as a DNA probe for both detection of Salmonella sp. and characterization of virulence plasmids from numerous field isolates. This virA probe hybridizes to a highly conserved gene carried on the large virulence plasmids of invasive Salmonella isolates. Colony blots prepared from feces directly plated onto MaConkey's agar failed to detect low numbers of Salmonella sp. However, hybridization of the VirA probe to vacuum blots or colony blots prepared from feces in tetrathionate enrichment broth incubated for 16 hours at 37 C was effective for detecting Salmonella sp. and resulted in an 85.9% correlation with culture results. The probe also demonstrated the highly conserved nature (96%) of the virulence gene among S. cholerae-suis isolate plasmids detected using Southern blot analysis.     

Detection of bovine herpesvirus-1 nucleic acid sequences, using a dot-blot hybridization procedure

A sensitive and specific procedure for the detection of Argentine isolates of bovine herpesvirus-1 was developed. The procedure was based on a dot-blot, nucleic acid hybridization, using 32P, nick-translated, plasmidic probes. The probes contained cloned Bam H1 restriction fragments in the left half of the viral genome. The detection limit of the procedure was 10 pg of viral DNA.     

Novel iridovirus in a nautilus (Nautilus spp.).

Intracytoplasmic inclusion bodies suggestive of iridovirus infection were observed in formalin-fixed, paraffin-embedded tissues from a nautilus (Nautilus spp.) that died without premonitory signs. Transmission electron microscopy revealed enveloped, hexagonal, viral particles that measured approximately 176 nm in diameter. Virions contained a dense central core and morphology typical of iridoviruses. Extracted DNA was amplified using primers homologous to conserved iridovirus sequences. The amplicons were cloned, sequenced, and determined to be approximately 60% similar to reported amphibian iridovirus sequences. A polymerase chain reaction-generated digoxigenin probe was used to detect viral nucleic acid in tissue sections by DNA in situ hybridization and high-affinity cytochemistry. The detected nucleic acid corresponded to the inclusion bodies observed microscopically. This represents a novel iridovirus of mollusks.     

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。     

Prevalence of antibodies to alcelaphine herpesvirus-1 and nucleic acid hybridization analysis of viruses isolated from captive exotic ruminants

A serologic survey was conducted to determine the prevalence of antibodies to alcelaphine herpesvirus-1 (AHV-1) in captive exotic ruminants within the United States. Forty-six percent of the members of the subfamily Alcelaphinae (wildebeest, topi, hartebeest) in the family Bovidae had virus-neutralizing antibody to AHV-1. Other subfamilies of Bovidae with high prevalence of virus-neutralizing antibodies to AHV-1 included Hippotraginae (oryx and addax) and Caprinae (sheep and goats), with prevalence of 45% and 29%, respectively. Herpesviruses that have been isolated from captive exotic ruminant species, including healthy animals and those with clinical malignant catarrhal fever at the Oklahoma City Zoo and the San Diego Zoo/Wild Animal Park, were analyzed by DNA restriction enzyme analysis and blot hybridization. Variation has been detected among the genomes of several malignant catarrhal fever virus isolates obtained from various exotic species of ruminants, using the DNA restriction enzymes BamHI and HindIII. The DNA of these virus isolates is distinct from that of bovine herpesviruses 1, 2, and 4, as demonstrated by restriction enzyme analysis and nucleic acid hybridization. On the basis of restriction enzyme analysis and nucleic acid hybridization data, the DNA from each of the putative alcelaphine herpesvirus isolates examined, except for the topi virus isolate, had a high degree of DNA sequence similarity with the original AHV-1 isolate, WC-11, from a blue wildebeest.     

Restriction fragment length polymorphism for the Yc subunit gene of rat liver glutathione S-transferase

An altered expression of the Yc subunit gene of rat glutathione S-transferase (GST) in the liver of the LEC rat, which is a mutant strain with spontaneous hereditary hepatitis associated with severe jaundice, has been reported. To provide further information concerning the structure of the Yc subunit gene, we carried out the Southern blot hybridization analysis of DNA samples from rats of eight different inbred strains including LEC with cDNA complementary to mRNA specific for the Yc subunit of rat liver GST as a probe. The hybridization patterns of the DNA samples from rats belonging to the different inbred strains showed interstrain variation in the length of restriction fragments with four restriction endonucleases. Since the DNA samples prepared from several rats of one inbred strain gave an identical hybridization pattern, the restriction fragment patterns for the Yc gene could be used as markers for genetic monitoring of inbred rat strains. Although the altered expression of Yc-Yc activity of GST has been observed in the liver of the LEC rat, the characteristic changes in the gene structure of the Yc subunit of LEC rat were not detected in the present hybridization analysis.     

Detection of phocine distemper virus using the polymerase chain reaction

During the fatal seal epizootics in the North and Baltic Seas in summer 1988 a virus was isolated which was shown to be the causal agent. It was subsequently classified as morbillivirus by neutralization assays, reaction with monoclonal antibodies and nucleic acid hybridization studies. The virus (tentatively called Phocine Distemper Virus, PDV) is difficult to grow in culture making rapid diagnosis difficult. We have used the Polymerase Chain Reaction (PCR) as an alternative and fast method to detect the presence of virus-specific nucleic acid and we describe here the amplification of cell culture derived PDV RNA in a "one-tube" reaction using heterologous (Rinderpest Virus cDNA derived) F gene primers. The resulting 370 bp DNA fragment was shown to be morbillivirus derived by Southern blot hybridization using cloned RPV F gene as probe.     

Proto-oncogenes of genomic DNA in clinically normal animals of various species

To provide information about oncogenes for molecular biological studies of tumors in domestic animals, the proto-oncogenes homologous to the c-myc, c-erbB-2, c-ros-1, c-yes-1, v-myc, v-Ki-ras, and v-Ha-ras oncogenes of genomic DNA in cattle, horses, pigs, dogs, cats, and chickens were investigated by Southern blot hybridization. High molecular weight genomic DNA in each of the animals contained proto-oncogenes that had a certain homology with the oncogenes used, but the extent of nucleotide homology of the proto-oncogenes differed in number and molecular weight: ie, 1 or 2 bands at 1.6 to 22.0 kilobase (kb) in the c-myc probe, 1 or 2 bands at 1.1 to 16.0 kb in the c-ros-1 probe, 1 to 3 bands at 0.7 to 23.0 kb in the c-erbB-2 probe, 1 to 4 bands at 0.6 to 18.0 kb in the c-yes-1 probe, 1 to 3 bands at 1.6 to 30.0 kb in the v-myc probe, 1 to 7 bands at 1.0 to 36.0 kb in the v-Ki-ras probe, and 1 to 4 bands at 1.0 to 27.0 kb in the v-Ha-ras probe. Furthermore, signal strength of each band, as determined by autoradiography, was not always the same for each probe in the various animals. Our findings indicate that these proto-oncogenes are well conserved with species specificities in each animal.     

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.     

地高辛标记P菌毛基因检测探针鸡大肠杆菌

用地高辛标记鸡大肠杆菌Ｐ型菌毛结构基因（ｐａｐＡ）制成核酸探针,作斑点杂交试验,检测２８个血清型鸡大肠杆菌５０个菌株与该探针的同源性。结果表明有５个菌株被检测为阳性,阳性率为１０％。     

Polymerase chain reaction amplification of pseudorabies virus DNA from acutely and latently infected cells

A characteristic of alphaherpesviruses, including pseudorabies virus (PRV), is that the acute phase of the disease is followed by lifelong latency. Latently infected animals are asymptomatic but can transmit reactivated virus. Corticosteroid administration, tissue explanation, blot- and in situ hybridizations have been used to demonstrate the presence of latent PRV infections. The use of blot hybridization as a convenient method for defining the incidence of PRV infections in swine herds has been hampered by the detection limit of this method. The objective of this study was to increase this sensitivity of blot hybridization by polymerase chain reaction (PCR) amplification of target sequences. Two sets of 20-mer primers were synthesized and used to amplify gX and gII glycoprotein gene sequences in two different strains of PRV. The specificity of the amplification was verified by Southern blot hybridization and restriction endonuclease analysis of the amplified fragments. Amplification of target sequences by PRC increased their detection limit by a factor of at least 10(5). Porcine ganglion samples, in which latency had been demonstrated by in vitro explanation, were analyzed by PCR together with positive and negative controls. Duplicate slot blot analyses of a portion of the amplified products were used to demonstrate latency in seven of eight samples. It was concluded that blot hybridization of PCR amplified DNA appears to be both a sensitive and convenient method for the detection of PRV induced latency.