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.     

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.     

Molecular cloning of complementary DNA from a pneumopathic strain of bovine viral diarrhea virus and its diagnostic application.

A pneumopathic strain of bovine viral diarrhea virus was grown in cell culture and purified. Genomic ribonucleic acid was extracted, polyadenylated at the 3' end, and copied into complementary DNA after oligo-dT priming. Complementary DNA was male double stranded and cloned into the pUC9 plasmid. Approximately 200 complementary DNA clones varying in length from 0.5 to 2.5 kilobases were obtained. Hybridization assays indicated that the sequences isolated were specific for bovine viral diarrhea virus and that at least 5.5 kilobases of bovine viral diarrhea virus genome was represented in the library of complementary DNA clones, the majority of which may have originated from the 3' end of the virus genome. One cloned complementary DNA sequence was used as a 32P-labelled hybridization probe for bovine viral diarrhea virus detection. The probe hybridized with all cytopathic and noncytopathic strains of bovine viral diarrhea virus tested and was 100 times more sensitive than infectivity assays for the detection of bovine viral diarrhea virus. Hybridization did not occur with nucleic acids from bovine coronavirus, bluetongue virus, bovine adenovirus or uninfected cell cultures. Native plasmid DNA sequences, labelled with 32P, did not hybridize with bovine viral diarrhea virus ribonucleic acid.     

Somatic cell mapping and restriction fragment length polymorphism analysis of bovine insulin-like growth factor I.

The DNA isolated from cow-hamster hybrid somatic cells segregating bovine chromosomes was analyzed by Southern blotting and hybridization with a heterologous [32P]-labeled porcine cDNA probe encoding insulin-like growth factor I (IGF-I). Thirteen of 25 cow-hamster hybrid cell lines exhibited the bovine-specific IGF-I fragment. Analysis for the retention or loss of bovine IGF-I with markers previously screened against the same panel of hybrid cells revealed a 100% concordance with lactate dehydrogenase B of bovine syntenic group U3 located on bovine chromosome 5. Restriction fragment length analyses of genomic DNA from animals representing five breeds (Angus, Polled Hereford, Simmental, Gelbvieh, and Belgian Blue) and from seven half-sib Angus calves indicated that polymorphisms for the genomic composition of the bovine IGF-I gene may exist in cattle populations.     

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.     

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。     

Specific DNA probe for the detection of Theileria sergenti infection in cattle

A simple procedure was developed for detection of Theileria sergenti infection on the basis of hybridization of parasite DNA with a specific probe. A genomic DNA library of T. sergenti constructed in pUC-18 was screened to detect clones containing the parasite's DNA sequences by colony and Southern hybridizations. Two positive DNA inserts were purified from the recombinant plasmids and used as probes labelled with 32P or non-isotopic reagent, biotin-11-dUTP. 32P-radiolabelled and non-radioactive probes appear to be sensitive enough to detect 15 pg (equivalent to 1,200 parasites) and 125 pg (equivalent to 10,000 parasites) of purified T. sergenti DNA, and in diluted T. sergenti-infected red blood cells, they are able to detect 8,000 parasites and 16,000 parasites, respectively.     

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.     

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.     

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)     

Detection of porcine parvovirus using nonradioactive nucleic acid hybridization

Nonradioactive slot blot hybridization assays were established for the detection of porcine parvovirus (PPV), using either a digoxigenin-labeled DNA probe or a biotinylated RNA probe. All probes were prepared from a 3.3-kb Pst1-EcoR1 DNA fragment of the NADL8 isolate of PPV. The sensitivity and specificity of the probes in a slot blot system were evaluated in comparison with a 32P-radiolabeled RNA probe. Using an anti-digoxigenin alkaline phosphatase detection system, at least 1 ng of viral replicative form (RF) DNA, or the equivalent of 100 plaque forming units (PFU) of infectious virus, could be detected by the digoxigenin-labeled DNA probe. When the biotinylated RNA probe and a strepavidin-alkaline phosphatase detection system were employed, 0.1 ng of RF DNA, or the equivalent of 10 PFU of infectious virus, were detected, comparable to the sensitivity of the 32P-radiolabeled RNA probe. Hybridization was not observed with control DNA samples extracted from swine testicle cells, porcine kidney (PK-15) cells, uninfected mixed swine fetal tissue, or from an unrelated DNA virus (pseudorabies virus) infected PK-15 cells. Different isolates of PPV, namely NADL8, NADL2, KBSH, and Kresse, reacted on an equimolar basis in sensitivity and specificity to the biotinlyated probe. Extraction of DNA directly on the filter membrane (direct filter hybridization) was employed in an attempt to reduce processing time by eliminating DNA extraction steps. Direct filter hybridization was indeed less time consuming; it was also comparable in sensitivity and specificity to those methods employing purified DNA.     

A human major histocompatibility complex (MHC) DNA probe recognizes goat genes

A human major histocompatibility complex (MHC) class II (DR-β) probe was hybridized with restriction enzyme digest of genomic DNA from 14 goats. Nine of the animals belonged to one family. Digestion of the DNA with the restriction enzyme Eco RI gave 7 fragments in 13 animals and 6 fragments in the last animal. No other polymorphism could be detected. Bam HI digestion gave from 3 to 6 fragments which displayed a considerable polymorphism. In the family studied, polymorphic fragments were inherited together with serologically defined lymphocyte antigen specificities believed to be coded for by MHC class I genes.     

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.     

Putative immunoglobulin VH genes of the goldfish, Carassius auratus, detected by heterologous cross-hybridization with a murine VH probe

By using a defined cDNA probe for the VH region of a murine phosphocholine-binding myeloma protein (S107) we have defined a family of distinct cross-hybridizing DNA sequences in genomic DNA of the goldfish. The estimated number of the goldfish putative VH family detectable by the S107 probe is about 36. By using two putative goldfish VH probes to analyze, by hybridization, the relationships among seven of the goldfish genomic clones, we have determined that the putative goldfish VH genes recognized by the S107 probe comprise at least several distinct families that are not closely related.     

Restriction enzyme maps for equine adenovirus 1 genome.

Physical maps were constructed for the genome of equine adenovirus 1 (EAV1) using the restriction enzymes; DraI, EcoRV, NotI and SfiI. The total size of the EAV1 genome was 34.4 kb estimated by comparison with known DNA standards and the polarity of the fragment order, with respect to the left and right molecular ends, was determined by hybridization with known regions of the human adenovirus 2 (HAV2) genome.     

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.     

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.     

Expression of fowl adenovirus type 10 antigens in Escherichia coli

In an attempt to construct a genetic map of the fowl adenovirus (FAV) and to determine which viral proteins are natural immunogens in chickens and hence may be relevant to protective immunity we have constructed an expression library of FAV type 10 DNA. The genomic DNA was partially digested with the restriction endonuclease Sau3A, and this DNA was inserted into the 3' terminal end of the beta-galactosidase gene in a plasmid vector. To date, approximately 600 clones have been identified that express FAV type 10 antigens as determined by immunological screening with rabbit antisera to purified virus, including one that has amino acid homology with the 100 kDa protein of human adenovirus type 5. These antigen positive clones were found to contain DNA from FAV type 10 genome as determined by hybridisation to FAV DNA. These clones will allow the further characterisation of FAV and possibly the identification of potential vaccine molecules.