Dichelobacter nodosus: differentiation of virulent and benign strains by gene probe based dot blot hybridisation.

Gene probes specific for benign and virulent strains of Dichelobacter nodosus were used in a dot blot hybridisation procedure involving 96 strains of D. nodosus isolated from cases of ovine footrot. The performance of the probes was compared with that of the elastase test. All 27 strains with elastase activity at 7 days and 12 of 25 strains with elastase activity at 14 days reacted with the virulent-specific probe. Twenty-four strains with elastase activity between 21-28 days, and 20 strains with negative elastase activity up to 28 days did not bind with this probe. On the other hand, the benign-specific probe failed to bind with the 27 strains with elastase activity at 7 days, and 13 of the 25 strains with elastase activity at 14 days but reacted with 20 of the 24 strains with elastase activity at 21-28 days, and 19 of the 20 strains with negative elastase activity up to 28 days. The 12 strains with elastase activity at 14 days which were detected by the virulent-specific probe were not those recognised by the benign-specific probe. The use of virulent and benign specific gene probes in combination provides a rapid and precise screening system for differentiation of virulent and higher intermediate footrot from benign and lower intermediate footrot.

Transmission of proliferative enteritis to swine by use of embryonating chicken eggs.

Embryonating eggs were inoculated with filtered porcine ileal mucosa containing intracellular curved rods (ICR) and incubated for 4 to 6 days. Three of 12 pigs given the eggs per os developed microscopic lesions of proliferative enteritis (PE). Nonchallenge-exposed control pigs did not develop lesions of PE. Four of six positive control pigs given ileal mucosa from pigs with PE also developed microscopic lesions of PE. All of the PE lesions were found in pigs necropsied 10 to 29 days after challenge exposure. None of the swine in the study had clinical signs or gross lesions of PE. Campylobacter spp were isolated from pigs with and without exposure to the ileal mucosa from pigs with PE. There was no relationship between Campylobacter spp isolation and development of lesions. Deoxyribonucleic acids extracted from embryonating chicken eggs injected with the equivalent of 0.5 mg of mucosal lesions and incubated for 4 days hybridized to a DNA probe specific for the ICR, whereas DNA extracted from 1.5 mg of mucosal homogenates of the same proliferative tissue did not hybridize with the same probe. Results of these experiments indicated that ICR injected into eggs remained infective for pigs and suggest replication of ICR in the first-passage eggs.

Characteristics of Vero cytotoxin producing Escherichia coli associated with intestinal colonization and diarrhea in calves.

Isolates of Escherichia coli which produce Vero cytotoxin (VTEC) were obtained during 1983-1989 from calves raised in 5 north-central states of the USA. All of the calves experienced intestinal epithelial colonization by VTEC, diarrhea or both; twelve of the calves had bloody diarrhea. Twenty one isolates were serogroup O111 and the others were O103, O69, O45, 026, O5, or non-typable (4 isolates). All but one of the isolates hybridized with the CVD419 probe which identifies most VTEC strains. Thirty two isolates hybridized with the VT1 probe, 3 with both the VT1 and VT2 probes, and one with neither probe. The culture filtrate of the VT probe negative isolate was partially neutralized by SLT I monoclonal antibody. For the other isolates, the results of toxin neutralization by anti-SLT I and anti-SLT II monoclonal antibodies corresponded exactly with the VT1 and VT2 probe hybridization results. Three of the strains adhered in a localized manner to HEp-2 cells and Intestine 407 cells.

Detection and identification of equine herpesvirus-1 and -4 by polymerase chain reaction.

A rapid method for detection and identification of equine herpesvirus-1 and -4 (EHV-1 and EHV-4) was developed using polymerase chain reaction (PCR). Primers for PCR were designed from aligned nucleotide sequences of glycoprotein B genes of EHV-1 and EHV-4 to amplify specific regions for EHV-1 or EHV-4 or a common region of both viruses. By using type specific primer mixture, amplified fragments were identified as EHV-1 or EHV-4 in a one-step reaction. We have applied this technique on specimens from aborted fetuses. The samples contained only EHV-1 and there was complete accordance between the results of PCR and virus isolation. Our PCR system could differentiate the two virus types rapidly in a one-step reaction.

Comparative hybridization and nucleotide sequence information from two noncytopathic isolates of bovine viral diarrhea virus.

Comparative hybridizations of 8 cytopathic (CP) and 11 noncytopathic (NCP) isolates of bovine viral diarrhea virus (BVDV) were done using 4 different cDNA hybridization probes. The hybridization probes were prepared from cDNA synthesized from 1 CP BVDV (NADL) and 2 NCP BVDV isolates (SD-1 and NY-1) within the p80 region and from cDNA spanning the 5' untranslated region of NCP SD-1. Hybridization with the 5'/SD-1 probe detected 19 out of 19 isolates, whereas the p80/NADL, p80/NY-1 and p80/SD-1 hybridization probes detected only 12, 16 and 13 isolates, respectively. To determine the basis for the different patterns of hybridization, the nucleotide sequence was determined for the p80/NY-1. The nucleotide sequence was compared with the published CP NADL and CP Osloss and NCP SD-1 and NCP1 nucleotide sequences and in 45% of the base differences between NY-1 and NADL, NY-1 and Osloss were divergent from NADL, SD-1 and NCP1. Based on comparative nucleotide sequence data and the different reactivities of the p80/NADL, p80/NY-1 and p80/SD-1 hybridization probes the relationships of the various test isolates were characterized.

The application of biotechnology to the diagnosis and control of animal diseases.

Methodologies for animal disease diagnosis and the analysis and control of animal health currently make much use of immunoassay techniques, in particular ELISA, and tests based on nucleic acid measurement (nucleic acid hybridisation, polymerase chain reaction [PCR]). In the latter area, the recently developed PCRs have become most useful. Recombinant technologies have yielded defined and extremely pure antigens, which can give more accurate interpretations of results obtained using diagnostic tests; in addition, such antigens obviate the need to handle live pathogens, except where these may be contained within the sample for diagnosis. The application of monoclonal antibodies (MAbs) to many tests, in particular those which use immunoassay bases or perform molecular fingerprinting of isolates, has increased the specificity and usefulness of these methodologies. Modern technology has permitted a considerable reduction in the use of animals for MAb generation through in vitro immunisation methods and the application of serum-free media with bio-reactors. These in vitro immunisation procedures have an additional application in the analysis of vaccine efficacy without recourse to animal experimentation. Furthermore, in vitro methods can be used to monitor the immune response in an animal and, in particular, to measure the development of immunological memory. It is now possible to estimate more accurately the quality and duration of an induced immune response without using animals for testing. Not only are the assay systems developed through biotechnology more sensitive and less hazardous than previous techniques (especially with regard to the necessity of handling live pathogens), but they can also determine more accurately the immune responsiveness or immune status of the animals under study.

Nucleic acid hybridisation and polymerase chain reaction in the diagnosis of infectious animal diseases.

The authors describe and summarise the use of nucleic acid hybridisation and polymerase chain reaction (PCR) technologies in the diagnosis of animal diseases. PCR is a powerful biological tool which enables exponential enzymatic amplification in vitro of a given deoxyribonucleic acid sequence. This technique is currently used to study the molecular pathogenesis of many infectious diseases and also for diagnosis. PCR is usually more sensitive than conventional methods and does not require complex facilities, and will therefore soon become the preferred technology of laboratory diagnosticians, field veterinarians and health officers.

Changes in the hybridization patterns of populations of Theileria sergenti during infection.

Restriction fragment length polymorphisms (RFLPs) of Theileria sergenti DNA were analysed using probes of a genomic DNA fragment (pTs 2) and a cDNA corresponding to this genomic probe (C-Ts 2). Each of the probes detected RFLPs in DNA from different stocks of Theileria sergenti. Additionally, using these probes, alterations in hybridization patterns were observed in samples of the parasites harvested at different times after individual calves had been infected with Theileria sergenti. This result suggests that the Theileria sergenti stocks used were mixed parasite populations.

Chemiluminescent detection of infectious bursal disease virus with a PCR-generated nonradiolabeled probe.

A polymerase chain reaction (PCR)-generated digoxigenin-labeled nonradioactive oligonucleotide probe was developed and utilized in slot-blot hybridization coupled with chemiluminescence for the detection of infectious bursal disease virus (IBDV). The probe was prepared from the RNA of the standard challenge strain (STC) of IBDV serotype 1 by reverse transcription followed by 2 PCR amplifications with 2 separate sets of primers. RNA of STC viruses prepared from bursae infected with STC viruses was subjected to the first PCR with the outer primers V8 and V9 that amplified a 607-base pair (bp) segment. The PCR product from the first PCR was eluted following agarose gel electrophoresis and subjected to the second PCR with the nested primers V6 and V7 that flanked a 351-bp segment. In the second PCR, dTTP was substituted by digoxigenin-11-dUTP in the PCR reaction mixture so that the amplified 351-bp DNA products were labeled with digoxigenin. The specificity of the PCR-generated digoxigenin-labeled probe was tested on different strains of IBDV, several unrelated avian viruses, and bacteria by slot-blot hybridization assay. Hybridization was detected by chemiluminescence. The sensitivity of the probe was assayed using 10-fold serial dilutions of purified RNA from the STC strain of IBDV. The PCR-generated digoxigenin-labeled probe hybridized with genomic RNA of STC and variant strains A, D, E, G, and GLS-5 of IBDV serotype 1 but not OH strain of IBDV serotype 2. The probe did not react with avian reovirus, infectious bronchitis virus, Salmonella enteritidis, Escherichia coli, or Staphylococcus aureus.(ABSTRACT TRUNCATED AT 250 WORDS)

Further characterization of Pasteurella haemolytica-like bacteria isolated from swine enteritis.

DNA-DNA hybridization studies were conducted on six Pasteurella haemolytica-like (PHL) organisms recovered from cases of swine enteritis. Chromosomal-enriched fractions of PHL organisms served as the source of DNA for Southern blots or as whole-chromosomal DNA probes. Under stringent hybridization conditions, chromosomal DNA probes of a prototype PHL (strain 6213A) organism distinguished other PHL organisms from Pasteurella haemolytica types A1 and T3, Pasteurella multiocida types A:1 and A:3, Escherichia coli, Pseudomonas aeruginosa, Actinobacillus pleuropneumoniae type 1, and Salmonella cholerasuis. The guanine-cytosine content of the DNA of three PHL strains was 41.2 to 42.8 mol % as calculated from the thermal denaturation midpoint temperatures. The PHL strains are Gram-negative, nonmotile, beta-hemolytic, pleomorphic, oxidase-positive, urease- and indole-negative, fermentative rods with the key characteristics of the species Pasteurella haemolytica. None of the PHL strains reacted with the type-specific antisera of P. haemolytica types 1 through 12 as tested by an agglutination procedure. These swine strains differed in their biochemical differentiation from P. haemolytica types A1 and T3 in that all produced acid from M-inositol and failed to grow on MacConkey agar. Acid production from trehalose and L-arabinose was variable with PHL strains. Leukotoxicity of PHL strains was evaluated by a colorimetric micro-titration assay. Sterile culture supernatants of three of five PHL strains were toxic to bovine neutrophils. Results of these studies suggest that the PHL organisms may belong to a new group of organisms under the genus Pasteurella.

Type I interferon genes in cattle: restriction fragment length polymorphisms, gene numbers and physical organization on bovine chromosome 8.

Multiple, superimposed Type I interferon (IFN) restriction fragments were resolved following 72-92 h of horizontal electrophoresis. Restriction fragment length polymorphisms (RFLPs) for alpha IFN (IFNA), beta IFN (IFNB), omega IFN (IFNW) and trophoblast IFN (IFNT) genes were identified in HindIII, EcoRI and TaqI digestions from 313 cattle. RFLPs with codominant segregation in cattle pedigrees were considered alleles, and 19 distinct polymorphic Type I IFN loci (5 IFNA, 4 IFNB, 8 IFNW and 2 IFNT) were identified. Allele frequencies and observed heterozygosity values were calculated for each locus and several loci were considered highly informative for linkage analysis. Bovine IFN gene numbers (10 IFNA, 6 IFNB, 20 IFNW and 6 IFNT) were estimated from the number of polymorphic loci plus additional monomorphic hybridizing bands present in EcoRI and HindIII digestions. Physical linkage of the Type I IFN gene families on bovine chromosome 8 was demonstrated by pulsed field gel electrophoresis (PFGE). Hybridization of two or more IFN probes to similarly sized PFGE fragments suggested the tentative gene family order: IFNA/IFNW-IFNT-IFNB. These studies provide a basis for the development of more detailed genetic and physical maps of the bovine Type I IFNs.

Determination of the relative abundance of skeletal muscle alpha actin mRNA in muscle of livestock species.

Three market-weight animals of meat-producing livestock species were slaughtered to obtain porcine (barrows), bovine (steers), ovine (wethers), and avian (cockerels) tissue samples. The four tissues of interest were skeletal muscle, heart, smooth muscle (stomach or gizzard), and liver. Total RNA was isolated from each tissue and then hybridized to a human skeletal (sk)-alpha-actin [32P]cDNA probe using both dot blot and Northern blot hybridization. No hybridization was observed with RNA from liver or smooth muscle from any of the species, suggesting little or no hybridization to nonmuscle and smooth muscle beta- and gamma-actin isoforms. The human sk-alpha-actin probe hybridized to RNA from skeletal muscle of pigs, cattle, sheep, and chickens, although relative hybridization was 75% less with chicken RNA. The hybridization was limited specifically to a band at 1.6 kb (kilobases), the known length of sk-alpha-actin mRNA. Hybridization was observed with RNA from pig heart (1.6 kb) and the relative abundance was consistently 7 to 10% of that observed with porcine skeletal muscle, even as stringency conditions were increased. These results indicate that the human sk-alpha-actin probe can be used to determine alpha-actin mRNA expression in skeletal muscle for pigs, cattle, and sheep.