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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.

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.

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.

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.

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.