Anthony J. Radford

Construction and characterization of a Mycobacterium-Escherichia coli shuttle vector

We have constructed a set of novel Mycobacterium-Escherichia coli shuttle vectors using either a kanamycin- or hygromycin-resistance gene and the replication region from a Corynebacterium plasmid. Important features of these new vectors (pEP2 and pEP3) are that they are small, contain multiple cloning sites, and replicate to high copy number in various Mycobacterium species and E. coli. These vectors are unusual in that plasmid replication in gram-negative and gram-positive bacteria appears to be controlled from a single region. These plasmids will be useful for the genetic analysis of Mycobacterium and gene expression in this genus, particularly Mycobacterium bovis BCG.

Diagnosis and epidemiology of bovine tuberculosis using molecular biological approaches

Conventional methods for diagnosis and epidemiological studies of bovine tuberculosis and other mycobacterial diseases are far from ideal. In the last twelve years, molecular biology has provided new approaches which have enabled detailed studies to be made of the molecular characteristics of Mycobacterium bovis, the causative organism of bovine tuberculosis. These characteristics have been investigated for their potential use in diagnosis and epidemiological studies. Restriction fragment analysis of genomic DNA from isolates of M. bovis has provided a highly discriminatory typing system which has been used extensively for epidemiological studies. DNA elements in M. bovis have been investigated for their potential use in diagnostic assays based on the polymerase chain reaction. Individual antigens of M. bovis and their reactive epitopes have been identified and investigated for their usefulness in both serological and T cell assays of animal infection. Serological assays have proven disappointing and the full potential of T cell assays has yet to be determined. Currently, T cell assays based on recombinant antigens have not been as sensitive as those based on natural antigens.

Foreign gene expression in Corynebacterium pseudotuberculosis : development of a live vaccine vector

A defined phospholipase D mutant of Corynebacterium pseudotuberculosis, designated Toxminus, was used as a live vector to express and deliver a range of candidate vaccine antigens to sheep. Expression levels of the foreign genes in Toxminus were evaluated when directed from a number of different promoters, both constitutively expressed and inducible, as fusions with expressed genes including a signal sequence, and from chromosomal and episomal loci. In general expression levels were low and it appeared that some of the recombinant proteins were tolerated by C. pseudotuberculosis Toxminus better than others. Gene expression was however sufficiently high for three of the genes to elicit antibody responses specific to the recombinant protein following a single dose of the live Toxminus vector vaccine. This work suggests that C. pseudotuberculosis Toxminus has potential for development as a live veterinary vaccine vector.

NUCLEOTIDE SEQUENCE OF THE REPLICATION REGION FROM THE MYCOBACTERIUM-ESCHERICHIA COLI SHUTTLE VECTOR PEP2

The replication region derived from the Corynebacterium diphtheriae-Escherichia coli plasmid pNG2 was sequenced. This 1.85-kb segment contains a single origin of DNA replication, one major open reading frame and shares no sequence homology with previously described plasmids.

Conventional and Contemporary Bacterial Veterinary Vaccines

The aim of this chapter is to review some of the conventional and contemporary methods used to vaccinate production animals against bacterial diseases. We confine our discussion to those veterinary diseases where recombinant DNA technology is beginning to play a part in vaccine development. The general objective of vaccinology is to achieve increasingly efficacious vaccines. An emerging trend toward this target is to identify those antigens capable of eliciting protective immunity then to present them to the animal in the optimal fashion. This concept has led first to the purification and presentation of single or mixed antigens, then to the production of recombinant antigens. Here we review the progress in bacterial veterinary vaccine development, from the use of simple bacterin preparations through to live delivery of recombinant antigens.