Oral and sub-cutaneous vaccination of commercial pigs with a recombinant porcine adenovirus expressing the classical swine fever virus gp55 gene.

A recombinant porcine adenovirus expressing the classical swine fever virus (CSFV) gp55/E2 gene was administered to commercially available pigs via oral or subcutaneous routes and their susceptibility to oral and subcutaneous challenge with CSFV was determined. 100% of animals vaccinated and challenged subcutaneously were protected. In the groups of pigs vaccinated either orally or subcutaneously and then challenged orally, 60% of animals were protected. Before challenge, neutralising antibodies to CSFV were detected in 60% of pigs vaccinated subcutaneously, but in none of those given the vaccine orally. CSFV antigen was found in the spleens of surviving pigs that had been vaccinated orally. In contrast, subcutaneous vaccination was shown to preclude the presence of CSFV in the spleen of animals that survived challenge.

Vaccination against ovine footrot using a live bacterial vector to deliver basic protease antigen.

A strain of Corynebacterium pseudotuberculosis, designated Toxminus, that has been rationally attenuated by deletion of the phospholipase D gene, is being developed as a live vaccine vector for the delivery of veterinary vaccine antigens. In the present study a recombinant form of the basic protease gene of the ovine footrot causative bacterium, Dichelobacter nodosus, was introduced into the vector strain using the high copy number plasmid pEP2. This strain secreted the basic protease protein. Vaccination trials in sheep with the recombinant strain demonstrated that, although an IgG immune response was elicited, the animals were not protected from footrot following artificial challenge under pen conditions. Although the sheep were not protected there was evidence that the progression of the disease was slowed.

Protection of pigs against classical swine fever with DNA-delivered gp55.

Classical swine fever virus causes significant mortality and morbidity in commercial piggeries in many countries in Europe and Asia. The protective antigen, gp55, is highly conformation-dependent and thus killed virus or bacterially produced proteins are not protective. This report demonstrates that DNA vaccination with the gene encoding gp55 can provide protective immunity with inoculation of two doses of 25 microg DNA or a single shot of 200 microg. Furthermore, the DNA can be delivered intramuscularly or by a simple spring-loaded needleless inoculator. In addition it is shown that inoculation of the DNA at a single site conveys the same level of immunity as division of the dose between two sites.

Vaccination with a single dose of a recombinant porcine adenovirus expressing the classical swine fever virus gp55 (E2) gene protects pigs against classical swine fever.

A recombinant porcine adenovirus (rPAV) with the gp55 (E2) gene from the classical swine fever virus (CSFV) 'Weybridge' strain inserted into the right hand end of the PAV serotype 3 (PAV3) genome was constructed. Expression of gp55 was directed by the major late promoter and tri-partite leader sequences located and cloned from PAV3. No compensatory deletions of PAV DNA sequences were made. Vaccination of outbred pigs with a single dose of the recombinant virus (rPAV-gp55) resulted in complete protection from lethal challenge with CSFV. No adverse clinical signs were observed in vaccinated animals following administration of rPAV-gp55 and following challenge, no clinical signs of CSF were observed prior to, or at, post mortem. The insert made into the rPAV increased the genome length to 106.8% of wild type and therefore exceeded the expected maximum insert size for a stable recombinant by almost 2%. Thus rPAV-gp55 contains the largest stable insertion made into a non-deleted Mastadeno virus recombinant so far reported.

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.

Vaccination and protection of pigs against pleuropneumonia with a vaccine strain of Actinobacillus pleuropneumoniae produced by site-specific mutagenesis of the ApxII operon.

The production of toxin (Apx)-neutralizing antibodies during infection plays a major role in the induction of protective immunity to Actinobacillus pleuropneumoniae reinfection. In the present study, the gene encoding the ApxII-activating protein, apxIIC, was insertionally inactivated on the chromosome of a serovar 7 strain, HS93. Expression of the structural toxin, ApxIIA, and of the two genes required for its secretion, apxIB and apxID, still occurs in this strain. The resulting mutant strain, HS93C- Ampr, was found to secrete the unactivated toxin. Pigs vaccinated with live HS93C- Ampr via the intranasal route were protected against a cross-serovar challenge with a virulent serovar 1 strain of A. pleuropneumoniae. This is the first reported vaccine strain of A. pleuropneumoniae which can be delivered live to pigs and offers cross-serovar protection against porcine pleuropneumonia.

Efficacy of an ovine caseous lymphadenitis vaccine formulated using a genetically inactive form of the Corynebacterium pseudotuberculosis phospholipase D.

Caseous lymphadenitis (CLA) is an economically significant disease of sheep caused by the gram-positive bacterium Corynebacterium pseudotuberculosis. CLA vaccines are currently formulated using formalin inactivated culture supernatants that are rich in the C. pseudotuberculosis phospholipase D (PLD) exotoxin. One alternative to chemical detoxification is to inactivate the PLD genetically. This procedure not only provides a means to remove an onerous chemical treatment step but also the opportunity to increase gene expression, therefore improve protein yields. Using site-specific mutagenesis the C. pseudotuberculosis PLD was inactivated by substituting a serine residue at histidine 20 within the enzyme active site. CLA vaccine formulated using genetically inactivated PLD protected 44% of sheep against C. pseudotuberculosis challenge compared with 95% protection offered by the formalin inactivated preparation. Since there was no apparent difference in immune response mounted by vaccinated sheep the reason for this variation in vaccine efficacy remains unclear. Although genetic inactivation can be a convenient means to produce toxoid vaccines its use to develop a new CLA vaccine provided no net benefit over the conventional formulation.

Protection of mice against challenge with homologous and heterologous serovars of Actinobacillus pleuropneumoniae after live vaccination.

Protective immune responses and the virulence of Actinobacillus pleuropneumoniae (APP) have been attributed, in part, to toxins (Apx) produced by the bacterium. A mutant of the serovar 7 strain HS93 (HS93Tox-), lacking the genes encoding the structural toxin ApxA and the post-translational activating protein ApxC, but retaining the genes required for secretion ApxB and ApxD, was isolated and shown to be attenuated in a mouse model. A plasmid vector system was developed and used to express the ApxA gene from within the HS93Tox- strain. The resulting strain, HS93Tox-/pIG-T1K, expresses the Apx structural protein in a non-activated form. HS93Tox-/pIG-T1K was shown to be attenuated in a mouse model and to be capable of inducing Apx-specific antibodies, which were boosted on re-inoculation. Live vaccination of mice with HS93Tox-/pIG-T1K offered protection against homologous wild-type serovar 7 challenge, and also heterologous challenge with a serovar 1 strain. This is in contrast to vaccination with the HS93Tox- strain, which failed to protect mice against a heterologous challenge.

Vaccine potential of attenuated mutants of Corynebacterium pseudotuberculosis in sheep.

Corynebacterium pseudotuberculosis, a gram-positive facultative intracellular bacterial pathogen, is the etiological agent of the economically important disease caseous lymphadenitis (CLA) in both sheep and goats. Attenuated mutants of C. pseudotuberculosis have the potential to act as novel vaccines against CLA and as veterinary vaccine vectors. In this report, we have assessed the virulence of both aroQ and pld mutants of C. pseudotuberculosis in sheep and concurrently their capacity to act as vaccines against homologous challenge. The results suggest that aroQ mutants of C. pseudotuberculosis are attenuated with regard to both lymph node persistence and vaccination site reactogenicity. Immunologically, aroQ mutants failed to elicit detectable specific gamma interferon (IFN-gamma)-secreting lymphocytes and induced low levels of antibodies to C. pseudotuberculosis culture supernatant antigens. Following subcutaneous vaccination, the immune responses induced by aroQ mutants did not protect sheep from infection with the wild-type strain but did appear to reduce the clinical severity of disease resulting from challenge. Conversely, an attenuated C. pseudotuberculosis strain expressing an enzymatically inactive phospholipase D exotoxin, when used as a vaccine, elicited a protective immune response. Protection appeared to correlate with in vivo persistence of the vaccine strain, the induction of IFN-gamma-secreting lymphocytes, and relatively high levels of antibodies to culture supernatant antigens. The results suggest that aroQ mutants of C. pseudotuberculosis may be overly attenuated for use as a CLA vaccines or as vaccine vectors.

Attenuation and vaccine potential of aroQ mutants of Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis, a gram-positive intracellular bacterial pathogen, is the etiological agent of the disease caseous lymphadenitis (CLA) in both sheep and goats. Attenuated mutants of C. pseudotuberculosis have the potential to act as novel live veterinary vaccine vectors. We have cloned and sequenced the aroB and aroQ genes from C. pseudotuberculosis C231. By allelic exchange, aroQ mutants of both C231, designated CS100, and a pld mutant strain TB521, designated CS200, were constructed. Infection of BALB/c mice indicated that introduction of the aroQ mutation into C231 and TB521 attenuated both strains. In sublethally infected BALB/c mice, both CS100 and CS200 were cleared from spleens and livers by day 8 postinfection. The in vivo persistence of these strains was increased when the intact aroQ gene was supplied on a plasmid in trans. Mice infected with TB521 harbored bacteria in organs at least till day 8 postinfection without ill effect. When used as a vaccine, only the maximum tolerated dose of CS100 had the capacity to protect mice from homologous challenge. Vaccination with TB521 also elicited protective immunity, and this was associated with gamma interferon (IFN-gamma) production from splenocytes stimulated 7 days postvaccination. The role of IFN-gamma in controlling primary infections with C. pseudotuberculosis was examined in mice deficient for the IFN-gamma receptor (IFN-gammaR(-/-) mice). IFN-gammaR(-/-) mice cleared an infection with CS100 but were significantly more susceptible than control littermates to infection with C231 or TB521. These studies support an important role for IFN-gamma in control of primary C. pseudotuberculosis infections and indicate that aroQ mutants remain attenuated even in immunocompromised animals. This is the first report of an aroQ mutant of a bacterial pathogen, and the results may have implications for the construction of aromatic mutants of Mycobacterium tuberculosis for use as vaccines.

Cytokine gene expression in sheep following experimental infection with various strains of Corynebacterium pseudotuberculosis differing in virulence.

Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis in sheep and goats. This disease is characterized by the development of pyogranulomas in the lymph nodes and lung tissue. To measure the cytokine gene expression in C pseudotuberculosis lesions, sheep were inoculated with two attenuated strains (Tox- and PLD-t) and a wild-type (WT) strain of C pseudotuberculosis and were necropsied at 7 or 28 days post-inoculation. The Tox- strain showed a strong reduction in virulence as assessed by the absence of disseminating lesions in the lymph nodes draining the inoculation site in contrast with the WT strain. The PLD-t strain showed an intermediate reduction in virulence. The two attenuated strains, however, induced the same amount of antibodies and IFN-gamma production as the WT strain. Using a semi-quantitative RT-PCR technique, the expression of inflammatory cytokines was found to be higher in the inoculation site, whereas expression of T-cell associated cytokines was more intense in the draining lymph node. On the whole, the infected sheep produced high levels of cytokines in at least one organ on days 7 or 28 post-inoculation. No significant differences in cytokine gene expression were shown between sheep infected with strains differing in virulence. Higher cytokine expression was measured in sheep with pyogranulomas in the draining lymph nodes as compared to those without, especially for interleukin-1 beta and interleukin-8. Overall, these results taken together confirmed the attenuation of virulence in Tox- and PLD-t strains of C pseudotuberculosis and showed the important role of PLD in disseminating the bacteria from the inoculation site to the draining lymph nodes. The pathogenesis of ovine caseious lymphadenitis was shown to be associated with production of cytokines at the pyogranuloma level, but the local cytokine patterns associated with different courses of infection were not distinguished.

Characterization of a Pasteurella multocida plasmid and its use to express recombinant proteins in P. multocida.

The complete nucleotide sequence of a naturally occurring 5.36-kb streptomycin and sulphonamide resistance plasmid, designated pIG1, isolated from type D Pasteurella multocida was determined. A 1.6-kb noncoding region and a 1.4-kb region encoding three putative proteins were shown by sequence homologies and functional characterizations to be involved in the replication and mobilization of pIG1, respectively. The remaining sequence carried an unusual arrangement of streptomycin- and sulphonamide-resistant genes when compared to various other plasmids. It appears that the antibiotic resistance region of pIG1 may have evolved by recombination between three different short direct repeat DNA sequences. A 4.5-kb recombinant plasmid was constructed by replacing the antibiotic resistance genes of pIG1 with a kanamycin resistance gene and seven unique restriction sites. The resulting plasmid, designated pIG112, stably replicates in P. multocida, Pasteurella haemolytica, Actinobacillus pleuropneumoniae, and Escherichia coli and can be introduced into these organisms by either transformation or conjugation. This vector exists at approximately 70 copies per cell in P. multocida and approximately 20 copies per cell in E. coli. To demonstrate plasmid-borne gene expression in P. multocida, the P. multocida dermonecrotic toxin gene, toxA, and a genetically modified form of this gene were cloned into pIG112 and expressed in high amounts in a nontoxigenic P. multocida strain. Cell culture assays demonstrated that nontoxigenic P. multocida expressing toxA was cytopathic, whereas a strain expressing the modified toxA derivative was not.

Cloning and manipulation of the Corynebacterium pseudotuberculosis recA gene for live vaccine vector development.

Corynebacterium pseudotuberculosis is an intracellular bacterial pathogen causing a chronic abscessing disease in sheep and goats called caseous lymphadenitis. We are developing this bacterial species as a live vector system to deliver vaccine antigens to the animal immune system. Foreign genes expressed in bacterial hosts can be unstable so we undertook to delete the C. pseudotuberculosis chromosomal recA gene to determine whether a recA- background would reduce the frequency of recombination in cloned DNA. Homologous DNA recombination within an isogenic recA- C. pseudotuberculosis was 10-12-fold lower than that in the recA+ parental strain. Importantly, the recA mutation had no detectable affect upon the virulence of C. pseudotuberculosis in a mouse model. Taken together these results suggest that a recA- background may be useful in the further development of C. pseudotuberculosis as a vaccine vector.

Caseous lymphadenitis vaccine development: site-specific inactivation of the Corynebacterium pseudotuberculosis phospholipase D gene.

Vaccines for ovine caseous lymphadenitis (CLA) are currently formulated using partially purified, formalin inactivated phospholipase D (PLD) derived from Corynebacterium pseudotuberculosis culture supernatants. Chemical treatment has been a common and effective way of inactivating bacterial toxins for use in toxoid vaccines. Genetic inactivation of toxin genes using site-specific mutagenesis has the potential to improve this process by providing a safer and more cost-effective product. In the present study amino acid substitutions at the putative catalytic site and metal binding domain of the PLD protein had a profound affect upon PLD activity and secretion from C. pseudotuberculosis. Two mutated PLD analogues that were secreted to a level of 40% compared to the wild-type and retained minimal activity showed promise for development as recombinant CLA vaccines. Further work will be required to establish their suitability for commercialization.

Protection of sheep against caseous lymphadenitis by use of a single oral dose of live recombinant Corynebacterium pseudotuberculosis.

An inactive form of the Corynebacterium pseudotuberculosis phospholipase D (PLD) gene was constructed and expressed in a PLD-negative strain (designated Toxminus) of C. pseudotuberculosis. Antibody responses specific to Toxminus and both Toxminus and PLD proteins were detected in sheep following oral administration of Toxminus or Toxminus expressing the PLD toxoid, respectively. However, only those sheep vaccinated with Toxminus expressing PLD toxoid were protected against wild-type challenge. These results confirm the importance of PLD as a protective antigen and demonstrate both the potential for developing an oral caseous lymphadenitis vaccine and C. pseudotuberculosis Toxminus as a live vaccine vector.

Putative functional domain within ORF2 on the Mycobacterium insertion sequences IS900 and IS902.

Repeated DNA sequences have been identified in a range of mycobacterial species and have been implicated in the increased virulence of some of these species, namely, Mycobacterium paratuberculosis and M. avium subsp. silvaticum. Here we present a case to suggest that the insertion sequences IS900 and IS902 encode a protein from a putative gene positioned on the complementary strand to their transposase genes. Based on amino acid homology analyses, this open reading frame (ORF2) could encode a transport protein. The ORF2 protein thus IS900 and IS902, may have a role in the increased pathogenicity of M. paratuberculosis and M. avium subsp. silvaticum from an M. avium background.

Expression of ovine gamma interferon in Escherichia coli and Corynebacterium glutamicum.

Bacteria of two species, Escherichia coli and Corynebacterium glutamicum, were used as hosts to express recombinant ovine gamma interferon as a fusion protein with glutathione S-transferase. The recombinant gamma interferon produced by both bacteria was biologically active in vitro and was recognized by anti-gamma interferon monoclonal antibodies. E. coli produced large amounts of soluble recombinant protein which could be purified by a simple affinity chromatography method. Only a small fraction of the recombinant protein made by C. glutamicum was recovered by this method. Expression of recombinant protein in C. glutamicum was unstable but could be controlled by increased regulation of the tac promoter. Both hosts expressed ovine gamma interferon at high levels, with the recombinant protein making up a significant proportion of the cellular protein content.

Phylogenetic relationship of Fusobacterium necrophorum A, AB, and B biotypes based upon 16S rRNA gene sequence analysis.

The 16S rRNA genes from virulent (AB) and benign (B) biotype strains of Fusobacterium necrophorum, the causative agent of ovine foot abscess, were cloned and sequenced. Notwithstanding the distinct phenotypic differences between the AB and B biotypes, a phylogenetic analysis in which the 16S rRNA gene sequences were used revealed the close relationship of these taxa. Comparison of the virulent and benign biotypes of F. necrophorum may therefore be a legitimate way to identify key virulence factors useful in vaccine development.

Characterisation of a highly repeated DNA sequence from Mycobacterium bovis.

We report characterisation of a novel repeat sequence from a Mycobacterium bovis genomic library. The highly repeated sequence belongs to a family consisting of a 24 base pair (bp) direct repeat (DR), that appears to be organized into clusters on the chromosome. We classify the 24-bp DR into the group of prokaryotic DNA repeats known as the interspersed repetitive sequence elements. The 24-bp DR will be of potential use as a DNA fingerprinting tool in epidemiological studies of M. bovis.

Restriction fragment length polymorphism analysis of Fusobacterium necrophorum using a novel repeat DNA sequence and a 16S rRNA gene probe.

A repeated DNA sequence was isolated from Fusobacterium necrophorum biotype AB, strain FnS1. The repeated sequence shared considerable homology with the transposase gene from the Pseudomonas syringiae insertion sequence IS801. The repeat sequence was used together with a 16S ribosomal RNA gene probe to type F. necrophorum isolates using restriction fragment length polymorphisms. The probes revealed differences between several clinical isolates and will be useful tools to study the epidemiology of ovine foot abscess and other diseases caused by F. necrophorum.