Neutralization of Apx toxicity as an alternative to antibiotics for control of contagious porcine pleuropneumonia.

Contagious pleuropneumonia is a severe acute disease that kills many growing pigs and causes lifelong damage to the lungs of those that survive. This impacts on the profitability of the production system. Pig farms regularly use antibiotics to control this disease because there is very little that can otherwise be used.

Before the advising veterinary surgeon on a pig unit can reduce the use of antibiotics, we need alternatives for them to use. Apart from improving husbandry practices such as increasing ventilation and reducing the number of animals held together, there is little that can be offered. This is a problem throughout the world and is one of the primary reasons for using prescription antibiotics in pigs throughout Europe. In the UK, there is no effective vaccine because these have failed to protect pigs from disease, or the vaccine was itself toxic. Efforts to make on-farm vaccines (emergency vaccines) have not solved the problem because they are not efficacious and antibiotics, in feed, in water and by injection, are used in an attempt to avoid catastrophic losses.

The disease is caused by a bacterium, Actinobacillus pleuroneumoniae, which produces two of three different protein toxins (ApxI, II and III). Production of these toxins by the pathogen is key to the disease process. Pigs that recover from disease have antibodies which neutralize the toxins and evidence suggests this is crucial in protecting pigs from the disease. It needs to be replicated in a successful vaccine. However, simply using the toxin(s) as a vaccine does not protect pigs. Despite stimulating production of antibodies these are not toxin-neutralizing antibodies. It appears that these bacteria have evolved to synthesise toxin molecules with irrelevant but highly immunogenic regions to distract the immune response to the wrong part of the toxin so that the antibody response is ineffective allowing the bacteria to spread and the disease continue.

In this project we will eliminate those parts of the toxin that are distracting the immune response and which appear to be causing failure of the toxin molecules to generate a neutralizing response when used as a vaccine. These small fragments will be joined to a carrier protein, modified diphtheria toxin. This will enhance the immune response and make the antigen large enough to be recognised by the pigs' immune system as a vaccine antigen. We will immunize pigs with these modified toxins and measure the immune response and the neutralizing effect against the active toxins. To improve the method of testing the toxins and the effect of neutralizing antibody, we will develop and test a pig model of dermal oedema. This will be used to indicate the best vaccine antigens for use in the pig model of pleuropneumonia. We will then proceed to immunize pigs and test the efficacy of the vaccination by experimental challenge of the pigs with the virulent pathogen.

If this hypothesis is correct, and the immune response to the toxin fragment is effective, this could be the step needed for production of an effective vaccination against pleuropneumonia and the opportunity, finally, to offer the pig industry an alternative to antibiotics which would markedly reduce the quantity of these drugs used in controlling pig respiratory disease.

The aim of this project is to modify the Apx toxins of Actinobacillus pleuropneumoniae to remove the immunologically dominant part of the molecule, which is interfering with stimulation of a neutralizing antibody response that is needed for vaccination to be effective. In this project we will develop a dermal oedema test for Apx toxin and its neutralization. We will also test the effect of those forms of modified Apx found to be able to generate an appropriate response in protection against pleuropneumonia in the target species.

Contagious porcine pleuropneumonia (CPP) is a severe disease that kills growing pigs and causes lesions in the pleura which require trimming on the slaughter line. Pig herds can and do become uneconomic and are regularly closed down as a result of losses incurred through morbidity and mortality following infection. Efforts to control CPP by vaccination have been disappointing and vaccines have not yet demonstrated adequate levels of protection. This reults in farmers resorting to use of in-feed prescription antimicrobials, including lincomycin, tylosin, tilmicosin, amoxicillin, chlortetracycline, together with injectables such as florfenicol, the floroquinolone enrofloxacin, and the macrolide tulathromycin. It is essential that to halt this widespread use of prescription antimicrobials in the pig industry, both in Europe and worldwide, and develop alternative strategies for control of this disease.

This project is designed to test the hypothesis that neutralizing epitopes of the Apx toxins will be more effective in generating a protective antibody response in pigs compared with the whole toxoid. Recombinant proteins, containing ApxI-III fragments will be generated fused to diphtheria toxoid CRP197. These will be used to immunize pigs, the response measured and the effect of vaccination tested in a novel model of dermal oedema and then in a well-refined challenge model of CPP in the pig.

Who will benefit from this research? How will they benefit?

1. Pig production.

Approximately 20 million tons of pig meat is slaughtered in the EU every year. Approximately 35% of these animals will have had or been exposed to pleuropneumonia. The large majority of these will have been treated with antimicrobial drugs on veterinary prescription. The direct beneficiaries of this research would therefore be those associated with the pig industry who are involved in producing healthy livestock in the face of enzootic disease. This is the farmers, veterinary surgeons who advise farmers and the veterinary pharmaceutical industry seeking the means to offer "biologicals" in the control of infectious disease.

Contagious pleuropneumonia has a substantial influence on production costs such that pig farming can become unprofitable when it is not controlled. Disease such as this is, in my own experience, responsible for the regular loss of pig farms in the UK and elsewhere in Europe. Estimating the cost of the disease has had varying results. Costs of medication, fatalities, reduced performance and carcase trimming in the abattoir are all contributory. Furthermore, it is a major welfare problem when animals are suffering the deep pain of pleural and lung lesions and dying due to pulmonary oedema and haemorrhage.

2. The wider community by reduction of antimicrobial use in pig farming.

Realistically, it is essential that for a substantial reduction of antimicrobial use in pig & poultry farming, alternatives to these drugs are made available.

It is not uncommon in parts of Europe (though not publicised), to have pigs on a rolling programme of prescription antimicrobials from the first days of life to shortly before the withdrawal period before slaughter. I have seen for myself, on farms, the many 25 kg bags of chlortetracycline, lincomycin, amoxicillin, tylosin, tilmicosin and others for inclusion in pig feed as a means of trying to control pig respiratory disease. While this is considered bad practice, and is strongly discouraged in the veterinary profession, it is often seen as the only means of keeping intensive pig production financially viable in the face of losses from infection. Lowering the use of antimicrobials would contribute to reducing contamination of the environment as a whole and the selection pressure and transmission, among commensal bacteria and pathogens alike, of genetic elements conferring resistance. Organisms carrying such genetic elements are distributed in the environment via wildlife: birds, rodents, insects that cohabit the pig farm. They are also probably distributed by human contacts and by the meat following slaughter. Vaccines are already used widely to protect pigs against other bacterial (atrophic rhinitis, swine erysipelas) and viral diseases (circovirus, parvovirus) and an effective vaccine to control A. pleuropneumoniae would be readily used in the industry if shown to be effective.

3. Veterinary pharmaceutical industry.

A vaccine to protect against porcine pleuropneumonia has been sought since the 1980s. The only commercial vaccines (PorcilisApp; Intervet and Pleurostar; Norvartis) have included Apx toxins in their formulation, but both have problems and neither are registered and available in the UK. Vaccines which have not included Apx have not protected against the disease. Those emulating the successful human vaccines for HIB, pneumococcal disease and Neisseria have simply failed. The veterinary pharmaceutical industry needs to have a clear approach to a successful vaccine and they will exploit the idea with alacrity. We are in regular contact with a number of major companies and have tested candidate vaccines for them (including A. pleuropneumoniae) in vivo, over 2 decades. Should the outcome of this research be positive, Veterinary pharma will benefit by producing an effective, commercially valuable vaccine to be used in several millions of pigs annually.