The virulence for gnotobiotic pigs of live attenuated vaccine strains of Salmonella enterica serovars Typhimurium and Enteritidis
Introduction
Salmonella-associated food poisoning remains one of the most important food borne zoonoses in the western world. Poultry and pigs are generally regarded as being the major sources of infection for man [1], [2].
Control of infection in enclosed poultry houses may be carried out by a combination of improved hygiene and management. However, high costs suggest that their implementation could be financially disadvantageous. In addition, there is less scope for such an approach, where extensive farming allows environmental infection. It is more likely, therefore, that given the problems associated with chemo-therapy and -prophylaxis, vaccination will become more widespread. Killed, whole cell autologous bactrins are used extensively on an ad hoc basis and one commercially produced killed vaccine is already used extensively in Europe in laying hens. However, it is generally regarded that these are less effective than live, attenuated vaccines against both systemic [3], [4], [5] and intestinal salmonellosis [6].
The use of live vaccines against Salmonella in food-animals has recently been reviewed extensively [7], [8], [9], [10]. A number of attenuations have been tested experimentally and found to be protective. These include mutations in the pathway to produce shikimate (aro) required for the production of para amino benzoic acid [11] and in the genes encoding adenylate cyclase and the cAMP receptor protein (cya and crp, respectively) resulting in an inability to grow on carbon sources other than glucose [12]. Purine-requiring mutants are also attenuated but, at least in mice, are poorly immunogenic [13]. Other live vaccines have been produced, which are being used in poultry in different countries [9]. These have been produced by chemical mutagenesis and although they are known to be purine-requiring the exact nature of the defining attenuating lesion is not known.
A number of criteria have been considered against which candidate vaccine strains can be measured [10], [14], [15]. Amongst essential characteristics are avirulence, not only for the host animal, but also for man. Ideally, vaccine strains should not be more virulent than the commensal strains of Escherichia coli, which are also consumed as a result of preparing and eating meat. Most of the mutations considered so far attenuate virulence in murine and avian typhoid caused by Salmonella enterica serovars Typhimurium, S. Enteritidis, S. Dublin [16], [17] and S. Gallinarum [17] respectively. However, systemic disease is rarely a sequela of the gastro-enteritis associated with infection by food poisoning strains and few animal models have been available to test their capacity to produce gastro-enteritis. There is some evidence that aroA mutants of S. Typhimurium may induce transient diarrhoea in calves [18], [19].
A number of model systems may be used to assess virulence in the absence of a full understanding of the pathogenesis of gastro-enteritis. Ligated ileal loops in animals may be used [20] and Salmonella strains also produce gastro-enteritis in young animals such as cats and dogs [21] and pigs [2]. Gnotobiotic pigs are highly susceptible to S. Typhimurium, oral infection resulting in diarrhoea and dehydration with associated invasion of tissues, the extent of this depending on the virulence of the strain [21], [22]. Since a proportion of the human population is immuno-compromised for different reasons and may therefore show reduced resistance to intestinal pathogens, an assessment of the virulence of these strains in a model such as this could be regarded as an essential prerequisite to their widespread use. We have therefore tested the effects on the virulence for gnotobiotic pigs of introducing attenuating mutations into S. Typhimurium and S. Enteritidis, mutations which are being considered for use in live vaccines for use with food animals.
Section snippets
Bacterial strains
S. enterica serovar Typhimurium F98 [6] and S. Enteritidis P125109 [24] are virulent for chickens and mice. Mutations in aroA were introduced from LT2 aroA::Tn10 by transduction using bacteriophage P22. This was followed by selection for loss of the transposon and associated deletion of part of the gene, as described by the same authors [6]. In newly hatched chickens the parent strains typically produce a typhoid like infection with associated high mortality. The mutants were completely
Clinical outcome of infections
All pigs (four or six for each strain) inoculated with the parent strains, S. Typhimurium F98 or χ3761 or S. Enteritidis P125109 showed evidence of diarrhoea 12–24 h later. Clinical signs were a liquid, yellow diarrhoea associated with increasing lethargy and, less frequently, vomiting. Pigs frequently lost their appetite as the infection progressed. The condition of one or two pigs in each group deteriorated quickly, possibly due to rapid systemic involvement, as occurs in Salmonella and E.
Discussion
A degree of virulence was observed in the Salmonella strains into which mutations had been introduced which were being considered for use with poultry. The extent of the attenuation was greater with the aroA strains (although there was variation between individual pigs, than with the cya crp mutants), which were highly virulent in this model.
The residual virulence seen with these potential vaccine strains in the young gnotobiotic pig model is in contrast to the almost total attenuation seen
Acknowledgements
The authors would like to acknowledge the financial support of the Ministry of Agriculture, Fisheries and Food, UK and the European Union (CT 98-4006).
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