Bacterial and Host Genes in Salmonella Colonisation in Poultry

Poultry remain the major source of Salmonella for man. Carcass contamination arises largely from the ability of food-poisoning serotypes of Salmonella to colonise the alimentary tract of poultry in the absence of disease. Egg infection can also result from surface contamination of the egg during passage through the cloaca of the infected bird. Elucidation of the microbiological and molecular basis of intestinal colonisation by Salmonella will underpin the development of biological control measures. Such measures will become increasingly important with (a) the economic constraints of upgrading housing and (b) the costs of improving feed and management quality and (c) problems associated with the use of antibiotics.
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This project seeks to provide information on the major bacterial and host factors which determine colonisation, knowledge of which might be used in the design of non-pharmaceutical, biological control measures. It also seeks to gain a further understanding of the mechanism of genus-specific exclusion of colonisation (in which one Salmonella strain may inhibit another in vivo) which may be used to enhance non-immune resistance in the chick intestine.
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The major aim of the project is to identify the mechanisms used by Salmonella strains in the intestine to obtain their energy, from major carbon sources and through selected electron acceptors other than oxygen.. There is evidence that most growth of Salmonella occurs at the boundary of the mucosa and the contents since Salmonella organisms are under starvation stress in the lumen where they are greatly outnumbered by other bacteria. We also have evidence that the mucus layer is an important carbon source for colonising Salmonella. Catabolism of these compounds in the gut does not involve oxygen as electron acceptor and it is likely that others are used in this anaerobic environment. Identification of the genes required for major carbon source utilisation and electron acceptor usage and incorporation of these mutated genes into live vaccines would induce a non-colonising vaccine phenotype which would be shed in the faeces of vaccinated birds for a shorter period than the live vaccines currently available and thus not be present in birds sent to slaughter.
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Colonisation of the alimentary tract of the newly hatched chicken by Salmonella results in massive multiplication and faecal shedding. This may be largely prevented by pre-inoculation with an avirulent Salmonella strain. This multiplies and excludes other strains within a matter of hours, by a purely microbiological phenomenon. This genus-specific inhibition of colonisation (a novel, genus-specific form of competitive exclusion) was originally identified and has been investigated extensively at IAH, Compton and modelled in vitro using stationary-phase nutrient broth cultures. One of the central controlling mechanisms relating to growth in vitro and in the intestine is the rate of initiation of chromosomal replication. It is likely that one component of this is quorum sensing, which is the ability of a bacterial culture to sense itself when the cells are present at high density by the constitutive production of a signalling molecule. The identification of products in stationary-phase broth cultures which regulate this will allow the possibility of oral administration of products to reduce bacterial growth rates in the intestine. The practical consequence is that live attenuated vaccines could be administered orally to newly hatched chicks such that exclusion occurs in the gut. The vaccine strain would persist long enough to induce immunity in the usual way. A combination of this characteristic and the non-colonising trait described above should lead to the development of vaccines which will fulfil the necessary criteria for live enteric vaccines, which include the requirement that vaccine strains should not enter the human food chain.
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We have identified differences in colonisation ability in different inbred lines of chickens housed at the IAH. The increased resistance seen in some lines is inherited as a dominant trait and is not related to sex, MHC (major histocompatibility complex), NRAMP1 (the ity gene responsible for resistance in some mouse breeds) or SAL1 (the major chicken gene identified at IAH, Compton, responsible for resistance to systemic salmonellosis). We will begin to identify the biological basis of this. A major possibility is a quantitative and/or qualitative difference in the alpha- and beta-defensins and the mucus produced by the mucosa. We will explore the synergy between the inhibition of colonisation as a result of this genetic component and the use of killed vaccines to control intestinal colonisation and faecal shedding. Incorporation of this genotype into commercial birds by a natural breeding programme should increase resistance to infection and thus reduce colonisation if applied to commercial poultry flocks. <P>

Progress: The 2,400 serological types of Salmonella enterica can be divided into two groups according to the extent that they colonise the alimentary tract of food animals and thereby enter the human food chain and cause enteritis. Serovars such as Salmonella Enteritidis and S. Typhimurium colonise well whereas other serovars that more characteristically produce typhoid-like infections in different animals are generally unable to colonise the gut. The basis of this difference is poorly understood. The reason for examining these genes is that these genes could be mutated in a live attenuated vaccine such that the vaccine also does not colonise the alimentary tract of food animals and itself is unlikely to enter the food chain. Examination of genome sequences has begun to reveal differences that might be responsible for some of these differences. These genomic differences include the presence of defective genes (pseudogenes) in sets of genes in typhoid strains which suggests that they might be involved in colonisation in the serovars in which these genes are functional.
<P>Some of these genes include those involved in respiration in which carbon sources are oxidised with a terminal external electron acceptor, such as oxygen, nitrate etc. We have thus produced mutations in a number of genes affecting the respiration phenotype and measured colonisation ability. We compared this ability with that of a pig typhoid serovars, S. Choleraesuis, which colonises very poorly. None of the respiration systems using thiosulphate, nitrate or tetrathionate appeared to be major electron acceptors. Tetrathionate is also used for the utilisation of propanediol, a carbon source that is metabolised via vitamin B12-dependent pathway. Again mutation of these systems produced small effects but nothing major despite the fact that some of the genes affected are also pseudogenes in non-colonisers.
<P>We have evidence from a microscopical study that Salmonella bacteria multiply preferentially close to the gut mucosa where oxygen levels will be higher and where nutrient concentrations will also be higher. It seems likely therefore that sialic acid, which is present as a major surface antigen on eukaryotic cells, will be an important and freely available carbon source for bacteria. We therefore sought to mutate nanH which is central to its utilisation. However, this gene is not possessed by several Salmonella strains that colonise indicating that it is not vital to this phenotype.
Since bacteria are likely to be under conditions of nutrient starvation in the gut lumen we also looked at energy storage compounds and their role in virulence. One of these, polyphosphate appears to be very important in regulating internal cellular conditions and bacterial growth and initiation of chromosomal replication.
<P>In addition to bacterial factors associated with colonisation, we have sought to define some of the host factors associated with genetic resistance to colonisation that occurs in certain in-bred lines of chickens. We have examined expression of several Gallinacins (avian intestinal defensin) in several parts of the intestine and other tissues. We have found differences in levels of expression between lines, with age and also after infection with Salmonella. This is being examined further through a PhD student.
<P>An extensions of a year has been awarded during which two further topics will be examined, namely the immune responses, both quantitative and protective, induced by serogroup C Salmonella strains and the possibility of eliminating the tissue carrier state observed with S. Pullorum and certain strain of S. Enteritidis by the use of live attenuated vaccines, with the aim of pushing the Th2-type responses observed with these tow organisms towards a more Th1-type response normally observed with S. Typhimurium. A separate report will be made on this area of work next summer. <P>