Microbiologist
Epitopix, Willmar, MN
U.S.A.
An essential factor required for Salmonella to induce clinical disease is the ability to proliferate successfully in the host (Braun and Killmann 1999, Ratledge and Dover 2000, Rosenberg and Young 1974). Iron is an essential nutrient for the growth of gram-negative bacteria, however, it has long been recognized that the free iron normally present in a vertebrate host is in to low of a concentration to support the growth of bacteria (Crichton 1991, Bullen and Griffiths 1999).
The majority of iron in the host is found intracellularly, or extracellularly complexed with high affinity iron binding proteins such as transferrin and lactoferrin. To circumvent this low iron environment, pathogenic bacteria utilize a high affinity iron transport system, consisting of siderophores and siderophore receptor proteins. Siderophores are low molecular weight compounds secreted by gram-negative bacteria under low iron conditions, which scavenge or steal extracellular iron away from the host. Siderophore receptor proteins are found on the outer membrane of the bacterial cell and are involved in the active transport of the iron siderophore complex into the bacterial cell (Neilands 1981, 1995) (see Figure 1).
Siderophore receptor systems are so crucial to bacterial survival within a host that successful pathogens often have receptors for siderophore proteins that are produced by other bacteria (Crosa 1984, 1989, 1999). This ability to utilize "exogenous" siderophores (those produced by microbes of other species or genera) illustrates the highly conserved nature of siderophore receptors. Protein analyses, shown in Figure 2, illustrate the homology of siderophore receptor and porin proteins among various isolates of Salmonella.
There are at least three features of the iron acquisition system of bacterial pathogens that open a window of opportunity for disease prevention by active immunization against siderophore receptors and porins:
- The large molecular weight (30-110 kDa) of common siderophore receptor and porin proteins causes them to be immunogenic; they are recognized as foreign proteins by the host immune system.
- Siderophore receptors and porins are located on the outer membrane surface of bacteria, so they are susceptible to circulating antibodies produced by the host.
- Because siderophore receptors are highly conserved proteins, specific antibodies against them cross react with widely varied bacterial species.
A novel vaccine technology developed by Epitopix (Willmar, MN) exploits the pathogenic bacteria's intrinsic need for iron. SRP technology uses purified siderophore receptor proteins (SRP) from Salmonella as an immunogen. Vaccinated animals recognize the bacterial SRP as foreign and mount an appropriate immune response. The culmination of this immune response is the development of memory cells which upon challenge will induce the production of antibodies against bacterial SRP. The anti-SRP antibodies bind siderophore receptors proteins present on the bacteria's outer membrane, blocking the transport of siderophores-iron complex and encouraging opsonization of the bacterial cell. This results in the animal's immune system recognizing and responding, to the invading bacteria, ultimately leading to destruction and clearance of the bacteria.
Willmar Poultry Company (WPC), the parent company of Epitopix has used SRP vaccine technology in Breeder Turkeys since 1996. SRP technology has been a very effective tool in reducing the incident of Salmonella isolated from breeder flocks as while as salmonella isolation from Hatchery debris. WPC routinely monitors for Salmonella in the breeder birds and in the hatchery debris. From 1986 to 1996, the flocks were consistently > 90% positive. This was despite the following prevention steps: Salmonella autogenous bacterins, shower in/shower out facilities, professional rodent control, wild bird exclusion, semi-annual formaldehyde barn washes, formaldehyde in feed, no animal protein in feeds, extruded feed, separate feed trucks and service trucks for breeder operation, use of direct feed microbial and separate breeder hatching facility. The same serotypes of Salmonella found in the breeder flocks could also be found contaminating the hatchery debris.
In 1996, 94 % of the Willmar Poultry breeder flocks (25.1% of individual samples) were positive for Salmonella. The veterinarians then implemented SRP technology Salmonella vaccines in the breeder flocks in 1996 and it took 18 months for the complete operation to be vaccinated. Vaccination along with continued biosecurity procedures reduced the Salmonella prevalence in the breeder flocks to as low as 31.4 % in 2005 (0.86% individual samples). This dramatic reduction in Salmonella isolation also resulted in a dramatic reduction in the percent positive isolation of Salmonella found in the hatchery debris, from 21 % in 1996 to 9.8% in 2005.
The success of SRP in the turkey industry prompted Epitopix to investigate the use of SRP technology for the control of numerous disease causing agents over a large range of animal species. Recently, Epitopix applied SRP technology to a vaccine directed in controlling Salmonella Enteritidis (SE).
Salmonella Enteritidis is a foodborne pathogen that has been widely implicated and associated with the consumption of contaminated eggs, and poultry products. Epitopix has demonstrated the efficacious nature of a vaccine utilizing SRP technology for controlling Salmonella Enteritidis in chickens. A vaccine formulation utilizing SRP from Salmonella Enteritidis was evaluated against a live SE challenge in SPF chickens.
This trial was divided into two challenge studies: Challenge trial number one evaluated mortality (see Figure 3). Challenge trial number two evaluated the quantitative clearance of SE from internal organs and faecal shedding differences between vaccinated and non-vaccinated birds after challenge (see Figures 4 and Figures 5). The results of these trials will be reviewed and discussed in detail.
References
1. Bullen, J. J., and E. Griffiths. 1999. Iron binding proteins and host defense, p. 327–368. In J. J. Bullen and E. Griffiths (ed.), Iron and infection, 2nd ed. John Wiley and Sons Ltd., New York, N.Y.
2. Crichton, R.R. 1991. Inorganic biochemistry of iron metabolism. Ellis Horwood Limited, Chichester, West Sussex, England.
3. Crosa, J. H. 1999. Molecular genetics of iron transport as a component of bacterial virulence, p. 255–288. In J. J. Bullen and E. Griffiths (ed.), Iron and infection, 2nd ed. John Wiley and Sons Ltd., New York, N.Y.
4. Crosa, J. H. 1984. The relationship of plasmid-mediated iron transport and bacterial virulence. Annu. Rev. Microbiol. 38:69–89.
5. Neilands, J. B. 1981. Microbial iron compounds. Annu. Rev. Biochem. 50:715–731.
6. Neilands, J. B. 1995. Siderophores: structure and function of microbial iron transport compounds. J. Biol. Chem. 270:26723–26726.
7. Ratledge, C., and L. G. Dover. 2000. Iron metabolism in pathogenic bacteria. Annu. Rev. Microbiol. 54:881–941.
From Proceedings of the "Midwest Poultry Federation Convention", St. Paul, Minnesota, U.S.A.
