Role of AKT1 & SIVA1 in resistance to avian salmonellosis

Poultry are a key reservoir of human Salmonella infections owing to the ability of some strains to colonise the avian intestines and reproductive tract. Birds often carry the bacteria in the absence of overt symptoms; however some types of Salmonella cause severe typhoid-like diseases in poultry that exert substantial welfare and economic costs. Global population growth and rising affluence are fuelling demand for poultry meat and eggs, and a need exists to enhance the supply and safety of such. Though vaccines are used in layers in some countries, most of the 55 billion chickens reared annually worldwide lack protection against Salmonella infection. We and others have discovered that some chickens exhibit heritable differences in resistance to Salmonella. It may be feasible to selectively breed for birds with improved resilience to Salmonella infection; however this requires the identification of resistance-associated factors and knowledge of how they act.

By analysing the genetic material of birds that differ in resistance, we have located a region of the chicken chromosome that confers protection against typhoidal salmonellosis, both in laboratory studies and commercial poultry populations. Recent studies have now resolved the region associated with resistance to just a handful of genes. It is highly plausible that variation affecting two genes in this region (AKT1 and SIVA1) explains why birds react to Salmonella in different ways, as the encoded proteins control host processes that impact on the fate of bacteria. For example, AKT1 and SIVA1 control the death of infected cells and the induction of immune responses, but have opposing activities. It is not possible for us to predict how the genetic changes affecting these genes will alter their expression or activity. Moreover, it is unclear how such factors may control the growth and spread of Salmonella in birds. We therefore propose to:

1. Examine if birds normally respond to Salmonella infection by activating the expression or function of AKT1 and SIVA1. We will examine this in cells cultured from chickens, as well as in intact birds, and associate any differences with host responses and the fate of the bacteria.

2. Examine if lines of chicken known to differ in resistance to Salmonella infection vary in the levels or activation of AKT1 and SIVA1.

3. Use specific inhibitors and bacterial strains to establish that AKT1 activation is necessary for Salmonella to grow and spread in birds.

4. Define the nature, frequency and consequences of genetic changes affecting AKT1 and SIVA1 in commercial poultry populations. This will aid the selective breeding of chickens that show improved resilience to Salmonella infection.

We are fortunate to have the support of one of the world's largest poultry breeding companies (Erich Wesjohann Group), who will provide birds, genome sequences, expertise and 10% of total project costs. This reflects the value of the proposed studies to the industry. The consortium has productively collaborated and the proposed studies are a timely, logical and feasible extension of our recent joint research.

Avian resistance to systemic salmonellosis is largely mediated by the SAL1 locus. Analysis of the segregation of SNPs in the progeny of a sixth generation back-cross of inbred lines that differ in resistance (6 and 15I), as well as F(13) inter-cross lines, has fine-mapped the QTL to a 0.4Mb region encoding AKT1 and SIVA1. In murine and human cells, these proteins have opposing effects on apoptosis and induction of innate immunity. In mammalian cells, Salmonella activates AKT via injection of SopB. This delays apoptosis and promotes net intracellular replication in vitro and in mice, as evidenced by studies with sopB mutants, null mice and specific inhibitors. By contrast, SIVA1 is pro-apoptotic and is proposed to control Salmonella via release of heterophil extracellular traps upon cell death. No evidence exists that these mechanisms are relevant in chickens and a need exists to unravel how resistance-associated SNPs exert their effect.

We propose to define the kinetics of AKT1 and SIVA1 activation upon Salmonella infection using specific antisera, and associate this with the fate of infected cells ex vivo and in chickens by flow cytometry and immuno-staining. We will examine if levels or activities of AKT1 and SIVA1 explain the differential resistance of lines 6 and 15I. Net replication of Salmonella in such birds will be quantified in cells and tissues by fluorescence dilution, and visualised relative to AKT1 activation and apoptosis. We will further define if AKT1 plays a protective role in avian responses to Salmonella by use of sopB mutants, inhibitors and siRNA-mediated knock-down. Toward marker-assisted selection, we will survey the frequency of informative SNPs in a population of Hy-Line layers directly related to that exposed to a recent fowl typhoid outbreak. Birds of defined genotype will be challenged to associate genetic variation with AKT1/SIVA1 levels or activation, host responses and Salmonella replication.

Poultry are vital to global food security, yet producers of meat and eggs face many challenges. Infectious diseases are a recalcitrant threat to avian health and vaccines are often expensive, ineffective or absent. Prophylatic use of antibiotics is prohibited in many countries and therapeutic use is restricted owing to the potential for entry of residues into the food chain. Resistance to antibiotics and anthelmintics, as well as the potential withdrawal of coccidiostats, is expected to lead to resurgence of diseases once considered under control. These issues, coupled with expansion of free-range and intensive production systems, threaten bird welfare and the sustainable supply of safe nutritious food. In this context, selective-breeding of poultry that are resilient to infection offers many advantages. This is considered feasible as the genetics of the >55 billion chickens reared worldwide each year are mostly controlled by just four companies. The research proposed herein has the potential to rapidly inform commercial breeding strategies to produce birds that are more resilient to fowl typhoid, as evidenced by the financial and in-kind support of the Erich Wesjohann Group. In addition to such direct impacts, it will be feasible to assess if layers bred for resistance to fowl typhoid also lay fewer Salmonella-positive eggs, given that oviduct and egg contamination may involve systemic translocation and SAL1 polymorphisms mediate control of S. Enteritidis ex vivo. The resistance-associated loci to be studied also support replication of other intracellular bacterial pathogens and viruses, therefore the proposed research will add value to efforts to control other avian diseases. Intellectual property arising from the project will be identified, protected and exploited as described in Pathways to Impact.

Though we will primarily focus our impact activities on bird health via links with the EW group, the project will also benefit academics studying varied aspects of host-pathogen interactions and instil training in diverse areas. Academics and Policy Makers will be informed of the nature and implications of the research via scientific and lay publications, presentations at symposia, invited lectures and via the comprehensive websites of the investigators. Moreover, the applicants actively engage with varied societies and forums to discuss issues affecting poultry health and strategies for disease control. Materials and data arising from the project will be made available to academia and industry for legitimate uses on request, subject to publication and scrutiny for IP. Exchange of staff and students will promote knowledge transfer.

The project will also raise issues and data of importance to the public. Prof. Stevens and Kaiser have used short films, public speeches and lay articles to convey the purpose and importance of their work. This has included public engagement on ethical aspects of genetic modification and methods to reduce, refine and replace animal use in the development of veterinary medicines. The investigators will also explore ways to educate school children, for example by extending established links with the National Centre for Biotechnology Education.