Role of AKT1 & SIVA1 in resistance to avian salmonellosis

Lead Research Organisation: The Pirbright Institute
Department Name: UNLISTED

Abstract

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.

Technical Summary

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.

Planned Impact

Please see document submitted by lead applicant (joint ref. M1529404)

Publications

10 25 50
 
Description This research is an ongoing collabroation with the Roslin Insititute and has identfied a gene involved in Salmonella disease resistance in chickens. We have identified the host gene in chickens that interacts with the bacterial protien SopB. Patent filed and continuing research with indutrial support.
Exploitation Route Role of AKT1 and SIVA1 in resistance to avian salmonellosis is progressing well. We have identified the host gene in chickens that interacts with the bacterial protien SopB. Patent filed and continuing research with indutrial support.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://www.pirbright.ac.uk/research/GeneticsGenomics/Default.aspx
 
Description This research is an ongoing collabroation with the Roslin Insititute and has identfied a gene involved in Salmonella disease resistance in chickens. "SAL1 Patent for Salmonella resistance in chickens"-. PCT/GB2010/000850. The Pirbright Institute.
Sector Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Contributed to Chief Medical Officers report- Genomics section
Geographic Reach Europe 
Policy Influence Type Implementation circular/rapid advice/letter to e.g. Ministry of Health
URL https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/6310...
 
Description Commercial Partner
Amount £30,000 (GBP)
Organisation HyLine 
Sector Private
Country United States
Start 08/2014 
End 06/2015
 
Title A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR 
Description Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes can be used to control random technical variation between samples. It is necessary to select reference genes with the lowest variation in representative samples. The candidate reference gene assays must be reliable. In particular, they should have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts. The panel was tested with TaqMan and SYBR Green systems in two experimental scenarios, a tissue collection, and virus infection of cultured fibroblasts. GeNorm and NormFinder algorithms were able to select appropriate reference gene sets in each case. We show the effects of using the selected genes on the detection of statistically significant differences in expression. The results are compared with those obtained using 28s ribosomal RNA, the present most widely accepted reference gene in chicken work, identifying circumstances where the use of this gene might provide misleading results. Widely used methods for eliminating DNA contamination of RNA reduced, but did not completely remove, detectable DNA. We therefore attached special importance to testing each qPCR assay for absence of signal using DNA template. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact The assays and analyses developed here provide a useful resource for selecting reference genes for investigations of avian biology. 
 
Title SAL Locus 
Description SAL1 Patent for Salmonella resistance in chickens"-. PCT/GB2010/000850. The Pirbright Institute. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2013 
Provided To Others? Yes  
Impact SAL1 Patent for Salmonella resistance in chickens"-. PCT/GB2010/000850. The Pirbright Institute. 
 
Title Avian 600k SNP Array 
Description Development of a high density 600K SNP genotyping array for chicken. 
Type Of Material Data analysis technique 
Year Produced 2013 
Provided To Others? Yes  
Impact Publication: Kranis A, Almas A Gheyas, Boschiero C, Turner F, Yu L, Smith S, Talbot R, Pirani A, Brew F, Kaiser P, Hocking P, Fife M et al. Development of a high density 600K SNP genotyping array for chicken. (2013) BMC Genomics, 14:59 
 
Description Roslin 
Organisation University of Edinburgh
Department The Roslin Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Joint Grant: 'Role of AKT1 & SIVA1 in resistance to avian salmonellosis' Stevens, Kaiser & Fife
Collaborator Contribution 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.
Impact SAL1 Patent for Salmonella resistance in chickens"-. PCT/GB2010/000850. The Pirbright Institute.
Start Year 2012
 
Title SAL1 Patent for Salmonella resistance in chickens 
Description SAL1 Patent for Salmonella resistance in chickens 
IP Reference PCT/GB2010/000850 
Protection Patent application published
Year Protection Granted
Licensed No
Impact Secured further funding and allowed Link grant with commercial company.
 
Description AB International Nidovirus Symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Batra, A., Maier, H.J., Britton P., Hiscox, J.A., Fife, M.S., 2014. AKT activation during infectious bronchitis virus infection. XIIIth International Nidovirus Symposium, Salamanca, Spain (Poster presentation)
Year(s) Of Engagement Activity 2014
 
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Part Of Official Scheme? No
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Primary Audience Professional Practitioners
Results and Impact This meeting brought together specialists working on the interface between genomics, genetic engineering and infectious disease with the aims of improving animal and human health and welfare.

Scientific sessions included:
Genetics of immune responses and disease resistance
Genetically engineered livestock (including genome editing)
Quantitative genetics and epigenetics applied to disease
Epidemiology and pathogen evolution
Bioinformatics, comparative and functional genomics
Precision medicine of animal companions
Year(s) Of Engagement Activity 2017
URL https://coursesandconferences.wellcomegenomecampus.org/events/item.aspx?e=635&dm_i=2SUU,HOGH,4R4AW1,...
 
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Geographic Reach Local
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Primary Audience Professional Practitioners
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Year(s) Of Engagement Activity 2014,2016
 
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Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I am on the organising committee for this conference. The second conference in this series will highlight recent advances in animal genetics and genomic technologies. It will bring together specialists working on the interface of genomics, genetic engineering and infectious disease with the aims of improving animal and human health and welfare.

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This year's conference will not only put the spotlight on the immune response of host animals and epidemiology but also cover the genetics and genomics of pathogens and the impact of animal-human relationships.

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Year(s) Of Engagement Activity 2019
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Results and Impact AS attended and presented at this event to stimulate increased interest in science and research.
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