Curli fimbriae of Salmonella Typhimurium induce an immune response in chickens producing IgY detectable
in serum and yolk.
Nicole Groves
A thesis submitted as partial fulfilment
of the requirements for the degree of
Masters of Philosophy (BABS)
School of Biotechnology and Biomolecular Sciences
Faculty of Science
March 2017 i
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Abstract
Curli fimbriae of Salmonella spp., also known as GVVPQ fimbriae due to a highly conserved amino acid sequence at the N-terminus, play an important role in bacterial virulence in the host gut environment. Curli are essential in Salmonella biofilm formation, environmental resilience, and bacterial attachment and persistence in the host. The genes encoding curli fimbriae are found in the vast majority of Salmonella serovars, and expression can be induced in most of these serovars under stressful environmental conditions. The development of a curli fimbriae-based immunoassay to detect seroconversion in poultry would allow for quick identification of Salmonella colonisation of most serovars, and could be incorporated into current routine detection and monitoring programs in agricultural industries.
Curli fimbriae were isolated from Salmonella Typhimurium phage type 135a and characterised by ELISA and mass spectrometry. Purified fimbriae were incorporated into a vaccine, based on an aluminium hydroxide adjuvant, which was administered to
13-week-old ISA Brown hens. Booster injections were given at 18 weeks and blood and egg samples were collected after this point. Crude IgY was purified from egg yolk samples using ammonium sulphate precipitation.
The efficacy of the immunisation was determined by building a purified fimbriae-based indirect ELISA. Blood samples taken at 23 weeks had a significantly higher level of anti- curli fimbriae antibodies compared to the control birds and the samples taken at 18 weeks. Egg yolks from vaccinated birds contained elevated levels of antibodies to the fimbriae from weeks 21-24, after which the titre slowly declined to week 28. Western
iv blotting using crude IgY purified from yolk showed that birds which had received the vaccine produced IgY specific to a wide array of Salmonella proteins, while the control birds did not.
These results suggest that the vaccine elicited an immune response of birds to curli fimbriae, and, while no challenge studies were performed, the ELISA prototype was able to detect seroconversion. This implies that curli fimbriae are immunogenic, and this ELISA, if developed further, could be used to diagnose hypervirulent Salmonella infection in poultry, and potentially other agricultural animals.
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Table of Contents
1 Introduction 1
1.1 Salmonella and Society 1
1.1.1Bacteriology of Salmonella 1
1.2 Salmonella and Disease 4
1.2.1 Virulence Factors 6
1.2.2 Biofilm Formation 13
1.2.3 Curli Fimbriae 16
1.3 Control and Detection of Salmonella 21
1.3.1 Prevention and Monitoring 21
1.3.2 Vaccination 24
1.4 Antibodies 28
1.4.1 Mammalian and Avian Antibodies 29
1.4.2 Research and Medicine 33
1.5 Aims and Significance 36
2 Materials and Methods 38
2.1 Purification of Curli Fimbriae 38
2.1.1 Expression of Curli 38
2.1.2 Digestion of Whole Cells 39
2.1.3 Differential Centrifugation 40
2.1.4 Lyophilisation and Quantification 40
2.2 Analysis of Extracted Fimbriae 41
2.2.1 Assessment of Purity by SDS-PAGE 41
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2.2.2 Mass Spectrometry 42
2.3 Vaccine Formulation 45
2.3.1 Vaccine Production 45
2.3.2 Vaccine Delivery 45
2.3.3 Sample Collection 46
2.3.4 Standard ELISA Blood Testing 47
2.4 Analysis of IgY 48
2.4.1 Crude Yolk IgY Purification 48
2.4.2 ELISA Development 49
2.4.3 Western Blotting 51
3 Results 56
3.1 Purification of Curli Fimbriae 56
3.1.1 Expression of Curli 56
3.1.2 Digestion of Whole Cells 58
3.1.3 Differential Centrifugation 58
3.1.4 Lyophilisation and Quantification 59
3.2 Analysis of Extracted Fimbriae 59
3.2.1 Assessment of Purity by SDS-PAGE 59
3.2.2 Mass Spectrometry 61
3.3 Vaccine Formulation 62
3.3.1 Vaccine Production and Delivery 62
3.3.2 Sample Collection 63
3.3.3 Standard ELISA Blood Testing 63
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3.4 Analysis of IgY 64
3.4.1 Crude Yolk IgY Purification 64
3.4.2 ELISA Development 65
3.4.3 Western Blotting 72
4 Discussion 80
4.1 Purification of Curli Fimbriae 80
4.1.1 Expression of Curli 80
4.1.2 Extraction of Fimbriae from Whole Cells 83
4.2 Analysis of Extracted Fimbriae 86
4.2.1 Assessment of Purity by SDS-PAGE and MS 86
4.3 Vaccine Formulation 92
4.3.1 Vaccine Production and Delivery 92
4.3.2 Standard ELISA Blood Testing 96
4.4 Analysis of IgY 96
4.4.1 Crude Yolk IgY Purification 96
4.4.2 ELISA Development 101
4.4.3 Western Blotting 106
5 Conclusions 111
References 113
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Acknowledgements
I would like to acknowledge all the people who supported me through this project, who helped me in my stressful moments, and celebrated my successes. In particular I would like to thank my parents, who gave me every kind of support I could need for the past two years.
I also would like to thank my supervisors, Julian Cox and Chris Marquis, for giving me this opportunity, and for all of their invaluable guidance and advice over the last two years. Thank you for helping me develop as a scientist and encouraging me to think deeply and creatively about the questions and problems I encountered.
Finally, thank you to all the staff at Birling Avian Laboratories, for your help over the years and for welcoming me even when time and bench space were limited. To Tony, who gave me the chance to work at Birling, for always pushing me to be better, and for always believing in me. And to Sue and Sarah, without whom I could not have gotten through the challenges I faced in this project, and who always had time to listen and offer advice no matter how busy they were.
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Abbreviations
BCA Bicinchoninic acid BSA Bovine serum albumin CFA Colonisation factor antigen DTT Dithiothreitol ELISA Enzyme-linked immunosorbent assay EPS Exopolysaccharide FITC Fluorescein isothicyanate H-chain Heavy chain HRP Horseradish peroxidase L-chain Light chain LDS Lithium dodecyl sulfate LPS Lipopolysaccharide MES 2-(N-morpholino)ethanesulfonic acid MS Mass spectrometry m/z Mass-to-charge ratio
OD650nm Optical density at 650nm OMP Outer membrane protein PBS Phosphate buffered saline RNA Ribonucleic acid RO Reverse osmosis SDS Sodium dodecyl sulfate SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis SPI Salmonella pathogenicity island spp. Species S/P Sample to Positive ratio TBST Tris-buffered saline with Tween 20 TGX Tris/glycine/SDS buffer Th1-cells Type 1 helper T-cells Th2-cells Type 2 helper T-cells TMB Tetramethylbenzidine T3SS Type 3 secretion system
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1 Introduction
1.1 Salmonella and Society
Salmonella species (spp.) are responsible for 1.3 billion infections and approximately 3 million human deaths annually (Cox et al., 2011; Yue et al., 2012). The vast majority of this morbidity and mortality (between 55-96% depending on local food production standards) is caused through ingestion of contaminated food and water (World Health
Organisation, 2007; Cox et al., 2011). While control and detection methods have been widely implemented through the food chain, their efficacy is questionable. There is a pressing need in the poultry industry for faster, reliable detection methods, and a wider variety of preventative methods to minimise the impact of Salmonella contamination on both human and animal health (Cox et al., 2011). To develop measures to reduce this mortality rate, the mechanisms and consequences of the disease must first be understood.
1.1.1 Bacteriology of Salmonella
Salmonella is a mesophilic, rod-shaped, Gram-negative facultative bacterium from the
Enterobacteriaceae family (Threlfall, 2008; Zhang et al., 2011). A typical Salmonella cell is shown schematically in Figure 1.1. Salmonella spp. are classified by species, subspecies, and serovar, for example Salmonella enterica subspecies enterica serovar
Typhimurium (abbreviated to S. Typhimurium). There are two recognised species of
Salmonella, S. enterica and S. bongori, though subspecies have only been identified for
S. enterica (Hald, 2013). Between the Salmonella spp., over 2,500 serovars have been
1 identified and recognised internationally, with this number rising every year (World
Health Organisation, 2013; Cosby et al., 2015).
Some serovars are host-specific; for example, S. Typhi, S. Paratyphi and S. Sendai are specific to humans, while S. Gallinarum and S. Pullorum are specific to poultry. Other serovars may act as pathogens in a much wider range of hosts, while being best adapted to one in particular. Laying hens, for example, are particularly good reservoirs for S. Typhimurium and S. Infantis (Yue et al., 2012; Desin et al., 2013; Hald, 2013).
Different serovars may also affect the same species differently; for example, S.
Virchow in young children occasionally causes septicaemia, while elderly patients are more likely to experience septicaemia due to S. Enteritidis (Cox et al., 2011).
Figure 1.1 – Typical Salmonella Typhimurium cell. (Source: de Jong et al., 2012a)
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The cause of this specificity, while still not fully understood, may lie partially in the differing survivability of Salmonella serovars in certain environments. This survivability is influenced by the ability of a specific serovar to handle temperature stress and nutrient availability, and their capacity to attach to biotic and abiotic surfaces. It has been shown, for example, that S. Enteritidis is better suited to survival in food processing environments such as stainless steel surfaces than S. Typhimurium or S.
Infantis (Cox et al., 2011). Additionally, some clusters of genes responsible for adhesins and fimbriae, proteins which assist in attachment to different surfaces, are only expressed in certain host species (Yue et al., 2012). This indicates that perhaps the internal environments of different host animals affect the ability of Salmonella serovars to attach to tissues and multiply sufficiently in different ways, leading to host- specificity of some serovars.
Salmonella enterica subspecies enterica and its associated serovars are responsible for
99% of human Salmonella infections worldwide (Cosby et al., 2015; Rivera-Chavez &
Baulmer, 2015). A large number of the studies carried out on Salmonella in the past have been focused on S. Enteritidis and S. Typhimurium, after significant outbreaks occurred in the 1980s in the UK. While these are still the most important serovars in human disease at present, other serovars may become more relevant in future, especially as antibiotic resistant strains are emerging in many regions of the globe
(World Health Organisation, 2007; Fabrega & Vila, 2013; Cosby et al., 2015). More populous regions, with less rigorous disease screening and hygiene standards in food production and preparation, including many developing countries, show much higher levels of Salmonella infection in humans (Lax et al., 1995; Threlfall, 2008).
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Salmonella enterica subspecies enterica serovar Typhimurium phage type 135a
(abbreviated to S. Typhimurium 135a) is of particular concern in Australia. While this phage type is not yet recognised internationally, a large number of outbreaks causing gastroenteritis in humans in the last twenty years have been epidemiologically linked to contaminated chicken meat or eggs. This particular phage type is a slight variation of the more widely recognised S. Typhimurium phage type 135 (McCall et al., 2003;
Hawkey et al., 2013).
1.2 Salmonella and Disease
Salmonellosis is the general term applied to disease caused by Salmonella spp. It occurs when Salmonella cells are ingested and adhere to the epithelial cells of the host gut. Evidence has shown that, depending on the serovar, ingestion of as few as ten
Salmonella cells may cause infectious disease in humans (Lax et al., 1995; Food
Standards Australia New Zealand, 2013). Toxins from the bacterial cytoplasm, which will be discussed in further detail later, are translocated to the host gut cells to cause the internalisation of the pathogen via endocytosis (Lahiri et al., 2014; Costa et al.,
2015). This leads to diarrhoea and dehydration, among other symptoms, in addition to local inflammation. In more severe instances, bacterial cells may invade other tissues and cause systemic infections or chronic conditions such as reactive arthritis and ankylosing spondylitis (D'Aoust, 1991; Cosby et al., 2015).
Salmonellosis, in the form of gastroenteritis, produces symptoms typical of food poisoning, including nausea and diarrhoea (Zhang et al., 2011; Yeung et al., 2013). If the infected individual is particularly at risk - for example, the elderly, very young, or those with compromised immune systems - severe dehydration resulting from these 4 symptoms may be fatal (World Health Organisation, 2013). Antibiotic treatment of uncomplicated gastroenteritis is not recommended as, in most cases, maintaining proper hydration is adequate, and overuse of antibiotics facilitates the evolution of resistant strains of bacteria (Fabrega & Vila, 2013; Yeung et al., 2013). Additionally, it has been suggested that treating gastroenteritis with antibiotics may increase the length of the carrier state, a condition in which the individual is asymptomatic but still contagious due to continued colonisation of the gut epithelium (D'Aoust, 1991;
Threlfall, 2008; Fabrega & Vila, 2013). The major serovars which cause this type of disease in humans are S. Typhimurium and S. Enteritidis (Suzuki, 1994; Lax et al., 1995;
Desin et al., 2013).
In rare cases, particularly invasive serovars usually involved in gastroenteritis (including
S. Virchow and S. Dublin) disseminate from the gut and invade other tissues such as the meninges, joints and bones as systemic infections. If left untreated these infections may develop into serious chronic conditions (Threlfall, 2008; Yue et al., 2012; Fabrega
& Vila, 2013; Yeung et al., 2013).
Finally, certain serovars, particularly S. Typhi, may cause typhoidal salmonellosis, characterised by a prolonged fever (D'Aoust, 1991; World Health Organisation, 2007;
Threlfall, 2008). Between 200,000 and 600,000 people die from typhoidal Salmonella infections annually, primarily young children in underdeveloped regions of Asia (World
Health Organisation, 2007), and treatment with antibiotics such as ciprofloxacin is essential (D’Aoust, 1991; Threlfall, 2008).
The variety of illnesses, and the increase and decrease of importance in human health between serovars points to the evolution of the genus over time. A prime example of 5 this is the rising rate of systemic disease caused by invasive non-typhoidal serovars such as S. Typhimurium in developing countries (Threlfall, 2008). This and other occurrences of increased virulence within the Salmonella spp., can likely be attributed in part to the genetic transfer of virulence factors between isolates (Threlfall, 2008;
Cosby et al., 2015).
1.2.1 Virulence Factors
The ability of a particular pathogen to cause disease is mediated by its virulence factors, such as toxins, hydrolysing enzymes and cell surface proteins. Virulence factors are involved in every stage of infection, from bacterial adhesion to host cells, to cell replication and release from host cells, enhancing resistance to host defence mechanisms and increasing the efficiency of infection. These factors vary between
Salmonella serovars and even strains, depending on the presence of genetic pathogenicity islands and virulence plasmids (Marcus et al., 2000; Yue et al., 2012;
Lopez et al., 2012).
At present, five main Salmonella pathogenicity islands (SPIs) in the bacterial chromosome have been identified and described, consisting of clusters of functionally related genes which contribute to a specific virulence phenotype (Marcus et al., 2000;
Ly & Casanova, 2007; Fabrega & Vila, 2013). The widespread distribution of similar pathogenicity islands in Salmonella indicates that these sections of genetic information may have been obtained and spread through horizontal gene transfer during the evolution of the pathogen (van Asten & van Dijk, 2005). Proteins encoded by the SPIs mostly consist of effector proteins and secretion systems designed to deliver these effectors to host cells during infection (Marcus et al., 2000). 6
Major contributors to the virulence of Salmonella are the Type III Secretion Systems
(T3SS) encoded by SPI-1 and SPI-2. T3SS are comprised of a large number of proteins
(often over twenty) which, when combined, form a syringe-like structure embedded in the cell membrane (Marcus et al., 2000; Puhar & Sansonetti, 2014). Once in contact with host epithelial cell surfaces, the T3SS assembles a machinery that injects bacterial effector proteins directly from the cytosol of the bacterial cells to the cytoplasm of the target (Ly & Casanova, 2007; Costa et al., 2015). These effector proteins trigger the reorganisation of the cytoskeletal structure of the tissue, allowing for internalisation of the pathogen within minutes of first contact with the host (Zhang et al., 2002; Ly &
Casanova, 2007; Lahiri et al., 2014). This mechanism greatly improves the efficiency of the bacterium to act on surrounding host tissue compared to other secretion systems, through which proteins are simply released by the cell into the extracellular environment to act on any host cells displaying the appropriate receptor (Puhar &
Sansonetti, 2014). Bacteria are then internalised in a membrane-bound vacuole which is modified by SPI-2 encoded T3SS to prevent degradation by the host cell. This vacuole moves closer to the host Golgi to obtain better access to nutrients, and is then able to replicate in a protected environment within the host (Ly & Casanova, 2007; Lopez et al., 2012; Lahiri et al., 2014). This sequence of events that leads to invasion of the gut epithelium is summarised in Figure 1.2. T3SS are so important to Salmonella virulence that when these systems are impaired in individual isolates, virulence decreases, and in some cases is completely neutralised (Puhar & Sansonetti, 2014).
However, the function of T3SS is dependent on the adhesion of Salmonella cells to the host epithelial cells (Fabrega & Vila, 2013). This is the function of several other
7 important virulence factors found on the surface of all Salmonella cells, namely, lipopolysaccharide (LPS), and a variety of fimbrial and non-fimbrial adhesis (Thorns &
Woodward, 2000; Malcova et al., 2008; Lopez et al., 2012; Yue et al., 2012;).
LPS is an endotoxin extending from the cell wall, and is comprised of three domains: the hydrophobic Lipid A, a non-repeating, short oligosaccharide core, and a long polysaccharide comprised mostly of mannose, galactose and rhamnose called the O- antigen (Lopez et al., 2012; Fabrega & Vila, 2013). Other, more unique, 3,6- dideoxysugars such as abequose and ascarylose are also found in LPS O-antigen, giving them a particularly distinctive structure. Individual serovars of Salmonella each display a different array of LPS, presenting a unique profile of O-antigens which help determine immunogenicity and serogroup identity (He & Liu, 2002; Murray et al.,
2003).
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Figure 1.2 – Invasion of gut epithelial tissue by Salmonella. (Source: Giannella, 1996).
LPS endotoxins have multiple roles in virulence, including protection of the bacteria from neutrophils, and aiding in gut colonisation (Yeung et al., 2013). LPS is essential for the persistence of the pathogen in serum, as it causes the complement system to be activated at a distance from the cell surface, preventing activated C1 molecules from reaching the cell membrane. This inhibits the classical pathway of the complement system, preventing activation of the complement cascade and protecting Salmonella from lysis caused by membrane attack protein (Murray et al., 2003). Similarly to T3SS encoded by SPI-1, LPS also affects intestinal mast cells, causing effector protein
9 release, which leads to a significant change in gut epithelium permeability
(Aschenbach et al., 2003; Yeung et al., 2013).
While LPS aids in gut colonisation and attachment to host cell surfaces, it is not the main attachment organelle of Salmonella. This role is played by several classes of fimbriae which have been identified on the bacterial cell surface (Thorns & Woodward,
2000; Yue et al., 2012). The fimbriae of Salmonella have been studied in depth only relatively recently, likely due to the poor expression of these structures under standard laboratory conditions. As such, information surrounding their functionality and mechanisms is limited; even the binding specificity of many fimbrial types present on
Salmonella cells is not yet fully elucidated (Chessa et al., 2008; Yue et al., 2012;
Fabrega & Vila, 2013). Fimbrial types were initially classified based on their ability to mediate haemagglutination of erythrocytes, however this classification has been reconsidered due to the discovery of morphologically similar types with no discernible role in haemagglutination (Thorns & Woodward, 2000). The current knowledge of the major types of Salmonella fimbriae is summarised in Table 1.1.
Comprised mostly of repeating subunits termed fimbrin, these filamentous protein structures mediate binding to particular carbohydrates of the host extracellular matrix
(Collinson et al., 1991; Collinson et al., 1992; Collinson et al., 1993; Thorns &
Woodward, 2000). The combined action of all the types of fimbriae present on the bacterial cell surface works to form a strong attachment between the bacterium and the host cell surface, where a biofilm is formed to enhance survival (Malcova et al.,
2008; Smirnova et al., 2010; Fabrega & Vila, 2013).
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Table 1.1 - Fimbriae types found on Salmonella cells.
Fimbriae Type Gene Structure Function Type I – Fim1 Two subunits (20kDa and 22kDa) with hollow Mediate haemagglutination. Participate in lectin-based chaperone- core, producing “channelled” appearance binding to laminin in host epithelial tissue to form biofilms. usher fimbriae under electron microscopy. Rod shaped, 7- Most intensively studied fimbrial type.1,2,5-7 8nm diameter, up to 100nm length. Includes SEF21.2-4 Allelic variant of Morphologically identical to Type I2-4,9 Do not mediate haemagglutination. Mediate attachment to fimH8 laminin in host tissue to form biofilms.2-4 Particularly enhances adhesion to chicken leukocytes.8 ND Thin and flexible. 3-5nm diameter.4, 9 Mediate haemagglutination of erythrocytes treated with tannic acid.4 Mediate attachment to host tissue for biofilm formation. Adhere to type V collagen. Not well distributed amongst Salmonella serovars.10 Sef9 <3nm diameter, 14kDa molecular weight. Only Do not mediate haemagglutination.11 present in S. Enteritidis and closely related serovars.11Filamentous. Peritrichous distribution.9 9,12 Lpf (Long polar 7nm diameter, 10-20μm length. Morphology Necessary for bacterial multiplication in early stages of biofilm fimbriae) similar to Type IV fimbriae. Polar expression.9 formation.6
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Pef9,12 (Plasmid- Similar to long polar fimbriae, but expressed Aid in accumulation of fluid during host invasion.6 Mediate encoded peritrichously.9 adhesion to host epithelial tissue, and target Lex blood group fimbriae antigen.12 Type IV Pil13 3nm diameter, 10-20μm length. Peritrichous Mediate haemagglutination. Involved in invasion of tissue, distribution4 particularly in typhoidal serovars.14,15 Bfp16 Bundle-forming pili. Polar expression. 7nm Do not mediate haemagglutination. Motile organelle used to diameter.9,15,17 propel cells toward host surfaces by twitching motility (retraction and extension of fimbriae at poles of cell).13,17 Facilitate binding between bacterial cells in biofilm formation.13,18,19 Curli agf OR csg9,20 3nm diameter, approximately 17kDa Attachment to fibronectin of extracellular matrix in host (Also known as molecular mass.6,21 Two subunits, AgfA and epithelial tissue.6,21,26 Facilitate autoaggregation to allow GVVPQ, SEF17) AgfB.22-24 Peritrichous distribution. Named clumping together of cells in a biofilm, forming three- after highly conserved sequence at N- dimesional mass.5,25,27 terminus.1,4,22,25 1 – Yue et al., 2012; 2 – Chessa et al., 2008; 3 – Muller et al., 1991; 4 – Thorns & Woodward, 2000; 5 – Ledeboer et al., 2006; 6 – Fabrega & Vila, 2013; 7 – Klemm, 1994; 8 – Guo et al., 2009; 9 – Baulmer & Heffron, 1995; 10 – Klemm et al., 2010; 11 – Edwards et al., 2000; 12 – Althouse et al., 2003; 13 Shi & Sun, 2002; 14 – Dougan & Baker, 2014; 15 – Zhang et al., 2000; 16 – Townsend et al., 2001; 17 – Semmler et al., 2000; 18 – Smirnova et al., 2010; 19 – Watnick & Kolter, 2000; 20 – Rhen, 2007; 21 – Norris, 2000; 22 – Li et al., 2004; 23 – Collinson et al., 1999; 24 – Gibson et al., 2007; 25 – Collinson et al., 1992; 26 – Collinson et al., 1991; 27 – Collinson et al., 1993; ND – no data.
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1.2.2 Biofilm Formation
Biofilm formation is an extremely advantageous element of bacterial virulence.
Individual cells approach the host cell surfaces using motile organelles (such as Type IV fimbriae in S. Typhi, and flagella in all Salmonella serovars) to establish temporary contact (Semmler et al., 2000; Yue et al., 2012; Fabrega & Vila,2013). Initial binding of cells to the host epithelium is mediated mostly by Type I fimbriae (Ledeboer et al.,
2006). These cells then begin to divide, forming microcolonies of bacterial sibling cells, as shown in Figure 1.3. A matrix of bacterial cells forms on the colonised surface as more individual cells approach the host tissue and use their motile organs, including
Type IV-like fimbriae, searching the surface for other bacteria and microcolonies to which they can adhere (Watnick & Kolter, 2000; Smirnova et al., 2010). This adherence to target tissue and aggregation of cells is initially mediated by the attachment fimbriae of the cell – in the case of Salmonella, Type I and curli fimbriae – preventing the infectious bacteria from being swept away by gut clearance mechanisms (Watnick
& Kolter, 2000; Fabrega & Vila, 2013).
The biofilm continues to grow three-dimensionally to cover more gut surface and build up a large cellular mass (Ledeboer et al., 2006). Mucoid exopolysaccharide (EPS) extending from the surface of the cell stabilises the biofilm, forming a well-hydrated, mostly anionic matrix of cells (Costerton et al., 1987; Watnick & Kolter, 2000; Ledeboer
& Jones, 2005; Malcova et al., 2008). The charged environment conferred by the EPS matrix and other surface molecules like LPS results in the binding of many antibacterial agents, including peptide and aminoglycoside antibiotics, at a protective distance from the colonies, providing an enclosed environment in which the majority of cells may
13 thrive. As such, the internal environment of the biofilm is highly protected from external factors such as antimicrobial agents, surfactants and host defences such as antibodies and phagocytes, and acts as a source of bacteria for persistent infection of the host (Costerton et al., 1987; Smirnova et al., 2010).
Nutrients and other molecules are often trapped within the biofilm during formation or are bound to the charged external surface, and are readily absorbed and broken down to be used by the cells. In an environment such as the gut epithelium, environmental nutrients are plentiful, enabling an increased metabolic rate among biofilm cells compared to their planktonic cell equivalents (Costerton et al., 1987;
Fabrega & Vila, 2013). Components of dead cells are easily retained and recycled by younger cells, and genetic information is quickly passed between individuals, supporting the suggestion that horizontal gene transfer has greatly influenced the evolution of virulence factors between Salmonella serovars (Costerton et al., 1987;
Watnick & Kolter, 2000; van Asten & van Dijk, 2005; Cosby et al., 2015). Additionally, biofilms may be formed on abiotic surfaces such as metal, glass or plastic. This is particularly problematic for the poultry industry as, during processing, contaminated poultry products make contact with surfaces which, if not cleaned properly, may become a source of further contamination of other products (Smirnova et al., 2010;
Chia et al., 2011).
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Figure 1.3 – Transmission electron microscopy of biofilm formation. (Source: Watnick & Kolter, 2000).
A study by Ledeboer et al. (2006) revealed that Salmonella mutants that could not produce curli fimbriae were less capable of forming biofilms with a high degree of three-dimensional growth. It was therefore suggested that the autoaggregative nature of curli fimbriae is a major contributor to the establishment of a large biofilm. A more recent study by Li et al. (2016) found that biofilm formation in S. Typhimurium was related to the increased expression of curli fimbrial genes. Additionally, curli fimbriae are unique in their ability to bind both fibronectin and laminin, major components of the host extracellular matrix, and therefore important targets for the adherence of cells to host tissue (Collinson et al., 1992; Gibson et al., 2007; Ledeboer et al., 2006).
Several studies have shown that mutants with no curli fimbriae are unable to bind these matrix components, and form far less autoaggregative colonies in vitro (Collinson
15 et al., 1991; Collinson et al., 1993; Watnick & Kolter, 2000; Gibson et al., 2007; Fabrega
& Vila, 2013). As such, biofilm formation by bacteria expressing curli fimbriae will be far more successful than by bacteria which do not.
1.2.3 Curli Fimbriae
Salmonella curli fimbriae have homology to other curli fimbriae present as part of the extracellular matrix of other species of Enterobacteriaceae, particularly in the highly conserved GVVPQ N-terminus sequence (Collinson et al., 1992; Norris, 2000). Isolates expressing curli grown on solid medium exhibit such a high level of aggregation between cells that even when individual colonies are scraped from the medium and roughly moved around, the colonial shape is maintained. Even resuspension of these colonies is difficult, as cells tend to remain in a clumped mass when added to buffer.
Mutants deficient in curli, however, tend to form more mucoid colonies which are easily resuspended (Collinson et al., 1991; Collinson et al., 1993). It has been suggested that this aggregative nature may contribute to the many instances of untypeable
Salmonella isolates found in tissue samples. The autoaggregation of large numbers of curli may make it very difficult to identify the flagellar and LPS antigens on the cell surface, interfering with standard serotyping techniques (Doran et al., 1993; Lee et al.,
2015).
The fimbrin subunit of curli is a 17kDa protein composed of two domains: major subunit AgfA, an amino acid protease-resistant subunit comprised of 109 amino acid residues, and AgfB, a protease-sensitive region at the N-terminus estimated at around
17-22 amino acids in length which acts as a nucleus for the aggregation of many AgfA subunits. Due to the molecular weight of the protein, these fimbriae are also known as 16
SEF17. The structure of this subunit is predicted as a series of parallel β-helixes linked by 4-6 amino acid residues, forming a five-coil right handed helix compacted into an oval-shaped molecule. A highly hydrophobic rectangular core is formed between β- sheet layers, while hydrogen bonds between individual β-sheets stabilise the molecule overall (Tian et al., 2015; Collinson et al., 1999). This predicted conformation allows for interaction of the fimbriae with the solvent, between the subunits, in part due to hydrophobic residues, and between fimbrial fibres (Collinson et al., 1999). It has been suggested that the smaller domain, AgfB, is localised on the surface of the bacteria, and acts as a nucleus for the polymerisation of AgfA to form the full fimbriae (Gibson et al., 2007; Li et al., 2016). In a study by Doran et al. (1993), a DNA probe for the structural gene of the fimbrial subunit, agfA, was used to detect the presence of the
SEF17 fimbriae-forming gene of 604 Salmonella isolates. Of these isolates, 603 hybridised strongly with the DNA probe, indicating that almost all Salmonella serovars are capable of producing SEF17, even if it is not always expressed. A further 266 other
Enterobacteriaceae isolates were tested by the same method, the vast majority of which did not hybridise at all with the probe, and those isolates that did hybridise only did so very weakly. As such, the evidence implies that the SEF17 subunit is unique to the genus Salmonella. A more recent study by White & Surette (2006) revealed that, when incubated below 30°C with low osmolarity and nutrient availability, it was possible to induce the production of SEF17 in 90% of isolates studied representing all groups of Salmonella spp. The almost universal presence of SEF17 therefore makes it a good potential target for genus-specific diagnostics and, potentially, vaccination for prevention of colonisation and pathogenesis of the bacteria (Doran et al., 1993).
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Curli expression can be detected by incubation with Congo Red dye, a molecule routinely used in diagnostics since the 1920s. Congo Red binds to the hydrophobic domains of amyloid fibrils, aggregative masses of β-sheet-rich protein which have been incorrectly folded in the body. It is thought that Congo Red binds to the loops between
β-sheets and, as such, SEF17 will also bind the dye due to its hydrophobicity and the predominantly β structure of the subunits (White et al., 2003; Schütz et al., 2011).
Colonies expressing curli on solid medium supplemented with Congo Red will be stained dark red. Additionally, the binding of Congo Red to curli does not affect the functionality of the fimbriae, as the 3-dimensional protein structure is highly conserved, meaning that fimbriae purified from screened colonies are still biologically active and useful for diagnostic purposes (Collinson et al., 1993; Schütz et al., 2011).
When grown on medium containing Congo Red dye, colonies usually present one of two main colonial morphologies: red, dry and rough (rdar), and smooth and white
(saw), as shown in Figure 1.4. Two other minor morphologies have also been identified, but are less easily distinguished: brown, dry and rough (bdar), and pink, dry and rough (pdar) (Malcova et al., 2008). It has been observed that the presence or absence of SEF17 is an important determinant of the colonial morphology of these bacteria (Ledeboer et al., 2006; White & Surette, 2006; Li et al., 2016). Both rdar and bdar morphologies occur in the presence of SEF17. The rdar morphology occurs when both SEF17 and cellulose comprise the extracellular matrix, while bdar colonies lack cellulose. Pdar colonies, however, produce cellulose but not SEF17. Finally, saw colonies produce neither cellulose nor SEF17 (Römling et al., 2000; Zogaj et al., 2003;
Malcova et al., 2008). The rdar morphology generally results in the greatest expression
18 of curli, but is usually not expressed until the colony reaches the stationary phase of growth (White & Surette, 2006; Ledeboer et al., 2006; Malcova et al., 2008).
Interestingly, the rdar morphotype also correlates to biofilm formation in the
Enterobacteriaceae family more broadly, further emphasising their important role in virulence (Malcova et al., 2008). Colonies grown in vitro present an rdar morphology best at temperatures lower than typical internal environments – around 20°C – and when nutrients are low in supply (Römling et al., 2000; Ledeboer et al., 2006; Eguale et al., 2014).
Figure 1.4 – Morphology of Salmonella cells grown on congo red agar with and without expression of curli. A – rdar morphology of colonies expressing curli; B – saw morphology of colonies not expressing curli. (Source: Malcova, 2008)
Unfortunately, the extraordinarily autoaggregative and insoluble properties of curli make separating and purifying these peptides much more challenging than other
19 fimbriae (Collinson et al., 1991; White et al., 2003). The usual method for extracting fimbriae from whole cells involves blending to shear fimbriae from the cells, followed by centrifugation to separate cells from the fimbrial proteins (Curtis et al., 2016). The aggregation of SEF17, however, prevents the fimbriae from being separated from the whole cell during the blending step. The curli will then be discarded with the rest of the whole cell after centrifugation, preventing their detection by conventional methods (Collinson et al., 1991; Collinson et al., 1992; Collinson et al., 1993). A more complicated method has previously been employed by Collinson et al. (1991) to detach and purify SEF17 – digestion with the enzymes RNase, DNase and lysozyme to lyse and degrade the whole cell, followed by SDS-PAGE electrophoresis. Insoluble fimbriae do not enter the stacking gel during electrophoresis, separating them from the rest of the cell contents. Additionally, Salmonella cultures grown in vitro show a different pattern of SEF17 expression, whereby curli are rarely expressed and are heavily influenced by growth conditions such as temperature and culture medium (Doran et al., 1993;
Ledeboer et al., 2006). This has contributed to the difficulty of research on SEF17 and therefore the lack of specific information around their structure and function. Further research into their function, structure and interactions with the host could allow curli to be incorporated into the techniques currently used for the prevention and control of Salmonella disease in humans and animals. To fully understand where curli may fit into the overall picture of Salmonella control, however, we must first explore the current practices in place to prevent and monitor infection, and their impact on animal and human health.
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1.3 Control and Detection of Salmonella
Risk reduction during the production of food products typically contaminated with
Salmonella is of great importance in both preventing infection, and detecting and controlling potential outbreaks. While Salmonella control varies globally due to differing national guidelines, there is a particular emphasis on the monitoring of bacterial populations in flocks, the prevention of infection and contamination during growing and processing, and strict vaccination regimes to minimise colonisation
(Giessen et al., 2006; Desin et al., 2013).
1.3.1 Prevention and Monitoring
Particular emphasis is placed on the prevention of human infection via a top-down strategy of quarantine and hygienic practices during primary production, the production of meat flocks (Doran et al., 1993; Hald, 2013). The passage of Salmonella spp. between flocks is most often facilitated by contaminated surfaces and equipment, feed and water and other infected organisms such as birds, rodents and insects (Zhang et al., 2011). As such, poultry farmers have adopted a series of risk minimisation practises to reduce the potential of infection (Cox et al., 2011). Risk is greatly reduced by cleaning habitats and equipment between housing different flocks, and rigorous cleaning of surfaces during processing (Chia et al., 2011). Provision of clean water, and feed treated by heat or with organic acids, decreases the likelihood of infection via the faecal-oral route, particularly if the feed is pelleted rather than given as a loose mix or mash (Zhang et al., 2011; Koyuncu et al., 2013). Pests may be controlled by fumigating sheds periodically, and replacing litter between flocks to eliminate any insects and
21 bacteria (Payne et al., 2007; Chen & Jiang, 2014). Similarly, any dead birds are ideally removed from the rest of the flock as quickly as possible (Zhang et al., 2011).
Finally, risk minimisation continues during processing of carcasses and eggs before sale. Decontamination treatments may be employed during the production of meat products, including treatment with chemical biocides such as lactic acid or chlorine, or hot steam (Hald, 2013). Refrigeration of meat and eggs is maintained at all stages of processing to reduce the rate of bacterial replication (Howard et al., 2012).
However, the efficacy of these methods is debatable, as studies between the 1960s and early 2000s show similar levels of Salmonella in meat products despite improvements in hygiene and processing over time (Cox et al., 2011). Additionally, most agricultural animals do not present with disease, rather serving as asymptomatic carriers of the bacteria (Hald, 2013). Therefore, routine monitoring of flocks by serological and bacteriological methods is recommended, if not required by law, in many countries (Feld et al., 2001).
Microbiological culture is widely used to identify infected birds and sheds, often forming the basis of Salmonella testing in diagnostic laboratories. Unfortunately, culturing requires multiple enrichment steps, and the use of multiple selective media, which becomes time-consuming and does not provide reliable results for a number of days (Lee et al., 2015). More rapid testing methods are available, for example, polymerase chain reaction (PCR), which can be routinely carried out on dust, faeces, swabs, and meat and egg products to ensure the removal of Salmonella contaminated products from the market (Threlfall, 2008; Cox et al., 2011; World Organisation for
Animal Health, 2011). While PCR has been shown to provide 100% of the sensitivity 22 and specificity of traditional culture methods, occasionally the matrix of the sample can lower the sensitivity of the test and require additional enrichment of the sample
(Malorny et al., 2007; Lee et al., 2015). Enzyme-linked immunosorbent assays (ELISAs), frequently carried out on blood samples, have also been shown to be equivalent to traditional culture methods (Lee et al., 2015).
There is still, however, a distinct need within the poultry industry for improved monitoring and prevention of Salmonella infection. A rapid, inexpensive test method which will reliably detect a broad range of Salmonella serovars, in particular, isolates with a particularly high level of virulence, would be invaluable to the control of disease worldwide.
Control of Salmonella infections is of increasing importance globally as rising resistance of the bacteria to standard antibiotics, including ampicillin and chloramphenicol, becomes evident (D'Aoust, 1991; Stanley et al., 2014). This issue has been exacerbated by the routine use of antibiotics in animal feed to control bacterial outbreaks. In the
1950s, the US government legalised the use of over 30 antimicrobial agents in feed by broiler farmers, even with no consultation with a veterinarian (Cosby et al., 2015). This global overuse of antibiotics has led to the evolution of a multitude of antibiotic resistance genes present in Salmonella isolates. In Ethiopia, for example, resistance to antibiotics widely utilised in agriculture, including tetracycline, sulphonamides and streptomycin, is rising (Eugale et al., 2014). This resistance pattern has been observed globally, and also includes fluoroquinolones, β-Lactams and phenicols. Resistance varies by country and continent depending on the primary antibiotics used in agriculture (de Jong et al., 2012b; Cosby et al., 2015). Even more concerning is the
23 evolution of multidrug resistant strains of Salmonella, which may only be treatable using newer antibiotics, which in turn allows for the development of resistance to these more effective drugs (Giamarellou, 2006; Lopez et al., 2012). While control measures are currently in place to limit the overuse of antibiotics – for example, a complete ban on antibiotics used as growth promoters in the EU – the spread of resistance via horizontal gene transfer is still a great threat to animal and human health (Stoll et al., 2012; Stanley et al., 2014). In light of this, other methods to control outbreaks, and prevent infection through the consumption of contaminated food must be implemented and further developed. Currently, the most promising prevention method is vaccination against Salmonella, using either subunits or whole cells (Bearson et al., 2016).
1.3.2 Vaccination
Several types of Salmonella vaccines are currently used for poultry, some more effective than others. Vaccines to protect against both S. Enteritidis and S.
Typhimurium, among several other important serovars, are currently in use to reduce the level of infection in agricultural animals (Desin et al., 2013). An ideal vaccine is developed to provide immunity to the vaccinated individual by provoking the production of antibodies specific to the infectious agent, which will be retained as part of the host’s immune defences (Zinkernagel, 2003). Three main types of vaccines are currently in use – live-attenuated, killed, and subunit vaccines (Desin et al., 2013).
Live-attenuated bacteria are widely used for vaccination in both broiler and layer flocks. These bacteria are generally treated with heat or chemicals to limit their survivability and virulence in the host, allowing for the development of immunity
24 without the risk of prolonged infection (Woodrow et al., 2012). Strains with genetic alterations to produce metabolic defects, which limit the bacteria to very few cycles of growth in vivo, have also produced good levels of immunity (Lopez et al., 2012; Desin et al., 2013). For example, removal of genes to reduce production and use of ATP produced strains of Salmonella which provide protection from wild type bacteria, but present little risk when injected live (Northern et al., 2009). S. Enteritidis cells with genetic defects are often administered orally to day-old chicks, reducing both rate of infection and contamination of eggs produced (Howard et al., 2012). One vaccine developed using live-attenuated S. Gallinarum, which causes fowl typhoid with a mortality rate up to 100%, also provides immunity to S. Enteritidis due to high antigenic similarity between the serovars (Desin et al., 2013).
A very recent study by Bearson et al. (2016) showed the success of a vaccine for pigs, developed as a live-attenuated strain of S. Typhimurium, with deletions in genes which regulate the expression of several outer membrane proteins. The resultant increased expression of these non-serovar-specific proteins allowed for much more cross- protection between serovars from one vaccine than standard live-attenuated
Salmonella vaccines in current use. These results suggest that highly conserved, ubiquitous external proteins may be ideal targets for future food animal vaccine development.
Killed bacteria may also be used as vaccines, and while these are safer than live pathogens, they are generally less effective. To provoke equivalent levels of immunity to a live-attenuated vaccine of the same serovar, a killed vaccine must be administered
25 in multiple doses, as the bacteria cannot multiply in the body as do live bacteria
(D’Aoust, 1991; Desin et al., 2013).
These principles apply to Salmonella vaccines. Killed S. Typhi vaccines require multiple doses to produce the same level of immunity as a live-attenuated S. Typhi vaccine
(Joneja & Bielory, 1990; D'Aoust, 1991). Furthermore, relevant antigens may not be produced in the same quantities, or at all, when bacteria are grown in vitro for killed vaccines, while live-attenuated bacteria are able to reproduce and express their full range of antigens in vivo (Woodrow et al., 2012). Certain antigens may not be present in killed vaccines at all due to the growth conditions of the original culture, for example, in the context of curli, which are only expressed at specific points during infection (Mika et al., 2012). Nevertheless, killed vaccines against S. Enteritidis and S.
Typhimurium have significantly reduced shedding of bacteria in layer birds in the US and Europe (Desin et al., 2013).
Recently, research into Salmonella subunit vaccines has been focused on common surface molecules such as fimbriae, flagella, and LPS (Ledeboer et al., 2006). Subunit vaccines, similar to killed vaccines, are far safer than live-attenuated bacteria, as isolated peptides, sugars, or nucleic acids are unable to cause disease (Desin et al.,
2013). Outer membrane proteins of S. Enteritidis used as subunit vaccines have been shown to significantly reduce the population of the bacterium in the caeca and lower intestine of chickens (Khan et al., 2003). However, expression levels of certain proteins in vitro may not always match their expression in vivo due to the differing environmental conditions such as temperature, pH and nutrient availability. Phase of growth may also influence protein expression, with some best expressed in the
26 exponential phase, and others in the more stressful stationary phase. The latter is the case for many virulence factors, including curli, as they are fundamental to the survival of the bacterial population in adverse environments such as the host gut, or on abiotic surfaces (Ledeboer et al., 2006).
Antibodies specific for antigens on the surface of the pathogen, including attachment proteins such as fimbriae, may inhibit binding to host surfaces and therefore reduce pathogenicity and infectious potential (Forthal, 2014). Zhang & Zhang (2010) found that subunit vaccines containing bacterial adhesion proteins could be used to prevent attachment of enterotoxigenic E. coli to the human small intestine. Additionally,
Krachler & Orth (2013) suggested that Salmonella Type 1 fimbrial proteins could also be used as vaccination targets to prevent attachment of the bacteria to host tissues. As such, a vaccine developed against curli to prevent attachment and growth of biofilms has the potential to influence the future control and treatment of Salmonella infection.
There is a clear trade-off between quality of immune response produced by a vaccine and the safety of the host. Live-attenuated pathogens will produce the best immune response, but are the least safe for the recipient (Pasquale et al., 2015). However, the immune response to safer subunit- or purified antigen-based vaccines can be improved with the addition of an adjuvant, a substance which greatly augments the level of antibody production and overall immune memory to the antigen compared to injecting the antigen alone (Jolles & Paraf, 1973; McKee et al., 2007).
Typically, adjuvants will stimulate a greater humoral response by interacting with antigen-presenting cells such as dendritic cells and macrophages, for example, by affecting their maturation, migration, or levels of antigen presentation (McKee et al., 27
2007; Li et al., 2014). When the efficiency of these antigen-presenting cells is increased, the response of the lymphocytes is in turn improved, resulting in greater production of antibodies by the B-cells. B-cells may also be more effectively converted into memory cells to provide long-term immunity (McKee et al., 2007).
Adjuvants may affect the stimulation of Type 1 helper T-cells (Th1-cells) by macrophages, to help mediate future response to infection. However, these adjuvants often also cause the development of delayed hypersensitivity, an unusually intense immune response to a particular antigen, which may cause tissue damage. An unnecessary inflammatory reaction caused by Th1-cells, the helper cells in control of cell-mediated immunity, is typical of adjuvants such as Freund’s complete adjuvant
(Joneja & Bielory, 1990; Karande & Mitragotri, 2010). Other adjuvants, such as alum salts, are designed to stimulate Type 2 helper T-cells (Th2-cells), which are primarily responsible for the production of antibodies, inducing immunological memory, and are not associated with hypersensitivity (Petrovsky & Aguilar, 2004).
1.4 Antibodies
The most basic immunoglobulin molecule is comprised of two separate chain subunits, a heavy and light chain (H-chain and L-chain, respectively), so named because of their comparative molecular weights. Two subunits of each chain type are joined together by disulphide linkages in a rough Y-shape, with the lower halves of the heavy chains forming the ‘stem’, or Fc region of the antibody, and the upper halves of the heavy chains combined with the light chains forming the ‘branches’, or Fab regions (Sela-
Culang et al., 2013). These subunits are conjugated so that each region has a high degree of flexibility and can move relatively independently of the other subunits, due 28 to a ‘hinge’ region located between the two heavy chains in the Fc region (Hansel et al., 2010). Each branch of the Fab region contains a variable binding site unique to the particular antigen to which the antibody is sensitive. As such, antibodies may bind to and neutralise one or more antigens at a time, while being able to twist and stretch to accommodate larger molecules via the hinge region (Nezlin, 1970; Chalghoumi et al.,
2009, Dias da Silva & Tambourgi 2010). Antibodies are classed depending on their particular combination of subunits, ranging from just one H-chain and L-chain, to a large network of linked chains (Franek, 1975; Schroeder & Cavacini, 2010).
1.4.1 Mammalian and Avian Antibodies
Three main classes of immunoglobulins exist in mammalian serum: IgG, the predominant fraction of serum antibodies and the only immunoglobulin to be transferred between mother and developing foetus in humans; IgM, a larger, complex- forming immunoglobulin with a much higher degree of asymmetry; and IgA, a class of variable size often found in secretions such as sputum and colostrum (Nezlin, 1970;
Schroeder & Cavacini, 2010). The structure and function of these immunoglobulins, and their avian counterparts, are described in Table 1.2. When a new antigen is encountered in the body, IgM is the first antibody released by the B cells into the blood to trigger the complement cascade. IgM is able to bind to multiple antigens or epitopes at any one time, allowing for greater efficiency of each individual immunoglobulin molecule, and crosslinking of antibody-antigen complexes to form larger particles
(Joneja & Bielory 1990; Schroeder & Cavacini, 2010). This is followed by a much greater production of IgG. The IgG then attaches to either invading cell walls, triggering the complement cascade and causing the lysis of the cell, or the toxins released by the
29 pathogen, inactivating them. Synthesis of antibodies by the B cells will continue until the antigen is neutralised or eradicated from the body, after which the immune response will be suppressed and only B memory cells will remain in circulation. Upon encountering the same antigen in the future, these B memory cells will rapidly divide and produce large amounts of IgG and IgM in a much shorter period of time
(Zinkernagel, 2003). Consequently, the host will have a level of immunity towards the pathogen and subsequent infections will be overcome in a much shorter time (Joneja
& Bielory, 1990).
Birds and other egg-laying animals, however, produce a different main antibody: IgY.
At first, IgY was referred to as IgG, with similar function and serum concentration in birds to that of true IgG in mammals, but further research has demonstrated important differences in the structure and function of these molecules (Chalghoumi et al., 2009; Spillner et al., 2012). Mammals are able to transfer antibodies and other immune system proteins to their young, after birth, via secretions in colostrum and milk, conferring passive immunity to many common pathogens that the infant might encounter before the full development of its own immune system (Schroeder &
Cavacini, 2010). Birds, such as chickens, however, must transfer these proteins to the embryo before the egg is laid. IgA and IgM are included in the white of the egg as it forms, while IgY moves into the already-formed egg yolk through specific transport receptors on the yolk membrane (Polanowski et al., 2012).
The main difference between IgY and IgG lies in the heavy chain of the antibodies, as displayed in Figure 1.6. While IgG has one variable region and three constant regions, chicken IgY has one variable region and four constant regions. The extra region of the
30
IgY heavy chain is present in the Fc region (Dias da Silva & Tambourgi, 2010; Spillner et al., 2012). Additionally, IgY does not possess a typical hinge region due to the extra Fc domain, resulting in low flexibility compared to IgG. It has been suggested that IgY may be an evolutionary precursor to mammalian IgG and IgE, and this Fc region may be a precursor to the hinge (Munhoz et al., 2014). Some flexibility of IgY arises from several proline and glycine residues contained in the first, second and third domains of the Fc region (Chalghoumi et al., 2009). Additionally, the β-sheet content of IgY is significantly lower than that of IgG. IgY does not cross-react with mammalian immunoglobulins, bind mammalian proteins A or G, or cause the activation of the mammalian complement system, further demonstrating the marked differences between the two molecules (Spillner et al., 2012; Munhoz et al., 2014; Nafea et al., 2015). It is interesting to note, however, that such distinctive differences are not present in IgA or
IgM of mammals and birds: the size, structure and function of these two antibody classes are very similar (Polanowski, et al., 2012).
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Table 1.2 - Summary of different antibodies present in mammalian and avian systems, comparing structure and function.
Molecule Species Structure Function IgG Mammalian Rough Y-shape. Two heavy chains (4 domains each) Second response antibody released by B cells when novel antigen is and two light chains (2 domains each)1,2 encountered. Forms the majority of the immune response when antigen is encountered in future.1 Attaches to viruses and toxins to neutralise them, or to cells to signal cell death. Triggers complement cascade.1-3 Active in blood and lymphatic system.4 IgM Mammalian & Avian Form complexes of five, rarely six Y-shaped First response antibody released by B cells when novel antigen monomers. Complexes contain polypeptide J-chain encountered.1 May react with multiple antigens due to complex structure, bound to two monomers to assist in secretion.1 allowing efficient recognition of multiple epitopes of novel antigen.1,3 Triggers complement cascade.S Present in egg white in birds. Active in blood and lymphatic system.4 Can cause aggregation of pathogens to facilitate removal by peristalsis.1,3 IgA Mammalian & Avian Y-shaped monomer in serum, dimer when secreted Present in serum, however main function is to confer immunity to young by mucosa. Dimer includes polypeptide J-chain through colostrum and breast milk. Also present at other mucosal surfaces, similar to IgM to assist in secretion, and another for example in saliva.1,4 Present in egg white in birds. While not as effective polypeptide termed the secretory component.1 as IgM, can also cause aggregation of pathogens to aid in clearance from the body.1,3 IgY Avian Rough Y-shape, similar to IgG. Two heavy chains (5 Very similar role to IgG in mammals. Produced after IgM when novel domains each) and two light chains (2 domains antigen first encountered, then in much greater amounts when antigen is each)5 encountered in future. Transferred to egg yolk during yolk formation to confer immunity to offspring.5,6 1 - Schroeder & Cavacini, 2010; 2 –Hansel et al., 2010; 3 - Forthal, 2014; 4 – Zinkernagel, 2003; 5 – Dias da Silva & Tambourgi, 2010; 6 – Polanowski et al., 2012.
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Figure 1.6 - Structure of mammalian IgG compared to avian IgY. Black denotes the immunoglobulin heavy chain, grey denotes the light chain. Domains denoted as “V” are variable regions which change depending on the antigen recognised by the antibody, domains denoted as “C” are constant regions. (A): mammalian IgG; (B): avian IgY. (Dias da Silva & Tambourgi, 2010).
1.4.2 Antibodies in Research and Medicine
Antibodies have a broad range of uses in both research and treatment of disease. At present, IgG is largely the antibody type of choice for laboratory and clinical use, generally produced in mammals such as mice, rats and rabbits, or horses and sheep for larger scale production (Schroeder & Cavacini, 2010), although specific IgY is also available. When conjugated to a fluorescent, chemiluminiscent or colourimetric label, antibodies can be used for specific detection of antigens in western blots, ELISAs, and
33 even in vivo for imaging of specific tissues (Nafea et al., 2015). Targeted drug delivery, to cancerous tissue, for example, can be accomplished using antibody-conjugated toxic compounds such as Ricin toxin A chain or Pseudomonas endotoxin (Bearson et al.,
2016). This increases the ratio of uptake of toxic drug in the targeted tissue to normal tissues, reducing the dose required for the drug to be effective, and decreasing the side effects caused by interaction with healthy tissue (Mao et al., 2004; Winnall et al.,
2014). Similarly, antibodies without conjugated drugs have been used to stimulate cell- mediated immune responses to pathogens including HIV (Narasimhan et al., 2016).
IgY is an efficient and ethical solution to the rising need for antibodies in modern science. One obvious advantage arises from the constant production of IgY by female chickens as it is synthesized, carried through the blood, and transferred to the egg yolk
(Dias da Silva & Tambourgi, 2010). Harvesting the IgY from an unfertilised egg yolk rather than the blood, as is the case for mammalian antibodies, results in no loss of animal life, and no additional pain or distress for the chicken itself (Kovacs-Nolan &
Mine, 2012). An average hen will lay approximately one egg a day once it reaches maturity, for around ten months, each with 50-100mg of IgY included in the yolk.
Overall this yields at least 1.5-2g of IgY per month per hen (Vega et al., 2012; Munhoz et al., 2014; Nafea et al., 2015). Approximately 2mg of IgY from egg yolk correlates to
300mL of mammalian blood serum or 600mL of whole blood, obtainable only from larger mammals, which are more difficult and expensive to farm, given the increased need for housing and feed (Dias da Silva & Tambourgi, 2010).
Additionally, chicken egg yolks contain IgY, and no other isotype of antibody, greatly simplifying the process of extraction and purification. Due to the significant structural
34 differences between avian IgY and other mammalian antibodies, IgY does not bind other proteins such as protein A or protein G, and does not activate the mammalian complement system (Vega et al., 2012). Further, due to the evolutionary distance between birds and mammals, and the structural differences between IgY and IgG, significantly less cross-reactivity occurs with mammalian proteins similar, but not identical to the target antigen when using IgY (Dias da Silva & Tambourgi, 2010). As such, IgY has a much higher immunogenicity towards mammalian proteins, making them far more efficient than antibodies produced against the same proteins in mammals such as rabbits or mice. This leads to more sensitive diagnosis and identification of proteins, and a much lower number of false-positive results (Tan et al.,
2012).
Antibodies are also used in passive vaccination, where specific immunoglobulins are collected from immunised individuals, and then delivered to an infected individual in order to confer temporary immunity to a toxin or pathogen. At present, passive vaccination is widely used in the treatment of snakebite, and diseases such as measles, rabies and cow mastitis (Harrison, 2004; Aizenshtein et al., 2013). The use of IgY in passive vaccination has also shown positive results in conferring some degree of immunity to pathogens such as Campylobacter jejuni in broiler chickens, E. coli in pigs, calves and rabbits, and rotavirus in mice and calves. Similar success has been observed in the treatment of Salmonella infections in chickens, calves and mice. Due to the highly stable nature of IgY over a wide pH range, oral administration is highly effective in the treatment of gastrointestinal infections like Salmonella, E. coli and
35
Campylobacter, as it allows the antibodies to reach the site of infection efficiently
(Chalghoumi et al., 2009; Rahman et al., 2013).
This form of treatment shows potential as a measure to minimise the evolution and spread of antibiotic-resistant pathogens in agricultural animals, alongside the neutralisation of pathogens which are already resistant to antibiotic therapy
(Chalghoumi et al., 2009; Polanowski et al., 2012). Further, the use of antibiotics in agricultural animals often results in economic losses within industry due to antibiotic residues remaining in products such as meat and milk (Zhen et al., 2009). Non- antibiotic-based treatments for infections are therefore worth further research and development. As such, an IgY-based approach to the control of Salmonella and other gastrointestinal pathogens in the poultry industry is an important area of future investigation.
1.5 Aims and Significance
This project aims to both elicit and detect an immune response in chickens to the subunit of curli purified from Salmonella Typhimurium PT135a, using a curli fimbriae- based ELISA. Fimbriae will be purified using a digestion method described previously by Collinson et al. (1991), which is expected to extract fimbriae with a high enough purity to be used for vaccine development. The vaccine will be developed using aluminium hydroxide (Al(OH)3) as an adjuvant, as it was hypothesised that this adjuvant will promote a detectable immune response while causing minimal distress to the animal. The IgY from both serum and egg yolk from vaccinated hens will be collected and purified via ammonium sulphate precipitation. The purified fimbriae will be used to develop a sandwich ELISA, which should allow for the detection of 36 seroconversion in birds. It is hypothesised that there will be a difference in the response of immunised bird sera and yolk samples to the developed ELISA compared to the same samples from non-immunised birds.
This ELISA will provide a basis for screening for evidence of colonisation or infection with particularly virulent salmonellae. Salmonella cells expressing curli are more likely to cause persistent, more severe disease than those without (Collinson et al., 1991;
Collinson et al., 1993; Watnick & Kolter, 2000; Gibson et al., 2007; Fabrega & Vila,
2013; Li et al., 2016). While determining the specific serovar of Salmonella would still require culture, the quicker identification of hypervirulent Salmonella outbreaks would allow for fast quarantine and treatment of the disease. Additionally, the curli subunit may be a useful addition to current subunit vaccines, if it confers good immunity to
Salmonella spp. or has the potential to inhibit adherence of Salmonella cells to the intestinal surface. Alternatively, if curli-specific IgY could be isolated from blood or yolk, it may have potential as a therapeutic agent to prevent or decrease colonisation in gut tissue.
37
2 Materials and Methods
2.1 Purification of Curli fimbriae
2.1.1 Expression of Curli by Salmonella Typhimurium 135a
A poultry field isolate of Salmonella enterica, subsp. enterica Typhimurium phage type
135a was obtained from Birling Avian Laboratories culture collection. This culture was originally stored on Cryobeads (Cryobank, MAST Group, UK) and was incubated on sheep blood agar (BioMerieux, Australia) under microaerophilic conditions (5-10% CO2) at 37°C for two days. Cells were scraped from the agar and suspended in 0.1% peptone water (Thermo Fischer Scientific, USA) at a concentration of approximately 108cfu/mL.
The culture was stored at 4°C.
Colonisation Factor Antigen (CFA) agar was prepared as described by Evans et al.
(1977) as 1% casamino acids (Bacto, Australia), 0.15% yeast extract (Oxoid, Australia),
0.005% MgSO4 (Fischer Scientific, USA), 0.0005% MnCl2 (Fischer Scientific, USA), and
2% agar (Difco, USA). Media were dissolved in reverse osmosis (RO) water and melted at 100°C for 20 minutes. Media were then mixed by hand briefly and sterilised at 121°C for 20 minutes. Cooled molten medium was poured into sterile Petri dishes and allowed to set at room temperature. Set plates were stored at 4°C until used.
The Salmonella Typhimurium suspension was streaked on CFA agar plates and incubated aerobically at 37°C for 24h, then moved to room temperature for a further 6 days. Identical plates were grown at room temperature for 7 days. Colonies were observed after this time for the rough, dry morphology indicative of the presence of fimbriae. 38
Nutrient T-medium with Congo Red was made up as described by Collinson et al (1991) as 1.5% nutrient agar (Oxoid, Australia) containing 1% tryptone (Oxoid, Australia) and
100mM Congo Red (Chem Supply, Australia). Media were dissolved in RO water and melted and sterilised as above. Plates were stored at 4°C until used. Plates were incubated aerobically at 37°C for 24h and then room temperature for a further 6 days.
Colonial morphologies were observed after 24h and after 6 days.
2.1.2 Digestion of whole cells
Cells were digested for curli isolation according to Collinson et al (Collinson et al.,
1991). Colonies presenting the rough, dry morphotype typical of curli expression were scraped from approximately 130 CFA plates and suspended in 70mL of 100mM Tris HCl
(MP Biomedicals, USA) with an adjusted pH of approximately 8.0. A pH probe (Eutech
Instruments, USA) was used to measure pH, and small amounts of concentrated HCl
(Sigma-Aldrich, USA) (approximately 500μL at a time) was added to lower the pH without drastically changing the volume and concentration of Tris. This suspension was supplemented with 0.1mg/mL of both RNase A (Sigma-Aldrich, USA) and DNase I (from bovine pancreas) (Roche, Australia). Cells were ruptured by sonication in a water bath at room temperature for three minutes, following which MgCl2 (Sigma-Aldrich, USA) was added to a concentration of 1mmol/L. The solution was incubated at 37°C for 20 minutes. Lysozyme (Sigma-Aldrich, USA) was added to a concentration of 1mg/mL before further incubation at 37°C for 40 minutes. Sodium dodecyl sulphate (SDS)
(Invitrogen, USA) was added to a final 1% concentration and the solution was incubated at 37°C for a further 30 minutes.
39
After incubation the cells were centrifuged (Centrifuge 5810R, Eppendorf, Germany) at
12,100 x g for 15 minutes at 25°C and the insoluble pellet was collected and washed with 100 mM pH 8.0 Tris buffer. The pellet portion included an insoluble liquid layer with a higher density and viscosity than that of the Tris buffer supernatant.
The pellet was resuspended in Tris buffer and the previous digestion with RNase A,
DNase I and lysozyme was repeated, after which cell digests were washed by centrifuging with Tris buffer. The pellet was resuspended in Tris buffer and an aliquot was taken for SDS-PAGE. The solution was centrifuged again and the pellet resuspended in 20mL SDS-PAGE sample buffer (10% glycerol (Sigma-Aldrich, USA),
100mM dithiothreitol (DTT) (Sigma-Aldrich, USA), 2% SDS, 62.5mM Tris HCl, with a pH of 6.8).
2.1.3 Differential centrifugation
SDS-PAGE samples were boiled using a heat block (Dry Block Heater, Rowe, Australia) at 100°C for 15 minutes to denature and dissolve any remaining protein present, and then centrifuged for 15 minutes at room temperature. Centrifuge speeds were varied to determine the effect of g-force on the amount of soluble protein removed from the final pellet. Aliquots of the samples (1mL) were individually spun at 600 x g and 21100 x g respectively. Pellets not taken for lyophilisation were resuspended in SDS-PAGE buffer without DTT.
2.1.4 Lyophilisation and quantification
Aliquots of protein spun at 21,100 x g (from 2.1.3) were resuspended in 1mL of 96% formic acid (Sigma-Aldrich, USA), vortexed until fully dissolved, and then frozen at -
40
80°C. Frozen protein was lyophilised in a freeze-dryer to produce a more aqueous- soluble white powder. This powder was diluted (1:180) in SDS buffer, sonicated for three minutes and vortexed to assist dissolution. Protein concentration was estimated in the resultant solution by recording absorbance at 280nm with ten replicates in a spectrophotometer (Synergy H1, Biotek, USA) along with blanks consisting of solvent only. The estimated molecular weight and extinction coefficient were calculated by entering the protein sequence into the ProtParam tool (Swiss Institute of
Bioinformatics, 2016), and these parameters were used to calculate the approximate concentration of protein in the original aliquot.
For comparison, another 1mL aliquot of lyophilised protein was weighed on an analytical balance to ensure the results from the spectrophotometer were not affected by insoluble clumps in the mixture. The theoretical absorbance value for this weight of protein was calculated using Beer’s law and used as a rough accepted value to compare to the measured absorbance values. A Student’s t-test was conducted on the absorbance data to find if there was a significant difference between the measured amounts of protein.
2.2 Analysis of extracted fimbriae
2.2.1 Assessment of purity by SDS-PAGE
Samples subjected to different centrifuge speeds (from 2.1.3) and the aliquot taken from the original cell digest (2.1.2) were subjected to SDS-PAGE to qualitatively assess the efficacy of the purification method. Samples (6.5µL) were mixed with lithium dodecyl sulphate (LDS) Sample Buffer (2.5μL) (Life Technologies, USA) and DTT Sample
41
Reducing Agent (1μL) (Life Technologies, USA), and were heated at 70°C for 10 minutes in a heat block. They were then loaded onto a pre-cast NuPAGE 12% polyacrylamide gel (Thermo Fischer Scientific, USA) in 2-(N-morpholino)ethanesulfonic acid (MES) SDS-
PAGE buffer (Life Technologies, USA). Samples were subjected to electrophoresis (Mini
Gel Tank, Thermo Fischer Scientific, USA) for 35 minutes at a constant 200V alongside
SeeBlue Plus2 prestained protein standard (range 4kDa to 198kDa) (Invitrogen, USA).
Lyophilised material obtained in 2.1.4 was also subjected to electrophoresis using the same method.
Following electrophoresis, the gel was stained with SimplyBlue Safestain (Invitrogen,
USA). The gel was first rinsed three times with 100mL of RO water for 5 minutes each to remove buffer salts and SDS, and then covered with SimplyBlue Safestain (approx.
20mL) and incubated at room temperature for one hour. The gel was then washed with 100mL of RO water for another hour at room temperature. For best results, gel was de-stained with a further 100mL of RO water overnight. The tray used for de- staining was covered with Parafilm (Sigma-Aldrich, UA) to prevent evaporation. The gel was visualised using a light box.
Visible bands were excised using a sterile scalpel. Solid white material that remained in the gel well after electrophoresis was collected using a 23 gauge needle and syringe
(Terumo, Australia) and by rinsing the wells with RO water.
2.2.2 Mass spectrometry
All mass spectrometric analysis to determine the identity of purified proteins was carried out at the Bioanalytical Mass Spectrometry Facility, at the University of New
42
South Wales, Australia. Excised gel bands were incubated in 25mM ammonium bicarbonate in acetonitrile to remove the Coomassie stain. Once visibly clear, bands were incubated with 10mM DTT in 50mM ammonium bicarbonate at 37°C for 30 minutes, followed by 25mM iodoacetamide in 50mM ammonium bicarbonate at 37°C for 30 minutes. Bands were washed with acetonitrile twice for 10 minutes each, and then incubated with 20mM ammonium bicarbonate containing 2.5μg/mL trypsin for 14 hours at 37°C. Bands were washed for 15 minutes with a 1:2 (v/v) mixture of deionised water with 1% (v/v) formic acid and acetonitrile.
Extracted peptides were dried, and dissolved in deionised water containing 0.05% (v/v) heptafluorobutyric acid and 0.1% (v/v) formic acid (Shevchenko et al., 1996).
A minimum of sodium deoxycholate buffer (2%) was used to dissolve the lyophilised powder sample from 2.1.4. Proteins were reduced by incubating with 5mM DTT at
37°C for 30 minutes, followed by 10mM iodoacetamide at 37°C for 30 minutes in the dark. Trypsin was added to 0.02µg/μL and incubated for 14 hours at 37°C. Sodium deoxycholate buffer was removed by adding 0.1% trifluoroacetic acid, then centrifuging the sample for 5 minutes at 12,100 x g.
Nano-liquid chromatography was utilised to separate proteins present in the gel bands and lyophilised powder samples. An Ultimate 3000 HPLC (High Performance Liquid
Chromatography) and autosampler system (Dionex, Netherlands) were used. Samples were concentrated and desalted onto a micro C18 precolumn (300µm x 5mm, Dionex) using a 98:2 ratio of deionised water to acetonitrile containing 0.5% trifluoroacetic acid at 15μL/min. Samples were washed for 4 minutes. The precolumn was then switched
(Valco 10 port valve, Dionex) into line with a fritless nano column (75µ x 10cm) containing C18 media (1.9µ, 120Å, Dr Maisch, Germany). A linear gradient of 49:1 43 deionised water to acetonitrile containing 0.1% formic acid, to 16:9 deionised water to acetonitrile (0.1% formic acid) at 200nL/min over 30 minutes, was used to elute the peptides from the column (Gatlin et al., 1998).
A high voltage of 2000V was applied to the low volume tee (Upchurch Scientific). The column tip was positioned approximately 0.5cm from the heated capillary (heated to
257°C) of an Orbitrap Velos (Thermo Electron, Germany) mass spectrometer. The mass spectrometer was operated in data-dependent acquisition mode. Electrospray was used to generate positive ions.
The Orbitrap was used to run a survey scan of m/s 350-1750, with lockmass enabled, at a resolution of 30,000 at m/z 400 and an accumulation target value of 1,000,000 ions. The ten most abundant ions with counts of greater than 4,000 and charge states of greater than +2, were sequentially isolated and fragmented within the linear ion trap. Collisionally induced dissociation was used for this process, with an activation q=0.25 and an activation time of 30ms at a target value of 30,000 ions. M/z ratios selected for MS/MS were dynamically excluded for 30 seconds.
The spectra obtained from the MS/MS analysis were searched against the UniProt database using MASCOT (version 2.5.1). Search criteria used included: trypsin as the enzyme specificity, precursor ion tolerance set to 4ppm, product ion tolerance set to
+0.4 Da, variable modification of methionine oxidation, and finally, one missed cleavage was allowed. The proteins analysed were all provided with a probability based Molecular Weight Search score, and the ions score significance threshold was set to 0.5.
44
2.3 Vaccine formulation
2.3.1 Vaccine production
The fimbriae vaccine was formulated in a UV sterilised laminar flow hood to avoid contamination. Purified fimbriae were re-taken in phosphate-buffered saline (PBS)
(137mM NaCl, 2.7mM KCl, 10.1mM Na2HPO4, 1.8mM KH2PO4) with a pH of 7.2, at a concentration of approximately 400µg/mL. The suspension was then added to
Alhydrogel® adjuvant (Invivogen, USA) in a 1:1 (v:v) ratio. Resultant concentration of protein in the adjuvant-antigen solution was 200µg/mL. The solution was mixed by inversion for 2 minutes and stored at 4°C.
The solution was tested for sterility by streaking onto separate sheep blood agar plates and incubating one at 37°C for two days, and one at room temperature for 4 days.
Plates were observed for any growth after this time, and samples were considered to be sterile if no growth of microorganisms was visible.
2.3.2 Vaccine delivery
Animal Ethics approval for animal testing was granted by the Birling Animal Ethics
Committee (approval number BAEC1061_07_16AU). Twenty female ISA Brown chickens were purchased from Baiada Poultry. Birds had been orally vaccinated at one day old, and at 6 weeks, against S. Typhimurium using Vaxsafe Salmonella
Typhimurium (Bioproperties, Australia). The birds were kept in five pens of four birds each. Each pen consisted of a cage with a perch, access to food and water and an enclosed, dark nesting box. Four pens, totalling sixteen birds, were injected with 0.5mL of curli vaccine via the intramuscular route, in the breast, with a sterile 28 gauge
45 needle and syringe at 13 weeks of age. Skin was disinfected with alcohol spray prior to injection to prevent infection. Two birds were observed for 90 minutes after injection for any signs of distress before the remaining birds were immunised. Another injection of freshly made adjuvant-antigen solution was administered in the same way five weeks later. Chickens were monitored for distress daily by physical examination around the injection site for signs of inflammation, including swelling or heat. The remaining four birds in pen 5 were not immunised, and were used as a control. These birds were handled and moved between cages the same as the immunised birds so that they experienced the same degree of stress.
Birds were also observed visually for any sign of difficulty in standing or moving, abnormal posture, depression, weight loss or lack of appetite. Any birds exhibiting these signs were euthanized by a veterinarian by atlanto-cervical dislocation.
2.3.3 Sample collection
Blood was taken from two randomly selected birds in each pen at 18 weeks old and 23 weeks old using a sterile needle and Vacuette clot activator blood collection tubes
(Greiner Bio-One, Austria). The blood was allowed to clot at room temperature and then centrifuged for 10 minutes at 1811 x g, after which the separated serum was poured off and stored at 4°C. The blood clot was discarded as contaminated waste.
Eggs were collected from each pen at 20 weeks, 21 weeks, 23 weeks, 24 weeks, 27 weeks and 28 weeks of age. One day’s worth of eggs was collected at each time point.
Eggs were labelled by writing in pencil on the shells and were stored at 4°C.
46
2.3.4 Standard ELISA blood testing
Blood samples taken at 18 and 23 weeks from all pens (immunised and non- immunised) were tested for Salmonella Typhimurium LPS antibodies, to determine whether there was a difference in seroconversion of LPS between vaccinated and unvaccinated birds. As the fimbriae preparation contained small amounts of highly immunogenic LPS, vaccinated birds may have experienced an increase in anti-LPS IgY compared to control birds. If this were the case, a difference observed in reaction to the fimbriae-based ELISA may have simply been caused by the difference in anti-LPS titre between groups, rather than the anti-curli antibody titre.
Testing was carried out using an X-OVO Flockscreen Salmonella Typhimurium (St) –
Chicken Antibody ELISA Kit (X-OVO, United Kingdom) as per the manufacturer’s instructions (X-OVO, 2016). Blood sera were diluted 1:500 in sample diluent (X-OVO),
United Kingdom) and 50μL of the resultant solution were added to the wells of the pre-coated plates along with positive and negative controls. The well contents were mixed by gentle agitation of the plate, and incubated at 37°C for 30 minutes, after which the plate was washed with wash buffer (X-OVO, United Kingdom) four times using a plate washer. To each well, 50μL of Enzyme Conjugate Reagent (X-OVO, United
Kingdom) was added, and the plate was again agitated briefly, incubated at 37°C for 30 minutes and washed as before. Finally, 50μL of ELISA Substrate Reagent (X-OVO,
United Kingdom) was added to each well, and the plate was incubated at 37°C for 15 minutes, during which a pale pink colour developed in positive wells. After this time,
50μL of ELISA Stop Solution (X-OVO, United Kingdom) was added to each well and the plate was briefly mixed. The plate was immediately read in a plate reader at 550nm.
47
The test was considered valid if the mean negative control absorbance was less than
0.2, and the mean positive control was at least 0.2 OD units greater than the negative mean. Sample results were determined using the Sample to Positive (S/P) ratio as per
Equation 2.1. Samples with an S/P greater than 0.25 were considered positive, those from 0.15 – 0.25 were considered a suspect positive, and those less than 0.15 were considered negative.
푆푎푚푝푙푒 퐴푏푠표푟푏푎푛푐푒 − 푁푒푔푎푡푖푣푒 퐶표푛푡푟표푙 퐴푏푠표푟푏푎푛푐푒 = 푆⁄ 푃표푠푖푡푖푣푒 퐶표푛푡푟표푙 퐴푏푠표푟푏푎푛푐푒 − 푁푒푔푎푡푖푣푒 퐶표푛푡푟표푙 퐴푏푠표푟푏푎푛푐푒 푃
Equation 2.1 – S/P ratio calculation
2.4 Analysis of IgY
2.4.1 Crude yolk IgY purification
IgY was extracted from egg yolk as described previously (Pavic 2010). Yolks were separated from egg white using a commercial separation device, and were diluted with distilled water in a 1:5 ratio (w/v). Samples were vortexed and frozen for 72h at -20°C.
Following this, yolk samples were thawed gradually at 4°C and centrifuged at 2,800 x g at room temperature for 20 minutes.
Ammonium sulphate (MP Biomedicals, USA) was added to the supernatant to 2molL-1 and the resulting solution was vortexed and incubated at room temperature for 2h.
The suspension was again centrifuged at 2,800 x g for 20 minutes at room temperature. The precipitate was collected and resuspended in 24mL of 2molL-1 ammonium sulphate, vortexed and incubated for 40 minutes at room temperature.
48
The solution was centrifuged as above and the precipitate was resuspended in PBS with vortexing. Purified IgY was then stored at 4°C.
SDS-PAGE was carried out as per 2.2.1 on the following samples: raw diluted egg yolk, solid fraction after freeze-thaw process, soluble fraction after freeze-thaw process, and the final purified IgY preparation. This was done to visualise the effectiveness of the purification process.
2.4.2 ELISA development
An ELISA was developed to detect the presence of IgY specific to curli in blood and digested yolk samples. The method used was adapted from Abcam (Abcam, 2016).
Freeze dried curli were diluted to 10μg/mL in coating buffer. Coating buffer consisted of 0.029mol/L sodium carbonate (Na2CO3) (BDH) and 0.071mol/L sodium bicarbonate
(Na2HCO3) in RO water with a pH of approximately 9.6. To each well of a microtitre plate, 50µL of fimbriae in coating buffer was added. The plate was incubated at room temperature for two hours, after which the solution was removed by inversion and the plate was washed four times with PBS using a plate washer (TECAN Hydroflex plate washer).
The wells were blocked using 200μL of blocking buffer, consisting of 1% bovine serum albumin (BSA) (Sigma-Aldrich, USA) in PBS. The plate was incubated at 4°C overnight.
The buffer was again removed and the plate was washed as before. Dry plates could then be stored at 4°C.
Initial ELISAs were carried out to determine the optimal concentration of sample tested. Yolk extract and blood serum samples were added to the plate at a 1:10 49 dilution in PBS. Samples were then doubly diluted in PBS down the plate so that the total volume in each well was 100μL. Plates were briefly rocked by hand, then incubated at room temperature for two hours, after which the liquid was removed and the plates were washed as before.
A 1:30,000 dilution of the secondary antibody, rabbit anti-IgY IgG conjugated to horseradish peroxidase (HRP) (Sigma-Aldrich, USA) was made up and 200μL was added to each well. Plates were left at room temperature for two hours. The liquid was removed and the plate was washed.
Tetramethylbenzidine (TMB) (IDEXX, Australia) was used as the substrate. To each well, 50µL of TMB was added and allowed to react for 20 minutes. Following this, 50μL of stop solution (IDEXX, Australia) was added, and the optical density was read immediately at 650nm using a plate reader (Hydroflex, TECAN, Switzerland).
The optimal concentration of serum and yolk extract for this specific ELISA was most likely in the region between saturation and extinction, where the graph of the first
ELISA began to curve. An ELISA in this range was carried out using sera from 18 weeks and 23 weeks. Sera were diluted 1:100 and doubly diluted down the plate in PBS as described earlier. The ELISA was carried out as before and the results graphed to determine the dilution which produced the greatest separation between positive and negative samples. This was repeated for the five yolk samples from 24 weeks.
Finally, an analysis of all yolk samples collected was carried out at the optimal concentration determined earlier, which was estimated to be 4mg digested yolk/mL.
Yolk samples from 24 weeks were used as the controls: Pen 1 (vaccinated birds) was
50 used as a positive control, while Pen 5 (unvaccinated birds) was used as a negative. A blank consisting of PBS only was also included in the test. The absence of a commercially available specific antibody against curli made the addition of a known positive control difficult. All yolk samples were tested in duplicate. Results were graphed according to the age of the birds and the measured optical density to visualise the immune reaction to the vaccine over time.
A Student’s T-test was used to compare the optical density of each blood and crude IgY sample to determine whether there was a significant difference between the vaccinated and unvaccinated birds at each time point.
2.4.3 Western Blotting
Both fimbriated and non-fimbriated cells were lysed in preparation for western
Blotting. Fimbriated cells were grown as per 2.1.1 while non-fimbriated cells were grown on CFA agar at 37°C for 24 hours. A plate with no culture added was incubated with both sets of samples to assess sterility of the media, as the inclusion of components of other bacteria could skew results. Two colonies were scraped from the plate and stored in sterile RO water for SDS-PAGE analysis. Remaining cells were scraped from the medium and were resuspended in lysis buffer (50mM Tris-HCl,
100mM NaCl, 2% SDS, 1mM DTT, 5% glycerol, 0.1mg/mL DNase, 1mM MgCl2). The suspension was briefly shaken and incubated at 37°C for 45 minutes. The digest was then boiled at 95°C for 35 minutes to improve dissolution. Aliquots of 500μL were taken from both the fimbriated and non-fimbriated cell preparations for SDS-PAGE analysis.
51
The digest was centrifuged at 21,100 x g for 15 minutes at room temperature. Aliquots of both the pellet and the supernatant from both fimbriated and non-fimbriated cultures were collected for SDS-PAGE analysis. The pellet samples were resuspended in
1mL of 96% formic acid and vortexed until fully dissolved, while the supernatant samples were combined 1:1 with 96% formic acid and vortexed briefly. This was done in order to solubilise any curli present in the digest. All samples were immediately frozen at -80°C. Frozen pellet and supernatant samples were lyophilised as per 2.1.4.
Lyophilised samples were resuspended in a minimum of PBS.
Samples were subjected to SDS-PAGE and stained alongside molecular markers as per
2.2.1. Of particular interest was the presence or absence of the band at approximately
17kDa representative of curli.
A bicinchoninic acid (BCA) protein assay was conducted to determine the concentration of protein in each lysate sample. A 25μL aliquot of each sample was added to 200μL of BCA assay buffer (copper (II) sulphate (Sigma-Aldrich, USA) and
235.2μL bicinchoninic acid solution (Sigma-Aldrich, USA)) in a microtitre plate, which was shaken briefly and incubated at 37°C for 30 minutes. The plate was then read at
650nm and the absorbances compared to standard protein solutions to determine protein concentration.
SDS-PAGE for western Blotting was run in duplicate on the same gel, including both fimbriated and non-fimbriated lyophilised lysates (soluble and insoluble fractions).
Another SDS-PAGE gel was run simultaneously with the identical samples for
Coomassie staining. Approximately 10μg of each protein mixture (as determined by the BCA assay) was added to an equal volume of Laemmli sample buffer (Bio-Rad, 52
Australia) and heated at 95°C for 10 minutes. Tris/Glycine/SDS (TGX) buffer (Bio-Rad,
Australia) was used as the running buffer, and samples were run alongside Precision
Plus Protein Dual Colour Standards (Bio-Rad, Australia) ranging from 250kDa to 10kDa.
Gels were subjected to electrophoresis for 25 minutes at a constant 200V in a Mini-
Protean Tetra Cell electrophoresis unit (Bio-Rad, Australia). Coomassie staining of the second gel was carried out as per 2.2.1.
The gel with samples run in duplicate was rinsed briefly in Towbin buffer (25mM Tris,
192mM glycine (Sigma-Aldrich, USA), 20% (v/v) methanol (Sigma-Aldrich, USA), pH 8.3) for transfer of the proteins to the western Blotting membrane. Whatman 3mm chromatography paper (GE Life Sciences, Australia) was cut to approximately the same size as the gel and was soaked in Towbin buffer. Ten pieces of soaked chromatography paper were placed on the anode of an Owl Semi-Dry Transfer system (Thermo Fischer
Scientific, USA), followed by the rinsed gel, one Biotrace NT pure nitrocellulose blotting membrane (GE Life Sciences, Australia) soaked in Towbin buffer, and another ten pieces of soaked chromatography paper. The cathode was placed on top to assemble the semi-dry transfer apparatus and the proteins were transferred at a constant
1.9mA/cm2 (total 97mA) membrane for one hour.
Following this, the membrane was separated from the gel and chromatography paper, and rinsed briefly in Towbin buffer. A Ponceau stain was used to ensure that the protein had been transferred successfully. Enough Ponceau stain (Sigma-Aldrich, USA) to cover the membrane was added, and incubated at room temperature for 10 minutes with rocking. The Ponceau stain was poured off and the membrane was rinsed with RO water for a few seconds, until the lanes and protein bands were visible. After
53 visualising the transfer, the remaining stain was removed by rinsing with RO water for several minutes, until no more stain could be observed.
A 10% skim milk solution was made up using Tris-buffered saline with Tween 20 (TBST) buffer (Sigma-Aldrich, USA), and the membrane was blocked using this solution for one hour at room temperature with rocking. The membrane was cut in half to separate the duplicate samples using a pair of scissors rinsed with 70% ethanol and RO water. The primary antibody solution was made up using crude IgY purified from egg yolks from both immunised and non-immunised birds, diluted 1:1000 in TBST with 5% skim milk.
Yolk extracts from 24 weeks were used as the largest difference in response to the curli ELISA was observed at this time point. Membrane halves were separately incubated with the different primary antibodies for one hour at room temperature with rocking.
The membrane halves were washed with TBST three times for five minutes each, after which they were incubated with the secondary antibody solution, consisting of a
1:20,000 dilution of the rabbit anti-IgY IgG conjugated to HRP in TBST. The membrane halves were incubated at room temperature for 1 hour with rocking, after which they were washed three times as before. A 1:1 mixture of peroxide and luminol substrate
(Millipore Immobilon Western, GE Life Sciences, Australia) was prepared and immediately pipetted over the membrane. Approximately 3mL of substrate solution was used, and was run over the membrane for approximately 1 minute.
Membranes were imaged immediately and simultaneously using an ImageQuant LAS
500 (GE Life Sciences, Australia) to produce a chemiluminescence image combined
54 with colourimetric markers. Chemiluminescent imaging was run on the automatic setting for 50 seconds.
Any bands observed to be strongly reactive were excised from the corresponding location of the Coomassie stained gel. Bands were frozen, treated and submitted for
MS analysis as per 2.2.2.
55
3 Results
3.1 Purification of Curli fimbriae
3.1.1 Expression of Curli
The expression of curli on the surface of Salmonella Typhimurium 135a was assessed by observing the morphology of colonies grown on both nutrient T-medium and CFA agar, with different temperature profiles. A rough, dry morphology was considered positive for curli, while the mucoid morphology was considered negative, as shown in
Figure 3.1. The results of growing S. Typhimurium 135a cultures in different conditions are summarised in Table 3.1. It was observed that no fimbriae were present on cells grown on either nutrient T-medium or CFA medium after 24 hours at 37°C. The
Nutrient T-medium also yielded no curli expression after a further 6 days at room temperature, while the CFA medium showed very good expression of fimbriae under the same incubation conditions. The CFA medium that was left at room temperature for 7 days showed only a slight degree of fimbriation.
The degree of fimbriation was uniform between plates subjected to the same growth conditions. Fimbriated colonies were much more difficult to scrape from the medium than non-fimbriated colonies.
56
Figure 3.1 – Expression of curli by Salmonella Typhimurium 135a. Left: rough, dry colonial morphology representative of a high degree of expression of curli in Salmonella Typhimurium 135a. Sample was grown on CFA medium for 24h at 37°C, then 6 days at room temperature. Right: mucoid colonial morphology representative of no expression of curli in Salmonella Typhimurium 135a. Sample was grown on nutrient agar at 37°C for 24h.
Table 3.1 – Salmonella Typhimurium 135a Expression of Curli on Nutrient T-Medium and CFA agar.
Medium Type Growth Conditions Fimbriae Expression1
Nutrient T-Medium 37°C for 24 h None
37°C for 24 h + 6 days at RT None
CFA Agar 37°C for 24 h None
37°C for 24 h + 6 days at RT High
RT for 7 days Slight
1 - Highly fimbriated samples exhibited a rough, dry morphology while samples with no fimbriae presented a mucoid morphology. Slightly fimbriated samples produced a mixture of both morphologies on a single plate, with less pronounced “wrinkles” present on the fimbriated colonies.
57
3.1.2 Digestion of whole cells
The first digestion of the cell suspension with RNase, DNase, Lysozyme and SDS produced a two-layered solution after centrifugation. The upper layer was considered to be the supernatant consisting of Tris-HCl buffer and dissolved proteins, while the immiscible lower layer was considered to be part of the pellet of larger proteins and cell components. This pale yellow lower layer was highly viscous and contained brown and white solids collected at the bottom of the tube after centrifugation. Some brown particulate matter was also observed suspended in the liquid, which was mostly broken up by sonication.
Upon the addition of more RNase and DNase, the immiscible layer almost instantly became more soluble in the Tris-HCl buffer. A similar, but slower effect was observed after the second addition of lysozyme. After the second digestion and centrifugation, the pellet appeared significantly smaller, the most notable change being in the liquid, which became slightly less viscous and reduced to a smaller volume. The solid layers appeared unchanged, with the same brown and white particles at the bottom of the tube.
3.1.3 Differential centrifugation
There was no obvious difference in size or physical appearance between the pellets produced using different spin speeds. Pellets consisted of a white solid which would not dissolve when resuspended in SDS. Pellets were difficult to resuspend in 96% formic acid, but after vortexing for several minutes the pellet appeared to dissolve.
58
3.1.4 Lyophilisation and quantification
Lyophilisation of the purified fimbriae produced a white powder which was soluble in
LDS buffer and SDS buffer, and partially soluble in PBS. The protein sequence was entered into ProtParam and the molecular weight was calculated to be
13362.12g/mol, not including the signal sequence of the peptide. The extinction coefficient was estimated to be 18450 units M-1 cm-1 at 280nm in PBS, and the isoelectric point was 5.1. The lyophilised powder was diluted by a factor of 180 in PBS and the optical density at 280nm was measured. After correcting for the blank and the dilution factor, the concentration of the protein in the original aliquot, based on the ten duplicates tested, was determined to be approximately 1.9 + 0.3 mg/mL (with standard error).
A Student’s T-test was used to compare the results of the average concentration of the ten duplicate samples tested in the spectrophotometer. The T-test found that there was no significant difference between the ten measured values at a 95% confidence level.
3.2 Analysis of extracted fimbriae
3.2.1 Assessment of purity by SDS-PAGE
The original cell digest was subjected to SDS-PAGE alongside molecular markers and the centrifuged samples to assess their purity, as shown in Figure 3.2. After staining, the original cell digest yielded bands at approximately 40kDa, 30kDa, 17kDa and
14kDa, as well as a band in the gel well associated with an insoluble white solid which did not enter the gel. The sample spun at 21,100 x g yielded only the band in the gel
59 well with the insoluble solid, and no bands in the gel itself. The sample spun at 600 x g yielded a similar gel well band, and the same 40kDa band observed in the original cell digest.
1 2 3 4 5
Figure 3.2 – SDS-PAGE gel of S. Typhimurium 135a digest comparing purification of original digest to samples centrifuged at different speeds. Lane 1 – molecular markers ranged 4-198kDa; Lane 2 – original cell digest before boiling in SDS buffer; Lane 3 – resuspended digest sample boiled in SDS buffer and centrifuged at 21,100 x g; Lane 4 – resuspended digest sample boiled in SDS buffer and centrifuged at 600 x g; Lane 5 – digest sample centrifuged at 21,100 x g after lyophilisation in 96% formic acid. Arrow indicates approximately 17kDa band suspected to be curli monomers.
The cell digest centrifuged at 21,100 x g was therefore considered to be the most pure sample; as such, this method was utilised in all further fimbrial purification. A sample
60 of the most pure digest was lyophilised in 96% formic acid was also subjected to SDS-
PAGE, producing a band in the gel well and a band at approximately 17kDa. Minimal solid material was observed in the gel well of the lyophilised sample compared to the original digest.
3.2.2 Mass spectrometry
The bands excised from the gels in 3.2.1, and the lyophilised protein from 3.1.4, were submitted for mass spectrometry to facilitate identification. The main component of each of these samples was determined to be AgfA, the main subunit of SEF17 fimbriae.
Minor components of the samples include AgfB, the minor subunit of SEF17, as wel as human keratin, lysozyme from Gallus gallus, and Salmonella outer membrane lipoprotein. The protein sequences identified as AgfA are displayed in Figure 3.3. All samples yielded the same protein sequences for AgfA. The minor components of all the samples tested were also the same.
It was observed that the lyophilised protein did not completely dissolve during the trypsin digest. As such, only the dissolved portion of the protein was loaded onto the column for mass spectrometry to avoid damaging the apparatus.
61
1 mkllkvaafa aivvsgsala gvvpqwgggg nhngggnssg pdstlsiyqy gsanaalalq
61 sdarksetti tqsgygngad vgqgadnsti eltqngfrnn atidqwnakn sditvgqygg |------1------| |------2------|------3------|
121 nnaalvnqta sdssvmvrqv gfgnnatanq y |------4------| ------2------|
Figure 3.3 – Protein sequence of agfA, major subunit of SEF17 fimbriae, with hits from mass spectrometry analysis. Section of protein detected by mass spectrometry is highlighted. Protein sequence is broken into four sections, denoting four peptide sequence hits. Hits were identical between all three samples tested. Arrow indicates cleavage point for signal sequence (amino acids 1-20).
3.3 Vaccine formulation
3.3.1 Vaccine production and delivery
The Alhydrogel curli vaccine was made fresh before each immunisation, and was cultured before administration to ensure sterility. The cultures showed no growth of microorganisms, and the vaccines were therefore considered to be sterile and safe to deliver to the birds. Vaccine was stored at 4°C until administered.
When left at 4°C, the vaccine separated as the heavier components settled at the bottom of the container. The solution was therefore inverted several times before drawing up into syringes to be administered, to ensure that the mixture was homogenous and each bird received the same dose of fimbriae and adjuvant. Birds did not appear distressed by the vaccination process.
Birds were monitored for signs of distress such as depression, appetite loss, difficulty standing, and abnormal posture, after the first immunisation, and on a daily basis. 62
During this time, no distress or discomfort in the birds was observed. Physical examination around the injection site also showed no evidence of adverse reactions.
All birds grew at a regular rate, were vocal and appeared healthy at all times. Birds began laying eggs at approximately 18 weeks of age.
3.3.2 Sample collection
Blood was collected from two randomly selected birds in each pen at 18 and 23 weeks of age, totalling ten blood samples per collection. Eggs were collected from all pens at
21, 23, 24, 27 and 28 weeks. Three to five eggs were collected from each pen each week.
3.3.3 Standard ELISA blood testing
Bloods taken at 18 and 23 weeks were tested using an X-OVO Flockscreen Salmonella
Typhimurium (St) – Chicken Antibody ELISA Kit to detect antibodies to S. Typhimurium
LPS. Six positive samples were detected among the sera from 18 weeks, five of which were vaccinated birds, and one unvaccinated bird. The remaining three samples were all suspect positive, including two immunised birds from different pens and one non- immunised bird. All pens had at least one bird positive for S. Typhimurium LPS antibodies.
The sera from 23 weeks showed similar results, with eight positive samples, seven from vaccinated birds, and one from the unvaccinated birds. The remaining two samples were also suspect positive, with one sample from the vaccinated birds, and the other from the unvaccinated birds. Again, all pens presented at least one positive sample.
63
No serum samples tested were negative for S. Typhimurium LPS antibodies.
3.4 Analysis of IgY
3.4.1 Crude yolk IgY purification
Egg yolk IgY was extracted by freeze-thaw and ammonium sulphate precipitation, which yielded a large amount of a white solid that was soluble in PBS.
The stained SDS-PAGE gel of the purification process, shown in Figure 3.4, showed the progressive removal of proteins from the IgY preparation. The whole egg yolk showed many bands between 25kDa and 198kDa. The solid fraction produced a smear with several distinct, high molecular weight bands from 198kDa to approximately 30kDa.
The soluble fraction produced much sharper, clearer bands, with three quite large bands at 25kDa, 40kDa and 60kDa, and number of smaller, higher molecular weight bands. Finally, the purified IgY preparation showed only the major bands at 25kDa,
40kDa and 60kDa. It was predicted that the bands at 25kDa and 60kDa corresponded to IgY subunits, as these were close to the typical molecular weights of IgY L-chains and
H-chains respectively.
64
1 2 3 4 5 kDa 198 98 62 49 38 28 17
14
6
3
Figure 3.4 - SDS-PAGE gel showing purification of IgY from yolk samples. Lane 1: molecular markers from 198kDa to 6kDa; Lane 2: whole egg yolk; Lane 3: solid fraction following freeze-thaw and centrifugation; Lane 4: soluble fraction following freeze- thaw and centrifugation; Lane 5: crude IgY preparation following ammonium sulphate precipitation. Arrows highlight bands at approximately 65kDa and 25kDa suspected to represent IgY H-chain and L-chain respectively.
3.4.2 ELISA development
Development of this ELISA posed multiple challenges, the majority of which were caused by the extreme insolubility of the curli. While lyophilisation in 96% formic acid did somewhat improve the solubility of the fimbriae, it did not allow complete dissolution of the protein in any standard buffers, including PBS, RO water, a solution of 2% SDS in PBS, or SDS PAGE buffer. While some protein dissolved in each solution, the greatest degree of dissolution being observed in the buffers containing SDS, visible solid particles of curli remained. Adding more solvent to the mixture did not appear to 65 decrease the amount of solid particles, nor did heating at 37°C, boiling at 90°C, or sonication for several minutes, indicating that the issue was more likely caused by insolubility rather than saturation of the buffers.
This same insolubility was observed when lyophilised curli were added to the carbonate coating buffer. After sonication and vortexing, each for several minutes, solid particles remained floating on the surface of the solution. The solution was mixed overnight by inversion at room temperature, however this also appeared to have minimal effect on the degree of dissolution of the fimbriae. As such, the concentration of antigen in the coating buffer could not be determined with accuracy. These particles were allowed to settle so they would not be added to the plate during the coating process, as they may have interfered with the binding of soluble particles to the plate surface. Once the particles were settled, plates were coated with 50μL of the curli in coating buffer at an approximate concentration of 10μg/mL for two hours at room temperature. Plates were blocked with 1% BSA overnight at 4°C, and the remainder of the antigen solution was stored at 4°C for future use.
Initial assays were carried out over a range of dilutions from 1:10 to 1:1280 for both the blood and 0.4g/mL yolk samples from 21 weeks, as shown in Figures 3.5 and 3.6 respectively. The curves for both sample types showed saturation until approximately
5mg digested yolk/mL and 12.5μL serum/mL, at which point the response appeared to decrease. Serum samples from 23 weeks were tested on a separate plate to those from 18 weeks. An unusual peak appeared in the 23 week bloods at approximately
6.25µL/mL. It was thought that this unusual peak may have been caused by
66 irregularities in the binding of the antigen to these particular wells during coating, or by inadequate washing caused by a blockage in one of the nozzles of the plate washer.
1.8
1.6
1.4
1.2
1 23 Weeks Vaccinated
0.8 23 Weeks Control
OD OD at 650nm 18 Weeks Vaccinated 0.6 18 Weeks Control 0.4
0.2
0 -1 0 1 2 3 4 5 Natural Log blood concentration (uL/mL)
Figure 3.5 – Initial ELISA carried out on serum samples taken from vaccinated and control birds at 18 and 23 weeks. A broad range of dilutions from 100µL serum/mL PBS to 0.8μL/mL was tested to determine the saturation point of the assay and to find an ideal range of testing for differences in titre. Assay was carried out on a microtitre plate coated with purified curli from Salmonella Typhimurium phage type 135a and blocked with 1% BSA. Samples were serially diluted down the plate. Error bars represent calculated standard error for each data point (n = 8).
67
1.4
1.2
1
0.8
20 Weeks Vaccinated 0.6
OD OD at 650nm 20 Weeks Control 0.4
0.2
0 -2 -1 0 1 2 3 4 Natural log yolk concentration (mg/mL)
Figure 3.6 – Initial ELISA carried out on crude IgY samples purified from yolk taken from eggs laid at 21 weeks by vaccinated and control birds. A broad range of concentrations from 40mg digested yolk/mL PBS to 0.3mg/mL were tested to determine the saturation point of the assay and to find an ideal range of testing for differences in titre. These concentrations were produced by serially diluting the samples down the plate. The assay was carried out on a microtitre plate coated with purified curli from Salmonella Typhimurium phage type 135a and blocked with 1% BSA.
The saturation point of the assay was determined by the initial ELISA, after which
ELISAs focused on smaller ranges of dilutions were carried out on sera from both time points to determine at which dilution the test and control sample responses were the most separated. The results are shown in Figure 3.7. New ELISA plates were prepared using the antigen solution prepared previously. It was found that there was no significant difference at the 95% confidence level between test and control sera at 18 weeks. There was, however, a significant difference in the response of vaccinated sera
68 at 23 weeks compared to control sera at 23 weeks, and both control and vaccinated sera at 18 weeks. There was no significant difference between the response of the control birds at 18 and 23 weeks at the same dilution, at the 95% confidence level. IgY from 24 weeks was similarly tested as shown in Figure 3.8, as the IgY detected in the sera at 23 weeks would have been included in the yolk at 24 weeks. A significant difference in response was found between the IgY of the vaccinated birds and the control birds at the 4mg/mL dilution.
It was observed that the optical density of sera of similar dilutions was different between experiments. Figure 3.5 shows a range of optical densities from 0.6 to 1.2 units for samples diluted between approximately 1μL serum/mL and 10μL serum/mL, while Figure 3.7 shows an optical density range of 0.6 to 0.25 for the same range of dilutions. This was most likely an effect of the unpredictable coating antigen concentration. A similar result was observed in the yolk samples, where concentrations of yolk between 4mg digested yolk/mL and 0.3mg digested yolk/mL in the initial and more precise ELISAs produced optical densities between approximately
1 and 0.3 units, and 0.5 and 0.1 units respectively. However, the two plates used in each experiment produced comparable results, as the optical density of each sample in
Figures 3.7 and 3.8 decreased to very similar points. As it was not possible to fully dissolve the antigen in coating buffer, it was noted that plates used in future to run
ELISAs between samples needed to be made up from the same stock solution at the same time to ensure consistency between the plates.
69
0.7
0.6
0.5
0.4 18 Weeks Vaccinated 18 Weeks Control 0.3 OD OD (650nm) 23 Weeks Vaccinated
0.2 23 Weeks Control
0.1
0 0 2 4 6 8 10 Blood Serum (µL/mL)
Figure 3.7 – ELISA of blood sera collected from vaccinated and control birds at 18 weeks and 23 weeks. Samples were serially diluted in a range of 10µL serum/mL PBS to 0.08µL/mL. Assay was carried out on a microtitre plate coated with purified curli from Salmonella Typhimurium phage type 135a and blocked with 1% BSA. Error bars represent calculated standard error for each data point (n = 8).
70
0.6
0.5
0.4
0.3 Vaccinated
OD OD (650nm) Control 0.2
0.1
0 0 0.5 1 1.5 2 2.5 3 3.5 4 Yolk (mg/mL)
Figure 3.8 – ELISA of crude IgY isolated from yolks of eggs laid by vaccinated and control birds at 24 weeks. Assay was carried out on a microtitre plate coated with purified curli from Salmonella Typhimurium phage type 135a and blocked with 1% BSA. Error bars represent calculated standard error for each data point (n=8).
The 4mg/mL dilution of the IgY samples produced the widest range of responses between test and control samples in Figure 3.8, and was chosen as a standard dilution to establish an overall image of immune response to the vaccine. All crude IgY from eggs between 21 and 28 weeks were tested at this dilution on the same microtitre plate coated with curli, as shown in Figure 3.9. It was observed that while the control
IgY samples remained between approximately 0.3-0.4 OD650nm at all time points, the vaccinated samples had quite a high response (approximately 0.6 OD650nm) at 21 weeks, which slowly decreased over time to approach a level similar to the control samples. A Student’s t-test was used to determine that there was a significant
71 difference at the 95% confidence level between vaccinated and control samples at 21,
23 and 24 weeks, while there was no significant difference found between test and control samples at 27 and 28 weeks.
0.7
0.6
0.5
0.4
Vaccinated 0.3 OD OD (650nm) Unvaccinated 0.2
0.1
0 18 20 22 24 26 28 30 Age (weeks)
Figure 3.9 – ELISA of all crude IgY samples purified from yolks collected from vaccinated and control birds from weeks 21 to 28. Error bars represent calculated standard error for each data point (n=8). Crude IgY was tested at a concentration of 4mg digested yolk/mL PBS.
3.4.3 Western Blotting
Fimbriated and non-fimbriated cells were lysed and digested in order to observe the reactivity of the purified yolk IgY against Salmonella proteins. Both fimbriated and non- fimbriated cells were difficult to dissolve in the lysis buffer, and in RO water for the eventual SDS-PAGE and western blot analysis. After the incubation for 45 minutes at
72
37°C in lysis buffer, the suspensions of both cell types were still quite viscous and incompletely resuspended. After 35 minutes at 95°C the digest appeared to be appropriately resuspended to continue the digest.
Both the supernatants and pellets of both cell type digests yielded a white powder when freeze-dried, which was easily resuspended in a minimum of PBS. Lyophilised samples, whole cells and digested samples were subjected to SDS-PAGE to visualise the digest, as shown in Figure 3.10.
Non-fimbriated whole cell and entire digest samples were difficult to load onto the gel and did not run well, forming thin smears down the lane. This was consistent after multiple repeat runs of electrophoresis. However, all other samples ran well. All samples produced a large number of protein bands, mostly at molecular weights of
40kDa and higher, with the exception of the insoluble fraction of non-fimbriated cells, which appeared to contain no protein at all. Of particular interest was a band that appeared in only the insoluble fraction of the fimbriated cells, at approximately 15kDa.
73
kDa 1 2 3 4 5 6 7 8 9
198
98
62 49
38
28
17
14
6
3
Figure 3.10 – SDS-PAGE gel of cell digestion process. All freeze-dried samples were first dissolved in formic acid. Lane 1: Molecular markers from 198kDa to 3kDa; Lane 2: fimbriated whole cells; Lane 3: non-fimbriated whole cells; Lane 4: whole digest of fimbriated cells; Lane 5: whole digest of non-fimbriated cells; Lane 6: Freeze-dried soluble fraction of fimbriated cells; Lane 7: freeze-dried soluble fraction of non- fimbriated cells; Lane 8: freeze-dried insoluble fraction of fimbriated cells; Lane 9: freeze-dried insoluble fraction of non-fimbriated cells. Band of interest at approximately 15kDa in insoluble fraction of fimbriated cells circled in red.
After this initial SDS-PAGE analysis, it was reasoned that samples that did not run well
would not produce a useful western blot. As such, only the lyophilised soluble and
insoluble fractions of both cell types were included in the western blot. A BCA assay
74 was used to determine the concentration of protein in each lyophilised sample to be used for the western blot. The values obtained showed that the highest concentration of protein was present in the soluble fraction of fimbriated cells, while no protein was detected in the insoluble fraction of the non-fimbriated cells. Volumes equal to 10μg of protein were used for SDS-PAGE, except for the insoluble non-fimbriated fraction, for which a volume of 25μL of sample was used, in order to add as much as possible to the gel well.
After electrophoresis, a Ponceau stain was used to visualise the membrane and ensure proteins had been adequately transferred, as shown in Figure 3.11. Bands were observed in both fimbriated cell lanes, and the soluble non-fimbriated cell lanes, however it appeared that minimal material had been transferred across from the insoluble non-fimbriated sample. This was similar to the appearance of the Coomassie stained gel (Figure 3.12). Also, the highest molecular markers (150kDa and 250kDa) had not been fully transferred. However, the proteins of interest were not located within this range, and therefore the transfer was considered to be successful.
Chemiluminescence imaging showed strong reactions to the crude IgY preparation taken from eggs laid by vaccinated birds, and no reaction to the IgY taken from eggs laid by unvaccinated birds. The membrane treated with immune IgY showed a dark smear from approximately 98kDa to 38kDa, followed by two more distinct bands at approximately 20kDa and 16kDa. One large, very dark band at 6kDa was also observed.
This was consistent for soluble fractions of both cell types, and the insoluble fraction of fimbriated cells. The insoluble fraction of the non-fimbriated cells, however, showed only a faint smear from 98kDa to 49kDa, and a faint reaction at the 6kDa band. No
75 other bands were observed in this sample. The membrane treated with unvaccinated bird IgY showed no reaction at all.
The chemiluminescence image of the membrane (Figure 3.13) was compared with the
Coomassie stained gel. The membrane showed several bands at lower molecular weights, including one very strongly reactive band at 10kDa, which matched the
Coomassie gel. However, instead of a large number of distinct bands at higher weights, the western blot produced a long smear. The strongly reactive band at approximately
10kDa in the western blot using vaccinated bird IgY as the primary antibody was excised from its corresponding location on the Coomassie stained gel and was submitted for MS analysis. This showed that the band was mainly comprised of 50S ribosomal protein. Several other uncharacterised Salmonella enterica proteins were also present, suggesting that the band also contained fractions of many different, much larger proteins.
76
kDa 1 2 3 4 5 1 2 3 4 5
150 100 75
50
37
25 20
15
10
Figure 3.11 – Ponceau stain of nitrocellulose membrane after transfer from SDS-PAGE gel. Halves of membrane are duplicated, containing identical samples. Lane 1 – molecular markers 250kDa to 10kDa; Lane 2 – soluble fraction of fimbriated cells; Lane 3 – soluble fraction of non-fimbriated cells; Lane 4 – insoluble fraction of fimbriated cells; Lane 5 – insoluble fraction of non-fimbriated cells.
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kDa 1 2 3 4 5
250 150 100 75
50
37
25
20
15
10
Figure 3.12 – Coomassie stain of Salmonella Typhimrium 135a cell digests, both fimbriated and non-fimbirated. Lane 1 – molecular markers 250kDa to 10kDa; Lane 2 – soluble fraction of fimbriated cells; Lane 3 – soluble fraction of non-fimbriated cells; Lane 4 – insoluble fraction of fimbriated cells; Lane 5 – insoluble fraction of non- fimbriated cells.
78
kDa 1 2 3 4 5 1 2 3 4 5
150 100 75
50
37
25 20
15
10 A B
Figure 3.13 –Western blot of Salmonella Typhimurium 135a cell digests, both fimbriated and non-fimbriated. All freeze-dried samples were first dissolved in formic acid. Image A – western blot of digests using crude IgY from vaccinated birds as primary antibody; Image B – western blot of digests using crude IgY from unvaccinated birds as primary antibody. For both images: Lane 1 – molecular markers 250kDa to 10kDa; Lane 2 – freeze-dried soluble fraction of fimbriated cells; Lane 3 – freeze-dried soluble fraction of non-fimbriated cells; Lane 4 – freeze-dried insoluble fraction of fimbriated cells; Lane 5 – freeze-dried insoluble fraction of non-fimbriated cells.
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4 Discussion
The purpose of this study was to investigate the potential of curli fimbriae as an immunogenic protein, and to develop an ELISA to detect anti-curli IgY in serum or yolk, and therefore seroconversion in birds to a wide range of hypervirulent Salmonella infections. A curli-based ELISA could quickly and accurately detect infection using both blood and yolk samples, and save precious time in the treatment and control of outbreaks among poultry flocks. Also, as the vast majority of Salmonella isolates are capable of producing curli, these structures could be a useful addition to current subunit vaccines in order to confer immunity to a wider variety of Salmonella serovars.
4.1 Purification of Curli fimbriae
4.1.1 Expression of Curli
The first objective of this project was to produce and harvest large amounts of curli for further purification. Expression of curli was difficult to induce in vitro, as the environmental and nutritional requirements for the transcription of this protein are quite specific. The curli promoter gene, csgD, has been shown to be transcribed only during the stationary phase of growth, and is increased by environmental stresses such as low oxygen availability (Gerstel & Romling, 2003; Rhen, 2007; Grantcharova et al.,
2010; Mika et al., 2012). Considering this, it was not surprising that the differing growth conditions showed that both the environmental conditions and agar composition greatly affect the expression of curli of Salmonella Typhimurium 135a. It is clear the expression of fimbriae required some days of growth to occur, as evidenced by the absence of fimbriae on either set of cultures after 24 hours of incubation.
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However, after seven days, some fimbriation had occurred on both the CFA plates left at room temperature for the entire period, and those downshifted from 37°C.
Expression of fimbriae was likely coincident with onset of stationary phase (Gerstel &
Römling, 2003; Mika et al., 2012).
The downshift in temperature from 37°C to room temperature also had a clear effect on the degree of fimbriation. The bacterial cultures grown on CFA agar at constant room temperature appeared to produce fewer fimbriae than the identical cultures moved from 37°C. Rhen (2007) showed that, when provided with plentiful nutrients,
Salmonella cultures grown under microaerophilic conditions produced more fimbriae than those under aerobic conditions. In light of this, a comparison between colonies grown under aerobic and anaerobic conditions, with and without the downshift in temperature may have provided more information on curli expression, and could be an important aspect of future work. It was also shown by Grantcharova et al. (2010) that environmental stress and genetic stress factors play a role in the mediation of csgD expression. As previously demonstrated by White & Surette (2006), temperatures not optimal for Salmonella growth (below 30°C) also promote curli production. The downshift in temperature in this experiment evidently triggered an increase in fimbrial production, to aid survival under these adverse conditions.
Previous studies have shown that CFA agar and nutrient agar are equivalent in promoting the expression of most fimbrial antigens in E. coli, as determined by measuring the degree of haemagglutination caused by the cells (Ghosh et al., 1993).
However, fimbriae such as SEF14 and SEF17 do not cause haemagglutination, and are not detected by this method. As such it could not be assumed that SEF17 would be
81 expressed on these media, or that it would be produced in adequate amounts for vaccine production. Studies have also shown that curli fimbriae are not expressed on nutrient agar unless tryptone (usually at 1%) is included as an additional amino acid and nitrogen source (Collinson et al., 1991; Atlas, 2010). This is a partial explanation as to why curli have been studied very little in the past – the lack of expression on more traditional media has meant the protein has not been available for study when analysing Salmonella cell structure and function.
No fimbrial expression was observed in any of the colonies grown on nutrient T- medium, regardless of the temperature downshift. This was an unexpected result, as it was not consistent with previous studies by Collinson et al. (1991, 1992). This could have occurred because previous studies of curli have been focussed primarily on the
Salmonella serovar Enteritidis, while this study used S. Typhimurium. A previous study by Encheva et al. (2007) showed that despite their very similar genome, there are differences in protein expression between serovars and even strains of S. enterica. It is possible that there is a slight variation in expression pattern and nutritional requirements in S. Typhimurium 135a for the production of curli, meaning that nutrient T-medium was not sufficient, while CFA medium was an adequate source of amino acids and nitrogen (Atlas, 2010).
The combination of these results provides evidence as to why the gut is an ideal environment for the production of these novel fimbriae. Fimbriae were produced when the bacteria were under stress, but had an adequate supply of nutrients. This mimics the gut environment, as the acidic pH in the gut presents environmental stress
82 to the bacteria, while the food being digested by the host provides the high concentration of nutrients required.
4.1.2 Extraction of fimbriae from whole cells
After harvesting large numbers of cells presenting the rdar morphology, a method was required to purify the curli from other cellular components. Most fimbriae of
Salmonella are separated from the cell by standard methods which utilise shear forces and differential centrifugation (Curtis et al., 2016). Conversely, curli remain cell wall- associated during these processes, attached to larger outer membrane proteins, and therefore cannot be purified in this manner (Karch et al., 1985; Collinson et al., 1991;
White et al., 2003). The insolubility and resistance to degradation of these fimbriae is, however, key to their separation; the method first described by Collinson et al. (1991) in 2.2.1 is designed to digest and remove almost all proteins except the target fimbriae. The enzymes and detergents used are generally very effective in breaking down cellular components, but can only act on soluble proteins. Therefore, the repeated centrifugation to remove any soluble components of the digest gradually removes almost all contaminants (Collinson et al., 1991; White et al., 2003).
Previous studies have shown that SDS-PAGE of the digested fimbriae result in the majority of remaining molecules entering the stacking gel, while the aggregative fimbriae remain as a solid in the gel well. This has previously been used as a separation method for the isolation of pure curli (Collinson et al., 1991; White et al., 2003;
Oppong et al., 2015). However, only very small amounts of protein can be isolated from each gel due to the volumes of standard SDS-PAGE gels. Preparative gels could be
83 used as an alternative to increase protein separation, however, this process would still be too time consuming and expensive to purify large amounts of fimbriae.
Since proteins are only able to enter an electrophoresis gel once dissolved in the SDS-
PAGE buffer, it was reasoned that the fimbriae alone would remain insoluble in the buffer. The majority of contaminating proteins would be solubilised by treatment with the loading buffer and heat, and could therefore be separated out by centrifugation prior to loading the samples. Thus, the centrifugation method described in 2.2.2 was developed to separate the insoluble aggregates from the contaminants without using large numbers of electrophoresis gels. If the levels of contaminants in the final products were comparable, this method would then be far more efficient for purification of curli.
Another important issue throughout this process was the digestion of the fimbriae into monomers which could be analysed by SDS-PAGE and MS. The autoaggregative nature of curli renders more conventional protein disruption treatments with urea, sodium hydroxide or SDS buffers ineffective (Reichhardt et al., 2015). To date, the only technique which yields a monomerised, soluble protein is treatment with very concentrated formic acid (van Gerven et al., 2015). The resistance of the aggregative polymers to all but the harshest of monomerisation treatments exemplifies how the presence of these fimbriae on cell surfaces in vivo protects cells and colonies from host antimicrobial defences. Phagocytes, surfactants, and even antibodies to other cell surface proteins can be kept at a distance by the impenetrable and unreactive mass of fimbriae, and other supporting extracellular matrix proteins (Costerton et al., 1987;
Ledeboer & Jones, 2005).
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An explanation for this unusual insolubility can be found through studies of other aggregative protein masses, especially those associated with degenerative diseases such as Parkinson’s disease and Alzheimer’s disease. These protein masses are generally composed of native proteins which have been misfolded during their synthesis (Reichhardt et al., 2015). Once large enough, these aggregates will precipitate out of solution and become very difficult to break down (Dueholm et al.,
2011). Curli are formed via a similar mechanism, and have been shown to share several physical, biochemical and structural properties with disease-related amyloids
(Reichhardt et al., 2015). A soluble monomer of AgfA is added to membrane-bound fimbrial subunit AgfB. This first AgfA subunit acts as a nucleus for the continued growth of the fibre, as more soluble monomers are attached to eventually produce an insoluble mass (Gibson et al., 2007; van Gerven et al., 2015). These fimbrial structures also share several physical similarities with aggregative amyloid proteins which cause degenerative diseases. Both types of protein include a high number of glutamine and asparagine residues, which contain sites with a high potential for hydrogen-bonding, allowing for the formation of strong bonds between amino acids of similar peptides when folded. The tertiary structure of each AgfA unit also resembles that of typical amyloid aggregates; AgfA contains a strand-loop-strand structure very similar to a cross-β fold appearing in several disease-causing aggregates, including β-amyloid, the protein responsible for Alzheimer’s disease (Dueholm et al., 2011; Reichhardt et al.,
2015).
The similarities between these two types of insoluble protein aggregates suggested that similar methods for disaggregation of these compounds may be successful.
85
Disease-causing amyloid aggregates have been shown to form when the native protein is closer to a neutral charge, as the pH of their environment approaches their isoelectric point. Similarly, they tend to disaggregate when exposed to pH levels distant from their isoelectric points (Picotti et al., 2007). Curli form very stable aggregates in the acidic environment of the host gut and have an isoelectric point of approximately 5.1 (Swiss Institute of Bioinformatics, 2016). As such, a very harsh acid treatment is required to disaggregate the protein to form soluble monomers. This appears to be the most likely reason more traditional treatments fail to solubilise the protein and only the extremely concentrated formic acid treatment described in 2.2.3 has been successful. This also suggests that the use of concentrated alkaline solutions with very high pH may also improve dissolution; however, previous work by Collinson et al. (1991) showed that fimbriae remain insoluble in 8M urea and 5M NaOH solutions. Future studies of Salmonella curli could include an investigation of the effect of much higher concentrations of strong bases on the solubility of AgfA.
4.2 Analysis of extracted fimbriae
4.2.1 Assessment of purity by SDS-PAGE and MS
When used as a vaccine, other immunogenic proteins contaminating the fimbrial preparation could compete with the curli and reduce the chicken immune response to the target antigen (Joneja & Bielory, 1990; Hunt et al., 2001; Waryah et al., 2016). It was therefore important to analyse the contaminants present in the protein preparation, as it was highly unlikely that a sample would ever be comprised entirely of the target protein (Hodge et al., 2013).
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The Coomassie stain of digested cells, pre-SDS buffer treatment, showed four clear bands at approximately 40kDa, 30kDa, 17kDa and 14kDa. This indicates that while the majority of proteins from the original whole cell culture had been removed, there were still several major contaminants within the sample. The band present at 14kDa most likely represented the lysozyme (molecular weight 14.3kDa) used in the digestion process which may not have been completely washed from the sample (Sigma-Aldrich,
2015). However, the other bands were more likely other Salmonella proteins left over after digestion.
The band at approximately 30kDa may have contained Salmonella LPS molecules, which have molecular weights of roughly 34kDa (Galdiero et al., 1999), but may vary depending on strain and serovar (He & Liu, 2002; Murray et al., 2003). The band at
40kDa was possibly Outer Membrane Protein (OMP) S3, a 37.9kDa protein detected as a trace contaminant in the final product by mass spectrometry. As curli often remain associated with the cell wall during purification, it is reasonable to suggest that the major contaminants of the digest would include outer membrane proteins from the original cell culture. The mass spectrometry data obtained from the final digested protein supports this, as lysozyme, OMP, and LPS were all detected in small amounts.
It was originally hypothesised that the band at 17kDa in the original sample represented a minimal amount of AgfA that had been monomerised by the SDS-PAGE preparation. However, this hypothesis was rejected after not observing the same band in the further digested samples. Instead, this 17kDa band is more likely another OMP, possibly Rck, a 17kDa OMP, or PagC, an 18kDa OMP (Heffernan et al., 1992).
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Finally, a dark band was observed in the gel well itself in all samples tested. This band likely represented curli and any other large or insoluble materials that did not enter the stacking gel. The darkness of this band indicated that there was a large amount of protein present, which was reasonable considering the wide variety of large proteins present in Salmonella. The mass spectrometry data obtained by testing the excised gel well confirmed that this material was primarily curli.
Interestingly, the bands at 17kDa and 40kDa are the only clearly visible bands remaining in the post-lyophilisation SDS-PAGE of the sample centrifuged at 600 x g. If, as suggested earlier, these bands represent OMPs attached to the aggregative fimbrial particles, they would be harder to separate from the digest compared to the lysozyme and LPS, which no longer appear as major contaminants. The lysozyme and LPS would readily dissolve in heated SDS buffer, and would be easily retained in the supernatant and removed from the sample. The OMPs, however, could still be bound to the curli aggregates in small amounts, lowering their solubility even when the sample was boiled (Collinson et al., 1991; Collinson et al., 1992; Collinson et al., 1993).
While the four bands for each of these contaminants are no longer visible in the sample centrifuged at 21,100 x g, lysozyme, OMPs and LPS were all detected in the mass spectrometry data. This is because mass spectrometry is an incredibly sensitive and precise technique (Hodge et al., 2013), while SDS-PAGE relies on the naked eye to observe any stained protein on the gel. It is highly likely that there is other protein present on the gel, but the amount of this protein was too small to visualise with staining. The absence of any contaminating bands on the SDS-PAGE gel, however, does provide evidence that the sample centrifuged at the faster speed of 21,100 x g was
88 more pure than the sample centrifuged at 600 x g. The reason for this is probably quite simple: the higher speed caused the pellet to be packed more firmly into the bottom of the microfuge tube than the sample centrifuged at 600 x g. This made it easier to completely remove the supernatant from the higher speed sample without losing part of the pellet, meaning that more of the contaminating proteins were eliminated from the fimbriae preparation.
The SDS-PAGE analysis of the final lyophilised material revealed both the original band of protein in the gel well, in addition to a new band at approximately 17kDa. Bands at this molecular weight have previously been associated with the AgfA (Collinson et al.,
1991). The actual molecular weight of AgfA is approximately 15kDa (as calculated using the ProtParam tool), and as such the band would normally be expected to migrate slightly further down the gel. However, the structure and variety of residues in individual proteins respond differently to electrophoresis, and the molecular markers can only be used as a relative guide. Additionally, the very little solid material observed in the well was indicative of the protein having dissolved and entered the gel, after monomerisation by the formic acid treatment.
Both the 17kDa band and the gel well were excised and taken for mass spectrometry, along with the whole lyophilised material sample, which was to be used for the vaccine preparation (without lyophilisation). This showed that the main component of all samples tested was indeed the curli subunit AgfA. Over half of the SEF17 peptide sequence was accounted for in the mass spectrometry results, including one sequence over 40 amino acids long. It is highly unlikely that other proteins would have contributed these specific sequences and so it can be stated with a great deal of
89 certainty that the major protein present was SEF17. Interestingly, the sequence hits were located closer to the C-terminus than the N-terminus. This is possibly a result of the incomplete dissolution of the lyophilised protein during the digestion process. The most aggregative, hydrophobic regions of the protein may not have been fully digested and would therefore have been unavailable for analysis by mass spectrometry.
The main contaminants present in the final preparation were determined by mass spectroscopy to be chicken lysozyme and Salmonella OMP. As discussed earlier, chicken lysozyme was required during the purification of the target protein and was observed as a band on the SDS-PAGE gel. However, this protein is of no concern if included in the vaccine, as it will be recognised as a self-protein and will not influence the immune response (Sela-Culang et al., 2013).
The contaminant with the highest potential to influence the immune response of the birds was the Salmonella OMP. This protein was likely not completely removed from the membrane bound end of the fimbriae. As an external protein, OMP is exposed to the immune system and induces an antibody response, giving it the potential to compete with SEF17 in antigenic potential (Joneja & Bielory, 1990; Maripandi & Al-
Salamah, 2010). While this may have decreased the effect of SEF17 in the vaccine, the amount of OMP present in the sample was very minor compared to the fimbriae. As such, the effect of this contaminant would most likely have been minimal. Importantly, the absence of other significant Salmonella proteins indicates that the digestion was very successful in degrading and removing all other cell contents from the fimbriae. If there had been other major contaminants from Salmonella in the preparation, further digestion of proteins and nucleic acids with detergents, and possibly more washings
90 may need to be carried out. However, it is unrealistic to expect the protein preparation to be completely pure.
These contaminants provided valuable information in regards to the efficacy of the purification method. During the centrifugation purification method, the antigen remains in a single tube while it is washed with sterile buffers. During electrophoresis, however, the antigen is moved between multiple tubes and the gel, providing more opportunities for contamination as it is handled. The protein would also come into contact with many more surfaces, including the non-sterile electrophoresis unit. There is a much higher possibility that the sample will become contaminated with environmental substances using the electrophoresis method.
The centrifugation method used here also has several other advantages over the original purification method utilised by Collinson et al. (1991). The lack of constraints imposed by maximum gel volumes makes it possible to process much larger amounts of protein at a time. Electrophoresis requires more time and a great amount of precision to avoid destroying the gel and losing the sample when collecting the fimbriae. It is also impossible to collect all of the fimbriae from the wells as it is often not visible, and larger solid particles will not easily enter a needle or pipette tip.
Additionally, the electrophoresis method requires transfer of the sample between multiple vessels, leaving protein on the sides of the tubes each time and reducing yield. Meanwhile, the centrifugation method only requires the use of one microfuge tube, resulting in minimal product loss. As such, it was concluded that this method was more effective and more efficient than the method described by Collinson et al.
(1991). A more feasible method for the purification of these fimbriae is an important
91 step in facilitating the research of their function, structure, and potential as an immunogenic target for diagnosis and vaccination.
In future, contamination could be further reduced through the use of more personal protective equipment at every stage of the digestion. Considering the nature of the contaminants in the protein preparation, and the low levels present, the purity was considered high enough to use as the antigen for the vaccine.
4.3 Vaccine formulation
4.3.1 Vaccine production and delivery
It is important to acknowledge that the vaccine produced in this study was not subjected to any challenge studies to determine its efficacy. As such, it was not a true vaccine in that there was no evidence produced that birds were protected against disease. Overall, each dose of the curli vaccine contained approximately 100μg of protein. This dose rate was determined based on typical dose rates of other subunit vaccines. Immunisation with too large or small a dose of antigen may cause issues with its immunogenicity, resulting in over-sensitisation or tolerance of the bird to the antigen. While studies have shown that the optimal amount of antigen for vaccination of chickens generally lies between 10µg and 1mg per dose, protein antigens are usually administered in the range of 10-100µg. This is because proteins are considered among the most efficient antigens, especially when compared to some other cell components such as nucleic acids (Chalghoumi et al., 2009). In order to not cause over-sensitisation of the birds to the antigen, but also give the best chance of stimulating an immune response to an antigen with unknown immunogenicity, 100μg per dose was chosen. It
92 has also been documented recently that exposure to curli fimbriae may carry DNA fragments and induce autoimmunity in the host, which would also have caused discomfort to the birds after vaccination (Tursi et al., 2017; Gallo et al., 2015).
Fortunately, the lack of adverse reactions to the second dose implied that no over- sensitisation had occurred.
It is possible that curli are not included in standard killed Salmonella vaccines, as they are not expressed in large quantities until fairly late in the growth cycle (Mika et al.,
2012). Studies involving killed vaccines often harvest cells for inactivation after growth for 24 hours or less (Coward et al., 2014; Ferreira et al., 2015), meaning, as shown by the growth experiment performed in this trial, that expression levels of curli will be relatively low compared to cells in vivo. This unintentional exclusion of such an important virulence factor may be limiting the efficacy of these vaccines, particularly in limiting adhesion of the bacteria to the host gut and the build-up of a biofilm to establish long-term infection (Watnick & Kolter, 2000; Ledeboer et al., 2006; Fabrega &
Vila, 2013; Li et al., 2016). Should curli elicit a strong immune response, the inclusion of this protein in vaccines as a subunit, or as a protein expressed on whole cells, may greatly enhance the immunogenicity and efficacy of existing vaccines.
It was important to monitor the birds for any adverse reactions to the immunisation, which would most likely be caused by the inclusion of Salmonella endotoxin, LPS. LPS is involved in a change in gut epithelium permeability during salmonellosis, causing diarrhoea in the host (Aschenbach et al., 2003; Yeung et al., 2013). While LPS was detected in the mass spectrometry of the protein preparation, there was evidently too small an amount present in the vaccine to cause any discernible reaction.
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It was also necessary to ensure that the vaccine was completely sterile. An intramuscular injection of a live pathogen would cause a systemic infection in the birds and, depending on the pathogen, quickly cause disease or death. In the case of live
Salmonella spp., birds most likely die within three to seven days (Gast, 2008). As a precaution, vaccine was not all made at the first immunisation and stored for five weeks, but instead separate batches were made and tested for sterility for each dose.
This required additional time and equipment. However, if the same batch had been used, it would have been exposed to air in the chicken shed, dust and other particles in the air, and multiple syringes and needles, even with good technique. It is highly likely that such exposure would lead to microbial contamination.
Another potential cause of adverse reactions may have been the adjuvant used. While aluminium salts are relatively safe compounds, approved for use in animal vaccines, their purpose is to provoke a more intense immune response (Jolles & Paraf, 1973;
Woodrow et al., 2012; Li et al., 2014). Thus, they may have caused an unpleasant inflammatory response at the injection site. The monitoring of the birds for pain and swelling on a daily basis was important to ensure that any adverse reactions were detected and appropriate action could be taken in accordance with the animal ethics approval granted. The absence of adverse reactions, however, suggests that the 1:1 ratio of antigen solution to adjuvant was appropriate.
Despite the risk of adverse reactions, the addition of the adjuvant to the curli antigen was considered necessary. Aluminium salt adjuvants have been proven to increase the titre of antibody produced after vaccination without compromising antibody quality.
When combined with aluminium salt adjuvants, the antigen adsorbs strongly to the
94 surface of the salt, using it as a depot and forming an antigen-adjuvant complex
(Morefield et al., 2005; Foged et al., 2014). This complex holds the antigen in tissues, including the lymphoid tissue, making the antigen more available and easier for antigen presenting cells to internalise. It is thought that some antigen will also be slowly released from the adjuvant into the body after injection, exposing it to the antigen-specific lymphocytes for a much greater period of time, prolonging the half-life of the antigen (Morefield et al., 2005; Li et al., 2014). As the curli used for immunisation had not been treated with formic acid or lyophilised, the majority of the protein remained insoluble. Aluminium salt adjuvant was the best choice for this antigen, as it will typically precipitate when combined with insoluble protein, forming particulate matter that can easily be engulfed by macrophages, thereby increasing the immune response (Joneja & Bielory, 1990; Morefield et al., 2005; McKee et al., 2007).
Additionally, aluminium salts cause a rush of neutrophils and eosinophils to the area, and strongly bias the immune system towards the production of Th2-cells over Th1- cells. Thus, future antibody production in the event of a repeat infection is enhanced without causing delayed hypersensitivity (Romagnani, 1999; Desin et al., 2013; Li et al.,
2014). While an aluminium salt adjuvant was considered to be the best option for this study, it may be useful in future to assess the impact of different adjuvants on the immune response of birds to the curli, such as Freund’s complete or incomplete adjuvant, or other aluminium salts besides Alhydrogel.
Route of administration was also a factor of both the immune response, and any potential adverse reactions. The route could affect the spread and clearance of the vaccine in the body, influencing whether or not the antigen will reach the secondary
95 lymphoid organs to react with the lymphocytes. While research into non-invasive routes of administration have showed some success in inducing immune responses via mucosal surfaces, for example, via oral or ocular administration (Woodrow et al.,
2012), the subcutaneous and intramuscular routes are still the most widely used.
Vaccines containing adjuvants are generally injected intramuscularly, as a reaction between the skin and the adjuvant may be quite intense, causing unnecessary pain and swelling if injected subcutaneously (Foged et al., 2014). For these reasons, only the intramuscular route was used in this study.
4.3.2 Standard ELISA Blood Testing
The sera samples were expected to contain anti-LPS IgY, due to the live oral vaccine administered to the birds at one day old, and again at 6 weeks. However, a small amount of Salmonella LPS was detected in the MS analysis of the curli vaccine preparation. The standard Salmonella Typhimurium LPS IgY test was carried out to ensure that there was not a difference between the anti-LPS IgY content of vaccinated and control sera. As such, any significant difference detected in the curli ELISA could be attributed to anti-curli IgY, rather than a difference in anti-LPS IgY levels. All sera were either positive or suspect positive at both 18 and 23 weeks, suggesting that a similar level of LPS specific IgY was present in the collected sera.
4.4 Analysis of IgY
4.4.1 Crude yolk IgY purification
Crude IgY was separated from eggs laid by both immunised and non-immunised birds by a freeze-thaw process and ammonium sulphate precipitation. Crude IgY needed to
96 be of sufficient purity for use in ELISA and western blotting, though separation of specific IgY was considered unnecessary.
Chicken egg yolk consists of two major fractions, the water-soluble fraction, and the granular, water-insoluble fraction. The water-soluble fraction contains mostly serum albumin, some glycoproteins, and IgY, while the granular fraction contains both high- density and low-density lipoproteins (Mann & Mann, 2008). Approximately 68% of yolk dry weight consists of low-density lipoproteins, and 16% of high-density lipoproteins
(Navidghasemizad et al., 2014). These proteins interact with the surfaces of oil droplets in suspension and with each other, forming a colloidal network to prevent the separation of oil and aqueous substances (Somasundaran, 2006).
This colloidal suspension needed to be broken down before the IgY could be purified and separated from the other fats and proteins of the yolk. This was the purpose of the freezing and slow thawing of the yolk in water described in 2.2.1. Other chemicals such as polyethylene glycol, or sodium sulphate may produce more pure protein preparations; however, extraction with water was sufficient at this stage, as the subsequent ammonium sulphate precipitation further improves purity (Kwan et al.,
1991; Pauly et al., 2011). Additionally, simply using water instead of organic solvents was both more environmentally friendly, and more cost effective.
As IgY is part of the water-soluble yolk fraction, centrifugation of the thawed suspension allowed the removal of the majority of larger proteins from the antibody preparation. This could be observed in the Coomassie stain of the separated fractions as the whole yolk sample contains bands across a wide range of molecular weights, but the separation of these proteins is clearly visible in the samples after freeze-thawing. 97
The granular fraction contained bands, mostly at high molecular weights, most likely representing both high and low density lipoproteins, while the soluble fraction contained mostly lower molecular weight proteins.
A typical IgY molecule has a molecular weight of 180kDa. This weight is comprised of two light chains of approximately 25kDa each, and two heavy chains of approximately
65kDa each. These subunits were expected to be visible after Coomassie staining, as opposed to one protein at 180kDa, because SDS-PAGE digestion breaks the disulfide bonds between the chains. Major bands in the final crude IgY preparation appeared at approximately 25kDa and 60kDa, approximately where the IgY subunits would be expected to appear. While the 60kDa band is not at the exact location on the gel it was expected to be, the running of some proteins may be affected by other components of the mixture, and the heavy chain may have migrated a little further than it would from a pure IgY sample. Additionally, molecular marker protein ladders are only an approximate guide to protein migration patterns.
Another band in the final crude IgY appeared at approximately 40kDa, and was most likely a combination of chicken serum albumin (molecular weight 44kDa), and yolk plasma glycoprotein (YGP), a very abundant protein found in the granular fraction of egg yolk. Typical chicken egg yolks contain high levels of two types of these proteins,
YGP40 and YGP42, with molecular weights of 40kDa and 42kDa respectively. The precursors of these proteins, vitellogenins, are synthesised in the liver, and are transported to the yolk, where they are cleaved into individual proteins. There is also possibly some contribution to this band by ovalbumin, a 42kDa protein abundant in egg white (Mann & Mann, 2008). While the majority of the granular fraction and egg
98 white was removed by the centrifugation and ammonium sulphate precipitation, small amounts of protein would have inevitably been left behind, and appeared on the SDS-
PAGE gel. These speculations could be confirmed if mass spectrometry or western blot analysis was performed on the band in question in a future study.
While ammonium sulphate precipitation is a reliable, efficient method to remove the majority of contaminating proteins from a sample based on their solubilities, this method is not as precise as more complicated chromatography methods. At any given concentration of ammonium sulphate, there are a large number of proteins that will precipitate (Duong-Ly & Gabelli, 2014). As such, while IgY can be purified from the majority of yolk proteins, other proteins with similar solubilities will remain in the sample.
Ammonium sulphate precipitation exploits the differences in protein solubility at different ionic strengths. The ionic strength of a solution determines the charges on the surface of suspended proteins; at an ideal ionic strength, the outer charges of the protein will be neutralised and the protein will dissolve readily. If the ionic strength is too high or too low for a given protein, the charges will cause the protein to aggregate and precipitate out of solution (Duong-Ly & Gabelli, 2014). The 2 molL-1 concentration of ammonium sulphate used in this study generally provides a high enough ionic strength to precipitate most IgY (Duong-Ly & Gabelli, 2014). However, other studies have shown that IgY is further purified when precipitated using 3 - 4.5molL-1 ammonium sulphate (Akita & Nakai, 1992; Ko & Ahn, 2006). This concentration was also shown to reduce a major protein contaminant of approximately 40kDa (Akita &
Nakai, 1992). It could be suggested that this higher concentration of salt would remove
99 the 40kDa contaminant observed here. Alternatively, a step-wise addition of ammonium sulphate to salt out various contaminants gradually would also improve purification. However, due to the great number of proteins present in egg yolk, it is unlikely that the bands in these different studies represent exactly the same protein.
Regardless, a higher concentration of ammonium sulphate may have increased the degree of purity of the IgY extracted. If the ELISA results showed significant interference of other proteins in the sample, then perhaps the higher concentration during purification would yield more precise results.
It would also have been possible to further purify the crude IgY using chromatography columns. Ion-exchange chromatography could be used to separate IgY based on their electrical charge. Alternatively, running the crude extract through an affinity chromatography column bound with anti-chicken IgY antibodies would allow all contaminating egg proteins to run out of the sample. The IgY would then be removed from the column using a low pH elution step, producing a much more pure solution of
IgY. This IgY would be a mixture of all the IgY produced by the hen, with a large range of antigens and epitopes represented. With fewer contaminating proteins present in the sample, the ELISA may work more precisely. Similarly, an affinity column could be coupled with purified curli to extract pure IgY specific only for curli. However, the insolubility of the fimbriae presents problems with this chromatographic method, as insoluble particles would be likely to block the column, and solutions with a very low pH such as 96% formic acid may degrade the column packing material.
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4.4.2 ELISA development
The development of a curli-based ELISA was the main goal of this project. This would allow quick identification of hypervirulent Salmonella infections of all strains and serovars. A quick and accurate method which could be used for both blood and yolk samples would help improve response times to Salmonella outbreak, and improve current screening methods for Salmonella infection, which at present are focussed on only a few serovars, excluding many hypervirulent strains.
The biggest impediment to the development of the curli-based ELISA was the lack of a commercially available IgY specific for curli. Hence, the preceding work on isolation of curli, bird immunization and purification of IgY from immunised birds allowed the preliminary development of an ELISA protocol. The ELISA developed in this study focussed on determining whether there was a significant difference in response to the curli-based ELISA between immunised and non-immunised birds. This provided valuable information on whether the vaccine increased the chicken immune response to Salmonella, and paved the way to the future development of a more quantitative assay.
The ELISA was first tested on the blood samples taken from birds at 18 and 23 weeks.
The results showed that there was no significant difference between the test samples and the control samples taken at 18 weeks. This was expected, as the first blood samples taken after the first immunisation should be low in specific IgY titre. This is because of the nature of the acquired immune response; the first response to a novel antigen introduced to the immune system is a large production of IgM, followed by a less intense production of IgY (Schroeder & Cavacini, 2010). However, the second 101 immunisation would have triggered the secondary antibody response to a known antigen, resulting in a mass production of IgY. Thus the second blood samples from test birds showed a significant difference in response to the ELISA when compared to the second control samples, and both test and control samples from 18 weeks.
Further, there was no significant difference found between the blood samples taken from control birds at 18 and 23 weeks. The immunised birds showed an increase in immune response to curli over time, while the control birds produced the same response throughout the trial. This suggests that the vaccine provoked a specific immune response in the birds, which allowed for a large production of anti-curli IgY when the antigen was encountered for a second time. It is, however, important to note that the difference in response to the ELISA between vaccinated and non- vaccinated birds was, while statistically significant, not large enough to be considered biologically relevant.
One major concern with the ELISA protocol was the need to freeze dry the antigen before it was coated on the plate. The purpose of freeze drying was to partially denature the fimbriae to expose some hydrophilic residues, allowing it to dissolve in aqueous coating buffer. However, denaturation of the protein may have changed the structure of the epitope to which the IgY bound. Fortunately, as the expected response to the ELISA was observed, the binding site for the IgY was clearly not affected.
There are two main types of antigenic epitopes bound by antibodies: conformational and linear. Conformational epitopes are the result of protein folding and the combining of several different sections of peptide to form the tertiary structure of the protein. Linear epitopes, however, arise from the series of amino acids in a single
102 section of peptide, and are not as dependent on secondary or tertiary structure
(Janeway et al., 2001). It is likely that the antibodies detected in this study are specific for linear epitopes, as the formic acid and lyophilisation treatment of the fimbriae before coating the plate was designed to cause denaturation of tertiary structure, but not degradation of the protein sequence itself. As such there is a high probability that any conformational epitopes were destroyed during this process. This would likely impact any further studies of the immunogenicity of curli conducted in vivo. As such, future work in this area could be focussed on the study of IgY specific for all immunogenic epitopes of curli, rather than just linear epitopes. Alternatively, further study into the immunogenicity of linear epitopes could be useful, to address the possibility of immunisation with shorter linear peptides.
Following the success of the blood testing, the ELISA was tested on the crude IgY preparation from egg yolk from 23 weeks. This time-point was chosen due to the timing of the blood sampling, as IgY could only be present in the yolk once it was circulating in the blood. As 23 weeks was the first point specific IgY was detected in the blood, the IgY from the same week was used. The range of dilutions tested revealed an approximate saturation point and extinction point of the assay. This initial testing was used to determine the best range of serum dilutions to give the most distinction between test and control samples. This smaller range of dilutions was then tested more extensively to produce a curve from which negative and positive samples could be easily distinguished.
The digested yolk samples tested using the ELISA showed the most difference in positive and negative samples at 4mg digested yolk/mL. This dilution was therefore
103 determined to be the best testing dilution, and all yolk samples taken over the trial were tested at this dilution. Significant differences were found between crude IgY taken from immunised and non-immunised birds from 21 weeks to 24 weeks, but not from 27 weeks to 28 weeks, at the 95% confidence level. This result demonstrates the expected immune response to a known antigen, where IgY levels increase dramatically in response to the antigen, and then slowly decline over time once the antigen is cleared from the system. It can therefore be concluded that the fimbriae subunit vaccine administered to the test birds provoked an immune response leading to seroconversion. As a result, anti-curli IgY was circulating in the blood and transferred to the yolks, and was detectable in the IgY purified from these yolks.
The background observed in the ELISA was high in this study. There are several possible causes for this, including non-specific binding, and possibly the addition of too high a concentration of secondary IgY. The insolubility of the antigen also likely contributed to the background, as the “sticky” fimbriae may have trapped other molecules in the aggregative protein mass. Inconsistent coating levels due to some antigen remaining undissolved, unintentional inclusion of the undissolved antigen, or perhaps even re-precipitation of fimbriae during coating may have also caused background noise, or differences in signal level between ELISA runs.
Also, IgY may have been raised against curli and LPS when the birds were exposed to S.
Typhimurium independent of the fimbriae vaccine, when vaccinated orally at one day old, and if they were exposed to wild S. Typhimurium during the study. Because of this previous exposure, the more important factor was the difference between test and control samples, rather than the level of background. Nevertheless, future work should
104 focus on reducing this background binding or at least identifying the cause of the high level of background reaction.
These results offer several possibilities for future research and development, including applications for purified specific IgY, as extracting IgY from egg yolk is an efficient and ethical method to mass-produce specific antibodies. Specific IgY could be used as a passive vaccination against hypervirulent Salmonella to help prevent and clear infections, if it could be delivered to the intestine without being digested. Curli-specific
IgY has potential as a disease prevention tool not only within the poultry industry, but in other agricultural industries as well, as IgY is able to bind to specific antigens in mammalian contexts with minimal cross-reactions and no activation of complement
(Vega et al., 2012).
The immunoassay developed in this project could be reversed to produce an ELISA capable of detecting hypervirulent Salmonella species in food or tissue samples, expediting identification of Salmonella infection to reduce response time to outbreaks.
Coating a microtitre plate with antibodies specific for the fimbriae, as produced in this study, would capture fimbriated cells. These could then be detected using the same antibodies conjugated to a label. One label that could be particularly useful is the streptavidin-biotin system, frequently used to amplify the signal in ELISAs. Streptavidin is conjugated with the secondary antibody before addition to the plate. Following this, biotin, usually bound to another enzyme or a fluorochrome, binds to multiple sites on the streptavidin molecule, producing a much more intense signal than an ELISA using a secondary antibody simply conjugated to the enzyme (Sigma-Aldrich, 2016). The use of the biotin-streptavidin system would allow for the detection of very small amounts of
105 binding of Salmonella cells to the specific antibodies bound to the plate. As such it would be possible to detect populations of multiple serovars of Salmonella in one test.
As antibodies were produced in vivo, there is a possibility that the vaccine used in this trial may confer a degree of immunity to Salmonella spp. This exciting possibility warrants further exploration, as current research is focussed on developing vaccines with a broad range of cross-protective immunity among serovars, rather than against only one or a few (Lee, 2015). As curli are, under the right conditions, produced in almost all Salmonella strains, this subunit could be used to develop a broader vaccine which could protect against a large number of serovars. Having a single, multi-valent vaccine effective against hypervirulent Salmonella would reduce the total amount of medication to which agricultural animals need to be exposed when they are young, reducing stress on the animals, and human labour.
Alternatively, if the vaccine used confers some immunity, but not enough to entirely protect from infection, the curli antigen could be added to current Salmonella subunit vaccines to improve resistance to colonisation.
4.4.3 Western Blotting
A western blot was performed using the crude IgY preparations as the primary antibody to visualise the reaction between specific antibody and AgfA. The Coomassie stains, the Ponceau stain and the western blot were all considered together to gain an understanding of the antibodies present in the yolks of control and test birds.
The membrane was stained with Ponceau stain immediately after the transfer process to assess the effectiveness of the transfer. The presence of red stain in a smear in all
106 the sample lanes indicates that a large amount of protein was successfully transferred from the gel to the membrane, and therefore would be available for western blotting.
It was noted, however, that the highest molecular marker did not transfer across, meaning that some higher molecular weight compounds may not have been transferred effectively. This could be reduced in future by changing the current or the duration of the transfer. However, the incomplete transfer of higher molecular weight proteins would not have impacted the transfer of the 17kDa curli proteins, and instead would have only affected other unidentified Salmonella proteins in the digest in that size range. As such, this had no impact on the experiment, and the transfer was considered successful.
The lanes containing the insoluble fraction of the non-fimbriated cells appeared to have little to no protein in them. This can be explained through the original difficulties encountered when purifying curli from the whole cells. When cells are lysed by shear forces, the majority of fimbriae separate from the cell surface and are easily purified.
Curli, however, remain associated with larger molecules from the cell wall. These fimbriae are extremely insoluble and only the harshest of treatments will cause their dissolution (Collinson et al., 1991; Collinson et al., 1992). Because of this, the lysis buffer and heat treatments were not able to solubilise all the proteins of the fimbriated cells, while the non-fimbriated cells were much more susceptible to this digestion. There was therefore very little protein remaining in the insoluble fraction of non-fimbriated cells. The fimbriated cells, however, had a significant amount of protein after centrifugation, most likely comprised of curli and proteins still attached to the fimbriae after digestion.
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The western blot results showed an obvious difference between the membrane halves, when IgY from immunised and non-immunised bird yolks was used as the primary antibody. The membrane incubated with positive crude IgY produced bands along both fimbriated cell lanes, and the soluble non-fimbriated lane. These lanes all contained a highly reactive band at approximately 10kDa. The insoluble non-fimbriated lane showed a slight reaction at the top and bottom of the sample run. The highly reactive band at approximately 10kDa on the positive membrane was excised from its corresponding location on the Coomassie stained gel and submitted for MS analysis.
Results showed that the band contained a number of small molecules, in particular, ribosomal subunits. This reactivity is possibly a result of bacterial cell lysis caused by antibodies in vivo, which would have released intracellular proteins, and ribonucleic acids (RNA) from ribosomes and protein translation. However, the strength of the reaction in this band, and the small size of the other proteins found, suggests that the band may also have contained multiple fractions of RNAs and much larger proteins, possibly including fimbriae, LPS and other immunogenic Salmonella proteins.
However, the membrane incubated with negative crude IgY showed no reaction at all.
This is again most likely due to the aggregative nature of the fimbriae, and their tendency to remain associated with cell wall and other cellular components during cell lysis (Karch et al., 1985; Collinson et al., 1991; White et al., 2003). While there is not an obvious curli band visible in the fimbriated lysate fractions, the difference between reactions suggests that curli were incompletely disassembled into discrete subunits and instead formed a ladder of subunit fractions of different sizes. The non-fimbriated lysate also reacting to the IgY preparation is not surprising, as some SEF17 protein was
108 likely present on the cells, but not in the same amount as the fimbriated cells displaying the rdar morphotype. Additionally, the dark band at the bottom of the western blot samples is darker in the lanes containing fimbriated cell lysate compared to the non-fimbriated cell lysate. These results support this hypothesis, as more of the curli antigenic epitopes would be present in the many digested proteins and RNAs of cells expressing large amounts of SEF17. The very minimal reaction observed in the negative western blot also shows that the birds not vaccinated against curli do not possess the antibodies to react to the fimbriae. These results indicate that the vaccination was successful, and a specific immune response was mounted against curli fimbriae. Future studies into the immunogenicity of this protein could focus on more in-depth proteomic analysis of the contents of the highly reactive sites in the western
Blot.
In light of these results, curli could prove to be a useful addition to current subunit vaccines. Previous studies have shown that fimbrial antigens of Salmonella induce some level of protective immunity when used as purified protein vaccines, and have been effective in reducing Salmonella colonisation (Thorns, 1995; Lopez et al., 2012). It has also been shown that vaccines targeting bacterial adhesive proteins inhibit the binding of bacteria to host tissue and other bacterial cells, greatly reducing biofilm formation, and thus virulence (Zhang & Zhang, 2010; Krachler & Orth, 2013; Forthal,
2014). As the results of this trial suggest that curli are immunogenic, and cause seroconversion, this protein could enhance the effectiveness of current subunit vaccines in preventing infection, which is a possibility worth further investigation.
109
110
5 Conclusions
This study aimed to immunise birds against curli to stimulate the production of specific
IgY, which could be detected by a curli-based ELISA. Specific IgY was detected in both the sera and yolks of vaccinated birds. However, background noise in the ELISA was relatively high, possibly due to the insolubility of the antigen used. Research into further developing this ELISA and lowering the background to increase reliability is needed before it could be used as a diagnostic tool to detect seroconversion in sera and yolk.
The curli-based ELISA developed in this study shows potential as a diagnostic tool for a wide range of hypervirulent Salmonella serovars. Future work to further develop this
ELISA would need to incorporate the testing of other important serovars, in particular,
Salmonella Enteritidis. Additionally, a source of pure curli-specific IgY would need to be found or established. This could potentially be sourced from the eggs of vaccinated birds, as collected IgY from egg yolks is an ethical, efficient solution, with a constant level of production over several months. The difficulty, however, would lie in the purification of specific IgY from the variety of antibodies present in yolk, as most IgY present in chicken serum is included in yolk.
Availability of specific IgY would facilitate more research into the structure and function of curli, to develop a full understanding of the impact of these proteins on
Salmonella virulence. Further study into the expression pattern of these unique structures may increase our understanding of Salmonella pathogenesis, which may lead to the development of better prevention and control methods. Particular
111 emphasis on developing new ways to solubilise this incredibly insoluble protein would greatly impact the versatility of the tools developed in this study. Alternatively, a recombinant section of the fimbriae, excluding the less soluble sections of the protein, could be used to increase solubility while still including the majority of conformational and linear epitopes in the vaccine.
Based on the results of the ELISA, it is reasonable to conclude that the vaccine was successful in stimulating the production of IgY in chicken blood against curli. Two immunisations were required for this process. This suggested that immune memory was established after the first dose, causing a production of mainly IgM with some IgY.
Then, after the second dose, the chicken immune system recognised the curli and produced large amounts of specific IgY. It is possible that a third booster vaccine could provoke another rise in IgY levels, which would be useful if, as mentioned earlier, the eggs were used as a specific IgY source.
The immune response provoked by the vaccine may have some untapped potential to protect birds against Salmonella infection. However, no challenge studies were performed to determine whether this vaccine conferred any level of protection from
Salmonella colonisation; to be truly utilised as a vaccine, studies would need to be carried out to determine the effect on disease in the host rather than just antibody production. It is possible that, due to the essential role of curli in the formation of biofilms, this vaccine could inhibit the initial colonisation of hosts. This possibility is worth future testing, alone and perhaps in combination with other effective subunits.
Investigating this with a much larger scale trial may provide valuable information about the prevention of Salmonella infection in the poultry industry.
112
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