Salmonella typing and colonisation of chickens by characterised Salmonella Sofia

Transcription

1 Salmonella typing and colonisation of chickens by characterised Salmonella Sofia A report for the Rural Industries Research and Development Corporation by M. W. Heuzenroeder, C. J. Murray, D. Davos and I.L. Ross December 2004 RIRDC Publication No 04/138 RIRDC Project No IMV-3A

2 2004 Rural Industries Research and Development Corporation. All rights reserved. ISBN ISSN Salmonella typing and colonisation of chickens by characterised Salmonella Sofia Publication No. 04/138 Project No. IMV-3A The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone Researcher Contact Details Dr Michael W. Heuzenroeder Infectious Diseases Laboratories Institute of Medical and Veterinary Science Frome Road ADELAIDE SA 5000 Phone: Fax: heuzenroeder@imvs.sa.gov.au In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: Fax: rirdc@rirdc.gov.au Website: Published in December 2004 Printed on environmentally friendly paper by Canprint ii

3 Foreword Contamination of poultry products by bacteria can lead to public health problems. Adverse, often uninformed and sensational media coverage of food poisoning incidents has heightened consumer awareness of food safety. These reports can often unnecessarily affect retail consumption of implicated products. In view of the risks that the above concerns pose, molecularly based rapid typing methods for Salmonella have been developed in previous RIRDC funded studies, so that the source of a human outbreak of Salmonella can be rapidly and accurately traced. It has recently become apparent that molecular methods that are routinely used at IMVS and by other laboratories, such as pulsed field gel electrophoresis (PFGE), are of limited use with some clonal (genetically closely related) serovars of Salmonella. In response to this problem a new method, amplified-fragment length polymorphism (AFLP) analysis, has been established to type strains from industry and other sources. This method combines universal applicability with high powers of discrimination and reproducibility. It can offer greater discriminating power between human and poultry isolates and can be used for epidemiological studies of disease transmission. Bacterial viruses or bacteriophage (phage, for short) are ubiquitous in the environment and have been implicated in the evolution of Salmonella and in the acquisition of genes encoding virulence factors. Bacteriophages are also used in the typing of Salmonella strains and the carriage of endogenous or temperate bacteriophage influences these typing results. Despite the fact that bacteriophage play a major role in the ecology of these organisms, few studies have been carried out to determine the influence these viruses have upon the distribution, typing and virulence of Salmonella. The genetic characterisation of two temperate phages that are found in a common Salmonella Typhimurium chicken associated strain forms a major part of this report. Salmonella Sofia currently represents around 50% of all chicken isolates of Salmonella submitted for typing by the poultry industry to the Australian Salmonella Reference Centre at IMVS. Despite the ubiquitous nature of this organism it is almost never associated with human disease. Database searches also confirm that S. Sofia is seldom seen elsewhere as associated with human disease. This compelling circumstantial evidence suggests that the organism is avirulent and a very efficient coloniser of chickens. Research was therefore undertaken to investigate whether S. Sofia can exclude virulent serovars using genetically characterised natural and mutant strains. Genetic characterisation of S. Sofia isolates from both Australia and overseas was also undertaken to determine whether S. Sofia from Australia form a unique genetic clone that might explain the extent of colonisation of Australian chickens by S. Sofia. This project was funded from industry revenue, which is matched, by funds provided by the Australian Government. This report, an addition to RIRDC s diverse range of over 1,000 research publications, forms part of our Chicken Meat R&D program, which aims to support increased sustainability and profitability in the chicken meat industry. Most of our publications are available for viewing, downloading or purchasing online through our website: downloads at purchases at Simon Hearn Managing Director Rural Industries Research and Development Corporation iii

4 Acknowledgments We would like to thank the RIRDC Chicken Meat Program for its generous support over many years, especially Vivien Kite for help, encouragement and support. Thanks also to Rina Willmore and Horst Schmieger for their work and help in the development of AFLP and genetic characterisation of the bacteriophages respectively. We would also like to thank staff at The Victorian Institute of Animal Science at Attwood for their help and suggestions in carrying out the chicken colonisation studies, particularly Catherine Ainsworth and Susan Bibby. Finally, and certainly not least, heartfelt thanks to Princess Mmolawa whose mammoth PhD study and thesis has provided data for a large part of this report. Her untimely death was a great loss to the field of microbiology and a cause of great sadness to all her colleagues and friends. iv

5 Abbreviations AFLP ASRC bp CDC CFU CsCl DNA dir. DT enr. ES FAFLP IMVS kb LB MLST nt O/N ORF PAI PCR PE pfu PIV PT PFGE Phage RAPD RDNC RFLP SDS SPI TBE TE tntc Tris U UPGMA UV VIAS v/v w/v XLD amplified fragment length polymorphism Australian Salmonella Reference Centre base pair Centers for Disease Control and Prevention colony forming units caesium chloride deoxyribose nucleic acid direct plating definitive phage type plating after enrichment PFGE protease K buffer fluorescent amplified fragment length polymorphism Institute of Medical and Veterinary Science kilobase Luria broth multilocus sequence typing nucleotide over night open reading frame pathogenicity island polymerase chain reaction Perkin Elmer plaque forming units PFGE tris-nacl buffer phage type pulsed field gel electrophoresis bacteriophage randomly amplified polymorphic DNA reactive, does not conform restriction fragment length polymorphism sodium dodecyl sulphate Salmonella pathogenicity island tris-borate-edta tris-edta too numerous to count tris (hydroxymethyl)aminomethane units unweighted pair group method with arithmetic averages ultra violet light Victorian Institute of Animal Science volume per volume weight per volume xylose lysine desoxycholate v

6 Contents FOREWORD... III ACKNOWLEDGMENTS... IV ABBREVIATIONS... V CONTENTS... VI EXECUTIVE SUMMARY... VIII 1. BACKGROUND AND OBJECTIVES Objectives How do the objectives relate to each other? Background to the project Typing systems Bacteriophage and Salmonella enterica S. Sofia and Australian chickens TYPING OF SALMONELLA ISOLATES Conventional typing Improvement of PFGE Random amplification of polymorphic DNA (RAPD) AMPLIFIED FRAGMENT LENGTH POLYMORPHISM TYPING Introduction Materials and methods Strains Fluorescent AFLP (FALP) Data analysis of FAFLP Pulsed-field gel electrophoresis Computer analysis of PFGE Results FAFLP of S. Sofia PFGE of S. Sofia FAFLP of S. Typhimurium DT PFGE of S. Typhimurium DT Comparison of similarity between FAFLP and PFGE for DT Discussion S. Sofia S. Typhimurium DT S. Typhimurium DT Conclusions CHARACTERISATION OF LYSOGENIC PHAGE IN SALMONELLA ENTERICA SEROVAR TYPHIMURIUM DT Introduction Methods Strains and phages Induction of prophages Caesium chloride gradient centrifugation Phage type conversion Southern hybridisation analysis vi

7 4.2.6 Generalised transduction of phage ST64T DNA sequence determination of ST64B DNA sequence determination of ST64T Sequence assembly and analysis Nucleotide accession numbers Results Caesium chloride gradient centrifugation ST64B is a defective phage? Phage type conversion mediated by phage ST64T Detection of phages ST64T and ST64B lysogens in other serovars Molecular typing of phage type convertants Amplified fragment length polymorphism (AFLP) analysis of phage type convertants Demonstration of generalised transduction by ST64T Genomic sequence of ST64T and ST64B Discussion SALMONELLA SOFIA COLONISATION STUDIES Introduction Methods Results Conclusions DISCUSSION Classical and molecular typing S. Sofia S. Sofia co-colonises with S. Typhimurium The significance of bacteriophage in Salmonella What has the DNA sequence of ST64T told us? What has the DNA sequence of ST64B told us? Molecular phage-typing MLST typing using phage immunity genes Summary of project outcomes IMPLICATIONS RECOMMENDATIONS REFERENCES PUBLICATIONS ARISING FROM THIS PROJECT APPENDIX vii

8 Executive Summary Conventional typing of Australian Salmonella isolates During the course of the project the chicken industry sent an average of 2,670 samples per annum ( inclusive) strains to The Australian Salmonella Reference Centre in Adelaide for conventional typing. S. Sofia is still the predominant isolate from chickens. It first appeared in significant numbers in 1980 and steadily increased to the point where it currently represents around 50% of all Salmonella isolates from chicken and now appears to have stabilised at this level of isolation after some variation in previous years. There have been no significant changes in Salmonella serovar distribution or incidence in isolation from humans and food animals, particularly chickens, over the course of this project. A major industry outbreak due to Salmonella Typhimurium phage type 126 occurred between May and November While commercially cooked products were not implicated, the outbreak once again focussed attention on raw chicken as a potential source of Salmonella into the community. Over the course of the project, the phage type distribution within S. Typhimurium human and chicken isolates changed dramatically. Most notably, the previously important DT 64 is now a relatively minor isolate from both chickens and humans. In 2001 phage type 126 was implicated in a large outbreak and has become a frequently isolated phage type in both humans and chickens. This trend has continued into In contrast to S. Typhimurium, S. Virchow phage type distribution did not markedly changed in the period and, for both chickens and humans, phage type 8 has predominated, together with phage type 34. Although other phage types are seen, they are not consistently isolated from year to year. Amplified fragment length polymorphism typing (AFLP) The underlying principle of phage typing is the host specificity of bacteriophages in the typing panel. Phage typing schemes for Salmonella enterica serovars are based on patterns of lysis produced by distinct phages isolated from a variety of sources. Phage typing has been used to subdivide isolates within serovars Typhi, Typhimurium, Enteritidis, Virchow, Hadar and Heidelberg. Although phage typing is essential for the subdivision of Salmonella serovars, the method can prove inadequate for serovars in which a small number of phage types predominate. Molecular typing is used when conventional methods fail to give sufficient discrimination between isolates. Pulsed field gel electrophoresis (PFGE) has been in use for some considerable time and is the accepted gold standard adopted by organisations such as Centers for Disease Control in Atlanta. Newer methods of molecular typing such as amplified fragment length polymorphism (AFLP) may offer significant advantages in speed and discriminatory power over established methods such as PFGE. The potential use of this method as a regular typing method was therefore investigated in this project. AFLP was used in conjunction both with PFGE and classical typing methods (i.e. serotyping and phage typing) to establish levels of discrimination between isolates, both individual strains and those suspected to belong to outbreaks. The most common serotype isolated from chickens, S. Sofia, was studied in this respect. It was found that S. Sofia was quite variable by AFLP, but no specific clone could be correlated to the time or place of isolation. It was concluded that there does not appear to be an Australian specific clone of S. Sofia that could account for its persistence in Australian chickens for such a long period of time. viii

9 S. Typhimurium DT126, the most common isolate from humans in 2001, was also examined by AFLP. The strains investigated ranged from epidemiologically unrelated strains to isolates associated with three outbreaks. Comparison with PFGE suggested that AFLP would provide greater discrimination between non-epidemiologically related isolates when compared to PFGE. Significance of bacteriophages in S. Typhimurium It has been observed for some time that certain phage types tend to predominate in S. Typhimurium in Australia. In 1998 work was begun to examine any temperate phages that could be carried by S. Typhimurium, in particular phage type or definitive type (DT) 64 which was at the time this project commenced one of the most common phage types from S. Typhimurium in chickens. The carriage of lysogenic (or temperate) bacteriophages can strongly influence phage type designation. In 1998, in work undertaken by this group, two lysogenic phages were induced from S. Typhimurium DT64. The induced phages were capable of mediating phage type conversion. The two phages have been designated ST64T and ST64B. An objective of this project was to determine whether genes carried by these phages might influence the virulence or distribution of the bacteria and this report describes the genetic characterisation of these two previously unknown temperate phages carried by S. Typhimurium DT 64. It was demonstrated that ST64B is a defective phage, and could not mediate phage type conversion. In contrast, phage type conversion could be mediated by phage ST64T and all converted phage types were shown to be lysogens of ST64T. Phage type conversion can have serious epidemiological consequences. In addition ST64T, like it s close relative, P22 (another Salmonella phage) also mediates generalised transduction, which is a method of bacterial genetic exchange and has the potential to transfer genes between bacteria for traits like virulence and antibiotic resistance. Although the ST64B and ST64B phages are carried by the same S. Typhimurium DT 64 isolate and are morphologically indistinguishable, they share very little DNA sequence similarity and this proves that both phages are distinct. Southern hybridisation analysis using both phage genomes (ST64T and ST64B) as probes indicated that ST64B genome was found in most S. Typhimurium definitive phage types tested whereas ST64T genome was found only in DTs 64 and 29. Interestingly, when ST64B phage was used as a probe to screen other Salmonella enterica serovars, the results indicated that sequences hybridising to ST64B are found in a number of non-typhimurium Salmonella serovars. Since temperate phage in Salmonella account for much of the genetic diversity between closely related Salmonella, the possibility exists that this diversity an be exploited to develop new, rapid typing systems. Colonisation studies with S. Sofia Salmonella Sofia currently represents 53.4% (2002 figures) of the approximately 2,500-3,000 Salmonella chickens isolates annually submitted to the IMVS for typing from the poultry industry. However, it is almost never associated with human disease. Therefore S. Sofia is not a pathogen for humans or chickens but it is an effective coloniser of chickens. It has been demonstrated in a previous project that certain S. Sofia strains are better colonisers of chickens and appear to be able to persist in the chicken even when challenged with S. Typhimurium. This raises the possibility that S. Sofia may be able to be used to decrease the level of colonisation of chickens with S. Typhimurium. Consequently, this possibility was investigated further in the current study. The molecular mechanism of S. Sofia colonisation was investigated. Filamentous fimbrial structures on the cell surface of bacteria can be essential for efficient colonisation of a host. The presence of fimbrial genes on the S. Sofia chromosome was confirmed by PCR and sequence analysis. The agfa gene appears to be universally found in S. Sofia isolates, where other fimbrial genes were less widespread. The agfa gene encodes the SEF17 fimbriae. ix

10 Studies were undertaken to determine what effect a mutation in the agfa gene has on the ability of S. Sofia to colonise chickens. A mutant strain, K4, developed in a previous RIRDC project was used and its colonising ability tested in an experimental protocol also developed in that project. In these experiments, birds initially colonised with S. Sofia (day 0) were exposed by direct contact with S. Typhimurium DT64 infected birds at day 5, rather than the usual protocol where in-contact exposure occurs at day 8. The rationale behind earlier exposure was that exposure to S. Typhimurium when S. Sofia numbers were higher (as they often are early in colonisation) might prevent or lessen subsequent S. Typhimurium colonisation of the birds that were initially colonised with S. Sofia on day 0. The results obtained suggest that strain K4 is no worse at colonising chickens than the nonmutant parental strain. Earlier exposure (day 5) to S. Typhimurium DT64 infected birds had no effect on the colonisation of the previously S. Sofia colonised birds by S. Typhimurium DT64 than those exposed at day 8. The more than two decade persistence of S. Sofia in Australian chickens as the predominant Salmonella isolate is a unique situation and not seen elsewhere in the world. Examination of S. Sofia strains isolated over 20 years from both here and overseas by AFLP indicated that there does not appear to be a specific dominant clone of this organism in Australia. It is also clear that S. Sofia can coexist in chickens with S. Typhimurium, but is unlikely to influence colonisation by that serovar. Implications AFLP was found to be a useful typing tool but will not replace other established methods for typing, although in some cases it is clearly superior to established methods and should be retained. A molecular phage-typing scheme can be developed using genomic data gathered in this project, which will have the advantage of greater and less subjective discrimination and be more rapid. Additional work on S. Sofia is considered unlikely to demonstrate a significant potential for its role in displacing more pathogenic strains from the chicken production environment, and it is therefore felt that no further work in this area is warranted. Recommendations Despite the fact that PFGE has been adopted as an international gold standard for molecular subtyping within serovars, this method still contains a number of serious deficiencies that are outlined in this report. It is becoming clear that DNA sequence based methods will eventually replace the older RFLP based methods. These newer methods are more amenable to automation and are more rapid. It is recommended that a molecular based typing system should be investigated to complement rather than replace classical phage typing. This would ensure continuity between the old system and the new. x

11 1. Background and Objectives 1.1 Objectives The project had four major objectives. These were: 1. To provide conventional and molecular rapid typing systems to the chicken meat industry on an ongoing basis for the purposes of organism tracing and for the monitoring of Salmonella serovars for public health and research objectives. 2. To test the feasibility of AFLP typing as an epidemiological tool to replace PFGE as the preferred molecular typing method. The AFLP method may offer greater discriminatory power and be less laborious than PFGE. 3. To characterise, by DNA sequence analysis, the defective lysogenic phage (bacteriophage) found in S. Typhimurium phage type 64. It is possible that this could lead to development of an alternative to bacteriophage typing of the most common phage-types of S. Typhimurium based upon PCR amplification of unique sequences and to an explanation as to why particular phage types predominate in humans and chickens. 4. To further study S. Sofia MH76 colonisation of chickens using different methods of inoculation and S. Typhimurium challenge. 1.2 How do the objectives relate to each other? The relationship between objectives 1 and 2 is clear, since both involve the typing of Salmonella isolates. Molecular typing is used when conventional methods fail to give sufficient discrimination between isolates. However, on some occasions there is a need for a method that provides discrimination above the established methods. Objective 2 involves the establishment of a new typing method (AFLP) and evaluation of its advantages and usefulness. The AFLP technique was also used to answer questions related to bacteriophage carriage by Salmonella strains in objective 3. This objective also attempts to discover the significance of bacteriophage found in certain phage types with regards to host pathogenicity and phage distribution. The AFLP technique is also used to determine whether there is something unique about Australian strains of S. Sofia i.e. is there an Australian clone of this organism? In this regard it also relates to the Objective 4. The experiments of objective 4 seek to clarify whether S. Sofia can reduce the colonisation of chickens with pathogenic serovars and whether certain bacterial structures on S. Sofia aid in its ability to colonise chickens. 1.3 Background to the project Typing systems Typing systems are used to define specific characteristics of the bacterium (van Belkum et al., 2001). Factors such as typeability, reproducibility, power of discrimination, ease of interpretation and ease of performance are considered in the evaluation of typing methods (Maslow et al., 1993a). Typing systems may be categorised as phenotypic techniques (those that detect characteristics expressed by the microorganisms (eg. serotyping and phage typing) or genotypic techniques (those involving DNA-based analyses of chromosomal or extra-chromosomal genetic elements, eg. PFGE, AFLP) (Maslow et al., 1993a). 1

12 Classical typing of Salmonella is carried out by serotype analysis of O (lipopolysaccharide) and H (flagella) antigens according to the scheme developed by Kauffmann and White (Kauffmann, 1954). The Kauffmann and White scheme is based on the immunologically distinct variations in somatic O antigens of the cell wall and flagellar H antigens which provide each Salmonella serovar with its own unique antigenic combination (Kauffmann, 1954). A limitation of this system is that a small number of S. enterica serovars is responsible for a majority of infections (Harvey et al., 1993). For organism tracing during an outbreak of salmonellosis, the need for a method to subdivide isolates within a serovar is essential. The classical method used has been phage typing (Callow, 1959; Hickman- Brenner et al., 1991). The underlying principle of phage typing is the host specificity of bacteriophages (Threlfall and Frost, 1990). Phage typing schemes for Salmonella serovars are based on patterns of lysis produced by distinct phages isolated from a variety of sources (Threlfall and Frost, 1990). Phage typing has been used to subdivide isolates within serovars Typhi (Anderson and Williams, 1956), Typhimurium (Callow, 1959; Anderson et al., 1977), Enteritidis (Ward et al., 1987), Virchow (Chambers et al., 1987), Hadar (De Sa et al., 1980) and Heidelberg (Harvey et al., 1993). Additional phage typing schemes are available for other Salmonella; for example, the phage typing scheme of S. enterica serovar Bovismorbificans has been available from the Australian Salmonella Reference Centre for over a decade. Phage typing is based on the assumption that strains which are epidemiologically related will exhibit the same phage type (PT) while unrelated strains will have different PTs. For this to be valid, the phage typing system must have a high discriminatory power in the bacterial population where the system is applied and the PTs must demonstrate high stability within the population (Hunter, 1990; Olsen et al., 1993). Although phage typing is essential for the subdivision of Salmonella serovars, the method can prove inadequate for serovars in which a small number of phage types predominate. For example, phage type 8 tends to predominate in S. Virchow in Australia (Australian Salmonella Reference Centre, Annual Reports ). Phage typing can also be subjective in nature and relies on the experience of the operator and uniformity between laboratories (Harvey et al., 1993). Although phage typing is still used widely for epidemiological purposes, several studies including this one have shown that some PTs convert from one type to another. The mechanism for this is usually the acquisition of a temperate bacteriophage or a plasmid. Therefore phage type conversion could potentially have serious implications for epidemiology and organism tracing. The likelihood of this occurring is hard to determine, but it should be noted that bacteriophage are the most abundant biological entities. Phage typing relies on three factors: Host controlled restriction/modification. This is enzymic methylation of host DNA that protects the host genome from cleavage by host restriction endonucleases. It is a defence against incoming phage DNA can be seen as non-host and cleaved by restriction enzymes. Adsorption properties of the infecting phage (depending on the base plate); and Susceptibility of the infecting phage to repressors and to various superinfection exclusion systems controlled by residing pro-phages (Schmieger, 1999). It is this third factor that has been the focus of this project, since it is well documented that Salmonella often carry temperate or lysogenic phage Bacteriophage and Salmonella enterica Large numbers of temperate bacteriophage can be released from logarithmic cultures by addition or application of an inducing agent (Bradley, 1967). Mitomycin C and UV light are probably the best known of all inducing agents. Induction of bacteriophage has been correlated with the release of toxins (Isaacson and Moon, 1975; Farkas-Himsley et al., 1977; Gemski et al., 1978; Takeda et al., 1979; Yee et al., 1993). Head et al. (1988) demonstrated that induction of a bacteriophage carrying toxin genes resulted in increased copy number of the toxin genes and a 100 to 200 fold increase in toxin synthesis. It has been shown that 2

13 the stx genes of enterohaemorhagic E. coli are co expressed with genes of the bacteriophage and an increase in toxin production during bacteriophage induction has been observed (Head et al., 1988). Bacteriophages have played a critical role in the evolution and acquisition of virulence factors in many bacterial pathogens, including Salmonella enterica. Temperate bacteriophages often encode properties that alter the host bacterium following the establishment of lysogeny. Figueroa Bossi and Bossi 1999 discovered the Gifsy 2 prophage, which carries a sodc gene from S. Typhimurium, coding for a periplasmic superoxide (Cu, Zn) dismutase previously implicated in Salmonella virulence (De Groote et al., 1997; Farrant et al., 1997). Using a mouse model, the study demonstrated the ability of Gifsy 2 phage to transfer its virulence traits to formerly attenuated strains (Figueroa-Bossi and Bossi, 1999). Similarly, another S. Typhimurium P2-like SopE-encoding phage, designated SopEφ, has been discovered (Mirold et al., 1999). SopE is one of the effector proteins, translocated into the host cell by the SPI-1 (Salmonella Pathogenicity Island- I) type III secretion system. A temperate bacteriophage encoding type III effector protein SopE has been isolated in S. Typhimurium (Mirold et al., 1999). Even though SopEφ prophage could not be induced from all isolates tested, it was carried by all natural SopE + Salmonella isolates using Southern blot analysis (Mirold et al., 1999). Interestingly, S. Typhimurium strains DT49, DT204 and DT204c which have a history of causing major outbreaks in the United Kingdom and East Germany during the 1970s and 1980s, were amongst the natural SopE + isolates tested (Threlfall et al., 1978a; Threlfall et al., 1978b; Wray et al., 1998). Unlike other strains which were also widespread at the time, these three definitive type strains persisted over a long period of time causing massive infections in cattle and humans (Threlfall et al., 1978a; Threlfall et al., 1978b; Wray et al., 1998). The authors speculated that lysogenic conversion with bacteriophage SopEφ could have contributed to the persistence of S. Typhimurium DT49, DT204 and DT204c (Mirold et al., 1999). Furthermore, the discovery of the SopEφ prophage suggests the mechanism by which the effector proteins may be transferred to a susceptible bacterial pathogen bearing a type III secretion system, such as those found in all Salmonella enterica. There is a wealth of evidence that the evolution of pathogenic variants in several bacterial species results from the acquisition of defined elements, including bacteriophages. However, bacteriophages are limited in their capacity to carry exogenous DNA. The release of a phage particle and the formation of an active bacteriophage decreases with an increase in the size of the genome, which in turn can lead to defective phages (Dobrindt and Reidl, 2000). Such an event may lead to the formation of PAIs (pathogenicity associated islands). The research group at IMVS had observed for some time that certain phage types tended to predominate in S. Typhimurium and in 1998 work was begun to examine any temperate phages that could be carried by S. Typhimurium, in particular phage type or Definitive Type (DT) 64. It had been observed that S. Typhimurium DT 64 was often the most common phage type isolated from S. Typhimurium in chickens (29.3% in 1996 (second most common) 51.7% in 1997, 51.6% in 1998 and 49.1% in 1999). It was also a commonly isolated from humans (7.9% in 1996, 29.2% in 1997, 14.6% in 1998 and 2.6% in 1999). These figures are from The Australian Salmonella Reference Laboratory Annual Report As mentioned above, the carriage of lysogenic (or temperate) bacteriophages largely determines phage type. In 1998, the IMVS research team induced two lysogenic phages from S. Typhimurium DT64. The induced phage was capable of mediating phage type conversion. The two phages were related genetically, but also have unique sequence as judged by hybridisation. These phages were found in a number of other S. Typhimurium phage types. The two phages have been designated ST64T (top) and ST64B (bottom) according to where the phage particles band in a caesium chloride gradient. The ST64B phage was partially genetically characterised at the commencement of this project. The ST64T phage was yet to be characterised at the commencement of the current project. 3

14 1.3.3 S. Sofia and Australian chickens Salmonella Sofia currently represents 53.4% (2002 figures) of the approximately 2,500-3,000 chicken isolates of Salmonella submitted to the IMVS from the chicken industry for typing. However, it is almost never associated with human disease. Database searches also confirm that S. Sofia is seldom seen elsewhere as associated with human disease. Therefore Salmonella Sofia is not a pathogen for humans or chickens but it is an effective coloniser of chickens. ( Annual Report, Australian Salmonella Reference Centre, Institute of Medical and Veterinary Science, Adelaide). In a previous project (IMVS-1A) the IMVS research team has demonstrated that certain S. Sofia strains are better colonisers of chickens and appear to be able to persist in the chicken even when challenged with S. Typhimurium. This raises the possibility that S. Sofia may decrease the level of colonisation of chickens with S. Typhimurium. Indeed, one strain S. Sofia MH76 was able consistently to colonise chickens up to day 36 even when challenged with S. Typhimurium. It is possible that earlier colonisation of chicks with this strain might result in a decrease in S. Typhimurium colonisation. The testing of this idea was one of the objectives of the current study. In the previous study, the question was asked: what is the molecular mechanism of S. Sofia colonisation? A PCR survey (carried out in previous project IMVS-1A) of the fimbrial genes in S. Sofia hoped to partially answer this question. The PCR survey identified the fimbrial genes that are widespread among S. Sofia strains. Filamentous fimbrial structures on the cell surface of bacteria can be essential for efficient colonisation of a host. This PCR evidence coupled with sequence data confirmed the presence of fimbrial genes on the S. Sofia chromosome. The agfa gene appears to be universally found in S. Sofia isolates, where other genes are less widespread. Therefore the agfa gene was inactivated by insertion of a kanamycin resistance cassette (880 bp). This results in the disruption of the gene preventing its expression. The colonising ability of the resulting strain K4 was tested in the current project for its colonising ability. The more than two decade persistence of S. Sofia in Australian chickens as the predominant Salmonella is a unique situation and not seen elsewhere in the world. However, it has been reported that strains from Israel are human pathogens, although this observation has never been published and must be treated with caution. This begs the question: is there something special about Australian S. Sofia that can explain what is seen in Australia? Is Australian S. Sofia different from the Israeli strains? Modern genetic fingerprinting methods such as PFGE and AFLP can answer this question. Examination of S. Sofia strains isolated over 20 years from both here and overseas forms part of the second objective of the current project in the validation and implementation of AFLP as a sub-typing tool. 4

15 2. Typing of Salmonella Isolates 2.1 Conventional typing During the course of the project the chicken industry has sent strains to The Australian Salmonella Reference Centre in Adelaide for conventional typing. The numbers received are listed below: July 2000 June ,641 July 2001 June ,137 July 2002 June ,233 This represents an average of 2,670 samples per annum over the course of the project. This is a similar number to the average 2,844 samples per annum submitted for the three years as presented in the previous project IMVS-1A. This suggests that the numbers of isolates received by the reference centre are reasonably stable. It is known that industry laboratories are carrying out initial screening for S. Sofia to reduce the number of strains sent to the reference centre. In spite of this, S. Sofia remains the most common serovar isolated from Australian chickens as determined by the reference laboratory. S. Sofia first appeared in significant numbers in 1980 and steadily increased to the point where it represents around 50% of all Salmonella isolates and now appears to have stabilised at this level. The reasons for this are unclear but are examined elsewhere in this report. Figure 2.1 illustrates the two-decade persistence of this organism in chickens. Percentage of S. Sofia of total Salmonella in chickens Figure 2.1 The percentage of S. Sofia in the total Salmonella isolates from chickens submitted to The Australian Salmonella Reference Centre from There is nothing of significance to report with respect to changes in Salmonella serovar distribution or incidence in isolation from humans and food animals, in particular, chickens. This is illustrated in Table 2.1. In previous reports it has been reported that S. Virchow, a serovar traditionally associated with chickens in Queensland, is being isolated with increasing frequency in the southern states. This is being investigated further. One large Salmonella outbreak during 2001, in which chicken was implicated, impacted upon the poultry industry during the course of the project. A South Australia outbreak due to Salmonella Typhimurium phage type 126, totalling 87 cases occurred between May and November A number of the cases had mixed infections with both Salmonella and Campylobacter. Human cases within the human population were traced to raw poultry from one major supplier. While Year 5

16 commercially cooked products were not implicated, the outbreak has once again raised the industry s and regulator s awareness of chicken meat as a potential source of Salmonella in the food chain. It has been a considerable time since the last outbreak identified from this source. Outbreaks associated with products used or produced in catering establishments have occurred with S. Typhimurium phage types 8, 64var, 99 and 135a in Eggs were investigated as a source of Salmonella in these outbreaks. However, no epidemiological links to the outbreak were established. In 2002 surveys of raw chicken meat collected from retail premises in South Australia confirmed the ongoing dominance of S. Sofia in raw chicken meat. S. Typhimurium phage type 126, which had been the cause of a large community outbreak in South Australia in 2001 (discussed above), was not isolated. Table 2.1 Most common Salmonella serovars from major sources HUMAN (1796) (1772) (1453) Typhimurium 30.6% Typhimurium 33.6% Typhimurium 29.2% Bovismorbificans 10.3 Bovismorbificans 11.3 Bovismorbificans 8.3 Enteritidis 8.7 Enteritidis 6.4 Enteriditis 7.5 Heidelberg 8.2 Heidelberg 5.3 Saintpaul 3.9 Ball 3.1 Chester 2.5 Heidelberg 3.6 Saintpaul 2.8 Virchowl 2.4 Mgulani 3.2 Chester 2.6 Stanley 2.2 Virchow 3.1 Virchow 1.7 Infantis 2.1 Muenchen 2.8 CHICKEN (2204) (2190) (1925) II Sofia 53.4% II Sofia 36.2% II Sofia 56.9% Typhimurium 24.0 Typhimurium 35.8 Typhimurium 20.1 Infantis 4.4 Bovismorbificans 4.4 Kiambu 5.1 Chester 2.2 Kiambu 4.0 Virchow 4.5 Agona 1.8 Virchow 3.8 Saintpaul 1.9 Bovismorbificans 1.6 Infantis 2.9 Bovismorbificans 1.4 Kiambu 1.6 Mbandaka 2.6 Agona 1.1 BOVINE (705) (251) (199) Anatum ** 34.0% Bovismorbificans 46.6% Bovismorbificans 40.7% Bovismorbificans 11.6 Typhimurium 25.1 Typhimurium 28.1 Give** 8.7 Dublin 17.1 Dublin 18.1 Senftenberg 8.5 Newport 2.4 Muenchen 2.0 Typhimurium 7.0 Orienbergr 1.2 Kottbus 2.00 PORCINE (105) (238) (21) Infantis 21.9% Bredeney** 48.7% Heidelberg 23.8 Derby 20.0 Derby** 31.5 Anatum 14.3 Bredeney 14.3 Senftenberg** 5.9 Ohio 9.5 Anatum 10.5 Heidelberg 2.1 Typhimurium 9.5 Typhimurium 7.6 Typhimurium 2.1 Anatum var Anatum 1.3 RAW MEATS (112) (29) (27) Muenchen 21.4% Bovismorbificans 31.0% Chester 25.5% Havana 17.0 Heidelberg 13.8 Derby 17.0 Bovismorbificans 8.9 Typhimurium 10.3 Bovismorbificans 12.8 II Fremantle 7.1 Orion 6.9 Bredeney 8.5 Chester 5.4 Rubislaw 6.9 Typhimurium 6.4 Typhimurium 5.4 Notes: ( ) = total isolates. ** These strains are from large surveys and will influence strain distribution. Most common serovars are given, hence totals in some cases do not equal 100%. These figures are from the 2002 annual report of the Australian Salmonella Reference Laboratory. Over the course of the project, the phage type distribution within S. Typhimurium human and chicken isolates has changed dramatically. This is demonstrated in Table 2.2. When this study was 6

17 initiated in 2000, S. Typhimurium DT64 (phage-type 64) was the most common phage type in chickens and a significant phage type in humans. Other phage types of significance in both humans and chickens were types 9 and 135. These phage types had been significant also in the years preceding 2000 and because of this, the study of these phage types and the relationships between them forms part of this report (see Chapter 3). In 2001, as previously mentioned, phage type 126 was implicated in a large outbreak and has become a frequently isolated phage type in both humans and chickens, this trend has continued into Most notably, the previously important phage type 64 is a relatively minor isolate from both chickens and humans. Table 2.2 Percentage of commonly identified S. Typhimurium phage types isolated from chickens from , as a percentage of total Salmonella isolated from all sources (Source: ASRC Annual Report 2002). Year Source and Phage Type Human Chicken < < < <6.1 < < < < < < <2.1 < <2.5 Notes: In 2002, phage type 126 was the second most common S. Typhimurium phage type isolated from chicken; phage type 41 (not shown) was the most common S. Typhimurium phage type in chickens, occurring at a frequency of 16.3%. In contrast to S. Typhimurium, S. Virchow phage type distribution has not markedly changed in the period For both chickens and humans phage type 8 has predominated, together with phage type 34. Although other phage types are seen, they are not consistently isolated from year to year. In 2001 The Australian Salmonella Reference Centre began testing for antibiotic resistance in Salmonella isolates. A table that illustrates the percentage of serovars resistant to four or more antibiotics isolated from chickens is shown in Table 2.3. More detailed analyses can be found in the annual reports of The Australian Salmonella Reference Centre 2001 and 2002 Annual Reports. 7

18 Table 2.3 Most common Salmonella isolates from chicken (2001) and percentage of each resistant to four or more antibiotics. Serovar % of total isolates % strains resistant to four or more antibiotics Subsp II Sofia Typhimurium Bovismorbificans Kiambu Virchow Infantis Mbandaka Agona Singapore Saintpaul Notes: From Davos "Antibiotic Sensitivity Profiles of Salmonella", in the proceedings of the 7th WPSA Asian Pacific Federation / 12th Australian Poultry and Feed Convention, Gold Coast, Qld Improvement of PFGE During this and previous projects a reliable PFGE (Pulsed Field Gel Electrophoresis) analysis procedure has been instituted, which has been used extensively in the analyses of Salmonella related outbreaks. This is a lengthy, labour intensive procedure that takes up to six days to complete. In the current project an alternative methodology designed to obtain a result in three days was investigated. This rapid procedure is based upon work by Matushek et al. (1996). The RFLP banding patterns obtained using the more rapid procedure were of comparable quality to those obtained using the original and slower procedure, but in general proved less reliable than the method of Maslow et al. (1993b). In view of these disadvantages this method has been abandoned in favour of the Maslow et al. (1993b) method described in Section 3 of this report. 2.3 Random amplification of polymorphic DNA (RAPD) The RAPD procedure involves the use of short primers of arbitrary sequence in a rapid PCR reaction conducted at low stringency to generate a band pattern which is visible on an agarose gel after gel electrophoresis. Amersham-Pharmacia Ready to Go RAPD analysis beads were tested to determine whether they could be used as a rapid alternative to phage and PFGE typing. The advantage in using this commercial kit is that all reagents, excluding the primers, are in tablet form and this makes the procedure very simple and easy to perform. The pilot study included S. Virchow strains for testing. It was found that there was no correlation between phage type and RAPD pattern. This is a similar observation to what had been observed with PFGE, where there was no correlation between phage type and PFGE pattern (see Final Report for RIRDC project IMVS-1A). In both cases for example, multiple patterns were observed for phage type 8 (the most common S. Virchow phage type). It was also shown that distinct phage types could have identical RAPD and PFGE patterns. As with all RAPD techniques, reproducibility is a significant problem over time. In view of this drawback, the RAPD technique, despite the convenience of the tablet form of the reagents, was not retained. 8

19 3. Amplified Fragment Length Polymorphism Typing 3.1 Introduction The purpose of this component of the project was to investigate the feasibility of amplified fragment length polymorphism (AFLP) typing as an epidemiological tool to replace or complement PFGE as a routine molecular typing method. AFLP was to be used in conjunction both with PFGE and classical typing methods (ie, serotyping and phage typing) to establish levels of discrimination between isolates, both individual strains and those suspected to belong to outbreaks. AFLP was tested on serovars of Salmonella of importance to the chicken industry, either serovars that are routinely associated with chicken products, or serovars associated with outbreaks. The most common serotype isolated from chickens in Australia is the presumably non-pathogenic Salmonella enterica subspecies II serovar Sofia (S. Sofia). In terms of public health, Salmonella enterica subspecies I serovar Typhimurium (S. Typhimurium) is often associated with food-borne gastroenteritis caused by Salmonella. The more than two decade persistence of S. Sofia in Australian chickens as the predominant Salmonella is a unique situation and not seen elsewhere in the world. However, it has been reported that strains from Israel are human pathogens, although this observation has never been published and must be treated with caution. The research undertaken sought to address the question whether there is something special about Australian S. Sofia that can explain what is seen in Australia and also whether Australian S. Sofia is different from the Israeli strains. As an initial study, fluorescent AFLP (FAFLP) was performed on 68 isolates of S. Sofia that had been isolated over a 30-year period. The results obtained from this study were compared to PFGE results in order to ascertain levels of discrimination between the two systems and also to determine whether a unique Australian clone of S. Sofia exists. The study was then extended to include serovar Typhimurium DT126 strains from an outbreak. 3.2 Materials and methods Strains All Salmonella isolates were obtained from the Australian Salmonella Reference Centre (ASRC), Institute of Medical and Veterinary Science, Adelaide, South Australia. Serotyping had previously been undertaken using the Kaufmann-White scheme and bacteriophage typing was performed using the Anderson scheme of 31 phages both by the ASRC Fluorescent AFLP (FALP) Overnight cultures of isolates were prepared and total genomic DNA was extracted from 10 mls of bacterial culture. The FAFLP was based on the MseI/EcoRI protocol developed by Vos et al. (1995). The FAFLP restriction endonuclease digestion/ligation and preselective and selective PCR steps were undertaken by the method described in the AFLP Microbial Fingerprinting Protocol Handbook (PE Applied Biosystems, Foster City, California). Adaptor sequences with MseI and EcoRI ends as well as primers for the preselective and selective PCR reactions including the 5 - labelled EcoRI selective primer are described in Table 3.1. PCR fragments were analysed with an ABI Prism 377 Sequencer (Applied Biosystems). Briefly, 1.8 µl of selective PCR product was added to an equal volume of GeneScan-1000 size standard and run under denaturing conditions on the sequencer. Fragments were resolved on a 36 cm, 5% Long Range/6M urea gel at 51 o C. Computer analysis of the resolved fragments was undertaken with the GeneScan Analysis v3.1 program (Applied Biosystems). 9

20 Table 3.1 Oligonucleotides and primers used in this study. Oligonucleotide Restriction Endonuclease Sequence (5-3 ) Ligation Adaptors Preselective Primers Selective Primers EcoRI (forward) EcoRI (reverse) MseI (forward) MseI (reverse) EcoRI MseI EcoRI MseI CTCGTAGACTGCGTACC AATTGGTACGCAGTCTAC GACGATGAGTCCTGAC TACTCAGGACTCAT GACTGCGTACCAATTC GATGAGTCCTGAGTAA (FAM)-GACTGCGTACCAATTCA GATGAGTCCTGAGTAAA Notes: The primers used in this study are based upon Vos et al. (1995). The selection of specific fragments for 2 nd round (selective) amplification and labelling is determined by the addition of a single A (bold) on the 3 end of each selective primer as well as the fluorescent 5- carboxyfluroscein label (FAM) linked to the 5 end of the EcoRI selective primer Data analysis of FAFLP Spreadsheet data generated by the GeneScan Analysis program was converted to PC format and opened in Microsoft Excel with file origin as DOS or OS/2 (PC-8). The Minutes and Data Point columns were deleted and the files reformatted for eventual analysis by GelCompar IV software. These files were then saved as Text (OS/2 or MS-DOS) files. Files were then converted via the Molecular Weight to Gel program and saved as.int files prior to analysis in GelCompar IV. Comparison or relatedness of isolates was undertaken by a dendrogram generated by Cluster by Bands with relatedness between isolates determined by Dice coefficient and clustering by UPMGA. Chromatogram files generated by the sequencer in Macintosh format were converted to PC format and given a.fsa extension. Files were imported into Genotyper ver. 3.6 NT (PE Biosytems) (Windows 2000 NT operating system) for detailed analysis of peaks Pulsed-field gel electrophoresis The protocol for PFGE followed that of Maslow et al. (1993b). Plugs were prepared by mixing washed cells from an overnight culture with an equal volume of melted 1.5% low melting point agarose. An aliquot was poured into a mould and allowed to set at 4 o C. Cells were lysed by incubating the plug in 4 mls lysis buffer supplemented with 4 mg lysozyme and 80 µg.ml -1 RNAse and incubating overnight at 37 o C with gentle shaking. Plugs were then proteinase K-digested by transferring the plug to 4 mls ES buffer supplemented with 100 µg.ml -1 proteinase K and incubating for approximately 12 hours at 50 o C with gentle shaking. The buffer was then removed and replaced by 4 mls fresh ESP buffer and incubated as before for another 12 hours. A small piece of plug suitable to fit into the well of the pulsed-field gel was washed four times with 1 ml fresh TE buffer at 37 o C (2 x 2 hour washes followed by 2 x 1 hour washes). A restriction digest solution was prepared comprising (per 100 µl) 20 U XbaI restriction endonuclease, 1 µl 10 mg,ml-1 bovine serum albumin, 10 µl 10x appropriate buffer and the volume taken up to 100 µl with water. The plugs were placed in 100 µl restriction digest solution and incubated overnight at 37 o C. The next day the plugs were placed into the wells of a 1% pulsed-field gel made with PFGE-grade agarose in 0.5 x TBE buffer. Staphylococcus aureus strain NCTC 8325 digested with SmaI was used as a control and marker strain for normalisation of gels during post-electrophoresis analysis. The pulsed-field gel electrophoresis was run on a BIO-RAD CHEF-DR III System for 19 hours at 6.0 V.cm -1 at 4 o C, initial switch time 2 seconds, final switch time 50 seconds. After running the gel was stained in ethidium bromide destained in water and photographed under UV light. 10

21 3.2.5 Computer analysis of PFGE A photograph of the gel was scanned into a computer and a tif file generated. The image was optimised in GelCompar IV software (Applied Maths, Kortrijk, Belgium) and normalised using the S. aureus marker control as per the program guidelines. A dendrogram showing relatedness of isolates was generated with similarity by Dice coefficient and clustering determined by UPMGA as for FAFLP analysis described above. 3.3 Results FAFLP of S. Sofia All 68 isolates of S. Sofia were successfully typed by the FAFLP method. A dendrogram of relatedness indicated that most isolates clustered into four separate groups (A-D) with >90% similarity between isolates within each group (Figure 3.1). All but seven isolates exhibited >85% similarity to the other S. Sofia isolates. The remaining seven isolates showed >70% homology to all other isolates. To test the reproducibility of this method, a subset of 15 isolates were selected at random and retested from the pre-selective PCR step onwards. Amplified fragment patterns generated in each instance exhibited >90% similarity to the original patterns for each isolate retested (data not shown). This was a result similar to that reported by Huys et al. (1996). For the 68 isolates there was no unique group that could be assigned to a specific geographical, temporal or source of isolation. Groups B and C contained the most isolates (26% and 47% of total isolates respectively) PFGE of S. Sofia The 68 S. Sofia isolates were also subjected to PFGE analysis (Figure 3.2). Less than 50% of the S. Sofia isolates tested were typable by this method FAFLP of S. Typhimurium DT126 A total of 92 S. Typhimurium DT126 isolates were analysed by the EcoRI(A) and MseI(A) FAFLP selective primer combination. All isolates were typable by this method. Isolates which clustered with >90% similarity as determined by Dice coefficient and UPMGA were considered to be genetically closely related (Figure 3.3). Three main clusters containing at least three isolates each were extrapolated from the dendogram. The cluster with the most isolates was group A1 (34 isolates) with the very closely related group A2 with 31 isolates. These two closely related groups contained most of the isolates attributed to two outbreaks that occurred in South Australia; one which occurred in the early months of 2001 (subsequently referred to as outbreak 1) and the second which occurred in the middle of the same year (subsequently referred to as outbreak 2). As well as containing most of the outbreak isolates these two clusters contained 69 epidemiologically unrelated isolates from most populated regions of Australia. These isolates were predominantly obtained from clinical patients or from chickens and chicken meat-related industries although five were from nonpoultry domestic animals. 11

22 % Similarity Isolate Date Source Locale i Sofia 45 pre 1992 Chicken S.A. Sofia 91 pre 1992 Chicken S.A. Sofia Chicken Israel Oct 2001 Chicken litter N.S.W Oct 2001 Chicken litter N.S.W Oct 2001 Chicken litter N.S.W Oct 2001 Chicken litter N.S.W. Sofia 44 pre 1992 Human N.T. Sofia Chicken W.A. Sofia 50 pre 1992 Avian unknown Sofia Chicken S.A. Sofia Chicken Vic. Sofia Chicken S.A. Sofia Human Israel Sofia Chicken S.A. MH89 pre 1992 Chicken S.A. Sofia 74 pre 1992 Dog N.S.W. Sofia Chicken S.A. Sofia 20 pre 1992 Human S.A Oct 2001 Chicken litter N.S.W. Sofia Chicken N.S.W. Sofia 51 pre 1992 Avian unknown Sofia Chicken Israel Sofia 121 pre 1992 Avian unknown Sofia Human Israel Oct 2001 Chicken litter N.S.W. Sofia 23 pre 1992 Meatworks N.T. Sofia 4 pre 1992 Chicken S.A Oct 2001 Chicken litter N.S.W. Sofia 79 pre 1992 Cooked N.S.W. Sofia Human Israel Oct 2001 Chicken meat N.S.W. Sofia Chicken Israel Oct 2001 Chicken meat N.S.W. Sofia 41 pre 1992 Water S.A. Sofia 78 pre 1992 Crocodile N.T. Sofia 17 pre 1992 Human N.T. Sofia Chicken S.A Oct 2001 Chicken litter N.S.W. MH76 pre 1992 Human S.A. MH76NA Lab-generated Nx r mutant of MH76 Sofia 71 pre 1992 Chicken Vic. Sofia Human Israel Sofia Chicken Vic Oct 2001 Chicken N.S.W. Sofia 43 pre 1992 Abbatoirs N.T. Sofia Chicken Vic. Sofia 77 pre 1992 Human S.A. Sofia 19 pre 1992 Human W.A. Sofia 16 pre 1992 Chicken meal Vic. Sofia Chicken Vic. Sofia Chicken Israel Sofia VIC pre 1992 unknown unknown Sofia 107 pre 1992 Chicken S.A. Sofia Chicken Vic. Sofia Chicken N.S.W. Sofia 22 pre 1992 Human S.A Oct 2001 Chicken litter N.S.W. Sofia Chicken unknown Sofia Human Israel Oct 2001 Chicken litter N.S.W Oct 2001 Chicken litter N.S.W. Sofia Chicken Qld. Sofia Chicken N.S.W. Sofia Chicken S.A. Sofia 122 pre 1992 unknown unknown Sofia 75 pre 1992 Effluent N.S.W. Sofia Chicken Israel A B C D Figure 3.1 A dendrogram showing the relationship between isolates of S. Sofia as determined by FAFLP. Each group of isolates with >90% identity are indicated on the right. Note: (i) letters in brackets after locale indicate isolation from a specific farm or processor. 12

23 Figure 3.2 Degradation of S. Sofia DNA during pulsed-field gel electrophoresis. Notes: When DNA from S. Sofia strains is resolved by PFGE there is a high frequency of isolates where DNA is degraded. The degradation may be due to a number of reasons but is often attributed to DNAase activity during cell lysis or by the action of TRIS radicals during the electrophoresis step. The three marker lanes (S. aureus) show what a good banding should look like. Based on these criteria, only two S. Sofia isolates, Sofia 139 and Sofia Vic would be typable by this method (isolate MH75 may be typable but the level of background material may mean that weaker bands may be missed). 13