Annex 2: Form for Project

Annex 2:

CSG7 FORM FOR PROJECT

Department for Environment, Food & Rural Affairs CSG 7 (Rev 6/01) 1 For DEFRA Use Only Chief Scientist’s Group, Cromwell House, Dean Stanley Street, London SW1P Proposal Code OZ0321ac 3JH Date received Telephone No. 020 7238 6000 Application for a Research Contract with DEFRA  Applicants should complete each part of the form as fully and clearly as possible.  To move from one fill-in location (field) to another, press the UP or DOWN arrow or click the location, unless directed otherwise.

GENERAL

Proposer’s full Tel. No. (incl. Dr Robert Davies 01932 357361 1. name and title STD code)

Position held Leader: Enterobacteriaceae Surveillance Fax No. 01932 357595 Section

E-mail address [email protected]

2. Name and Veterinary Laboratories Agency address of Woodham Lane organisation New Haw Addlestone Surrey Postcode KT15 3NB

3. (a) Project title (maximum 120 characters) Investigation of the role of environmental contamination in the epidemiology of Salmonella infection in egg-laying flocks

(b) Abstract of research. To include the main objective, policy relevance and intended use of results. This table will expand to accommodate the information you wish to enter. To move to the next field, press the DOWN arrow TWICE. In response to Research Requirement R9 and communications re extension to allow longitudinal studies of positive farms identified in the EU Layer Survey.

Salmonella has been shown to be capable of prolonged survival in the environment and has been associated with persistent and recurrent infections on poultry (especially on multistage sites for egg production) and pig farms as well as in hatcheries and feedmills. Although Salmonella contamination may be present this is not always associated with acquisition of infection by animals placed in the contaminated housing and other vectors such as rodents and arthropods may also be involved. There may also be as yet unknown management and stress factors, which could contribute to the risk. Although reported cases of Salmonella have been falling in humans and in poultry it is not certain if this will be sustainable, especially if a new epidemic strain similar to S.Enteritidis or S.Typhimurium DT104 emerges in the egg industry. Achievement of Government targets for a 20% reduction in foodborne disease could be compromised by a new major egg-borne epidemic. The objective of this work is to gather as much information as possible about the risks of carry-over of Salmonella infection in the various sectors of the layer industry by a combination of approaches: 1. Investigations of carry-over of Salmonella into flocks where infection has been found in previous flocks. This work will include the various production categories in the commercial sectors of egg production. These investigations will be supported by the use of molecular genetic typing of strains. Persistently infected sites will be visited and sampled both qualitatively in an intensive manor and quantitatively for key sample types. 2. Laboratory investigation of the phenotypic and genetic characteristics of persistent strains of Salmonella in terms of characteristics such as survivability, disinfectant resistance, acid and heat tolerance and virulence. 3. Exposure experiments in which chickens would be exposed to known levels of Salmonella on environmental materials and the influence of different periods of environmental survival, under different conditions, on infectivity. The results of this work will be used to formulate improved advice for DEFRA on the need for changes in policy or Codes of Practice on egg production, and practical advice for poultry company veterinarians and quality managers. Results will be communicated via internal reports, scientific papers, presentations at specialist poultry disease meetings and targeted articles in the poultry farming press so that new knowledge can be distributed as widely and effectively as possible. (c) Total cost to DEFRA (ex. VAT) £464,532 (d) Date submitted to DEFRA 4th January 2005

CSG 7 (Rev 6/01) 2 SECTION ONE - SUMMARY

4. (a) Sub-contractor’s name(s) and address(es) (if applicable):

Postcode Postcode

(b) Joint contractor’s name(s) and address(es) (if applicable: Dr Vivien Allen Department of Clinical Veterinary Science Division of Food Animal Science University of Bristol, Langford, Avon

Postcode BS40 5DU

CSG 7 (Rev 6/01) 3

Postcode

5 (a) DEFRA reference under which proposal has been submitted e.g. DEFRA Animal Health and Welfare Central Strategic Research Fund or other open competition Requirements Document advertisements, Food Research Requirements or ROAME A 2002-2003 : Open Competition R9 forms (i.e. DEFRA customer guidance on the problems to be addressed and the required objectives of the research).

(b) Duration in years (or months 5 years (d) Proposed start date 01 October 2002 if less than one year)

6. Summary of total estimated costs (excluding VAT). This should include the costs of the research work which will be funded by (a) DEFRA, (b) Bodies other than DEFRA, (c) ‘in kind’ contributions as a cash value, as appropriate. Funding bodies Project Project Project Project Project Total Year 1 Year 2 Year 3 Year 4 Year 5 (£) (a) DEFRA £91,134 £98,808 £108,659 £79,864 £86,067 £464,532 (b) other than DEFRA

TOTAL COST £91,134 £98,808 £108,659 £79,864 £86,067 £464,532 N.B. Applicants are advised to clear the costs at question 21 with their respective Finance departments and to agree the value of any ‘in kind’ contributions with those involved with the work before completing this summary.

CSG 7 (Rev 6/01) 4 SECTION TWO - SCIENCE DEFRA funds research in support of its policy requirements. These are described in the DEFRA R & D Strategies, and individual programme objectives may be described in more details in ROAME A’s or in documentation supporting advertised calls for proposals. 7. Purpose. Summarise the scientific or technical problem which you propose to address and give reasons why DEFRA support should be given. This table will expand to accommodate the information you wish to enter. To move to the next field, press the DOWN arrow TWICE. Salmonella, particularly S.Enteritidis PT4, contamination of shell eggs has been a major problem in the egg industry in most developed countries where an intensive egg industry has developed. Although the origin of the epidemic strain of PT4 is uncertain it is thought most likely to have originated as a feed contaminant which became established within primary poultry breeding companies and was then disseminated world-wide by international trade. Since the introduction of the Poultry Breeding Flocks and Hatcheries Orders 1989 and 1993 there has been minimal evidence of S.Enteritidis infection at elite and grandparent level in chicken breeding flocks but the industry has been left with widespread persistent environmental contamination of commercial flocks. The practical and economic difficulties of controlling infection are substantial, particularly on large multi-age cage layer sites. Prompted by a worsening of the infection level in commercial laying flocks after these were removed from legislation (Poultry Laying Flocks Order 1989) in 1993, the egg industry, largely in response to commercial pressure from key major retailers introduced the 'Lion' and 'Laid in Britain' codes. The former code involving improved laying house and egg hygiene combined with vaccination appears to have been associated with a fall in S.Enteritidis reports from laying flocks and in human S.Enteritidis cases and outbreaks. Unfortunately it has not so far been possible to independently verify this improvement but planned surveys of shell eggs at retail and of commercial laying flocks, if set up appropriately using techniques and laboratories comparable with earlier surveys, should assist with quantifying the current problem. Studies at VLA in MAFF Project OZ0317: Epidemiological Investigations of Salmonella contamination in table egg production; have focussed on detailed investigations of commercial laying flocks and layer breeder flocks where S.Enteritidis has been identified by passive surveillance or as a result of human food poisoning outbreaks. These studies have shown that although vaccination can help reduce the level of S.Enteritidis contamination in laying flocks and their environment it is not fully effective in multi-age cage layer sites. Moreover the standard of cleaning and disinfection and rodent control on most farms leaves a great deal to be desired and is inferior to that of the broiler industry. The work planned in this proposal will supply much needed additional information on the role of persistent environmental contamination in egg production by building on the strong foundation of experience VLA has gained in project OZ0317. The complementary experience of Prof. Tom Humphrey, who is the leading authority on Salmonella contamination of eggs, will provide additional added value. The experience of Dr Vivien Allen with controlled poultry Salmonella infections will also be extremely valuable. The work will gather quantitative evidence on Salmonella levels in the environment of cage layer, barn egg production and free-range flocks. The work will evaluate more fully the role of mice and flies, will use powerful molecular genetic tools to map the dissemination of particular clones of S.Enteritidis around the farm environment and will supplement this fieldwork with experimental work on the strains involved. In particular their ability to survive on environmental surfaces and in competition with other bacteria in the environment, resistance to disinfection and other adverse conditions, phenotypic and genotypic stress survival response characteristics and virulence potential after exposure to environmental stresses will be evaluated. The work will also allow more prolonged follow-up of persistently infected farms identified in project OZO317, in particular the effect of the introduction of the new live vaccine on these farms. Purpose in relation to extension work Whilst Project OZ0321 has been in progress there have been a number of developments which have taken place which affect the situation re Salmonella in table egg production. Firstly, several new live vaccine options have been introduced and vaccinated progeny are in rear or early production now. The current end date of OZ0321 does not permit an evaluation of the effect of introduction of these vaccines into persistently infected farms. Secondly, at relatively short notice, the EU baseline prevalence survey for Salmonella in laying flocks began end October 2004. This will run for one year and should identify many more infected flocks than current monitoring. This offers an opportunity to investigate positive farms, especially non-cage systems, which have been underrepresented in the current study to determine the nature of persistence of infection and the effectiveness of control measures. Thirdly, the EU survey is compulsory, and has the backing of the main egg industry representative bodies (BEIC, UKEPRA) which should improve the acceptance of farm based research studies to egg producers. To investigate persistence in two consequent flock cycles at least two years is needed since a typical laying flock is maintained for 52 weeks or longer. For this reason an extension to OZ0321 of 2 years has been requested to complete these studies. This package of work conforms with the recommendations of the ACMSF Second Report on Salmonella in Eggs and should provide DEFRA with the information required to formulate improved control policies, advice and Codes of Practice for Salmonella in egg production.

CSG 7 (Rev 6/01) 5 8. Scientific context. Please describe how your proposal relates to the current state of knowledge (full reference, see Annex B) and in which ways the results will advance scientific/technical understanding. To move to the next field, press the DOWN arrow TWICE. In the mid 1980s a rapid rise in human cases of S.Enteritidis Phage Type (PT) 4 and a limited range of other, largely related, phage types associated with the consumption of eggs occurred in many counties (St Louis 1988, Anon 1989). Despite controls introduced in the late 1980s and early 1990s the number of human cases continued to rise (Anon 1990, Ebel and others 1992, Hogue and others 1997b) and there was no improvement in the prevalence of eggs contaminated with S.Enteritidis between 1991 and 1995/6 (De Louvois 1997). The ability of certain more invasive S.Enteritidis strains to selectively colonise and persist in the ovary and oviduct has been the predominant factor in the development of the epidemic (Humphrey et al 1996, Jorgensen et al 2000, Timoney and others 1989, Barrow 1991, Gast 1994, Keller and others 1997, Gast and Holt 1998). Transovarian infection and vertical transmission of infection was also highly instrumental in the rapid spread of the organism within poultry breeding pyramids and by international trade in breeding stock (Lister 1988, Nakamura and others 1993, Baumler and others 2000, Laconcha and others 2000), although the current controls implemented in the United Kingdom on breeding flocks should ensure that the commercial day old chicks are free from S.Enteritidis when placed on farm. Although the prevalence of contaminated eggs on retail sale has been low, very large numbers are eaten (Anon 1993, Gast and Holt 2000a) in a lightly cooked or raw state. Similarly, numbers of Salmonella inside eggs are usually low but occasional eggs with higher numbers are laid, especially in the early stages of flock infection (Bichler and others 1996) or when birds are under stress, especially at the end of the life of the flock (Nakamura and others 1994). Low numbers of S.Enteritidis within eggs may also multiply at higher temperatures, (Humphrey 1990, 1994, Humphrey and Whitehead 1993, Kim and others 1989, Saeed and Koons 1993) so a combination of high initial Salmonella numbers in an egg, poor temperature control and use of such eggs in a mixed food that is consumed by several people is likely to be the cause of most outbreaks (Wall and Ward 1999). In Great Britain improved biosecurity and hygiene in the poultry industry and vaccination of the majority of commercial laying birds and broiler breeders, introduced in the mid to late 1990s, has been followed by a large reduction in reported incidents of S.Enteritidis in poultry and in humans (Anon 2000a). Despite this S.Enteritidis associated with consumption of contaminated eggs is still the predominant cause of Salmonella outbreaks in humans in Great Britain and many other countries (Kist and Frietag 2000, Palmer and others 2000). The cost to society in terms of illness, mortality and lost productivity associated with egg-borne S.Enteritidis is considerable (Bender and Ebel 1992, Anon 2000b). Despite this there has been little research into the extent and characteristics of S.Enteritidis infection on laying farms in Great Britain. In the United States of America it has been estimated that 45% of laying flocks may be infected with S.Enteritidis (Hogue and others 1997a). Anecdotal evidence suggests that a similarly high level of flock infection may have occurred in Great Britain before the start of the voluntary vaccination policy, but it has not been possible to independently assess this. To maximise the control of S.Enteritidis in egg production it is necessary to institute effective measures at each stage of the egg production and distribution chain (Anon 1998), however there has been relatively little on- farm research, especially in Great Britain (Leslie 1996). The accurate identification of infected flocks is a pre- requisite for the control of S.Enteritidis (Hoop 1997). Various methods of monitoring poultry flocks for S.Enteritidis have been described. In laying flocks the use of an ELISA to identify antibodies in serum or egg yolk offers a convenient means of detection but this cannot be used in vaccinated flocks (Nicholas and Cullen 1991, Gast and others 1997, Feld and others 2000). It is possible to sample individual birds but cloacal swabbing is not effective for detecting a low prevalence of infection (Bichler and others 1996). Post-mortem examination of cull birds or spent hens is laborious and expensive and small scale environmental sampling has been shown to give equivalent sensitivity to testing 50 caecal samples at slaughter (Kingston 1981). Environmental sampling has been shown to be an accurate indicator of the presence of Salmonella in poultry flocks (Kradel and Miller 1991, Poppe and others 1992, Waltman and others 1992). A high level of contamination of certain elements of the environment can also act as an indicator of factors which may contribute to the spread of infection, eg. high available water levels in litter due to poor drainage and humidity control may lead to increased spread of Salmonella, cloacal infection and egg contamination (Keller and others 1995, Mallinson and others 1997, Smith and others 2000). Similarly a high level of Salmonella in accumulated dust, due to inefficient ventilation may be an indicator of an increased risk of spread of infection by respiratory or conjunctival routes, which increases the risk of systemic disease and egg infection (Chart and others 1992, Humphrey and others 1992, Leach and others 1999). The work described in this proposal will assess these risks by quantifying the numbers of Salmonella persisting in the environment of laying flocks and exposing chickens to these materials. Environmental monitoring must be carried out correctly however (Davies and Wray 1996), as the distribution of Salmonella contamination within a poultry house is not uniform (Hayes and others 2000) so small scale limited sampling may fail to detect Salmonella infections which have just begun. Similarly, sampling which relies purely on faecal samples, drag swabs or boot swabs may miss flocks which have passed the peak of infection but which are still producing contaminated eggs (Kinde and others 1996, Riemann and others 1998). Although there appears to be a poor relationship between the prevalence of infected eggs and cloacal excretion of S.Enteritidis or the anti S.Enteritidis antibody titre (Humphrey and others 1991), there is a closer relationship between internal egg contamination and the level of long term infection, as judged by caecal carriage (Methner and others 1995). Similarly, there is good agreement between the level of environmental contamination and the prevalence of caecal infection, the S.Enteritidis phage types present and the level of internal egg contamination and associated human disease (Altekruse and others 1993, Henzler and others 1994 & 1998), Schlosser and others 1995, Mallinson and others 2000). Although the size of the sample taken is important for optimising Salmonella detection (Funk and

CSG 7 (Rev 6/01) 6 others 2000) it is also important not to pool too many subsamples together or a low prevalence of localised Salmonella may be missed (Rolfe and others 2000). More sensitive sampling methods than those currently used are required (Muller & Korber 1992, Awad-Masalmeh and Thiemann 1993, Nief and Hoop 1998). The work planned in this project will quantify Salmonella numbers in various sample types to assess the sensitivity of the methods and relate this to the risk of infection in subsequently housed flocks of birds. Once the appropriate samples have been taken it is essential to handle them correctly, to begin tests as soon as possible after collection and not to add liquid media unless testing is to begin on the day of collection (Davison and others 1995). Selecting the best culture techniques to use for faecal and environmental samples is also very important (Davies and Wray 1994). Many standard Salmonella culture methods have evolved from those used for human foodstuffs or clinical investigations so lack the selectivity to inhibit the competitive faecal flora which greatly outnumbers Salmonella in farm samples (Cox and Berrang 2000). Multiple culture methods carried out in parallel or repeated fractional enrichments have been shown to be more sensitive than single standard methods (Geue and Schluter 1998) but better results still can be obtained for S.enterica subspecies enterica by testing a larger number of samples using a single sensitive culture technique (Davies 1996). Even when a suitable method is used there may still be considerable variations between laboratories in the effectiveness of its application (Edel and Kampelmacher 1968, Raes and others 2000). The method used for the work detailed in this proposal has been shown in EU ring trials to give superior results for the isolation of S.Enteritidis and other serovars from faecal and environmental samples (Voogt and others 2000). The important role of mice in the dissemination and persistence of S.Enteritidis on laying and other poultry farms has been described many times (Henzler and Opitz 1992, Davies and Wray 1995a, Kinde and others 1996). It is possible that the virulence of S.Enteritidis may be enhanced by selection or passage in the mouse population (Guard-Petter and others 1997). The significance of infected/contaminated arthropod vectors has been more uncertain (Davies and Wray 1995b, Gray and others 1999) and more work is needed to define the role of invertebrate vectors and larval forms which may harbour more persistent Salmonella (Aballay and others 2000). Houseflies and other flies have been found to be carrying S.Enteritidis on laying farms (Olsen and Hammack 2000). Flies may leave poultry houses in daytime and return at night so there is an opportunity for transmission of infection both ways (Anderson and Poorbaugh 1964). The role of these wildlife vectors in the persistence of Salmonella on laying farms will be investigated in detail in this proposed study. In free-range farms there is also a constant risk of transmission of Salmonella from human or livestock waste to chickens via the movement of wild birds (Ferns and Mudge 2000, Palmgren and others 2000). This aspect will be included in the research plans for the proposed study. The failure of insufficiently thorough biosecurity, pest control and terminal hygiene to control S.Enteritidis infection on laying farms (Hogue and others 1997b, Hoop 1997, Henzler and others 1998, Schlosser and others 1999, Sommer and Vasicek 2000) will be investigated in detail in this project. This problem has led to the widespread use of vaccination in many countries. Not all vaccines are equally effective (Davison and others 1999, Zhang-Barber and others 1999) but in general both live and killed vaccines give some protection (Cooper and others 1994, Lillehoj and others 2000, Parker and others 2001) and an adjuvented bacterin, which is available in the UK, has been shown to reduce the risk of infection (Timms and others 1990, Feberwee and others 2000, Tenk and others 2000, Yamane and others 2000). Vaccination or competitive exclusion treatment typically reduces the prevalence of Salmonella in most vaccinated flocks. This makes detection of infected flocks more difficult and therefore requires extremely intensive sampling regimes to assess the significance of persistent contamination. Such sampling will be used in the proposed study and the work will assist in assessing the effectiveness of the new live vaccine, which will be in widespread use at the start of this project. Contamination of egg packing plants has been an area where there has been little study and the significance of Salmonella found on grading equipment and the packing plant environment is uncertain (Jones and others 1995, Koidis and Bori 1999). The dry environment in packing plants and the subsequent cooling of eggs (Migamoto and others 1998, Gast and Holt 2000b, Thompson and others 2000) makes multiplication of Salmonella unlikely but on farm shared egg transport systems and packing plants could represent a focus of Salmonella contamination which could be relevant in introducing infections into newly housed flocks. This aspect will be investigated in detail during the proposed work in this project. Techniques to assess the potential virulence and survivability of Salmonella strains found to be persisting in the environment (Gast and Beard 1992, Guard-Petter et al 1996, Humphrey et al 1995, 1996, 1998, Jorgensen et al 2000) will be used in this study. Sensitive molecular epidemiological typing methods are now available for S.Enteritidis and other serovars (Liebana et al 2000). These will be used to track sources of Salmonella from within the farm environment as well as Salmonella strains newly introduced from outside the farm. The synergistic package of proposed approaches and collaboration of leading UK researchers on Salmonella in this project offer a powerful team which should generate results which can be used to considerably enhance control of Salmonella in egg production. Scientific context in relation to proposed extension work Vaccination for S.Enteritidis has been a major factor which has contributed to the virtual elimination of S.Enteritidis for broiler breeding and production (Wall and Ward 1999). This has been achieved by a combination of vaccination, which has increased the threshold for infection, and improved farm hygiene, and biosecurity in an all in-all out system. This is not possible to operate on most commercial laying farms as these are set up for continuous multi-age production to supply a range of grades of egg. This means that the opportunity for reducing the challenge to vaccinated birds housed on an infected farm is reduced as there is always an on-going source of infection on site. It is therefore very important to use

CSG 7 (Rev 6/01) 7 the best possible practical option for vaccination. Currently there are available killed injectable vaccines for S.Enteritidis and S.Enteritidis/S.Typhimurium combined; two different live vaccines for S.Enteritidis; one for S.Typhimurium, and a live S.Gallinarum 9R strain vaccine intended to protect against S.Enteritidis. Other vaccines are licensed elsewhere in the EU and Salmonella Group C vaccines are under development. Initial results from OZ0321 suggest that some live vaccine combinations may be producing a better result than other options but this needs to be confirmed across a wider range of farms. The latest revision of the EU Zoonoses Directive and regulations requires targets to be set for Salmonella infection in the major livestock species. The first EU survey, involving commercial laying flocks, began October 2004 but most UK farms are due to be sampled during 2005. This survey involves a random selection of farms and includes a high proportion of non-cage flocks. This is a valuable opportunity to investigate the distribution and persistence of Salmonella on non-cage layer farms identified during the survey. It will also provide an opportunity to evaluate control measures such as vaccination, improved pest control/biosecurity and cleaning and disinfection. This aspect will benefit from experience gained during OZ0321. A further advantage of recruiting farms from the survey is that they will have been selected for sampling by a randomised process, sampled using a standardised protocol and tested using a standardised method at a single reference laboratory. This means that Egg Industry accusations of study farms recruited for OZ0321 through human disease outbreak tracebacks and clinical investigations being supposedly unrepresentative would be addressed and a comparison of results from existing OZ0321 farms and those recruited via the survey could validate the results of the former. The extension work in this proposal will therefore aim to recruit a range of study farms from the EU Layer Survey and investigate these in detail over a two flock cycle period.

9. Objective(s). Please give details of (a) each scientific objective, (b) to what extent theses objective(s) are interdependent; and (c) whether any factors exist to delay achievement of the objective(s). Where there is more than one contractor, please show clearly below the roles of each. (a) Scientific objective(s). (Technical and Scientific aims of the research which must be measurable and timebound, please number the objectives). If your application is accepted, these Scientific objectives will be included in the agreement between you and the Department. Please, therefore, restrict your entry to the salient points and set these out clearly and concisely. To move to the next field, press the DOWN arrow TWICE. 01 Investigative visits to infected laying flocks 02 Quantitative estimation of environmental Salmonella levels on infected farms 03 Molecular genetic typing for Epidemiological Analysis of Salmonella isolates from field studies 04 Investigation of the infectivity of environmental materials for chicks and laying hens 05 Simulation of environmental contamination with known levels of Salmonella 06 Correlation between survivability of Salmonella strains, their physiology and infectivity 07 Production of advisory material based on other study and other practical research 08 Longitudinal study of Salmonella distribution, persistence and control on farms recruited from the EU Layer Survey.

CSG 7 (Rev 6/01) 8 (b) Interdependence of objective(s). To what extent does the success of one scientific objective depend on the successful completion of another? How essential is each scientific objective in achieving the overall objective. move to the next field, press the DOWN arrow TWICE. Objectives 02, 03, 04, 06 and 07 are largely dependent on work carried out in 01, which will be the major part of the study. 05 and 06 could possibly be carried out in isolation and 07 could be derived from study trips and close observation of similar work in other countries where researchers are not penalised by the need to report Salmonella isolations determined purely from research studies. 08 is independent of the other objectives.

(c) Please give details of any particular factors which might cause delays in the achievement of these objective(s). What are the chances of this happening; what are the probable consequences; and what steps will you take to prevent this happening? To move to the next field, press the DOWN arrow TWICE. Recruitment and retention of study farms has been problematic with the egg industry. VLA currently has contacts with a range of persistently infected flocks which can be used to initiate these more detailed studies. University of Bristol (UB) has agreement with an egg company to work on a more limited basis with flocks at the end of lay and after depopulation only. It will be necessary to do a large amount of extra screening visits to identify infected flocks from this company as effective monitoring is not in place. Further flocks may be recruited during the course of the project and during the remainder of the current VLA project. If the proposed national survey of egg producers is initiated (unlikely in the short term unless reporting issues are resolved) it is likely that about 10% of these will be identified as infected and it may be possible to recruit further farms from within this group. A partial relaxation of the Salmonella reporting requirements whereby research isolates did not have to be reported to EHOs and CCDCs would greatly assist recruitment of further farms. It is anticipated that recruitment of farms from the EU Layer Survey will be easier than previous recruitment as all farms will already have been visited by SVS and the VLA Surveillance nominated officer – in fact it is planned to carry out the first sampling visit for OZ0321 at the same time as the nominated officer visit where possible. The first positive farm from the EU survey has already been recruited in anticipation of this extension work.

10. (a) Approaches and Research Plan. Outline the experimental approaches to be used in realising the scientific objectives and set out the work plan for the life of the project stating clearly how you intend to proceed. Please number the Approaches in the same way as the Objectives. Where there is more than one contractor, please show clearly below the roles of each. If your application is accepted, the Approaches and Research Plan will be included in the agreement between you and the Department. Please therefore, restrict your entry to the salient points and set these out clearly and concisely. To move to the next field, press the DOWN arrow TWICE. The overall aim of the project is to gather detailed information of the behaviour of Salmonella, particularly S.Enteritidis in the environment of the various forms of egg production and assess the risk of such contamination for infecting laying hens or rearing chicks. This will be achieved by fulfilling the following scientific objectives: 01 Regular investigative visits to infected laying flocks (90% VLA, 10% University of Bristol (UB)) Commercial laying farms where S.Enteritidis or any other type of Salmonella has been identified will be approached to request their collaboration with the study. The British Egg Industry Council will also be consulted in an attempt to recruit more farms for the study. Assistance from AHEG will also be requested to help identify and recruit notified study sites. If the proposed laying farm survey is begun then it may also be possible to use some of the farms identified in the survey for further study. Initially the project will concentrate on sites already involved with VLA studies but further sites will be recruited as the project progresses. Up to 6 infected or previously infected flocks from each of the main production sectors (cage, free range, barn), as available, will be visited on a regular basis before and after re-population to assess the level of environmental contamination in relation to the stage of production and faecal excretion of Salmonella. Further flocks will be surveyed at the end of lay and at depopulation. It will be necessary to carry out an extensive screening of flocks in the UB company as no effective monitoring is in place on which to base identification of infection and the prevalence is likely to be low as a result of vaccination. The VLA study flocks will mostly be available during the whole of the production cycle but the UB flock owners will only permit sampling at the end of lay and in empty houses. Phenotypic and molecular genetic comparisons will therefore be made to link organisms from birds and the environment where regular sampling is not possible. Where available, samples will be examined from feed and delivery crates (or rearing accommodation) to confirm that birds are not carrying Salmonella on entry to the laying houses. Flocks recruited at the start of the project will be followed through at least two whole laying cycles and flocks recruited subsequently will be followed for as long as remaining project time allows. Data on the distribution of contamination will be related to management, biosecurity and cleansing and disinfection practices. The effect of the recent introduction and widespread use of an auxotrophic live S.Enteritidis vaccine, (TADVacE) which should have reached a large proportion of laying flocks by the start of the project, will be of particular interest. Where possible advice will be given on improved hygiene and the effects of this, if implemented, will be evaluated.

CSG 7 (Rev 6/01) 9 counts because of the overwhelming burden of other organisms in the samples. A dilution and enrichment technique will therefore be adopted to quantify Salmonella levels in multiple samples of faeces, litter, dust, mouse droppings and viscera, potential arthropod vectors etc. from selected study farms. A similar approach will be used to estimate the numbers of Salmonella surviving on cleaned and disinfected surfaces. Levels of Salmonella in the air during cleaning and disinfection and after restocking will be estimated using settlement plates and selective culture.

03 Molecular genetic typing for epidemiological analysis of Salmonella isolates from field studies (100% VLA) A cascade approach to determining genetic relatedness and differences in strains removed from the field investigations will be used. All isolates will be fully serotyped and selected isolates will be subjected to phagetyping and anti-biogram typing, molecular genetic techniques such as plasmid profile analysis, Pst1/Sph1 ribotyping and pulsed field gel electrophoresis will be used to compare strains and confirm the persistence of particular clones versus introduction of new strains from outside the farm or elsewhere on the farm.

04 Investigation of the infectivity of environmental materials for chicks and laying hens (90% UB, 10% VLA) In order to assess the infectivity of potentially contaminated environmental materials, for example, dried faeces and egg residues, dust, mouse droppings, flies, arthropods etc. will be gathered during farm visits, the level of Salmonella quantified and the material exposed to chicks, laying hens and mice (because of their potential to amplify environmental contamination). The materials will be presented by direct exposure, mixing with feed or with water. In the first instance the material will be placed in the environment of groups of 5 commercially obtained day-old chicks housed under controlled conditions. This infection model has been shown to be extremely sensitive and has been used extensively in earlier studies by the UB team (Allen et al. 1997) including past MAFF (e.g. CSA1551 Salmonella infection in the laying hen) and an ongoing DEFERA project (CSA 5519 An in vivo poultry model to study the effect of growth promoters on resistance to human antibiotics in mixed bacterial populations). The chicks will be humanely killed after 7-10 days and liver and caecal content cultured for salmonellas. The numbers of salmonellas in each sample is likely to be low and this level is unlikely to induce any clinical signs in the animals. Given the uneven distribution of salmonellas in naturally contaminated materials it may be necessary to repeat any particular challenge experiment up to three times and with different flocks. The assessment of potential infectivity will be less sensitive in mice and considerably so in adult hens since the infective dose is higher. Therefore only when particular sample types have proved to be infective with the chick model will mice and adults hens at point of lay be challenged. If little infection of chicks occurs in the initial studies point of lay birds and mice will challenged to check for enhanced susceptibility and subsequent amplification of infection.

05 Simulation of environmental contamination with known levels of Salmonella (100% UB) This would involve approaches similar to 03 but would use known levels of Salmonella and would also include simulated contamination of floor, wall and equipment with different levels of Salmonella in a dilute faecal suspension.

06 Correlation between survivability of Salmonella strains, their physiology and infectivity (80% UB, 20% VLA) Salmonella strains isolated from the field studies which either persisted or failed to persist in the environment will be compared according to their ability to survive on surfaces, in the presence of noxious agents such as detergents, disinfectants, acids and alkaline conditions using previously published techniques (Humphrey et al 1995). The genetic basis of the phenotypic behaviour will also be investigated by genetic determination of strains response mechanisms such as rpoS, the multiple antibiotic resistance (MAR) locus, generalised efflux pumps as well as virulence determinants.

The proposer is also Project Leader for the EU Layer Survey so is in close touch with arrangements and developments in this. Lines of communication with SVS and VLA Surveillance Division nominated officers already exist and guidance documents for sampling and statutory visits have been produced. The aim will be to recruit from the positive farms a representative range of up to 40 non- cage flocks and up to 10 cage flocks. The non-cage flocks will provide study sites to boost the generation of data on distribution and persistence of Salmonella in free-range and barn flocks gathered from the more limited number of farms currently participating in OZ0321. The cage flocks from the survey will be used for comparative purposes to see if there is any difference in the behaviour of Salmonella in randomly identified cage layer farms compared with those currently recruited in OZ0321. Flocks will be sampled using pooled faeces and environmental samples at key

CSG 7 (Rev 6/01) 10 and disinfection and data gathered on the methods used for decontamination so that the most effective methods can be identified. Where infection persists between flocks farmers will be advised on improvements and the results of this monitored at the turnaround of subsequent flocks. The sensitivity of the standard methods used for the EU Layer Survey will be determined by comparison with culture of 60 spent hens from a range of flocks and with more intensive samples within the houses. Quantification of Salmonella in pooled faeces using a dilution-enrichment method will also be carried out to facilitate comparisons with previous work and give a more accurate indication of the effectiveness of vaccination. The role of wildlife pests will be investigated by culturing faeces and carcases, especially during the period between cleaning and disinfection and re-stocking. Farms will be selected from the positive farms in the EU survey to represent as many types of vaccination programmes as are available. Where vaccination programs are found to be more effective then farmers and veterinary surgeons will be advised so that more prospective studies can be carried out in the subsequent flocks where possible. The potential for contamination of eggs will be assessed by samples taken from grading and packing equipment when this does not compromise trade. Evidence of persistence of specific strains of Salmonella will be confirmed by further typing of representative panels of isolates by serotyping, phage typing, antimicrobial resistance testing and molecular genetic techniques as proposed for the current OZ0321 studies. In addition a highly promising sequence based technique for subdivision of strains (VNTR typing) will be assessed for subdivision of S.Enteritidis PT4, S.Typhimurium DT104 and other highly clonal Salmonella serovars and strains from the current OZ0321 work and the extension project. A selection of strains will also be examined by micro-array to determine associations between virulence and survival characteristics and genetic characteristics. At the end of the extension work advisory documents, in particular an updated draft of the Lion Code, will be produced and submitted to egg industry representatives for comment and consideration.

(b) Will the research require a survey to be carried out, or a questionnaire to be used? Enter YES or NO No (Surveys are only acceptable if they form an essential part of the project. Ministerial approval is required, and time must be allowed for this before any agreement is signed.)

CSG 7 (Rev 6/01) 11 11. Milestones. Based on your research plan, please give milestones (i.e. points at which progress can be assessed) with targets for monitoring progress of the research towards the scientific objectives. Where work is seasonal, please express milestones in day, month and year form e.g. 01:07:99. If work is not seasonal, please express milestones in day, month and year form and in terms of numbers of months from the proposed start date e.g. month 15.. Each milestone should relate to one scientific objective, i.e. the milestones for objective 1 should be numbered 01/01, 01/02 etc. Each milestone title should not be more than 120 characters, a description is optional. (a) Primary milestones. (These must number no more than four in each project year. Achievement of each must be essential if the objectives of the project are to be met. If your application is accepted, they will form part of the agreement between you and the Department.) To exit this field, press the DOWN arrow TWICE. Milestone Target date Title 01/01 30/12/02 Planning meeting of collaborators (Month 3) 01/02 30/01/03 Consultation with egg industry representatives and specialist veterinarians (Month 4) 01/03 28/02/03 Commence regular longitudinal visits to study farms (Month 5) 02/01 30/04/03 Pilot and optimise environmental Salmonella quantification techniques (Month 7) 03/01 30/10/03 Pilot application of molecular typing techniques (Month 13) 04/01 30/10/03 Commence infectivity of environmental materials study (Month 13) 05/01 30/10/03 Commence infectivity of simulated environmental contamination studies (Month 13) 06/01 30/08/04 Commence physiology and infectivity studies (Month 23) 02/02 30/06/05 Complete quantitative Salmonella field studies and report (Month 33) 06/02 30/07/05 Complete laboratory survivability and infectivity studies and report (Month 34) 03/02 30/08/05 Complete molecular epidemiological typing and report (Month 35) 07/01 30/09/05 Complete advisory material, draft publications and draft code of practice (Month 36) updates 08/01 30/11/05 Recruitment of study farms from the layer survey and completion of (Month 38) initial Salmonella status assessments 08/02 30/12/05 Start of spent hen port-mortem culture investigations (Month 39) 08/03 30/05/06 Evaluation of sensitivity of layer survey sampling methods (Month 44) 08/04 30/09/06 Report on investigations of infection in first replacement flocks (Month 48) 08/05 30/05/07 Commence evaluation of VNTR typing for clonality of S.Enteritidis on study farms 08/06 30/08/07 Report on effectiveness of vaccination and other control measures on study farms 08/07 30/09/07 Report on microarray analysis of selected strains from study farms 08/08 30/09/07 Report on comparison of survey and non-survey farms and updated advisory document on improved monitoring and control measures

CSG 7 (Rev 6/01) 12 (b) Secondary milestones. (These are unrestricted in number. They should be helpful to the management of the project but not essential to the achievement of the objectives. If your application is accepted, they will not form part of the agreement between you and the Department. Please prefix number of milestones with an S to indicate that it is a Secondary milestone.) To exit this field, press the DOWN arrow TWICE.

Milestone Target date Title

12. Quality Assurance. Please state what procedures you operate for Quality Assurance, including registration to BS 5750/ISO 9000, NAMAS or GLP.To exit this field, press the DOWN arrow TWICE. Quality Assurance Statement for VLA: The principal assurance of quality in research in the VLA is based on external assessment by a Visiting Group of independent scientists held every four years. The last visit in Bacteriology was in 1997, when a satisfactory report was obtained. VLA Weybridge is the EU/CRL and OIE National Reference Laboratory for Salmonella.

The Salmonella and E. coli typing sections and Regional laboratories of VLA are UKAS-accredited and the rest of the Bacteriology Department is working towards full UKAS accreditation. All test work is conducted according to Standard Operating Procedures. The T.B. Section is GLP-compliant. External Quality Control is undertaken where available and appropriate. QA for Salmonella isolation and serotyping is also conducted under the DEFRA Approved Laboratories Schemes and the European National Reference Laboratory Network. Practices and risk assessments for work concerning genetic manipulation are scrutinised by the VLA Local Genetic Manipulation Committee, which has delegated responsibility from the HSE.

Quality Assurance Statement for University of Bristol: The Food Microbiology Laboratory is working towards UKAS accreditation and participates in the PHLS External Food EQA Scheme as well as the MAFF (DEFRA) Salmonella QA Scheme. Scores for the PHLS Pathogen, Indicator Organism and Aerobic Colony Count Schemes are currently 0.54, 1.2 and 0.91 standard errors above the mean, respectively. They have had no erroneous results for the MAFF Salmonella QA Scheme since they began to participate several years ago.

13. Use of animals (a) Does any of the work outlined in the proposal require a licence from Tick as appropriate the Home Secretary under the Animal Scientific Procedures Act 1986? Enter YES or NO YES NO (b) If ‘Yes’, please give an estimate of the numbers of each species to be used. To move to the next field, press the DOWN arrow TWICE Up to 60 mice, 1080 day old chicks, 108 laying hens

(c) DEFRA requires full implementation of 3R principles in any research project using animals, whether or not requiring a licence under the 1986 Act. If your proposal involves the use of animals, please confirm that you have read the statement and will seek to implement it in full (the statement is available on the DEFRA website at http://www.maff.gov.uk/research/publications) Yes, I have read the statement and will implement it in full 

14. Equipment devoted to project (a) Please list the existing capital equipment which you will use for this project. To move to the next field, press the DOWN arrow TWICE VLA will make available for this project vehicles and equipment for farm investigations, laboratory equipment including extensive range of molecular genetic typing and bionumerics equipment.

UB will make available equipment for farm investigations and laboratory equipment for microbiological diagnostic purposes for this project. Equipment and accommodation will also be made available for the on site animal trials.

(b) Give justification for, and estimated cost of any new capital equipment which will have to be purchased for this project for which you expect DEFRA to contribute. N.B. DEFRA will not normally contribute to the cost of any

CSG 7 (Rev 6/01) 13 new item that will duplicate one already in your possession. (See Section 3, note 20(d).) To move to the next field, press the DOWN arrow TWICE

CSG 7 (Rev 6/01) 14 CSG 7 (Rev 6/01) 15 15. Staff effort (a) Please list the names and grades of staff who will work on the project together with details of their specialism (including relevant papers published). To move to the next field, press the DOWN arrow TWICE VLA Name Grade Specialism Dr Rob Davies Pay Band B - Veterinary Microbiology/Field Investigations

Dr Felicity Clifton-Hadley Pay Band B - Veterinary Microbiology Dr Ernesto Liebana Pay Band C - Molecular epidemiology Prof. Martin Woodward Pay Band A - Molecular genetics/pathogenesis

University of Bristol Prof. Tom Humphrey Professorial scale - Food Microbiology/Food Safety Dr Viv Allen Research Fellow 2/2 - Zoonotic Pathogens/Food Safety

TBA Research Assistant RIA2

Please refer to Annex A for relevant publications

(b) Please state how many working days equals one staff year VLA 213 days UB 220 days

CSG 7 (Rev 6/01) 16 (c) Summary of staff time involved You should show here the staff years (to first decimal place only) expected to be spent on the project for each grade of staff involved, including both scientists and assistants, during each year of the project. STAFF TIME Grade Year 1 Year 2 Year 3 Year 4 Year 5 TOTAL TIME VLA Time Breakdown Band A 0.1 0.1 0.1 0 0 0.30 Band B 0.1 0.1 0.1 0.09 0.05 0.44 Band C 0.2 0.2 0.2 0 0.05 0.65 Band D 0.1 0.1 0.1 0.01 0.01 0.32 Band E 0.2 0.2 0.2 0.47 0.56 1.63 Band F 0.2 0.2 0.2 0.33 0.29 1.22

University of Bristol Time Breakdown Professorial* 0.02 0.02 0.02 0.06 R2/2 0.4 0.4 0.4 1.2 RIA2 0 0 0.3 0.3 *funded by UB

Extension – to be added later VLA time only

To create a new entry line, press the TAB key. To exit press DOWN arrow TWICE

TOTAL STAFF YEARS 1.32 1.32 1.62 0.90 0.96 6.12

16. Communication of results (a) How will the results be communicated? Please list anticipated numbers and if possible expected dates for submission of e.g. publication in refereed journals, trade journals or the press, presentations or demonstrations to the scientific community in trade organisations and internal reports or publications. To move to the next field, press the DOWN arrow TWICE It is anticipated that at least 4 scientific papers, to be submitted to journals such as Epidemiology and Infection, Applied and Environmental Microbiology and Veterinary Microbiology will be produced on the basis of this work. At least a similar number of presentations will be given at major national and international scientific conferences. Opportunities will also be taken to present the work at specialist poultry meetings such as British Veterinary Poultry Association. Results will also be communicated directly to participating poultry companies so that they can improve their levels of Salmonella control. Advisory material including a revised code of practice for prevention and control of Salmonella in egg production will also be produced and corresponding articles will be produced for the poultry press.

N.B. In any publication including press articles, the financial support of the Department MUST be acknowledged. (b) What measures will be taken to encourage technology transfer? To move to the next field, press the DOWN arrow TWICE Information will be provided direct to participating poultry companies and their poultry veterinarians. Practical guidance documents will also be produced. Sampling and culture protocols will be made available to participating poultry companies and their private laboratories.

CSG 7 (Rev 6/01) 17 17. Benefits (a) Please describe and quantify the benefits which may arise from this project, how the results will be used and who will make use of the results of this research (e.g. Department, industry or consumers). To move to the next field, press the DOWN arrow TWICE This project will generate to information which should prompt the egg industry to adopt higher standards of biosecurity and cleaning and disinfection. This will reduce the environmental challenge for birds and assist the performance of the Salmonella vaccine. Quantitative data on environmental contamination could be fed into risk assessments being developed elsewhere. Any reduction in the potential for contamination of eggs will have positive benefits for human health and the costs of the NHS and lost working/leisure spend days. The work will also have a side benefit of bringing together the two leading research groups working on Salmonella in egg production in the UK and this should be the start of a powerful alliance to address future practical problems of foodborne zoonoses.

(b) Do you think further research or development will be needed before these benefits can be realised? To move to the next field, press the DOWN arrow TWICE It is likely that the work will identify further areas and development of practical Salmonella control measures. Identifying and prioritising these will be an important output of this project.

(i) protectable results (e.g. patents, design Rights etc)? (Enter YES or NO) No

(ii) other commercially negotiable Results (such as ‘know-how’)? (Enter YES or NO) Probably

If YES, please give details including interest already expressed To move to the next field, press the DOWN arrow TWICE A major retailer has requested assistance from two of the applicants with their strategies for control of Salmonella and other foodborne zoonoses in poultry. Work carried out in the project is likely to enhance the contributors consultancy involvement and so indirectly benefit public health through the influence major retailers have with their suppliers.

18. Other details

(a) Is this work currently or about to be submitted in another application elsewhere? (Enter YES or NO) No

If YES:  to which organisation? to which organisation

 and by what date is a decision expected? (b) With reference to questions 6(b) and (c) please give a brief description of the nature of their contribution. (A letter agreeing to the collaboration should be attached to this application.) (i) Funding contributions other than DEFRA To move to the next field, press the DOWN arrow TWICE

(ii) ‘In kind’ contributions To move to the next field, press the DOWN arrow TWICE

CSG 7 (Rev 6/01) 18 19. Referees Applicants should be aware that their application may be submitted to external referees considered appropriate by DEFRA for comment. Applicants may suggest below up to three external referees, although any decision as to whether any name suggested is approached will lie with DEFRA.

Name Address Telephone number

Please note that applicants must not nominate collaborators in any current project or research paper, or experts from their own organisation

CSG 7 (Rev 6/01) 19 SECTION THREE - RESOURCES ????? FINANCIAL GUIDELINES FOR PROJECT COST ESTIMATES To read these Notes move down the page using the scroll bar on the right and then TAB. Once a price for the project has been agreed with the Department, and an agreement signed, no increase in price can be considered. Please note that any over or underspends in any one project year cannot be carried over into the next project year. The following Notes are to help you provide all the details necessary for the project costs. 20. (a) Pay costs You should include the costs of personnel working directly on the project. Your costings must be supported by a detailed breakdown showing for each person separately: (i) the amount of staff time (e.g. number of days, months or years) by grade / salary bands for each year of the project including staff to be recruited; N.B. An explanation should be given where the staff effort increases or decreases during the life of the project. (ii) the proposed annual salary (including London (or other town) Weighting Allowances, employers NI and Superannuation) and salary spine point (i.e. pay band) of each person during each year of the project. In appropriate cases, the Department is willing to accept pay calculations on the basis of average pay costs. In this event you should indicate the average pay used for the grade(s) in question. (b) Inflation (i) If the project is submitted under a competition, a percentage to cover inflation can be built into the price, but please bear in mind that overall cost is a factor in the selection process. (ii) If the project is not submitted under a competition, costings must be submitted at current prices, and DEFRA will add an allowance for inflation in line with the Treasury's forecast of GDP deflator. (c) Consumables These will be essentially scientific laboratory supplies, (e.g. glassware, chemicals) costing individually up to £2,000 in value which are purchased from third parties. Please list separately all consumables to be used, including, if possible, quantities. (d) Equipment Capital equipment is a fixed asset costing over £2,000 in value which is expected to yield continuous service beyond the year in which it is purchased. It includes items such as scientific and information technology equipment. The equipment must be essential to the carrying out the project. Three quotations must be obtained for each item of equipment. (See note (ii) below.) For new equipment the Department will only fund that proportion of its working life (normally 5 years) to which it is used solely on the project (i.e. if a project is of 3 years duration the Department will fund 3 th /5 of the cost at the rate of 1/5 each year. Where equipment has a useful life of more than 5 years and/or is used for other purposes, you should make an appropriate reduction in the annual rental charged to the Department. Where new equipment is required please give details of the make, model, price and the year when each item is to be purchased and its purpose. Likewise, please indicate when equipment is to be leased from the manufacturer and give details of the costs of rental for each year. A piece of equipment may need to be allocated full-time to a project. In such a case, the fact that an organisation owns a similar piece of equipment for use on other projects does not remove the need here for that equipment to be either purchased or hired, although the usual rules on the amount to be paid will apply. It is however for the contractor to justify such a purchase. You may be asked by the Department to provide the following as appropriate: (i) the original purchasing invoice or top copy of the rental agreement. This will be returned immediately after a copy has been taken; and (ii) the original written quotations obtained from three different suppliers.

CSG 7 (Rev 6/01) 20 N.B. In appropriate cases e.g. where it can be shown that the technical specification of equipment precludes all but a single supplier, a single oral/written quotation will be acceptable. (e) Travel Visits to conferences and similar functions in the U.K. or elsewhere and any foreign visits will not normally be regarded as an eligible cost. Exceptionally, however, such costs may be funded where you can demonstrate to the Department’s satisfaction that the visits are essential to the project. Where travel costs are necessary, details of their frequency, purpose, destination, the mileage and rate per mile (for road travel), air/rail fares, and number of persons travelling should be given. (f) Overheads Central and departmental costs (direct) that underpin the research activities and costs (indirect) which cannot readily be uniquely assigned to particular research projects. These may include the following: ● financial services (finance, accounting, tendering, marketing); ● personnel services; ● staff facilities (transport, health and safety, training, welfare, laundry); ● departmental services (administration, library, secretarial, printing, minor stores items and laboratory and workshop support); ● staff management, and cover for maternity and long-term sickness benefits. You should include details of the method of calculation of the overhead rate, (to be expressed as a percentage of direct salary costs (excluding Superannuation and NI) plus consumables) and list separately the items covered. (g) Sub-contracts, consultancy fees, etc. You should show that this work is essential to the success of the project. Any costs under this heading must be identified separately. Please detail separately the component parts of any consultancy or sub-contract, including pay costs, consumables, equipment, travel, overheads and other costs which have been included. (h) Other costs You should include here items which do not readily fit under the headings provided e.g. laboratory/analytical services, laboratory animals, servicing of equipment, any non-equipment rental charges, recruitment costs, computer software, stationery items, student registration fees and glasshouse heating. You should also provide a short explanation of the need for all the items you list here. (i) VAT Please follow these notes carefully because, in certain circumstances, VAT can be reclaimed from HM Customs and Excise, thereby lowering the cost of the research. (I) DEFRA is an eligible body under the VAT (Education) Regulations 1994. If your organisation is also an eligible body, you should not charge VAT on the total price of the research services you provide to DEFRA. You may, however, include in your price, any VAT in respect of services/items purchased in order to carry out the research, provided you are ineligible to reclaim this VAT from HM Customs and Excise. This also applies if you are not a registered trader. (II) If your organisation is not an eligible body, and is registered for VAT, you must charge VAT at the standard rate on the total price of the research done for DEFRA. DEFRA can recover this VAT under S41 of the VAT Act 1994. Completing the costs section at question 21. ● Organisations in category (I) above Include the VAT that your organisation cannot recover from HM Customs and Excise within the price for the service/item under the appropriate section. For example, The price under ‘Sub- Contractor’ should include any VAT charged by the sub-contractor (do not separately identify the VAT element). You should enter nothing in the VAT line at the foot of the cost table as this would result in double-counting.

● Organisations in category (II) above

CSG 7 (Rev 6/01) 21 Insert the VAT to be charged in the VAT line at the foot of the cost table. Include no VAT charges elsewhere in the cost table as this would result in double-counting. (j) Ineligible costs The following are excluded from eligible costs: ● interest charges; ● hire purchase interest and any associated service charges; ● profit earned by a subsidiary or by an associated undertaking on work sub-contracted under the project;

N.B. Contingency allowances expressed as an arbitrary percentage overall addition to eligible costs are excluded.

CSG 7 (Rev 6/01) 22 SECTION THREE - Continued

21. Estimated total project (all funding bodies) - detail Before completing this section you should read carefully the Notes above which explain what project costs the Department is prepared to consider. These must be project year figures, not financial year costs.

Year1 Year2 Year3 Year4 Year5 TOTAL Project year £ £ £ £ £ £ Pay costs (see note a) 27,700 29,095 30,412 35,647 38,376 161,230 Cost Breakdown for VLA Consumables (see note c)(specify) 3,000 4,000 2,500 4,700 5,900 20,100 Laboratory reagents and disposable equipment

Equipment (see note d)(specify) (Existing equipment will be allocated to the project)

Travel expenses (see note e) (Travel to meetings and study sites) 1,000 1,500 1,000 2,000 1,200 6,700

Overheads (see note f) (specify) (Financial services, personnel, staff 28,086 28,086 27,033 34,517 37591 155,313 facilities, management services, test materials)

Sub contracts, consultancy (see note g) [University of Bristol Joint Contract [26,812] [28,144] [41,308] 0 0 [96,264] costs]

Other costs (see note h) Sample collection and despatch 3,000 3,000 1,500 0 0 7,500 Animal costs 0 134 0 3,000 3,000 6,134 Inflation at 2.5% 1,536 4,849 4,906 0 0 11,291 TOTAL PROJECT COSTS* 91,134 98,808 108,659 79,864 86,067 464,532

VAT (see note i)

(i) Are you registered for VAT? (Enter YES or NO) NO

If YES what is your VAT registration number? * Excluding VAT : See also note 20(b) - non-competitive work must be costed at current prices.

CSG 7 (Rev 6/01) 23 SECTION THREE - Continued

Year Year Year Year Year TOTAL Project year £ £ £ £ £ £ Pay costs (see note a) 13,238 14,184 23,701 51,123 Cost Breakdown for University of Bristol 4,000 4,000 4,000 12,000 Consumables (see note c)(specify) , laboratory media, reagents and disposable equipment e.g. gloves, swabs, plastic containers

Equipment (see note d)(specify)

Travel expenses (see note e) 2,000 2,000 2,000 6,000

Overheads (see note f) (specify)

(46% of salaries - NIS/Pen) 5,021 5,377 8,994 24,911 (46% of consumables) 1,840 1,840 1,840

Sub contracts, consultancy (see note g)

Other costs (see note h)

Animal Costs 713 743 773 2,230

TOTAL PROJECT COSTS* 26,812 28,144 41,308 96,263

VAT (see note i)

(i) Are you registered for VAT? (Enter YES or NO) Yes

If YES what is your VAT registration number? GB139085946 * Excluding VAT : See also note 20(b) - non-competitive work must be costed at current prices.

CSG 7 (Rev 6/01) 24 SECTION FOUR - DECLARATION Declaration I confirm that I have read this application and Department’s standard contractual terms and conditions and that: (a) DEFRA may show this application to third parties for the purposes of obtaining expert opinion on its scientific merits; and (b) if granted, the work will be accommodated and administered in our Organisation in accordance with DEFRA’s contractual arrangements. The staff gradings and salaries quoted are correct and in accordance with the normal practice of this Organisation. 22. (a) Head of Department Signature Date

Name and initials Dr Sarah Evans

Organisation Veterinary Laboratories Agency

. (b) Administrative Authority Signature Date

Name and initials Dianne Bunce

Position Research Account Manager

Organisation Veterinary Laboratories Agency

Full postal address Veterinary Laboratories Agency Weybridge Woodham Lane New Haw Addlestone Surrey Postcode KT15 3NB Telephone No. (including STD code) 01932 357978 Ext. 2894

23. Name of project leader (if different to 1)

Full postal address of project leader

Postcode Telephone No. (including STD code) Ext. Fax No. (including STD code)

Note: This application should be submitted by/through: (a) the Head of Department; and (b) the officer who will be responsible for administering any funds that may be awarded. Each should sign the above declaration. You now need to complete a separate ANNEX A for each person who is to be engaged on the research. Please also complete ANNEX B. To do this, please use the CSG7A.dot provided.

CSG 7 (Rev 6/01) 25

SECTION FOUR - DECLARATION Declaration I confirm that I have read this application and Department’s standard contractual terms and conditions and that: (a) DEFRA may show this application to third parties for the purposes of obtaining expert opinion on its scientific merits; and (b) if granted, the work will be accommodated and administered in our Organisation in accordance with DEFRA’s contractual arrangements. The staff gradings and salaries quoted are correct and in accordance with the normal practice of this Organisation. 22. (a) Head of Department Signature Date 17/07/01

Name and initials Dr G Perry

Organisation University of Bristol, Department of Clinical Veterinary Science

. (b) Administrative Authority Signature Date

Name and initials Mrs Rachel Fleetwood

Position Faculty Accountant

Organisation University of Bristol

Full postal address University of Bristol Finance Office Senate House Tyndall Avenue Bristol

Postcode BS40 5DU Telephone No. (including STD code) 01179 287910 Ext.

23. Name of project leader (if different to 1) Dr Vivien Allen

Full postal address University of Bristol of project leader Department of Clinical Veterinay Science Division of Food Animal Science Langford North Somerset

Postcode BS40 5DU Telephone No. (including STD code) 01179 289430 Ext. Fax No. (including STD code) 01179 289324

CSG 7 (Rev 6/01) 26 ANNEX A CURRICULUM VITAE OF STAFF TO BE ENGAGED ON THE RESEARCH

Please complete a separate form for each person to be engaged in the scientific aspects of the work

1. Surname Davies Forename(s) Robert Harold 2. Degrees: BVSc (Bristol, July 1979) PhD (Bristol Feb 1997)

This table will expand to accommodate the information you wish to enter. To move to the next field, press the DOWN arrow TWICE. Feb. 1990 - date - Veterinary Researcher (Head of Enterobacteriaceae Surveillance Testing Section), Department of Bacterial Diseases, VLA Weybridge

Aug. 1979 - Feb. 1990 - Veterinary surgeon in agricultural general practice, partner for three years in last practice before joining VLA

This table will expand to accommodate the information you wish to enter. To move to the next field, press the DOWN arrow TWICE. Davies RH (1994). Salmonella pollution in poultry units and associated enterprises. In: Pollution in Livestock Systems. (Eds.) I. ApDewi, R.F.M. Arfod, I.F.M. Marai, H. Orned. CAB International, Oxford. pp137-169.

Davies RH, McLaren IM, Wray C (1994). Environmental aspects of salmonella on animal production units. Proceedings of the Livestock Environment IV. 4th International Symposium. (Eds.) E. Collins, C. Boon. University of Warwick, Coventry, England, 6-9 July 1993. pp111-115.

Davies RH, Wray C (1995). Mice as carries of Salmonella enteritidis on persistently infected poultry units. Veterinary Record 137:337-341.

Davies RH, Wray C (1996). Determination of an effective sampling regime to detect Salmonella enteritidis in the environment of poultry units. Veterinary Microbiology 50:117-127.

Davies RH, Wray C (1996). Persistence of Salmonella enteritidis in poultry units and poultry feed. British Poultry Science 37:589-596.

Davies RH, Wray C (1996). Seasonal variations in the isolation of Salmonella typhimurium, Salmonella enteritidis, Bacillus cereus and Clostridium perfringens from environmental samples. Journal of Veterinary Medicine Series B, 43:119-127.

Davies RH (1997) A two year study of Salmonella typhimurium DT104 infection and contamination on cattle farms. Cattle Practice. 5: 189-194.

Davies RH, Nicholas RAJ, McLaren IM, Corkish JD, Lanning DG, Wray C. (1997) Bacteriological and serological investigations of persistent Salmonella enteritidis infection in an integrated poultry company. Veterinary Microbiology 58, 277-293

Davies RH, Wray C. (1997) Distribution of salmonella contamination in 10 animal feed mills. Veterinary Microbiology 51: 159-169

Davies RH, Teale CJ, Wray C, McLaren IM, Jones YE, Chappell S & Kidd S (1998) Nalidixic acid resistance in salmonellas isolated from livestock in Great Britain, with particular reference to turkeys Veterinary Record 144, 320-322

Davies RH (1998) Salmonella typhimurium DT104 in the United Kingdom. Proc. Ann. Conf. Danish Vet. Assoc. 1-4 Sept. 1998, Nyborg, Denmark p.1-14

CSG 7 (Rev 6/97) 27 Davies RH, Bedford S & C Wray (1998) A semi-quantitative study of the effectiveness of disinfection of broiler breeder houses contaminated with salmonella. Proc. 4th World Congress on Foodborne Infections and intoxications. 7-12 June 1998, Berlin. 414-424

Davies RH, McLaren IM & S Bedford (1999) Observations on the distribution of Salmonella in a high throughput pig abattoir. Veterinary Record. 145, 655-661

Davies RH (2001) Salmonella Typhimurium DT104 in Great Britain. Zoonose-Nyt (Denmark) 8, (1) 9-12

Davies RH, Breslin M, Corry JEL, Hudson W, Allen VM (2001) Observations on the distribution and control of Salmonella in two integrated broiler companies. Veterinary Record, (in press)

Davies RH, Bedford S, Shankster S (2001) Enhanced culture techniques for detection of Salmonella. Veterinary Record 17, 539-540

Gibbens JC, Pascoe SJS, Evans SJ, Davies RH, Sayers AR (2001) A trial of biosecurity as a means to control Campylobacter infection in broiler chickens. Preventive Veterinary Medicine 48, 85-99

Randall LP, Wray C & RH Davies (1999) Survival of verocytotoxin-producing Escherichia coli O157 under simulated farm conditions. Veterinary Record 145 (17) 500-501

CSG 7 (Rev 6/97) 28 ANNEX A CURRICULUM VITAE OF STAFF TO BE ENGAGED ON THE RESEARCH

Please complete a separate form for each person to be engaged in the scientific aspects of the work

1. Surname Prof. Woodward Forename(s) Martin John 2. Degrees: BSc (Hons) Microbiology with Chemistry (Reading University 1973-1976) PhD Microbial Genetics (Reading University 1976-1979)

This table will expand to accommodate the information you wish to enter. To move to the next field, press the DOWN arrow TWICE. 1989 - date Head of Molecular Genetics, Bacteriology, CVL. Senior Research Officer II 1997 - date Visiting Professor in Veterinary Microbiology Royal Holloway University of London 1992 - 1997 Non-stipendiary senior lecturer, Royal Holloway University of London 1986 - 1989 Research Officer, Bacteriology, CVL. 1984 - 1986 Lecturer in Genetics, Oxford Polytechnic. 1982 - 1984 Lecturer In Genetics and Microbiology, Central London Polytechnic. 1980 - 1982 Postdoctoral researcher, Prof. Mandelstam Microbiology Unit Oxford University

recent funding sources include:- (1) MAFF (CSG/AHFS), (2) DoH, (3) British Council, (4) Wellcome [visiting students] (5) British Egg Marketing Board, (6) Royal Society [visiting students], (7) Intervet Animal Health, (8) Pfizer (9) Pharmacia (10) Inveresk (11) Novartis This table will expand to accommodate the information you wish to enter. To move to the next field, press the DOWN arrow TWICE.

CSG 7 (Rev 6/97) 29 1. M Hinton, G R Pearson, E J Threlfall, B Rowe, M J Woodward and C Wray (1989) Experimental S. enteritidis infection in chicks. Vet. Rec. 124:223-224.

2. R G Hewinson, S J Cartwright, M P E Slack, R D Whipp, M J Woodward and W W Nichols (1989) Permeability to cefsulodin of the outer membrane of Ps. aeruginosa and discrimination between B- lactamase-mediated trapping and hydrolysis as mechanisms of resistance. Eur. J. Biochem. 179:667-675.

3. M J Woodward, I McLaren and C Wray (1989) Genetic evidence for a chromosomally integrated multiresistance plasmid in S. dublin. J. Med. Microbiol. 28:205-210.

4. M J Woodward, I McLaren and C Wray (1989) Distribution of virulence plasmids within Salmonellae. J. Gen. Microbiol. 135:503-511.

5. M J Woodward (1989) DNA probes for diagnosis: An introduction. State Vet. J. 4:28-41.

6. P Klaasen, M J Woodward, F G van Zijderveld and F K de Graaf (1990) The 987P gene cluster in enterotoxigenic E.coli contains an STpa transposon that activates 987P expression. Infect. Immun. 58:801-807.

7. M J Woodward, C Wray, G A Ridha and J R Walton (1990) Plasmid and chromosomal related toxin polymorphism of E.coli serogroup 0138; plasmid transfer and co-integration with pRP4. J. Med. Microbiol. 31:241-249.

8. M J Woodward, R Kearsley, C Wray and P L Roeder (1990) DNA probes for the detection of toxin genes in E.coli isolated from diarrhoeal disease in cattle and pigs. Vet. Microbiol. 22:227-290.

9. M E Collins, A Patki, S Wall, A Nolan, J Goodger, M J Woodward and J W Dale (1990) Cloning and characterisation of the gene for the 19kDa antigen of M.bovis. J. Gen. Microbiol. 136:1429- 1436.

10. C Wray and M J Woodward (1990) Biotechnology and veterinary science: production of veterinary vaccines. Rev. Sci. Tech. Off. Int. Epiz. 9:779-794.

11. P J Carroll, M J Woodward and C Wray (1990) Detection of LT and STIa toxins by latex and EIA tests. Vet. Rec. 29:335-336.

12. M J Woodward and C Wray (1990) Nine DNA probes for the detection of toxin and adhesin genes in E.coli isolated from diarrhoeal disease in animals. Vet. Microbiol. 25:55-65.

13. R G Hewinson, S E S Rankin, B J Bevan and M J Woodward (1991) Detection of C. psittaci from avian field samples using the PCR. Vet. Rec. 128:129-130.

14. R G Hewinson, P C Griffiths, S E S Rankin, M Dawson and M J Woodward (1991) Towards a differential polymerase chain reaction test for C. psittaci. Vet.Rec. 128:381-382.

15. M J Woodward and G J Sullivan (1991) Nucleotide sequence of a repetitive element isolated from Leptospira hardjo-bovis. J. Gen. Microbiol. 137:1101-1109.

16. M J Woodward, G J Sullivan, N M A Palmer, J C Woolley and J S Redstone (1991) Development of a PCR test specific for L. hardjo-bovis. Vet. Rec. 128:281-283

17. R G Hewinson, J P Lowings, M D Dawson and M J Woodward (1991) Prions progress. Nature. 351:291

18. P M Roehe and M J Woodward (1991) Polymerase Chain Reaction amplification of segments of pestivirus genomes. Arch.Virol. Suppl. 3:231-238.

19. P M Roehe, M J Woodward and S Edwards (1992) Characterisation of p20 gene sequences from a border disease-like pestivirus isolated from pigs. Vet. Microbiol. 33:231-238.

CSG 7 (Rev 6/97) 1 20. C Wray, I M McLaren and M J Woodward (1992) The use of plasmid analysis to study the epidemiology of Salmonella. Flair 6:906:67-71.

21. M J Woodward, P J Carroll and C Wray (1992) Detection of entero- and verocyto-toxin genes in Escherichia coli from diarrhoeal disease in animals using the polymerase chain reaction. Vet. Microbiol. 31:251-261.

22. M J Woodward and J S Redstone (1993) Differentiation of Leptospira serovars by PCR and RFLP analysis. Vet. Rec. 132:325-326.

23. C Turcotte and M J Woodward (1993) Cloning, DNA Nucleotide sequence and distribution of the gene encoding SEF14, a fimbrial antigen of Salmonella enteritidis. J. Gen. Microbiol. 139:1477- 1485.

24. K A Webster, J D E Pow, M Giles and M J Woodward (1993) Detection of Cryptosporidium parvum using a specific Polymerase Chain Reaction. Vet. Para. 50:35-44.

25. M J Woodward, C J Thorns and C Turcotte (1993) Fimbriae of Salmonella enteritidis: Molecular analysis of SEF14 and vaccine development potential. In: The Biology of Salmonella. Ed. F. Cabello et al (1993) Plenum Press, New York. pp79-82.

26. J B Bashiruddin, R A J Nicholas, R A Ready, F G Santini, M J Woodward and T K Taylor (1993) Detection of Mycoplasma DNA from CBPP-affected cattle in Italy by PCR. Vet.Rec. 134:240-241

27. M J Woodward and J S Redstone (1994) Deoxynucleotide sequence conservation of the endoflagellin subunit protein, FlaB, within the genus Leptospira. Vet. Microbiol. 40:239-251.

28. C Wray and M J Woodward (1994) Laboratory Diagnosis of E.coli Diseases. In: Escherichia coli in domestic animals and humans. Ed. C L Gyles. CAB International, Wallingford [Oxon], UK.

29. G L Cooper, L M Venables, M J Woodward and C E Hormaeche (1994) The invasiveness and persistence of S. typhimurium and a genetically-defined S. enteritidis aroA strain in young chickens. Infect. Immun. 62:4739-4746.

30. G L Cooper, L M Venables, M J Woodward and C E Hormaeche (1994) Vaccination of chickens with strain CVL30, a genetically defined Salmonella enteritidis aroA live oral vaccine candidate. Infect. Immun. 62:4747-4754.

31. C Wray, M J Woodward and I M McLaren (1994) Escherichia coli infection in farm animals - 25 years of progress. J. Med. Microbiol. 41:156-158.

32. C Wray, L P Randall, I M McLaren and M J Woodward (1994) Verocytotoxigenic Escherichia coli from animals, their incidence and detection. In: Recent Advances in Verocytotoxin-producing Escherichia coli Infections. Ed M A Karmali and A G Goglio. Elsevier Science B. V., Amsterdam.

33. G Cooper, L M Venables, M J Woodward and C E Hormaeche (1995) The invasiveness, persistence and efficacy of Salmonella enteritidis aroA strain CVL30 in chickens. In: COST 97; Protection of poultry from foodborne pathogens. Ed B Nagy, E Nurmi and RWA Mulder. EC Luxembourg.

34. C J Thorns, C Turcotte and M J Woodward (1995) Role of the SEF14 fimbrial antigen in the colonisation and persistence of Salmonella enteritidis infection in poultry. In COST 97; Protection of poultry from foodbourne pathogens. Eds B Nagy, E Nurmi and RWA Mulder. EC Luxembourg.

35. E Allen-Vercoe, S Cawthraw, D G Newell and M J Woodward (1996) Expression of C.jejuni flaA epitopes within a modified flagellin expressed in S.enteritidis. In Campylobacters Helicobacters, and related organisms Eds Newell et al Plenum Press, New York p667-672.

36. C J Thorns, C Turcotte, S Gemmell and M J Woodward (1996) Studies into the SEF14 fimbrial antigen in the pathogenesis of Salmonella enteritidis infection. Microbiol. Path. 20:235-246.

CSG 7 (Rev 6/97) 2 37. M J Woodward and S E S Kirwan (1996) Detection of Salmonella enteritidis in eggs by PCR. Vet. Rec. 138:411-413.

38. R D Ayling, M J Woodward, S Evans and D G Newell (1996) Restriction fragment length polymorphism of polymerase chain reaction products applied to the differentiation of poultry Campylobacters for epidemiological investigations. Res. Vet. Sci. 60:168-172

39. M J Woodward, E Allen-Vercoe and J S Redstone (1996) Distribution, gene sequence and expression in vivo of the plasmid encoded fimbriae of Salmonella serotype Enteritidis. Epidem. Infect. 117:17-28

40. J S Redstone and M J Woodward (1996) The application of the ligase mediated PCR to the differentiation of Leptospira serogroups. Vet. Microbiol. 51:251-362

41. S Rijpkema, S Hughes, G David and M J Woodward (1997) Partial identification of spirochaetes from two dairy cows with digital dermatitis by polymerase chain reaction analysis of the 16S ribosomal RNA gene. Vet. Rec. 140:257-259.

42. R G Hewinson, P C Griffiths, B J Bevan, S E S Kirwan, M E Field, M J Woodward and M Dawson (1997) Detection of Chlamydia psittaci in avian clinical samples by polymerase chain reaction. Vet. Microbiol. 54:155-166.

43. C Wray and M J Woodward (1997) Escherichia coli in Animal Disease. In: Escherichia coli: Mechanisms of virulence. Ed. M. Sussman. SGM special publication, Cambridge, UK.

44. M.J.Woodward (1997) Escherichia coli; an old friend revisited. The Pig Journal 39:54-62.

45. A J Woolford, R G Hewinson, M J Woodward and J W Dale (1997) Sequence heterogeneity of an MPB70 gene homologue in Mycobacterium kansasii. FEMS Microbiol. Letts. 148:43-48

46. E Allen-Vercoe, M Dibb-Fuller, C J Thorns and M J Woodward. (1997) SEF17 fimbriae are essential fior the convoluted colony morphology of S. enteritidis. FEMS Microbiol. Letts. 153:33-42.

47. Collighan R and M J Woodward (1997) Sequence analysis and distribution of an IS3-like insertion element isolated from Salmonella enteritidis. FEMS Microbiol Letts 154:207-213.

48. Collighan R and M J Woodward (1997) Spirochaetes and other bacterial species associated with digital dermatitis. FEMS Microbiol. Letts. 156:37-41.

49. Dibb-Fuller M, E Allen-Vercoe, M J Woodward and C J Thorns (1997) Expression of SEF17 fimbriae by S. enteritidis. Letts. Appl. Bacteriol. 25:447-452

50. M J Woodward, C Swallow, A Kitching, C Dalley and A R Sayers (1997) Serodiagnosis of Leptospira hardjo-bovis by MAT, ELISA and IMMUNOCOMB; a comparative exercise using field sera. Vet. Rec. 141:603-604.

51. M J Woodward, E Allen-Vercoe, R La Ragione, M Dibb-Fuller, M Carter and C J Thorns (1997) Molecular approaches to the study of the role of fimbriae in the biology of Salmonella enteritidis and other salmonellas. In: Proceedings of the XIth International Congress of the World Veterinary Poultry Association p.109ff ed., Szekely, Budapest, Hungary.

52. Demirkan I, S D Carter, R D Murray, R W Blowey and M J Woodward (1998) The frequent detection of a Treponeme in bovine digital dermatitis by immunocytochemistry and polymerase chain detection. Vet Microbiol 60:285-292.

53. M J Woodward, E Allen-Vercoe, R La Ragione, M Dibb-Fuller, M Carter and C J Thorns (1998) Molecular approaches to the study of the role of fimbriae in the biology of Salmonella enteritidis and Escheirchia coli. In EU Cost Action 97 Pathogenic micro-organisms in poultry and eggs. 5. Poultry and Food Safety. 93-102.

54. E Allen-Vercoe, R Collighan, M J Woodward. (1998) The variant rpoS allele S. enteritidis strain

CSG 7 (Rev 6/97) 3 27655R does not affect virulence in the chick model nor constitutive curliation but does generate a cold sensitive phenotype. FEMS Microbiol Letts 167:245-253.

55. L Petrovska, R G Hewinson, G Dougan D Maskell, and M J Woodward (1999). Brucella melitensis 16M; Characterisation of the galE gene and mouse immunisation studies with a galE deficient mutant. Vet. Microbiol. 65: 21-36.

56. S L Walker, M Sojka, M Dibb-Fuller and M J Woodward (1999). The effect of pH, temperature and surface contact upon the elaboration of fimbriae and flagella by Salmonella enteritidis. J. Med Microbiol. 48: 253-261.

57. E Allen-Vercoe and M J Woodward (1999) Adherence of Salmonella enterica serovar Enteriditis to chick gut explant; the role of flagella but not fimbriae. J. Med. Microbiol 48:771-780.

58. M J Woodward, D Gavier-Widen, I McLaren, C Wray, M Sozmen and G R Pearson (1999) Infection of gnotobiotic calves with Escherichia coli O157:H7 strain A84. Vet. Rec. 144: 466-470

59. M Dibb-Fuller, E Allen-Vercoe, C J Thorns and M J Woodward (1999) Characterisation of fimbrial and flagella mediated adherence to and invasion of INT407 monolayers by Salmonella enterica serotype Enteritidis. Microbiol 145: 1023-1131

60. R M La Ragione, R J Collighan, M J Woodward (1999). Non-curliation of Escherichia coli O78:K80 isolates associated with IS1 insertion in csgB and reduced persistence in poultry infection. FEMS Microbiol Letts 174: 247-253

61. E Allen-Vercoe, R Sayers and M J Woodward. (1999) Virulence of Salmonella enterica serovar Enteritidis aflagellate and afimbriate mutants in the day old chick. Epidem Infect 122: 395-402.

62. G R Pearson, K Bazeley, J R Jones, J R Gunning, M J Green, A Cookson and M J Woodward. (1999) Attaching and effacing lesions in the large intestine of an 8 month old heifer associated with a naturally occurring, verocytotoxigenic E. coli O26:K60 infection in a groups of animals with dysentry. Vet Rec 145: 370-373.

63. E Allen-Vercoe, M J Woodward. (1999) Colonisation of the chicken caecum by afimbriate and aflagellate derivatives of Salmonella enterica serotype Enteritidis. Vet Microbiol 69: 265-275.

64. I Demirkan, S D Carter, C A Hart, M J Woodward. (1999) Isolation and cultivation of a spirochaete from bovine digital dermatitis. Vet Rec 145: 497-498.

65. M J Woodward. Digital Dermatitis; what role spirochaetes? (1999) Cattle Practice 7: 345-348.

66. A Cookson and M J Woodward (1999) The role of fimbriae in adhesion of verocytotoxigenic Escherchia coli (VTEC) Biofilm

67. R M La Ragione, W A Cooley and M J Woodward. (2000) Adherence of avian Escherichia coli O78:K80 to tissue culture, tracheal and gut explants; the role of fimbriae and flagella J Med Microbiol 49: 327-338

68. C J Thorns and M J Woodward. (2000) The molecular biology of the fimbriae of Salmonellas. In Salmonella in the Domestic Animals Ed C. Wray and A. Wray. CAB International Wallingford Oxfordshire UK. Pp 35-56.

69. M J Woodward. M Sojka, K Sprigings and T J Humphrey. (2000) The role of SEF14 and SEF17 fimbriae of Salmonella enterica serotype Enteritidis in adherence to inanimate surfaces. J Med Microbiol 49, 481-487.

70. R J Collighan, R D Naylor, P K Martin, W A Cooley, N Buller, M J Woodward. (2000) A spirochaete isolated from a case of severe virulent ovine foot-rot is closely related to treponemes isolated from bovine digital dermatitis and human periodontitis. Vet. Microbiol. 74, 249-257.

71. J M C Robertson, G Grant, E. Allen-Vercoe, M J Woodward, A Putzai and H J Flint. (2000)

CSG 7 (Rev 6/97) 4 Adhesion of S. enteritidis strains lacking fimbriae and flagella to rat ileal explants cultured at the air interface or submerged in tissue culture medium. J. Med. Microbiol. 49, 691-696.

72. R M La Ragione, Sayers A R and M J Woodward. (2000) The role of flagella and fimbriae in the invasion, colonisation and persistence of Escherichia coli O78:K80 in the chick. Epidem Infect 124, 351-363.

73. M Dibb-Fuller and M J Woodward. (2000) The contribution of fimbriae and flagella Characterisation of fimbrial and flagella of Salmonella enterica serotype Enteritidis in the colonisation, invasion, persistence and lateral transfer in chicks. Avian Pathol 29, 295-304.

74. K N Ganeshram, A Ross, R P Cowell, C Cefai and M J Woodward. (2000) Recurring febrile illness in a slaughter house worker. Postgrad. Med. J. 76, 790-791.

75. H Chart, H R Smith, R M La Ragione and M J Woodward. (2000) An investigation into the pathogenic properties of Escherichia coli strains BLR, BL21, DH5a and EQ1. J. Appl. Microbiol. 89: 1048-1058

76. R. M. La Ragione, K. E. Coles, F. Jørgensen, T. J. Humphrey and M. J. Woodward. (2000) Virulence in the chick model and stress tolerance of Salmonella enterica serovar Orion 15+. Int. J. Med. Microbiol. 290, 121-130.

77. M Sojka and M J Woodward. Analysis of expression of flagella by Salmonella enterica serotype Typhimurium by monoclonal antibodies recognising both phase specific and common epitopes. (2001) Vet. Microbiol. 78, 61-77.

78. P J Naughton, G Grant, S Bardocz, E Allen-Vercoe, M J Woodward A Putzai. Expression of Salmonella enteritidis type 1 fimbriae (SEF21) is an advantage in the early colonisation of the rat gut in vivo but presents a competitive disadvantage in the long term (2001) J. Med. Microbiol. 50, 191-197.

79. E Liebana, L Garcia-Migura, M F Breslin, R Davies and M J Woodward. Diversity of Salmonella enterica Serovar Enteritidis from English poultry farms by multiple genetic fingerprinting. (2001) J. Clin. Microbiol. 39, 154-161.

80. R J Collighan, S L Walker, and M J Woodward. Sequence analysis and distribution in Salmonella enterica serotypes of IS1230, an IS3-like insertion element. (2001) Int. J. Med. Microbiol. 290, 619- 626.

81. D. Wales, F. A. Clifton-Hadley, A. L. Cookson, M. P. Dibb-Fuller, C. M. Hayes, R. M. La Ragione, J. M. Roe, K. A. Sprigings, G. R. Pearson and M. J. Woodward. Preliminary observations on E. coli O157:H7 in sheep. Epidemiology of Verocytotoxigenic E. coli (2001) Pp105-113 In Verocytotoxigenic E. coli in Europe, 5; Concerted Action CT98-3935. Eds. Duffy, Garvey, Coia, Wasteson and MacDowell, Published by Teagasc, The National Food Centre, Castleknock, Dublin, Ireland.

82. L. P. Randall and M. J. Woodward. (2001)The Multiple Antibiotic Resistance (mar) Locus in Salmonella enterica serovar Typhimurium DT104. Appl. Environ. Microbiol. 67, 1190-1197.

83. R M La Ragione, G Casulla, S M Cutting and M J Woodward. (2001) Bacillus subtilis spores competitively exclude Escherichia coli O70:K80 in poultry. Vet. Microbiol. 79, 133-142.

84. R A Walker, N Saunders, A J Lawson, E A Lindsay, M Dassama, L R Ward, M J Woodward, R H Davies and E J Trelfall. (2001) Use of a LightCyclerTM gyrA mutation assay (GAMA) for the rapid identification of mutations conferring decreased susceptibility to ciprofloxacin in multiresistant Salmonella enterica serotype Typhimurium DT104. J. Clin. Microbiol. 39, 1443-1448. 85. D. Wales, F. A. Clifton-Hadley, A. L. Cookson, M. P. Dibb-Fuller, R. M. La Ragione, K. A. Sprigings, G. R. Pearson and M. J. Woodward. (2001) Experimental infection of six-month old lambs with Escherichia coli O157:H7 Vet. Rec. 148, 630-631.

86. R J Collighan, and M J Woodward. The SEF14 fimbrial antigen of Salmonella enterica serovar

CSG 7 (Rev 6/97) 5 Enteritidis is encoded within a pathogenicity islet. (2001) Vet. Microbiol. 80, 235-245.

87. R Walker, E Lindsay, M J Woodward, L R Ward, J Trelfall. Variation in antibiotic resistance genes amongst multiresistant Salmonella enterica serotype Typhmurium phage type U302 (MR U302) from humans, animals and foods. (2001) Microb Drug Resist. 7, 13-21.

88. L P Randall and M J Woodward. The mar locus is involved in virulence of Salmonella Typhimurium DT104 in chickens (J. Med Microbiol accepted)

89. D. Wales, G. R. Pearson, A. M. Skuse, J. M. Roe, C. M. Hayes, A. L. Cookson, M. J. Woodward. Attaching and effacing lesions in neonatal lambs experimentally inoculated with Escherichia coli O157:H7 (J. Med. Microbiol. accepted)

90. L P Randall, S Cooles, A R Sayers and M J Woodward. Cyclohexane Resistance in Salmonella of different serotypes : Increased Resistance to Multiple Antibiotics, Disinfectants and Dyes. (J. Med Microbiol. accepted)

91. T A Cogan, H M Lappin-Scott, C E Benson, M J Woodward, M R W Brown, A W Smith and T J Humphrey An assessment of models used to study the growth of Salmonella enterica serovar Enteritidis in egg contents. (Appl. Environ. Microbiol. accepted)

92. M P Dibb-Fuller, A Best, D A Stagg, W A Cooley and M J Woodward. Bovine primary cell cultures; an in vitro model for studying the attachment of Escherichia coli O157:H7 and other enteropathogens. (J. Med. Mircrobiol. accepted)

93. E Gubbels, H de Greve, I Dewerte, E Allen-Vercoe. M J Woodward and J-P Hernalsteens. Identification of a Salmonella enteritidis pathogenicity islet invovled in systemic disease. (submitted to Mol. Microbiol.)

94. M De Fillette, E Allen-Vercoe, I Dewerte, H Bouchet, M J Woodward and P Pohl Role of AgfA subunit protein of SEF17 fimbriae in the adherence and pathogenesis of Salmonella enterica serotype Enteritidis (submitted to Infect Immun.)

95. M J Woodward. Leptospires. (Molecular Medical Microbiology; invited author, in press)

96. A Papconstantinopouloul, G Frankell, B Wren, J Parkhill, M J Woodward D Pickard, G Dougan. The sivH genes of Salmonella share homology with the Invasin (inv) / Intimin (eaeA) gene family. (submitted to Microbiol)

97. J M C Robertson, G Grant, E. Allen-Vercoe, M J Woodward, A Putzai and H J Flint. Flagella appear more important than fimbriae for the pathogenesis of oral Salmonella enterica serovar Enteritidis in the of rat. (submitted to Microbiol.)

98. L. P. Randall and M. J. Woodward. Cyclohexane Resistant Salmonella from Animals; Antibiotic and Disinfectant Resistance, OmpF analysis, Acid and Heat Tolerance and Cell Surface Hydrophobicity. (Vet. Microbiol. submitted)

99. F A Clifton-Hadley, M Breslin, L M Venables, K A Sprigings, S W Cooles and M J Woodward. A laboratory investigation of an inactivated, bivalent Salmonella enterica serovar Enteritidis/Typhimurium vaccine (Salenvac T) against Typhimurium challenge in poultry. (in preparation)

100.E. Liebana, D Guns, L Garcia-Migura, M J Woodward, F A Clifton-Hadley and R H Davies. Molecular typing of Salmonella serotypes prevalent in animals in England: Assessment of methodology. (Submitted to J. Clin. Microbiol.)

CSG 7 (Rev 6/97) 6 ANNEX A CURRICULUM VITAE OF STAFF TO BE ENGAGED ON THE RESEARCH

Please complete a separate form for each person to be engaged in the scientific aspects of the work

1. Surname Clifton Hadley Forename(s) Felicity Ann 2. Degrees: B.A.,M.A., Vet.M.B.,Ph.D.

This table will expand to accommodate the information you wish to enter. To move to the next field, press the DOWN arrow TWICE. Oct. 1977 - Dec. 1980 A.R.C. Veterinary Fellow, University of Cambridge

Jan.1981 - Sept. 1986 Research Associate, University of Cambridge

Oct. 1986 - Nov. 1996Career break

Dec. 1996 – March 2000 Veterinary Research Officer, Department of Bacterial Diseases, VLA,Weybridge March 2000 to date Joint workgroup leader in DBD Food Safety and Zoonoses Programme This table will expand to accommodate the information you wish to enter. To move to the next field, press the DOWN arrow TWICE.

CSG 7 (Rev 6/97) 7 2000 Clifton-Hadley, F.A. (2000). Detection and diagnosis of Escherichia coli O157 and other verocytotoxigenic E. coli in animal faeces. Reviews in Medical Microbiology, 11(1), 47-58.

Clifton-Hadley, F.A., Wales, A., Dibb-Fuller M.P., Cookson, A.L., La Ragione, R., Sprigings, K. and Woodward, M.J. (2000). Pathogenesis of Escherichia coli O157:H7 in experimentally- inoculated sheep: 2 dosing and sampling procedures. Research in Veterinary Science

Wales, A.D., Clifton-Hadley, F.A., Cookson, A.L., Dibb-Fuller, M.P., LaRagione, R.L., Pearson, G.R. and Woodward, M.J. (2000). Oral inoculation of sheep with E. coli O157:H7: Experimental findings. 4th International Symposium and Workshop on “Shiga toxin (verotoxin) -producing Escherichia coli infections” VTEC 2000. Kyoto, Japan Oct 29-Nov 2, 2000. p.168 no.378. Chairman Y. Takeda.

2001 Wales, A.D. Clifton-Hadley, F.A. Cookson, A.L. Dibb-Fuller, M.P. La Ragione, R.M. Sprigings, K.A., Pearson, G.R. and Woodward, M.J. (2001). Experimental infection of six-month-old lambs with Escherichia coli O157:H7 Veterinary Record 148, 630-631.

Submitted Liebana, E., Garcia-Migura, L., Guard-Petter, J., McDowell, S.W.J., Rankin, S., Opitz, H.M., Clifton- Hadley, F.A. and Davies, R.H. (2001). Salmonella enterica sero-type Enteritidis phage types 4, 7, 6, 8, 13a, 29, and 34 : A comparative analysis of genomic fingerprints from geographically distant isolates. Journal of Applied Microbiology

Liebana, E., Guns, D., Garcia-Migura, L., Clifton-Hadley, F.A., Woodward, M.J. and Davies, R.H. (2001) Molecular Typing of Salmonella serotypes prevalent in animals in England: Assessment of methodology. Journal of Clinical Microbiology

See attached list for previous publications

1975 Christian, FA and Gordon, JL (1975) Platelet function in C6-deficient rabbits. Aggregation and secretion induced by collagen and zymosan. Immunology, 29,131-141

1980 Clifton-Hadley, FA and Alexander, TJL (1980). The carrier site and carrier rate of Streptococcus suis type II in pigs. Veterinary Record, 107: 40-41

Elliott, SD, Clifton-Hadley, F and Tai, J (1980). Streptococcal infection in young pigs. V. An immunogenic polysaccharide from Streptococcus suis type 2 with particular reference to vaccination against streptococcal meningitis in pigs. Journal of Hygiene, 85: 275-285

1981 Alexander, TJL and Clifton-Hadley, FA (1981) Epidemiology and control of streptococcal meningitis. Veterinary Record, 109, 224

Clifton-Hadley, FA (1982). Studies of Streptococcus suis type 2 infection in pigs. Titles of dissertations approved in the University of Cambridge during the academical year 1981 1982. PhD Thesis, Department of Clinical Veterinary Medicine. Cambridge, UK; Board of Graduate Studies, 4 Mill Lane

Clifton-Hadley, FA (1981). Studies of Streptococcus suis type 2 infection in pigs. Index to Theses, 31, 103; Ph.D. Thesis, Univ. Cambridge

Clifton-Hadley, FA and Alexander, TJL (1981). Studies of Streptococcus suis type 2 infection. Pig Veterinary Society Proceedings, 8, 8-17

1982 Clements, MR, Hamilton, DV, Clifton-Hadley, FA and O'Reilly, JF (1982) Streptococcus suis type II infection. A new industrial disease?The Practitioner, 226, 323-325

Clifton-Hadley, FA (1982) Streptococcal meningitis. Pig Farming Autumn Health Supplement

1983

CSG 7 (Rev 6/97) 1 Clifton-Hadley, FA (1983). Review series Zoonoses in practice: Streptococcus suis type 2 infections. British Veterinary Journal, 139, 1-5

Clifton-Hadley, FA (1983) Epidemiology of Streptococcus suis type 2 infections.Commission of the European Communities proceedings of a seminar on "Some diseases of emerging importance to Community trade" [edited by J.R. Walton, E.G. White and S.A. Hall] 33-39 Report EUR 8515 EN

Clifton-Hadley, FA and Alexander, TJL (1983). Further studies of Streptococcus suis type 2 infection. Pig Veterinary Society Proceedings, 10, 14-19

1984 Clifton-Hadley, FA (1984). Studies of Streptococcus suis type 2 infection in pigs. Veterinary Research Communications, 8, 217-227

Clifton-Hadley, FA and Alexander, TJL (1984). Epidemiology of Streptococcus suis type 2. Proceedings of the 8th International Pig Veterinary Society Congress. p135. Ghent, Belgium

Clifton-Hadley, FA, Alexander, TJL, Enright, MR and Guise, J (1984). Monitoring herds for Streptococcus suis type 2 by sampling tonsils of slaughter pigs. Veterinary Record, 115, 562-564

Clifton-Hadley, FA, Alexander, TJL, Upton, I and Duffus, WPH (1984). Further studies on the subclinical carrier state of Streptococcus suis type 2 in pigs. Veterinary Record, 114, 513-518

Clifton-Hadley, FA and Enright, MR (1984). Factors affecting the survival of Streptococcus suis type 2. Veterinary Record, 114, 584-586

1985 Alexander, TJL, Clifton-Hadley, FA and Enright, MR (1985) The aetiology, epidemiology, diagnosis and control of Streptococcus suis type 2 infection. Proceedings of the Academia de Ciencias Veterinarias de Cataluna, IV Simposio Internacional Sobre Produccion, Barcelona 7 & 8 June 1985

Clifton-Hadley, FA (1985) Epidemiology of Streptococcus suis type 2 infection.Veterinary Annual, 25, 161-166.

Clifton-Hadley, FA, Alexander, TJL and Enright, MR (1985). Diagnosis of Streptococcus suis type 2 infection in pigs. Pig Veterinary Society Proceedings, 14, 27-34

1986 Clifton-Hadley, FA, Alexander, TJL and Enright,MR (1986) The epidemiology, diagnosis, treatment and control of Streptococcus suis type 2 infection. Proceedings of the American Association of Swine Practitioners, Minneapolis, March 16-18 1986, pp. 471-491

Clifton-Hadley, FA, Alexander, TJL and Enright, MR (1986). Monitoring slaughter pigs for Streptococcus suis type 2:Studies of chance contamination. Veterinary Record, 118, 274

Clifton-Hadley, FA, Alexander, TJL, Enright, MR and Lindsay, HJ (1986) Monitoring herds for Streptococcus suis type 2: cross-reactions and variations in virulence. Proceedings of the International Pig Veterinary Society Congress (Barcelona), 9,359

Clifton-Hadley, FA, Enright, MR and Alexander, TJL (1986). Survival of Streptococcus suis type 2 in pig carcases. Veterinary Record, 118, 275

1987 Enright, MR, Alexander, TJL and Clifton-Hadley, FA (1987). Role of houseflies (Musca domestica) in the epidemiology of Streptococcus suis type 2. Veterinary Record, 121, 132-133

1988 Clifton-Hadley, F and Alexander, T (1988). Diagnosis of Streptococcus suis infection in pigs. In Practice, 10, 185-187

1989 Clifton-Hadley, FA (1989). Streptococcal meningitis in pigs. Pig News and Information, 10 9-12

1991 CSG 7 (Rev 6/97) 2 Clifton-Hadley, F. and Alexander, T. (1991). Diagnosis of Streptococcus suis infection in pigs. In: E. Boden (ed.) Swine Practice, (Ballière Tindall, London) 115-126

CSG 7 (Rev 6/97) 3 ANNEX A CURRICULUM VITAE OF STAFF TO BE ENGAGED ON THE RESEARCH

Please complete a separate form for each person to be engaged in the scientific aspects of the work

1. Surname Allen Forename(s) Vivien Mary 2. Degrees: PhD University of Bristol , Faculty of Medicine, by thesis entitled Salmonella infections in broiler chickens: epidemiology and control during incubation and brooding (1997)

Professional qualifications: Fellow of the Institute of Biomedical Sciences

This table will expand to accommodate the information you wish to enter. To move to the next field, press the DOWN arrow TWICE. 1992-present Research Fellow/Associate/Assistant, University of Bristol 1979-1992 Laboratory Manager/ microbiology technician, University of Bristol 1964.1971 Medical Laboratory Scientific Officer, PHLS, Bristol Funded at present on MAFF project CSA 5519 (An in vivo poultry model to study the effect of growth promotors on resistance to human antibiotics in mixed bacterial populations) and FSA project BO3008 (Studies to identify critical points for infection of live birds or contamination of poultry carcasses with Campylobacter and Salmonella spp)

This table will expand to accommodate the information you wish to enter. To move to the next field, press the DOWN arrow TWICE. Relevant papers; CHERRINGTON, C.A., ALLEN, V. & HINTON, M. (1992) The influence of temperature and organic matter of the bacteriacidal activity of short-chain acids on salmonella. Journal of Applied Bacteriology 72, 500-503. HINTON, M., ALLEN, V.M. & WRAY, C. (1992) The influence of growth promoting antibiotics on the colonisation of the caecum of young chicks following consumption of feed artificially contaminated with salmonellas. In: Prevention and Control of Pathogenic Micro-organisms in Poultry and Poultry Meat Processing. 7. The Role of Antibiotics in the Control of Food Borne Pathogens. Edited by M. Hinton & R.W.A.W. Mulder. pp. 69-75. Beekbergen: Agricultural Research Service (DLO-NL). [ISBN 90 71463 60 5; ISSN 0925 7217] GUDMUNSDOTTIR, K.B., MARIN, M.L., ALLEN, V.M., CORRY, J.E.L. & HINTON, M. (1993) The antibacterial activity of inorganic phosphates. In: Prevention and Control of Pathogenic Micro-organisms in Poultry and Poultry Meat Processing. 11. Contamination with pathogens in relation to processing and marketing. Edited by J. Löpfe, C.A. Kan & R.W.A.W. Mulder. pp. 95-100. Beekbergen: Agricultural Research Service (DLO-NL). [ISBN 90 71463 65 6; ISSN 0925 7217] ALLEN, V.M. & HINTON, M. (1993) Factors effecting the survival and growth of salmonellas in the environment of broiler chickens. In: Livestock Environment IV. Edited by E.collins & C. Boon. pp.124-128. Michigan: American Society of Agriculure Engineers. HINTON, M., ALLEN, V.M. & LINTON, A.H. (1994) The effect of the management of calves on the prevalence of antibiotic resistant strains of Escherichia coli in their faeces. Letters in Applied Microbiology 19, 197-200. DESMIDT, M.,DUCATELLE, R., HAESEBROUCK, F., DE GROOT, P.A., VERLINDEN, M., WIJFFELLS, R., HINTON, M., BALE, J. & ALLEN, V. (1996) Detection of antibodies to Salmonella Enteritidis in sera and egg yolks obtained from experimentaly and naturally infected chickens. Veterinary Record 138, 223-226. HINTON, M.H., ALLEN, V.M., TINKER, D.B., GIBSON, C. and WATHES, C.M. (1996) The dispersal of bacteria during the defeathering of poultry. In: Factors Affecting the Microbiology Quality of Meat 2. Slaughter and Dressing. Edited by M.H. Hinton and C. Rowlings. Bristol: Bristol University Press. pp. 113-121 TINKER, D.B., GIBSON, C., HINTON, M.H., ALLEN, V.M. and WATHES, C.M. (1996) Reduction of cross-contamination in defeathering machinery. World Poultry 12, 13-15 ALLEN, V.M. & HINTON, M. (1997) Dye reduction tests for the assesssment of microbial contamination

CSG 7 (Rev 6/97) 4 in meat production. In: Factors Affecting the Microbiology Quality of Meat 4. Microbial Methods for the Meat Industry. Edited by M.H. Hinton and C. Rowlings. Bristol: Bristol University Press. pp. 161-171 ALLEN, V.M., FERNANDEZ, F., HINTON, M.H. (1997) Evaluation of the influence of supplementing the diet with mannose or palm kernal meal on Salmonella colonisation in poultry. British Poultry Science 38, 485-488 ALLEN, V.M. (1999) Salmonella infections in broiler flocks: A European perspective. China Agro 6, 103-108 MEAD, G.C., ALLEN, V.M., BURTON, C.H., CORRY J.E.L. (2000) Microbial cross-contamination during air-chilling of poultry. British Poultry Science, 41, 158-162 ALLEN, V., CORRY, J.E.L., BURTON, C.H., WHYTE, R.T., MEAD, G.C. (2000) Hygiene aspects of modern poultry chilling. International Journal of Food Microbiology 58, 39-48 ALLEN, V.M., BURTON, C.H., CORRY J.E.L., MEAD, G.C., TINKER, D.B. (2000) Investigation of hygiene aspects during air chilling of poultry carcasses using a model rig. British Poultry Science 41, 575-583 DAVIES, R.H., BRESLIN, M., CORRY, E.L., HUDSON, W., ALLEN, V.M. (2001) Observations on the distribution and control of Salmonella in two integrated broiler companies. Veterinary Record, (in press)

CSG 7 (Rev 6/97) 5 ANNEX A CURRICULUM VITAE OF STAFF TO BE ENGAGED ON THE RESEARCH

Please complete a separate form for each person to be engaged in the scientific aspects of the work

1. Surname Humphrey Forename(s) Thomas John 2. Degrees: BSc 1st Class Honours Microbiology, Hatfield Polytechnic, June 1971 PhD Microbial Physiology, University of East Anglia, April 1975 FRCPath, 1998

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Oct 1976 - Apr 1981 Lecturer/Senior Lecturer in Food and Agricultural Microbiology, Natural Sciences Department, Seale-Hayne College, University of Plymouth

Apr 1981 - Mar 2001 Grade C Clinical Scientist, Head of PHLS Food Microbiology Research Unit, Exeter.

Apr 2001 - to date Professor of Food Safety Department of Clinical Veterinary Science, University of Bristol

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CSG 7 (Rev 6/97) 6 Humphrey, T.J. (1994). Contamination of egg shell and contents with Salmonella enteritidis: a review. International Journal of Food Microbiology, 21, 31-40.

Williams, A., Davies, A.C., Wilson, J., Marsh, P.D., Leach, S., Humphrey, T.J. (1998). Contamination of the contents of intact eggs by Salmonella typhimurium DT104. Veterinary Record, 143, 562- 563.

Leach, S.A., Williams, A., Davies, A.C., Wilson, J., Marsh, P.D., Humphrey, T.J. (1999). Aerosol route enhances the contamination of intact eggs and muscle of experimentally infected laying hens by Salmonella typhimurium DT104. FEMS Microbiology Letters, 171, 203-207.

Humphrey, T.J., Williams, A., McAlpine, K., Lever, S., Guard-Petter, J., Cox, J.M. (1996). Isolates of Salmonella enterica Enteritidis PT4 with enhanced heat and acid tolerance are more virulent in mice and more invasive in chickens. Epidemiology & Infection, 117, 79-88.

Humphrey, T.J., Williams, A., McAlpine, K., Jørgensen, F., O'Byrne, C. (1998). Pathogenicity in isolates of Salmonella enterica serotype Enteritidis PT4 which differ in RpoS expression: effects of growth phase and low temperature. Epidemiology & Infection, 121, 295-301.

Jørgensen, F., Leach, S., Wilde, S., Mackey, B., Stewart, G.S.A.B., Humphrey, T.J. (2000). Invasiveness in chickens, stress resistance and RpoS status of wild type Salmonella enterica Typhimurium definitive type 104 and Enteritidis phage type 4 isolates. Microbiology, 146, 3227-3235.

La Ragione, R.M., Coles, K.E., Jørgensen, F., Humphrey, T.J., Woodward, M.J. (2001). Virulence in the chick model and stress tolerance of Salmonella enterica serovar Orion var. 15+. International Journal of Medical Microbiology, 290, 707-718.

CSG 7 (Rev 6/97) 1 ANNEX A CURRICULUM VITAE OF STAFF TO BE ENGAGED ON THE RESEARCH

Please complete a separate form for each person to be engaged in the scientific aspects of the work

1. Surname Liebana Forename(s) Ernesto 2. Degrees: BVSc (UCM, Universidad Complutense Madrid, 1991) Spanish National Award for Veterinary Medicine Studies (1991).

Honours project, grade A (1992).

PhD in Veterinary Science (UCM, May 1996) Special Mention for Doctorate Studies Veterinary School of Madrid (1997) Royal Academy of PhDs UCM Extraordinary Award (1997) National Award for PhD studies University of Leon / SYVA Laboratories (1998)

This table will expand to accommodate the information you wish to enter. To move to the next field, press the DOWN arrow TWICE. # October 1999 -Present, Veterinary Researcher Grade C Department of Bacterial Diseases, VLA Weybridge.

# October 1998 - September 1999, Postdoctoral Fellow Veterinary Sciences Division DANI-Queen's University of Belfast funded by the European Union (TMR/Marie Curie felloship).

# October 1997 - September 1998, Postdoctoral Fellow (Honorary Research Fellow) in the Veterinary Sciences Division DANI-Queen's University of Belfast. Funded by the Ramón Areces Foundation.

# 1997, from January to September, Postdoctoral Research Fellowship assigned to project I+D 0030/94 Veterinary School of Madrid, Spain.

# 1996, Laboratory Team Manager for the Eradication Campaigns in the Comunidad Autónoma de Madrid, Regional Agricultural Laboratory of Madrid, Spain.

# 1993, 1994 and 1995, Spanish Ministry of Education and Science short term fellowships for visiting the Australian Reference Laboratory for Bovine Tuberculosis.

# 1992 to 1995, Research Scholarship for Ph.D. Studies granted by the Spanish Ministry of Education and Science. Department of Animal Pathology I, School of Veterinary Medicine, Complutense University of Madrid, Spain.

# 1991, Research Scholarship INIA (Spanish National Institute for Animal and Agricultural Research). Department of Fish Pathology

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CSG 7 (Rev 6/97) 2 1) Simple and Rapid Detection of Mycobacterium tuberculosis Complex Organisms in Bovine Tissue Samples by PCR. Liébana, E. et al. Journal of Clinical Microbiology. 1995, vol. 33 (1):: 33-36.

2) Eficacia comparativa de la intradermoreacción y de la prueba de liberación de gamma interferón para el diagnóstico de la tuberculosis bovina en una prueba de campo. Domingo, M.; Liébana, E. et al. Medicina Veterinaria. 1995, vol. 12 (5):: 307-317.

3) La tuberculosis felina: un riesgo para la salud humana y animal. Lesiones y diagnóstico. Novoa, C., Pickering, X., Sánchez, B., García, P., Aranaz, A., Liébana, E. et al. Medicina Veterinaria. 1995, vol. 12 (5):: 279-284. Awarded with the Félidos Medicina Veterinaria/Pierre-Richard Dick (VIRBAC) Award.

4) The use of a PCR assay for the rapid diagnosis of canine and feline tuberculosis. Aranaz, A.; Liébana, E. et al. Veterinary Record. 1996, 138: 276-280.

5) Direct detection of Mycobacterium bovis from tissue samples: improvement of a DNA extraction method for PCR amplification. Aranaz, A.; Liébana, E. et al. Tuberculosis in Wildlife and Domestic Animals. Otago Conference Series No.3. 1996. Pages 60-63. Ed. Univ. of Otago Press, Dunedin, New Zealand.

6) A field evaluation of the interferon-gamma assay and the intradermal tuberculin test in dairy cattle in Spain. Domingo, M.; Liébana, E. et al. Tuberculosis in Wildlife and Domestic Animals. Otago Conference Series No.3. 1996. Pages 304-306. Ed. Univ. of Otago Press, Dunedin, New Zealand.

7) Eradication of tuberculosis from goat herds by means of the gamma-IFN assay and the single intradermal comparative tuberculinization test. Vidal, D.; Domingo, M.; Aranaz, A.; Liébana, E. et al. Tuberculosis in Wildlife and Domestic Animals. Otago Conference Series No.3. 1996. Pages 328-330. Ed. Univ. of Otago Press, Dunedin, New Zealand.

8) Assessment of genetic markers for species differentiation within the Mycobacterium tuberculosis complex. Liébana, E. et al. Journal of Clinical Microbiology. 1996, vol 34 (4):: 933-938.

9) Infecciones por M. bovis en el hombre: ¿Una realidad infravalorada en nuestro país? Liébana, E. et al. Boletín Epidemiológico Semanal. 1996, vol 3 (17):: 181-188.

10) Mycobacterium genavense infection in canaries. Ramis, A.; Ferrer, L.; Aranaz, A.; Liébana, E. et al. Avian Diseases, 1996. 40: 246-251.

11) Spacer oligonucleotide typing of Mycobacterium bovis strains from cattle and other animals: a tool for studying epidemiology of tuberculosis. Aranaz, A.; Liébana, E. et al. Journal of Clinical Microbiology, 1996. 34(11):: 2734-2740.

12) Laboratory diagnosis of avian mycobacteriosis. Aranaz, A.; Liébana, E et al. Seminars in Avian and Exotic Pet Medicine, 1997. 6 (1):: 9-17.

13) The insertion element IS6110 is a useful tool for DNA fingerprinting of Mycobacterium bovis isolates from cattle and goats in Spain. Liébana, E. et al. Veterinary Microbiology, 1997. 54 (3-4):: 223-233.

14) Tuberculosis respiratoria en bóvidos. Aranaz, A.; Liébana, E. et al. Patología del aparato respiratorio de los bóvidos: Formación continuada en Veterinaria. 1997. Ed. Pulso Ediciones, Barcelona, España.

15) Evaluation of the gamma-interferon assay for eradication of tuberculosis in a goat herd. Liébana, E. et al. Australian Veterinary Journal. 1998. 76 (1) : 50-53.

16) Evaluation of four DNA typing techniques in epidemiological investigations of bovine tuberculosis.

CSG 7 (Rev 6/97) 1 Cousins, D.V.; Williams, S.; Liébana, E. et al. Journal of Clinical Microbiology, 1998. 36(1):: 168-178.

17) Restriction fragment length polymorphism and Spacer oligonucleotide typing: a comparative analysis of fingerprinting strategies for Mycobacterium bovis strains. Aranaz, A.; Liébana, E. et al.. Veterinary Microbiology, 1998. 61: 311-324.

18) Generation of CD8+T-cell responses to Mycobacterium bovis and mycobacterial antigen in experimental bovine tuberculosis. Liébana, E. et al. Infection and Immunity. 1999 67(3):1034-1044.

19) Mycobacterium caprae sp. nov.: a taxonomic study of a new member of the Mycobacterium tuberculosis complex isolated from goats in Spain. Aranaz, A.; Liébana, E. et al. International Journal of Systematic Bacteriology, 1999.49: 1263-1273.

20) Comparison of different methods for diagnosis of bovine tuberculosis from tuberculin or interferon- reacting cattle in Spain. Gonzalez-Llamazares, O.R., Gutierrez-Martin, C.B., Aranaz-Martin, A., Liebana, E. et al. Journal of Applied Microbiology. 1999. 87: 465-471.

21) Cellular interactions in bovine tuberculosis: release of active mycobacteria from infected macrophages by antigen-stimulated T-cells. Liébana, E. et al. Immunology. 2000. 99:1-8.

22) In vitro T-cell activation of monocyte-derived macrophages by soluble messengers or cell-to-cell contact in bovine tuberculosis. Liébana, E. et al. Immunology. 2000. 100:1-13.

23) Diversity of Salmonella enterica serovar Enteritidis from English Poultry Farms Assessed by Multiple Genetic Fingerprinting Liebana, E. et al. Journal of Clinical Microbiology. 2001.39(1):154-161.

24) The use of LigthCyclerTM gyrA mutation assay (GAMA) for the rapid identification of mutations conferring decreased susceptibility to ciprofloxacin in multiresistant Salmonella enterica serotype Typhimurium DT104. Walker, R.A., Saunders, N., Lawson, A.J., Lindsay E.A., Dassama, M., Ward, L.R., Woodward, M.J., Davies, R.H., Liebana, E., and Threlfall, E.J. Journal of Clinical Microbiology. 2001. 39(4):1443- 1448.

25) Lipopolysaccharide microheterogeneity of Salmonella serotypes Typhimurium, Enteritidis, and Schwarzengrund. Parker, C.T., Liebana E., Henzler, D.J., and Guard-Petter, J. Environmental Microbiology. 2001.3(5):332-342.

26) Use of molecular fingerprinting to assist the understanding of the epidemiology of Salmonella contamination within broiler production. Liebana, E. et al., British Poultry Science. 2001. Accepted for publication..

27) Salmonella enterica serovar Enteritidis phage types 4, 7, 6, 8, 13a, 29, and 34 : A comparative analysis of genomic fingerprints from geographically distant isolates. Liebana, E. et al. Journal of Applied Microbiology. 2001. Submitted.

28) Molecular Typing of Salmonella serotypes prevalent in animals in England: Assessment of methodology. Liebana, E. et al. Journal of Clinical Microbiology. 2001. Submitted.

29) Rapid screening of molecular markers suggestive of antibiotic resistance in Salmonella enterica isolates of veterinary interest using Light-CyclerTM technology. Liebana, E. et al. Manuscript in preparation.

30) Ciprofloxacin resistance in Cylohexane resistant Salmonella of animal origin: Efflux or Mutations in gyrA

CSG 7 (Rev 6/97) 2 Randall, L., Liebana E. et al. Manuscript in preparation.

CONFERENCE PRESENTATIONS

1) Comparative studies of different methods for the diagnosis of bovine tuberculosis. Aranaz, A.; Liébana, E. et al. XIV Congreso Nacional de Microbiología. Zaragoza, Spain September 1993.

2) Evaluation of the technique RAPDs for typing of microorganisms belonging to the Mycobacterium tuberculosis complex. Aranaz, A., Liébana, E and Cousins, D.V.. JBC, 1st Bioscience Symposium. Madrid, Spain. April 1995.

3) Conference: "Tuberculosis: emergent disease within pet animals". IV Jornada técnica de AVEDILA. Madrid, Spain. May 1995.

4) Influence of the decontamination procedure and culture media in the isolation of Mycobacterium bovis from animal tissues. Liébana, E. et al. XV Congreso Nacional de Microbiología. Madrid, Spain. September 1995.

5) Eradication of tuberculosis from caprine herds by means of the tuberculin test and determination of IFN- in blood samples. Vidal, D.; Domingo, M.; Casal, J.; Liébana, E et al.. Congreso Nacional de Anatomía Patológica. León, Spain. June 1995.

6) Conference: "Importance of epidemiology in planning the tuberculosis eradication campaigns". Jornada sobre Detección de IFN- y su aplicación al diagnóstico de la tuberculosis en rumiantes. Madrid, Spain. October 1995.

7) Bovine and caprine subpopulations of Mycobacterium bovis. Liébana, E. et al. Congreso Nacional de Taxonomía Bacteriana. La Rábida, Spain. June 1996.

8) Conference: "Molecular typing of Mycobacterium bovis: a powerful tool in the epidemiology of the disease". Reunión Científica sobre el Impacto de las enfermedades transmisibles de los animales en la Salud Humana. Madrid, Spain. October 1996.

9) Conference: "Prevalence of tuberculosis in goats" Curso de Manejo, producción y patología de ovino y caprino. Ilustre Colegio Oficial de Veterinarios de Madrid. Madrid, Spain. March 1997.

10) A new menber of the Mycobacterium tuberculosis complex isolated from goats in Spain. Suárez, G.; Liébana, E. et al. XVI Congreso de la Sociedad Española de Microbiología. Madrid, Spain. July 1997.

11) Development of a DNA Amplification Method and its Application to Diagnosis of Bovine Tuberculosis. Liébana, E. et al. IUMS. 7th International Congress of Bacteriology and Applied Microbiology Division. Prague, July 1994.

12) Diagnosis of Tuberculosis in Dogs and Cats by PCR. Liébana, E et al. The Australian Society for Microbiology Scientific Meeting. Melbourne, September, 1994.

13) Isolation of an Aerobic Actinomycete from an Outbreak of Bovine Mastitis in the North of Spain . Aranaz, A.; Liébana, E. et al. The Australian Society for Microbiology Scientific Meeting. Melbourne, September, 1994.

14) Application of a [3H]-Uracil incorporation assay for detection of cytotoxic responses in bovine tuberculosis. Liébana E. et al. 6th Annual Congress of the British Society for Immunology. Harrogate International Centre, U.K., 1-4 December 1998.

CSG 7 (Rev 6/97) 3 15) Activation of bovine T-cell subpopulations in bovine tuberculosis. Welsh, M.; Smyth, A.; Liébana, E. et al. Association of Veterinary Teachers and Research Workers (Irish Branch) annual meeting. Veterinary Sciences Division, Belfast, U.K., 19 March 1999.

16) Activation of bovine T-cell subpopulations in bovine tuberculosis. Welsh, M.; Smyth, A.; Liébana, E. et al. Association of Veterinary Teachers and Research Workers (British Branch) annual meeting. Scarborough, U.K. 31 March 1999.

17) Requirement of an endogenous pathway of antigen processing for presentation of soluble antigens to CD8+ T-cells in bovine tuberculosis. Liébana, E. et al.ASM conference "A Cell Biology Approach to Microbial Pathogenesis". Portland (Oregon USA). 25-27 April 1999.

18) Effect of the paratuberculosis vaccine on the IFN- test for tuberculosis in goats Aranaz, A., del Rio, R., Ruiz, J., Liebana, E., et al. Third International Conference on Mycobacterium bovis. Cambridge (UK). 14-17 August 2000.

19) Discriminatory power of multiple genetic fingerprinting in the analysis of Salmonella enterica strains from poultry farms in the United Kingdom. Liebana, E. Et al. 5th International Meeting on Bacterial Epidemiological Markers. Noordwijkerhout (The Netherlands) 6-9 September 2000.

20) The use of plasmid profile analysis to study the epidemiology of Salmonella in broiler chicken flocks. Crowley, C., Corry, J., Liebana E, Davies R. Euroconference, Food safety assurance and veterinary public health .September 2000. University of Veterinary Medicine Vienna, Veterinarplatz 1, 1210 Vienna, Austria.

21) Conference: Molecular tools for detection and epidemiological studies of Salmonella spp. Liebana, E. 41st Meeting of the Poultry Disease Group, British Poultry Meat Federation, London (UK) 16 November 2000.

22) Conference: Molecular tools for epidemiological studies of Salmonella. Foodborne zoonosis Discussion group:6th Meeting. VLA Weybridge, UK. January 2001.

23) Conference:Clonal lines of Salmonella enterica serovar Enteritidis phage types 4, 7 and 6 determined by multiple fingerprinting of genomic DNA. Liebana, E. et al. Association of Veterinary Teachers and Research Workers (British Branch) annual meeting. Scarborough, U.K. April. 2001.

24) Pulsed field electrophoresis typing of animal Escherichia coli O157 isolates. Avery, S.M., Liebana, E. et al. Epidemiology of VTEC (Animal, food, abd biomedical aspects of verocytotoxigenic E. coli (CT98-3935) EU Concerted Action. Dublin. Ireland. April 2001.

25) Rapid screening of mutations conferring fluoroquinolone resistance in Salmonella enterica isolates of veterinary interest using a Light-Cycler gyr-A (GAMA) mutation assay. Liebana, E. et al. Nucleic acid-based technologies DNA/RNA/PNA diagnostics. Advances in assays, molecular labels, signaling and detection. Washington D.C. USA. May 2001.

26) Molecular typing of Salmonella serotypes prevalent in animals in England: Assessment of methodologies. Liebana, E. et al. X International Symposium of Veterinary Laboratory Diagnosticians and OIE Seminar on Biotechnology. Salsomaggiore, Parma. Italy. July 2001.

CSG 7 (Rev 6/97) 4 ANNEX B BIBLIOGRAPHY This table will expand to accommodate the information you wish to enter. To move to the next field, press the DOWN arrow TWICE.

CSG 7 (Rev 6/97) 1 Press enter ABALLAY A., YORGEY P., AUSUBEL F.M. (2000) Salmonella typhimurium proliferates and establishes a

persistent infection in the intestine of Caenorhabditis elegans. Current Biology, 10, 1539-1542.

ACMSF (2001) Advisory Committee on the Microbiological Safety of Food Second Report on Salmonella

in Eggs. London. The Stationary Office.

ALLEN, V.M., FERNANDEZ, F., HINTON, M.H. (1997) Evaluation of the influence of supplementing

the diet with mannose or palm kernal meal on Salmonella colonisation in poultry. British Poultry

Science 38, 485-488

ALTEKRUSE S., KOEHLER J., HICKMAN-BRENNER F., TAUX R.V. & FERRIS K. (1993) A comparison

of Salmonella Enteritidis phage types from egg-associated outbreaks and implicated laying flocks.

Epidemiology and Infection, 110, 17-22.

ANDERSON J.R. & POORBAUGH J.H. (1964) Observations on the ethology and ecology of various

Diptera associated with Northern California poultry ranches. Journal of Medical Entomology, 1, 131-147.

ANON (1989) Salmonella in eggs. Report of the House of Commons Agriculture Committee (HMSO,

London).

ANON (1990) The Committee on the Microbiological Safety of Food. The Microbiological Safety of Food:

Part 1: Report of the Richmond Committee on the Microbiological Safety of Food to the Secretary of State

for Health, The Minister of Agriculture, Fisheries and Food and the Secretaries of State for Wales,

Scotland and Northern Ireland. London: HMSO.

ANON (1993) Advisory Committee on the Microbiology Safety of Food: Report on Salmonella in Eggs.

London HMSO.

ANON (1998) Salmonella enteritidis risk assessment: shell eggs and egg products. Final Report: US Food

Safety and Inspection Service.

ANON (2000a) Salmonella in Livestock Production 1999. MAFF. Published by Veterinary Laboratories

Agency.

ANON (2000b) A report of the Study of Infectious Intestinal Disease in England. Food Standards Agency.

London: The Stationery Office.

AWAD-MASALMEH M. & THIEMANN G. (1993) Salmonella monitoring and related parameters in laying

hen farms and hatcheries in Austria. Tieraztliche-Umschau, 48, 706-713.

BARROW P.A. (1991) Experimental infection of chickens with Salmonella enteritidis. Avian Pathology, 20,

145-153.

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CSG 7 (Rev 11/99) 28 CSG7 - Research Proposal - Executive Summary

Proposal Title: Investigation of the role of environmental contamination in the epidemiology of Salmonella (in response to R9) Proposing Veterinary Laboratories Agency Organisation: Duration: 3 years Start Date: 01.10.2002 Total cost to MAFF: £298,601

Background:  Continued S.Enteritidis infection associated with UK produced shell eggs and egg production despite industry initiatives  Work in USA, Denmark, France and ongoing VLA work shows poor disinfection and rodent control on laying farms with infection overwhelming the protective effect of vaccination in many cases  Shortage of data on Salmonella in UK egg production because of industry sensitivity  Need to obtain quantitative data and exposure-infection data to inform future quantitative risk assessment models and policy

Objectives  To carry out detailed farm investigations to identify sources of infection and nature of persistence of and Salmonella in the environment  To quantify Salmonella levels in a range of environmental samples, including surfaces after Methodology: cleansing and disinfectant and rodent droppings etc.  To apply powerful molecular genetic typing methods to help interpret the epidemiology of the persistence and spread of Salmonella on the farm  To investigate the infectivity of residual contaminated material taken from laying farms for chicks and laying hens  To construct controlled and quantified environmental exposure methods to further evaluate environmental risks to chickens  To correlate phenotypic and genotypic characteristics of persistent and non-persistent strains isolated from farms with survival and infectivity characteristics  To produce advisory material for the egg industry and Government Methodology  Intensive sampling techniques and sensitive culture techniques capable of identifying a low prevalence of infection in vaccinated flocks will be used to determine persistence and dissimilar of Salmonella  Sensitive dilution-enrichment techniques will be used to quantify Salmonella before and after cleansing and disinfection  To apply a hierarchical approach to molecular epidemiological typing of isolates based primarily on PSTI/Sph1 ribotyping and PFGE  Controlled exposure of chicks and POL birds to contaminated materials gathered from farms and laboratory simulations  Phenotypic and genotypic testing of Salmonella strains in relation to environmental and virulence characteristics  Production of codes of practice, advisory leaflets and trade journal articles on improved control of Salmonella in laying flocks Collaboration: Prof. Tom Humphrey and Dr Vivien Allen, University of Bristol British Poultry Veterinary Association Benefits:  Improved data on risk of environmental persistence and compromise of the effectiveness of vaccination  Production of quantitative field and controlled laboratory simulation model data which can be introduced into later risk assessment models  Close working relationship with egg producers and specialist poultry veterinary advisors will enhance future control Other points: Ongoing work at VLA has shown that on multistage laying sites vaccination is only partially successful in controlling Salmonella, mainly because of poor rodent and fly control and substandard cleansing and disinfection. If this is allowed to persist it is possible that S.Enteritidis mutations with antigenic shifts rendering them less responsive to control by vaccination will be selected. This aspect should be examined in detail in another project but is outside the scope of this proposal as currently written.

CSG 7 (Rev 11/99) 30