Review of the Food-borne Zoonoses Research ... - ARCHIVE: Defra

Contents
Section A – Introduction...................................................................................... 2
1. Introduction................................................................................................ 3
2. Defra‘s aim and strategic priorities............................................................
3. The Animal Health and Welfare Strategy..................................................
4. The FBZ research programme..................................................................
4.1 Samonella..................................................................................
4.2 VTEC O157..............................................................................
4.3 Campylobacter.........................................................................
4.4 Cryptosporidium.......................................................................
5. Costs of FBZ research............................................................................
6. Aims of the FBZ research review............................................................
7. Terms of reference for external referees.................................................
Section B – Review Summaries........................................................................
1. Salmonella...............................................................................................
2. Campylobacter.........................................................................................
3. E. coli.......................................................................................................
4. Other zoonotic pathogens of interest.......................................................
Annex 1 – Index of projects reviewed..................................................................
Annex 2 – Project abstracts and project review summaries................................
Annex3 – List of external reviewers....................................................................
Annex 4 – Assessment form templates...............................................................
Annex 5 – Review Timetable................................................................................
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Section A – Introduction
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Review of the Food-borne Zoonoses (FBZ) Research
Programme 2003 – 2007.
(26 th to 27 th November 2007).
1. Introduction
Defra is a major funder of research, spending in the order of £150 million per year. The
research commissioned supports Defra in meeting its overarching aim and strategic
priorities (see section 2). Approximately £40 million of the annual budget is spent on
animal health and welfare research, which also serves to support the goals of the Animal
Health and Welfare Strategy for Great Britain (see section 3). In general, the results
generated from research are used to ensure existing Defra policies are based on sound
evidence, to identify the need to develop new policy, and to support Defra‘s regulatory
roles.
As part of an ongoing process of evaluation, Defra research programmes are subject to a
formal review process every three to five years.
The remit of the Food-borne Zoonoses (FBZ) research programme covers a number of
specific diseases. With this in mind, the review process not only considers the merits of
individual research projects, but also considers the balance of the programme against its
objectives by examining whether there has been an appropriate prioritisation of resource
to meet most effectively the objectives of disease prevention and control. In addition,
given the continuing threat foodborne diseases pose to the UK, the review will address
how the research programme could best meet future evidence needs of the Department
and if there is sufficient resource allocated to do so.
In consideration of resource management it is important to recognise opportunities for
working in partnership, an underpinning element of the Animal Health and Welfare
Strategy, and identifying areas where cost and responsibility sharing with stakeholders
are appropriate.
In broadening the scope and mechanisms by which the R&D programme may be
managed, it is also necessary to identify research and evidence that may be needed on
top of the current portfolio. Studies, for example, that better inform the Department and
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stakeholders on economics, social science and behaviour change, with respect to
disease management, may be included by such a process.
2. Defra’s aim and strategic priorities
At the time of this review Defra's overarching aim was sustainable development, which is
defined as ‘development which enables all people throughout the world to satisfy their
basic needs and enjoy a better quality of life without compromising the quality of life of
future generations.‘
Under this overarching aim, five strategic priorities were identified, including:
1. Climate change and energy: Making a full contribution, domestically and
internationally, to addressing the long-term threats presented by climate change and
unsustainable energy use, and to ensure adequate mitigation of the consequences
which are already unavoidable.
2. Sustainable consumption and production: Breaking the link between economic growth
and environmental degradation and resource use through promoting and enabling
more sustainable patterns of consumption and production.
3. Protecting the countryside and natural resource protection: Creating a robust policy
framework and evidence base in order to promote the sustainable use and
enhancement of the country's natural heritage and ecosystems.
4. Sustainable rural communities: Encouraging sustainable regeneration in
disadvantaged rural areas, promoting social inclusion and reducing deprivation.
Ensuring higher quality, more accessible public services to rural communities.
5. Sustainable farming and food, including animal health and welfare: Helping to create
a sustainable food and farming supply chain serving the market and the environment;
putting in place systems to reduce risks of animal diseases, and being ready to
control them when they occur.
In addition to this, Defra has an ongoing responsibility for emergency preparedness,
including planning for emergencies in animal and plant diseases, flooding, food supply,
water supply and dealing with the consequences of a chemical, biological, radiological or
nuclear incident.
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Plans for taking forward these priorities are described in more detail in Defra‘s five year
strategy (Delivering the Essentials of Life), which was published in 2004. However it
should be noted that Defra was undergoing a strategy refresh focusing on the mission of
‗one planet living‘ at the time of this review.
3. The Animal Health and Welfare Strategy for Great Britain
The Animal Health and Welfare Strategy for Great Britain, was published in 2004, with
the overall aim of ‗developing a new partnership to make a lasting and continuous
improvement in the health and welfare of kept animals, while protecting society, the
economy, and the environment from the effect of animal diseases‘. To bring about this
aim, the following strategic outcomes were identified:
1. Working in partnership
2. Promoting the benefits of animal health and welfare, particularly emphasising
prevention is better than cure
3. Ensuring a clearer understanding of the costs and benefits of animal health and
welfare practices
4. Understanding and accepting roles and responsibilities
5. Delivering and enforcing animal health and welfare standards effectively.
4. The FBZ research programme
The FBZ research programme forms part of a wider body of research on animal health
and welfare. This research portfolio is managed on behalf of the Chief Veterinary Officer
and is closely associated with the FBZ policy division to which it provides a substantial
part of the evidence base. From the current Animal Health and Welfare research
portfolio, approximately 6% of the budget is allocated to the FBZ research programme
(Figure 1).
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Figure 1. Spend on Animal Health and Welfare research against policy area for
2007/2008
To note: approx. £3million pa of the total budget are sourced directly from policy programmes, including
£2million of the TB spend and £1million of the Veterinary Training and Research Initiative (VTRI) spend.
The FBZ research programme covers research projects on non-notifiable zoonotic
diseases. Zoonoses are defined by the World Health Organisation (OIE) as "Diseases
and infections which are naturally transmitted between vertebrate animals and man".
Between 2002 and 2007, the programme has included research on Salmonella species;
E. coli O157; Campylobacter species; Yersinia enterocolitica; Cryptosporidium species;
and Bovine neosporosis. Key themes within diseases include diagnostics, intervention
strategies, epidemiology, pathogenesis, and source identification. It should be noted that
though Salmonella is non-notifiable disease, it is ‗reportable‘. This means that should it
be detected in samples tested in a laboratory, this must be reported to the local
Veterinary Laboratories Agency (VLA) laboratory (In England and Wales), and to the
local Divisional Veterinary Manager in Scotland.
The rationale for funding research on FBZ is set out in the policy and scientific objectives
of the research defined below:
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Policy Objectives
The key policy objectives are as follows:
help minimise the risk of the zoonotic infections being transmitted to man;
reduce the use of medicinal products for the control of zoonotic infections and
thereby minimise any risk to the consumer from food and the environment arising
from their residues
reduce the threat to animal welfare and economic performance posed by these
diseases and any long term disadvantages of current treatments for these
diseases;
To maintain nuclei of expertise at the strategic level on a range of zoonotically
important diseases to promote food safety
To encourage projects co-funded with industry and the European Union which
meet the objectives above.
Government intervenes in animal health and welfare for four reasons where the market
on its own cannot deliver some or all of the objectives:
To protect human health
To protect and promote the welfare of animals
To protect the interests of the wider economy, environment and society
International Trade
Government will use these reasons as the starting point for any considerations as to
whether intervention should take place. It is the role of Government to balance these
interests and resolve them to the greatest advantage of all those affected.
Scientific Objectives
A number of scientific objectives are apparent which will support the policy objectives set
out above and the overall rationale. These are not intended to be comprehensive nor
mutually exclusive.
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The scientific objectives are as follows:
To identify the potential hazards posed to human health by animal diseases and
human diseases which are believed to have an animal reservoir, and identify
measures that can be taken to reduce the risk from these hazards.
To maintain nuclei of expertise that can be used to promote sustainable genetic
and immunological approaches to zoonotic disease control.
To increase knowledge of the epidemiology and pathogenesis of foodborne
zoonoses using molecular genetic approaches with the aim of improving diagnosis
and disease control by sustainable approaches which do not include
chemotherapy.
To maintain nuclei of expertise at the strategic level on a range of important
zoonotic diseases. These should act as foci for research co-funded with industry
with the aim minimising the microbiological hazards of UK agriculture and
promoting the production of safe food.
4.1 Salmonella
Salmonellae have been recognised as important pathogens for many years. S. Enteritidis
and S. Typhimurium have accounted for the majority of cases of human salmonellosis
since the 1980s and have consistently been the most commonly implicated pathogens in
general outbreaks of foodborne disease. Under-ascertainment of infectious intestinal
disease (IID) is well recognised, and the true population burden is greater than that given
by national surveillance. For every report to national surveillance for Salmonella spp.
there are approximately three cases in the community. In 2005, 12,652 laboratory
confirmed cases of salmonellosis were reported in the UK, (Figure 2) a decrease of 14%
on the 14,729 confirmed cases recorded in 2004. The total number of reports in 2005 are
approximately a third of the number recorded in 1997 1 .
The salmonellae are a group of organisms with a diverse range of host species including
mammals, birds, reptiles and fish. Investigations have shown that infection can be
acquired through the consumption of a large variety of different foods if they become
contaminated, as well as through direct contact with a wide range of animal species and
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Figure 2. Laboratory reports of Salmonella in people, UK 1983 – 2005.
contact with faecally contaminated environments. The serotyping and phage typing
schemes that have been developed have enabled microbiologists to differentiate
between thousands of Salmonella strains with widely varying natural histories, which
helps to identify the source of infections 1 .
Epidemiological and microbiological investigations have demonstrated that poultry and
eggs are the main source for the most important disease-causing strains of S. Enteritidis.
Outbreaks of infection are most commonly associated with the consumption of poultry
and eggs. In 2005, there was a marked decline in the number of outbreaks of S.
Enteritidis (non PT4) associated with catering establishments in England and Wales,
which followed a decline in the purchasing of non-UK eggs by the catering sector 1 .
Strains of S. Typhimurium have been found to be associated with the consumption of a
variety of foods including beef, dairy produce, pork, lamb, chicken and turkey. A range of
vehicles of infection has been found to be associated with the other serotypes of
Salmonella. Most are of animal origin, however a wide variety of spices, herbs and other
produce have also been implicated in general outbreaks of infection 1 .
1 Data sourced from Zoonoses Report UK – available via
http://defraweb/animalh/diseases/zoonoses/zoonoses_reports/zoonoses2005.pdf
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4.2 VTEC O157
Vero cytotoxin-producing Escherichia coli (VTEC) have emerged in the last 25 years as a
group of pathogens of worldwide importance. The number of laboratory confirmed cases
of VTEC O157 infection in the UK rose by 25% from 926 in 2004 to 1155 in 2005.
However, overall the general trend has been stable in the last few years (see Figure 3) 1 .
Figure 3. Laboratory reports of VTEC 0157 in people, UK 1987 – 2005.
The main reservoir of VTEC O157 in livestock is ruminants, particularly cattle, sheep and
goats but the organism has been found in faeces from a wide range of animals including
horses, pigs, dogs, domestic geese and wild rabbits. Information from outbreak
surveillance in the UK demonstrates that VTEC O157 infection can be transmitted via
consumption of contaminated food or water; person to person spread; contact with
livestock; and environmental exposure 1 .
4.3 Campylobacter
The number of laboratory confirmed cases reached a peak of 65,209 cases in 1998.
Since then there has been a general decline in disease incidence. However between
2004 and 2005 the number of reported cases of campylobacteriosis in the UK again rose
from 47,466 to 49,803 (see figure 4) 1 .
1 Data sourced from Zoonoses Report UK – available via
http://defraweb/animalh/diseases/zoonoses/zoonoses_reports/zoonoses2005.pdf
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Figure 4. Laboratory reports of Campylobacter in the UK 1992 – 2005.
Most cases of Campylobacter infection are thought to be sporadic and the routes of
transmission remain unclear. Poultry meat may be an important vehicle of infection and
surveys have shown that a significant proportion of raw poultry meat for human
consumption is contaminated. Evidence from experimental studies suggests that
Campylobacter has a low infectious dose and thus cross-contamination of ready-to eat
foods by raw meat may be an important route of infection. The roles of other animal
products, other foods, water and non-foodborne exposures are still under investigation 1 .
4.4 Cryptosporidium
Cryptosporidia are protozoan parasites with a widespread distribution in farm and wild
animals. The parasite can cause clinical disease (cryptosporidiosis) in animals, usually
neo-natal diarrhoea, although subclinical infection is common particularly in lambs.
Typing for Cryptosporidium parvum is now becoming available and has enabled
improved identification of the different species. C. hominis (previously C. parvum
genotype 1) is normally only recovered from humans although there have been
exceptional reports of isolates reported from animals. C. parvum (previously C. parvum
genotype 2) is found in both animals and humans 1 .
In the UK in 2005 there were 5,288 cases reported compared with 4,197 cases in 2004
(see figure 5) 1 .
1 Data sourced from Zoonoses Report UK – available via
http://defraweb/animalh/diseases/zoonoses/zoonoses_reports/zoonoses2005.pdf
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Figure 5. Laboratory reports of Cryptosporidium in the UK, 2005.
5. Costs of FBZ research
The total Defra funding for research on Foodborne Zoonoses in 2007/08 was £2.6 million
and was allocated as shown in Figure 6. A further £2million per annum is being invested
(from Defra) in the Veterinary Training and Research initiative (VTRI), which although
focused on the development and training to support veterinary research, consists of
many elements that are complementary to the FBZ research programme. The VTRI
projects are reviewed under a separate process and will not be subject to scrutiny within
this review.
Figure 6. Allocation of funding within the FBZ research programme 2007/08
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6. Aims of the FBZ research review
The current and completed FBZ research projects from 2002 to 2007 were evaluated as
part of the review (see Annex A).
The FBZ research programme was reviewed by a panel consisting of external referees
and Defra officials and the aims were:
to evaluate completed and current research projects in relation to:
o their scientific quality
o their usefulness to policy and contribution to the evidence base
o the delivery of the overall objectives of the FBZ research programme
to assess the size, scope and balance of the current FBZ research programme in
relation to current policy needs
to consider the future direction of the FBZ research programme and identify future
priorities, taking into account the size, scope and balance of the current research
programme, as well as research funded in the field by other sponsors
7. Terms of reference for external referees
On the basis of project information provided prior to the review meeting, as well as
presentations and discussions at the review meeting, the terms of reference are:
To consider the relevance and appropriateness of the research for funding by Defra
To consider the soundness and appropriateness of the scientific approaches used
and if they are being taken forward competently
To examine the progress being made towards the objectives and the likelihood of
success
To consider if the findings from the research are based on sound evidence
To consider the collaboration with other institutes and universities both nationally and
internationally
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To assess the effectiveness with which appropriate opportunities for technology
transfer are being addressed, and in particular, whether reports and publications are
being delivered to an appropriate standard
To consider the value for money of the research
To consider the future scientific direction of the work
To prepare a written report (i.e. assessment form for external referees) on the
research area and to comment verbally at the review meeting in November 2007 (if
attending)
To confirm that any views expressed are entirely objective
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Section B – Review Summaries
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1. Salmonella Review Summary
1.1 Success of research in providing value to Defra
Salmonellosis is one of the most important bacterial diseases of man and animals
worldwide and has important implications for human and animal health and welfare. At
present Salmonella Enteritidis and S. Typhimurium account for the majority of cases of
human salmonellosis.
Defra is responsible for the protection of animal health and has a major role in protecting
human health from animal diseases. The panel felt that it was therefore appropriate for
Defra to fund work in this area and agreed that Defra‘s portfolio of Salmonella research
has contributed significantly to the Salmonella evidence base, to developing scientific
methodology and maintaining essential expertise.
Development of Molecular Methods
DNA techniques have been employed to develop molecular subtyping methods for
Salmonella. This has helped to improve strain identification and comparison of
Salmonella species.
Research is currently in place to develop rapid screening tools to improve the detection
and control of Salmonella, by providing an early warning system for the emergence of the
next epidemic Salmonella strain or clone.
Poultry Research – Broilers
Valuable research has been undertaken investigating bacterial and host genes involved
in Salmonella colonisation in poultry. This has contributed to our understanding of the
genetic basis of host resistance to colonisation which could be exploited using genetic
selection.
Commissioned research aimed at assisting the turkey industry with reducing Salmonella
and antimicrobial resistance is at an early stage. It was thought that this had the potential
be extremely important to Defra in providing evidence on the occurrence and
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epidemiology of Salmonella and antimicrobial-resistant E. coli. This information is a
necessary requirement for the formulation of future monitoring and control policies.
Poultry Research – Layers
The research on non-specific and innate resistance to Salmonella infection was
considered to have advanced the understanding of some of the fundamental
mechanisms for establishment and clearance of Salmonella infections in chickens and
pigs. This research has, therefore, contributed to the knowledge in area of innate
responses to infection and the exploitation of these responses to control infection.
Although at an early stage, commissioned research investigating the effect of
immunosuppression at the point-of-lay on salmonella infection and immunity in laying
hens will provide information at a particularly important period in the development of the
laying hen, at a time when it is known that Salmonella breakdowns tend to occur.
The series of epidemiological studies investigating Salmonella contamination in layer
flocks and eggs has been fundamental in assisting development of policy both within UK
and within EU, as it has allowed Defra to negotiate a UK position based on best available
science. This ongoing research has enabled Defra to build better relationships with the
industry, assisting both subsequent research and also policy development involving
partnership with industry.
Work is also in place to help manage the challenges to the egg industry posed by new S.
Enteritidis phage types and it is hoped that this work will eventually lead to improved
vaccines to control potential new strains.
Porcine and Bovine Research
A study of multiple-resistant S. Typhimurium DT 104 undertaken in cattle has provided a
unique body of evidence on the epidemiology of Salmonella infection in cattle herds in
Great Britain. It was an opportunity to study the dynamics of an epidemic Salmonella
serotype in the national cattle population.
Research has provided useful information on the molecular basis of Salmonella
pathogenesis in cattle and pigs and has the potential to assist with control through
providing data for the rational development of live vaccines. A series of epidemiological
studies of Salmonella infection in pigs has contributed to the development of diagnostic
tests, sampling methodology and in improving methods of control.
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In summary, the pig research was considered to be worthwhile and should clarify many
questions concerning salmonellosis in pigs, from which future control strategies can be
determined.
1.2 Issues and areas of concern relating to this research
Concerns were raised with one of the research projects which generated no publications.
From the outset, contractors should consider how best to communicate research findings
to Defra and the wider scientific community.
It was noted that Defra should scrutinise proposals particularly carefully when there may
be a sole supplier of materials that are essential to the success of the project.
1.3 Gaps in Research
Development of Molecular Methods
The improvement of typing methods is an important research area, particularly since
there is a need to have a better understanding of Salmonella epidemiology. The industry
needs highly reproducible, rapid, cheap methods which are specific for Salmonella and
reasonably sensitive. There is a need for a more rapid but highly specific test for
confirmation of Salmonella of public health significance.
Poultry Research - Layers
EU legislative changes will impact on the industry and Defra needs the UK negotiating
stance to be based on sound science. It is likely that there will be a need for further work
to address some of the new issues that will arise out of the legislation implementation.
Salmonella Enteritidis remains the most important Salmonella of public health
significance in the poultry industry. Historically outbreaks in poultry in UK have been
associated with S. Enteritidis phage types 4, 6 and 7 which are related. It is important to
the UK poultry industry to establish if the vaccines currently available are effective
against other S. Enteritidis phage types prevalent in the rest of Europe. The work would
contribute significantly to the evidence base and should result in improved methods of
control.
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There is a need to objectively assess all practical interventions which might obviate the
need for heat treatment of eggs as, for many producers, this will not be economically
feasible.
It was also thought useful to develop a cost-effective screening test to distinguish
between flocks which are highly, moderately and minimally contaminated.
If the on-going research on avian immunological response during the point-of-lay period
proves successful there will be a need to understand how the information can be utilised
in the field to improve salmonella control. It was also suggested that it might be useful to
investigate changes at the end-of-lay when Salmonella breakdowns are also known to
occur.
Porcine Research
Control of Salmonella in pigs is highly complex and UK pig industry is significantly
different from that in other member states, with a higher percentage of outdoor units and
a broad breeder base in the UK. Research from other EU member states or third
countries is therefore not always directly applicable. Salmonella control in UK for this
sector remains problematic. There appear to be regional differences that need to be
understood better.
The lack of a satisfactory vaccine for use in pigs is an issue, however there may well be
sufficient information at this point for pharmaceutical companies to make a satisfactory
vaccine. It is more a case of companies being persuaded that there is a sufficient market
for a vaccine.
Bovine Research
With regards to cattle it is probably reasonable to believe that a new epidemic of
Salmonella in cattle will occur at some time in the future as there have been a series of
epidemics in the past. Should such an epidemic occur, research will need to be put in
place at an early stage to establish the epidemiology of the disease in order to attempt to
reduce the size of the epidemic. Research is underway to try to assist with the prediction
of a new epidemic.
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Currently there is more concern about Salmonella controls for other species and, despite
the high level of herd breakdowns, it is not clear that they are resulting in real public
health significance.
1.4 Balance of funding in this FBZ programme.
It was felt that it would not be appropriate to cut back on Salmonella research while the
new EU regulations are being implemented.
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2. Campylobacter review summary
2.1 Success of research in providing value to Defra
Campylobacter species is the most common cause of bacterial food-borne illness in the
UK. While there are a number of routes by which humans are exposed to
Campylobacter, poultry meat is thought to be an important source of infection. A
reduction in Campylobacter colonisation of poultry flocks is therefore a Defra priority, and
a substantial portion of the Animal Health and Welfare research budget has been
directed to this subject.
At the time of commissioning some of the research covered by this review, the role of
poultry as a source for human campylobacteriosis was still uncertain. This uncertainty
stemmed from a poor understanding of Campylobacter sp. and their epidemiology.
Research within this programme has increased our understanding of this organism
significantly, and revealed the difficulties in typing due to the genetic instability of
campylobacters. An MLST method for Campylobacter sp., developed under Defra
funding, is of fundamental importance in providing a robust method for typing this genus.
In addition, the MLST method has been adopted by clinical laboratories for use in typing
human isolates, and the tool has been adopted at international level demonstrating the
value of the work performed.
A project investigating carriage of Campylobacter spp. in pet dogs indicated that the
common causes of human infection, C. jejuni and C. coli, are not often isolated from
dogs. Veterinarians are regularly asked about pets as sources of human infection and
this project was valuable in providing reassurance on this issue.
Investment in research into the bacterium and its survival, persistence and epidemiology
in poultry flocks has increased the knowledge base of this human pathogen which, it is
anticipated, will be of value in developing effective controls for this organism.
A number of research projects within this programme have investigated non-biosecurity
interventions including, competitive exclusion, vaccination, maternal antibodies and
innate immunity. This has been a difficult area of research, however the widespread
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nature of Campylobacter, and lack of success in controlling this organism through
biosecurity, highlights the necessity for progressing other potential approaches to control.
2.2 Issues and areas of concern relating to this research
A number of concerns were voiced by the reviewers in relation to the Campylobacter
research programme:
1. It was felt that teams working on Campylobacter research often did so in isolation
from each other. Increasing collaboration, and bringing the complementary
expertise of different teams together would be beneficial to progressing the
science in this area.
2. At present Defra, FSA and BBSRC each hold separate reviews of their research
programmes. While each are kept abreast of work being funded by the others and
there was not a problem in duplication of effort, it was suggested that a more
joined up approach to research reviews would be useful in identifying gaps in
research.
3. Reviewers highlighted the importance of contractors alerting Defra when project
objectives are found to no longer be appropriate. This enables an alternative
project plan to be agreed so that the contract still delivers information that is of
value to Defra.
4. Research has shown a small seasonal effect on Campylobacter colonisation in
chickens in the UK. It is therefore important to ensure any research into
interventions is properly designed to prevent conclusions being drawn on natural
fluctuations in Campylobacter levels.
5. For vaccination or competitive exclusion to be of significant use in the future it is
important to build links with industry to take work to the next stage and test
findings under field conditions.
2.3 Gaps in research
While the development of MLST for Campylobacter sp. was acknowledged to be of
fundamental importance, reviewers agreed that it was not the perfect epidemiological tool
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for this organism. Research within the programme had shown that a variety of typing
methods may be used depending on the question being asked. A good routine tool for
typing Campylobacter sp. would therefore be of value to enable source attribution of
infection. Developments in genetic chips may be particularly useful in typing
Campylobacter sp. in the future. Due to the unpredictable nature of where genetic
change occurs, a chip carrying a large number of genetic elements and their variants
may be the key to typing this unstable genus.
A lot of resources have been invested in developing the MLST database. It is vital that
analysis of this database now takes place to identify the sources of Campylobacter that
are responsible for human infection. It is also important to ascertain whether the
database has been populated in an un-biased manner, or whether there are sample
types that are under-represented and need to be included.
The interrelationships between strains found in poultry units, processing units, the
environment, wild life and domestic animals is unknown. This information would help to
address the question of the contribution of contamination in the abattoir, food processing
environment, and household strains to human disease.
Reviewers noted that there are still many fundamental questions that need to be
addressed by basic research, such as the relationship between strain type and virulence
potential. Basic research is also needed to further understand the interaction between
genomics and physiology i.e. the behaviour of the organism in vitro and in vivo. This work
was considered to be more appropriate for funding by the BBSRC or Wellcome Trust.
Fundamental questions also remain unanswered in relation to the role of maternal
antibodies in chick immunity. Despite the lack of success in identifying suitable vaccine
candidates to-date, the reviewers felt that pursuing the vaccination approach was
worthwhile. They noted, however, the need to expand expertise on Defra-funded projects
to include immunologists and industry representatives with vaccine expertise. LINK
projects, where Defra and private companies share the cost of research, were thought to
be ideal for progressing research in this area.
One of the flaws in some of the research undertaken is the uncertainty over whether
enough colonies are examined to be confident that a single Campylobacter strain is
present rather than multiple strains. Reviewers suggested that a project examining large
numbers of isolates would be useful in determining whether co-infection with multiple
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strains is common, and how many colonies should be selected to be reasonably
confident of only one Campylobacter strain being present.
A question that intrigues many researchers, the answer to which could be valuable in
control of Campylobacter, is why some poultry farms are consistently negative for the
organism. If the factors involved in a farm remaining uninfected could be elucidated this
may reveal potential control strategies.
Biosecurity measures that have been successful in controlling Salmonella infection on
poultry farms and other diseases have not had the same success in reducing
Campylobacter infection. Reviewers suggested that a critical review of on-farm
interventions to control Campylobacter would be useful in identifying which biosecurity
measures were most successful. It was noted that a critical review of both biosecurity
and non-biosecurity interventions in the control of Campylobacter was in the process of
being commissioned by FSA and Defra.
Other studies suggested were:
studies to determine most effective biosecurity measures e.g. is dipping boots
more or less effective than changing them?
investigations on stocking densities, general health and welfare and
Campylobacter colonisation
studies to determine the effect of on farm control measures with campylobacters
present in humans
2.4 Balance of funding in this research area
Reviewers felt that with the introduction of new EU legislation on Salmonella control,
Salmonella research should take priority for research in the short-term. With Salmonella
levels being successfully reduced, more of the budget should be targeted towards
Campylobacter control in the longer-term. The need to co-ordinate Defra-funded
research on Campylobacter with work supported by other funding bodies was also
highlighted
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3. E. coli review summary
3.1 Success of research in providing value to Defra
In recent years, research projects including those supported by Defra have contributed
greatly to a better understanding of E. coli O157 infections in animals and man. The
series of Defra funded projects has led to significant progress being made in several
areas, from gaining better knowledge of the prevalence of infection in cattle and other
livestock species through to the development of possible approaches to the control of the
infection in cattle.
3.2 Issues and areas of concern relating to this research
A wide range of research projects were reviewed and while some areas of research had
not been particularly productive there were other areas where excellent progress had
been made. Research related to the predilection site for E. coli O157 colonisation in
cattle and the progress towards developing control methods based on this knowledge
were rated highly. It was noted that VTECs other than E. coli O157 also are a disease
risk and the research programme should not focus entirely on E. coli O157.
3.3 Gaps in research
A better understanding is still required of the epidemiology of the infection particularly as
it is now clear that infection can be acquired from environmental and other sources in
addition to the well described route through eating infected meat products. An on-going
review project funded by the FSA on past and current research on verocytotoxin
producing Escherichia coli (VTEC) in relation to public health protection is expected to
provide some useful indications of research gaps and requirements.
It was considered that further research on vaccination as a possible approach to control
was justified and it was noted that there is a current LINK project looking at vaccination
strategies in cattle. Although research has shown that other livestock species apart from
cattle can be colonised to a varying extent by E. coli O157, it was suggested that a better
26

understanding is needed of why some animals such as pigs and poultry are not acting as
a significant source of infection.
The research had contributed to the formulation of practical strategies that could reduce
the risk of carriage of VTEC O157 in cattle but there is a need to determine how these
findings can be more effectively disseminated to the farming industry.
3.4 Balance of funding in this research area
Compared to the Salmonella and Campylobacter research programmes, the VTEC
programme is relatively small. It was considered that this is appropriate and that the on-
going studies on possible control methods were justified. It was recommended that the
focus on control should not be limited to VTEC but that a broader approach to improved
biosecurity on farm should be considered. It was noted that such a project was currently
under consideration for funding
27

4. Other zoonotic pathogens of interest review summary
4.1 Success of research in providing value to Defra
Defra has funded research projects on Cryptosporidium, Yersinia and Neospora. Each
project was thought to have improved understanding of the zoonotic risk from these
pathogens and therefore funding in this area has been of value to Defra.
Studies of Cryptosporidium infection confirmed that pets are rarely a source of infection
for humans but that some human infections could be attributed to farm animals.
Challenge experiments demonstrated for the first time that C. hominis could infect a wide
range of farm species, including calves, lambs piglets and poultry. The research on
Neospora did not provide evidence that there is a zoonotic risk from cattle. Yersinia
enterocolitica strains present in livestock were shown to be a potential threat to human
health.
4.2 Issues and areas of concern relating to this research
There were no generic issues of concern in this research area. The Yersinia research
had generated valuable publications, but it was considered disappointing that more had
not been published from the Cryptosporidium work.
4.3 Gaps in research
Although additional work could always be carried out, no significant gaps were identified
in the research.
It was felt that the research agenda in this area should largely be driven by policy needs.
There was potential for more work on Cryptosporidium sp. and Yersinia sp. There was
not a strong case for Defra to fund further studies on Neospora sp. but a case could be
made for working in partnership with industry on Neospora as an endemic disease.
Surveillance or prevalence studies would be useful to determine the importance of
diseases such as Q fever, toxoplasmosis or Lyme disease in UK livestock. Research to
28

investigate the vulnerability of the UK to infections brought in by exotic pets should also
be considered.
Other related areas of work in the future might also benefit research on zoonoses. For
example, work on detection by array-based technologies would help identify new
pathogens and strains emerging in the UK.
4.4 Balance of funding in this research area
It was considered necessary for Defra to monitor this area and to have a budget
available to enable a rapid response to emerging needs as assessed by Defra and the
various specialist advisory groups.
29

Annex 1 – Projects Reviewed
30

Project
Code
Title
LK0665 Improving gut health and nutrient capture of broiler
chickens through selection for innate immune
function
LK0666
OZ0135
OZ0138C
Vaccination strategies for control of
enterohaemorrhagic Escherichia coli O157:H7 in
cattle
Epidemiological studies of multiple-resistant
Salmonella Typhimurium DT104 in cattle
A longitudinal study of faecal excretion of VTEC
O157 in cattle to determine epidemiological
patterns and risk factors associated with excretion
OZ0144 Constraints to uptake of adequate biosecurity on
UK cattle and sheep farms, with special reference
to zoonotic diseases
OZ0145 VTEC O157 on farm control: Effective measures,
perception and risk communication
Index of Projects Reviewed
Start
Date
01/01/06
01/10/05
01/04/97
01/10/98
01/06/02
01/10/05
End
Date
31/12/08
30/09/08
31/03/02
31/01/05
31/12/03
31/03/08
Contractor(s)/ Subcontractor(s)
Institute for Animal Health
University of Edinburgh
Scottish Agricultural College &
Moredun Research Institute
(sub-contractors)
Veterinary Laboratories
Agency
Veterinary Laboratories
Agency
Laboratory of Enteric
Pathogens, Central Public
Health Laboratory,
University of Liverpool &
University of Reading
(subcontractors)
The University of Reading
Scottish Agricultural College
(sub-contractor)
Veterinary Laboratories
Agency
Cost (£)
351,814
(Defra:
£174,813, Other
funders:
£177,001)
628,763 (Defra:
£328,762; Other
funders:
£300,001)
523,530
2,242,139
113,279
210,125
Page
Number
83
94
56
95
97
95
31

OZ0311 Conventional versus capillary electrophoresis of
RFLP fragments and PCR products for sensitive
OZ0312
and rapid subtyping of Salmonella.
Development of a sensitive and specific molecular
typing method for the epidemiological study of
Salmonella
OZ0313 Non-specific and innate resistance to Salmonella
infection in chickens and pigs
OZ0314 The role of defined bacterial genes and host
genetic background in intestinal colonisation of
poultry by Salmonella
OZ0315 Salmonella pathogenesis and immunity in cattle
and pigs
OZ0316 Epidemiological studies of Salmonella in pigs and
control by intervention.
OZ0317 Epidemiological investigations of Salmonella
contamination in table egg production
OZ0318 Understanding the dynamics of endemic and
epidemic Salmonella infections in cattle: A
comparative modelling approach
OZ0319
OZ0320
OZ0321
01/06/99
03/01/00
01/04/99
01/04/99
01/04/99
01/08/00
01/08/00
01/01/03
Salmonella pathogenesis in cattle and pigs 01/07/02
Bacterial and host genes in Salmonella
colonisation in poultry
Investigation of the role of environmental
contamination in the epidemiology of Salmonella
infection in egg-laying flocks
01/07/02
01/10/02
31/03/04
31/01/03
30/06/02
30/06/02
30/06/02
31/12/06
31/01/03
30/04/06
30/09/06
30/06/05
30/09/07
Central Science Laboratory
Laboratory of the Government
Chemist
Stobhill NHS Trust & Poultry
Health Services (subcontractors)
Institute for Animal Health
Institute for Animal Health
Institute for Animal Health
Veterinary Laboratories
Agency
London School of Hygiene &
Tropical Medicine (subcontractor)
Veterinary Laboratories
Agency
University of Liverpool
373,347
228,700
689,400
679,725
656,100
1,222,749
227,692
399,343
Veterinary Laboratories
Agency & University of
Lancaster (sub-contractors)
Institute for Animal Health 709,200
Institute for Animal Health
Veterinary Laboratories
Agency
956,279
464,534
43
38
54
44
58
59
49
61
62
44
49
32

OZ0323
OZ0324
OZ0325
OZ0326
OZ0327
OZ0328
An integrated risk based approach to the control of
Salmonella in UK pig farms.
New and emerging Salmonella serovars;
epidemiological, risk-based and molecular
approaches to their identification and control: set
up studies.
A monitoring, control and education package to
assist the egg industry with Salmonella reduction
and achieving EU targets
Managing the challenges to the egg industry posed
by new SE PTs
Effect of immunosuppression associated with
point-of-lay on Salmonella infection and immunity
in laying hens
A monitoring, control and education package to
assist the turkey industry with reduction of
Salmonella and antimicrobial resistance and
achieving EU targets
01/04/05
01/10/05
01/04/06
01/04/06
01/07/06
01/04/07
31/10/08
31/01/09
31/03/09
31/03/09
20/06/09
31/03/10
University of Bristol (subcontractor)
Veterinary Laboratories
Agency
Health Protection Agency,
University of Liverpool,
Imperial College,
London School of Hygiene &
Tropical Medicine & University
of Wageningen (sub-
contractors)
Veterinary Laboratories
Agency
University of Liverpool (subcontractor)
Veterinary Laboratories
Agency
University of Bristol, Natural
England & Central Science
Laboratory (sub-contractors)
University of Bristol
Veterinary Laboratories
Agency (sub-contractor)
University of Liverpool
Veterinary Laboratories
Agency
Agricultural Development
Advisory Service & University
1,053,974
1,009,045
753,006
403,472
198,000 (Defra:
£37205;
BBSRC:
£160795)
1,138,946
59
41
49
52
48
46
33

OZ0402
OZ0404
OZ0405
OZ0407
OZ0604
OZ0605
What is the potential for human isolates of both
genotypes of C. parvum to infect, colonise and be
excreted by farm animals
Bovine neosporosis: the evaluation of zoonotic risk
and the development of evidence-based control
strategies
Genotypic and phenotypic comparison of Yersinia
enterocolitica from humans and animals
Evaluation and risk assessment of zoonotic
transmission of Cryptosporidium
Characterisation of strain variation in
Campylobacter jejuni.
Genotypic and Phenotypic Instability of
Campylobacters from Environmental, Animal and
Human Sources
01/04/00
01/11/02
01/05/02
01/04/03
01/04/99
01/08/99
31/03/03
31/12/05
30/04/05
31/01/08
30/09/04
31/07/02
of Bristol (sub-contractors)
Veterinary Laboratories
Agency
Cryptosporidium Reference
Group, Health Protection
Agency (sub-contractor)
University of Liverpool
Veterinary Laboratories
Agency
London School of Hygiene &
Tropical Medicine (sub-
contractor)
Veterinary Laboratories
Agency, CREH Analytical
Limited & PHLS
Cryptosporidium Reference
Unit, Swansea Public Health
Laboratory (sub-contractors)
University of Oxford
Health Protection Agency (subcontractor)
Veterinary Laboratories
Agency
Scottish Reference Laboratory
for Campylobacters, Aberdeen,
Queens University, Belfast,
Campylobacter Reference Unit,
Health Protection Agency &
Institute for Animal Science
and Health, The Netherlands
406,684
242,267
245,488
783,631
774,549
298,279
64
68
66
64
70
72
34

OZ0606
OZ0607
OZ0608
OZ0609
OZ0610
OZ0611
OZ0612
OZ0613
OZ0614
OZ0703
Protective immunity and competitive exclusion in
development of effective intervention products for
poultry.
An investigation of the distinguishing features of C.
jejuni strains that have host/disease associations.
Epidemiological studies and development of
practical control measures for Campylobacter in
broiler flocks
Determining the role of maternal antibodies in the
lag phase of Campylobacter jejuni infection in
chickens.
Survival and persistence of campylobacters in
poultry farm environments and suggested control
measures
Development of a comprehensive MLST database
for assessment of Campylobacter risk factors
The epidemiology of campylobacter infection in
dogs in the context of the risk to humans
Towards risk-based control of Campylobacter:
developing the evidence base using
epidemiological and bacteriological approaches
To develop vaccination approaches to the control
of Campylobacter in poultry.
Plant antibody delivery of passive immunisation
against E. coli O157:H7: a novel means of control
in the animal
01/04/01
01/07/01
01/07/02
01/05/05
01/09/05
01/07/05
18/07/05
01/01/06
01/04/06
01/09/99
31/03/04
30/06/04
30/06/06
31/05/06
31/01/09
30/06/08
16/07/08
30/09/10
31/03/09
01/06/03
(sub-contractors)
Veterinary Laboratories
Agency
Veterinary Laboratories
Agency
Veterinary Laboratories
Agency & University of Bristol
(co-contractors)
Imperial College (subcontractor)
Veterinary Laboratories
Agency
University of Bristol &
Veterinary Laboratories
Agency (co-contractors)
Scottish Agricultural College &
DTA Ltd (sub-contractors)
University of Oxford
University of Liverpool
Veterinary Laboratories
Agency
Veterinary Laboratories
Agency
Veterinary Laboratories
Agency
ADAS Consulting & University
232,145
508,987
859,393
50,012
746,102
503,312
228,351
1,558,693
(Defra:
1,495,693; Other
funders:
£200,000)
267,629
354,108
76
74
78
82
78
70
84
78
76
86
35

OZ0704
OZ0705
OZ0706
OZ0707
Quantification of E. coli O157:H7 virulence factors
in vivo using real-time RT-PCR
A Proteomic Approach to Identify Virulence
Determinants of EHEC 0157.
EHEC O157 pathogenesis: Ovine and other animal
model studies
Identification of factors mediating intestinal
colonisation of cattle by enterohaemorrhagic
Escherichia coli O157:H7
OZ0708 A Systems Analysis Methodology to Elucidate and
Evaluate the Critical Control Points for E. coli
0157:H7 in Cattle and sheep from farm to abattoir
OZ0709
OZ0710
Epidemiology of VTEC 0157 and other VTECs
likely to be pathogenic to man in farm wastes
The role of goats and pigs in the maintenance and
transmission of VTEC including EHEC O157:H7
01/06/99
01/08/99
01/04/99
01/10/99
01/01/00
01/04/02
01/10/02
30/09/02
12/09/02
30/03/04
11/04/07
30/06/02
31/03/05
30/09/05
of Leicester (sub-contractors)
University of Glasgow 200,764
University of Southampton
Veterinary Laboratories
Agency
University of Bristol Veterinary
School (sub-contractor)
Institute for Animal Health
Silsoe Research Institute
Veterinary Laboratories
Agency (sub-contractor)
Veterinary Laboratories
Agency,
Health Protection Agency,
Silsoe Research Institute &
University of Liverpool (sub-
contractors)
Veterinary Laboratories
Agency
University of Bristol Veterinary
237,995
374,377
603,124
151,494
365,493
399,546
88
90
91
98
100
91
101
36

OZ0711
Incidence and control of VTEC in animal feeds 01/08/02
OZ0712 Escherichia coli O157 interventions and control 01/04/03
OZ0713
The role of the native host gut flora and innate
immune status upon the colonisation of E. coli
O157 in ruminants
01/10/04
31/01/05
30/09/06
30/09/07
School (sub-contractor)
ADAS UK Ltd
Direct Laboratory Services Ltd
(co-contractor)
Scottish Agricultural College
University of Edinburgh and
Biomathematics & Statistics
Scotland (sub-contractors)
Veterinary Laboratories
Agency
Moredun Research Institute
(sub-contractor)
132,343
503,523
272,863
92
93
101
37

Annex 2 – Project Abstracts and
Project Review Summaries
This annex contains an abstract prepared by the contractor for each project reviewed,
and a brief summary of the review discussion on each project.
Further details on each project, including the final report for completed projects, can be
accessed at http://randd.defra.gov.uk by searching for the project code or a keyword.
38

Salmonella Research Projects
Project code: OZ0312
Project title: Development of a sensitive and
specific molecular typing
method for the epidemiological
study of Salmonella
Start date (dd/mm/yy): 03/01/2000
End date (dd/mm/yy): 31/01/2003
£228,700
Total cost:
Affiliation: LGC
Sub-contractor(s):
Abstract of research
Stobhill NHS Trust
Poultry Health Services
Bacterial pathogens such as Salmonella and Campylobacter are a major cause of
infection in the UK, and present a significant public health problem world-wide. Identifying
the source of infection is central to control the spread of disease, but can be hampered
by the inability to discriminate between isolates within the most common Salmonella
phage and serotypes using conventional typing methods. In this project the use of a
molecular typing method, fluorescent AFLP profiling (AFLP), as a tool for highly
discriminatory characterisation of food borne pathogens was evaluated, using Salmonella
as a model organism.
Until recently, conventional bacteriological methods such as serotyping and phage typing
have been used for the characterisation of Salmonella.
Molecular methods such as AFLP, plasmid analysis and macro-restriction fragment
polymorphism typing by pulsed-field gel electrophoresis (PFGE) can provide improved
discrimination and sensitivity in bacterial typing. AFLP analysis generates a detailed
genomic profile from an organism, consisting of a number of accurately sized DNA
fragments. The characteristic fragments within the profile are produced by selective PCR
amplification of total bacterial DNA, after initial restriction enzyme digestion. Profiles
produced from bacterial isolates are then be compared to assess the similarity of strains.
The genomic profiles provide a good indication of the relatedness of isolates, and
mathematical methods of evaluating evolutionary relationships from AFLP profiling data
have been developed.
39

To test the suitability of the AFLP method for sensitive characterisation of Salmonella
species, over 400 isolates were selected for investigation, from the collection of the
Scottish Salmonella Reference Laboratory at Stobhill Hospital in Glasgow. A catalogue of
representative strains was produced, including 243 isolates of human origin, 83 animal
isolates, 37 from a variety of foods and 26 strains acquired from environmental sources.
The isolates had already been characterised by classical typing methods by the Scottish
Reference Laboratory. In addition, the majority of the strains had been typed using
PFGE, and their antibiotic resistance profile determined. DNA extracts from the
resuscitated Salmonella strains were provided by the Scottish Salmonella Reference
Laboratory for AFLP analysis at LGC.
The first stage of the project focused on development of a robust protocol for the
generation of AFLP profiles. To facilitate inter-laboratory comparison it was decided to
utilise commercially available AFLP reagents provided as a Microbial Fingerprinting Kit.
However, in the last year of the project, the highly variable quality of commercially
available reagents necessitated a change to independently-sourced materials. The effect
of a variety of parameters on the robustness and quality of AFLP profiles was assessed,
and an optimised protocol was developed. To promote reproducible data interpretation
and comparability of results between analysts and laboratories, methods for
normalisation of data and criteria for AFLP profile acceptance were developed. However,
the inherent variability in profile intensity was a significant challenge to the reproducibility
of the method, with subjective calling of signals on the borderline of automated detection.
The variation in fragment intensity is one of the main problems with robust isolate
characterisation by AFLP analysis.
An extensive range of commercially available PCR primer and restriction enzyme
combinations were evaluated for sensitivity against a set of closely related isolates, and
novel combinations targeting hyper-variable regions of the Salmonella genome were also
developed and tested. Genomic digestion with EcoRI and Taq I, followed by amplification
using EcoRI-T and TaqI-0 selective primers was identified as the most discriminatory
approach, and this was used to profile the extensive catalogue of isolates. The AFLP
profiles produced were then compared with known phenotypic characteristics of the
isolates to identify markers correlating with particular traits.
To facilitate identification of unique markers for particular characteristics, a searchable
database was developed at LGC using Microsoft Access. The database allowed both
storage of isolate profiles and comparison of molecular and phenotypic characteristics of
strains, enabling AFLP profiles of unknown samples to be matched with related profiles
already entered into the database. The effectiveness of the database system was
demonstrated in a blind trial, although in contrast to previous studies characterising
smaller numbers of isolates, robust correlation between molecular markers and phage
type or other phenotypic characteristics could not be established.
In conclusion, the AFLP typing approach offers several advantages over currently used
typing methods, including accuracy and precision of fragment sizing, and the suitability of
the profile information for database storage. However, the complex, multi-step nature of
the method can lead to high failure rates, and the relatively high cost of commercial
reagents and equipment are further drawbacks to widespread uptake of the AFLP
approach. Overall, the discriminatory power of the technique was not found to be
sufficiently high to warrant extensive further effort in overcoming the very real drawbacks
of the method, particularly with the advent of newer techniques including proteomic and
microarray methods, and the widespread adoption of PFGE analysis for international
surveillance initiatives. .
40

Review Summary
This project aimed to improve 1) the speed of salmonella typing and 2) the ability to type
some of the epidemic serotypes and subtypes (for example, Salmonella Enteritidis and
Salmonella Typhimurium DT 104) to improve outbreak tracing and epidemiology in
general. The use of AFLPs (amplified fragment length polymorphisms) was assessed as
a typing method with these aims in view.
At the time of funding, this was important methodology to try to develop. The work was
appropriately carried out and, although it was reasonably successful, AFLP was not
sufficiently robust to be taken further when other typing methods showed more promise.
41

Project code: OZ0324
Project title: New and emerging Salmonella
serovars; epidemiological, riskbased
and molecular
approaches to their
identification and control: set up
studies.
Start date (dd/mm/yy): 01/10/2005
End date (dd/mm/yy): 31/01/2009
Total cost:
£1,009,045
Affiliation: Veterinary Laboratories Agency
Sub-contractor(s): University of Liverpool
Abstract of research
Salmonella enterica is a notifiable disease agent. The genus comprises of some 2500
serovars that are defined by serological reactions recognising surface antigens including
the Vi capsular antigen, the somatic O antigen and the often phase variable flagella H
antigen and can be is divided into six subspecies. S. enterica subsp. enterica affects
warm blooded animals including man whereas the other subspecies are very rarely
associated with disease. Furthermore, certain serovars within subspecies I are
associated with specific hosts. Host adaptation and the form of disease mediated by
Salmonella is serovar specific. S. Typhi and Paratyphi cause severe systemic (typhoidal)
disease in man. Other serovars such as S. Gallinarum are adapted to cause a systemic
disease in poultry and rarely affects man. On the other hand, serovars such as S.
Enteritidis and S. Typhimurium are promiscuous causing disease in man and animals.
However, the mode of disease is largely gastro-intestinal (non-typhoidal). These two
serovars cause the significant majority of human Salmonellosis. Within the EU, 100,000
cases were recorded in 1997 reducing to 73,000 in 2001. In the United States, estimates
of 30,000 to 40,000 cases per year have been made.
The epidemiology of non-typhoidal Salmonellosis in man over the past decades
worldwide has been dominated by S. Typhimurium, S. Enteritidis, S. Newport, S,
Heidelberg and S. Hadar. However, there are geographical variances with Hadar more
pronounced in Europe than either Newport or Heidelberg. Why S. Typhimurium,
particularly the multiresistant clones such DT104, and S. Enteritidis, notably phage type
4, emerged so dramatically in recent years are still unclear although hypotheses abound.
Review Summary
The rationale behind this project was considered important. Historical examination of
salmonella data from humans and animals suggests that, despite the measures already
put in place, a future epidemic in humans is likely to arise from infections in the animal
population. Since eradication of salmonella from animals is unlikely to be feasible, it will
42

e important to identify an epidemic strain early, so that measures can be taken to
reduce the size and cost of any resulting epidemic. It is also important to identify the
factors that make a strain of salmonella epidemic, and also why such strains disappear in
due course.
This work helps maintain an important section of expertise at VLA and keeps the VLA at
the forefront of scientific methodology in this area, particularly with regard to array work
and developments in GIS. The potential benefits outweigh the risks associated with the
development and application of new techniques. The panel felt that the work would yield
valuable data when completed.
43

Project code: OZ0311
Project title: Conventional versus capillary electrophoresis of
RFLP fragments and PCR products for sensitive and
rapid subtyping of Salmonella.
Start date (dd/mm/yy): 01/06/99
End date (dd/mm/yy): 31/03/04
£373,347
Total cost:
Affiliation: Central Science Laboratory
Abstract of research
We compared different molecular typing techniques in combination with two
electrophoretic techniques (conventional gel and capillary electrophoresis) for molecular
subtyping of a wide range of Salmonella enterica serovars from animal, human and
food or feed sources. These included Pulsed-field Gel Electrophoresis (PFGE),
Restriction Fragment Length Polymorhism (RFLP) and polymerase chain (PCR) based
techniques such as Fluorescent Amplified Restriction Fragment Length Polymorphism (f-
AFLP), Random Amplification of Polymorphic DNA (RAPD), Repetitive Extragenic
Palindromic (REP) and Enterobacterial Repetitive Intergenic Consensus (ERIC)
PCR.Comparisons between gel based and capillary electrophoresis showed that gel
electrophoresis was more reliable and gave greater band resolution than capillary
electrophoresis based the equipment available. Most methods gave good but not perfect
serovar identification. PFGE and f-AFLP were comparable and were the most promising
techniques for differentiation and characterisation at strain level within serovars. f-AFLP
analysis requires improved standardisation including use of internal standards and
triplicate analysis.
Review Summary
This was an important comparison of techniques aimed at improving typing methods for
salmonella, with the approaches used valid at the time of funding.
Unfortunately, none of the methods delivered the sensitivity required for epidemiological
studies. Some useful information was obtained which informed future approaches to
molecular typing.
A major criticism of the study was that the work carried had not been published in the
scientific literature as the findings would have been useful to other scientists in the field.
44

Project code: OZ0314
Project title: The role of defined bacterial genes and host genetic
background in intestinal colonisation of poultry by
Salmonella
Start date
01/04/99
(dd/mm/yy):
End date
(dd/mm/yy):
30/06/02
Affiliation: Institute for Animal Health
Project code: OZ0320
Project title: Bacterial and host genes in Salmonella colonisation in
poultry
Start date
01/07/02
(dd/mm/yy):
End date
30/06/05
(dd/mm/yy):
£956,279
Total cost:
Affiliation: Institute for Animal Health
Abstract of research
We have demonstrated that reproducible differences exist in the extent to which several
Salmonella serovars colonise the alimentrary tract of genetically different inbred
populations of chickens. This is not associated with the MHC type, is not sex linked and
not the result of SAL1 or NRAMP1 haplotypes but is a dominant trait. Similar patterns are
found when chickens are also infected with Campylobacter jejuni indicating a common
mechanism. Considerable differences in the patterns of expression of selected defensin
have been found in the intestinal tissues. No major differences could be found in the
extent to which mucin obtained from resistant or susceptible lines of chickens were able
to act as nutrient sources for colonising Salmonella serovars. The resistance
characteristic has enormous potential for selective breeding and for use with vaccines for
infection control.
Bacterial mutational studies indicated that ability to utilise sialic acid was not essential to
colonisation. A number of electron acceptors used under anaerobic conditions were
tested and none was found to be essential. Greater reductions were seen when mutants
were unable to use substrate level phosphorylation suggesting that fermentation may be
of greatest importance in the large gut. The energy storage compounds glycogen and
polyphosphate contributed little to colonisation whereas they and particularly
polyphosphate was involved in survival outside the body suggesting a very important role
in homeostasis.
45

Review Summary
The knowledge that there is a genetic basis to resistance to intestinal colonisation by
salmonella in poultry is important. It was felt, however, the work would have benefited
from a greater involvement with the poultry industry, since without this, the findings could
not be applied. It was noted that follow-on work (LK0665) was being undertaken under
the LINK programme, which is jointly funded by Defra and industry.
The work also contributed to maintaining an expert research team that has contributed,
and subsequently published, high quality research.
46

Project code: OZ0328
Project title: A monitoring, control and education package to assist
the turkey industry with reduction of Salmonella and
antimicrobial resistance and achieving EU targets
Start date (dd/mm/yy): 01/04/07
End date (dd/mm/yy): 31/03/10
£1,138,946.00
Total cost:
Affiliation: Veterinary Laboratories Agency (VLA)
Sub-contractor(s): 1. Agricultural Development Advisory Service (ADAS)
2. University of Bristol
Abstract of research
The work proposed in this large multidisciplinary project aims to rapidly gather
information from turkey breeding and production. Initially by means of the EU baseline
Salmonella survey which has already been largely completed as a separate contract.
Fortunately the samples taken for that survey can, by agreement with the flock owner, be
used for isolation of antimicrobial (fluoroquinolone and third generation cephalosporin)
resistant E.coli and non-selected isolates. This, together with the further characterisation
of the isolates in terms of bacterial identity MIC and resistance mechanisms, and a risk
factor analysis for occurrence of resistance, will be the first objective (01) of the project.
This will be followed by a detailed status report on the British turkey industry together
with a publication on Salmonella trends (02).
Control of Salmonella depends on effective identification of its occurrence and Objective
03 provides a package of work designed to optimise and quantify the sensitivity of
detection as well as achieving practical economies. It is necessary to carry out detailed
investigations (04) of the occurrence of Salmonella in the complex network of turkey
breeding and production to define where the problems are occurring and to identify 'bad'
practice which can be corrected and 'best practice' which can be extended more widely.
These investigations will also intercalate studies on the dynamics of resistant E.coli with
and without fluoroquinolone treatment and will lead to the development of improved
objective auditing procedures for control of Salmonella (05). In Objective 06 successful
management methods which have been shown to control Salmonella will be applied to
problem farms as trial interventions for both Salmonella and resistant E.coli, and for more
difficult cases competitive exclusion and organic acid treatment will also be used (07).
The costs and benefits of these control options will be assessed as part of an economic
analysis (08) and finally the lessons learned from the totality of the research, and other
relevant current sources, will be consolidated in an industry education package (09)
which will include a roadshow campaign and a training DVD and e-mail information
network. The project involves major players in Salmonella field research and
consultancy and major turkey breeding and production integrations and their veterinary
consultants to form a powerful project task-force. The package of objectives therefore
fully supports industry and Defra priorities and public health interests and will provide the
turkey industry with effective methods to meet and exceed future EU requirements.
47

Review Summary
The project was felt to have the potential to provide extremely important information for
Defra‘s policies. The findings were likely to assist the turkey industry in meeting
European Community salmonella targets and to cement the joint industry/government
approach to salmonella control. Furthermore, the data collected is relevant to the
sustainable use of important antimicrobials in this sector and the long-term health of the
UK turkey industry. The project also fits well with the Defra policy on working in
partnership with the relevant groups.
At this early stage, there are no conclusions, dissemination, or reports on progress to
assess the project. However, it was noted to be a well thought-out project, by an
impressive team, which should produce very valuable information for government and
industry if it continues to receive full support from the industry.
48

Project code: OZ0327
Project title: Effect of immunosuppression associated with pointof-lay
on Salmonella infection and immunity in laying
hens
Start date (dd/mm/yy): 01/07/06
End date (dd/mm/yy): 20/06/09
£198,000 (Defra: £37205; BBSRC: £160795)
Total cost:
Affiliation: Department of Veterinary Pathology,
University of Liverpool
Abstract of research
Previously we have shown that suppression of cell-mediated immunity at point-of-lay in
hens is a critical factor in reproductive tract infection by Salmonella enterica and
subsequent transmission to eggs. This project will characterise the immunolgical
structure of the avian reproductive tract, which immunological changes take place at
point-of-lay and determine whether these affect the immune response to Salmonella and
in particular whether suppression of immune responses reduce the efficacy of
vaccination, leading to a gap in immune protection. This will be achieved through
investigating changes in cell populations, expression of key cytokines and chemokines
and antibody levels. We will assess changes in protection through in vivo challenge
experiments. We will then look at how changes in vaccination or stimulation of the
immune system through recombinant cytokines can improve protection and prevent any
gap in immunity.
Review summary
Research on the avian immunological response during the point-of-lay period, when
salmonella breakdowns tend to occur, should provide valuable information for salmonella
control. The overall plan for the work is clearly laid out and the researchers are making
good progress towards the stated objectives.
If this approach proves to be successful, there will be a need to understand how the
information can be utilised in the field and possible to investigate changes at the end-oflay
when salmonella breakdowns are also known to occur.
It is appropriate that this work is jointly funded with BBSRC as it may take some years for
results of the work to benefit UK poultry industry.
49

Project code: OZ0317
Project title: Epidemiological investigations of Salmonella
contamination in table egg production
Start date (dd/mm/yy): 01/08/00
End date (dd/mm/yy): 31/01/03
£227,692.00
Total cost:
Affiliation: Veterinary Laboratories Agency (VLA)
Project code: OZ0321
Project title: Investigation of the role of environmental
contamination in the epidemiology of Salmonella
infection in egg-laying flock
Start date (dd/mm/yy): 01/10/2002
End date (dd/mm/yy): 30/09/2007
Total cost:
£464,534.00
Affiliation: Veterinary Laboratories Agency (VLA)
Sub-contractor(s): University of Bristol
Project code: OZ0325
Project title: A monitoring, control and education package to assist
the egg industry with Salmonella reduction and
achieving EU targets
Start date (dd/mm/yy): 01/04/06
End date (dd/mm/yy): 31/03/09
Total cost:
£753,006.00
Affiliation: Veterinary Laboratories Agency (VLA)
Sub-contractor(s): University of Bristol
Natural England
Central Science Laboratory
50

Abstract of research
The VLA research work on Salmonella in commercial laying farms began officially in
2000 with Project OZ0317, although some pilot investigations had been carried out prior
to that. Voluntary investigations on laying farms where S.Enteritidis (SE) had been
identified as a result of trace back investigations from food poisoning outbreaks,
investigations of clinical disease or (rarely) identification of SE as a result of voluntary
monitoring under the British Egg Industry Council (BEIC) Lion Code scheme showed that
the principal problem was horizontal infection resulting from persistence of contamination
in poorly cleaned houses and particularly in breeding mice or rat populations in the
houses. Occasional new introductions of infection due to infection in breeding flocks or
contamination of hatcheries were also investigated but these incidents were uncommon.
Persistence of SE appeared to be independent of vaccination status, except in barn or
free-range farms where there were no rodent problems which became free of infection
when new vaccinated flocks were introduced. Contamination of eggs from infected
vaccinated flock birds reduced by around 70% compared with those from non-vaccinated
flocks. Cross-contamination during egg packing was also identified. The scope of the
work was widened with increased funding in Project OZ0321 and various investigations
involving quantification of SE demonstrated high numbers of organisms associated with
rodents and flies. Exposure studies at Bristol University demonstrated the high risk
associated with contaminated drinking equipment (a common finding in layer flocks)
regardless of vaccination. Vaccine transition studies suggested that there may be
significant differences between the efficacy of the various vaccination programmes.
Randomly recruited farms from the EU Baseline Survey showed identical problems to the
original study cohort and sampling comparisons identified significant differences in the
sensitivity of various monitoring options, especially in large barn or free-range flocks
where subdivision of floor spaces led to a dilution effect and false negative results when
limited numbers of boot swabs were used. In Project OZ0325 good progress has been
made on problem farms by including specialist pest control and decontamination services
in the project team and the project leader has been involved in redrafting various codes
of practice and SVS guidance documents as well as the BEIC Lion Code and together
with Natural England has been involved in a nationwide road show campaign for the egg
industry.
Review Summary
The work covers a very wide spectrum of issues of key relevance to the commercial egg
industry and extends the fundamental knowledge of the practical epidemiology in this
area. Different parts of the work have contributed to the evidence base, to the
development of diagnostic tests and sampling methodology and to improvement in
methods of control. It was particularly encouraging to find that salmonella-positive flocks
can turn negative, and that infection levels can be reduced.
The work was fundamental to building better relationships with the industry, which has
assisted both subsequent research and also policy development involving partnership
with industry. The current research is crucial to Defra in developing with industry a
salmonella control programme that meets EC salmonella requirements and keeps the UK
egg industry at the forefront in terms of quality. It has allowed Defra to negotiate a UK
position based on best available science and the main research worker has acted as an
independent adviser to the EC. The results of the research have been successfully
disseminated to industry by means of a road-show led by the British Egg Industry
Council.
51

It is clear that the EC legislative changes, which began to be introduced from 2007, will
impact on the industry, possibly quite severely, and it is likely that there will be a need for
further work to address some of the new issues that will arise out of the implementation
of the legislation.
52

Project code: OZ0326
Project title: Managing the challenges to the egg industry posed
by new SE PTs
Start date (dd/mm/yy): 01/04/06
End date (dd/mm/yy): 31/03/09
Total cost:
£403,472.00
Affiliation: University of Bristol
Sub-contractor(s): VLA
Abstract of research
Salmonella spp. are highly important zoonotic pathogens causing over one billion cases
of infection worldwide each year. One Salmonella serovar, in particular, is most
important. For the last 20 years there has been a pandemic of Salmonella Enteritidis
(SE) infection in almost all parts of the world, including Europe and this has largely been
egg-associated. The pandemic continues and SE remains a highly important zoonotic
pathogen. The longevity of the pandemic is unique and is associated with the invasive
behaviour of SE in chickens and the fact that infection in laying hens is largely subclinical.
The pandemic does have dynamic aspects, however, and the last few years the
UK, and some other EU member states, has seen a marked shift in phage types of SE
from human cases. Prior to this, one PT, PT4 dominated the epidemiological picture in
Western Europe. More outbreaks in the UK are now caused by PTs other than 4. For
example, PT 1 has risen from c.850 cases in 1999 to c.2000 in 2003. Similarly, cases
caused by PT 14b rose from c.150 to c.950 in the same period. The new PTs may
become established in the UK and it is important to establish the risk posed by these new
and potentially highly dangerous PTs, which threaten animal and public health in the UK.
Changes in Salmonella type, as manifested by PT alterations, occur with high frequency
and must challenge control measures put in place to deal with different strains.
The research is identifying how a range of relevant SE PTs differ in surface structures,
particularly LPS, and how these changes influence the growth and survival of the
bacteria in whole eggs and egg components. The work will also encompass interaction of
SE PTs with immune sera from vaccinated hens and infection biology in immunised (with
PT4) and non-immune hens. Identification of surface structures and how these affect
virulence will eventually enable improved vaccines to be produced. The investigators are
already interacting with vaccine companies.
Review Summary
Understanding differences between phage types of S. Enteriditis was considered
important, particularly if it shed light on understanding the risk to public health associated
with table eggs. Vaccination of poultry with a vaccine based on phage type 4 has been a
significant plank in the control of S. Enteritidis in the UK and it was important to establish
53

if the vaccines currently available are effective against other S. Enteritidis phage types
prevalent in the rest of Europe. The work will contribute significantly to the evidence base
and should result in improved methods of control. The project is intended to assist the
poultry layer industry to reduce levels of salmonella and it is important that the industry
remains involved.
54

Project code: OZ0313
Project title: Non-specific and innate resistance to Salmonella
infection in chickens and pigs
Start date (dd/mm/yy): 01/04/99
End date (dd/mm/yy): 30/06/02
£689,400
Total cost:
Affiliation: Institute for Animal Health
Abstract of research
We have studied the biological and cellular basis of the difference in in vivo killing of
Salmonella strain by chicken lines which are either resistant or susceptible to systemic
salmonellosis. Resistance correlated with killing by macrophages cultured in vitro from
the blood of the respective chicken lines. This also correlated with the extent to which
antibacterial oxidative radicals were produced in response to in vitro infection. This is a
useful biological marker of this important genetic trait.
Infection of epithelial cells by different Salmonella serovars provoked different responses.
S. Typhimurium induced high levels of the pro-inflammatory chemokines IL-1 and IL-6
whereas S. Gallinarum was able to suppress their production. The suppressive
capabilities of Salmonella strains was also demonstrated by virulent strains of S.
Typhimurium in murine macrophages which were able to down-regulate the production of
nitric oxide through the NF kappa B pathway. The induction of innate responses by live
vaccines and their protective ability has been demonstrated in gnotobiotic pigs which has
been found to be mediated by the induction of IL-8 induced neutrophils into the villus.
The stimulation of the innate response by live vaccines is a useful attribute. We have
also found the neutrophils have the potential to act as antigen presenting cells through
the expression of the B17 co stimulatory molecule.
Review Summary
The project was appropriate at the time it was commissioned. It was carried out to a high
standard and produced interesting results. It has considerably advanced the
understanding of some of the fundamental mechanisms for establishment and clearance
of salmonella infections in chickens and pigs. The goals of the research, namely 1) to
determine the cellular basis for the difference in susceptibility to systemic salmonellosis
in inbred chicken lines, and 2) to determine the basis for the stimulation of non-specific
immunity in the pig intestine following intestinal colonisation by an avirulent rough
Salmonella Infantis strain, have been achieved.
The pig work demonstrated effective activation of innate immune response by one
serotype (S. Infantis) to protect from disease induced by another serotype (S.
Typhimurium). However, this protection did not reduce the colonisation of the intestine
so, as in the chicken work, it is more relevant to control of animal disease than to the
zoonoses.
55

Four published papers have come out of this project, two on poultry and two on pigs. The
poultry work has rapidly been overtaken by alternative vaccine approaches. The pig work
is still continuing, with the aim of solving problems specific to the UK. Any future work
should be done with full involvement of the industry.
56

Project code: OZ0135
Project title: Epidemiological studies of multiple-resistant
Salmonella Typhimurium DT104 in cattle
Start date (dd/mm/yy): 01/04/1997
End date (dd/mm/yy): 31/03/2002
£523,530
Total cost:
Affiliation: Veterinary Laboratories Agency
Abstract of research
This 5-year epidemiological project aimed to provide a better understanding of the
epidemiology of multiple-resistant (MR) Salmonella Typhimurium DT 104 and other
Salmonella infections in cattle herds. The project started during an epidemic of MR S.
Typhimurium DT 104 in cattle, and increasing numbers of human infections with this
organism, in the 1990s in Great Britain. The original proposal included an intervention
study to test the effectiveness of control measures in cattle herds, but this was replaced
with additional epidemiological studies because there was a marked decline in reported
cases of this organism in cattle during the study.
The studies demonstrated:
1. Salmonella infection of dairy herds in England and Wales was common but most
herds were infected with serotypes unusual in human cases.
2. Infection was clustered geographically and more common in large herds
3. Management risk factors were identified providing evidence for control
4. Herds with clinical outbreaks of S Typhimurium DT104 did not usually experience
further outbreaks but risk was higher than the general population
5. Biosecurity on dairy farms was generally poor and, at the time, it was considered
difficult to improve sufficiently to control Salmonella
6 The standardised environmental sampling protocol used had many advantages in
terms of cost and feasibility for the detection of Salmonella at the farm level and could be
adapted for other livestock systems and organisms
7. Many Salmonella infections in cattle herds will be unreported by routine surveillance
of laboratory submissions for clinical diagnoses.
It is unlikely that Salmonella will be eradicated from dairy farms in the short to medium
term, and therefore human exposure to this organism originating from dairy farms should
be minimised, for example by ensuring the proper pasteurisation of milk and provision of
advice to all farm workers and visitors.
There were numerous potential transmission routes for Salmonella, and other infections,
within the structure of G.B dairy farms (e.g., high number of animal movements) and the
industry (e.g., network of markets and dealers). Emerging serotypes (eg MR S. Newport)
are likely to spread rapidly after introduction and effective intervention to limit their spread
will be difficult. Emerging serotypes not associated with disease will not be readily
identified by current surveillance.
57

Review Summary
This epidemiological study was carefully planned and executed and the results can be
used to inform salmonella surveillance/control and basic biosecurity extension work.
Care would need to be taken in using the information since the epidemiological
information was gained while the S. Typhimurium DT 104 epidemic was waning. There
was also some discussion as whether the selection of herds for the recurrence study had
potential for bias.
Several peer-reviewed scientific publications were reported but these had not in fact
been published as of November 2007. More might have been done to disseminate the
results among farmers, for example, through the farming press.
It is probably reasonable to believe that a new epidemic of Salmonella in cattle will occur
at some time in the future, as there has been a series of epidemics in the past. Research
will need to be put in place at an early stage to establish the epidemiology of the disease
during the rise in the epidemic, in order to attempt to reduce the size of the epidemic.
Research is underway to try to assist with the prediction of a new epidemic.
58

Project code: OZ0315
Project title: Salmonella pathogenesis and immunity in cattle and
pigs
Start date (dd/mm/yy): 01/04/1999
End date (dd/mm/yy): 30/06/2002
£656,100
Total cost:
Affiliation: Institute for Animal Health, Compton
Abstract of research
Salmonella enterica subspecies I is an important cause of enteric disease in man and
farmed livestock. Infections often involve acute enteritis and may be complicated by lifethreatening
systemic sequelae depending on serovar- and host-specific factors. In the
absence of effective vaccines, control and therapy of Salmonellosis in livestock relies on
fluid replacement and antibiotic use. Widespread concern exists that the latter may lead
to an increase in transmissible antibiotic resistance. Toward the development of
intervention strategies not reliant on antibiotic use, Defra project OZ0315 aimed to
identify and characterize factors influencing Salmonella pathogenesis and host
responses in cattle and pigs. We characterized a panel of S. enterica serovars of
differing virulence in calves and pigs and observed that systemic virulence correlated
with enhanced persistence in mesenteric lymph nodes but not the response of primary
macrophages infected ex vivo. The cell tropism of S. enterica serovars in the bovine
intestinal mucosa was also examined and host cell-killing by a novel caspase-dependent
mechanism distinct from apoptosis characterized. Systemic translocation of S. Dublin in
calves was observed to occur via efferent lymphatics, but not venous blood, in a cell-free
niche. By targeted and genome-wide mutagenesis factors influencing both the enteric
and systemic virulence of Salmonella in calves and pigs were identified, providing
valuable data for the rational attenuation of Salmonella for use as live vaccines.
Review Summary
This was a good, practical project with an excellent output of 18 published papers. It had
provided information on the molecular basis of salmonella pathogenesis in cattle and
pigs and had the potential to assist with control by providing data for the rational
development of live vaccines. However, it was noted that while this was high-quality
science, it was, for Defra‘s purposes, quite distant from practical applications.
The contractors had much expertise in the area and used novel techniques to further
develop the work. There was progress towards identifying suitable vaccine candidates,
but the project leader has now left. Project OZ0319 was set up to address some of the
questions raised by this study.
59

Project code: OZO316
Project title: Epidemiological studies of Salmonella in pigs and
control by intervention.
Start date (dd/mm/yy): 01/08/2000
End date (dd/mm/yy): 31/12/2006
£1,222,749
Total cost:
Affiliation: VLA
Sub-contractor(s):
Project code: OZ0323
London School of Hygiene and Tropical Medicine
Project title: An integrated risk based approach to the control of
Salmonella in UK pig farms.
Start date (dd/mm/yy): 01/04/2005
End date (dd/mm/yy): 31/10/2008
£1,053,974.00
Total cost:
Affiliation: VLA
Sub-contractor(s): Health Protection Agency
University of Liverpool,
Imperial College,
London School of Hygiene and Tropical Medicine,
University of Wageningen
Abstract of research
Salmonella bacteria live in the gut of all animals including man and some 15,000 human
cases are reported annually. About 1500 cases are caused by Salmonella Typhimurium
(STM), which is found in cattle, poultry and pigs. STM occurs in 1/8th of British slaughter
pigs potentially leading to human disease. The Industry established the ZAP control
programme and the Food Standards Agency aims to reduce salmonella in pigs by 50%.
By understanding the epidemiology of salmonella infection in pigs and through the
foodchain we can devise control measures to reduce the human health risk.
Salmonella infection can be detected by culture of the bacteria, which shows status
today, or by testing for circulating antibodies which measures lifetime exposure and we
used both methods. A survey of 107 farms showed that most (>50%) have a low
prevalence of infection (

We created a farm-fork quantitative risk assessment model that indicates reducing
between-pen transmission, e.g. by not mixing pigs, is crucial. No more than one third of
human STM cases may be attributable to GB pigs and a reduction in prevalence on farm
will result in some public health benefit.
Review Summary
This project is considered to have produced good basic work, which needed to be done.
In order to reduce the incidence of salmonella in pigs, it is vital to look at the basic
epidemiology of the infection in these animals, which is complex. Progress has been
painfully slow but a lot of basic work needed to be done. This project has produced
important basic information about the prevalence of salmonella in finishing pig farms in
UK and identified risk factors for herds becoming infected.
Project OZO316 provided an early and important attempt at joint Defra/industry
partnership. The poor support for the intervention trial was disappointing and affected the
scientific value of that part of the project. Useful lessons were learned about projects
involving collaboration with industry, which have been helpful in delivering subsequent
Defra/industry research on the poultry side.
This work has led into the next project OZ0323 and this work is still on-going. OZ0323
includes extensions to include the potential impact from farm to retailer. OZ0323 is a
worthwhile, multi-disciplinary project and should clarify many questions concerning
salmonellosis in pigs, from which future control strategies can be determined, although
farm-level risk factors can be difficult to identify for a multi-factorial disease. The study
highlighted the difficulties in successfully implementing interventions to decrease the
prevalence of salmonella. One query that arose was the difference between test results
and prevalence.
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Project code: OZ0318
Project title: Understanding the dynamics of endemic and
epidemic Salmonella infections in cattle: A
comparative modelling approach
Start date (dd/mm/yy): 01/01/03
End date (dd/mm/yy): 30/04/06
£399,343.00
Total cost:
Affiliation: University of Liverpool
Sub-contractor(s): Veterinary Laboratories Agency
University of Lancaster
Abstract of research
There were four interrelated aspects to this study of endemic and epidemic Salmonella
infections in dairy cattle, as follows:
1. Mathematical modeling. Considered both within herd and between herd transmission.
Focused on teasing out features underlying the variation in the infection dynamics of
different serotypes.
2. Immunological studies and quantitative microbiology. Began with the objective of
developing methods for measuring immunological responses to Salmonella infections in
cattle. Concerns arose over validity in very young animals and suitability for determining
long-term immunity. Alternative approach then taken (2a), involving on-farm longitudinal
studies and experimental Salmonella survival studies.
3. Space-time statistical analysis. Largely concerned with investigating the evidence for
clustering, as this could provide evidence relating to the transmissibility of infection (or its
origin in localised environmental processes). 4. Systematic literature review.
Review Summary
It was felt it should be useful to develop a model for salmonella in cattle. Although the
mathematics behind the modelling is complex and difficult to understand, the researchers
had done a good job of presenting their approach openly and clearly, including an openaccess
web page. The model had only been developed for cattle because of time
restraints and it may be worth considering whether it could be applied to pigs.
There was concern that the model would not be satisfactory if it did not take account of
the carrier state associated with S. Dublin, which is what makes S. Dublin so difficult to
control in the British population. There was also uncertainty about how the model had
been developed—it started as a generic salmonella model, which was then applied to
Salmonella Typhimurium, Salmonella Agama, and Salmonella Dublin, and then labels of
endemic and epidemic were applied, with no account taken of the carrier state.
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Project code: OZ0319
Project title: Salmonella pathogenesis in cattle and pigs
Start date (dd/mm/yy): 01/07/2002
End date (dd/mm/yy): 30/09/2006
£709,200
Total cost:
Affiliation: Institute for Animal Health, Compton
Sub-contractor(s): None
Abstract of research
Salmonella enterica is an enteric bacterial pathogen of worldwide importance. Over
2500 distinct serovars of S. enterica exist and these can be divided into three broad
types; ubiquitous serovars that can cause acute but self-limiting gastroenteritis in a broad
range of hosts (e.g. S. Typhimurium), host-restricted serovars that exhibit a preference
for a narrower range of hosts (e.g. S. Dublin and S. Choleraesuis) and host-specific
serovars that produce disease in only one host (e.g. S. Typhi in humans). Host-restricted
and -specific serovars often cause only mild enteritis but possess the ability to
disseminate from the intestines to the organs of the body, causing typhoid fever-like
illnesses in animals that may be severe and life-threatening. Non-typhoidal Salmonellosis
caused by ubiquitous serovars also poses serious welfare and economic problems for
livestock producers. Moreover, Salmonella infection of pigs and cattle can lead to entry of
the pathogen into the food chain and environment, providing an important reservoir of
human infection. The molecular mechanisms underlying the ability of Salmonella enterica
to colonise the intestines of food-producing animals, induce enteritis and in some cases
translocate to distal sites, are poorly understood. Defra-funded research conducted
under project OZ0319 aimed to understand these processes in greater detail and to use
the knowledge to develop, test and refine novel methods of disease control. Such
research led to the testing of subunit vaccines in pigs, live-attenuated vaccines in calves
and analysis of the protective efficacy of small molecule inhibitors that disarm a key
Salmonella virulence factor.
Review Summary
Innovative and novel methodologies were used to examine the pathogenesis of
salmonella in cattle and pigs, including factors that play conserved roles in colonisation of
calves, pigs and chickens, and host-specific colonisation factors. The researchers
identified cytokine responses of the intestinal mucosa following infection with S.
Typhimurium and the host-specific serovars, S. Dublin and S. Choleraesuis. The project
included an examination of the basis of attenuation of mutant strains in vitro and in
animals. The researchers further examined the efficacy of a subunit vaccine that
comprised S. Typhimurium Type III secreted proteins in pigs, and the ability of smallmolecule
inhibitors of Type III secretion to block Salmonella-induced Enteritis.
Although the study has increased background knowledge, for this project to be really
relevant to Defra, it also needed to attract the interest and funding of the wider
63

agricultural industry/public health community. Without this link, it might be more
appropriate for further funding to come from BBSRC than Defra.
An experienced team with a good track record for producing high-quality science worked
on this project. The work is sound and appropriate, and dissemination of findings was
impressive, with the production of several excellent scientific papers.
64

7. n
Project code: OZ0402
Other zoonotic pathogens of interest
Project title: What is the potential for human isolates of both
genotypes of C. parvum to infect, colonise and be
excreted by farm animals
Start date (dd/mm/yy): 01/04/2000
End date (dd/mm/yy): 31/03/2003
£406,684
Total cost:
Affiliation: Veterinary Laboratories Agency,
Sub-contractor(s):
Project code: OZ0407
Cryptosporidium Reference Group,
HPA
Project title: Evaluation and risk assessment of zoonotic
transmission of Cryptosporidium
Start date (dd/mm/yy): 01/04/2003
End date (dd/mm/yy): 31/01/2008
£783,631
Total cost:
Affiliation: VLA
Sub-contractor(s):
Abstract of research
CREH Analytical Limited,
PHLS Cryptosporidium Reference Unit, Swansea
Public Health Laboratory
Cryptosporidium infection in people can result in severe clinical disease, which in
immuno-compromised patients can be fatal. Most human infection is with either of two
predominant species, C. hominis (thought until recently to be human specific) and C.
parvum. Both cause sporadic cases and outbreaks of disease. However, the Health
Protection Agency (HPA) and UK Cryptosporidium Reference Unit (CRU) have identified
other species and genotypes previously associated with animals, including C.
meleagridis, C. felis and C. canis in patients. Species identification is currently only
possible by molecular analyses and is not undertaken during routine diagnosis in primary
65

testing laboratories. In addition, as more discriminatory sub-typing methods are
developed and applied, evidence for heterogeneity within species is increasing.
The studies undertaken in OZ0402 and OZ0407 aimed to investigate the relationship
between isolates of Cryptosporidium in human disease and in animal and environmental
contacts using molecular analyses to enhance characterisation and discrimination
between sub-types. Studies were designed to establish zoonotic potential using
infectivity experiments in calves, lambs, pigs, turkeys and chickens to assess whether
different genotypes can infect, colonise and be excreted by farm animals. The host range
of Cryptosporidium species, genotypes and subtypes was investigated using enhanced
surveillance of human cases in defined geographical areas and following up potential
sources of zoonotic infection for humans by sampling key pet, farm animal and/or
environmental contacts. Although transmission of Cryptosporidium to humans by direct
animal contact has been demonstrated and reported, the risk of disease through direct or
indirect contact with animals or animal faeces during occupational or recreational
activities is unknown. Quantitative risk assessment modeling was used to assess the
contribution of farm animals and pets to human infection using data derived from these
studies and to suggest intervention strategies for the prevention and control of zoonotic
disease.
Review Summary
This research has substantially improved our understanding of cryptosporidiosis in
animals and the linkage with disease in humans. It has allowed the development of
centre of expertise at the VLA and has also provided a basis for excellent collaboration
between VLA and HPA in this important area.
The projects were generally well devised and the methodologies employed in the
laboratory and the environment were appropriate. However, there were differing opinions
on the importance of the conclusions obtained. The number of novel and pertinent
findings were fewer than had been expected from such a large project, which was using
new molecular tools to identify genetic differences between C. parvum (which infects
calves) and C. hominis (which was thought to infect only humans).
One key finding of the studies was that isolates of the putative human-specific species C.
hominis could readily reproduce in neonatal animals of three different mammalian
livestock species: sheep, cattle and pigs. Furthermore, infections were observed in
chickens and poultry. Matches were obtained between C. parvum sub-types isolated
from humans and isolates from environmental samples from farms that the patient had
visited. Significantly, particular species sub-types may be commonly associated with
human infection. This shows that, in principle, molecular typing systems can be
employed in epidemiological tracing studies. However, no link was obtained between
infection of humans and infection in domestic pets including dogs.
The risk assessment part of the project was criticised on the grounds that the sample
size was too small, and because a hypothesis had not been properly identified. Overall,
the risk assessment work had been difficult to assess because too little detail was
provided.
Some disappointment was expressed at the rate at which peer-reviewed publications
were being produced. In particular, the discovery that C. hominis can grow in other
species, including chickens, is a major finding that warranted rapid publication.
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Project code: OZ0405
Project title: Genotypic and phenotypic comparison of Yersinia
enterocolitica from humans and animals
Start date (dd/mm/yy): 01/05/2002
End date (dd/mm/yy): 30/04/2005
£245,488
Total cost:
Affiliation: Veterinary Laboratories Agency
Sub-contractor(s): Department of Infectious and Tropical Diseases,
London School of Hygiene and Tropical Medicine
Abstract of research
Many cattle, sheep and pigs in the UK are colonised with Yersinia enterocolitica, which
can cause diarrhoea in humans but is also associated with more serious diseases. To
investigate the sources of human yersiniosis, a phenotypic and genotypic comparison of
human and livestock strains was undertaken in collaboration with the HPA. Results
indicated that 58% of the animal isolates and 53% of the human isolates were Biotype
(BT)1a, which is considered ―non-pathogenic‖. BTs1b and 2-5 are considered
―pathogenic‖. BT3 (O:5,27) was isolated from sheep (35%), pigs (22%) and cattle (4%)
but was not detected in human isolates. Among the human strains, 24% were BT3 (O:9)
and 19% BT4 (O:3). Only pigs (11%) carried BT3 (O:9) strains. To further investigate the
relationship between human and animal isolates a AFLP-based genotyping technique
was developed in collaboration with DFVF and applied to 88 representative strains. The
strains separated into two distinct AFLP clusters; cluster A and cluster B (largely BTs2-4
and BT1a respectively). Serotype-associated sub-clusters were observed. AFLP profiles
indicated pigs and sheep as sources of human BT3 (O:9) and BT4 (O:3) infections
respectively. Some strains causing human disease were undetected in livestock. Further
genotyping was undertaken using a pan-Yersinia DNA micro-array in collaboration with
LSHTM. Two distinct clades were observed reflecting the AFLP results and providing
evidence of evolutionary trends in the Yersinia genus.
The representative strains were also assayed for a range of virulence-associated
properties. The results indicated that most isolates had the capacity to cause disease in
humans on the basis of adherence, invasiveness and survival in macrophages but
differences were observed in cytokine secretion induced in vitro between the ―nonpathogenic‖
and ―pathogenic‖ strains.
Only 3 non-invasiveness strains, were detected. All were BT1a were non-motile. This
was further investigated using site directed mutagenesis, for the first time, in a BT1a
strain to construct a flagella-deficient mutant. The results suggest that flagella are
important virulence factors for BT1a strains.
Overall this project has significantly increased understanding of the role of Y.
enterocolitica from livestock in human yersiniosis and indicated that BT1a strains are
potentially pathogenic to humans.
Review Summary
The project was developed following the collection of Yersinia isolates during the sheep,
cattle and pig abattoir survey in 1999/2000. Yersinia enterocolitica is not a significant
67

animal pathogen but is a human pathogen. The survey in 1999/2000 had indicated quite
high prevalence of Yersinia enterocolitica in pigs (26%), less so in sheep (13%) and
cattle (6%).The project has clarified which animal species were excreting isolates which
are also pathogenic serotypes for humans. The research has developed a testing facility
for Yersinia enterocolitica and contributed to the evidence base.
The most important output has been the suggestion that more of the Yersinia
enterocolitica strains present in livestock have the potential to pose a threat to human
public health than was previously thought. The identification of the potential virulence of
BT1a strains is important as these strains colonise a significant proportion of livestock.
The study has generated a number of valuable publications, which advance knowledge
in the subject area.
Further research is not currently required in the absence of clear direction from
medical/public health authorities that this is a sufficiently important area that merits
additional work in animals. It would be more appropriate for FSA or DH to fund further
work on understanding the pathogenicity for humans.
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Project code: OZ0404
Project title: Bovine neosporosis: the evaluation of zoonotic risk
and the development of evidence-based control
strategies
Start date (dd/mm/yy): 01/11/2002
End date (dd/mm/yy): 31/12/2005
£242,267.00
Total cost:
Affiliation: University of Liverpool
Abstract of research
The newly recognised protozoan parasite Neospora caninum has recently emerged as a
major cause of disease in cattle and dogs worldwide (1). In cattle, research by various
international groups, including our own laboratory in a previous DEFRA funded research
project (Contract CSA 3618, 1996 - 1999), has shown the importance of the infection in
the etiology of abortion and the significance of vertical transmission in its epidemiology.
This project concentrated on the recently discovered oocyst stage of N. caninum. It
sought to improve rational control measures in cattle by determining if, and under what
circumstances, oocysts could infect pregnant cattle and cause disease, what risk factors
exist and how likely are oocysts to infect the environment. The existence of
environmental contamination by oocysts also creates a potential zoonotic hazard which
was evaluated in a large scale human serological study.
The project failed to find any evidence of exposure to infection in over 3700 human
serum samples rigorously selected and examined by two assays. The results show that
the risk of human infection in the UK is extremely low and that millk and meat from UK
cattle can be regarded as safe in this respect.
In cattle, the project showed that oocyst infection in pregnancy can lead to abortion in
cattle if exposure occurs around mid-pregnancy but not if it occurs early or late in
pregnancy. However, exposure at 210 days in pregnancy led to high rates of congenital
infection in healthy calves. Thus oocyst infection is likely to be extremely important in
maintaining the basic reproductive rate of the parasite at >1 by creating infected females
which are then presumed to endogenously transplacentally infect all their offspring.
Review Summary
Overall the project has generated some important findings, specifically the lack of
evidence for human infection and zoonotic transmission, and the lack of associated
evidence that dogs were a major source of oocyst infection to cattle. Also important was
that although oocysts can infect pregnant cows and cause abortion or congenital
infection, epidemiological risk factors suggested that the likelihood of this occurring was
low.
The results of this research have clearly answered the policy question on zoonotic risk
and have shown that, if a zoonotic risk to humans exists in the UK, it is very low. The
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esearch has also substantially improved our understanding of the epidemiology of
Neospora caninum.
There is no requirement for further funding from this programme because the results
have shown that the organism has no real zoonotic significance.
The team have built up significant expertise on this parasite and it is for another
programme to consider whether further funding on purely animal health/animal welfare
grounds might be justified.
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Campylobacter Research Projects
Project code: OZ0604
Project title: Characterisation of strain variation in Campylobacter
jejuni.
Start date (dd/mm/yy): 01/04/1999
End date (dd/mm/yy): 30/09/2004
£774,549
Total cost:
Affiliation: University of Oxford
Sub-contractor(s): HPA
Project code: OZ0611
Project title: Development of a comprehensive MLST database for
assessment of Campylobacter risk factors
Start date (dd/mm/yy): 01/07/2005
End date (dd/mm/yy): 30/06/2008
£503,312.00
Total cost:
Affiliation: University of Oxford
Sub-contractor(s):
Abstract of research
OZ0604
Campylobacter species, particularly Campylobacter jejuni, are a major cause of human
gastro-enteritis in the UK and elsewhere. In the majority of infections, which are
sporadic, the precise epidemiology is not determined, although several food producing
animals have been implicated as important sources of infection. The epidemiology of
zoonotic transmission of C. jejuni to man remains obscure due to inconsistencies within
and between the various phenotyping and molecular typing methods used to
characterise C. jejuni isolates. These inconsistencies arise from the inability of the
current methods to unambiguously define strain types and genetic lineages, and the lack
of precise understanding of the variation which they index. This work will address these
problems, definitively establishing the range and mechanisms of genetic variability in
populations of C. jejuni, by the application of the most recently developed sequence
typing technologies to a minimum of 1500 isolates, representative of those obtained from
human infections, farm animals, the human food chain, and the environment. Multilocus
sequence typing (MLST), which identifies strain relationships on the basis of neutral
variation in housekeeping genes, and antigen gene sequence typing, which will be used
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to elucidate the antigenic repertoire of this bacterium at one antigen locus, will provide an
accurate, comprehensive, and portable typing system for C. jejuni and related organisms,
with a firm theoretical basis in bacterial population biology.
OZ0611
Campylobacter is the most common cause of bacterial infectious intestinal disease in the
UK. Although various risk factors for infection, including consumption of undercooked
poultry or unpasteurised milk have been identified, the epidemiology of this disease
remains poorly understood. This is because (i) outbreaks are rarely detected, hence
valuable information on sources and transmission routes which is obtained from outbreak
investigations is generally lacking, and (ii) Campylobacter genomes are genetically
diverse, and unstable, with frequent inter and intragenomic recombination, together with
phase variation, which complicates the interpretation of data from many typing methods.
However, a suitable typing techniqe is multilocus sequence typing (MLST). MLST for
Campylobacter jejuni, together with a comprehensive internet accessible MLST database
(http://pubmlst.org/campylobacter/) was developed with DEFRA support (contract
number: OZ0604). MLST offers advantages over previous typing techniques including
100% typeability, reproducibility, and the straightforward sharing of data via the internet.
The Campylobacter MLST database now contains 4434 isolates (29 th May 2007) and
provides a resource for the scientific, public health, and veterinary communities as well
as the food industry. Database submissions are received from laboratories
internationally. The database is curated to ensure quality control, and additional MLST
schemes and databases have been set up for other Campylobacter species including C.
coli, C. fetus and C. lari. If it is to continue, and undergo further expansion, this program
of work will require continued support.
Review Summary
The work performed in OZ0604 and OZ0611 has been of fundamental importance in
developing and applying a robust molecular typing method to Campylobacter. Adoption
of MLST for Campylobacter at an international level demonstrates the strength of the
work performed.
As OZ0611 comes to an end and further work is considered, it is important that the MLST
database is populated with campylobacters of public health importance. In addition, when
selecting environmental isolates, quantity, source, geographical, and temporal factors
must all be taken into account to ensure the database is not biased. The database must
then be interrogated to identify sources of human infection and therefore where
interventions will have the greatest impact.
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Project code: OZ0605
Project title: Genotypic and Phenotypic Instability of
Campylobacters from Environmental, Animal and
Human Sources
Start date (dd/mm/yy): 01/08/1999
End date (dd/mm/yy): 31/07/2002
£298,279
Total cost:
Affiliation: Veterinary Laboratories Agency
Sub-contractor(s): Scottish Reference Laboratory for Campylobacters,
Aberdeen
Department of Food Science, Queens University,
Belfast
Campylobacter Reference Unit, Laboratory of Enteric
Pathogens, Central Public Health Laboratory (now
Health Protection Agency), London
Institute for Animal Science and Health, The
Netherlands
Abstract of research
Campylobacters can only grow in a host, usually in the gut, and are considered very
fragile organisms. However, they are ubiquitous in the general environment, where they
survive a wide range of stresses, which may be potentially lethal. This conundrum is
confounding for the investigation of the epidemiology of campylobacters in the poultry
farm and meat production environments, particularly given the absence of known
mechanisms of stress adaptation in campylobacters. Genetic instability is a wellrecognised
property of Campylobacters and has been proposed as a survival
mechanism. In this project the role of genetic instability as a mechanism for stress
survival and adaptation during exposure to the host gut was investigated.
Genotypic and phenotypic instability occurred in about 20% of outbreak-related strains
investigated and was most readily observed using the typing technique of PFGE. Such
instability was detected as a change in banding patterns consistent with point mutations.
However, the apparent loss or gain of DNA fragments, including fragments of up to 50kb,
was also detected, suggesting DNA insertion/deletion events. Nevertheless, some
genotypically stable clones were also detectable. The mechanisms and role of genetic
instability were investigated in a clonally-derived set of strains previously recovered from
a single batch of poultry meat. Instability was induced by in vitro exposure to heat and
cold stresses as well as frozen storage. Using a competition model of chick colonisation
following oral challenge with multiple variants, evidence was obtained for a selective
benefit in vivo for some variants. These results demonstrate that genetic instability is an
important ecological property of campylobacters and that understanding of this property
will be essential to the detection and control of this foodborne pathogen.
Review Summary
OZ0605 commenced at a time when little was known about the genetic variability of
Campylobacter spp. The researchers looked at a wide range of typing methodologies
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and have contributed to our understanding of the difficulties in typing campylobacters.
This was important work which needed to be done. The project has confirmed that there
is no single ideal typing method that can be applied to every situation; a combination of
methods is often needed depending on the questions to be answered.
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Project code: OZ0607
Project title: An investigation of the distinguishing features of C.
jejuni strains that have host/disease associations.
Start date (dd/mm/yy): 01/07/01
End date (dd/mm/yy): 30/06/04
£508,987
Total cost:
Affiliation: Veterinary Laboratories Agency (Weybridge)
Sub-contractor(s):
Abstract of research
The attribution of poultry as the major source of human campylobacteriosis remains
debatable. In order to inform risk assessment models, this project aimed to investigate
the role of other livestock as potential sources and the possibility that all strains of
campylobacter were pathogenic for humans. A multi-pronged approach was therefore
adopted to investigate the relationships between C.jejuni pathogenicity and host. Initial
studies using higher disciminatory MLST-based approaches demonstrated that the
populations of C. jejuni from human cases and from colonised animals overlap
significantly, indicating all veterinary campylobacters are potentially pathogenic to man.
However, at least one host-associated cluster was evident in pigs. Additionally, genomic
stability and instability were important factors in the ecology of this bacterial species.
A second approach investigated the molecular pathogenesis of disease, in order to
identify virulent and non-virulent organisms. In vitro assays indicated that there were no
obvious associations between genotype and putative campylobacter virulence properties,
therefore, attempts were made to identify specific genetic markers of virulence. A
transposon mutant library was produced and invasion-deficient mutants selected, which
may provide future markers of invasiveness. Rapid molecular assays for screening CDT
activity were developed and for the first time the immunogenicity of CDT was
investigated using a novel neutralisation assay. Human anti-CDT antibodies were
detectable in infected humans but no antibodies were detected in colonised chickens
indicating a host-related specificity in response. Additionally significant progress was
made on the identification of a fimbrial surface structure.
Finally, a sentinel study was undertaken which demonstrated passage-associated
changes in the genome sequence strain, NCTC11168, resulting in differences in chick
colonisation, motility, morphology and invasiveness. Transcript analysis demonstrated
changes in expression of a number of genes involved with the adaptation to microaerobic
growth. This work is of huge significance to the research community by clarifying the
importance of strain origin in virulence and physiological studies.
Review summary
At the start of project OZ0607 it was beginning to be recognised that poultry is not the
only source for human campylobacter infection, and not all strains of campylobacter are
pathogenic to humans. In order to more accurately attribute disease source and identify
potential control measures, indicators of the relationships between strains, pathogenicity
and host were required.
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Fundamental issues concerning the function of various parts of the Campylobacter
genome were addressed in this project. The researchers explored many avenues, but
due to the complexity of the genome it was difficult to draw definitive conclusions. The
project demonstrated that the relationships between strains, pathogenicity, and host are
complex, and it was not possible to identify unique markers. This type of research is
essential, however, to improve our understanding gene function, which will assist in more
focused typing systems in the future.
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Project code: OZ0606
Project title: Protective immunity and competitive exclusion in
development of effective intervention products for
poultry.
Start date (dd/mm/yy): 01/04/2001
End date (dd/mm/yy): 31/03/2004
£232,145.00
Total cost:
Affiliation: Veterinary Laboratories Agency
Sub-contractor(s):
Project code: OZ0614
Project title: To develop vaccination approaches to the control of
Campylobacter in poultry.
Start date (dd/mm/yy): 01/04/2006
End date (dd/mm/yy): 31/03/2009
£267,629.00
Total cost:
Affiliation: Veterinary Laboratories Agency
Sub-contractor(s):
Abstract of research
Campylobacter jejuni is the leading cause of bacterial enteritis in the UK. The handling
and/or consumption of contaminated poultry meat is believed to be a risk factor. The
avian intestine is considered the natural environment for the organism, and up to 90% of
poultry flocks in the UK are colonised asymptomatically. Thus, reduction or elimination of
the organism in poultry in order to reduce the number of human cases is an objective of
Defra and FSA. A number of intervention strategies have been proposed, including
increased bio-security, vaccination and competitive exclusion (CE). Increased biosecurity
alone has so far failed to consistently reduce the extent of colonisation, so a
multi-pronged approach would seem to offer the most promise. Understanding host and
bacterial factors associated with colonisation should enable the development of defined,
targeted interventions. Early Defra-funded work at VLA (project OZ0129) demonstrated
that, despite being considered a commensal in chickens, C. jejuni elicits strong humoral
responses and that exposure can lead to a protective immunity. This work was furthered
in project OZ0606 by investigating the feasibility of vaccination. Work was also
undertaken to identify bacterial factors essential for colonisation. In addition, following on
from an earlier project (OZ0603) the potential for CE by homologous and heterologous
organisms was investigated. Results from this project indicated that vaccination was an
area worthy of further research, leading to the funding of OZ0614 which focuses entirely
on this area of intervention.
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Review summary
Project OZ0606 focused on the dynamics of intestinal colonisation of chickens with
campylobacter, including the effect of competitive exclusion and immune responses
during colonisation. This project has demonstrated a clear link between maternal
immunity and the colonisation lag phase in chicks, and also identified factors that could
be used in control of campylobacter. OZ0606 has also increased knowledge of the use of
competitive exclusion products in the laboratory environment. The results from this
project showed promise but unfortunately the researchers have had difficulty replicating
their work in the subsequent project (OZ0614). This is a difficult research area, however
the widespread nature of campylobacter, and the lack of success in controlling this
organism through biosecurity, highlights the necessity of progressing the vaccination
approach.
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Project code: OZ0608
Project title: Epidemiological studies and development of practical
control measures for Campylobacter in broiler flocks
Start date (dd/mm/yy): 01/07/02
End date (dd/mm/yy): 30/06/06
£859,393
Total cost:
Affiliation: Veterinary Laboratories Agency
Sub-contractor(s): Co-contractor - University of Bristol
Sub-contractor - Imperial College
Project code: OZ0610
Project title: Survival and persistence of campylobacters in poultry
farm environments and suggested control measures
Start date (dd/mm/yy): 01/09/05
End date (dd/mm/yy): 31/01/09
£746,102.00
Total cost:
Affiliation: University of Bristol
Sub-contractor(s): Co-contractor - VLA
Sub-contractors - SAC, DTA Ltd
Project code: OZ0613
Project title: Towards risk-based control of Campylobacter:
developing the evidence base using epidemiological
and bacteriological approaches
Start date (dd/mm/yy): 01/01/06
End date (dd/mm/yy): 30/09/10
Total cost:
£1,558,693 (Defra: £1,495,693; Other funders:
£200,000)
Affiliation: Veterinary Laboratories Agency
Sub-contractor(s):
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Abstract of research
OZ0608
The adoption of on-farm control measures by the poultry industry is needed to reduce the
number of Campylobacter-infected birds that enter processing plants, and therefore the
number of human cases of campylobacteriosis associated with poultry meat
consumption. Enhanced biosecurity on poultry farms would be expected to control other
zoonoses, for example Salmonella, and improve the health, productivity and welfare of
poultry flocks. However, the epidemiology of Campylobacter in poultry is complex and
the development of suitable control strategies is problematic. This project was designed
to meet this challenge by bringing together scientists from a range of disciplines including
bacteriology, epidemiology, statistics and socio-economics. A close collaboration with
industry will ensure timely communication of the research outputs and that a sustainable,
practical and cost-effective control package which is more likely to be adopted is
developed. The diversity of poultry production systems (e.g., conventional, organic and
free-range) will be addressed by the project.
In project OZ0608 a novel intensive, retrospective environmental testing strategy was
adopted, based on a design developed by DG Newell. A real-time PCR-based test was
developed and validated to allow rapid identification of flock colonising C. jejuni/coli
strains from DNA prepared from recovered colonies and aliquots of enriched cultures
from flock and environmental samples. This test was implemented during a preliminary
field study. Using a standardised sampling strategy, samples taken from the poultry
house and surrounding farm environment at each visit during the crop cycle were
enriched and plated, but in this new approach isolates and enriched cultures were then
stored at –80 o C until isolates were recovered from the faeces or caeca of the target flock.
A strain-specific probe was designed against the flock strain and used to screen DNA
prepared from the strain(s) first isolated from the chickens and compared with traditional
cultural isolation and characterisation of isolated colonies to determine potential sources
of flock colonising campylobacters. This approach was then used to determine potential
sources of flock colonisation in a large scale study involving 26 target flocks on 15 farms.
A practical control package was developed progressively throughout the project. A
qualitative farmer survey to determine the attitudes to the control of Campylobacter in
broiler flocks was conducted followed by a quantitative study of farmers‘ attitudes. Both
surveys were carried out to gain information on (i) current farm practices (ii) biosecurity
control measures currently in place and (iii) those that could be adopted as well as
constraints to adoption. The cost-effectiveness of a set of control measures were
evaluated using standard techniques of cost-effectiveness analysis. Ten interventions,
including hygiene barriers in anterooms, visitor restrictions, and the use of handwash and
alcohol sprays were put into place on two farms to help provide information on adoption
of control measures, effects on Campylobacter prevalence and time to establishment of
colonisation. Finally, a cross-sectional and longitudinal study was performed to
determine differences in time to colonisation between conventional flocks and organic
and free-range flocks. The information generated by these activities, together with the
Quantitative Risk Assessment and field studies were all used to inform the development
of a workable and cost-effective control package.
OZ0610
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Although enhanced biosecurity measures on farms appear to reduce colonisation of
flocks, the proportion of Campylobacter positive flocks entering the processing plant
remains high. Therefore it is necessary to identify strategies for on-farm interventions in
order to reduce the total campylobacter load on farms and their ability to persist in the
bird until slaughter. To achieve this the reservoirs of campylobacters surviving or
recycling within or re-entering the farm are being investigated. The impact of both abiotic
and biotic environmental factors e.g bacteriophage, on campylobacter persistence in the
environment and their influence on colonisation and seasonality of campylobacter
carriage in broiler flocks is being studied to improve the understanding of the survival
mechanisms. In addition the ability to detect, identify and classify campylobacter strains
in poultry farm environments is being advanced. Scientists from a range of disciplines
including microbiology, with expertise in aerobiology, bacteriophage and biofilms in the
poultry environment, and poultry epidemiologists, specialising in veterinary, molecular
characterisation, modelling, and risk analysis are involved in this project. This team is
being aided by advice and collaboration with consultants of international standing from
Denmark, Sweden, Netherlands, USA and the UK. In-vivo chick modelling is being used
to investigate the susceptibility of broilers to colonisation by campylobacters in
environmental samples identified as potential reservoirs of infection. The studies are
benefiting from the probe approach developed for project OZ0608 to identify sources
associated with caecal campylobacter strains colonising the broiler flocks. Links
identified by SVR sequence are being explored further using MLST. On-going research
projects at both participating institutes are informing this project while close collaboration
with the poultry industry is ensuring rapid communication of research outputs and that
strategies recommended will be adopted.
OZ0613
The work undertaken by project OZ0613 is a comprehensive package of multidisciplinary
research aimed at supplying data on which to base practical policy decisions
on the control of Campylobacter in broiler production.
The laboratory and in-field validation work carried out in Objective 1 will provide
sensitivity and specificity data on the Campylobacter Survey methodology (Objective 2).
By determining these data suitable correction factors can be applied to the apparent
survey prevalence, via a modelling approach, to obtain an estimate of the true
prevalence.
The survey carried out in Objective 2 is the first survey on Campylobacter in broiler flocks
at slaughter to be carried out in the UK on a national-scale. The survey has been based
on the EU specifications and as such will meet the main requirements of the EU survey
when it becomes mandatory (which is likely to be January 2008). By commencing the
survey in January 2007 the VLA has been able to trial the EU methodology and identify a
number of issues which have been fed back to Defra. In addition to determining the
prevalence of Campylobacter in broiler flocks at slaughter, the research undertaken by
the VLA will provide a valuable data set for investigating risk factors associated with flock
prevalence. The survey also provides a valuable, epidemiologically robust, source of
samples which can be used for molecular epidemiological studies, particularly
comparison with human strains by MLST or other suitable methods, and for antimicrobial
resistance testing.
Objective 3 focuses on more detailed epidemiological investigations of the depopulation
process. This work is linked with an existing University of Bristol FSA project and will
provide substantial added value by virtue of provision of strains from Bristol studies of the
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thinning process for molecular tracking to elucidate the most important sources
associated with flock colonisation. In addition, the whole chain of events from farm
through to the slaughter process will be followed in a separate study to investigate the
sources of infection and contribution to Campylobacter load associated with clearance
and transport. A number of exposure experiments will also be carried out in Objective 3
to investigate the survival and colonisation potential of environmental strains.
All of the above work will provide good quality data with which to update the VLA risk
assessment model (Objective 4).
Review Summary
OZ0608 was an ambitious project which has moved our understanding of the
environmental sources of Campylobacter on the poultry farm forward considerably. The
team also developed a PCR screening test for strain-specific detection of
Campylobacter, enabling detection of the organism where traditional methods failed.
While there were data deficiencies in the Quantitative Risk Assessment, it proved useful
in the development of practical interventions, and data gathered from OZ0610 and
OZ0613 will be used to update the model.
Following on from OZ0608, project OZ0610 aims to investigate the role of environmental
survival, reservoirs of infection, and recycling systems, both on or entering the farm, in
the colonisation of broiler flocks with C. jejuni. A vast amount of data has been collected
by the team, which has yet to be fully analysed, in order to identify badly needed
intervention measures for the poultry industry.
Building on both OZ0608 and OZ0610, project OZ0613 will continue to advance
knowledge of the farm situation, environmental stresses, and seasonality that may
adversely affect campylobacters and be used in control. The project includes the first UK
survey of campylobacters in broilers at slaughter meeting recommendations of the
Advisory Committee on the Microbiological Safety of Food, and the EU requirement for a
survey from January 2008. It is anticipated that this project will significantly contribute to
the campylobacter evidence base and provide valuable data on which future costeffective
controls can be based.
All three projects in this cluster have benefited from a close working relationship with the
FSA and industry. Such collaboration is also invaluable in ensuring findings and
recommendations from research projects are implemented on farms.
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Project code: OZ0609
Project title: Determining the role of maternal antibodies in the lag
phase of Campylobacter jejuni infection in chickens.
Start date (dd/mm/yy): 01/05/2005
End date (dd/mm/yy): 31/05/2006
£50,012
Total cost:
Affiliation: Veterinary Laboratories Agency
Sub-contractor(s):
Abstract of research
Campylobacter jejuni is the leading cause of bacterial enteritis in the UK. The handling
and/or consumption of contaminated poultry meat is believed to be a risk factor. The
avian intestine is considered the natural environment for the organism, and up to 90% of
poultry flocks in the UK are colonised asymptomatically. Thus, reduction or elimination of
the organism in poultry in order to reduce the number of human cases is an objective of
DEFRA and FSA. A number of intervention strategies have been proposed, including
increased bio-security, vaccination and competitive exclusion. Increased bio-security
alone has so far failed to consistently reduce the extent of colonisation, so a multipronged
approach would seem to offer the most promise. Commercial birds tend to
exhibit a lag-phase of infection, wherein they usually remain uncolonised during the first
2-3 weeks of life. Thereafter the organism spreads rapidly through the flock. The lagphase
represents a window-of-opportunity to apply intervention strategies. Moreover,
understanding the under-lying mechanisms could help in the development of successful
strategies. One likely contributory factor to the lag-phase, maternally-derived immunity,
was investigated. Previous DEFRA-funded work indicated that the susceptibility of
commercial birds to colonisation is inversely proportional to the levels of anti-C. jejuni
antibodies present in the chicks. These antibodies are vertically transmitted from infected
breeder hens. Their levels decline to background during the first 3-4 weeks of life. The
role of maternal immunity was studied by looking at the susceptibilities to colonisation of
chicks derived from SPF and commercial flocks and from experimental hens colonised
and uncolonised by C. jejuni.
Review summary
The role of the humoral antibody response, including passive transfer of maternal
antibodies, remains unclear, making development of a vaccine to prevent poultry
colonisation with Campylobacter more difficult. This project has contributed to filling this
knowledge gap, confirming that maternal antibodies play a role in the colonisation lag
phase, but that other unknown factors are also involved. It has also shown that any
exposure of very young chicks to campylobacter will result in colonisation. The results of
this work have assisted in formulation of policy, suggesting that attempting to keep
breeding stock free of campylobacter may not be a useful strategy in the effort to reduce
human campylobacteriosis.
83

Project code: LK0665
Project title: Improving gut health and nutrient capture of broiler
chickens through selection for innate immune function
Start date (dd/mm/yy): 01/01/2006
End date (dd/mm/yy): 31/12/2008
Total cost:
£351, 814 ( Defra: £174,813, Other funders:
£177,001)
Affiliation: Institute for Animal Health, Compton
Sub-contractor(s):
Abstract of research
The UK poultry industry faces numerous challenges in order to remain sustainable.
These include the imminent move to more extensive rearing systems; the withdrawal of
prophylactic and many therapeutic antibiotics, and other drugs such as anti-coccidials;
resistance and residue problems with anti-helminthics. These challenges will all have an
impact on poultry health, but also have the potential to impact on human health, as
increased incidence of food-borne zoonotic pathogens in chickens, for example, has the
potential to lead to an increase in these diseases in man, It is important that poultry
breeders are able to select for genetic improvement in performance when birds are
reared in such environments, which should also lead to improvements in nutrient capture.
This programme will seek to
- provide a bridge between conventional positional (QTL) and physiological candidate
gene approaches to identify genes controlling variation in innate immune responses
- define the extent of genetic variability in loci that influence the innate immune response
of the chicken and relate this to resistance/susceptibility to Salmonella and
Campylobacter, and also the impact of these variations on nutrient capture
- determine the relevance in commercial broiler populations of genetic variation in
immune response found in inbred research lines
- provide new opportunities for selective breeding of commercial broilers for improved
innate resistance to enteric disease and hence improved nutrient capture and food safety
Review summary
The main aim of this project is to increase innate immunity in broilers, and thus increase
resistance to low-level pathogens in the environment. This supports the sustainable
approach to disease control. If commercial lines of chickens which are resistant to
salmonella and/or campylobacter infection become available it could have a major impact
on this reservoir of infection.
It is too early to say whether this approach will be a success. At the time of review this
project was only 50% completed and had suffered some delays, however, access to new
equipment should enable the time to be recovered.
84

Project code: OZ0612
Project title: The epidemiology of campylobacter infection in dogs
in the context of the risk to humans
Start date (dd/mm/yy): 18/07/2005
End date (dd/mm/yy): 16/07/2008
£228,351
Total cost:
Affiliation: University of Liverpool
Sub-contractor(s):
Abstract of research
Campylobacter is the most common bacterial cause of human gastroenteritis in England
and Wales. Most cases are due to Campylobacter jejuni infection, although C. coli and
other species including C. upsaliensis may also be involved. Most cases of
campylobacteriosis in man are non-epidemic, and there tends to be a higher prevalence
in children. Poultry meat has always been considered to be a major source of human
campylobacteriosis, but campylobacter are also found in a number of other species,
including dogs. The prevalence of infection in dogs appears to be high (up to 56%) and
there is evidence that dogs may be a risk factor for human infection. Humans have
frequent contact with dogs and dog faeces, and clearly where faecal pathogens such as
campylobacter are present there may be considerable opportunities for zoonotic
transmission.
This proposal addresses the risk of human campylobacter infection from dogs. Although
there is epidemiological evidence of an association between campylobacter in dogs and
disease in humans, there is little information on specific risk factors for this. Specifically,
we will
• determine the prevalence of campylobacter carriage in dogs in a community-based
census study, in selected kennel populations and in the wider population. • identify risk
factors associated with campylobacter carriage in dogs, including human, household,
and management variables.
• determine the population structure of the campylobacter isolates from dogs in relation
to human strains. C.jejuni isolates will be examined in collaboration with Dr K Dingle,
University of Oxford.
The study will also provide a resource for small animal infectious disease surveillance.
More specifically we will
• create a microbiological and epidemiological data archive which could be used, with
further funding, as a surveillance resource for other zoonotic, or potentially zoonotic
canine faecal pathogens. In this context it has been agreed with Defra that as part of this
project the samples will be screened for Salmonella spp.
• provide added value to the contact network studies between dogs, and dogs and
humans already funded by VLA/Defra (PU/T/PSC/04(22)).
• produce a template for future surveillance studies on small animal pathogens, both in
kennelled populations and in the community.
• provide a small animal project input into the proposed National Centre for Zoonosis
Research, to be based at the University of Liverpool.
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Review summary
While poultry meat is generally accepted as the primary source of infection for cases of
human campylobacteriosis, there is also evidence to suggest that a significant proportion
of these infections originate from alternative sources. The primary aim of this project was
to determine carriage of Campylobacter spp. by the UK dog population, and any risk this
may cause to human health.
Results to-date indicate that dogs are not an important source of C. jejuni or C. coli, the
common cause of infection in humans. C. upsaliensis was the most common
campylobacter species detected in dogs. While this organism is zoonotic, it is recovered
much less frequently from human campylobacter infections.
86

Project code:
VTEC O157 Research Projects
OZ0703
Project title: Plant antibody delivery of passive immunisation
against E. coli O157:H7: a novel means of control in
the animal
Start date (dd/mm/yy): 01/09/1999
End date (dd/mm/yy): 01/06/2003
£354,108
Total cost:
Affiliation: Veterinary Laboratories Agency
Sub-contractor(s): ADAS Consulting
Abstract of research
Biology Department, University of Leicester
Intimin, TIR and EspA proteins are expressed by Enterohaemorrhagic E. coli O157:H7.
EspA proteins are part of the type three secretion system needle complex that delivers
TIR to the host epithelial cell whilst surface arrayed intimin docks the bacterium to the
translocated TIR. This process leads to intimate attachment resulting in attaching and
effacing lesions which are essential for colonisation and persistence in ruminants.
Recombinant forms of these effector proteins from E. coli O157:H7 were produced and
used to elicit immune responses in rabbits and immune phage-display antibody libraries
were produced. Screening of these immune libraries by phage-antibody panning and
colony filter screening produced a panel of antibodies with specificity for EspA or intimin.
Antibodies recognising different C-terminal epitopes on intimin bound specifically to the
gamma intimin of O157:H7 and not to other classes of intimin whereas antibodies raised
against EspA recognised EspA analogues from other serotypes also. Anti-intimin
antibodies were also produced as fusion proteins coupled to the reporter molecule
alkaline phosphatase, allowing the one-step detection of γ-intimin. The isolated
recombinant monoclonal antibodies were functional in a range of assay formats including
ELISA, Western blot and dot blots demonstrating diagnostic potential.
We assessed whether these antibody constructs could be used to disrupt the intimate
association of E. coli O157:H7 with the host cell but neither anti-γ intimin or anti-EspA
antibodies did so although the anti-EspA antibodies significantly reduced the extent of E.
coli O157:H7-induced host cell actin rearrangement. In tests with traditionally made
antibodies, both monoclonal and polyclonal antibodies completely blocked adherence
and cytoskeletal changes within the host cell. Both polyclonal and monoclonal antibodies
could be used to label E. coli O157 EspA filaments and these immunoreagents did not
inhibit the formation of such filaments. This is the first report of monoclonal antibodies to
EspA capable of disrupting the TTSS function of E. coli O157:H7.
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Review summary
A key aim of this project was to express recombinant antibodies in edible crop plants, to
facilitate the delivery of passive immunisation against Escherichia coli O157:H7 to
mucosal surfaces of livestock. Recombinant antibodies were produced, however these
were less protective against E. coli O157 adherence factors than traditionally produced
antibodies. As a consequence, the assays with transgenic plants were not performed.
This was an ambitious project which was more in the area of pure research rather than
applied research, however, it fitted well with the Defra request at the time for novel
control methodologies. The work was useful preliminary research to determine whether
immunisation might be an option.
88

Project code: OZ 0704
Project title: Quantification of E. coli O157:H7 virulence factors in
vivo using real-time RT-PCR
Start date (dd/mm/yy): 01/06/1999
End date (dd/mm/yy): 30/09/2002
£200,764
Total cost:
Affiliation: Dept. Vet. Pathology, University of Glasgow
Sub-contractor(s): None
Abstract of research
The asymtomatic carrier bovine is a major reservoir of the food borne zoonotic E.coli
O157:H7. Previous studies have shown that an individual animal may shed organisms
intermittently throughout life and that infection/colonisation can be cleared but that the
same animal can also become reinfected. The main site of carriage in the adult bovine is
the colon although organisms have been described colonising other regions of the
alimentary tract in the adult carrier animal. E.coli O157:H7 has been reported to cause
disease in young calves and we have recently identified O157 organisms attached to
intestinal epithelium in an adult cow with profuse bloody diarrhoea.
Various virulence determinants - VT1, VT2, intimin, fimbrae - have been identified by
culturing E.coli O157 in vitro and in studies using mutated organisms. However, whilst
the role of some of these determinants in pathogenicity has been established, the precise
molecular events associated with colonisation, persistence of infection/carriage and
clearance of carriage have not been determined. In this project we will apply the new
and exciting technology of real-time, convential PCR and RT-PCR to quantitate the
number of organisms and the expression of mRNAs for various virulence factor produced
by E.coli O157:H7 organisms in vivo. This will be combined with in situ RT-PCR
techniques to obtain a detailed understanding of microbial gene expression during
colonisation/ attachment/carriage in the bovine alimentary tract.
Thus we will use modern molecular techniques to obtain a clearer understanding of the
distribution of infection and pathogenesis (expression of virulence determinants in vivo)
of E.coli O157:H7 in cattle. These data would underpin future studies investigating in
detail host responses in the carrier animal, relating these to microbial virulence factors
and host-pathogen interactions. The overall aim of this approach is to devise strategies
leading to reduction/elimination of the E.coli O157:H7 carrier state in cattle.
These studies therefore address MAFF policy relating to MAFF Strategic Research
Requirements to obtain a clearer understanding of the distrubution of infection,
pathology, colonisation, host specificity and virulence of the E.coli O157:H7 bacterium in
animals. Such knowledge is essential to the development of preventative and control
measures for O157 infection of animals, thereby reducing food contamination and the
incidence of O157 food poisoning in man.
Development of these techniques for E.coli O157:H7 could also form the basis of
investigating host-pathogen interactions of other food borne zoonoses at the molecular
level.
89

Review summary
This project aimed to develop real time PCR techniques to identify and quantify virulence
factors produced by E. coli O157 in the animal, and to study interactions between the
organism and host cells at the molecular level.
The project suffered from a number of delays and difficulties, not least the outbreak of
FMD in 2001. Only a minor part of this project was realised in the development of real
time PCR, however this was not tested in animals and the method has not been
published. While reviewers acknowledged some genuine setbacks for the research team
they felt alternative approaches could have been used. OZ0704 did not therefore rate
well with the reviewers.
90

Project code: OZ0705
Project title: A Proteomic Approach to Identify Virulence
Determinants of EHEC 0157.
Start date (dd/mm/yy): 01/08/1999
End date (dd/mm/yy): 12/09/2002
£237,995
Total cost:
Affiliation: University of Southampton
Sub-contractor(s): VLA
Abstract of research
Escherichia coli O157:H7 (EHECO157) is a major food-borne pathogen in the UK,
potentially fatal to the young, elderly and immunocompromised, and the leading cause of
acute kidney failure in children. The cited sources are bovine-derived meat and milk,
direct contact with infected bovines and human-to-human contact while other sources,
especially ovine ones are becoming a cause for concern. Epidemiological studies
indicate a significant prevalence of E. coli O157:H7 in healthy cattle, with evidence for
persistence resulting in long-term sporadic excretion and seasonal infection. The
mechanisms by which cattle are colonised and the factors affecting persistence are not
understood. Oral infection of neonate calves results in so-called attaching and effacing
gut lesions but this is strain-dependant. Moreover, older animals do not display such
lesions implying that other genetic determinants are likely to be responsible for
persistence. Given that the genome of EHEC O157 is some 20 % larger than that of the
laboratory strain K-12, there is considerable genetic capacity for novel pathogenic
determinants and some of these loci are likely to contribute to persistence. The key aim
of this proposal was to utilise a sensitive and rapid proteomic approach to identify novel
proteins and their encoding genes that are induced in the bovine gut. This information
will enable the development of preventative and control measures against this pathogen.
The key aim of this proposal was to utilise a sensitive and rapid proteomic approach to
identify novel proteins and their encoding genes that are induced in the bovine gut. This
information will enable the development of preventative and control measures against
this pathogen.
Review summary
This proposal used the proteomic approach to identify novel proteins and their encoding
genes that are induced in the bovine gut, with the aim of developing preventative and
control measures against EHEC O157.
The project developed and applied a number of methods for the investigation of the
virulence and physiology of EHEC in the gut. While the results were not applicable to
Defra policy, the research progressed the use of proteomics in this area.
91

Project code: OZ0709
Project title: Epidemiology of VTEC 0157 and other VTECs likely
to be pathogenic to man in farm wastes
Start date (dd/mm/yy): 01/04/2002
End date (dd/mm/yy): 31/03/2005
£365,493
Total cost:
Affiliation: Veterinary Laboratories Agency
Sub-contractor(s): Health Protection Agency, Silsoe Research Institute,
University of Liverpool
Abstract of research
Possible routes of human infection of Verocytotoxin-producing Escherichia coli (VTEC)
serogroup O157 include contaminated food or beverages, direct and indirect contact with
farm animals and person-to-person. In particular, cattle have been identified as a major
domestic animal reservoir of VTEC O157 and therefore their manure can be a source of
pathogens entering the environment and food chain. Consequently, the epidemiology of
VTEC O157 in farmyard manure (FYM), slurry and dirty water on both dairy and beef
cattle farms was investigated. The overall aim of Project OZ0709 was to devise a set of
recommendations that can be adopted by farmers in order to reduce the risk of human
VTEC infection that is attributable to farm waste. To formulate such recommendations, a
multi-disciplinary approach was adopted which included a review of existing farm waste
management methods; an epidemiological study; engineering visits, risk assessment &
mathematical modelling.
From the project it was concluded that the management of waste significantly varies
between farms, however there are recommendations/guidelines available to which
farmers should adhere. Such recommendations are often with particular reference to the
transfer of food-borne pathogens on to crops and are therefore not VTEC O157 specific.
By identifying specific VTEC O157 recommendations from within the project it was
concluded that farmers adhering to existing guidelines are managing their waste in such
a way that minimises the risk of environmental contamination with VTEC and hence
maintaining a low risk of humans becoming infected with VTEC O157 due to direct or
indirect exposure to cattle waste.
Review summary
Project OZ0709 aimed to produce guidelines for farmers on ways to minimise the risk of
environmental contamination with VTEC, and consequently reduce the risk of humans
becoming infected with VTEC O157 through direct or indirect exposure to cattle waste.
The results back up what had been previously thought. This project produced valuable
data in providing assurance that current guidelines for management of farm waste are
appropriate.
92

Project code: OZ 0711
Project title: Incidence and control of VTEC in animal feeds
Start date (dd/mm/yy): 01/08/2002
End date (dd/mm/yy): 31/01/2005
£132,343
Total cost:
Affiliation: ADAS UK Ltd
Sub-contractor(s): Direct Laboratory Services Ltd
Abstract of research
This study consisted of the following tasks:
Task 1: Scientific literature review. This summarised published and unpublished
information on the incidence of VTEC and stx-harbouring phages in animal feeds. The
report was submitted to Defra in December 2003.
Task 2: An examination of imported feeds to determine the extent of their contamination
with VTEC. Imported feeds were supplied from importing mills or feed manufacturers.
Task 3: An assessment of the potential effect of manufacture on the viability of E. coli
O157. The effects of heat-treatment – reflecting conditions under commercial compound
feed manufacture - on a mixture of E. coli O157 isolates existing in ground and pelleted
cattle feed were studied. This task was completed under laboratory-controlled conditions.
Task 4: Determine the prevalence of VTEC in farm-stored and trough-fed feeds.
Feeds taken from stores and feeding troughs on farms were analysed for E. coli O157
and other VTEC strains to assess the risk these pose to the spread of VTEC. Analysis of
feeds from troughs and on-ground feed passages, which were recently accessed by
livestock, indicated that VTEC are likely to be spread via animal‘s mouths.
Task 5: Determine the prevalence of VTEC in forages fed fresh on farms
Forage crops (e.g. fresh grass, maize silage and grass silage), which were grown in
fields where livestock had been grazing, were sampled and analysed for VTEC strains. A
mini-silo study was undertaken using forage maize to study the effects of ensiling on the
presence of VTEC isolates.
Review summary
This study aimed to determine any impact which animal feeds may make to the
transmission of VTEC, and to identify practices in feed production that may contribute to
or reduce the levels of VTEC in livestock feeds. The results are what might have been
expected, indicating that feeds are unlikely to be a significant transmission route for
VTEC in the UK. It was, however, important to fund this work to obtain this evidence.
While the work has contributed to the evidence base, it would have been of more value if
the problems in obtaining representative feeds had been overcome and more feeds had
been investigated.
93

Project code: OZ0712
Project title: Escherichia coli O157 interventions and control
Start date (dd/mm/yy): 01/04/2003
End date (dd/mm/yy): 30/09/2006
£503,523
Total cost:
Affiliation: SAC
Sub-contractor(s): University of Edinburgh
Abstract of research
BioSS
Verocytotoxin producing Escherichia coli (VTEC) serotype O157:H7 is a significant
infectious intestinal disease pathogen that can cause a severe and potentially fatal illness
in humans. The main reservoir of E. coli O157 is the gastrointestinal tract of cattle, and
the overall size of this animal reservoir is a major determinant of the threat that the
organism poses to public health.
Our group have made significant advances in understanding the persistence of the
organism in the bovine host and identified the terminal rectum as the primary site for
colonisation associated with persistent and high level excretion of the organism. The
primary objective of study OZ0712 is to understand this colonisation of the terminal
rectum and to examine treatments or interventions to reduce or prevent carriage at the
site. Practical control strategies may be developed from the understanding of the
bacterial-host interactions that allow E. coli O157:H7 to persist on the rectal mucosa or
from the development of methods that treat or prevent colonisation. Opportunity will also
be taken to validate simple screening tests that could be used in the field or abattoir to
identify colonised animals.
Review summary
This project aimed to acquire knowledge of how E. coli O157:H7 persists in the GI tract
of cattle, and to use this information to develop practical methods for detection and
control.
The research team have significantly contributed to our understanding of the persistence
of E. coli O157:H7 in the bovine host, and in identifying the terminal rectum as the
primary site for colonisation. A simple dip stick method was also developed for detection
of high level faecal carriage of this organism. Direct application of chlorhexidine to the
terminal rectal mucosa was found to be the most effective treatment investigated. This
treatment was able to reduce or completely eliminate E. coli O157:H7 and has the
potential to be an important control option. The development of this practical intervention
is being taken forward in project OZ0714 (To develop a cost effective and practical
method to reduce E. coli O157 infection in cattle prior to slaughter).
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Project code: LK0666
Project title: Vaccination strategies for control of
enterohaemorrhagic Escherichia coli O157:H7 in
cattle
Start date (dd/mm/yy): 01/10/2005
End date (dd/mm/yy): 30/09/2008
Total cost:
£628,763.00 (Defra: £328,762; Other funders:
£300,001)
Affiliation: University of Edinburgh
Sub-contractor(s): SAC, MRI
Abstract of research
A consortium of scientists based the University of Edinburgh, the Moredun Research
Institute and the Scottish Agricultural College working in partnership with Novartis Animal
Vaccines Limited propose a three year LINK research programme that has two related
aims. The first is to develop an effective and inexpensive vaccine to limit EHEC
colonisation of cattle and therefore reduce the threat to human health from this pathogen.
The second is to develop systems for delivery of antigens to gastrointestinal follicleassociated
epithelium in order to generate appropriate mucosal responses.
The two aims are intrinsically linked as the antigens to be trialed in the vaccines are likely
also to target FAE and will be tested to compare intra-muscular injection with mucosal
delivery. This research builds on five years of research into EHEC colonisation of cattle
by the research consortium and two years of investment by NAVL into adhesins
expressed by EHEC O157:H7. The current identified antigens are the Loc8 fimbrial
adhesin, the H7 flagellin and EspA type III translocation filaments. These will be tested
alone and in combination.
The research will exploit well developed in vivo and in vitro systems to investigate the
interaction of EHEC O157:H7 with the bovine host including a colonisation protocol for
calves developed by the grouping that has led to the identification of the terminal rectum
as the principal site of colonisation of cattle by EHEC O157:H7. This protocol is being
applied to test direct antimicrobial interventions by our grouping. The research combines
the expertise of scientists in immunology, FAE biology, animal infectious diseases,
vaccine development and molecular biology with excellent microbiology laboratories and
large animal containment facilities.
Review summary
Non-foodborne infections are estimated to make up more than 50% of EHEC cases. It is
important to work toward the development of a vaccine for cattle to reduce the impact of
agricultural sources on this source of human disease.
The main aim of project LK0666 is to develop an effective, inexpensive vaccine to limit
EHEC colonisation of cattle, thereby reducing risk to public health from this pathogen.
This is a difficult subject but is being tackled well by the research team. The high quality
work continues to increase the E. coli O157 evidence base.
95

Project code: OZ0138C
Project title: A longitudinal study of faecal excretion of VTEC O157
in cattle to determine epidemiological patterns and
risk factors associated with excretion
Start date (dd/mm/yy): 01/10/1998
End date (dd/mm/yy): 31/01/2005
£2,242,139
Total cost:
Affiliation: VLA
Sub-contractor(s): Laboratory of Enteric Pathogens, Central Public
Health Laboratory, London
Project code: OZ0145
University of Liverpool
Veterinary Epidemiology and Economics Research
Unit (VEERU), School of Agriculture and Policy
Development, University of Reading
Project title: VTEC O157 on farm control: Effective measures,
perception and risk communication
Start date (dd/mm/yy): 01/10/05
End date (dd/mm/yy): 31/03/08
£210,125
Total cost:
Affiliation: VLA
Sub-contractor(s): N/A
Abstract of research
OZ0138
The objective of the project was to map and explore the epidemiology of VTEC O157 in
cattle, which at the beginning of the project was unknown. The aim was to develop
strategies for control of VTEC O157 in cattle to ultimately reduce disease in humans. The
project revealed that 38.7% of English and Welsh cattle herds were infected with VTEC
O157 and young-stock between 3 and 18 months of age were high-risk animals. VTEC
O157 was intermittently excreted by individual animals and the group status also
appeared to be interchangeable, which highlighted the importance of environmental
reservoirs. An individual animal is more likely to be infected if it is dirty, fed milk, VTEC is
detected in the drinking water or if persistent shedding animals are in the same group.
The risk for a group increases if it is Campylobacter positive, the bedding is wet, fed
straw or the group is large, whereas assessing the bedding daily, presence of springs
and poultry as well as raised awareness about VTEC O157 reduced the prevalence in
96

young-stock. The effect of control measures, that targeted at various combinations of the
above risk factors and potential environmental reservoirs, was assessed in a randomised
controlled trial. A visual effect of a package consisting of clean and dry near-environment
and animals, avoiding introduction into groups and overall farm-level biosecurity
measures was identified, albeit the cost of implementation of the whole package was
much higher than the economic benefits to society.
OZ0145
OZ0145 provided additional value of the data collected under OZ0138 by identifying and
describing compliance and motivation factors present during the trial and adjusting for
these in the statistical models, yielding evidence that dry bedding, closed rearing groups,
closed herd policy and no contact with other cattle reduced VTEC in a group of cattle
over a period of 4.5 months. These results were disseminated via presentations aimed at
farmers, private veterinarians, articles in trade and scientific journals and presentations at
international conferences. The data was also used to examine excretion patterns, coexistence
and risk factors of VTEC, Campylobacter and Coccidia in young cattle for a
period of 6-8 months and revealed that Campylobacter increased the risk of VTEC
excretion, changing water in troughs reduced both Campylobacter and Coccidia and that
large herds were more likely to harbor all three pathogens. The prevalence over time was
also used to validate a stochastic model of transmission of VTEC within a beef herd,
which was produced to test potential future interventions. The farmers from the trial were
revisited and interviewed about control of zoonses present on their farms, their concerns
and their attitudes and constraints in applying control measures and their opinions of
responsibility and financing safe cattle products. The main constraints were lack of
knowledge, lack of belief in self-efficacy and no financial flexibility within the enterprise,
but in general the attitudes towards improving food safety were very positive. The project
will proceed to review risk factors and control strategies for several zoonotic agents
important for cattle aged 3-24 months of age to work towards a combined zoonotic
control package.
Review Summary
Project OZ0138 produced valuable data on the prevalence of E. coli O157 in cattle in
England and Wales. At the time of commissioning, the incidence of human VTEC O157
disease had been increasing and this research provided essential data on the
epidemiology of this organism in the UK. Results also complemented the work carried
out by the Scottish Agricultural College, showing colonisation of calves occurs ‗postrumen‘
for E. coli O157. Risk factors for cattle herds becoming infected with E. coli O157
were identified; while this indicated that some measures could be taken to reduce the risk
of cattle excreting E. coli O157, elimination of the organism is not currently possible.
However, the work contributed to the clean livestock at slaughter policy.
Interventions identified in project OZ0138 were trialled in the follow-on project, OZ0145,
and the motivators for uptake of control measures by farmers assessed. The project
showed that changes to young-stock management, including keeping bedding dry and
avoiding introduction of new animals to established herds can reduce VTEC O157 on
cattle farms. Integration of measures to reduce risk of E. coli O157 infection with those to
reduce the risk from other pathogens may be more likely to be taken up by farmers than
a series of individual packages. This indicates that a more holistic approach to
communication may be more appropriate than the current approach of communicating
one aspect of food production at a time.
97

Project code: OZ0144
Project title: Constraints to uptake of adequate biosecurity on UK
cattle and sheep farms, with special reference to
zoonotic diseases
Start date (dd/mm/yy): 01/06/2002
End date (dd/mm/yy): 31/12/2003
Total cost: £113,279
Affiliation: The University of Reading
Sub-contractor(s): The Scottish Agricultural College
Inverness
Abstract of research
The overall objective of the project is to identify and rank the main constraints, including
economic constraints, to the implementation of adequate biosecurity measures on UK
cattle and sheep farms. The measures we consider are mainly aimed at the control of
potentially zoonotic diseases, but also include biosecurity for other animal infections, in
terms of the spread to human population and within and between livestock populations
on UK cattle and sheep farms. Our final objective is to make recommendations that
would enhance such implementation.
One of the aims of the Defra Research and Development Strategy is to guarantee the
safety of human food that originates from livestock. It is envisaged that an improved
understanding of biosecurity constraints will lead to the formulation of recommendations
at both farm and policy level, on how to increase the implementation of biosecurity
measures on these farm. These recommendations will, in turn, contribute to the aims of
Defra‘s R&D policy by enhancing food safety.
Review Summary
At the time of commissioning, this was a novel study aiming to identify the constraints to
implementation of biosecurity on UK cattle and sheep farms. The major barriers and
drivers for implementation of biosecurity were determined through interviews with both
farmers and veterinarians.
This project highlighted the difficulties in getting across the biosecurity message and
supports the view that true partnership is required to deliver this message.
The identification of costs and benefits of controls have been incorporated in other
projects within the overall zoonoses research programme to address some of the
knowledge gaps.
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Project code: OZ0707
Project title: Identification of factors mediating intestinal
colonisation of cattle by enterohaemorrhagic
Escherichia coli O157:H7.
Start date (dd/mm/yy): 01/10/1999
End date (dd/mm/yy): 11/04/2007
£603,124
Total cost:
Affiliation: Institute for Animal Health, Compton.
Sub-contractor(s): None
Abstract of research
Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of zoonotic diarrhoeal
pathogens of worldwide importance. Human infections are frequently caused by direct or
indirect contact with ruminant faeces and may be complicated by haemorrhagic colitis
and life-threatening renal and neurological sequelae. Strategies to reduce the prevalence
of EHEC in ruminants are predicted to lower the incidence of human infections; however
the molecular mechanisms influencing intestinal colonisation of food-producing hosts by
EHEC are ill-defined. Defra-funded research conducted under project OZ0707 aimed to
understand this process in greater detail and to exploit the knowledge to develop, test
and refine novel methods of disease control. The research involved the most
comprehensive survey for EHEC virulence factors performed to date and yielded
valuable insights that could not have been obtained in surrogate rodent or cell-based
assays. The project involved targeted and genome-wide mutagenesis of E. coli O157:H7
and analysis of wild-type and mutant strains in vitro and in bovine oral challenge, ligated
intestinal loop and ex vivo organ culture assays. The information led to the testing of
several subunit vaccines in cattle and has been disseminated via numerous high-impact
publications, review articles and invited lectures.
Review Summary
This research has contributed to the evidence base for VTEC O157 and to the
development of improved methods of control. The study provided baseline data on the
mechanisms of colonization in cattle with E. coli O157 and showed that it might be
difficult to develop functional vaccines. The work was carried out to a high scientific
standard and resulted in several publications in high-impact journals.
Major colonization factors of E. coli O157 were identified but vaccination studies with
purified antigen indicated, however, that colonization was not inhibited even in the
presence of high-titre antibodies against these adhesion factors. The results indicated
that the way forward may be to construct non-pathogenic probiotic strains that might
occupy the niche which is currently used by pathogenic E. coli O157.
99

Project code: OZ0708
Project title: A Systems Analysis Methodology to Elucidate and
Evaluate the Critical Control Points for E. coli
0157:H7 in Cattle and sheep from farm to abattoir
Start date (dd/mm/yy): 01/01/2000
End date (dd/mm/yy): 30/06/2002
£151,494.00
Total cost:
Affiliation: Silsoe Research Institute
Sub-contractor(s): Veterinary Laboratories Agency
Abstract of research
An extensive literature review on the epidemiology of E. coli O157:H7 on farms was
carried out. On the basis of this a conceptual model was proposed. This was reviewed by
an expert workshop attended by other scientists and Defra representatives, and some
revisions were made.
The original objective was to develop a network model of the system. This was found to
be inappropriate because of several cyclic features, so a simulation approach was used.
This had two aspects: a deterministic simulation of the herd or flock and a stochastic
simulation of the transmission of E. coli O157:H7 within the herd/flock.
Limited testing was possible with the available survey data. The model was found to
produce estimates of animal prevalence (the overall proportion of animals shedding) that
were consistent with the data. However, for cattle it was found to overestimate the withinherd
prevalence and underestimate the herd prevalence (the proportion of herds
containing shedding animals).
Sensitivity analyses found that the most sensitive parameters were those related to
transmission from grass and enclosures to animals, contact between animals, and
pathogen survival on grass, in slurry and in barns.
The model was used to investigate the possibility of reducing prevalence by a range of
management interventions. Among the most important risk factors were: mixing sheep
and cattle, keeping animals in large groups, high stocking density, introducing positive
animals and reducing the interval between slurry spreading and grazing.
Review Summary
This project developed simulation models for interaction between farm animals and a
large number of farm management factors, including farm waste management. The
models use data from the literature on E. coli epidemiology.
Some of the conclusions are of practical importance for a possible reduction of E. coli
O157 carriage and transmission. They are in line with what might have been expected
and do contribute to improving methods of control and the evidence base.
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Project code: OZ0706
Project title: EHEC O157 pathogenesis: Ovine and other animal
model studies
Start date (dd/mm/yy): 01/04/99
End date (dd/mm/yy): 30/03/04
£374,377
Total cost:
Affiliation: Veterinary Laboratories Agency,
Sub-contractor(s): Department of Pathology and Microbiology
University of Bristol Veterinary School
Project code: OZ0713
Project title: The role of the native host gut flora and innate
immune status upon the colonisation of E. coli
O157 in ruminants
Start date (dd/mm/yy): 01/10/04
End date (dd/mm/yy): 30/09/07
£272,863
Total cost:
Affiliation: Veterinary Laboratories Agency,
Sub-contractor(s): Comparative and Functional Genomics
Moredun Research Institute
Project code: OZ0710
Project title: The role of goats and pigs in the maintenance
and transmission of VTEC including EHEC
O157:H7
Start date (dd/mm/yy): 01/10/02
End date (dd/mm/yy): 30/09/05
£399,546
Total cost:
Affiliation: Veterinary Laboratories Agency,
Sub-contractor(s): University of Bristol Veterinary School
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Abstract of research
Projects OZ0706, OZ0710 and OZ0713 have run over the past ten years with VLA as the
lead organisation within a highly collaborative network involving numerous partners,
notably Bristol University and the Moredun, and more recently with the Defra Fellowship
at Edinburgh University, the IAH and Imperial College. The overall goal was to
understand the mechanisms of colonisation and persistence of Escherichia coli O157:H7
in sheep (the second most prevalent affected species) and other at risk food producing
animals. It is not possible to summarise these three highly successful projects within 250
words as the studies generated 40 peer reviewed papers (see section 9) to which the
reader has access, as all are in the public domain.
Various O157 strains have been used in deliberate oral inoculation studies in
conventionally reared ovines, porcines, caprines and poultry. All these animals are
susceptible to persistent colonisation.
In ovines, very young animals (six days old) are susceptible to colonisation by O157 by
intimate attachment as AE lesions were observed in the large intestine, although very
rare and very small. In older animals (six weeks and six months old) O157 induced
lesions were exceedingly rare and were only seen in the caecum. However, intimin
deficient mutants were cleared more quickly than wild type strains and interestingly a tir
mutant was cleared even more rapidly in the same models. These findings confirmed
persistent colonisation is dependent upon intimin and translocation of the Tir into host
cells.
In caprines, conventionally reared goats aged 6 days are susceptible to colonisation by
O157 by intimate attachment as AE lesions were observed ileum, caecum, colon and
rectum from all animals. Eight-week-old conventionally-reared goats were inoculated
orally in separate experiments with O157:H7 and an intimin deficient derivative.
Significantly fewer initimin deficient bacteria were shed only on days 2 (p=0.003) and 4
(p=0.014) whereas from day 7 to 29 days there were no differences. Tissues taken from
this study group were examined histologically. AE lesions were not observed at any
intestinal site of the animals except for one animal, which uniquely shed approximately, 1
x 10 7 O157:H7 per gram of faeces for the preceding three days and which showed a
heavy, diffuse infection with cryptosporidia and abundant, multifocal AE lesions in the
distal colon, rectum and at the terminal rectum and recto-anal junction. These AE lesions
were confirmed by immunohistochemistry to be associated with E. coli O157:H7. We
conclude that co-infection may be predisposing factor for colonization in caprines (and
therefore probably other at risk species). Five-year-old nannies with kids at foot were
dosed orally with O157:H7 at about six days after parturition. Faecal shedding was
variable up to day 11 after dosing (~1 x 10 4 E. coli O157:H7 cfu per gram faeces) but no
kids became positive for E. coli O157:H7 organisms. Thus, sucking kids are refractive to
colonisation by E. coli O157:H7 probably because the challenge dose was low and the
kids had access to milk that is known to be highly protective.
In porcines, we showed intimin was not required for persistence although in neonates AE
lesions were observed readily after oral inoculation. Indeed, intimin deficient mutants
adhered equally well to IPI-21 (porcine) cells and IVOC (all sections of the gut) as the
wild type. Also, histological analysis of pig tissues at post mortem examination revealed
that E. coli O157 specifically stained bacteria were associated with the mucosa of the
ascending and spiral colon but no AE lesions were detected.
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In poultry, until recently considered free of O157 infection, hatchlings inoculated orally
with 10 3 cfu were colonised persistently up to and beyond point of lay (20+ weeks of
age). Shedding can be significant (~10 5 cfu g -1 faeces) for this entire period and egg
shells but not egg contents can be contaminated. Eight strains have been tested in the
poultry model and variable colonisation was shown with 2 strains being cleared by 80
days whilst other persisted for 213 days. Intimin was not essential for colonization and no
AE lesions were induced in any section of the gut.
These data point to the role of intimin/Tir in ovines but colonisation of caprines, porcines
and poultry was less dependent upon intimin/Tir as in ovines. Indeed, the data indicated
that long term persistence may be dependent on factors other than intimin and Tir as well
as host factors such as provision of colostrum in the young (negative effect) or prior
colonization by other pathogens (positive effect). To study bacterial factors, we focused
upon those genes identified by analysis of the O157 genome that may contribute to
persistent colonisation of ruminants and sheep in particular. Defined targeted mutants
were constructed (e.g. eae, tir, efa/toxB, waaZ, fliC, lpf, nleD, tccP, ‗O‘ island [O-7, O-36
and O-50] etc) made primarily in E. coli O157:H7 strain NCTC12900 that is a nontoxigenic
strain and other model strains also. Mutants lacking LPS were uniformly
attenutated in all species as might be anticipated. Flagella played a key role in poultry but
no other animals species tested. Of the other factors tested, small contributions to
colonization and persistence were detected for most. To study host factors, we focused
on the general health status and demonstrated that animals pre-inoculated with C.
parvum prior to challenge with O157:H7 developed multifocal attaching and effacing
lesions in the caecum, colon, rectum and at the recto-anal junction, confirmed by
immunohistochemistry to be associated with O157:H7, and shed very high numbers of
O157. Also, neonates deprived of colostrum (sheep and goats) are highly susceptible to
colonization and very high shedding.
From the large animal studies, it was clear that O157 has a tropism for the large intestine
but there was no concrete evidence to support the hypothesis that the terminal rectum or
recto-anal junction was the preferred site of colonisation, as suggested for bovines. What
was interesting was the observation that animals colonised beyond about 28 days p.i.
(and therefore defined as persistent shedders) had O157 organisms throughput the
entire gastro-intestinal tract rather than just the large bowel. The significance of this is
unclear and experiments are in hand now to answer this question.
Another observation from the animal studies was the frequent occurrence of AE lesions
caused of non-O157 bacteria, some of which were identified as belonging to O26, O45,
O86 and O115 serogroups. In a brief survey of E. coli from an abattoir survey, it was
noted that AEEC comprised up to 10% of all the isolates and each was shown to
possess a functional eae (intimin) gene. A selection of these strains has been studied in
detail in the various models in this project and of 33 strains tested in gut loops, all formed
AE lesions. Whether the presence of these AEC interfered with O157 colonisation is
under assessment currently. In one experiment, shedding of O157 after oral challenge by
ovines pre-dosed with O26 showed no differences compared with control animals does
with O157 alone. Surprisingly, O157 suppressed shedding of the resident O26
organisms, an interaction that requires analysis.
Review Summary
These inter-related projects sought to explore the potential for non-bovine food-producing
animals to become colonised with E. coli O157. It was acknowledged that the results
103

produced were both relevant and important and had contributed significantly to the
evidence base for VTEC O157. A large number of scientific publications had been
generated.
The work on sheep and goats has been of value in the control of transfer of infection to
humans from animals on open farms. It has also provided new information on the
mechanism of long term colonisation of E. coli O157 and the potential role of other
strains as natural antagonists against infection. The results from challenge studies in pigs
and poultry have raised many questions as these indicate that both species are
susceptible to E. coli O157. In practice, poultry meat and pig meat and also contact with
poultry and pigs have not proved to be a risk for human infection, despite the large
population of free range pigs and poultry in England. This contradiction needs to be
better understood, for example by studying the influence of natural intestinal flora on as
potential antagonists for E. coli O157.
104

Annex 3 – External Reviewer List
105

List of External Reviewers.
Mr. Paul Gayford, formerly of Defra
Prof. John Threlfall, Health Protection Agency, UK
Prof. Will Waites, University of Nottingham, UK
Dr. Paul McMullin, Poultry Health Centre, UK
Mr. David Burch, Octagon Services Ltd, UK
Dr. Helene Wahlström, National Veterinary Institute, Sweden
Dr. Cornelius Pope, Public Health Agency, Canada
Dr. Mike Taylor, Central Science Laboratory, UK
Prof. Dirk Pfeiffer, Royal Veterinary College, UK
Dr. Vincent McDonald, Barts and the London School of Medicine and Dentistry, UK
Dr. Huw Smith, Scottish Parasite Diagnostic Laboratory, UK
Dr. Nick Dorell, London School of Hygiene and Tropical Medicine, UK
Prof. David McDowell, University of Ulster, UK
Dr. Elizabeth Innes, Moredun Research Institute, UK
Dr. Stephen On, ESR Christchurch Science Centre, New Zealand
Prof. Cecil McMurray, Sci-Tec Consultancy, UK
Mr. Keith Gooderham, Poultry health and welfare specialist, UK
Dr. Mogens Madsen, Dianova, Denmark
Dr. Barti Synge, Scottish Agriculture College, UK
Dr. Helge Karch, Institut fur Hygiene, Germany
Dr. Lothar Beutin, NRL-E. coli, Federal Institute for Risk Assessment, UK
Prof. Mac Johnson, retired from the Royal Veterinary College, UK
106

Annex 4 – Assessment Forms
107

Review of Defra’s FBZ Research Programme, 26 th – 27 th November 2007
ASSESSMENT FORM - POLICY
PROJECT CODE(S):
POLICY GROUP(S) APPRAISED BY:
DATE:
Instructions: Please assign a score and provide written comments where necessary.
Scores should be based on a 1-5 scale, where:
1 = Not at all 2 = Partial 3 = Satisfactory 4 = Good 5 = Very Good
1. How useful has the research been to Policy?
(This may include helping to answer an urgent policy question, informing the formulation
of a policy, contributing to the evidence base, maintaining scientific expertise,
development of rapid and accurate diagnostic tests, development of improved methods of
control, other etc.)
Score
Comments
2. Has the research raised further questions that need addressing with Defra
R&D funding?
Yes / No
If yes, please provide details
3. Please state any questions you wish to be forwarded to the contractor for
comment -
108

109

Review of Defra’s FBZ Research Programme, 26 th – 27 th November 2007
ASSESSMENT FORM – EXTERNAL REFEREES
PROJECT CODE(S):
APPRAISED BY:
DATE:
The scores and comments you provide will be used for a range of purposes, including (1) to inform Defra
personnel (2) for feed back to the project leader(s) and (3) for possible inclusion in the review output
document (which will be published on the Defra website). Please note that while a list of the review panel
members will be publicly available, with reference to points 2 and 3, the scores and comments provided by
each referee will not be directly attributed to them, but rather referee 1, referee 2 etc. However, you should
be aware that Departmental correspondence, including peer review processes, fall within the remit of
regulations that permit greater access to information, including Freedom of Information, Environmental
Information Regulations and the code of practise on access to government information. In the event that
peer review information (such as the names of peer reviewers and the comments they made) should
become the subject of such a request, the Department will seek to protect the interests of peer reviewers in
the light of legal requirements.
Instructions: Please assign a score and provide written comments where necessary.
Scores should be based on a 1-5 scale, where:
1 = Not at all 2 = Partial 3 = Satisfactory 4 = Good 5 = Very Good
1. Relevance and appropriateness for R&D funding by Defra
2. Soundness and appropriateness of the scientific approaches
and methods
3. Appropriateness of the contractors, sub-contractors and
collaborators (e.g. personnel and facilities)
4. Rate of progress to date in achieving the aims and objectives
of the research
5. Probability of success (if the research is ongoing)
6. Conclusions based on sound evidence
7. Dissemination of findings
8. Quality of science
9. Value for money
10. Overall rating (it is important to provide a score here)
110

11. Overall opinions on the research area (including your overall views, as well as any
comments you have on questions 1-10)*
12. Has the research raised further questions that need addressing with Defra R&D
funding?
YES/NO
If yes, please provide details*
13. Are the areas within the current research addressing topics of highest priority?
YES/NO
If not, which topics would you identify as high?*
14. Please state any questions you wish to be forwarded to the contractor for
comment –
*Please expand boxes as required
111

Annex 5 – Review Timetable
112

Salmonella, Cryptosporidium species, Yersinia enterocolitica and
Bovine neosporosis
Monday 26 th November 2007
Venue: Defra, Room LG04/05/06, 1a Page Street, Westminster, London SW1P 4PQ
09:00 – 09:10 Registration and coffee
9.10 – 9.30 Introductions
Molecular Methods
Salmonella
09.30 – 09.45 Project OZ0312: Development of a sensitive and
specific molecular typing method for the epidemiological
study of Salmonella.
09.45 – 10:00 Project OZ0324: New and emerging Salmonella
serovars; epidemiological, risk-based and molecular
approaches to their identification and control.
10.00 – 10.15 Project OZ0311: Conventional versus capillary
electrophoresis of RFLP fragments and PCR products
for sensitive and rapid subtyping of Salmonella.
Poultry – Broilers
10.15 -10.35 Project OZ0314: Role of defined bacterial genes and
host genetic background in intestinal colonisation of
poultry by Salmonella.
Follow-up Project OZ0320: Bacterial and host genes in
Salmonella colonisation in poultry.
10.35 – 10.50 Project OZ0328: A monitoring, control and education
package to assist the turkey industry with reduction of
Salmonella and antimicrobial resistance and achieving
targets.
10.50 – 11.10 Coffee break
Poultry – Layers
11.10 – 11.25
11.25 – 11.45
Project OZ0327: Effect of immunosuppression
associated with point-of-lay on Salmonella infection and
immunity in laying hens.
Project OZ0317: Epidemiological investigations of
Salmonella contamination in table egg production.
Follow-up Project OZ0321: Investigation of the role of
environmental contamination in the epidemiology of
Salmonella infection in egg-laying flock.
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11.45 – 12.00
Bovine and Porcine
Follow-up Project OZ0325: A monitoring, control and
education package to assist the egg industry with
Salmonella reduction and achieving EU targets.
Project OZ0326: Managing the challenges to the egg
industry posed by new SE PTs.
12.00 – 12.15 Project OZ0313: Non-specific and innate resistance to
Salmonella infection in chicken and pigs.
12.15 – 12.30 Project OZ0135: Epidemiological studies of multiple
resistant S. typhimurium DT 104 infection in cattle.
12.30 – 12.45 Project OZ0315: Salmonella pathogenesis and
immunity in cattle and pigs.
12.45 – 13.05 Project OZ0316: Epidemiological studies of Salmonella
in pigs and control by intervention.
Follow-up Project OZ0323: An integrated risk based
approach to the control of Salmonella in UK pig farms.
13.05 – 13.20 Project OZ0318: Understanding the dynamics of
endemic and epidemic Salmonella infections in cattle
and pigs.
13.20 – 13.35 Project OZ0319: Salmonella pathogenesis in cattle and
pigs.
13.35 – 14.30 Lunch
Other
14.30 – 14.50
14.50 – 15.05
15.05 – 15.20
15.20 End of meeting
Project OZ0402: What is the potential for human
isolates of C. parvum to infect, colonise and be excreted
by farm animals.
Follow-up Project OZ0407: Evaluation of Zoonotic
Transmission of Cryptosporidium Spp. and C. Parvum
genotypes between humans and animals.
Project OZ0405: The genotypic and phenotypic
comparison of virulent Yersinia enterocolitica from
humans and animals.
Project OZ0404: Bovine neosporosis: the development
of evidence-based control systems.
114

Campylobacter and E. coli
Tuesday 27 th November 2007
Venue: Defra, Room LG04/05/06, 1a Page Street, Westminster, London SW1P 4PQ
09.00 – 09.10 Registration and coffee
09.10 – 09.30 Introductions
Campylobacter
Molecular Methods
09.30 – 09.50 Project OZ0604: Characterisation of strain variation in
Campylobacter jejuni.
Follow-up Project OZ0611: Development of a
comprehensive MLST database for assessment of
Campylobacter risk factors.
09.50 – 10.05 Project OZ0605: Genotypic and phenotypic instability of
Campylobacters from animal and human sources.
10.05 – 10.20 Project OZ0607: An investigation of the distinguishing
features of C. jejuni strains that have host/disease
implications.
Poultry broilers
10.20 – 10.40 Project OZ0606: Protective immunity and competitive
exclusion in development of effective intervention
products for poultry.
Follow-up Project OZ0614: To develop vaccination
approaches to the control of Campylobacter in poultry.
10.40 – 11.00 Project OZ0608: Epidemiological studies and
development of practical control measures for
Campylobacter in broiler flocks.
Follow-up Project OZ0610: Survival and persistence of
Campylobacters in poultry farm environments and
identification of control measures.
Follow-up Project OZ0613: Towards risk-based control
of Campylobacter: developing the evidence base using
epidemiological and bacteriological approaches.
11.00 – 11.15 Project OZ0609: Determining the role of maternal
antibodies in the lag phase of Campylobacter jejuni
infection in chickens.
11.15 – 11.30 Project LK0665: Improving gut health and nutrient
capture of broiler chickens through selection for innate
immune function.
11.30 – 11.50 Coffee break
115

Campylobacter Other
11.50 – 12.05 Project OZ0612: The epidemiology of Campylobacter
infection in dogs in context of the risk of infection to
humans.
E. coli
12.05 – 12.20 Project OZ0703: Plant antibody delivery of passive
immunisation against E. coli 0157:H7: a novel means of
control in the animal.
12.20 – 12.35 Project OZ0704: Quantification of E. coli 0157:H7
virulence factors in vivo using real-time RT-PCR and in
situ RT-PCR.
12.35– 12.50 Project OZ0705: A proteomic approach to identify
virulence determinants of EHEC 0157.
13.05– 13.20 Project OZ0709: Epidemiology of VTEC 0157 and other
VTECs likely to be pathogenic to man in farm wastes.
13.20– 13.35 Project OZ0711: Incidence and control of VTEC in
animal feeds.
13.35– 14.30 Lunch
14.30 – 14.45 Project OZ0712: Escherichia coli O157 interventions
and control.
14.45 – 15.00 Project LK0666: Vaccination strategies for control of
enterohaemorrhagic Escherichia coli O157:H7 in cattle.
15.00 – 15.20 Project OZ0138C: A longitudinal study of faecal
excretion of VTEC 0157 in cattle to determine
epidemiological patterns and risk factors.
Follow-up Project OZ0145: VTEC O157 on farm control:
effective measures, perception and risk communication.
15.20 – 15:35 Project OZ0144: Constraints to uptake of adequate
biosecurity on UK cattle and sheep farms, with special
reference to zoonotic diseases.
15.35 – 15.50 Project OZ0707: Identification of factors mediating
colonisation of cattle by enterohaemorrhagic
Escherichia coli
15.50 – 16.05 Project OZ0708: A systems analysis methodology to
elucidate and evaluate the critical control points for E.
coli 0157:H7 in cattle and sheep from farm to abattoir.
16.05 - 16.25 Project OZ0706: EHEC 0157 Pathogenesis; ovine and
animal model studies.
Follow-up Project OZ0710: The role of goats and pigs in
the maintenance and transmission of VTEC including
EHEC 0157:H7.
Follow-up Project OZ0713: The role of native host gut
flora and innate immune status upon the colonisation of
E. coli O157 in ruminants.
16.25 End of meeting
116