SYNOPSES FROM THESES
of recently-qualified BEMB-sponsored PhD Students
(in lay language)
| Student |
Dr
Elizabeth Haygreen |
| Institution/Supervisor |
IAH,Compton.
Dr Fred Davison |
| Subject |
Enhancing
in ovo vaccination against MDV and IBDV
using novel DNA-vaccines |
| Submitted |
September
2004 |
Enhancing in ovo vaccination
using novel DNA vaccines, 2000-2004.
Infectious bursal disease (IBD) or Gumboro disease
is an economically important disease of chickens
caused by a small and widespread RNA virus,
infectious bursal disease virus (IBDV). Chicks
are protected by high levels of maternally-derived
antibodies, and early vaccination with live
vaccines is an integral part of health management
of commercial flocks. However, over the last
25 years new strains of IBDV have emerged that
can overcome high levels of maternally-derived
antibodies and can break vaccine-induced protection.
To combat this 'hotter' strains of IBDV are
being used as vaccines but some of these can
cause bursal damage, and risk causing immunodepression.
This has led to an interest in developing novel
vaccination strategies to produce better protection
and, at the same time, eliminate damaging side-effects.
One such approach is to vaccinate with DNA that
encodes only a part of a virus but has not sufficient
information to construct the infectious agent.
Once administered to the chicken, the part of
the virus is synthesized and is then recognized
by the chicken's immune system, stimulating
immunity to the virus. A further advantage could
be achieved if such a vaccine were given very
early in life and was not affected by maternally-derived
antibodies. In ovo vaccination is a relatively
new method to administer vaccines to chickens
and has many advantages such as stimulation
of earlier immunity, precise and uniform injection,
reduction in bird stress and reduced labour
costs.
A DNA vaccine was constructed (called pCI-Vp2)
containing the gene coding for the IBDV protein,
VP2 from the virulent strain F52/70. In ovo
injection successfully delivered pCI-Vp2 to
the embryo and the vaccine was found to localize
to the proventriculus and thymus. Protection
against IBD-induced mortality as well as bursal
pathology was stimulated after in ovo injection
of pCI-Vp2 using a prime-boost strategy, described
as follows. pCI-Vp2 was administered once at
18 days of incubation and was then boosted with
another vaccine containing the same VP2 gene
but this time in a fowlpox vector (fpIBD1).
Strikingly, unlike the majority of successful
IBDV vaccines, no antibody response was stimulated,
indicating that cell-mediated immune responses
were providing the protective effects. This
suggests that the prime-boost strategy can be
used to circumvent the problem of maternally-derived
antibodies neutralizing a vaccine virus. Although
boosting with the fowlpox recombinant fpIBD1
was required, these results show DNA vaccination
via the in ovo route has much potential and
is a promising area for research.
Dr Haygreen is now working at the Edward Jenner
Institute for Vaccine Research.
| Student |
Dr Paul Carroll |
| Institution/Supervisor |
CVL.
Professor M J Woodward |
| Subject |
Immune responses of chicks
and layers to Salmonella enteritidis |
| Submitted |
September 2003 |
A major threat to both the poultry
industry and the consumer is the food-borne
bacterium Salmonella. Pathogenic Salmonella
strains are divided into those that cause typhoidal
and enteric infections. The two main typhoidal
serotypes in poultry are S. Gallinarum and S.
Pullorum, which are the causative agents of
fowl typhoid and pullorum disease respectively.
The two commonly isolated enteritis-causing
serotypes are S. Enteritidis and S. Typhimurium.
These serotypes can be isolated from the faeces,
eggshells and poultry meat of infected birds.
Furthermore, Salmonella can be spread
to uninfected birds en route to and at the abattoir.
However, what is of even greater concern to
human health is that S. Enteritidis is
able to invade gastro-intestinal tissues, migrate
to and colonise the ovary and oviduct of the
hen. This leads to the colonisation of the egg
contents and the potential of transmission.
Therefore, it is in our interests to understand
pathogen-poultry interactions as it allows us
to develop strategies to combat Salmonella
infections.
Several antigens expressed on
the surface of Salmonella have been shown
to play a role in Salmonella pathogensis
and lipopolysaccharide (LPS), a predominant
surface structure plays an important role in
bacterial survival, adaptation to the environment
and pathogenesis in Salmonella, and many
other bacterial pathogens. However, the precise
role of this bacterial structure in the infection
of S. Enteritidis in the chicken is still
little understood. Thus, in this study, the
LPS structure of S. Enteritidis was modified
so a truncated form was expressed on the surface
of the bacteria. The LPS defective strain was
then compared to the LPS proficient strain under
various conditions. The study showed that LPS
plays an important role in many aspects of S.
Enteritidis poultry infections including colonisation,
invasion and persistence in both the day-old
and four-week-old chick.
Recognition by the immune system
of important epitopes on the bacteria leads
to efficient host clearance responses. Currently,
the S. Enteritidis vaccination strategy
targets surface antigens such as LPS that are
able to stimulate both the innate and adaptive
immune responses. And since LPS has been shown
to play an important role in colonisation and
persistence in poultry it can be stated that
the current vaccination is on the right track
for controlling S. Enteritidis infection.
Paul Carroll recently commenced
a research appointment with Queen Mary College,
University of London.
| Student |
Dr Tristan Cogan |
| Institution/Supervisor |
PHLS,
Exeter. Professor T Humphrey |
| Subject |
Factors affecting the Growth
of Salmonella Enteritidis in Eggs |
| Submitted |
March 2002 |
The reasons for the prevalence
and success of Salmonella enterica serovar Enteritidis
are poorly understood. The aim of this study
was to identify the factors that allow Enteritidis
to survive and multiply within the egg and to
compare these to other serovars of Salmonella.
As well as helping to explain the success of
Enteritidis, this allows an assessment of the
risk posed by other Salmonella serovars to be
made now that vaccination has removed Enteritidis
from laying hens.
-
This study examined the
ability of a range of different isolates
of Salmonella to survive and multiply in
eggs.
-
The role of bacterial surface
structures involved in movement, attachment
and cell invasion in growth within the egg
was examined.
-
The ability of Salmonella
strains to use the albumen as a source of
nutrients for growth was also investigated.
Salmonella Enteritidis was found
to have a number of abilities that allow single
cells to survive in the albumen and move into
the yolk of the egg and then grow to a high
level, frequently greater than one billion bacteria
per egg. Firstly, it was able to survive well
in the albumen at hen body temperature. Other
strains of Salmonella did not have this ability.
Secondly, it could use the glucose present in
fresh eggs as an energy source and to make the
albumen less alkaline and so more suitable for
bacterial growth.
Work on bacterial surface structures
showed that bacteria must be able to move in
order to multiply in the egg and that adhesive
surface structures, fimbriae, are used by Salmonella
to attach to and enter the yolk.
This combination of attributes
is not shared by other Salmonella serovars that
have been widespread in the past, such as Gallinarum,
Pullorum and Typhimurium. Future work will concentrate
on the possession of these attributes by other
poultry-associated strains of Salmonella.
Tristan Cogan is now working
as a Clinical Scientist for the Health
Protection Agency based at the Unversity
of Bristol.
| Student |
Dr Karen Hackney |
| Institution/Supervisor |
IAH,
Compton. Drs Peter Kaiser and Paul Britton |
| Subject |
In ovo vaccination using
infectious bronchitis virus defective RNAs
expressing chicken cytokine and heterologous
pathogen genes |
| Submitted |
October 2002 |
Poultry producers must constantly
vaccinate their birds against common pathogens
endemic in poultry producing areas to minimise
the threat of disease outbreak. Considering
the vast numbers of birds to be vaccinated,
the most beneficial vaccines are those that
can be given by mass administration, thus requiring
a minimum amount of labour. Such routes include
in ovo vaccination or delivery by aerosol or
drinking water. In ovo vaccination offers many
benefits over post-hatch vaccinations including
the potential for earlier immunity, reduced
contamination, precise and uniform injection,
reduction in bird stress and reduced labour
costs.
Novel adjuvants are being investigated
as a means of enhancing the immune response
to a vaccine. Cytokines are potent regulatory
proteins that act as a communication network
between cells throughout immunological development
and during immune responses, directing the immune
response to that required to combat a particular
pathogen. Using cytokines as adjuvants therefore
may augment vaccine efficacy. For example, certain
cytokines (interferon-g (IFN-g), interleukin-2
(IL-2) and IL-18) control anti-viral responses,
and therefore may be useful as adjuvants with
an anti-viral vaccine.
Novel delivery systems are also
being investigated. Defective RNAs (D-RNAs)
are naturally generated by RNA viruses and require
a helper virus in order to replicate and package.
Coronavirus D-RNAs, including infectious bronchitis
virus (IBV) D-RNAs, have been used as vectors
to express foreign genes. D-RNA CD-61, derived
from the Beaudette strain of IBV, was used to
successfully express chicken IFN-g, IL-2 and
IL-18, both in vitro and in ovo, proof of principle
that cytokines could be functionally delivered
in this manner. However, attempts to deliver
both the cytokine gene and another foreign gene
in the same vector were less successful - science,
like life, is seldom simple. Nevertheless, our
results indicate that delivery of cytokines
using a D-RNA vector system has adjuvant potential
in an in ovo vaccination system.
Karen Hackney is now working
with Bayer Ltd in Newbury.
| Student |
Dr Giles Barkley |
| Institution/Supervisor |
Leeds/
Professor M Forbes and Dr Helen Miller |
| Subject |
Phosphorus
Nutrition of Laying Hens: Phytase and Diet
Selection |
| Submitted |
2001 |
The
phosphorus (P) nutrition of laying hens was
considered using microbial phytase supplementation
of plant-based diets and diet selection methodology.
Five experiments were conducted to establish
the optimal level of dietary available phosphorous
(AP) necessary to efficiently meet the metabolic
requirements of the laying hen. Published data
on AP requirements were statistically analysed
from which a model was developed to predict
requirements. Access to limestone granules significantly
increased egg weights. Food was eaten at a constant
rate throughout the photoperiod whereas limestone
granules were eaten in the final four hours
of the photoperiod. There was sufficient AP
in wheat-soybean diet (with neither DCP nor
microbial phytase supplementation ) providing
180 mg/b/d to meet the P requirement of the
laying hen throughout a 10 week trial. The lowest
level of AP fed throughout the laying year provided
290 mg/b/d and was more than sufficient to meet
the hens' P requirements. Supplementation with
microbial phytase of a maize-soybean diet was
efficacious in releasing phytate-P in order
to increase egg production relative to an unsupplemented
diet. The AP requirement established using a
maize-soybean diet was determined to be 180
+/- 40 mg/b/d. The determined AP requirement
was in the same range as the prediction model's
requirement of 170 mg/b/d for a laying hen aged
26 weeks fed a diet with an energy concentration
of 12.1 MJ/kg (as in the diet selection trial).
Some of the published literature tended to overestimate
the AP requirement of the laying hen as when
there was no production response to AP levels
the lowest AP level in the trial was recommended
as sufficient. It may be possible to reduce
AP levels from those fed commercially. This
would reduce costs, improve feed efficiency,
reduce excretion and potentially reduce P pollution
in the environment.
Giles
Barkley is now working as Senior Poultry Scientist
with ADAS, Gleadthorpe.
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