THE UNIVERSITY OF MELBOURNE
ANIMAL ETHICS COMMITTEE
APPLICATION FOR APPROVAL TO USE ANIMALS FOR SCIENTIFIC PURPOSES RESEARCH OR TEACHING ACTIVITY
Before completing an application, applicants should familiarise themselves with all relevant guidelines and
legislation, including the NHMRC Australian code for the care and use of animals for scientific purposes
(2013).
Applicants should visit the OREI website and download the Application Form each time a new application is
prepared, in order to ensure the most up-to-date version of the form is used. Applicants should also read the
separate Additional Information sheet for specific guidelines on how to complete the application.
To ensure that all AEC members are provided with sufficient information to participate effectively in the
assessment of the application, all responses should be clear, concise and written in plain English.
*Please save your application as a Word file (not a PDF) and use the Ethics ID number as the file name*
1.
ADMINISTRATIVE DETAILS
Project ID and Title:
ID No:
Example3
Title:
The role of gut-resident T cells in protecting against enteric Listeria infection
Contacts:
Name
Department/School
Project Supervisor
Investigator 1
Microbiology and Immunology
Primary Contact
Investigator 2
Microbiology and Immunology
2.
PROJECT BACKGROUND & AIMS
2.1
Provide a plain English summary of the background to the proposed work
*Please note, terms defined in the Glossary are indicated by a hyperlink.
T lymphocytes or T cells are white blood cells that eliminate infected cells from the body. Given this capacity,
new generation vaccines are being designed to expand the numbers of pathogen-specific T cells and in so
doing, generate immunity against infection.
Pathogens can invade humans through a variety of different tissues, usually body surfaces. For example,
Influenza virus and Mycobacterium tuberculosis infect through the lung; Salmonella and Rotavirus through the
gut; and a variety of bacteria, fungi and viruses can infect the skin. It is now realised that for T cells to provide
the best protection against infection of the skin and lung, they must be located, or resident, within the skin and
lung at the time of re-infection. In this way, "tissue-resident" T cells will encounter the pathogen at the point of
entry and thus be able to quickly eliminate the microbe before it can spread. However, whether T cells located
within the gut are necessary and/or capable of similarly controlling enteric infections remains unclear. Given that
infectious diarrhoea is one of the leading causes of mortality within developing countries, understanding the
requirements for vaccine-mediated protection against enteric infections is paramount.
In these studies, mice will be inoculated with Listeria monocytogenes (LM), a food borne bacterium that invades
the lining of the gut and causes wide spread disease within humans. The mice will be inoculated either orally to
elicit a population of gut-resident T cells, intravenously to elicit T cells in the blood, or subcutaneously to elicit T
cells in the skin. At various times after inoculation, mice will be (a) orally infected with LM to determine whether
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the gut-resident T cells provide superior protection against enteric infection; and/or (b) killed to analyse the
migration of T cells to the gut. In some experiments, prior to infection with LM, we will inject mice with small
numbers of T cells derived from T cell receptor (TCR) transgenic (Tg) mice, specific for the LM strains we will
use. This will aid our ability to track and observe T cell migration to the gut.
All infections will use a sub-lethal dose of LM. Infection may result in transient, moderate symptoms, including
ruffled fur, weight loss up to 15% of starting body weight, and reduced activity. These symptoms are not
expected to last beyond 5-7 days after infection, and LM is completely cleared within 10-14 days after infection.
Mice will be closely monitored using the attached monitoring checklist (daily for 14 days then twice weekly), and
death will never be used as an endpoint.
2.2
Outline the overall aim and any specific aims of the proposed work
Using a well-established mouse model, we aim to examine whether pathogen-specific T cells lodged within the
intestine by vaccination can provide protection against subsequent infection with Listeria monocytogenes (LM).
Specifically, our experiments will address whether vaccines that establish gut-resident T cell populations provide
superior protection against subsequent Listeria infection. Secondly, we will examine how T cells migrate from
the blood into the gut during infection. Thirdly, we will attempt to develop means to enhance T cell migration to
the gut, and thereby enhance the number of gut-resident T cells capable of controlling subsequent gut infection.
2.3
If applicable, provide details of the relationship of the proposed work to other work, e.g. AEC approved
research or teaching, and clinical or agricultural activities. Include, as relevant, application ID numbers.
This work relates to that of Ethics ID 1234567, where we are examining the formation of resident T cell
populations in tissues such as the skin following virus infection or induction of inflammation, and how these cells
can be manipulated to provide protection against infection.
3.
EXPERIMENTAL OR COURSE DESIGN
Provide the details of your proposed research or teaching activity. It should be clear and concise, but
must contain enough detail that a proper assessment of the impacts of the protocols/procedures on
each animal (or group of animals) can be made.
You should make reference to: the number and types of animals, relative to specific procedures; the
details of each protocol/procedure; the details of pain or distress experienced by the animals; the
details of monitoring; and the fate of the animals.
CONTENTS
A. Experiment Outlines
E.1
Do vaccines that induce gut-resident T cells provide protection against enteric Listeria infection?
E.2
Examining the migration of T cells from the blood to the gut following enteric Listeria infection
E.3
Enhancing migration of T cells to the gut following enteric Listeria infection
B. Procedures (incorporating animal welfare and monitoring)
i. Standard monitoring
ii. Standard techniques
Anaesthesia by isoflurane inhalation
Oral inoculation
Injections (intravenous, subcutaneous)
Killing mice by carbon dioxide administration
iii. Experimental procedures
P1: Infection with Listeria monocytogenes
P2: T cell transfer
C. Mouse strains used in the project
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A. EXPERIMENT OUTLINES
Experiment 1 (E.1): Do vaccines that induce gut-resident T cells provide protection against enteric
Listeria infection?
In this experiment, we will compare the protection provided by different routes of vaccination against subsequent
Listeria infection. These routes are as follows:
• Oral – induces gut-resident T cells
• Intravenous – induces T cells in the blood
• Subcutaneous – induces T cells in the skin
1. C57BL/6 mice will be infected with Listeria monocytogenes (LM) either by oral, intravenous or subcutaneous
inoculation (Procedure 1). This primary infection will act here as a vaccination. A control group of C57BL/6 mice
will be uninfected.
2. Mice will be orally challenged with LM (secondary infection; Procedure 1) at one of the following time points
following vaccination: either during active Listeria infection (days 5-10 post-infection (p.i.)); when Listeria is
cleared and memory T cells are developing (days 10-30 p.i.); or during late memory (days 40-100 p.i.).
3. Between 2-10 days following the second (challenge) LM infection, mice will be killed by carbon dioxide
administration, and organs (intestine, spleen and liver) will be harvested to measure the bacterial load.
Listeria infection
- Oral
- Intravenous or
- Subcutaneous
Kill for
organ
harvest
5-100 days
2-10 days
Experiment 2 (E.2): Examining the migration of T cells from the blood to the gut following enteric
Listeria infection
Chemokines are a large family (~50 members) of proteins that are released from tissues to recruit T cells from
the circulation into tissues such as the gut lining. In this experiment we will measure which chemokines are
released by the intestine following Listeria infection. Secondly, we will assess which chemokine receptors T cells
use to migrate into the intestine during an enteric Listeria infection. From these experiments we will understand
what molecules are involved in recruiting T cells into the intestine during infection.
E.2.1 Chemokine analysis:
1. C57BL/6 mice will be infected orally with LM (Procedure 1).
2. Mice will be killed by carbon dioxide administration either during active Listeria infection (days 5-10 p.i.), when
Listeria is cleared and memory T cells are developing (days 10-30 p.i.), or during late memory (days 40-100
p.i.). A control, uninfected group of C57BL/6 mice will also be killed. Organs will be harvested for analysis.
Oral inoculation
with LIsteria
Kill for organ
harvest
5-100 days
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E.2.2 T cell chemokine receptor analysis:
1. C57BL/6 mice will be injected intravenously with TCR Tg cells (Procedure 2) that will act as a readily
detectable population of pathogen-specific T cells.
2. Next day, mice will be infected orally with LM (Procedure 1).
3. Mice will be killed by carbon dioxide administration either during active Listeria infection (days 5-10 p.i.), when
Listeria is cleared and memory T cells are developing (days 10-30 p.i.), or during late memory (days 40-100
p.i.). A control group of C57BL/6 mice (injected with TCR Tg cells but uninfected) will also be killed. Organs will
be harvested for analysis.
Oral
inoculation
with Listeria
Inject
TCR Tg
cells iv
Kill for organ
harvest
1 day
5-100 days
Experiment 3 (E.3): Enhancing migration of T cells to the gut following enteric Listeria infection
Dendritic cells (DCs) are white blood cells that activate T cells within the lymph nodes of the body, directing
them to migrate to the site of infection. Here, we will examine whether DCs from the intestine selectively direct T
cells to migrate to the gut during intestinal Listeria infection. If this is the case, vaccines can employ intestinal
DCs, or replicate their mode-of-action to enhance T cell migration to the gut, and thereby enhance the number
of gut-resident T cells that will in turn be able to control subsequent gut infections.
1. C57BL/6 mice will be orally infected with LM (Procedure 1).
2. At a time point during active infection (5-10 days post infection), mice will be injected intravenously with TCR
Tg cells (Procedure 2). A control, uninfected group of C57BL/6 mice will also be injected with TCR Tg cells. The
TCR Tg cells will have been cultured in the laboratory with dendritic cells isolated from uninfected C57BL/6 mice
or from C57BL/6 mice actively infected with LM.
3. Mice will be killed by carbon dioxide administration 12-48 hours later and organs harvested for analysis.
Inject
TCR Tg
cells iv
Oral
inoculation
with Listeria
5-10 days
Kill for
organ
harvest
12-48 hours
B.
B. PROCEDURES
This section describes the individual procedures that are combined together to form the experiments described
in Part A. The individual procedures are have been broken down into "Standard techniques" and "Experimental
procedures". Standard techniques include methods of inoculation and anaesthesia that are common to the
Experimental procedures.
Immediately following each procedure are listed:
• Any welfare issues that may arise for the mice undergoing that procedure, and
• The appropriate mouse monitoring that will be undertaken during the acute period following the procedure.
Acute experimental monitoring is performed by investigators as dictated by the particular procedure being
performed and the relevant Intervention Criteria (see attachments). Following the acute period, monitoring then
reverts to Standard Monitoring, described below.
All procedures will be performed in the laboratory of the Microbiology and Immunology Biological Research
Facility, adjacent to the room where the mice are housed.
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STANDARD MONITORING
Routine monitoring for mice will be a visual inspection for appearance and behaviour. For appearance, mice
should have a smooth coat (no ruffling of fur), regular breathing and absence of hunching or back arching. For
behaviour, mice should be alert, interacting with cage mates and moving around the cage. All mice included in
an experimental cohort are monitored weekly by the researchers, in addition to weekly monitoring by the animal
technicians.
STANDARD TECHNIQUES
Isoflurane inhalation administered by an anaesthetic machine
This anaesthetic can be used to render mice unconscious for approximately 2-3 minutes. Mice will be
transferred to a dedicated mouse box connected to an anaesthetic machine generating an isoflurane/oxygen
mix. Anaesthesia will be confirmed by slow breathing and the absence of a foot pinch reflex and eye-blink reflex.
Following the treatment procedure, the mice will be returned to a housing cage and monitored until they have
regained full consciousness and are moving normally about the cage (approximately 2-3 minutes).
WELFARE: Mice appear healthy and do not exhibit any signs or symptoms of distress during this procedure.
MONITORING: Mice will be monitored for 10 minutes after anaesthesia for signs of normal behaviour and
movement.
Oral inoculation
Mice will be anaesthetized using inhalation anaesthetic isoflurane. A maximum 200ul inoculum will be
administered to the mice using a 21G gavage needle (blunt ended) attached to a 1ml syringe. Care will be taken
to ensure the gavage needle is placed in the oesophagus and not the trachea. Prior to insertion of the gavage
needle, the distance from the nose to the stomach will be measured to determine the depth to which the needle
can be safely inserted.
WELFARE: Mice appear healthy and do not exhibit any signs or symptoms of distress during this procedure
when performed correctly.
MONITORING: Mice will be monitored for 10 minutes after inoculation and again the following day for signs of
normal behaviour and movement. If the mouse coughs or displays breathing difficulties, indicating that the
inoculum has been injected into the trachea by accident, the mouse will be immediately euthanized by carbon
dioxide administration.
Injections
Intravenous injections (iv) will be in the tail vein using a 26G needle, 1ml syringe, 200ul volume. A 200ul
volume is necessary to prevent cells from clumping together in the inoculum. Mice will be warmed for 5-15
minutes on a light box calibrated to ensure the cage temperature does not exceed 40°C in a 30 minute period,
and monitored with a temperature probe. The mouse will be restrained using a purpose designed brass or
plastic restraint.
Subcutaneous injections (sc) will be in the non-weight bearing area of both hind limbs (foot hock) using a 30G
needle, 0.5ml insulin syringe and maximum 25ul volume. Mice will be anaesthetized using isoflurane.
WELFARE: Mice appear healthy and do not exhibit any signs or symptoms of distress during the injection
procedures.
MONITORING: Mice will be monitored for 2 or 10 min following iv or sc injection, respectively, and again the
following day for signs of normal behaviour and movement and that the site of injection is not red or inflamed.
Killing mice by carbon dioxide administration
Using a CO2 cylinder fitted with a regulator and flow meter, carbon dioxide will be administered into the mouse
home cage at a slow fill rate of 20% volume per chamber size per minute. Where only some mice in a cage are
to be killed, they will be transferred to a clean IVC cage. Mice will be observed during the procedure. Mice that
will not subsequently be dissected will be subjected to a second procedure to ensure they are dead, i.e. cervical
dislocation or diaphragm rupture.
EXPERIMENTAL PROCEDURES
Procedure 1: Infection with Listeria monocytogenes (LM)
Strains of LM used in this project will be wild type LM and a recombinant LM that has been genetically
engineered to express a portion of another agent (e.g. HSV or chicken ovalbumin) that will stimulate the TCR Tg
T cells used in this project.
Mice will be weighed prior to LM infection to determine an experimental starting weight.
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Oral infection
The investigator performing this technique will remove food and water from the home cage for up to 5 hours
prior to oral infection of mice. Deprivation is required to reduce stomach acids, leading to increased
animal staff need to contact the researcher during the deprivation period. Mice will then be anaesthetised using
inhalation anaesthetic isoflurane. The anaesthetized mouse will be transferred to a Class II biosafety cabinet.
An inoculum containing 2 x 10^9 cfu LM in 200ul saline for primary infection or 2 x 10^10 cfu LM in 200ul saline
for secondary infection will be administered to the mice using a 21G gavage needle (blunt ended) attached to a
1ml syringe. The mice will be placed back into the home cage and ad lib food and water will be replaced by the
investigator. Mice will be monitored until they are fully conscious and have recovered, which usually does not
take more than a few minutes.
Intravenous infection
Following warming, mice will be placed in a tube restrainer. The tail vein will be located and 1000 cfu LM in
200ul saline will be injected using a 26G needle and a 1ml syringe. The mice will be placed back in the home
cage and monitored for 2 min to ensure they are moving and behaving normally.
Subcutaneous infection
Mice will be anaesthetised using inhalation anaesthetic isoflurane. The anaesthetized mouse will be transferred
to a Class II biosafety cabinet and injected subcutaneously into the non-weight bearing area of both hind limbs
with 10,000 cfu LM in 25ul saline using a 30G needle and 0.5ml insulin syringe. The mice will be placed back
into the home cage and monitored until they are fully conscious and have recovered, which usually does not
take more than a few minutes.
WELFARE: Infection of C57BL/6 mice with the stated doses of LM may cause transient, moderate symptoms of
infection, including ruffled fur, weight loss up to 15% of starting body weight, and reduced activity. These
symptoms do not last beyond 5-7 days after infection and mice recover fully.
MONITORING: Infected mice will be visually checked daily for 14 days for signs of ill health such as ruffled fur,
hunched up appearance, lethargy and loss of body condition. Should any of the mice show signs of ill health, the
mouse will be visually checked and weighed twice daily while symptoms last or until mice are killed (as
described above). After 14 days, LM will be completely cleared and mice will then be checked by standard
monitoring.
Procedure 2: T cell transfer
In order to study the T cell response we routinely transfer a small number of pathogen-specific T cells from TCR
Tg mice into other mice and subsequently examine the behaviour of these cells against a defined agent (e.g.
Listeria monocytogenes). This is useful because in a normal mouse the numbers of specific T cells are too low
to be detected.
- TCR Tg mice will be killed by carbon dioxide administration and the spleen and lymph nodes removed.
- In the laboratory, T cells will be enriched and, in some cases, activated by in vitro culture with dendritic cells
isolated from uninfected C57BL/6 mice or from mice actively infected with LM. In some cases the T cells will be
labelled in vitro with a fluorescent dye.
- Typically, up to 50 x 10^6 T cells will be transferred by intravenous tail vein injection into recipient C57BL/6
mice, which will then be used in experiments as detailed in Part A.
- T cells will be transferred either 1 day before infection (naïve T cells) or 5-10 days after infection (activated T
cells).
- T cells may be derived from one or more TCR Tg mice and co-transferred into recipient mice to compare
different types of T cells.
WELFARE: Our long experience with this procedure has proven that the injection of T cells is well tolerated by
recipient mice. In vitro manipulations of T cells prior to their injection does not lead to any detrimental effect on
the health or welfare of the recipient mice.
MONITORING: Mice will be monitored immediately after their injection and again the next day for signs of
normal behaviour and movement.
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C. MOUSE STRAINS USED IN THE PROJECT
Mouse strain
C57BL/6
TCR Tg
Used in Procedure:
1, 2
2 (killed for organs only)
Number requested
720
24
Description of genetic modification:
TCR Tg mice have been genetically modified so that all T cells have a T cell receptor (TCR) with the same
specificity. Some mice may express a second genetic modification such as a fluorescent tag (dsRed, Cyan,
GFP, YFP) or congenic mutation (Ly5.1) so the cells can be tracked, eg. gBT-I/dsRED. The generation of mice
with multiple combinations of the above genes has no additional implications for animal welfare. These mice are
only used to isolate T cells which are then used for experiments.
TCR Tg mice to be used in this project:
gBT-I: TCR for glycoprotein B of herpes simplex virus (HSV)
gDT-II: TCR for glycoprotein D of herpes simplex virus (HSV)
OT-I: TCR for chicken ovalbumin/H-2Kb
OT-II: TCR for chicken ovalbumin/I-Ab
4.
INVESTIGATORS & COMPETENCY
In the table below, identify individual investigators against the protocols/procedures that they will be
performing. For each protocol/procedure, indicate whether the investigator is ‘C’ (competent) or ‘T’
(needs training). Where an investigator is not performing a particular protocol/procedure, the associated
cell should be left blank. All Animal Facility Managers need to be named here if animals are to be located
in University animal facility premises. In addition, animal facility staff must be included if they will be
involved in performing procedures.
INVESTIGATOR
Investigator 1
Investigator 2
Investigator 3
Investigator 4
Animal facility
staff
Animal Facility
manager
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
T
T
C
C
C
C
C
C
C
C
N/A
N/A
N/A
C
N/A
N/A
N/A
5.
HOUSING OF ANIMALS
5.1
For each species/strain requested, complete a row in the table.
Species / Strain
Mouse, all animals
5.2
Procedure 2: T cell
transfer
Procedure 1:
Infection with
Listeria – IV, SC
Procedure 1:
Infection with
Listeria – oral
Killing mice by
carbon dioxide
administration
Anaesthesia by
isoflurane inhalation
Monitoring
PROTOCOL/PROCEDURE
Location
Microbiology & Immunology
Animal Facility, PDI
Housing {select one}
Standard
Grouping {select one}
Grouped
If relevant, provide further details of housing, including, as relevant, details of outdoor housing, any
special housing requirements, details of enrichment, etc. Where housing is not applicable, please
explain why.
Mice will be housed in the Techniplast GreenlineTM independently ventilated cage (IVC) system. These cages
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have a solid floor, filtered air and a food rack. Water is provided by a central system integrated into the rack or
sipper sacs™ (purpose designed plastic bags) which both use the same style nozzle for delivery. There will be a
maximum of five mice per cage. Environmental enrichment for nesting and play will include shredded paper,
tissues, cardboard cylinders, plastic boxes and wooden bars.
6.
REPLACEMENT
Replacement refers to methods that avoid or replace the use of animals.
6.1
Explain why it is necessary to use animals for the proposed work.
Currently, it is not possible to accurately measure a wide range of cellular immune responses in infected organs
of human individuals. Experimental mice are necessary for this research because the complex genetic and
immunological events that determine the outcome of bacterial infection can only be studied in vivo.
6.2
Provide evidence for the consideration of alternatives to animal use.
In order to study the peripheral T cell immune response, this work requires all the components of an intact
immune system including multiple cell subsets and physiological cell microenvironments that cannot be
recapitulated in vitro. The complexity of the immune response cannot be reproduced in vitro with human cells or
cell lines.
6.3
Provide justification for the choice of animal/s (species, strain, genetic modification, sex and age)
The immune response to Listeria monocytogenes has been well-characterised in C57BL/6 mice (by our
collaborators in the USA and in our department) so these mice are our animal of choice for investigating the role
of gut-resident T cells following enteric infection. Furthermore, the TCR Tg mice included in this study are on a
C57BL/6 background. The TCR Tg mice provide a population of easily detectable T cells that can be tracked
during infection through the presence of markers such as Ly5.1/Ly5.2 and fluorescent proteins. Both male and
female mice will be used, from 6 weeks of age.
7.
REDUCTION
Reduction refers to methods that minimise the number of animals required to achieve the aims of the work.
7.1
Provide statistical or other justification for the number of animals requested. Break down the total
number by procedures, treatments, repeats, groups, etc.
To examine the role of gut-resident T cells following infection with LM, a group of 5 animals per time point will be
used. Each experiment will be performed three times to confirm reproducibility. This will provide 15 data points
for each time point and ensure statistical validity with a two-way ANOVA.
In most experiments following infection with LM, up to three different time points will be examined. These
include:
Day 5-10 post infection - during active infection with LM
Day 10-30 post infection - when LM has been cleared and long-lived "memory" T cells (that protect against
reinfection) are forming and are circulating in the blood and/or are present in tissues.
Day 40-100 post infection - to determine whether memory T cells are still circulating and/or present in tissues.
Each experiment will also include a control group of uninfected mice.
In Experiments E.2.2 and E.3, TCR Tg cells will be isolated from different TCR Tg mice (gBT-I, gDT-II, OT-I or
OT-II) or TCR Tg cells will be cultured in the laboratory under different conditions prior to transfer into C57BL/6
mice. In our experience, one TCR Tg mouse is required to provide enough T cells for transfer into 10 recipient
C57BL/6 mice.
Experiment E.1:
C57BL/6 mice will be infected with LM by three different routes to induce T cells in different parts of the body
(gut, blood or skin). A control group will be uninfected. At three different time points following primary infection,
mice will be infected orally with LM (secondary infection) and will be killed at two different time points following
this infection for analysis.
Thus we request 5 mice x 4 modes of infection (3 routes; 1 uninfected) x 3 secondary infection time points x 2
analysis time points x 3 (times the experiment will be performed) = 360 C57BL/6 mice.
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Experiment E.2.1:
C57BL/6 mice will be infected orally with LM and will be killed for analysis at three different time points following
infection. A control, uninfected group will also be killed for comparison.
Thus we request 5 mice x 4 analysis time points (3 infection; 1 uninfected) x 3 (times the expt will be performed)
= 60 C57BL/6 mice.
Experiment E.2.2:
C57BL/6 mice will be transferred with TCR Tg T cells and infected orally with LM. C57BL/6 mice will be killed for
analysis at three different time points following infection. A control, uninfected group will also be killed for
comparison. Two different kinds of TCR Tg cells will be analysed and compared in this experiment.
Thus we request 5 mice x 4 analysis time points (3 infection; 1 uninfected) x 2 kinds of TCR Tg cells x 3 (times
the expt will be performed) = 120 C57BL/6 mice and 12 TCR Tg mice for transfer of T cells into C57BL/6 mice.
Experiment E.3:
C57BL/6 mice will be infected orally with LM and, during active infection, transferred with TCR Tg T cells. A
control, uninfected group will also be transferred with TCR Tg cells. The TCR Tg T cells will have been cultured
in the laboratory with dendritic cells under four different conditions. Dendritic cells will have been isolated from
either uninfected C57BL/6 mice or from C57BL/6 mice actively infected with LM.
Thus we request 5 mice x 2 analysis time points x 4 culturing conditions for TCR Tg cells x 3 (times the expt will
be performed) = 120 C57BL/6 mice and 12 TCR Tg mice for transfer of T cells into C57BL/6 mice.
In addition we will require 10 uninfected and 10 infected C57BL/6 mice for the isolation of dendritic cells x 3
(times the expt will be performed) = 60 C57BL/6 mice. Dendritic cells are a rare cell population hence it is
necessary to use 10 mice to obtain sufficient cells.
Table 1: Summary of numbers of mice required
Experiment
E.1
E.2.1
E.2.2
E.3
TOTAL
7.2
C57BL/6
360
60
120
180
720
TCR Tg
0
0
12
12
24
Have the animals been used in another project? If so, outline the cumulative burden and provide
justification for re-use.
Not applicable.
8.
REFINEMENT
How have techniques been refined to minimise impacts on animal welfare?
Investigators will be trained and competent in all animal techniques, including monitoring. All procedures have
been refined through previous projects to minimise pain and distress, and stringent Intervention Criteria have
been developed to manage welfare issues. Anaesthesia and inoculation procedures (oral, iv, sc) are relatively
quick procedures and mice appear to recover normal behaviour and movement very quickly. Mice will be
monitored immediately following these procedures and again the next day to ensure no detrimental effects.
Infection of mice with Listeria may cause transient, moderate symptoms of disease but mice will be monitored
daily according to the attached Intervention Criteria until Listeria infection is cleared (by day 14 post inoculation).
Disease symptoms are not expected to last beyond 5-7 days after infection. Should mice show signs of
moderate severity according to the Intervention Criteria, frequency of monitoring will increase to twice daily,
additional monitoring criteria will be included (weighing), and investigators will seek AFM/AWO advice where
appropriate for animal care. If symptoms progress to Severe (unexpected), mice will be immediately killed. Ad
libitum food and water will be provided at all times (except for a 5 hour period prior to oral gavaging) and home
cages will be enriched with materials for nesting and play, such as shredded paper, tissues, cardboard
cylinders, plastic boxes and wooden bars.
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9.
OVERALL JUSTIFICATION
Explain how the potential impacts on the wellbeing of animals in this project are justified by the potential
benefits of the proposed work.
Infectious diarrhoea, caused by bacteria and viruses that infect through and replicate within the intestine, is a
major cause of mortality worldwide. Herein, we wish to develop a vaccination strategy centred on T cells that will
protect against intestinal infection by Listeria monocytogenes, a food-borne bacteria that causes wide-spread
disease in humans. Specifically, we will investigate if bacteria-specific T cells lodged within the gut-lining can
provide effective protection against re-infection, and thereby limit subsequent bacteria spread and disease. This
work, using a well-established mouse model, will provide information about what is needed to protect against gut
infections. Experimental mice are necessary for this research rather than cell lines because the complex genetic
and immunological events that determine the outcome of bacterial infection can only be studied in vivo. Our
study will examine if targeting immune T cells to the intestine provides optimum protection against intestinal
infection and determine what molecules contribute to intestinal targeting and residency. Given that current
vaccines do not elicit such "gut-resident" immune cells, our findings will have implications in rethinking the
strategy for vaccines against enteric infections.
As described in this application and in question 8, appropriate training, monitoring, and husbandry will minimize
the impact on the animals used in this study.
10.
GLOSSARY
Provide descriptions/definitions of abbreviations and scientific terms in language that can easily be
understood by the lay reader.
Scientific Term
T cells
Pathogen
Enteric infection
TCR
transgenic
Memory T cells
Dendritic cell
in vitro
in vivo
cfu
Chemokines
Chemokine receptor
Lay Description
An immune cell type that provides antigen (bacteria) specific immunity
and drives clearance of a bacterial infection
An infectious agent such as a virus, bacterium, prion, fungus, viroid, or
parasite that causes disease in its host
Infection of the intestine
T cell receptor - a molecule found on the surface of T lymphocytes (or
T cells) that is responsible for recognizing antigens
Containing a gene or genes transferred from another species
A persisting pool of T cells that are generated after initial encounter
with a microbial agent or vaccine that are subsequently able to respond
faster and provide protection against subsequent encounters with the
original microbial agent
Rare cell of the immune system that engulfs microorganisms and
activates T cells
Refers to a biological function or event that occurs outside of a living
organism, typically in test tubes, culture dishes or elsewhere
Refers to a biological function or event that occurs inside of a living
organism
Abbreviation for "colony forming units", a standard unit to express live
bacterial number
Proteins that are released from tissues to recruit T cells from the
circulation into tissues, such as the gut lining
Chemokine-specific receptor expressed on the surface of T cells
Office for Research Ethics and Integrity | Animal Ethics
New Project Application form | Version 1.1 | July 2015
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