GNOTOBIOTIC ANIMALS

Gnotobiosis (from Greek roots gnostos "known" and bios "life") refers to an engineered state of an organism in which all forms of life (i.e., microorganisms) in or on it, including its microbiota, have been identified.[1] The term gnotobiotic organism, or gnotobiote, can refer to a model organism that is colonized with a specific community of known microorganisms (isobiotic or defined flora animal) or that contains no microorganisms (germ-free) often for experimental purposes.[2][3][4][5] The study of gnotobiosis and the generation of various types of gnotobiotic model organisms as tools for studying interactions between host organisms and microorganisms is referred to as gnotobiology.
Gnotobiotics is the scientific study of animals or other organisms that are raised in germ free environments or ones that contain only specifically known germs. The gnotobiotic laboratory animal is potentially a very valuable tool for investigating any suspected interaction between the host and its associated microflora or between different components of that flora. However, like many other good ideas, the production of gnotobiotes is simple in concept but complicated in execution. In the early stages the greatest obstacles to the general use of germ free animals were the expense and the restricted amount of space that could be maintained free from contaminants. Nowadays, with modern isolators and facilities it is easier to produce gnotobiotic animals at relatively modest price.
Gnotobiotic animals or Gnotobiote are an animal stock or strain in which only certain known strains of bacteria and other microorganisms are present. Technically the term also includes germ free animals as the status of their microbial communities is also known (Reyniers, 1959). Gnotobiotic animals are derived by aseptic hysterotomy or hysterectomy, embryo transfer or sterile hatching of eggs and are continuously maintained using aseptic technique where the microbial status of the animal is fully defined; includes both germ free and defined flora animals. Animals reared in a gnotobiotic colony are devoid of normal flora, has poorly developed immune systems, lower cardiac output, thin intestinal walls, low antibody titers low metabolism rate and high susceptibility to infectious pathogens (Wostmann et al., 1996). Lower amounts of serum gamma globulins have been observed in germ free animals of several species and the quantity increases on association with microorganisms. Nuttall and Thierfelder are considered pioneers of gnotobiotics and germ free research. Germ free mice have adapted anatomically and physiologically to life without microbes
The gnotobiotic principles used in the production of infection free laboratory animals evolved from the efforts to rear and study animals in the absence of microbes or in association with one or more pure cultures of microbes . The gnotobiotic animal is potentially a very valuable tool for Scientitist.

1. Presented by
Dr. Subhrajitdas.
B.V.SC & AH, CAU, Mizoram
M.V.Sc(LPM), DSVCKV, Chattisgarh

2. INTRODUCTION
1.Gnotobiosis(gnotos+bios)
condition of life in which only one type of organism are
present
2.Gnotobioticanimals(gnotos+biota=known flora or
fauna)
Those animals with certain strains of bacteria & other
microorganism are present.
3.Laboratoryanimal
Those animals are exclusively used for research purpose.

3. Classification
Gnotobiotic
animals
Axenic
animals
Asociated
animals

4. Cont...
1.Axenic animals = Germ free.
Free of all detectable microorganism
Pups obtaineswith sterile hysterectomy or hysterotomy
into sterile atmosphere of the isolator.
2.Asociated animals:
Derived from axenicanimas colonising them artificially
with one or more species of micro organism.
Monoxenic,dixenic,polyxenic

5. HISTORY
The concept of germ-free was recognized more than a century ago by Louis
pasteur(1885),although he concluded that bacteria –free existence is
impossible.
Ten year later in 1895,Nuttle and Thierfelderat berlinUniversity produced
the first GF animal(guinea pigs) which survived for as long as 13days.
However ,Due to the lack of knowledge concerning nutrition, it took 50 more
year to produce first GF rat colonies were established in the late 1940s.

6. Technique
Caesarianin the sterile environment.
Transfer the newborn in a sterile isolator.
Provide sterilized air,water,food.
Exposeto those organism that resercher
wish to have in the animal

7. Equipments
Sterile isolator and set up for rearing germ free mice
Transfer chamber compatible with the isolator
Autoclave with validated programs for the sterilizatiionof
supplies and drinking water inside the transfer chamber
Surgical equipment :
LuteolilVet, 50 mg/ml, 0.1 ml, room temperature, CeVa,
Holland
VirkonS, 1% solution, room temperature, AntecInt. Ltd.

8. Isolator Technology
Isolators provide physical barriers that allow creation of a sterile
environment. These devices have an air supply, air inlet and
outlet, transfer port, and arm-length gloves, as well as a special
tank filled with disinfectant and used for the transfer of mice in and
out (Figs. 1 and 2).
Bedding, food, water, and equipment, including cages, must first
be sterilized (autoclaved) and are then put into the isolator through
the so-called sterile lock.
Air is sterilized upon entry and exhaust by mechanical filtration
under positive pressure.
The transferof animals in and out of the isolator is usually carried
out via autoclave jars (Fig.3).
The common practice is to separate multiple mouse strains and
multiple inoculation experimental groups in separate isolators [3],
altogether increasing the cost and space for such experimentation.

9. Fig 1-Gustafsson steel isolator

10. Fig 2-composition of plastic isolator

11. Fig 3-Transfer of mice from inside the isolator

12. STEPS
Ensure availability of isolator reared,germ-free surrogate mother with newborn
pups(<5 days old)
Day 0
Set up the relevant mating of mouse strain to be converted to germ-free
status.
Day 1
Check for mating plug and identify the donor female(s) for the experiment.
Day 17
Give pregnant donor female(s) a subcutaneous injection of Promon(5 mg/0.1
ml).
Day 18
Carefully following the protocol for isolator entry procedures, transfer the
sterile instruments and supplies required for surgery into the isolator in
which the surrogate female(s) are housed. Prepare the hysterectomy
suite/surgical transfer chamber: fill up the reservoir with 1% VirkonS, sterilize
the surgical compartment and ventilate it overnight.
Day 20
Transfer water, paper towels and surgical instruments from the isolator to the
sterilized compartment of the transfer chamber.

13. Method…continued…
Working in the Non-sterile compartment of the surgical
transfer chamber.
Sacrifice of the donor female by cervical dislocation &
submerge the whole animal in the 1% virkonssolution for 1
minute.
Use sterile scissors to open the abdomen ,wear sterile
gloves.
Clamp the top of each uterine horn & the base of the uterus
close to the cervix.
Cut out the uterine package & place it in the transfer
chamber reservoir filled with 1% virkonsfpr1 min.

14. Method….continued…
Inside the sterile compartment of the transfer chamber;
Rinse the ‘uterine package’ with sterile water to remove the VirkonS.
Open the ‘uterine package’ with scissors and take out the pups.
Stimulate breathing of the pups while cleaning them with dry paper
towel.
When pups are breathing normally and have gained a ‘healthy’ skin
colortransfer them to the isolator housing the surrogate mother.
Take out her own litter and mix pups from both litters outside the
cage.
Remove some of the original pups so that the surrogate mother has
the same number of pups to feed. Gently rub the pups with bedding
material from the surrogate mother’s cage soaked in water. Put the
mixed litter together with the foster mother.
Check for adoption not earlier than 24 hours after transfer.
Monitor a microbiological status of the isolator and the animals it
houses 3 weeks after transfer.

15. Usesof gnotobioticanimals :
To study the effect of symbiosisbetween animal &
microorganism.
To study the reaction on thediet& its development on
the diet.’
Acts as a source of sterileorgans, tissues for cultivation.
To study thedefencemechanism.
To study the aetiologyof infectious diseases.
To study the process of physiological ageing.
To study the wound healing process.
Used in toxicology, pollution control & vaccinetest.

16. Disadvantages
There are some necessary microorganism which doesn’t
exist in gnotobioticanimal. Example-gut flora.
Maintenance of these animal is costly.
Any lack of dietary nutrition may lead to death of the
animal.
Germ free animal tend to have a short life.
These animal lack immunity to pathogen.
The animals have a low cardiac output, which makes
them very sensitive to shock.
Introducing several microorganism at once may result in
shock.

17. Conclusion
Gnotobioticanimal studies and experiments
provide opportunities for understanding how
Alzheimer's disease affects the brain, and for
studying potential new treatments.

18. Reference
EMMA protocol -production of germ-free mice
Protocol for generating germ-free (axenic) mice
using caesarean section rederivation.
Use of Germ-Free Animal Models in Microbiota-
Related Research MahaAl-Asmakh1,2* and Fahad
Zadjali3
Book:-Care & Management of Laboratory & Pet
Animals –Y.B.Rajeshwari

19. THANK YOU
To contact
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