Closed colony of source animals
Fetal and newborn rabbits used for preparation of fetal precursor cell transplants in the described method have to originate from closed colony of rabbits, with documented lineages for >3 years (>30 generations), bred and reared in captivity, and not exposed to vectors of infectious agents.
All rabbits have to be marked by an identification number for life, that relates to their ancestors and to its genotype group.
In order to qualify as a source of fetal and newborn rabbits to be used for preparation of fetal precursor cell transplants, a closed colony has to be set up in accordance with guidelines of WHO and AAALAC, have an adequate surveillance program for infectious agents for >3years (if less than 3 years, then immunological assays for all known infections of rabbits have to be carried out, and their results must be negative), and live attenuated vaccines must not have been utilized for infection prevention .
Guidelines of WHO and AAALAC for closed colonies, that are usually duplicated by the laws and regulations in individual countries, consist of:
1/ Criteria for animal admission:
New rabbits are admitted into the closed colony only from another closed colony that is proven to
- not contain animals with any sign of any disease, infectious or non-infectious;
- contain only animals that are marked and identified for a minimum of 25 generations of ancestors;
- have documentation about the useful features of kept animals, and that of their ancestors;
- maintain acceptable nutritional and microclimactic conditions.
All new rabbits are placed under a quarantine for 4 weeks, and if no sign of disease appears, then introduced into a closed colony.
2/ Disease monitoring programm:
Veterinary technicians observe rabbits daily. They watch for the amount of feed used, vitality and the behaviour of animals. If anything untoward is observed, the veterinarian is immediately called in to make a decision about the quarantine.
The rooms for quarantine are separate from the main rabbit pavillons. (Healthy animals transferred from other colonies are not mixed up in the same quarantine room with diseased animals under observation.)
After the transfer, in the quarantine room, specimens of blood for hematology, and possibly toxicology, nasal swab and feces specimen for microbiology, etc., are taken.
No animal is ever returned from quarantine for diseased rabbits back to the colony.
In case of death, a full autopsy is carried out and specimens taken for bacteriological and toxicological testing.
Rabbit epizootiology of the country, or ecologic region of the country, has to be well studied, so that the disease monitoring program is targeted.
Every 6 months a nasal swab is obtained from randomly selected group of female rabbits, which is sent for a microbiological examination to the accredited veterinary laboratory.
3/ Criteria for the isolation or elimination of diseased animals:
At the first sign of any disease the animal is quarantined, and from quarantine never ever returned back to the colony.
As a matter of policy the use of antibiotics and other medications must be minimal. The organizational, zootechnical and nutritional regimen has to be set-up so that there is no need for a regular medicamentous treatment.
4/ Criteria for health screening and surveillance of humans entering the facility: Veterinary technicians in immediate contact with rabbits change before entering the rabbit hall completely from underwear up into work clothes and shoes. Upon departure they change again into street clothes/shoes. Other people not working in the rabbit hall are prohibited from entering.
All personnel undergoes yearly physicals with chest x-rays, complete blood count and blood chemistry.
5/ Facility cleaning arrangements:
There must be a continuous cleaning of cages and rabbit halls. A scheme of cleaning of individual sectors has to be devised, whereby the on-going programms is not interfered with. The empty space is mechanically cleansed by water under pressure, disinfected chemically and difficult to reach corners by propan flame.
The quality of cleansing is controlled by regular collection of swabs from the surfaces critical from the viewpoint of contact with animals.
6/ The source and delivery of feed, water and supplies:
Feed must be made exclusively from components of plant origin. Although its composition is controlled by manufacturer, it has to be re-checked in the closed colony by appropriate analytic procedures.
Rabbit halls have to be connected to a source of drinking water for human consumption, that fulfills all public health criteria, bacteriological and chemical.
Feed components for the entire colony must be documented. No recycled or rendered animal material must be used in the feed. All components of feed (peas, barley, dried grass, rye, etc.) have to be produced without chemical insecticides, or herbicides, and with only a minimal use of chemical fertilizers. A vitamin/mineral mixture of prescribed combination, guaranteed by the manufacturer, must be added to the feed pellets.
All feed should be from local production companies, imports are to be avoided.
7/ Measures to exclude arthropods and other animals:
Rabbit halls must be protected by mechanical barriers against insects and rodents.
8/ Animal transportation:
There is no transportation of rabbits while in closed colony, with the exception of being taken to the quarantine room for diseased animals.
The quarantine rooms have similar technological, micro-climactic and nutritional conditions as the main rabbit halls, but are isolated and separate from rabbit halls.
For the transportation of newborn rabbits, or pregnant rabbit females to be used as a source of rabbit fetuses, to the manufacturing facility, see below.
9/ Dead animal disposition:
Dead animals must be placed in transport cages, that are moved out of the rabbit hall and handed to the transportation personnel in such a way that the transportation personnel does not enter the interior of the rabbit hall at any time. Before any further use, the transport cages are mechanically cleaned and chemically disinfected.
Hormonal stimulation for impregnation must be avoided if fetuses / newborns are to be used for manufacture of fetal precursor cell transplants.
Pregnant females deliver newborns in the delivery boxes, filled with micro-biologically verified straw.
The movement of rabbits is always ’one way’, i.e. ‘all in - all out’. A necessary quantity of rabbit females is ‘pre-scheduled’ to deliver within 24 hours of the starting day of the primary organ cultures.
On the next day all newborns of these females are taken out of closed colony and transported to the manufacturing facility.
The pregnant female(s) designated to be a source of rabbit fetuses for the production of the same batch of fetal cell transplants are taken out of colony and transported to the manufacturing facility in the same fashion.
Transportation of newborn rabbits to the production facility is carried out in the closed boxes, in a passenger car. Newborn rabbits are taken out of their delivery boxes and immediately placed into the transportation boxes, that are opened only in the manufacturing facility, by the personnel attired in sterile gowns, with masks and sterile gloves.
Pregnant rabbit females, designated to be a source of rabbit fetuses, are removed from closed colony one to two days before delivery date and taken to the manufacturing laboratory in transport cages by car.
The distance from the closed colony to the manufacturing laboratory must not exceed 20 - 30 km (12 - 18 miles). Too long transportation is extremely stressful for rabbits, as is evident during the autopsy, that disqualifies too many of them as animal source for fetal precursor cell transplantation.
No rabbit taken out of closed colony is ever returned back.
All age groups of rabbits in the colony are individually followed insofar the health and production usefulness is concerned. Identification numbers of each animal are interconnected with the system of dual evidence. Each animal has a record card on its cage, which contains all data observed during its life. The same information is entered into the computerized data base, which contains also the genetic relationships of individual animals within the same generation and between generations. Notes of all visits by the veterinarian, and all other medically important data are entered in these records as well.