Screening for infectious agents / Individual qualification of animal sources

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Screening for known infectious agents before and during manufacture of fetal cell transplants consists of:

1/ continuous evaluation of health status of the entire closed colony of rabbits, all marked and identified by number since birth, with a complete documentation;

2/ continuous evaluation of all ancestors (for at least 3 generations) of ‘pre-scheduled’ rabbit females, ‘sources’ of all fetal and newborn rabbits needed for manufacture of a batch of fetal cell transplants, marked by a batch number

3/ continuous evaluation of health status of ‘pre-scheduled’ pregnant rabbit female

4/ observation of health status of fetal and newborn rabbits;

5/ daily macro- and microscopic evaluation of each primary tissue culture;

6/ final assay of each fetal cell transplant by:

a/ histologic evaluation, whereby bacteria can be identified as well,

b/ bacteriological testing,

c/ bacterial endotoxin test.

The screening program relies on the following data:

1/ in-depth knowledge of epizootiology of country (or ecologic region) of manufacture,

2/ in countries with established rabbit farming, the pathogens are found in adult rabbits only (there are no pathogens in fetal or newborn rabbits that are used in BCRO method of preparation of fetal precursor cell transplants,

3/ fetal rabbits are actually sterile,

4/no retroviruses have been found, to date, in rabbits,

5/ there is no known transmission of viruses from rabbit to man.

Monitoring of closed colony for infectious agents that are ‘not apparent clinically’ is based on utilization of all adult rabbit females as ‘sentinel animals’ for recognition of any new infectious agent or disease not known today, as per ‘PHS Guidelines on Infectious Disease Issues in Xenotransplantation’ of January 19, 2001:

A/ All adult rabbit females are examined by the doctor of veterinary medicine at the beginning of a breeding cycle, and every 6 months thereafter, and the following specimens are collected:

1/ blood samples of serum, and leukocytes, to be kept in liquid nitrogen for 5 years for future microbiological testing,

2/ specimen for blood count and peripheral blood smear,

3/ specimen of feces to test for parasites,

4/ nasal swab for microbiological culture.

B/ After death all adult rabbit females are subjected to a full autopsy, and specimens of blood, liver, spleen, bone marrow, brain, are obtained, to be stored for 5 years in liquid nitrogen, for the future microbiological and other tests necessary for identification of viruses capable of recombination, complementation or pseudotyping, in an investigation of new unexpected disease or of infectious agents that may not be apparent clinically today. The results of the autopsy are placed in the animal’s health records, and archived. In case that autopsy reveals an infection pertinent to the health of the fetal precusor cell transplants’ recipient, this finding is immediately communicated to a clinical center, where fetal cell transplants originating from the fetuses & newborns delivered by this rabbit female were implanted in the named patient’s body.

The daily evaluation of primary tissue cultures, macroscopically and under inverted microscope, and the final assesment of the quality of culture by a histological examination, identifies a presence of microrganisms as well. Routine bacteriological cultures on fluid thioglycollate medium and soybean-casein digest medium and a bacterial endotoxin test are done for each fetal cell transplant. In case of any suspicion of contamination the respective fetal cell transplant must be discarded without delay. In order to maintain their clinical effectiveness, fetal cell transplants have to be shipped immediately after their release and there is no time to make any extensive investigation and testing sufficient for making the decision whether to release the particular fetal precursor cell transplant for an implantation..

Colony Health Maintenance and Surveillance.

The qualification of closed colony as a source of newborn and fetal rabbits for the preparation of fetal precursor cell transplants is based on the facts that

- it fulfills all criteria of a closed colony, and

- an adequate surveillance programm for infectious agents have been in place for at least 3 years.

Documentation of the colony health maintenance and surveillance programm has to be maintained for at least 5 years at the closed colony, the longer the better. All incidents that could have affected the colony health have to be recorded, such as breaks in the environmental barriers, diseases outbreaks or sudden animal deaths .

Individual Source Animal Screening and Qualification.

The qualification of individual fetal and newborn rabbits as individual source animals for preparation of fetal precursor cell transplants are based on the facts that

- they originate from a closed colony;

- they originate from documented pure lines;

- their health is documented;

- the health of their immediate ancestors is documented;

- the health of the colony they originate from is documented.