APPENDIX 12
INJECTING AND BLEEDING RABBITS
Rabbits used for antiserum production should be healthy and 6-12 months old. Label each animal with an ear tattoo or tag. Maintain individual records for each rabbit of all treatments to which the rabbit is subjected.
During ear (intravenous) injections, intramuscular injections, and trial bleedings, the rabbit must be restrained (immobilized). The recommended method is to roll the rabbit tightly into a large towel. The fore and rear limbs must be well secured by the towel. For intraperitoneal injections, the animal may be strapped to a rack or held on its side by another person. During cardiac puncture, a bleeding rack is used to hold the rabbit on its back (Figure A.12). Another approach is to sedate the rabbit with an injected tranquilizer such as Rompun [(Xylazine), Haver‑Lockhart Bayvet Division, Cutter Laboratories, Inc. Shawnee, Kansas, USA]. The use of ether or chloroform should be avoided.
The following schedules have been used successfully for antisera development in rabbits.
Schedule 1: (Schmidt, Bankole, and Bohlool, 1968)
DAY PROCEDURE
1 Inject 0.5 ml intravenously (IV)
2 Inject 1.0 ml IV
3 Inject 1.5 ml IV
7 Inject 1.5 ml IV
8 Inject 2.0 ml IV
9 Inject 2.0 ml IV and 2.0 ml subcutaneously (SC)
16 Test bleeding and titer determination
18 Cardiac bleed (30‑50 ml)
25 Inject 2.0 ml SC
32 Cardiac bleed (30‑50 ml)
39 Inject 2.0 ml SC
46 Cardiac bleed (30‑50 ml)
Schedule 2: (Dudman, 1964)
DAY PROCEDURE
1 Inject 1 ml of mixture of equal parts culture suspension and Freund's complete adjuvant (IM)
28 1 ml IV (antigen alone)
30 Bleed from ear 10‑20 ml
32 Bleed from ear 10‑20 ml
34 Bleed from ear 10‑20 ml
Schedule 3: (Somasegaran, unpublished)
DAY PROCEDURE
1 Inject SC 1 ml of emulsion of equal parts of antigen suspension and Freund's complete adjuvant
14 1 ml IV (antigen suspension alone)
28 Test bleeding and titer determination
30 Cardiac bleed (30‑50 ml)
37 Inject 1.0 ml IV
44 Cardiac bleed (30‑50 ml)
An intramuscular injection (IM) is used to start the immunization schedule (Exercise 6). Immobilize the rabbit by rolling it tightly into a large towel. Free one of the rear legs, and use alcohol to swab a small area of the skin covering the thigh muscle. Insert the needle about 1.5 cm into the muscle and inject. A large gauge needle (20G) is recommended to introduce the emulsion quickly and reduce the animal's discomfort.
Subcutaneous (SC) booster injections are usually given to maintain the antibody titer. Inject the antigen under the skin in the shoulder area. Use a 3‑5 ml syringe fitted with a 22 gauge needle.
Intravenous injections are given into the marginal ear vein of one ear. Expose the vein by shaving a small section of the ear with a razor blade. Swab the shaved area with alcohol (70%) and inject the antigen with a 1‑2 ml syringe fitted with a narrow gauge (25G) needle. If the schedule calls for several consecutive injections, make the first injection at the distal end of the ear. Progress toward the base of the ear with each successive injection.
For test bleeding, extract blood from the ear not used for injections. Shave a small area along the marginal ear vein, and swab the area with alcohol (70%). To prevent blood from spreading into the fur, apply vaseline around the area to be nicked. Use a scalpel with a small pointed blade (#11) and make a small nick in the vein. Collect 1‑2 ml of blood in a test tube. Stop the bleeding by applying light pressure to the injury with the thumb and forefinger. If additional bleedings are found necessary, progressively nick the ear closer to its base.
Alternatively, blood may be drawn from the marginal ear vein with a 1‑2 ml syringe equipped with a 26G needle.
There are various methods of extracting larger volumes of blood from rabbits. Among those frequently practiced are the cutting of the jugular vein, ear bleeding with the help of a vacuum, and cardiac puncture. Cardiac puncture (Figure A.13) is recommended here because it is fast and efficient.
The rabbit is tied to the inclining bleeding rack. The area above the sternum is shaved and swabbed with 70% alcohol. The blood is extracted with a large syringe (50 ml) fitted with a 18G needle and emptied into a sterile screw capped tube. About 50 ml blood can be taken from a ten to twelve pound rabbit without endangering the animal's life.
A bleeding rack may be built by nailing two wooden rails to a board (Figure A.12) and elevating one side with a wooden support to provide an incline of approximately 12°. The distance between the rails should be 4‑6 cm, depending on the neck size of the rabbits used. The rabbit's head is held by the rails at the upper end, while the legs are tied to a cleat at the lower end.
The Bellco rabbit bleeding apparatus (Figure A.13) is another convenient means of obtaining large quantities of blood from a rabbit. Bellco's instructions provide the following information:
Equipment required: Vacuum pump (or line), a sharp razor blade, receptacle (culture tube or flask with appropriate size rubber stopper), short piece of heavy rubber or plastic hose for attachment to vacuum line.
The ear of the animal is disinfected, a single slit is made through the marginal ear vein, and the ear is inserted into the large opening of the apparatus. The vacuum line is opened gradually until a vacuum lock is obtained on the head of the animal. Immediately the blood begins to flow in a steady stream. As much as 50 ml can be obtained in one minute without any sign of trauma to the animal.
The entire rabbit bleeding apparatus is autoclaved, the ear of the rabbit is treated with a disinfectant, and only one tube is used for each animal.
Figure A.12. A bleeding rack
Figure A.13. The BELLCO #5640-1111 rabbit bleeding apparatus as shown on the manufacturer's instruction sheet.
Figure A.14. Collecting blood from a rabbit by cardiac puncture:
a) Rabbit secured to bleeding rack
b) Drawing the blood
c) Close-up of draw