Before diluting vaccines, each vial should be carefully inspected, particularly regarding the name, expiration date, dosage, seal integrity, damage, and moisture absorption. For vaccines requiring special diluents, the specified diluent must be used. Other vaccines can generally be diluted with physiological saline or distilled water. The diluent should be cool, which is especially important during hot weather. The amount of diluent used must be calculated and measured carefully and accurately. The dilution process should be protected from light, dust, and performed under aseptic conditions, especially for injectable vaccines, which require strict aseptic techniques. The dilution process should generally be carried out in stages, and the vaccine vial should be rinsed 2–3 times with the diluent. Diluted vaccines should be used as soon as possible, and unused vaccines should be refrigerated in a refrigerator or ice water bucket.

For diluting Marek’s disease vaccines stored in liquid nitrogen, extra caution is required. If the manufacturer provides an operating procedure, it must be strictly followed. If no procedure is available, the following precautions may be referenced:

I. General Requirements  

1. Vaccines stored in liquid nitrogen must be managed and diluted by designated specialized technical personnel.  

2. Regularly measure and record the liquid nitrogen level in the tank, and replenish it promptly when insufficient.  

3. Liquid nitrogen tanks should be stored in safe locations, maintaining a certain distance from dormitories, offices, warehouses, etc.  

4. When transporting vaccines with liquid nitrogen tanks, dedicated vehicles must be used. Passenger transport vehicles are not permitted. If long-distance transport such as by train is required, relevant departments must be notified, and corresponding preventive measures should be taken before transport.

II. Vaccine Dilution Process  

1. Operators should first wear protective masks and gloves.  

2. Diluents should normally be stored at 4℃, and the temperature of the diluent before dilution should be 15–27°C (follow the manufacturer’s instructions).  

3. Prepare a 15–27℃ water bath (bucket) as required by the vaccine manufacturer, along with 1–2 long-handled tongs, ice cubes, trays, buckets, tap water, syringes, 18-gauge needles, etc.  

4. Open the liquid nitrogen tank, remove one vaccine vial, and quickly return the remaining vials to the tank.  

5. Immediately place the removed vaccine vial into the prepared warm water bath to thaw it quickly.  

6. After the vaccine is completely dissolved, immediately dry it with a cloth, shake the vial to ensure no vaccine liquid remains in the neck, and break the vial at the neck as far away from the operator’s face and body as possible.  

7. Take a syringe with an 18-gauge needle, draw 1–2 ml of diluent at 15–27℃ (follow the manufacturer’s instructions), then draw the vaccine liquid into the syringe. Gently mix and inject it into the diluent bottle. Then, draw diluent to rinse the vaccine vial 2–3 times, and add the rinse liquid to the diluent bottle.  

8. Gently shake the diluent bottle containing the vaccine to ensure even distribution of the vaccine.  

9. Keep the diluted vaccine at 15–27℃ (follow the manufacturer’s instructions), and maintain this temperature range during injection.  

10. Diluted vaccines must be used within one hour of dilution.

V. Vaccination Routes  

Operational errors during immunization are one of the common causes of immunization failure. The advantages, disadvantages, and precautions for different vaccination routes are as follows:

I. Drinking Water  

Drinking water immunization avoids the need to catch each bird, reducing labor and stress. However, this method is influenced by many factors. Key operational precautions include:  

1. The vaccine must be a high-efficacy live vaccine.  

2. The water used should be cool and must not contain any substances that could inactivate the vaccine virus or bacteria.  

3. During drinking water immunization, feed should not contain any drugs that could inactivate the vaccine virus or bacteria.  

4. Add 0.1%–0.3% skimmed milk or sorbitol to the water to protect the vaccine’s potency.  

5. To ensure each chicken consumes a sufficient amount of vaccine uniformly within a short time, water should be withheld for 2–4 hours before providing the vaccine water (depending on ambient temperature).  

6. The amount of water used to dilute the vaccine should be determined based on the chickens’ age and room temperature, ensuring the diluted vaccine is consumed within 1–2 hours.  

7. Ensure sufficient drinkers are available so that over two-thirds of the flock can drink simultaneously.  

8. Drinkers should not be placed in direct sunlight. In windy or dusty conditions, all drinkers should be moved indoors.  

9. During hot summer weather, drinking water immunization is best completed in the morning.

II. Eye and Nose Drops  

If performed correctly, eye and nose drop immunization can be highly effective, particularly for vaccines against respiratory diseases. However, this method requires more labor and causes some stress. Slight carelessness can lead to failure. Key precautions include:  

1. The diluent must be distilled water or physiological saline, or at a minimum, cooled boiled water. Do not arbitrarily add antibiotics or other chemical drugs.  

2. The amount of diluent must be accurate. It is best to pre-test the dropper or needle to determine the number of drops per ml before calculating the actual amount of diluent needed.  

3. For accuracy, handle one chicken at a time; do not handle multiple chickens simultaneously.  

4. Before administering the drops, position the chicken’s head and neck horizontally (with one eye and nostril facing up and the other down), and use a finger to block the downward-facing nostril.  

5. After administering the drops, pause briefly to ensure the vaccine liquid is fully absorbed before gently releasing the chicken.  

6. Ensure separation between vaccinated and unvaccinated birds to avoid mixing.  

7. To reduce stress, administer the vaccine in dim light at night or during the day with doors and windows closed and lighting reduced.

III. Intramuscular or Subcutaneous Injection  

Intramuscular or subcutaneous injection ensures accurate dosing and reliable results but requires more labor and causes greater stress. Key operational precautions include:  

1. The vaccine diluent must be sterilized. Do not arbitrarily add antibacterial drugs.  

2. The dilution and injection volume should be appropriate. Too small a volume increases operational error, while too large a volume complicates the process. Generally, 0.2–1 ml per bird is suitable.  

3. When using automatic syringes, frequently verify the discrepancy between the scale volume and the actual volume to avoid significant dosing errors.  

4. Syringes and needles must be sterilized before use.  

5. Subcutaneous injections are generally administered in the dorsal neck region, while intramuscular injections are given in the breast muscle or the muscle-rich area near the shoulder joint.  

6. The direction and depth of needle insertion should be appropriate. For subcutaneous neck injections, the needle should be directed backward and downward, parallel to the longitudinal axis of the neck. The insertion depth for chicks is 0.5–1 cm, and for older birds, 1–2 cm. For breast muscle injections, the needle should be roughly parallel to the breastbone, with an insertion depth of 0.5–1 cm for chicks and 1–2 cm for older birds.  

7. After injecting the vaccine, slowly withdraw the needle to prevent leakage.  

8. During injection, shake the vaccine bottle continuously to ensure uniformity.  

9. Vaccinate healthy flocks first, followed by potentially healthy flocks, and finally diseased flocks.  

10. The need for one needle per bird and disinfection of injection sites can be determined based on practical circumstances. However, needles used to draw the vaccine and those used for injection must be strictly separated. Hygiene should be emphasized to prevent the spread of infectious diseases or local infections at the injection site.

IV. Aerosol Immunization  

Aerosol immunization saves significant labor and can be highly effective if performed correctly, especially for vaccines targeting respiratory diseases. However, it can cause stress and potentially trigger chronic respiratory diseases. Key precautions for aerosol immunization include:  

1. Before aerosol immunization, test the aerosol machine’s performance to determine droplet size, diluent volume, distance (height) from the spray nozzle to the flock, and operator movement speed for reference during implementation.  

2. The vaccine must be highly efficacious.  

3. Several days before and after aerosol immunization, add antibacterial drugs to feed or drinking water to prevent chronic respiratory diseases.  

4. Use deionized water or distilled water to dilute the vaccine. Do not use tap water, boiled water, or well water.  

5. Add 0.1% skimmed milk or 3%–5% glycerol to the diluent.  

6. The amount of diluent depends on the aerosol machine and the density of the flock (cage or floor rearing). Strictly follow the manufacturer’s recommended dosage.  

7.Strictly control droplet size: Use droplets of 30–100 μm for chicks and 5–30 μm for adult chickens.  

8. During aerosol immunization, close all doors and windows in the poultry house and turn off fans or exhaust systems. Wait 20–30 minutes after spraying before opening doors and windows and restarting fans (depending on room temperature).  

9. The temperature inside the poultry house during aerosol immunization should be appropriate. Immunization should not be conducted when temperatures are too low or too high. In hot weather, perform aerosol immunization during the cooler evening hours.  

10. Relative humidity in the poultry house also affects aerosol immunization. Generally, a relative humidity of around 70% is ideal.  

11. During aerosol immunization, move the spray nozzle 50–80 cm above the flock, targeting the chickens’ heads back and forth to ensure full coverage. The chickens’ head and back feathers should feel slightly damp after immunization.

V. Wing Web Stab  

Wing web stab immunization is used for fowl pox vaccination. Generally, dilute 1,000 doses of vaccine with 25 ml of physiological saline. Use a vaccination needle (or syringe) to draw the diluted vaccine and stab the inner wing web where there are no blood vessels. Stab once for chicks and twice for older birds. Ensure the vaccination needle has drawn the diluted vaccine to guarantee each chicken receives an adequate dose.

VI. Vent Brush or Drop  

Vent brush or drop immunization is currently only used for virulent infectious laryngotracheitis vaccines. For emergency vaccination in affected flocks, dilute 1,000 doses of vaccine in 25–30 ml of physiological saline. Hold the chicken with its head down and vent up, and use a vaccination brush (small brush or cotton swab) dipped in the vaccine to brush the vent mucosa 3–4 times. Ensure the vaccine liquid only contacts the vent and does not touch the chicken’s skin or feathers or fall to the ground, causing environmental contamination and disease spread.