Cultivation of new castle disease virus (NDV) in chicken embryo

Virulent strains of NDV are

1. Velogenic strain

Velogenic strain MDT is less than 60 hours.

2. Mesogenic strain

Mesogenic strain MDT is between 60-90 hours.

3. Lentogenic strain

 Lentogenic strain MDT is more than 90 hours.

Precaution

The lab should be free from NDV related vaccine.

Sample collection

When there is a history of respiratory problems, diarrhea, or death, we can collect NDV from the brain, spleen, liver, and from the bone marrow of long bones of birds.

Material required for the cultivation of NDV in chicken embryo

  • Homogenizer
  • Centrifuge machine
  • Centrifuge tube
  • Filter paper of pore size 0.45 um.
  • 9-10 days old embryonated egg (It should be specific pathogen-free (SPP)).
  • Egg incubator
  • Candle lamp
  • Antibiotic solution (PSG: Phosphate buffer saline glucose)
  • All requirements for the HA test (Haemagglutination test)
Approach: Allantoic cavity

Procedure

  1. Take a sample and homogenize it with 2-3 ml of PSG and antibiotic solution (Antibiotics will kill any bacteria present, and we centrifuge at 2000 Xg for 10 minutes).
  2. Safe supernatant  (it contains a virus) and discard pellet. Transfer it into another tube through 0.45um pore sized filter paper (No bacteria will come to infiltrate).
  3. After the filtration, inject supernatant (0.1 ml) to 9-10 days old embryonated egg.
  4. Incubate egg.
  5. Candle egg after post inoculation twice a day daily.
  6. Observe the death of an embryo.
  7. Record the death of an embryo.
  8. Record the time of the embryo.
  9. Death time tells the strain type.
  10. HA basically determine the virus titer.
  11. Make a hole in the shell and collect allantoic fluid for the HA test.
  12. It tells only virus presence and quantity.
  13. HI (haemagglutination inhibition test) contains Ab for NDV.
  14. Mix them and the reaction will start.

Calculation of standard MDT

MDT: It stands for mean death time. It is the meantime in hours for the minimum lethal dose to kill embryos.
  • Take allantoic fluid.
  • Make 10 fold serial dilution (up to 0.00000001 or 10-8)
  • Incubate each dilution in 5 separate eggs.

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