Impact of diluent pH and holding conditions on Newcastle Disease vaccine virus infectivity titer

Newcastle disease is a viral disease causing severe economic losses worldwide. Despite routine vaccination, severe outbreaks continue to occur. Vaccine failures can result from mutations in the virus; however, virus inactivation may also occur in lyophilized Newcastle disease virus (NDV) live vaccines due to diluent properties at the time of administration. The present study evaluated the virus infectivity titer of NDV live vaccine reconstituted in diluent at different pH levels and holding temperatures. Two 3x2 factorial trials were conducted, using 3 diluent pH levels (pH 5, 7, and 9). At each pH level, reconstituted virus was held one hour at room temperature or on ice. The first trial utilized 124 fertilized SPF eggs (20 eggs per group, 4 eggs for negative control). The second trial utilized 217 fertilized eggs (35 eggs per group, 7 eggs for negative control). The second trial’s eggs came from a commercial laying hen flock with known low titers of anti-NDV antibodies. Comparison of the two trials verified that the commercial laying hen flock low level of anti-NDV antibodies did not affect the virus infectivity titer measurement. All eggs were incubated under standard conditions. Eggs were inoculated with 100 µl NDV vaccine on day 9 of incubation. Dilutions from 10-3x to 10-7x were used to measure the virus infectivity titer. Four days after inoculation, all eggs were chilled for at least 2 hours. Allantoic fluid was harvested from the 13-day old embryonated eggs and virus infectivity titer was measured by hemagglutination test. Minimum reduction in virus infectivity titer was recorded in pH 7 diluent held on ice (1st trial: 108.00EID50/dose, 2nd trial: 107.69EID50/dose). The virus was less stable at pH 5 dileunt held on ice (1st trial: 106.67EID50/dose, 2nd trial: 106.84EID50/dose). Maximum reduction of infectivity titer was observed in pH 9 diluent held at room temperature (1st trial: 106.25EID50/dose, 2nd trial: 106.13EID50/dose). The results of these studies indicate that optimum diluent pH for effective delivery of the NDV live vaccines for administration is 7. However, holding virus on ice may have beneficial effects on preserving the infectivity titer of the virus, even at pH 5 or 9 conditions.

Key Words: Newcastle disease, vaccine, diluent pH, Infectivity titer.

Poster presented at the International Poultry Scientific Forum during IPPE 2020.