Research at UAF - Preparation and field evaluation of cell culture adapted thermostable Newcastle disease V4 vaccine

Name of Principal Investigator: Dr. M. Shahid Mahmood
Cost: Rs. 60,000/-
Duration: 12 Months
Funding Agency: University of Agriculture,  Faisalabad.

Progress Reports


INTRODUCTION Newcastle disease (ND) is a major constraint to village poultry production throughout developing countries, frequently causing mortality rates of 75% to 100% in unvaccinated flocks. According to Jordan and Pattison (1996), ND is caused by a group of closely related viruses that form the avian paramyxovirus type (PMV-1). Nine serogroups of avian paramyxoviruses have been recognized: [APMV-1 to APMV-9]. APMV-1 remains the most important pathogen for poultry (Alexander, 2003). The incubation period of ND after natural exposure has been reported to vary from 2 to 15 days. The time for appearance of the symptoms varies, depending on the infecting paramyxovirus, host species and its age and immune status, infection with other organisms, environmental conditions, the route of exposure, and the dose (Alexander, 2003). Transmission can occur by direct contact with feces and respiratory discharges or by contamination of the environment including food, water, equipment and human clothing. Newcastle disease viruses can survive for long periods in the environment, especially in feces. Strains of NDV have been grouped into five pathotypes on the basis of the clinical signs seen in infected chickens (Jordan & Pattison, 1996; OIE, 2000). Chickens infected with virulent NDV may die without showing any signs of illness. But if signs appears then these are fluffy feathers and having coat dragging on the ground, sleepy appearance and do not eat. Slight difficulties in breathing during infection but may show severe difficulty in breathing with distress and gasping. Other signs include swelling of the head & neck, marked decrease in egg production, shaking, twisted neck but paralysis of wings and legs may sometimes be seen in advanced stages of the disease. Lethargy, weakness, muscle tremors, conjunctivitis & nasal discharge, sternal & lateral recumbency (Alders and Spradbrow, 2001; Olivier, 2004). Vaccination has been reported as the only safeguard against endemic ND (Usman, 2002). Inactivated vaccines give very good immunity without vaccinal reactions and have been widely used, but are relatively expensive and require considerable attention to training when used by non-veterinary personnel. Live vaccines are easy to apply and relatively inexpensive, and give moderately good immunity. Among the live vaccines, the heat resistant vaccines require less stringent transport requirements in the field, and they have also been widely used in villages. Recombinant vaccines have the advantage that they can be serologically detected (Bell, 2001). The selection of a ND vaccine for use in rural chicken depends on the local conditions in each country. A selection criterion includes ease of use, cost, thermostability, immunogenicity, availability and transportability. In circumstances where the cold chain is weak or absent, the only reliable option is the use of thermostable ND vaccines, i.e., the live vaccines NDV4-HR and I-2 (Bensink and Spradbrow, 1999). In most cases where farmers are to contribute wholly or partially to the cost of the vaccine, the price of the vaccine is a major factor. The lower price of the vaccine, the greater the number of farmers who are able to afford to pay for it and, consequently, the greater the vaccination coverage. Many strains of Newcastle disease virus other than velogenic strains are used in the production of live vaccines. A thermostable vaccine enables distributors and users to reduce the problems associated with inadequate cold chains in the field. It is essential that users understand that a thermostable vaccine must still be treated with some of the respect due to a biological product, that is the vaccine cannot be exposed to sunlight and frequent shifts in temperature and still expect it to remain active (Alders and Spradbrow, 2001) The heat resistant V4 (NDV4-HR) vaccine against ND has yielded encouraging results in many countries of Africa (Alders and Spradbrow, 2001) and Southeast Asia (Spradbrow, 1993-94). NDV4-HR vaccine is a live vaccine having characteristics including thermostability, retaining its activity for 12 weeks at a temperature of 28°C in freeze-dried form. It can be administered via intraocular, intranasal, oral through drinking water; mixed with certain feeds or by injection (Spradbrow, 1993–94; Anon, 1991). Its ease of administration makes it suitable candidate for use in rural as well as commercial poultry (Spradbrow, 1993-94); it is avirulent strain and can be safely administered to chickens of any age from day-old chicks to adult birds (Spradbrow, 1993-94; Anon, 1991) its biological safety is superior to that of other living ND vaccine strains such as B1 or La Sota etc (Anon, 1991).


To isolate indigenous V4 strain of NDV from ND field outbreaks To characterize the NDV using molecular biology techniques To develop cell culture adapted V4 vaccine To evaluate the potential of indigenous V4 strain through humoral and cellular immune response and protection against challenge using an economical feed delivered vaccine To replace the problem of heat inactivation by the use of thermostable vaccine

Characterization of Thermostable V4 strain of NDV:
The samples were collected from ND field outbreaks. These samples were characterized using PCR and nucleotide sequencing techniques to find out maximum homologous thermostable strain of NDV.

Adaptation of V4 NDV on Vero cell line:
Cells and Media:
Vero cell line was purchased from Sigma Aldrich.  Glasgow minimal essential medium (GMEM) with 5% fetal calf serum was used as growth medium while GMEM with 2% fetal calf serum as maintenance medium. Dulbecco’s Phosphate buffer saline (PBS) was used for washing the monolayers and 0.25% trypsin-EDTA solution for sub-culturing.

Adaptation on Vero cell line:

Vero cell line was sub-cultured and 0.2 ml of vaccine was used for infection using 0.2µm pore size filters. These flasks were incubated at 37oC in the presence of 5% CO2 in CO2 incubator. Culture fluid was harvested each time by three freeze-thaw cycles and clarified by centrifugation @ 5000 rpm for 5 mints. Serial passagings were done for adaptation and attenuation (Rasool and Hussain 2006). This was identified on Vero cells through characteristic cytopathic effects (CPEs), including degeneration, roundening, granularity, vacuolation and formation of synctia (Ahamed et al., 2004).

Identification and titration of virus:
For confirmation and identification, culture supernatant from each passage was subjected to Spot Haemagglutination test (HA) (Khan, 1995), Micro Haemagglutination test (Summera 2006) and Virus Neutralization Test (Chimeno Zoth et al., 2008).

Preparation of vaccine:

Cell culture adapted NDV4 supernatant was centrifuged @ 5000 × g for 5 minutes. Then supernatant was further passed through filter having 0.2 µm pore size. To check the infectivity titre EID50 was calculated as described by Simone (2005). Sterility and safety tests were performed as described by Summera (2006) and Rasool and Hussain (2006), respectively.

Treatment of vaccine with wheat, rice and crumbs coated feed:

Treatment of vaccine with wheat, rice and crumbs coated feed were conducted as described by Summera (2006). The thermostable vaccines thus prepared were inoculated to rural as commercial poultry birds. Humoral immune response was checked by using HAI and ELISA as described by Barileng (2006) and cell mediated immune response was checked by macrophage engulfment test as described by Summera (2006).