Seroconversion of a Thermostable Live Attenuated Lentogenic Strain Newcastle Disease Vaccine (Local Isolate) in Chicken

Vaccine was prepared by using the isolate, exposed to 430C for 12 days. The vaccination was performed in two thousand four hundred breeder flocks (Banoraja) in 3 different flocks @106.5 EID50 per bird oronasally at the age of day 5 (primary vaccination) followed by vaccination through drinking water on day 26 (boostering) in farm condition and thereafter every 45 days interval. The vaccine was also inoculated intranasally @106.5 EID50 in Two thousand backyard birds (Haringhata Black and non-descriptive) and were boostered after 21 days of 1st vaccination with the same dose and subsequent vaccination was performed at every 45 days interval. Serum samples were collected for HI titre at regular intervals and it was observed that the mean antibody both in backyard birds and breeder flocks was above 1.50 ± 00. Therefore, it was concluded that the thermostable live attenuated lentogenic strain Newcastle Disease vaccine after inoculation into backyard birds and breeder flocks, provides satisfactory level of immunity.

India has emerged on the world poultry map as the 3 rd largest egg (56 billion eggs) and 5 th largest poultry meat (2.6 million tons) producer. Total chicken population has registered an annual growth of 7.3% in the last decade. While farm chicken grew at the rate of 12.4%, desi chicken showed much lower growth rate of about 2%. Other poultry species showed reduction of 2.3 % per annum between 2003 and 2007. Newcastle disease (ND) is the main killer of these chickens in many developing countries (Alexander, 1991). Ten serogroups of avian paramyxoviruses have been recognized: [APMV-1 toAPMV-10] and APMV-1 remains the most important pathogen for poultry while others are known to cause disease in poultry and other types of birds (Alexander, 2003). To counter this deadly disease, proper control methods are needed. Vaccination has been reported as the only safeguard against endemic ND (Usman, 2002). It has been very difficult to vaccinate village chickens against ND using conventional vaccines. However, vaccines with the specific properties of thermostability have become a solution to these problems (Spradbrow, 1996). The Australian Centre for International Agricultural Research (ACIAR) sponsored project for vaccination against ND by using Australian V 4 and I 2 strains (ND asymptomatic pathotypes) in thirty countries in South East Asia, Africa and Australia. A thermostable vaccine enables distributors and users to reduce the problems associated with inadequate cold chains in the field.

Source of the virus for the vaccine production
Vi r u s e s u s e d i n t h i s s t u d y f o r thermostability testing were isolated, characterised and provided by the Department of Veterinary Epidemiology and Preventive Medicine, Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery Sciences, Kolkata-37.

Embryonated Specific Pathogen Free (SPF) fowl eggs
Embryonated SPF fowl eggs were procured from Venky's (India) Ltd., SPF Eggs Division, Pune for propagation of virus as recommended by OIE, 2009.

Spot/slide agglutination test
One drop of allantoic fluid, collected during harvesting of virus, was dropped on a grease free glass slide. Freshly prepared 0.5% chick RBC suspension was added to the allantoic fluid in 1:1 ratio (50% v/v). Both the suspensions were mixed thoroughly by rotating the slide gently. The slide was examined by diffused light to see any haemagglutination.

Standard Plate Haemagglutination Test (HA) as per OIE (2009) Determination of Embryo Infective Dose fifty (EID50) of the virus as per Reed and Muench (1938) and FAO (2002)
The HA titre was estimated as per OIE (2009) and the Embryo Infective Dose fifty (EID 50 ) was determined as per the FAO(2002) and the result was calculated as per Reed and Muench (1938).

Source of the vaccine
The thermostable virus was serially passaged in 9-11 days old embryonated SPF fowl eggs and observed no embryopathy on 45 th passage. For confirmation of attenuation, the passaged virus was inoculated @ 10 6.5 EID 50 oro-nasally to 10, five days old SPF chicks and observed no abnormality/ mortality for 21 days post inoculation. The 44 th passaged virus was inoculated into five 9-11 days old embryonated SPF fowl eggs and allantoic fluids were harvested after 5 days of post inoculation. After estimation of HA titre separately the allantoic fluid was pooled together considered as master seed virus and the HA titre and EID 50 was estimated. The master seed virus was given three serial passages in SPF fowl eggs. Allantoic fluid was harvested after 5 days of incubation and HA titre was estimated. Finally, the allantoic fluids of 3 passages were pooled together considered as working seed virus and the HA titre and EID 50 was estimated. The working seed virus was serially inoculated for three passages in SPF fowl eggs. After 5 days of incubation, allantoic fluids were harvested from every passage of each inoculated egg and HA titre was determined as per the OIE (2009). Finally, the allantoic fluids of the three passages were pooled together considered as vaccine and HA titre was determined as per the OIE (2009). The vaccine dose was calculated by determining the EID 50 as per the method of FAO (2002) and Reed and Muench (1938). For confirmation of vaccine virus as APMV-1, HI test was performed using known positive antisera of APMV-1.

Determination of EID 50 of locally isolated velogenic pathotype ND challenge virus
EID 50 of the locally isolated velogenic pathotype of NDV was determined in 9-11 day old embryonated SPF fowl eggs as per the standard method Reed and Muench, 1938.

Experimental design
Study of the seroconversion of the thermostable live attenuated lentogenic strain (local isolate) ND vaccine in layer and backyard birds.

For layer bird
Two thousand four hundred commercial layer chicks (Banoraja) in3 different flocks were vaccinated with live attenuated thermostable lentogenic strain (local isolate) ND vaccine @10 6.5 EID 50 per bird oronasally at the age of day 5 (primary vaccination) followed by vaccination through drinking water on day 26 (boostering) in farm condition and thereafter every 45 days interval .

For backyard bird
Two thousand backyard birds (Haringhata Black and non-descriptive) were inoculated oronasally @ 10 6.5 EID 50 of prepared vaccine to each bird ignoring the age of the bird and were boostered after 21 days of 1 st vaccination with the same dose and subsequent vaccination was performed at every 45 days interval.

Serology
Serum samples from 10% of the vaccinated birds were assayed by haemaggluntination inhibition (HI) antibody test as per OIE (2009) before primary vaccination/1 st vaccination, before boostering and then every 45 days interval prior to vaccination.

Challenge study
Twelve vaccinated birds were subdivided into two groups i.e. vaccinated experimental group  e. a, b, c and d) in the rows did not differ significantly with each other) time of prior to challenge and then weekly for 3 weeks. HI antibody titre was determined from each sample as per the method OIE (2009).

Statistical analysis
The results were analyzed statistically by one way ANOVA to study the seroconversion and protectivity of live lentogenic strain (local isolate) ND vaccine through drinking water in commercial broiler birds.

Antibody response in layer birds
Similar finding was also observed by Iroegbu et al. (2014) who reported that unvaccinated laboratory-raised, five week old cockerels were fed V 4 Newcastle disease vaccine in cassava once, twice or thrice; and tested for hemagglutination inhibition (HI) antibody response and observed that the immune status of the chicken flocks improved with number of vaccinations.
During the two years studies the mean antibody titre of one of the three flocks decreased (i.e. 1.634±0.02) but not below protective level at the age of 12 th month (11 months, 26 days). Investigation in the farm it was revealed that E. coli and M. gallisepticum organism have been isolated and identified from that flock as no outbreak of NDV or no clinical signs of ND was seen and even no APMV-1 virus was isolated from the lesion of the dead birds during that period.

Antibody response in Haringhata Black backyard birds
After regular revaccination, the mean antibody titre increased significantly (P<0.05) to protective level (i.e.1.5±0.04) and afterwards again increased significantly (P<0.01) above protective level throughout the trial period. By thorough investigation in the village, coccidia infection was isolated and identified along with tapeworm infestation among those backyard birds at that time. Therefore, the slight decline in antibody titre might be due to secondary infection of coccidiosis and tapeworm infestation. Similar finding was also observed by Hassan et al. (2012).

Antibody response in non-descriptive backyard birds
After revaccination, the mean titre increased significantly (P<0.01) i.e. 1.820±0.05and 1.890±0.06 and persisted above protective level. Thus, it can be concluded that the thermostable vaccine produced significant protective titre when inoculated to the backyard birds. Bouzari and Spardbrow (2006) also reported that the Heat-resistant strains of Newcastle disease virus, such as strain V 4 are being used as vaccine to protect flocks of rural chicken in developing countries.

Challenge study
From the table, it was observed that before challenge the mean HI titres of both the groups were same and very good i.e. 2.11±0.04. After challenge the mean antibody titres of the vaccinated experimental group decreased significantly (P<0.01) i.e. 1.53±0.03 on 7 th day with no mortality/abnormality. Afterwards, the mean antibody titres increased significantly (P<0.05) on 14 th day (i.e. 1.68±0.04) and at the end of the observation period (i.e. 1.76±0.03) with no mortality/abnormality.
On the other hand, the mean antibody titre of the vaccinated control group decreased gradually with the advancement of time and reached to 0.42±0.03 at the end of the experiment with no mortality/abnormality. Similar finding was also described by Rahman and Khan (2004).

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The live attenuated lentogenic strain thermostable ND vaccine (local isolate) was absolutely safe and showed desirable potency in layer chicks.

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The thermostable live attenuated lentogenic ND vaccine (local isolate) was highly potent and generated sufficient immune response when administered through drinking water in farm condition. But the immune response was not optimum when the flocks were concomitantly infected with other diseases.

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The vaccine is also highly efficacious and produces optimal immune response in backyard birds when administered intranasally in village level.

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The vaccinated backyard birds became able to withstand the challenge infection of velogenic pathotype of NDV (local isolate).

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The vaccine without any additive (stabilizer) when kept at room temperature, could retain its viability for at least three months.

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The vaccine can be used in field level.