IMMUNOGENICITY OF FORMALDYDE INACTIVATED NEWCASTLE DISEASE VIRUS FIELD ISOLATE IN MATERNAL ANTIBODY FREE CHICKENS Anak Agung Ayu Mirah Adi 1 *, IGusti Agung Arta Putra 2, Nyoman Mantik Astawa 3, I Made Kardena 1, Yasunobu Matsumoto 4 1 Laboratory of Veterinary Pathology, 3 Laboratory of Veterinary Virology Department of Animal Disease, Faculty of Veterinary Medicine, 2 Laboratory of Anatomy and Physiology Faculty of Animal Husbandry Udayana University, Jln. PB Sudirman Denpasar, Bali 80232, Indonesia. 4 Laboratory of Global Animal Resource Science, Department of Global Agricultural Sciences, Graduate School of Agricultural and Life Sciences, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. *Corresponding author : aaa_mirahadi@unud.ac.id Abstract Newcastle disease (ND) is one of the most devastating diseases that affect chickens and has caused severe economic losses in the poultry industry worldwide. In this experiment, a viscerotropic velogenic NDV-/Bali-1/07 was inactivated with formaldehyde 0.1% and was used experimentally as a vaccine. The inactivated NDV and commercial NDV LaSota vaccines were tested for their efficiency in generating humoral immune response in different groups of undetectable anti-ndv maternal antibody chickens. Two group of chickens consisting of 10 chickens each groups received 0.2 ml formaldehyde inactivated NDV (A group) and commercial NDV LaSota accine (B group) intramuscularly respectively. The HI titer of each group were monitored weekly for the presence of NDV antibody, starting from 1 week post vaccination until 4 week post vaccination. Result of this study showed that the mean of HI titer in the A group was similar than that of the B groups during all study weeks. At the end of experiment, the HI Log 2 mean titer of NDVantibody in A group (8.8 ± 1.09) was not significantly different to that of B group (8.5 ± 0.9) (p>0.05). In conclusion, the antibody response induced by inactivated NDV vaccine in chickens is similar to those induced by commercial NDV LaSota vaccine. Key words: NDV, Maternal antibodies, HI titers, LaSota Vaccine. Background Chickens are susceptible to many infectious diseases. One of the most important of these is the viral disease known as Newcastle disease (ND), which causes devastating losses in both commercial and village chickens. Indonesia in one of the country where virulent strains of Newcastle disease (ND) virus are endemic (OIE, 2004). The genotype of virulent NDV strain found in Asean countries including Indonesia is genotype VII (Adi et al., 2010; Xiao et al.,2012). Vacccination for ND is routinely practiced in countries where ND is endemic (Senne,et.al.,
2004;OIE,2004). The types of vaccines and vaccination schedules used vary depending on the potential threat, virulence of the field challenge virus, host and host immune status. Most of viral vaccines with inactivated antigen were prepared using formaldehyde. In the present study Balinese native viscerotropiv velogenic NDV was inactivated with formaldehyde. The potency of both prepared vaccines and a commercial vaccine were established by HI test in undetectable anti-ndv maternal antibody chickens. This research was conducted in order to know the profile of anti-ndv MaAb village chickens and the immunogenicity of formaldehyde inactivated NDV field isolate in MaAb free chickens. MATERIALS AND METHODS Virus preparations. NDV/Bali-1/-07 isolate was propagated in 10-day old embryonated chicken eggs. The eggs were observed for 24-72 hrs post inoculation. Allantoic fluid of the inoculated eggs was harvested, centrifuged 1200 rpm, 30 min and supernatant was collected. Hemagglutination (HA) assay was performed in V-bottom 96-well plates with 1% chicken red blood cell as described with Adi, et al (2010). Formalin treatment. Formaldehyde (Merck, Germany) solution was diluted in double distilled water (DDW). The final concentration of 0.1% (King 1991) was attended by virus solution. The viruses were mixed and incubated for 16 hrs at 37 C. Anti-NDV Maternal antibody (MaAb) free chicken. Thirty one day old chicken were housed in one isolated cage. The presence of anti-ndv MaAb, were tested every week by performing Hemagglutination inhibition (HI) test as are described below. Chicken with undetectable HI titer (HI titer < 2 1 ) were defined as free anti- NDV MaAb and were used in the experiment. HI test for detection anti-ndv MaAbs. The HI test was performed according to the procedure of OIE (2014). Briefly two fold serial dilution of 25 l serum was made with PBS in V bottom microtiter plate up to 10 th well. Then 25 l of 4 HA unit antigen was added up to 11 th well. The plates were kept at room temperature (RT) for 30 minutes to facilitate antigen antibody reaction. Finally 50 l 1% (v/v) chicken red blood cells (RBCs) suspension was added to each well. The 11 th well contains antigen and RBCs as the positive control and the 12 th well contains RBCs only as negative control. After mixing thoroughly, the RBCs were allowed to settle at RT for 40 minute and agglutination was assessed by tilting the plates. The HI titers were expressed as log2 values of the highest reciprocal of the dilution which showed hemagglutination inhibition.
HI titer log 2 Experimental design. Chicken with undetectable anti-ndv MaAb (n=20) were used in this study. The chickens were randomly divided in 2 groups consist of 10 chickens each. A group received 0.2 ml formaldehyde inactivated NDV and B group received commercial vaccine; Medivac ND LaSota (Medion, Bandung-Indonesia) at the doses of 10 7 EID 50 by the intramuscular route. Blood samples were collected directly from heart at week 1, 2, 3 and 4 post inoculation. Serum was separated, heat inactivated and stored at -20 C for future use. Serology. Serum titers to NDV post vaccination were determined by HI tests. The HI data titer were then analyses. Statistical analyses. Data were examined using a commercially available statistical package (SPSS version 17 for Windows) and comparisons were made using the descriptive statistics and one way ANOVA RESULTS. MaAb profile of the village chicken of 1-8 weeks old are shown in figure 1. The mean anti- NDV MaAb at 1,3,6 and 8 week old were 4.68+ 0.85; 3.8+ 0.86; 2.16+ 0.62; 0.16+ 0.47 respectively. The HI titer reach undetectable level at 8 weeks old. 7 6 5 4 3 2 1 0-1 -2 1 3 6 8 Weeks Figure 1. Profile of anti-ndv maternal antibodies (MaAb) at 1 to 8 weeks old. The anti MaAb declined to undetectable level or below 2 1 at 8 weeks old.(n=30) HI Log 2 total mean titer in the study groups following vaccination are shown in Figure 2. The mean of HI titer in the A group at 1 to 4 week post vaccination were 5 + 1.5; 8,4+ 1.8; 8.8 + 1.6 and 8.8 + 1.1 respectively. Whereas the mean of HI titer in the B group were: 4.6 + 0.9; 6.8+ 0.8; 8.4 + 0.5 and 8.2+ 0.8 respectively. From the mean HI titer it is able to see that at 2 week post vaccination the mean HI titer in the A group seem to be higher than that of B group however, statistically it was not significant different (p=0.1). No significant differences were found for mean HI titer, between A and B groups during all study weeks (p=0.6). As well as, at
the end of experiment 4 weeks post vaccination the HI Log 2 mean titer of NDV-antibody in A group (8.8 ± 1.09) was not significantly different to that of B group (8.5 ± 0.9) (p>0.05). Figure 2: Anti-NDV antibodies profile after vaccination with field isolate inactivated formalin (A) and commercial vaccine (B) during 1-4 weeks post vaccination. Note Mean HI log2 titers in the serum. Discussion Reducing losses of large numbers of village chickens to virulent Newcastle disease is an essential first step to improving their productivity. In Indonesia vaccination program in commercial chicken have been done for many decades, however in village chickens, the owner not so care about vaccination due to the small scale farming system and the availability of the vaccine. Allan et al. (1978) reported that the lowest protective level of anti-ndv HI antibody in chickens is 2 4 HI units. In this study we found that the anti-nv Mab of village chicken declined to be un-protective level at 3 weeks old. It means at 3 weeks old the village chicken under threaten of ND as the virulent NDV circulating in the endemic area. Vaccination is the most important action for preventing this disease. In this study we tried to make vaccine using NDV field isolate that was isolated from ND outbreak of village chicken in Karangasem Bali, by formalin inactivated. The antibody response induced by inactivated NDV vaccine in chickens is similar to those induced by commercial NDV LaSota vaccine. It mean that field isolate formalin inactivated could be use as vaccine candidate and could induce antibody as good as commercial vaccine. In conclusion, based on the profile of anti-ndv MaAb, village chickens under threaten of ND starting from 3 weeks old as the MaAb titers were declined under the non protective levels. NDVinactivated NDV vaccine in the future might be possible to use in village chicken instead of commercial vaccines to overcome the availability of vaccine for small scale farmer. References
1. Adi, A. A. A. M., Astawa,N.M.,Putra, K.S.A.,Hayashi,Y. and Matsumoto,Y. 2010. Isolation and characterization of a pathogenic Newcastle disease virus from a natural case in Indonesia. J. Vet. Med. Sci.72:313 319. 2. Alexander, D. J.2013. Newcastle disease, other Paramyxoviruses and Pneumovirus infections. pp. 63-100. In: Diseases of Poultry. 11 th edn (Saif, Y. M., Barnes,H.J., Glossons,G.R., Fadly, M.A., McDougald,D.J., and Swayne,D.E.).Iowa State University press, Ames,I.A. 3. Allan, W. H., Lancaster, J. A. and Toth, B. 1978. Newcastle disease vaccines: their production and use. FAO animal production and health series, No. 10, FAO, Rome. 4. King, D.J. (1991). Evaluation of different methods of inactivation of Newcastle disease virus and avian influenza virus in egg fluids and serum. Avian diseases 35:505-514. 5 Nakamura, K., Ohtsu, N., Nakamura, T., Yamamoto, Y., Yamada, M., Mase, M. and Imai, K. 2008. Pathologic and immunohistochemical studies of Newcastle disease (ND) in broiler chickens vaccinated with ND: severe nonpurulent encephalitis and necrotizing pancreatitis. Vet Pathol.45:928 933. 6. OIE. 2004. Manual of diagnostic tests and vaccines for terrestrial animals. 5 th Edition, Chapter 2.1.15. Newcastle disease. Accesed on January 27 th, 2007. Sally,E.,G. 2002.A basic laboratory manual for the small-scale roduction and testing of I-2-Newcaste disease vaccine:newcastle disease vaccines: an overview: http://www.fao.org/docrep/005/ac802e/ac802e04.htm. Accesed on August 23 th, 2016. 7. Senne,D.A., KingD.J., Kapczynski,D.2004. Control of Newcastle disease by vaccination.dev Biol(Basel). 119:165-170. 8. Xiao S, Paldurai A, Nayak B, Samuel A, Bharoto EE, et al. 2012. Complete genome sequences of Newcastle disease virus strains circulating in chicken populations of Indonesia. J.Virol 86(10): 5969 5970.