AVAS Annals of Veterinary and Animal Science RESEARCH ARTICLE V:4(3) OPEN ACCESS

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1 Rahman et al. Annals of Veterinary and Animal Science [Received: 10 Jan 17, Accepted: 13 Jun 17, Published: 30 Jun 17] ] eissn: pissn: AVAS Annals of Veterinary and Animal Science RESEARCH ARTICLE V:4(3) OPEN ACCESS Evaluation of serum antibody titer level against Newcastle disease virus in vaccinated broiler chickens Rahman, Mohammad Mostafijur 2* ; Sarker, Rahul Deb 1 and Mohammed Nooruzzaman 1 ABSTRACT The research was carried out to evaluate the serum antibody titer level against Newcastle disease virus (ND) in vaccinated broiler chickens from two commercial broiler farms in Mymensingh district in Bangladesh. Serum sample were collected during 7 th, 14 th and 21 st of post vaccination and antibody (Ab) titer was determined by Haemagglutination Inhibition (HI) test. A total of 92 serum samples (n=46/each farm) were tested. The Ab titer of 85.9 % (n=79) serum were found at protective level log 2 4 ; 7.6 % (n=7) were found below the protective level (log 2 1 to log 2 2 ). In farm A 7 th of post vaccination Ab titers varies from log 2 5 to log 2 9 with log 2.35 and geometric mean titers 121.8; In 14 th Ab titers ranges from log 2 3 to log 2 7 with log 2.72 and geometric mean titers 38.1 and 21 st day of post vaccination Ab titer varies from log 2 3 to log 2 7 with log 2.35 and geometric mean titers In farm B 7 th of post vaccination Ab titers varies from log 2 4 to log 2 9 with log 1.96 and geometric mean titers 66.6; In 14 th titers varies from log 2 4 to log 2 6 with log 5.31 and geometric mean titers 30.6 and at 21 st of post vaccination Ab titers varies from log 2 2 to log 2 4 with log 4.38 and geometric mean titers 6.6. Broilers at the age 2-3 week were more immune against NDV. Keyword: Newcastle disease, HI test, antibody titer, broiler. INTRODUCTION Newcastle disease (ND) or Ranikhet disease is considered as one of the major problem towards the development of poultry industry in Bangladesh. Newcastle disease virus (NDV), also known as virulent forms of Avian Paramyxovirus serotype 1 (APMV- 1), is a single-stranded, negative-sense RNA virus within the genus of Avulavirus in the family of Paramyxoviridae (Lamb et al., 2005). Newcastle disease (ND) also known as Ranikhet disease (RD) is a highly contagious viral disease that attacks many species of domestic and wild bird (Mozaffor Hossain et al., 2010). Newcastle disease has been recognized as one of the major problems of the large and small poultry industries in Bangladesh (Islam et al., 1998). No other poultry virus comes close due to its high mortality, morbidity, stress, decreased egg production and hatchability resulting high economic losses (Alexander, 2000). Isolation * Corresponding author: mrrana@pstu.ac.bd 1 Department of Pathology, Bangladesh Agricultural University, Mymensingh, Bangladesh. 2 Department of Pathology and Parasitology, Faculty of Animal Science and Veterinary Medicine, Patuakhali Science and Technology University, Barisal-8210 of NDV in embryonated chickens eggs and their identification by haemagglutination (HA) and hemagglutination inhibition (HI) test with a NDV mono-specific antiserum is a gold standard for the detection of NDV (OIE, 2012). The efficacy of vaccinations can be estimated best with challenge experiments but they are expensive and time consuming (Czifra et al., 1998). Therefore, serological tests are frequently used to assess protective response; Haemagglutinationinhibition (HI) test is the method of choice. It detects antibodies against the haemagglutinating epitopes of the avian paramyxo-virus 1 (PMV-1) (Czifra et al., 1998). Serum antibody titers against Newcastle disease indicate the protective immune response against Newcastle disease of poultry. The present study was conducted to evaluate the serum antibody titers level against Newcastle disease virus after vaccination of broiler with lasota strains of ND vaccine in two commercial broiler farms under Mymensingh district in Bangladesh. MATERIALS AND METHODS Study Design The study was conducted in the department of pathology, faculty of veterinary science, naturepub academics Inc. Natural Science Research Forum (nsrf) Sylhet Agricultaral University, Sylhet

2 Bangladesh Agricultural University, Mymensingh. Two commercial broiler farms were randomly selected from two different upazilla of Mymensingh district namely Balipara broiler farm (Farm A) and Muktagacha broiler farm (Farm B). At the beginning of the study birds from both experimental farms were vaccinated with Newcastle disease (Lasota strain with EID 50) vaccine at the age of 3-5. Blood sample were collected randomly from both experimental farms at three episodes i.e. 7 th, 14 th and 21 st of post vaccination. A total of 92 serum samples were collected and tested to evaluate serum antibody titer level against Newcastle disease of poultry by Haemagglutination Inhibition (HI) test. Antigen The antigen (Newcastle disease vaccine Lasota strains with EID 50) used for HI test was collected from a reputed pharmaceutical company of Bangladesh. Collection of Serum Blood sample were collected aseptically in a 3 ml disposable syringe directly from the heart at 7 th of post vaccination. Blood samples were collected either from wings veins or right jugular vein of broilers at 14 th and 21 st of post vaccination. Blood samples were kept undisturbed for about minutes at room temperature. Then clear serum were collected in Eppendorf tube and stored at C freezer. Preparation of Chickens Red Blood Cell (1 % v/v) Suspension Chickens blood samples were collected from a live bird market. Blood was collected in a 15ml falcon tube containing 5 ml Alsever s solution as anticoagulant. Collected blood was then centrifuge at 1500 rpm for 5-7 minutes and the supernatant was poured off. PBS (1X) was added into the falcon tube containing blood and centrifuge at 1500 rpm for 5-7 minutes. This step is repeated for 4-5 times for washing chickens blood. 1% v/v suspension of chicken RBC was prepared by adding PBS. Haemagglutination Test and determination of 4HA unit virus for HI test The v-bottomed 96 wells plastic plate was placed. 25 l of PBS was dispended into each well of one raw. 25 l of antigen suspension placed into first well and made two fold dilutions by transferring 25 l of fluid from each well to next and lastly discard 50 l from the last well. A negative control well was prepared by using only PBS. Then 25 l of 1% chickens RBC suspension was added tilted gently and allowed to stand for 40 minutes. The result was recorded, the last wells to show hemagglutination which indicate 1 HA unit. Haemagglutination Inhibition (HI) Test PBS (25 l) was dispended into 12 wells of one raw of the plastic v-bottomed 96 plate. Two fold serial dilution of field serum (25 l) was made up to 11 th plate. Equal volume of 4HA unit of ND virus added into each well up to 12 th wells. Then the mixture was kept for 30 min. at room temperature then 25 l of 1 % (v/v) chickens RBC suspension was added to all wells. The RBC is allowed to settle down for 40 minute. Agglutination was assessed by tilting the plates. The samples showing peculiar central button shaped settling of RBCs were recorded as positive. The last wells which had a complete inhibition, was consider as the HI antibody titer (OIE, 2002). Statistical analysis All data were collected and summarized in the computer program MS EXCEL (Microsoft Co.). The statistical data analysis (log based) was performed by STATA 13.0 (STATA COP.) special edition software. Values were expressed as mean ± SD and geometric mean titer. RESULTS AND DISCUSSION A total of 92 serum samples (n=46/each farm) were collected and tested to evaluate the serum antibody HI titer against NDV 95

3 from two vaccinated broilers farms. Among all the samples, 79 samples (85.9%) were found at protective level log 2 4 ; 6 samples (6.5%) were found moderate protection level Log 2 3 and 7 samples (7.6%) were below protective level Log log2 2 (Table In 7 th of post vaccination antibody titers levels against NDV varies from log 2 4 to log 2 9 (respective percentage were 6.25, 25.0, 43.75, 12.5, 6.25 and 6.25) with log based 1.96 and geometric mean titers In 14 th of post vaccination HI Table 1: Protective (%) antibody titer level against NDV in vaccinated broiler chickens ND HI antibody titers level (%) Total Sample Non protective level ( ) Moderate protective level (2 3 ) Protective level 2 4* 92 cases 7.6 (n=7) 6.5 (n=6) 85.9 (n=79) HI- Haemagglutination Inhibition test; *- Protective level 1). In 7 th of post vaccination antibody titers levels against NDV varies from log 2 5 to log 2 9 (their respective percentage were 14.3, 14.3, 42.9, 21.4 and 7.1) with log based 2.35 and geometric mean titers In 14 th of post vaccination HI antibody titers varies log 2 3 to log 2 7 (their respective percentage were 6.3, 18.8, 31.25, 31.25and 12.5) with log based 2.72 and geometric mean titers In sera collected at 21 st day of post vaccination the titer varies from log 2 3 to log 2 7 (respective percentage was 6.25, 6.25, 37.5, 37.5, and 12.5) with log based 2.35 and geometric mean titers 43.3 (Table 2). titers varies from log2 4 to log2 6 (respective percentage were 37.5, and 31.25) with log 5.31 and geometric mean titers Whereas sera collected at 21 st of post vaccination HI antibody titers varies from log 2 2 to log 2 4 (respective percentage were 50.0, 28.6 and 21.4) with log 4.38 and geometric mean titers 6.6 (Table 3). A Newcastle disease HI antibody titer log 2 3 (i.e. 1:8) and above is consider as positive for specific immunity (Allan and Gough, 1974; Numan et al., 2005; Mozaffor Hossain et al., 2010). (Ghaniei A and Mohammadzadeh N, 2012; OIE, 2012) reported sera with antibody titers log2 4 Table 2: HI antibody titer level against NDV in vaccinated broiler chickens (Farm A) HI 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 Log titer (2 1 ) (2 2 ) (2 3 ) (2 4 ) (2 5 ) (2 6 ) (2 7 ) (2 8 ) (2 9 ) 7 th 14 th 21 st Mean ± SD 2.8 ± ± ±2.58 HI- Haemagglutination Inhibition test; - Geometric Mean titer; SD- Standard deviation Key: - Geometric mean titer ( = n x1x2x3...xn) Farm B against ND were considered positive. On the other hand, (Jalil et al., 2009) reported HI titer log 2 7 as positive. In the present 96

4 Table 03: HI antibody titer level against NDV in vaccinated broiler chickens (Farm B) HI titer 1:2 (2 1 ) 1:4 (2 2 ) 1:8 (2 3 ) 1:16 (2 4 ) 1:32 (2 5 ) 1:64 (2 6 ) 1:128 (2 7 ) 1:256 (2 8 ) 1:512 (2 9 ) Log Mean ± SD 7 th th 21 st Key: - Geometric mean titer ( = n x1x2x3...xn) 2.67 ± ± ±2.08 study serum HI antibody titer log 2 4 was considered as the protective threshold and titer below log 2 3 against ND was categorized as non-protective % serum samples were at protective level against NDV after vaccination with ND vaccine (lasota strains). Bell and Moulodi (1988) recorded antibody titer levels against NDV in different regions of Morocco ranging from 5 to 83% (average 35%) in vaccinated chickens. Husna et al. (2016) recorded 70.9 % birds had antibody titer at protective level. Mozaffor Hossain et al. (2010) found that 78.04% samples of broilers were positive for NDV antibodies in Bangladesh which is slightly lower from the present findings. On the other hand, findings of Numan et al. (2005) disagree with the present findings. Numan et al. (2005) found 98.07% of serum samples were positive for specific immunity against NDV for broilers in Pakistan which is higher than the present findings. In the present research sera collected at 7 th of post vaccination show higher HI antibody titer and against NDV. The serum antibody titer against NDV and geometric mean titers were found higher at 7 th of post vaccination (2-3 weeks of age) in both experimental broiler farms. This finding was supported by some previous observation (Husna et al., 2016; Mozaffor Hossain et al., 2010) who found vaccinated broiler chickens at the age above 2-3 weeks are more protective against Newcastle disease. In some cases serum antibody titer against NDV were found below the protective level. Lower serum antibody titer may due to either vaccination failure or may be due to presence of maternal antibody which neutralize vaccine virus. The higher serum HI antibody titers levels and geometric mean was resulting decrease susceptibility to clinical infection to NDV. CONCLUSION In conclusion, 85.9 % sample was found at protective level of serum HI antibody titer against Newcastle disease virus. The HI antibody titers against NDV were higher in sera collected at 7 th of post vaccination while serum antibody titer level declining in sera collected at 14 th and 21 st of post vaccination. The findings of present study revealed that lasota strains EID 50 vaccine of NDV has significance potency against Newcastle diseases of broilers. ACKNOWLEDGEMENT Authors would like to express special gratitude to staff of the department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh Bangladesh for their cordial assistance and support during the research. Conflict of Interest The authors declare that no conflict of interest exists. 97

5 REFERENCES 1. Alexander DJ (2000). Newcastle disease and other avian Paramyxoviruses. In: Revue Scientifiqueet Technique de l OIE, pp: Allan WH, Gough RE (1974). A standard Haemagglutination Inhibition test for Newcastle disease a comparison of macro and micro methods. Veterinary Record, 95: Bell JG, Moulodi S (1988). A reservoir of virulent Newcastle disease virus in village chicken flocks. Preventive Veterinary Medicine, 6: Czifra G, Mészáros J, Horváth E, Moving V, Engström BE (1998). Detection of NDV specific Antibodies and the Level of Protection Provided by a Single Vaccination in Young Chickens. Avian Pathology, 27(6): Ghaniei A, Mohammadzadeh N (2012). Detection of Newcastle disease virus antibodies in serum of broiler chickens of Iran. Journal of Animal and Poultry Sciences, 1(1): Husna A, Badruzzaman ATM, Islam MS, Runa NS, Runa NY, Yesmin S, Noor M, Alam KJ, Islam MN, Rahman MM (2016). Evaluation of humoral immunity against Newcastle disease virus in vaccinated chickens at Dhaka and Narayanganj districts of Bangladesh. Annals of Veterinary and Animal Science 3(4): Islam MR, Khan MAHNA, Das PM, Bari ASM (1998). Poultry diseases diagnosed at necropsy in 1997 and 1998 in the Department of Pathology of Bangladesh Agricultural University, Mymensingh. Proceedings of 5th BSVER Annual Scientific Conference held on 3-4 at Bangladesh Agricultural University, Mymensingh. 8. Jalil MA, Samad MA, Islam MT (2009). Evaluation of maternally derived antibodies against Newcastle disease virus and its effect on vaccination in broiler chicks. Bangladesh Journal of Veterinary Medicine, 7(2): Lamb RA, Collins PL, Kolakofsky D, Melero JA, Nagai Y, Oldstone MBA, Pringle CR, Rima BK (2005). Family Paramyxoviridae. In: Virus Taxonomy, VIIIth Report of the International Committee on Taxonomyof Viruses. Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA, (eds.), Elsevier Academic Press, San Diego, pp: Mozaffor Hossain KM, Ali MY, Yamato I (2010). Antibody levels against Newcastle disease virus in chickens in Rajshahi and surrounding districts of Bangladesh, International Journal of Biology, 2(2): Numan M, Zahoor MA, Khan HA, Siddique M (2005). Serological status of Newcastle disease in broilers and layers in Faisalabad and surrounding districts. Pakistan Veterinary Journal, 25(2): OIE (2002). Office International Des Epizooties Manual of Standards for Diagnostic Tests and Vaccines. 4th Eds., Paris, France. 13. OIE (2012). Newcastle disease. Manual of diagnostic tests and vaccines for terrestrial animals. Chapter international-standardetting/terrestrial-manual/accessonline 98

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