Isolation and Biological Characterization of Newcastle Disease Virus (NDV) Field

Background: Avian Paramyxovirus (APMV) type-1 is the leading cause of Newcastle Disease (ND) and taxonomically belongs to the family Paramyxoviridae, genus Avulavirus. Due to its high transmission rate, Newcastle Disease (ND) is included in the A list by the OIE. Purpose: To determine the biological characterize the Newcastle Disease Virus (NDV) field isolate of pigeons (Columba livia) using Mean Death Time (MDT), Intracerebral Pathogenicity Index (ICPI), and Intravenous Pathogenicity Index (IVPI). Methods: Twenty pigeon organ samples were obtained from bird markets in East Java, and one was used as a positive control (LaSota). Organs were isolated from embryonated chicken eggs, identified by the HA test, and then confirmed by the HI test. Furthermore, positive samples were tested for MDT with a 10-1-10-18 dilution (0.1 ml and observed for eight days. The ICPI test used a fresh NDV suspension (0.05 ml and was observed for eight days. The IVPI test used a dose of 0.1 ml and was observed for ten days. Result: The MDT values of isolates MB1/NDV/19, MB2/NDV/19, MB3/NDV/19, and MG1/NDV/19 were 91.2 hours, 112.8 hours, 110.4 hours, and 124,8 hours. The ICPI values of isolate MB1/NDV/19 was 0.2375, MB2/NDV/19 was 0.375, MB3/NDV/19 was 0.5375, and MG1/NDV/19 was 0.3. The IVPI value of isolate MB1/NDV/19 was 0, MB2/NDV/19 was 0, MB3/NDV/19 was 0, and MG1/NDV/19 was 0. Conclusion: All four field samples were positive for NDV as a lentogenic strain based on the MDT, ICPI, and IVPI tests.

Avian Paramyxovirus (APMV) type-1 is the main cause of Newcastle Disease (ND) and taxonomically belongs to the family Paramyxoviridae, genus Avulavirus.Newcastle Disease (ND) is included in the A list by the OIE owing to its high transmission rate.
To determine the biological, characterize the Newcastle Disease Virus (NDV) field isolate of pigeons (Columba livia) using Mean Death Time (MDT), Intracerebral Pathogenicity Index (ICPI), and Intravenous Pathogenicity Index (IVPI).Twenty pigeon organ samples were obtained from bird markets in East Java and one was used as a positive control (LaSota).Organs were isolated from embryonated chicken eggs, identified by the HA test, and then confirmed by the HI test.Furthermore, positive samples were tested for MDT with a 10-1-10-18 dilution (0.1 ml and observed for eight days.The ICPI test used a fresh NDV suspension (0.05 ml and was observed for 8 days.The IVPI test used a dose of 0.1 ml and was observed for 10 days.
All four field samples were positive for NDV as a lentogenic strain based on the MDT, ICPI, and IVPI tests.

INTRODUCTION
Avian Paramyxovirus (APMV) type-1 is the main cause of Newcastle Disease (ND).Taxonomically, the ND virus belongs to the Paramyxoviridae family, Avulavirinae subfamily, and genus Avulavirus.Newcastle Disease (ND) has been identi ed as having 21 serotypes, such as APMV 1-21 and is known to infect more than 200 species of birds, including the Columbidae family (Columbiformis) (Amarasinghe et al., 2019;OIE, 2021).e ND virus is divided into several strains based on the degree of virulence, ordered from high to low pathogenicity, namely Viscerotropic Velogenic, Neurotropic Velogenic, Mesogenic, Lentogenic or respiratory, and subclinical (OIE 2021).e structure of the ND virus is circular, pleomorphic, diameter 100-500 nm, enveloped, negative polarity, non-segmented genome, ss-RNA with six main proteins: NP, P, M, F, HN, L, and two non-structural proteins, namely V and W (Snoeck et al., 2013;Ashraf and Shah, 2014;He et al., 2018; ).
e O ce International des Epizooties (OIE, 2009) has classi ed the ND virus as an A-list disease because of its high infectivity, high mortality, and morbidity in poultry (OIE, 2009).Pigeons (Columbidae) are usually unvaccinated, live wild in nature, and act as reservoirs for ND virus antigens.In the Columbidae bird group, the ND virus is known as Pigeon Paramyxoviruses 1 (PPMV-1) (Wang et al., 2021).PPMV-1 has a morbidity rate of up to 100% and a mortality rate of approximately 70%, with torticollis being the characteristic clinical feature (Guo et al., 2014).e genus Columba is the main host species; however, several studies have shown that cross-infection occurs in chickens, leading to malignant traits (Brown and Bevins, 2017;Xiang et al., 2019).e pathogenicity of the ND virus in causing death in embryonated chicken eggs or Mean Death Time (MDT) is divided into velogenic strains (<60 h), mesogenic strains (60-90 hours), and lentogenic strains (>90 h) (Alazawy and Al Ajeeli, 2020;OIE, 2009).e ability of the ND virus to cause death of 1-Day-Old Chick (DOC) through the Intracerebral Pathogenicity Index (ICPI) test, with an assessment category that ICPI>1.5 was declared a velogenic strain, 0.7<ICPI<1.5 was declared a mesogenic strain, ICPI<0.7 was declared a lentogenic strain (Hossain et al., 2017;OIE, 2009).e ability of the ND virus to cause death of 6-week-old chickens was assessed through the Intravenous Pathogenicity Index (IVPI) test, with the following assessment categories IVPI>1.45 was declared, velogenic strain; 0<IVPI<1.45was declared, mesogenic strain; and IVPI=0, lentogenic strain (Awad et al., 2015;OIE, 2009).Based on the above background, this study aimed to explain the biological characteristics of eld isolates of pigeon (Columba livia) ND virus using pathogenicity tests, including MDT, ICPI, and IVPI.

Sample Collection
e samples were collected using a purposive sampling method.In this study, several organ samples of non-vaccinated pigeon (Columba livia) collected from bird markets in East
A total of 20 samples of pigeons (Columba livia) from bird markets in East Java (four samples from the Bratang Bird Market, Surabaya City; ve samples from Candi District, Sidoarjo Regency; three samples from Sidoarjo District, Java were used. e samples collected in this study were obtained from necropsy pigeons (Columba livia).Several organs were collected, such as the brain, trachea, lungs, liver, proventriculus, and intestine

Hemagglutination Test
Hemagglutination test (HA) was performed according to the OIE protocol.Positive HA samples were then tested using the hemagglutination inhibition test (HI) to con rm the presence of the Newcastle Disease (ND) virus (OIE, 2021).

Mean Death Time
e Mean Death Time (MDT) was calculated to test the pathogenicity of ND virus in Embryonated Chicken Eggs.Inoculate 0.1 ml suspension on ve Embryonated Chicken Eggs per dilution.Observations were made every 12 h for seven days (OIE, 2009;Roohani et al., 2015).e suspension was titrated to obtain a 10-1-10-18 dilution MDT = ( Ʃ Eggs dead at n 1 hours) x (n 1 hours) + (Ʃ Eggs dead at n 2 hours) x (n2 hours)..etc

Ʃ Total Dead Eggs
Intracerebral Pathogenicity Index e Intracerebral Pathogenicity Index (ICPI) was used to test the pathogenicity of the ND virus in 10 DOCs aged 1 day.A fresh suspension of ND virus with a titer of not less than 2 was injected intracerebral into 10 DOCs at as much as 0.05 ml.Observations were made for 8 days every 24 h for eight days (OIE, 2009;Roohani et al., 2015).(a = normal, b = sick, c = dead).

Intravenous Pathogenicity Index
Intravenous Pathogenicity Index (IVPI) was performed to test the pathogenicity of the ND virus in 10 chickens aged 6 weeks.A fresh suspension of ND virus with a titer of not less than 2 was intravenously injected into 10 chickens aged 6 weeks at a dose of 0.1 ml.Observations were made every 24 h for 10 d (OIE, 2009).Sidoarjo Regency; three samples from Gresik Regency; four samples from Krian District, Sidoarjo Regency, and one sample from Pasuruan Regency), and ve organs were taken for each sample.e organs examined in this study were the brain, respiratory organs (trachea-pulmo), hepar, and digestive organs (proventriculus and intestine).e organs were crushed and placed in 1.5 mL centrifuge tubes containing 1.0 mL of transport medium and labeled.e total number of organs used in this study comprised 100 organs from six regions.
e organs were processed into a 10% suspension and then inoculated into 9 days old Embryonated Chicken Eggs for 120 h, with three Embryonated Chicken Eggs per organ.A total of 300 eggs were used.Egg candling once per day.If the eggs died during the incubation period (<120 h), the eggs were placed in the refrigerator at 4°C for 24 h, the allantoic uid was collected from the eggs, and checked through the HA-HI test using the microtechnical method.Eggs that were still alive on the last day of the incubation period (120 h) were placed in a refrigerator at 4oC °C for 24 h, and the allantoic liquid was collected and examined using a microtechnical HA-HI test.e rst step was the HA test, followed by the HI test using NDV antiserum.Based on the results of the HA-HI test, four out of 20 samples (11 organs) were positive for the ND virus, as described in Table 1.

Mean Death Time (MDT) Test
e Mean Death Time (MDT) was calculated for 90 Embryonated Chicken Eggs, with each dilution of ve eggs.Before inoculating the eggs, the suspension was titrated until a dilution was obtained starting from 10-1-10-18.e dilution is based on the fact that the results of a 10-10 dilution still result in complete death (100%).A total of 0.1 ml (10-1 dilution) was inoculated into 5 eggs aged 9-11 days.e same procedure was performed for the 10-2 to 10-18 dilutions.Egg observations were carried out every 12 h for ± 7 days using an egg candler to check whether dead embryos were present (OIE, 2009).In this study, 100% above 10-10 dilutions.Dead eggs were stored in a refrigerator.At the end of the observation period, the remaining live eggs were placed in the refrigerator for further HA testing.e data collected from the Mean Death Time (MDT) observations of all positive isolates are shown in the following graph (Picture 1).

Intracerebral Pathogenicity Index (ICPI) Test
e Intracerebral Pathogenicity Index (ICPI) was calculated for 10 DOCs aged 1 day (OIE, 2009;Roohani et al., 2015).A fresh suspension of Newcastle disease (ND) virus with a titer of not less than 2 was injected intracerebral as much as 0.05 ml.Observations were made for 8 days every 24 h for eight days, and the DOC conditions were observed.In this study,

Intravenous Pathogenicity Index (IVPI) Test
Intravenous Pathogenicity Index (IVPI) was performed on 10 chickens aged 6 weeks.A fresh suspension of Newcastle disease virus (ND) with a titer not less than 2 mL was injected intravenously as much as 0.1 ml (OIE, 2009).Observations were made every 24 h for 10 days, and the condition of the chickens was monitored.In this study, chicken isolates  HA testing was carried out on the four isolates to check for the presence of hemagglutinin produced by the Newcastle disease (ND) virus, which was inoculated in allantoic uid.e HA test results for the four positive isolates were shown by the presence of complete hemagglutination (di use) and without erythrocyte precipitate.
e Mean Death Time (MDT) was calculated based on the number of embryonated chicken eggs that died at the highest dilution tested.
Newcastle Disease (ND) virus pathogenicity test through the Intracerebral Pathogenicity Index (ICPI), using a fresh suspension with a titer of not less than 2 intracerebral doses of 0. ) belong to the lentogenic pathotype.e Intracerebral Pathogenicity Index (ICPI) test category of ≤0.7 was declared a lentogenic strain (Almubarak, 2019).
Clinical symptoms are associated with the young age of DOC, when the immune system is not working properly (Schijns et al., 2013).
e most visible clinical symptoms during the incubation period on day 1 for DOC isolate MB3/NDV/19 were airsacculitis and facial swelling, followed by death on day 2. Clinical symptoms of airsacculitis for DOC isolates MB2/NDV/19 and MG1/NDV/19 were observed on the 4th day and followed by death the next day.Clinical symptoms of airsacculitis caused by DOC isolate MB1/NDV/19 were observed on day 5, followed by death on day 7. e IVPI test rating category that causes death in 6-week-old chickens was equal to 0 (IVPI=0) and was declared lentogenic (Awad et al., 2015;OIE, 2009).
e immune system of 6-week-old chickens generally operates well.erefore, clinical symptoms did not appear a er the ND virus injection.e bursa of Fabrisius develops rapidly in young chicks and reaches its maximum size at 4-12 weeks of age.e largest mass in the bursa of Fabrisius is due to the large population of B lymphocyte cells that produce IgM (lymphoblasts), which undergo a maturation process.Young birds aged 2-8 weeks have a very active bursa of Fabrisius (Palya et al., 2014).
the DOC isolates MB1/NDV/19 died on the 7th day, MB2/NDV/19 died on the 5th day, and MB3/NDV/19 died on the 2nd day, whereas isolates MG1/NDV/19 died on the 6th day.Data collected from ICPI observations of all positive isolates are shown in the following graph (Picture 2).

e
Intravenous Pathogenicity Index (IVPI) test uses a fresh suspension titer of not less than 2 intravenously in the brachial vein at a dose of 0.1 ml. e results of this study show the same value (IVPI Graph A-D).e results obtained from the observation of the Intravenous Pathogenicity Index (IVPI) of isolate MB1/NDV/19 was 0. e Intravenous Pathogenicity Index (IVPI) value of the isolate MB2/NDV/19 was 0. e Intravenous Pathogenicity Index (IVPI) value of the isolate MB3/NDV/19 was 0. e Intravenous Pathogenic-ity Index (IVPI) of isolate MG1/NDV/19 was 0. From these results, it was concluded that the Bratang isolates (MB1/ND-V/19, MB2/NDV/19, and MB3/ NDV/19) and the Gresik isolate (MG1/NDV/19) belonged to the lentogenic pathotype.

Table 1 .
The HA-HI test results were positive from pigeons (Columba livia) isolate.