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Meskipun semua virus ND milik dari tipe serotip tunggal, mereka tidak pernah secara genetik
terpisah. Berdasarkan kepada fusi sekuens gen, strain virus ND dibagi menjadi dua Kelas, I dan II.
Setidaknya, 18 genotip dan berbagai subgenotif telah diketahui, [2-4], tetapi keragaman ini terus
menerus berkembang

Although allNDV strains belong to a single serotype, they are nevertheless genetically highly diverse.
Based onthe complete fusion gene sequences, NDV strains are divided into two classes, I and II. In
the latter, atleast 18 genotypes and multiple subgenotypes have been dened [24], but the
diversity continues toincrease as surveillance improves. NDV has a spacious diapason of host,
including approximately241species of 27 orders, of the known 50 orders ofbirds[1]. More commonly
species include chickens,turkeys, ducks, pigeons, guinea fowl, Japanese quail and many wild birds of
all ages [5]. Themostsusceptibleavian species to this disease are chickens and also some mammals
like humans, cats and dogs[6]. The disease transmits through droppings and secretions from the
nose, mouth and eyes of infected birds. The disease spreads by contaminated water,feed and
transport. Airborne transmission ofthevirus is also an important route of transmissionforND [7].
Mechanical transfer of infected faecesoccursby rodents, insects, dogs, eas,or scavenginganimals
[7]. Infection takes place by virus inhalation,ingestion or by contact with conjunctiva.

2. Aldous E.W., Mynn J.K., Banks J., et al. A molecular epidemiological study of avian paramyxovirus
type 1 (Newcastledisease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of
the fusion protein gene Avian Pathol. 2003. Vol. 32. P. 239256.

3. Liu M., Shen X., Cheng X.., Li J., Dai Y. Characterization and Sequencing of a Genotype VIId
Newcastle Disease Virus Isolated from Laying Ducks in Jiangsu, China // Genome Announc. 2015.
3(6). P. e01412e01415.

4. Miller P.J., Haddas R., Simanov L., Lublin A., Rehmani S.F., Wajid A., Bibi T., Khan T.A., Yaqub T.,
Setiyaningsih S., Afonso C.L. Identi Cation of new sub-genotypes of virulent Newcastle disease virus
with potential panzootic features // Infect Genet Evol. 2015. Vol. 29. P. 216229.

5. Miller P.J., Estevez C., Yu Q., Suarez D.L., King D.J. Comparison of viral shedding following
vaccination with inactivated and live Newcastle disease vaccines formulated with wild type and
recombinant viruses // Avian Dis. 2009. Vol. 53. P. 3949.

6. Nanthakumar T., Kataria R.S., Tiwari A.K., Butchaiah G., Kataria J.M. Pathotyping of newcastle
disease viruses by RT-PCR and restriction enzyme analysis // J. Vet. Res. Commun. 2000. Vol. 24.
P. 275286.

7. Ullah S., Ashfaque M., Rahman S.U., Akhtar M., Rehman A. Newcastle disease virus in the
intestinal contents of broilers and layers // Pak. Vet. J. 2004. 24(1). P. 2830.

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to evaluate the prevalence of the disease in areas where vaccination does not take place, and to
verify the antigenic response to vaccination.

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The causal agent is the Newcastle disease virus (NDV) which is a negative-sense single-stranded RNA
virus belonging to the family Paramyxoviridae. Through restriction site mapping and sequence
analysis of the fusion gene (F-gene), NDV strains have been divided into eight genotypes (Ballagi et
al., 1996). The strains are also classified into highly virulent (velogenic), intermediate (mesogenic) or
avirulent (lentogenic) based on their pathogenicity in chickens (Beard and Hanson, 1984).

NDV infections of poultry range from latent to rapidly fatal depending upon the pathotype of virus
involved (Alexander, 2003). The transmission of NDV occurs through newly introduced birds, selling
or giving away sick birds, exposure to fecal and other excretions from infected birds and contact with
contaminated feed, water, equipment and clothing (Tu et al., 1998). The disease causes high
economic losses due to high mortality, morbidity, stress, decreased egg production and hatchability
(Alexander, 2000).

Preparation of chicken red blood cell (RBC) suspension

A total of 5 ml of chicken blood was collected aseptically in a disposable syringe containing 1 ml of

sodium citrate (4% solution) as anticoagulant. The blood was centrifuged at 1500 rpm for 15
minutes and the plasma and buffy coat were removed with a pipette. After washing three times with
phosphate buffered saline (PBS), 1% suspension in PBS was prepared for HI test.

Haemagglutination inhibition (HI) test

HI test was done according to the procedure of OIE (2002). Briefly, two fold serial dilution of 25l
serum was made with PBS in V-bottomed microtiter plates (Nunc) up to 10 th well. Twenty five l
of 4 haemagglutinating (HA) units of Newcastle disease virus or antigen was added up to 11 Th well.
The plates were kept at room temperature for more than 30 minutes to facilate antigen antibody
reaction. Then 50l of 1% (v/v) chicken RBC suspension was added to each well. The 11 th well
contains antigen and RBCs as the positive control and the 12 well contains only RBCs as the negative
control. After gentle mixing, the RBCs were allowed to settle at room temperature for 40 minutes
and agglutination was assessed by tilting the plates. The samples showing peculiar central button
shaped settling of RBCs were recorded as positive and maximum dilution of each sample causing
haemagglutination inhibition was considered as the end point, which was used to estimate the HI
titer. The HI titer of each serum sample was expressed as reciprocal of the serum dilution.

Statistical analysis

The seroprevalence of NDV infection in relation to age of birds and seasons of year was compared by
means of the Chi-square test. A significant level of 5% was used.

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Newcastle disease (ND) is an acute, rapidly spreading, contagious, nervous and respiratory disease of
birds of all ages caused by the avian Paramyxovirus serotype 1 (APMV-1) (Okeke and Lamorde,
1988). Transmission of the virus is most common through bird to bird contacts (Hugh-Jones et al.,
1973), faecal exposure, respiratory discharges or other discharges from infected birds and through
contact with contaminated feeds, water, equipment, poultry attendants and clothing, leading to
high morbidity and mortality (Oladele et al., 2003; Saidu et al., 2006)

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.Basedonseverityofclinicaldisease, the strains of NDV were originally classified into 3 groups
basedontheirvirulencesuchaslentogenic,mesogenic,and velogenic. Lentogenic strains, especially in
adult chickens, may cause minimal or no clinical signs. However, the disease produced by mesogenic
strains may cause mortality that can reach 25% and those by in velogenic strains may reach up to
100% [3] Differences in virulence of the virus occur during any disease outbreak, so determining
virulence is essential for theeffectivecontrolofthedisease