Newcastle Disease Virus Detection From Chicken Organ Samples Using Reverse Transcriptase Polymerase Chain Reaction

Newcastle disease (ND) is a systemic, viral respiratory disease that is acute and easily transmitted which affects various types of poultry, especially chickens. Diagnosis of ND which generally involves virus isolation and subsequent identification with serological assays has limitations that needs more time. This research was aimed to detect Newcastle Disease virus (NDV) in chickens suspected with ND using the Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) technique. Nine chicken organ samples such as lien, trachea, and lungs were collected from chicken farms diagnosed with ND. The organ samples were processed and the targeted viral RNA was extracted using the RNA extraction kit. Genome amplification was performed with RT-PCR using specificprimers to target the F gene. Amplification results produced an amplicon product of 565 base pairs (bp). PCR product samples were then visualised using agar gel electrophoresis and viewed using the unified gel documentation system. Amplification results show nine samples positive for the DNA bands corresponding to the targeted band of the NDV F gene fragment. The results of this research confirm that the RT-PCR method is applicable for NDV detection from chicken organ samples.


Introduction
Newcastle disease (ND) is a systemic, respiratory disease that is acute and easily transmitted which is caused by a virus and affects various types of poultry, especially chickens (Tabbu, 2000). ND was first recognized in Indonesia in 1926 and classified as a viral disease (Alexander et al., 2004). The virus is endemic in most regions of the country as it has been isolated frequently in outbreaks (Dharmayanti et al., 2014;Wibowo et al., 2012). Wibowo et al. (2012) reports three types of Newcastle Disease virus (NDV) virulence found in the field, which are low, moderate, and virulent strains. According to Alexander (2003), the most virulent NDV strain is the Asiatic type which causes a 100% mortality rate in susceptible poultry, causing significant loss in Indonesia's poultry industry.
According to FAO (2010), poultry farming facilities throughout Indonesia range from backyard productions (sector 4) to high or low biosecurity commercial poultry production (sectors 2 and 3) to integrated, industrial productions (sector 1); with sector 2 commercial poultry productions dominating the national market. Regardless the level of biosecurity applied, all four sectors are in threat of Tetelo disease -local name for ND (Wibowo & Amanu, 2010). This poses major problem to the nation as the disease is a sporadic epizootics despite implementation of routine vaccination programmes (Kryger et al., 2010). The consequences of the disease are low growth rate and production, high expense on prevention and treatment, and high mortality rate (Amanu & Rohi, 2005).
The Office of Internationale des Epizooties (OIE) classified ND as list A disease (Alexander, 2000). According to OIE (1996), list A diseases are defined as "transmissible diseases that have the potential for very serious and rapid spread, irrespective of national borders, that are of serious socio-economic or public health consequence and that are of major importance in the international trade of animals and animal products." The virus belongs to the genus of Avulavirus and Paramyxoviridae family.
It has an enveloped, non-segmented, negativesense, single stranded RNA genome that encodes six proteins (Rabalski et al., 2014;Shirvan & Mardani, 2014). Avian paramyxovirus type 1 (APMV-1) or ND is a virus that occurs in filamentous or spherical forms (pleomorphic) and consists of a single molecule of negative sense, single-stranded RNA enclosed in an envelope with prominent glycoprotein spikes (MacLachlan & Dubovi, 2010).
The RNA genome is about 15-18 kb in size and can consist up to at least three genome lengths; 15,186, 15,192 and 15,198 nucleotides (Miller et al., 2010). These nucleotides make up six genes which encode the following six structural proteins: nucleocapsid protein (NP/N), phosphoprotein (P), matrix protein (M), fusion protein (F), haemagglutinin-neuraminidase (HN) and large protein (L) in the order of 3'leader-N-P-M-F-HN-L-5'trailer (Kho et al., 2003). Two additional non- proteins contribute to the virulence characteristic of NDV, but the primary determinant is the cleavability of the F protein (Cattoli et al., 2011). The F protein is synthesized as an inactive precursor (F0) and is only fusogenic after cleavage into two biologically active F1 and F2 polypeptide subunits, by host cell proteases (de Leeuw et al., 2005). The cleavage site of the F protein differs in arrangement of multiple basic amino acids depending on the degree of virulence. NDV is highly contagious, with reports stating that even low pathogenic viral strains (lentogens) that cause mild infections of the respiratory tract can greatly increase its pathogenicity even during a single passage (Rabalski et al., 2014).
A definitive diagnosis of ND requires virus isolation and subsequent laboratoric characterization.
Virus isolation is carried out by injecting and incubation antibiotic suspensions of samples retrieved from live birds into the allantoic cavity of embryonated chicken eggs. After the incubation period, the allantoic fluid is then harvested and tested (Alexander et al., 2004). Clinical signs and lesions may be highly suggestive; however this method proved impracticable as a routine tool (Haque et al., 2010). Instead conventional techniques such as agar gel precipitation test (AGPT), haemagglutination (HA) and haemagglutination inhibition (HI) tests were employed to assess antibody levels in birds. The AGPT has been found to be simple, inexpensive and faster in assessing ND infection in birds, nevertheless at present the HI test is most widely used technique (Alexander et al., 2004).  (OIE, 1996). The RT-PCR method is superior to other NDV confirmatory diagnostic methods (Singh et al., 2005). This method uses a gene-specific primer to generate singlestranded cDNA copies of NDV RNA gene, with the help of reverse transcriptase (RT) enzyme. The cDNA copies are used as PCR templates to produce exponential amplification of the viral nucleic acids, even if it is present in small amounts (Rio et al., 2011).
The analysis study on NDV isolates by Angeliya et al. (2014) Table 2

Results and Discussion
This research applied RT-PCR technique to detect the presence of NDV genome in chickens organ samples suspected with NDV. RT-PCR results are summarised in Table 3 and Figure 1.
In Table 3, the RT-PCR results were recorded as positive with a '+' sign, and negative with a '-' sign, and both positive and negative controls serve as reference. The La Sota live ND vaccine used as the positive control contains the targeted 565 bp NDV genome and GeneRuler 100bp DNA Ladder helps estimate the size of the nucleotides separated and detect whether the desired gene sequence was amplified.
Band formations along lanes 2 to 10 in   NDV detection with RT-PCR primarily targets the F gene because NDV virulence is principally dependent on the amino acid sequence at the F0 cleavage site (Alexander, 2000). In comparison, Dharmayanti et al. (2014) applied another set of primers to target different portion of the F gene with a 535 bp product Likewise, Singh et al. (2005) used the same primers for the 356 bp amplicon, along with another pair of primers targeting the 216 bp internal sequences of the NDV F gene cleavage activation. Results from these studies show that RT-PCR amplification applying F gene as the target sequence to be effective for NDV detection. NDV sequencing using RT-PCR has been established as an effective method, especially after prior isolation and serology tests as prescribed by the OIE Standards Commission (Oberdorfer & Werner, 1998). A research by Kencana et al. (2012) used this technique for confirmatory diagnosis of ND in native Bali chickens. Kencana (2012) carried out propagation of samples in embryonated chicken eggs and subsequent confirmatory serology tests prior to RT-PCR amplification. The same method was reported by Dharmayanti et al. (2014).
However, in this research RT-PCR was performed to detect NDV directly from chicken organ samples. According to Kant et al. (1997) found direct RT-PCR detection from organ samples containing virulent NDV to be possible, but failed to detect NDV in some tissue samples containing nonvirulent NDV due to the assay sensitivity. Another report by Gohm et al. (2000) states that rapid NDV detection is achieved by employing RT-PCR directly on samples from affected birds without prior virus isolation. These findings are in close agreement with the present study.

Conclusion
All nine samples were confirmed positive as NDV by RT-PCR using specific primers of targeted F gene fragment and produced of 565 base pairs amplicon product. The result showed that this method could be used for NDV detection from chicken organ samples without prior isolation and propagation in embryonated eggs. Hence, the RT-PCR method is applicable for detection of NDV in chicken organ samples.