Molecular Detection and Pathotyping of Newcastle Disease Virus in Pigeons from Karnataka, India

Newcastle disease (ND) is an economically important disease affecting the poultry industry across the globe (Cattoli et al., 2011, Alexander et al., 2012 and OIE, 2012). The ND is caused by Avian avulavirus type 1 (AAvV-1), that belongs to genus Avian orthoavulavirus 1 (AOAV-1) within the subfamily Avulavirinae of the family Paramyxoviridae (ICTV, 2019). This enveloped virus has a negative-sense single-stranded genome of approximately 15 kb in size, which codes for six proteins (Tirumurugaan et al., 2011). The world organization for Animal health recognized ND as “notifiable” (Dimitrov et al., 2016). The Newcastle disease virus (NDV), broadly categorized into four major pathotypes, depending on the severity of the disease as velogenic, mesogenic, lentogenic and asymptomatic. The velogenic strains are associated with high mortality and are further classified as neurotropic or viscerotropic and mesogenic strains cause primarily respiratory disease (Naveen et al., 2014). Lentogenic and asymptomatic strains usually cause mild respiratory and enteric infections, respectively (OIE, 2012). Newcastle disease virus, is a serotype 1 of avian paramyxovirus (APMV-1). It has two distinct clades; class I and class II and class II is further divided into 18 recognized genotypes (Diel et al., 2012). The most common methods of patho typing of NDV involve the determination of intra-cerebral pathogenicity index (ICPI) and Mean death time (MDT) (Roohani et al., 2015). Due to expansion of the poultry industry and usage of live virus vaccines may lead to the probability of spillover of vaccine strains (Swayne et al., 2014 and Devlin et al., 2016).
       
As mentioned, a wide range of wild bird species can contact the varying degree of infection. Pigeons are susceptible to infection with velogenic NDV and act as potential carriers in the transmission of velogenic NDV among poultry flocks (Deepthi et al., 2020).

Study area and sample collection
 
Karnataka Veterinary, Animal and Fisheries Sciences University, Nandinagar, Bidar, is very close to Chitta forest and found dead pigeons (n=5) at college premises and performed postmortem to collect tissue samples from march to may 2021. The tissues of the lungs, spleen, trachea, proventriculus and brain with typical lesions were collected and pooled in sterile containers with sterile phosphate buffered saline (PBS) pH 7.0 -7.4 and samples were transported on ice to the laboratory and stored at -80°C until processing.
 
Preparation of sample
 
Propagation of ND virus
 
Tissue lysates were inoculated in 9 days old embryonated SPF chicken Eggs (6 eggs per each sample) (OIE, 2012)  (Supplied by Venkey’s, Venkateshwara hatcheries Pvt. Ltd. Pune) by allantoic cavity route and found the MDT of embryos (Shan et al., 2021) and ICPI in day-old chicks (Damena et al., 2016).
 
HA and HI tests
 
Spleen, trachea and proventriculus were processed as 10% homogenates in 500 μl of PBS supplemented with antibiotics (100 units of penicillin G, 100 µg of streptomycin and 0.25 µg of amphotericin B/ ml) and tissue homogenates were clarified by centrifugation at 4°C for 8 min at 2500 rpm and the supernatant was further filtered through a syringe filter (0.45 µm) and subjected to Hemagglutination (HA) test; Hemagglutination Inhibition (HI) test was employed as per standard procedures mentioned in the Terrestrial Manual of OIE, 2012, by using polyclonal chicken antiserum raised against LaSota (Chowdhary et al., 2020).
 
Molecular detection of NDV
 
Viral RNA extraction
 
The HI positive samples were further processed for RNA isolation employing TRIzol® Reagent (Invitrogen, USA, Cat # 15596026) following the manufacturer’s instructions.
 
Reverse transcriptase polymerase chain reaction (RT-PCR)
 
Extracted RNA was used to get DNA by One step RT-PCR kit (Cat # 12574018) to amplify a 356 bp fragment of the NDV Fusion gene. Samples were confirmed as ND by Reverse Transcriptase Polymerase chain reaction (RT-PCR) using primers targeting fusion protein cleavage site (FPCS). Partial sequences of F gene (356 bp) of the isolates are amplified by using the following published primers NDVF-5'-GCAGCTGCAGGGAT TGTGGT-3' and NDVR-5'-TCTTTGA GCAGGAGGATGTTG-3' targeting FPCS of a fusion gene (Toyoda et al., 1989, Nanthakumar et al., 2000, Naveen et al., 2014 and Roohani et al., 2015). The primers generate an expected amplicon size of 356 bp. Briefly, a total of 25 μl reaction mixture is prepared using 2 μl of RNA, 12.5 μl of 2X master mix with 1 μl primer NDVF, 1 μl primer NDVR, 1 μl of Superscript-III Taq Polymerase enzyme and 7.5 μl nuclease free water. The reaction conditions were as follows: Reverse transcriptase step 55°C/ 30 min followed by One initial PCR activation cycle at 94°C for 2 min followed by 39 three-step cycles of 94°C for 15 sec, 58°C for 45 sec and 68°C for 1 min 30 sec; then 68°C for 5 min. The amplified PCR products were analysed by using 1% agarose gel containing ethidium bromide and were observed under UV illumination. 

Postmortem lesions
 
The post-mortem examination of pigeons showed congestion and haemorrhages noticed on crop and trachea, also seen enteritis, whitish-green intestinal contents and cloudiness of the air sacs of dead pigeons Fig 1 and similar findings were examined by Chowdhary et al., (2020).
 

       
Out of five collected pooled samples, three were positive for the HI test in which LaSota vaccine was used as a positive control Ojasvita et al., (2019). Samples confirmed by HI (titers >1/16) test (Fig 2) and similar results of titers were reported by Zhan et al., (2021). Positive samples were further confirmed by RT-PCR targeting partial fusion gene encompassing FPCS as like Nanthkumar et al., (2000).
 

 
Pathotyping
 
All three HI positive samples were identified pathotype as Velogenic based on the MDT of embryos and ICPI values in day-old chicks. ND virus isolates reported at 42 hours, similar findings reported by Chowdhary et al., (2020) where 89 hours as per results reported by Shan et al., (2021) in Mesogenic pathotype.
       
ND Virus isolated from an outbreak in racing pigeons in India was found to be velogenic since the MDT was 60 hours as per Roy et al., (2000) and MDT recorded in our study was 42 hours which indicated Velogenic pathotype.
       
The ICPI values in this study obtained as 1.8 (Velogenic) and similar findings were reported by Chowdhary et al., (2020) and reported almost similar findings (1.78) by Nanthakumar et al., (2000), where it was reported 1.4 by Roy et al., (2000) as Mesogenic type and 0.7 reported by Damena et al., (2016) as is Lentogenic type.
       
The 28% of domestic pigeon population grown in the kanyakumari were reservoirs of NDV and may produce outbreaks at any time, when the suitable climate to epidemics (Kumar and Rajan 2016).
 
Reverse transcriptase polymerase chain reaction
 
Extracted RNA was used to get DNA by One step RT-PCR kit (Invitrogen, USA with  Cat # 12574018) to amplify a 356 bp fragment of the NDV Fusion gene from positive samples of pigeons (Naveen et al., 2014). Standard vaccine (LaSota) was used as a positive control; Chowdhary et al., 2020 also used the LaSota vaccine as a positive control. RT-PCR system is used to amplify a specific portion of a Fusion genome that will give added value, contains F0 cleavage site to assess the virulence and detection of the NDV (Maqbool et al., 2017) (Fig 3). F gene sequences encode for the cleavage activation site of the fusion protein with restriction enzymes (Nanthakumar et al., 2000).
 

       
From an epidemiological view point, pigeon presents a unique of free ranging nature favouring contact with other wils birds and commercial poultry which increase the risk of exposure to virus (Alexander et al., 2012).
       
In this current study, the causative agent of Velogenic NDV can cause outbreaks in unvaccinated poultry flocks of the area transmitted by feed and water sources of the contaminated fecal-oral route from the pigeon. This study is supported by the findings of Cattoli et al., (2011).

In conclusion, as per our knowledge, this is the first report of molecular detection and pathotyping of NDV in pigeon from Karnataka. Still, more molecular and epidemiological studies have to be carried out to reveal the Antigenic characterization and control strategies of Newcastle disease in pigeons.

I greatly thankful to Dr. Vijay Kumar Anumolu, Assistant professor, Department of Veterinary Public Health and Epidemiology, College of Veterinary Science, PVNRTVU, Hyderabad, Telangana, for providing one step RT-PCR kit (Invitrogen, USA).

The authours declare that they have no conflict of interests.

The Institutional animal ethics committee approved all the experimental animal protocols performed in the study of Veterinary College, KVAFSU, Bidar, Karnataka (Approval number: 13/2020/VCB/VMC, dated: 13/3/2020).