Molecular epidemiology of respiratory virus - Newcastle disease in backyard poultry

Introduction

Backyard poultry production plays a vital role in sustaining rural livelihoods in India by providing nutritional security and supplementary income to smallholder farmers (Sonaiya, 2007; FAO, 2010) [11,6] . However, the unorganized nature of backyard poultry rearing, coupled with minimal biosecurity and irregular vaccination practices, predisposes birds to various infectious diseases. Among these, Newcastle Disease (ND) is considered one of the most economically significant viral diseases affecting poultry, capable of causing high morbidity and mortality (Alexander, 2000; Saif et al., 2008) [2,10] . Newcastle Disease Virus (NDV), also known as avian orthoavulavirus-1, belongs to the genus Orthoavulavirus within the family Paramyxoviridae (Alexander & Senne, 2008) [4] . NDV strains vary widely in virulence and are classified as lentogenic, mesogenic, or velogenic based on clinical severity and molecular characteristics. Respiratory involvement is a hallmark of ND, particularly in lentogenic and mesogenic strains, and is frequently observed in backyard poultry due to continuous exposure to environmental stressors and infected birds. Backyard poultry often acts as a silent reservoir of NDV, facilitating virus persistence and transmission to commercial flocks (Biswas et al., 2005; Hamilton-West et al., 2012) [5,7] . Conventional diagnostic methods based on clinical signs and postmortem lesions are insufficient for precise characterization of circulating NDV strains. Molecular techniques such as RT-PCR offer sensitive and specific detection and enable epidemiological tracking of NDV in field conditions. The present study was therefore undertaken to investigate the molecular epidemiology of respiratory NDV infections in backyard poultry in Namakkal district, Tamil Nadu.

Materials and Methods

Study area and period

The study was conducted in four taluks of Namakkal district, Tamil Nadu Tiruchengode, Rasipuram, Paramathivelur, and Namakkal between June 2016 and April 2017. The region is characterized by a high density of both commercial and backyard poultry populations.

Sampling strategy

A total of 117 backyard poultry farms rearing between 5 and 100 birds were surveyed. Farms were categorized into small (5-20 birds), medium (21-50 birds), and large (>50 birds) holdings. Epidemiological data including age, vaccination status, management practices, and proximity to commercial farms were recorded using a structured questionnaire.

Sample collection

A total of 239 samples were collected, comprising 110 tracheal swabs, 107 faecal swabs, 17 necropsy specimens, and 5 pox lesions from clinically affected and apparently healthy birds. Samples were transported to the laboratory in phosphate-buffered saline under cold chain conditions.

Virus isolation in SPF embryonated eggs

Processed samples were inoculated into the allantoic cavity of 9-11-day-old specific pathogen-free embryonated chicken eggs. Eggs were incubated at 37°C and candled daily for five days. Embryos exhibiting characteristic ND lesions such as haemorrhages were harvested, and allantoic fluid was collected for further analysis.

RNA extraction and RT-PCR

Viral RNA was extracted from allantoic fluid using the TRIzol LS method. RT-PCR was performed using NDVspecific primers targeting the fusion (F) protein gene, yielding a 356 bp amplicon, a widely used molecular marker for NDV detection and characterization (Aldous & Alexander, 2001; Wise et al., 2004) [3,12] . The LaSota vaccine strain served as a positive control.

Statistical analysis

Incidence rates were calculated, and associations between NDV occurrence and epidemiological risk factors were assessed using chi-square analysis. A p-value <0.05 was considered statistically significant.

Results

Virus isolation and gross pathology

Out of the total samples processed, fourteen NDV field isolates were successfully propagated in SPF embryonated eggs. Embryos showed characteristic occipital and generalized haemorrhages consistent with NDV infection (Figure 1). [8,10] .

Risk factor analysis

NDV incidence was higher in unvaccinated flocks and in freeranging management systems. Farms located within 1 km of commercial poultry units showed a slightly higher prevalence, although the difference was not statistically significant (Table 2).

Discussion

The present study provides molecular evidence of NDV circulation in backyard poultry in Namakkal district. The detection of NDV in apparently healthy birds underscores the role of backyard poultry as reservoirs facilitating virus persistence. Higher susceptibility in younger birds may be attributed to immature immune responses and lack of vaccination. Molecular detection targeting the F gene remains a reliable approach for NDV identification, as demonstrated by consistent amplification of the 356 bp product, as also reported by Aldous and Alexander (2001) [3] and Wise et al. (2004) [12] . The similarity in amplicon size between field isolates and vaccine strains highlights the need for further sequencing studies to determine virulence and genotype distribution. The absence of a structured vaccination schedule and low biosecurity practices in backyard systems significantly contribute to NDV endemicity. Regular vaccination, farmer awareness, and integration of backyard poultry into regional disease surveillance programs are essential to mitigate ND outbreaks.

Conclusion

This study confirms the endemic presence of respiratory Newcastle disease virus in backyard poultry through molecular epidemiological investigation. Backyard poultry populations serve as important reservoirs for NDV, posing a continuous risk to commercial poultry. Implementation of regular vaccination programs, improved management practices, and molecular surveillance is imperative for effective control of ND in mixed poultry production systems (Alexander, 2001; OIE, 2009) [3,13] .

Acknowledgement

AcknowledgementsThe authors acknowledge the support of the Department of Veterinary Preventive Medicine, Veterinary College and Research Institute, Namakkal, for providing facilities to carry out this research.

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