Molecular epidemiological insights into respiratory fowl pox virus circulation in backyard poultry of Tamil Nadu, India

Introduction

Backyard poultry farming plays a vital role in rural economies by contributing to household income, nutritional security, and livelihood sustainability. In India, indigenous poultry are commonly reared under scavenging or semi-intensive systems, often without structured vaccination schedules or biosecurity practices. Such production systems increase vulnerability to infectious diseases and facilitate the maintenance of pathogens that may threaten commercial poultry enterprises (Sonaiya, 2007;Biswas et al., 2005) [3, 1] . Fowl pox is a viral disease caused by Fowl Pox Virus (FPV), a member of the genus Avipox virus under the family Poxviridae. The disease manifests primarily in two clinical forms: the cutaneous form and the diphtheritic or respiratory form. The respiratory form is characterized by proliferative lesions involving the oral cavity, pharynx, larynx, and trachea, frequently resulting in respiratory distress and compromised performance (Tripathy & Reed, 2020) [5] . FPV is transmitted through direct contact, contaminated materials, and mechanical vectors such as mosquitoes and other biting insects. Backyard poultry, due to free-range management and increased vector exposure, are particularly susceptible to infection. Despite the endemic nature of the disease, limited molecular epidemiological information is available on respiratory fowl pox virus in backyard poultry populations. Polymerase Chain Reaction (PCR) assays targeting conserved genes such as P4b have emerged as reliable tools for sensitive and specific detection of FPV. This study was therefore undertaken to investigate the molecular epidemiology of respiratory fowl pox virus in backyard poultry of Namakkal district and to identify associated risk factors.

Materials and Methods

Study area and duration

The study was carried out in four taluks of Namakkal district, Tamil Nadu, India, over a period extending from June 2016 to April 2017. The area is characterized by dense poultry activity, including both backyard and commercial poultry farming.

Study population and sampling

A total of 117 backyard poultry farms rearing desi birds were included. Farms were classified based on flock size as small (5-20 birds), medium (21-50 birds), and large (≥51 birds). Epidemiological information pertaining to age, vaccination status, management practices, and proximity to commercial farms was collected using a structured questionnaire. Altogether, 239 samples were obtained, including tracheal swabs (n = 110), faecal swabs (n = 107), necropsy tissues (n = 17), and pox lesion scabs (n = 5), from both clinically affected and apparently healthy birds. Samples were transported to the laboratory in chilled conditions using phosphate-buffered saline.

Virus isolation

Homogenates prepared from respiratory and lymphoid tissues were inoculated onto the Chorioallantoic Membrane (CAM) of 9-11-day-old specific pathogen-free embryonated chicken eggs. Inoculated eggs were incubated at 37°C and examined daily. CAMs exhibiting typical pock lesions were harvested for molecular analysis.

DNA extraction and PCR reaction mixture

Viral DNA was extracted from CAM tissues and pox scab samples using the QIAamp® DNA Mini Kit according to the manufacturer's instructions then DNA was prepared for PCR reaction (Table 1 &1a). PCR amplification was performed using FPV-specific primers targeting the P4b gene (Table 2), yielding an expected product size of approximately 578 bp. The BM strain fowl pox vaccine was used as a positive control. Volume 50 units/ml 400µM 400µM 400µM 400µM 3mM Holding temperature 4⁰C

Submarine gel electrophoresis

The electrophoresis tank was filled with 1X Tris acetate buffer until the gel tray was completely submerged. A volume of 5 µl of RT-PCR product was mixed with gel loading buffer and loaded into the wells along with the PCR-amplified samples, positive control (reference vaccine strain), negative control, and a 100 bp DNA ladder. After loading, the lid of the electrophoresis unit was closed and connected to the electrical leads. DNA migration occurred towards the positive anode (red lead). Electrophoresis was performed at 80 V for 45 minutes.

Visualization of PCR products

Following electrophoresis, the gel was visualized under a UV transilluminator. The amplified products were identified by comparing the band sizes with the 100 bp DNA ladder. Samples were considered positive only when NDV, IBV, FPV, and ILT amplicons produced bands corresponding to 356, 410, 580, and 588 bp, respectively, running parallel to the expected DNA fragments on the gel, with no amplification observed in the negative control. Gel images were documented using a gel documentation system (Bio-Rad, Germany).

Statistical analysis

Farm-level incidence was calculated, and associations between FPV occurrence and risk factors such as age, vaccination status, management system, and proximity to commercial farms were evaluated using chi-square analysis. Statistical significance was determined at p<0.05. The vaccine strain used as control also showed specific amplification (Figure 3).

Results

Pathological findings and virus isolation

Incidence of FPV

The overall farm-level positivity for fowl pox among suspected farms was 62.5%. FPV DNA was detected in both clinically affected birds and apparently healthy birds, indicating the presence of subclinical infections within backyard poultry populations (Table 1)

Analysis of risk factors

A higher frequency of FPV detection was recorded in birds below 24 weeks of age compared to older birds. Unvaccinated flocks exhibited greater positivity than vaccinated flocks. Birds maintained under free-range systems showed significantly higher FPV incidence than those reared under semi-intensive conditions (p<0.05). No statistically significant association was observed between FPV occurrence and proximity to commercial poultry farms (Table 2).

Discussion

The findings of the present study demonstrate the endemic presence of respiratory fowl pox virus in backyard poultry farms of Namakkal district. PCR-based detection targeting the P4b gene proved effective in identifying FPV infections, including subclinical cases that may otherwise go unnoticed. The increased incidence observed in younger birds could be attributed to underdeveloped immune responses and heightened susceptibility to infection. Higher detection rates in unvaccinated flocks highlight the absence of systematic vaccination programs in backyard poultry systems. Free-range birds are more frequently exposed to insect vectors and contaminated environments, which likely explains the significantly higher incidence recorded under this management system. The lack of a significant association with proximity to commercial farms suggests that FPV transmission in backyard poultry is predominantly influenced by local ecological factors and vector activity rather than direct transmission from commercial flocks. Comparable observations have been reported in other endemic settings where vector-mediated spread plays a major role (Bolte et al., 1999;Tripathy, 1993) [2,4] . Detection of FPV in apparently healthy birds further emphasizes the role of backyard poultry as silent carriers, facilitating viral persistence and sporadic outbreaks.

Conclusion

This study provides molecular confirmation of respiratory fowl pox virus circulation in backyard poultry of Tamil Nadu. The higher occurrence among young, unvaccinated birds maintained under free-range conditions underscores the need for enhanced surveillance, vaccination awareness, and vector control measures. Integration of molecular diagnostic tools into routine monitoring programs is essential to minimize the impact of fowl pox and to prevent wider dissemination of the virus.

Acknowledgement

AcknowledgementsThe authors gratefully acknowledge the support and facilities provided by the Department of Veterinary Preventive Medicine, Veterinary College and Research Institute, Namakkal. The cooperation extended by backyard poultry farmers during sample collection is sincerely appreciated.

References

1. Biswas PK, Christensen JP, Ahmed SSU, Barua H, Das A, Rahman MH, et al. A longitudinal study of the incidence of major endemic and epidemic diseases affecting semi-scavenging chickens. Avian Pathol. 2005;34(4):303-312.
2. Bolte AL, Meurer J, Kaleta EF. Avian host spectrum of avipoxviruses. Avian Pathol. 1999;28(5):415-432.
3. Sonaiya EB. Family poultry, food security and the impact of HPAI. World's Poult Sci J. 2007;63(1):132-138.
4. Tripathy DN, Reed WM. Pox. In: Swayne DE, editor. Diseases of poultry. 14th ed. Hoboken (NJ): WileyBlackwell; 2020.