Samples were collected from suspected cases observed in Veterinary Clinics and reported by pigeon owners in Basrah city center. The owner’s name, bird’s tag, and color were recorded. The study duration was from May to June 2022. A comprehensive examination of 120 domestic pigeons revealed 20 that displayed the clinical symptoms of fowl pox. The focus on pigeons with skin lesions, a prevalent and diagnostically valuable indication of the disease, underscored the importance of accurate sample collection and analysis. This endeavor was crucial for the well-being of the pigeon population and the overall management of potential disease outbreaks within the district.

The suspected cases were sent to the Department of Veterinary Pathology and Poultry Diseases at the University of Basrah for examination. Tissue samples from the skin around the eye and peck were pinched and pulled out using a scalpel, and each piece was divided into portions. The first portion was pooled in a sterile container with 5 ml of a 50% glycerol phosphate buffer solution and stored at -20°C for the polymerase chain reaction (PCR). The second portion was fixed in a 10% formalin solution for the histopathological examination, according to Luna [10].

Virus DNA was extracted from dermal lesions and a vaccine for fowl pox using a commercial kit (Wizard Genomic DNA Purification Kit; Promega, USA) according to the manufacturer’s instructions. DNA was eluted and stored at -20°C until used. For all PCR reactions, the mixture contained 25 μL of GoTaq G2 Colorless PCR Master Mix (Promega), 5 μL of the extracted DNA, 1.5 μL of each primer, and nuclease-free water that was added to a final volume of 50 μL. A control positive tube (DNA extracted from a standard Fowl Pox vaccine) and a negative control tube (DNA extracted from a control negative sample) were included. The presence of pox virus in the DNA extracted from clinical samples was confirmed by performing poxvirus-specific PCR with the primers designed for amplifying the 578 bp pox virus 4b gene (virus core protein) (4b forward, 5' -CAGCAGGTGCTAAACAACAA-3', and 4b reverse, 5'-CGGTAGCTTAACGCCGAATA-3') [11,12]. Macrogen (Korea) was then used to purify and sequence the PCR products of the pox virus 4b gene. The PCR cycles for the Avipoxvirus 4b gene consisted of one cycle at 94°C for 5 minutes, 35 cycles at 94°C for one minute, 61°C for one minute, and 72°C for one minute. One cycle was performed at 72°C for 10 minutes after these cycles. The clade of the poxvirus-positive sample was determined by amplifying the locus fpv140 by PCR using the forward primer (5'-GAAGTAGAGTTATCGGTTC-3') and the reverse primer M2912 ( 5'-GGTGATCCATTTCCATTTC-3'), as described elsewhere [13,14]. The fpv140 PCR distinguishes between clades A and B, which are 1,800 and 2,400 base pairs long, respectively. Amplification was performed after initial denaturation for 5 minutes at 94°C for 35 cycles and consisted of one minute of denaturation at 94°C, one minute of annealing at 55°C, and 2 minutes of extension at 72°C. A final extension step was performed for 15 minutes at 72°C. Subsequently, 2% (for P4b) and 1.2% (for fpv140) agarose gel electrophoresis was used to separate 5 microliters of the amplified PCR products. Ethidium bromide was then used to stain the samples, and an ultraviolet transilluminator was used to observe them next to a 1 K DNA ladder (Promega). Evolutionary analyses were conducted using MEGA11, and the evolutionary history was inferred using the neighbor-joining method.

In this study, 120 pigeons were examined. Twenty suspected cases of pigeon pox showed the cutaneous forms of pox infection, characterized by the development of wart-like growths on the skin and mucous membranes of the bird. These growths were observed on the head, face, beak, eyes, legs, feet, and wings. The growths were grayish-white in color and ranged in size from small papules to large nodules. These lesions were associated with swelling, redness, and ocular discharge, which led to impaired vision and difficulty in feeding in some cases (Fig. 1). These signs were more common in young pigeons. The suspected cases were lethargic and less active, tended to spend more time resting or sleeping, and showed less interest in their surroundings.

Histopathology revealed severe epidermal hyperkeratosis, and there was epidermal maculopapular evidence of an erosive epidermal lesion (Fig. 2). In addition, epidermal acanthotic papular lesions with basal vacuolation and superficial and deep dermal perivascular lymphocytic infiltration were observed (Fig. 3). An epidermal acanthotic papular lesion with basal vacuolation and superficial and deep dermal perivascular lymphocytic infiltration were noted (Fig. 4). An area of keratinocyte necrosis was observed beneath the hyperkeratotic epidermal layer, as shown in Fig. 5.

Pox virus-specific DNA (amplification of the 578 bp region of the highly conserved P4b gene of the pox virus core protein) was detected in the vaccine and the 20 suspected samples. Five random positive PCR samples were extracted and sent for sequencing to acquire a cross-sectional representation of the observed cutaneous symptoms and enable a more precise and nuanced analysis of the underlying disease.

Sequence analysis showed that all 5 strains were closely related, with relatively minor differences. Five representative sequences were selected and deposited in the NCBI database (OR187728, OR187729, OR187730, OR187731, and OR187732). Fig. 6 shows the derived phylogenetic trees. All the strains of this study were included in clade A (fowl pox virus) according to the phylogenetic classification by Jarmin et al. [15] (Fig. 7). Consequently, amplification of the gene fpv140 gave 1800 bp PCR-positive results, including the vaccine-positive control (Fig. 8).