Detection of parasitic infection status
To rapidly identify these two parasite species, PCR amplification was performed using the nematode universal primer COX1 sequence and positive bands were sent to BGI for sequencing. The obtained COX1 sequence was analysed by Blast and tentatively identified as Sphaerirostris picae and Ascaridia galli. Representative nucleotide sequences from this study have been deposited in the GenBank database. The COX1 genome of Sphaerirostris picae is registered as OP881432. The COXl sequence of Ascaridia galli is registered as OP881429. Among the 11 Oriental Magpie, 4 magpies were infected with Sphaerirostris picae, and a total of 23 of Sphaerirostris picae were detected. There was Ascaridia galli infection in 7 magpies, and a total of 17 Ascaridia galli were detected. Among them, 2 magpies found co-infection between Sphaerirostris picae and Ascaridia galli. In order to verify the infection status, the fecal DNA of 11 magpies was extracted for retesting, which was consistent with the above results.
Molecular genetic characterization
In order to determine the phylogenetic position of Sphaerirostris picae and Ascaridia galli, an evolutionary analysis was performed. The phylogenetic tree constructed for Sphaerirostris picae based on the COX1 gene (Fig. 1) shows that the sequences obtained in this study cluster with those of Sphaerirostris picae (MK471355.1). And at present, the COX1 data on the Sphaerirostris picae species on GenBank is only one isolated from Pakistan, and these two sequences are similar to Sphaerirostris Lanceoides (MT476588.1) are most closely related. The phylogenetic tree of Ascaridia galli based on the COX1 gene is shown in Fig. 2. The sequences of the Ascaridia galli obtained in this study clustered with those of the reference chicken ascaris and were most closely related to Perostrongylus falciformis (KY365437.1) and Oslerus osleri (MT229983.1).
Sphaerirostris picae stereoscopic microscope and scanning electron microscope observations
The specimens were observed under a stereoscopic microscope, one by one, and the trunk length was measured with a vernier caliper and the body width was measured with a micrometer. Trunk length ranges from 12.4–37.8 mm, width 0.9–5.6 mm, with characters of Sphaerirostri. The larvae are creamy white and the adults are yellowish-brown. Trunk spindle-shaped, gradually tapering toward both extremities, rounded posteriorly (Fig. 3A, B, C, D, G). Proboscis divided into two parts separated by constriction, bare and flat apically. Anterior proboscis nearly spherical, Posterior proboscis cylindrical, with more widely-spaced armature. (Fig. 3E, F). All hooks emerged from elevated round rims on proboscis surface (Fig. 3H).
Ascaridia galli stereoscopic microscope and scanning electron microscope observations
All specimens were yellowish, linear, tapering at both ends under the stereoscopic microscope (Fig. 4, A, B, C, D). The transverse lines of the cuticle covering the trunk surface are clearly visible (Fig. 4, G), with an average length of 67.2 mm, a trunk length of 41.6–74.8 mm and a width of 2-4.6 mm. A thickened dilatation, the neural ring, is visible distally near the head end (Fig. 4, E, F). The cloaca is located at the lower end of the preanal sucker (Fig. 4, H). When observed by light and electron microscopy, the diameter of the worms became larger as they increased in size during processing, which led to some discrepancies with the initial size.