First reported Porrocaecum angusticolle infection in Griffon vulture (Gyps fulvus) in China

This present study is the first case of a Porrocaecum angusticolle (P. angusticolle) infection reported in Griffon vulture (Gyps fulvus) in China. This study aimed to identify the nematode species and explore the genetic evolution of worms infecting Gyps fulvus (G.fulvus). Clinical examination revealed several milky white parasites in the stomach and intestinal tract. Polymerase chain reaction and partial 18S gene sequencing analyses identified these worms to be P. angusticolle (SD isolates). Further phylogenetic analyses revealed that they shared the highest genetic identity (99.9%) with a P. angusticolle isolate (EU004820.1) from Germany. Our study is the first report on the identification and characterization of P. angusticolle infecting G.fulvus in China, based on clinical findings and molecular diagnosis. Therefore, our study provides novel insights for the diagnosis of P. angusticolle infections and the prevention of nematode transmission in wild and domestic animals.

Notably, a P. angusticolle infection is typically diagnosed based on clinical symptoms, such as pathological lesions, and molecular diagnosis. To the best of our knowledge, only 16 nucleotide sequences of the P. angusticolle genome have been submitted to the GenBank database, including those of the 18S, 28S, COX, and ITS genes. In the present study, we used primers targeting the 18S gene to perform sequencing of avian samples for the diagnosis of infection and identification of nematode worms. Interestingly, by combining the evaluation of clinical symptoms and molecular identification, we diagnosed, for the first time, a case of P. angusticolle infection of G.fulvus in China.

Case presentation
In January 2022, we were notified that a male G.fulvus older than 10 years of age had died. Following dissection, we found dozens of milky white parasites in its stomach and intestinal tract, with the size of worms ranging from 7 to 15 cm. We then collected these parasitic worms from the deceased vulture for further studies. We also examined the bird and observed clinical signs of the intestinal tract showing hemorrhagic spots and anabrosis.

Gene sequence and analysis
Following recovery and purification using the Agarose Gel DNA Extraction Kit, PCR products were sequenced by Biosune Biotechnology Co., Ltd. We analyzed the obtained sequencing results using the MEGA and DNAstar software and compared the identified sequences of 18S, COX1, and ITS genes of P. angusticolle with those of other nematodes. First, we used the GenBank and PubMed online websites to perform a comparative analysis of the sequences of highly homologous geographical parasite strains, especially their 18S sequences. In addition, using the DNAstar MegAlign Pro with the Clustral W algorithm, we analyzed the distance and divergence of these sequences. Subsequently, we used the Kimura 2-parameter model and maximum likelihood method in MEGA 6.0 software to draw the phylogenetic tree.

Results and discussion
Decreased immune function following parasite infection might be one of the potential risk factors leading to the death of G.fulvus birds (Figure 1) (Guivier et al., 2017;Lima and Lodoen, 2019). In the present study, we report the first case of an identified P. angusticolle infection of G.fulvus, confirmed by clinical findings and molecular diagnosis (Cabezoń et al., 2011;Darwich et al., 2012;Chakarov and Blanco, 2021). Notably, we identified adult worms in the stomach and intestinal tract of the bird instead of nematode eggs (Figure 2). Previous studies have reported the occurrence of approximately 40 nematode species worldwide (Li and Scholz, 2019), including Porrocaecum semiteres (Syrota and Kharchenko, 2015), Porrocaecum ensicaudatum (Kijewska et al., 2002), Porrocaecum aridae, Porrocaecum crissum deslong, Porrocaecum praelongum, Image of a G.fulvus. This is partial head photograph of G.fulvus in animal zoo.
Porrocaecum reticulatum (Li et al., 2015), Contracaecum multipapillatum (Navone et al., 2000;Valles-Vega et al., 2017), Contracaecum micropapillatum, Contracaecum bancrofti, Contracaecum variegatum, Contracaecum eudyptulae, and Contracaecum ogmorhini (Shamsi et al., 2009). To date, the complete lifecycle of most Porrocaecum and Contracaecum species remains unclear. Only a few species have been reported to include intermediate hosts, such as earthworms (Moravec, 1971), insectivores (Portoleś et al., 2004), and fish (Moravec, 2009). Under suitable environmental temperatures at the range of 22-32°C, eggs are hatched into active larvae, which enter the earthworm, developing into invasive larvae and cysts after 2 months. Following feeding of the terminal host on an infected worm-containing carcass, the invasive larvae in the worm develop into adults in the small intestine of the terminal host over 3 weeks. For example, in the case of C. multipapillatum, the first intermediate host is the cyclops, while the second intermediate host is fish (Moravec, 2009).
Previous studies have mainly focused on the morphological and genetic characterization of cases of P. angusticolle infections in B. buteo, Strigiformes, A. clanga, A. chrysaetos, and other birds (Kijewska et al., 2002;Santoro et al., 2010;Santoro et al., 2012;Tomaś et al., 2017;Guo et al., 2021). Therefore, information on the lifecycle of P. angusticolle is still lacking. In these previous studies, adult P. angusticolle worms were typically found in the superficial layer of the stomach and intestinal tract. Interestingly, both Porrocaecum larvae and adult worms can drill into the mucous membrane of the gastric wall of the glandular stomach to produce hemorrhagic spots, bruises, and erosive ulcers, which affect the growth and development of birds, and can lead to death in cases of severe infection (Mozgovoi, 1953;Guo et al., 2021).
In the present study, genetic analyses revealed that the partial sequence of the identified 18S rRNA fragment of P. angusticolle in our study (Figure 2) exhibited the highest phylogenetic identity (99.9%) with that of an isolate from Germany (Germany: EU004820.1). Subsequently, we analyzed the phylogenetic relations of our P. angusticolle strain to that of other Porrocaecum species, such as P. reticulatum (China HB: MF072700.1), P. depressum (USA: U94379.1), Porrocaecum sp. (Italy: MT141136.1), and P. streperae (USA: EF180074.1), and found that they ranged from 99.6 to 99.9% (Figure 3). The generated phylogenetic tree further confirmed the evolutionary relationship between Porrocaecum and other nematode species (Figure 3). In conclusion, in our study, we performed 18S gene analysis to identify and characterize a P. angusticolle infection in G.fulvus in China. We also examined the COX and ITS genes; however, limited information on the phylogenetic relationships of these genes is available in PubMed and GenBank.
The G.fulvus is a bird species belonging to the Accipitridae family of vultures. It inhabits a wide range of habitats, reaching up to 2500 m above sea level, such as rocky alpine and plateau areas, grasslands, and scrub and semi-desert areas (Davidovićet al., 2020;Pirastru et al., 2021). Owing to its excellent night vision, G.fulvus forages for dead animals during the night while feeding on wild animals such as goats, deer, and gazelles in the daytime, relying on its sensitive smell to locate decaying animal carcasses (Marin et al., 2014;Sevilla et al., 2020). It is widely distributed throughout Europe, the Middle East, and North Africa, as well as in India and the Himalayas. However, it is most common in countries bordering the Mediterranean Sea, with the largest population number detected in Spain, accounting for three-quarters of the European population (Davidovićet al., 2022). To the best of our knowledge, this study is the first report on the identification and genetic characterization of P. angusticolle infection in G.fulvus in China.
Although this study is the first report of P. angusticolle infection in G.fulvus, it has some limitations. First, although we combined PCR methods, gene sequencing, and clinical factors to diagnose the P. angusticolle infection, there are no completely set diagnostic criteria for P. angusticolle. In addition, we did not record enough clinical pictures and symptoms to support our diagnosis, and few studies have reported P. angusticolle infection in birds.
Nevertheless, our study extends the current geographical distribution and host species of P. angusticolle, confirming the spread and genetic evolution of this nematode in Asia and highlighting the importance of the molecular diagnosis of P. angusticolle infections in domestic and wild animals.

Ethics statement
Written informed consent was obtained from the participant/ patient(s) for the publication of this Brief Research Report.

Author contributions
GL designed the study and drafted the manuscript. QL, WZ and XS collected the animal specimens and supported the experiment. All persons who have made substantial contributions to the work are reported in the manuscript. All authors contributed to the article and approved the submitted version.

Funding
This study was supported by National Natural Science Foundation of China (No.31502057) and Linyi University Highlevel Talent Funding Support (No. Z6122016).

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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