Birds play an important role for several arthropod-borne viruses such as Usutu virus (USUV) and Sindbis virus (SNV) in the old world, and Saint Louis encephalitis virus (SLEV), Eastern Equine encephalitis virus (EEEV) in the new world, and West Nile virus (WNV) worldwide. The transmission cycles of these viruses include arthropod species as vectors and birds as reservoirs. When the vector feeds on infected birds, which are amplifying hosts, they get infected and later on the vector supports virus replication with virions accumulation in the salivary glands to perpetuate transmission to vertebrates (Chan et al., 2015).

Toscana virus (TOSV) and Sandfly Fever Sicilian virus (SFSV) are both sandfly-borne phleboviruses that belong to the Phlebovirus genus in the Phenuiviridae family (order Bunyavirales). Both TOSV and SFSV can infect humans and can cause diseases. SFSV can cause 3-day fever that is characterized by a brutal onset with short but high fever with companion signs such as cutaneous rash headache, photophobia, retro-orbital pain, myalgia and general malaise. TOSV can cause the same manifestations as SFSV, but it can also be responsible for various central and/or peripheral neurological signs, most commonly meningitis and encephalitis (Alkan et al., 2013).

Although antibodies against TOSV and SFSV were found in horses, cats, dogs, cattle, goat and bats (Alwassouf et al., 2016a,b; Ayhan et al., 2017a,2021), the amplifying host (if any) remains undiscovered. Since dogs are the reservoir host for certain species of Leishmania, it has been hypothesized that they could also play this role for TOSV and/or SFSV. In addition, recent findings have pointed in this direction (Dincer et al., 2015). However, an experimental study conducted with beagle dogs, with two doses of TOSV and SFSV independently, showed absence of clinical manifestation, and viral RNA/infectious virus was not detected in a large variety of clinical samples (blood, saliva, stools, urine, sperm, and bone marrow). Also, the immune response was very limited. The authors conclusions were that dogs are not likely to play a decisive role in the natural transmission and maintenance of TOSV or SFSV (Muñoz et al., 2021). Bats also have been suggested as a reservoir of phleboviruses. However, a study carried out in eight European bat species suggests that bats are not likely to play a major role in the natural cycle of these two viruses (Ayhan et al., 2021).

The knowledge on the role of birds in the ecology and natural cycle of phleboviruses transmitted by sandflies is limited. Recently, TOSV RNA was detected in brain and kidney tissues from three different wild bird species; a greater flamingo (Phoenicopterus roseus), a great white pelican (Pelecanus onocrotalus), and a black stork (Ciconia nigra) (Hacioglu et al., 2017). Interestingly, genetic analysis indicated that RNA sequences belonged to different strains corresponding to each of the two phylogenetic lineages A and B (Hacioglu et al., 2017). In contrast, there is no data supporting an interplay between SFSV and wild birds. Nonetheless, several sandflies species (mostly Phlebotomus sergenti) feed on blood of poultry animals (Bongiorno et al., 2003; Svobodová et al., 2003).

In Spain, twelve species of sandflies have been described (Muñoz et al., 2021), including Phlebotomus perniciosus and Phlebotomus papatasi, which are a proven vector of TOSV (Remoli et al., 2016). SFSV have been identified in P. papatasi and Phlebotomus ariasi species since now (Izri et al., 2008; Alkan et al., 2013). The presence of the antibodies against TOSV has also been demonstrated in a number of domestic animals (cats, dogs, goats, sheep, cows, pigs, and horses) in Spain (Navarro-Marí et al., 2011). Even, cases of infection due to TOSV were recorded in humans in the country (Eitrem et al., 1991; Mendoza-Montero et al., 1998; Echevarría et al., 2003; Sanbonmatsu-Gámez et al., 2009). In contrast, human cases of infection with SFSV have not been documented in Spain (García San Miguel et al., 2021) and seroprevalence studies reported only a 2.2% rate of positivity (Eitrem et al., 1991).

Migratory birds play an important role in the transmission of diseases due to their great mobility from one area to another, which makes them potential vectors of diseases that can affect domestic animals and human health (Abulreesh et al., 2007). The Common Quail adds to its migratory status the fact of being a game species, which enhances the possible transmission of diseases by direct contact through the food chain. Therefore, the periodic detection of pathogens is of great importance to predict future disease risks for both wildlife and humans (Youssef and Mansour, 2014).

In this study, we investigated if the Common Quail bird could play a role in the natural cycle of TOSV and SFSV. We have collected and analyzed sera from wild quails in Spain to determine the presence of TOSV and SFSV neutralizing antibodies.

The “Global Vector Control Response (GVCR) 2017–2030” was approved by the World Health Assembly in 2017 and provided strategic guidance to countries for urgent strengthening of vector control to prevent diseases and outbreaks (World Health Organization [WHO], 2022). However, phleboviruses transmitted to humans and animals by sandflies are relatively neglected and are not on the priority list of national and international public health agencies. Even though the problem is neglected, the public health impact of sandfly-borne infections is relevant in countries along the Mediterranean and expanding northwards. The problem is developing in Europe because, due to climate and environmental changes and human activity, phlebovirus vectors have increased in frequency (Maroli et al., 2013; Alten et al., 2016). The niche modeling approach predicts climatically suitable areas for sandflies as being not only in the Mediterranean region but also in Central and Northern Europe, e.g., countries like Austria, Switzerland, and Germany, and even regions in Scandinavia and the mainland of Great Britain (Koch et al., 2017). On the other hand, the great mobility of people and trade also favors the dispersion of sandflies. Considering the growing expansion of phlebovirus vectors and the millions of people that are potentially exposed, it is important to find phlebovirus reservoirs and to understand their cycles in order to propose preventative measures. Our study aims to contribute to knowledge on the circulation of SFSV and TOSV in animals that have so far been studied infrequently.

Several studies demonstrated the circulation of SFSV in a wide geographical area, including Asia, south Europe, and Africa; in contrast TOSV circulation is restricted within the Mediterranean basin (Ayhan et al., 2017b). If the maintenance and the transmission of these viruses appear highly dependent on the abundance and the distribution of the relevant sandfly species, there still are in our knowledge of rural and/or urban cycles because to date, the vertebrate reservoir host for TOSV and SFSV, if any, remains undiscovered.

In this study, we found for the first time SFSV and TOSV neutralizing antibodies in wild birds. Our results show rates of neutralizing antibodies that were among the highest ever detected in vertebrates (Alwassouf et al., 2016b; Ayhan et al., 2021). Moreover, if the cut-off is decreased and moved from 40 to 20, NT Abs against SFSV and TOSV would be observed in 59% and 58.5% of quails, respectively. These results contrast with the unbalanced NT Abs rates previously observed in dogs from Portugal, Cyprus, Crete, and mainland Greece (Alwassouf et al., 2016b). Interestingly, the same quail serum often contained NT Abs against TOSV and SFSV. On the other hand, we found significant interannual differences in seroprevalence in quails in concordance with the dynamics of viruses in reservoir species (Serra-Cobo and López-Roig, 2017; Table 2 and Figure 2). We also found NT Abs against TOSV and SFSV in some common swift (Apus apus) from Spain (unpublished data). Although SFSV RNA and TOSV RNA have been rarely detected in vertebrates, TOSV RNA has only been recently detected for the first time in birds. Taking into account our results and that birds can be reservoir hosts for other zoonotic arboviruses, we can say that they could be also reservoirs of the natural cycle of SFSV and TOSV.

The quails analyzed are migratory birds who come from Maghreb or Sub-Saharan Africa into Europe in April to breed. In October, they leave Europe for Africa where they overwinter (Figure 3; Perennou, 2009). Accordingly, they stay in the Mediterranean area at least from April to October, which is the period of activity of the sandfly. Entomological surveys in several countries of the Mediterranean basin showed the presence of two sandfly species: P. perniciosus and P. papatasi, which are the known vectors of TOSV and SFSV, respectively. The presence and abundance of P. perniciosus were shown on the island of Mallorca and the region close to the Pyrenees of Catalonia (Alcover et al., 2014; Gálvez et al., 2020). However, the low seroprevalence described in vertebrates tested for the presence of antibodies against SFSV suggests that this virus might circulate at very low levels in European regions where quails are breeding. In addition, during the last decade, SFSV-like viruses were detected by molecular methods in sandflies collected in Tunisia (Utique virus and Saddaguia virus) (Fares et al., 2015; Dachraoui et al., 2016); accordingly, it is plausible that quails get infected by SFSV or SFS-like viruses. When we consider the known distribution of SFSV in Africa and low seroprevalence of SFSV in Spain, we can speculate Africa as a probable infection region. Entomological surveys introduced two predominance sandfly species: P. papatasi and P. perniciosus in Algeria and Morocco, of the genus Phlebotomus, from which P. papatasi is a known vector of SFSV (Boudrissa et al., 2012; Boussaa et al., 2016; Muñoz et al., 2021). In addition, SFSV was detected from P. ariasi in field-collected sandflies in Algeria, indicating the virus circulation in North Africa (Izri et al., 2008). The wide distribution of these sandfly species indicate that both viruses circulate in the Mediterranean region and that common quails are likely to become infected on the two shores of the Mediterranean.

Several studies have shown that bird species can be a potential blood source for a number of sandfly species, since they display the lowest defensive behavior compared to other animals (Svobodová et al., 2003). In this sense, it is likely that when different types of vertebrate hosts are available, sandflies tend to choose poultry over mammals. This behavior has been observed repeatedly in rural areas of Maghreb where poultry, donkey, goats, and sheep are sheltered together.

Taking into account the results obtained and the recent detection of TOSV in large migrating birds (Hacioglu et al., 2017), it should be considered that quails (and possibly other birds) can be a reservoir or amplifying hosts for TOSV and SFSV, and possibly other sandfly-borne phleboviruses. Quails are considerably more mobile than terrestrial mammals and can spread pathogens over long distances. In this sense, they are possibly good reservoirs and have the potential to rapidly and widely spread viruses. Within the European Union, at least 1,607,964 quail are shot each year, which is equivalent to 40% of the average breeding population of the EU countries (Hirschfeld et al., 2019). This adds a degree of concern regarding the risk of spreading viruses, since, during hunting activities, both dogs and humans are in direct contact with quail.

Our results, together with previous data, constitute a body of evidence that shows that the common quail plays a prominent role in the ecology of these viruses. Future research should address this hypothesis using both experimental studies and wildlife based studies.

The original contributions presented in this study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

The animal study was reviewed and approved by the ethical recommendations of the European Union and Spanish Legislation (Spanish Law 5/1995 and Decree 214/1997) and was approved by the Ethics Committee on Animal Experimentation from the University of Barcelona.

NA, RC, and JS-C conceived the study. NA, JR-T, ML-R, DV, JF, and AM developed the methodology and analyzed and interpreted the data. NA performed serological assays and wrote the manuscript. JR-T, ML-R, DV, JF, and AM designed, conducted, and provided the bird serum samples the clinical samples. All authors edited successive drafts and approved the final version.