This is the first experiment which demonstrates that European quail (Coturnix c. coturnix) can be infected with both HPAIV and LPAIV. This quail subspecies can be found not only in the wild all over the Palearctic region, but also in many parts of Europe as a game bird species raised in outdoor operations. Despite the relevance of this game bird species, no studies had previously investigated its AIV infection dynamics. The aim of the present study was to elucidate the putative role of European quail in the ecology of influenza A viruses by assessing the pathogenesis, transmissibility, and viral shedding of quail experimentally infected with two different HPAIV subtypes (H7N1 and H5N1) and one LPAIV (H7N2).
Quail exhibited a high susceptibility to both HPAIV used in this study, as demonstrated by severe clinical signs and high mortality rates. With the earliest onset, most rapid progression of disease, and shortest MDT in H5N1/HP-infected quail, it is apparent that this virus is more virulent for this species than the H7N1/HP . The high pathogenicity observed for both HPAIV is in accordance with natural and experimental H5N1 HPAIV infections in chickens and other gallinaceous species, among which are Japanese quail [3, 4, 14]. Although previous studies with Japanese quail reported minimal clinical signs or even sudden deaths without apparent symptoms [3, 14, 15], clinically neurological dysfunction was an evident sign in most of the HPAIV-infected quail of the present study. Certain gross findings indicative of AI were not as extensive and obvious as for chickens (e.g., presence of edematous, hemorrhagic, and necrotic cutaneous lesions), but affected tissues were known target organs for influenza A viruses in other gallinaceous species, including Japanese quail [3, 4, 14, 44]. Interestingly, H5N1/HP showed a broader tissue distribution compared with H7N1/HP, suggesting that virus replication in a particular target organ other than respiratory or intestinal organs may contribute to the virulence of the HPAIV in quail, as previously stated . Particularly, neurotropism is considered one of the main factors for the fatal course of AI in birds [45, 46], evidenced in our study by the higher virulence of the H5N1/HP compared with H7N1/HP. Our findings in H7N2/LP correlate well with those of Makarova et al. , in which a wide range of LPAIV subtypes could replicate efficiently in Japanese quail, predominantly in the respiratory tract. In our study, European quail could also maintain the infection without clinical involvement, and shed the virus mainly orally during a substantial period.
Effective viral transmission from inoculated quail to naive contact birds was confirmed for the three studied viruses, even though their origin avian hosts were as diverse as chicken, mallard, and great crested grebe. This finding suggests that adaptation may not be needed to allow AIV to replicate and transmit in European quail, confirming the substantial role that this species may play in AI epidemiology. As in a previous work with H5N1 HPAIV in Japanese quail , both HPAIV used in our study confirmed to be able to transmit among European quail. Moreover, transmission of H7 isolates (both H7N1/HP and H7N2/LP) is of great importance because: I) this is the first transmission evidence of an H7 HPAIV in quail so far; and II) previous experiments with LPAIV failed to confirm this capability in Japanese quail . Not only had the onset of clinical signs proved infection in contact birds, but also their antibody responses (in the case of H7N2/LP challenge) and their efficient viral shedding. Given that quail shed virus mainly orally, contact birds might have been infected by the oral-oral route. In fact, such viral shedding predominance, also stated in previous studies with Japanese quail [14–16], is already known to differ from that observed in LPAIV waterfowl reservoirs .
Ingestion of contaminated water has already been suggested as a possible transmission route . Interestingly, the earlier detection of viral H5N1/HP RNA in water followed by H7N1/HP and finally by H7N2/LP could mirror the initial ability of the virus to replicate in host cells, be shed, and thus, be more likely transmissible to naive birds. Drinking water should be particularly taken into account for quail and other game birds raised in outdoor operations, where AI viruses from wild birds could be introduced to the poultry flock. Furthermore, contamination of the environment by respiratory secretions and infected carcasses likely would result in indirect oral transmission of the virus. Although minor, cloacal shedding was consistently detected in HPAIV-infected quail, confirming that European quail might have functional binding receptors in both trachea and intestine, as already confirmed for both quail subspecies [24, 25, 49, 50]. Besides, feathers could likely act as potential source for virus transmission in European quail, especially in recently dead birds susceptible to feather picking. To date, the relevance of feathers as a location for viral replication and potential origin of dissemination in HPAIV infection has been evidenced in certain bird species [4, 51, 52], but had not yet been demonstrated in quail.
The high degree of correlation between C-ELISA and HI results suggests that such tests seem to be equally sensitive and specific when assessing quail serological responses, as previously stated for Japanese quail . Antibody response in HPAIV-inoculated quail started as early as 5 dpi, further confirming infection of the birds and an early humoral immune response. Seroconversion in H7N2/LP-infected quail at the end of the experiment proved effective infection not only among inoculated birds but also among contacts. In general, antibody titers in the present study were akin to those previously observed in AIV-infected Japanese quail [5, 53, 54] and gradually increased throughout the experiment, as already observed in H9N2 LPAIV-infected Japanese quail .
The high susceptibility of European quail to H7N1/HP and H5N1/HP would make this species a good sentinel of the presence of HPAIV in the environment, both in the wild or in semi-extensive farms. On the other hand, infected quail can shed a considerable amount of AIV before the appearance of overt clinical signs, death or both (around four days in the present experiment). Therefore, spreading disease into the wild by releasing apparently healthy farm-reared quail for hunting purposes could represent a substantial threat, even higher if assuming that this species could act as a mixing vessel like already stated for the Japanese quail. Furthermore, European quail may be considered sentinels (both for HPAIV and LPAIV) and reservoirs (for LPAIV), which is of special interest as most wild individuals are migratory . The application of surveillance measures on quail flocks before and after release is of importance to avoid introduction of HPAIV, as well as other pathogens, in the natural ecosystem.
Current active AI surveillance activities include sampling of both OS and CS, as well as blood [55, 56]. Passive surveillance of dead or moribund birds involves the same samples as for active surveillance (when possible) along with tissue collection through necropsy [55, 56]. On the basis of our findings, OS could be used as a unique tool for successful virus detection in active AI surveillance programs in quail, as it has been assessed for other minor species in which pathogenesis is still poorly understood . In addition, brain, pancreas, and heart specimens would be suitable in passive surveillance when HPAIV is suspected. Our results suggest that European quail, like Japanese quail, could play a key role in AI epidemiology because of the high susceptibility to HPAIV and the noteworthy spread of both HPAIV and LPAIV. Taking into account the similarities in viral dynamics between Japanese and European quail, the latter would also presumably have the capability to act as an intermediate host for avian/mammalian reassortant viruses, although further experiments are needed to address this issue. In addition, future studies comparing AI infection dynamics between Japanese and European quail by experimental infections with the same AIV strains would strengthen the present data. Altogether, our results underline the complexity of managing AI outbreaks when different susceptible species are involved.