Evidence of antibodies against SARS‐CoV‐2 in wild mustelids from Brittany (France)

Abstract In the French region of Brittany, mainly in the department of the Côtes d'Armor, during the first half of 2021, seropositivity for SARS‐CoV‐2 was detected in five wild mustelids out of 33 animals tested (15.6%). Anti‐SARS‐CoV‐2 IgG was detected against at least four out of five recombinant viral proteins (S1 receptor binding domain, nucleocapsid, S1 subunit, S2 subunit and spike) in three pine martens (Martes martes) and in two badgers (Meles meles) using the automated western blot technique. An ELISA test also identified seropositive cases, although these did not align with western blot results. Although the 171 qPCRs carried out on samples from the 33 mustelids were all negative, these preliminary results from this observational study nevertheless bear witness to infections of unknown origin. The epidemiological surveillance of Covid‐19 in wildlife must continue, in particular with effective serology tools.


INTRODUCTION
Human infection with a newly identified coronavirus, SARS-CoV-2, was reported in China at the end of 2019 (Huang et al., 2020). This pathogenic coronavirus is responsible for the COVID-19 pandemic which has caused 483 million cases of infection and 6 million deaths.
Despite the health measures taken and the extensive use of vaccines in developed countries, SARS-CoV-2 continues to spread, particularly due to the appearance of new genetic variants. The precise origin of this virus has not yet been firmly established, but the fact that the coronavirus closest to SARS-CoV-2 (BatCoV RaTG13) has been identified in the intermediate horseshoe bat (Rhinolophus affinis) enables us to hypothesize that this coronavirus is a zoonotic pathogen (Zhou et al., 2020). It is possible that an animal coronavirus may have crossed possibility of reservoirs of SARS-CoV-2 in wild animals with the potential for virus mutation and transmission from wildlife to humans and are nonetheless of great interest in the context of the prevention of Covid-19 . Infection was found in white-tailed deer (Odocoileus virginianus) first in the United States and then in Canada, and this species is a possible reservoir host for SARS-CoV-2 (Hale et al., 2022;Palermo et al., 2022;WOAH, 2022). In addition, in feral American mink (Neovison vison) from Utah (USA) and Spain, SARS-CoV-2 infection has been detected (Aguiló-Gisbert et al., 2021;Shriner et al., 2021). It is well known that two well-studied species of mustelids (ferret and mink) are very receptive and sensitive to the point that the ferret has become a useful model of Covid-19 for experimental infections (Alluwaimi et al., 2020;Boklund et al., 2021). In a farm in western France, mink were infected and then euthanized at the request of the health authority (Anses, 2021). In this context, an observational study was conducted in the French region of Brittany to detect SARS-CoV-2 infection and seroprevalence in wild mustelids.

Animals and samples
Following an agreement with the hunting federations of two French departments in Brittany, Morbihan and Côtes dt'Armor, we were able to take samples from the corpses of 33 mustelids, just after their death. males. There were 23 animals from the department of the Côtes dt'Armor and 10 from the Morbihan (Data S1). The sites where the mustelids were found dead or shot were identified ( Figure 1).
In the field, we performed nasal, skin and rectal swabs and blood sampled from the heart (one tube of blood with EDTA and one dry tube with serum separator gel). These samples were transported at +4 The test was validated when the OD of positive control (OD PC ) was ≥0.35 and a mean ratio of positive (OD PC ) and negative (OD NC ) control was higher than 3. The OD of each sample (OD N ) was used to calculate the sample to positive ratio (S/P) (expressed as a %) where S/P = 100 × (OD N -OD NC )/(OD PC -OD NC ). When the S/P score was lower than 50% by ELISA, samples were considered negative. They were considered as positive when it was higher than 60% and doubtful when 50% < S/P score < 60%.
amplification and absence of RT-PCR contamination, respectively.
Phage RNA internal control was added in each sample to validate RNA extraction (Amrane et al., 2020). This qPCR test was performed on the nasal, rectal and cutaneous swabs and blood sample collected from the 33 mustelids and on biopsies taken from the nine frozen carcasses. and 20) showed high reactivity for RBD, nucleocapsid, S1 subunit, S2

RESULTS AND DISCUSSION
subunit and/or spike (Figure 1). These two badgers were killed at the same place, in Perret (Côte d'Armor), on the same day in May 2021. The two positive martens were from the Côte d'Armor and the third came from the Morbihan (Data S1). In addition, two of the five AWB positive mustelids (pine martens MU3 and 14) were also ELISA positive.
Serum from eight mustelids showed reactivity against only one or two proteins out of five viral proteins targeted by AWB. The badger MU10 showed reactivity only against the nucleocapsid protein; the polecat MU32 and the pine marten MU13 showed reactivity only against the RBD. The beech marten MU7, the pine marten MU25 and the badger MU17 showed reactivity against both spike and S2 protein, the American mink MU29 against S1 and S2 subunits and the pine marten MU15 only against RBD and nucleocapsid proteins. Furthermore, there were four ELISA positive animals, only two of which were also positive on AWB (MU11 and MU13).
All the swabs (nasal, rectal and cutaneous) and the blood samples taken in the field from the 33 mustelids were negative to the specific SARS-CoV-2 RT-PCR test. Likewise, all the RT-PCRs carried out on the samples taken from nine carcasses kept frozen were negative.
Initially, serological screening was carried out using the ELISA test.
Due to the positivity of several sera, additional investigations were implemented with the AWB to confirm SARS-CoV-2 seropositivity.
AWB detects IgG against five different viral antigens (including spike subunit and nucleocapsid proteins) and therefore is of higher specificity than the ELISA which only detects total immunoglobulin against the nucleocapsid protein. AWB is a reliable technique to confirm the presence of SARS-CoV-2 antibodies and has been successfully employed on human and dog sera (Edouard et al., 2021;Laidoudi et al., 2021). As a secondary antibody, we used an anti-ferret IgG that may cross-react with IgG from other closely related species as indicated by the manufacturer. In fact, we found three positive martens and two badgers suggesting that anti-ferret IgG can also detect marten and badger IgG antibodies efficiently but it is possible that seroprevalence was underestimated in our study. The strong serological reactivity against four or five different antigens of the virus using AWB con-  our result using a neutralization assay because of the low quantity of sera available.
For eight mustelids, AWB results were inconclusive and therefore were not considered positive for SARS-CoV-2. The AWB profiles showed reactivity against only one or two proteins suggesting crossreactivity with a different coronavirus to SARS-CoV-2 or an incomplete serological response, that is to say that some individuals produce antibodies only against the spike (or certain subunits) or the nucleocapsid but not against all five viral antigens (Li & Li, 2021;Lv et al., 2020). Unfortunately, cross-reactivity with another coronavirus could not be evaluated by AWB because pre-pandemic mustelid sera were not available. However, cross-reactivity was detected using AWB and recombinant antigen on pre-pandemic human sera, but a low value of chemiluminescence was observed and reactivity could be seen for only one to three antigens out of the five (data not shown). Therefore, we chose stringent criteria to define western blot positivity and selected sera as positive if they were reactive against at least four antigens.
The ELISA kit uses a truncated nucleocapsid protein in order to limit cross-reactions with other coronaviruses (Spada et al., 2021). The diagnostic specificity of this test based on double antigens is >99% in dogs (Laidoudi et al., 2021). The sera from badgers MU11 and MU33, which were positive only by ELISA, were too hemolysed to be able to draw reliable conclusions of AWB because capillaries could be blocked by red blood cells. However, three mustelids presented negative ELISA results despite being positive by AWB (MU19, 20, and 24). These results are consistent with the AWB having greater sensitivity than the ELISA test (Cortes et al, 2006;Vola et al., 2019). All samples taken were negative using the highly sensitive RT-PCR assay. However, internal controls were positive for all the specimens tested attesting to the good quality of the RNA extraction.
It was not our intention to conduct representative sampling from wild mustelids. Instead, we conducted an opportunistic study of a deliberately small number of wild mustelids, which benefited from the should also be suspected in each case. Initial transmission of SARS-CoV-2 to wild mustelids may have occurred through indirect contact with an infected human through environmental contamination (e.g. wastewater, household waste etc.). All the mustelids studied lived in agricultural areas with human settlements. It is possible that direct transmission amongst mustelids may have occurred, although we have no direct evidence for this. Nevertheless, it is interesting that the two infected badgers (MU19 and 20) were from the same location and that this species is known to be more sociable than other mustelids (Wang, 2011).
Viral circulation amongst mink is rapid and they are highly susceptible to the virus (Shuai et al., 2020). Like humans, they express the angiotensin-converting enzyme 2 (ACE2) receptor on the cells of the respiratory tract, which facilitates viral penetration (via the spike protein) and infection, depending on their abundance and distribution (upper vs. lower respiratory tract)  The infection of wild animals with SARS-CoV-2 has been increasingly studied in North America since the discovery of infected whitetailed deer (Hale et al., 2022;Palermo et al., 2022;Palmer et al., 2021).
With the identification of cases in wild animals, the World Organisation for Animal Health has proposed recommendations for people working on wild animals (WOAH, 2020).

CONCLUSION
This study has demonstrated for the first time SARS 10-IAHU-03, the Region Provence-Alpes-Côte d'Azur and European funding FEDER PRIMI.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

ETHICS STATEMENT
In seven cases, the animals died as a result of road collision, 11 mustelids were shot dead in accordance with current hunting regulations and 15 others were trapped and euthanized in accordance with article R 427-6 of the French Environment Code. For ethical reasons of biodiversity protection, strict limits were imposed on the number of animals studied.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.