key: cord-0426333-5331c0c8
authors: Davoust, Bernard; Guérin, Patrick; Orain, Nicolas; Fligny, Camille; Flirden, Fabien; Fenollar, Florence; Mediannikov, Oleg; Edouard, Sophie
title: Evidence of antibodies against SARS-CoV-2 in wild mustelids from Brittany (France)
date: 2022-01-20
journal: bioRxiv
DOI: 10.1101/2022.01.20.477038
sha: 135da512d03fa8c3da0b57dc43a9cf12dfc30f45
doc_id: 426333
cord_uid: 5331c0c8

In the French region of Brittany, mainly in the department of the Côtes d’Armor, during the first semester of 2021, seropositivity for SARS-CoV-2 was detected in five wild mustelids out of 32 animals tested. Anti-SARS-CoV-2 IgG against at least four out of five recombinant viral proteins (S1 receptor binding domain, nucleocapsid, S1 subunit, S2 subunit and spike) were detected using automated western blot technique in three martens (Martes martes) and two badgers (Meles meles). An ELISA test also objectified seropositivities. Although the 171 qPCRs carried out on samples from the 33 mustelids were all negative, these preliminary results (observational study) nevertheless bear witness to infections of unknown origin. The epidemiological surveillance of Covid-19 in wildlife must continue, in particular with the tools of efficient serology.

Human infection by a newly identified coronavirus, SARS-CoV-2, was reported in China, end of 2019 (Huang et al., 2020) . This pathogenic coronavirus is responsible for the COVID-19 pandemic which, over two years, has caused, 288 million cases of infection and 5.45 million deaths [www.worldometers.info/coronavirus/]. Despite the health measures taken and the massive use of vaccines, 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 Chinese horseshoe bats (Rhinolophus affinis), enables us to hypothesize that it is a zoonotic pathogen (Zhou et al., 2020 ). An animal coronavirus has crossed the barrier passing from bats to humans via, possibly, another close-to-human animal acting as a vector or even a secondary reservoir. This passage from an animal coronavirus to humans has resulted in adaptation to the host through viral mutations. Today SARS-CoV-2 spreads primarily from person to person all over the world. Animals seems low implicated in the spread of Covid-19 . First, animals can be infected by asymptomatic infected or sick people. This has been well described in domestic animals (cats, dogs, ferrets), animals raised for fur (minks) and zoo animals (felines, primates, etc.) Fenollar et al., 2021; Jemeršić et al., 2021; Pomorska-Mól et al., 2021; OIE, 2022) . To date, cases of human infection with SARS-CoV-2 from an animal (reverse zoonosis) have proved exceptional and limited to the particular ecosystem of mink farms (Hammer et al., 2021; Oude Munnink et al., 2021) . The latest epidemiological studies show that there are now wild animals naturally infected with SARS-CoV-2. These observations of a reservoir of pathogens, susceptible to mutations, and potentially responsible for transmission from wildlife to humans, are of great interest in the context of the prevention of Covid-19 (Delahay et al., 2021) . Thus, white-tailed deer (Odocoileus virginianus) in the USA and then in Canada are reservoir hosts for SARS-CoV-2 (Palermo et al., 2021; Hale et al., 2021; Palmer et al., 2021) . In addition, in wild American minks (Neovison vison) from Utah (USA) and Spain, a SARS-CoV-2 infection has been detected (Shriner et al., 2021; Aguiló-Gisbert et al., 2021) . It is well known that mustelids are very receptive and sensitive to the point that the ferret has become the best model of Covid-19 for experimental infections (Alluwaimi et al., 2020; Boklund et al., 2021) . In a farm in western France, minks were infected and then euthanised at the request of the health authority (Anses, 2021). In this context, an observational study, limited in time and space, was conducted to detect SARS-CoV-2 infection in wild mustelids collected in the French region of Brittany.

Following an agreement with the hunting federations of two French departments in Brittany, Morbihan and Côtes d´Armor, we were able to take samples from the corpses of 33 mustelids, just after their death. From April to June 2021, we sampled: 14 martens (Martes martes), 10 badgers (Meles meles), 4 American minks (Neovison vison), 3 polecats (Mustela putorius) and 2 beech martens (Martes foina). In seven cases, the animals died as a result of a road collision. In addition, 11 mustelids were shot dead in accordance with current hunting regulations and 15 others were trapped and shot down in application of 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. The total group of 33 mustelids studied was made up of 19 females and 14 males. There were 23 animals from the department of the Côtes d´Armor (No. 22) and 10 from the Morbihan (No. 56) . The sites where the mustelids were found dead or shot were located by their latitude and longitude (Table 1) .

In the field, we performed nasal, skin and rectal swabs as well as a blood sample from the heart (one tube of blood on EDTA and one dry tube with serum separator gel). of positive control (ODPC) was ≥ 0.35 and a mean ratio of positive (ODPC) and negative (ODNC) control was higher than three. The optical density of each sample (ODN) was used to calculate the sample to positive (S/P) ratio (expressed as a %) where S/P= 100 * (ODN -ODNC)/(ODPC -ODNC). 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< P/S score< 60%.

The Jess TM Simple Western automated nano-immunoassay system (ProteinSimple, San Jose, CA, USA, a Bio-Techne Brand), a capillary-based size separation of proteins was used to evaluate the absolute serological response to five viral antigens from sera (Edouard et al., 2021) . SARS-CoV-2 Multi-Antigen Serology Module® including S1

receptor binding domain (RBD) (48-kDa), nucleocapsid (58-kDa), S1 subunit (105-kDa), 

All the results are presented in Table 1 28.21]) sera. Five mustelids (four females and one male) were seropositive using AWB and showed high reactivity for RBD, nucleocapsid, S1 subunit, S2 subunit and/or spike ( Figure 1 ). Positive mustelids were three martens (MU3, 14, 24) and two badgers (MU19 and 20). These two badgers were slaughtered at the same place, in Perret (Côte d'Armor), the same day in May 2021. Two martens were from the Côtes d'Armor and the third from Morbihan but from a town, La Gacilly, located on the southern edge of the Côtes d'Armor. In addition, two of these five mustelids (MU3 and 14) were also positive in ELISA. The sera of eight mustelids showed reactivity only against one and/or two proteins out of five viral proteins of AWB. The badger MU10 showed reactivity only against the nucleocapsid protein, MU32 and MU13 showed reactivity only against RBD.

MU7, MU25 and MU17 showed reactivity against both spike and S2 protein, MU29

against S1 and S2 subunits and MU15 only against RBD and nucleocapsid. For the polecat (MU4), the AWB test was not performed due to insufficient serum. Furthermore, there were three false negative animals with the ELISA test (MU19, 20, 24) and two positive animals (MU11 and MU33) with ELISA for which AWB remained negative.

All the swabs (nasal, rectal and cutaneous) and the blood sample taken in the field on the 33 mustelids were negative in the specific SARS-CoV-2 qPCR test. Likewise, all the qPCRs carried out, a posteriori, on the samples taken from nine corpses kept frozen were negative.

After discussing the reliability of our results, we will move on to their epidemiological significance. The potential impact on public health of transmission of SARS-CoV-2 to wild mustelids will be the subject of recommendations for active epidemiological surveillance.

Initially, serological screening was carried out with the ELISA test. Due to the positivity of several sera, additional investigations were implemented with the western blot technique which gives more precise results. The strong serological reactivity against four or five different antigens of the virus confirm specificity of antibodies to this coronavirus, indicating a humoral immune response linked to contact with the agent of the Covid-19 pandemic. All five AWB positive animals (MU3, 14, 19, 20, 24) were definitely infected with SARS-CoV-2. For eight mustelids, the AWBs were doubtful and were not considered positive for SARS-CoV-2. The AWB profiles showed reactivity against only one or two proteins suggesting cross reactivity with another coronavirus than SARS-CoV-2 or an uncompleted serological response (Lv et al., 2020; Li and Li, 2021) . It is known that minks can be infected with an Alphacoronavirus, which is not zoonotic (Stout et al., 2021) . Moreover, one ferret enteric coronavirus (FRECV) is similar to feline coronaviruses (Haake et al., 2020) . 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) . This ELISA test is useful to investigate SARS-CoV-2 antibodies in minks (Chaintoutis et al., 2021 The mustelids in our study are common in France ("Least Concern" status of the International union for the protection of nature) and considered as likely to cause damage in the two departments. Badgers are slaughtered by administrative order of the prefects. In France, the American mink population is not indigenous. It is due to escapes from mink farms for fur, especially in Brittany, in the 1960s (Léger et al., 2018) .

Mustelids adapt to a varied diet depending on the seasons (small mammals, worms, insects, fruits, etc.). Their way of life is discreet with preferentially nocturnal outings from their burrows. They live in fields and forest areas and approach human dwellings only occasionally. Pine martens live mostly in woods and are generally solitary. They move along the ground with nosy behavior. They frequent places of human passage such as woodpiles and forest roads in search of prey (Schwanz, 2000) . The transmission of SARS-CoV-2 to wild mustelids may have occurred, initially, through indirect contact with an infected human through environmental contamination (wastewater? household waste? aerosols?). All the mustelids studied lived in anthropized and non-isolated rural areas. From one or more index cases, transmission spread directly between mustelids. This is certain for Perret's two badgers (MU19 and 20). Badgers have more social contact than martens (Wang, 2011) . Another hypothesis regarding the origin of the infection can be made but has not been demonstrated: it is known that in November 2020, a mink farm in Eure-et-Loir was widely infected with SARS-CoV-2, which led to the slaughter of all the animals (Anses, 2021). It seems possible that an infected mink escaped from the farm and subsequently infected wild mustelids, via an epidemiological chain of transmission.

The epizootic could have spread in a few weeks, in particular, as far as the Côtes d'Armor, located some 300 km away. This distance renders this hypothesis rather improbable. Nevertheless, escapes of this type have been strongly suspected for the outbreaks in Utah (USA) and Spain (Shriner et al., 2021; Aguiló-Gisbert et al., 2021) .

Viral circulation between mustelids is rapid. Indeed, they are the most receptive animals to SARS-CoV-2 (Shuai et al., 2020) . Like humans, they have the angiotensin-converting enzyme 2 (ACE2) receptor on the cells of the respiratory tract, which facilitates viral penetration (via the spike protein) and infection (Covid-19) (Lean et al., 2021) . Infection with SARS-CoV-2 of ferrets (Mustela putorius furo), laboratory animals, shows that they remain carriers of the virus for 14 days while the specific antibodies persist for several months (Monchatre-Leroy et al., 2021) . In addition, in infected farms, minks were generally asymptomatic, some of them present with cough and fever. Excess mortality can be observed (Boklund et al., 2021; Pomorska-Mól et al. 2021 ).

The seropositivities that we have highlighted are proof that wild mustelids are good epidemiological sentinels for Covid-19. The question of their role as a reservoir for SARS-CoV-2 must be asked even if the five seropositive animals were no longer carriers of the virus. The problematic with mustelid coronaviruses is their mutagenic power, which produces potentially zoonotic viral variants. This has been well documented in a mink farm in Denmark (Oude Munnink et al., 2021; Hammer et al., 2021) . Twelve people in contact with mink carrying SARS-CoV-2 were infected with an entirely new emerging variant (cluster 5) (Lassaunière et al., 2021) . To protect public health, reinforced epidemiological surveillance and biosecurity measures have been taken, at the request of the health authorities, in the three mink farms remaining in France (Anses, 2021). In addition, it is likely that mink farming will be banned in France in the future. However, our study shows that the pandemic virus circulates in wildlife in mustelids of two species (marten and badgers). It is therefore important to extend our one-off investigation to an active epidemiological surveillance of mustelids (injured or slaughtered) from corpses collected by the departmental hunting federations. Implementing this behoves the ministries responsible for wildlife and animal diseases.

The infection of wildlife with SARS-CoV-2 has been increasingly studied in the United States since the discovery of infected white-tailed deer (Odocoileus virginianus) (Palermo et al., 2021; Hale et al., 2021) . Antibodies were detected in 152 samples (40%) from 2021 using a surrogate virus neutralization test (Chandler et al., 2021) . The role of these deer in the evolution of the pandemic is not known. From the outset of the pandemic, domestic animals (dogs and cats) were found to be carriers of SARS-CoV-2 and have been the subject of numerous studies and case reports (OIE, 2022) . With the identification of cases in wildlife, the OIE has proposed specific recommendations (OIE, 2020) . Their application will make it possible in the future to better understand the epidemiological situation in several countries.

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The authors are grateful to the staff of OpenHealth Company and the Côtes d´Armor and Morbihan hunter federations who made the collection of samples possible. The authors would like to thank Innovative Diagnostics which provide them the ELISA ID Screen® SARS-CoV-2 kit. We are grateful also to Raphaël Tola, Laurence Thomas, Naomie Canard and Marie-Charlotte Mati for their technical help.