Monitoring of SARS‐CoV‐2 infection in mustelids – – 2021 – EFSA Journal

3.2.1 SARS‐CoV‐2 virus detections in the EU/EEA and UK in mustelids

As of 29 January 2021, SARS‐CoV‐2 has been reported in 400 mink farms for fur production in eight countries in the EU/EEA, namely 290 farms in Denmark, one in France, 21 in Greece, one in Italy, two in Lithuania, 69 in the Netherlands, three in Spain and 13 in Sweden (Figure 4 and Table 1). Sporadic SARS‐CoV‐2 cases (virus detected by reverse transcriptase‐polymerase chain reaction (RT‐PCR) have been recorded in kept ferrets in Slovenia (reported to OIE13) and in Spain (Gortázar et al., 2021).

The legal basis for reporting SARS‐CoV‐2 infection in the EU in mustelids and raccoon dogs is the COMMISSION IMPLEMENTING DECISION (EU) 2020/2183 laying down certain protective measures in relation to reporting infection with SARS‐CoV‐2 in mink and other animals of the family Mustelidae, as well as in raccoon dogs that entered into force on 21 December 202014 and according to which Member States shall submit a report to the Commission within three days after the first confirmation within their territory of the infection of mink, and other animals of the family Mustelidae and of raccoon dogs with SARS‐CoV‐2.

Denmark

In total, 290 of approximately 1,140 mink farms have been reported with SARS‐CoV‐2 in Denmark, since the first case was confirmed on 15 June 2020. In June, SARS‐CoV‐2 was detected in mink on another two farms. From July to the beginning of August, no further outbreaks in mink were registered. On 14 August, the first outbreak in the second phase of the epidemic was registered, with a slow increase in numbers of affected farms in August, and a steep increase from September onwards. Until October, all the outbreaks were detected in farms located in two municipalities in Northern Jutland while, from the beginning of October, outbreaks started occurring in neighbouring municipalities (Boklund et al., 2021). Overall, 186 infected farms were detected in Northern Jutland, seven in North‐West Jutland, 25 in Mid‐West Jutland, 15 in Mid Jutland, 37 in West Jutland, 6 in South‐West Jutland, 9 in South‐East Jutland, 1 in South Jutland and 4 in an island 40 km from Northern Jutland. Already at the first sampling date, 100% virus prevalence among tested mink was observed in 65% of the infected farms, and in 45% of the farms where no clinical signs of infection were observed (Boklund et al., 2021). At this stage of the production cycle, the autumn, the total of 1,140 Danish mink farms included a total of approximately 15 million mink. On 4 November, it was decided to cull all mink in the country. Most mink were culled and most farms closed, at least until the end of 2021. One or two mink farms remain in the country (12 January 2021). The new Act on the killing of and temporary ban on keeping mink will takes effect on 15 January 2021. The Act imposes a ban on keeping of mink until 31 December 2021. No new SARS‐CoV‐2 occurrences have been detected since 7 December 2020, when the most recent outbreak was detected. The reason for suspicion were: clinical signs in 33% of the farms, persons tested positive in 24% of the farms, surveillance of dead mink in 32% of the farms, and tracing of contacts to infected farms in 9% of the farms. Mink‐associated SARS‐CoV‐2 strains, characterised by the spike protein change Y453F, were initially observed in mink and humans in June (Hammer et al., 2020). Almost all infected mink farms were infected by mink‐associated variant strains and until now 5 clusters of mink‐associated variant strains have been identified. These strains have spilled back to the human population and spread in the communities. However, after the culling of mink farms, the incidence of these strains in the human population has decreased significantly (Hammer et al., 2020).

France

On 20 November, one of the four mink farms present in France tested positive to SARS‐CoV‐2 (OIE15). Out of 4,100 animals, 180 were sampled. On those samples, 180 serological tests (ELISA) were performed with 174 positive results, and 110 virological tests (RT‐PCR), with 33 positive results. No clinical signs of SARS‐CoV‐2 infection were reported in the infected farm. Results coming from these other breeder farms were negative (ELISA and/or seroneutralisation and RT‐PCR of oropharyngeal swabs).

Greece

As of 29 January 2021, SARS‐CoV‐2 has been detected on 21 out of the 91 mink farms present in Greece. On 13 November, the first case of SARS‐CoV‐2 was confirmed on a mink farm in the northern part of the country, in Kozani regional unit (Western Macedonia region). The suspicion was raised on 11 November by the local veterinarian due to the presence of respiratory symptoms, reduced feed intake and increased mortality. As a precautionary measure and based on increased morbidity/mortality, it was decided to cull the animals. A control zone of 10 km radius around the affected farm, where almost 40 mink farms were located, was established. Movement restrictions and strict biosecurity measures were implemented in the control zone. The epidemiological investigation and virus genome sequencing from animals and humans identified two family members working at the farm, who both tested positive for SARS‐CoV‐2, as the origin of the virus. At the same time, it was decided to test all workers/owners for SARS‐CoV‐2 in the 91 mink farms present in Greece and to repeat the testing procedure at least every 7 days until vaccination of human population in direct contact with mink (farm staff, owners, veterinarians) is concluded. The first cycle was completed on 13 November 2020 and testing still continues. Between 13 November and 3 December, 11 additional SARS‐CoV‐2 positive mink farm outbreaks were detected. They are all located in Western Macedonia: five in Kozani, four in Kastoria and two in Grevena regional unit. In 10 out of these 11 farms, suspicion was raised following notification of confirmed human cases as part of the repeated weekly surveillance conducted in all mink farm workers/owners. The 11th infected farm, was detected as the owner observed and reported increased morbidity and mortality; however, also in this case, there was a link with a human case as the owner tested positive to SARS‐CoV‐2. In total, in 3 out of the 11 farms, increased morbidity and mortality was observed. At this stage, available information indicates a human‐to‐animal transmission in most of the farms, where the presence of SARS‐CoV‐2 has been confirmed up to 3 December.

Genetic sequencing analysis was performed on the detected positive samples for the variants in aminoacidic positions found in sequenced genomes in Denmark and UK and discussed in the Rapid Risk Assessment by ECDC (ECDC, 2020a,b,c). From these mutations only the Y453F mutation has been detected in the analysed mink genomes from Greek farms indicating that these variants are unrelated to the Danish and Dutch variants.

Following the confirmation of the 11 new outbreaks, it was decided to stop culling animals. Stamping out and official disposal of carcasses were only applied to the first confirmed farm mentioned above. Strict biosecurity measures (e.g. mandatory use of personal protective equipment) and movement restrictions have been implemented in all mink farms (not only in the infected ones) since then. From 3 December 2020 to 8 January 2021, SARS‐CoV‐2 was confirmed in another nine mink farms in the Western Macedonia. These were located in the regional units of Kozani (5), Kastoria (2) and Grevena (2). In four of those farms, increased morbidity and mortality was observed. Enhanced biosecurity measures and special protocols are also implemented in the establishments where pelting and pelt drying take place. The pelting period concluded in the week 6—12 December. Animals from infected farms were pelted last, under official surveillance, right after pelting for all mink from non‐infected farms was completed. All dried raw skins remained stored for at least 4 weeks at the highest possible room temperature. Around 400.000 breeding animals are currently kept on farms. Possible SARS‐CoV‐2 circulation in the breeding population will continue to be monitored in the following months by repeated clinical and laboratory examinations.

Italy

SARS‐CoV‐2 was confirmed in a mink farm with 26,600 animals in the Cremona province, Lombardy region on 10 August 2020. After the detection of a human case among workers in the farm on 31 July 2020, 20 dead mink were tested, one gave a weakly positive result to RT‐PCR without any anatomopathological findings. All the animals in the farm were in a good health conditions without any suspect signs of infection. After these first positive results, an intensified surveillance was carried on in the whole farm, which encompassed two weekly clinical inspections in each shed, two weekly samplings of 30 oropharyngeal and faecal swabs and virological testing of all dead animals. Overall, 1,124 tests were carried out, leading to the identification of another weak positive RT‐PCR result from a faecal swab on 10 October 2020. In both positive samples, the genetic material was insufficient to carry out genetic analysis of the virus. On 6 November 2020, a third weakly positive case from the same farm was confirmed in a faecal swab sampled in a different shed than the one giving the previous positive result. Also, in this occasion, neither clinical signs of infection nor abnormal mortality were observed. On the basis of the diagnostic result, the entire herd was culled in compliance with a Ministerial Order which provides the mandatory notification of SARS‐CoV‐2 infections in mustelids and the killing and destruction of all the mink present in herds where the presence of SARS‐CoV‐2 is laboratory confirmed. The same Order also provides, as a precautionary measure, by 23 November, the ban of the breeding activities over the whole territory until the end of February 2021 providing only the maintenance of the breeders without any reproductive activities. The ordinance introduced also the ban on the introduction of new animals in mink farms and the activation of enhanced surveillance in Italian mink farms based on clinical checks, diagnostic tests (serological and virological) on a weekly basis and the application of strict biosecurity measures. In February 2021, an assessment of the surveillance activities carried out on the national territory, also on the basis of the evolution of the epidemiological situation in both humans and animals, will be carried out and it will be decided whether to continue to apply the restrictive measures in the future months or to restart of the breeding activity).

Lithuania

As of 29 January 2021, SARS‐CoV‐2 has been confirmed in two commercial mink farms in Lithuania. The first outbreak was detected in a farm with 60,000 animals in Varpių village, in the municipalities of Jonava, located in Kaunas County in central Lithuania. On 24 November, the local veterinary authority received a notification about an increase in animal mortality from a mink farm (169 mink from all age groups were found dead, but mainly young animals). Ten samples were randomly taken from dead mink and pooled in groups of five in the National Food and Veterinary Risk Assessment Institute (SFVS). The results were positive for SARS‐CoV‐2. On 25 November, out of 155 mink found dead, 22 additional samples were taken from different cages with sick mink and were analysed individually by RT‐PCR for SARS‐CoV‐2. On 26 November, all samples tested positive. The SFVS started an epidemiological investigation. According to the preliminary information, it is most likely that the introduction of the virus originated from a farm worker. On 24 November, one worker tested positive for SARS‐CoV‐2, and additionally five farm workers were later detected positive. Passive surveillance of COVID‐19 is carried out by the SFVS at mink farms. In addition to passive surveillance, up to 26 November, 86 samples were taken from dead mink from all mink farms in Lithuania for SARS‐CoV‐2 testing (one dead mink from each of 86 mink farms in Lithuania). All samples, apart those taken from the infected farm, gave negative results. On 30 December, a second farm, raising 55,000 animals, was detected to be infected in Pavartyciu village, located in Radviliškis municipality, Šiauliai County. As part of passive surveillance and due to the COVID‐19 detection in five workers, five dead mink were sampled and SARS‐CoV‐2 was detected in four mink (OIE16).

The Netherlands

In total, 69 mink farms (out of 126) were reported as SARS‐CoV‐2 infected in the Netherlands, 44 in the province of Noord Brabant, 23 in the province of Limburg, and two in the province of Gelderland. The first outbreak was diagnosed on 24 April 2020 and the last on 4 November 2020. On 29 farms (42%), the owner noticed clinical signs and reported the suspicion to the competent authority, whereas 39 (57%) infections were detected through the early warning system (EWS) in place (the obligation to test dead animals on a weekly basis). One farm (1%) was detected based on a human link with a previously infected farm. All the infected farms were inspected by an official veterinarian to collect samples for confirmation and only in seven out of the 69 farms (10%) no clinical signs of SARS‐CoV‐2 infection were seen. All the animals in affected farms were culled. A longitudinal study on the first four outbreaks revealed that the virus was efficiently circulating among mink with variable morbidity and mortality (Molenaar et al., 2020; Oreshkova et al., 2020). At the time of writing this report, the routes of transmission are still unclear, although transmission through infected humans explains most of the infections, and, therefore, epidemiological investigations to give more insight in the sources of virus introduction are still ongoing. Information available indicates at least five different introductions from humans into mink farms (Oude Munnink et al., 2020b). In addition, mink‐to‐human infection was demonstrated and the overall percentage of SARS‐CoV‐2‐infected humans present on infected mink farms was 66%, where the human virus is always genetically similar to that of the mink (Oude Munnink et al., 2020b). Therefore, information available indicates that most of the outbreaks has been caused by (infected) humans going from farm to farm, but in addition there was a strong spatial clustering of which the transmission route has not yet been elucidated. Around mid‐December, all mink have been pelted, implying that currently no farmed mink are bred in the Netherlands and no new infections will occur. Originally, the Netherlands passed a ban on fur farming in 2012 that would phase‐out mink fur production entirely by 2024. Following the coronavirus outbreaks on mink farms, the government declared an early shutdown of the industry. This means that from 8 January 2021 onwards, it is no longer allowed to keep farmed mink in the Netherlands.

Poland

Considering the reports of SARS‐CoV‐2 infections in mink in other European countries and the high incidence of SARS‐CoV‐2 human infections in Poland, a SARS‐CoV‐2 private, unofficial sampling in mink was conducted on one farm located in Pomorskie Voivodeship, North Poland. Throat swabs were collected from 91 minks culled for pelting on 17 November 2020. In the laboratory of Gdańsk Medical University SARS‐CoV‐2 was initially identified in the farmed mink with a prevalence of 16.5% (8.4–28.6%). Following the media reports of COVID‐19 infection on the mink farm, the Veterinary Inspection authority, the State Veterinary Institute in Puławy and the State Sanitary Inspectorate (PIS) took immediate action to investigate the farm health status, including daily clinical examinations of the mink on the farm and taking samples for laboratory tests. As a result of the investigation mentioned above, the Veterinary Inspection authorities unequivocally stated that: (i) the results of official laboratory tests showed neither the presence of genetic material of SARS‐CoV‐2 nor specific antibodies against this virus in samples taken; (ii) the clinically observed flock did not show any symptoms that might indicate SARS‐CoV‐2 infection in the animals; and (iii) diagnostic tests carried out by the PIS in humans did not confirm SARS‐CoV‐2 infection in persons professionally linked to the farm mentioned above. The Veterinary Inspection authorities concluded that the media reports about the first confirmation of SARS‐CoV‐2 infection on a mink farm published by the Medical University of Gdańsk have been verified negatively.

Slovenia

A ferret kept as pet animal in a COVID‐19 positive household was confirmed as SARS‐CoV‐2 positive by RT‐PCR on 1 December 2020. The animal showed clinical gastrointestinal signs and the source of infection was the contact with COVID‐19 positive people (OIE13).

Spain

One mink farm in the Autonomous Community of Aragon and one in the Autonomous Community of Galicia out of 29 farms in the country were reported as SARS‐CoV‐2 positive. From the first cases of SARS‐CoV‐2 reported in mink farms in the Netherlands in April, hygiene and biosecurity measures in mink farms, as well as surveillance on them were reinforced in Spain. During the first month, two reports of clinical suspicions were received on 27 and 28 April, from farms located in the provinces of Ávila and Guipúzcoa, both cases were discarded after analysis of samples in the laboratory. During the second half of May, a human outbreak of COVID‐19 was detected in the province of Teruel. Some of those individuals were working on a mink farm with around 19,500 adult and 73,200 young animals. The farm has a feed factory, where food for the animals is produced, as well as its own pelting facility. This is the only mink farm in the Autonomous Community of Aragon that did not have epidemiological links to other mink farms. Since the outbreak of COVID‐19 was reported in people related to the farm, on 27 May, samples from lung, spleen, liver and small intestine from four mink from the farm were analysed by RT‐PCR and tested negative for SARS‐CoV‐2. On 8 June, oropharyngeal and rectal swabs and exudates from internal organs from 20 additional animals were sampled. An inconclusive RT‐PCR result, in one oropharyngeal swab only, led to a further sample collection at the mink farm. On 22 June, samples of oropharyngeal and rectal swabs from 30 live animals, as well as swab and lung parenchyma samples from six dead animals were collected. One of the oropharyngeal swabs tested positive for SARS‐CoV‐2 by RT‐PCR, and samples from seven other animals gave non‐conclusive results (all of them asymptomatic). On 7 July, oropharyngeal and rectal swabs were collected from 90 live animals (30 adults and 60 young animals); 86.67% of animals tested positive for SARS‐CoV‐2 by RT‐PCR. No mortality nor clinical signs compatible with SARS‐CoV‐2 infection or drop in feed consumption were observed at farm level. A stamping out policy in the farm, including official cleaning and disinfection of the premise after culling was implemented. Genetic analysis from the complete sequence of 34 viruses and the sequence of the gene encoding the spike protein of four more viruses revealed: i) the presence of the D614G mutation, that defines the predominant clade in Spain and Europe, in the spike protein; ii) the absence of the Y453F mutation described in Cluster 5, as well as the rest of the changes that define this cluster that appeared in some of the mink farms in the Netherlands; iii) the presence of the N501T mutation, which is a site in all the sequences analysed; this is a site of host adaptation and antigenic drift that is part of the group of mutations that have been identified in the recent UK viral variant (N501Y); and iv) the F486V and D796H mutations have been always identified together in 22 of the 38 sequences; these changes have not been identified in the GISAID database in either human or mink samples.

On 19 January 2021 a second SARS‐CoV‐2 outbreak was confirmed in a mink farm in Galicia, in the municipality of A Baña. The farm had a total of 3,100 animals (2,500 females and 600 males) and all were slaughtered few days after the confirmation of the outbreak, in application of the current legal regulations and with the aim of preserving public health. The outbreak had been suspected since the middle of December when, out of 20 oropharyngeal swabs collected on 10 December as part of the active surveillance in place, four gave positive results and three were inconclusive for SARS‐CoV‐2 by RT‐PCR. On 15 and 18 December, an additional 30 and 90 oropharyngeal swabs were taken respectively, and all gave negative results by RT‐PCR. On 4 January 2021, 90 oropharyngeal swabs were sampled with four giving an inconclusive result by RT‐PCR. On 18 January four samples were collected from the manure of the mink farm and one sample tested positive for SARS‐CoV‐2 by RT‐PCR. Neither clinical signs nor abnormal mortality had been observed among the animals. The origin of the infection remains under investigation, although preliminary information shows that the most probable origin of the introduction of the virus into the farm could has been through two workers from the holding. Both workers, although having no clinical signs of SARS‐CoV‐2 and being PCR‐negative, were tested serologically positive for SARS‐CoV‐2 antibodies in samples collected on 11 December. The results of the genetic analysis revealed the presence of the pD614G mutation in spike protein (in four samples) and of N501T (in one sample). Neither Y453F nor other mutations and deletions described in the Cluster 5 isolate have been detected. Neither the strain from the United Kingdom VOC 202012/01, nor the South‐African 501Y.V2, nor the Brazilian variant included in lineage B.1.1.248 have been detected.

On 22 January, a third SARS‐CoV‐2 outbreak was confirmed on a farm rearing 1,010 mink (800 females and 210 males) in the Municipality of Navatalgordo, in the region of Castille and León. No signs of SARS‐CoV‐2 infection were observed at the farm. The origin of the virus remains under investigation, although preliminary information shows that the most probable source could have been the owner of the holding as he had clinical symptoms on 4 January and gave a positive result by antigen test on 8 January 8. Also, the owner and two workers gave positive results for SARS‐CoV‐2 by RT‐PCR in samples taken on 14 January 2021. Genetic analysis of the viruses is ongoing.

Sporadic cases have also been recorded in ferrets in Ciudad Real province, detecting SARS‐CoV‐2 RNA in six out of 71 ferrets (8.4%) and isolating the virus from one rectal swab (Gortázar et al., 2021). This confirms that natural SARS‐CoV‐2 infection does sporadically occur in kept ferrets, at least under circumstances of high viral circulation in the human population. However, none of the 20 ferrets resampled 2 months later was PCR positive, including one individual that had given a positive result two months earlier. Therefore, small ferret collections are probably unable to maintain prolonged virus circulation (Gortázar et al., 2021).

Sweden

The Swedish mink fur sector is composed of ~ 35 farms with in total 600–650,000 animals. Eighteen of these farms are located in the municipality of Sölvesborg, the County of Blekinge, in the south‐eastern part of the country. In May to June 2020, a dialogue between the competent authority and representatives from the Swedish mink sector was initiated to raise awareness, to ensure enhanced on‐farm biosecurity practices and to raise vigilance regarding increased morbidity and mortality. In early October, given the absence of any reports of morbidity/mortality from the Swedish mink farms, an active surveillance scheme, based on sampling up to five dead mink per farm per week, was initiated. Within the scheme, mink found dead were submitted to the National Veterinary Institute, SVA, in Uppsala, where they were sampled with swabs from the oral cavity and pharynx and analysed by RT‐PCR. The surveillance scheme was voluntary and organised in close collaboration with the industry. Between mid‐October and mid‐November, SVA received 74 submissions of dead mink, representing between 1 and 4 submissions per farm. Thirteen farms gave positive results for SARS‐CoV‐2. All positive farms were located in Sölvesborg, the County of Blekinge, in the south‐eastern part of the country. None of the positive farms had reported increased morbidity or mortality before testing positive but, retrospectively, a slight increase in daily mortalities could be observed in the records from several of the farms.

Movement restrictions and strict biosecurity measures were imposed on all mink farms in Sweden after the confirmation of the first affected farm. None of the affected farms was culled. The Swedish outbreak coincided with annual pelting, when approximately 80% of the mink are killed as part of the production cycle. According to the assessment made, culling of affected farms as part of disease control measures would not speed up the process of reducing the number of susceptible animals compared with the annual pelting and killing, and would therefore not contribute to any significant reduction in the risk of further spread of the disease. Pelting was carried out from mid‐November to early December, under strict biosecurity recommendations, to prevent mink‐to‐human SARS‐CoV‐2 transmission. After pelting, approximately 90,000 breeding animals remain in Sweden. The situation for SARS‐CoV‐2 in the mink that remain will be further monitored using serology in live animals, as well as RT‐PCR on a sample of animals found dead.

Sequences from humans and mink from the same mink farms cluster closely together, suggesting within‐farm human‐to‐mink and/or mink‐to‐human transmission.

3.2.2 SARS‐CoV‐2 virus detections outside the EU/EEA and UK in mustelids

From 29 January 2021, outside Europe SARS‐CoV‐2 virus has been detected in American mink in Canada and the United States.

Canada

From 29 January 2021, SARS‐CoV‐2 has been detected in two mink farms in Canada. On 8 December, SARS‐CoV‐2 was confirmed in a mink farm rearing 15,000 animals in British Columbia. On 3 December, workers of the mink farm were diagnosed as COVID‐19 positive. When sampling of the mink took place, on 4 December, the animals did not display any signs of infection, but an increased mortality (1%) was observed in the days following sampling. Human‐to‐animal transmission was suspected as introduction of the infection into mink farms (OIE17). On 23 December, a second outbreak was confirmed in a farm rearing 950 mink in British Columbia. Samples were taken on 21 December as the animals had shown clinical signs of diarrhoea and increased mortality (3%). Initial testing has revealed no evidence of human SARS‐CoV‐2 infection in farm workers. Source of the outbreak is still under investigation. On both farms, animals were euthanised and frozen for pelting (OIE18).

United States

From 29 January 2021, the United States Department of Agriculture Animal and Plant Health Inspection Service (USDA‐APHIS) confirmed 16 SARS‐CoV‐2 outbreaks in mink farms in the US (APHIS USDA, 2020a), 11 in Utah, 2 in Wisconsin, 2 in Michigan and 1 in Oregon. The first detection was in Utah and, according to OIE,19 the outbreak started on 26 July; the last outbreak in a mink farm was detected in Oregon and started 22 October (OIE²⁰). Overall, 177,357 animals were bred in those farms and 16,130 died due to infection (OIE²⁰). Because some workers on these farms had COVID‐19, it is likely that infected farm workers were the initial source of the mink infections (APHIS USDA, 2020a).

On 13 December, USDA communicated the first SARS‐CoV‐2 virus detection in a free‐living American mink. The animal was trapped under the monitoring activities carried out around an infected farm in Utah. The animal was confirmed by genetic testing as a wild mink, not an escaped farmed mink.

3.2.3 Genetic analysis of SARS‐CoV‐2 in mustelids

To date, published genetic data on SARS‐CoV‐2 from mustelids are very scarce and limited to that reported in two studies from the Netherlands and Denmark, respectively, investigating possible transmission between mink and humans (Hammer et al., 2020; Oude Munnink et al., 2020b), and one national report describing the evolution of the SARS‐CoV‐2 epidemic in mink in Denmark (Boklund et al., 2020). The strains from the Netherlands and Denmark are not closely related and fall into different genetic clades, which excludes a direct link between farms in the two affected countries (ECDC, 2020a). However, in both countries, SARS‐CoV‐2 strains related to mink farms fall into some separate clusters, suggesting several introductions from humans to mink, followed by transmission chains from farm to farm. The mutation Y453F in the receptor‐binding domain (RBD) of the spike protein, suggested as an adaptation of the virus to mink, has been observed in many of the SARS‐CoV‐2 strains in both countries, independently of the clustering (ECDC, 2020a; Oude Munnink et al., 2020b; Welkers et al., 2020). This mutation has also been observed in SARS‐CoV‐2 strains associated with mink in, e.g. Greece and Sweden. However the Y453F has also been observed in human cases not related to mink (e.g. South Africa, Belarus, Russia, Switzerland).

In September 2020 in North Jutland, Denmark, a variant referred to as Cluster 5 was detected in five mink farms and in 12 human cases. This Cluster 5 variant contains four mutations (Y453F, I692V, M1229I, S1147L), and one deletion (69–70 del) in the S protein.20

The deletion of two amino acids (AAs; 69–70) in the S protein is not present in any of the mink strains from the Netherlands, but has been widely reported in human cases unrelated to mink without Y453F.21

Due to its characteristics, and the role of S protein in the virus, i.e. host–cell interaction, it has been hypothesised that the Cluster 5 variant may affect the immune response, probability of reinfection, vaccine efficacy and treatment with convalescent plasma (ECDC, 2020a; Mallapaty, 2020) and may have an increased binding affinity to the human angiotensin‐converting enzyme 2 (ACE2) receptor. However, scattered information is only available so far and it is not possible to infer any definitive conclusion. Therefore, along with the lineage B.1.1.7, the Cluster 5 variant would merit a mutation surveillance effort (Genomics UK Consortium, 2020).

In conclusion, it is envisaged to monitor the genetic characteristics of virus from all infected mink farms to evaluate the progression of the evolution of the virus, which seems to find in mink a suitable model to evolve and to ascertain that variants of public health concern, such as the Cluster 5, are not circulating.

3.2.4 Susceptibility of mustelids and other animals to SARS‐CoV‐2

Information on susceptibility to SARS‐CoV‐2 in animals is reported in Section 3.3.2.

3.2.5 Pathogenesis, clinical signs and immune response

SARS‐CoV‐2 is an enveloped beta‐coronavirus sharing 79% genome sequence identity with SARS‐CoV and 96.2% with bat coronavirus RatG13 (Yan et al., 2020). The viral envelope is coated by spike (S) glycoprotein, envelope (E) and membrane (M) proteins. The S protein is composed of two subunits, S₁ and S₂. The S₁ subunit comprises an amino‐terminal domain and a RBD (Xiao et al., 2003; Babcock et al., 2004; Wong et al., 2004). The RBD is the ligand for the cellular receptor ACE2. The S₂ subunit is characterised by a fusion peptide (FP) region, which is involved in the release of the viral genome into the host cell cytoplasm (Liu et al., 2004).

The multiple sequence alignments of ACE2 revealed high homology and high conservation in the binding region among different animal species. It has been shown that the 3D structure of human and mink ACE2 is highly conserved and that the binding region of mink ACE2 complements perfectly the SARS‐CoV‐2 ligand (Hayashi et al., 2020). In addition, ACE2 is most abundantly expressed on type II alveolar and glandular epithelial cells in the trachea and bronchi of ferrets (Van den Brand et al., 2008).

Ferrets (Mustela putorius furo) have been shown to be a suitable model for the pathogenicity of SARS‐CoV‐2 (Johansen et al., 2020; Muñoz‐Fontela et al., 2020). Natural infection can occur, presumably after contact with infected humans.13 In experimental infections, ferrets are highly susceptible to SARS‐CoV‐2, with efficient virus replication especially occurring in nasal turbinates, soft palate and tonsils. Following mucosal exposure to SARS‐CoV‐2, clinical signs are generally absent or mild. When clinical signs are observed, these are characterised by lethargy, nasal discharge, wheezing, sneezing and loose stools. In some cases, mild lymphopenia and neutrophilia have also been observed. Virus replication occurs in the upper respiratory tract as soon as 2 days after infection and lasts for few days after infection (Schlottau et al., 2020; Shi et al., 2020). Virus replication in ferrets appears to be restricted to the respiratory and gastrointestinal tracts.

American mink is the other species belonging to the family Mustelidae known to be susceptible to SARS‐CoV‐2.

Pathogenesis in mink is largely unknown and mostly limited to information from studies evaluating natural infections in farmed mink (Hammer et al., 2020; Molenaar et al., 2020; Oreshkova et al., 2020; Oude Munnink et al., 2020). The available information demonstrates that SARS‐CoV‐2 can replicate efficiently in the upper and lower respiratory tracts of mink and that host–pathogen interaction resembles that observed in humans and other susceptible animals.

Infection in mink has been associated most commonly to the observation of a reduced feed intake, followed by respiratory signs, nasal discharge and sneezing. Increased mortality in a small percentage of adult mink was reported in different outbreaks in the EU. Post‐mortem analyses revealed signs of pneumonia with lung lobes swollen, dark red and without any tendency to collapse. A few animals showed free blood in the upper respiratory tract.

Histological findings of tissues from deceased animals revealed interstitial pneumonia, multifocal areas with thickening and degeneration of alveolar septa with type II pneumocyte proliferation and diffuse alveolar damage. Alveolar lumina were filled with inflammatory leucocytes and desquamated cells. The epithelial cells of bronchioles in affected areas showed severe necrosis and formation of syncytial cells. Trachea showed loss of cilia with swollen epithelial cells. Nasal conchae showed multifocal swelling and degeneration of epithelial cells with diffuse loss of cilia.

The presence of SARS‐CoV‐2 antigen was found in nasal conchae, trachea, bronchioles and alveolar epithelial cells (Molenaar et al., 2020).

Very recently, results from an experimental infection in mink were reported (Shuai et al., 2020). Mink were inoculated intranasally and killed 4 days afterwards. Viral RNA was detected in the nasal washes and from the ear swabs and rectal swabs of two animals. Infectious virus was detected in the nasal washes, but not from the conchae swabs or rectal swabs of any animals at any time. Viral RNA was detected in the nasal turbinates, soft palates, tonsils, all lung lobes and submaxillary lymph nodes, in the trachea and in the ileum, but was not detected in heart, kidneys, spleen, liver, pancreas and brain. Virus was detected to a lesser degree in most viral RNA‐positive samples. The respiratory tract of the virus‐inoculated mink was severely compromised, with mucinous‐purulent secretion, containing neutrophil debris and mucus, inflammatory infiltrates, epithelial degeneration and necrosis.

SARS‐CoV‐2 nucleoprotein antigen was verified in nasal mucinous‐purulent secretions, in the epithelium of the nasal mucosa of the vestibular region, respiratory region and olfactory region and that of the tracheal mucosa, suggesting an involvement of nasal secretion in transmission and olfactory function impairment, as also observed in humans (Luers et al., 2020).

The above‐mentioned results, along with those described in natural outbreaks, demonstrate that the host‐pathogen interaction of SARS‐CoV‐2 in mink and ferret resembles that observed in humans.

Symptomatic SARS‐CoV‐2 infections in humans in fact, can progress in the more severe cases in acute respiratory distress syndrome (ARDS) (McGonagle et al., 2020). Viral infection and replication can cause virus linked pyroptosis and vascular leakage. Pyroptosis is a highly inflammatory form of programmed cell death that triggers inflammation with the recruitment of cells and the production of pro‐inflammatory cytokines and chemokines IL‐1β, IL‐6, IFN‐γ, MCP1 and IP‐10, which in turn prime T and B lymphocytes evoking an adaptive immune response. This response is effective in controlling and resolving the infection, but in some cases, an uncontrolled immune response occurs with the release of a cytokine storm that causes severe tissues damage and multi‐organ failure (Tay et al., 2020).

3.2.6 Infection dynamics and transmission routes

As described in Section 3.3.2, some animal species are susceptible to SARS‐CoV‐2 and have the capacity to transmit the virus as shown by experimental or natural infections (Freuling et al., 2020; Schlottau et al., 2020). However, to date significant between‐animal spread in field settings has only been described for farmed mink.

The high animal density that is present in a typical mink farm (generally 5,000–20,000 animals), provides ideal conditions for viral transmission (Hobbs and Reid, 2020). Once introduced, usually by infected humans as suggested also by outbreak investigations in the affected MSs (Section 3.2.1), SARS‐CoV‐2 appears to spread efficiently among the animals within the farm, as indicated by high prevalence of antibody and PCR‐positive animals (commonly up to 100%) detected in affected farms, in which in‐depth investigations have been carried out (Hammer et al., 2020; Oreshkova et al., 2020). In mink, the virus can be found in secretions from the respiratory tract and in faeces (Shuai et al., 2020; Boklund et al., 2021). Extensive virus replication also results in local environmental contamination as shown by detection of virus RNA in air and dust samples collected within (but not outside) the affected premises (Hammer et al., 2020), as observed in the epidemics occurred in mink farms in the Netherlands (Oreshkova et al., 2020). Also, in Denmark, viral RNA has been identified 2–3 metres from mink in two out of eight infected mink farms, while there was no detection beyond 3 metres from the mink in any of the 17 farms where samples were collected in this distance (Boklund et al., 2021). In the Netherlands, viral RNA was observed in the inhalable dust in mink farms and outside, close to the entrance, but not outside the premises of infected mink farms (de Rooij et al., 2021). This indicates that SARS‐CoV‐2 could be transmitted between animals mainly by direct or close contact via infectious droplets and aerosols, but also via indirect contact via feed or bedding, or in air dust containing faecal matter up to short distance (few metres). Additionally, experimentally transmission was shown in ferrets via air flow between cages (Richard et al., 2020). Further investigation under field conditions is needed to confirm this transmission pathway. Experience from affected MSs demonstrates that once SARS‐CoV‐2 has been introduced into an area with high density of mink farms, farm‐to‐farm transmission is likely to occur, despite enhanced biosecurity implemented to halt the spread (Boklund et al., 2020; Oude Munnink et al., 2020b). The possible transmission chain probably starts with the introduction of the virus into a farm through infected personnel, then it can quickly spread through the animal population, further personnel may acquire the infection from animals and transmit this further to other farms. SARS‐CoV‐2 has been detected in other susceptible animals, such as cats (Boklund et al., 2021), although evidence for transmission through this pathway is lacking up to date.

Although virus RNA has to date only been found within infected premises, and not outside (de Rooij et al., 2021), aerosol spread can be considered as a possible pathway for between‐farm transmission. In the Netherlands, whole genome sequencing indicated that five different virus clusters were circulating, two of which remained limited to a single farm. Markedly, each of these five clusters had been detected by June and no new clusters were seen in mink farms even though SARS‐CoV‐2 outbreaks were observed until 4 November. In addition, virus from these five clusters has only been observed in mink and persons associated with mink farms, not in any other person, even though efforts have been made to sequence samples from people living in the neighbourhood of mink farms. This suggests between‐farm transmission of SARS‐CoV‐2 virus in the Dutch situation, either through people associated with mink farms or some other unknown mechanisms. A strong spatial clustering of infected farms was observed in Denmark as well as in the Netherlands.

To date, however, the main routes of between farm transmissions remain unknown. From the experience gathered in Denmark and in the Netherlands (Boklund et al., 2021, Fischer et al., in preparation), the only risk factor identified is short distance to the nearest SARS‐CoV‐2 positive farm and in Denmark also large farm size (Boklund et al., 2021). This pattern of close proximity between affected farms was observed also during the epidemics of porcine epidemic diarrhoea (PED) caused by another coronavirus with a certain ability to survive in the environment, particularly during cold seasons, in the US and Japan in 2013–2014. Despite high biosecurity measures in place the spread could not be halted (Alvarez et al., 2016; Sasaki et al., 2017).