Incident 1

At the Bronx Zoo in New York, USA, in late March 2020, two Malayan tigers (Panthera tigris jacksoni), two Amur tigers (Panthera tigris altaica), and three Transvaal African lions (Panthera leo krugeri) developed clinical signs including coughing and wheezing.^1,7 One tiger and one lion had periods of decreased appetite, and the lion had vomiting that lasted for 3 d (Table 2). Respiratory signs lasted an average of 4.5 d (range, 1–16 d). Signs were considered mild to moderate. One Malayan tiger with more prolonged clinical signs progressing to anorexia was anesthetized for examination, treatment, and sample collection for diagnostics. Polymerase chain reaction (PCR) testing was positive for SARS-CoV-2 on nasal swabs, oral swabs, tracheal wash fluid, and feces. Virus was isolated from tracheal wash fluid, and serum was positive for antibodies on virus neutralization (titer = 64). Virus neutralization opportunistically performed on the other Malayan tiger 7 mon after infection was positive (titer = 32). Virus neutralization performed on one lion 18 mon after infection was also positive (titer = 32). All felids, as well as a third asymptomatic Amur tiger housed in the same building as the other tigers, were confirmed positive by fecal PCR. Virus was isolated from fecal samples of one Amur tiger and one lion. Prolonged fecal shedding of viral RNA was confirmed. Average duration of shedding (from onset of clinical signs to last positive sample) was 23 d (range, 12–39 d; Table 3). The lions were housed in a separate building from the tigers. Viral genotyping was performed and confirmed that tigers and lions had different strains of the virus (Fig. 1). The zoo was closed to the public at the time, and no staff were allowed to report to work if ill with clinical signs consistent with COVID-19. Health department investigations confirmed that tiger keepers had the same strain of the virus as the tigers and likely infected the animals during a phase of asymptomatic or presymptomatic shedding. Although lion keepers tested serologically positive for SARS-CoV-2, none were PCR positive at the time of testing, so it could not be confirmed whether lions and the keepers had the same strain of the virus. No PPE was used by staff working around the animals before the animals developing clinical signs, because transmission of SARS-CoV-2 from humans to nondomestic felids had never previously been documented, and there were shortages of PPE for use by human medical professionals in New York City at that time. After infection was confirmed, personnel access to the tigers and lions was restricted to those necessary to care for them. In addition, staff used surgical facemasks, face shields, coveralls, and disposable gloves when working around the felids as well as during food preparation. Attempts were made to maintain a minimum of 6 ft (1.8 m) between animals and staff, and voluntary training was suspended. To the extent possible, dry cleaning of enclosures after spraying with a hydrogen peroxide product was performed. To avoid aerosolizing potentially infectious particles, pressure hoses were not used when cleaning enclosures. Note that the keepers that cared for the infected tigers also cared for nine snow leopards (Panthera uncia). However, the snow leopards were not housed in the same building; instead, they were housed in outdoor enclosures. One snow leopard with a chronic recurring cough tested negative by fecal PCR 3 mon after the confirmed infection in the tigers; the other snow leopards were not tested.

Table 2.

Summary of clinical signs in nondomestic felids infected with SARS-CoV-2 between March 2020 and February 2021.^{1,3,4,6,7,10,12}

Table 3.

Summary of duration of clinical signs versus duration of shedding of SARS-CoV-2 RNA in nondomestic felids.^{1,3,4,6,7,10,12}

Figure 1.

(A) Timeline and location of SARS-CoV-2 infections in nondomestic felids between March 2020 and February 2021. (B) SARS-CoV-2 isolates from nondomestic felids in the case series compared with human isolates and isolates from subsequent infections in nondomestic felids.^{1,3,4,6–8,10,12,13}

Incident 2

At the Lory Park Zoo and Owl Sanctuary in Midrand, South Africa, in July 2020, two cohoused cougars (Puma concolor) developed clinical signs including anorexia, diarrhea, and oculonasal discharge. One cougar had a persistent dry cough. Clinical signs lasted between 8 and 13 d. The cougar with the persistent cough was immobilized for examination and diagnostic testing. SARS-CoV-2 infection was confirmed by PCR testing of a nasal swab collected during the examination. Additional PCR tests were performed on nasal swabs collected from the positive cougar at 1, 3, 5, and 7 wk and fecal swabs at 3, 5, and 7 wk after the initial presentation. At week 5, the nasal swab was still positive, but the fecal swab only showed a weak positive response. The cougar tested negative on both nasal and fecal swabs 7 wk postinfection.⁶ The zoo was closed to the public at the time of infection; the cougars were housed in an off-display area, and only staff members could approach within 6 ft of the animals. Staff members with possible signs of COVID-19 were prohibited from working, and all staff were screened daily with health questionnaires and temperature checks. However, presymptomatic staff members working with the cougars 10 d before the cougars developing clinical signs eventually became ill and tested positive for SARS-CoV-2. Prior to the cougars developing signs, PPE included cloth masks that were worn around the cougars and during food preparation. Quaternary ammonium and biguanide footbaths were used. After SARS-CoV-2 infections were confirmed, cougars were serviced by one keeper who wore a disposable surgical mask, disposable surgical gloves, rubber boots, and protective clothing that could be removed and disinfected. Footbaths were changed to a potassium peroxymonosulfate product. Because of a chronic medical condition in one of the cougars necessitating close contact with the keeper, it was elected to continue working within 6 ft of the cat. Disposable enrichment items were used. Note that two tigers that were housed in the same building as the cougars never developed clinical signs.

Incident 3

At the Knoxville Zoo in Tennessee, USA, in October 2020, three Malayan tigers housed individually but within the same building developed clinical signs including coughing, lethargy, and anorexia.³ One tiger also developed mucoid ocular discharge. Clinical signs ranged from mild to severe and lasted for an average of 8 d (range, 5–11 d). Each tiger was sedated for examination and diagnostics. Two of the tigers tested positive for SARS-CoV-2 by PCR on oral and/or nasal swabs on the day of examinations; the other tiger was not swabbed. On subsequent swabs collected voluntarily from the nose and/or mouth of two tigers over the next 40 d, none were positive. Fecal PCR was positive in two of three tigers that remained positive for 11 and 23 d, respectively. Serology by enzyme-linked immunosorbent assay (ELISA) and serum neutralization was positive on the two tigers that had blood repeatedly collected voluntarily over the course of 2–3 mon. Serum neutralization titers peaked at 181 for one tiger around the 1-mon mark and at >4,096 for the other tiger around the 2-mon mark. Staff members had to complete a daily health questionnaire, and those with possible signs of COVID-19 were prohibited from working. However, no staff members working with the felids were ever confirmed to be infected with SARS-CoV-2 prior to the tigers developing clinical signs. Despite intensive investigations by the Department of Public Health, a source of infection for the Malayan tigers was not identified. The zoo was open to the public at the time of infection, and patrons could traverse a walkway approximately 6 ft over the exhibit. This may have served as the source of infection, but it cannot be confirmed. Keepers working with the tigers developed clinical signs after the tigers did, which may indicate possible transmission from the tigers to the staff, although the evidence was not conclusive.⁵ Prior to the felids developing clinical signs, staff wore gloves and cloth facemasks when around the cats and for food preparation. After SARS-CoV-2 was confirmed in the tigers, PPE was switched to Tyvek^® (Dupont, Wilmington, DE 19805, USA) suits, N95 masks (minimum filtration percentage, 95%), boot covers, and face shields. Only limited personnel were allowed near the Malayan tigers. Hand feeding and nonmedically necessary training were suspended.

Incident 4

At the Barcelona Zoo in Spain in November 2020, four Southwest African lions (Panthera leo bleyenbergui) housed together developed clinical signs including coughing, sneezing, and nasal discharge.⁴ Two of these felids also showed apathy and decreased appetite. Generally, signs were considered mild, although one lion had a severe cough. Clinical signs lasted for an average of 17 d (range, 14–19 d). Nasal swabs were collected from each awake lion every few days by opportunistically swabbing while the cat targeted on an object. Then lions remained positive by the antigen test (COVID-19 Ag Test reference 243103N-20, 47445 Moers, Germany) for an average of 13 d (range, 8–16 d). PCR testing of nasal swabs remained positive for an average of 19 d (range, 16–26 d). Fecal PCR testing of combined samples from the group remained positive for approximately 18 d. Virus was isolated from the nasal swabs of two lions, but from none of the fecal samples. Within 2–5 wk of the initial diagnosis, each lion was anesthetized for blood collection for serology, including virus neutralization and ELISA. Seropositivity was confirmed by both tests on both sampling dates. Serum neutralization titers ranged from 32 to 128 on the initial test. Sampling was repeated on three lions 4 mon after clinical presentation; titers were sustained in two of the felids, but decreased in the third felid. The lion with the decreased titer was confirmed seronegative 7 mon after clinical presentation. The other two lions sustained high titers (32–64) 10 mon after infection. The zoo was closed to the public when the lions developed clinical signs. Staff members with possible signs of COVID-19 were prohibited from working. Two lion keepers who worked with the lions 2–4 d before they became ill tested positive for SARS-CoV-2. They were asymptomatic when working with the lions. Before confirming infections in the lions, PPE included cloth masks while working inside the building as well as when preparing food and enrichment items. After confirming SARS-CoV-2 infections, PPE requirements were increased to include a staff changing area with footbaths containing aldehyde-based disinfectant, FFP3 masks (minimum filtration percentage, 99%), face shields, Tyvek coveralls, and rubber boots. No changes were implemented in training routines; however, enrichment items donated by the public were not introduced to the lions for 24 h after donation.

Incident 5

At the Louisville Zoo in Kentucky, USA, in November 2020, three snow leopards housed individually but within the same building developed mild clinical signs including coughing, wheezing, and sneezing.¹² Clinical signs lasted an average of 7 d (range, 2–16 d). Infection with SARS-CoV-2 was confirmed by fecal PCR. Daily fecal sampling was performed until no viral RNA was detected for at least a week. The snow leopards shed viral RNA intermittently for an average of 31.5 d (range, 30–33 d). A snow leopard keeper tested positive for SARS-CoV-2 approximately 1 wk before the onset of clinical signs in the snow leopards. The strain of virus from the keeper was not sequenced, but it was assumed the virus was transmitted from the staff to the snow leopards. The zoo was open at the time of the snow leopards' infections, but no members of the public were allowed within 6 ft of the animals at any time. No staff members with clinical signs of COVID-19 were allowed to report to work and were expected to monitor themselves for fever before reporting. Before confirming infections in the snow leopards, PPE included facemasks (cloth or surgical) while working near the leopards and when preparing food. Disposable gloves were to be worn during food preparation, but staff compliance was not consistent. After SARS-CoV-2 was documented in felids at the Bronx Zoo, the Louisville Zoo implemented social distancing with susceptible species including the snow leopards, maintaining at least a 6-ft distance from the animals when possible, as well as minimizing any training and feeding by stick. After strongly suspecting SARS-CoV-2 infections in the snow leopards, staff PPE requirements were increased to include face shields, dedicated Tyvek coveralls, plastic shoe coverings, and footbaths containing phenolic-based disinfectant. Stalls were disinfected with a quaternary ammonia product. Note that two tigers were housed in the same building as the snow leopards, and the same keeper staff cared for a jaguar (Panthera onca), a cougar, and two Canada lynx (Lynx canadensis); only the snow leopards developed clinical signs.

Incident 6

At The Wildcat Sanctuary in Minnesota, USA, in January 2021, 12 Bengal tigers (Panthera tigris tigris), one Sumatran × Bengal cross tiger, three African lions, a white lion, and eight cougars developed clinical signs including coughing, wheezing, and deceased appetite. Of the 127 felids housed at the sanctuary, 47 felids spread throughout five buildings were potentially exposed by being housed within the same building, sharing a fenceline with sick felids, and/or by use of shared tools. Twenty-five felids developed clinical signs that lasted an average of 6 d (range, 1–16 d). One tiger was anesthetized and confirmed positive for SARS-CoV-2 by PCR on a nasopharyngeal swab. Infection with SARS-CoV-2 was presumed, but not confirmed, for the other clinically affected felids. Staff members were confirmed to have COVID-19 infections before the felids becoming ill; however, they likely transmitted the virus during an asymptomatic or presymptomatic period. Sanctuary policy prevented staff members with clinical signs of COVID-19 from working. The sanctuary was closed to the public during the time when the felids developed clinical signs. Before confirming infection in the tiger, PPE requirements included wearing cloth masks around the felids and using gloves during food preparation. After the felids developed clinical signs of SARS-CoV-2, staff wore N95 masks. When working with cats with clinical signs, staff also wore goggles and Tyvek suits or other designated clothing. Staff stopped sharing utensils between cats or, when necessary, disinfected utensils between cats. Bleach footbaths were instituted. Enclosures were spot cleaned instead of cleaned with high-pressure hosing. Voluntary examinations of felids were suspended. Training was limited in an attempt to maintain a minimum of a 6-ft distance from the felids when possible, although feeding by tongs was maintained.

Incident 7

At the Borås Zoo in Sweden in January 2021, two Amur tigers and five African lions housed within the same building developed clinical signs including coughing, sneezing, and dyspnea (severe in the tigers, mild-to-moderate signs in the lions). The male tiger and one lion had nasal discharge. Wheezing was noted in both tigers. Clinical signs lasted 5–10 d in the lions and 3 d in the male tiger. The 17-yr-old female tiger developed a decreased appetite, and 2 d later was found moribund with severe respiratory symptoms and neurologic deficits. Because of a poor prognosis and geriatric status, euthanasia was elected. Necropsy revealed distal tracheobronchitis with intraluminal mucohemorrhagic-purulent exudate, necrotizing bronchiolitis, and mucosal adenitis as well as focal acute pneumonia. Immunohistochemistry confirmed the presence of SARS-CoV-2 antigen associated with these lesions, and the tiger tested positive for SARS-CoV-2 on PCR of oropharyngeal swab and feces. Virus isolation was positive. Neoplasia of the liver and cervix were identified along with pyometra and hemometra as significant comorbidities. Repeated fecal PCR testing for SARS-CoV-2 was performed on the male tiger and pooled fecal samples from the lion group. All tested positive on the first test, but they were negative when retested twice approximately 1 mon after clinical signs were first noted. A keeper tested positive for COVID-19 infection before the felids becoming ill; however, the keeper likely transmitted the virus during an asymptomatic or presymptomatic period. Two additional staff members became ill soon after clinical signs were seen in the felids. The same strain of virus was confirmed in the female tiger and three staff members. Zoo policy prevented staff members with clinical signs of COVID-19 from working. The zoo was closed to the public when the felids developed clinical signs. Before confirming SARS-CoV-2 in the felids, safety precautions and PPE consisted of good hand hygiene with disinfection and common (although not consistent) use of disposable gloves. After infection was confirmed, PPE requirements included consistent use of gloves, three-layer medically approved reusable facemasks, a potassium peroxymonosulfate–based footbath, and coveralls. A reduced number of staff prepared the felids' food and cleaned the enclosures. Efforts were made to maintain a 6-ft distance from the felids, and training sessions were suspended. Enrichment items were no longer shared between felids. Upon receiving two negative fecal PCR tests, the use of footbaths and coveralls was discontinued.

Incident 8

At the Fort Wayne Children's Zoo in Indiana, USA, in February 2021, two cohoused Sumatran tigers (Panthera tigris sumatrae) developed mild clinical signs including coughing, wheezing, deceased appetite, and occasional sneezing. One tiger had a chronic intermittent unilateral nasal discharge that worsened as the other clinical signs ensued. Clinical signs lasted 5.5 d. Infection with SARS-CoV-2 was confirmed by fecal PCR testing. Daily fecal samples were collected from both tigers until both were confirmed negative for seven consecutive tests. The tigers remained positive for 22 and 26 d, respectively, after the onset of clinical signs. One tiger shed intermittently, sometimes going 5 d without shedding viral RNA. The other tiger shed consistently every day until shedding abruptly stopped. Repeated voluntary blood collection was performed for serology with a mix-and-read (fluorescence-based) assay. Seroconversion was confirmed in both tigers. The source of the infection was never confirmed; all tiger keepers were tested soon after the tigers developed signs and were confirmed to be negative. No keepers reported any symptoms. One keeper that worked in the same building as the tigers tested positive for SARS-CoV-2, but did not have close contact with the tigers or the tiger keepers. Zoo policy prevented staff members with clinical signs of COVID-19 from working. Daily health questionnaires had to be completed before arriving at work. The zoo was closed to the public during the time when the felids developed clinical signs. Before confirming SARS-CoV-2 infection in the felids, PPE consisted of cloth facemasks when around the felids, although compliance was inconsistent. Disposable gloves were used during food preparation. Efforts were made to maintain a 6-ft distance from the felids, but due to the dimensions of the indoor holding space, this was difficult to maintain. Training sessions were limited to 10 min in duration. After infection was suspected PPE included N95 masks, face shield or goggles, scrubs or coveralls over clothing, dedicated footwear or boot covers, two pairs of disposable gloves, quaternary ammonium–based footbaths, and double bagging of fecal waste for disposal. Stalls were generally dry cleaned or, when hosing was needed, the stalls were disinfected with a quaternary ammonium product 10 min before gentle hosing. The outer layer of gloves and mask were disposed after hosing. Voluntary training sessions were minimized, except for blood collection to measure antibody response. Sessions were limited to just one keeper and veterinarian.

Incident 9

At the Prague Zoo in the Czech Republic in February–March 2021, two Asiatic lions (Panthera leo leo), two Malayan tigers, one Sumatran tiger, and one Amur leopard cat (Prionailurus bengalensis euptilurus) housed in the same building developed clinical signs of illness.¹⁰ Of the six felids, all had nasal discharge, four had coughing, four had lethargy, three had sneezing, and one had a decreased appetite. Duration of clinical signs lasted 2–11 d. Infection with SARS-CoV-2 was confirmed by fecal PCR. Testing was repeated 14 d later, at which time all felids were negative. One to 2 d before the onset of clinical signs in the felids, two keepers were diagnosed with COVID-19. The felids and one keeper were confirmed to have infection with the same variant (Alpha). The zoo was closed to the public at the time of infection. Before confirming SARS-CoV-2 in the felids, PPE consisted of KN95, FFP2/FFP3 masks, gloves, hand disinfectants, and disinfection mats. Despite this precaution, it is suspected that the infection transmitted from the staff to the felids.

Viral sequencing

Viral sequencing was attempted, but not successful, for all cases. Generally, the sequence was consistent with the strain that was circulating in the human population at the time (Fig. 1). Phylogenetic tree analysis of the SARS-CoV-2 spike gene showed that isolates from Incidents 1 and 3–9 were closely related to contemporary human isolates and belonged to seven different lineages: B1, B.1, B.1.177, B.1.564, B.1.1.7, B.1.177.21, and B.1.2 (Fig. 1B). These large cat strains shared from 99.8 to 100% identities with corresponding human SARS-CoV-2 strains in the spike gene. All these outbreaks were caused by a single introduction except Incident 1 at the Bronx Zoo, where two different lineages were involved with human-to-felid transmissions. The SARS-CoV-2 zoo animal infections that occurred in the early period of the pandemic including Incidents 1–8 were nonvariant strains, whereas Incident 9 was caused by the Alpha variant (Fig. 1B). None of the felids were positive for the Delta variant.