key: cord-0796524-84pvyhzg
authors: Goryoka, Grace W.; Cossaboom, Caitlin M.; Gharpure, Radhika; Dawson, Patrick; Tansey, Cassandra; Rossow, John; Mrotz, Victoria; Rooney, Jane; Torchetti, Mia; Loiacono, Christina M.; Killian, Mary L.; Jenkins-Moore, Melinda; Lim, Ailam; Poulsen, Keith; Christensen, Dan; Sweet, Emma; Peterson, Dallin; Sangster, Anna L.; Young, Erin L.; Oakeson, Kelly F.; Taylor, Dean; Price, Amanda; Kiphibane, Tair; Klos, Rachel; Konkle, Darlene; Bhattacharyya, Sanjib; Dasu, Trivikram; Chu, Victoria T.; Lewis, Nathaniel M.; Queen, Krista; Zhang, Jing; Uehara, Anna; Dietrich, Elizabeth A.; Tong, Suxiang; Kirking, Hannah L.; Doty, Jeffrey R.; Murrell, Laura S.; Spengler, Jessica R.; Straily, Anne; Wallace, Ryan; Behravesh, Casey Barton
title: One Health Investigation of SARS-CoV-2 Infection and Seropositivity among Pets in Households with Confirmed Human COVID-19 Cases — Utah and Wisconsin, 2020
date: 2021-04-13
journal: bioRxiv
DOI: 10.1101/2021.04.11.439379
sha: 160a459df9c3efe9454b697b5d1c7cf90b4a0018
doc_id: 796524
cord_uid: 84pvyhzg

Background Approximately 67% of U.S. households have pets. Limited data are available on SARS-CoV-2 in pets. We assessed SARS-CoV-2 infection in pet cohabitants as a sub-study of an ongoing COVID-19 household transmission investigation. Methods Mammalian pets from households with ≥1 person with laboratory-confirmed COVID-19 were eligible for inclusion from April–May 2020. Demographic/exposure information, oropharyngeal, nasal, rectal, and fur swabs, feces, and blood were collected from enrolled pets and tested by rRT-PCR and virus neutralization assays. Findings We enrolled 37 dogs and 19 cats from 34 of 41 eligible households. All oropharyngeal, nasal, and rectal swabs tested negative by rRT-PCR; one dog’s fur swabs (2%) tested positive by rRT-PCR at the first animal sampling. Among 47 pets with serological results from 30 households, eight (17%) pets (4 dogs, 4 cats) from 6 (20%) households had detectable SARS-CoV-2 neutralizing antibodies. In households with a seropositive pet, the proportion of people with laboratory-confirmed COVID-19 was greater (median 79%; range: 40–100%) compared to households with no seropositive pet (median 37%; range: 13–100%) (p=0.01). Thirty-three pets with serologic results had frequent daily contact (≥1 hour) with the human index patient before the person’s COVID-19 diagnosis. Of these 33 pets, 14 (42%) had decreased contact with the human index patient after diagnosis and none (0%) were seropositive; of the 19 (58%) pets with continued contact, 4 (21%) were seropositive. Interpretations Seropositive pets likely acquired infection from humans, which may occur more frequently than previously recognized. People with COVID-19 should restrict contact with animals. Funding Centers for Disease Control and Prevention, U.S. Department of Agriculture

Background 43 Approximately 67% of U.S. households have pets. Limited data are available on SARS-CoV-2 in pets. 44 We assessed SARS-CoV-2 infection in pet cohabitants as a sub-study of an ongoing COVID-19 45 household transmission investigation. 46 

Mammalian pets from households with ≥1 person with laboratory-confirmed COVID-19 were eligible 48 for inclusion from April-May 2020. Demographic/exposure information, oropharyngeal, nasal, rectal, 49 and fur swabs, feces, and blood were collected from enrolled pets and tested by rRT-PCR and virus 50 neutralization assays.

Findings 52 We enrolled 37 dogs and 19 cats from 34 of 41 eligible households. All oropharyngeal, nasal, and rectal 53 swabs tested negative by rRT-PCR; one dog's fur swabs (2%) tested positive by rRT-PCR at the first 54 animal sampling. Among 47 pets with serological results from 30 households, eight (17%) pets (4 dogs, 55 4 cats) from 6 (20%) households had detectable SARS-CoV-2 neutralizing antibodies. In households 56 with a seropositive pet, the proportion of people with laboratory-confirmed COVID-19 was greater 57 (median 79%; range: 40-100%) compared to households with no seropositive pet (median 37%; range: 58 13-100%) (p=0.01). Thirty-three pets with serologic results had frequent daily contact (≥1 hour) with 59 the human index patient before the person's COVID-19 diagnosis. Of these 33 pets, 14 (42%) had 60 decreased contact with the human index patient after diagnosis and none (0%) were seropositive; of the 61 19 (58%) pets with continued contact, 4 (21%) were seropositive. bats. 1 Threats from pathogens shared by humans and animals highlight the need for a One Health 70 approach for detection, prevention, and control. 2 One Health is a collaborative, multisectoral, and 71 transdisciplinary approach with the goal of achieving optimal health outcomes recognizing the 72 interconnection between people, animals, plants, and their shared environment. 73 In the United States (U.S.), approximately 85 million households (67%) own ≥1 pet, with dogs (63 74 million households) and cats (43 million households) being most popular. 3 Human-animal interactions 75 are associated with improved mental, social, and physiologic health 4 and are critical for people with 76 service and working animals 5 . 77 Some animals, including pets, have been naturally infected with SARS-CoV-2, almost exclusively after 78 exposure to an infected person. 6-8 Dogs, cats, ferrets, hamsters, and rabbits are pet species with 79 demonstrated susceptibility to SARS-CoV-2 infection under experimental conditions. Cats, ferrets, and 80 hamsters can transmit the virus to naïve cohabitants of the same species. 9-14 Additionally, no virus, 81 including SARS-CoV-2, has ever been reported as a contaminant on pet fur. However, animal health and 82 welfare concerns have been reported, 15,16 including reports of misuse of cleaning products on pets to the 83 Pet Poison Hotline (R. Schmid, personal communication). 84 We conducted a One Health household transmission investigation to better characterize SARS-CoV-2 85 infection in mammalian pets living in households with people with COVID-19 to inform guidance and 86 decision-making during this pandemic and for future preparedness efforts.

Evidence before this study 89 Both natural and experimental infections with SARS-CoV-2 have been reported in multiple species of 90 companion animals, including dogs, cats, ferrets, hamsters, and rabbits. Cats, ferrets, and hamsters can 91 transmit SARS-CoV-2 to naïve members of the same species. Natural infections of companion animals 92 have occurred almost exclusively after contact with a person with COVID-19. 94 This is one of the earliest studies to assess risk and behavioral factors related to SARS-CoV-2 95 transmission between people and pets. In households with humans with laboratory-confirmed COVID-96 19 and pets, 20% had pets with serological evidence of past SARS-CoV-2 infection. SARS-CoV-2 97 seropositivity in pets was more prevalent among households with higher rate of human COVID-19 98 infections, and less prevalent among households where owners limited interactions with pets when the 99 owner's COVID-19 symptoms began. To our knowledge, this is the first study to detect RNA of SARS-100 CoV-2, or any virus, on animal fur. 102 Understanding the epidemiologic role that animals may play in the COVID-19 pandemic is crucial to 103 inform guidance and decision making for public health and animal health officials. Our findings add to 104 the growing body of evidence demonstrating SARS-CoV-2 transmission can occur between people and 105 pets-most often from people to pets-and suggest this transmission may occur more frequently than 106 previously recognized. These data highlight the importance of further research, including identification 107 of specific risk factors for human-to-pet transmission, addressing pets in public health guidance during 108 pandemics, and including pets in future pandemic preparedness planning. for all human household members; most human household members had respiratory specimens collected 124 for SARS-CoV-2 viral testing and blood for serology testing at ≥2 time points. Physical characteristics 125 of each residence, including size, were also described. 17

Of 62 enrolled households, 41 households with ≥1 mammalian pet living in the household were eligible 127 for inclusion in this One Health investigation ( Figure S1 ). Eligible households were contacted by phone 128 during March-April 2020. Pets were enrolled if owners consented, a questionnaire was completed, and 129 ≥1 sample was collected from each pet. Phone interviews were conducted prior to initial home visits to 

During household visits, veterinarians attempted to collect oropharyngeal, nasal, rectal, and fur swabs, 142 feces, and blood from pets. Bilateral deep nasal, oropharyngeal, and rectal swabs were collected, when 143 possible, using sterile polyester tipped swabs (tip diameter, 1.981 mm for nasal, 5.2 mm for oral and 144 rectal). Swabs were placed into 3mL of brain heart infusion broth. Fur swabs were collected in duplicate 145 using 2x2-inch sterile gauze pads rubbed across the back and the abdomen, as well as the dorsal and 146 ventral paws and between the metacarpal and digital pads of each pet. One sample was stored dry and 147 one was stored in RNAlater (Thermo Fisher Scientific, Waltham, Massachusetts). All samples, except 148 for dry fur swabs and fecal samples, were stored on ice packs for immediate shipping and were 149 processed for testing upon arrival at WVDL (Madison, Wisconsin). Dry fur swabs and fecal samples 150 were placed in containers without media and were frozen immediately at -80°C until testing. Serum 151 samples were obtained from venous blood (1-3mL) collected and processed in serum separator tubes; 152 sera were decanted and stored at -80°C until testing. Table 2 ). The median proportion of human household members with laboratory-183 confirmed COVID-19 was 45% (range: 13%-100%); of 72 total people with confirmed infection, 71 184 (99%) ever experienced symptoms. Additional household characteristics are described in Table 2. 185 Fifty-six pets (100%) had oral and fur swabs, 55 (98%) had nasal swabs, 54 (96%) had rectal swabs, 14 186 (25%) provided fecal samples, and 47 (84%) provided blood samples. Fourteen pets had repeat oral, 187 nasal, rectal, and fur swabs, 6 had repeat fecal samples, and 11 had repeat blood samples.

The median time from symptom onset of the index patient to first date of pet sampling was 27 days 189 (range: 3-46 days; Table 2 ). The median time from first positive diagnostic result of the index patient to 190 first date of pet sampling was 20·5 days (range: 3-42 days) and was similar between households with 191 and without seropositive pets (21·5 vs. 20 days).

All oropharyngeal, nasal, and rectal swabs and fecal specimens tested negative by rRT-PCR, except one 193 rectal swab sample from a cat was presumptive positive that was not confirmed (Supplementary 194 Materials; Table S1 ). Among 47 pets with serological results from 30 households, eight pets (17%; 4 195 dogs, 4 cats) from 6 (20%) households, had detectable SARS-CoV-2 neutralizing antibodies. Three pets 196 from these 6 households had seronegative results. The neutralizing titers for all seropositive dog samples 197 were 32 while cat titers ranged from 32 to 128 (Table S1 ). Demographic pet data by serology result are 198 presented in Table 1 . Timelines for human and animal sample collection among households with 199 seropositive pets, as well as symptom onset and duration in people in those households, are depicted in person who was initially not tested and the one who was initially negative were tested two days later, 206 both were positive. (Figure 2 ). Seven near-complete or complete-genomes were generated from this 207 household; one each from humans 1-3, three from human 4 collected at three time-points, and one 208 consensus sequence from the dog fur swabs. High sequence similarity suggests one introduction from 209 the community and subsequent internal household transmission (Figure 2; Figure S2) . Notably, the dog 210 had no evidence of infection; all samples were negative by rRT-PCR and the dog was also seronegative 211 ( Figure 2 ). Viral culture was attempted on the rRT-PCR positive fur swab, but was negative.

Owners reported clinical signs consistent with SARS-CoV-2 infection among 14 (25%) pets during the 213 time from symptom onset of the index patient until time of sampling (Table S2 ). The most reported 214 clinical signs were respiratory (16%), including sneezing (7%), coughing (7%) and nasal discharge 215 (5%). Among the 8 seropositive pets, clinical signs were reported in only 2 (25%); one dog had nasal 216 discharge and one dog had decreased appetite. Among 39 seronegative pets, clinical signs were reported 217 in 8 (21%) ( Table S2) .

Forty-six (98%) of 47 pets with serological results were primarily indoor pets; one pet, an 8-year-old 219 seropositive cat, spent ≥50% time outdoors (Table 1) . Seropositivity among pets occurred more 220 commonly among households with higher rates of secondary transmission among people; the median 221 proportion of people with laboratory-confirmed COVID-19 in households with a seropositive pet was 222 79% (range: 40-100%) compared to 37% in households with no seropositive pet (range: 13-100%) 223 (p=0.01) ( Table 2) . Overall, owners reported pets had fewer daily interactions lasting ≥1 hour and fewer 224 types of interaction with the index patient after their COVID-19 diagnosis; interactions included petting, 225 cuddling, feeding, sleeping in the same location, pets licking the index patient's face or hands, taking for 226 walks, sharing food, and grooming ( Figure 3 ). Among the 47 pets with serologic results, 33 (70%) pets 227 were reported to have frequent daily contact (≥1 hour) with the index patient before the person's 228 diagnosis. Of 14 pets with decreased interactions, none (0%) were seropositive. Nineteen pets continued 229 to have frequent contact with the index patient after their diagnosis; of these, 4 (21%) were seropositive.

Five (15%) of 34 households, comprising 12 (21%) pets, reported that, after their COVID-19 diagnosis, 231 the index patient began wearing face masks and 2 (6%) also reported glove use around pets. In 232 households using face masks, among pets with serological results, one of eight (13%) pets was 233 seropositive, while in households not using face masks, seven of 39 (18%) pets were seropositive.

Of 34 households, 10 (29%) identified a household member familiar with CDC recommendations for 235 people with suspected or confirmed COVID-19 restricting contact with pets 18 ; three (30%) of the 10 236 households had a seropositive pet. Of the 10 households familiar with CDC recommendations, 237 implementation of precautions was low; the index patient in one (10%) household reduced interactions 238 with pets after the person's diagnosis, one (10%) household used masks and gloves while interacting 239 with pets, and one (10%) household reported both reduced interaction and mask and glove use. CoV-2 transmission to pets.

Several seropositive animals identified roamed freely in the yard or neighborhood during their likely 266 infectious window, which raises concern for potential transmission of virus from infected pets to people 267 and susceptible animals, which is biologically plausible, but has not yet been documented. One 268 seropositive cat spent ≥50% of its time outdoors. Experimental studies have documented that cats with 269 SARS-CoV-2 can transmit SARS-CoV-2 to other cats, 13,24 leading to concerns of transmission between 270 cats that roam outdoors; however, this was not assessed in this investigation. 271 We detected SARS-CoV-2 RNA in fur swabs collected from only one dog but were not able to culture 272 the virus from these samples. Thirty (54%) pets were sampled at a time when at least one household 273 member was symptomatic and 14 (25%) pets at a time when at least one household member tested 274 positive; therefore, some environmental contamination from human viral shedding may have been 275 missed. Our findings suggest that viral RNA on the fur was due to environmental contamination from 276 human household members. Fomite transmission from pet fur seems unlikely although more studies are 277 needed to determine the potential of pet fur to serve as a fomite for SARS-CoV-2 transmission.

In households where the index patient decreased duration of interaction with pets after the person's 279 diagnosis, no pets in this study were seropositive. In two households with seropositive pets, the index 280 patient increased their duration of interaction with pets after their diagnosis (Figure 3 ). This finding 281 highlights the importance of people with suspected or confirmed COVID-19 restricting contact with pets 282 and other animals to prevent person-to-animal transmission, in accordance with CDC 283 recommendations 25 . 284 We identified 10 households with awareness of CDC's recommendations of restricting interactions with 285 pets for people with COVID-19 25 before enrollment. While this metric was captured only at a single 286 time point, it emphasizes the importance of providing accurate and timely health protection messaging 287 for pets during a pandemic caused by an emerging zoonotic disease.

Our findings provide additional characterization of potential SARS-CoV-2 transmission from people 289 with laboratory-confirmed COVID-19 to pets in households; however, several limitations are noted.

While directionality cannot be proven based on these results, the epidemiological information gathered, Hall, Scott Nabity, Jacqueline Tate, and others on the CDC COVID-19 Epidemiology Task Force.

Additionally, we would like to thank all our local, state, and federal human and animal health/laboratory 337 partners. We also thank Hannah Rettler and colleagues from the Utah Department of Health and Dr.

Stephen Welch for graphic design assistance with the figures. Lastly, a sincere thank you to the 339 participating households in the Salt Lake City and Milwaukee metropolitan areas for their interest in this 340 investigation and willingness to involve their pets, which made this One Health investigation possible. which all tested negative by rRT-PCR, and a blood sample which was negative by virus neutralization.

Panel B. Enhanced view of branch-tip from comprehensive phylogram (see Figure S2 ), depicting here 440 the seven study sequences (red) alongside selected Utah complete genome sequences available 441 from Global Initiative on Sharing All Influenza Data. Branch length is by divergence. See Figure S2 for 442 zoomed-out dendrogram depicting additional available sequences from Utah. 

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