key: cord-0833287-lxbqi5qu
authors: Villanueva‐Saz, Sergio; Giner, Jacobo; Tobajas, Ana Pilar; Pérez, María Dolores; González‐Ramírez, Andrés Manuel; Macías‐León, Javier; González, Ana; Verde, Maite; Yzuel, Andrés; Hurtado‐Guerrero, Ramón; Pardo, Julián; Santiago, Llipsy; Paño‐Pardo, José Ramón; Ruíz, Héctor; Lacasta, Delia María; Sánchez, Lourdes; Marteles, Diana; Gracia, Ana Pilar; Fernández, Antonio
title: Serological evidence of SARS‐CoV‐2 and co‐infections in stray cats in Spain
date: 2021-03-23
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.14062
sha: a29532dd374ee415d240628893d97c195dea55c9
doc_id: 833287
cord_uid: lxbqi5qu

A new coronavirus known as SARS‐CoV‐2 emerged in Wuhan in 2019 and spread rapidly to the rest of the world causing the pandemic disease named coronavirus disease of 2019 (COVID‐19). Little information is known about the impact this virus can cause upon domestic and stray animals. The potential impact of SARS‐CoV‐2 has become of great interest in cats due to transmission among domestic cats and the severe phenotypes described recently in a domestic cat. In this context, there is a public health warning that needs to be investigated in relation with the epidemiological role of this virus in stray cats. Consequently, in order to know the impact of the possible transmission chain, blood samples were obtained from 114 stray cats in the city of Zaragoza (Spain) and tested for SARS‐CoV‐2 and other selected pathogens susceptible to immunosuppression including Toxoplasma gondii, Leishmania infantum, feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) from January to October 2020. Four cats (3.51%), based on enzyme‐linked immunosorbent assay (ELISA) using the receptor binding domain (RBD) of Spike antigen, were seroreactive to SARS‐CoV‐2. T. gondii, L. infantum, FeLV and FIV seroprevalence was 12.28%, 16.67%, 4.39% and 19.30%, respectively. Among seropositive cats to SARS‐CoV‐2, three cats were also seropositive to other pathogens including antibodies detected against T. gondii and FIV (n = 1); T. gondii (n = 1); and FIV and L. infantum (n = 1). The subjects giving positive for SARS‐CoV‐2 were captured in urban areas of the city in different months: January 2020 (2/4), February 2020 (1/4) and July 2020 (1/4). This study revealed, for the first time, the exposure of stray cats to SARS‐CoV‐2 in Spain and the existence of concomitant infections with other pathogens including T. gondii, L. infantum and FIV, suggesting that immunosuppressed animals might be especially susceptible to SARS‐CoV‐2 infection.

Coronaviruses (Covs) are a group of zoonotic viruses classified into four different genera including alpha, beta, gamma and delta coronavirus (Tiwari et al., 2020) , which affect humans and animals equally.

In December 2019, a novel betacoronavirus pathogen called Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) was described in China as the causative agent of coronavirus disease 2019 . This new virus belongs to the same family and genus as SARS-CoV and MERS-CoV, sharing more genetic similarity with SARS-CoV, and it is considered a zoonotic agent mainly transmitted human to human (Petrosillo et al., 2020) .

In animals, these viruses cause different clinical patterns. Two different types of feline coronaviruses have been described including type I and type II Covs that belong to alphacoronavirus, affecting both domestic and wild felines (Stout et al., 2020) . The virus exists in a form that is responsible for the severe and frequently lethal disease named feline infectious peritonitis (FIP) (Paltrinieri et al., 2020) . In experimental conditions, SARS-CoV-2 infection of cats occurs by respiratory droplets resulting in mild respiratory signs , but in most of the cases, cats remain asymptomatic (Bosco-Lauth et al., 2020; Ruiz-Arrando et al., 2020) , capable of transmitting SARS-CoV-2 to other cats (Gaudreault et al., 2020) .

Nevertheless, SARS-CoV-2 and feline coronavirus are taxonomically distant viruses with different clinical and pathological features (Paltrinieri et al., 2020) .

Coronaviruses enter into host cells through a combination of interactions between the viral Spike protein with the mammalian heparan proteoglycans and the angiotensin-converting enzyme 2 protein (ACE2) (Clausen et al., 2020) . Among mammalian species, simian ACE2 is the closest homologue to human ACE2, followed by cat ACE2 with an 85.2% overall identity (amino acid) compared to human ACE2. Feline ACE2 differs only 4 out of a total of 20 contacting residues from human ACE2 (Stout et al., 2020) . This variation can affect the efficiency of RBD binding to ACE2 and is the major determinant of a species' susceptibility to SARS-CoV-2 (Gryseels et al., 2020) .

Stray cats in Europe and other world regions are a potential source of zoonotic diseases posing a risk for human health, they can be used as sentinels for the presence of infection in a given geographic area. The presence of SARS-CoV-2 in cats and other felids have been described in recent reports (Gryseels et al., 2020) .

Recently, in northern Italy, 3.9% of cats had measurable SARS-CoV-2 neutralizing antibody response that prevented re-infection to coronavirus (Patterson et al., 2020) . Transmission of SARS-CoV-2 from humans to domestic cats, tigers and lions has also been detected and cat-to-cat transmission has been demonstrated in experimental infections (Bosco-Lauth et al., 2020; Shi et al., 2020) . This situation should be taken into account as a possible warning towards the population of stray cats in urban areas. The role of domestic animals including pets and stray animals in the context of COVID-19 is not well determined and a human-to-animal zoonosis could be occurring (Hobbs & Reid, 2020; Leroy et al., 2020; Tiwari et al., 2020) . However, the level of natural infections within this scope is largely unknown owing to a marked knowledge gap which requires urgent attention. The aim of this study is to contribute to the knowledge of the role played by stray cats in the context of SARS-CoV-2 and analyse if the presence of concomitant infections with other pathogens, including Toxoplasma gondii, Leishmania infantum, feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV), may predispose the SARS-CoV-2 infection in this species.

The study was carried out in the city of Zaragoza (41° 38' 58.8948'' N and 0° 53' 15.7632'' W, Aragon region, Spain) from January to October 2020 (Table 1) . The study population comprised 114 stray cats captured in urban areas of Zaragoza within a trap, neuter and release sterilization program run locally to control stray feline colonies.

The procedure took place within 36 hr from the capture. 16.67%, 4.39% and 19.30%, respectively. Among seropositive cats to SARS-CoV-2, three cats were also seropositive to other pathogens including antibodies detected against T. gondii and FIV (n = 1); T. gondii (n = 1); and FIV and L. infantum (n = 1). The | 3

Cats were anaesthetized by subcutaneous injection with a combination of dexmedetomidine (Dexdomitor ® ) 15 µg/kg), ketamine (Anaestamine ® , 5 mg/kg) and methadone (Semfortan ® , 0.3 mg/kg).

Information about breed, age and gender was recorded and a complete physical examination was carried out before sampling.

Prior to collecting blood, the fur of the cats was trimmed around the jugular region. One ml of blood was collected aseptically by jugular venipuncture to obtain the serum. Blood and separated serum were stored at −20°C until processing. Routine laboratory tests such as a complete blood count and biochemical profile were not 

The DNA sequence encoding amino acid residues 319-541

PATVCGPKKSTNLVKNKCVNF) of the RBD was codon optimized and synthesized by Gen-Script for expression in HEK293 cells. The DNA, containing at the 5′-end a recognition sequence for KpnI, and at the 3′end a stop codon and a recognition sequence for XhoI, was cloned into a modified pHLSec containing after the secretion signal sequence a 12xHis tag, a superfolder GFP and a Tobacco Etch Virus (TEV) cleavage site, rendering the vector pHLSec-12His-GFP-TEV-SRBD. Both the synthesis of the RBD construct and the engineered pHLSec together with the cloning of RBD into pHLSec-12His-GFP-TEV were performed by GenScript. pHLSec-12His-GFP-TEV-RBD was transfected into HEK293F cell line (Thermo Fisher Scientific) as described below. Cells were grown in suspension in a humidified 37°C and 8% CO 2 incubator with rotation at 125 r.p.m.

Transfection was performed at a cell density of 2.5 × 106 cell/ml in fresh F17 serum-free media with 2% Glutamax and 0.1% P188.

For each 150 ml of culture, 450 μg of the plasmid (1 μg/μl) was diluted to 135 μl with sterilized 1.5 M NaCl. This mixture was added to each 150-ml cell culture flask and incubated for 5 min in the incubator. After that 1.35 mg of PEI-MAX (1 mg/ml) was mixed to 135 μl with sterilized 1.5 M NaCl and added to the cell culture flask. Cells were diluted 1:1 with pre-warmed media supplemented with valproic acid 24 hr post-transfection to a final concentration of 2.2 mM.

Cells were harvested 6-day post-transfection by spinning down at 300× g for 5 min, after which the supernatants were collected and centrifuged at 4,000×g for 15 min. Supernatant was dialyzed against buffer A (25 mM TRIS pH 7.5, 300 mM NaCl) and loaded into a His-Trap Column (GE Healthcare). Protein was eluted with an imidazol gradient in buffer A from 10 mM up to 500 mM. Buffer exchange to 25 mM TRIS pH 7.5, 150 mM NaCl (buffer B) was carried out using a HiPrep 26/10 Desalting Column (GE Healthcare).

TEV protease was then added in a ratio 1:50 (TEV:RBD) to the fusion construct in order to cleavage the His-GFP. After 20 hr of reaction at 18°C, the cleavage was satisfactorily verified through SDS-PAGE.

TEV protease and GFP were removed from the solution using a His-TrapColumn (GE Healthcare), and the SRBD was collected from the flow-through. Quantification of protein was carried out by absorbance at 280 nm using the theoretical extinction coefficient, ε280 nm

Antibodies to SARS-CoV-2 were determined by an indirect ELISA for the detection of IgG specific for RBD. Ninety-six-well plates were coated overnight, at 4°C with 50 µl/well of RBD protein at 1 µg/ml in phosphate-buffered saline (PBS). Subsequently, the coating solution was removed and the plate was washed three times with 200 μl per well of PBS containing 0.05% Tween 20 (PBST). Later, 300 μl of PBST and 3% dry skimmed milk was added to each well as blocking solution.

Plate was incubated with blocking solution for 1 hr at 37°C in a moist chamber. 100 µl of cat sera, diluted 1:100 in PBST and 1% dry skimmed milk (PBST-M), was added to each well. The plates were incubated for 1 hr at 37°C in a moist chamber. After washing the plates for 30 s six times with PBST followed by one wash with PBS for 1 min, 100 µl/ well of multi-species horseradish peroxidase conjugate (Thermo Fisher Scientific) was added per well. The plates were incubated for 1 hr at 37°C in the moist chamber and were washed again with PBST and PBS as described above. The substrate solution (ortho-phenylene-diamine) and stable peroxide substrate buffer (Thermo Fisher Scientific) were 

Female ND January 2020 30 6 13 11

February 2020 11 5 6

June 2020 21 11 10

July 2020 12 6 6

August 2020 10 3 5 2

September 2020 29 17 12

October 2020 1 1

Total of samples 114 49 52 13

Hipo MPP-96). As a positive control, each plate included serum from a human patient diagnosed with COVID, confirmed by a molecular test and a commercial quantitative ELISA, and serum from a healthy, non-infected cat obtained prior to pandemic COVID-19 situation as negative control. The same positive and negative sera were used for all assays and plates, with a constant inter-assay variation of <10%. Plates with an inter-assay variation of >10% were discarded. All samples were run in duplicate. The cut-off was set to 0.30 Optical Density units (OD units) (mean + 3 standard deviations of values from 92 cats obtained prior the COVID-19 situation in 2015), and the results above this value were considered positive.

For IFAT, the antigen was obtained as described previously (Goldman, 1957) . Briefly, purified tachyzoites were resuspended in 

The ELISA was performed on all sera as described previously 

The rapid test (Uranotest FeLV-FIV, URANOVET) was performed following the instructions of the manufacturer. All tests were stored at room temperature and were performed as described in the instructions supplied with the test kit.

FASTest ® FIP (MEGACOR Diagnostik) is a rapid immunochromatographic test for the qualitative detection of antibodies against the FCoV in whole blood, plasma, serum and effusion of the cat.

This rapid test was performed following the instructions of the manufacturer. All tests were stored at room temperature and were performed as described in the instructions supplied with the test kit.

Data collected for the entire population were analysed using descriptive statistics. Univariate analysis of categorical data was performed to determine possible associations between SARS-CoV-2 positivity and the following variables: sex and seropositivity for L. 

All the tested cats (52 females, 49 males and 13 non-determined)

were shorthaired type, adults (more than 1-year-old) and classified as apparently healthy, with no evident systemic signs found in the general physical examination.

No significant association (data not shown, p >.05) was detected between the positivity for anti-SARS-CoV-2 antibodies and gender as well as between the positivity for T. gondii, FeLV, FIV and L. infantum. A significant association was detected between gender and seropositivity for FIV (p =.0117), being higher percentages in males (75% higher than females, 25%).

The seroprevalence of SARS-CoV-2 infection was 3.51%. Among the 114 cats, four cats were seropositive by ELISA (Table 2) None of the seropositive cats to SARS-CoV-2 were also positive to FCoV (Table 2 ). In this sense, no serological cross-reactivity was detected between the SARS-CoV-2 and FCoV. 

+ + - - + 1 Male: 1 + - + - + 1 Male: 1 + - + - - 1 Female: 1 + - - - - 1 Non determined - + + - - 3 Male:3 - - + - + 3 Male:1 Female: 1 Non determined:1 - - + - - 6 Female 4 Male 2 - + - - - 8 Male 2 Female 6 - + - - + 5 Male 4 Female 1 - + - + - 1 Male - + - + + 1 male - - - + - 2 Female 2 - - - + + 1 Female 1 - - - - + 10

Co Table 2 .

In this study, 114 stray cats in the city of Zaragoza, in Aragon Region (Chia et al., 2020; Klumpp-Thomas et al., 2020) . Therefore, it was decided to use RBD as antigen in the in-house ELISA described in the previous section.

Furthermore, anti-RDB antibodies can be considered as neutralizing antibodies because in most cases they disrupt the RBD-ACE2

interaction (Barnes et al., 2020; Yuan, Liu, et al., 2020; . This has been supported by studies in animal models ing cat-to-cat transmission or mink-to-cat transmission (Oreshkova et al., 2020) , as well as animal (mink)-to-human transmission that has been also described (Oreshkova et al., 2020) . Since SARS-CoV-2 is able to infect a wide range of mammal species and it is not possible to determine individual susceptibility for all mammal species, people interacting with any wild mammal species should take sanitary precautions to prevent transmission to wildlife (Gryseels et al., 2020) .

Also, people who are suspected or confirmed to be infected with SARS-CoV-2 and to prevent anthropogenic transmission should limit contact with animals altogether (Hobbs & Reid, 2020) .

Among pets, cats are more likely than dogs to shed infectious virus, implying that they are more prone to transmitting SARS-CoV-2 cat-to-cat (Halfmann et al., 2020; Shi et al., 2020) . In Wuhan, a survey identified that the seroprevalence of SARS-CoV-2 in stray cats was 14.7% . On the other hand, our study reveals that 3.51% of the cats were seropositive for SARS-CoV-2, according to the in-house ELISA analysis. The differences in the results between these studies could be explained because,

in Wuhan, samples were collected from January to March 2020 when probably the circulation of the virus was superior than in Zaragoza. It is important to remark that samples from most of the seropositive cats detected in our study were collected in January and February 2020, when the rapid outbreak of the disease was not detected yet in Spain. Therefore, it could be considered that an important transmission could have happened in this period of time in Zaragoza. Recently, the presence of SARS-CoV-2 infection in Europe has been detected prior to pandemic time in Italy (La Rosa et al., 2020) and France (Deslandes et al., 2020) in December

September 2019 (Apolone et al., 2020) . In Spain, SARS-CoV-2 RNA was detected in metropolitan wastewater samples taken in late

February 2020 in the Region of Valencia and Murcia being the samples collected from 12 March to 14 April 2020 (Randazzo, Truchado, et al., 2020) . All these studies and our results could indicate that SARS-CoV-2 extended by European countries early 2020 and people could transmit infections to the cats in the moment of feeding or cuddling, kissing or being licked or sharing food with them.

We against the viral protein p24, whilst the identification of FeLV infection is focused on the detection of p27 antigen. The prevalence rate in our study for FeLV (4.39%) was slightly lower than that reported in previous studies in Catalonia (Spain) where prevalence of selected infectious disease agents in stray cats was 6% (Ravicini et al., 2016) and 8.5% in a previous study performed in Catalonia and Mallorca (Solano-Gallego et al., 2006) . By contrast, the prevalence of FIV in- -Garrido et al., 1972; Miro et al., 2004) . This type of serological analysis is important to be performed because the level of seroprevalence could be an alternative to measure the spread of T. gondii in the environment (Braga et al., 2012) , being considered stray cats as sentinels of the exposure to oocysts of T. gondii from the environment and through ingestion of tissue cysts. The presence of T. gondii infection is more likely to occur in cats with suppressed immune systems, including young kittens and cats affected with FeLV or FIV (Sousa et al., 2014) . In our study, co-infections were de- Further prevalence surveys in stray cats, also using other diagnostic methods, are warranted to clarify the role of these hosts in the epidemiology of SARS-CoV-2 transmission as well as the potential of immunocompromised stray cats as a potential reservoir for SARS-CoV-2.

RH and JP are supported by ARAID Foundation, Work in the RH laboratory is funded by MCNU (CTQ2013-44367-C2-2-P, BFU2016-75633-P and PID2019-105451GB-I00) and Gobierno de Aragón 

The authors declare that they have no competing interests.

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

Sergio Villanueva-Saz https://orcid.org/0000-0001-6209-4282

Delia María Lacasta https://orcid.org/0000-0002-7822-6646

Estudios sobre la epidemiologia de la toxoplasmosis. La infeccion del gato domestico en los alrededores de Madrid. Encuestas serologica y coproparasitologica [Study on the epidemiology of toxoplasmosis. Infection in the domestic cat in suburbs of Madrid

Unexpected detection of SARS-CoV-2 antibodies in the prepandemic period in Italy. Tumori, 300891620974755. Advance online publication

SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies

Evaluation of the EUROIMMUN Anti-SARS-CoV-2 ELISA Assay for detection of IgA and IgG antibodies

Experimental infection of domestic dogs and cats with SARS-CoV-2: Pathogenesis, transmission, and response to reexposure in cats

Occurrence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in cats with outdoor access in

Serological differentiation between COVID-19 and SARS infections. Emerging Microbes & Infections

SARS-CoV-2 infection depends on cellular heparan sulfate and ACE2

Serological survey of SARS-CoV-2 for experimental, domestic, companion and wild animals excludes intermediate hosts of 35 different species of animals

SARS-CoV-2 was already spreading in France in late

The role of passive immunization in the age of SARS-CoV-2: An update

SARS-CoV-2 infection, disease and transmission in domestic cats

Staining Toxoplasma gondii with fluorescin labelled antibody: A new serologic test for antibodies to Toxoplasma based upon inhibition specific staining

Risk of human-to-wildlife transmission of SARS-CoV-2. Preprints

Transmission of SARS-CoV-2 in domestic cats

Animals and SARS-CoV-2: Species susceptibility and viral transmission in experimental and natural conditions, and the potential implications for community transmission

Standardization of enzyme-linked immunosorbent assays for serosurveys of the SARS-CoV-2 pandemic using clinical and at-home blood sampling. medRxiv: the Preprint Server for Health Sciences

SARS-CoV-2 has been circulating in northern Italy since december 2019: Evidence from environmental monitoring. The Science of the Total Environment

The risk of SARS-CoV-2 transmission to pets and other wild and domestic animals strongly mandates a one-health strategy to control the COVID-19 pandemic. One Health

From people to Panthera: Natural SARS-CoV-2 infection in tigers and lions at the Bronx Zoo

Prevalence of antibodies to Toxoplasma gondii and intestinal parasites in stray, farm and household cats in Spain

Clinical and pathological findings in SARS-CoV-2 disease outbreaks in farmed mink (Neovison vison)

SARS-CoV-2 infection in farmed minks, the Netherlands

Feline infectious peritonitis (FIP) and coronavirus disease 19 (COVID-19): Are they similar

Evidence of exposure to SARS-CoV-2 in cats and dogs from households in Italy. bioRxiv: the Preprint Server for Biology

COVID-19, SARS and MERS: Are they closely related? Clinical Microbiology and Infection: The Official Publication of the European Society of Clinical Microbiology and Infectious Diseases

Metropolitan wastewater analysis for COVID-19 epidemiological surveillance

SARS-CoV-2 RNA in wastewater anticipated COVID-19 occurrence in a low prevalence area

Prevalence of selected infectious disease agents in stray cats in Catalonia

Detection of SARS-CoV-2 in pets living with COVID-19 owners diagnosed during the COVID-19 lockdown in Spain: A case of an asymptomatic cat with SARS-CoV-2 in Europe

Detection of SARS-CoV-2 in a cat owned by a COVID-19-affected patient in Spain

Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARScoronavirus 2

Serological and molecular evidence of exposure to arthropod-borne organisms in cats from northeastern Spain

Serological detection of Toxoplasma gondii, Leishmania infantum and Neospora caninum in cats from an area endemic for leishmaniasis in Brazil

Coronaviruses in cats and other companion animals: Where does SARS-CoV-2/COVID-19 fit? Veterinary Microbiology

COVID-19: Animals, veterinary and zoonotic links. The Veterinary Quarterly

Analytical and clinical validation of an ELISA for specific SARS-CoV-2 IgG, IgA, and IgM antibodies

Comparison of a qualitative immunochromatographic test with two quantitative serological assays for the detection of antibodies to Leishmania infantum in dogs

Structural basis of a shared antibody response to SARS-CoV-2

A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV

Serological evidence of leishmaniasis in cats in Aragon, Spain

A serological survey of SARS-CoV-2 in cat in Wuhan