key: cord-0824438-ceskhcb3
authors: Stranieri, Angelica; Lauzi, Stefania; Giordano, Alessia; Galimberti, Luigi; Ratti, Gabriele; Decaro, Nicola; Brioschi, Federica; Lelli, Davide; Gabba, Silvia; Amarachi, Ndiana Linda; Lorusso, Eleonora; Moreno, Ana; Trogu, Tiziana; Paltrinieri, Saverio
title: Absence of SARS‐CoV‐2 RNA and anti‐SARS‐CoV‐2 antibodies in stray cats
date: 2021-06-30
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.14200
sha: 6264e10660673371614ac44d1ba4987ce0a4fbd8
doc_id: 824438
cord_uid: ceskhcb3

SARS‐CoV‐2 positive or seropositive owned cats have been reported worldwide. The detection of seropositive stray cats in the proximity of farms of infected minks, coupled with the demonstration of cat‐to‐cat transmission in experimental settings, raise the question whether stray cats may have an epidemiological role in the COVID‐19 pandemic and may act as sentinel for the circulation of SARS‐CoV‐2. The aim of this study was to evaluate the presence of SARS‐CoV‐2 RNA and anti‐SARS‐CoV‐2 antibodies in free roaming cats belonging to colonies located in an area highly affected by the COVID‐19 pandemic and to correlate the results with the positivity rate in people sharing the same area. Interdigital, cutaneous, oropharyngeal, nasal and rectal swabs, as well as blood samples, were collected from 99 cats living in colonies and admitted to our hospital for neutering. This caseload corresponds to the 24.2% of the feline population living in the 25 sampled colonies and to the 5.6% of all the free‐roaming registered cats. The presence of SARS‐CoV‐2 RNA in swabs was assessed using real time RT‐PCR. Anti‐SARS‐CoV‐2 serum antibodies were assessed using commercially available ELISA kits and confirmed by serum virus neutralization. In people, the SARS‐CoV‐2 positivity rate ranged from 3.0% to 5.1% (mean rate: 4.1%) and the seropositive rate from 12.1% to 16.3% (mean rate: 14.2%). Most of the colonies were in urban areas and resident cats had frequent contacts with external cats or people. A COVID‐19 positive caretaker was found, whereas all the cats were negative for SARS‐CoV‐2 RNA and seronegative. Although the negative results cannot exclude previous infections followed by decrease of antibodies, this study suggests that colony cats do not have an important epidemiological role in SARS‐CoV‐2 transmission dynamics. Further studies on larger caseloads are warranted, also in the light of the emerging new viral variants, on a One Health perspective.

are warranted, also in the light of the emerging new viral variants, on a One Health perspective.

The coronavirus disease 2019 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led, at the time of writing, to more than two million and a half of deaths worldwide (Du Toit, 2020; https://covid19.who.int). The potential role of domestic and non-domestic animals in the pandemic has been gaining increasing interest, since many positive results at both molecular and serological tests have been recorded in different animal species (Tazerji et al., 2020) . Dogs and cats have been intensively investigated because of their close contact with humans. In experimental settings, dogs showed low susceptibility to SARS-CoV-2 infection, whereas in cats the virus replicated more efficiently and was transmitted via airborne route between cats (Halfmann et al., 2020; Shi et al., 2020) .

Despite sporadic cases of SARS-CoV-2 positive and seropositive cats, presumably infected by their owners, have been reported worldwide (Hosie et al., 2021; Pagani et al., 2021; Patterson et al., 2020) , the role of domestic cats as source of infection for humans seems unlikely also because cats can clear the infection in a short time (Neira et al., 2021; Patterson et al., 2020; Temmam et al., 2020) .

Nevertheless, the presence of seropositive stray cats and one cat harboring viral RNA in the proximity of SARS-CoV-2 infected mink farms (Oreshkova et al., 2020) has raised the question whether stray cats may have an epidemiological role in the transmission of the virus and may act as sentinel animals for the circulation of SARS-CoV-2 in specific areas. A recent report on stray cats in Spain evidenced a low seropositivity rate (3.5%), mostly in cats with co-infections (Villanueva-Saz et al., 2021) .

Thus, the aim of this study was to evaluate the presence of SARS-CoV-2 RNA and anti-SARS-CoV-2 antibodies in free roaming cats belonging to several feline colonies located in one Veterinary District of the Regional Health Protection Agency (Agenzia di Tutela della Salute, ATS Città metropolitana di Milano-Distretto Alto Lodigiano) of Lodi province (ATS-AL), which was firstly and highly affected at the beginning of the COVID-19 pandemic and is still recording a high number of human cases (Dashboard COVID-19), and to correlate the possible presence of SARS-CoV-2-positive or of seropositive cats with the rate of infection in people sharing the same area.

Feline colonies were included in this study if the following informa- Based on these data, the possible correlation between the number of positive people and numbers of inhabitants or population density was also calculated using a Spearman's correlation test run on the Analyseit statistical software (Analyse-it Ltd, Leeds, UK).

Cats were admitted to the Veterinary Teaching Hospital of the University of Milan to undergo orchiectomy or ovariectomy, as part of an agreement between the University and the ATS-AL regarding the demographic control of stray animals. During admission, the information about the colony as well as signalment (sex, breed and presumptive age) and any information regarding the clinical status of the cats was recorded. The cats were administered intramuscular dexmedetomidine (10 µg kg −1 ; Dexdomitor, Vetoquinol, Italy), ketamine (2 mg kg −1 ; Lobotor, Acme srl, Italy) and methadone (0.2 mg kg −1 ; Semfortan, Eurovet Animal Health B.V., Italy). After 15 min, a blood sample was taken from the jugular vein and placed in a plain tube, and the following swabs were taken: interdigital swabs, collected by rotating the swabs between the digits of one front and one hind paws; cutaneous swabs, collected by rubbing the swabs on the skin and hair of the back and abdomen; oropharyngeal swabs, collected by inserting and gently rotating the swab into the posterior pharynx and tonsillar areas; nasal swabs, inserted into each nostril and gently rotated in order to harvest the nasal fluid; rectal swabs, collected by inserting the swab through the anal sphincter and gently rotating it.

Shortly after the collection, blood samples were centrifuged, serum was separated and divided in two aliquots placed in Eppendorf tubes.

The aliquots were frozen at −20 • C until shipping, in cold chain, to the Virology Department of the University of Bari (UniBA, Bari, Italy) and to the Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (IZSLER, Brescia, Italy) for further analyses, as below described. Each swab was dipped in a 1 ml solution based on phosphate buffered saline (PBS pH 7.2) supplemented with 10% glycerol and antibiotics (1%) and frozen at −80 • C until shipping in cold chain to the IZSLER upon further analyses, as described below.

Sample preparation, RNA extraction and the SARS-CoV-2 real time RT- Positive and negative SARS-CoV-2 respiratory specimens from human patients sampled for COVID-19 diagnostic purposes and routinely processed at the IZSLER were used as positive and negative controls, respectively.

All the 99 serum samples were screened with the multispecies enzyme- (SVN) assay, performed by IZSLER as previously described (Meyer et al., 2020) . Positive controls for all the serological tests included canine and feline sera that had tested positive by plaque reduction neutralization assay (Decaro et al., 2021a; Patterson et al., 2020) . Briefly, sera were tested in two-fold serial dilutions (starting from 1:10) of heat-inactivated sera (30 min, 56 • C) and were then incubated with 100

TCID50 of the SARS-CoV-2 strain HCoV-19/Italy/310904/46/2020 at 37 • C and 5% CO 2 , for 1 h in 96-wells plates. Vero-E6 cells were added in a concentration of 2 × 10 4 cells per well and incubated for 72 h at 37 • C with 5% CO 2 . The serum virus neutralization titer (VNT50) was defined as the reciprocal value of the sample dilution that showed a 50% protection of virus growth. Sera with titres ≥ 10 were considered as positive for SARS-CoV-2 antibodies. SVN was not performed on 9 samples due to low volume or sera, hemolysis or cytotoxicity.

In the presence of negative results, the maximum possible prevalence in the total cat population was calculated using WinEpi (http://www.

winepi.net).

The ATS-AL includes 35 out of the 60 (58.3%) municipalities comprised by the province of Lodi.

Feline colonies are present in 34 out of the 35 municipalities of the Veterinary District, for a total of 221 colonies and a total of approximately 1768 registered cats.

This study involved 25/221 (11.3%) of the colonies belonging to the ATS-AL, which are distributed in 12 municipalities ( Figure 1, Table 1 ).

These colonies include 2-35 animals (mean and median: 16.4 and 15 cats per colony, respectively), for a total of 409 cats (23.1% of the colony cats registered at the ATS-AL).

From each colony, one to 19 cats were enrolled after being admitted for neutering (mean and median: four and two cats/colony, respectively) for a total of 99 cats, corresponding to the 24.2% of the 409 cats living in the 25 sampled colonies and the 5.6% of all the freeroaming registered cats of the Lodi province. Cats per colony ranged between two and 35 animals and the percentage of cats enrolled from each colony ranged from 3.3% to 76.0% of the total number of cats living in each colony. Of the 99 cats enrolled, 53 were female and 46 were male (53.5 and 46.4%, respectively). The presumptive age could be determined in 88/99 cats, and it ranged from 6 to 60 months (mean:

18.8 months, median: 12 months).

The information on the site of the colonies and on the interactions with cats and people other than those of the colony are summarized in Table 2 . As seen in the table, most of the colonies were in urban areas and resident cats had frequent contacts with external cats (i.e., cats living in proximity of the colonies but not registered by the public Authorities, which may occasionally come into contact with colony cats) or people. The number of caretakers varied from 1-4 per colony 

From the 99 examined cats, 98 nasal and 99 each of the other swabs were collected, for a total of 494 swabs. Serum was available for all the 99 cats.

Despite an in-depth clinical examination was not performed, all nondomesticated animals during the pre-surgical physical examination did not show abnormal clinical findings.

All the swabs tested negative for SARS-CoV-2 RNA.

All sera tested negative using the ELISA tests and negative results were confirmed by SVN, except for one sample that tested positive using the indirect ELISA but resulted negative using the double antigen ELISA tests, the Surrogate VNT ELISA test and SVN and was then categorized as a false positive result.

A 2.7% and a 2.9% maximum possible prevalence in the total cat population living in the 25 sampled colonies from the 12 municipalities or in the whole Lodi province was calculated for SARS-CoV-2 with negative RT-qPCR and serological results in all the samples tested, respectively.

in the context of the current pandemic and the elucidation of the role of such animals, especially cats, in the epidemiology of the infection is needed (Hosie et al., 2021; Pagani et al., 2021; Patterson et al., 2020) .

Stray cats consist of a peculiar population of cats that can serve as a good indicator of pathogen propagation between cats and outside the domestic environment (Halfmann et al., 2020; Oreshkova et al., 2020; Shi et al., 2020) . (Bosco-Lauth et al., 2020; Gaudreault et al., 2020; Shi et al., 2020) , and may be found positive up to 54 days after owner diagnosis (Hamer et al., 2020) . The few data on duration of seroconversion in natural settings suggest that antibodies in cats decrease below detection limits in 110 days (Pagani et al., 2021; Zhang et al., 2020) . It may be possible that infection occurred months before sampling, followed by complete serological negativization, indicating that this possible infection was not followed by persistent cat-to-cat transmission and formation of SARS-CoV-2 endemic feline populations. In any case, also this scenario would support the hypothesis that colony cats do not play an epidemiological role in the COVID-19 pandemic.

However, the fact that all our samples were negative for SARS-CoV-2 may be indicative, in the total cat population of Lodi province, of the absence of infection or of a low (2.9%) maximum possible prevalence. (Patterson et al., 2020) . However, in one study no association between age and seropositivity was found, but information on the mean age were not provided (Fritz et al., 2021 

This work was supported by a grant of Fondazione CARIPLO-Misura a sostegno dello sviluppo di collaborazioni per l'identificazione di terapie e sistemi di diagnostica, protezione e analisi per contrastare l'emergenza Coronavirus e altre emergenze virali del futuro, project "Genetic characterization of SARS-CoV2 and serological investigation in humans and pets to define cats and dogs' role in the COVID-19 pandemic (COVIDinPET).

The authors declare that there is no conflict of interest regarding the publication of this article.

The study was approved by the Institutional Animal Care and Use

Committee and by the Institutional Ethical Committee (approval num-bers_31/20 and 43/20, respectively). Ana Moreno https://orcid.org/0000-0002-8497-9708

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