Results and discussion
All dogs were found SARS-CoV-2 PCR negative. Out of the 22 cats tested, one was SARS-CoV-2 Rt-qPCR positive with a Ct of 29 from the rectal swab whereas nasopharyngeal swabs were negative. Another cat was found positive for the feline coronavirus (Ct 25.6).
The RNA was sent to Institut Pasteur for confirmation of SARS-CoV-2 and was confirmed PCR positive (CT 43, 30 and 32 with RdRp IP2, RdRp IP4 and E genes).
The rectal swab extract found positive by RT-qPCR was subjected to targeted iSeq100 system sequencing, and the run produced 3,858,853 raw reads with a Phred quality score of 28. By using BWA-MEM, the mapping against SARS-CoV-2 reference (NC_045512) allowed the retrieval of high quality viral genomic sequences, with a read depth of 30x, covering around 94% of the full length genome (sequence data has been deposited in GISAID with accession number EPI_ISL_437349).
Sequence analysis of the cat SARS-CoV-2 genome showed that it belongs to the Nextstrain (https://nextstrain.org/ ) proposed phylogenetic clade A2a, which is consistent with the phylogenetic analysis of the recent human French SARS-CoV-2. Compared to the Wuhan reference sequence, and on the basis of the 94% covered genome, the cat SARS-CoV-2 sequence displays eight nucleotide mutations, with two not yet described in ORF1ab (T10800C and A13394G). Among these mutations, five have led to amino-acids mutations, of which four in ORF1ab (T265I, I3512T, K4377E and P4715L). It should also be noted that the cat SARS-CoV-2 genome presents the amino-acid mutation D614G in the Spike glycoprotein, specific of the clade A2 and encompassing most of the French SARS-CoV-2 sequences (figure 1).
The confirmed positive cat was a 9-year-old female of European breed. It has been hospitalised on 13 April due to anorexia, vomiting and cough. These clinical signs occurred 17 days after her owner has been sick from COVID-19. The first samples (NP and R swabs, serum) were taken on 17 of April, eight days after the beginning of symptoms. A second set of samples was taken on cat that was healed (on 27 April) and the PCR results were negative for both swabs. These results suggest a rapid clearance of the virus from the intestinal tract, which is consistent with experimental infection of cats suggesting presence of infectious virus only until day 6 post-infection (Shi et al, 2020). Serological analysis by MIA showed a strong positive signal against SARS-CoV-2 for the two sera taken at 10 days apart (figure 2). The value of fluorescence emitted was 36 times higher than the average of the values emitted by control cats (sera sampled before the appearance of COVID-19). A cross-reactivity was observed with the N SARS-CoV antigen but given the high sequence identity between the two antigens, this result was expected. Both sera were also confirmed positive by ELISA. The mean value of the sixteen cats sampled before 2019 and the apparition of the SARS-CoV-2 virus gave a mean OD value of 0,074, with a standard deviation of 0,0116. Therefore, the cut-off was OD450nm : 0,109 (mean of negative values + 3x SD). The two sera taken at 10 days apart gave a mean value of 0.682 and 0.356 respectively. These OD values, clearly above the standard cut-off criteria (OD450 nm 0,109), reveal the presence of specific antibodies to the SARS-CoV-2. The serological results (MIA + ELISA) confirm that the cat was able to replicate SARS-CoV-2.
Cats had already been tested positive for the SARS-CoV-2 in the United States, Belgium, Hong Kong and Wuhan, China, where outbreaks occurred. In the cases reported in Belgium and in the USA, clinical signs were reported (diarrhoea, vomiting and breathing difficulty for the first and respiratory illness for the second) whereas no clinical sign was detected in Hong Kong. Experimentally, pulmonary lesions have been described in young cats (Shi at al, 2020). Serological tests carried out on cats in Wuhan demonstrated that that SARS-CoV-2 has infected cat populations in Wuhan during the outbreak (15 of 102 tested were positive in ELISA and 11 had neutralizing antibodies with titers ranging from 1/20 to 1/1080). The highest titers correspond to cats living with owners with confirmed COVID-19 infection. No information on clinical signs in any of the tested cats is available. Recently, Halfmann et al (2020) showed the possible transmission of SARS-CoV-2 between domestic cats in close contact.
As in previously identified cases worldwide, the French cat was living with a person who was confirmed positive for COVID-19. However, a previous study carried out on the animals of veterinary students of the Alfort veterinary school (Enva, Maisons Alfort), also infected by SARS-CoV-2 suggested that cats and dogs are not easily infected by the SARS-CoV-2, even when they are in close contact with infected owners (Temmam et al, 2020). Large serological analyses will be necessary to evaluate the real circulation of the SARS-CoV-2 among pets.
Concerning the infection of dogs with SARS-CoV-2, two cases have been reported in Honk- Kong in February and March 2020 (Sit, et al, 2020). In both cases, dogs were placed under quarantine after their owner was hospitalised due to COVID-19. Samples taken from the dogs were tested positive for SARS-CoV-2 by RT-PCR and serological analysis. Both animals did not exhibit any specific clinical signs. Shi et al, 2020 have shown by experimental infection that dogs have low susceptibility to SARS-CoV-2. In our investigations, none of the 11 dogs tested were PCR positive despite having been in close contact with their infected owners and exhibiting clinical signs.
Even though the risk of transmission of the SARS-CoV-2 to the cat appears to be low, the authors therefore advise people with COVID-19 to limit close contact with their cats, without compromising their welfare.