Coronavirus (SARS-CoV-2) in Antillean Manatees (Trichechus Manatusmanatus)

For two years, the world has been experiencing a coronavirus (SARS-CoV-2) pandemic. Non-human animals are susceptible to the virus, including marine mammals. Here we aimed to test Antillean manatees, Trichechus manatus manatus, for the coronavirus (SARS-CoV-2). We collected samples from 19 individuals kept under the responsibility of the Brazilian centre for research and conservation of aquatic mammals (ICMBio/CMA). We analysed the samples through RT-PCR and RT-LAMP-PCR and found that two of the 19 manatees tested positive for SARS-CoV-2. Such a result led to a new biosecurity protocol in the ICMBio/CMA to avoid potential human-manatee coronavirus contamination, showing how we can use simple genetic tools to improve the care and conservation of manatees. The technique isolates puries amplied a diagnose experimental for


Introduction
Since late 2019, the world has been experiencing a coronavirus (SARS-CoV-2) pandemic, with the rst case reported in Brazil in February 2020 (Werneck and Carvalho 2020). The transmission of the virus from humans to animals is possible, especially mammals (including marine ones), which are already known to be susceptible to the disease (IUCN SSC 2020; Gryseels et al. 2020). Infected wastewater discharged into natural water systems are supposed to contaminate aquatic species (Mathavarajah et al. 2020; Audino et al. 2021; Charlie-Silva et al. 2021). West Indian manatees (Trichechus manatus) are marine mammals categorised worldwide as Vulnerable due to the sharp decline in their populations (Deutsch et al. 2008). The subspecies Trichechus manatus manatus (Antillean manatee) occurs in Brazil, being categorised as Endangered in the Brazilian list of threatened species (ICMBio 2018). The species is targeted by a conservation action plan and a long-term reintroduction program in Brazil (Luna and Passavant 2010; Ordinance ICMBio N° 249/2018). The National Centre for Research and Conservation of Aquatic Mammals (ICMBio/CMA) is a Brazilian Government unit responsible for caring for Antillean manatees in captivity and rehabilitation and reintroduction processes (Ordinance No. 554 2020, May 25th, 2020). The centre carries out periodic health assessments of the manatees. Given the current coronavirus pandemic (SARS-CoV-2), the centre felt the need to verify whether the Antillean manatees were susceptible to the virus. Therefore, the present study aimed to test Antillean manatees for the coronavirus (SARS-CoV-2).

Sample collection
We collected samples from 19 Antillean manatees (Supplementary Information - Table 1) kept under the responsibility of the Centro de Pesquisa e Conservação de Mamíferos Aquáticos do Instituto Chico Mendes de Biodiversidade (ICMBio/CMA). We collected samples from the nostrils of the manatees with swabs ( Figure 1) and preserved them in the eld in Phosphate-buffered saline PBS (1ml of solution and 0,1X Phosphate-buffered saline PBS) for up to 24h before freezing the samples at -20C. We also collected biometrics measures and blood samples from all the animals to perform blood and serum biochemicals counts (urea, creatinine, GOT, GPT, glucose, triglycerides). This study complied with the Brazilian law (Permit number: SISBIO/ICMBio N°77116-1).

Sample Analysis
We obtained the viral RNAs from the swab samples using the ReliaPrep™ Viral Total Nucleic Acid Puri cation Kit-Promega (extractions and puri cations were done following the kit protocol). The RNAs were then analysed through RT-PCR and RT-LAMP and stored at -80°C. The RT-PCR technique isolates and puri es viral RNA from the samples, which is then reverse transcribed to cDNA and subsequently ampli ed in a real-time PCR thermocycler. RT-PCR is the test recommended by WHO (gold standard) to diagnose and monitor individuals with active infections. The RT-LAMP-PCR technique is a new experimental technique for fast and sensitive RNA detection. The RT-LAMP-PCR method is performed in simple isothermal conditions using four or six speci c oligonucleotide primers for the target sequence of the COVID-19.

RT-PCR assay
We used the MOLECULAR SARS-CoV2 (E) -Bio-Manguinhos kit for the diagnosis by quantitative reverse transcription PCR (RT-PCR). This kit uses the primer and probe sequences from the Berlin Protocol were used in the reaction. A total of 5 uL of RNA, previously extracted from the animals, was added to the LAMP reaction. It was incubated in the thermocycler (Veriti™ 96-Well Thermal Cycler) at 72°C for 60 minutes, followed by 5 min at 80°C. Con rmation of a positive RT-LAMP reaction was achieved by observing the LAMP ampli ed products by the naked eye immediately after the addition of 2uL of Sybr Green TM I nucleic acid gel stain/Invitrogen Dilution 1:10) into the LAMP tube. LAMP products were exposed to UV light with a UV Lamp UVL-56, 6-watt, 365 nm Handheld (UVP, Upland, CA, USA) to observe the uorescence. The results were con rmed by electrophoresis (Agarose Gel 2%). RT-LAMP primers: The primers used in this study were previously published by Lamb et al. (2020). Four primers were used in the LAMP reaction, two inner primers (FIP-BIP) and two outer primers (F3-B3). The primer sequences are shown in Supplementary Information - Table 2. The target of the primers is a non-structural protein 3 (NSP3) of SARS-CoV-2.

Results & Discussion
Despite the presumed susceptibility for SARS-CoV-2 in marine mammals (Nabi 2020; Mathavarajah et al. 2021), to date, the infection of aquatic mammals had not been con rmed. Our results show evidence that Antillean manatees can be contaminated by SARS-CoV-2, and therefore, we con rmed the rst case of Our results of the RT-LAMP reaction con rmed that the same two animals were COVID-19 positive ( Figure  3). The RT-LAMP assays con rmed the presence of the NSP3 coding region of open reading frame (ORF) 1Ab. Previously published works suggest that the NSP3 protein is essential for SARS-CoV-2 replication, translation of the mRNA transcripts, and suppression of the immune response (Raj 2021;Emam et al. 2021).
Blood counts were unaltered and biochemical serum tests results did not show any common abnormality between both contaminated individuals (Supplementary information -table 1). Furthermore, none of the animals presented clinical symptoms that could indicate COVID-19 contamination.
After SARS-CoV-2 detection in the study manatees, all 24 staff at the Itamaracá captive centre were tested. Six staff members tested positive for COVID-19, from which two had direct contact with the animals. Although the keepers could have transmitted COVID-19 to the manatees, the actual source of infection is still uncertain. The water in the manatee pools come directly from the sea, and they receive a freshwater source for hydration and bottle feeding. Mathavarajah et al. (2021) suggested that the lack of treatment of wastewater in common sewers could transfer SARS-CoV-2 to natural waters. Furthermore, Charlie-Silva et al. (2021) found that fragments of the COVID-19 virus present in wastewater were capable of infecting tadpoles (Physalaemus cuvieri). Therefore, we highlight the potential risk of this transmission route to our study animals.
Many species, including manatees, requires human interventions to guarantee their populations' survival.
Our results led to a new security protocol in the ICMBio/CMA captive centre to avoid potential humanmanatee coronavirus contamination, showing how we can use genetic tools to improve the care and ultimately the conservation of the threatened Antillean manatees. We suggest prioritizing vaccination and Author Consent -All authors have read the nal version of the manuscript and consent to its publication.
Author Contributions -FLNA and KL were responsible for the sample collections; FLM, NANB and RMLA were responsible for the genetic analyses; FL and BB were responsible for the elaboration of a rst draft, and all authors reviewed, adjusted, and commented on the nal draft. RU reviewed the grammar and language. All authors contributed to the study conception, design and execution.
Con icts of Interest -The authors have no con icts of interest to declare that are relevant to this article.
Ethics approval -The study complied with Brazilian law and was conducted under the SISBIO license 77116-1 held by Fernanda Attademo.
Data Availability -The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.  Figure 1 Method showing nasal swab sample collected from an Antillean manatee. The arrows point to the nasal opening. We introduced the swab into the nostril once it opened for breath. a-d: steps of the procedure