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 confirmed. Our results show evidence that Antillean manatees can be contaminated by SARS-CoV-2, and therefore, we confirmed the first case of COVID-19 in a Sirenia species. Our RT-PCR analysis showed that the viral target envelope protein gene (E gene) was reported in two of the 19 investigated samples: the Cycle of quantification (Ct) for sample 4 was 37.04, and the Ct of sample 14 was 38.58 (Figure 2). No other samples had amplification that was detectable by the Kit used in this study. The reaction controls were analyzed and were within the parameters established by the Kit manufacturer, where the negative control was undetermined, and the Ct of the positive control was less than or equal to 37, which in this case, had a Ct of 30.80. The E gene of SARS-CoV-2 detected in the Antillean manatees by Real-Time PCR is used as a first-line screening tool. This gene encodes the envelope (E) protein of SARS-CoV-2 playing a fundamental role in the viral assembly, envelope formation, pathogenesis, and viral replication. The E protein is mostly expressed during the virus replication cycle (Rahman et al. 2021).
Our results of the RT-LAMP reaction confirmed that the same two animals were COVID-19 positive (Figure 3). The RT-LAMP assays confirmed 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 human-manatee 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 regular COVID-19 testing of the staff and animals in all institutions that keep manatees and other animals in captivity.