Title : Potential sars-cov-2 susceptibility of cetaceans stranded along the italian coastline
Abstract:
Due to marine mammals’ demonstrated susceptibility to SARS-CoV-2, based upon the homology level of their angiotensin-converting enzyme 2 (ACE2) viral receptor with the human one, alongside the global SARS-CoV-2 occurrence and faecal contamination of the river and marine ecosystems, SARS-CoV-2 infection may be plausibly expected to occur also in cetaceans, with special emphasis on inshore species like bottlenose dolphins (Tursiops truncatus). Moreover, based on immune and inflammatory responses to SARS-CoV-2 infection in humans, macrophages could also play an important role in antiviral defence mechanisms. In order to provide a more in-depth insight into SARS-CoV-2 susceptibility in marine mammals, we evaluated the presence of SARS-CoV-2 and the expression of ACE2 and the pan-macrophage marker CD68. Aliquots of tissue samples, belonging to cetaceans stranded along the Italian coastline during 2020-2021-2022, were collected for SARS-CoV-2 analysis by real-time PCR (RT-PCR) (N=61 tissue aliquots and N=25 swabs) and Immunohistochemistry (IHC) (N = 59); thirty-two aliquots of pulmonary tissue sample (N = 17 Tursiops truncatus, N = 15 Stenella coeruleoalba) available at the Mediterranean Marine Mammal Tissue Bank (MMMTB) of the University of Padua (Legnaro, Padua, Italy) were analyzed to investigate ACE2 expression by IHC. In addition, ACE2 and CD68 were also investigated by Double-Labeling Immunofluorescence (IF) Confocal Laser Microscopy. No SARS-CoV-2 positivity was found in samples analyzed for the survey while ACE2 protein was detected in the lower respiratory tract albeit heterogeneously for age, sex and species, suggesting that ACE2 expression can vary between different lung regions and among individuals. Finally, double IF analysis showed elevated colocalization of ACE2 and CD68 in macrophages only when an evident inflammatory reaction was present, such as in human SARS-CoV-2 infection.
Keywords: SARS-CoV-2; ACE2; CD68; marine mammals
Audience take away:
Because the demonstrated susceptibility of marine mammals to SARS-CoV-2 (Audino et al.,2021), this study underlines the importance, from a One Health perspective, of monitoring stranded specimens for systematic surveillance of SARS-CoV-2 infection in marine mammals; this is essential to protect human health and that of endangered marine mammal species.
Furthermore, has been previously studied how cetaceans deal with some pathogens of anthropogenic origin (e.g. Salmonella spp, Toxoplasma gondii), which can adversely affect both their individual health and the conservation status of the population.
The presence of SARS-CoV-2 has been demonstrated in the wastewater and rivers of countries with high COVID-19 caseloads; viruses in raw wastewater are not readily removed by treatment and thus become environmental pollutants; although the ocean provides for rapid dilution of sewage, its self-depuration capacity is finite, especially in coastal areas. In addition, as the Mediterranean is a “closed sea basin”, a “concentrating activity” towards chemical pollutants as well as towards infectious pathogens is possible. Although we found no evidence of SARS-CoV-2 spillover in cetaceans stranded on the Italian coast and monitored by our network, ACE2 expression in lung tissues suggests a potential susceptibility of marine mammals to SARS-CoV-2 infection.
From the evaluation of the expression and the effect of age, sex, and species on the expression of the viral host cell receptor ACE2 in the lung tissue of S. coeruleoalba and T. truncates, to assess whether, as in humans, there are differences in the expression, we found no difference.
Furthermore, neutrophil granulocytes and macrophages play a key role in inflammatory and immune response in terrestrial and aquatic mammals. We noted in the cetacean lung tissues a peculiar mode of compartmentalization of macrophages that suggested different functional specialization. The macrophage cytotype, the so-called pulmonary intravascular macrophage (PIM), probably involved in the uptake and subsequent phagocytosis of foreign elements (particulate matter of physical or biological origin) plays a role complementary to the other macrophage cytotype residing in the lung, namely, the alveolar macrophage. As revealed by immunofluorescence analyses, ACE2/CD68 co-localization was higher when an infectious agent was present and was probably on activated type 1 macrophages (M1). These findings indicate that in cetaceans alveolar macrophages and PIM, as frontline immune cells, may be directly targeted in the course of SARS-CoV-2 infection.
