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 fecal 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 defense 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, were collected for SARS-CoV-2 analysis by real-time PCR (RT-PCRT) (N = 43) 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, gender/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: ACE2; CD68; SARS-CoV-2; marine mammals.

Figure 1

Immunohistochemical analysis with anti-SARS-CoV-2 polyclonal antibodies (3A, 5A) (A) Human lung (positive control): positive immunoreactivity within type I pneumocytes from alveolar respiratory epithelium, 3A polyclonal Ab. (B) Hamster lung (positive control): positive labeling of alveolar macrophages, 5A polyclonal Ab. (C) Bottlenose dolphin (T. truncatus) lung: absence of staining, 5A polyclonal Ab. (D) Bottlenose dolphin lung: absence of staining, 3A polyclonal Ab.

Figure 2

Immunohistochemical analysis by means of an anti-ACE2 polyclonal antibody on lung tissues from bottlenose dolphins (T. truncatus). (A) Lung, adult (ID 133–Table S1). Positive labeling on the surface of alveolar (type I pneumocytes) and bronchiolar epithelial cells. (B) Lung, adult (ID 201–Table S1). Absence of staining. (C) Lung, juvenile (ID 349–Table S1). Type I pneumocytes and bronchiolar epithelial cells are positive for ACE2. (D) Lung, calf (ID 359–Table S1). Absence of staining.

Figure 3

Immunohistochemical analysis by means of an anti-ACE-2 polyclonal antibody on lung tissues from striped dolphins (S. coeruleoalba). (A) Lung, adult (ID 167–Table S1). Positive labeling in type I pneumocytes from the alveolar respiratory epithelium and in alveolar macrophages. (B) Lung, adult (ID 447–Table S1). Absence of staining. (C) Lung, juvenile (ID 255–Table S1). Type I pneumocytes and alveolar macrophage are positive for ACE-2. (D) Lung, calf (ID 374–Table S1). Absence of staining.

Figure 4

Immunofluorescence analysis of ACE2 and CD68 (macrophage) in lungs from T. truncatus. (A) ID 123 ACE2 (green) positive cells. (B) ID 123 CD68 (red) positive cells. (C) ID 123 Merge ACE2 (green) and CD68 (red) with co-localization in yellow. (D) ID 133 ACE2 (green) positive cells. (E) ID 133 CD68 (red) positive cells. (F) ID 133 Merge ACE2 (green) and CD68 (red) No co-localization. (G) ID 142 ACE2 (green) positive cells. (H) ID 142 CD68 (red) positive cells. (I) ID 142 Merge ACE2 (green) and CD68 (red) with co-localization in yellow. (J) ID 343 ACE2 (green) positive cells. (K) ID 343 CD68 (red) positive cells. (L) ID 343 Merge ACE2 (green) and CD68 (red) No co-localization. Blue: 4,6-diamidino-2-phenylindole (DAPI). Scale bars, 10 µm.

Figure 5

Western Blot of ACE2 and CD68 protein expression on S. coeruleoalba and T. truncatus dolphin lung samples: (A) ACE2 in S. coeruleoalba lung sample; (B) CD68 in S. coeruleoalba lung sample; (C) ACE2 in T. truncatus lung sample; (D) CD68 in T. truncatus lung sample. Arrows indicates the correct bands. MW, molecular size markers (in kilodaltons).

Figure 6

Map of stranding sites.