Scanning Electron Microscopic Findings on Respiratory Organs of Some Naturally Infected Dromedary Camels with the Lineage-B of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Saudi Arabia-2018

Abstract

The currently known animal reservoir for MERS-CoV is the dromedary camel. The clinical pattern of the MERS-CoV field infection in dromedary camels is not yet fully studied well. Some pathological changes and the detection of the MERS-CoV antigens by immunohistochemistry have been recently reported. However, the nature of these changes by the scanning electron microscope (SEM) was not revealed. The objective of this study was to document some changes in the respiratory organs induced by the natural MERS-CoV infection using the SEM. We previously identified three positive animals naturally infected with MERS-CoV and two other negative animals. Previous pathological studies on the positive animals showed varying degrees of alterations. MERS-CoV-S and MERS-CoV-Nc proteins were detected in the organs of positive animals. In the current study, we used the same tissues and sections for the SEM examination. We established a histopathology lesion scoring system by the SEM for the nasal turbinate and trachea. Our results showed various degrees of involvement per animal. The main observed characteristic findings are massive ciliary loss, ciliary disorientation, and goblet cell hyperplasia, especially in the respiratory organs, particularly the nasal turbinate and trachea in some animals. The lungs of some affected animals showed signs of marked interstitial pneumonia with damage to the alveolar walls. The partial MERS-CoV-S gene sequencing from the nasal swabs of some dromedary camels admitted to this slaughterhouse confirms the circulating strains belong to clade-B of MERS-CoV. These results confirm the respiratory tropism of the virus and the detection of the virus in the nasal cavity. Further studies are needed to explore the pathological alterations induced by MERS-CoV infection in various body organs of the MERS-CoV naturally infected dromedary camels.

Keywords: MERS-CoV; SEM; ciliary loss; dromedary camel; lesion scoring.

Figure 1

Histopathological and immunohistochemical changes in some MER-CoV naturally infected dromedary camels: (A): Nasal turbinate showed a denuded area of epithelial layer (arrow), H&E, ×100. (B): Bronchus showed a denuded area of epithelia (arrow) with many cells losing their cilia (arrowhead), H&E, ×400. (C): Nasal turbinate showed marked epithelial hyperplasia (arrow), H&E, ×100. (D): Lung showed interstitial pneumonia with alveolar wall thickening (arrow), H&E, ×100. (E): Nasal turbinate showed no IHC signal (negative control). (F): Bronchus showed detection of MER-CoV antigen in the epithelial layer and appears as a brown color (arrow). (G): Nasal turbinate showed IHC detection of MER-CoV antigen in the epithelial and glandular layer and appears as a brown color (arrows). (H): Lung showed detection of MER-CoV antigen in the thickened alveolar wall and appears as a brown color (arrow).

Figure 2

Scanning electron microscopy of the epithelium of the nasal turbinate of some MERS-CoV naturally infected dromedary camels compared to the non-infected animals: (A): Epithelium of nasal turbinate of a non-infected MERS-CoV dromedary camel showed the normal distribution of cilia giving the carpet shape appearance (asterisks) and normal goblet cells distribution across the section (arrows), ×2000. (B): Epithelium of Nasal turbinate of a positive MERS-CoV naturally infected dromedary camels showed an area of marked ciliary loss (asterisks) (×450). (C): Epithelium of nasal turbinate of a positive MERS-CoV naturally infected dromedary camel showed goblet cell proliferation (triple merged arrows), (× = 900). (D): Epithelium of nasal turbinate of a positive MERS-CoV naturally infected dromedary camel showed extensive goblet cell proliferation in higher magnification (triple merged arrows), ×1400.

Figure 3

Scanning electron microscopy of the epithelium of the trachea of MERS-CoV naturally infected dromedary camels compared to the non-infected animals: (A): Epithelium of the trachea of a non-infected camel showed the normal distribution of cilia giving the carpet shape appearance (asterisks) and normal goblet cells distribution across the section (arrows), ×900. (B): Higher magnification of A (×3000). (C): The epithelium of the trachea of an infected camel showed areas of mild ciliary loss (asterisks), areas of ruptured proliferated goblet cell (triple merged arrows), and areas of mucous secretion (× = 900). (D): Epithelium of the trachea of an infected camel showed moderate ciliary loss areas (asterisks), moderate goblet cell proliferation areas (triple merged arrows) covered with mucous secretion (× = 1400). (E): Epithelium of the trachea of one infected camel showed complete damage of the apical portion of the luminal cell surface with complete ciliary loss and absence of the carpet shape appearance except for a small ciliated area on the upper right area of the scanning micrograph (arrows), ×750. (F): Epithelium of the trachea of one infected camel showed eroded epithelial proliferating cells interspersed with less ciliated areas (asterisks), ×900.

Figure 4

Scanning electron microscopy of the lungs of MERS-CoV naturally infected dromedary camels compared to the non-infected animals: (A): Lung tissues of a non-infected camel showed normal distribution of thin wall alveoli (×50). (B): Lung tissues of a non-infected camel showed normal distribution of thin wall alveoli and alveolar duct connected to the respiratory bronchioles (×50). (C): Lung tissues of a non-infected camel showed normal distribution of thin wall alveoli and normal bronchiole with two blood vessels (×100). (D): Lung tissues of an infected camel showed thickening of the walls of the alveoli as well as thickening of the inter-alveolar septa (×55). (E): Lung tissues of an infected camel showed thick walls of alveolar ducts (AD) connected to thick walls alveoli and respiratory bronchioles (RB), ×50. (F): Lung tissues of an infected camel showed thickening in the alveolar walls, inter-alveolar septa, and peribronchiolar tissue (×100).

Figure 5

Identification of the circulating strains of MERS-CoV. The maximum likelihood analysis using the Mega-7 program of the partial MERS-CoV-S gene sequences from the nasal swabs of some dromedary camels received at this slaughterhouse. The obtained sequences (highlighted in black triangles) are clustered together with clade-B of MERS-CoV.