Detection of MERS-CoV antigen on formalin-fixed paraffin-embedded nasal tissue of alpacas by immunohistochemistry using human monoclonal antibodies directed against different epitopes of the spike protein

Middle East respiratory syndrome (MERS) represents an important respiratory disease accompanied by lethal outcome in one third of human patients. In recent years, several investigators developed protective antibodies which could be used as prophylaxis in prospective human epidemics. In the current study, eight human monoclonal antibodies (mAbs) with neutralizing and non-neutralizing capabilities, directed against different epitopes of the MERS-coronavirus (MERS-CoV) spike (MERS-S) protein, were investigated with regard to their ability to immunohistochemically detect respective epitopes on formalin-fixed paraffin-embedded (FFPE) nasal tissue sections of MERS-CoV experimentally infected alpacas. The most intense immunoreaction was detected using a neutralizing antibody directed against the receptor binding domain S1B of the MERS-S protein, which produced an immunosignal in the cytoplasm of ciliated respiratory epithelium and along the apical membranous region. A similar staining was obtained by two other mAbs which recognize the sialic acid-binding domain and the ectodomain of the membrane fusion subunit S2, respectively. Five mAbs lacked immunoreactivity for MERS-CoV antigen on FFPE tissue, even though they belong, at least in part, to the same epitope group. In summary, three tested human mAbs demonstrated capacity for detection of MERS-CoV antigen on FFPE samples and may be implemented in double or triple immunohistochemical methods.info:eu-repo/semantics/acceptedVersio

Co‐localization of Middle East respiratory syndrome coronavirus (MERS‐CoV) and dipeptidyl peptidase‐4 in the respiratory tract and lymphoid tissues of pigs and llamas

This study investigated the co‐localization of the Middle East respiratory syndrome coronavirus (MERS‐CoV) and its receptor dipeptidyl peptidase‐4 (DPP4) by immunohistochemistry (IHC) across respiratory and lymphoid organs of experimentally MERS‐CoV infected pigs and llamas. Also, scanning electron microscopy was performed to assess the ciliary integrity of respiratory epithelial cells in both species. In pigs, on day 2 post‐inoculation (p.i.), DPP4‐MERS‐CoV co‐localization was detected in medial turbinate epithelium. On day 4 p.i., the virus/receptor co‐localized in frontal and medial turbinate epithelial cells in pigs, and epithelial cells distributed unevenly through the whole nasal cavity and in the cervical lymph node in llamas. MERS‐CoV viral nucleocapsid was mainly detected in upper respiratory tract sites on days 2 and 4 p.i. in pigs and day 4 p.i. in llamas. No MERS‐CoV was detected on day 24 p.i. in any tissue by IHC. While pigs showed severe ciliary loss in the nasal mucosa both on days 2 and 4 p.i. and moderate loss in the trachea on days 4 and 24 p.i., ciliation of respiratory organs in llamas was not significantly affected. Obtained data confirm the role of DPP4 for MERS‐CoV entry in respiratory epithelial cells of llamas. Notably, several nasal epithelial cells in pigs were found to express viral antigen but not DPP4, suggesting the possible existence of other molecule/s facilitating virus entry or down regulation of DPP4 upon infection.info:eu-repo/semantics/publishedVersio

Co-localization of Middle East respiratory syndrome coronavirus (MERS-CoV) and dipeptidyl peptidase-4 in the respiratory tract and lymphoid tissues of pigs and llamas

This study investigated the co-localization of the Middle East respiratory syndrome coronavirus (MERS-CoV) and its receptor dipeptidyl peptidase-4 (DPP4) by immunohistochemistry (IHC) across respiratory and lymphoid organs of experimentally MERS-CoV infected pigs and llamas. Also, scanning electron microscopy was performed to assess the ciliary integrity of respiratory epithelial cells in both species. In pigs, on day 2 post-inoculation (p.i.), DPP4-MERS-CoV co-localization was detected in medial turbinate epithelium. On day 4 p.i., the virus/receptor co-localized in frontal and medial turbinate epithelial cells in pigs, and epithelial cells distributed unevenly through the whole nasal cavity and in the cervical lymph node in llamas. MERS-CoV viral nucleocapsid was mainly detected in upper respiratory tract sites on days 2 and 4 p.i. in pigs and day 4 p.i. in llamas. No MERS-CoV was detected on day 24 p.i. in any tissue by IHC. While pigs showed severe ciliary loss in the nasal mucosa both on days 2 and 4 p.i. and moderate loss in the trachea on days 4 and 24 p.i., ciliation of respiratory organs in llamas was not significantly affected. Obtained data confirm the role of DPP4 for MERS-CoV entry in respiratory epithelial cells of llamas. Notably, several nasal epithelial cells in pigs were found to express viral antigen but not DPP4, suggesting the possible existence of other molecule/s facilitating virus entry or down regulation of DPP4 upon infection

Experimental infection of dromedaries with Middle East respiratory syndrome-Coronavirus is accompanied by massive ciliary loss and depletion of the cell surface receptor dipeptidyl peptidase 4

Middle East respiratory syndrome (MERS) represents an important respiratory disease accompanied by lethal outcome in one-third of human patients. Recent data indicate that dromedaries represent an important source of infection, although information regarding viral cell tropism and pathogenesis is sparse. In the current study, tissues of eight dromedaries receiving inoculation of MERS-Coronavirus (MERS-CoV) after recombinant Modified-Vaccinia-Virus-Ankara (MVA-S)-vaccination (n = 4), MVA-vaccination (mock vaccination, n = 2) and PBS application (mock vaccination, n = 2), respectively, were investigated. Tissues were analyzed by histology, immunohistochemistry, immunofluorescence, and scanning electron microscopy. MERS-CoV infection in mock-vaccinated dromedaries revealed high numbers of MERS-CoV-nucleocapsid positive cells, T cells, and macrophages within nasal turbinates and trachea at day four post infection. Double immunolabeling demonstrated cytokeratin (CK) 18 expressing epithelial cells to be the prevailing target cell of MERS-CoV, while CK5/6 and CK14 expressing cells did not co-localize with virus. In addition, virus was occasionally detected in macrophages. The acute disease was further accompanied by ciliary loss along with a lack of dipeptidyl peptidase 4 (DPP4), known to mediate virus entry. DPP4 was mainly expressed by human lymphocytes and dromedary monocytes, but overall the expression level was lower in dromedaries. The present study underlines significant species-specific manifestations of MERS and highlights ciliary loss as an important finding in dromedaries. The obtained results promote a better understanding of coronavirus infections, which pose major health challenges.info:eu-repo/semantics/publishedVersio

Cryopreservation of Canine Primary Dorsal Root Ganglion Neurons and Its Impact upon Susceptibility to Paramyxovirus Infection

Canine dorsal root ganglion (DRG) neurons, isolated post mortem from adult dogs, could provide a promising tool to study neuropathogenesis of neurotropic virus infections with a non-rodent host spectrum. However, access to canine DRG is limited due to lack of donor tissue and the cryopreservation of DRG neurons would greatly facilitate experiments. The present study aimed (i) to establish canine DRG neurons as an in vitro model for canine distemper virus (CDV) infection; and (ii) to determine whether DRG neurons are cryopreservable and remain infectable with CDV. Neurons were characterized morphologically and phenotypically by light microscopy, immunofluorescence, and functionally, by studying their neurite outgrowth and infectability with CDV. Cryopreserved canine DRG neurons remained in culture for at least 12 days. Furthermore, both non-cryopreserved and cryopreserved DRG neurons were susceptible to infection with two different strains of CDV, albeit only one of the two strains (CDV R252) provided sufficient absolute numbers of infected neurons. However, cryopreserved DRG neurons showed reduced cell yield, neurite outgrowth, neurite branching, and soma size and reduced susceptibility to CDV infection. In conclusion, canine primary DRG neurons represent a suitable tool for investigations upon the pathogenesis of neuronal CDV infection. Moreover, despite certain limitations, cryopreserved canine DRG neurons generally provide a useful and practicable alternative to address questions regarding virus tropism and neuropathogenesis

Calcinosis in a roe deer fawn (Capreolus capreolus) in northern Germany

BACKGROUND: Calcinosis has been reported for a broad range of different animals. Causes for calcinosis include metabolic disorders due to kidney failure, intoxication with calcinogenic plants, or iatrogenic overdose of vitamin D. Especially young animals seem to be very susceptible to developing calcinosis. Currently, however, there is a lack of information on calcinosis in wildlife. CASE PRESENTATION: The following case report describes a roe deer fawn admitted to a clinic due to general weakness and myiasis. Plasma levels for creatinine, urea and phosphate were highly elevated, whereas the total calcium level was decreased. Necropsy revealed calcinosis due to calcification in many organs. The reason for calcinosis in this particular case might be kidney failure. Plasma samples from other hunted roe deer fawns also showed high phosphate levels. CONCLUSIONS: Roe deer fawns might be susceptible to calcinosis due to high plasma phosphate, which could be a result of kidney failure or different feed. Further research into calcium and phosphate homeostasis in roe deer is necessary

Pathology in captive wild felids at German zoological gardens

This retrospective study provides an overview on spontaneous diseases occurring in 38 captive wild felids submitted for necropsy by German zoological gardens between 2004 and 2013. Species included 18 tigers, 8 leopards, 7 lions, 3 cheetahs and 2 cougars with an age ranging from 0.5 to 22 years. Renal lesions, predominantly tubular alterations (intra-tubular concrements, tubular degeneration, necrosis, intra-tubular cellular debris, proteinaceous casts, dilated tubuli) followed by interstitial (lympho-plasmacytic inflammation, fibrosis, metastatic-suppurative inflammation, eosinophilic inflammation) and glomerular lesions (glomerulonephritis, glomerulosclerosis, amyloidosis) were detected in 33 out of 38 animals (87%). Tumors were found in 19 of 38 felids (50%) with 12 animals showing more than one neoplasm. The tumor prevalence increased with age. Neoplasms originated from endocrine (11), genital (8), lympho-hematopoietic (5) and alimentary organs (4) as well as the mesothelium (3). Most common neoplasms comprised uterine/ovarian leiomyomas (5/2), thyroid adenomas/adenocarcinoma (5/1), pleural mesotheliomas (3), hemangiosarcomas (2) and glossal papillomas (2). Inflammatory changes were frequently encountered in the intestine and the lung. Two young animals displayed metastatic mineralization suggestive of a vitamin D- or calcium intoxication. One tiger exhibited degenerative white matter changes consistent with an entity termed large felid leukoencephalomyelopathy. Various hyperplastic, degenerative and inflammatory changes with minor clinical significance were found in several organs. Summarized, renal lesions followed by neoplastic changes as well as inflammatory changes in lung and gastrointestinal tract represent the most frequent findings in captive wild felids living in German zoological gardens

Co-localization of Middle East respiratory syndrome coronavirus (-CoV) and dipeptidyl peptidase-4 in the respiratory tract and lymphoid tissues of pigs and llamas

This study investigated the co-localization of the Middle East respiratory syndrome coronavirus (-CoV) and its receptor dipeptidyl peptidase-4 (4) by immunohistochemistry (IHC) across respiratory and lymphoid organs of experimentally -CoV infected pigs and llamas. Also, scanning electron microscopy was performed to assess the ciliary integrity of respiratory epithelial cells in both species. In pigs, on day 2 post-inoculation (p.i.), 4--CoV co-localization was detected in medial turbinate epithelium. On day 4 p.i., the virus/receptor co-localized in frontal and medial turbinate epithelial cells in pigs, and epithelial cells distributed unevenly through the whole nasal cavity and in the cervical lymph node in llamas. -CoV viral nucleocapsid was mainly detected in upper respiratory tract sites on days 2 and 4 p.i. in pigs and day 4 p.i. in llamas. No -CoV was detected on day 24 p.i. in any tissue by . While pigs showed severe ciliary loss in the nasal mucosa both on days 2 and 4 p.i. and moderate loss in the trachea on days 4 and 24 p.i., ciliation of respiratory organs in llamas was not significantly affected. Obtained data confirm the role of 4 for -CoV entry in respiratory epithelial cells of llamas. Notably, several nasal epithelial cells in pigs were found to express viral antigen but not 4, suggesting the possible existence of other molecule/s facilitating virus entry or down regulation of 4 upon infection

Porcine Forebrain Vacuolization Associated with Wasting in Pigs : A Novel Pathological Outcome Associated with Vitamin-Mineral Deficiency?

The term wasting is a clinical name describing a physical condition characterized by growth retardation, usually of multifactorial origin. The present study describes an apparently new condition of pigs characterized clinically by wasting and pathologically by vacuolization of the brain. During 2016-2018, animals from eight farms were weaned in good body condition, and after 1-2 weeks, they started losing weight. To investigate potential causes of this condition, apparently sick and healthy pigs from each herd of eight affected farms were studied by means of histopathology, transmission electron microscopy and detection of usual infectious agents. Histopathologically, the most consistent lesion was neuropil vacuolization of the prosencephalon, mainly located in the thalamic nuclei and in the transition between the white and grey matter of the neocortex. Electron microscopy of some of these sick animals showed preserved axons, with dilated myelin sheaths (interpreted as edema of myelin sheath). The literature suggests this lesion type is linked to congenital or metabolic (toxic/deficiency) scenarios. The present case was probably a vitamin/mineral deficiency since supplementation with nutritional complexes solved the problem. The term wasting refers to a clinical sign used to describe a physical condition characterized by growth retardation, usually of multifactorial origin. The objective of the present study was to describe for the first time a pathological process characterized by forebrain neuropil vacuolization in pigs showing wasting without conspicuous neurological signs. To characterize the lesions pathologically, affected and non-affected pigs from eight of these farms were investigated. Histologically, the most consistent lesion was neuropil vacuolization of the prosencephalon, mainly located in the thalamic nuclei and in the transition between the white and grey matter of the neocortex (40/56 in sick and 4/30 in healthy pigs). In the most severe cases, the vacuolation also involved the midbrain, cerebellar nuclei and, to a lesser extent, the medulla oblongata. Vacuolization of the forebrain was associated with pigs experiencing marked emaciation and growth retardation. Although the specific cause of the present case remained unknown, the preventive use of multivitamin and mineral complexes in drinking water ameliorated the condition, strongly suggesting a metabolic origin of the observed condition