Ophidian Paramyxovirus in Snakes in the Canary Islands: An Immunohistochemical Study

J. Orós, J. Sicilia, A. Torrent, P. Castro, S. Déniz¹, A. B. Casal, E. R. Jacobson² and B. L. Homer³

Department of Histology and Pathology, Veterinary Faculty ULPGC, Trasmontaña s/n, 35416 Arucas (Las Palmas), Spain; ¹Department of Infectious Diseases, Veterinary Faculty ULPGC; ²Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, PO Box 100126, Gainesville, FL 32610-0126, USA; ³Department of Pathobiology, College of Veterinary Medicine, University of Florida

Abstract. Ophidian paramyxovirus (OPMV) has surfaced as a very important pathogen in both viperid and nonviperid snakes. This short communication reports a retrospective immunohistochemical study in order to confirm OPMV infection as cause of mortality in several collections of snakes in the Canary Islands. Tissues from 17 snakes belonging to 4 private collections of Las Palmas de G. C. that experienced mortalities attributed to respiratory infection between 1995 and 1999 were selected. For immunohistochemical labelling, sections were exposed to a polyclonal antiserum against an Aruba Island rattlesnake (Crotalus unicolor) isolate of OPMV. Viral antigen was immunohistochemically detected in the lungs of 6 snakes belonging to three different reptile collections. These are believed to be the first reported cases of OPMV infection in Spain and the first reported case of immunohistochemical detection of OPMV antigen in the liver of a snake suffering pulmonary infection by paramyxovirus.

Key words. Immunohistochemistry, ophidian paramyxovirus, snake.

Introduction

In 1972, a respiratory epizootic caused by a paramyxo-like virus spread through a collection of fer-de-lance snakes (Bothrops atrox) at a serpentarium in Switzerland (Clark and others 1979). Since then, numerous outbreaks have been described in collections of snakes in the United States, Mexico and Germany (Jacobson and others 1981, Blahak and others 1991, Jacobson and others 1992).

The disease is characterised by proliferative pneumonia, with or without interstitial inflammation (Jacobson and others 1992). Other lesions that have been described in OPMV infections include encephalitis and pancreatic hyperplasia (Jacobson and others 1980, 1992). Recently, a transmission study was carried out at the University of Florida and Koch’s postulates were fulfilled (Jacobson and others 1997). Diagnosis is based upon isolation of the virus in tissue culture, demonstration of the virus by electron microscopy, and demonstration of rising titers of antiviral antibodies by haemagglutination inhibition tests and, most recently, by immunoperoxidase staining of viral antigen in sections of infected lungs (Homer and others 1995).

This short communication reports a retrospective immunohistochemical study in order to confirm OPMV infection as cause of mortality in several collections of snakes in the Canary Islands. These are believed to be the first reported cases of OPMV infection in Spain and the first reported case of immunohistochemical detection of OPMV antigen in the liver of a snake suffering pulmonary infection by paramyxovirus.

Material and methods

We selected tissues from 17 snakes belonging to 4 private collections of Las Palmas de G. C. that experienced mortalities attributed to respiratory infection between 1995 and 1999. After necropsy, tissue samples were fixed in 10 % neutral buffered formalin and embedded in paraffin.

For immunohistochemical labelling, sequential 4-µm sections of lung, liver, spleen and pancreas were adhered to poly-L-lysine-coated slides. Sections were deparaffinized and immersed in 3% H₂O₂ in absolute methanol for 30 min. They were then rinsed in phosphate buffered saline (PBS) and placed in a solution of 0.1% protease, pH 7.2, for 5 min at room temperature. The sections were washed three times in PBS, blocked with 5% normal goat serum for 30 min and exposed to a polyclonal antiserum against an Aruba Island rattlesnake (Crotalus unicolor) isolate of OPMV (Richter and others 1996) applied at dilution 1:500 for 2 hr. The sections were rinsed in PBS and incubated for 30 min with a 1:5 dilution of biotinylated antibody to mouse and rabbit IgG (LSAB Kit; Dako, Burlingame, CA, USA), rinsed again in PBS, and incubated for 30 min with a 1:5 dilution of streptavidin-peroxidase complex reagent (LSAB Kit; Dako). All incubations were at room temperature. After rinsing in PBS the sections were exposed to 3-amino-9-ethylcarbazole (AEC) (Sigma, St Louis, MO, USA) for 5 min. They were the counterstained with Mayer’s haematoxyline and mounted using an aqueous mounting medium (Immu-mount; Shandon, Pittsburgh, PA, USA). Paraffin-embedded normal tissues from two healthy boa constrictor and nonimmune serum from a rabbit served as negative procedure controls. Tissues from an Aruba Island rattlesnake, Crotalus unicolor, experimentally inoculated with OPMV (Jacobson and others 1997) were used as positive controls.

Results

At necropsy, a variety of pulmonary gross lesions were detected in all selected snakes, including diffuse to focal accumulations of caseous necrotic cellular debris, diffuse hemorrhage of the lung and air sac system, and oedema and severe diffuse congestion of the lung.

Histological changes in affected lungs included hyperplasia and hypertrophy of septal and faveolar epithelial cells, loss of ciliated cells, mixed leukocytic interstitial infiltrates, and fibrinonecrotic exudate in the lumen of proximal and distal faveolar compartments. Macrophages and gram-negative microorganisms were often detected within this exudate. No intraepithelial intracytoplasmic inclusion bodies nor epithelial syncytial cell formation were observed. Some of the lungs showed moderate multifocal granulomatous pneumonia with gram-negative bacteria and deposits of calcium in the granulomata. Microbiological study had confirmed isolation of Aeromonas sp. , Proteus sp. and Morganella morganii from the lungs of 8 snakes. No significant lesions were observed in other organs.

Viral antigen was immunohistochemically detected in the lungs of 6 snakes belonging to three different reptile collections. There was strong multifocal to diffuse linear red labelling of the luminal surface of bronchial and faveolar epithelium (Figures 1 and 2). In cases where was a hyperplastic epithelium, cytoplasmic labelling was observed within the superficial cells lining air passageways. None of the negative controls stained positively for paramyxovirus antigens.

Figure 1. Immunoperoxidase red labelling for OPMV antigen of the luminal bronchial epithelium. Bitis gabonica rhinoceros. x 20	Figure 2. Immunoperoxidase red labelling for OPMV antigen of the faveolar epithelium. Crotalus atrox. x 40

Immunoreactivity was also detected in some hepatocytes and Kupffer cells of the liver of a western diamondback rattlesnake (Crotalus atrox) (Figure 3). No immunoreaction was observed in other organs.

Figure 3. Hepatocytes and Kupffer cell showing immunoreaction for OPMV antigen. Crotalus atrox. x 40

Discussion

The pulmonary lesions observed in this study were similar to those described previously. A proliferative interstitial pneumonia was seen in all cases showing positive immunoreactivity. However, although eosinophilic intracytoplasmic inclusions have been described within pneumocytes in other reports (Jacobson and others 1981, 1992, Homer and others 1995), inclusions were not seen in any of our cases. In a transmission study intracytoplasmic inclusions were seen only in epithelial cells of the lung of an Aruba Island rattlesnake (Crotalus unicolor) that died at day 19 postinoculation (Jacobson and others 1997). Syncytial cell formation is not frequent in OPMV infections (Jacobson and others 1997). In mammals, the ability to form syncytial cells in the lungs appears to vary among different animals infected with paramyxoviruses (Summers and Appel 1985, Brown and others 1991, Kennedy and others 1991). We did not observe syncytial cell formation in any of the snakes.

The pulmonary lesions associated with OPMV infection are highly suggestive of the infection but are not considered pathognomonic (Homer and others 1995). In this study approximately 40% of the sections that had proliferative pneumonia did not show immunoreactivity. The possibility that other pathogens cause similar lesions could explain these results, although antigen denaturation associated with the formalin fixation (Rickert and Maliniak 1989), and possible antigen variability among strains of OPMV (Blahak and others 1991, Homer and others 1995) should be considered.

This study confirms the usefulness of this polyclonal anti-OPMV antiserum to detect OPMV antigens in formalin-fixed paraffin-embedded lung of infected snakes. The advantages of using an immunohistochemical technique to detect viral antigens in formalin-fixed tissue sections compared to virus isolation from fresh tissue include: ability to perform retrospective studies on paraffin-embedded tissues, reduced time for a definitive diagnosis, low cost, and the possibility to detect the virus associated with specific cells and thus to study the pathogenesis.

In a similar study developed at the University of Florida the authors used the avidin-biotin-peroxidase complex (ABC) method and the diaminobenzidine (DAB) as chromogen (Homer and others 1995). We used the LSAB method because it has been described as more sensitive than the ABC method with other pathogens (Orós and others 1996). The use of AEC as substrate of the immunoreaction has been evaluated more efficient than DAB in previous studies with other pathogens because of the strong red colour provided by AEC facilitating the interpretation of results (Orós and others 1996).

Gram-negative microorganisms have been commonly isolated from lungs of dead snakes during epizootics of OPMV (Jacobson and others 1981, 1992). Although primary lesions of paramyxoviral infections in snakes are confined to respiratory system, particularly lung, lesions in other organs have been previously described. Moderate chronic hepatitis, diffuse hepatic necrosis and/or multifocal pyogranulomatous hepatitis are often observed, but gram-negative microorganisms are usually associated with these lesions (Jacobson and others 1981, 1992). Immunoreactivity for OPMV was observed in the liver of a western diamondback rattlesnake. Although isolation of OPMV from the liver has been described (Jacobson and others 1981), there are no descriptions of immunohistochemical detection of OPMV antigen in this organ. A viraemic status of the snake could explain this finding.

This is believed to be the first report of OPMV in Spain. Because serological studies have not been carried out in Spain, the diagnosis of OPMV is generally made postmortem. Therefore the prevalence in private collections in Spain is unknown. Serology, necropsy and histopathology of snakes showing clinical signs consistent with OPMV infection would be needed to establish the extent of infection in captive snakes in Spain. The reptile trade probably contributes to propagation of OPMV. Because there is only effective treatment against secondary bacterial infections, strict quarantine of newly acquired snakes should be followed to prevent the infection from spreading (Jacobson and others 1992). Currently, there is no vaccine available for protecting snakes against OPMV infection. A killed vaccine was developed but the response was both variable and transient (Jacobson and others 1991).

Acknowledgements

The authors would like to thank A. Afonso, Deparment of Morphology, University of Las Palmas de Gran Canaria, and S. Tucker, Department of Small Animal Clinical Sciences, University of Florida, for technical assistance.

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