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(1987) Dissociative anesthesia. In Principles and Practice of Veterinary Anesthesia. Ed C. E. Short. Baltimore, Williams & Wilkins. pp 158- 169 Immunohistochemical detection of ophidian paramyxovirus in snakes in the Canary Islands J. OROS, J. SICILIA, A. TORRENT, P. CASTRO, S. DENIZ, A. ARENCIBIA, E. R. JACOBSON, B. L. HOMER Veterinary Record (2001) 149, 21-23 J. Or6s, DVM, PhD, J. Sicilia, DVM, A. Torrent, DVM, P. Castro, A. Arencibia, DVM, PhD, Department of Histology and Pathology, S. Deniz, DVM, PhD, Department of Infectious Diseases, Veteriniary Faculty ULPGC, Trasmontana s/n, 35416 Arucas (Las Palmas), Spain E. R. Jacobson, DVM, PhD, Department of Small Animal Clinical Sciences, B. L. Homer, DVM, PhD, Department of Pathobiology, College of Veterinary Medicine, University of Florida, PO Box 100126, Gainesville, FL 32610-0126, USA IN 1972, a respiratory epizootic spread through a collection of fer- de-lance snakes (Bothrops atrox) at a serpentarium in Switzerland. Although the disease was originally thought to be bacterial in origin, a paramyxo-like virus was isolated (Clark and others 1979). Subsequently, it was reported that the affected species was Bothrops moojeni, not B atrox (Foelsch and Leloup 1982). In 1979, the first outbreak of the disease was reported in viperids in the USA, in a private collection of rock rattlesnakes (Crotalus lepidus) in Tallahassee, Florida, and since that time, numerous outbreaks have been described in collec- tions of snakes in the USA, Mexico and Germany (Jacobson and others 1981, 1992, Blahak and others 1991 ). Since then, ophid- ian paramyxovirus (oPMv) has been identified as a very important pathogen in both viperid and non-viperid snakes. The disease is characterised by proliferative pneumonia, with or without interstitial inflammation (Jacobson and oth- ers 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 cul- ture, demonstration of the virus by electron microscopy, demonstration of rising titres 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 undertaken to confirm OPMV infection as the 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 from pulmonary infection as a result of paramyxovirus. Tissues were selected from 17 snakes belonging to four pri- vate collections in Las Palmas de Grand Canaria that experi- enced mortalities attributed to respiratory infection between 1995 and 1999. The species studied were a western diamond- back rattlesnake (Crotalus atrox), pygmy rattlesnake (Sistrurus miliarius), rhinoceros viper (Bitis gabonica rhinoceros), palm viper (Bothrops species), anaconda (Eunectes murinus), brown tree snake (Boiga irregularis), indigo snake (Drymarchon corais), Asian rat snake (Gonyosoma oxycephala), emerald tree boa (Corallus canina), black rat snake (Elaphe obsoleta), two ball pythons (Python regius), two Burmese pythons (Python molu- rus bivittatus) and three king snakes (Lampropeltis getulus). At necropsy, a variety of pulmonary gross lesions were detected in all the snakes, including diffuse to focal accumu- lations of caseous necrotic cellular debris, diffuse haemor- rhage of the lung and air sac system, and oedema and severe diffuse congestion of the lung. Moderate multifocal exudative to granulomatous pneumonia was observed in five snakes, including a ball python, the palm viper, the pygmy rattle- snake, the Asian rat snake and the anaconda. The liver was enlarged and pale in the western diamondback rattlesnake, brown tree snake, rhinoceros viper and palm viper. Tissue samples were fixed in 10 per cent neutral buffered formalin and embedded in paraffin. Histological changes in affected lungs included hyperplasia and hypertrophy of septal and faveolar epithelial cells, loss of ciliated cells, mixed leucocytic 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 intracyto- plasmic inclusion bodies or 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. Micro- biological analysis had confirmed isolation of Aeromonas species, Proteus species and Morganella morganii from the lungs of the anaconda, emerald tree boa, pygmy rattlesnake, palm viper, Asian rat snake, black rat snake, a king snake and a ball python. A mild diffuse depletion of splenic lymphoid tissue was also observed in a king snake, a ball python and the anaconda. No significant lesions were observed in other organs. For immunohistochemical labelling, sequential 4 Mtm sec- tions of lung, liver, spleen and pancreas were adhered to poly- L-lysine-coated slides. Sections were deparaffinised and immersed in 3 per cent hydrogen peroxide in absolute methanol for 30 minutes. They were then rinsed in phosphate- buffered saline (PBS) and placed in a solution of 0-1 per cent protease at pH 7-2 for five minutes at room temperature. The FIG 1: Immunoperoxidase red labelling for ophidian paramyxovirus antigen of the luminal bronchial epithelium of a rhinoceros viper (Bitis gabonica rhinoceros). x 20 The Veterinary Record, July 7, 2001 group.bmj.com on August 18, 2011 - Published by veterinaryrecord.bmj.com Downloaded from