Detection of herpesvirus-like and papillomavirus-like particles associated with diseases of tortoises

DURING September and October of 1996, the Central Veterinary Laboratory (CVL) received samples of liver and spleen tissue from Hermann's tortoises (Testudo hermanni) and leopard tortoises (Geochelone pardalis) from a tortoise breeding colony in south Yorkshire. The colony had experienced an outbreak of disease associated with clinical signs of ocular and nasal discharge, and intermittent paralysis eventually resulting in 100 per cent mortality in one group of 29 individuals, some of which had succumbed to the disease and some of which were killed humanely on welfare grounds. Gross postmortem findings included emaciation, muscle wasting, thickening of coelomic membranes and enlargement of the liver and spleen. Histopathology revealed evidence of widespread lymphoproliferative changes affecting various organs and also pancreatitis and tracheitis. These findings will be reported in detail elsewhere. The samples reported on here were all submitted to the CVL by the owner of the tortoise colony on the advice of his veterinary surgeon. In November of the same year, samples of lung wash and oral scrapings from a Horsfield's tortoise (Testudo horsfieldii) with stomatitis were submitted by a practice in Mid Glamorgan for examination by negative-contrast electron microscopy. This tortoise subsequently died. All samples were processed for electron microscopy. If immediate processing was not possible, the samples were stored frozen at -70'C. Processing was carried out using standard procedures as follows. Twenty per cent (w/v) homogenised tissue suspensions were prepared in deionised water. Following clarification by centrifugation at 720 g for five minutes, the supernatants were concentrated by ultracentrifugation at 32,000 g for one hour at 4°C. The resulting pellets were resuspended in a minimal volume of deionised water before being examined by electron microscopy. Herpesvirus-like particles were seen in nine of the 12 samples submitted from the south Yorkshire colony-(Fig 1). All the submitted spleen samples demonstrated the presence of these particles. No particles were seen in three of the six submitted liver samples. The average size of the particles was 105 nm in diameter and the typical surface structure and icosahedral symmetry of herpesviruses was clearly visible. In the lung wash sample, papillomavirus-like particles were seen (Fig 2) with an average particle diameter of 49 nm. No virions were detected in the oral scrapings. Tissues for attempted virus isolation were prepared from the liver and spleen samples by making 20 per cent (w/v) suspensions in antibiotic phosphate buffered saline. After being allowed to stand for 60 minutes these were clarified by centrifugation at 720 g for five minutes for inoculation. The filtered supematants were inoculated onto a variety of cell cultures: tortoise embryo fibroblast, chick embryo fibroblast, chick embryo liver, and cultures of a cell line of Fat Head Minnow (purchased from the European Cell Culture Collection). The inoculated cultures were incubated at a temperature of 31°C for seven days for each of three passages. The tortoise embryo fibroblast cultures were prepared from 51-day-old leopard tortoise embryos cultured by standard procedures (Schat and Purchase 1989). No bacteria were cultured from the inoculum and no visible cytopathic effects were noted in any of the culture systems used.