Enzyme-Linked Immunosorbent Assay for Detecting Herpesvirus Exposure in Mediterranean Tortoises (Spur-Thighed Tortoise [Testudo graeca] and Hermann's Tortoise [Testudo hermanni])

Abstract

Herpesviruses are well-known infectious agents with remarkably wide host ranges. Starting in 1975 (33), several reports have documented the presence of herpesvirus-like particles in land tortoises and freshwater and marine turtles (5, 7-9, 11, 15, 17, 19-23, 27-29, 30, 38). Recent investigations have revealed an association between the presence of herpesvirus and an upper respiratory tract disease in Mediterranean tortoises [spur-thighed tortoise (Testudo graeca) and Hermann's tortoise (T. hermanni)] (5,8,9,17,20,23,(27)(28)(29)30).In tortoises with herpesvirus infection, clinical signs range from a mild conjunctivitis to a severe stomatitis-glossitis and pharyngitis. Diphtheritic plaques can be observed on the dorsal surface of the tongue and on the hard palate of infected tortoises. Frequently, a clear serous to a mucopurulent nasal discharge is present. Signs of central nervous system disease have also been reported in Mediterranean tortoises with herpesvirus infection (17).Eosinophilic intranuclear inclusions, often seen in multiple tissues, are particularly prominent in tortoises with pharyngitis and glossitis. As seen with transmission electron microscopy, inclusions consist of numerous viral particles. The morphology and morphogenesis have been used to categorize the virus as herpesvirus.A diagnosis of herpesvirus infection is often made based solely upon light or electron microscopy findings. Antemortem diagnosis can be made using biopsy specimens of oral lesions. A serum neutralization (SN) test has been developed but is limited in its application since it is only available in a few research laboratories in Europe (10). In addition, time is a limiting factor with the SN test. Ten to 14 days are required to obtain the final reading and a laborious procedure is required. An easier and faster but equally reliable serodiagnostic test is needed. In this report, we describe the development of an enzyme-linked immunosorbent assay (ELISA) that can be used to monitor the exposure to herpesvirus of free-range, private, and zoo collections of tortoises. MATERIALS AND METHODSViruses. Herpesvirus isolates HV1976 and HV4295/7R/95 were used as antigens in the ELISAs and immunoblotting. HV1976 was isolated from a captive Hermann's tortoise from the United States (Washington), while HV4295/7R/95 was isolated from a captive Hermann's tortoise in Germany during a herpesvirus outbreak in a private collection (27).Antigen preparation for ELISA. The herpesvirus isolates were grown in terrapene heart cell monolayers (TH-1; ATCC-CCL 50 Sub-line B1; American Type Culture Collection, Rockville, Md.) in T-150 plastic flasks with ventilated caps (Corning, Rochester, N.Y.) for use as ELISA antigens. The TH-1 cells were grown in Dulbecco's modified Eagle's medium F12 (Gibco BRL, Grand Island, N.Y.) with 5% fetal bovine serum (Sigma, St. Louis, Mo.), gentamicin (60 mg/liter) (Sigma), penicillin G (120,000 U/liter), streptomycin (120,000 U/liter), and amphotericin B (300 g/liter) (ABAM; Sigma). Infected cell monolayers were scraped a...

Mycoplasma and Herpesvirus PCR Detection in Tortoises with Rhinitis-stomatitis Complex in Spain

“…It has been suggested that distinct herpesviruses are adapted to different species of tortoises, and Testudo spp. seem to be the most vulnerable (Origgi and Jacobson, 2000). Bacteria have also been suggested to play a role in respiratory disease in tortoises (Biberstein and Fowler, 1980;Snipes and Biberstein, 1982;Lawrence and Needham, 1985), although these have also been recovered from apparently healthy tortoises (Martinez-Silvestre and Mateu, 1997).…”

“…1). Seroconversion of tortoises to both types of ChHV (I and II) has been demonstrated 4 wk following infection (Origgi et al, 2001) but coinfection had not been previously described. Interspecific coinfection (M. agassiziiChHV) was described by Soares et al (2004) but the type of ChHV was not identified.…”

“…Additionally, 40% of samples from France and Morocco were collected from the wild. Despite reports of M. agassizii in free-living individuals Feldman et al, 2006), there are only anecdotal reports of ChHV from wild tortoises (Origgi et al, 2001;Marschang and Schneider, 2007). These results suggest that M. agassizii could be more frequent in wild and asymptomatic tortoises, while ChHV may be associated more frequently with disease in captive tortoises.…”

“…Tortoises suffer loss of weight and nasal discharge that results in a weakened physical condition and mortality (Martinez-Silvestre and Mateu, 1997). Disease is highly transmissible because foamy exudate from the external nares provides a significant source of infection for horizontal transmission (Muro et al, 1998;Origgi and Jacobson, 2000). Viral agents associated with significant pathology include iridovirus (Westhouse et al, 1996;Marschang et al, 1998;Cunningham, 2000), papilloma-like virus (Jacobson et al, 1982), poxvirus (Oró s et al, 1998) and a picorna-like virus termed virus ''x'' (Marschang and Ruemenapf, 2002).…”

“…In addition, M. agassizii has been involved in the decline of wild tortoise populations (Brown et al, 1994). Although ChHV outbreaks have been documented only in captive tortoises, this pathogen could potentially emerge in wild populations (Origgi et al, 2001;Marschang and Schneider, 2007). Captive and wild populations are connected by the popularity of tortoises as pets (Pé rez et al, 2004) and the collection and release of wild individuals is a frequent practice, mostly in the Mediterranean basin (Ló pez-Jurado et al, 1979;Curl et al, 1985;Fritts and Jennings, 1994;Waller, 1997;Pujol, 1998;Gimé nez et al, 2004;Pé rez et al, 2004).…”