PROTEIN ELECTROPHORESIS OF PLASMA SAMPLES FROM BOA CONSTRICTORS WITH AND WITHOUT REPTARENAVIRUS INFECTION.

Reptarenaviruses infect a variety of boid and pythonid snake species worldwide and have been shown to be the cause of inclusion body disease (IBD). Little is known about the correlations between virus infection and clinical disease, as well as the effects of viral infection on the immune system and the blood protein fractions. The goal of this study was to examine the differences in the plasma protein fractions in reptarenavirus reverse transcription polymerase chain reaction (RT-PCR)-negative and -positive tested snakes with and without clinical signs of disease. Blood from a total of 111 boa constrictors (Boa constrictor) was evaluated. Reverse transcription PCRs and H&E staining for inclusion bodies were carried out on each sample for the detection of reptarenavirus, and the plasma protein fractions were evaluated by capillary zone electrophoresis (CZE). Thirty four of the 111 evaluated snakes were positive by RT-PCR and 19 of the 34 showed clinical signs of disease. In comparison with IBD-negative healthy boa constrictors, the positive snakes with clinical signs had significantly lower albumin levels (P = 0.0052), lower A: G ratios (P = 0.0037), and lower α-globulin levels (P = 0.0073), while their γ-globulin levels were significantly higher (P = 0.0004). In the same comparison, clinically healthy arenavirus-positive boas showed only significantly lower α-globulin (P = 0.0124) and higher γ-globulin levels (P = 0.0394). The results of the present study indicate that reptarenavirus infection may influence plasma protein fractions in boa constrictors.

DETECTION OF AN ARENAVIRUS IN A GROUP OF CAPTIVE WAGLER'S PIT VIPERS (TROPIDOLAEMUS WAGLERI).

A group of eight Wagler's pit vipers (Tropidolaemus wagleri) from a private collection died with respiratory signs within 6 mo of one another. The group consisted of an adult breeding pair that was wild caught and six offspring from this pair. Four of the dead snakes were submitted for gross and histopathology. Signs of bacterial pneumonia were detected in all four examined snakes. No inclusion bodies suggestive of viral infection were found in any of the examined tissues. Polymerase chain reactions for the detection of ferla-, adeno-, reo-, and nidoviruses were all negative, but reptarenaviruses closely related to viruses previously described in boa constrictors (Boa constrictor) with inclusion body disease were detected in two of the four snakes. This is the first description of reptarenaviruses in viperid snakes. The pathogenic role of the virus in illness is unknown.

Comparison of three different PCR protocols for the detection of ferlaviruses.

BACKGROUND: Ferlaviruses are important pathogens in snakes often associated with respiratory and neurological disease. The detection of ferlaviral RNA by PCR is considered to be the most reliable method for the diagnosis of infection. The PCRs that have been used most commonly for this purpose have not been properly assessed to determine their sensitivity, specificity and ability to detect the known genetic diversity of this group of viruses. The aim of this study was to compare three published PCR protocols so that a single method could be recommended to laboratories that perform this testing. RESULTS: Comparisons were carried out using cell culture isolates and tissues from snakes infected with specific virus genotypes. A single round PCR targeting a short segment of the viral polymerase (L) gene provided the highest sensitivity and specificity, and detected isolated ferlaviruses from all four described genogroups, as well as from tissues of infected snakes. CONCLUSION: A broadly-reactive PCR for the detection of all known ferlaviruses was found to provide a good combination of detection limit, specificity and speed. Based on these criteria, this method is recommended for the diagnosis of ferlavirus infections.

DETECTION OF OPHIDIOMYCES OPHIODIICOLA IN TWO CAPTIVE BOCOURT WATER SNAKES ( SUBSESSOR BOCOURTI) AND ONE CAPTIVE PUEBLAN MILK SNAKE ( LAMPROPELTIS TRIANGULUM CAMPBELLI).

Two captive Bocourt water snakes ( Subsessor bocourti) presented with chronic white skin lesions on their heads; Ophidiomyces ophiodiicola was identified by culture and polymerase chain reaction (PCR) in skin scrapings from both snakes. Histopathology performed in one Bocourt water snake revealed fungal hyphae in epidermal structures of lesions. One Pueblan milk snake ( Lampropeltis triangulum campbelli) from the same zoologic institution presented with yellow crusts and white blisters on its body, from which O. ophiodiicola was identified by culture and PCR. Two of the three snakes apparently recovered from lesions after multiple natural sheds, whereas the third snake died. This is the first report of O. ophiodiicola infection in Bocourt water snakes and in a Pueblan milk snake, as well as the first report of O. ophiodiicola in France.

DETECTION OF INTRANUCLEAR COCCIDIOSIS IN TORTOISES IN EUROPE AND CHINA.

Intranuclear coccidiosis of tortoises (TINC) has been described in association with systemic disease in various species of tortoises. TINC has been detected in numerous tortoises from the United States, but there are only a few reports from tropical tortoises in Germany and no reports from Asia. Using a real-time polymerase chain reaction assay, samples from 1,011 tortoises were screened for the presence of TINC. Samples originated from animals kept in captivity in Europe and in China. Coccidia were detected in a total of 27 chelonians (2.7%), including the first description of TINC in a marginated tortoise ( Testudo marginata ), Hermann's tortoise ( Testudo hermanni ), African spurred tortoise (Centrochelys sulcata), and yellow-footed tortoise (Chelonoidis denticulatus). The highest percentage of positive animals was found in radiated tortoises ( Astrochelys radiata ). Although the percentage of positive animals was relatively low, this study demonstrates the global distribution of TINC in captive chelonians as well as expanding the known host range for these pathogens.

Detection of nidoviruses in live pythons and boas. / Nachweis von Nidoviren bei lebenden Pythons und Boas.

OBJECTIVES: Nidoviruses have recently been described as a putative cause of severe respiratory disease in pythons in the USA and Europe. The objective of this study was to establish the use of a conventional PCR for the detection of nidoviruses in samples from live animals and to extend the list of susceptible species. MATERIALS AND METHODS: A PCR targeting a portion of ORF1a of python nidoviruses was used to detect nidoviruses in diagnostic samples from live boas and pythons. A total of 95 pythons, 84 boas and 22 snakes of unknown species were included in the study. Samples tested included oral swabs and whole blood. RESULTS: Nidoviruses were detected in 27.4% of the pythons and 2.4% of the boas tested. They were most commonly detected in ball pythons (Python [P.] regius) and Indian rock pythons (P. molurus), but were also detected for the first time in other python species, including Morelia spp. and Boa constrictor. Oral swabs were most commonly tested positive. CONCLUSION: The PCR described here can be used for the detection of nidoviruses in oral swabs from live snakes. These viruses appear to be relatively common among snakes in captivity in Europe and screening for these viruses should be considered in the clinical work-up. CLINICAL RELEVANCE: Nidoviruses are believed to be an important cause of respiratory disease in pythons, but can also infect boas. Detection of these viruses in live animals is now possible and can be of interest both in diseased animals as well as in quarantine situations.

Detection of Mycoplasma spp., herpesviruses, topiviruses, and ferlaviruses in samples from chelonians in Europe.

We tested samples from 1,015 chelonians in Europe for Mycoplasma spp., herpesviruses, ranaviruses, picornaviruses, and ferlaviruses by PCR. Mycoplasma spp. were detected in 42.1% and herpesviruses were detected in 8.0% of tested chelonians. Differentiation of the herpesviruses revealed that 46.9% of the detected chelonian viruses were testudinid herpesvirus 1 (TeHV-1) and 54.3% were TeHV-3, including co-detections of TeHV-1 and -3 in 3 tortoises. TeHV-4 was detected in a leopard tortoise ( Stigmochelys pardalis), and a herpesvirus that could not be further characterized was found in a pond slider ( Trachemys scripta). Picornaviruses (topiviruses) were detected in 2.2% of the tested animals; ferlaviruses were found in 0.6%; no ranaviruses were detected in any of the animals tested. Mycoplasma spp. were detected significantly more often in Horsfield's tortoises ( Testudo horsfieldii), leopard tortoises, and Indian star tortoises ( Geochelone elegans) than in other species. Horsfield's tortoises were also significantly more often positive for TeHV-1. Mycoplasma and TeHV-1 were co-detected in 3.0%, and mycoplasma and TeHV-3 in 2.3%. The TeHV-4-positive tortoise was also positive for mycoplasma. Mycoplasma and picornaviruses were co-detected in 1.2% of the tortoises. A spur-thighed tortoise ( Testudo graeca) was positive for mycoplasma and a ferlavirus. In some cases, >2 pathogens were detected. A significant correlation between mycoplasma and herpesvirus detection was found. Of all tested animals, 47.6% were positive for at least one pathogen, demonstrating the importance of pathogen detection in captive chelonians.

Detection of testudinid herpesvirus type 4 in a leopard tortoise (Stigmochelys pardalis). / Nachweis von Testudinid-Herpesvirus Typ 4 bei einer Pantherschildkröte (Stigmochelys pardalis).

Several animals from a mixed species collection of tortoises in Germany died unexpectedly. Some of the affected leopard tortoises (Stigmochelys pardalis) from this group showed respiratory signs. Samples were collected from one of the ill tortoises, and a Mycoplasma spp. and a herpesvirus were detected by PCR. Sequencing of a portion of the DNA polymerase gene of the herpesvirus showed 99% identity with testudinid herpesvirus 4, previously described only once in a bowsprit tortoise (Chersina angulata) in the United States.