April L. Childress

Detection and Analysis of Six Lizard Adenoviruses by Consensus Primer PCR Provides Further Evidence of a Reptilian Origin for the Atadenoviruses

ABSTRACT A consensus nested-PCR method was designed for investigation of the DNA polymerase gene of adenoviruses. Gene fragments were amplified and sequenced from six novel adenoviruses from seven lizard species, including four species from which adenoviruses had not previously been reported. Host species included Gila monster, leopard gecko, fat-tail gecko, blue-tongued skink, Tokay gecko, bearded dragon, and mountain chameleon. This is the first sequence information from lizard adenoviruses. Phylogenetic analysis indicated that these viruses belong to the genus Atadenovirus, supporting the reptilian origin of atadenoviruses. This PCR method may be useful for obtaining templates for initial sequencing of novel adenoviruses.

RANAVIRUS INFECTION OF FREE-RANGING AND CAPTIVE BOX TURTLES AND TORTOISES IN THE UNITED STATES

Iridoviruses of the genus Ranavirus are well known for causing mass mortality events of fish and amphibians with sporadic reports of infection in reptiles. This article describes five instances of Ranavirus infection in chelonians between 2003 and 2005 in Georgia, Florida, New York, and Pennsylvania, USA. Affected species included captive Burmese star tortoises (Geochelone platynota), a free-ranging gopher tortoise (Gopherus polyphemus), free-ranging eastern box turtles (Terrapene carolina carolina), and a Florida box turtle (Terrepene carolina bauri). Evidence for Ranavirus infection was also found in archived material from previously unexplained mass mortality events of eastern box turtles from Georgia in 1991 and from Texas in 1998. Consistent lesions in affected animals included necrotizing stomatitis and/or esophagitis, fibrinous and necrotizing splenitis, and multicentric fibrinoid vasculitis. Intracytoplasmic inclusion bodies were rarely observed in affected tissues. A portion of the major capsid protein (MCP) gene was sequenced from each case in 2003–2005 and found to be identical to each other and to Frog virus 3 (FV3) across 420 base pairs. Ranavirus infections were also documented in sympatric species of amphibians at two locations with infected chelonians. The fragment profiles of HindIII-digested whole genomic DNA of Ranavirus, isolated from a dead Burmese star tortoise and a southern leopard frog (Rana utricularia) found nearby, were similar. The box turtle isolate had a low molecular weight fragment that was not seen in the digestion profiles for the other isolates. These results suggest that certain amphibians and chelonians are infected with a similar virus and that different viruses exist among different chelonians. Amphibians may serve as a reservoir host for susceptible chelonians. This report also demonstrated that significant disease associated with Ranavirus infections are likely more widespread in chelonians than previously suspected.

Consensus nested PCR amplification and sequencing of diverse reptilian, avian, and mammalian orthoreoviruses.

The orthoreoviruses are segmented RNA viruses that infect diverse vertebrate host species. While the most common human orthoreovirus, Mammalian Reovirus, is not typically associated with significant disease, the majority of Orthoreovirus species have been shown to cause significant and often fatal disease in reptiles, birds, and primates. There is significant potential for jumping species. A consensus nested-PCR method was designed for investigation of the RNA-dependent RNA polymerase gene of Orthoreovirus and Aquareovirus. This protocol was used to obtain sequencing template from reoviruses of three different vertebrate classes. Bayesian and maximum likelihood phylogenetic analysis found that all viruses analyzed clustered in the genus Orthoreovirus, that reptile reoviruses formed three distinct clusters, and that an African grey parrot reovirus clustered with Nelson Bay virus from bats. This PCR method may be useful for obtaining templates for initial sequencing of novel orthoreoviruses from diverse vertebrate hosts.

Characterization of an outbreak of astroviral diarrhea in a group of cheetahs (Acinonyx jubatus)

Abstract A Mamastrovirus was identified in an outbreak of diarrhea in cheetahs (Acinonyx jubatus). Five young adult and two adult cheetahs presented with lethargy, anorexia, watery diarrhea and regurgitation over an 11-day period. Fecal samples were submitted for electron microscopy and culture. Electron microscopy results revealed particles morphologically consistent with an astrovirus, and no other viral pathogens or significant bacterial pathogens were identified. The astrovirus was confirmed and sequenced using consensus astroviral PCR, resulting in a 367 base pair partial RNA-dependent-RNA polymerase (RdRp) product and a 628 base pair partial capsid product. Bayesian and maximum likelihood phylogenetic analyses were performed on both the RdRp and the capsid protein segments. All animals were monitored and treated with bismuth subsalicylate tablets (524mg PO BID for 5 days), and recovered without additional intervention. This is the first report we are aware of documenting an astrovirus outbreak in cheetah.

Systemic adenovirus infection in Sulawesi tortoises (Indotestudo forsteni) caused by a novel siadenovirus

A novel siadenovirus was identified in the Sulawesi tortoise (Indotestudo forsteni). A group of 105 Sulawesi tortoises was obtained by the Turtle Survival Alliance. Many of the tortoises were in poor health. Clinical signs included anorexia, lethargy, mucosal ulcerations and palatine erosions of the oral cavity, nasal and ocular discharge, and diarrhea. Initial diagnostic tests included fecal testing for parasites, complete blood count and plasma biochemical analysis, mycoplasma serology, and polymerase chain reaction (PCR) testing for intranuclear coccidia and chelonian herpesvirus. Treatment included administration of antibiotics, antiparasitic medications, parenteral fluids, and nutritional support. Tissue samples from animals that died were submitted for histopathologic evaluation. Histopathologic examination revealed systemic inflammation and necrosis associated with intranuclear inclusions consistent with a systemic viral infection in 35 tortoises out of 50 examined. Fecal testing results and histopathologic findings revealed intestinal and hepatic amoebiasis and nematodiasis in 31 animals. Two of 5 tortoises tested by PCR were positive for Chlamydophila sp. Aeromonas hydrophila and Escherichia coli were cultured from multiple organs of 2 animals. The mycoplasma serology and PCR results for intranuclear coccidia and chelonian herpesvirus were negative. Polymerase chain reaction testing of tissues, plasma, and choanal/cloacal samples from 41 out of 42 tortoises tested were positive for an adenovirus, which was characterized by sequence analysis and molecular phylogenetic inference as a novel adenovirus of the genus Siadenovirus. The present report details the clinical and anatomic pathologic findings associated with systemic infection of Sulawesi tortoises by this novel Siadenovirus, which extends the known reptilian adenoviruses to the chelonians and extends the known genera of reptilian Adenoviridae beyond Atadenovirus to include the genus Siadenovirus.

Intranuclear Coccidiosis in Tortoises: Nine Cases

Chelonian intranuclear coccidiosis has been reported once, in two radiated tortoises (Geochelone radiata), and is apparently rare. We describe intranuclear coccidiosis diagnosed histologically in two radiated tortoises, three Travancore tortoises (Indotestudo forstenii), two leopard tortoises (Geochelone pardalis), one bowsprit tortoise (Chersina angulata), and one impressed tortoise (Manouria impressa). Infection was systemic and involved alimentary, urogenital, respiratory, lymphoid, endocrine, and integumentary systems. Trophozoites, meronts, merozoites, macrogametocytes, microgametocytes, and nonsporulated oocysts were seen histologically or by electron microscopy. intracytoplasmic and extracellular stages of parasite development also were identified histologically. Sequencing of a coccidial 18S rRNA consensus polymerase chain reaction (PCR) product revealed a novel sequence that provided phylogenetic information and may be useful for further diagnostic test design. intranuclear coccidiosis was associated with variable degrees of inflammation in all cases, was considered the cause of death in six tortoises, and was a substantial contributing factor to the cause of death in two tortoises.

Massive visceral pentastomiasis caused by Porocephalus crotali in a dog.

The testes of a 5-year-old, male, crossbred Schnauzer dog were the indicator organs for detection of massive pentastomiasis. Necropsy revealed numerous additional encysted parasites within the mesenteric lymph nodes, omentum, liver, sub-serosa of the small and large intestines, mesentery, and lungs. The nymphs had a pseudosegmented body, containing large eosinophilic glands and a chitinous cuticle with characteristic pores. Their hook configuration was consistent with that of Porocephalus. A pentastomid-specific 18S rRNA polymerase chain reaction (PCR) was designed and used to amplify template for sequencing. The sequence of the PCR product was 99.7% homologous with the reference sequence for P. crotali. This pentastomid parasite has been reported in North American snakes of genera Crotalus and Agkistrodon. Mammals are intermediate hosts, and snakes are the definitive hosts. Porocephalus crotali has been reported in dogs only once, and molecular methods have not been used previously to identify the species in clinical pentastomiasis.

Serologic and molecular evidence for Testudinid herpesvirus 2 infection in wild Agassiz's desert tortoises, Gopherus agassizii.

Following field observations of wild Agassizs desert tortoises (Gopherus agassizii) with oral lesions similar to those seen in captive tortoises with herpesvirus infection, we measured the prevalence of antibodies to Testudinid herpesvirus (TeHV) 3 in wild populations of desert tortoises in California. The survey revealed 30.9% antibody prevalence. In 2009 and 2010, two wild adult male desert tortoises, with gross lesions consistent with trauma and puncture wounds, respectively, were necropsied. Tortoise 1 was from the central Mojave Desert and tortoise 2 was from the northeastern Mojave Desert. We extracted DNA from the tongue of tortoise 1 and from the tongue and nasal mucosa of tortoise 2. Sequencing of polymerase chain reaction products of the herpesviral DNA-dependent DNA polymerase gene and the UL39 gene respectively showed 100% nucleotide identity with TeHV2, which was previously detected in an ill captive desert tortoise in California. Although several cases of herpesvirus infection have been described in captive desert tortoises, our findings represent the first conclusive molecular evidence of TeHV2 infection in wild desert tortoises. The serologic findings support cross-reactivity between TeHV2 and TeHV3. Further studies to determine the ecology, prevalence, and clinical significance of this virus in tortoise populations are needed.

A Novel Herpesvirus of the Proposed Genus Chelonivirus from an Asymptomatic Bowsprit Tortoise (Chersina angulata)

Abstract A wild-caught Bowsprit tortoise (Chersina angulata) was received into quarantine and appeared clinically normal. Oral swabs for consensus herpesvirus polymerase chain reaction (PCR) and sequencing were obtained during routine quarantine, and a novel herpesvirus was identified. Comparative sequence analysis shows that this virus is a member of the subfamily Alphaherpesvirinae in the proposed genus Chelonivirus. Host/virus co-evolution appears to be common amongst herpesviruses and their hosts, and the most significant disease is typically seen when herpesviruses jump to related host species. Previous studies have found some diversity of herpesviruses in tortoises. This report expands the number of known herpesviruses of tortoises. It is reasonable to expect that there will be significantly different clinical consequences of different tortoise herpesviruses in different species, and that identification of host/virus relationships will aid in clinical management of tortoise collections. Further work is needed to determine the clinical implications of this and other tortoise herpesviruses in different tortoise species.

Partial characterization of a new adenovirus lineage discovered in testudinoid turtles.

In the USA and in Hungary, almost simultaneously, adenoviruses of a putative novel lineage were detected by PCR and sequencing in turtles belonging to four different species (including two subspecies) of the superfamily Testudinoidea. In the USA, partial sequence of the adenoviral DNA-dependent DNA polymerase was obtained from samples of a captive pancake tortoise (Malacochersus tornieri), four eastern box turtles (Terrapene carolina carolina) and two red-eared sliders (Trachemys scripta elegans). In Hungary, several individuals of the latter subspecies as well as some yellow-bellied sliders (T. scripta scripta) were found to harbor identical, or closely related, putative new adenoviruses. From numerous attempts to amplify any other genomic fragment by PCR, only a nested method was successful, in which a 476-bp fragment of the hexon gene could be obtained from several samples. In phylogeny reconstructions, based on either DNA polymerase or hexon partial sequences, the putative new adenoviruses formed a clade distinct from the five accepted genera of the family Adenoviridae. Three viral sub-clades corresponding to the three host genera (Malacochersus, Terrapene, Trachemys) were observed. Attempts to isolate the new adenoviruses on turtle heart (TH-1) cells were unsuccessful. Targeted PCR screening of live and dead specimens revealed a prevalence of approximately 25% in small shelter colonies of red-eared and yellow-bellied sliders in Hungary. The potential pathology of these viruses needs further investigation; clinically healthy sliders were found to shed the viral DNA in detectable amounts. Based on the phylogenetic distance, the new adenovirus lineage seems to merit the rank of a novel genus.