James F. X. Wellehan

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.

Characterization of phylogenetically diverse astroviruses of marine mammals

Astroviruses are small, non-enveloped, positive-stranded RNA viruses. Previously studied mammalian astroviruses have been associated with diarrhoeal disease. Knowledge of astrovirus diversity is very limited, with only six officially recognized astrovirus species from mammalian hosts and, in addition, one human and some bat astroviruses were recently described. We used consensus PCR techniques for initial identification of five astroviruses of marine mammals: three from California sea lions (Zalophus californianus), one from a Steller sea lion (Eumetopias jubatus) and one from a bottlenose dolphin (Tursiops truncatus). Bayesian and maximum-likelihood phylogenetic analysis found that these viruses showed significant diversity at a level consistent with novel species. Astroviruses that we identified from marine mammals were found across the mamastrovirus tree and did not form a monophyletic group. Recombination analysis found that a recombination event may have occurred between a human and a California sea lion astrovirus, suggesting that both lineages may have been capable of infecting the same host at one point. The diversity found amongst marine mammal astroviruses and their similarity to terrestrial astroviruses suggests that the marine environment plays an important role in astrovirus ecology.

Cetacean Morbillivirus: Current Knowledge and Future Directions

We review the molecular and epidemiological characteristics of cetacean morbillivirus (CeMV) and the diagnosis and pathogenesis of associated disease, with six different strains detected in cetaceans worldwide. CeMV has caused epidemics with high mortality in odontocetes in Europe, the USA and Australia. It represents a distinct species within the Morbillivirus genus. Although most CeMV strains are phylogenetically closely related, recent data indicate that morbilliviruses recovered from Indo-Pacific bottlenose dolphins (Tursiops aduncus), from Western Australia, and a Guiana dolphin (Sotalia guianensis), from Brazil, are divergent. The signaling lymphocyte activation molecule (SLAM) cell receptor for CeMV has been characterized in cetaceans. It shares higher amino acid identity with the ruminant SLAM than with the receptors of carnivores or humans, reflecting the evolutionary history of these mammalian taxa. In Delphinidae, three amino acid substitutions may result in a higher affinity for the virus. Infection is diagnosed by histology, immunohistochemistry, virus isolation, RT-PCR, and serology. Classical CeMV-associated lesions include bronchointerstitial pneumonia, encephalitis, syncytia, and lymphoid depletion associated with immunosuppression. Cetaceans that survive the acute disease may develop fatal secondary infections and chronic encephalitis. Endemically infected, gregarious odontocetes probably serve as reservoirs and vectors. Transmission likely occurs through the inhalation of aerosolized virus but mother to fetus transmission was also reported.

Marine mammal zoonoses: a review of disease manifestations.

Marine mammals evoke strong public affection as well as considerable scientific interest. However, the resultant close contact with marine wildlife poses human health risks, including traumatic injury and zoonotic disease transmission. The majority of zoonotic marine mammal diseases result in localized skin infections in man that resolve spontaneously or with appropriate medical therapy. However, other marine mammal zoonoses, if left untreated, induce life‐threatening systemic diseases that could pose public health risks. As the number of zoonotic diseases rises, the diagnosis of and treatment for these emerging pathogens pose special challenges requiring the expertise of physicians, veterinarians and wildlife biologists. Here, we provide a comprehensive review of the bacterial, viral and fungal marine mammal zoonotic diseases that we hope will be utilized by public health professionals, physicians, veterinarians and wildlife biologists to better understand, diagnose and prevent marine mammal zoonotic diseases.

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.

DNA Fingerprinting of Pasteurella multocida Recovered from Avian Sources

ABSTRACT Repetitive sequence-based PCR (rep-PCR) and amplified fragment length polymorphism (AFLP) were used to characterize a sample of 43 field isolates and 4 attenuated vaccine strains of Pasteurella multocida recovered from multiple avian species. Both rep-PCR and AFLP assays were rapid and reproducible, with high indices of discrimination. Concordance analyses of rep-PCR and AFLP with somatic serotyping indicate that, in general, somatic serotyping is a poor indicator of genetic relatedness among isolates of P. multocida. In addition, the data provide evidence of host specificity of P. multocida clones. Overall, the results of our study indicate that the rep-PCR and AFLP techniques enable rapid fingerprinting of P. multocida isolates from multiple avian species and enhance the investigation of fowl cholera outbreaks.

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.

IRON STORAGE DISEASE IN CAPTIVE EGYPTIAN FRUIT BATS (ROUSETTUS AEGYPTIACUS): RELATIONSHIP OF BLOOD IRON PARAMETERS TO HEPATIC IRON CONCENTRATIONS AND HEPATIC HISTOPATHOLOGY

Abstract This study evaluated the relationship between blood iron parameters and hepatic iron concentrations, and correlation of histologic findings with hepatic iron concentrations in a captive population of Egyptian fruit bats (Rousettus aegyptiacus) and island flying foxes (Pteropus hypomelanus). Blood samples were collected for complete blood counts, plasma biochemical profiles, serum iron concentrations, total iron-binding capacity, whole-blood lead concentrations, and plasma ferritin assays. Liver samples obtained by laparotomy were divided, with one half processed for histologic examination and the other half frozen and submitted for tissue mineral analysis. The histologic sections were scored by two blinded observers for iron deposition, necrosis, and fibrosis. The Egyptian fruit bats had significantly higher liver iron (mean = 3,669 ± 1,823 ppm) and lead (mean = 8.9 ± 5.8 ppm) concentrations than the island flying foxes (mean [Fe] = 174 ± 173 ppm, mean [Pb] = 1.9 ± 0.5 ppm). Hepatic iron concentrations significantly correlated with tissue lead concentrations, histologic grading for iron and necrosis, serum iron, transferrin saturation, and plasma ferritin (P < 0.001). Blood lead concentrations negatively correlated with tissue lead concentrations (P < 0.001). When the product of transferrin saturation and serum iron was greater than 51, an individual animal had a high probability of having iron overload. When the product of these two variables was greater than 90, there was a high probability that the animal had hemochromatosis. On the basis of this study, it appears that evaluation of serum iron, transferrin saturation, and plasma ferritin are useful and noninvasive methods for diagnosis of hemochromatosis in Egyptian fruit bats.

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.