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Ellen Ariel - One of the best experts on this subject based on the ideXlab platform.
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Dose-dependent morbidity of freshwater turtle hatchlings, Emydura macquarii krefftii, inoculated with Ranavirus isolate (Bohle Iridovirus, Iridoviridae).
Journal of General Virology, 2019Co-Authors: Wytamma Wirth, Lin Schwarzkopf, Lee F Skerratt, Anna Tzamouzaki, Ellen ArielAbstract:Ranaviral infections cause mass die-offs in wild and captive turtle populations. Two experimental studies were performed to first determine the susceptibility of an Australian turtle species (Emydura macquarii krefftii) to different routes of infection and second examine the effect of viral titre on the morbidity in hatchlings. All inoculation routes (intracoelomic, intramuscular and oral) produced disease, but the clinical signs, histopathology and time to onset of disease varied with the route. The median infectious and lethal doses for intramuscularly inoculated hatchlings were 10(2.52) ((1.98-2.93)) and 10(4.43) ((3.81-5.19)) TCID50 ml(-1), respectively. Clinical signs began 14 to 29 days post-inoculation and the median survival time was 22 days (16-25) across all dose groups. For every 10-fold increase in dose, the odds of developing any clinical signs or severe clinical signs increased by 3.39 [P
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dose dependent morbidity of freshwater turtle hatchlings emydura macquarii krefftii inoculated with ranavirus isolate Bohle Iridovirus iridoviridae
Journal of General Virology, 2019Co-Authors: Wytamma Wirth, Lin Schwarzkopf, Lee F Skerratt, Anna Tzamouzaki, Ellen ArielAbstract:Ranaviral infections cause mass die-offs in wild and captive turtle populations. Two experimental studies were performed to first determine the susceptibility of an Australian turtle species (Emydura macquarii krefftii) to different routes of infection and second examine the effect of viral titre on the morbidity in hatchlings. All inoculation routes (intracoelomic, intramuscular and oral) produced disease, but the clinical signs, histopathology and time to onset of disease varied with the route. The median infectious and lethal doses for intramuscularly inoculated hatchlings were 10(2.52) ((1.98-2.93)) and 10(4.43) ((3.81-5.19)) TCID50 ml(-1), respectively. Clinical signs began 14 to 29 days post-inoculation and the median survival time was 22 days (16-25) across all dose groups. For every 10-fold increase in dose, the odds of developing any clinical signs or severe clinical signs increased by 3.39 [P<0.01, 95 % confidence interval (CI): 1.81-6.36] and 3.71 (P<0.01, 95 % CI: 1.76-7.80), respectively. Skin lesions, previously only reported in ranaviral infection in lizards, were observed in the majority of intramuscularly inoculated hatchlings that developed ranaviral disease. The histological changes were consistent with those in previous reports for reptiles and consisted of necrosis at or near the site of injection, in the spleen, liver and oral cavity. Systemic inflammation was also observed, predominantly affecting necrotic organs. The estimates reported here can be used to model ranaviral disease and quantify and manage at-risk populations.
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pathogenesis of Bohle Iridovirus genus ranavirus in experimentally infected juvenile eastern water dragons intellagama lesueurii lesueurii
Veterinary Pathology, 2019Co-Authors: Alicia Maclaine, Maria J Forzan, Narges Mashkour, Jennifer L Scott, Ellen ArielAbstract:Juvenile eastern water dragons (Intellagama lesueurii lesueurii) are highly susceptible to infection with Bohle Iridovirus (BIV), a species of ranavirus first isolated from ornate burrowing frogs i...
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Discovery of an Australian Chelonia mydas papillomavirus via green turtle primary cell culture and qPCR.
Journal of Virological Methods, 2018Co-Authors: Narges Mashkour, Alicia Maclaine, G W Burgess, Ellen ArielAbstract:The number of reptilian viruses detected are continuously increasing due to improvements and developments of new diagnostic techniques. In this case we used primary cell culture and qPCR to describe the first Australian Chelonia mydas papillomavirus. Commercial chelonian cell lines are limited to one cell line from a terrestrial turtle (Terrapene Carolina). To establish primary cultures from green turtles (Chelonia mydas), turtle eggs were collected from Heron Island, Queensland, Australia. From day 35 of incubation at 29°, the embryos were harvested to establish primary cultures. The primary cell cultures were grown in Dulbecco's Modified Eagle Medium, 90% and foetal bovine serum, 10%. The cells became uniformly fibroblastic-shaped after 15 passages. The growth rate resembled that of cells originating from other cold-blooded animals and the average doubling time was ∼5 days from the 20th passage. Karyotyping and molecular analysis of mitochondrial DNA D-loop gene were carried out for cell authentication. The primary cell cultures were screened to exclude mycoplasma contamination. Two primary cell lineages were found to be susceptible to Bohle Iridovirus. The primary cell cultures were used to screen samples from green turtles foraging along the East Coast of Queensland for the presence of viruses. Homogenates from eight skin tumour samples caused cytopathic effects and were confirmed by qPCR to be infected with papillomavirus.
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Serological survey of Australian native reptiles for exposure to ranavirus
Diseases of Aquatic Organisms, 2017Co-Authors: Ellen Ariel, E. Elliott, Jonathan Meddings, J. Miller, M.b. Santos, Leigh OwensAbstract:Ranaviruses have been isolated from many ectothermic vertebrates, and serological surveys of both amphibians and reptiles have shown the presence of ranaviral antibodies in a proportion of these populations. An enzyme-linked immunosorbent assay (ELISA) was developed to measure serum antibodies against ranavirus in Australian reptiles. The ELISA was validated with serum from challenge trials with Bohle Iridovirus (BIV) in 6 reptilian species. A preliminary serosurvey of northern Queensland riparian reptile fauna (saw-shelled turtles Myuchelys latisternum, Krefft's river turtles Emydura macquarii krefftii, freshwater crocodiles Crocodylus johnstoni, as well as the snakes Boiga irregularis, Dendrelaphis punctulatus, Tropidonophis mairii, Morelia spilota, Liasis childreni and L. fuscus) revealed evidence of past exposure to Bohle iridoviral antigens in part of the population at several locations sampled. Furthermore, in Krefft's river turtles and freshwater crocodiles, a statistically significant trend was apparent for larger reptiles to be more likely to have BIV-reactive sera than smaller individuals. The use of adult tortoise populations as sentinels can assist in monitoring the presence of BIV in northern Australian freshwater streams, and thereby the potential dangers to native fauna from this agent.
Alex D Hyatt - One of the best experts on this subject based on the ideXlab platform.
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Assessment of virally vectored autoimmunity as a biocontrol strategy for cane toads
PLOS ONE, 2011Co-Authors: Jackie A. Pallister, Nicole A Siddon, Damien C.t. Halliday, Anthony J. Robinson, Daryl Venables, Rhonda D. Voysey, Donna G. Boyle, Thayalini Shanmuganathan, Christopher M. Hardy, Alex D HyattAbstract:Background The cane toad, Bufo (Chaunus) marinus, is one of the most notorious vertebrate pests introduced into Australia over the last 200 years and, so far, efforts to identify a naturally occurring B. marinus-specific pathogen for use as a biological control agent have been unsuccessful. We explored an alternative approach that entailed genetically modifying a pathogen with broad host specificity so that it no longer caused disease, but carried a gene to disrupt the cane toad life cycle in a species specific manner. Methodology/Principal Findings The adult beta globin gene was selected as the model gene for proof of concept of autoimmunity as a biocontrol method for cane toads. A previous report showed injection of bullfrog tadpoles with adult beta globin resulted in an alteration in the form of beta globin expressed in metamorphs as well as reduced survival. In B. marinus we established for the first time that the switch from tadpole to adult globin exists. The effect of injecting B. marinus tadpoles with purified recombinant adult globin protein was then assessed using behavioural (swim speed in tadpoles and jump length in metamorphs), developmental (time to metamorphosis, weight and length at various developmental stages, protein profile of adult globin) and genetic (adult globin mRNA levels) measures. However, we were unable to detect any differences between treated and control animals. Further, globin delivery using Bohle Iridovirus, an Australian ranavirus isolate belonging to the Iridovirus family, did not reduce the survival of metamorphs or alter the form of beta globin expressed in metamorphs. Conclusions/Significance While we were able to show for the first time that the switch from tadpole to adult globin does occur in B. marinus, we were not able to induce autoimmunity and disrupt metamorphosis. The short development time of B. marinus tadpoles may preclude this approach.
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Bohle Iridovirus as a vector for heterologous gene expression
Journal of Virological Methods, 2007Co-Authors: Jackie Pallister, Sarah Goldie, Barbara E H Coupar, Brian J Shiell, Wojtek P Michalski, Nicole A Siddon, Alex D HyattAbstract:Abstract The large double-stranded DNA (ds DNA) viruses were among the first to be used to construct recombinant viruses, but to date this has not been achieved with any members of the ds DNA virus family, Iridoviridae. We identified a non-essential gene, the viral homologue of eukaryotic initiation factor 2α (eIF-2α), in Bohle Iridovirus (BIV, genus Ranavirus). A recombinant BIV was constructed with the neomycin resistance gene and the Bufo marinus (cane toad) adult globin gene inserted into the BIV eIF-2α region. Adult globin expressed by the virus was detected on western blot, demonstrating that foreign genes can be expressed by the recombinant BIV in vitro and suggesting the possibility of using a recombinant BIV in the biological control of cane toads.
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Development of real-time PCR assays for the detection and differentiation of Australian and European ranaviruses.
Journal of Fish Diseases, 2007Co-Authors: Jackie Pallister, Alex D Hyatt, A Gould, D Harrison, James K. Jancovich, Hans G. HeineAbstract:Serious systemic disease in fish and amphibians is associated with the ranaviruses, epizootic haernatopoietic necrosis virus (EHNV) and Bohle Iridovirus (BIV) in Australia, and European sheattish virus (ESV) and European catfish virus (ECV) in Europe. EHNV, ESV and ECV are recognized causative agents of the OlE (Office International des Epizooties) notifiable systemic necrotizing Iridovirus syndrome and are currently identified by proteinbased assays, none of which are able to rapidly identify the specific agents. The aim of this study was to develop T aqMan real-time PCR assays that differentiated these viruses using nucleotide sequence variation in two ranavirus genes. A conserved probe representing 100% sequence homology was used as a reference for virus-specific probes. The virus-specific probes produced a similar signal level to the conserved probe while those probes binding to non-target viral DNA produced an altered fluorescent curve. The pattern of probe binding was characteristic for each virus. Sensitivity, specificity and dynamic range of the assay were assessed. The test is currently useful as a research and initial screening tool, with the potential to become a sensitive and specific method for detection and differentiation of ranaviruses with further development.
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promoter activity in the 5 flanking regions of the Bohle Iridovirus icp 18 icp 46 and major capsid protein genes
Archives of Virology, 2005Co-Authors: Jackie Pallister, Barbara E H Coupar, S. Goldie, Alex D HyattAbstract:Bohle Iridovirus (BIV) belongs to the genus Ranavirus, of which Frog virus 3 (FV-3) is the type species. We are developing BIV as a recombinant viral delivery vector, and as a first step we located specific BIV promoter sequences to drive foreign gene expression in the recombinant virus. By comparison with FV-3 sequences, the genes encoding ICP 18 and ICP 46 in BIV were identified and sequenced. Putative promoter regions of these two early genes and of the major capsid protein (MCP) gene were identified, cloned into pSFM21, and luciferase production was then used to assess the promoter activity of these regions.
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Special topic review: Iridoviruses associated with epizootic haematopoietic necrosis (EHN) in aquaculture : Virus diseases in aquaculture
World Journal of Microbiology & Biotechnology, 1997Co-Authors: W. Ahne, Alex D Hyatt, M. Bremont, R.p. Hedrick, Richard J WhittingtonAbstract:Systemic infections of teleost fishes caused by Iridoviruses have recently been recognized in Australia, Asia, Europe and the USA. These Iridoviruses are different from those of the established genera Lymphocystivirus and Goldfish Virus 1-like Viruses of the family Iridoviridae. The agents exhibit similar physicochemical properties, are antigenically related and prove to be of high virulence to different teleost fishes in aquaculture. The first Iridovirus, epizootic haematopoietic necrosis virus, responsible for an epizootic outbreak of haematopoietic necrosis in redfin perch, was reported in Australia. Some years later, similar Iridovirus epizootics occurred in sheatfish and catfish in Europe. The Australian and the European isolates proved to be antigenically related and showed properties in common with frog virus 3, the type species of the genus Ranavirus of the Iridoviridae. Further Iridovirus isolates from fish, amphibians and reptiles exhibited a close relationship with each other and with frog virus 3. It is important to note that the Australian amphibian Iridovirus, Bohle Iridovirus, was experimentally transmitted to teleost fish inducing high mortalities. The occurrence of similar viruses in different host species in the aquatic environment and their inter-species transmission emphasize the importance of health control in aquaculture.
Leigh Owens - One of the best experts on this subject based on the ideXlab platform.
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Serological survey of Australian native reptiles for exposure to ranavirus
Diseases of Aquatic Organisms, 2017Co-Authors: Ellen Ariel, E. Elliott, Jonathan Meddings, J. Miller, M.b. Santos, Leigh OwensAbstract:Ranaviruses have been isolated from many ectothermic vertebrates, and serological surveys of both amphibians and reptiles have shown the presence of ranaviral antibodies in a proportion of these populations. An enzyme-linked immunosorbent assay (ELISA) was developed to measure serum antibodies against ranavirus in Australian reptiles. The ELISA was validated with serum from challenge trials with Bohle Iridovirus (BIV) in 6 reptilian species. A preliminary serosurvey of northern Queensland riparian reptile fauna (saw-shelled turtles Myuchelys latisternum, Krefft's river turtles Emydura macquarii krefftii, freshwater crocodiles Crocodylus johnstoni, as well as the snakes Boiga irregularis, Dendrelaphis punctulatus, Tropidonophis mairii, Morelia spilota, Liasis childreni and L. fuscus) revealed evidence of past exposure to Bohle iridoviral antigens in part of the population at several locations sampled. Furthermore, in Krefft's river turtles and freshwater crocodiles, a statistically significant trend was apparent for larger reptiles to be more likely to have BIV-reactive sera than smaller individuals. The use of adult tortoise populations as sentinels can assist in monitoring the presence of BIV in northern Australian freshwater streams, and thereby the potential dangers to native fauna from this agent.
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pathogenicity in six australian reptile species following experimental inoculation with Bohle Iridovirus
Diseases of Aquatic Organisms, 2015Co-Authors: Ellen Ariel, Jennifer L Scott, Wytamma Wirth, G W Burgess, Leigh OwensAbstract:Ranaviruses are able to infect multiple species of fish, amphibian and reptile, and some strains are capable of interclass transmission. These numerous potential carriers and reservoir species compound efforts to control and contain infections in cultured and wild populations, and a comprehensive knowledge of susceptible species and life stage is necessary to inform such processes. Here we report on the challenge of 6 water-associated reptiles with Bohle Iridovirus (BIV) to investigate its potential pathogenicity in common native reptiles of the aquatic and riparian fauna of northern Queensland, Australia. Adult tortoises Elseya latisternum and Emydura krefftii, snakes Boiga irregularis, Dendrelaphis punctulatus and Amphiesma mairii, and yearling crocodiles Crocodylus johnstoni were exposed via intracoelomic inoculation or co-habitation with infected con-specifics, but none were adversely affected by the challenge conditions applied here. Bohle Iridovirus was found to be extremely virulent in hatchling tortoises E. latisternum and E. krefftii via intracoelomic challenge, as demonstrated by distinct lesions in multiple organs associated with specific immunohistochemistry staining and a lethal outcome (10/17) of the challenge. Virus was re-isolated from 2/5 E. latisternum, 4/12 E. krefftii and 1/3 brown tree snakes B. irregularis. Focal necrosis, haemorrhage and infiltration of granulocytes were frequently observed histologically in the pancreas, liver and sub-mucosa of the intestine of challenged tortoise hatchlings. Immunohistochemistry demonstrated the presence of ranavirus antigens in the necrotic lesions and in individual cells of the vascular endothelium, the connective tissue and in granulocytes associated with necrosis or present along serosal surfaces. The outcome of this study confirms hatchling tortoises are susceptible to BIV, thereby adding Australian reptiles to the host range of ranaviruses. Additionally, given that BIV was originally isolated from an amphibian, our study provides additional evidence that interclass transmission of ranavirus may occur in the wild.
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influence of temperature and exposure time on the infectivity of Bohle Iridovirus a ranavirus
Aquaculture, 2012Co-Authors: K La Fauce, Ellen Ariel, Suzanne L Munns, Catherine M Rush, Leigh OwensAbstract:Abstract This study examined the functional temperature range of a ranavirus outside host cells over increasing temperatures and exposure times and subsequently tested infectivity in cell culture. Initially, cell susceptibility was determined by incubating Bohle Iridovirus (BIV) at 30 °C, 40 °C, 50 °C and 60 °C for 5, 30 and 60 min and subsequently titrating samples in one epithelioma papulosum cyprinid (EPC) and two Bluegill fry 2 (BF2) lineages at 28 °C. Titres obtained in the three cell lines were similar and EPC cells were subsequently used to further investigate ranavirus infectivity with two degree increments in temperature between 40 °C and 60 °C for 5, 30 and 60 min. The rate of inactivation was found to be dependent on temperature and time of exposure. Bohle Iridovirus could replicate in EPC cells following exposure to most temperatures and prolonged time, but titers were reduced as temperature and time of exposure increased. Viral titres were greatest (10 8 TCID 50 /ml) after exposure to 30 °C and declined with increasing time of exposure and increasing temperature. Declines in BIV infectivity were largely between 40 °C (10 8 TCID 50 /ml) and 44 °C (10 5 TCID 50 /ml at 5 and 30 min and 10 3.5 TCID 50 /ml at 60 min) and secondly at temperatures greater than 52 °C (from 10 3.5 TCID 50 /ml and approaching zero with increasing temperature and time). Treatment at 58 °C for 60 min and 60 °C for 30 and 60 min resulted in complete loss of BIV infectivity. The results from this study show that ranavirus can withstand much higher temperatures than previously thought, which is fundamental for understanding ranavirus epidemiology, indirect transmission dynamics and for biosecurity purposes.
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experimental challenge and clinical cases of Bohle Iridovirus biv in native australian anurans
Diseases of Aquatic Organisms, 2002Co-Authors: Bryan R Cullen, Leigh OwensAbstract:Ranaviruses have been observed with increasing frequency amongst poikilothermic vertebrate hosts. The impact of ranaviruses upon amphibian populations has remained largely unknown. A gene probe for Bohle Iridovirus (BIV) based upon primers designed to detect epizootic haematopoietic necrosis virus (EHNV) was constructed. A PCR and dot-blot system was used suc- cessfully in screening for the presence of BIV nucleic acid in digested formalin-fixed, paraffin- embedded amphibian tissues. Juvenile frogs were more susceptible to BIV than adults. In experi- mental challenges and epizootics in captive frogs, juvenile Litoria caerulea, L. alboguttata, Cyclorana brevipes and Pseudophryne coriacea were acutely susceptible. High mortality (at or near 100%) resulted, usually occurring within 5 to 25 d depending on dose and method of exposure. Histopatho- logical changes included mainly hepatic, renal and splenic necroses. Significant haemosiderosis was encountered in more chronically infected frogs. BIV could be reisolated from juvenile L. caerulea >40 d after inoculation, and >200 d after the first mortalities occurred in an epizootic in L. albogut- tata. Adult L. rubella, L. inermis, L. caerulea, Cophixalus ornatus and Taudactylus acutirostris were less susceptible in trials ranging from 30 to >100 d. There was some evidence of chronic infection, and BIV could be detected by PCR. Wild moribund adult L. caerulea from Townsville and captive juvenile Pseudophryne corieacea from Sydney undergoing mortality tested positive with the BIV PCR. PCR and dot blot was more sensitive than viral isolation. PCR could detect BIV in amphibians long after BIV challenge, and in amphibians which appeared healthy. Ranaviruses could be having an impact on Australian herpetofauna.
Jackie Pallister - One of the best experts on this subject based on the ideXlab platform.
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Bohle Iridovirus as a vector for heterologous gene expression
Journal of Virological Methods, 2007Co-Authors: Jackie Pallister, Sarah Goldie, Barbara E H Coupar, Brian J Shiell, Wojtek P Michalski, Nicole A Siddon, Alex D HyattAbstract:Abstract The large double-stranded DNA (ds DNA) viruses were among the first to be used to construct recombinant viruses, but to date this has not been achieved with any members of the ds DNA virus family, Iridoviridae. We identified a non-essential gene, the viral homologue of eukaryotic initiation factor 2α (eIF-2α), in Bohle Iridovirus (BIV, genus Ranavirus). A recombinant BIV was constructed with the neomycin resistance gene and the Bufo marinus (cane toad) adult globin gene inserted into the BIV eIF-2α region. Adult globin expressed by the virus was detected on western blot, demonstrating that foreign genes can be expressed by the recombinant BIV in vitro and suggesting the possibility of using a recombinant BIV in the biological control of cane toads.
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Development of real-time PCR assays for the detection and differentiation of Australian and European ranaviruses.
Journal of Fish Diseases, 2007Co-Authors: Jackie Pallister, Alex D Hyatt, A Gould, D Harrison, James K. Jancovich, Hans G. HeineAbstract:Serious systemic disease in fish and amphibians is associated with the ranaviruses, epizootic haernatopoietic necrosis virus (EHNV) and Bohle Iridovirus (BIV) in Australia, and European sheattish virus (ESV) and European catfish virus (ECV) in Europe. EHNV, ESV and ECV are recognized causative agents of the OlE (Office International des Epizooties) notifiable systemic necrotizing Iridovirus syndrome and are currently identified by proteinbased assays, none of which are able to rapidly identify the specific agents. The aim of this study was to develop T aqMan real-time PCR assays that differentiated these viruses using nucleotide sequence variation in two ranavirus genes. A conserved probe representing 100% sequence homology was used as a reference for virus-specific probes. The virus-specific probes produced a similar signal level to the conserved probe while those probes binding to non-target viral DNA produced an altered fluorescent curve. The pattern of probe binding was characteristic for each virus. Sensitivity, specificity and dynamic range of the assay were assessed. The test is currently useful as a research and initial screening tool, with the potential to become a sensitive and specific method for detection and differentiation of ranaviruses with further development.
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promoter activity in the 5 flanking regions of the Bohle Iridovirus icp 18 icp 46 and major capsid protein genes
Archives of Virology, 2005Co-Authors: Jackie Pallister, Barbara E H Coupar, S. Goldie, Alex D HyattAbstract:Bohle Iridovirus (BIV) belongs to the genus Ranavirus, of which Frog virus 3 (FV-3) is the type species. We are developing BIV as a recombinant viral delivery vector, and as a first step we located specific BIV promoter sequences to drive foreign gene expression in the recombinant virus. By comparison with FV-3 sequences, the genes encoding ICP 18 and ICP 46 in BIV were identified and sequenced. Putative promoter regions of these two early genes and of the major capsid protein (MCP) gene were identified, cloned into pSFM21, and luciferase production was then used to assess the promoter activity of these regions.
Alicia Maclaine - One of the best experts on this subject based on the ideXlab platform.
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Characterisation of ranaviral infection and its management in Australian lizards
2020Co-Authors: Alicia MaclaineAbstract:Background Reptiles are considered to be one of the most evolutionary and ecologically remarkable groups of living organisms, having successfully inhabited most of the planet including the oceans. Despite this, reptile species worldwide are on the decline due to threats such as residential and commercial development, agriculture and aquaculture, climate change, and introduction of invasive species and diseases. Approximately 19% of all assessed reptiles globally are listed as 'threatened' by the International Union for Conservation of Nature (IUCN) Red List of Threatened Species. Infectious diseases are listed as one of the top five causes of global species extinctions and one of the biggest causes of morbidity and mortality in reptiles. Ranaviruses (family Iridoviridae) have been identified as emerging pathogens of ecological significance in ectothermic vertebrates due to their expanding host and geographic range. This group of viruses infects over 175 species of ectothermic vertebrates worldwide and is listed as notifiable to the The World Organization for Animal Health (Office International des Epizootics, OIE) in amphibians and fish. The majority of ranaviral research has been conducted in amphibians with only a few surveys targeting wild reptiles despite several reported mortality events in captive lizards and turtles. Hence the aims of this thesis were to investigate the susceptibility and pathogenesis of Ranavirus sp. in juvenile eastern water dragons (Intellagama lesueurii lesueurii); determine if Ranavirus sp. is present in Australian lizards; and to identify and understand Australian reptile owners experience and management of disease in captive reptile collections. The susceptibility of an Australian semi-aquatic lizard to Bohle Iridovirus In Chapter 2 we investigated the susceptibility of juvenile eastern water dragons to a local ranavirus isolate (Bohle Iridovirus, BIV) via oral inoculation, intramuscular injection, and cohabitation with orally infected lizards. This lizard species was investigated as they share habitat with several fish, amphibians and reptiles shown to be susceptible to BIV. A range of tissues (spleen, kidney, lung, liver, kidney, gastrointestinal tract, heart, tongue, brain, and bone marrow) were collected for histopathology, and liver and kidney samples were also collected for viral isolation and polymerase chain reaction (PCR). The outcome of this study demonstrated that juvenile eastern water dragons are susceptible to BIV via all exposure methods and have the ability to infect naive individuals. These findings add another ectotherm to the list of species susceptible to ranavirus. The pathogenesis of Bohle Iridovirus infection in juvenile eastern water dragons In order to investigate the pathogenesis of BIV in this host, juvenile eastern water dragons were orally infected with BIV and euthanized at pre-determined time-points (Chapter 3). Tissue samples were collected for histopathology, immunohistochemistry (ISH), in-situ hybridization (ISH), viral isolation and PCR. The findings from this study identified the progression of BIV infection which appeared to start in the spleen, followed by the liver, then the other organs. Ranaviral DNA was detected by PCR in liver, kidney and cloacal swabs at 3 days post infection, suggesting cloacal swabs could be a reliable source of diagnostic sampling in BIV-infected lizards. Histopathology changes were observed in the liver and tongue at 3 days post infection and IHC identified viral antigen in the spleen at 6 days post infection. The ISH labelling of skin, bone marrow, liver, pancreas, stomach, intestine and spleen matched the location and pattern detected by IHC. Infection was well underway before clinical signs were observed. Molecular detection of Ranavirus sp. in captive and wild Australian lizards Wild and captive Australian lizards from northern Queensland, New South Wales and Australian Capital Territory were surveyed for ranaviral DNA using combined oral-cloacal swabs and PCR (Chapter 4). Ranaviral DNA was detected in samples from 4/123 asymptomatic captive lizards and 5/63 asymptomatic wild lizards. These PCR-positive samples belonged to three central bearded dragons (Pogona vitticeps) and one frilled neck lizard (Chlamydosaurus kingii) from two different captive collections, and five wild eastern water dragons from Paluma Range National Park, Queensland. Amplicons from this study shared 100% nucleotide identity with the cognate regions of BIV and four other ranaviruses and were only one base different to the cognate region of epizootic haematopoietic necrosis virus, an Australian ranavirus that affects fish and is listed as notifiable to the OIE. The detection of ranavirus in asymptomatic lizards in both captivity and in the wild introduces the possibility of carrier lizards and highlights importance of disease management strategies (e.g. quarantine). The health and wellbeing of Australian pet reptiles An online survey (SurveyMonkey®) of Australian reptile owners was conducted between November and December 2017 (Chapter 5). This cross-sectional study consisted of multiple choice and open-ended questions. Quantitative data were analysed descriptively using frequencies, mean, median, standard deviation, range, and interquartile range. Open-ended question responses were analysed thematically and grouped into themes. The average age of participants was 34 years old with snakes and lizards the most popular reptile kept in captivity. Most participants cleaned enclosures weekly, disinfected enclosures monthly, and used UVA/UVB lights, heat lamps and multivitamin supplements to prevent health problems within their collection. Quarantine periods were employed by 72% of participants for an average of 4 weeks, with only 30% physically isolating the animal. Disease knowledge was limited to non-infectious diseases such as metabolic bone disease. Barriers to seeking veterinary assistance for unwell reptiles included cost and perceived lack of knowledge/experience on the veterinarians' part. Findings from this survey identified the need for more readily available resources for Australian reptile keepers including access to information on diseases and experienced veterinarians. Outcomes This research has identified eastern water dragons as a susceptible species to ranaviral infection and provides further evidence of the ability of ranaviruses to infect a wide range of ectothermic vertebrates. The detection of ranavirus in asymptomatic wild and captive lizards suggests the possibility that ranavirus is circulating in the wild and is part of the normal microflora of Australian lizards. This also identifies lizards as a potential host that can spread and amplify ranaviruses in the wild. Further investigation is required to characterize the ranavirus found in this study, and molecular and serum surveys of wild and captive populations. Furthermore, the detection of ranavirus in captive lizards combined with the results from the survey of Australian reptile owners highlights the need for more readily available resources on disease identification, prevention, and treatment.
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pathogenesis of Bohle Iridovirus genus ranavirus in experimentally infected juvenile eastern water dragons intellagama lesueurii lesueurii
Veterinary Pathology, 2019Co-Authors: Alicia Maclaine, Maria J Forzan, Narges Mashkour, Jennifer L Scott, Ellen ArielAbstract:Juvenile eastern water dragons (Intellagama lesueurii lesueurii) are highly susceptible to infection with Bohle Iridovirus (BIV), a species of ranavirus first isolated from ornate burrowing frogs i...
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Discovery of an Australian Chelonia mydas papillomavirus via green turtle primary cell culture and qPCR.
Journal of Virological Methods, 2018Co-Authors: Narges Mashkour, Alicia Maclaine, G W Burgess, Ellen ArielAbstract:The number of reptilian viruses detected are continuously increasing due to improvements and developments of new diagnostic techniques. In this case we used primary cell culture and qPCR to describe the first Australian Chelonia mydas papillomavirus. Commercial chelonian cell lines are limited to one cell line from a terrestrial turtle (Terrapene Carolina). To establish primary cultures from green turtles (Chelonia mydas), turtle eggs were collected from Heron Island, Queensland, Australia. From day 35 of incubation at 29°, the embryos were harvested to establish primary cultures. The primary cell cultures were grown in Dulbecco's Modified Eagle Medium, 90% and foetal bovine serum, 10%. The cells became uniformly fibroblastic-shaped after 15 passages. The growth rate resembled that of cells originating from other cold-blooded animals and the average doubling time was ∼5 days from the 20th passage. Karyotyping and molecular analysis of mitochondrial DNA D-loop gene were carried out for cell authentication. The primary cell cultures were screened to exclude mycoplasma contamination. Two primary cell lineages were found to be susceptible to Bohle Iridovirus. The primary cell cultures were used to screen samples from green turtles foraging along the East Coast of Queensland for the presence of viruses. Homogenates from eight skin tumour samples caused cytopathic effects and were confirmed by qPCR to be infected with papillomavirus.
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susceptibility of eastern water dragons intellagama lesueurii lesueurii to Bohle Iridovirus
Diseases of Aquatic Organisms, 2017Co-Authors: Alicia Maclaine, Narges Mashkour, Jennifer L Scott, Ellen ArielAbstract:Ranaviruses infect and have been associated with mass mortality events in fish, amphibians and reptiles and are capable of interclass transmission. Eastern water dragons (EWDs), a semi-aquatic squamate, have an overlapping distribution with several species shown to be susceptible to Bohle Iridovirus (BIV). However, this species has not been previously investigated, and no known mass mortalities have occurred in wild populations. Here we report the experimental infection of juvenile EWDs with BIV to investigate a water-dwelling lizards’ susceptibility to a ranaviral strain present in northern Queensland, Australia. Lizards were exposed via oral inoculation, intramuscular injection, or cohabitation with orally infected lizards. All exposure methods were effective in establishing an infection as demonstrated by skin lesions and pathological changes in the internal organs. Necrosis, haemorrhage and inflammation were observed histologically in the pancreas, liver, spleen, kidney and submucosa of the gastrointestinal tract of BIV-exposed lizards. Variably sized basophilic intracytoplasmic inclusion bodies were observed in the liver of 6/14 BIV-exposed lizards. Virus was isolated from the liver and kidney of all BIVinfected lizards and confirmed with quantitative PCR (qPCR). The outcome of this study demonstrates that juvenile EWDs are susceptible to BIV, thereby adding Australian lizards to the broad host range of ranaviruses. Furthermore, this study provides additional evidence of BIV’s ability to infect different classes of ecothermic vertebrates.
