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Detection of dwarf gourami iridovirus (Infectious spleen and kidney necrosis virus) in populations of ornamental fish prior to and after importation into Australia, with the first evidence of infection in domestically farmed Platy (Xiphophorus maculatus)

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Title: Detection of dwarf gourami iridovirus (Infectious spleen and kidney necrosis virus) in populations of ornamental fish prior to and after importation into Australia, with the first evidence of infection in domestically farmed Platy (Xiphophorus maculatus) Detection of dwarf gourami iridovirus (Infectious spleen and kidney necrosis virus) in populations of ornamental fish prior to and after importation into Australia, with the first evidence of infection in domestically farmed Platy (Xiphophorus maculatus).Preventive Veterinary Medicine http://dx.

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... All of the ornamental fish species tested were positive for iridovirus through two-step polymerase chain reaction (PCR). Paradise fish (Macropodus opercularis) imported from Indonesia [31], common platy, pearl gourami, zebrafish, swordtail, ram cichlid from major ornamental fish breeding states in peninsular Malaysia [32], platys X. maculatus from Australia [33], angelfish Pterophyllum altum imported from Colombia, platys X. maculatus Available at www.veterinaryworld.org/Vol.13/November-2020/37.pdf of unknown origin [5], X. hellerii, X. maculatus, P. sphenops, T. trichopterus [4], Poecilia reticulata, T. leeri, Apistogramma ramirezi [3] from Southern Malaysia, zebrafish Danio rerio from a research facility in Spain [34], and three marine ornamental fish, Banggai cardinalfish P. kauderni [21], orbiculate batfish Platax orbicularis [35], and majestic angelfish Pomacanthus navarchus [2] have reported positive for megalocytiviruses. Other ornamental fish species from around the world that have been detected for megalocytiviruses infection since 1989 until 2019 are listed in Table-1 [1][2][3][4][5]15,16,[20][21][22][25][26][27][30][31][32]34,[36][37][38][39][40][41]. ...
... Megalocytivirus infection is known to cause non-specific clinical signs in infected ornamental fish, similar to clinical signs that are common for other diseases [13,42]. The most common external clinical signs observed due to Megalocytivirus infection (Table-2) [1][2][3][4][5]13,15,16,[21][22][23]25,28,31,38,42] and mortality [5,13,35,42] have been reported in many studies. An unusual white fecal was produced by P. kauderni from California before the fish died [21]. ...
... Megalocytivirus-infected fish have been reported to be asymptomatic or apparently healthy [1,3,4,30,34,35]. The absence of specific clinical signs in ornamental fish has made it difficult for farmers to detect Megalocytivirus; therefore, Megalocytivirus-positive fish often exported overseas during the asymptomatic carrier stage [3,4]. ...
Article
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Iridoviruses, especially megalocytiviruses, are related to severe disease resulting in high economic losses in the aquaculture industry worldwide. The ornamental fish industry has been affected severely due to Megalocytivirus infections. Megalocytivirus is a DNA virus that has three genera; including red sea bream iridovirus, infectious spleen and kidney necrosis virus, and turbot reddish body iridovirus. Megalocytivirus causes non-specific clinical signs in ornamental fish. Cell culture, histology, immunofluorescence test, polymerase chain reaction (PCR) assay, and loop-mediated isothermal amplification assay have been used to diagnose megalocytiviruses. Risk factors such as temperature, transportation (export and import), and life stages of ornamental fish have been reported for the previous cases due to Megalocytivirus infections. In addition, other prevention and control methods also have been practiced in farms to prevent Megalocytivirus outbreaks. This is the first review of megalocytiviruses in ornamental fish since its first detection in 1989. This review discusses the occurrences of Megalocytivirus in ornamental fish, including the history, clinical signs, detection method, risk factors, and prevention measures.
... Outbreaks have occurred most frequently in East and South-East Asia (Subramaniam et al. 2012). The international trade of ornamental fish has aided the spread of these viruses to Europe (Sriwanayos et al. 2013) and Australia (Rimmer et al. 2015). Disease is more common in summer when the water temperature is higher; market-sized fish are susceptible, but disease is more severe in juveniles where mortality can reach 100% (Kurita & Nakajima 2012). ...
... This evidence confirmed links between the origin of infection and the trade in live ornamental fish in Australia . Although considered exotic to Australia, ISKNV-like megalocytivirus was detected by qPCR in both imported dwarf gourami prior to quarantine and in domestic ornamental fish at wholesaler premises and at one ornamental fish farm (Rimmer et al. 2015). ...
... All reactions were in duplicate, and a positive and notemplate control was included in each run. For paraffin-embedded tissue samples, DNA was tested neat and diluted 1:10 as described by Rimmer et al. (2015). Threshold cycle (Ct) values were determined with MxPro quantitative PCR software (Stratagene). ...
Article
Megalocytiviruses cause high mortality diseases that have seriously impacted aquaculture, with the most frequent outbreaks occurring in East and South-East Asia. The international trade of juvenile fish for food and ornamental aquaculture has aided the spread of these viruses, which have spread to Europe and Australia and other regions. Australian freshwater fishes were examined for susceptibility to infection with the exotic megalocytivirus, dwarf gourami iridovirus (DGIV), which belongs to a group with the type species, Infectious spleen and kidney necrosis virus (ISKNV). Fish were held at 23 ± 1 °C and challenged by intraperitoneal (IP) injection or by cohabitation with Murray cod, Maccullochella peelii (Mitchell) infected with DGIV. A species was deemed to be susceptible to DGIV based on evidence of viral replication, as determined by qPCR, and megalocytic inclusion bodies observed histologically. Horizontal transmission occurred between infected Murray cod and golden perch, Macquaria ambigua (Richardson), Macquarie perch, Macquaria australasica (Cuvier) and Murray cod. This indicated that DGIV shed from infected fish held at 23 °C can survive in fresh water and subsequently infect these naïve fish. Further, DGIV administered IP was highly pathogenic to golden perch, Macquarie perch and Murray cod. Compared to these species, the susceptibility of southern pygmy perch, Nannoperca australis (Gunther) was lower. Freshwater catfish (dewfish), Tandanus tandanus (Mitchell), were not susceptible under the experimental conditions based on the absence of clinical disease, mortality and virus replication. This study showed the potential risks associated with naïve and DGIV-infected fish sharing a common water source.
... In the affected gouramis, there were areas of degeneration and necrosis in the spleen, kidney and liver. Diffuse necrosis along with a relative increase in the red pulp of the spleen, as observed in the present study, has been reported in ISKNV-affected fishes previously (Bermúdez et al., 2018;Fraser et al., 1993;Ramírez-Paredes et al., 2020;Rimmer et al., 2015). Although hypertrophied basophilic cells were observed in the spleen of affected gourami, these were not observed frequently in contrast to earlier reports (Fraser et al., 1993;Sukenda et al., 2020). ...
... The degeneration of renal tubules with cast formation, as observed in the present study, has earlier been reported in Asian seabass (Dong et al., 2017). However, in contrast, renal tubules have been reported to largely remain unaffected in gourami and zebrafish (Bermúdez et al., 2018;Rimmer et al., 2015). Although a few basophilic cells were observed in the kidney, hypertrophied cells typical of ISKNV (Bermúdez et al., 2018;Jung-Schroers et al., 2016;Sukenda et al., 2020) were not observed in the present study, in accordance with an earlier report (Dong et al., 2017). ...
... The international trade of fish for food and ornamental aquaculture has facilitated the spread of viruses like SVCV (Goodwin, 2002), KHV (Haenen et al., 2004), CyHV-2 (Becker et al., 2014), DGIV (Rimmer et al., 2015) and ISKNV (Nolan et al., 2015) to distant regions. Thus, the live fish trade has been identified as a biosecurity risk despite relatively stringent import quarantine measures being applied in various countries. ...
Article
Megalocytivirus cause diseases that have serious economic impacts on aquaculture, mainly in East and South-East Asia. Five primary genotypes are known: infectious spleen and kidney necrosis virus (ISKNV), red sea bream iridovirus (RSIV), turbot reddish body iridovirus (TRBIV), threespine stickleback iridovirus (TSIV) and scale drop disease virus (SDDV). ISKNV-mediated infectious spleen and kidney necrosis disease (ISKND) is a major viral disease in both freshwater and marine fish species. In this study, we report the isolation of ISKNV from diseased giant gourami, Osphronemus goramy, in India. Transmission electron microscopy of ultrathin sections of kidney and spleen revealed the presence of numerous polygonal naked viral particles having an outer nucleocapsid layer within the cytoplasm of enlarged cells (115–125 nm). Molecular and phylogenetic analyses confirmed the presence of ISKNV and the major capsid protein (MCP) (1,362 bp) gene in the infected fish had a high similarity to the other ISKNV-I isolates. Moreover, ISKNV was propagated in the Astronotus ocellatus fin (AOF) cell line and further confirmed genotypically. A high mortality rate (60%) was observed in gourami fish injected with ISKNV-positive tissue homogenate through challenge studies. Considering the lethal nature of ISKNV, the present study spotlights the implementation of stringent biosecurity practices for the proper control of the disease in the country.
... Australia is considered to have a highly regulated biosecurity system with stringent controls, compared with global standards for importing live fish (Whittington & Chong, 2007). However, despite these controls, there have been several incursions of exotic aquatic pathogens of international significance, including Infectious spleen and kidney necrosis virus (ISKNV) (Rimmer et al., 2015), Cyprinid herpesvirus 2 (Becker et al., 2014;Stephens, Raidal, & Jones, 2004) and ...
... Real-time PCR (qPCR) assays provide the high sensitivity required for surveillance and are compatible with rapid, high-throughput laboratory methods although there is insufficient validation data to estimate the diagnostic sensitivity and diagnostic specificity of these tests (Gias, Johnston, Keeling, Spence, & McDonald, 2011;Lin et al., 2017;Rimmer, Becker, Tweedie, & Whittington, 2012b). These assays were considered the best available surveillance tools and were required as part of an emergency disease response situation in Australia (Mohr et al., 2015;Rimmer et al., 2015). ...
... an exotic pathogen to Australia following intensive biosecurity containment measures undertaken in response to (a) an epidemic mortality event at a land-based recirculating aquaculture facility producing food fish (Lancaster, Williamson, & Schroen, 2003) and (b) numerous subclinical detections of the virus at domestic retailers and one ornamental fish breeding facility (Rimmer et al., 2015). In response to these virus detections, the Australian Department of Agriculture and Water Resources (DAWR) published a risk assessment stating the import controls for the importation of freshwater fish belonging to the cichlid, gourami and poeciliid families did not meet Australia's appropriate level of protection for ISKNV (Australian Department of Agriculture, 2014b). ...
Article
Movements of large volumes and species varieties make the ornamental fish industry a high-risk pathway for the transfer of aquatic pathogens to new geographical regions and naïve hosts, potentially resulting in emergency disease events. Infectious spleen and kidney necrosis virus (genus Megalocytivirus) is considered exotic to Australia despite documented incursions since 2003. There are current import controls requiring freedom from infection for entry to Australia. The objective was to evaluate the effect of tissue pooling strategies for qPCR testing using a SYBR® assay for freedom from ISKNV at 2% expected prevalence with 95% confidence. Tissue homogenates from apparently healthy imported ornamental fish were tested as individuals and in pools of 5 and 10. Analytical sensitivity of the qPCR assay was reduced by two orders of magnitude when the nucleic acid extraction process was accounted for by spiking the plasmid in fish tissues and compared with molecular grade water. Diagnostic sensitivity of the assay was substantially reduced when testing tissues in pools compared with individual testing. For Population 1 (66% positive for ISKNV with moderate viral loads), surveillance sensitivity was only achieved using individual testing. For Population 2 (100% positive ISKNV with high viral loads), surveillance sensitivity was achieved using 260 fish in pools of 10 for a total of 26 tests or 200 fish in pools of 5 for 40 tests. Surveillance sensitivity could be maximized even when there was a reduction in pooled diagnostic sensitivity compared with diagnostic sensitivity for individual fish by increasing the sample size. Pooled sensitivity was influenced by the prevalence and variable virus load among fish with subclinical infections. Pooled testing is highly effective when the prevalence is >10% which should be informed by prior knowledge or pooling can be used for a screening test to rapidly identify populations with high prevalence.
... The live ornamental fish trade is a known pathway for the introduction of invasive fish, parasites, and pathogens, globally (Whittington and Chong, 2007;Lymbery et al., 2014;Becker et al., 2014;Rimmer et al., 2015;Trujillo-Gonzaĺez et al., 2018a). Globalization, advances in technology and transport capability have facilitated the translocation of fish from remote locations and increased the number and volume of species traded. ...
... Trade of live animals at this scale presents the potential for introduction of invasive fish species and their associated parasites to endemic ecosystems (Lymbery et al., 2014;Sheath et al., 2015;Trujillo-Gonzaĺez et al., 2018a), with important impacts on endemic wildlife, farmed fish species and natural resources (Knight, 2010;Lymbery et al., 2014;Gallardo et al., 2016). For this reason, governments may establish biosecurity protocols to detect and prevent the translocation of hazardous parasites and pathogens though the live ornamental trade (Whittington and Chong, 2007;Rimmer et al., 2015;Trujillo-Gonzaĺez et al., 2018b). ...
Article
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The ornamental fish trade provides a pathway for the global translocation of aquatic parasites. Myxozoa is comprised of highly specialized metazoan parasites of aquatic hosts with a wide host range. Interest in the group has intensified along with the development of aquaculture due to emergent pathogenic myxozoan species in both freshwater and marine environments. However, little is known on myxozoan diversity in the ornamental fish trade. We examined 630 ornamental fish imported from Asia to Australia (representing 24 fish populations, including freshwater and wild caught marine fish species) for myxozoan parasites during 2015. Fish were sampled under Australian quarantine following veterinary certification that they showed no clinical signs of pests and diseases from the exporting country and visual inspection at Australian border control. Myxozoan parasites infected 8 of 12 freshwater populations and 8 of 12 marine fish populations. A total of 12 morphologically distinct Myxobolus spores were detected amongst all goldfish, Carassius auratus populations. Myxidium spores were detected in kissing gourami, Helostoma temminckii, and Ceratomyxa sp. spores were detected in cardinal fishes, Cheilodipterus quinquelineatus, Pterapogon kauderni, and Zoramia leptocantha. Kudoa sp. spores were detected in C. quinquelineatus, Sphaeramia nematoptera and Z. leptocantha. Results of this study show that Australian pre-export health requirements and visual inspections do not reliably detect myxozoan infections. Inspection prior to exportation and at border control should account for the highly cryptic nature of myxozoan parasites and consider alternative detection methods to complement inspections at border control.
... Many species important to aquaculture are susceptible to infection and in surveys, PCR test results have revealed an average prevalence of 15% across 100 species of apparently healthy wild caught marine fish in China (Wang et al., 2007). These viruses have also been detected in apparently healthy freshwater fish in Australia, Malaysia and Korea, including important ornamental species from families: Cichlidae and Poeciliidaes (Jeong et al., 2008;Rimmer et al., 2015;Subramaniam et al., 2014a). Although extensive surveys have not been reported for TRBIV, the potential for persistent subclinical infection was indicated by a transmission study with several susceptible species of fish (Oh et al., 2006). ...
... Movement of subclinically infected fish is a pathway for introduction of disease to new regions, and regulation of trade requires strategies to detect this state of infection. For example, DGIV from subclinically infected gourmai was transmissible to Murray cod; these ornamental fish had passed quarantine inspection prior to importation into Australia, and the same virus caused a disease outbreak in farmed Murray cod Lancaster et al., 2003;Rimmer et al., 2015). Subclinical infection of marine ornamental fish originating from Indonesia, Banggai cardinalfish (Pterapogon kauderni) and orbiculate batfish (Platax orbicularis) also succumbed to disease shortly after international transport (Sriwanayos et al., 2013;Weber et al., 2009). ...
Chapter
Iridoviruses are large, double-stranded DNA viruses with a host range restricted to insects and ectothermic vertebrates. Viruses of the family Iridoviridae are emerging pathogens that are widespread in a diverse range of environments and hosts. The family includes numerous virus species belonging to the genera Ranavirus, Lymphocystivirus and Megalocytivirus. These species are the cause of important diseases characterized by mortality with impacts on the production, conservation and welfare of fish. These pathogens are the focus of biosecurity and regulatory controls because the threat to naïve fish populations with an expanded geographic distribution would have severe impacts on aquaculture production. The nomenclature of this family is confusing and the taxonomy is under review as knowledge of the epidemiology and techniques for diagnosis and control is rapidly developed.
... In 2015, Rimmer et al. [13] found a positive result in several species of fish. The majority of the animals with positive results in the PCR were apparently healthy and had a low viral load, which indicates that certain species are potential reservoirs of the virus [13]. ...
... In 2015, Rimmer et al. [13] found a positive result in several species of fish. The majority of the animals with positive results in the PCR were apparently healthy and had a low viral load, which indicates that certain species are potential reservoirs of the virus [13]. ...
Article
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The genus Megalocytivirus belongs to the family Iridoviridae and due to the high mortality rates among infected animals, represents a significant threat to world aquaculture. The affected fish present general and nonspecific symptoms, which makes the early diagnosis of the infection difficult. Nine samples from ornamental fish of the species Poecilia reticulata and Pygocentrus nattereri with nonspecific symptomatology were submitted to molecular diagnosis for Megalocytivirus through the PCR technique. Infection by this genus was detected in 100% of the analyzed samples, and three of them were sent for sequencing. All samples showed identity values of 100% with the Megalocytivirus Sabah/RAA1/2012 strain BMGIV48 described in Malaysia. These results indicate, for the fir
... The worldwide market for live ornamental fish accounts for 1.5 billion fish yearly, including 2000 different species [15,19]. The extensive trade of a large variety of fish has increased the introduction of pathogens to new geographical regions and hosts [19]. ...
... The worldwide market for live ornamental fish accounts for 1.5 billion fish yearly, including 2000 different species [15,19]. The extensive trade of a large variety of fish has increased the introduction of pathogens to new geographical regions and hosts [19]. ...
Article
Full-text available
Megalocytiviruses have a worldwide distribution, causing serious economic loss to the global aquaculture industry. They also present a threat to ornamental fish trade because megalocytiviral infections have unspecified symptoms, making early diagnosis difficult. In this study, 100 ornamental fish from 24 different species were tested by PCR for megalocytivirus, with a 47% positive rate being identified. Phylogenetic reconstruction, based on the major capsid protein (MCP) gene, clustered all Brazilian samples into a single clade, showing identity values ranging from 99% to 100% when compared to each other. This is the first report of megalocytivirus infection in some ornamental fish species in Brazil.
... non-native; Lymbery et al. 2014) fishes and diseases (e.g. Lintermans 2004; Whittington and Chong 2007;Albins and Hixon 2008;Rimmer et al. 2015). ...
... A database of historical aquarium fish imports to Australia with a high degree of taxonomic resolution would be of benefit where an emergent biosecurity concern is identified among species of a risk group (e.g. Becker et al. 2014;Rimmer et al. 2015;Trujillo-González et al. 2018b). This would allow DAWR to retrospectively determine the total quantity of consignments imported to Australia containing the species from the pathway of concern. ...
Article
Context Biological resource use represents the most common direct threat to biodiversity. Despite this, there is a paucity of comprehensive and overarching data relating to the biological resource use. The global aquarium trade encompasses millions of individual live fishes representing thousands of marine and freshwater species traded on an annual basis. The lack of specific data systems for recording information where fish are exported or imported has resulted in limited accessible trade data. An evaluation of the data-reporting frameworks presently employed by countries engaged in the aquarium trade is warranted to better understand the means by which comprehensive data on the aquarium trade can be made more accessible. Aims This study examines the data-reporting framework of The Australian Government Department of Agriculture and Water Resources (DAWR) used to collate aquarium fish import data, and its capacity to inform on the aquarium trade biodiversity imported to Australia. Methods Aquarium import records from 2010–16 were provided by DAWR and used to determine the quantity of individual fishes and consignments imported to Australia. The potential biodiversity of imports was determined from the Australian Government’s List of Permitted Live Freshwater/Marine Fish Suitable for Import 2018 (Number 69, F2017C00079), the legislative document identifying species permitted import to Australia for the aquarium trade. Species permitted import were cross-referenced with the International Union for Conservation of Nature (IUCN) Red List to address whether the Australian aquarium trade is importing threatened species. Key results A total of 10320 consignments encompassing more than 78.6 million aquarium fishes were imported to Australia between 2010 and 2016. A total of 4628 species of fishes were permitted import to Australia for the aquarium trade with 73 of the marine species (2.0%) and 81 of the freshwater species (7.5%) found to be threatened with some degree of extinction risk. The data-reporting framework for aquarium fish imports offered limited capacity to taxonomically differentiate imports and only 12.5% of all aquarium fishes imported could be identified to species. Conclusions The aquarium fish import records provided by DAWR had limited taxonomic resolution and, consequently, limited capacity to contribute to an improved understanding of the biodiversity imported to Australia for the aquarium fish trade. While more detailed information is available than is presently collated by DAWR, the availability of this information is constrained by the laws around protected information and the resources available to DAWR. Implications Accessible, detailed information on aquarium fish imports is necessary to support comprehensive research capable of addressing threats to biodiversity loss from the aquarium trade. To this end, the means by which Australian aquarium import data can be reported at greater taxonomic resolution under the existing legislative and resource restraints should be explored further.
... parasitic nematode Spirocamallanus istiblenni Noble, 1966, in Gaither et al. 2013, and multiple examples in Lymbery et al. 2014) and aquaculture industries (e.g. Cyprinid herpesvirus 2 in Becker et al. 2014; Infectious spleen and kidney necrosis virus in Rimmer et al. 2015). ...
... If no signs of disease or infection are visually detected and all documentation is in order, fish are then transported to Approved Arrangement Facilities for mandatory pre-import quarantine periods before they are released by Australian quarantine and sold in the aquarium market (DAWR 2018). Nonetheless, pathogens considered to present a risk to biosecurity can go undetected despite stringent biosecurity (Rimmer et al. 2015;Trujillo-González et al. 2018), highlighting the need for more sensitive screening methods that can identify high risk shipments and subclinical infections. ...
Article
Full-text available
The global ornamental fish trade enables translocation of exotic aquatic pathogens. In many countries, health certification and visual inspection of imported fish are key components of biosecurity to prevent the introduction of aquatic diseases. However, infected fish do not always exhibit clinical or behavioural signs of disease, and alternatives to visual inspection must be validated. This study examined the use of environmental DNA (eDNA) to detect sub-clinical parasite infections at border control. We simulated the export process of live ornamental fish in which non-infected fish, infected fish, treated fish, and non-infected fish held in contaminated water were packaged and delivered in 48 h. Quantitative PCR (qPCR) was used to detect eDNA of an ectoparasitic monogenean, Neobenedenia girellae, infecting barramundi, Lates calcarifer. The qPCR assay did not reliably detect parasite eDNA under 2 copies/µL from fish with sub-clinical infections (mean parasite intensity = 6.80 ± 4.78 S.D.), suggesting parasite infection loads tested in this study may be too low for reliable detection within the timeframe used to export live ornamental fish. Quantitative PCR tests detected parasite eDNA in 50% of infected fish and 70% of non-infected fish in contaminated transport water. This indicated a high plausibility of false negative detections because of low eDNA concentrations in transport water and false positive detections of DNA from dead parasites in the water. Environmental DNA screening methods for border control biosecurity must overcome limitations posed by low eDNA concentrations in the water, limited timeframes for sample processing, and the essential differentiation between live parasite infections and dead, non-viable parasites.
... However, SACIV and TSGIV have unique genome compositions, such as a duplicated MCP gene, and so are classified as belonging to TRBIV genotype Clade 2 (Go et al. 2016, Koda et al. 2018). Since the late 1990s, MCVs derived from ornamental fish have been reported as ISKNV genotype Clade 1 or 2 (Go et al. 2016), but due to the limited number of genes used for comparison, such as MCP and/or ATPase, MCVs in ornamental fish, especially those from ISKNV genotype Clade 1, have been difficult to differentiate from ISKNV derived from food-fish species (Sudthongkong et al. 2002b, Go et al. 2006, Jeong et al. 2008b, Mohr et al. 2015, Rimmer et al. 2015. Previously, RSIV-Ku derived from red sea bream had been classified as ISKNV genotype Clade 1 using the MCP and ATPase gene sequences, but subsequent whole genome studies indicate that this virus is an ISKNV/RSIV recombinant containing a limited genome region (representing approximately 7% of the whole genome) that is most closely related to RSIV genotype Clade 2 (Shiu et al. 2018), thus demonstrating the importance of obtaining the whole genome sequence. ...
... Finally, since sub-clinical MCV infections have been regularly detected in apparently healthy ornamental fish (Rimmer et al. 2015, Mohr et al. 2015, it could be speculated that ornamental fish represent the natural hosts/reservoirs of MCVs and the mass mortalities of food-fish species observed over the past several decades originated from MCV infections of these natural reservoirs. Thus MCV-infected ornamental fish could pose a risk to an extremely broad range of fish species as suggested by others (Go & Whittington 2006, Jeong et al. 2008a, Rimmer et al. 2017. ...
Article
Using cultures of the SKF-9 cell line, megalocytivirus AFIV-16 was isolated from imported angelfish Pterophyllum scalare held in quarantine at the Australian border. The cytopathic effect caused by isolate AFIV-16 presented as cell rounding and enlargement, but complete destruction of the infected cell cultures did not occur. The infected cells demonstrated immunocytochemical reactivity with monoclonal antibody M10, which is used for diagnosis of OIE-listed red sea bream iridoviral disease. Using electron microscopy, the virus particles, consisting of hexagonal nucleocapsids, were observed in the cytoplasm of SKF-9 cells. The replication of AFIV-16 in cultured SKF-9 cells was significantly greater at 28°C incubation than at 22 and 25°C incubation, whereas no difference in growth characteristics was observed for red sea bream iridovirus (RSIV) isolate KagYT-96 across this temperature range. Whole genome sequencing demonstrated that AFIV-16 has a 99.96% similarity to infectious spleen and kidney necrosis virus (ISKNV), the type species in the genus Megalocytivirus. AFIV-16 was classified into ISKNV genotype Clade 1 by phylogenetic analysis of the major capsid protein gene nucleotide sequence. This is the first report of whole genome sequencing of an ISKNV genotype megalocytivirus isolated from ornamental fish.
... All fish used were considered free of megalocytiviruses on the basis that this group of viruses remains exotic to Australia in cultured food fish and in wild fish species (Go & Whittington 2006, Rimmer et al. 2015. Additionally, for all species, samples of kidney and/or splenic samples from all fish that died during quarantine were tested for megalocytivirus using quantitative real-time polymerase chain reaction (qPCR) to further verify the absence of megalocytivirus. ...
... Consequently, megalocytiviruses from ornamental fish have the potential to pose a much greater biosecurity risk than has currently been accepted. There is the potential for release of megalocytiviruses from ornamental fish culture facilities such as the facility in which ISKNV was detected in ornamental farmed platies Xiphophorus maculatus (Rimmer et al. 2015) to surrounding freshwater environ-ments, and, through their capacity to infect euryhaline species, such as Australian bass, to spread megalocytivirus to marine ecosystems and marine aquaculture facilities. ...
Article
The Australian native marine fish species, silver sweep Scorpis lineolata, is susceptible to the megalocytivirus Infectious spleen and kidney necrosis virus (strain DGIV-10) obtained from a freshwater ornamental fish, dwarf gourami Trichogaster lalius. This was demonstrated by direct inoculation and through cohabitation. Transmission by cohabitation was also demonstrated from inoculated freshwater Murray cod Maccullochella peelii to euryhaline Australian bass Macquaria novemaculeata and to marine silver sweep. The virus was also transmitted from infected marine silver sweep to euryhaline Australian bass and then to freshwater Murray cod. This study is the first to demonstrate the virulence of a megalocytivirus derived from ornamental fish in an Australian marine species and the first to show a feasible pathway for the exchange of megalocytiviruses between freshwater and marine finfish hosts. These results demonstrate that megalocytiviruses from freshwater ornamental fish have the potential to spread to diverse aquatic environments.
... Recently, ISKNV was reported from exotic ornamental fishes with subclinical infection from India (Girisha et al., 2021). Global live ornamental fish trade without the compliance of appropriate pathogen screening and quarantine serves as the major reason for the transboundary spread of ISKNV (Jung-Schroers et al., 2016;Rimmer et al., 2015). (Armstrong & Ferguson, 1989). ...
... Regardless of the genetic diversity that exists among ISKNV strains present in India, its detection in wild pearlspot underpins the importance of biosecurity in aquaculture to evade the probable spreading of the virus in the wild.Etroplus suratensis is one of the three cichlid species endemic to the Indian peninsular region. ISKNV is known to infect other exotic cichlids such as Oscar (Astronotus ocellatus), angel fish (Pterophyllum scalare) and Nile tilapia (Oreochromis niloticus)(Ramírez-Paredes et al., 2021;Rimmer et al., 2015). Some of these exotic cichlid species are cultured and traded in India for ornamental and food purposes. ...
Article
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A large‐scale mortality of pearlspot, Etroplus suratensis was reported from Peechi Dam, an artificial tropical lake made for irrigation and drinking water supply in Kerala, India during 2018. This dam is located in the premises of Western Ghats, recognized as one of the biodiversity hotspots of the world. The objective of this study was to identify the aetiological agent of this large‐scale mortality of E. suratensis by systematic diagnostic investigation and identification of pathogen. Virus isolation was carried out on a species‐specific pearlspot fin (PSF) cell line. Infected PSF cells showed cytopathic effects (CPE) like cell shrinkage, rounding, enlargement, clustering, and subsequent detachment of cells with a high viral titre of 106⋅95 TCID50 mL−1 at 8 days post inoculation (dpi). Histopathological examination of the fish showed the presence of numerous abnormal enlarged basophilic cells and intracytoplasmic eosinophilic inclusions in the liver. Moreover, transmission electron microscopy (TEM) analysis revealed the presence of large numbers of 125–132 nm viral particles in the spleen tissues. PCR amplification and phylogenetic analysis of the major capsid protein (MCP) gene sequence confirmed that the causative agent was Infectious spleen and kidney necrosis virus (ISKNV) of the genus Megalocytivirus. The experimental infection recorded 86.7±2.7% mortality in the E. suratensis (body weight ‐ 11.01±2.7 g; body length 8.01±2.23 cm) injected with 1 × 104⋅25 TCID50 mL−1 ISKNV per fish. Our detailed investigation provided definitive diagnosis of ISKNV in the severe mass mortality event in wild E. suratensis in Peechi Dam, India, adding one more species to expanding host range of ISKNV infection. The high mortality rate of ISKNV infection in pearlspot suggests the perilous nature of this disease, particularly among the wild fish population. This article is protected by copyright. All rights reserved
... RSIV and TRBIV) are aquatic pathogens with increasing global distribution and a broad host range (Hick et al., 2016). The host range includes fish species from a variety of habitats (Wang et al., 2007;Dong et al., 2017;Rimmer et al., 2017;Tsai et al., 2020) and with a variety of uses including species critical to food security (He et al., 2001;Subramaniam et al., 2016), those used for pets (Rimmer et al., 2015;Koda et al., 2018) and those of high conservation value (Rimmer et al., 2017;Go and Whittington, 2019). It is now known that MCVs were circulating in ornamental fish species in the late 1980s, which precedes the first outbreak in mandarin fish in 1994 ca. ...
... The early detection of MCVs from ornamental fish mostly originated from South East Asia (Go et al., 2016). The international spread of MCVs may have been aided by the subclinical nature of infections (Joon et al., 2008;Rimmer et al., 2015) and a lack of effective pre-border disease surveillance polices (Whittington and Chong, 2007;Johnson et al., 2019). ...
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In 2016, famers reported mass mortality events in hybrid grouper within 2–4 weeks following transfer to sea cages on the northern coast of Bali. The objective was to obtain an aetiological diagnosis using a broad range of traditional and emerging diagnostic approaches. A group of 24 and 12 fish with and without clinical signs were sampled from 10 affected populations at 9 farms. Samples for histopathology, ectoparasites evaluation and molecular approaches for microbiology were obtained with a diagnostic post-mortem examination. Fish with clinical signs had a significantly higher likelihood of having pale anterior and posterior kidneys and a liver that was pale and reduced in size compared to fish without clinical signs. There were no differences in the prevalence and quantity of Megalocytivus (MCV) or nervous necrosis virus (NNV) in tissues observed from fish with and without clinical signs. Nearly 55 % of fish were infected with NNV irrespective of clinical signs. There were no histopathological lesions consistent with virial nervous necrosis and the NNV infections were considered subclinical. 80 % of grouper were infected with MCV, irrespective of clinical signs. A significant proportion of fish with clinical signs (true prevalence 94.4 %; 95 % CI 79–100) had observed megalocytes and pathology consistent with disease caused by ISKNV compared to those without clinical signs (true prevalence 47.2 %; 95 % CI 27–70). Metagenomic sequences generated using Illumina Miseq and taxonomically labelled using BlastN + revealed that the Megalocytivirus was 99.9 % similar to Infectious spleen and kidney necrosis virus (ISKNV). The unbiased sequencing did not detect any novel DNA viruses or bacterial pathogens of clinical significance. The monogenean, Benedenia epinepheli and a leech, Zeylanicobdela arugamensis were detected at 6/9 and 9/9 farms, respectively. Our approach identified several pathogens reported in grouper aquaculture with histopathology showing that ISKNV was a necessary cause for the mass mortality events.
... In Australia, an iridovirus was detected in farmed murray cod Maccullochella peelii peelii that has been identified as a strain of ISKNV (Lancaster et al. 2003, Go & Whittington 2006 ). In addition, the presence of ISKNV was reported in 97 of 111 ornamental fish imported into Australia from Singapore, Malaysia, and Sri Lanka (Nolan et al. 2015) and in 10 of 14 species or varieties of ornamental fish that were imported to Australia (Rimmer et al. 2015). ISKNV infections occur in a great number of fishes, including freshwater, brackish , and marine species. ...
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In 2014, infectious spleen and kidney necrosis virus (ISKNV), a member of the genus Megalocytivirus, was detected for the first time in ornamental fish in Germany. Since 2013, angelfish Pterophyllum spp. originating from Colombia have experienced significant epizootics in a number of German retailers' facilities. The diseased fish showed symptoms such as increased ventilation, swollen gills, and ulcerations of the skin. In 2014, diseased angelfish P. altum and platys Xiphophorus maculatus maintained in the same recirculating system were examined. Histopathological lesions included hypertrophic cells, single-cell necrosis, and an inflammatory infiltration of granulocytes, lymphocytes, and macrophages in liver, spleen, and kidney. Transmission electron microscopy revealed the presence of numerous poly gonal viral particles (150 nm in diameter) within the cytoplasm of enlarged cells. A PCR assay for the detection of megalocytiviruses amplified 777 bp of major capsid protein gene that was 100% identical to ISKNV. This is the first report of an ISKNV outbreak in Germany that most probably was introduced by infected angelfish from Colombia. To our knowledge, this is the first report of ISKNV detected in fish imported from South America. Given the lethal nature of megalocytiviruses, proper biosecurity would seem prudent in countries like Germany where these emerging pathogens are not established.
... Besides, giant gourami are considered as a source of transmission of lymphocystis disease virus to other vulnerable fish (Whittington and Chong, 2007). Furthermore, ornamental gourami could carry a strain of infectious spleen and kidney necrosis virus (ISKNV) without developing clinical signs of infection, which has raised a concern of biosecurity risk in Australia (Anderson et al., 1993;Rimmer et al., 2015). Such examples highlight the importance of this fish species as a carrier for multiple piscine viruses. ...
Article
Tilapia lake virus (TiLV) is a novel orthomyxo-like virus that causes high mortality in tilapia and its hybrid species. Currently, no clinical infection of TiLV in other fish has been reported. In this study, a laboratory controlled TiLV infection was investigated in 10 warm water fish species consisting of giant gourami (Osphronemus goramy), snakeskin gourami (Trichogaster pectoralis), iridescent shark (Pangasianodon hypophtthalmus), walking catfish (Clarias macrocephalus), striped snake-head fish (Channa striata), climbing perch (Anabas testudineus), common carp (Cyprinus carpio), silver barb (Barbodes gonionotus), Asian sea bass (Lates calcarifer), and red hybrid tilapia (Oreochromis spp.). Our results revealed that only red hybrid tilapia and giant gourami developed clinical signs of infection including skin erosion, pale skin, exophthalmos and skin haemorrhage with a cumulative mortality rate of 60–100%. Amplification of TiLV using RT-qPCR confirmed the presence of the virus in the livers of challenged fish at 7 and 21 days post infection (dpi) with threshold cycle (Ct) values of 19.57 to 31.18, and 27.78 to ND (not detected) for red hybrid tilapia and giant gourami, respectively. Histopathological lesions were observed including hepatocellular necrosis and syncytial formation in the liver and lymphoid depletion in the anterior kidney of giant gourami and red hybrid tilapia. Moreover, the presence of TiLV in the brain and liver of giant gourami was further confirmed using in situ hybridization. A cohabitation challenge study indicated that the virus could be transmitted from infected red hybrid tilapia to naïve giant gourami with low mortality compared to an intraperitoneal injection. As polyculture systems are common practice in large parts of the aquaculture sector and multiple fish species share natural water resources, it is likely that direct and indirect contacts of infected fish could spread the disease in a farm with polyculture of multiple species. Therefore, the awareness of the spread of TiLV should be given. As such, biosecurity measures should be implemented if rearing tilapia with other fish species. Nevertheless, our study highlighted that most warm water fish species except giant gourami are resilient to TiLV infection.
... For example, infectious spleen and kidney necrosis virus (ISKNV), one of the genotypes in Megalocytivirus in the family iridoviridae, was isolated from ornamental fishes, such as angelfish and dwarf gourami(Kawato et al., 2020;Rimmer et al., 2017). An additional study revealed that indigenous fishes in Australia, silver sweep and Murray cod, were also susceptible to the virus(Go and Whittington, 2019;Rimmer et al., 2015).The data indicated that transmission of the virus may have occurred possibly from ornamental ...
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Ornamental and laboratory fish populations are part of the global trade and can spread pathogens around the world. Laboratory fish are widely used as model for biomedical research, which can be impacted by underestimated health conditions affecting the fish model. The global ornamental fish industry deals with a huge diversity of fish species. High mortality rate often causes significant losses, linked to polymicrobial infections facilitated by stressful conditions compromising host health, although accurate data on ornamental fish trade losses remain difficult to retrieve. Pet fish diseases can spread undetected between artificially recreated ecosystems, posing threats difficult to eradicate once established or when contaminating natural water systems. This 3-hour virtual workshop aimed at highlighting novel aspects of the pathobiology and diagnostics of infectious threats that could be spread through ornamental fishes and impact research using laboratory fish as biological models. In total, 108 participants from various countries all over the world joined this virtual workshop during the EAFP conference live streaming.
... Iridovirus infections show complex clinical symptoms with a wide geographical distribution and host variety (Rimmer et al., 2015;Sriwanayos et al., 2013;Subramaniam et al., 2012;Williams, 2008). ...
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Members of the Iridoviridae family have been considered as aetiological agents of iridovirus diseases, causing fish mortalities and economic losses all over the world. Virus identification based on candidate gene sequencing is faster, more accurate and more reliable than other traditional phenotype methodologies. Iridoviridae viruses are covered by a protein shell (capsid) encoded by the important candidate gene, major capsid protein (MCP). In this study, we investigated the potential of the MCP gene for use in the diagnosis and identification of infections caused Megalocytivirus of the Iridoviridae family. We selected data of 66 Iridoviridae family isolates (53 strains of Megalocytivirus, eight strains of iridoviruses and five strains of Ranavirus) infecting various species of fish distributed all over the world. A total of 53 strains of Megalocytivirus were used for designing the complete primer sets for identifying the most hypervariable region of the MCP gene. Further, our in silico analysis of 102 sequences of related and unrelated viruses reconfirms that primer sets could identify strains more specifically and offers a useful and fast alternative for routine clinical laboratory testing. Our findings suggest that phenotype observation along with diagnosis using universal primer sets can help detect infection or carriers at an early stage.
... These species are also often associated with the infection of Megalocytivirus, which is one of the genera within the family Iridoviridae and can be distinguished by the 120-300 nm diameter of naked or enveloped icosahedral virions (Chinchar et al 2005). Mostly, the occurrence of Megalocytivirus infection in ornamental fish has been reported in the fish imported from Asian countries such as Malaysia and other similar countries to Australia (Rimmer et al 2015;Nolan et al 2015). Until 2013, 97 species of ornamental fish from 111 samples (variety of species) in Australia tested positive for the presence of Megalocytivirus (Nolan et al 2015). ...
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Malaysia has more than 500 culturists that are involved in the ornamental fish industry for culturing 250 species of 550 varieties including local and exotic species. However, Megalocytiviruses have been associated globally with severe systemic disease and economic loss in farmed food fish and ornamental fish. The aim of this study was to detect the presence or absence of Megalocytivirus in a few ornamental species including Xiphophorus hellerii, Xiphophorus maculatus, Poecilia sphenops and Trichopodus trichopterus from Southern Malaysia. Out of 195 samples, the PCR analysis demonstrated 26 positive pooled samples (n = 130) for the presence of Megalocytivirus. The positive samples did not show any clinical sign of Megalocytivirus except pale gills and enlarged liver in T. trichopterus and distended body in X. hellerii. Sequencing analysis of Megalocytivirus major capsid protein (MCP) revealed that the ISKNV strains in this study demonstrated high nucleotide identity to each other and reference ISKNV, 97% to 100%. Based on the phylogenetic tree, the ISKNV strains were closely related to ISKNV and ISKNV strain RSIV-Ku and can be classified into Megalocytivirus genotype I.
... ISKNVs are widely distributed in marine and freshwater fishes in Southeast Asian countries; while RSIVs are distributed mainly among marine fish species in East and Southeast Asian countries; and TRBIVs are almost completely restricted to flatfish in Korea and China. Megalocytiviruses, particularly the ISKNV genotype, have been transmitted throughout the world due to international trade of live ornamental fish [5,8,15,17,34], and outbreaks of the disease have occurred in Australia, America, and Europe [5,8,23,25]. ...
Article
Megalocytiviruses are classified into three genotypes, infectious spleen and kidney necrosis virus (ISKNV), red seabream virus (RSIV), and turbo reddish body iridovirus (TRBIV), based on the major capsid protein and ATPase genes. However, only a few complete genome sequences have been obtained. This paper reports the complete genome sequence and phylogenetic analysis of an RSIV-Ku strain megalocytivirus. The genome sequence comprises 111,154 bp, has 132 putative open reading frames, and is homologous mostly to ISKNV, except for the sequence in the region 58981–66830, which is more closely related to that of the RSIV genotype. The results imply that RSIV-Ku is actually a natural recombinant virus.
... among marine and freshwater fishes in Southeast Asian countries, and TRBIV was only found among flatfish in Korea and China (Fu et al., 2011;Song et al., 2008). Nevertheless, megalocytiviruses, particularly ISKNV, have spread through the international trade of live ornamental fishes (Go Lancaster, Deece, Dhungyel, & Whittington, 2006;Jung-Schroers et al., 2016;Nolan Stephens, Crockford, Jones, & Snow, 2015;Rimmer et al., 2015;Whittington & Chong, 2007). ...
Article
Cell cultures derived from the brain tissues of Aequidens rivulatus (Günther) have been characterized previously. In this study, a continuous cell line ARB8 was further established, and its growth characteristics, transcription and susceptibility to fish viruses—including chum salmon reovirus (CSV), marbled eel infectious pancreative necrosis virus (MEIPNV), grouper nervous necrosis virus (GNNV), giant seaperch iridovirus (GSIV), red seabream iridovirus (RSIV), koi herpesvirus (KHV), herpesvirus anguilla (HVA) and marbled eel polyoma-like virus (MEPyV)—were examined. ARB8 cells that showed epithelioid morphology and were passaged >80 times grew well at temperatures ranging from 25°C to 30°C in L-15 medium containing 5%–15% foetal bovine serum. The cells constitutively transcribed connexion 43, glutamine synthetase, nestin and nkx6-2, which are markers for neural progenitor cells. The cells were highly susceptible to CSV, MEIPNV, GSIV and RSIV and showed the typical cytopathic effect (CPE). However, the cells were resistant to GNNV, KHV, HVA and MEPyV because no significant CPE was noted after infection. Optimal temperatures for virus production ranged from 25°C to 30°C. The results revealed that the neural progenitor cell line ARB8 can potentially serve as a useful tool for investigating fish viruses and isolating new viruses in ornamental cichlid fishes.
... On arrival to Australia, fish are visually inspected by the Australian Quarantine Service for signs of infection and disease. Nonetheless, visual inspections do not account for cryptic pests, with cases of alien viruses going undetected at quarantine and entering Australia via the ornamental fish trade (Becker et al. 2014;Rimmer et al. 2015). For this reason, the Department of Agriculture and Water Resources, Australia, is reforming current biosecurity protocols by placing greater emphasis on managing biosecurity risks off-shore at exporting countries (Hood and Perera 2016). ...
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The ornamental fish trade provides a pathway for the global translocation of aquatic parasites. We examined a total of 1020 fish imported from Singapore, Malaysia, Thailand, or Sri Lanka to Australia (including freshwater and marine fish species) for monogenean ectoparasites. Fish were received following veterinary certification that they showed no clinical signs of pests and diseases from the exporting country and visual inspection at Australian border control. Australian import conditions require mandatory treatment for goldfish with parasiticides (e.g. trichlorfon, formaldehyde, sodium chloride) for the presence of gill flukes (Dactylogyrus vastator Nybelin, 1924 and Dactylogyrus extensus Mueller and Van Cleave, 1932) prior to export. Over 950 individual parasites were detected in five imported fish species, representing 14 monogenean species. Seven Dactylogyrus spp. including D. vastator and three Gyrodactylus spp. infected goldfish, Carassius auratus Linnaeus, 1758, from Malaysia, Singapore, and Thailand. Dactylogyrus ostraviensis Řehulka, 1988, infected rosy barb, Pethia conchonius Hamilton, 1822, from Singapore, Sri Lanka, and Thailand while two Trianchoratus spp. infected three spot gourami, Trichopodus trichopterus Pallas, 1970 and pearl gourami Trichopodus leerii Bleeker, 1852, from Sri Lanka. Urocleidoides reticulatus Mizelle & Price, 1964, infected guppy, Poecilia reticulata Peters, 1859, from Sri Lanka. The discovery of D. vastator in goldfish, as well as 13 other monogenean species, shows that pre-export health requirements, which include chemical treatment of goldfish, and inspection of all ornamental fish species did not prevent infection by monogeneans. Inspection prior to exportation and at border control must account for the highly cryptic nature of monogenean parasites and consider alternatives to current pre-export conditions and visual inspection at border control.
... Globally, the ornamental fish trade is a known route of exotic pathogen and parasite translocations (Trujillo-González et al., 2018;Whittington & Chong, 2007), with detrimental ecological (e.g., parasitic nematode Spirocamallanus istiblenni Noble, 1966 in Louisa E Wood and James Guilder contributed equally to the writing of this manuscript. Gaither et al., 2013) and economic [e.g., the dwarf gourami iridovirus (DGIV) in Rimmer et al., 2015] consequences. Furthermore, intentional release or escape of ornamental fish species from secure systems represent major pathways for non-native fish introductions and translocations globally (see Chan et al., 2019 for a global review). ...
Article
The freshwater and marine ornamental fish industry is a primary pathway for hazard introduction and emergence, including aquatic animal health diseases and non-native species. Prevention measures are key to reducing the risk of hazard incursion and establishment, however, there is currently little understanding of the biosecurity practices and hazard responses implemented at post-border stages of the ornamental fish supply chain. This study addressed this knowledge gap, using questionnaires to collate information on actual biosecurity behaviours and hazard responses practised by ornamental fish retailers and hobbyist communities in England. Actual behaviours varied considerably within retailers and hobbyists, suggesting that reliance on preventative practices by individuals in the post-border stages of the ornamental fish supply chain is likely to be ineffective in minimising the risk of hazard incursion and establishment. Resources should be allocated towards improving and enforcing robust pre-and at-border control measures, such as risk-based surveillance of ornamental fish imports at border controls. In addition, these findings should be used to implement targeted awareness-raising campaigns and help create directed training on biosecurity practices for individuals involved in the post-border stages of the ornamental supply chain. This article is protected by copyright. All rights reserved.
... Molecular techniques have the potential to provide rapid and efficient detection for quarantine inspection and border control. Molecular tech- niques have been used to detect viral infections in ornamental species, with highly sensitive and accurate results (see Becker et al., 2014;Rimmer et al., 2015) and may prove to be highly efficient tools in parasite detection and parasitology research for biosecurity ( Bass et al., 2015). Environmental DNA (eDNA) for example offers noninvasive and comprehensive methods for assessing parasite diversity in imported fish consignments by testing the water used to transport the fish ( Collins et al., 2013). ...
Chapter
Goldfish, Carassius auratus Linnaeus, 1758, are immensely popular ornamental cyprinid fish, traded in more than 100 countries. For more than 500 years, human translocation has facilitated the spread of goldfish globally, which has enabled numerous and repeated introductions of parasite taxa that infect them. The parasite fauna assemblage of goldfish is generally well documented, but few studies provide evidence of parasite coinvasion following the release of goldfish. This review provides a comprehensive synopsis of parasites that infect goldfish in farmed, aquarium-held, native, and invasive populations globally and summarises evidence for the cointroduction and coinvasion of goldfish parasites. More than 113 species infect goldfish in their native range, of which 26 species have probably coinvaded with the international trade of goldfish. Of these, Schyzocotyle acheilognathi (Cestoda: Bothriocephalidae), Ichthyophthirius multifiliis (Ciliophora: Ichthyophthiriidae), Argulus japonicus (Crustacea: Argulidae), Lernaea cyprinacea (Crustacea: Ergasilidae), Dactylogyrus anchoratus, Dactylogyrus vastator and Dactylogyrus formosus (Monogenea: Dactylogyridae) are common to invasive goldfish populations in more than four countries and are considered a high risk of continued spread. Coinvasive parasites include species with direct and complex life cycles, which have successfully colonised new environments through utilisation of either new native hosts or suitable invasive hosts. Specifically, I. multifiliis, A. japonicus and L. cyprinacea can cause harm to farmed freshwater fish species and are important parasites to consider for biosecurity. These species may threaten other aquatic animal industries given their low host specificity and adaptable life histories. Future attention to biosecurity, management and border detection methods could limit the continued spread of exotic parasites from the ornamental trade of goldfish.
... In a histopathological manner, hypertrophic cells with large basophilic cytoplasmic inclusions can be found in internal organs ( Gibson-Kueh et al., 2003;He et al., 2002). Outbreaks have occurred most frequently in East and South-East Asia (Subramaniam, Shariff, Omar, & Hair-Bejo, 2012), although the international trade of ornamental fish has aided the spread of these viruses to Europe (Jung-Schroers et al., 2016;Sriwanayos et al., 2013) and Australia ( Mohr et al., 2015;Nolan, Stephens, Crockford, Jones, & Snow, 2015;Rimmer, Whittington, Tweedie, & Becker, 2017;Rimmer et al., 2015). ...
Article
Zebrafish has become a popular research model in the last years, and several diseases affecting zebrafish research facilities have been reported. However, only one case of naturally occurring viral infections was described for this species. In 2015, infectious spleen and kidney necrosis virus (ISKNV) was detected in zebrafish from a research facility in Spain. Affected fish showed lethargy, loss of appetite, abnormal swimming, distention of the coelomic cavity and, in the most severe cases, respiratory distress, pale gills and petechial haemorrhages at the base of fins. Cytomegaly was the most relevant histopathological finding in organs and tissues, sometimes associated to degenerative and necrotic changes. ISKNV belongs to the relatively newly defined genus Megalocytivirus, family Iridoviridae, comprising large, icosahedral cytoplasmic DNA viruses. This is the first case of naturally occurring Megalocytivirus infection in zebrafish research facilities, associated with morbidity. The virus has been identified based on both pathologic and genetic evidence, to better understand the pathogenesis of the infection in zebrafish and the phylogenetic relationship with other iridoviruses. Given the ability of megalocytiviruses to cross‐species boundaries, it seems necessary to implement stringent biosecurity practices as these infections may invalidate experimental data and have major impact on laboratory and cultured fish.
... On arrival to Australia, fish are visually inspected by the Australian Quarantine Service for signs of infection and disease. Nonetheless, visual inspections do not account for cryptic pests, with cases of alien viruses going undetected at quarantine and entering Australia via the ornamental fish trade (Becker et al. 2014;Rimmer et al. 2015). For this reason, the Department of Agriculture and Water Resources, Australia, is reforming current biosecurity protocols by placing greater emphasis on managing biosecurity risks off-shore at exporting countries (Hood and Perera 2016). ...
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The phylogenetic tree (Figure 7) in the published document has incorrect Bayesian analysis posterior probabilities. This error prevents accurate analysis by future research in parasitology. The figure is therefore replaced by the corrected figure below.
... Of these, only P. latipinna is readily available through commercial sources. However, as these would need to be sourced through the ornamental fish trade, which contains both a mix of locally bred and imported individuals, and the preexisting prevalence of megalocytivirus likely to be high in imported stocks (Rimmer et al. 2015), this species was not considered further due to the potential difficulty in obtaining megalocytivirus-free stock. ...
Article
Megalocytiviruses, particularly red seabream iridovirus, infect a broad range of fish including both freshwater and marine species. Although a limited number of infectious spleen and kidney necrosis virus (ISKNV) strains have been reported in association with mortality events in marine aquaculture species, the potential host range for ISKNV strains, particularly of those that have been detected in ornamental fish, has not been well characterised. There have also been few reports on the susceptibility of euryhaline fish species that could potentially transmit megalocytiviruses between freshwater and marine environments. We found that the euryhaline Australian native percichthyid fish, Australian bass Macquaria novemaculeata, is susceptible experimentally to ISKNV (strain DGIV-10), obtained from a freshwater ornamental fish, dwarf gourami Trichogaster lalius. Australian bass developed clinical disease following direct inoculation and also following cohabitation with infected fish, and were able to transmit DGIV-10 to naïve Murray cod Maccullochella peelii. This study demonstrated the potential for a euryhaline species to become infected with, and transmit, the megalocytivirus ISKNV between fish populations.
... A total of 22 pooled samples of X. maculatus, P. reticulata, T. leeri and A. ramirezi demonstrated positive results for the presence of ISKNV. The result from this study is consistent with previous studies which showed Megalocytivirus infection in freshwater ornamental fish (Subramaniam et al., 2014;Nolan et al., 2015;Rimmer et al., 2015;Mohr et al., 2015;Jung-Schroers et al., 2016). In Malaysia, ISKNV was present in ornamental fish species from four different families' including common platy (Poeciliidae), swordtail (Poeciliidae), pearl gourami (Osphronemidae), ram cichlid (Cichlidae) and zebrafish (Cyprinidae) (Subramaniam et al., 2014). ...
Article
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Malaysia is the 8 th largest world producer of freshwater ornamental fish. Ornamental fish are commonly associated with Megalocytiviruses infection, which has led to severe diseases and economic loss. The purpose of this study was to detect the presence or absence of Infectious Spleen and Kidney Necrosis Virus (ISKNV) in four ornamental fish species namely Xiphophorus maculatus, Poecilia reticulata, Trichogaster leeri and Apistogramma ramirezi from Malaysia. A total of 175 samples were analysed using PCR analysis for detection of ISKNV. The PCR analysis demonstrated 22 positive pooled samples (n = 110) for the presence of ISKNV. No clinical signs were observed in positive samples except darkened body in X. maculatus. Sequencing analysis of Megalocytivirus major capsid protein (MCP) revealed that the ISKNV strains in this study demonstrated high nucleotide identity to each other and reference ISKNV (96% to 100%). Based on the phylogenetic tree, the ISKNV strains were closely related to reference ISKNV and can be classified into Megalocytivirus genotype I.
... Although VHSV had been present in European waters for a long time, it is presumed that it did not cause disease outbreaks until rainbow trout was introduced for farming from North America [84]. Mohr et al. [85] and Rimmer et al. [86] isolated infectious spleen and kidney necrosis virus (ISKNV) from imported ornamental fish into Australia that led to ISKV outbreaks in farmed Platy (Xiphophorus maculatus). Similarly, Gomez et al. [87] detected NNV from imported fish into South Korea. ...
Article
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Aquaculture is the fastest food-producing sector in the world, accounting for one-third of global food production. As is the case with all intensive farming systems, increase in infectious diseases has adversely impacted the growth of marine fish farming worldwide. Viral diseases cause high economic losses in marine aquaculture. We provide an overview of the major challenges limiting the control and prevention of viral diseases in marine fish farming, as well as highlight potential solutions. The major challenges include increase in the number of emerging viral diseases, wild reservoirs, migratory species, anthropogenic activities, limitations in diagnostic tools and expertise, transportation of virus contaminated ballast water, and international trade. The proposed solutions to these problems include developing biosecurity policies at global and national levels, implementation of biosecurity measures, vaccine development, use of antiviral drugs and probiotics to combat viral infections, selective breeding of disease-resistant fish, use of improved diagnostic tools, disease surveillance, as well as promoting the use of good husbandry and management practices. A multifaceted approach combining several control strategies would provide more effective long-lasting solutions to reduction in viral infections in marine aquaculture than using a single disease control approach like vaccination alone.
... All studies were secondary data sources: their lists of captive ornamental species in Australia were a by-product of scientific research. Research aims were varied and included reports on zoonosis infections from ornamental fish (Musto et al. 2006;Senanayake et al. 2004;Robson et al. 1998;Moulsdale et al. 1983), outcomes of ornamental fish surgery (Chin et al. 2017;O'Hagan and Raidal 2006;Raidal et al. 2006), welfare of captive fish (Sullivan et al. 2016;Sullivan 2014), and diseased fish assessments in 1.) import lots (Trujillo-Gonzalez et al. 2018;Nolan et al. 2015;Rimmer et al. 2015;Stephens et al. 2009;Evans and Lester 2001;Anderson et al. 1993;Humphrey et al. 1986;Ashburner 1975), 2.) fish farms (Mohr et al. 2015;Becker et al. 2014;Humphrey and Ashburner 1993), and 3.) pet stores (Palermo 2016;Morine et al. 2012;Wickins et al. 2011;Zanguee et al. 2010;Go et al. 2006). Data were predominately recorded from private collections (73.7%), with data also sourced from import and export lists (14.5%), pet stores (7.1%), wholesaler/ornamental fish dealers (4.5%), ...
Article
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Invasive species represent one of the greatest biological threats to Australian ecosystems this century. Facilitated by global interdependence, increased connectivity, and established trade routes, the dissemination of non-native ornamental species has led to substantial establishments in Australian waterways. Despite this, recent and ongoing research into the trade and invasive potential of non-native ornamental fish species in Australia is lacking and well behind the global standard. Hampered by a shortage of adequate funding and an inability to make rapid policy-based decisions due to industry influence, restrictions on trade have been slow or non-existent in recent years. Further, the development and maintenance of accurate species trade lists as well as dedicated funding and a coordinated approach to compliance is currently inadequate across all Australian jurisdictions. Here we aimed to identify if existing ornamental freshwater fish records from scientific literature in Australia, including veterinary reports and zoonoses studies, were an appropriate alternative to direct industry monitoring necessary in producing comprehensive trade lists. To test this alternative approach, we identified and collated scientific literature that had recorded captive freshwater fish in the Australian ornamental industry. Our review identified a still inchoate scientific body of literature that is a poor substitute for direct survey approaches, with minimal reporting evident in Australia on the freshwater ornamental fish in trade. Assessment of available species records indicated unassessed, greylisted freshwater fish form a substantial part of the Australian ornamental industry. Nomenclature issues and potential exploitation by the ornamental fish industry were also identified. Given the paucity of contemporary literature on the presence and abundance of traded species within Australia, initiatives including pet store surveys and e-commerce monitoring are vital to collate a complete list of traded species necessary for management of this non-native community. We highlight key research priorities and provide recommendations on the future management needs of the Australian freshwater ornamental fish industry.
... ytridiomycosis; Bergmann et al., 2010, koi herpes virus [KHV];Hall et al., 2013; Hall, Soje, et al., 2014b, ISAV and SAV, respectively; Monaghan et al., 2015, KHV). Persistent subclinical infection with megalocytivirus (MCV) illustrates the variability in analyte distribution within a population that will alter PSe in a population-dependent manner.Rimmer et al. (2015) identified populations of ornamental fish ...
Article
Samples from multiple animals may be pooled and tested to reduce costs of surveillance for infectious agents in aquatic animal populations. The primary advantage of pooling is increased population‐level coverage when prevalence is low (<10%) and the number of tests is fixed, because of increased likelihood of including target analyte from at least one infected animal in a tested pool. Important questions and a priori design considerations need to be addressed. Unfortunately, pooling recommendations in disease‐specific chapters of the 2018 OIE Aquatic Manual are incomplete and, except for amphibian chytrid fungus, are not supported by peer‐reviewed research. A systematic review identified only 12 peer‐reviewed aquatic diagnostic accuracy and surveillance studies using pooled samples. No clear patterns for pooling methods and characteristics were evident across reviewed studies, although most authors agreed there is a negative effect on detection. Therefore, our purpose was to review pooling procedures used in published aquatic infectious disease research, present evidence‐based guidelines, and provide simulated data examples for white spot syndrome virus in shrimp. A decision tree of pooling guidelines was developed for use by peer‐reviewed journals and research institutions for the design, statistical analysis and reporting of comparative accuracy studies of individual and pooled tests for surveillance purposes.
... More recently, infectious spleen and kidney necrosis virus (ISKNV) was documented in a zebrafish facility in Spain (Bermúdez et al., 2018). The latter is of particular concern as it has broad host specificity, is very common in aquarium fishes (Rimmer et al., 2015) and is lethal to zebrafish (Crim & Riley, 2012). ...
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The use of zebrafish (Danio rerio) in biomedical research has expanded at a tremendous rate over the last two decades. Along with increases in laboratories using this model, we are discovering new and important diseases. We review here the important pathogens and diseases based on some 20 years of research and findings from our diagnostic service at the NIH‐funded Zebrafish International Resource Center. Descriptions of the present status of biosecurity programmes and diagnostic and treatment approaches are included. The most common and important diseases and pathogens are two parasites, Pseudoloma neurophilia and Pseudocapillaria tomentosa, and mycobacteriosis caused by Mycobacterium chelonae, M. marinum and M. haemophilum. Less common but deadly diseases are caused by Edwardsiella ictaluri and infectious spleen and kidney necrosis virus (ISKNV). Hepatic megalocytosis and egg‐associated inflammation and fibroplasia are common, apparently non‐infectious, in zebrafish laboratories. Water quality diseases include supersaturation and nephrocalcinosis. Common neoplasms are spindle cell sarcomas, ultimobranchial tumours, spermatocytic seminomas and a small‐cell carcinoma that is caused by a transmissible agent. Despite the clear biosecurity risk, researchers continue to use fish from pet stores, and here, we document two novel coccidia associated with significant lesions in zebrafish from one of these stores.
... However, any test that reacts directly with the pathogen (e.g., pathogen DNA or antigens) or host responses (e.g., antibodies) is necessarily dose-dependent ( Fig. 3; Eq. (11)). The variation in the amount of the analyte between infected individuals or among laboratories, protocols, etc., may be small enough to ignore (but see e.g., Rimmer et al. 76 ), or the concentrations generally large enough that detection is assured (e.g., with clearly diseased animals), in which case it may be reasonable to assume diagnostic sensitivity is constant. At the other extreme, recently infected individuals, sub-clinical carriers 16 , or otherwise inapparent infections tend to produce or shed little analyte and are thus much less easily detected than clinically infected animals. ...
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The aim of this study is to report the occurrence of Lymphocystivirus in Brazilian ornamental fish. From 25 ornamental fish species submitted for molecular diagnosis, only one sample (Pomacanthus imperator) was positive, and its viral nucleotide sequence obtained clustered with sequences of genotype VII. To our knowledge, this is the first report on the genetic characterization of Lymphocystivirus in Brazil.
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Infectious spleen and kidney necrosis virus (ISKNV), a member of family iridoviridae, reported for the first time in a wide range of ornamental fish species in India. Significant mortalities during the year 2018‐19 were reported from a number of retailers in the region with various clinical signs. The samples of moribund, dead and apparently healthy ornamental fishes were collected from retailers, located in three districts of Karnataka, India. Out of 140 fish samples, 16 samples (11.42 %) representing 10 different fish species were found positive to ISKNV by OIE listed primers and same samples were reported to amplify the major capsid protein (MCP) gene of ISKNV. Further, sequence analysis of MCP gene showed that all strains detected in this study were closely related to other documented isolates from different countries with an identity ranging from 98.76 to 100 %. Further, they clustered in the clade of ISKNV, during the phylogenetic analysis. The sequence similarity was high (99.94%) to ISKNV strains from Japan, Australia, and Malaysia. This is the first report of an ISKNV infection in India. Moreover, out of 10 ISKNV positive fish species; three species were reported positive to ISKNV for the first time in the world. Further, the in‐vitro experiment showed the growth of virus in Asian seabass cell line, which is a natural host of ISKNV. Therefore, considering the lethal nature of megalocytiviruses to infect a vast range of species, proper biosecurity measures needs to be taken to control these emerging pathogens.
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Mass mortality occurred in 2-year-old koi (fancy carp) Cyprinus carpio in Taiwan in 2002. Affected fish did not show any external signs except swollen gills. The consistent histological changes were observed in the gills; hyperplasia of epithelial cells, infiltration of eosinophilic granular cells and fusion of the secondary lamellae. Negatively stained nucleocapsids were icosahedral and 112 ± 1 nm in diameter. Koi herpesvirus (KHV) was detected in the diseased fish by PCR using specific primers for KHV, and sequence of the amplicon showed a 99% identity with the published data. This is the first report of KHV infection in Taiwan.
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Mass mortality among common carp Cyprinus carpio, especially in large-sized fish cultured in Lake Kasumigaura, has occurred since early October 2003. The affected fish swam lethargically near the surface. A histopathological examination revealed hyperplasia and necrosis of the epithelial cells in the gills. DNA of the koi herpesvirus (KHV) was detected in the gills and kidneys of moribund fish by polymerase chain reaction (PCR) assay. The virus could not be isolated from the tissues, but the inoculation of gill homogenate from affected fish into healthy carp reproduced the disease conditions and killed the fish. KHV was detected from the gills of the experimentally infected fish by PCR, and the virus was isolated from those fish with KF-1 cells.
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A herpesvirus was isolated from adult koi, a strain of common carp Cyprinus carpio, suffering mass mortality in two outbreaks—one in the mid-Atlantic region of the United States and the second in Israel. The principal external signs of dying fish were pale and irregularly colored gills. There were few consistent internal signs in either outbreak. The most prominent microscopic lesions were in the gills, where hyperplasia and necrosis of the epithelium were severe. Other lesions included interstitial nephritis, splenitis, and enteritis. Affected cells often contained nuclei with marginated chromatin and faint intranuclear inclusions. Typical herpesvirus particles were present in branchial epithelial cells, hepatocytes, and among circulating leukocytes. Inoculations of the koi fin (KF-1) cell line with tissue extracts from the gill and kidney–spleen resulted in cytopathic effects characterized by severe vacuolation first detected after 7 d incubation at 20°C. Exposures of adult koi to the herpesvirus as propagated in KF-1 cells by bath or intraperitoneal injections resulted in 80–100% mortality during a 26-d period, and the virus was reisolated from the gill, kidney, liver, spleen, intestine, and brain of dead fish. The viral agents from koi in Israel and the United States appear to be similar if not identical; both could be distinguished from Herpesvirus cyprini by indirect fluorescent antibody tests with rabbit anti-H. cyprini serum. Other factors should be examined but we strongly suspect that this newly recognized koi herpesvirus (KHV) has the potential to be a significant cause of mortality among koi and presumably common carp.
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Megalocytiviruses have been associated with epizootics resulting in significant economic losses in public aquaria and food-fish and ornamental fish industries, as well as threatening wild fish stocks. The present report describes characteristics of the first megalocytivirus from a wild temperate North American fish, the threespine stickleback Gasterosteus aculeatus. Moribund and dead fish sampled after transfer to quarantine for an aquarium exhibit had amphophilic to basophilic intracytoplasmic inclusions (histopathology) and icosahedral virions (transmission electron microscopy) consistent with an iridovirus infection. Phylogenetic analyses of the major capsid, ATPase, and DNA polymerase genes confirmed the virus as the first known member of the genus Megalocytivirus (family Iridoviridae) from a gasterosteid fish. The unique biologic and genetic properties of this virus are sufficient to establish a new Megalocytivirus species to be formally known as the threespine stickleback iridovirus (TSIV). The threespine stickleback is widely distributed throughout the northern hemisphere in both freshwater and estuarine environments. The presence of megalocytiviruses with broad host specificity and detrimental economic and ecologic impacts among such a widely dispersed fish species indicates the need for sampling of other stickleback populations as well as other North American sympatric marine and freshwater ichthyofauna.
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The objective of this study was to describe the frequency of histopathological lesions and categorize histopathologically evident infections in sick ornamental fish from pet shops in New South Wales, Australia. We examined 108 fish that had evidence of morbidity or mortality, including 67 cyprinids, 25 osphronemids, 11 poeciliids, 4 characids and 1 cichlid, sourced from 24 retail outlets. Conditions frequently observed in the study population included branchitis (62/86, 72.1%), visceral granulomas (41/108, 38.0%), dermatitis (17/55, 30.9%), wasting (31/108, 28.7%), and intestinal coccidiosis (18/104, 17.4 %). Branchitis and dermatitis were usually due to monogenean flukes, or flagellate or ciliate protozoa. Intralesional Microsporidia (16/41, 39.0%), mycobacteria (7/41, 17.%), or Myxosporidia (5/41, 12.2%) were identified in the majority of fish with visceral granulomas; however, special stains were critical in their identification. The proportion of histologically evident infections was remarkably high (77/108, 71.3%), and parasitic infections predominated. Many pathogens identified in the study have low host specificity and/or direct life cycles which would facilitate transmission to exposed naive fish populations, potentially posing a threat to native and commercial fish populations. Those caring for sick ornamental fish should take appropriate steps to investigate infectious disease and should take precautions that prevent the spread of pathogens.
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Comparative analysis of molecular sequence data is essential for reconstructing the evolutionary histories of species and inferring the nature and extent of selective forces shaping the evolution of genes and species. Here, we announce the release of Molecular Evolutionary Genetics Analysis version 5 (MEGA5), which is a user-friendly software for mining online databases, building sequence alignments and phylogenetic trees, and using methods of evolutionary bioinformatics in basic biology, biomedicine, and evolution. The newest addition in MEGA5 is a collection of maximum likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models (nucleotide or amino acid), inferring ancestral states and sequences (along with probabilities), and estimating evolutionary rates site-by-site. In computer simulation analyses, ML tree inference algorithms in MEGA5 compared favorably with other software packages in terms of computational efficiency and the accuracy of the estimates of phylogenetic trees, substitution parameters, and rate variation among sites. The MEGA user interface has now been enhanced to be activity driven to make it easier for the use of both beginners and experienced scientists. This version of MEGA is intended for the Windows platform, and it has been configured for effective use on Mac OS X and Linux desktops. It is available free of charge from http://www.megasoftware.net.
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Iridoviruses infect food and ornamental fish species from a wide range of freshwater to marine habitats across the globe. The objective of the current study was to characterize an iridovirus causing systemic infection of wild-caught Pterapogon kauderni Koumans 1933 (Banggai cardinalfish). Freshly frozen and fixed specimens were processed for histopathologic evaluation, transmission electron microscopic examination, virus culture, molecular virologic testing, microbiology, and in situ hybridization (ISH) using riboprobes. Basophilic granular cytoplasmic inclusions were identified in cytomegalic cells often found beneath endothelium, and hexagonal virus particles typical of iridovirus were identified in the cytoplasm of enlarged cells by transmission electron microscopy. Attempts at virus isolation in cell culture were unsuccessful; however, polymerase chain reaction (PCR)-based molecular testing resulted in amplification and sequencing of regions of the DNA polymerase and major capsid protein genes, along with the full-length ATPase gene of the putative iridovirus. Virus gene sequences were then used to infer phylogenetic relationships of the P. kauderni agent to other known systemic iridoviruses from fishes. Riboprobes, which were transcribed from a cloned PCR amplification product from the viral genome generated hybridization signals from inclusions within cytomegalic cells in histologic sections tested in ISH experiments. To the authors' knowledge, this is the first report of a systemic iridovirus from P. kauderni. The pathologic changes induced and the genomic sequence data confirm placement of the Banggai cardinalfish iridovirus in the genus Megalocytivirus family Iridoviridae. The ISH provides an additional molecular diagnostic technique for confirmation of presumptive infections detected in histologic sections from infected fish.
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Megalocytivirus is causing economically serious mass mortality by infecting fish in and around the Pacific region of Asia. The recent emergence of many new iridoviruses has drawn attention to the marked taxonomic variation within this virus family. Most studies of these viruses have not included extensive study of these emergent species. We explored the emergence of red sea bream iridovirus (RSIV) on a fish farm in Japan, and we specifically endeavored to quantify genetic and phenotypic differences between RSIV isolates using in vitro and in vivo methods. The three isolates had identical major capsid protein sequences, and they were closely related to Korean RSIV isolates. In vitro studies revealed that the isolates differed in replication rate, which was determined by real-time quantitative PCR of viral genomes in infected cells and cell culture supernatant, and in cell viability, estimated by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay for infected cells. In vivo studies showed that the isolates exhibit different virulence characteristics: infected red sea bream showed either acute death or subacute death according to infection with different isolates. Significant differences were seen in the antigenicity of isolates by a formalin-inactivated vaccine test. These results revealed that variant characteristics exist in the same phylogenetic location in emergent iridoviruses. We suggest that this strain variation would expand the host range in iridoviral epidemics.
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A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
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A systemic viral infection in both gourami Trichogaster spp. and swordtail Xiphophorus hellerii and an outbreak of lymphocystis in scalare Pterophyllum scalarae and gourami are reported to have occurred in fish reared in ornamental fish farms in Israel. The systemic infection developed in endothelial cells that became hypertrophic and their contents were modified. The presence of such cells in light-microscopically examined stained smears and sections provides an initial indication for this systemic viral infection. Infection in gourami caused hemorrhagic dropsy. Transmission electron microscopic (TEM) images of iridovirus-like particles recovered from gouramies showed them to be 138 to 201 nm from vertex to vertex (v-v); those from swordtails were 170 to 188 nm v-v. TEM images of lymphocystis virions from scalare were 312 to 342 nm v-v and from gourami 292 to 341 nm v-v. Lymphocystis cells from the gourami were joined by a solid hyaline plate, which was lacking in the infection in scalare where the intercellular spaces between the lymphocystis cells consisted of loose connective tissue.
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The first outbreaks of a disease connected with high mortality of common carp and koi carp caused by koi herpesvirus (KHV) were reported in 1998 in Israel and in the United States. Since then, several cases have been confirmed all over the world. At present, this viral disease is considered to be one of the most risky factors affecting populations of common carp and koi carp. Affected fish become disoriented and swim erratically with high breathing frequency, swollen gills and partially local skin lesions. The virus was isolated from the tissues of fish showing signs of the disease and subsequently cultured on koi fin (KF-1) cells. Electron microscopic examinations revealed morphological signs identical with viruses of the family Herpesviridae. Analysis of virion polypeptides and gene DNA showed the differences between KHV and the well-known herpesvirus of cyprinids, Herpesvirus cyprini (CHV), and Channel catfish virus (CCV). Water temperature is a factor influencing the onset and severity of disease. Fish seem most susceptible at water temperatures of 18-28°C, no morbidities occur at 13°C and 30°C. At present, diagnosis of KHV is mainly based on detection of viral DNA by PCR method.
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In 1997 two and in 1998 six cases of mass mortality in Koi carp (Cyprinus carpio L.) with similar symptoms were examined. Gross examination revealed in each of the cases moderate to extensive epidermal lesions, gill necrosis and enopthalmus. Different ectoparasites were found on the skin only in low numbers, on the gills slight to heavy infestations, mainly with protozoan parasites encountered. Pathological anatomy revealed pale internal organs and an enlarged anterior kidney. Different opportunistic bacteria were isolated from inner organs, with Aeromonas sobria in 7/8 cases. Cohabitation trials confirm the infectious nature of the disease to different breeds of Cyprinus carpio. Transmission electron microscopy (TEM) revealed the presence of virus-like particles in the nuclei and cytoplasm of respiratory epithelial cells of gills.
Article
1990年秋に初めて発生し, 1991年に西日本の主要養殖県に発生域が拡大した養殖マダイの大量斃死の原因究明を行った結果, 以下のことが明らかとなった。 1.病魚の外観は体色の黒化や褪色, 体表や鰭の出血性のスレを特徴とし, 剖検すると鯉の褪色, 鰓弁の点状出血や先端部からの出血, 囲心腔内の出血等が観察された。 2.病理組織学的には脾臓組織のほか, 心臓の心内膜下, 鰓の中心静脈洞内皮および中肋の軟骨膜に接する組織間隙, 腎臓の間質組織と糸球体に肥大球形化した細胞が多数観察されるのが特徴であった。肝臓ではその出現頻度は低かった。 3.電顕観察ではこれらの異形肥大細胞の細胞質内に平面的には6角形を呈する, 直径が200~240nmのウイルス粒子の増殖像が観察された。組織切片のフォイルゲン反応による核酸染色では異形肥大細胞の細胞質にDNAが検出され, 本ウイルスはDNAウイルスの一種のイリドウイルス科に属すると考えられた。 4.病魚の脾臓砕磨濾液をRTG-2, CHSE-214, FHM, BF-2およKRE-3細胞に接種すると, 球形化を特徴とするCPEが発現したが, いずれも感染力価は低かった。 5.病魚の脾臓磨砕液を用いてマダイとブリ稚魚に実験感染した結果, 両魚種ともマダイの自然発病魚と同様の症状を呈して死亡し, これらの魚種に対する病原性が確認された。 以上の一連の試験結果から, マダイに発生した大量斃死はイリドウイルスの感染に起因すると結論された。
Article
The polymerase chain reaction (PCR) was used to amplify DNA of the red sea bream iridovirus (RSIV). Four oligonucleotide primer sets based on the ATPase gene, DNA polymerase gene, and a Pst I-restriction fragment of RSIV genomic DNA were synthesized. PCR products of the expected size were amplified with each primer set after 30 cycles using template DNA which was extracted from the supernatant of tissue-cultured GF cells infected with RSIV. These amplified products were shown to be specific for the genomic DNA of RSIV by Southern blot hybridization. In addition, PCR products were obtained from the DNAs of the spleen and blood of red sea bream, Pagrus major, artificially infected with RSIV. Furthermore, the PCR products were detected from the tissues of cultured marine fish naturally infected with RSIV and from the supernatant of tissue-cultured GF cells infected with RSIV isolated from cultured marine fish. However, PCR products were not obtained at 3 months post-challenge from the spleens of red sea bream infected by intraperitoneal inoculation of RSIV. None of the PCR products were obtained from the supernatant of tissue-cultured FHM cells infected with frog virus 3 or from the lymphocystis tissue of Japanese flounder, Paralichthys olivaceus, infected with fish lymphocystis disease virus.
Article
Occurrences of red sea bream iridoviral disease (RSIVD) in cultured marine fish were investigated by our diagnostic works and by collecting information from prefectural fisheries research stations. The infection was identified by using indirect immunofluorescence test with a monoclonal antibody against red sea bream iridovirus (RSIV). In total, RSIVD was confirmed in 29 cultured marine fish species from 1996 to 2000. After 1995, 11 fish species were newly confirmed as susceptible hosts to RSIV. Since the first occurrence in 1990, RSIVD has been recorded in a total of 31 cultured marine fish species in 18 prefectures located in south-western part of Japan.
Article
This report documents an emerging trend of identification of Megalocytivirus-like inclusions in a range of ornamental fish species intercepted during quarantine detention at the Australian border. From September 2012 to February 2013, 5 species of fish that had suffered mortality levels in excess of 25% whilst in the post-entry quarantine and had Megalocytivirus-like inclusion bodies in histological sections were examined by PCR. The fish had been imported from Singapore, Malaysia and Sri Lanka. Ninety-seven of 111 individual fish from affected tanks of fish tested were positive for the presence of Megalocytivirus by PCR. Sequence analysis of representative PCR products revealed an identical sequence of 621 bp in all cases which was identical to a previously characterized Megalocytivirus (Sabah/RAA1/2012 strain BMGIV48). Phylogenetic analysis of available Megalocytivirus major capsid protein (MCP) sequences confirmed the existence of 3 major clades of Megalocytivirus. The virus detected in this study was identified as a member of Genotype II. The broad host range and pathogenicity of megalocytiviruses, coupled to the documented spread of ornamental fish into the environment, render this a significant and emerging biosecurity threat to Australia.
Article
We revisit the problem of interval estimation of a binomial proportion. The erratic behavior of the coverage probability of the standard Wald confidence interval has previously been remarked on in the literature (Blyth and Still, Agresti and Coull, Santner and others). We begin by showing that the chaotic coverage properties of the Wald interval are far more persistent than is appreciated. Furthermore, common textbook prescriptions regarding its safety are misleading and defective in several respects and cannot be trusted. This leads us to consideration of alternative intervals. A number of natural alternatives are presented, each with its motivation and context. Each interval is examined for its coverage probability and its length. Based on this analysis, we recommend the Wilson interval or the equal-tailed Jeffreys prior interval for small n and the interval suggested in Agresti and Coull for larger n. We also provide an additional frequentist justification for use of the Jeffreys interval.
Article
Viruses in the genus Megalocytivirus (Family Iridoviridae) have emerged as a source of significant disease and mortality in fish worldwide. Although regarded as exotic in Australia, the importation of ornamental fish provides a transmission pathway for the introduction of a megalocytivirus, dwarf gourami iridovirus (DGIV), which is a strain of infectious spleen and kidney necrosis virus (ISKNV). There are no effective cell lines for isolation of ISKNV-like viruses, particularly those from ornamental fish. Consequently a quantitative polymerase chain reaction (qPCR) assay was developed for the detection and quantification of known megalocytiviruses from fish tissues. A plasmid containing a target DGIV sequence was developed for use as a positive control and also for quantification of the virus in qPCR, and is suitable for inter-laboratory standardisation of the assay. The limit of detection for the qPCR assay was 100 copies and the assay did not cross-react with ranaviruses or other aquatic viral pathogens tested. The method of DNA extraction (magnetic bead or spin column based) had no effect on the sensitivity of the qPCR assay. Analytical sensitivity was comparable to a nested PCR assay which employed the same internal primers; however, compared to the Office International des Epizooties (OIE) reference PCR protocol, sensitivity was approximately 3–4 logs greater. The OIE reference PCR protocol detected only 8% of the samples identified to be positive by qPCR. This rapid, sensitive and specific test with the accompanying plasmid as a reference standard will be of benefit to the national quarantine system as it can be used to screen fish prior to importation as well as while fish are in a quarantine approved premises, as a way of limiting the inadvertent release of DGIV and related megalocytiviruses from Australian quarantine. The assay also detects red sea bream iridovirus (RSIV).
Article
Viruses in the family Iridoviridae cause severe disease and economic loss in a wide range of cultured, wild and ornamental fish species in many countries, justifying the development and validation of diagnostic tests. In an effort to improve the efficiency of test protocols, in this study manual and high throughput semi-automated methods for tissue homogenisation and nucleic acid purification were compared for the detection of Epizootic Haematopoeitic Necrosis Virus (EHNV). The effectiveness of these methods at releasing EHNV particles and obtaining DNA was evaluated by virus isolation in bluegill fry (BF-2) cells, enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction (qPCR). Samples were prepared using tissue from infected redfin perch (Perca fluviatilis). Antigen and DNA yield were greater after homogenisation by bead-beating than by manual grinding of tissues from experimentally infected fish, which contained low quantities of virus. Bead-beating was compatible with virus isolation. There was no difference between the manual and semi-automated methods using samples from naturally infected fish which contained large amounts of virus. There was no difference in DNA yield between manual and semi-automated nucleic acid extraction for experimentally infected fish, however nucleic acid yields were greater after manual extraction for samples from naturally infected fish. Semi-automated tissue homogenisation and nucleic acid extraction required the least amount of time and were the most cost effective. The results of this study can be used as a guide to the selection of sample preparation procedures for other ranaviruses and probably more widely.
Article
Spring viremia of carp virus (SVCV) is an important viral pathogen of common carp Cyprinus carpio and one of only five fish pathogens listed as notifiable in the 2002 International Aquatic Animal Health Code of the Office International des Epizooties (OIE). Spring viremia is most problematic in Europe. The disease has been reported in many regions in the world but never in North America. In April 2002, an epizootic with clinical signs consistent with SVCV occurred on a fish farm on the East Coast of the United States that raises koi, a strain of common carp. A virus was isolated on epithelioma papillosum cyprini cells and subsequently demonstrated by immunocytochemistry to be SVCV. The OIE reference laboratory for SVCV (Centre for Environment, Fisheries, and Aquaculture Science, Weymouth, England) has confirmed the identity of the virus.
Article
In February 2003 there were 90% losses of Murray cod (Maccullochella peelii peelii) fingerlings in a Victorian aquaculture facility. The disease was caused by a megalocytivirus (Family Iridoviridae) closely related to the recognized species Infectious Spleen and Kidney Necrosis Virus (ISKNV) and strain dwarf gourami iridovirus (DGIV), neither of which had previously been reported from farmed or wild fish in Australia. Experimental transmission trials were undertaken to test the hypothesis that the outbreak could have arisen through introduction of a virus with ornamental gouramis imported from South East Asia. Intraperitoneal injection of Murray cod fingerlings using filtered tissue homogenates from dwarf gourami (Colisa lalia) positive for megalocytivirus DNA by polymerase chain reaction (PCR) resulted in >90% mortality. Mortality was also induced by cohabitating Murray cod fingerlings and dwarf gourami; as the fish were physically separated, the virus spread between the two species via water. Histopathology revealed lesions identical to those reported in the Victorian outbreak, 125–130 nm icosahedral virions were observed in lesions and most exposed fish were PCR positive. DNA sequencing confirmed 99.9 to 100% homology of major capsid protein and ATPase nucleotide sequences between DGIV, ISKNV, the viral inoculum obtained from dwarf gourami and virus present in experimentally infected Murray cod. These findings confirm that Murray cod are highly susceptible to a megalocytivirus present in ornamental fish imported from South East Asia. The implications for aquaculture, conservation of native fish and quarantine policy are discussed.
Article
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Article
An iridovirus-like agent associated with the ‘reddish body syndrome’ (RBS) of farmed turbot Scophthalmus maximus was found in China. By light microscope, many enlarged cells were observed in the spleen and kidney of diseased turbot. Iridovirus-like particles were detected by transmission electron microscopic (TEM) examination in various organs of diseased turbot. The virion had an envelope and viral particles were only present in the cytoplasm of infected cells. TEM images of virions showed them to be icosahedral in symmetry, measuring 120–130 nm from vertex to vertex and 110–116 nm from face to face. Complete virions consisted of three layers: the outer layer was an icosahedral capsid 10–14 nm in thickness, the intermediate layer was an 11–15 nm thick translucent space and the inner spheroid core was a homogeneously electron-dense nucleoid measuring 64–70 nm in diameter. The virus particle acquired an envelope through budding from the cytoplasm into a cytoplasmic vesicle. Infected cells became hypertrophic and the cytoplasm was homogeneous. According to the TEM examination, the virions were mostly in cells of gill, intestinal submucosa, spleen and basement membranes of capillaries in glomeruli of the diseased fish. The spleen was the major target organ for the virus. Further analysis using PCR suggested that the molecular characterization of the virus was different from that of red sea bream iridovirus (RSIV) in Japan, Korea, Taiwan and infectious spleen and kidney necrosis virus (ISKNV) in China. This is the first report of iridovirus infection in turbot in China.
Article
This paper deals with some basis properties of screening tests. Such tests purport to separate people with disease from people without. Minimal criteria for such a process to be a test are discussed. Various ways of judging the goodness of a test are examined. A common use of tests is to estimate prevalence of disease; frequency of positive tests is shown to be a bad estimate, and the necessary adjustmants are given.
Article
The development of polymerase chain reaction (PCR) DNA amplification methods has afforded molecular studies of fixed paraffin-embedded tissue samples and other archival material. Some fixation methods damage DNA, and thus deleteriously affect subsequent PCR analysis. This study addressed the effect of short- and long-term storage (2 hr to 30 days) in a variety of fixatives (10% buffered-neutral formalin [BNF], 95% ethanol, acetone, and OmniFix) before paraffin embedding. We tested the ability of prepared tissue sections to yield DNA amplification products ranging from 268 to 1327 bp. Results indicated that tissues fixed for 8 days in BNF were able to amplify 536-bp but very little 989-bp DNA fragments; after 30 days of BNF fixation only a 268-bp fragment was amplifiable. Samples fixed in OmniFix and acetone yielded products of 989 and 1327 bp, respectively, after 8 days of fixation; both fixatives yielded 989-bp amplification products after 30 days of fixation. Tissues fixed in 95% ethanol for up to 30 days efficiently produced DNA amplification fragments of up to 1327 bp in length. The results provide important information for prospective studies that involve PCR analysis from archival material. Furthermore, fixation and long-term storage in ethanol should prove particularly useful in remote areas where refrigeration or immediate sample-processing is unavailable.