Partial validation of a TaqMan real-time quantitative PCR for the detection of ranaviruses

Stilwell, Natalie K.; Whittington, Richard J.; Hick, Paul; Becker, Joy A.; Ariel, Ellen; Beurden, Steven van; Vendramin, Niccoló; Olesen, Niels Jørgen; Waltzek, Thomas B.

doi:10.3354/dao03214

Cited by 30 publications

(28 citation statements)

References 52 publications

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“…DNA was extracted from the internal tissues using the DNeasy Blood and Tissue extraction kit (Qiagen, Valencia, California, USA) following the manufacturer's protocol and then screened for Ranavirus spp. DNA using a quantitative real‐time PCR assay that amplifies 96 bp of the major capsid protein (Stilwell et al, ). Each sample was run in triplicate 20 μL reactions with 5 μL of template DNA for 45 cycles.…”

Section: Methodsmentioning

confidence: 99%

Do scavengers prevent or promote disease transmission? The effect of invertebrate scavenging on Ranavirus transmission

2019

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Host–parasite interactions are shaped by the broader web of community interactions, from interspecific competition to predator–prey dynamics. Heterospecific scavengers might also affect parasite transmission from infectious carcasses, which can be an important source of infections for some wildlife diseases. A robust scavenger community can quickly remove carcasses and tissue and thus prevent secondary transmission by necrophagy or contact with infectious carcasses. Alternatively, by spreading infectious particles and tissues throughout the environment, scavengers may increase rates of casual contact with pathogens and thus overall transmission. However, there has been little empirical consideration of the contrasting roles that scavengers might play in infectious disease dynamics. We carried out a series of studies to determine the efficiency with which scavenging invertebrates remove carcasses of Long‐toed Salamander (Ambystoma macrodactylum) larvae and their role in the transmission of frog virus 3 (Genus: Ranavirus, Family: Iridoviridae) from carcasses. We then estimated the functional response of one efficient invertebrate scavenger (Family: Dytiscidae) to increasing carcass densities in field conditions in order to determine the capacity of scavenging invertebrates to consume large amounts of carcass tissue, as may be present at high prevalence sites. We found that removal of infectious carcasses by scavengers strongly reduced transmission to naïve larvae. Scavengers were as effective at reducing transmission from a carcass as a physical barrier preventing contact with the carcass. There was little evidence that scavenging released sufficient infectious tissues into the water column to rival direct contact as a route of infection. Moreover, while scavenging rates saturated at increasing carcass densities, consistent with a type II functional response, there were sufficient densities of dytiscid larvae, not to mention other scavenging invertebrates, in a surveyed pond to theoretically prevent transmission from carcasses. Our results suggest that at least in systems in which conspecific necrophagy is common, the scavenger community can play an important role in reducing transmission. A plain language summary is available for this article.

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Section: Methodsmentioning

confidence: 99%

Do scavengers prevent or promote disease transmission? The effect of invertebrate scavenging on Ranavirus transmission

2019

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“…All animals used in these experiments were confirmed negative for PaV1 DNA using the qPCR assay described in Clark et al (2018) [22]. The primers were PaV1nucleaseF (5’-CGTTGTACGGAATCGTTATTAAAGC-3’) and PaV1nucleaseR (5’-GACACGACCAATTGAAGAAAAACTAC-3’).…”

Section: Methodsmentioning

confidence: 99%

“…Amplification and detection were performed using a ThermoFisher Quant Studio 5 Real-Time PCR system. The reaction mix was subjected to an initial temperature of 95 °C for 20 s, then 40 cycles at 95 °C for 3 s and 60°C for 30 s. Quantification of the number of PaV1 copies were determined by measuring cycle threshold values and using the standard curve described in Clark et al (2018) [22]. Each sample was analyzed in triplicate, and the mean viral quantity calculated.…”

Section: Methodsmentioning

confidence: 99%

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White Spot Syndrome Virus and the Caribbean Spiny Lobster, Panulirus argus: Susceptibility and Behavioral Immunity

Ross

Behringer

Bojko

2018

Preprint

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2 The Caribbean spiny lobster Panulirus argus is susceptible to infection by Panulirus argus Virus 3 1 (PaV1), the only virus known to naturally infect any lobster species. However, P. argus is able 4 to mitigate PaV1 transmission risk by avoiding infected individuals. White Spot Syndrome Virus 5 (WSSV) has a particularly wide host range. WSSV has not been documented in wild 6 populations of spiny lobsters, but has been experimentally transmitted to six other lobster 7 species from the genus Panulirus spp. While WSSV has been detected intermittently in wild 8 populations of shrimp in the Caribbean region, the risk to P. argus has not been evaluated.9 Potential emergence of the disease could result in fisheries losses and ecological disruption.10 To assess the risk to P. argus, we tested its susceptibility to WSSV via injection and waterborne 11 transmission. We also tested whether healthy lobsters can detect and avoid conspecifics with 12 qPCR-quantifiable WSSV infections. We found P. argus to be highly susceptible to WSSV via 13 intramuscular injection, with mortality reaching 88% four weeks post inoculation. Panulirus 14 argus was also susceptible to WSSV via waterborne transmission, but WSSV burden was low 15 after four weeks via qPCR. Behavioral assays indicated that P. argus can detect and avoid 16 conspecifics infected with WSSV and the avoidance response was strongest for the most 17 heavily infected individuals -a response comparable to PaV1-infected conspecifics.18 Panulirus argus is the first spiny lobster found to be susceptible to WSSV in the Americas, but it 19 is possible that a generalized avoidance response by healthy lobsters against infected 20 conspecifics provides a behavioral defense and may reduce WSSV infection potential and 21 prevalence. Such avoidance may extend to other directly transmitted pathogens in spiny lobster 22 populations preventing them from becoming common in their population. 24Author Summary 25 Erica P. Ross is a PhD candidate at the University of Florida, studying the disease ecology of the 26 Caribbean spiny lobster, with a focus on chemosensory ecology. Donald C. Behringer is an 27 associate professor at the University of Florida and his research focuses on disease ecology, 28 epidemiology, and fishery ecology, with a focus on crustaceans and other marine invertebrates.29 Jamie Bojko received his PhD from the University of Leeds and is currently a post-doctorate 30 associate at the University of Florida studying experimental and systemic crustacean pathology. 31 35 globe. WSSV has resulted in mass mortality and economic loses of > 3 billion USD (US dollars), 36 annually [1, 2]. The virus can infect all species of penaeid shrimp and over 40 other species of 37 Crustacea (e.g., crabs, crayfish, and lobsters), with some studies showing successful 38 transmission to other arthropods, such as insects [3 -5]. WSSV has had its greatest impact on 39 aquaculture populations, but some screening studies have identified the virus in crustacean hosts 40 outside of aquaculture facili...

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“…EHNV can be detected by virus isolation in several cell lines [4,11], antigen-capture ELISA [12], and histological sections using specific anti-EHNV antibodies [2]. Further, there are several polymerase chain-reaction (PCR) assays targeting the major capsid protein [13,14,15] or polymerase gene [16,17].…”

Section: Introductionmentioning

confidence: 99%

Geographic Distribution of Epizootic haematopoietic necrosis virus (EHNV) in Freshwater Fish in South Eastern Australia: Lost Opportunity for a Notifiable Pathogen to Expand Its Geographic Range

Becker

Gilligan²,

Asmus³

et al. 2019

Viruses

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Epizootic haematopoietic necrosis virus (EHNV) was originally detected in Victoria, Australia in 1984. It spread rapidly over two decades with epidemic mortality events in wild redfin perch (Perca fluviatilis) and mild disease in farmed rainbow trout (Oncorhynchus mykiss) being documented across southeastern Australia in New South Wales (NSW), the Australian Capital Territory (ACT), Victoria, and South Australia. We conducted a survey for EHNV between July 2007 and June 2011. The disease occurred in juvenile redfin perch in ACT in December 2008, and in NSW in December 2009 and December 2010. Based on testing 3622 tissue and 492 blood samples collected from fish across southeastern Australia, it was concluded that EHNV was most likely absent from redfin perch outside the endemic area in the upper Murrumbidgee River catchment in the Murray–Darling Basin (MDB), and it was not detected in other fish species. The frequency of outbreaks in redfin perch has diminished over time, and there have been no reports since 2012. As the disease is notifiable and a range of fish species are known to be susceptible to EHNV, existing policies to reduce the likelihood of spreading out of the endemic area are justified.

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Partial validation of a TaqMan real-time quantitative PCR for the detection of ranaviruses

Cited by 30 publications

References 52 publications

Do scavengers prevent or promote disease transmission? The effect of invertebrate scavenging on Ranavirus transmission

Do scavengers prevent or promote disease transmission? The effect of invertebrate scavenging on Ranavirus transmission

White Spot Syndrome Virus and the Caribbean Spiny Lobster, Panulirus argus: Susceptibility and Behavioral Immunity

Geographic Distribution of Epizootic haematopoietic necrosis virus (EHNV) in Freshwater Fish in South Eastern Australia: Lost Opportunity for a Notifiable Pathogen to Expand Its Geographic Range

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