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Detection and reporting of ranavirus in amphibians

Citation for published version:Black, Y, Meredith, A & Price, SJ 2017, 'Detection and reporting of ranavirus in amphibians: Evaluation ofthe roles of the world organisation for animal health and the published literature', Journal of WildlifeDiseases, vol. 53, no. 3, pp. 509-520. https://doi.org/10.7589/2016-08-176

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DOI: 10.7589/2016-08-176 Journal of Wildlife Diseases, 53(3), 2017, pp. 000–000� Wildlife Disease Association 2017

DETECTION AND REPORTING OF RANAVIRUS IN AMPHIBIANS:

EVALUATION OF THE ROLES OF THE WORLD ORGANISATION FOR

ANIMAL HEALTH AND THE PUBLISHED LITERATURE

Yvonne Black,1,2,5 Anna Meredith,2 and Stephen J. Price3,4

1 Centre for Systems Studies, Hull University Business School, University of Hull, Hull HU6 7RX, UK2 Royal (Dick) School of Veterinary Studies and The Roslin Institute, University of Edinburgh, Easter Bush Campus,Midlothian EH25 9RG, UK3 Institute of Zoology, ZSL, Regents Park, London NW1 4RY, UK4 UCL Genetics Institute, Gower Street, London WC1E 6BT, UK5 Corresponding author (email: Y.Black@2016.hull.ac.uk)

ABSTRACT: Pathogens of wildlife can have direct impacts on human and livestock health as well as onbiodiversity, as causative factors in population declines and extinctions. The World Organization forAnimal Health (OIE) seeks to facilitate rapid sharing of information about animal diseases to enable up-to-date risk assessments of translocations of animals and animal products. The OIE also producesmanuals of recommended methods to standardize diagnostic testing. Ranaviruses are importantamphibian pathogens that may have spread through international trade, and infections becamenotifiable to OIE in 2009. We surveyed and reviewed published literature for data on sampling,diagnostic testing, and reporting of ranavirus during 2009–2014. We also investigated attitudes andawareness of the OIE and its recommendations for best practice. We found that sampling effort isuneven and concentrated in the northern hemisphere. We also identified citizen science projects thathave the potential to improve the quantity and quality of data on the incidence of ranavirus infectionand the circumstances surrounding disease outbreaks. We found reporting of infection to beinconsistent: reporting was split between the published literature (where it was subject to a 2-yr lag) andthe OIE with little overlap, results of negative diagnostic tests were underreported, and scientificresearchers lacked awareness of the role of the OIE. Approaches to diagnostic screening were poorlyharmonized and heavily reliant on molecular methods. These flaws in the mechanisms of ranavirusdetection and reporting hamper the construction of a comprehensive disease information database.

Key words: Molecular diagnostics, OIE, ranavirus, surveillance, wildlife disease, World Organiza-tion for Animal Health.

INTRODUCTION

Infectious diseases of wildlife can drivedeclines and extinctions of wildlife popula-tions and place significant burdens on thehealth of humans and domestic animals (Joneset al. 2008; MacPhee and Greenwood 2013).International cooperation in the control ofanimal diseases is facilitated by the WorldOrganization for Animal Health (OIE). Themain objective of OIE is to enable rapidinformation transfer about animal diseasesbetween its 180 member states as well asnonmembers (OIE 2017). The World AnimalHealth Information System (WAHIS) wasdeveloped to facilitate this transfer of infor-mation.

The OIE member states are required to fileregular updates of their national and regional

status with respect to listed diseases (OIE2016a). Laboratories undertaking diagnostictesting for notifiable diseases report positiveresults to their national veterinary authority,which then reports to the OIE (Jebara et al.2012). The frequency of reports depends onthe status (i.e., whether disease/infection ispresent or absent), with weekly updatesrequired immediately following a new out-break. The database of reports is intended toenable risk analysis of international trade inanimal products or sanitary methods in theinternational food trade—limiting pathogenpollution (Cunningham et al. 2003) andreducing the risk of new disease outbreaks.In addition to sharing disease information, theOIE produces the Terrestrial and AquaticAnimal Health Codes, setting out standardsfor the improvement of animal health and

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welfare worldwide, and for safe internationaltrade in animals and their products (OIE2016a). Manuals of Diagnostic Tests arepublished to standardize use diagnostic tech-niques for listed diseases (OIE 2016b),pointing laboratories to established methods.

Until recently, most OIE listed diseasescould be described as having direct impactson either human health (zoonotic pathogens)or food production (pathogens capable ofaffecting domestic food producing animals).However, two diseases linked to amphibiandeclines, and known to have been translocatedby humans, were listed as notifiable to theOIE in 2009: chytridiomycosis, caused by the‘‘chytrid’’ fungus Batrachochytrium dendro-batidis (Bd), and ranavirosis, caused by largedouble-stranded DNA viruses of the genusRanavirus (Schloegel et al. 2010). Neither ofthese pathogens has zoonotic potential, andalthough they can affect domestic amphibiansused in food production, they are primarily ofglobal concern because of impacts on wildpopulations and communities of amphibians(Cunningham et al. 2003; Price et al. 2014).

Ranaviruses (genus Ranavirus, family Iri-doviridae) have a patchy global distributionand broad host ranges, affecting amphibians,reptiles, and fish (Duffus et al. 2015). Inamphibians, they are important pathogenscapable of causing population decline andextinction, which may affect entire communi-ties (Earl and Gray 2014; Price et al. 2014).Humans have contributed to the ongoingemergence of ranavirosis through the move-ment of amphibians in trade (Picco andCollins 2008; Schloegel et al. 2009). Thepotential for very severe host impacts and alikely role for international trade in facilitatingemergence were major factors behind theOIE decision to make ranavirus notifiable(Schloegel et al. 2010).

We assessed the nature and extent of globaldetection and reporting of ranavirosis. Weevaluated the role of the OIE in ranavirussurveillance with respect to its main objec-tives: to facilitate rapid sharing of informationand to enable risk assessments of trade inanimal products. We also evaluated the role ofthe published literature in reporting informa-

tion about ranavirus distribution. We analyzeddata on the motivations for undertakingsampling amphibians for detection of ranavi-rus infection and the uptake and use of therecommended methods given in the OIE’sAquatic Manual. Finally, we used additionalsurvey data to assess the potential for anenhanced role for citizen science projects inranavirus surveillance.

MATERIALS AND METHODS

Systematic literature review

We searched the literature in January 2015 withthe use of the bibliographic databases: BioMedCentral, BIOSIS Citation Index, and CAB Ab-stracts. Variations of ‘‘amphibian’’ and relatedterms (amphib*, frog, toad, salamander, newt,caecilian, caudat*, anur*, gymnophion*) weresearched for in conjunction with variations of‘‘ranavirus’’ or ‘‘iridovirus’’ (ranavir* or iridovir*).Articles which did not describe original researchinvolving testing of naturally infected amphibiansfor ranaviruses sampled since 2009 (i.e., reviewarticles and articles describing testing of experi-mentally infected amphibians) were discarded.Data collection was restricted to 2009 or afterbecause ranavirus infection became notifiable tothe OIE in that year, and one of our objectiveswas to compare published studies and OIE data.All papers included in the review related tosampling carried out since 2009 and werepublished before January 2015.

To ensure that the literature search wasexhaustive we checked our article list againstresources made available by the Global RanavirusConsortium (GRC; GRC 2014) and a tablepublished by Miller et al. (2011) which detailedall instances of amphibian ranavirus infectionknown at that time by geographical region. Theseresources revealed only one additional reference,a thesis paper (Todd-Thompson 2010).

We collated data from published articles,including the geographic origin of the amphibiansamples, whether captive or free-ranging hostswere tested, the number of samples tested, thediagnostic method(s) used, and the number ofsamples positive for ranavirus infection. Paperswere categorized according to the period of timein which samples were collected (2009–2011 or2012–2014) to enable comparisons with question-naire data.

Questionnaires

We produced two surveys, approved by theRoyal (Dick) School of Veterinary Studies Human

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Ethical Review Committee at the University ofEdinburgh, with questions relating to ranavirussurveillance and diagnostic testing using Survey-Monkey (2015). The first survey targeted labora-tories or institutions undertaking diagnostictesting of amphibian samples for ranaviruses(‘‘laboratory survey’’) to gather data on theamount, motivation, and methods of ranavirussurveillance (Supplementary Materials S1). Thesecond survey targeted nongovernmental organi-zations and citizen science projects (‘‘NGOsurvey’’) and aimed to characterize the role andpotential for such projects in surveillance (Sup-plementary Material S2).

We contacted the GRC mailing list, Interna-tional Union for Conservation of Nature Amphib-ian Specialist Group (ASG) members, and authorsof published papers on the subject of amphibianranaviruses by email in late 2014 to invite them torespond to the questionnaires online. We alsoemailed links to the NGO survey to representa-tives of amphibian conservation groups. Links toboth online surveys were published in the ASGblog, and an article highlighting the surveys wasincluded in FrogLog (Black 2015). Maps summa-rizing data from both the systematic review andquestionnaire data were generated in R (R CoreTeam 2013) using the World Borders Dataset(Sandvick 2009).

Both questionnaires contained a section forindividual and organizational contact details, butrespondents were assured that data would becategorized only by geographical region andreported anonymously. Personal details of respon-dents were used to avoid duplication betweenquestionnaire data and published records.

The laboratory survey contained sections on thesources of samples, the purpose of testing, thenumber of samples tested between 2012 and 2014inclusive, the number of positive results, andreporting methods. Questions focused on thepreceding 3 yr (2012–2014) for practical reasonsrelating to ease of access to records. The NGOsurvey contained sections on the type of organi-zation, the recording of amphibian mortalityevents, and the facilitation of sample submissionfor diagnostic testing.

Participants in the laboratory survey were alsoasked about the specifics of diagnostic tests usedas well as attitudes towards and use of the OIEAquatic Manual. The Aquatic Manual currentlyrecommends two molecular approaches for de-tection and identification of ranaviruses: PCRtesting followed by either restriction endonucle-ase analysis (REA) or DNA sequencing (OIE2014). Follow-up of PCR with DNA sequencing isrecommended to confirm that positive resultsyield from amplification of the desired targetrather than some other nonspecific target andtherefore confirm that the assay is robust.

In addition to responses to survey questions, weassessed how frequently the OIE-recommendedPCR method was utilized via a search of the NCBInucleotide databases (Clark et al. 2016). Wecompared the OIE-recommended primer set toPCR primers 4 and 5 of Mao et al. (1997), whichtarget different regions of the same major capsidprotein (MCP) gene. To obtain sequences for thePCR products produced by both assays, weextracted the nucleotide sequence at the genomicregions amplified by each primer set from the typespecies of ranavirus, frog virus 3 (Tan et al. 2004).To check how many sequences from each assayhad been archived in GenBank, we used the Frogvirus 3 sequences as queries to search for similarsequences at the same genomic region. Weperformed megablast searches against the nr/ntdatabases with ‘‘max. target sequences’’ set to1000, ‘‘expect threshold’’ set to 0.1 and remainingsearch parameters set to defaults (McGinnis andMadden 2004). Searches were limited to entriestagged ‘‘Ranavirus (taxid:10492).’’ To recover onlysequences likely to originate from sequencing ofPCR products we removed those hits with lowerthan 75% coverage of the full PCR products as wellas removing hits to whole or partial genomesequences or the complete gene.

Phylogenetics

In addition to validating results of diagnostictests, we hypothesized that information gainedfrom DNA sequencing might convey importantinformation about virus genotype that could beused to make management decisions. We usedphylogenetics to assess the precision of virusgenotype information gained from sequencing theproduct of a positive PCR screen for the MCPgene of ranavirus (see Supplementary MaterialsS3 for detailed methods). We constructed aphylogenetic tree from sequences in the genomicregion amplified by the MCP PCR primers 4 and5 (Mao et al. 1997) and compared this to a treeconstructed from the sequences of 26 completegenes (considered more reliable than candidategene approaches for inferring relationships amongvirus isolates (Jancovich et al. 2010; Mavian et al.2012). Both phylogenetic trees used nucleotidedata from 18 ranavirus isolates with whole-genome data available in NCBI GenBank (isolatedetails in Table S1).

Statistical analysis

To evaluate the potential for bias in reportingthe absence ranavirus through the publishedliterature we performed a chi-squared (v2) teston the proportion of positive to negative recordscompared to the same type of data gatheredthrough the lab survey.

BLACK ET AL.—DETECTION AND REPORTING OF RANAVIRUS 3

RESULTS

Forty-three papers were included in thesystematic review of literature (Supplementa-ry Materials Table S2). The laboratory surveywas completed by 43 respondents and theNGO survey by 21 respondents. Not allrespondents answered all questions.

Surveillance effort

Our survey of ranavirus reporting over theperiod 2009–2014 showed that although thenumber of samples and sampling events pergeographic area was extremely variable, allcontinents where amphibians occur weresampled except Australia (Fig. 1). Fewsampling events were recorded in someregions. For example, Africa remained largelyunsampled apart from Cameroon and Mada-gascar. Most effort was expended in thenorthern hemisphere; 79% (34/43) of pub-lished papers recorded sampling from NorthAmerica and Europe but relatively few studiesreported sampling in the tropics and in thesouthern hemisphere. Most records from thesouthern hemisphere occurred in the last 3 yrexamined (Fig. 1c), suggesting that changemay be occurring.

Scientific researchers made up 88% (30/34)of laboratory survey respondents who an-swered a question on the source of amphibiansamples. Sixty-eight percent of them saidsamples were ‘‘always’’ or ‘‘often’’ the resultof their own sampling with samples submittedby members of the public, governmentagencies, and veterinarians contributing tothe remainder (Fig. 2a). Respondents listedongoing surveillance for a previously foundpathogen, routine scanning for a pathogen notpreviously present, investigations of massmortality events, and studies to answerspecific research questions as motivations forsampling amphibians for ranavirus with sim-ilar frequencies (Fig. 2b).

Citizen science

A total of 48% (10/21) of NGO surveyrespondents stated that they facilitated thesubmission of amphibian samples for labora-

tory testing. Data on amphibian mortality anddisease distribution was also collected bycitizen science projects: 67% of NGO surveyrespondents reported that their organizationreceived data concerning sick or dead am-phibians from members of the public.

Reporting

A total of 12% (5/43) of published papersreported no samples testing positive forranavirus. In comparison, 33% (5/15) ofrespondents to the laboratory survey reportedthat they had obtained no positive results. Asignificantly higher proportion of publishedpapers reported the presence of ranaviruscompared to unpublished laboratory surveys(v2¼9.11, P,0.01).

Only 14% (6/43) of published papersconducted sampling between 2011 and 2014,as compared with 86% between 2009 and

FIGURE 1. Geographical overview of samplingeffort by researchers who performed diagnostic testsfor Ranavirus using data from (a) published literature(in the period 2009–2011); (b) published literature(2012–2014); (c) responses to a survey of laboratories(2012–2014). n¼number of samples collected at eachlocation.

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2011. Some of these changes may reflectactual changes in ranavirus diagnostic testingefforts; however, they may also be a conse-quence of the considerable time lag inpublishing data. The mean lag time (date oflast sampling subtracted from publicationdate) for publication was 2 yr (standard

deviation¼1.2 yr), with some studies notpublished until 5 yr after the last samplingdate.

The WAHIS database is not a comprehen-sive source of distribution data for infectionwith ranaviruses. Our laboratory survey andliterature review revealed amphibian samplesfrom Germany, Russia, Costa Rica, Nicaragua,and Cameroon that tested positive since 2009(Fig. 3), but WAHIS has ‘‘no informationavailable’’ for these countries (WAHID 2015).The disease was also listed by WAHIS asnever having been reported in China, and‘‘not reported in this period’’ in Belgium orSpain, although the published literature de-tailed ranavirus infections in each of thesethree countries since 2009 (Fig. 3).

Survey data revealed a general lack ofawareness regarding the existence and func-tion of the OIE: only 21% (7/34) of respon-dents who answered a question on the subjectstated that they reported the results of theirtesting to the OIE. Results of testing werereported to bodies other than the OIE, with32% of laboratory questionnaire respondentsstating that they reported results to the samplesubmitter, national conservation bodies, orgovernment entities.

FIGURE 2. Sources of samples and reasons forundertaking diagnostic testing for ranaviruses. (a)Respondents to a survey of laboratories (n¼34)indicated the frequency with which a list of individualsand organizations submitted amphibian samples totheir laboratory (question 4, laboratory survey; seeSupplementary Materials S1). (b) Given a list ofpossible motivations, respondents (n¼22) indicated thefrequency with which amphibian ranavirus testing wascarried out at their laboratories (question 20, labora-tory survey; see Supplementary Materials S1). Sug-gested motivations were ‘‘Routine scanning (for apathogen not previously present),’’ ‘‘Ongoing surveil-lance (for a previously found pathogen),’’ ‘‘(Toinvestigate a) mass mortality event’’ or ‘‘(To answera) specific research question.’’ Respondents were notrequired to select a frequency (always, often, some-times, occasionally, or never) for every source/motivation on the lists. ‘‘Not selected’’ is used whereno selection was made and is expected to correspondclosely with ‘‘never’’ as, in all cases, respondents hadselected other options as applying always, often,sometimes, or occasionally.

FIGURE 3. Comparison of the World Organizationfor Animal Health (OIE) reporting system and thepublished literature as sources of information aboutspatial and temporal trends in reports of Ranavirusinfections and disease outbreaks between 2009 and2014. The OIE reporting occurs at a national level soreports are summarized at that level and indicated byflags.

BLACK ET AL.—DETECTION AND REPORTING OF RANAVIRUS 5

Diagnostic methods

In total, 56% (24/43) of published accountsand 68% (15/22) of survey respondents whoanswered questions on diagnostic methods,applied OIE recommended methods (cellculture, PCR with sequence analysis, or both).None described the use of PCR-REA. Moststudies used just one diagnostic method forranavirus diagnostic testing (58% of publishedaccounts and 45% of laboratory surveyrespondents). Both conventional and quanti-tative PCR approaches predominated. Onlyone study that applied a single technique useda non–PCR-based approach (cell culture; Fig.4a). The use of DNA sequencing was not doneto confirm specific amplification of PCR

targets in 47% (20/43) of published studies,and 50% (8/16) of laboratory survey respon-dents using conventional PCR also did notroutinely carry out sequencing of PCRproducts.

The OIE manual of diagnostic tests was notreferred to for best practice by 66% (22/33) oflaboratory survey respondents. The failure tofollow OIE recommendations stemmed froma lack of awareness, perceived capacity, andconfidence in the methods. Twelve respon-dents had never heard of the Aquatic Manual,and cost (three respondents), time (tworespondents), or lack of agreement with therecommendations (three respondents) wereother reasons given.

A variety of published PCR protocols(Table 1) were cited by published papersand by laboratory survey respondents. Noneof these publications contained details of thediagnostic sensitivity or specificity of theprotocol when used on amphibian samples.The most recent edition of the AquaticManual recommends the use of a specificPCR targeting the ranavirus MCP gene (580base pairs [bp] sequence; OIE 2016b).However, we found that most investigators(94% of respondents) using a conventionalPCR method followed the PCR protocol ofMao et al. (1997) which targets a differentregion of the same gene. An analysis ofsequences that were present in the NCBInucleotide sequence databases confirmedthis preference for the Mao et al. (1997)assay over the recommended OIE protocol:there were 81 entries for the Mao et al.(1997) PCR product compared with only 30entries for the OIE recommended PCRproduct (Supplementary Materials S3). Un-published protocols were used by 62% (8/13)of laboratory survey respondents who usedquantitative real-time PCR (qPCR) and halfof these stated that their protocol was either‘‘not validated’’ or that they were unsure howit was validated.

Phylogenetics

To assess the quality of information lostthrough failure to follow OIE recommenda-

FIGURE 4. Summary of common techniques usedin diagnostic testing for Ranavirus recorded (a) inpublished studies and (b) by respondents to a surveyof laboratories. Where more than one test was used bya single respondent or study, the different methodsused are each counted once—frequency is thereforeable to exceed the number (n) of individual respon-dents/studies that contributed. PCR¼conventionalpolymerase chain reaction assay; qPCR¼real-timequantitative PCR; VI¼virus isolation; Other¼any otherdiagnostic technique.

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tions regarding sequencing of PCR products,we compared phylogenies constructed from 1)sequences in the genomic region covered bythe product of the most commonly used PCRmethod (Mao et al. 1997), and 2) a concate-nated alignment of 26 conserved ranavirusgenes. The concatenated gene approachresulted in much more data for analysis: thesequence alignment was 38,793 bp in lengthversus 531 bp for the MCP PCR productalignment. However, both phylogenies sup-ported very similar topologies, with the lack ofmonophyly of common midwife toad virus–like (CMTV-like) viruses in the MCP treebeing the major difference. In spite of anoverall loss of resolution resulting from themuch smaller data set, the MCP PCR producttree offered a reasonable phylogenetic signal;that is, sequence data derived from this testcould be used to assign virus isolates to themain virus types (Fig. 5).

DISCUSSION

We used a systematic literature review andquestionnaire surveys to collect data onsampling of amphibians for ranaviruses, diag-nostic methods, and reporting of results since2009 when infection with these viruses was

made notifiable to the OIE. We examinedthese data in the context of the OIE’sobjective to facilitate rapid sharing of infor-mation about infections with important animal

TABLE 1. PCR methods used in diagnostic screening for Ranavirus infection reported through the publishedliterature and a questionnaire survey of laboratories. Details of original publication where method was describedand the availability of validation (diagnostic sensitivity/specificity) data.

Citation Validation data available?

Conventional PCR protocols

Mao et al. (1997) No

Hyatt et al. (2000) No

Holopainen et al. (2009) No

Bollinger et al. (1999) No

Kattenbelt et al. (2000) No

Real time PCR (qPCR) protocols

Picco et al. (2007) Cites Brunner (2004)

Pallister et al. (2007) No

Brunner and Collins (2009) No

Brunner (2004) Some validation data published later(Hoverman et al. 2011, citing Picco et al. 2007)

Forson and Storfer (2006) No

Allender et al. (2013) Yes (in turtles)

FIGURE 5. Phylogenetics was used to assess theamount of information on virus genotype (phyloge-netic signal) obtained through DNA sequencing of acommonly generated PCR product by comparison to amultigene method of tree construction from completevirus genomes. Both trees contain the same virusisolates and are presented side by side with majorclades highlighted. The phylogeny on the left utilizesdata from 26 conserved genes (total alignment lengthwas 38,793 base pairs [bp]). The phylogeny on theright utilizes sequences covering part of the majorcapsid protein gene corresponding to the regionamplified by primers 4 and 5 (531 bp) from Mao etal. (1997).

BLACK ET AL.—DETECTION AND REPORTING OF RANAVIRUS 7

pathogens. We found that OIE members wereunderreporting ranavirus infection to theOIE, resulting in a WAHIS database whichdid not reflect the full state of knowledge. Wealso found that recommendations set out inthe Aquatic Manual were not routinelyfollowed. Furthermore, failure to follow OIEadvice to sequence PCR products has resultedin a loss of genetic data that could be used tocharacterize viruses with direct implicationsfor management.

The published literature and OIE reportingsystem represent the two main reportingmechanisms for ranavirosis, but we foundthere to be very little overlap between thetwo. Of 19 nations reporting ranavirus infec-tions in the period 2009–2014, only threewere present in both WAHIS and thepublished literature (though the Frenchoutbreak has been published recently; Miaudet al. 2016). Ranavirus reporting for theremaining nations was split evenly betweenthe OIE system and the published literature.Efforts to control diseases with direct effectson domestic animals, coordinated by the OIE,have resulted in real successes such as theeradication of rinderpest (Njeumi et al. 2012).However, reporting of wildlife diseases can beopportunistic, as seen with avian influenzasurveillance in wild birds (Machalaba 2015).Only half of laboratory survey respondentsreported their results either directly to theOIE or to another body that may haveassumed this responsibility.

We found that most samples tested forranavirus were collected by research scien-tists, which in the context of the split inreporting between OIE reporting system andthe published literature, suggests a lack ofcommunication between the academic com-munity and government agencies. This meansthat timely reporting of results is likely toremain a challenge. The primary output ofacademic research is publication in peer-reviewed journals, but we found weaknessesin the scientific literature as a reportingmechanism, with an average time lag betweensampling and publication of 2 yr. We alsofound a significant bias towards the publica-tion of positive results. Despite a move toward

open-access data sharing, barriers to datapublishing and reuse remain, including a lackof the time and resources but also a lack ofexplicit career rewards for such sharing (Swanand Brown 2008).

It is not clear whether ranavirosis is atypical case for wildlife diseases listed by theOIE but it is reasonable to think there will becomparable cases, because some of thelaboratories we surveyed are centers fordiagnostic testing of pathogens from diversehost species, and many of the surveyed NGOswere amphibian and reptile groups with aninterest in multiple diseases. The fungaldisease, Bd and ranavirus became OIE listedat the same time, and diagnostic testing forboth pathogens often occurs in the samelaboratories, so many of the same issues withreporting may be relevant to reporting ofchytridiomycoses. A brief comparison of thedatabases of the open-source ‘‘Bd-maps’’project and the OIE appeared to confirmthis; e.g., Bd-maps recorded Bd in Chile after2009 (Bourke et al. 2010) but the OIEdatabase had no record from Chile.

In addition to these challenges surroundingreporting, we also found challenges aroundsampling for ranavirosis with a geographicalbias towards the northern hemisphere. Am-phibian mass-mortality events triggered sam-pling for ranaviruses (e.g., Reshetnikov et al.2014), but our survey respondents indicatedthat diagnostic testing, ongoing surveillanceand the pursuit of answers to specific researchquestions were equally common motivations.The availability of funding is likely to be a keyfactor affecting the geographical distributionof sampling effort (Jones et al. 2008). Howev-er, although some countries conduct wildlifedisease surveillance as a part of routinemanagement, most still only address eventsin postoutbreak scenarios (Schwind et al.2014).

The OIE has also been unsuccessful instandardizing diagnostic testing methodsthrough its manuals; rather, we found manydifferent tests in use. We found that a singleassay is typically used in testing for ranavi-ruses, almost always a molecular method.Molecular methods are hugely valuable, but

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they only provide information on the presenceof a pathogen rather than disease. Rijks et al.(2016) have taken a more thorough approach,utilizing pathology in combination with mo-lecular approaches. This is by no means aproblem specific to ranavirus research, but arigorous understanding of the limitations ofcommonly utilized molecular methods isrequired, especially the necessity for valida-tion. By comparison, diagnostic testing ofchytridiomycoses has been more standardizedbut similarly narrow in terms of the type ofdiagnostic approach used. For both B. den-drobatidis and Batrachochytrium salaman-dravorans, the rapid publication of amolecular method was followed by almostuniversal adoption (Boyle et al. 2004; Blooi etal. 2013). In the case of ranavirus, there havebeen periods of stability (e.g., the dominanceof the Mao et al. (1997) PCR method) butmany—often unvalidated—qPCR protocolsare now in use.

The OIE does not include a qPCR methodamong its recommended methods, but this islikely because of a lack of available protocolsat the time of the last release of its manual. Inthe absence of OIE recommendations, weurge caution in the interpretation of results.Methods are generally unvalidated or validat-ed only in a limited way (Jaramillo et al. 2012).Miller et al. (2015) warn against reliance onhigh cycle threshold (Ct) scores from qPCRassays in detection because of the risk of false-positive results. At the limit of detection (thelowest amount of the target detectable in asingle reaction), results for identical samplesfrom different laboratories may differ (Waib-linger et al. 2011). Despite this, recent studieshave made strong conclusions based only onhigh or unreported Ct scores from partiallyvalidated qPCR methods (Kolby et al. 2015;Warne et al. 2016).

The OIE recommends DNA sequencing ofPCR products to support results from diag-nostic testing, but we found that sequencingwas not carried out in half of cases. Inlocalities where infections have been con-firmed previously, such repeated confirmationmay be unnecessary. However, given theimperfect specificity of molecular methods

and potential for contamination of samples, itseems a highly desirable step when testing forinfections in new localities or hosts. We alsoshowed that even small amounts of DNAsequence data can yield useful informationabout virus genotype, which might haveimportant implications for management giventhat virus genotypes are somewhat spatiallyseparated (e.g. CMTV-like viruses in main-land Europe and Asia) and that genotype maypredict differences in virulence and hostrange (Teacher et al. 2010; Price et al.2014). In many cases, we found that evenwhen sequencing had occurred these datawere not always archived in a public database,which limits the capacity to deliver the OIEobjective of enabling the best possible riskassessments.

In addition to the OIE system of reporting,there are some disease-specific internationalreporting systems. The afore-mentioned ‘‘Bdmaps’’ is an open-source geographical data-base to collate the results of diagnostic testingfor Bd (Aanensen and Fisher 2016). TheGlobal Ranavirus Consortium recentlylaunched a similar project for ranavirusreporting: the Global Ranavirus ReportingSystem (GRRS; GRRS 2016). These systemsrely on voluntary reporting of disease infor-mation. The GRRS has the potential toprovide a more comprehensive source ofinformation on ranavirus infection than cur-rently exists. Certainly, the established Bd-maps project holds a large volume of data.The GRRS is heavily integrated within theacademic community (who currently under-take the bulk of ranavirus diagnostic testing)through the Global Ranavirus Consortium,and therefore might be able to cut the lag inreporting via academic literature by gainingpermission to share basic information aheadof publication. This online system also uses asimple, open-access form that delivers a morestraightforward reporting route than thatcurrently utilized by the OIE.

Citizen science projects can make impor-tant contributions to wildlife disease surveil-lance (Lawson 2015). Indeed, approximatelyhalf of the respondents to our NGO surveyhad facilitated the submission of dead am-

BLACK ET AL.—DETECTION AND REPORTING OF RANAVIRUS 9

phibians for diagnostic testing. Citizen scienceprojects can also co-ordinate targeted diseasesurveillance efforts (Griffiths et al. 2015) andenable large-scale reconstructions of diseaseemergence to test hypotheses about modes ofspread (North et al. 2015; Price et al. 2016).Our NGO survey also showed that manyamphibian groups recorded mortality andmorbidity events, which can be used as abasis for syndromic surveillance. Projects inthe Netherlands, the UK, and the US arecurrently monitoring the spatial and temporaldistribution of amphibians and their mortalityevents, as well as facilitating postmortemexamination and diagnostic testing of deadamphibians by liaising with members of thepublic (Lawson et al. 2015; Reptile, Amphib-ian, and Fish Conservation in The Nether-lands 2016; US Geological Survey 2016).

If we are to meet the challenges presentedby the rapidly evolving interactions betweenpathogens, wildlife, livestock, and humans,then the global scientific community mustcooperate wherever possible to adopt a robustapproach to diagnostic testing and rapid,consistent sharing of data. This should bedone utilizing the best available platform,which may be either WAHIS or a disease-specific network such as the GRRS.

ACKNOWLEDGMENTS

We thank Will Leung for helpful discussionsabout molecular methods and Trent Garner forreviewing a draft of the manuscript. We also thankall those who responded to the laboratory andNGO surveys. S.J.P. was funded by NaturalEnvironment Research Council grants (NE/M00080X/1, NE/M000338/1, and NE/M000591/1) and European Research Council grant 260801-BIG-IDEA.

SUPPLEMENTARY MATERIAL

Supplementary material for this article ison l ine a t h t tp : / /dx .do i .org /10 .7589/2016-08-176.

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