Risk analysis CMTV-like virus April 2013 1 - NVWA...Risk analysis CMTV-like virus April 2013 2 This document should be referred to as: Rijks JM, Sptizen-van der Sluijs A, Leuven RSEW,

_____________________________________________________________________________________________Risk analysis CMTV-like virus April 2013 1


This document should be referred to as:

Rijks JM, Sptizen-van der Sluijs A, Leuven RSEW, Martel A, Kik M, Pasmans, F, Zollinger R, Verbrugge LNH, Gröne

A (2012). Risk analysis of the common midwife toad-like virus (CMTV-like virus) in the Netherlands. NVWA, Min EZ

report 60000784-2012


Table of contents

Executive summary 5

Part 1 - General background information for risk analysis 9

Theme 1 - The virus 10

Theme 2 - Distribution of CMTV-like virus 12

Theme 3 - Known susceptible host species 14

Theme 4 - Host life stages affected 16

Theme 5 - Dispersal characteristics of host species 18

Theme 6 - Distribution of susceptible host species in the Netherlands 20

Theme 7 - Distribution of potential hosts across the border 22

Theme 8 - Accompanying amphibian species 24

Theme 9 - Water body types with CMTV-like virus 26

Theme 10 - Month of outbreak and water temperature 28

Theme 11 - Water quality at outbreak sites 30

Theme 12 - Inventory of trade of amphibians in Belgium 32

Theme 13 - Inventory of hobby kept amphibians in Belgium 34

Theme 14 - Legal re-introductions and translocations in the Netherlands 36

Theme 15 - Illegal introductions 38

Theme 16 - Flyways 39

Part 2 - Risk assessment 40

1. Method 40

2. Results 42

2.1 Probability of introduction 42

2.1.1 Introduction through human activities 42

2.1.1.1 Legal trade through Schiphol 42

2.1.1.2. Amphibian trade in Belgium 44

2.1.1.3 Entry of CMTV-like virus imported via live amphibians into nature 45

2.1.2. Introduction through natural dispersal 46

2.1.2.1 Introduction via infected amphibians crossing borders into the Netherlands 46

2.1.2.2 Introduction via migrating birds 46

2.2 Likelihood of establishment 47

2.2.1 Susceptible hosts are present 47

2.2.1.1 Susceptible species 47

2.2.1.2 Susceptible populations 47

2.2.1.3 Susceptible individuals 47

2.2.2 Dutch environment in general appears suitable 49

2.2.2.1 Temperature 49

2.2.2.2 Suitable sites 49

2.2.3 The virus has strategies to maintain itself 49

2.2.3.1 Probable lengthy survival outside the host 50

2.2.3.2 Reservoirs 50

2.3 Probability of spread 50

2.3.1 Spread through human activities 50

2.3.1.1 Legal re-introductions and translocations of threatened native species 50

2.3.1.2 Other human-mediated transfer of potentially infected hosts 52

2.3.1.3 Dispersal by fomites (equipment, transfer of water or sediment) 52

2.3.2 Natural spread 52

2.3.2.1 Dispersal of (sub-)clinically infected hosts 52

2.3.2.2 Birds or other animals 53


2.4 High risk areas 53

2.5 Impact 54

2.5.1 Ecological impact 54

2.5.1.1 Amphibians 54

2.5.1.2 Effects on biodiversity 55

2.5.2 Socio-economic impact 56

2.6 Risk classification using the ISEIA protocol 57

2.6.1. Expert consensus scores 57

2.6.2 Dispersion potential or invasiveness 57

2.6.3 Colonisation of high conservation value habitats 58

2.6.4 Adverse impacts on native species 58

2.6.5 Alteration of ecosystem functions 58

2.6.6 Risk classification 59

Part 3 - Risk management options 61

3.1. Prevention of introduction 61

3.1.1 Make sure that CMMTV-like virus can be detected in imported captive specimens

at the border

3.1.1.1 Obtain sufficient sight on amphibians imported 61

3.1.1.2 Improve the capacity to detect CMTV-like virus in imported specimens

at the border 61

3.1.2 Reduce the risk that CMTV-like virus enters nature via imported kept amphibians

and their waste 63

3.1.2.1 Obtain good sight on amphibian and vivarium waste disposal behavior 63

3.1.2.2 Make sure that people are aware of risky behavior and know how to

minimize it 63

3.1.3 Make sure that CMTV-like virus imported via natural dispersal can be detected early

on at its site of introduction, before it occurs widespread 63

3.1.3.1 Obtain a better understanding of the relative importance of introduction

via natural dispersal 63

3.1.3.2 Ensure monitoring for early detection and source tracing 63

3.2 Elimination 65

3.2.1 Assisted elimination – currently only applies to captive populations and small

free-living populations 65

3.2 1.1 Encourage destocking and disinfection for elimination of infection in

captive settings 65

3.2.1.2 Explore options for elimination in the case of (small) infected free-living

populations 65

3.2.2 Natural elimination – learn from it for larger scale assisted elimination 66

3.3 Control 68

3.3.1 Prevent human mediated spread of virus within the Netherlands 68

3.3.1.1 Prevent inadvertent to new sites via re-introduction projects 68

3.3.1.2 Prevent inadvertent spread to new sites by public or field biologists 68

3.3.2 Explore the possibility to take advantage of natural barriers to limit natural dispersal 68

3.3.3 Try to avoid further impact on threatened species and high conservation value habitats 68

3.3.4 Make sure the correct data is collected to be able to predict the effectiveness of control

And elimination measures 69

Glossary 71

List of abbreviations 72

Acknowledgements 73

References 73

Contributors 80


Executive summary

The term CMTV-like virus is used to indicate the common midwife toad virus (CMTV, GenBank accession no.

FM213466.1) and viruses with partial major capsid genes showing 99.8% to 100 % sequence homology. CMTV-like

virus infection is an emerging infectious disease in wild amphibians in the Netherlands. One possibility is that CMTV-

like virus is a recently introduced exotic pathogen.

Study aim and set-up The aim of this study is to perform a risk analysis for CMTV-like virus in the Netherlands. General background

information from the literature, field surveys and interviews, was compiled in 16 themes (Part 1). Subsequently, an

assessment was performed to identify 1) the possible routes of introduction of CMTV-like virus into the Netherlands,

2) the likelihood of its establishment, 3) the likelihood of its spread, and 4) the possible ecological and social impact of

CMTV-like virus. An ecological risk classification score was obtained using the Belgian Invasive Species

Environmental Impact Assessment (ISEIA) protocol (Part 2). Finally, opportunities for management were evaluated,

as well as aspects that require further understanding for appropriate decision-making (Part 3).

Part 1 - General background information for risk analysis (themes 1-16) CMTV-like virus is an amphibian ranavirus (Th.1). The virus has been detected in restricted areas in Spain in 2007

and 2008, in Belgium in 2010, and in the Netherlands in 2010, 2011 and 2012. In the Netherlands, the virus was first

detected in a high conservation value habitat, the National Park Dwingelderveld (Th.2).

Ranaviruses infect amphibians, reptiles or fish, or several of these (Th.1). CMTV-like virus has caused disease and

mortality in half of the amphibian species that are native to the Netherlands. Among these species is the vulnerable

common spadefoot Pelobates fuscus. The susceptible amphibian species belong to the order of frogs (Anura) or

salamanders (Caudata). In addition, CMTV-like virus infections have been observed naturally in amphibian species

exotic to the Netherlands, in particular the American bullfrog Lithobathes catesbianus and poison dart frogs

Dendrobates spp. The infected amphibians can show disease, but infection can also be subclinical. Evidence for

infection of fish is inconclusive: one specimen found dead tested positive by PCR but autolysis hampered

pathological investigation. Therefore, it remains unclear whether this case represented a true infection or

contamination from the aquatic environment (Th.3). Multiple life stages of amphibians were shown to be infected in

the Netherlands. The fact that not only larvae and juveniles but also (sub-)adult amphibians died during outbreaks, is

suggestive of disease emergence in immunologically naïve populations (Th.4).

A number of the susceptible host species are common species found in most of the country (Th.5), as well as in the

neighbouring countries Belgium and Germany (Th.6). Many are good dispersers, meaning they may move distances

up to several kilometres per year (Th.7). This information is relevant to the potential of the virus to spread. Further,

many of the susceptible host species are common and characteristic accompanying species for each other,

suggesting frequent opportunity for inter-species exposure (Th.8).

Themes 9 – 11 report environmental factors that are associated with disease occurrence. The water bodies where

CMTV-like outbreaks have been detected are both natural and man-made and vary in soil type, size, altitude, and

uses. However, they were generally permanent, small to medium sized and unconnected to other water bodies

(Th.9). Mass mortalities due to CMTV-like virus occurred in the period May to September 2011. Monitoring at infected

pools in the National Park Dwingelderveld showed that water temperature at monitored sites was on average ± 19°C,

and it followed mean daily air temperature. However, mortality can also occur in water that is several degrees colder

(Th.10). Measurements provided no clear evidence for poor water quality at sites with CMTV-like virus associated

amphibian mortality (Th.11).

Themes 12 – 16 cover aspects relevant to translocation of infection. For understanding of the ways how amphibians

could enter the Netherlands via Belgium, data were obtained on amphibians provided for trade by importing dealers

in Belgium. Most of these amphibians were captive bred or farmed, but some were wild-caught. Several thousands of

amphibians are imported into Belgium from outside the EU each year. In addition, numerous species of exotic and

native amphibians are kept and exchanged between Dutch and Belgian hobbyists. There is virtually no knowledge


regarding ranavirus infections occurring in such animals (Th.12 &13). Within the Netherlands, recently three native

endangered amphibian species have been legally translocated and re-introduced into new areas. These legally

authorized biological conservation activities have a phased implementation (activity identification, procurement of

stock, growth of stock in captivity, release at site and monitoring at site); however, the risk of transfer of CMTV-like

virus is not yet fully taken into account in these implementation steps (Th.14). There are other (un-)intentional

releases of amphibians in the Netherlands besides these authorized re-introduction activities. Most releases are

untraceable, and the frequency of occurrence of such events is unknown. However, some releases have been

documented, such as the release of alpine newts and common midwife toads from France and elsewhere into the

area where CMTV-like virus disease events are occurring in the Netherlands. The significance of these releases for

the introduction of CMTV-like virus infection is undetermined. (Th.15). Finally, birds have been suggested as cause of

spread of ranavirus. Migratory birds have flyways along the axe Spain-Belgium-the Netherlands. However, there is no

definite proof that dispersal of the virus via birds occurs (Th.16).

Part 2 - Risk assessment §2.1 Probability of introduction from abroad - CMTV-like virus is present in the Netherlands at least

since 2010, but there is no evidence suggesting that it is an endemic virus. Two possible pathways for introduction of

CMTV-like virus into the Netherlands are human-mediated introduction or introduction via natural dispersal of infected

or contaminated animals. With regards to human mediated introduction, it has to be noted that numerous amphibian

species from all over the world are traded. The traceability of imported specimens is poor, and data is lacking on

prevalence of CMTV-like infection in these amphibians. Further, the detection of CMTV-like virus infection during

import via the national airport Schiphol is currently highly unlikely, unless the animals are clinically ill or dead at the

moment of inspection. However, the chance that animals arrive clinically ill or dead is rather low due to short flight

and total transportation times. To the best of our knowledge, live amphibians are not imported for food production in

the Netherlands; many are imported to serve as pets. Wild caught specimens of unknown health status are likely to

be regularly introduced into facilities where captive-bred species are kept. There is significant captive-bred exotic

amphibian trade, also within the EU, but the volume is not quantified and there is poor insight into the occurrence of

ranavirus outbreaks in such collections. If ranavirus occurs in captive amphibians, there is a chance that it is

introduced into nature. It is relevant that CMTV-like virus exists in a free-living amphibian population of American

bullfrogs across the border in Belgium, as this could lead to introduction via natural dispersal. Documented evidence

for introduction of CMTV-like virus via natural dispersal of infected amphibinans or eventually via other animals is still

lacking. To conclude, there are several possible routes of introduction of CMTV-like virus, but species, numbers, and

prevalence of CMTV-like infection in imported specimen is unknown.

§2.2 Risk of establishment - CMTV-like virus has already persisted in the area where it was initially detected

in the Netherlands. The area where it is present includes a Natura 2000 area, National Park Dwingelderveld. For

establishment, the virus requires susceptible hosts and suitable environmental conditions, and needs to be able to

maintain itself from year to year. Species susceptible to CMTV-like virus include common and widespread amphibian

species, of which the populations so far appear immunologically naïve. The environmental conditions of water bodies

in which CMTV-like virus thrives, are diverse and commonly found throughout several water types in the Netherlands.

The water temperature of these water bodies is likely to be favourable to ranavirus replication in the summer and

virus survival in the winter. Finally, CMTV-like virus probably has several mechanisms to maintain itself after initial

introduction. These include interspecies transmission, co-evolution, lengthy survival outside the host under certain

conditions, and intra-species reservoirs. Taking all information together, the conclusion is that conditions for

establishment are present in the Netherlands, and so the probability of establishment is high.

§2.3 Risk of spread - The virus has been detected in a restricted area in the Netherlands, initially at sites

located in the Province of Drenthe, but in 2012 it was also detected in the Province of Overijssel. One possibility is

that it naturally spread after initial introduction. Short distance natural dispersal via (sub-)clinically infected hosts is

very likely. A number of susceptible amphibian host species are good dispersers, up to 15 km a year. However,

human-mediated introductions cannot be excluded. Short to long distance dispersal of CMTV-like virus within the

Netherlands via human-mediated activities is highly likely, as this has been documented for other ranaviruses

elsewhere.


§2.4 Area at risk - The area at risk is the whole of the Netherlands, in habitats where amphibians live. Very high

risk areas are areas in provinces where threatened species occur. For the common spadefoot these are located in

the provinces of Drenthe, Overijssel, Noord Brabant, Gelderland, and Limburg. For the common midwife toad these

are located throughout the entire country.

§2.5 Impact - A negative effect of emerging CMTV-like virus on amphibian numbers is seen at outbreak sites.

Elsewhere, the emergence of ranavirus infection at sites was transient, catastrophic, or persistent with recurrent

mortality events and populations declines of on average more than 81%. Follow-up at some CMTV-like virus infected

sites suggests that catastrophic and persistent effects on populations may also be occurring. However, the events are

too recent for final judgement and long-term monitoring will be required to corroborate these observations. Further, a

number of characteristics of the CMTV-like virus suggest that it has the potential to cause endangered species to go

(locally) extinct. There is an urgent need for more understanding into possible effects on fish. Other ecological and

socio-economic effects are very poorly documented.

§2.6 Score - Current - The risk classification of the CMTV-like virus for the current situation in the Netherlands is

B2, using the ISEIA protocol. This indicates a non-native species with a restricted distribution range and moderate

environmental hazard (i.e., ecological risk) that should be placed on a watch list. Future – If no measures are taken,

the future scenario shows a potential increase in the invasion of the CMTV-like virus resulting in a widespread

distribution and viral disease of amphibians in a significantly increasing number of high conservation value habitats,

resulting in highly adverse direct and indirect impacts on biodiversity and alterations of ecosystem functions at a

wider spatial scale. In particular, the expected increase in number of high conservation value habitats with adverse

effects would lead to an increase in the total risk score by one point according to the ISEIA protocol. Combined with

the widespread distribution this will result in a reclassification to a higher environmental risk (A3 - Black list

classification).

Part 3 - Risk management and risk communication Two important assumptions underlying the proposed risk management and risk communication measures are that 1)

CMTV-like virus is not widespread but emerging and 2) there are no other ranaviruses in the Netherlands.

§3.1 Prevention of introduction – To prevent the introduction three leads are provided. 1) The first lead is to

make sure that CMTV-like virus can be detected in imported captive specimens at the border. This requires more

insight into the species and numbers of amphibians traded commercially or exchanged internationally, and the

prevalence of infection in the imported specimen and in natural populations in other countries. Further the capacity to

detect and confirm cases at the border must be increased. 2) The second lead is to reduce the risk that CMTV-like

virus enters nature via imported kept amphibians or their waste. This requires obtaining good sight on amphibian and

vivaria waste disposal behavior, establishing how the risk of introducing the virus can be mitigated, and

communicating this to the public. 3) The third lead is to arrange that CMTV-like virus imported via nature can be

detected early on at its site of introduction, before it occurs widespread. To achieve this, monitoring through

investigation of unusual mortality events in amphibians was recommended. Further, specific attention for CMTV-like

virus in amphibians along the Dutch-Belgian border near Hoogstraten was suggested.

§3.2 Elimination –Assisted virus elimination, i.e., elimination undertaken by humans, is currently only

recommended in captive populations and in small free-living populations. Humane, timely and thorough destocking

and disinfection of premises is the most secure option if CMTV-like virus is found in captive amphibians. Acceptable

elimination measures for use in water bodies are virtually unidentified. Therefore some guidance in taking the

decision to opt for elimination or not in small free-living amphibian populations is recommended, and practical advice

for best practice should be provided. The need to obtain permission to destock should be known to the public and the

process speedy. Technical alternatives for achieving virus elimination from natural sites and side-effects should be

investigated, and official registration of disinfectants for such use encouraged. The effect of water body management

interventions on the occurrence of CMTV-like virus infections should be monitored, even those not primarily

undertaken with the view of elimination. The mechanisms and factors underlying natural elimination, i.e., elimination

achieved by natural processes in the wild, are likely to provide information useful for assisted elimination on a large

scale. These can be identified through long-term monitoring and should then be translated into practical measures

for use in the field.


§3.3 Control - Possibilities to control CMTV-like virus focus on minimizing spread and impact. Special effort should

be made to prevent sites where threatened species are found from becoming infected. Projects legally authorized to

translocate and re-introduce threatened species need to take the necessary precautions to avoid introducing disease

in the process. Veterinary advice to minimize this risk throughout the project’s implementation is required. Education

of the public to refrain from releasing amphibians into nature may reduce spread via intentionally released live

animals. Education of field biologists to properly clean and disinfect equipment, shoeing and clothes should reduce

spread via fomites, in particular if disinfection is made more practical. Absolute containment of wild animals in an area

is not possible, but possibly there are barriers in the landscape and land uses that can limit natural dispersal by

amphibians of which temporary use could be made. Vaccination of threatened species is not an option at this stage,

because there are no vaccines available for CMTV-like virus or other ranaviruses. Chemotherapy too is not available.

Finally, epidemiological models are very useful to properly assess the effectiveness of control measures. The

information in this document can be used to define which data is missing to make such models, so that the collection

of such data could then be a priority if the route of control and elimination are taken.

To conclude:

1. Risk assessment

1a) CMTV-like virus was detected for the first time in the Netherlands in a pond in National Park Dwingelderveld

in 2010, and there is no evidence suggesting it is an endemic virus. There are several potential routes of

introduction, both human-mediated and via nature, but it is currently not possible to determine probability of

introduction via these routes.

1b) CMTV-like virus has been detected in a restricted area of the Netherlands only. It has maintained itself in

amphibians in water bodies of Dwingelderveld since 2010. Suitable hosts and environmental conditions are

present, and the probability that it can establish itself is high.

1c) Initially observed only in the Province of Drenthe, CMTV-like virus infections were detected in the Province of

Overijssel in 2012. The probability that CMTV-like virus will spread is high, either short distances (up to 15

km a year) through infected amphibians, or short to long distances via humans and possibly birds.

1d) The area at risk is the whole of the Netherlands, in habitats where amphibians live.

1e) So far, CMTV-like virus has caused catastrophic and persistent effects on populations at infected sites. Other

ecological and socio-economic effects are very poorly documented.

1f) Currently the CMTV-like virus is assessed to be on the ‘Watch List’ (B2), being a non-native species with a

restricted distribution range and moderate environmental hazard. In the future, without interference, the

distribution of CMTV-like virus is likely to be more widespread, with more adverse effects on high

conservation habitats. This would place it on the ‘Black List’ (A3).

2. Risk management and communication

2a) To prevent the introduction of CMTV-like virus, three measures are recommended:

- make sure that CMTV-like virus can be detected in imported captive specimens at the border.

- reduce the risk that CMTV-like virus enters nature via imported kept amphibians or their waste.

- arrange that CMTV-like virus imported via nature can be detected early on at its site of introduction, before

it occurs widespread.

2b) Destocking and disinfection of premises should be used for virus elimination in captive populations. Options

for elimination of virus from free-living populations should be explored further, because there is still little

experience with such measures. Monitoring the effect of water body management interventions on CMTV-

like virus presence and a better understanding of the mechanisms and factors leading to ‘natural elimination’

can contribute to this.

2c) Control focuses on limiting the spread of the infection and the impact on threatened species through

education of the public and field biologists. Epidemiological models could help to assess the effectiveness of

suggested control measures.


Part 1

General background information for risk analysis (themes 1 – 16) General background information is provided as 16 theme pages (Th.). The relevance of theme for each component of

the risk assessment (introduction, establishment, spread or impact, or several) is indicated first. Then after defining

theme concepts and providing trends and patterns, the significance of the findings for CMTV-like virus infection

mentioned.

Sources of information - The sources of information were literature, field data, interview data and expert opinion.

Literature - Literature was searched using Scopus, Web of Science and Pubmed. No limit in year of publication was

added. ‘Ranavirus was used as search term, and yielded 250 records of papers and book chapters. Documents were

screened for suitability for this risk assessment by checking for relevant information. Additionally, for information on

host species, the library of RAVON (17,000 records) was accessed using ReferenceManager. Field data - In 2011,

an extensive survey in the aftermath of the 2010 outbreak of ranavirus in National Park (NP) Dwingelderveld was

conducted. This included post-mortem investigation of mortality events nationwide1, as well as monitoring of a

number of ponds in NP-Dwingelderveld regularly during the period June – October 20112. Per visit per pond, the

number of sighted individual amphibians were noted, as well as the species, their condition (healthy, sick or dead),

life stage (adult, sub-adult, juvenile or larvae). Additionally characteristics of the water body were noted, such as

water temperature, and water quality was measured. In 2012,as part of this assignment, two field visits (24/8/12 and

4/9/12) were made to this NP to obtain an update. Interview data and expert opinion - For information on

amphibian trade via Schiphol, DWHC consulted the Netherlands Food and Consumer Product Safety Authority and a

board member of the ATA (Animal Transportation Association). For information on legal re-introduction and

translocation programmes, DWHC consulted amphibian re-introduction/translocations files at Dutch Ministry of

Economic Affairs, Natuurbalans – Limes Divergens BV, and RAVON. To get an idea about the trade of amphibians in

Belgium, UGent consulted the FAVV (Federal Agency for the Safety of the Food Chain, Belgium), research

laboratories dealing with amphibians, and animal importers. About 40 research labs can perform research on

amphibians. Three of these labs have indicated that they, sometimes, import amphibians. In Belgium, 124 merchants

are registered as allowed to import amphibians. The five most important amphibian importers were contacted for this

survey. In addition, a questionnaire was sent to private amphibian collections in Belgium.


Theme 1 – The virus

Theme relevant to pathogen introduction, establishment, spread and impact

Definition – CMTV - Common midwife toad virus (CMTV; GenBank accession no. FM213466.1)3 is a double-

stranded DNA virus belonging to the genus Ranavirus, family Iridoviridae. The ranavirus genus - The identification

of the different virus species belonging to the genus Ranavirus is on-going. To date six (6) species are officially

recognized and there are also a number of tentative species, i.e., ranavirus isolates that may be classified as distinct

ranavirus species in the future. The six recognized species are Ambystoma tigrum virus (ATV), Bohle irido virus

(BIV), Frog virus 3 (FV3/TFV), Epizootic haematopoietic necrosis virus (EHNV), European catfish virus (ECV/ESV),

and Santee-Cooper ranavirus (SCRV, LMBV)4. CMTV is a tentative species, with an intermediate position in

evolution between EHNV-like viruses and FV3-like viruses5. CMTV-like virus - Besides CMTV, a number of CMTV-

like ranaviruses have been described6-8

. Genetic material of these viruses, amplified by PCR and then sequenced,

shows 99.8% to 100 % sequence homology with CMTV partial major capsid protein gene (GenBank accession no.

FM213466.1). Definition When referring to CMTV and such CMTV-like viruses collectively, we will further use the

term CMTV-like virus.

Patterns and trends in other ranaviruses – Ranaviruses are infective as naked particles and even more so as

enveloped particles. An envelope is acquired when the virion buds through the host cell membrane4. Transmission

with and without direct contact - Transmission is horizontal. It can be through direct through direct contact with

infected individuals, cannibalism and necrophagy of infected individuals, or exposure to water and fomites containing

virus 9-16

. There is no conclusive proof for vertical transmission to date, though it has been suggested 17

.High

infectivity - ATV needs about 1 s of direct contact for transmission via intact or damaged skin10

. FV3 released in

water by infected specimens infected others within 3 hours of exposure16

. Replication temperature fits

poikilothermic hosts - Ranaviral replication takes place at 12-32°C, with viral protein synthesis occurring within

hours of cell infection. Cell death can occur as quickly as a few hours following infection, by either necrosis or

apoptosis 18

. Survival in the environment is enhanced by cold temperatures - Water - Virions remain stable

in water for extended periods at 4°C4. For two amphibian and two reptilian ranaviruses, it was demonstrated that

about 10% of the initial virus load remained infectious for 2 months in water at +4°C and for 3weeks to a month at

+20°C19

. Cell-free EHNV remained infective after 97 days in water (no detail provided on temperature)14

. Sediment –

Wood frogs (Lithobates sylvaticus) could be infected by exposure to sediment from a site where a ranavirus die-off

was occurring13

. For two amphibian and two reptilian ranaviruses it was demonstrated that about 10% of the initial

virus load remained infectious for over a month in soil at +4°C19

. BIV survives desiccation for up to six weeks at

temperatures up to 42°C4, but ATV degrades quickly

9. In tissue - EHNV remained infectious in tissue of dead fish for

more than seven days at +4°C, and more than two years at -20°C and -70°C14

. Virus Inactivation - Heat –

Ranaviruses are inactivated within 30 minutes at 60°C4; ENHV was also inactivated after 24 hours at 40°C

14. Ultra-

violet-irradiation – FV3 is inactivated by UV-irradiation4. Ultraviolet sterilizing units were effective in inactivating

water-suspended iridovirus isolated from frogs at a flow rate comparable to that used in nurseries in aquaculture

(5000 L/h). With transmittance reduced to 27.7%, the minimum effective dose was 2.6 × 104 uW.sec/cm².

20. Extreme

pH: One hour of pH<4 or pH>12 inactivated EHNV14

. Disinfectants – In absence of organic matter, sodium

hypochlorite (200 mg / l) is effective14, 21

; Ranavirus is also inactivated by 70% ethanol14

. Surface application for one

(1) minute or more of chlorhexidine 0.015% (0.75% Nolvasan® 2.0), 0.180% sodium hypochlorite (3.0% bleach 6.0),

or potassium peroxymonosulfate 0.204% (1% Virkon S® 20.4) reduced ranavirus load by 8 log10, and was

recommended for disinfecting contaminated equipment 21

.

Significance for CMTV-like virus - Little research has been done specifically on CMTV-like viruses, other than the

complete genome sequencing by Mavian et al., 20125. Likely data on transmission modes, infectivity, replication

temperature, survival and inactivation is based on extrapolation of knowledge with regards to other ranaviruses.


Figure 1 – Electron Microscopy (EM) photograph of an array of CMTV-like virus. Ranaviruses have a distinctive icosahedral shape, approximately 150 nm in diameter, visible by EM as paracrystalline arrays in cytoplasma of cells

22. The genome of CMTV (106,878 bp) contains 104 open reading frames encoding putatively expressed

proteins5.


Theme 2 - Distribution of CMTV-like virus Theme relevant to pathogen introduction, establishment, spread and impact

Definition - The distribution of the virus is its occurrence in space and time.

Patterns and trends - CMTV-like virus has only been detected in Europe, namely in Spain3, 23

, Belgium8 and the

Netherlands1, 6

(Figure 2a, left). Spain 2007 & 2008 - The very first detection (and description) was in northern

Spain in 2007, when amphibian mortality occurred at a site in National Park (NP) Picos de Europa3. This was

followed in 2008 by amphibian mortality at a second site in the NP Picos de Europa, only 1 km away from the first23

.

In between these two events23

and in subsequent years (Balseiro A., pers. comm. 2012) no further CMTV-associated

mortality was observed. Belgium 2010 - In Belgium, CMTV-like virus was not detected in any of the amphibian

mortality events investigated between 2007 and 20106. However in 2010 it was found in healthy larvae of free-living

exotic species in Hoogstraten (Figure 6, Theme 6). The Netherlands 2010, 2011 & 2012 - In the Netherlands,

CMTV-like virus was first detected in 2010, at NP Dwingelderveld (Drenthe province; Figure 2a, right)6. Subsequently,

the situation in NP Dwingelderveld was monitored, and a nationwide request was put out to the public to report

significant amphibian mortality events. The public reported 20 significant amphibian mortality events outside NP in

2011. At 16 sites no CMTV-like virus or other ranavirus was detected. At the remaining four (4) sites, however,

mortality was shown to be associated with CMTV-like virus infection1. These sites were Wijster, Darp, Fluitenberg

and Ijhorst (Drenthe province). These are all located within 25 km of each other and NP Dwingelderveld, where

CMTV-like virus associated mortality continued in 2011 (Figure 2b) and 20121, 2

. In 2012, CMTV-like virus associated

amphibian mortality occurred at a sixth site, Staphorst (Overijssel province), located only 7 km to the south of IJhorst

(Figure 2a, right)1. Retrospectively, three amphibian mass mortality events were reported to have occurred in the

summer or autumn of 2009 and 2010 to the east-north- east of NP Dwingelderveld; ranavirus infection may have

been the cause but no material remained or has since been submitted to ascertain this1. Finally, CMTV(-like virus)

was detected in 2010 in exotic hosts belonging to a captive collection of which the location is not disclosed7.

Significance - Phylogenetic analyses shows ranaviruses generally group according to their geographic and their

host class origin24

. Because CMTV-like virus was detected in several European countries, it has been suggested that

it may have a wide geographic distribution across the European mainland5. Distribution suggests disease

emergence in the Netherlands Regardless of whether this is the case or not, the nationwide surveillance based

on investigation of unusual mortality events in the Netherlands in 2011 points towards focal occurrence of severe

disease and mortality due to CMTV-like virus in the Netherlands, i.e., a disease hotspot of about 500 km2. This

suggests infectious disease emergence1.


Figure 2a - Left – Countries (green surfaces) where CMTV-like virus has been detected to date (December 2012). CMTV-like virus has a focal distribution in each country (red dots) and is not known to occur widespread. Right – The sites where CMTV-like virus has been detected in the Netherlands, per year of first detection.

Figure2b – Ponds and pools monitored or visited after a mortality report, Dwingelderveld 2011. Those monitored were Kolenveen (A), Bezoekerscentrum (C & D), Achterlandseveen (E), and Smitsveen (F). Those visited were Drostenvbeen (H), Noorsterveen (I) and Holveenslenk (J). CMTV-like virus infected hosts were found at all sites except D and H.


Theme 3 - Known susceptible host species Theme relevant to pathogen introduction, establishment, spread and impact

Definition - A host species is considered susceptible to CMTV-like virus infection and disease if individuals

belonging to the species were found to have post-mortem lesions consistent with ranavirus infection associated with

ranavirus genetic material25

showing 99-100% sequence homology25

with gen bank Accession No. FM213466.1. A

host species is considered susceptible to CMTV-like virus infection if ranavirus genetic material showing 99-

100% sequence homology with gen bank Accession No. FM213466.1 was detected in internal organs of individuals

belonging to the species. A host species is considered possibly susceptible to CMTV-like virus infection if

ranavirus genetic material showing 99-100% sequence homology with gen bank Accession No. FM213466.1 was

detected in individuals belonging to the species without confirmation of infection.

Patterns and trends - There are eleven (11) host species susceptible to CMTV-like virus infection and

disease: - The edible frog (Pelophylax klepton esculentus,family Ranidae)6

⋅ The marsh frog (Pelophylax ridibundus,family Ranidae)1

⋅ The pool frog (Pelophylax lessonae,family Ranidae)6

⋅ The smooth newt (Lissotriton vulgaris, family Salamandridae)6

⋅ The common toad (Bufo bufo, family Bufonidae)1

⋅ The common spadefoot toad (Pelobates fuscus, family Pelobatidae)1

⋅ The common midwife toad (Alytes obstetricans, family Alytidae)3, 23

⋅ The Alpine newt, subspecies cyreni (Mesotriton alpestris cyreni, family Salamandridae)3, 23

⋅ The Golfo Dulce poison frog (Phyllobates vittatus,family Dendrobatidae)7

⋅ The black-legged poison frog (Phyllobates bicolor, family Dendrobatidae)7

⋅ The green and black poison dart frog (Dendrobates auratus, family Dendrobatidae)7.

The first three are collectively named water frogs. For the first eight host species, infection and disease was detected

in wild populations. These eight host species are native to the Netherlands (though the specific subspecies of the

eighth species that was affected in Spain is not the subspecies present in the Netherlands). For the last three host

species, infection and disease was detected in captive aquarium-kept frogs in the Netherlands. These last three host

species are all exotic species for the Netherlands There are two (2) host species susceptible to CMTV-like

virus infection. These are the common frog (Rana temporaria, family Ranidae)1 and the American bullfrog

(Lithobates catesbeianus, family Ranidae)8. Evidence for disease in the infected common frog examined was

inconclusive1, and the American bullfrogs were clinically healthy tadpoles

8. The American bullfrog, exotic to Europe,

may be a carrier of ranaviral disease8. There is one (1) host species possibly susceptible to CMTV-like virus

infection, the ten-spined stickleback (Pungitius pungitius, family Gasterosteidae). This is a species of fish that is

native to the Netherlands. One specimen of this species, originating from a pond where an outbreak of CMTV-like

virus disease had occurred, was found to be positive by PCR-test. However, this specimen was too decomposed for

post-mortem examination and due to the pooling of multiple organs in the sample tested, it is not possible to know if

the virus had infected the fish or simply occurred on the skin, gills or in the lumen of the intestines of the fish1, 2

.

Difference in host species susceptibility – The current data is too restricted for conclusions in this regard.

However, there is some evidence for the occurrence of subclinical infections.

Significance – CMTV-like virus has a broad host range. Multiple native amphibian species susceptible - At

least half the native amphibian species present in the Netherlands are susceptible to CMTV-like virus infection and

disease (Figure 3). Confirmed infection with CMTV-like virus has been demonstrated in the two orders of the

amphibian class, Anura (frogs and toads) and Caudata (salamanders and newts). Exotic amphibian species -

Exotic species of amphibians can also be infected, sometimes associated with disease. Species belonging to

other taxonomic classes - It is still unclear if hosts from other classes, such as fish, can be infected. Subclinical

infections occur.


Figure 3 – Overview of current knowledge on CMTV-like virus host species


Theme 4 – Host life stages affected

Theme relevant to pathogen establishment, spread and impact

Definitions - Amphibians have several life stages. After spawning/fertilisation, embryo’s develop into larvae, which

in turn develop into juveniles and then (sub)adults. Metamorphosis is the change in the form and in the habits of an

animal during normal development after the embryonic stage. It includes the full development from the larval stage up

till the moment the juveniles go on land. Species with aquatic hibernation are present year round in water bodies.

Patterns and trends – CMTV-like virus affects both pre and post metamorphic life stages. Life stages involved

in mass mortality events - Mass mortality events associated with CMTV-like virus in Spain involved only larvae

and juveniles, whereas in the Netherlands adult life stages also succumbed 1, 3, 6, 23

. Life stages in which mortality was

observed were in the aquatic phase, and often in or following periods of high visibility (Figure 4). The numbers of

animals observed to die ranged from several tens (+ in Figure 4) to hundreds or even thousands (+++ in Figure 4).

The larvae observed to die were in different stages of development. So far no spawn has been tested, but spawn

death was reported for Darp. Life stages involved in the aftermath of an outbreak – During the monitoring in

NP Dwingelderveld in 2011, disease and mortality were observed predominantly in the adult stages of the water frogs

(Table 1)2. Life stages involved in subclinical infections - The sub-clinically infected American bullfrogs in

Belgium were larvae8.

Table 1 – Amphibian host species and their life stages found sick or dead during the pool monitoring in NP Dwingelderveld from May to October 2011. CMTV-like virus was confirmed in many of the water frogs but not in the great crested newts.

Significance and discussion – Host life stages affected in the Netherlands suggestive of

immunologically naïve populations - CMTV-like virus outbreaks involve larvae, juveniles and (sub)adults, and

possibly spawn too. The fact that life stages are collectively affected, including adult life stages, is suggestive of

disease emergence in immunologically naïve populations. For comparison, mass mortality in adults is reported from

the United Kingdom (UK) where ranavirus disease is emerging since 198526

, while outbreaks with ranaviruses in the

United States of America (USA) typically involve larvae and recently metamorphosed juveniles only27

. CMTV-like

infection in spawn has not been demonstrated as yet but cannot be excluded at sites known to harbour the virus,

even though there is some evidence that amphibians are least susceptible to ranavirus infection during embryo

stage28

. It is still unknown if vertical transmission occurs with ranaviruses9, 22

. Aquatic phase - The stages affected

are observed in the aquatic phase. This can be observation bias (visibility) or an effect of increased transmission

opportunities during the aquatic phase. During the aquatic phase of the life cycle, there is congregation of individuals,

likely to increase direct contact transmission opportunities. Further, water is a ranavirus vehicle16

and it is likely there

will be more indirect transmission.


Figure 4 – Life stages involved in CMTV-like virus associated mass mortality events in the Netherlands plotted against life stage visibility (phenophase). The events were NP Dwingelderveld (NP), IJhorst (IJ), Wijster (W), Fluitenberg (F), Darp (D) and Staphorst (S). Stages observed to die in numbers of hundreds to thousands are indicated by +++, stages observed to die in numbers of tens by +. The phenophases were compiled using the phenological frequency diagrams provided per species in Creemers R & van Delft J, 2009

29.The blue shades indicate

the aquatic phenophase (the darker the shade the greater the relative proportion of sightings), the green the terrestrial phenophase.


Theme 5 - Distribution of susceptible host species in the

Netherlands Theme relevant to pathogen establishment, spread and impact

Definition – The spatial distribution maps of species of amphibians in the Netherlands are based on the observation

of the species in a given square kilometre. A species occurring widespread is often also common, but not necessarily.

Patterns and trends –Distribution and occurrence of native susceptible species (Figure 5) - The water

frogs, consisting of edible frogs, marsh frogs and pool frogs, can be detected in nearly the whole of the Netherlands

and currently the national distribution is known on 5 x 5 km base30

. Pool frogs are found in all provinces except

Zeeland and Flevoland. The edible frog is the commonest water frog in the Netherlands and can be found in all

provinces31

. Similar to the edible frog, the marsh frog inhabits all provinces in the Netherlands. The common frog

and the common toad are also present in all provinces, and are among the most common amphibian species in the

Netherlands32, 33

. The smooth newt is the most common of the four Dutch newt species, ranging throughout almost

the entire country34

. The subspecies of the Alpine newt (Mesotriton alpestris cyreni) affected in Spain is not present

in the Netherlands, but Ichthyosaura (or: Mesotriton) alpestris is present and inhabits the provinces Limburg, Noord-

Brabant, Zeeland, Gelderland and Drenthe. The common spadefoot, considered a threatened species in the

Netherlands35

, occurs in the provinces Drenthe, Overijssel, Gelderland, Noord-Brabant and Limburg. Its range has

decreased by 74% since 195036

. The natural range of the common midwife toad in the Netherlands is restricted to

the southernmost part of the province of Limburg, east of the river Meuse. This species is listed on the Red List as

‘vulnerable’ and its range has decreased with 42% since 195037

. Distribution and occurrence of exotic

susceptible species - Established populations of American bullfrog are rare and the policy is to eliminate them.

The map presented in figure 6 shows the situation up to 2007: one American bullfrog was sighted in 2002 in an

Amsterdam garden pond; a few lived in open air terrariums in Limburg, at least up to 2003; one was captured in ‘het

Wormdal’, Limburg province in 2006. Since then, there have been new findings. In particular, some American

bullfrogs have successfully reproduced in Baarlo, Noord-Brabant province29

, and there are 6 sites in and around

Baarlo where bullfrogs occur not yet figuring on the map. One of these sites was successfully eliminated in 201038

.

Another specimen was reportedly shot in Sint-Oedenrode, Noord Brabant province, near the valley of the Belgian-

Netherlands small river Dommel (2009). The origin of this latter specimen is unknown39

.

Significance –Common and widely distributed susceptible species - Common native host species susceptible

to CMTV-like virus have a nationwide distribution. Endangered species - The endangered common spadefoot

occurs within the CMTV-like virus disease hotspot. Potential source - Introduced Alpine newts and common

midwife toads occur in the CMTV-like virus disease hotspot.


Figure 5 - Distribution of susceptible amphibian host species in the Netherland, period 1996 – 2007 (Source: Creemers & van Delft, 2009

29). Red dots are natural populations, blue dots are introduced populations, maintaining

themselves. Larger dot sizes indicate a higher percentage of occupied squared kilometres. A grey background indicates the presence of elevated sandy soils or the hilly area in South Limburg.


Theme 6- Distribution of potential hosts across the border Theme relevant to introduction

Definition - Distribution maps indicate the area of spread of a host species, and do not reflect numbers.

Patterns and trends – Native susceptible species - As in the Netherlands, native species susceptible to CMTV-

like virus infection have a widespread distribution in Belgium and Germany. In particular the edible frog, the common

frog, the common toad and the smooth newt are distributed more or less throughout Flemish Belgium40

and

Germany41

. Exotic susceptible species - Further, the exotic American bullfrog occurs in the wild at least in

Belgium (Figure 6).

Significance – General - Given the presence of many susceptible host species along the borders, CMTV-like virus

is not likely to stop at the border, either way. Specific potential source - The most northern reproducing population

of the American bullfrog shown on the map is the population from which the American bullfrog larvae were taken that

were infected with CMTV-like virus8. This population is located close to the Dutch border (< 5 km).


Figure 6 - Distribution of the American bullfrog Lithobates catesbeianus in Flemish Belgium (1996-2012). In black the IFBL-square-kilometer-blocks where the American bullfrog has been observed. Those encircled by red represent regions where the American bullfrog is reproducing in the wild. IFBL is the mapping grid used by the Instituut voor Floristiek van België en Luxemburg. (Source: Robert Jooris, Hyala, Natuurpunt).


Theme 7 - Dispersal characteristics of host species Theme relevant to pathogen introduction and spread

Definition - Biological dispersal refers to species movement away from an existing population or the parent

organism.

Patterns and trends

Dispersal distance native species - All post metamorphic life stages disperse, in particular juveniles. Pool frogs

can be highly mobile. Within a season (March – October/November) they disperse several kilometres42

. In Austria,

they proved to be able to disperse distances of 15 km in ten days43

. The edible frog colonizes new water bodies

easily and in Germany, dispersal distances of approximately 2 km are recorded, mainly of juveniles41

. Literature

states that marsh frogs are relatively sedentary44

. The animals stay close to the water and rarely move over land41

.

However, they have been able to colonize the province Flevoland within a period of 30 years, which is indicative of

high dispersal ability45

. The greatest dispersal distances recorded for juvenile and adult common frogs are 2 – 4

km46-49

. During the migration of the common toad from hibernation sites towards reproduction sites, the toads may

disperse several kilometres50

. The smooth newt is reasonably mobile, and colonizes new habitats quickly51

, these

newts may disperse several hundreds of meters47

.The Alpine newt has an action radius of 400 m.47

The species

colonizes new ponds rapidly when these are situated close to already occupied ponds. Both young and adult

individuals disperse throughout the reproductive season52

. This is in contrast to the common spadefoot, which has a

limited distribution capacity, with the animals hibernating relatively close to their reproduction sites47, 53, 54

.

Comparable site fidelity is shown by the common midwife toad, where the distance between their summer habitat

and reproduction site measures less than 100 m.47, 55

. Dispersal distance exotic species - Post-metamorphic

stages of the American bullfrog are capable of dispersing long distances and are adept at colonizing new sites

(>1200 m)56

. A review of the dispersal distances of amphibians based on mark-and-capture studies and displacement

studies quotes three studies in which adult American bullfrogs moved maximum distances of 1600, 914 or 966 m {{}}.

Significance – Potential for dispersal via hosts - Amphibian hosts susceptible to CMTV-like infection can

disperse distances up to 15 km, mostly as juveniles or (sub-)adults (Figure 7). The effect of CMTV-like virus disease

on dispersal and dispersal distance is unknown. It seems reasonable to assume little effect of CMTV-like virus on the

dispersal of sub-clinically infected individuals.


Figure 7 – Host species classified according to dispersal speed


Theme 8 - Accompanying amphibian species Theme relevant to pathogen establishment, spread and impact

Definition - Accompanying amphibian species are those that occur remarkably often with a given other species.

Common accompanying species are those often seen in its proximity, characteristic accompanying species

are those that have significant overlap in their area of occurrence with it. Characteristic accompanying species cannot

be determined for rare species.

Patterns and trends - The Alpine newt is accompanied by all species of Dutch amphibians, some of the

characteristic species reflecting its occurrence in poor, humid elevated sandy soils with some forest cover in the

south of the Netherlands52

. The smooth newt, having a large distribution range and broad habitat choice, is

accompanied by all species of Dutch amphibians, the most important characteristic ones being the common frog, the

common toad, and the three water frog species34

.The common frog is accompanied by all native amphibian species,

especially the smooth newt, the common toad and water frogs32

. The common toad is accompanied by all species of

Dutch amphibians, of which the most important species are the smooth newt, common frog and the three water frog

species33

. Characteristic species accompanying the marsh frog are the edible frog and the natterjack toad (Epidalea

calamita)45

. The pool frog is accompanied by the crested newt (Triturus cristatus) and moor frog (Rana arvalis), all

indicative of good quality waters30

. The most important common and characteristic accompanying species of the

edible frog are the smooth newt, the pool frog, the common frog, and the common toad31

. The common midwife

toad is accompanied commonly by the Alpine newt, the smooth newt, yellow-bellied toad (Bombina variegata) and

the common frog, as well as by all other widely distributed amphibian species. It is the only species that has the

yellow-bellied toad as characteristic accompanying species37

. Rare susceptible species - The common spadefoot

is rare, so characteristic accompanying species cannot be determined. Besides commonly occurring species,

common accompanying species are the great crested newt and the moor frog36

.

Significance: Inter-species transmission - Concurrent CMTV-like virus infection in multiple species is observed

in single water bodies in the field, both in Spain23

and in the Netherlands (Dwingelderveld 2010, Wijster 2011,

Fluitenberg 2011, Staphorst 2012)1. It is therefore most likely that inter-species transmission of CMTV-like virus

occurs. Favorable conditions - Common susceptible host species are both common and characteristic

accompanying species of each other (Figure 8, left side). This suggests frequent occurrence of conditions favorable

to inter-species exposure and transmission events. Endangered species – Common susceptible host species are

also common accompanying species for endangered species.


Figure 8 - Amphibian species accompanying known susceptible host species. The percentage represents the probability of that a host species is found in the same square kilometre as the susceptible species (common accompanying species). A green background indicates that the accompanying species is among the five accompanying species having the highest area-of-occurrence overlap with the susceptible species (Source: compiled based on data in Creemers & Van Delft, 2009

29).


Theme 9 - Water body types with CMTV-like virus Theme relevant to establishment

Definition - A water body is any significant accumulation of water, covering the Earth or another planet.

Patterns and trends: Water body types – The water body types varied (Figure 9). Spain - The sites in Spain

where CMTV was detected were a permanent drinking trough (fishing not possible) and a pool, both located at

high altitude23

. NP Dwingelderveld - The 2010 mass mortality occurred in a shallow, 3000 m3 artificial pond

adjacent to the visitor centre of National Park Dwingelderveld (site C)6. The sites that were monitored in 2011 and

where incidental CMTV-like virus cases were detected were pools (e.g., sites A, E and F)2. IJhorst - The mortality

in 2011 occurred at a garden pond1.Wijster - The mortality in 2011 occurred at a garden pond (170 m

2) with water

plants1.Darp - The mortality in 2011 occurred at garden pond, measuring 10 x 10 m.

1. Fluitenberg - The mortality

in 2011 occurred in a garden pond was constructed in 1991. Staphorst - The site at which the common spadefoots

died in 2012 concerns a natural swimming pool with sandy shores, it is approximately 2 – 2.5 m. deep and at the

bottom there is some waterweed growing1.

Presence of fish - NP Dwingelderveld - Ten-spined sticklebacks occur in site C. IJhorst – The pond contained

ide Leuciscus idus melanotus (family Cyprinidae). Wijster – The pond had frozen the winter before the 2011

outbreak and all fish had died. The fish had been replaced in the spring by ide and goldfish Carassius auratus

auratus (family Cyprinidae). Some juvenile goldfish died at the same time as the CMTV-like virus infected amphibians

but the cause of death of the fish was not determined. Darp - The pond contains fish. Fluitenberg - The pond

contained ide until 2006. In the period 2007-2012, the pond harboured a healthy school of minnows (Pimephales

promelas auratus, family Cyprinidae). Minnows are exotic fish species originating from North America.

Significance and discussion – CMTV-like virus infections occur in a variety of water types - The water

bodies were pools and ponds, natural or man-made, located at different altitudes and different soil types. All were

permanent. The landscape setting found to play a role in the USA (high catchment positions in wetlands57

) may

possibly apply to Spain but not to the Netherlands. Generally in relatively small water bodies with standing

water - In general they were small- to medium-sized and not directly connected to other water bodies. They do not

to dry up naturally. Fish – Fish species were often present in these water bodies. The fish species could be native or

exotic, wild or cultured, and were often from the carp family. In water bodies with standing water, fish—if they are not

refractory to infection58

with CMTV-like virus—are unlikely to be natural dispersal vectors.


Figure 9 – Water bodies where CMTV-like virus associated mortality occurred.


Theme 10 - Month of outbreak and water temperature Theme relevant to establishment

Definition - Water temperature was measured using a HANNA, HI 98311 in 0.1°Celsius (Figure 10a). Mean daily air

temperature was obtained from KNMI (Hoogeveen). Mean water temperature per site is the mean of the water

temperatures measured during all the 17 monitoring moments (between June and October 2011).

Patterns and trends – Outbreaks occur late spring and in the summer – The initial mass mortality events at

all eight (8) known CMTV-like outbreak sites occurred between May and September. At the two sites in Spain, the

CMTV outbreaks occurred in August (2008) and September (2007)23

. At the six sites in the Netherlands where

CMTV-like virus outbreaks with mass mortality occurred, these outbreaks were detected between May and

September: May (2011, IJhorst), July (2011, Wijster; 2012, Staphorst), August (2011, Fluitenberg) and September

(NP Dwingelderveld, 2010; Darp, 2011)1, 6

. Timing mortality in outbreak aftermath - At the water bodies in NP

Dwingelderveld that were monitored from June to October 2011, the first confirmed ranavirus disease and mortality

cases of the year were detected in June and July (Table 2). Mortality continued to be observed up to September2. In

2012, dead infected common frogs were reported in NP Dwingelderveld as early as March in 2012, but it is unclear if

ranavirus disease contributed to their death (cf. Theme 3)1. Water temperature and air temperature (physical

stressors) – NP Dwingelderveld 2011 - Water temperatures were monitored simultaneously with amphibian

populations in NP Dwingelderveld in 2011. The water temperatures measured ranged between 9.9°C (site F on

October 24th

) and 25.4°C (sites C and E on August 5th

). Water temperature followed the air temperature, water

temperature being roughly 3 to 4°C higher than the mean daily air temperature (Figure 10, b and c). The mean

water temperature in the month of at which the first sighting of a PCR-positive animal was found, measured on

average 19.1 ± 1.5°C. This temperature is not significantly different from the mean water temperature of the four sites

(A,C,E and F) during the study period (19.0 ± 3.4°C). The mean daily air temperature during the study period was

15.0 ± 2.6°C. Air temperature IJhorst outbreak May 2011 - The water temperature at the start of the outbreak

was not measured, but the ambient air temperature at the day of the first case being 7.5°C. Air temperature NP

Dwingelderveld March 2012 - The water temperature was not measured where the common frogs were found, but

the ambient air temperature on 27/3/12 was 9.3°C.

Significance and discussion – CMTV-like virus outbreaks tend to occur in the spring and summer

months - This is consistent with findings other ranaviruses elsewhere22, 27

. Host life cycle factors (Th.4) and effects

of temperature on virus replication rate (Th.1) and on host immunity (this theme) could contribute to this observation.

Relation mortality and (water) temperature, possible effects on host immune function – The relation

between water temperature and ranavirus infections in poikilothermic hosts is still not fully understood. In Maine,

USA, there was no relation between temperature and onset of ranavirus-associated mortality, but sites that

experienced mortality were markedly warmer compared to those with unaffected populations59

. In Tennessee, USA,

there was a significant positive association between water temperature and ranavirus prevalence only in fall, and not

in the other three seasons60

. Experimentally, temperature strongly influenced both time to death and mortality rate in

larval Sonoran tiger salamanders (Amblyoma tigrum) exposed to via Amblyoma tigrum virus (ATV)61

. Most

salamanders survived when exposed at 26°C, nearly all died 10°C, and all died at 18°C. The experiment showed that

the salamanders’ immune function was negatively affected in the lower temperature range61

. In conclusion, water

temperature is a probable determinant in CMTV-like virus-associated mortality in amphibians, but its relation to

mortality and its importance relative to other environmental factors is not fully understood.


Table 2 – Water temperature data obtained in the period June to October 2011

Figure 10 – Left - Water temperature was measured using a HANNA thermometer. Right – Graphs of water temperature measured during monitoring moments in NP Dwingelderveld at Koleveen (Site A, top) and Smitsveen (Site F, bottom) and mean air temperature. All other monitored sites showed the same fluctuations.


Theme 11 - Water quality and habitat use at outbreak sites Theme relevant to establishment

Definition – Water quality was analyzed by measuring pH, NO3, NH4, O-PO4, Na, K, Cl, Al, Fe, Zn, t-P, t-S, Mn, Si,

Mo, and Ca. Acidity and conductivity were measured in the laboratory on the day following the sampling using a pH

electrode with a TIM800 pH meter and an ABU901 Autoburette from Radiometer Copenhagen.

Patterns and trends – Water quality (chemical stressors) - From the two sites in Spain, no information on the

water quality is available. Also, no water quality data is available from the NP Dwingelderveld, before or during the

2010 outbreak6. However, in 2011 and 2012 information was collected, and no significant changes in the chemical

composition were present2. Water quality proved to be sufficient and nutrients resemble values expected for pools

(pers. comm. Prof. J. Roelofs)2. Table 3 gives an overview of all measured variables in water bodies at the monitored

locations. The pH ranged between 3.9 – 7.6 (mean ± sd; 5.3 ± 0.8) for the pools and 6.5 – 8.1 for the garden pond

(site G). The Staphorst site is a natural swimming pool with sandy shores (Figure 11). The water quality is measured

regularly. Oxygen levels, EGV and acidity all gave normal values in 2012 before the outbreak (pers. comm. H. Hop –

district water board Groot Salland). Habitat use by cattle – There was no evidence for cattle in the surroundings of

the water bodies. Habitat use by humans - Several but not all of the infected sites were garden ponds.

Further, the initial outbreak site in NP Dwingelderveld was used for educating the public on amphibians and

excursions with fishing nets were organised for the children. The Staphorst site is used by the public for swimming

(Figure 11).

Significance and discussion – No clear evidence for chemical stressors, no cattle presence – In the

Netherlands, no strikingly abnormal features were detected with regards the water quality of the water bodies

monitored in 2011. There was no indication for high aluminum. The acidity in a number of pools was rather low.

Possibly conductivity was slightly low and ammonium slightly high. Elsewhere high aluminum has been associated

with sites with ranavirus infection 59

. Low acidity (pH <4.5) increased the likelihood of a site to be unaffected by

ranavirus elsewhere59

. Low conductivity had been the best predictor for ranavirus-affected sites (no ranavirus at sites

>60 µS/cm) in a study elsewhere 59

. Further, elsewhere, cattle and nitrogenous fertilizers have been associated with

higher prevalence of ranavirus infection and more severe ranavirus-associated disease62, 63

. Possible explanations

put forward were immune-suppression due to elevated un-ionized ammonia concentrations and high nitrate and

nitrite levels (water quality) and more transmission opportunities due to less vegetation and thus more clustering of

individuals62, 63

. Sometimes but not always habitat use by humans – Human exploitation occurred at a number

of sites but not at all of them. Elsewhere, anthropogenic disturbance of habitat has been associated with higher

prevalence of ranavirus infection64

. One possible explanation is that the host’s acquired immune response was

negatively affected, another that there is more risk of pathogen introduction64

.


Tables 3 a, b, and c – Water quality characteristics (mean ± standard deviation and ranges) for water bodies in NP Dwingederveld (site A-F) and IJhorst (Site G).Values in µmol/l.

Figure 11 – Was disturbance a stressor at Staphorst?


Theme 12 - Inventory of trade of amphibians in Belgium

Theme relevant to introduction

Definitions - Farm bred = wildlife farming. Definition of wildlife farming according to CITES: modified form of wild

harvest, habitat managed to enhance and maximize natural recruitment for the specific purpose of harvesting the

increased production, generally confined geographically and applied to segments of the population occurring on

privately-owned lands, monitoring impact of management on wild population(s) not critical.

Patterns and trends – Numbers imported as registered by FAVV - The number of imported amphibians via

airports and harbours registered by the Federal Agency for the Safety of the Food Chain (FAVV) increased from

2,612 in 2011 (January – December) to 4,170 in 2012 (January – October). The countries of origin of these animals

are summarized in Table 4. Additionally, in 2012, 190 wild caught Kassina and Hyperolius species (family

Hyperoliidae) imported from Burundi were confiscated. Numbers and species imported for research - Three of

40 research labs sometimes performing research on amphibians indicated that they import amphibians. African

clawed frogs Xenopus laevis and tropical clawed frogs Silurana tropicalis are the species most frequently used for

research in these three laboratories. In 2011, 550 X. laevis and 500 S. tropicalis were imported from the USA for

research use. It is not known how these animals were brought into Belgium. The participating research labs did not

import amphibians in 2012 due to breeding at their own facilities. Amphibians traded by importing dealers - In

Belgium, 124 merchants are registered as allowed to import amphibians. About 4,977 amphibians were offered in

2012 at the Belgian market by the three (3) of the 5 most important amphibian dealers. Table 5 gives an overview of

the different species and their numbers.

Table 4 - Numbers of amphibians imported in Belgium via airports and harbours

Significance – The above provides some insight into the volume of the trade in amphibians in Belgium.

*


Table 5 – Numbers of amphibians in animal shops in Belgium in 2012


Theme 13 - Inventory of hobby kept amphibians in

Belgium

Theme relevant to introduction

In 12 captive collections (including two zoos) in Belgium, who responded to the questionnaire sent out by UGent,

1,385 amphibians are kept. The table provides an overview of amphibian species kept by respondents. This list is

heavily biased towards urodelan amphibians. The response of urodelan keepers was much higher compared to that

of the anuran keepers. Despite dendrobatid frogs being the most widely kept amphibians in captivity, they are

severely underrepresented in the listing.

Table 6 - Captive kept amphibian species in Belgium in 2012.



Theme 14 - Legal re-introductions and translocations in

the Netherlands

Theme relevant to introduction and spread

Definition – Re-introduction - Re-introduction is an attempt to establish a species in an area which was once part

of its historical range, but from which it has been extirpated or become extinct65

. Translocation - Translocation is the

deliberate and mediated movement of wild individuals or populations from one part of their range to another65

.

Amphibian re-introduction/translocation activities require exemptions from the Flora and Fauna Act (FFW), the law

that protects native wildlife species in the Netherlands, in order to be legal. These may be exemptions to capture and

remove individuals (article 9 of the FFW) or egg masses (article 12) from the wild; to transport and keep eggs and

individuals in captivity (article 13); to release them back into nature in the Netherlands (article 14); and to handle

individuals at sites during subsequent monitoring of the population.

Patterns and trends – Three amphibian species recently re-introduced or translocated - From the global

perspective, the conservation status of the 16 amphibian species present in the Netherlands is of least concern

(IUCN; www.iucn.org). From the national perspective, however, half of these species are near-threatened (1),

vulnerable (2), endangered (3) or critically endangered (1) (Dutch Red list, 2007). Legal re-introductions and

translocations since the adoption of the FFW ten years ago have concerned the critically endangered species

(yellow-bellied toad Bombina variegata, (starting 2004) and two of the endangered species, the common

spadefoot Pelobates fuscus and the European tree frog Hyla arborea, (starting 2009). Implementation pattern -

Examination of the submitted re-introduction projects for these three species showed that all have an identification

phase (Step 0 in Figure) and an implementation phase (Steps 1-4 in Figure). Documents resulting from the

identification phase form the basis on which the Dutch Ministry of Economic Affairs (EZ) makes decisions regarding

the law exemptions. Typically, partial egg masses or young larvae are either removed from the wild in the

Netherlands or obtained from captive adult stock (Step 1). They are then raised in captivity (Step 2) until they are of

an age deemed appropriate for release at the re-introduction site (Step 3). The best age for release varies per

species and is a trade-off between survival chances (increases with age) and inclination to stay in place (decreases

with age for species that are not sedentary). Initially, step 2 was performed at one site in Nijmegen. Recently, step 2

is occurring at several sites, and the tendency is to move away from controlled conditions (plastic basins) to more

natural conditions (nets in outdoor ponds). An under-exposed feature of these re-introduction programs is that captive

parent stocks are being set-up. Pathogen-screening and hygiene - Recent re-introduction projects include testing

for Batrachochytrium dendrobatidis prior to release, and project documents include the ‘Hygiene protocol for handling

amphibians in the field’ 66

. However, structural screening for the presence of ranavirus before release into nature is

missing.

Significance – Risk per implementation step - Species being re-introduced can be susceptible for CMTV

infection. With nationwide exemptions, infected stock may be procured from areas where CMTV occurs. Also, under

current exemptions, captive parent stock may be legally imported from other European countries without any disease

control. Infection could thus be translocated to the sites where the stock is grown in captivity. The length of the stay in

captivity is in favour of CMTV detection, but raising stock in outdoor ponds rather than under controlled conditions

makes detection and especially elimination more difficult. If not detected, infection could be translocated to the site of

release. Regions at risk - Currently, this would imply introduction of the virus into the provinces of Noord-Brabant

and Limburg, also home to the endangered fire salamander Salamandra salamandra, the vulnerable palmate newt

Lissotrition helveticus and the vulnerable CMTV susceptible common midwife toad Alytes obstreticans.

Figure 12- Summary of the ten amphibian translocation projects since the ‘Flora and fauna wet’ was adopted.



Theme 15 - ‘Unofficial introductions’ Theme relevant to introduction and spread

Definition - Unofficial introductions – By ‘unofficial introductions’ are meant all human activity-mediated release

of amphibians into nature, which are not legal re-introductions or translocations.

Patterns and trends - Unfortunately many unofficial introductions have occurred either with amphibians that were

collected abroad (mainly France) or from natural Dutch populations. Introductions have occurred with nearly all

amphibian species, the focus here is on known susceptible host species (for maps, cf. Th.5). Two less common

host species with widespread unofficial introductions - Alpine newt - The Alpine newt has been introduced to

many sites in the Netherlands, even as early as the 1900s in Drenthe. Introductions occurred mostly around cities. In

Drenthe it was introduced in Rheebruggen in the 70s of the last century, and also around 1995 at the Utrechtse

Heuvelrug, the Veluwe and Arnhem52

. Common midwife toad - The midwife toad only occurs naturally in the

southern part of Limburg, but can be found throughout the country, mostly in or in the close vicinity of cities. They

include both offspring from specimens originating in France or Limburg37

. Since the 70s of the last century a

population survives in Rheebruggen (Drenthe), and these toads were collected in Northern France67

. Common

widespread host species unofficially introduced on Wadden Sea islands – Water frogs - Unofficial

introductions are known from the 1930s in Urk and Texel. In Texel around 1968 approximately 50 frogs were

released, this introduction failed. In the eighties another introduction occurred in Texel, and this introduction

succeeded. In Ameland in 2003 and Schiermonnikoog in 2009, water frogs popped up as well, all introduced. In the

beginning of July 1971 over 10,000 larvae were released in Rotterdam and in November of that year, 2,000 Bulgarian

water frogs were released near Rijsoord. It is unknown if these larvae grew successfully and if they mixed with the

native frogs30

. Common toad - The species was unsuccessfully introduced in Texel between 1979 –198568

. To our

knowledge, there is no more information on introductions of the common toad in the Netherlands. Smooth newt -

Even though the smooth newt is widely distributed in the Netherlands, it has been introduced in the first half of the

20th century on all Wadden islands, apart from Texel at which it occurs naturally. It is unknown whether there are

more recent introductions34

. No known unofficial introductions Common spadefoot - No information is known

about unofficial introductions of spadefoots in the Netherlands. Exotic species - Amercian bullfrog: cf. Th.5.

Significance - Widespread unofficial introductions Introduced Alpine newts and common midwife toads occur in

the CMTV-like virus disease hotspot (cf. Th.5).


Theme 16 - Flyways Theme relevant to introduction and spread

Definition - A flyway is the entire range of a migratory bird species (or groups of related species or distinct

populations of a single species) through which it moves on an annual basis from the breeding grounds to non-

breeding areas, including intermediate resting and feeding places as well as the area within which the birds migrate69

.

Patterns and trends – There are flyways along the CMTV-like virus axe Spain-Belgium-Netherlands.

Significance – Hypothetical dispersal via birds – It has been hypothesized that birds may act as vectors in the

spread of ranavirus infections, but to date there is no scientific evidence for this.

Figure 13 – Eurasian Flyways. In red the south-eastern European flyway, in blue other migration flyways (source: SE European Bird Migration Network, http://www.seen-net.eu/)


Part 2 Risk assessment Method Using the compiled background data and further information obtained as mentioned in Part 1, assessment was made

of the possible routes of introduction of CMTV-like virus into the Netherlands, the likelihood of its establishment and

spread, and the possible social and ecological impact of CMTV-like virus. Risk was then scored as detailed in next

paragraph.

Risk assessment scoring method - Dispersion potential, invasiveness and ecological impacts - The

Belgian Invasive Species Environmental Impact Assessment (ISEIA) protocol70

was used to assess risks associated

with dispersion potential, invasiveness and ecological impacts. The risk assessment was carried out by an expert

team. This team consisted of six (6) persons: Rob van Leuven, (RU), An Martel (U Gent), Annemarieke Spitzen-van

der Sluijs (RAVON), Ronald Zollinger (RAVON), Marja Kik (DWHC) and Jolianne Rijks (DWHC). Each expert

completed an assessment form independently, based on the content of a knowledge document. Following this

preliminary individual assessment, the entire project team met, elucidated differences in risk scores, discussed

diversity of risk scores and interpretations of key information. The results of these discussions were presented in a

draft report. Further discussion led to agreement on consensus scores and the level of risks relating to the four

sections contained within the ISEIA protocol (Table 7). The ISEIA protocol contains twelve criteria that match the last

steps of the invasion process (i.e., the potential for spread, establishment, adverse impacts on native species and

ecosystems). These criteria are divided over the following four risk sections: (1) dispersion potential or invasiveness,

(2) colonisation of high conservation habitats, (3) adverse impacts on native species, and (4) alteration of ecosystem

functions. Section 3 contains sub-sections referring to (i) predation / herbivory, (ii) interference and exploitation

competition, (iii) transmission of diseases to native species (parasites, pest organisms or pathogens) and (iv) genetic

effects such as hybridisation and introgression with native species. Section 4 contains sub-sections referring to (i)

modifications in nutrient cycling or resource pools, (ii) physical modifications to habitats (changes to hydrological

regimes, increase in water turbidity, light interception, alteration of river banks, destruction of fish nursery areas, etc.),

(iii) modifications to natural successions and (iv) disruption to food-webs, i.e. a modification to lower trophic levels

through herbivory or predation (top-down regulation) leading to ecosystem imbalance. Each criterion of the ISEIA

protocol was scored. Possible scores are 1 (low risk), 2 (medium risk) or 3 (high risk). Definitions for low, medium and

high risk, according to the four sections of the ISEIA protocol are given in table 7. If knowledge obtained from the

literature review was insufficient, then the assessment was based on expert judgement and field observation leading

to a score of 1 (unlikely) or 2 (likely). If no answer could be given to a particular question (no information) then no

score was given (DD - deficient data). Finally, the highest score within each section was used to calculate the total

score for the species. Consensus on the risk score of each section was reached using a hierarchical method where

evidence from within the Netherlands was given priority over evidence derived from impacts occurring outside the

Netherlands. It was also considered that the suitability of habitats in the Netherlands may change due to e.g. water

temperature rise due to climate change. Moreover, consideration was given to the future application or non-

application of management measures that will affect the invasiveness and impacts in the Netherlands. Subsequently,

the Belgian Forum Invasive Species (BFIS) list system for preventive and management actions was used to

categorise the species of concern70

. This list system was designed as a two dimensional ordination (Environmental

impact * Invasion stage; Figure 14). It is based on guidelines proposed by the Convention on Biological Diversity

(CBD decision VI/7) and the European Union strategy on invasive non-native species. Environmental impact of the

species was classified based on the total risk score (global environmental risk) which is converted to a letter / list:

score 4-8 (C), 9-10 (B - watch list) and 11-12 (A - black list). This letter is then combined with a number representing

invasion stage: absent (0), isolated populations (1), restricted range (2), and widespread (3). Socio-economic and

public health impacts - Potential socio-economic and public health impacts did not form a part in the risk analysis

according to the ISEIA protocol. However, these potential risks should be considered in an integrated risk analysis.

Socio-economic risks were examined as part of the literature study and in discussions with project partners. Socio-

economic risks occurring at present or in the future dependent on alterations in habitat suitability and management

interventions were considered.


Table 7 - Definitions of criteria for risk classifications per section used in the ecological risk assessment protocol (Branquart et al., 2009

70).

1. Dispersion potential or invasiveness risk

Low The species does not spread in the environment because of poor dispersal capacities and a

low reproduction potential.

Medium

Except when assisted by man, the species doesn’t colonise remote places. Natural dispersal

rarely exceeds more than 1 km per year. However, the species can become locally invasive

because of a strong reproduction potential.

High

The species is highly fecund, can easily disperse through active or passive means over

distances > 1km / year and initiate new populations. Are to be considered here plant species

that take advantage of anemochory, hydrochory and zoochory, insects like Harmonia axyridis or

Cemeraria ohridella and all bird species.

2. Colonisation of high conservation habitats risk

Low Population of the non-native species are restricted to man-made habitats (low conservation

value).

Medium Populations of the non-native species are usually confined to habitats with a low or a medium

conservation value and may occasionally colonise high conservation habitats.

High

The non-native species often colonises high conservation value habitats (i.e. most of the sites

of a given habitat are likely to be readily colonised by the species when source populations are

present in the vicinity) and makes therefore a potential threat for red-listed species.

3. Adverse impacts on native species risk

Low Data from invasion histories suggest that the negative impact on native populations is

negligible.

Medium

The non-native is known to cause local changes (<80%) in population abundance, growth or

distribution of one or several native species, especially amongst common and ruderal species.

The effect is usually considered as reversible.

High

The development of the non-native species often causes local severe (>80%) population

declines and the reduction of local species richness. At a regional scale, it can be considered

as a factor for precipitating (rare) species decline. Those non-native species form long standing

populations and their impacts on native biodiversity are considered as hardly reversible.

Examples: strong interspecific competition in plant communities mediated by allelopathic

chemicals, intra-guild predation leading to local extinction of native species, transmission of

new lethal diseases to native species.

4. Alteration of ecosystem functions risk

Low The impact on ecosystem processes and structures is considered negligible.

Medium The impact on ecosystem processes and structures is moderate and considered as easily

reversible.

High

The impact on ecosystem processes and structures is strong and difficult to reverse. Examples:

alterations of physico-chemical properties of water, facilitation of river bank erosion, prevention

of natural regeneration of trees, destruction of river banks, reed beds and / or fish nursery areas

and food web disruption.

Figure 14 - BFIS list system to identify species of most concern for preventive and mitigation action (Branquart et al., 2009

70).


Results 2.1 – Probability of introduction

Possible recent introduction - CMTV-like virus is already present in a restricted area of the Netherlands (Th.2),

where it is associated with disease emergence1, 6

. The cause of this disease emergence is not fully understood but

the most likely possibility is that CMTV-like virus was recently introduced from abroad, either once, or on multiple

occasions into the same area. Elsewhere, in the U.K., another ranavirus is causing disease among amphibians since

the mid-1980s. Phylogenetic analyses comparing multiple isolates of ranaviruses provides evidence that this virus

was probably introduced into the U.K. from the American continent24

. Potential geographical origin - Other

countries where CMTV-like virus has been detected incidentally and locally are Spain and Belgium (map in Th.2)3, 8

.

The global distribution of CMTV-like virus is unknown at the moment. Possibly it has a wider occurrence5, that has

not been detected due to under-surveillance71

. Assuming introduction from abroad, more insight into possible

pathways is helpful for tracing origin, for preventing future introductions of CMTV-like virus into susceptible host

populations, or both.

2.1.1 Introduction through human activities Among the possible pathways of introduction from abroad (Figure 15), there are those associated with human

activities. The focus has been on obtaining 1) data with regards to the legal trade in amphibians through Schiphol

airport, 2) data with regards to the legal trade in amphibians in Belgium and from Belgium to the Netherlands, and 3)

data on release of imported known susceptible hosts into nature. Entry of CMTV-like virus via fomites into the

Netherlands follows the same mechanisms as depicted for spread in §2.3.

2.1.1.1 Legal trade through Schiphol

Schiphol airport is one of the two airports in the Netherlands from where live animals are imported from abroad,

including from outside the European Union, and the main airport for import of wild animals. The other airport,

Maastricht, focuses on transport of equines. Rotterdam harbour is the main port of entry of animal products.

∗ Numbers and species imported via Schiphol airport – An ‘umbrella’ CN-code Worldwide, trade in

animals and products is regulated using CN (combined nomenclature; Council Regulation [EEC] No 2658/87 of 23

July 1987 on the tariff and statistical nomenclature and on the Common Customs Tariff). Live amphibians fall

under the CN-code 0106 ‘other living animals’ (specifically CN-code 0106 9000 ‘All live animals other than

mammals, birds and reptiles that have been named or considered elsewhere. Live frogs, to be kept alive in

vivaria, or to be slain for human consumption, fall under this CN code’). This ‘umbrella’ CN code provides the

veterinary inspection no insight into whether amphibians being imported with a shipment or not. Only 4

shipments of amphibians were identified via CN-code in TRACES Import of animals and products into the

EU are tracked through the Trade Control and Expert System (TRACES) database. Between 2006 and 2012,

there have been over 11,000 CN-code 0106 shipments from outside the EU into the EU via the Netherlands. Only

four (4) of these 11,000 shipments specified that the animals being shipped were ‘amphibians and reptiles’,

providing health certificates to the veterinary inspection. These shipments concerned between 150 and 350

animals, of 1-12 different species of frogs and reptiles, or of unspecified species. All four shipments entered the

EU from abroad (Singapore, Suriname) via Schiphol, and had other European countries (Spain, Czech Republic)

as final destination. It is quite possible that amphibians with the Netherlands as destination were present in some

of the other 10,996 shipments with CN-code 0106. For example, if a shipment contains mostly live insects, it may

be registered as ‘insects’, while amphibians travel on the same certificate. A number of CN-code 0106 9000

documents were opened randomly in TRACES, but none had amphibians on them. Search in the TRACES

database targeting the more likely importers and countries of origin also did not provide more data. Purpose of

trade It follows that no overview of the purposes for which amphibians are imported was obtained. However,

frogs are not being imported to harvest frog legs in the Netherlands (Antwoord 31/10/12 op kamervraag, NVWA).

Remark - For a subgroup of amphibians traded, namely the endangered species, some information on numbers

traded may be obtained from Customs, as Customs do ‘Convention on International Trade in Endangered species

of Wild Fauna and Flora (CITES)’ checks. CITES documents accompanying shipments indicate the purpose (e.g.,

T=for commercial purpose, Z=for zoos) and the source (e.g.,W=wild-caught) of animals transported. CITES export


Figure 15 – Possible pathways of introduction from abroad


and re-export permits may be detailed to number and species level. Information may be available at the

organization (Ministry of EZ, DR-Loket) that provides the CITES documents (lead not pursued in this study).

∗ Transport conditions and health check – Casualities during transport limited by IATA regulations

IATA Live Animal Regulations are set annually by the International Air Transport Association (IATA) and accepted

as guidelines in respect of transportation of animals by air by CITES and the OIE. The EU has adopted these

regulations as the minimum standard for transporting animals in containers, pens and stalls. IATA Live Animal

Regulations have evolved greatly, including container requirements. Airlines such as KLM inform shippers about

these requirements and actively coach the shippers throughout the whole process (even visiting the facilities of

regular shippers). Death of animals during air transport is very rare. Short transport time Transport duration is

generally kept short. For example, considering a trader in Suriname ships amphibians to a trader in the

Netherlands, the time frame is likely to resemble the following: a 3-hour drive to airport to arrive 3 – 5 hours before

the embarking, say that is at 17.45 in the evening. The flight would arrive around 9 hours later at Schiphol, 08.00

hours local time, undergo its health check at 11.00 and be picked up for delivery in the Netherlands at 12.00, i.e.,

a delivery from trader to trader within 24 hours. Subclinical cases may go unnoticed. Health check involves

visual inspection The health check is performed by the NVWA and consists of verification of the documents, in

particular if these are correctly completed, whether the contents of the shipment are conform the certificate

(verification often done in collaboration with Customs and the airline), and whether the animals appear healthy.

The latter is done by visual health inspection of a sample of the animals. At Schiphol, all boxes of shipments are

usually opened for inspection, unless the shipping concerns ornamental fish. In case of ornamental fish, a sample

consisting of at least 10% of the shipment and a minimum of 2 boxes is inspected. Importing parties can verify the

health conditions required for certification on the NVWA site (Veterinary Import Online [IVO],

http://wisdom.vwa.nl/ivo/Start.do). Ranavirus infection and health certification The health certificate used in

the context of CN-code 0106 shipments poses no requirements with regards to ‘infection with ranavirus’ for

amphibians, therefore specific certification is absent. This is different for EHNV and live ornamental fish

(VILS012001A, version 1.0.0). For ornamental fish it must be certified that the animals were in quarantine

(Beschikking 2008/946/EG), or that the country of origin of the fish is free of EHN and that EHN is notifiable in that

country, that known susceptible species are from areas where EHN does not occur, and that no vaccination

against EHNV was provided.

To conclude, it appears that for live amphibian traded, the numbers and species imported from outside the EU and

inside the EU through Schiphol can currently hardly be traced. Further, there is no specific control for ranaviruses in

amphibians via health certificate requirements. It seems unlikely that infection with ranavirus in amphibians will

currently be detected at Schiphol, because traders are unlikely to put individuals from a batch of amphibians clinically

infected with ranavirus on transport, transport time is so short cases incubating the disease may not develop into

clinical cases within it, and sub-clinical cases will not be detected by visual inspection. Present requirements for

documentation are too limited to allow answers to the following question: how many animals in what status of health

of what species are imported?

2.1.1.2 Amphibian trade in Belgium

Amphibians may also be imported into the Netherlands from European countries by other means than by air. For this

reason an inventory of trade was made of trade in amphibians in Belgium and from Belgium to the Netherlands, as

well as an inventory of privately kept amphibians and their possible exchange to the Netherlands.

∗ Inventory of trade of amphibians in Belgium - A wide variety of amphibian species is imported, sold en kept

in captivity in Belgium. Several thousand imported from outside the EU per year The Federal Agency for

the Safety of the Food Chain (FAVV) registered 2,612 amphibians imported into Belgium from outside the EU in

2011 and 4,170 in the first ten months of 2012. They arrived from Africa (Togo, Nigeria), Asia (Indonesia), South

America (Peru), Central America (Nicaragua, Panama) and North America (US). Also at least 190 amphibians

(Kassina and Hyperolius species imported from Burundi) were confiscated at customs (Th.12). The number of

amphibians imported is likely to be greater than these figures. The laboratories imported in 2011 550 African

clawed frogs (Xenopus laevis) and tropical clawed frogs (Silurana tropicalis) from the US for research use. The

fact that the laboratories indicate a number of amphibians imported from the US that is greater than that

mentioned by the FAVV again highlights the lack of traceability of amphibians with the current international

registration system as detailed previously (§ 2.1.1.1). Traded amphibians are often bred but some are

wild-caught. Among the amphibians offered by the three (3) Belgian importers in 2012, 52.8% were captive


bred , 5.4% were farm bred, 10.4% were wild-caught, and 31.4% were of unknown source (Th.12). It is

remarkable that a third of the animals were of unknown source, including two species (the Australian green tree

frog Litoria caerulea and the Amereega trivittata) that were imported from or through the Netherlands. Further, at

least 58% of the amphibians were captive or farm bred, indicating the importance of captive breeding for the

supply of exotic amphibians. The three consulted dealers do not sell imported amphibians directly to animal shops

in the Netherlands.

∗ Inventory of hobby kept amphibians – Many more captive kept species than native wild species -

The inventory (Th.13). provides insight into the diversity of amphibian species kept in captivity, already over six

times the number of native amphibian species in the Netherlands. Captive kept species include native

species - Among these are at least three (3) species that occur natively in the Netherlands and are susceptible

to CMTV-like virus (the common midwife toad Alytes obstetricans, the smooth newt Lissotriton vulgaris and the

common spadefoot toad Pelobates fuscus). Kept amphibians are often bred but some are wild-caught

The numbers presented in the inventory of hobby kept amphibians provide similar insight into the relative

importance of sources as the inventory at the traders: 60% were captive-bred, 13% were wild caught and 27%

were of unknown source, reinforcing the previous observation that captive breeding is the most common source of

amphibians kept in captivity. Ranavirus situation in kept amphibians is virtually unknown Virtually nothing

is known about the presence of ranavirus in captive collections in Europe, though CMTV-like virus infection is

described in a captive collection in the Netherlands (Th.3)7 and ranavirus infection was found in red tailed knobby

newts (Tylototriton kweichowensis) from three hobby collections in Belgium and the Netherlands72

. In the latter

case, the newts had been imported from abroad by a Belgian shop. A minority had shown limited skin ulceration

but otherwise they seemed healthy and they had been sold72

. This example shows how hobbyists in the

Netherlands can be supplied by shops outside the Netherlands and demonstrates how ranavirus infection may be

missed during import and spread, thus presenting a potential source for infection of wild animals. Deaths among

captive amphibians are not often investigated, so the frequency of occurrence of CMTV-like virus infections in

kept specimen remains unknown. Exchange of kept amphibians across the border - Exact data regarding

exchange of amphibians between Belgium and the Netherlands are not available since registration of animal

movements is generally not required. However, keepers of amphibians in both countries have very frequent

contacts and country borders do not play any limiting role regarding the exchange of amphibians between

keepers of both countries. Based on the questionnaire, an estimated minimum of 50% of the private owners in

Belgium exchange animals with keepers in the Netherlands. Captive offspring of Belgian amphibian breeders is

widely distributed among Dutch keepers (and vice versa), both for anurans (e.g. during ‘Dendrobatidae

Nederland’ meetings) and for urodelans (e.g. during ‘Salamandervereniging’ meetings).

To conclude, the data on amphibian trade in Belgium confirms 1) the difficulty of tracing imported live amphibians

and 2) the difficulty of detecting ranavirus infections during the actual import without quarantine, and 3) the frequent

import of amphibians from Belgium into the Netherlands. Further, it highlights 4) the diversity of amphibian species

being kept— both exotic and native species—, and 5) the fact that the market is largely supplied by these captive

bred animals while too little is known about ranavirus infections in captive collections. It is currently not possible to

quantify the risk of introducing CMTV-like virus through amphibians imported from Belgium.

2.1.1.3 Entry of CMTV-like virus imported via live amphibians into nature

In this paragraph we hypothesize on routes of entry of imported CMTV-like virus imported via live hosts into nature.

∗ Disposal of imported infected amphibians - CMTV-like virus from abroad could enter nature via the (un-)

intentional release of an imported infected host (exotic or native species). There is evidence for the release of

susceptible host species into nature. For example, of American bullfrogs (Th.3 and Th.5), common midwife toads

(imported from France and released in Rheebruggen, which lies within the CMTV disease hotspot; Th.5 and

Th.15), and water frogs (imported from Bulgaria; Th.15). But there is no data on the CMTV-like virus status of the

source populations or the released specimen.

∗ Via secondary cases - Further, imported infected hosts or their waste products could infect specimen of native

susceptible host species that are kept as pet in captivity in the same facility (e.g., Salamandridae, Bufonidae,

Genus Pelobates). The latter may be disposed of or even be legally introduced into nature via garden ponds.

Evidence for the occurrence of such disposal is the presence of introduced populations of Alpine newts in or close

to cities (Th.15). Again there is no data on the CMTV-like virus status of the released specimen.


Overall conclusion pathways of introduction

At the moment it is not possible to draw a quantitative conclusion because precise information on imported

amphibian species, numbers and prevalence of CMTV-like virus infection in these is largely lacking. However,

with regards to human-mediated import of infected specimen, it is clear that: 1) numerous species from all over

the world are traded and kept together; 2) traceability of specimens imported is poor; 3) captive-bred exotic

amphibian trade is significant and elusive, at least in terms of diseases occurring; 4) wild caught specimens of

unknown health status are regularly introduced into facilities where captive-bred species are kept; 5) disease

detection during import is unlikely. With regards to natural dispersal as possible route of introduction, it is noted

that CMTV-like virus infection was found to occur in a free-living population of amphibians across the Belgium

border and that spread into the Netherlands has to be considered.

To obtain quantitative data on species and numbers imported on short notice would require obtaining data from

customs, amphibian dealers and amphibian keepers; to obtain full and continuous insight into this requires a

means to trace the amphibians that are imported. To obtain data on the health status of imported specimen

requires surveillance for CMTV-like infection in the wild abroad and in captive collections. To obtain data on

amphibian disposal frequency would require obtaining data from amphibian keepers.

∗ Unintentional transport – Sometimes amphibians are unintentionally transported with other goods, such as

vegetables. The route is hard quantify.

∗ Via fomites - Finally, aquarium or vivarium water and sediment may contain infectious virus and be disposed of

in nature. In a study in the context of invasive water plants, 2% of the 230 persons with aquaria or garden ponds

that responded to a questionnaire confirmed they disposed of water plants in Dutch open waters. Motives given

were that it was a pity to throw away living plants, and that it nice to be able to see beautiful plants in open waters.

In a study in New Zeeland, at least two of 43 aquarium holders disposed aquarium wastes into out-door ponds or

storm-waters drains (risk for entering nature)73

.

To conclude, there are multiple routes through which human-mediated imported CMTV-like virus could in principle

enter nature, but quantification is difficult.

2.1.2. Introduction through nature Introduction of CMTV-like virus from abroad through nature can in theory occur via infected hosts from neighbouring

countries that cross the border into the Netherlands through natural dispersal, or via migrating birds.

2.1.2.1 Introduction via infected amphibians crossing borders into NL

American bullfrog - CMTV-like virus infection occurs in wild populations of American bullfrogs in Belgium8, less

than 5 km from the Dutch border, and less than 15 km from the centre of Breda (Th.3 and Th.6). American bullfrogs

have good dispersal abilities (Th.7)39

. On the Dutch side of the border, in Sint-Oedenrode, Noord Brabant province,

an American bullfrog was reportedly destroyed in 2009 (Th.5), but it was not submitted for examination so it could not

be determined if its origin was the Belgian wild population. Though there is active removal of exotic American

bullfrogs from nature in both countries, other susceptible species sharing their habitat may have been infected by

them and not detected to date. Native species - Many of the susceptible host species, that are native, common and

widespread species in the Netherlands are also native, common and widespread species in Germany and Belgium

(Th.6). However, to date—possibly due to under-surveillance—there have been no reports of CMTV-like virus in

native species from these two countries (Th.2).

To conclude, CMTV-like virus has been shown to occur in at least one amphibian species, which is a good

disperser, close to the Dutch border (Hoogstraten).

2.1.2.2 Introduction via migrating birds

Introduction of CMTV-like virus from abroad could in theory occur via migrating birds (Th.16). Birds have been

suggested as vehicle for ranavirus spread, by virus carriage on their feet or bills, or by regurgitation of ingested

infected material within a few hours of feeding74

. The presence of the amphibian chytrid fungus Batrachochytrium

dendrobatidis on feet of duck (Anatidae) has been demonstrated75

, but to date there is no hard evidence ranavirus

could indeed be spread in such a way.

To conclude, the relevance of this route of introduction is unknown.


2.2. – Probability of establishment

CMTV-like virus has persisted locally for at least 2 years - CMTV-like virus is known to have persisted for two

years in NP Dwingelderveld1, 2

. The area where CMTV-like virus is already present has a diameter of approximately

25 km (map in Th.2)1. Requirements for establishment - This section provides an overview of virus-, host- and

environment-related factors, and their interplay, which may influence (further) establishment of CMTV-like virus.

CMTV-like virus requires susceptible hosts for replication. This means that to establish itself, the virus needs to have

access host populations that belong to susceptible species, that have poor innate immunity to the virus and that have

not yet acquired sufficient immunity, or that have enough susceptible individuals in them. Further, the virus needs

suitable environmental conditions. Finally, it needs to be able to maintain itself in time.

2.2.1 Susceptible hosts are present 2.2.1.1 Susceptible species

Broad host range - CMTV-like virus has a broad host range, infecting at least 9 of the 16 native Dutch amphibian

species, and possibly infecting fish (Th.3)1, 3, 6, 8, 23

. Common species - Water frogs, common frogs, common toads

and smooth newts are susceptible to infection and among the most common species in the Netherlands. They have a

wide distribution and inhabit a great diversity of habitat types (Th.5). They are each other’s accompanying species

(Th.8). There is field evidence for inter-species transmission of CMTV-like virus (Th.8). Differences in species

susceptibility to CMTV-like virus are likely but have yet to be determined. Some species may be reservoir hosts, but

these still need to be determined.

To conclude, susceptible species occur commonly throughout the Netherlands, suggesting establishment of

infection could occur in suitable habitats for amphibians nationwide.

2.2.1.2 Susceptible populations

Immunologically naïve populations likely - The CMTV-like virus-associated massive mortality observed at NP

Dwingelderveld (2010)6, IJhorst (2011), Wijster (2011), Fluitenberg (2011), Darp (2011) and Staphorst (2012)

1

affected all life stages including adult specimens (Th.4). This suggests that the affected populations were

immunologically naïve or at least largely susceptible to CMTV-like virus infection6. Currently no evidence for co-

evolving CMTV-like virus in the Netherlands – Evidence from elsewhere with other ranaviruses shows newly

introduced ranavirus strains are generally more pathogenic than endemic strains76, 77

. Virulence appears to be related

to the genetic similarity of the introduced strain with coevolved strains and with host specificity22

. For example, ATV

(one of the other ranaviruses) appears to co-evolve with local tiger salamander populations in North America.

Disease emerged when novel strains were introduced into populations that had not co-evolved with these strains77, 78

.

There is no evidence for the presence of co-evolving CMTV-like virus in the Netherlands; however, this cannot be

excluded for certain due to under-surveillance and under-detection. No evidence for presence of other

ranaviruses that could possibly lead to cross-protection - There is some cross-protection between

ranaviruses. Antibody raised against the major capsid protein (MCP) of one ranavirus species often cross-reacts with

other members of the genus, as MCP is highly conserved79

. For this reason, CMTV-like virus may have difficulty

establishing itself if there are other ranaviruses present which infect the susceptible host species. No studies have

been performed to determine the level of acquired immunity against CMTV-like virus in amphibians in the wild in the

Netherlands or elsewhere. However, to date there is no evidence for other ranaviruses infecting amphibians in the

wild in the Netherlands.

To conclude, to date there no evidence for previous exposure to CMTV-like virus or another ranavirus and therefore

Dutch amphibians are considered to be susceptible to infection.

2.2.1.3 Susceptible individuals

Annual recruitment in water bodies - Many amphibian species reproduce in pools and ponds80

. The (sub)adults

congregate, and offspring is produced. Based on what is currently known about ranavirus transmission (Th.1), this

offspring is likely to be susceptible. In other words, hosts are pooled during the aquatic phase and some to all are

likely to be susceptible. Effects of relative host density/life stage –The life stage predominantly present in the

water depends on the time of the year (Th.4). The stages affected in the CMTV-like virus associated mass


Figure 16 - Probability of establishment


mortality events in the Netherlands were often stages present in peak numbers (relative high density) in the aquatic

phase at the time (Th.4, Figure 4). Experimentally, host density was a determinant of ranavirus infection12

. Adults –

Mostly (sub-)adult amphibians return to the same water body each year, a few will colonize new ponds (Th.7). If the

(sub-)adults have been decimated in the previous year, and they are not yet replaced by others, this will affect the

numbers of offspring produced in that water body. Other factors – The number of susceptible hosts available is

influenced by predation and other causes of amphibian death.

To conclude, at the level of water bodies there is annual recruitment of offspring likely to be susceptible to CMTV-

like virus infection. Recruitment of susceptible animals is important for a pathogen to maintain itself in a population.

2.2.2 Dutch environment in general appears suitable 2.2.2.1 Temperature

Replication - The suitable replication temperature of CMTV-like virus is not known but likely to be within the range

of 12°C-32°C as found for other ranaviruses (Th.1). This is consistent with water temperatures at sites where CMTV-

like virus disease and mortality have been observed (Th.10). Based on mean daily air temperatures (KNMI,

http://www.knmi.nl/klimatologie/grafieken/jaar), such water temperatures are common between May and September

in the Netherlands. Survival - On the other hand, survival of the virus outside the host is best at low temperatures

(Th.1). Low temperatures occur in winter. Whether survival of CMTV-like virus is long enough to overcome Dutch

winters in absence of sub-clinically infected hosts remains to be determined.

To conclude, temperatures likely to be suitable for virus replication occur for at least 6 months a year. In the

remaining part of the year, temperatures are favourable for virus survival outside of the host (Th.1 and §2.2.3.1).

2.2.2.2 Suitable sites

Water body type - The water bodies involved in the CMTV-like virus outbreaks in Spain and in the Netherlands

varied in nature. However, they were generally not connected to other surface water bodies (standing water),

permanent, small and rather shallow (Th.9). Such water bodies are suitable breeding sites for many amphibian

species. Further, ranavirus dose may be more likely to build up in small still standing water bodies. Landscape,

land use, water quality, pesticides and predation – Elsewhere, increased prevalence of ranavirus infection was

associated with 1) landscape factors such as high watercatchment areas57

, 2) land use factors such as grazing by

cattle62, 63

and intensive human exploitation64

, and 3) water quality parameters, in particular low conductivity; possibly

low pH and high aluminum59

. Further, reduced survival due to experimental ranavirus infection was associated with

4) presence of pesticides or 5) predators or both81

. Conductivity and pesticides were not measured in the context of

CMTV-like virus. Otherwise, apart eventually from human disturbance, the existing data do not suggest that such

factors are playing a role in the emergence of CMTV-like virus in the Netherlands (Th.9 and Th.11). In contrast, in

terms of perspectives for establishment of the virus, cattle and human disturbance are commonplace in the

Netherlands.

To conclude, multiple water bodies in the Netherlands would be suitable for CMTV-like virus infection to occur.

There is no clear evidence for enhancement of emergence by landscape, chemical or biological stressors known from

the literature, further supporting the concept that CMTV-like virus is likely to be an exotic virus. Some stressors are

definitely present in the Netherlands, e.g., cattle and human disturbance, and could enhance establishment

2.2.3 The virus has strategies to maintain itself Ranaviruses can cause severe disease and high mortality, in particular up to 100% of larvae can die, this stage being

the most susceptible developmental stage28, 82

. Pathogens that cause high mortality are at risk of burning themselves

out. However, ranaviruses have several mechanisms to be able to persist at infected sites. Two have been previously

mentioned, namely the broad host range (§ 2.2.1.1) and co-evolution (§ 2.2.1.2). Further there is the probable

lengthy survival in the environment and the possibility of intra-species reservoirs.

2.2.3.1 Probable lengthy survival outside the host

Specific data on survival of CMTV-like virus in the environment is lacking. However there is evidence that ranaviruses

in general can remain infective for extended periods of time in water, sediment and tissues, in particular when


temperatures are low, i.e. + 4°C or below zero (Th.1). This suggests long survival periods of ranaviruses in ponds in

winter and that amphibians can be (re-)infected when they return to an infected pond after hibernation.

2.2.3.2 Reservoirs

Sub-clinical or sub-lethal infections with CMTV-like virus occur8, but there is no information on duration of

infectiousness of such cases. Subclinical 14, 83, 84

or sub-lethal infections 9, 11, 85

have also been described for other

ranaviruses, and are considered likely to contribute to maintenance or re-introduction of the ranavirus infection at

sites when the next breeding season starts 22, 80

. Lower doses of ranavirus are more likely to lead to sub-lethal

infections86

.

This means that to maintain itself, it needs to have access host populations that 1) belong to susceptible species, 2)

have poor innate immunity to the virus, and 3) have not yet acquired sufficient immunity, i.e., are naïve, or have

enough susceptible individuals in them

2.3 Probability of spread

This section concerns possible routes of spread of CMTV-like virus from areas in the Netherlands where it is

established in host species to areas where it is not yet present. Distinction is made between spread through human

activities and spread through nature (Figure 17). It applies to the known disease hotspot, but also to possible future or

not yet detected sites of establishment.

2.3.1 Spread through human activities 2.3.1.1 Legal re-introductions and translocations of threatened native species

Currently three native amphibian host species are involved in re-introduction or translocation projects. These are the

common spadefoot, the yellow-bellied toad (Bombina variegata) and European tree frog (Hyla arborea) (Th.14).

∗ Common spadefoot - The common spadefoot is susceptible to CMTV-like virus infection and disease1. For re-

introduction purposes, this species is harvested as spawn from the wild. Until 2012, spawn was removed only

from sites in Limburg and Gelderland. But in spring 2012, as part of a large-scale attempt to strengthen the

remaining small natural populations, it became legal to collect spawn from sites nationwide. An experimental

study, with other ranavirus species and other host species, has shown that embryo stages (fertilized spawn) can

be infected with ranavirus. However, embryo stages have lower susceptibility to infection than larvae28

. No spawn

was found in 2012 in Staphorst, where CMTV-like virus was detected later in that season. Nonetheless spawn

was taken from other sites within the CMTV-like virus disease hotspot, including Valthe where the largest

common spadefoot population of the Netherlands is found. Spawn was raised to larvae in basins by

Natuurbalans-Limes Divergens BV (in Nijmegen) and by RAVON (at Artis). At both culture sites some mortality

occurred during the growth; dead larvae from Nijmegen were submitted for testing to exclude ranavirus infection.

The 2012 larvae were released in the provinces of Noord-Brabant and Limburg. As far as is known, CMTV-like

virus is currently not present in these provinces.

∗ Yellow-bellied toad - Contrary to the common spadefoot, yellow-bellied toads are not procured from the wild,

but captive bred. Risk of infection during captivity by cross contamination or parent stock import cannot be

excluded. However no cases of CMTV-like virus infection in this species have been described to date and its

susceptibility to CMTV-like virus is unknown.

Overall conclusion probability of establishment

The ecology of ranaviruses involves a complex interaction of reservoir species, transmission routes, environmental

persistence, stressors, and host immunity22

. Nevertheless the required susceptible hosts, the suitable environment

and mechanisms for persistence are all present in the Netherlands: 1) CMTV-like virus can infect a broad range of

amphibian species occurring in the Netherlands, and many of these are common and found nationwide; 2) the

populations affected appear immunologically naïve; 3) offspring is likely to be susceptible and recruited annually;

4) temperature appears to be favourable for replication in hosts in the summer and for virus survival in the winter;

5) the type of water bodies affected are commonly found throughout the Netherlands; and 6) the virus probably

has multiple mechanisms to maintain itself after initial introduction. Therefore without interfering, the risk of

permanent establishment of CMTV-like virus is high (e.g., in Drenthe Province).


Figure 17 - Potential routes of spread


∗ European tree-frog - Like the common spadefoot, the European tree frog is procured from the wild, but CMTV-

like virus has not been detected in the areas where European tree frog larvae are procured. Risk of infection

during captivity by cross contamination cannot be excluded. However no cases of CMTV-like virus infection in this

species have been described to date and its susceptibility to CMTV is unknown.

To conclude, there is currently no evidence that CMTV-like virus has been dispersed through legal re-introduction or

translocation projects. However, ranavirus infection risk is currently not being taken into account in the re-introduction

programs. With nationwide exemptions from the Dutch Flora- en fauna Act (FfWet), stock can be legally procured

from areas where CMTV-like virus occurs. Further, under current exemptions, captive parent stock of native species

may be legally imported from other European countries without any disease control. Transferring newly procured

stock directly to outdoor ponds may be good for animal welfare but will hamper early detection of disease and will

make elimination difficult and devastating (previously used individual basins provided a better ‘quarantine’). If not

detected, infection can be translocated to the site of release. Currently, this would imply introduction of the virus into

the provinces of Noord-Brabant and Limburg. These provinces are home to three other amphibian species that are

classified as endangered on the Dutch Red list, one of which is susceptible to CMTV-like virus (the common midwife

toad Alytes obstetricans).

2.3.1.2 Other human-mediated transfer of potentially infected hosts

We hypothesize that besides legal re-introduction and translocations, CMTV-like virus could be spread from an

infected area to another through transfer of infected (pet-trade) specimens between garden ponds. It seems likely

that such transfers occur in the Netherlands on a regular basis, and perhaps also covering greater distances. Further,

for example when garden owners who construct a pond may be impatient for amphibians to reach their pond at their

own pace, and transport egg masses or vegetation from nearby ponds or ditches. With the current data these routes

are hard to quantify.

2.3.1.3 Dispersal by fomites (equipment, transfer of water or sediment)

Transmission of ranaviruses through fomites has been reported for fish14

and amphibians10

. Ranaviruses can remain

infectious for a while in water and sediment. Harp & Petranka13

showed wood frogs (Lithobates sylvaticus) could be

infected by exposure to sediment from a site where a ranavirus die-off was occurring. Ranaviruses have thus the

potential to be dispersed by fomites, as boots, fishing gear, farm equipment and boats, cf. § 1.1.

To conclude, legal re-introduction or translocation projects present a risk. Other routes of spread are poorly

quantified as yet, but represent a certain risk. 2.3.2 Natural spread 2.3.2.1 Dispersal of (sub-)clinically infected hosts

Good dispersers among susceptible host species -In the Netherlands, some of the host species susceptible to

CMTV-like infection are good dispersers, being both commonly occurring species (§ 1.3) and dispersing hundreds of

meters up to 15 kilometres (§ 1.7). These host species are water frogs, common toads and smooth newts. Role

(sub)-clinical infected specimens? - Severely diseased individuals may be reluctant to move and not disperse.

However, this probably does not apply to individuals that are either sub-clinically infected, or in early stages of

disease or recovering. At the sites monitored in Dwingelderveld in 2011, incidental mortality was observed in different

life stages while the remaining individuals appeared healthy 2. Also, during the CMTV-like virus outbreak in Staphorst

in 2012, healthy looking juvenile common spadefoot toads were moving from water to terrestrial habitat, while other

juvenile spadefoot toads at the site were dying. Some of the healthy looking individuals may have been sub-lethally or

sub-clinically infected. Fish - In water bodies with standing water, fish—if they can be infected with CMTV-like virus—

are unlikely to be dispersers in natural spread.

To conclude, short distance dispersal via (sub-)clinically infected hosts appears to be a very likely route of spread.

2.3.2.2 Birds or other animals13- For details see 3.1.2.2.


2.4 High risk areas

A high risk area is an area at risk of being occupied by CMTV-like virus (the infection is currently absent but likely to

establish itself if introduced). As concluded in 2.2, it is likely that CMTV-like virus can spread over the whole of the

Netherlands. This implies the whole of the Netherlands is a high risk area, in habitats where amphibians live.

Zones where threatened species occur are very high risk areas. Based on the current knowledge of ranaviruses

and specifically of CMTV-like virus, very high risk areas on short notice are the sites with the spadefoot and common

midwife (Th.5). Common spadefoot - The outbreak in Staphorst is only 55 km away (as the crow flies) from Valthe.

Here lives the most viable population of spadefoot in the Netherlands of today. From Staphorst it is an even shorter

distance to the population around Zwolle and to the populations along the Overijsselse Vecht87

. In Overijssel several

isolated populations occur, all small and vulnerable for stochastic events. This indicates that especially in the

Northern provinces the spadefoot is potentially threatened by the arrival of CMTV-like virus. Further, due to the ease

by which ranaviruses are likely to be transported (§ 2.1 & 2.3.) over long distances, there is a possibility that more

southern populations of spadefoot in Gelderland, Noord-Brabant and Limburg may be wiped out by the introduction of

CMTV. Common midwife toad - In the eighties of the last century, it was feared that the common midwife toad

would disappear completely from the Netherlands. In order to prevent this over 500 ponds were constructed.

Subsequently a conservation plan88

and an action plan89

were implemented. The species prospered because of this,

but it is still considered ‘vulnerable’ 35

.

Overall conclusion with regards to high risk areas

Given that the broad host range includes common and widespread species, and that types of sites and the

environmental conditions at the sites at which currently CMTV-like ranavirus has been detected are widely present

in the Netherlands, all Dutch water bodies suitable for amphibians, and their surroundings, are high risk areas.

Very risk areas are areas in provinces where threatened species occur. For the common spadefoot these are

located in Drenthe, Overijssel, Noord Brabant, Gelderland, en Limburg province. For the common midwife toad

these are located throughout the Netherlands.

Overall conclusion with regards to pathways of spread

The overall conclusion with regards to pathways of spread is that short to long distance dispersal of CMTV-like via

human-mediated activities, and especially short distance dispersal via (sub-)clinically infected hosts are very likely,

though poorly quantified.


2.5 Impact

2.5.1 Ecological impact Data on the damage occurring in terms of amphibian mortality is largely described in Th.4. Here the focus is on the

probable long term impact if CMTV-like virus establishes itself permanently and spreads through high risk areas.

2.5.1.1 Amphibians

The probable long- term effects on amphibian population sizes, and the likelihood that species could become extinct,

are discussed.

∗ Impact on population size. Other ranaviruses elsewhere – Ranaviruses have the potential to impact

on amphibian populations11

. If ranavirus spreads in wild populations of amphibians it may have severe

consequences on the infected populations of amphibians, other susceptible species and their predators90

. A

major cause of mass mortality in the USA and the UK – In the USA, amphibian mortality events that

occurred in the period 1996 – 2001 were investigated, and it was found that over half of the mortality events were

associated with ranavirus infection and in most of these events ranavirus infection was the sole cause27

. Mortality

was very high and recruitment was consistently negligible or zero. Certain sites monitored showed annually

recurrent mortality. Despite the impact on numbers at the sites, the events were not associated with host species

declines, possibly because all of the events were occurring in fairly widespread and abundant species27

. In the

UK, significant local declines in numbers of adult common frogs have been recorded since the mid-1980s and

associated with ranavirus infection26, 91, 92

. Impact transient, catastrophic or persistent - A study on the

long-term impact of ranaviral disease (FV-3) in the UK showed that common frog populations at sites responded

in three different manners to the emergence of disease: emergence was transient, catastrophic, or persistent with

recurrent mortality events. At sites with transient emergence (16/38, 42%), mortalities were limited to the initial

mass mortality. At sites with catastrophic emergence (4/38, 11%), no frogs were seen at the pond since the

initial ranavirus-associated mortality. At sites with persistent emergence (18/38, 47%), at least one outbreak was

recorded every two years. The populations that experienced these recurring mortality events showed median

declines of 81% in the number of adult frogs, whereas comparable uninfected populations showed no change in

population size over the same time period92

. Also, the larger the initial frog population at site experiencing

persistent emergence was, the greater was the relative decline92

. Possible explanatory factors for these three

different outcomes include differences in virulence or pathogenicity of circulating virus lineages11

and differences

in the immune response of the host populations (§2.2). In general, when populations in affected ponds rebound, it

is not clear whether this reflects the presence of surviving immune animals or re-colonisation by naïve animals93

.

CMTV-like virus in the Netherlands –CMTV-like virus can also cause high mortality. At sites with mass

mortality events, the numbers that died were tens to thousands, and these often included adult specimens (Th.4).

NP Dwingelderveld –In the aftermath of the first detected outbreak, a number of pools and ponds (Th.2) were

visited with regular interval in NP Dwingelderveld, in total on 17 occasions between May and October 2011.

Amphibians were searched along the border of the water body, using a net. It was found that 2.1% of all

encountered amphibians—corresponding to 6.4% of the encountered water frogs—were sick or dead specimen.

Many of the dead specimens were confirmed to be infected and diseased with CMTV-like virus (Th.4, Table 1). In

2012, in the context of this study, the same ponds in NP Dwingelderveld were visited twice (24/8/12 and 4/9/12).

Comparison with data obtained in 2011 was possible for sites A, C, D, E and F (Table 8). The pH at the sites

ranged from 4.65 to 6.77 (mean 5.49 ± 0.37). The closest comparable date with pH measurement in 2011 was

17/8/2011; then it ranged from 4.05 to 5.94 (mean 4.96 ± 0.37). On both days in all ponds amphibians were

encountered. The species encountered were mainly water frogs, but also crested newts, smooth newts and one

common frog and one moor frog. Numbers encountered and proportions found sick or dead were comparable to

2011 (Table 8). IJhorst – The IJhorst was also visited on 24/8/12 and 4/9/12. During the two visits to the garden

pond in IJhorst, no amphibians were observed. The owner said that in 2012 no amphibians were seen at all.

Interpretation – Interpretation is seriously hampered by under-surveillance in all years but 2011. Taking this into

account, and the fact that information on amphibian population size in NP Dwingelderveld prior to the outbreak is

anecdotal, NP Dwingelderveld data could indicate persistent emergence. And the IJhorst may be an example of

catastrophic emergence.


Table 8 – Amphibian host species found sick or dead during monitoring at pools in NP Dwingelderveld on

comparable dates in 2011 and 2012

∗ Host species extinction risk– Density of hosts plays a role in ranavirus infection (§2.2.1.3)12

. Mathematical

models and epidemiological theory predict that pathogens do not drive their hosts to extinction when transmission

is density dependent. The host may suffer from local extinction, but overall the pathogen is lost before the host

species goes extinct94

. However, this does not apply 1) when the pathogen has a multiple hosts some of which

can act as reservoir hosts and in which the pathogen can remain viable, 2) when the pathogen alters population

fitness, i.e., pathogen-driven host selection as may occur in sites with recurrent mortality events, and 3) when

anthropogenic translocation events cause repeated introductions into populations where the pathogen would

normally fade out off94

. These three conditions are met for CMTV-like virus, and therefore in particular host

species classified as vulnerable may be at risk of extinction. Common spadefoot - The spadefoot population in

Staphorst that was infected with CMTV is a relatively large population. In Staphorst in June 2012 hundreds of

dead or dying spadefoot larvae and smooth newts were found. Sick or dead adult spadefoot toads were not

present, which is not odd since this life stage only occurs in the water during a few weeks in spring. The impact of

the mortalities may jeopardize the sustainable survival of this population, because in the Netherlands the species

occurs in only 38 areas, and in most of these there is only a single reproduction pond available. The Staphorst

location was visited again in September and no amphibians were seen.

To conclude, the impact on common amphibian populations is likely to vary between localized extinction,

persistence or recovery. Small populations of endangered species, such as the common spadefoot, may be prone to

(local) extinction.

2.5.1.2 Effects on biodiversity

Amphibians are an important component of the aquatic as well as terrestrial food web. Their loss can influence

predatory birds, macro-fauna, fish and water quality. The latter is because tadpoles and larvae are herbivorous.

Therefore (local) removal of amphibians in an ecosystem is likely to have severe implications for the stability of this

ecosystem. Effects of population declines or extinction of rare species on the local ecosystem – Literature

on the effects of amphibian population declines or extinction of rare species on the structure and function of

ecosystems is uncommon. One study removed tadpoles of two rare species from ponds and examined the effect of

species loss on the local ecosystem. This study found that while amphibians can influence food web dynamics, the

influence of the removal on system processes in temporary forest ponds was limited95

. Another study was a review

that focused on neotropical highland streams, where impacts will likely be greatest. It concluded that there was

evidence for ecological effects of catastrophic amphibian declines: “Amphibian declines will have large-scale and

lasting ecosystem-level effects, including changes in algal community structure and primary production, altered

organic matter dynamics, changes in other consumers such as aquatic insects and riparian predators, and reduced

energy transfers between streams and riparian habitats’96

. The authors also indicated that effects of loss of one

species of amphibian causes effects in aquatic as well as in terrestrial ecosystems due to habitat and functional

differences between larvae and adults in most amphibians species96

. Fish – The risk of CMTV-like virus infection is


not limited to amphibians. CMTV-like virus possibly infected fish at the site of the first outbreak (Th.3.). Elsewhere, an

outbreak involving another ranavirus provided clear evidence that it was affecting both fish and amphibian hosts in

that water body97

. Also, experimentally, other ranaviruses isolated from frogs elsewhere were able to cause infection

in fish14, 98

.

To conclude, the impact of large scale and continuing mortalities on ecosystem stability is likely but still uncertain, as

no studies on this topic have been conducted. The potential infectivity of CMTV-like virus for fish needs to be

determined.

2.5.2 Socio-economic impact 2.5.2.1 Current CMTV-like virus outbreak

The socio-economic impact of the emerging CMTV-like virus in its area of occurrence was not assessed

systematically. However, the outbreaks had direct consequences at the sites used for human recreation (Th.11).

Educational activities for children were suspended at the pond at the visitor centre in NP Dwingelderveld. At

Staphorst the public was distressed by the sight of dead toads and worried if there were any implications for the

health of pet animals and themselves. Physical measures are taken to try to prevent reoccurrence. Research, in

particular surveillance activities and diagnostic tests, have also already cost several hundred thousand Euros.

2.5.2.2 Other

Scientific literature on the financial costs of the emergence of a ranavirus is lacking. Literature data on the societal

costs of the emergence of a ranavirus are also lacking. At the emotional level- The idea that the human use of

amphibian habitat, not only nature reserves, but also ditches and garden ponds, could extirpate species, is a thought

that is likely to cause much distress among people with nature affection. This number is most likely very high

considering the number of people with membership of nature conservations agencies. Mosquitoes- In wetlands it

was shown that mosquitoes avoided laying eggs in habitats which contained larval salamanders and tadpoles. In

addition, the survival in the presence of salamanders was low99

. Hence, the suppression of amphibian populations by

disease most likely affects mosquito populations, which are inconvenient to people (and possibly vectors of disease).

Overall conclusion with regards to impact

A negative effect of emerging CMTV-like virus on amphibian numbers is seen at outbreak sites. Elsewhere, the

effect of emerging ranaviruses on amphibian populations has been transient, catastrophic, or persistent with

recurrent mortality events. Follow-up at some infected sites suggests that this may also be the case for

populations infected by CMTV-like virus. However, due to under-surveillance, no firm conclusions can be drawn.

With regards to risk of extinction of host species, a number of characteristics of CMTV-like virus suggest that it

may cause endangered species to go (locally) extinct. Other effects on biodiversity are very poorly documented.

Other amphibian ranaviruses elsewhere are able to infect fish. It is still unclear CMTV-like virus does. Given the

economic value of fish, more insight into potential consequences for fish species present in the Netherlands is

desirable.


2.6 Risk classification using the ISEIA protocol 2.6.1. Expert consensus scores The risk classifications attributed to the CMTV-like virus for criteria of the ISEIA protocol were determined based on

an evaluation of all available information in this report by a multidisciplinary panel of experts. They were all medium or

high (Table 9). The total risk score attributed to this species was 10 out of a maximum risk score of 12. This score

results in an overall classification to category B, indicating a moderate environmental risk on the basis of current

knowledge.

Table 9 – Risk classifications attributed to CMTV-like virus

2.6.2 Dispersion potential or invasiveness Classification – High risk. Currently, the CMTV-like virus appears to be locally present in a restricted range and is

already locally invasive in the Netherlands (Th.2 and §2.2). Ranaviruses show a high replication ability after infection

of host cells (Th.1). Clinically infected amphibians were found in urbanized areas, rural areas as well as remote areas

(Th.9). It is probable that sub-lethal and sub-clinical infections occur (§2.2.3.2). Introduction to remote areas will most

likely be restricted to human-assisted spread or passive dispersion via infested amphibians and/or eventually via

other animals (§2.1). This includes spread via waterfowl or other animals carrying mud from one water body to

another or predators of infected amphibians that vomit elsewhere their stomach content. However, documented

evidence for dispersal of CMTV-like virus and other ranaviruses elsewhere via other animals than amphibians is still

lacking (§2.1.2.2 and §2.3.2.2).

There is multiple evidence that the CMTV-like virus can easily disperse through the above mentioned means over

distances > 1 km per year. Firstly, clinical (and so possibly sub-clinical) infection is recorded in half of the native

amphibian species and several susceptible species are known to disperse > 1 km per year and even up to 15 km per

year (Th.3 and Th.7). In addition, available data over the year 2011 show that the CMTV-like virus disease reports

were clustered close to the location of first record in 2010, NP Dwingelderveld, and that in 2012 the new site

Staphorst was located at a distance of 7 km south of the nearest site known to have infected amphibians in 2011,

namely IJhorst (Th.2). This spread of the virus may be caused by passive dispersion via natural means and/or

multiple human assisted introductions. Retrospective proving of causality between certain vectors and the emergence

of this viral disease in this area is no longer possible because empirical data are lacking.

2.6.3 Colonisation of high conservation value habitats Classification – Medium risk. This criterion addresses the potential of the CMTV-like virus to spread and to

cause viral diseases in habitats with high conservation values (irrespective of its dispersal capacities). According to

the ISEIA protocol, habitats with a high conservation value are those where disturbance by man is minimal, thus

allowing specific natural communities and threatened native species to occur, such as listed in the European Union

Habitats Directive.

The available data for the Netherlands show that clinical (and possibly sub-clinical) infections of amphibians with

CMTV-like virus have been recorded in several water bodies in one area with high conservation values: i.e., NP

Dwingelderveld. This nature reserve contributes to the Dutch ecological main structure (EHS) and is designated as

Natura 2000 area according to the European Habitats Directive and Birds Directive100

. Relevant habitat types for

international classification of this area are acid moorland pools (H3160), peat bogs (H7110B and H7120) and water

ISEIA section Consensus score Risk classification

Dispersion potential or invasiveness 3 High risk

Colonization of high conservation value habitats 2 Medium risk

Adverse impacts on native species 3 High risk

Alteration of ecosystem functions 2 Medium risk

Global environmental risk 10 B-list category


bodies with pioneer vegetation (H7150). These habitat types are very important for biodiversity conservation,

including several threatened amphibian species. For the great crested newt (Triturus cristatus, H1166), an Annex II

protected species of the European Union Habitats Directive, there are species conservation goals explicitly

mentioned for this Natura 2000 area101

.

In addition, spread and emerging of disease has also been recorded at several sites with a low to medium

conservation value (Th.2). Although these water bodies lack a formal conservation status, some of them largely

contribute to habitat for endangered amphibian species in the Netherlands, such as the common spadefoot.

So, it can be concluded that the current spread and adverse impacts of the CMTV-like virus are confined to sites with

a low or medium conservation value and are recorded occasionally in high conservation value habitats, resulting in a

medium risk score. It should be explicitly noted that available data on current spread and effects of this virus in the

Netherlands allow multiple interpretations. We interpreted viral infections of amphibians in several water bodies within

one Natura 2000 area as occasionally occurring and not as often occurring in high conservation value habitats.

2.6.4 Adverse impacts on native species Classification – High risk. Several risk criteria of the ISEIA protocol related to this section are not straightforward

applicable in risk assessments of exotic viruses. This section mainly addresses the potential of the CMTV-like virus to

cause effects on amphibians.

The CMTV-like virus shows a large host range, among which several common amphibian species and some

endangered species, such as the common spadefoot toad and common midwife toad. Mortality at infected sites

suggests that the endangered great crested newt may also be a host for the virus, but this is still unconfirmed. In the

Netherlands, clinical infections and mass mortality are documented for all life stages of amphibian species. Effects on

species of other taxonomical groups, such as reptiles and fish, cannot be excluded based on evidence from abroad

(§2.5.1).

The potential risk of regional extinction of endangered amphibian species in Netherlands is relatively high due to

small and isolated populations of such species that are sensitive to the CMTV-like virus. Some common amphibian

species with high dispersal ability are also sensitive to the virus and sub-clinical infected individuals could easily

spread this virus.

A study on the long-term impact of ranaviral disease in the United Kingdom showed that populations of the common

frog showed different responses to introduction of a closely related virus (FV-3-like virus). The disease was transient,

catastrophic or persistent with recurrent mortality events. Declines of more than 80% in the number of adult frogs and

reduction of genetic variability were reported at sites where disease persisted92, 102

. There was no sign of recovery of

the common frog population over the ensuing five years. Other affected species included common toads, midwife

toads and natterjack (Bufo calamita). In contrast, pool frogs were scarcely affected.

Taking into account the high negative impact of the CMTV-like virus on amphibian species observed at several

locations in the Netherlands as well as severe effects of this virus and closely related viruses on amphibian

populations in other countries in temperate regions, potential risks causing severe population declines of amphibians

and the reduction of local species richness is therefore highly likely in the Netherlands. At a regional scale, the virus

can be considered as a factor precipitating endangered amphibian species decline, resulting in a high risk

classification in this category: adverse impact on native species.

2.6.5 Alteration of ecosystem functions Classification – Likely, medium risk. Risk classification within the section alteration of ecosystem functions

should be based on four sub-criteria: 1) Modification of nutrient cycling or resource pools; 2) Physical modifications of

habitat; 3) Modification to natural succession; and 4) Disruption to food webs. Viruses can not directly alter these

ecosystem functions, but may cause indirect effects in case of strong declines of their host populations. This impact

category is only poorly documented, therefore according to the ISEIA protocol, an adapted risk scoring system based

on expert judgment has been applied. Available literature and data on effects of CMTV-like or related viruses focuses

only on documenting and identifying causes of disease, mass mortality or population declines of amphibians.


Documented evidence for indirect alteration of ecosystem functioning observed within the Netherlands, countries with

a similar climate (e.g., Belgium, United Kingdom and Denmark) or other climate regions (e.g., Spain) is lacking. The

scoring system is adapted to scores 1 = unlikely (low risk) or 2 = likely (medium risk).

Amphibians are known to play a number of important roles in functioning of ecosystems96

. Evidence for alteration of

ecosystem functions presented here originates from studies quantifying impacts of strong amphibian population

declines by other types of environmental deterioration. Ongoing research in Central America, where declines of

amphibian populations are occurring in streams, suggests that these losses will have wide-ranging consequences for

ecosystem functioning. Such effects will most likely be large-scale and lasting including changes in algal community

structure and primary production, altered organic matter dynamics, changes in other consumers such as aquatic

insects and riparian predators, and reduced energy transfers between streams and riparian habitats96

.

Larval mosquitoes were less abundant in pools with higher densities of larval salamanders in studied ponds in the

USA99

. In addition, experiments on mosquito oviposition and survival showed that mosquitoes avoided ovipositing in

habitats containing larval salamanders and tadpoles and had low survival in the presence of salamanders. These

data indicate that predation by larval salamanders may influence the breeding distribution of mosquitoes by imposing

selective pressure on ovipositing adults. It was concluded that measures to protect amphibians may contribute to

controlling mosquito production in wetlands, potentially minimizing disease risk to humans99

.

It was considered that similar impacts on ecosystem functioning as described above could also occur in the

Netherlands, because the CMTV-like virus is also expected to cause strong declines of local populations (>80%) of

several sensitive amphibian species (Section 2.6.4). Therefore, expert judgement resulted in a risk score of 2: likely

for this impact parameter.

2.6.6 Risk classification Classification – B2, watch list. According to the Belgian Forum on Invasive Species (BFIS) list system, the risk

classification of a non-native species corresponds to the consensus score (ISEIA, 2009; Table 8) combined with the

current distribution within the country in question.

The risk classification of the CMTV-like virus for the current situation in the Netherlands is B2. This indicates a non-

native species with a restricted distribution range and moderate environmental hazard (i.e., ecological risk) that

should be placed on a watch list (Figure 18).

Figure 18 - CMTV-like virus risk classification for the current situation, according to the BFIS list system.


A re-grading of this risk classification (Table 10) will be required in case of future situations where no management

interventions follow to prevent introductions of CMTV-like virus in the Netherlands and secondary spread within the

country. Moreover, climate change may cause habitat alterations.

In case of absence of management interventions, the CMTV-like virus is expected to distribute wider. Spread and

emerging of disease in other areas with high conservation values (Natura 2000; EHS: Dutch ecological main

structure) can potentially occur if the virus was accessed by multiple vectors. Such vectors include visitors of nature

areas with mud on foot wear or sampling equipment, legal or illegal (re-)introductions of amphibians, dumping of

terrarium animals, mud on water fowl and natural dispersal of clinical and subclinical infested amphibians.

According to climate change scenarios developed by the Dutch Royal Meteorological Institute the average air

temperature in the Netherlands potentially will increase during winter by 0.9-2.3 oC and 1.8-4.6

oC over the years

1990-2050 and 1990-2100103

. The average summer temperature may increase over these periods with 0.9-2.8 oC

and 1.7-5.6 oC, respectively. As air and water temperature of shallow water bodies are strongly correlated (Th.10),

temperature of breeding and nursery sites of amphibians are also expected to increase over the coming decades

during winter as well as summer periods. This may affect multiple virus, host and environment-related factors and

their interactions. Based on the contemporary body of knowledge it is not possible to predict whether these climate-

related changes will enhance or impede establishment. Suitable replication temperatures for other ranaviruses lie in

between 12-32°C (Th. 1). Hypothetically, water temperatures, shown to be a few degrees higher than ambient air

temperatures (Th.10), may be warm enough for virus replication earlier and later in the season, while not becoming

too high. On the other hand, higher temperatures during winter and more freezing and thawing events may have a

negative effect on virus survival outside of the host (Th.1; §2.2). Scenarios could be examined using epidemiological

models. For example temperature optimal for CMTV-like virus replication is unknown. Also, it is not determined how

important virus survival in the environment in winter is for persistent emergence compared to for instance sub-

clinically infected hosts (§2.2).

The adverse effects of the CMTV-like virus on native amphibian species in the current situation already resulted in a

high risk classification. A wider distribution of this virus in the future scenario without management intervention will

likely result in a further increase of adverse impacts on sensitive species at a larger spatial scale and can be

considered as a factor precipitating regional species decline. Scientific documentation of alterations of ecosystem

functions by declines of amphibian populations is not available for the Netherlands and countries in similar or other

climate zones, leading to assessment of this risk criterion by expert judgement (§ 2.6.5). Alterations of ecosystem

functions in a future situation are judged to be likely at a regional scale due to the increase of adverse impacts on

populations of sensitive amphibian species.

Table 10 - Theoretical classification CMTV-like virus according to potential future situation without management measures.

Therefore, the future scenario shows a potential increase in the invasion of the CMTV-like virus resulting in a

widespread distribution and viral disease of amphibians in a significantly increasing number of high conservation

value habitats, resulting in highly adverse direct and indirect impacts on biodiversity and alterations of ecosystem

functions at a wider spatial scale. In particular, the expected increase in number of high conservation value habitats

with adverse effects would lead to an increase in the total risk score by one point according to the ISEIA protocol

(Table 10). Combined with the widespread distribution this will result in a reclassification to a higher environmental

risk (A3 - Black list classification).

ISEIA section Consensus score Risk classification

Dispersion potential or invasiveness 3 High risk

Colonization of high conservation value habitats 3 High risk

Adverse impacts on native species 3 High risk

Alteration of ecosystem functions 2 Likely, medium risk

Global environmental risk 11 A-list category


Part 3 Risk management and communication

Major assumptions underlie the risk management and communication proposals. The most important assumption is

that CMTV-like virus does not occur widespread in the Netherlands as of now. This is based on the focal distribution

of the 2011 outbreaks, for which no particular triggering factors could be found. We also assume that CMTV-like virus

in the Netherlands is a single virus, and there are no other ranaviruses. More surveillance including at non-outbreak

sites and virus sequencing would confirm or infirm these assumptions.

Most likely the economic costs of restoring a disrupted ecosystem are higher than the costs of preventive or curative

measures.

3.1. Prevention of introduction

Measures to prevent the introduction of CMTV-like virus from abroad into areas in the Netherlands that are presumed

naïve were elaborated based on risk assessment results (§2.1). Note that many measures will be useful also with

regards to import risk of other pathogens106

. Priority, feasibility and an indication of costs of measures were based on

expert judgment (Table 11).

3.1.1 Make sure that CMTV-like virus can be detected in imported captive specimens at the border

3.1.1.1 Obtain sufficient sight on amphibians imported

Currently there is no good sight on numbers, species, origin and destination of amphibians imported: a) Quantitative

data on amphibian trade (legal or illegal) is lacking. b) Traceability of imported specimens is poor. c) There is

significant captive-breeding of species exotic to the Netherlands for trade or sales within Europe and elsewhere. This

study made clear that to prevent the introduction of CMTV-like virus via traded amphibians and possibly other

poikilothermic hosts, more information is needed on (illegal) trade, the pet industry and international exchange of

animals among hobbyists.

∗ Obtain good real-time sight on amphibians imported from outside the EU into Europe and the

Netherlands in particular (numbers, species, origin, destination).

∗ Obtain better sight on amphibians imported from within the E.U. into the Netherlands via animal

dealers and via hobbyists (volume, species, origin, destination).

3.1.1.2 Improve the capacity to detect CMTV-like virus in imported specimens at the border

Currently health status monitoring opportunities of imported and traded specimens are poor: a) Disease detection

during import is unlikely. b) Wild caught specimens of unknown health status are regularly introduced into facilities

where captive-bred species are kept. c) Captive-bred exotic amphibian trade is elusive in terms of diseases occurring

in the facilities concerned. This requires improved capacity for detecting CMTV-like virus at the border. Wildlife trade

is a multibillion dollar industry107

and stakeholders should be involved in the process in order to ensure effectiveness

of chosen preventive measures107

.

∗ Increase the knowledge and awareness about host species that can be infected with CMTV-like

virus - Imported specimens of species belonging to the amphibian families Alytidae, Bufonidae, Dendrobatidae,

Pelobatidae, Ranidae, Salamandridae should be considered as possible hosts of CMTV-like virus80, 109

. The

situation regarding fish is at the moment unclear (Th.3). It is very likely that amphibian species from other families

are also susceptible, because the virus effectively caused disease in nearly all the amphibian species found in the

NP-Dwingelderveld and the surrounding area. The virus was also capable of infecting captive Dendrobatidae,

which are frogs native to a completely different continent, South America. Therefore this knowledge needs to be

updated continuously.

∗ Increase the knowledge and awareness about the range of occurrence of CMTV-like virus – The

global distribution of CMTV-like virus is unknown. Therefore, additional data is required to assess whether CMTV-

like virus is a European virus or originates from some other continent (Th.2). International exchange of information


on ranavirus disease should help to answer this question and help to identify areas which may present a risk that

the virus is introduced together with imported animals. Such information should be made widely available.

∗ Increase the capacity to detect and confirm cases at import - Diagnosis is usually based on a number of

findings (see textbox). Suspect clinical cases can be detected by veterinary authorities, traders and hobbyists.

Information on clinical signs and possibilities for laboratory testing including postmortem analysis should be given.

Development of a rapid detection test for use in the field would be beneficial. Further education about biosecurity

measures, proper disposal of dead animals and disinfection of the environment should be made readily available.

∗ Quarantine - Quarantine periods required to detect ranavirus may differ per species but should cover the length

of incubation period105

. The incubation period of CMTV-like virus is unknown but other ranaviruses have

incubation periods that can be as long as a month86, 105

. To be effective the quarantine period has therefore to be

quite long, which has the cost consequences. Water temperature during quarantine would need to be defined,

because it affects the duration of the incubation period and clinical manifestation104

. An alternative to quarantine is

the requirement to restrict the procurement of specimens from certified CMTV-like virus–free collections. There

exist no certification schemes for amphibians, therefore generally accepted principles should be followed and

adapted accordingly).

Diagnosis of CMTV-like virus

1. Clinical signs – Possible clinical signs include hemorrhages, edema, erythema, erratic swimming

behavior in larvae, lethargy, and sudden death1, 3, 6, 23

. These together with the numbers of dead

animals and the involvement of multiple life stages in outbreaks are suggestive but not pathognomonic

of CMTV-like infection1. In other words, such clinical signs can also be seen in other diseases and are

not specific enough for diagnosis.

2. Post-mortem examination and histopathology – Microscopy includes the examination of formalin-

fixed, paraffin-embedded and H&E stained tissue sections. Lesions include foci of necrosis in liver,

kidney, spleen, pancreas, gastro-intestinal tract or skin or several of these, associated with round

intracytoplasmic basophilic inclusions3, 6, 23

. Histopathological examination in combination with

laboratory tests allows distinction between infected specimens and specimens contaminated by the

environment.

3. Laboratory tests - Methods used in diagnosis of CMTV-like infection have been virus isolation in

cell culture and PCR with sequencing of products3, 6, 7, 25

. Organs of choice in dead animals are

liver, spleen and kidney. PCR tests on tissues provide information on the presence of genetic material

of the virus in organs. In live animals, tail clips and swabs collected from the cloaca or oral cavity can

be taken. Swabs will only be positive if the virus is present in mucosal epithelium, saliva, faeces or

urine. Real-time PCR was observed to be more sensitive than conventional PCR110

. Cytopathogenic

effects in cell culture can be used to confirm the virus is infective. Iridoviral particles can be detected by

electron microscopy (Th.1) 3, 6

. Immunohistochemistry has been performed, and is useful for

understanding pathogenesis23

. For other ranaviruses enzyme-linked immunosorbent assays (ELISA)

have been described, in particular antigen-capture ELISA for EHNV104

. Sensitivity and specificity of

tests are to be taken into account in interpretation. Note: There are official reference laboratories for

fish ranaviruses.

4. Limitations – Detection level - In particular in sub-lethally infected specimens, virus levels may be

below detectable levels by virus isolation, an observation made for ATV in tiger salamanders9. PRC,

generally considered very sensitive, was not as sensitive as virus isolation in one ranavirus study. In

vivo examination - PCR tests on tail clips (ante-mortem) underestimate the true prevalence of ATV

infection in the first days of infection, but increase with days after exposure111

.


3.1.2 Reduce the risk that CMTV-like virus enters nature via imported kept amphibians or their waste

3.1.2.1 Obtain good sight on amphibian and vivarium waste disposal behavior

Currently there is lack of data on amphibian and vivarium waste disposal behavior. Behavior of the general public

that increases the likelihood of the disease being introduced into nature must be identified. This is required to

determine the most risky behavior, and so to provide relevant practical advice.

3.1.2.2. Make sure that people are aware of risky behavior and know how to minimize it

It is important that people are aware that the virus can be introduced into nature through the release or disposal of

(sub-)clinically infected amphibians or their waste material, and that they are provided with information on how to

minimize this risk.

∗ Provide education for the public

∗ Identify possible technical solutions for minimizing infection via contaminated water and other

vivarium waste products

3.1.3 Make sure that CMTV-like virus imported via natural dispersal can be detected early on at its site of introduction, before it occurs widespread.

3.1.3.1 Obtain a better understanding of the relative importance of introduction via natural

dispersal

Prevention of introduction of CMTV-like virus from abroad through natural routes may be difficult, if amphibians, birds

and predators effectively and regularly introduce it. To date, no information indicates that this is the case. However

this situation may evolve.

∗ Consolidate international cooperation within Europe with regards to ranavirus infections - for updates

on CMTV-like virus occurrence, and ranavirus surveillance in general.

∗ Further investigate the role of (migrating) birds and predators in ranavirus transmission - for a better

understanding of long distance introduction through natural routes.

3.1.3.2. Ensure monitoring for early detection and source tracing

The aim is to detect introduction through nature at an early stage, so that there may be a possibility to eliminate the

virus before it becomes widespread.

∗ Ensure monitoring of unusual mortality in amphibians in the Netherlands - The aim is to detect

introduction through nature at an early stage, so that there may be a possibility to eliminate the virus before it

becomes widespread. Source tracing at sites of new introduction will improve introduction prevention measures.

∗ Establish monitoring for CMTV-like virus near Hoogstraten - There is a known local risk near the Belgian

border. The CMTV-like virus is found there in the American bullfrog, an exotic species, for which the Dutch policy

is removal from nature. The risk for the Netherlands is due to the likelihood of inter-species transmission, with

major risk for threatened species located in the south of the Netherlands. Monitoring of CMTV-like infection in

amphibians at the Dutch border is relevant. In addition, there is a need for close international cooperation to fully

understand the issue and assess its relative importance.


Table 11 – Measures to prevent the introduction of CMTV-like virus from abroad into Dutch nature. Priority, feasibility and costs are based on expert judgment only.

3.1 Options for prevention Priority Feasibility Costs

3.1.1 Make sure that CMTV-like virus can be detected in imported captive

specimens at the border

3.1.1.1 Obtain sufficient sight on amphibians imported

* Obtain good real-time sight on amphibians imported into the

Netherlands via animal dealers (numbers, species, origin, destination)

High Fair. Likely to be

long-term

Low, once put in place

* Obtain better sight on amphibians imported into the Netherlands via

hobbyists (volume, species, origin, destination)

High Fair Moderate

3.1.1.2 Improve the capacity to detect CMTV-like virus in imported

specimens at the border

* Increase the knowledge and awareness about host species that can be

infected with CMTV-like virus

High Good Low, basis laid in this

report, regular

updating needed

* Increase the knowledge and awareness about the range of occurrence

of CMTV-like virus

Moderate Fair, if international

cooperation and

funding is achieved

Moderate

* Increase the capacity to detect and confirm cases at import Very high Good Low-high

* Quarantine High Moderate Quarantine costs.

Pass on to consumer?

3.1.2 Reduce the risk that CMTV-like virus enters nature via in imported

captive amphibians or their waste

3.1.2.1 Obtain good sight on amphibian and vivarium waste disposal

behavior

High Good Moderate

3.1.2.2 Make sure that people are aware of risky behavior, and know

how to minimize this

* Provide education to the public High Moderate, needs to

be continuous

Moderate,

continuous

* Identify the possible technical solutions for minimizing infection via

contaminated water and other vivarium waste products

High Good Pass on to consumer

3.1.3 Make sure that CMTV-like virus imported via natural dispersal can be

detected early on at its site of introduction, before it occurs

widespread.

3.1.3.1 Obtain a better understanding of the relative importance of

introduction via natural dispersal

* Consolidate international cooperation within Europe with regards to

ranavirus infections

High Good Low

* Further investigate the role of (migrating) birds and predators in

ranavirus transmission

Low Good Moderate

3.1.3.2 Ensure monitoring for early detection and source tracing

* Ensure monitoring of unusual mortality in amphibians in the

Netherlands

Very high Good Moderate

* Establish monitoring for CMTV-like virus near Hoogstraten Very high Good Moderate


3.2 Elimination

This section deals with the current knowledge on the possibilities to eliminate CMTV-like virus infection from (small)

populations, i.e., measures to prevent establishment of the virus. These possibilities were elaborated based on risk

assessment results (§2.2) and literature. Priority, feasibility and an indication of costs of measures were based on

expert judgment (Table 12).

3.2.1 Assisted elimination – currently only applies to captive populations and small free-living populations Assisted elimination is elimination that is achieved by measures undertaken by humans. Distinction is made between

captive populations (such as stock of endangered species raised for re-introduction into the wild) and free-living

populations.

3.2.1.1 Encourage destocking and disinfection for elimination of infection in captive settings

In captive populations infected with CMTV-like virus the aim is to eliminate infection, to avoid establishment and

spread. The most secure option is destocking and disinfection of premises, similar to what has been recommended

for the elimination of other ranaviruses in captive facilities105

. The rationale behind destocking is to eliminate infected

specimens, including those sub-clinically infected, and the rationale behind disinfection is to inactivate virus present in

the environment of these infected hosts (see §2.2.3.). For disinfectants used on contaminated equipment and

containers, see section §3.3.

∗ Ensure destocking is done humanely, timely and thoroughly. Good information and conditions for

humane destruction need to be available. Euthanized stock should be properly disposed of (e.g., burning, or

buried and limed105

). Further, current knowledge suggests apparently healthy specimens could be sub-clinically

infected and therefore, until further notice, destocking of these too is recommended.

3.2.1.2 Explore options for elimination in the case of (small) infected free-living populations

Acceptable elimination measures for use in water bodies are virtually unidentified. If the infected population is small,

elimination of CMTV-like infection may be preferable over control. Elimination could be considered for infected

populations in small man-made water bodies such as garden ponds, or local populations at sites where CMTV-like

virus has been newly introduced and rapidly detected, before becoming widespread (cf. 3.1.3.2.).

* Ensure new areas to which the virus has spread are detected as early as possible – cf. 3.1.3.

* Provide help with the decision to opt for elimination or not in natural settings, and advice for best

practice - The decision to opt for elimination or control needs to be timely and informed. Guidelines could contain

criteria to aid decision-making and practical advice for best practice.

* Make sure the steps to be taken to obtain permission to destock are well-known to the public and

permissions are given in a timely fashion - Permission to destock is likely to be necessary, and the process

should be outlined and facilitated for timely action.

* Investigate the technical alternatives for achieving virus elimination from natural sites and their

side-effects; Encourage registration of disinfectants for inactivation of ranavirus in the environment

at Ctgb - Elimination of CMTV-like virus in free-living populations is difficult. Measures as destocking,

disinfection and physical alteration of sites have consequences for other flora and fauna, and are not always

effective. The problem with destocking in natural settings is that amphibians are mobile and full destocking may

not be achieved. The problem with disinfectants in natural settings is that they do not generally work in presence

of organic material, such a pond mud. Further, none has been registered for such use at Ctgb (‘College voor

toelating gewasbeschermingsmiddelen en biociden’), and it is very unlikely companies will invest in registration

because there is no profit to be made. For other ranaviruses, there is some experimental evidence that virus is

inactivated when water bodies dry up10

. There may also be other physicochemical ways than desiccation to

inactivate virus (UV, ozone, cf. Th.1) that possibly lead to elimination of virus from the site. Measures applicable to

wild settings may come from measures developed for the amphibian trade107

.

* Make sure that effect of water body management interventions on the occurrence of CMTV-like

virus is properly monitored - Experience with measures that could eliminate CMTV-like virus from free-living

populations is very limited:


- To the best of our knowledge, destocking and disinfection were put to practice only once with the aim of

eliminating CMTV-like virus from a site with a free-living population. This was at the first site where CMTV-like

virus was detected in Spain (2007). Here tadpoles were euthanized, and the site was disinfected with sodium

hypochlorite (bleach, cf. § 3.3.). The site was a permanent drinking trough, so it was small and man-made (cf.

photo in Th.9). CMTV-like virus infection has not occurred at this site again (Balseiro A., pers. comm. 2012).

However, the next year (2008), infected amphibians were found in a natural pool in close proximity to the first

site. It is unclear if this was due to spread from the first site prior to elimination efforts, or introduction from a

different source. This second site could not be disinfected (cf. photo in Th.9). The site was monitored for

occurrence of CMTV-like virus infections in amphibians in the subsequent years but the infection has not been

detected again (Balseiro A., pers. comm. 2012), indicating the virus did not establish itself in the second site.

Taken together, the usefulness of the destocking and disinfection at the first site remains unknown.

- In Belgium, there were efforts to remove American bullfrogs from nature because they are invasive exotic

species. The efforts were not specifically directed towards eliminating the CMTV-like virus infected populations

and the effect of American bullfrog removal on the local occurrence of CMTV-like virus infection was not

monitored. Therefore it is unknown whether the efforts to remove of American bullfrogs from the environment

were effective for clearing CMTV-like infection from the area or not.

- In the Netherlands two water bodies underwent physical modifications for other reasons. The pond in Wijster

(outbreak in 2011) underwent complete renovation in the spring 2012 because it leaked, and no dead

amphibians were reported in the summer of 2012. At Staphorst (outbreak in 2012), soil of the pond was dug

out and overturned and some of it was moved a short distance in the autumn of 2012. This is done every 3 to

5 years because it is a swimming pond. The effect on the occurrence of CMTV-like virus infection at Staphorst

is still unknown.

Because there is so little experience with eliminating CMTV-like virus from natural sites, effects of such

interventions on the presence of CMTV-like virus should be monitored, even if they not primarily undertaken for

elimination.

3.2.2 Natural elimination–learn from it for larger scale assisted elimination Natural elimination is elimination that is achieved by natural processes in free-living populations. The water bodies

used by amphibians are often not connected to other surface water bodies, and in the case of another ranavirus,

FV3, emergence of infection was transient in 42% of the ponds investigated92

. Understanding more of the

mechanisms and the factors involved in natural elimination of infection from sites may provide information on how to

assist elimination of infection in large areas and populations.

* Establish long-term monitoring for understanding the factors that contribute to transient emergence

at sites - CMTV-like virus is (at least) present in the Netherlands in an area of 25 km diameter (ca. 500 km2), and

highly likely to establish itself and spread without intervention (§2.6.). The area is too large to consider elimination

on short term, but—assuming transient emergence occurs at some sites—it does provide an opportunity to learn

about natural elimination processes. A better understanding of mechanisms and factors causing CMTV-like virus

disappearance rather than maintenance could be gained by long term monitoring of outbreak sites and their

surroundings. More insight into the role of different amphibian species and the role of other classes (fish, reptile,

invertebrate) in the epidemiology would be gained.

* Translate to acceptable eradication measures / environmentally friendly biosecurity measures for

use in water bodies.


Table 12 – Options and recommendations for elimination of CMTV-like virus infection in captive and free-living populations. Priority, feasibility and costs are based on expert judgment only.

3.2 Options and recommendations for elimination Priority Feasibility Costs

3.2.1 Assisted elimination - currently only applies to captive populations and

small free-living populations

3.2.1.1 Encourage destocking and disinfection for elimination of

infection in captive settings.

* Ensure destruction and disinfection can be done humanely, timely and

thouroughly. It is important that persons confronted with CMTV-like virus

infections in captive settings have ready access to guidelines for humane

destocking and good disinfection.

Very high Good Low (excluding the

possible value of

destocked

animals)

3.2.1.2 Explore options for elimination in the case of (small) infected

free-living populations and monitor the effect of interventions.

* Ensure new areas to which the virus has spread are detected as early

as possible

cf. 3.1.3

* Provide help with the decision to opt for elimination or not in natural

settings, and advice for best practice.

High Moderate Low

* Make sure the steps to be taken to obtain permission to destock are

well-known to public and speedy.

High Good Low

* Investigate the technical alternatives for achieving virus elimination from

natural sites and their side-effects.

Medium If economic.

Industry?

Low to high.

* Encourage registration of desinfectants for inactivation of ranavirus in

the environment at Ctgb.

High Low Moderate, for

industry

* Make sure that effect of water body management interventions on the

occurrence of CMTV-like virus is properly monitored. Even if such

measures are not primarily designed to eliminate CMTV-like virus, they

may provide valuable leads for elimination.

Very high Fair Low

3.2.2 Natural elimination - learn from it for larger scale assisted elimination

* Establish long-term monitoring for understanding the factors that

contribute to transient emergence at sites.

High Long-term Moderate

* Translate to acceptable eradication measures / environmentally friendly

biosecurity measures for use in water bodies.

Medium If economic.

Industry?

Low to high.


3.3 Control

This section deals with the current knowledge on the possibilities to control CMTV-like virus infection, i.e., measures

to prevent spread to new areas and diminish the impact on amphibian populations. They were elaborated based on

risk assessment results (§2.3-§2.5) and literature. Priority, feasibility and an indication of costs of measures were

based on expert judgment (Table 13).

3.3.1 Prevent human mediated spread of virus within the Netherlands Human mediated spread of virus within the Netherlands is highly likely without intervention. This can be by

translocation infected animals or through fomites such as landing nets.

3.3.1.1 Prevent spread to new areas via re-introduction projects

Officially authorized re-introduction and translocation activities concern threatened amphibian species. These re-

introduction activities do not yet fully take into account the occurrence of CMTV-like virus infection in the Netherlands

and elsewhere in Europe, while the impact of an introduction of CMTV-like virus via such activities into the destination

site could undo the intended effect of these activities, or worse. Re-introduction projects are implemented in steps,

and disease risk can be minimized in each step. For example, procuring stock from wild populations in the zone

known to harbor CMTV-like virus infections in amphibians is best avoided. And quarantine is advisable for new stock

(cf. 3.1.1.2.), as is the use of separate equipment for each tank. Biologists performing re-introduction and

translocation activities should obtain veterinary advice for disease prevention during the entire project and take action

accordingly. If infection occurs despite the preventive measures, elimination of the infected population is advisable

(cf. 3.2.1.1).

3.3.1.2 Prevent inadvertent spread to new sites by public or field biologists

∗ Increase awareness of the public (see 3.1.2.2.) - The public is often not aware of the risk of spreading

infection by moving (sub-clinically) infected specimens, and needs to be educated with regards to this (see

3.1.2.2.; Figure 19 left). This is particularly relevant at sites known to be infected.

∗ Increase awareness of field biologists - Field biologists are an important group to target when it comes to the

risk of dispersal by fomites112

.

∗ Ensure disinfecting in the field is made practical - Disinfection protocols for ranaviruses are based mostly

on the work of Langdon (1989)14

, Miocevic et al. (1993)20

and Bryan et al. (2009)21

. Based on these Phillot et

al.(2010)112

provided an overview of disinfectants to use for ranavirus disinfection (Table 14). Information on

hygiene protocols is already provided by RAVON, but field application can be improved. It would be preferable

that all products recommended are officially registered for such use at the Ctgb (cf. also 3.2.1.2; Figure 19, right).

For compliance to protocols and effectiveness, the application should be practical.

3.3.2 Explore the possibility to take advantage of natural barriers to limit natural dispersal Based on current knowledge, dispersal by (sub-clinically) infected amphibians is highly likely. Further,dispersal by

birds or other animals (vectors) may be possible.

* Obtain a better understanding of the relative importance of the species in natural dispersal - Which

species are important herein and at which rate? (cf. 3.1.3.1.)

* Identify possible natural barriers - Absolute containment of wild animals in an area is not possible. Also,

current conservation efforts are directed towards linking populations of wild animals to improve their fitness. On

the other hand, the fact that effort is put into infrastructure to link populations also means there are barriers in our

landscapes and land uses that can limit natural dispersal by amphibians. Temporary use could be made of them.

* Explore the benefits and downsides of containment

3.3.3 Try to avoid further impact on threatened species and high conservation value habitats All habitats where amphibians live in the Netherlands are at risk of being infected, including high conservation value

habitats (§2.4.). The virus represents a threat for host species on the Dutch Red list. Vaccination of threatened


species is not an option at this stage. There are no vaccines available for CMTV-like virus or other ranaviruses104, 105

,

and chemotherapy is not available104

. *

* Prevent spread to areas where threatened species are found (see 3.1, 3.3.1 and 3.3.2.) - Particular

focus should be on preventing sites where threatened species are found from becoming infected (e.g., the South

of the Netherlands).

* Gain additional information on the risk for threatened species that occur in the current area of

occurrence - Additional information should be gained on the impact of CMTV-like virus on threatened species.

Not only for species already known to be susceptible, but also for other threatened poikilothermic species present

in the area of occurrence of CMTV-like virus, such as the amphibian predating grass snake Natrix natrix.

3.3.4 Make sure the correct data is collected to be able to predict the effectiveness of control and elimination measures Epidemiological models are very useful to properly assess the effectiveness of control and elimination measures. The

quantitative parameters for the disease modelling lack partially. The information in this document can be used to

define which data is missing to make such models, so that the collection of such data could then be a priority if the

control and elimination are undertaken.

Figure 19– Left, Poster board warning the public not to remove amphibians from the infected site C in NP Dwingelderveld. Right - Disinfection of wetsuit and equipment.


Table 13 – Control options, measures to prevent spread to new areas and diminish the impact on (threatened) amphibian populations. Priority, feasibility and costs are based on expert judgment only.

Table 14 – Overview by Phillot et al.(2010)110

of disinfectants to use for inactivate ranavirus.

3.3 Options and recommendations for control Priority Feasibility Costs

3.3.1 Prevent human mediated spread of virus within the Netherlands

3.3.1.1 Prevent spread to new areas via re-introduction projects High Good Low

3.2.1.2 Prevent inadvertent spread to new sites by public or field

biologists

* Increase awareness of the public (see 3.1.2.2) Very high Moderate, needs to

be continuous

Moderate,

continuous

* Increase awareness of field biologists Very high Moderate, requires

change in behaviour

Low, continuous

* Ensure disinfecting in the field is made practical Very high Good Moderate

3.3.2 Explore the possibility to take advantage of natural barriers to limit

natural dispersal

* Obtain a better understanding of the relative importance of the species

in natural dispersal

Low Fair Moderate

* Identify possible natural barriers Low Fair Moderate

* Explore the benefits and downsides of containment Low Fair Moderate

3.3.3 Try to avoid further impact on threatened species and high

conservation value habitats

* Prevent spread to areas where threatened species are found

* Gain additional information on the risk for threatened species that occur

in the current area of occurrence

High Fair Moderate

3.3.4 Make sure the correct data is collected to be able to predict the

effectiveness of control and elimination measures

Very high Good Low

see 3.1, 3.3.1 and 3.3.2


Glossary

Biosecurity measures a set of preventive measures designed to reduce the risk of transmission of infectious

disease.

CMTV-like virus common midwife toad virus (CMTV)3 and viruses with partial major capsid genes showing

99.8% to 100 % sequence homology (GenBank accession no. FM213466.1)25

.

Co-evolution the evolution of two or more interdependent species, each adapting to changes in the

other.

Cross-protection the protection against infection by a pathogen given to the host by its prior infection with a

related pathogen

Density-dependent the likelihood of transmission increases with increasing host density.

Hazard a biological, chemical or physical agent in, or a condition of, an animal or animal product

with the potential to cause an adverse health effect113

.

Hazard identification the process of identifying the pathogenic agents which could potentially be introduced in

the commodity considered for importation113

.

Infectivity the ability to produce infection

Pathogen an agent of disease (microorganism capable of causing disease)

Pathogenicity the ability of a pathogen to cause disease in a host organism

Pathognomonic a specifically distinctive or characteristic of a disease or pathologic condition; denoting a

sign or symptom on which a diagnosis can be made.

Phenology the study of recurring phenomena (patterns of animal activity), such as animal migration,

especially as influenced by climatic conditions.

Ranavirus a virus belonging to the Ranavirus genus

Risk the likelihood of the occurrence and the likely magnitude of the biological and economic

consequences of an adverse event or effect to animal or human health113

.

Risk analysis the process composed of hazard identification, risk assessment, risk management and risk

communication113

.

Risk assessment the evaluation of the likelihood and the biological, economic and public health

consequences of entry, establishment and spread of a hazard within the territory of an

importing country113.

Risk communication the interactive transmission and exchange of information and opinions throughout the risk

analysis process concerning risk, risk-related factors and risk perceptions among risk

assessors, risk managers, risk communicators, the general public and other interested

parties113

.

Virulence the ability (of a pathogen) to overcome bodily defenses (of a host); the ability to cause

rapid, severe and destructive disease. In the narrow sense: the increased mortality rate in

infected individuals86


List of abbreviations

ATV Ambystoma tigrum virus

BIV Bohle irido virus

bp base pairs

BFIS Belgian Forum on Invasive species

CMTV Common midwife toad virus

Ctgb College voor toelating gewasbeschermingsmiddelen en biociden

DWHC Dutch Wildlife Health Centre

ECV European catfish virus

EHS Ecological Main Structure

EHNV Epizootic haematopoietic necrosis virus

EU European Union

FAVV Federal Agency for the Safety of the Food Chain (Belgium)

FfWet Flora- and fauna Act

FV3 FV3

IATA International Aviation Transport Association

ISEIA Invasive Species Environmental Impact Assessment

NVWA Nederlandse Voedsel en Waren Autoriteit

NP National park

OIE World Organization for Animal Health

RAVON Reptile, Amphibian and Fish Conservation Netherlands

RU Radboud University Nijmegen

SCRV Santee-Cooper ranavirus

Th. Theme


Acknowledgements

We would like to thank the NVWA for financial support of this study and Trix Rietveld for critical comments on an

earlier version of the report. Many persons contributed to this study. In particular we would like to acknowledge Lenie

Algra-Verkerk, Fons van Asten, Ana Balsiero, Roschong Boonyarittichaikij, Wilbert Bosman, Przemyslaw Busse,

Raymond Creemers, Ben Crombaghs, Jeroen van Delft, Anjolieke Dertien, Rinus Gerlofsma, Xavier Harduin, Jelger

Herder, Robert Jooris, Marc Schils, Ger van der Sluijs, Marisca Stege, Raymond Tilburg, Matt Whiles, Peter

Wohlsein, Maarten Zeylmans van Emmichoven. Last but not least we would like to acknowledge the owners or the

managers of the sites where outbreaks occurred in the Netherlands, and Dick Willems, without whom CMTV-like

virus may have gone undetected for a long time.

Photos

Title page – Joran Janse

Electron microscopy picture – Peter Wohlsein

Water bodies Spain – Ana Balsiero

Water body and disturbance Staphorst - Marc Schils

Thermometer, poster board – Jolianne Rijks

Disinfection of wetsuits and equipment – Annemarieke Spitzen-van-der-Sluijs

All other pictures - Jelger Herder, www.digitalnature.org

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Contributors

The consortium conducting this risk analysis consisted of four partners, three from the Netherlands and one from

Belgium. A list of contact details per organization is provided in Annex 1. Dutch Wildlife Health Centre DWHC

Expertise - The purpose of the Dutch Wildlife Health Centre (DWHC) is to enhance knowledge and expertise in

wildlife health in the Netherlands. This serves to provide scientifically based information for political and practical

decisions concerning public health, wild and domestic animal health, and nature conservation issues. The first

outbreak of ranavirus in free-living amphibians was detected through the general wildlife disease surveillance that is

one of the main activities of the DWHC. Subsequently, and together with RAVON, DWHC conducted the 2011

ranavirus surveillance project. Nature of contribution - Within this study, DWHC coordinated the activities of the

partners including final reporting. Focus was on CMTV-like virus infection occurring in amphibians. DWHC performed

a literature review in order to obtain an overview of CMTV-like virus, potential hosts, and potential environmental

factors. In addition, DWHC used data obtained from earlier and ongoing CMTV-like virus cases and studies. DWHC

obtained information from official sources, to try to be able to quantify risks. This included import data from Schiphol

airport with regards to amphibian species known to be susceptible to CMTV-like virus infection and information on

officially permitted translocations of potential hosts. DWHC participated in the risk assessment score workshop.

DWHC assessed possible detection methods with regard to amphibian hosts, and defined possible risk management

options. DWHC had contact with the pathologist experts in the UK. The experts involved:

∗ Prof. Dr. Andrea Gröne, DVM, Dipl. ECVP, Dipl. ACVP is a veterinary pathologist, the Director of the

DWHC and the Head of the Division Pathology in the Department of Pathobiology at the University of

Utrecht. She supervises DWHC activities and provided input in the final reporting.

∗ Dr. Marja Kik DVM, Dipl. Vet Path RNVA, Dipl. ECZM (Herpetology) is a staff veterinary pathologist working

in pathology of exotic animals and wildlife and is an expert in ranavirus-induced disease. She was involved

as pathologist in the 2011 ranavirus surveillance project.

∗ Dr. Jolianne Rijks, DVM, works at DWHC as an epidemiologist. She studied veterinary medicine and

obtained her PhD degree with research on the 2002 phocine distemper virus epidemic in seals. She has (co-

)authored 7 peer-reviewed papers on wildlife related topics. She has prepared and managed the 2011

ranavirus surveillance project on behalf of DWHC, and coordinated the present study. RAVON

Expertise - RAVON stands for Reptile, Amphibian and Fish Conservation Netherlands. The RAVON Foundation is a

non governmental organization (NGO) with 1800 volunteers, more than 1000 contributors and 28 professional staff

members with offices in Nijmegen and Amsterdam. Organization members inform and educate, conserve species

and their habitats, organize two national monitoring networks and collect distribution data for the conservation of

reptiles, amphibians and fish in the Netherlands. RAVON staff also gives advice and carries out research for a wide

range of clients. RAVON and DWHC have worked together on the ranavirus. Nature of contribution - RAVON

contributed in providing the host species related data relevant to natural pathways, establishment possibilities and

likelihood of dispersal for CMTV-like virus. RAVON provided information on the host species, vector species and

affected species necessary to define risk areas. RAVON provided data collected in 2011 in the National Park

Dwingelderveld, supplemented by data collected during two field visits in 2012 (August and September), to assess

the impact of CMTV-like virus on host populations and to define the ecological characteristics of the described

outbreaks, with special emphasis on defining the future scenarios that may occur. In this RAVON and RU

collaborated closely. RAVON contributed to define the ecological, economic and social consequences of ranavirus

outbreaks and participated in the risk assessment score workshop. The experts involved:

∗ Ir. A. M. Spitzen – van der Sluijs is project leader and professional staff member at RAVON. She is also a

PhD student at Ghent University under the supervision of professor Frank Pasmans and professor An

Martel, where she’s studying the impact of B. dendrobatidis on native amphibian species in relation to

environmental conditions. She has (co-)authored 9 peer-reviewed papers on diverse topics, and her main

interests are the effects of invasive pathogens on amphibian communities, the resistance of landscape

matrices on dispersal possibilities of reptiles and amphibians, and the auto-ecology of the legless lizard,

Anguis fragilis. She has conceived managed the 2011 ranavirus surveillance project on behalf of RAVON.

∗ Drs. R. Zollinger, team leader and manager/researcher at Research and Conservation department of

RAVON. He is a biologist (terrestrial ecology, population dynamics, nature conservation), published several


papers on biology of amphibians, birds and nature conservation topics. Involved in several interdisciplinary

projects and is active member of the European Herpetological Society. Radboud University Nijmegen

Expertise - The research focus of the Institute for Water and Wetland Research at the Radboud University Nijmegen

(RU) is the natural environment, in particular aquatic ecosystems and wetlands. The Department of Environmental

Science was founded in 1991. It focuses on understanding and predicting biological responses to physical and

chemical pressures. Nature of contribution - If there were specific environment related factors (e.g., climate, water

quality parameters) affecting the probability of establishment and spread, RU analyzed whether and in which water

systems these occur, and how these match now and in the future with species. RU assisted RAVON with defining the

ecological, economic and social consequences of CMTV-like virus outbreaks, supervising and warranting the quality

of the impact assessment. RU prepared, presided and wrote down the results of the risk assessment score

workshop. The experts involved:

∗ Dr. Rob S.E.W. Leuven (1957) studied biology (aquatic ecology, animal physiology, fisheries &

aquaculture). His PhD-thesis concerned the impacts of acidification on the biodiversity and functioning of

aquatic ecosystems. He was project leader of several large research projects commissioned by the World

Bank, European Commission, the Netherlands Organization for Scientific Research and several Dutch

governmental and non-governmental organizations. He successfully supervised 10 PhD-projects and

(co)authored more than 210 papers (among which 90 ISI-WOS articles), 18 scientific books and 50

professional reports. His main research interest is understanding the impact of multiple stressors on

biodiversity and functioning of aquatic ecosystems (rivers, floodplains, moorland pools and peat bogs).

Recent research focuses on effects of exotic invasive species on aquatic ecosystems. Special attention is

paid to the invasion process (including dispersal patterns, causes of establishment success, physiological

tolerances and biological traits of invaders), risk assessment of aquatic invasions, effects of climate change

on global redistribution of species, ecological and socio-economical impacts and invasion management.

∗ Laura N.H. Verbrugge (MSc) studied Environmental Sciences at Utrecht University (BSc) and Radboud

University Nijmegen (MSc - science communication track). At present she is working as a researcher on a

joined PhD project of the Institute for Science, Innovation and Society (ISIS) and the Institute of Water and

Wetland Research (IWWR), both stationed at the Radboud University. Her research activities include a

broad spectrum of topics concerning non-native species including (ecological) risks, public perception and

preventive measures and communication campaigns. She had published several papers and reports on risk

assessment of non-native species and has gained experience in moderating risk assessment workshops

held in the Netherlands. Currently she is also involved in the evaluation of the Dutch code of conduct for

invasive aquatic plants, a voluntary agreement between the government and horticulture sector with the

objective prevent further introductions of invasive species by increasing public awareness.

6.4. Faculty of Veterinary Medicine, Ghent University, Belgium

Expertise - The research group “Amphibian and reptile diseases” is hosted by the Division of Avian and Exotic

Animals (An Martel) and the Laboratory of Veterinary Bacteriology and Mycology (Frank Pasmans) at the Department

of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Belgium (UGent).

This research group focuses on amphibian diseases in the wild and captivity, has conducted several studies on

ranavirus infection (see reference list) and was involved in the diagnosis of ranavirus in the two ranavirus associated

mortality events in the Netherlands. Nature of contribution - The main contribution of UGent to this study was to

make an inventory of trade in amphibians in Belgium and from Belgium to the Netherlands, and an inventory of

privately kept amphibians in Belgium and their possible exchange to the Netherlands. The experts involved:

∗ Prof. Dr. An Martel, DVM, is the Head of the Division of Avian and Exotic Animals at the Department of

Pathology, Bacteriology and Avian Diseases at the Faculty. Her research focus is infectious diseases in

birds, reptiles and amphibians. She currently supervises 5 PhD students working on infectious diseases in

amphibians. She has been promoter of 4 PhD theses and (co-)authored more than 80 ISI listed publications.

She recently described a novel pathogen (the genus Amphibiichlamydia) in amphibians.

∗ Prof. Dr. Frank Pasmans, DVM, is the Director of the Laboratory of Veterinary Bacteriology and Mycology.

He has a lifelong interest in reptiles and amphibians, one of his major research topics being reptile and

amphibian diseases, with emphasis on host pathogen interactions. He has been promoter of 19 defended

PhD theses and (co-)authored 200 ISI listed publications.
