Amphibians are currently experiencing the great-est decline of all vertebrate taxa (Stuart et al 2004)and one of the major causes of decline is the chytridfungus Batrachochytrium dendrobatidis (Bd) the path -ogenic agent of amphibian chytridiomycosis (Sker-ratt et al 2007) Bd infects the keratinized tissue ofamphibian skin where it disrupts oxygen ion andwater transport and can lead to cardiac arrest andmortality (Voyles et al 2009)
Bd can infect a wide range of amphibian spe -cies though susceptibility to chytridiomycosis variesgreatly among species For example in Xenopus lae-vis Rana catesbeiana (Lithobates catesbeianus) andR pipiens Bd infection generally does not cause dis-ease symptoms (Parker et al 2002 Schloegel et al2010 Chatfield et al 2013) Researchers have begunto investigate the potential for non-amphibian taxa toact as alternative hosts which might carry and trans-mit Bd infection Several non-amphibian specieshave been demonstrated to harbor Bd including
Batrachochytrium dendrobatidis in natural andfarmed Louisiana crayfish populations prevalence
and implications
Laura A Brannelly1 Taegan A McMahon2 Mitchell Hinton3 Daniel Lenger4 Corinne L Richards-Zawacki4
1One Health Research Group College of Medical and Veterinary Sciences James Cook University Townsville Queensland Australia
2Department of Biology University of Tampa Tampa Florida USA3Department of Wildlife Fish and Conservation Biology University of California at Davis Davis California USA
4Department of Ecology and Evolutionary Biology Tulane University New Orleans Louisiana USA
ABSTRACT The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) has been linkedto global declines and extinctions of amphibians making it one of the most devastating wildlifepathogens known Understanding the factors that affect disease dynamics in this system is criticalfor mitigating infection and protecting threatened species Crayfish are hosts of this pathogen andcan transmit Bd to amphibians Because they co-occur with susceptible amphibian communitiescrayfish may be important alternative hosts for Bd Understanding the prevalence and seasonaldynamics of crayfish infections is of agricultural and ecological interest in areas where crayfish arefarmed and traded for human consumption We conducted a survey of Bd in farmed and naturalcrayfish (Procambarus spp) populations in Louisiana USA We found that Bd prevalence andinfection intensity was low in both farmed and native populations and that prevalence varied sea-sonally in wild Louisiana crayfish This seasonal pattern mirrors that seen in local amphibians Ascrayfish are an important globally traded freshwater taxon even with low prevalence they couldbe an important vector in the spread of Bd
KEY WORDS Alternative hosts middot Aquaculture middot Batrachochytrium dendrobatidis middot Enzootic middot Invasive species middot Procambarus clarkii
Resale or republication not permitted without written consent of the publisher
This authors personal copy may not be publicly or systematically copied or distributed or posted on the Open Web except with written permission of the copyright holder(s) It may be distributed to interested individuals on request
Dis Aquat Org 112 229ndash235 2015
Anolis lizards (Kilburn et al 2011) 3 species of snake(Kilburn et al 2011) Caenorhabditis elegans nema-todes (under laboratory conditions Shapard et al2012) and wading birds (Garmyn et al 2012) How-ever none of these studies have demonstrated theability of the pathogen to complete its life cycle on orin these non-amphibian taxa
Only 1 study to date has demonstrated that Bd cancomplete its life cycle in a non-amphibian host andtransmit Bd infection to amphibians McMahon et al(2013) found that crayfish (Procambarus spp andOrconectes spp) can be Bd infected in natural popu-lations can carry Bd for 3 mo in the laboratory andcan transmit infection to tadpoles under laboratoryconditions Crayfish exposed to Bd under laboratoryconditions also suffered gill damage and mortalitysuggesting that Bd may pose a health threat to thesecommercially important animals as well McMahonet al (2013) suggest that crayfish may be important inthe Bdminusamphibian disease dynamic because pres-ence of crayfish correlates positively with the pres-ence of Bd in amphibian communities crayfish pres-ence is a better predictor of Bd presence than thepresence of the North American bullfrog R cates-beiana a purported reservoir species The NorthAmerican bullfrog has been suggested as a diseasereservoir because it can harbor large Bd loads anddoes not usually develop symptoms of the diseasechytridiomycosis (Schloegel et al 2010)
Procambarus spp are common inthe southeastern USA and are one ofthe most widely traded freshwatertaxa globally with the majority of indi-viduals coming from Louisiana USA(Holdich 1993) The crayfish marketcontributes over $150 million annuallyto Louisianarsquos economy and over125 000 acres of land are devoted tofarming crayfish (McClain amp Romaire2007) With so much land devoted toaquaculture many amphibians comein close contact with farm populationsof crayfish Therefore farmed crayfishmay impact the Bd infection dynamicsof natural populations of the crayfishand amphibians with which they co-occur Additionally P clarkii is tradedinternationally and could be a vectorfor Bd spread globally
We conducted a seasonal field sur-vey for Bd infection prevalence in bothnatural and farmed populations ofnative crayfish species in Louisiana
Loui siana was chosen for this study because thestatersquos farms supply a large proportion of the globalcrayfish market (Holdich 1993 McClain amp Romaire2007) and natural crayfish populations are wide-spread and abundant permitting a comparison ofpathogen pre valence and load between farmed andwild animals Furthermore data on the prevalenceand seasonality of Bd in amphibian populationsexists for this region (Brannelly et al 2012) but simi-lar metrics for infection in co-occurring natural andfarmed crayfish populations are not available Aclearer understanding of the prevalence intensityand seasonality of Bd in crayfish is needed to betterunderstand the potential impact of Bd on crayfishpopulations as well as the importance of crayfish asan alternative host for this pathogen with potentialwidespread ramifications through the global crayfishtrade
MATERIALS AND METHODS
Crayfish collection
Natural populations
Crayfish Procambarus spp were collected fromsoutheastern Louisiana (Maurepas Wildlife Manage-ment Area n = 129 and Tulane Universityrsquos F
230
D
E
C
B A
N
40 km
Fig 1 Southeastern Louisiana showing sampling locations for this study Letters correspond with site names in Table 1 (A) Tulane Universityrsquos FEdward Herbert Research Center in Belle Chase (natural) (B) MaurepasWildlife Management Area (natural) (C) Lafayette (farm) (D) Morgan City
(farm) and (E) Belle River (farm)
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Brannelly et al Chytrid fungal infection in crayfish
Edward Herbert Research Center in Belle Chase n =142 Fig 1 and see Table 1) Collection occurred inthe spring (February to April) and fall (Septemberand November) of 2012 by sweep netting and usingbaited minnow traps Each crayfish was removedfrom the net or trap individually using a clean plasticbag and transported in the sealed bag to Tulane Uni-versity There crayfish were euthanized by freezingat minus20degC for a minimum of 2 h The crayfish sampledin this study were not sampled for any other study
Farmed populations
P clarkii were collected live from restaurants in theNew Orleans Louisiana metropolitan area in Febru-ary and April of 2012 These crayfish were suppliedby farms located in Morgan City Lafayette and BelleRiver Louisiana (Fig 1 and see Table 1) and from1 other farm of unknown location within the stateEach restaurant provided 20 to 22 crayfish (total n =82) Animals were held together in large mesh bagswith no water in a 4 to 10degC refrigerator at eachrestaurant for a maximum of 3 d after delivery fromthe farms and before we procured them As the ani-mals were kept communally before we procuredthem there was the potential for cross-contamina-tion When we received the animals each individualwas removed from the communal mesh bag using aclean inverted plastic bag which was then sealed fortransportation to Tulane University Animals wereeuthanized by freezing at minus20degC for a minimum of2 h We were not able to collect farmed crayfish in thefall because the artificial ponds used to farm theseanimals are drained during this season
Testing for Bd infection
Crayfish were thawed completely before process-ing The inside of the gastrointestinal (GI) tract ofeach animal was swabbed using a sterile MW113swab (Medical Wire and Equipment) and Bd loadwas quantified using a real-time polymerase chainreaction (qPCR analysis see lsquoDNA extraction andanalysisrsquo) The crayfish GI tract was chosen for patho-gen load analysis because the fungus is known toinfect the GI tract by implanting in the intestinal wall(McMahon et al 2013) While McMahon et al (2013)also found Bd on crayfish carapace the histopathol-ogy of carapace infection is unknown therefore forthe purpose of this study we chose to focus our sam-pling effort on the inside of the GI tract A positive
qPCR result indicates the presence of Bd DNA in thesample but cannot differentiate the presence of tran-sient DNA from an active infection of the GI tract tis-sue This would require histological examinationwhich was outside the scope of this study HoweverMcMahon et al (2013) previously demonstrated us -ing histopathological examinations of the GI tractthat crayfish of the same genus can become infectedwith Bd and can carry that infection for an extendedperiod of time While we cannot be certain that theindividuals that tested positive for Bd in our studyhad active infections (as opposed to harboring tran-sient Bd DNA due to ingestion of contaminated mate-rial) in either scenario the presence of Bd couldhave important implications for understanding therole of crayfish in the spread and dynamics of Bdinfection in amphibians As long as the pathogenremains viable on passage through the crayfish di -gestive system ingestion of Bd by crayfish destinedfor trade could permit transmission of the pathogento new areas even in the absence of an active GIinfection McMahon et al (2013) showed transmis-sion to amphibians from crayfish that were internallyinfected under laboratory conditions which suggeststhat Bd exiting the crayfish digestive system is viable
To access the GI tract the abdomen of each carcasswas separated from the thorax and the uropod wasseparated from the tail and slowly pulled away fromthe body with the GI tract still attached The fecalmatter inside the GI tract causes PCR inhibition so itwas removed with a sterile swab (MW113) Then theinside of the GI tract was swabbed using 30 strokeswith a second sterile MW113 swab GI swabs werekept frozen at minus20degC in 15 ml microtubes until qPCRanalysis The dissected animal was then placed in anew plastic bag and re-frozen Gloves were changedbetween handling the external surface of each cray-fish and the internal GI tract and between each newindividual
DNA extraction and analysis
To test for the presence of Bd we extractedgenomic DNA from the GI swabs using the QiagenDNeasy Blood and Tissue Kit We followed the man-ufacturersquos instructions for animal tissue using a finalelution volume of 200 microl Once extracted qPCR(Applied Biosystems 7500) was used to detect thequantity of Bd on each swab qPCR analysis was car-ried out following the protocol described by Boyle etal (2004) except that we added 07 microl of bovineserum albumin to each qPCR reaction to minimize
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Dis Aquat Org 112 229ndash235 2015
inhibition and all wells contai ned an internal positivecontrol (VICtrade IPC Applied Biosystems) Positiveand negative controls previously extracted fromswabs of known Bd-positive and Bd-negative captiveRana catesbeiana and a dilution series of Bd stan-dards (provided by A Hyatt) were included in eachqPCR run Samples were run in singlicate (Kriger etal 2006) and DNA was not diluted prior to analysis
Statistical analysis
Microsoft Excel was used to calculate 95 confi-dence intervals on the proportion of Bd-positive indi-
viduals following Newcombe (1998) Zoosporeequivalents (ZE) in the GI tract in Bd-positive indi-viduals were compared between farmed and naturalpopulations using a 2-tailed t-test in SPSS (v21)
RESULTS
Bd prevalence in Louisiana crayfish was seasonalwith higher prevalence in farmed and wild-caughtanimals in the spring than in wild-caught animals inthe fall (Fig 2 Table 1) This pattern mirrors seasonaltrends in Bd prevalence for amphibian species in theregion some of which were sampled from the samesites as our wild-caught crayfish samples (Fig 2modified from Brannelly et al 2012) Farmed crayfishtested positive for Bd infection in the spring (Febru-ary and April 2012) with a prevalence of 60 (load1074 plusmn 396 (SD) ZE per swab n = 5) and no farmedanimals were available for testing in the fall Bd waspresent in natural crayfish populations in the springwith a prevalence of 331 (load 2217 plusmn 815 ZE perswab n = 9) but no Bd was detected from those samesites in the fall Farmed crayfish had a significantlylower ZE than wild-caught animals (t-test t12 = 2914p = 0013)
DISCUSSION
The seasonal pattern of Bd prevalence in wild-caught Louisiana crayfish in 2012 mirrored that seenin amphibian species sampled in the same region in2010 and 2011 with higher prevalence during thespring breeding season and no infection detectedduring the fall season Prevalences were lower in the
232
000
010
020
030
040
050
060
Pro
por
tion
infe
cted
Janu
ary
Febr
uary
M
arch
Apr
il M
ay
June
Ju
ly Aug
ust
Septe
mbe
r Oct
ober
Nov
embe
r Dec
embe
r
Fig 2 Proportion of Batrachochytrium dendrobatidis (Bd)-positive crayfish sampled in 2012 in southeastern Louisiana(d) Wild-caught and (d) farm-collected animals The pro-portion of (s with dashed error bars) Bd-infected amphi -bians in southeastern Loui siana sampled in 2010 to 2011is also shown modified from Brannelly et al (2012 their
Fig 2) The error bars are 95 confidence intervals
Site Latitude Longitude Month No Bd + ind Average zoospore Site type(degN) (degW) total sampled load (plusmnSD)
A 2989 89953 February 6 35 2167 plusmn 1147 NaturalMarch 0 62April 0 40
September 0 5B 30108 90435 April 3 52 1354 plusmn 618 Natural
November 0 77C 30214 9203 April 3 22 1126 plusmn 549 FarmD 29701 91206 April 0 22 FarmE 29888 91206 April 2 20 479 plusmn 662 FarmF Unknown February 0 20 Farm
Table 1 Sampling of natural and farmed crayfish Sites are (A) Tulane Universityrsquos F Edward Herbert Research Center inBelle Chase (natural) (B) Maurepas Wildlife Management Area (natural) (C) Lafayette (farm) (D) Morgan City (farm) and (E)Belle River (farm) and (F) unknown location in Louisiana (farm) Bd Batrachochytrium dendrobatidis See Fig 1 for sampling
locations in Louisiana USA
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Brannelly et al Chytrid fungal infection in crayfish
crayfish than in the amphibians sampled in a givenmonth (Fig 2) However since crayfish and amphi -bians were sampled on different dates and in differ-ent years and in some cases also from different sitescaution should be used in comparing prevalence esti-mates between these 2 taxa In this study we did notfind Bd-infected crayfish in September (05 Bd+95 CI = 0 to 435 prevalence) or November (077Bd+ 95 CI = 0 to 48 prevalence) of 2012 butMcMahon et al (2013) found Bd at 173 prevalencein Louisiana crayfish in September of 2011 Our fail-ure to detect Bd in the fall could be due to yearly vari-ation in disease prevalence or an artefact of our smallsample size early in the fall Although our study didnot support the idea that crayfish act as disease reser-voirs during the late summer or fall months we diddetect Bd in crayfish during the spring and thereforecannot rule out the idea that these animals may playan important role in disease dynamics Summer tem-peratures in Louisiana typically exceed the thermaltolerance of Bd and amphibians may clear theirinfections during this time It is possible that crayfishare also able to clear Bd infections during high sum-mer temperatures but more research is needed totest this prediction
Farmed and wild-caught crayfish Bd prevalenceswere similar to each other in the spring when farmedcrayfish were available (Fig 2) It remains unclearwhether the effects of Bd on crayfish health shouldbe an important consideration for farmers Bd infec-tion has been shown to cause mortality in laboratory-exposed crayfish (McMahon et al 2013) but itsimpact on farmed and natural crayfish populationsremains unstudied While we did not examine ani-mals for pathology we did find that farmed crayfishhad significantly lower Bd loads than wild-caughtanimals The different conditions these animals ex -perienced directly prior to sampling could also havecontributed to the difference in pathogen load Wild-caught animals were separated upon capture andkept alive only for transport between the field andthe laboratory while the farmed crayfish were keptalive communally in a refrigerated room for up to 3 dbetween capture and sampling However beforeconcluding that farmed animals have lower patho-gen loads than natural populations a sample of cray-fish directly from the farms analogous to our naturalpopulation sampling methods is needed While com-munal refrigerated conditions might have impactedour Bd load results we found that farmed animalsheld in analogous conditions to those destined fortrade were positive for Bd at a similar prevalence towild-caught animals Six percent of the farmed indi-
viduals left the farm and were sold for consumptionwhile infected with Bd
Minimizing disease transmission between wildlifeand farmed animals is important and this topic hasbeen extensively studied in domesticated animalsglobally Wild animals can carry disease and infectindividuals in farmed populations which is particu-larly concerning for livestock and farm owners (egMycobacterium bovis Delahay et al 2001) Farmedanimals can also introduce spread and maintaininfection in natural populations which can be de -trimental to vulnerable species living near farms(eg chickens introducing avian malaria into nativeHawaiian bird populations Warner 1968) Bothfarmed and natural crayfish populations in south-eastern Louisiana were Bd positive in the springwhich is the time when they commonly share habi-tats with amphibians Crayfish infected with Bd inclose proximity with amphibian populations mayplay a role in amphibian disease dynamics espe-cially if infected crayfish are transported to areas thatwere previously Bd free
Our study found that farmed crayfish were Bdpositive during the trading season If farmed cray-fish that are globally traded are infected with Bdthen even a low prevalence of infection could haveimplications for global amphibian conservation Theinternational crayfish market is large with 40 000 to60 000 t traded per annum (Holdich 1993) whichequates to approximately 08 to 12 billion indivi -duals The North American species Procambarusclarkii makes up 85 of all crayfish trade (Holdich1993) Most of these globally traded crayfish origi-nate from farms in Louisiana (Holdich 1993) Cray-fish are the most widely introduced freshwatertaxon globally (Helms et al 2013) likely becomingestablished after escaping from farms (Gherardi2006) as farms rarely exercise biosecurity measuresto ensure isolation from natural populations Addi-tionally P clarkii is a highly successful invasivespecies once it escapes its native range It can re -produce by par thenogenesis can survive in ephe -meral ponds by burrowing deep into mud banks intimes of drought (which may help Bd persist) andtends to escape from farms and travel quickly tolocal ponds where it then reproduces (Gherardi2006) There is already unequivocal evidence thatthe invasive P clarkii has driven amphi bian declinethrough predation rather than pathogen spread inmany regions of Europe (Cruz et al 2006ab Cruz2008 Ficetola et al 2012) Although there is a clearcorrelation between amphi bian de clines and inva-sion by P clarkii in Europe the potential relation-
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Dis Aquat Org 112 229ndash235 2015
ship between amphibian declines crayfish invasionand disease has not been explored
Trade of diseased individuals whether it be amphi -bians or other aquatic organisms might explain thespread of the Bd pathogen globally (Fisher amp Garner2007 Garner et al 2009 Schloegel et al 2009 Kolbyet al 2014) While the origin of Bd is still unclear itappears that strains of Bd found in parts of Europeare most closely related to strains found in the east-ern USA (Rosenblum et al 2013) which could beexplained by the import of invasive North Americanspecies like Rana catesbeiana and P clarkii for con-sumer purposes Spain is one place where the role ofcrayfish as an important alternative host for Bd de -serves further study as this country has been experi-encing some of the greatest amphibian declines inEurope (Gherardi et al 2001 Garner et al 2006) Thedeclines have been attributed to both amphibian pre-dation by the invasive P clarkii as well as chytrid-iomycosis (Gherardi et al 2001 Garner et al 2006)P clarkii were introduced into Spain twice for thepurposes of farming for consumption from farms inLouisiana between 1973 and 1974 (Hasburgo-Lorena1986) Since then crayfish have spread into Portugaland other parts of Europe either through importationfrom Spain or migration out of farms It is possiblethat Bd was introduced into the area through im -ported crayfish and subsequently spread to naturalamphibian populations Although amphibian tradehas been implicated in the global spread of Bd(Fisher amp Garner 2007 Garner et al 2009 Schloegelet al 2009) even regions devoid of commercial amphi -bian importation have been exposed to this patho-gen For example Bd has recently been de tected forthe first time in amphibians coming from Madagas-car (Kolby 2014) where North American Procam-barus spp have been introduced into the wild andare now rapidly spreading (Jones et al 2009) Whilethe correlation between amphibian disease and cray-fish presence has not been studied in EuropeanMalagasy or other ecosystems our study suggeststhat farmed P clarkii can carry Bd and that createsthe risk of zoonotic spill-over if appropriate controlmeasures are not taken
Bd disease dynamics are challenging to understandand predict and when we consider non-amphi bianhosts the story becomes increasingly complex Thereare current biosecurity protocols enacted globally toreduce disease spread through amphibian trade andcontaminated field equipment (Green et al 2009Murray et al 2011) but little attention has beendirected towards the prevention of Bd spread by non-amphibian hosts In this study we found that both
natural and farmed crayfish populations carry Bdinfection in Louisiana and that infection is seasonalin wild-caught animals similar to the pattern of pre -valence seen in amphibian populations in the sameregion While prevalence in our study was low cray-fish species may still be an important part of the disease dynamic especially because they are mass-distributed globally We would argue that a 60Bd infection prevalence in a species that is as widelytraded as P clarkii represents a significant risk anddeserves to be considered in biosecurity measures
Acknowledgements We thank Matthew Robak for helprunning the qPCR analysis Matthew Chatfield for help inthe laboratory and the field and Jonathan Kolby for com-menting on the manuscript
LITERATURE CITED
Boyle DG Boyle DB Olsen V Morgan JAT Hyatt AD (2004)Rapid quantitative detection of chytridiomycosis (Batra-chochytrium dendrobatidis) in amphibian samples usingreal-time Taqman PCR assay Dis Aquat Org 60 141minus148
Brannelly LA Chatfield MWH Richards-Zawacki CL (2012)Field and laboratory studies of the susceptibility of thegreen treefrog (Hyla cinerea) to Batrachochytrium den-drobatidis infection PLoS ONE 7 e38473
Chatfield MWH Brannelly LA Robak MJ Freeborn L Lail-vaux SP Richards-Zawacki CL (2013) Fitness conse-quences of infection by Batrachochytrium dendrobatidisin northern leopard frogs (Lithobates pipiens) EcoHealth10 90minus98
Cruz J (2008) Collapse of the amphibian community of thePaul do Boquilobo Natural Reserve (central Portugal)after the arrival of the exotic American crayfish HerpetolJ 18 197minus204
Cruz MJ Pascoal S Tejedo M Rebelo R (2006a) Predationby an exotic crayfish Procambarus clarkii on natterjacktoad Bufo calamita embryos its role on the exclusion ofthis amphibian from its breeding ponds Copeia 2006 274minus280
Cruz MJ Rebelo R Crespo EG (2006b) Effects of an intro-duced crayfish Procambarus clarkii on the distributionof south-western Iberian amphibians in their breedinghabitats Ecography 29 329minus338
Delahay RJ Cheeseman CL Clifton-Hadley RS (2001)Wildlife disease reservoirs the epidemiology of Myco -bacterium bovis infection in the European badger (Melesmeles) and other British mammals Tuberculosis (Edinb)81 43minus49
Ficetola GF Siesa ME De Bernardi F Padoa-Schioppa E(2012) Complex impact of an invasive crayfish on fresh-water food webs Biodivers Conserv 21 2641minus2651
Fisher MC Garner TWJ (2007) The relationship betweenthe emergence of Batrachochytrium dendrobatidis theinternational trade in amphibians and introduced amphi -bian species Fungal Biol Rev 21 2minus9
Garmyn A Van Rooij P Pasmans F Hellebuyck T Van DenBroeck W Haesebrouck F Martel A (2012) Waterfowl potential environmental reservoirs of the chytrid fungusBatrachochytrium dendrobatidis PLoS ONE 7 e35038
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Brannelly et al Chytrid fungal infection in crayfish 235
Garner TWJ Perkins MW Govindarajulu P Seglie DWalker S Cunningham AA Fisher MC (2006) The emer -ging amphibian pathogen Batrachochytrium dendroba-tidis globally infects introduced populations of the NorthAmerican bullfrog Rana catesbeiana Biol Lett 2 455minus459
Garner TWJ Stephen I Wombwell E Fisher MC (2009) Theamphibian trade bans or best practice EcoHealth 6 148minus151
Gherardi F (2006) Crayfish invading Europe the case studyof Procambarus clarkii Mar Freshw Behav Physiol 39 175minus191
Gherardi F Renai B Corti C (2001) Crayfish predation ontadpoles a comparison between a native (Austropotamo-bius pallipes) and an alien species (Procambarus clarkii)Bull Fr Peche Piscicult 361 659minus668
Green DE Gray MJ Miller DL (2009) Disease monitoringand biosecurity Forestry Wildlife and Fisheries Publica-tions and Other Works Knoxville TN http traceten-nesseeedu utk_ forepubs2
Hasburgo-Lorena A (1986) The status of Procambarusclarkii population in Spain Freshw Crayfish 6 131minus136
Helms B Loughman ZJ Brown BL Stoeckel J (2013) Recentadvances in crayfish biology ecology and conservationFreshw Sci 32 1273minus1275
Holdich DM (1993) A review of astaciculture freshwatercrayfish farming Aquat Living Resour 6 307minus317
Jones JPG Rasamy JR Harvey A Toon A and others (2009)The perfect invader a parthenogenic crayfish poses anew threat to Madagascarrsquos freshwater biodiversity BiolInvasions 11 1475minus1482
Kilburn VL Ibaacutentildeez R Green DM (2011) Reptiles as potentialvectors and hosts of the amphibian pathogen Batra-chochytrium dendrobatidis in Panama Dis Aquat Org97 127minus134
Kolby JE (2014) Presence of the amphibian chytrid fungusBatrachochytrium dendrobatidis in native amphibiansexported from Madagascar PLoS ONE 9 e89660
Kolby JE Smith KM Berger L Karesh WB Preston APessier AP Skerratt LF (2014) First evidence of amphib-ian chytrid fungus (Batrachochytrium dendrobatidis) andranavirus in Hong Kong amphibian trade PLoS ONE 9 e90750
Kriger KM Hero JM Ashton KJ (2006) Cost efficiency in thedetection of chytridiomycosis using PCR assay Dis AquatOrg 71 149minus154
McClain WR Romaire RP (2007) Procambarid crawfish lifehistory and biology Publication No 2403 Southern Re -gional Aquaculture Center Mississippi State UniversityStoneville MS
McMahon TA Brannelly LA Chatfield MWH Johnson PTJand others (2013) Chytrid fungus Batrachochytrium den-drobatidis has nonamphibian hosts and releases chemi-cals that cause pathology in the absence of infectionProc Natl Acad Sci USA 110 210minus215
Murray K Skerratt LF Marantelli G Berger L Hunter DMahony MJ Hines H (2011) Hygiene protocols for thecontrol of diseases in Australian frogs A report for theAustralian Government Department of SustainabilityEnvironment Water Population and Communities Avail-able at wwwenvironmentgovau resource hygiene-protocols-control-diseases-australian-frogs
Newcombe RG (1998) Two-sided confidence intervals forthe single proportion comparison of seven methods StatMed 17 857minus872
Parker JM Mikaelian I Hahn N Diggs HE (2002) Clinicaldiagnosis and treatment of epidermal chytridiomycosisin African clawed frogs (Xenopus tropicalis) Comp Med52 265minus268
Rosenblum EB James TY Zamudio KR Poorten TJ and oth-ers (2013) Complex history of the amphibian-killingchytrid fungus revealed with genome resequencingdata Proc Natl Acad Sci USA 110 9385minus9390
Schloegel LM Picco AM Kilpatrick M Davies AJ HyattAD Daszak P (2009) Magnitude of the US trade inamphibians and presence of Batrachochytrium dendro-batidis and ranavirus infection in imported North Ameri-can bullfrogs (Rana catesbeiana) Biol Conserv 142 1420minus1426
Schloegel LM Ferreira CM James TY Hipolito M and oth-ers (2010) The North American bullfrog as a reservoir forthe spread of Batrachochytrium dendrobatidis in BrazilAnim Conserv 13 53minus61
Shapard EJ Moss AS San Francisco MJ (2012) Batra-chochytrium dendrobatidis can infect and cause mortal-ity in the nematode Caenorhabditis elegans Myco-pathologia 173 121minus126
Skerratt LF Berger L Speare R Cashins S and others (2007)Spread of chytridiomycosis has caused the rapid globaldecline and extinction of frogs EcoHealth 4 125minus134
Stuart SN Chanson JS Cox NA Young BE Rodrigues ASLFischman DL Waller RW (2004) Status and trends ofamphibian declines and extinctions worldwide Science306 1783minus1786
Voyles J Young S Berger L Campbell C and others (2009)Pathogenesis of chytridiomycosis a cause of catastrophicamphibian declines Science 326 582minus585
Warner RE (1968) The role of introduced diseases in theextinction of the endemic Hawaiian avifauna Condor 70 101minus120
Editorial responsibility Louise Rollins-Smith Nashville Tennessee USA
Submitted June 16 2014 Accepted October 30 2014Proofs received from author(s) December 22 2014
Aut
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Dis Aquat Org 112 229ndash235 2015
Anolis lizards (Kilburn et al 2011) 3 species of snake(Kilburn et al 2011) Caenorhabditis elegans nema-todes (under laboratory conditions Shapard et al2012) and wading birds (Garmyn et al 2012) How-ever none of these studies have demonstrated theability of the pathogen to complete its life cycle on orin these non-amphibian taxa
Only 1 study to date has demonstrated that Bd cancomplete its life cycle in a non-amphibian host andtransmit Bd infection to amphibians McMahon et al(2013) found that crayfish (Procambarus spp andOrconectes spp) can be Bd infected in natural popu-lations can carry Bd for 3 mo in the laboratory andcan transmit infection to tadpoles under laboratoryconditions Crayfish exposed to Bd under laboratoryconditions also suffered gill damage and mortalitysuggesting that Bd may pose a health threat to thesecommercially important animals as well McMahonet al (2013) suggest that crayfish may be important inthe Bdminusamphibian disease dynamic because pres-ence of crayfish correlates positively with the pres-ence of Bd in amphibian communities crayfish pres-ence is a better predictor of Bd presence than thepresence of the North American bullfrog R cates-beiana a purported reservoir species The NorthAmerican bullfrog has been suggested as a diseasereservoir because it can harbor large Bd loads anddoes not usually develop symptoms of the diseasechytridiomycosis (Schloegel et al 2010)
Procambarus spp are common inthe southeastern USA and are one ofthe most widely traded freshwatertaxa globally with the majority of indi-viduals coming from Louisiana USA(Holdich 1993) The crayfish marketcontributes over $150 million annuallyto Louisianarsquos economy and over125 000 acres of land are devoted tofarming crayfish (McClain amp Romaire2007) With so much land devoted toaquaculture many amphibians comein close contact with farm populationsof crayfish Therefore farmed crayfishmay impact the Bd infection dynamicsof natural populations of the crayfishand amphibians with which they co-occur Additionally P clarkii is tradedinternationally and could be a vectorfor Bd spread globally
We conducted a seasonal field sur-vey for Bd infection prevalence in bothnatural and farmed populations ofnative crayfish species in Louisiana
Loui siana was chosen for this study because thestatersquos farms supply a large proportion of the globalcrayfish market (Holdich 1993 McClain amp Romaire2007) and natural crayfish populations are wide-spread and abundant permitting a comparison ofpathogen pre valence and load between farmed andwild animals Furthermore data on the prevalenceand seasonality of Bd in amphibian populationsexists for this region (Brannelly et al 2012) but simi-lar metrics for infection in co-occurring natural andfarmed crayfish populations are not available Aclearer understanding of the prevalence intensityand seasonality of Bd in crayfish is needed to betterunderstand the potential impact of Bd on crayfishpopulations as well as the importance of crayfish asan alternative host for this pathogen with potentialwidespread ramifications through the global crayfishtrade
MATERIALS AND METHODS
Crayfish collection
Natural populations
Crayfish Procambarus spp were collected fromsoutheastern Louisiana (Maurepas Wildlife Manage-ment Area n = 129 and Tulane Universityrsquos F
230
D
E
C
B A
N
40 km
Fig 1 Southeastern Louisiana showing sampling locations for this study Letters correspond with site names in Table 1 (A) Tulane Universityrsquos FEdward Herbert Research Center in Belle Chase (natural) (B) MaurepasWildlife Management Area (natural) (C) Lafayette (farm) (D) Morgan City
(farm) and (E) Belle River (farm)
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opy
Brannelly et al Chytrid fungal infection in crayfish
Edward Herbert Research Center in Belle Chase n =142 Fig 1 and see Table 1) Collection occurred inthe spring (February to April) and fall (Septemberand November) of 2012 by sweep netting and usingbaited minnow traps Each crayfish was removedfrom the net or trap individually using a clean plasticbag and transported in the sealed bag to Tulane Uni-versity There crayfish were euthanized by freezingat minus20degC for a minimum of 2 h The crayfish sampledin this study were not sampled for any other study
Farmed populations
P clarkii were collected live from restaurants in theNew Orleans Louisiana metropolitan area in Febru-ary and April of 2012 These crayfish were suppliedby farms located in Morgan City Lafayette and BelleRiver Louisiana (Fig 1 and see Table 1) and from1 other farm of unknown location within the stateEach restaurant provided 20 to 22 crayfish (total n =82) Animals were held together in large mesh bagswith no water in a 4 to 10degC refrigerator at eachrestaurant for a maximum of 3 d after delivery fromthe farms and before we procured them As the ani-mals were kept communally before we procuredthem there was the potential for cross-contamina-tion When we received the animals each individualwas removed from the communal mesh bag using aclean inverted plastic bag which was then sealed fortransportation to Tulane University Animals wereeuthanized by freezing at minus20degC for a minimum of2 h We were not able to collect farmed crayfish in thefall because the artificial ponds used to farm theseanimals are drained during this season
Testing for Bd infection
Crayfish were thawed completely before process-ing The inside of the gastrointestinal (GI) tract ofeach animal was swabbed using a sterile MW113swab (Medical Wire and Equipment) and Bd loadwas quantified using a real-time polymerase chainreaction (qPCR analysis see lsquoDNA extraction andanalysisrsquo) The crayfish GI tract was chosen for patho-gen load analysis because the fungus is known toinfect the GI tract by implanting in the intestinal wall(McMahon et al 2013) While McMahon et al (2013)also found Bd on crayfish carapace the histopathol-ogy of carapace infection is unknown therefore forthe purpose of this study we chose to focus our sam-pling effort on the inside of the GI tract A positive
qPCR result indicates the presence of Bd DNA in thesample but cannot differentiate the presence of tran-sient DNA from an active infection of the GI tract tis-sue This would require histological examinationwhich was outside the scope of this study HoweverMcMahon et al (2013) previously demonstrated us -ing histopathological examinations of the GI tractthat crayfish of the same genus can become infectedwith Bd and can carry that infection for an extendedperiod of time While we cannot be certain that theindividuals that tested positive for Bd in our studyhad active infections (as opposed to harboring tran-sient Bd DNA due to ingestion of contaminated mate-rial) in either scenario the presence of Bd couldhave important implications for understanding therole of crayfish in the spread and dynamics of Bdinfection in amphibians As long as the pathogenremains viable on passage through the crayfish di -gestive system ingestion of Bd by crayfish destinedfor trade could permit transmission of the pathogento new areas even in the absence of an active GIinfection McMahon et al (2013) showed transmis-sion to amphibians from crayfish that were internallyinfected under laboratory conditions which suggeststhat Bd exiting the crayfish digestive system is viable
To access the GI tract the abdomen of each carcasswas separated from the thorax and the uropod wasseparated from the tail and slowly pulled away fromthe body with the GI tract still attached The fecalmatter inside the GI tract causes PCR inhibition so itwas removed with a sterile swab (MW113) Then theinside of the GI tract was swabbed using 30 strokeswith a second sterile MW113 swab GI swabs werekept frozen at minus20degC in 15 ml microtubes until qPCRanalysis The dissected animal was then placed in anew plastic bag and re-frozen Gloves were changedbetween handling the external surface of each cray-fish and the internal GI tract and between each newindividual
DNA extraction and analysis
To test for the presence of Bd we extractedgenomic DNA from the GI swabs using the QiagenDNeasy Blood and Tissue Kit We followed the man-ufacturersquos instructions for animal tissue using a finalelution volume of 200 microl Once extracted qPCR(Applied Biosystems 7500) was used to detect thequantity of Bd on each swab qPCR analysis was car-ried out following the protocol described by Boyle etal (2004) except that we added 07 microl of bovineserum albumin to each qPCR reaction to minimize
231A
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y
Dis Aquat Org 112 229ndash235 2015
inhibition and all wells contai ned an internal positivecontrol (VICtrade IPC Applied Biosystems) Positiveand negative controls previously extracted fromswabs of known Bd-positive and Bd-negative captiveRana catesbeiana and a dilution series of Bd stan-dards (provided by A Hyatt) were included in eachqPCR run Samples were run in singlicate (Kriger etal 2006) and DNA was not diluted prior to analysis
Statistical analysis
Microsoft Excel was used to calculate 95 confi-dence intervals on the proportion of Bd-positive indi-
viduals following Newcombe (1998) Zoosporeequivalents (ZE) in the GI tract in Bd-positive indi-viduals were compared between farmed and naturalpopulations using a 2-tailed t-test in SPSS (v21)
RESULTS
Bd prevalence in Louisiana crayfish was seasonalwith higher prevalence in farmed and wild-caughtanimals in the spring than in wild-caught animals inthe fall (Fig 2 Table 1) This pattern mirrors seasonaltrends in Bd prevalence for amphibian species in theregion some of which were sampled from the samesites as our wild-caught crayfish samples (Fig 2modified from Brannelly et al 2012) Farmed crayfishtested positive for Bd infection in the spring (Febru-ary and April 2012) with a prevalence of 60 (load1074 plusmn 396 (SD) ZE per swab n = 5) and no farmedanimals were available for testing in the fall Bd waspresent in natural crayfish populations in the springwith a prevalence of 331 (load 2217 plusmn 815 ZE perswab n = 9) but no Bd was detected from those samesites in the fall Farmed crayfish had a significantlylower ZE than wild-caught animals (t-test t12 = 2914p = 0013)
DISCUSSION
The seasonal pattern of Bd prevalence in wild-caught Louisiana crayfish in 2012 mirrored that seenin amphibian species sampled in the same region in2010 and 2011 with higher prevalence during thespring breeding season and no infection detectedduring the fall season Prevalences were lower in the
232
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010
020
030
040
050
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infe
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Janu
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arch
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ay
June
Ju
ly Aug
ust
Septe
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r Oct
ober
Nov
embe
r Dec
embe
r
Fig 2 Proportion of Batrachochytrium dendrobatidis (Bd)-positive crayfish sampled in 2012 in southeastern Louisiana(d) Wild-caught and (d) farm-collected animals The pro-portion of (s with dashed error bars) Bd-infected amphi -bians in southeastern Loui siana sampled in 2010 to 2011is also shown modified from Brannelly et al (2012 their
Fig 2) The error bars are 95 confidence intervals
Site Latitude Longitude Month No Bd + ind Average zoospore Site type(degN) (degW) total sampled load (plusmnSD)
A 2989 89953 February 6 35 2167 plusmn 1147 NaturalMarch 0 62April 0 40
September 0 5B 30108 90435 April 3 52 1354 plusmn 618 Natural
November 0 77C 30214 9203 April 3 22 1126 plusmn 549 FarmD 29701 91206 April 0 22 FarmE 29888 91206 April 2 20 479 plusmn 662 FarmF Unknown February 0 20 Farm
Table 1 Sampling of natural and farmed crayfish Sites are (A) Tulane Universityrsquos F Edward Herbert Research Center inBelle Chase (natural) (B) Maurepas Wildlife Management Area (natural) (C) Lafayette (farm) (D) Morgan City (farm) and (E)Belle River (farm) and (F) unknown location in Louisiana (farm) Bd Batrachochytrium dendrobatidis See Fig 1 for sampling
locations in Louisiana USA
Aut
hor c
opy
Brannelly et al Chytrid fungal infection in crayfish
crayfish than in the amphibians sampled in a givenmonth (Fig 2) However since crayfish and amphi -bians were sampled on different dates and in differ-ent years and in some cases also from different sitescaution should be used in comparing prevalence esti-mates between these 2 taxa In this study we did notfind Bd-infected crayfish in September (05 Bd+95 CI = 0 to 435 prevalence) or November (077Bd+ 95 CI = 0 to 48 prevalence) of 2012 butMcMahon et al (2013) found Bd at 173 prevalencein Louisiana crayfish in September of 2011 Our fail-ure to detect Bd in the fall could be due to yearly vari-ation in disease prevalence or an artefact of our smallsample size early in the fall Although our study didnot support the idea that crayfish act as disease reser-voirs during the late summer or fall months we diddetect Bd in crayfish during the spring and thereforecannot rule out the idea that these animals may playan important role in disease dynamics Summer tem-peratures in Louisiana typically exceed the thermaltolerance of Bd and amphibians may clear theirinfections during this time It is possible that crayfishare also able to clear Bd infections during high sum-mer temperatures but more research is needed totest this prediction
Farmed and wild-caught crayfish Bd prevalenceswere similar to each other in the spring when farmedcrayfish were available (Fig 2) It remains unclearwhether the effects of Bd on crayfish health shouldbe an important consideration for farmers Bd infec-tion has been shown to cause mortality in laboratory-exposed crayfish (McMahon et al 2013) but itsimpact on farmed and natural crayfish populationsremains unstudied While we did not examine ani-mals for pathology we did find that farmed crayfishhad significantly lower Bd loads than wild-caughtanimals The different conditions these animals ex -perienced directly prior to sampling could also havecontributed to the difference in pathogen load Wild-caught animals were separated upon capture andkept alive only for transport between the field andthe laboratory while the farmed crayfish were keptalive communally in a refrigerated room for up to 3 dbetween capture and sampling However beforeconcluding that farmed animals have lower patho-gen loads than natural populations a sample of cray-fish directly from the farms analogous to our naturalpopulation sampling methods is needed While com-munal refrigerated conditions might have impactedour Bd load results we found that farmed animalsheld in analogous conditions to those destined fortrade were positive for Bd at a similar prevalence towild-caught animals Six percent of the farmed indi-
viduals left the farm and were sold for consumptionwhile infected with Bd
Minimizing disease transmission between wildlifeand farmed animals is important and this topic hasbeen extensively studied in domesticated animalsglobally Wild animals can carry disease and infectindividuals in farmed populations which is particu-larly concerning for livestock and farm owners (egMycobacterium bovis Delahay et al 2001) Farmedanimals can also introduce spread and maintaininfection in natural populations which can be de -trimental to vulnerable species living near farms(eg chickens introducing avian malaria into nativeHawaiian bird populations Warner 1968) Bothfarmed and natural crayfish populations in south-eastern Louisiana were Bd positive in the springwhich is the time when they commonly share habi-tats with amphibians Crayfish infected with Bd inclose proximity with amphibian populations mayplay a role in amphibian disease dynamics espe-cially if infected crayfish are transported to areas thatwere previously Bd free
Our study found that farmed crayfish were Bdpositive during the trading season If farmed cray-fish that are globally traded are infected with Bdthen even a low prevalence of infection could haveimplications for global amphibian conservation Theinternational crayfish market is large with 40 000 to60 000 t traded per annum (Holdich 1993) whichequates to approximately 08 to 12 billion indivi -duals The North American species Procambarusclarkii makes up 85 of all crayfish trade (Holdich1993) Most of these globally traded crayfish origi-nate from farms in Louisiana (Holdich 1993) Cray-fish are the most widely introduced freshwatertaxon globally (Helms et al 2013) likely becomingestablished after escaping from farms (Gherardi2006) as farms rarely exercise biosecurity measuresto ensure isolation from natural populations Addi-tionally P clarkii is a highly successful invasivespecies once it escapes its native range It can re -produce by par thenogenesis can survive in ephe -meral ponds by burrowing deep into mud banks intimes of drought (which may help Bd persist) andtends to escape from farms and travel quickly tolocal ponds where it then reproduces (Gherardi2006) There is already unequivocal evidence thatthe invasive P clarkii has driven amphi bian declinethrough predation rather than pathogen spread inmany regions of Europe (Cruz et al 2006ab Cruz2008 Ficetola et al 2012) Although there is a clearcorrelation between amphi bian de clines and inva-sion by P clarkii in Europe the potential relation-
233A
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Dis Aquat Org 112 229ndash235 2015
ship between amphibian declines crayfish invasionand disease has not been explored
Trade of diseased individuals whether it be amphi -bians or other aquatic organisms might explain thespread of the Bd pathogen globally (Fisher amp Garner2007 Garner et al 2009 Schloegel et al 2009 Kolbyet al 2014) While the origin of Bd is still unclear itappears that strains of Bd found in parts of Europeare most closely related to strains found in the east-ern USA (Rosenblum et al 2013) which could beexplained by the import of invasive North Americanspecies like Rana catesbeiana and P clarkii for con-sumer purposes Spain is one place where the role ofcrayfish as an important alternative host for Bd de -serves further study as this country has been experi-encing some of the greatest amphibian declines inEurope (Gherardi et al 2001 Garner et al 2006) Thedeclines have been attributed to both amphibian pre-dation by the invasive P clarkii as well as chytrid-iomycosis (Gherardi et al 2001 Garner et al 2006)P clarkii were introduced into Spain twice for thepurposes of farming for consumption from farms inLouisiana between 1973 and 1974 (Hasburgo-Lorena1986) Since then crayfish have spread into Portugaland other parts of Europe either through importationfrom Spain or migration out of farms It is possiblethat Bd was introduced into the area through im -ported crayfish and subsequently spread to naturalamphibian populations Although amphibian tradehas been implicated in the global spread of Bd(Fisher amp Garner 2007 Garner et al 2009 Schloegelet al 2009) even regions devoid of commercial amphi -bian importation have been exposed to this patho-gen For example Bd has recently been de tected forthe first time in amphibians coming from Madagas-car (Kolby 2014) where North American Procam-barus spp have been introduced into the wild andare now rapidly spreading (Jones et al 2009) Whilethe correlation between amphibian disease and cray-fish presence has not been studied in EuropeanMalagasy or other ecosystems our study suggeststhat farmed P clarkii can carry Bd and that createsthe risk of zoonotic spill-over if appropriate controlmeasures are not taken
Bd disease dynamics are challenging to understandand predict and when we consider non-amphi bianhosts the story becomes increasingly complex Thereare current biosecurity protocols enacted globally toreduce disease spread through amphibian trade andcontaminated field equipment (Green et al 2009Murray et al 2011) but little attention has beendirected towards the prevention of Bd spread by non-amphibian hosts In this study we found that both
natural and farmed crayfish populations carry Bdinfection in Louisiana and that infection is seasonalin wild-caught animals similar to the pattern of pre -valence seen in amphibian populations in the sameregion While prevalence in our study was low cray-fish species may still be an important part of the disease dynamic especially because they are mass-distributed globally We would argue that a 60Bd infection prevalence in a species that is as widelytraded as P clarkii represents a significant risk anddeserves to be considered in biosecurity measures
Acknowledgements We thank Matthew Robak for helprunning the qPCR analysis Matthew Chatfield for help inthe laboratory and the field and Jonathan Kolby for com-menting on the manuscript
LITERATURE CITED
Boyle DG Boyle DB Olsen V Morgan JAT Hyatt AD (2004)Rapid quantitative detection of chytridiomycosis (Batra-chochytrium dendrobatidis) in amphibian samples usingreal-time Taqman PCR assay Dis Aquat Org 60 141minus148
Brannelly LA Chatfield MWH Richards-Zawacki CL (2012)Field and laboratory studies of the susceptibility of thegreen treefrog (Hyla cinerea) to Batrachochytrium den-drobatidis infection PLoS ONE 7 e38473
Chatfield MWH Brannelly LA Robak MJ Freeborn L Lail-vaux SP Richards-Zawacki CL (2013) Fitness conse-quences of infection by Batrachochytrium dendrobatidisin northern leopard frogs (Lithobates pipiens) EcoHealth10 90minus98
Cruz J (2008) Collapse of the amphibian community of thePaul do Boquilobo Natural Reserve (central Portugal)after the arrival of the exotic American crayfish HerpetolJ 18 197minus204
Cruz MJ Pascoal S Tejedo M Rebelo R (2006a) Predationby an exotic crayfish Procambarus clarkii on natterjacktoad Bufo calamita embryos its role on the exclusion ofthis amphibian from its breeding ponds Copeia 2006 274minus280
Cruz MJ Rebelo R Crespo EG (2006b) Effects of an intro-duced crayfish Procambarus clarkii on the distributionof south-western Iberian amphibians in their breedinghabitats Ecography 29 329minus338
Delahay RJ Cheeseman CL Clifton-Hadley RS (2001)Wildlife disease reservoirs the epidemiology of Myco -bacterium bovis infection in the European badger (Melesmeles) and other British mammals Tuberculosis (Edinb)81 43minus49
Ficetola GF Siesa ME De Bernardi F Padoa-Schioppa E(2012) Complex impact of an invasive crayfish on fresh-water food webs Biodivers Conserv 21 2641minus2651
Fisher MC Garner TWJ (2007) The relationship betweenthe emergence of Batrachochytrium dendrobatidis theinternational trade in amphibians and introduced amphi -bian species Fungal Biol Rev 21 2minus9
Garmyn A Van Rooij P Pasmans F Hellebuyck T Van DenBroeck W Haesebrouck F Martel A (2012) Waterfowl potential environmental reservoirs of the chytrid fungusBatrachochytrium dendrobatidis PLoS ONE 7 e35038
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Brannelly et al Chytrid fungal infection in crayfish 235
Garner TWJ Perkins MW Govindarajulu P Seglie DWalker S Cunningham AA Fisher MC (2006) The emer -ging amphibian pathogen Batrachochytrium dendroba-tidis globally infects introduced populations of the NorthAmerican bullfrog Rana catesbeiana Biol Lett 2 455minus459
Garner TWJ Stephen I Wombwell E Fisher MC (2009) Theamphibian trade bans or best practice EcoHealth 6 148minus151
Gherardi F (2006) Crayfish invading Europe the case studyof Procambarus clarkii Mar Freshw Behav Physiol 39 175minus191
Gherardi F Renai B Corti C (2001) Crayfish predation ontadpoles a comparison between a native (Austropotamo-bius pallipes) and an alien species (Procambarus clarkii)Bull Fr Peche Piscicult 361 659minus668
Green DE Gray MJ Miller DL (2009) Disease monitoringand biosecurity Forestry Wildlife and Fisheries Publica-tions and Other Works Knoxville TN http traceten-nesseeedu utk_ forepubs2
Hasburgo-Lorena A (1986) The status of Procambarusclarkii population in Spain Freshw Crayfish 6 131minus136
Helms B Loughman ZJ Brown BL Stoeckel J (2013) Recentadvances in crayfish biology ecology and conservationFreshw Sci 32 1273minus1275
Holdich DM (1993) A review of astaciculture freshwatercrayfish farming Aquat Living Resour 6 307minus317
Jones JPG Rasamy JR Harvey A Toon A and others (2009)The perfect invader a parthenogenic crayfish poses anew threat to Madagascarrsquos freshwater biodiversity BiolInvasions 11 1475minus1482
Kilburn VL Ibaacutentildeez R Green DM (2011) Reptiles as potentialvectors and hosts of the amphibian pathogen Batra-chochytrium dendrobatidis in Panama Dis Aquat Org97 127minus134
Kolby JE (2014) Presence of the amphibian chytrid fungusBatrachochytrium dendrobatidis in native amphibiansexported from Madagascar PLoS ONE 9 e89660
Kolby JE Smith KM Berger L Karesh WB Preston APessier AP Skerratt LF (2014) First evidence of amphib-ian chytrid fungus (Batrachochytrium dendrobatidis) andranavirus in Hong Kong amphibian trade PLoS ONE 9 e90750
Kriger KM Hero JM Ashton KJ (2006) Cost efficiency in thedetection of chytridiomycosis using PCR assay Dis AquatOrg 71 149minus154
McClain WR Romaire RP (2007) Procambarid crawfish lifehistory and biology Publication No 2403 Southern Re -gional Aquaculture Center Mississippi State UniversityStoneville MS
McMahon TA Brannelly LA Chatfield MWH Johnson PTJand others (2013) Chytrid fungus Batrachochytrium den-drobatidis has nonamphibian hosts and releases chemi-cals that cause pathology in the absence of infectionProc Natl Acad Sci USA 110 210minus215
Murray K Skerratt LF Marantelli G Berger L Hunter DMahony MJ Hines H (2011) Hygiene protocols for thecontrol of diseases in Australian frogs A report for theAustralian Government Department of SustainabilityEnvironment Water Population and Communities Avail-able at wwwenvironmentgovau resource hygiene-protocols-control-diseases-australian-frogs
Newcombe RG (1998) Two-sided confidence intervals forthe single proportion comparison of seven methods StatMed 17 857minus872
Parker JM Mikaelian I Hahn N Diggs HE (2002) Clinicaldiagnosis and treatment of epidermal chytridiomycosisin African clawed frogs (Xenopus tropicalis) Comp Med52 265minus268
Rosenblum EB James TY Zamudio KR Poorten TJ and oth-ers (2013) Complex history of the amphibian-killingchytrid fungus revealed with genome resequencingdata Proc Natl Acad Sci USA 110 9385minus9390
Schloegel LM Picco AM Kilpatrick M Davies AJ HyattAD Daszak P (2009) Magnitude of the US trade inamphibians and presence of Batrachochytrium dendro-batidis and ranavirus infection in imported North Ameri-can bullfrogs (Rana catesbeiana) Biol Conserv 142 1420minus1426
Schloegel LM Ferreira CM James TY Hipolito M and oth-ers (2010) The North American bullfrog as a reservoir forthe spread of Batrachochytrium dendrobatidis in BrazilAnim Conserv 13 53minus61
Shapard EJ Moss AS San Francisco MJ (2012) Batra-chochytrium dendrobatidis can infect and cause mortal-ity in the nematode Caenorhabditis elegans Myco-pathologia 173 121minus126
Skerratt LF Berger L Speare R Cashins S and others (2007)Spread of chytridiomycosis has caused the rapid globaldecline and extinction of frogs EcoHealth 4 125minus134
Stuart SN Chanson JS Cox NA Young BE Rodrigues ASLFischman DL Waller RW (2004) Status and trends ofamphibian declines and extinctions worldwide Science306 1783minus1786
Voyles J Young S Berger L Campbell C and others (2009)Pathogenesis of chytridiomycosis a cause of catastrophicamphibian declines Science 326 582minus585
Warner RE (1968) The role of introduced diseases in theextinction of the endemic Hawaiian avifauna Condor 70 101minus120
Editorial responsibility Louise Rollins-Smith Nashville Tennessee USA
Submitted June 16 2014 Accepted October 30 2014Proofs received from author(s) December 22 2014
Aut
hor c
opy
Brannelly et al Chytrid fungal infection in crayfish
Edward Herbert Research Center in Belle Chase n =142 Fig 1 and see Table 1) Collection occurred inthe spring (February to April) and fall (Septemberand November) of 2012 by sweep netting and usingbaited minnow traps Each crayfish was removedfrom the net or trap individually using a clean plasticbag and transported in the sealed bag to Tulane Uni-versity There crayfish were euthanized by freezingat minus20degC for a minimum of 2 h The crayfish sampledin this study were not sampled for any other study
Farmed populations
P clarkii were collected live from restaurants in theNew Orleans Louisiana metropolitan area in Febru-ary and April of 2012 These crayfish were suppliedby farms located in Morgan City Lafayette and BelleRiver Louisiana (Fig 1 and see Table 1) and from1 other farm of unknown location within the stateEach restaurant provided 20 to 22 crayfish (total n =82) Animals were held together in large mesh bagswith no water in a 4 to 10degC refrigerator at eachrestaurant for a maximum of 3 d after delivery fromthe farms and before we procured them As the ani-mals were kept communally before we procuredthem there was the potential for cross-contamina-tion When we received the animals each individualwas removed from the communal mesh bag using aclean inverted plastic bag which was then sealed fortransportation to Tulane University Animals wereeuthanized by freezing at minus20degC for a minimum of2 h We were not able to collect farmed crayfish in thefall because the artificial ponds used to farm theseanimals are drained during this season
Testing for Bd infection
Crayfish were thawed completely before process-ing The inside of the gastrointestinal (GI) tract ofeach animal was swabbed using a sterile MW113swab (Medical Wire and Equipment) and Bd loadwas quantified using a real-time polymerase chainreaction (qPCR analysis see lsquoDNA extraction andanalysisrsquo) The crayfish GI tract was chosen for patho-gen load analysis because the fungus is known toinfect the GI tract by implanting in the intestinal wall(McMahon et al 2013) While McMahon et al (2013)also found Bd on crayfish carapace the histopathol-ogy of carapace infection is unknown therefore forthe purpose of this study we chose to focus our sam-pling effort on the inside of the GI tract A positive
qPCR result indicates the presence of Bd DNA in thesample but cannot differentiate the presence of tran-sient DNA from an active infection of the GI tract tis-sue This would require histological examinationwhich was outside the scope of this study HoweverMcMahon et al (2013) previously demonstrated us -ing histopathological examinations of the GI tractthat crayfish of the same genus can become infectedwith Bd and can carry that infection for an extendedperiod of time While we cannot be certain that theindividuals that tested positive for Bd in our studyhad active infections (as opposed to harboring tran-sient Bd DNA due to ingestion of contaminated mate-rial) in either scenario the presence of Bd couldhave important implications for understanding therole of crayfish in the spread and dynamics of Bdinfection in amphibians As long as the pathogenremains viable on passage through the crayfish di -gestive system ingestion of Bd by crayfish destinedfor trade could permit transmission of the pathogento new areas even in the absence of an active GIinfection McMahon et al (2013) showed transmis-sion to amphibians from crayfish that were internallyinfected under laboratory conditions which suggeststhat Bd exiting the crayfish digestive system is viable
To access the GI tract the abdomen of each carcasswas separated from the thorax and the uropod wasseparated from the tail and slowly pulled away fromthe body with the GI tract still attached The fecalmatter inside the GI tract causes PCR inhibition so itwas removed with a sterile swab (MW113) Then theinside of the GI tract was swabbed using 30 strokeswith a second sterile MW113 swab GI swabs werekept frozen at minus20degC in 15 ml microtubes until qPCRanalysis The dissected animal was then placed in anew plastic bag and re-frozen Gloves were changedbetween handling the external surface of each cray-fish and the internal GI tract and between each newindividual
DNA extraction and analysis
To test for the presence of Bd we extractedgenomic DNA from the GI swabs using the QiagenDNeasy Blood and Tissue Kit We followed the man-ufacturersquos instructions for animal tissue using a finalelution volume of 200 microl Once extracted qPCR(Applied Biosystems 7500) was used to detect thequantity of Bd on each swab qPCR analysis was car-ried out following the protocol described by Boyle etal (2004) except that we added 07 microl of bovineserum albumin to each qPCR reaction to minimize
231A
utho
r cop
y
Dis Aquat Org 112 229ndash235 2015
inhibition and all wells contai ned an internal positivecontrol (VICtrade IPC Applied Biosystems) Positiveand negative controls previously extracted fromswabs of known Bd-positive and Bd-negative captiveRana catesbeiana and a dilution series of Bd stan-dards (provided by A Hyatt) were included in eachqPCR run Samples were run in singlicate (Kriger etal 2006) and DNA was not diluted prior to analysis
Statistical analysis
Microsoft Excel was used to calculate 95 confi-dence intervals on the proportion of Bd-positive indi-
viduals following Newcombe (1998) Zoosporeequivalents (ZE) in the GI tract in Bd-positive indi-viduals were compared between farmed and naturalpopulations using a 2-tailed t-test in SPSS (v21)
RESULTS
Bd prevalence in Louisiana crayfish was seasonalwith higher prevalence in farmed and wild-caughtanimals in the spring than in wild-caught animals inthe fall (Fig 2 Table 1) This pattern mirrors seasonaltrends in Bd prevalence for amphibian species in theregion some of which were sampled from the samesites as our wild-caught crayfish samples (Fig 2modified from Brannelly et al 2012) Farmed crayfishtested positive for Bd infection in the spring (Febru-ary and April 2012) with a prevalence of 60 (load1074 plusmn 396 (SD) ZE per swab n = 5) and no farmedanimals were available for testing in the fall Bd waspresent in natural crayfish populations in the springwith a prevalence of 331 (load 2217 plusmn 815 ZE perswab n = 9) but no Bd was detected from those samesites in the fall Farmed crayfish had a significantlylower ZE than wild-caught animals (t-test t12 = 2914p = 0013)
DISCUSSION
The seasonal pattern of Bd prevalence in wild-caught Louisiana crayfish in 2012 mirrored that seenin amphibian species sampled in the same region in2010 and 2011 with higher prevalence during thespring breeding season and no infection detectedduring the fall season Prevalences were lower in the
232
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ary
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arch
Apr
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ay
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ly Aug
ust
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embe
r Dec
embe
r
Fig 2 Proportion of Batrachochytrium dendrobatidis (Bd)-positive crayfish sampled in 2012 in southeastern Louisiana(d) Wild-caught and (d) farm-collected animals The pro-portion of (s with dashed error bars) Bd-infected amphi -bians in southeastern Loui siana sampled in 2010 to 2011is also shown modified from Brannelly et al (2012 their
Fig 2) The error bars are 95 confidence intervals
Site Latitude Longitude Month No Bd + ind Average zoospore Site type(degN) (degW) total sampled load (plusmnSD)
A 2989 89953 February 6 35 2167 plusmn 1147 NaturalMarch 0 62April 0 40
September 0 5B 30108 90435 April 3 52 1354 plusmn 618 Natural
November 0 77C 30214 9203 April 3 22 1126 plusmn 549 FarmD 29701 91206 April 0 22 FarmE 29888 91206 April 2 20 479 plusmn 662 FarmF Unknown February 0 20 Farm
Table 1 Sampling of natural and farmed crayfish Sites are (A) Tulane Universityrsquos F Edward Herbert Research Center inBelle Chase (natural) (B) Maurepas Wildlife Management Area (natural) (C) Lafayette (farm) (D) Morgan City (farm) and (E)Belle River (farm) and (F) unknown location in Louisiana (farm) Bd Batrachochytrium dendrobatidis See Fig 1 for sampling
locations in Louisiana USA
Aut
hor c
opy
Brannelly et al Chytrid fungal infection in crayfish
crayfish than in the amphibians sampled in a givenmonth (Fig 2) However since crayfish and amphi -bians were sampled on different dates and in differ-ent years and in some cases also from different sitescaution should be used in comparing prevalence esti-mates between these 2 taxa In this study we did notfind Bd-infected crayfish in September (05 Bd+95 CI = 0 to 435 prevalence) or November (077Bd+ 95 CI = 0 to 48 prevalence) of 2012 butMcMahon et al (2013) found Bd at 173 prevalencein Louisiana crayfish in September of 2011 Our fail-ure to detect Bd in the fall could be due to yearly vari-ation in disease prevalence or an artefact of our smallsample size early in the fall Although our study didnot support the idea that crayfish act as disease reser-voirs during the late summer or fall months we diddetect Bd in crayfish during the spring and thereforecannot rule out the idea that these animals may playan important role in disease dynamics Summer tem-peratures in Louisiana typically exceed the thermaltolerance of Bd and amphibians may clear theirinfections during this time It is possible that crayfishare also able to clear Bd infections during high sum-mer temperatures but more research is needed totest this prediction
Farmed and wild-caught crayfish Bd prevalenceswere similar to each other in the spring when farmedcrayfish were available (Fig 2) It remains unclearwhether the effects of Bd on crayfish health shouldbe an important consideration for farmers Bd infec-tion has been shown to cause mortality in laboratory-exposed crayfish (McMahon et al 2013) but itsimpact on farmed and natural crayfish populationsremains unstudied While we did not examine ani-mals for pathology we did find that farmed crayfishhad significantly lower Bd loads than wild-caughtanimals The different conditions these animals ex -perienced directly prior to sampling could also havecontributed to the difference in pathogen load Wild-caught animals were separated upon capture andkept alive only for transport between the field andthe laboratory while the farmed crayfish were keptalive communally in a refrigerated room for up to 3 dbetween capture and sampling However beforeconcluding that farmed animals have lower patho-gen loads than natural populations a sample of cray-fish directly from the farms analogous to our naturalpopulation sampling methods is needed While com-munal refrigerated conditions might have impactedour Bd load results we found that farmed animalsheld in analogous conditions to those destined fortrade were positive for Bd at a similar prevalence towild-caught animals Six percent of the farmed indi-
viduals left the farm and were sold for consumptionwhile infected with Bd
Minimizing disease transmission between wildlifeand farmed animals is important and this topic hasbeen extensively studied in domesticated animalsglobally Wild animals can carry disease and infectindividuals in farmed populations which is particu-larly concerning for livestock and farm owners (egMycobacterium bovis Delahay et al 2001) Farmedanimals can also introduce spread and maintaininfection in natural populations which can be de -trimental to vulnerable species living near farms(eg chickens introducing avian malaria into nativeHawaiian bird populations Warner 1968) Bothfarmed and natural crayfish populations in south-eastern Louisiana were Bd positive in the springwhich is the time when they commonly share habi-tats with amphibians Crayfish infected with Bd inclose proximity with amphibian populations mayplay a role in amphibian disease dynamics espe-cially if infected crayfish are transported to areas thatwere previously Bd free
Our study found that farmed crayfish were Bdpositive during the trading season If farmed cray-fish that are globally traded are infected with Bdthen even a low prevalence of infection could haveimplications for global amphibian conservation Theinternational crayfish market is large with 40 000 to60 000 t traded per annum (Holdich 1993) whichequates to approximately 08 to 12 billion indivi -duals The North American species Procambarusclarkii makes up 85 of all crayfish trade (Holdich1993) Most of these globally traded crayfish origi-nate from farms in Louisiana (Holdich 1993) Cray-fish are the most widely introduced freshwatertaxon globally (Helms et al 2013) likely becomingestablished after escaping from farms (Gherardi2006) as farms rarely exercise biosecurity measuresto ensure isolation from natural populations Addi-tionally P clarkii is a highly successful invasivespecies once it escapes its native range It can re -produce by par thenogenesis can survive in ephe -meral ponds by burrowing deep into mud banks intimes of drought (which may help Bd persist) andtends to escape from farms and travel quickly tolocal ponds where it then reproduces (Gherardi2006) There is already unequivocal evidence thatthe invasive P clarkii has driven amphi bian declinethrough predation rather than pathogen spread inmany regions of Europe (Cruz et al 2006ab Cruz2008 Ficetola et al 2012) Although there is a clearcorrelation between amphi bian de clines and inva-sion by P clarkii in Europe the potential relation-
233A
utho
r cop
y
Dis Aquat Org 112 229ndash235 2015
ship between amphibian declines crayfish invasionand disease has not been explored
Trade of diseased individuals whether it be amphi -bians or other aquatic organisms might explain thespread of the Bd pathogen globally (Fisher amp Garner2007 Garner et al 2009 Schloegel et al 2009 Kolbyet al 2014) While the origin of Bd is still unclear itappears that strains of Bd found in parts of Europeare most closely related to strains found in the east-ern USA (Rosenblum et al 2013) which could beexplained by the import of invasive North Americanspecies like Rana catesbeiana and P clarkii for con-sumer purposes Spain is one place where the role ofcrayfish as an important alternative host for Bd de -serves further study as this country has been experi-encing some of the greatest amphibian declines inEurope (Gherardi et al 2001 Garner et al 2006) Thedeclines have been attributed to both amphibian pre-dation by the invasive P clarkii as well as chytrid-iomycosis (Gherardi et al 2001 Garner et al 2006)P clarkii were introduced into Spain twice for thepurposes of farming for consumption from farms inLouisiana between 1973 and 1974 (Hasburgo-Lorena1986) Since then crayfish have spread into Portugaland other parts of Europe either through importationfrom Spain or migration out of farms It is possiblethat Bd was introduced into the area through im -ported crayfish and subsequently spread to naturalamphibian populations Although amphibian tradehas been implicated in the global spread of Bd(Fisher amp Garner 2007 Garner et al 2009 Schloegelet al 2009) even regions devoid of commercial amphi -bian importation have been exposed to this patho-gen For example Bd has recently been de tected forthe first time in amphibians coming from Madagas-car (Kolby 2014) where North American Procam-barus spp have been introduced into the wild andare now rapidly spreading (Jones et al 2009) Whilethe correlation between amphibian disease and cray-fish presence has not been studied in EuropeanMalagasy or other ecosystems our study suggeststhat farmed P clarkii can carry Bd and that createsthe risk of zoonotic spill-over if appropriate controlmeasures are not taken
Bd disease dynamics are challenging to understandand predict and when we consider non-amphi bianhosts the story becomes increasingly complex Thereare current biosecurity protocols enacted globally toreduce disease spread through amphibian trade andcontaminated field equipment (Green et al 2009Murray et al 2011) but little attention has beendirected towards the prevention of Bd spread by non-amphibian hosts In this study we found that both
natural and farmed crayfish populations carry Bdinfection in Louisiana and that infection is seasonalin wild-caught animals similar to the pattern of pre -valence seen in amphibian populations in the sameregion While prevalence in our study was low cray-fish species may still be an important part of the disease dynamic especially because they are mass-distributed globally We would argue that a 60Bd infection prevalence in a species that is as widelytraded as P clarkii represents a significant risk anddeserves to be considered in biosecurity measures
Acknowledgements We thank Matthew Robak for helprunning the qPCR analysis Matthew Chatfield for help inthe laboratory and the field and Jonathan Kolby for com-menting on the manuscript
LITERATURE CITED
Boyle DG Boyle DB Olsen V Morgan JAT Hyatt AD (2004)Rapid quantitative detection of chytridiomycosis (Batra-chochytrium dendrobatidis) in amphibian samples usingreal-time Taqman PCR assay Dis Aquat Org 60 141minus148
Brannelly LA Chatfield MWH Richards-Zawacki CL (2012)Field and laboratory studies of the susceptibility of thegreen treefrog (Hyla cinerea) to Batrachochytrium den-drobatidis infection PLoS ONE 7 e38473
Chatfield MWH Brannelly LA Robak MJ Freeborn L Lail-vaux SP Richards-Zawacki CL (2013) Fitness conse-quences of infection by Batrachochytrium dendrobatidisin northern leopard frogs (Lithobates pipiens) EcoHealth10 90minus98
Cruz J (2008) Collapse of the amphibian community of thePaul do Boquilobo Natural Reserve (central Portugal)after the arrival of the exotic American crayfish HerpetolJ 18 197minus204
Cruz MJ Pascoal S Tejedo M Rebelo R (2006a) Predationby an exotic crayfish Procambarus clarkii on natterjacktoad Bufo calamita embryos its role on the exclusion ofthis amphibian from its breeding ponds Copeia 2006 274minus280
Cruz MJ Rebelo R Crespo EG (2006b) Effects of an intro-duced crayfish Procambarus clarkii on the distributionof south-western Iberian amphibians in their breedinghabitats Ecography 29 329minus338
Delahay RJ Cheeseman CL Clifton-Hadley RS (2001)Wildlife disease reservoirs the epidemiology of Myco -bacterium bovis infection in the European badger (Melesmeles) and other British mammals Tuberculosis (Edinb)81 43minus49
Ficetola GF Siesa ME De Bernardi F Padoa-Schioppa E(2012) Complex impact of an invasive crayfish on fresh-water food webs Biodivers Conserv 21 2641minus2651
Fisher MC Garner TWJ (2007) The relationship betweenthe emergence of Batrachochytrium dendrobatidis theinternational trade in amphibians and introduced amphi -bian species Fungal Biol Rev 21 2minus9
Garmyn A Van Rooij P Pasmans F Hellebuyck T Van DenBroeck W Haesebrouck F Martel A (2012) Waterfowl potential environmental reservoirs of the chytrid fungusBatrachochytrium dendrobatidis PLoS ONE 7 e35038
234A
utho
r cop
y
Brannelly et al Chytrid fungal infection in crayfish 235
Garner TWJ Perkins MW Govindarajulu P Seglie DWalker S Cunningham AA Fisher MC (2006) The emer -ging amphibian pathogen Batrachochytrium dendroba-tidis globally infects introduced populations of the NorthAmerican bullfrog Rana catesbeiana Biol Lett 2 455minus459
Garner TWJ Stephen I Wombwell E Fisher MC (2009) Theamphibian trade bans or best practice EcoHealth 6 148minus151
Gherardi F (2006) Crayfish invading Europe the case studyof Procambarus clarkii Mar Freshw Behav Physiol 39 175minus191
Gherardi F Renai B Corti C (2001) Crayfish predation ontadpoles a comparison between a native (Austropotamo-bius pallipes) and an alien species (Procambarus clarkii)Bull Fr Peche Piscicult 361 659minus668
Green DE Gray MJ Miller DL (2009) Disease monitoringand biosecurity Forestry Wildlife and Fisheries Publica-tions and Other Works Knoxville TN http traceten-nesseeedu utk_ forepubs2
Hasburgo-Lorena A (1986) The status of Procambarusclarkii population in Spain Freshw Crayfish 6 131minus136
Helms B Loughman ZJ Brown BL Stoeckel J (2013) Recentadvances in crayfish biology ecology and conservationFreshw Sci 32 1273minus1275
Holdich DM (1993) A review of astaciculture freshwatercrayfish farming Aquat Living Resour 6 307minus317
Jones JPG Rasamy JR Harvey A Toon A and others (2009)The perfect invader a parthenogenic crayfish poses anew threat to Madagascarrsquos freshwater biodiversity BiolInvasions 11 1475minus1482
Kilburn VL Ibaacutentildeez R Green DM (2011) Reptiles as potentialvectors and hosts of the amphibian pathogen Batra-chochytrium dendrobatidis in Panama Dis Aquat Org97 127minus134
Kolby JE (2014) Presence of the amphibian chytrid fungusBatrachochytrium dendrobatidis in native amphibiansexported from Madagascar PLoS ONE 9 e89660
Kolby JE Smith KM Berger L Karesh WB Preston APessier AP Skerratt LF (2014) First evidence of amphib-ian chytrid fungus (Batrachochytrium dendrobatidis) andranavirus in Hong Kong amphibian trade PLoS ONE 9 e90750
Kriger KM Hero JM Ashton KJ (2006) Cost efficiency in thedetection of chytridiomycosis using PCR assay Dis AquatOrg 71 149minus154
McClain WR Romaire RP (2007) Procambarid crawfish lifehistory and biology Publication No 2403 Southern Re -gional Aquaculture Center Mississippi State UniversityStoneville MS
McMahon TA Brannelly LA Chatfield MWH Johnson PTJand others (2013) Chytrid fungus Batrachochytrium den-drobatidis has nonamphibian hosts and releases chemi-cals that cause pathology in the absence of infectionProc Natl Acad Sci USA 110 210minus215
Murray K Skerratt LF Marantelli G Berger L Hunter DMahony MJ Hines H (2011) Hygiene protocols for thecontrol of diseases in Australian frogs A report for theAustralian Government Department of SustainabilityEnvironment Water Population and Communities Avail-able at wwwenvironmentgovau resource hygiene-protocols-control-diseases-australian-frogs
Newcombe RG (1998) Two-sided confidence intervals forthe single proportion comparison of seven methods StatMed 17 857minus872
Parker JM Mikaelian I Hahn N Diggs HE (2002) Clinicaldiagnosis and treatment of epidermal chytridiomycosisin African clawed frogs (Xenopus tropicalis) Comp Med52 265minus268
Rosenblum EB James TY Zamudio KR Poorten TJ and oth-ers (2013) Complex history of the amphibian-killingchytrid fungus revealed with genome resequencingdata Proc Natl Acad Sci USA 110 9385minus9390
Schloegel LM Picco AM Kilpatrick M Davies AJ HyattAD Daszak P (2009) Magnitude of the US trade inamphibians and presence of Batrachochytrium dendro-batidis and ranavirus infection in imported North Ameri-can bullfrogs (Rana catesbeiana) Biol Conserv 142 1420minus1426
Schloegel LM Ferreira CM James TY Hipolito M and oth-ers (2010) The North American bullfrog as a reservoir forthe spread of Batrachochytrium dendrobatidis in BrazilAnim Conserv 13 53minus61
Shapard EJ Moss AS San Francisco MJ (2012) Batra-chochytrium dendrobatidis can infect and cause mortal-ity in the nematode Caenorhabditis elegans Myco-pathologia 173 121minus126
Skerratt LF Berger L Speare R Cashins S and others (2007)Spread of chytridiomycosis has caused the rapid globaldecline and extinction of frogs EcoHealth 4 125minus134
Stuart SN Chanson JS Cox NA Young BE Rodrigues ASLFischman DL Waller RW (2004) Status and trends ofamphibian declines and extinctions worldwide Science306 1783minus1786
Voyles J Young S Berger L Campbell C and others (2009)Pathogenesis of chytridiomycosis a cause of catastrophicamphibian declines Science 326 582minus585
Warner RE (1968) The role of introduced diseases in theextinction of the endemic Hawaiian avifauna Condor 70 101minus120
Editorial responsibility Louise Rollins-Smith Nashville Tennessee USA
Submitted June 16 2014 Accepted October 30 2014Proofs received from author(s) December 22 2014
Aut
hor c
opy
Dis Aquat Org 112 229ndash235 2015
inhibition and all wells contai ned an internal positivecontrol (VICtrade IPC Applied Biosystems) Positiveand negative controls previously extracted fromswabs of known Bd-positive and Bd-negative captiveRana catesbeiana and a dilution series of Bd stan-dards (provided by A Hyatt) were included in eachqPCR run Samples were run in singlicate (Kriger etal 2006) and DNA was not diluted prior to analysis
Statistical analysis
Microsoft Excel was used to calculate 95 confi-dence intervals on the proportion of Bd-positive indi-
viduals following Newcombe (1998) Zoosporeequivalents (ZE) in the GI tract in Bd-positive indi-viduals were compared between farmed and naturalpopulations using a 2-tailed t-test in SPSS (v21)
RESULTS
Bd prevalence in Louisiana crayfish was seasonalwith higher prevalence in farmed and wild-caughtanimals in the spring than in wild-caught animals inthe fall (Fig 2 Table 1) This pattern mirrors seasonaltrends in Bd prevalence for amphibian species in theregion some of which were sampled from the samesites as our wild-caught crayfish samples (Fig 2modified from Brannelly et al 2012) Farmed crayfishtested positive for Bd infection in the spring (Febru-ary and April 2012) with a prevalence of 60 (load1074 plusmn 396 (SD) ZE per swab n = 5) and no farmedanimals were available for testing in the fall Bd waspresent in natural crayfish populations in the springwith a prevalence of 331 (load 2217 plusmn 815 ZE perswab n = 9) but no Bd was detected from those samesites in the fall Farmed crayfish had a significantlylower ZE than wild-caught animals (t-test t12 = 2914p = 0013)
DISCUSSION
The seasonal pattern of Bd prevalence in wild-caught Louisiana crayfish in 2012 mirrored that seenin amphibian species sampled in the same region in2010 and 2011 with higher prevalence during thespring breeding season and no infection detectedduring the fall season Prevalences were lower in the
232
000
010
020
030
040
050
060
Pro
por
tion
infe
cted
Janu
ary
Febr
uary
M
arch
Apr
il M
ay
June
Ju
ly Aug
ust
Septe
mbe
r Oct
ober
Nov
embe
r Dec
embe
r
Fig 2 Proportion of Batrachochytrium dendrobatidis (Bd)-positive crayfish sampled in 2012 in southeastern Louisiana(d) Wild-caught and (d) farm-collected animals The pro-portion of (s with dashed error bars) Bd-infected amphi -bians in southeastern Loui siana sampled in 2010 to 2011is also shown modified from Brannelly et al (2012 their
Fig 2) The error bars are 95 confidence intervals
Site Latitude Longitude Month No Bd + ind Average zoospore Site type(degN) (degW) total sampled load (plusmnSD)
A 2989 89953 February 6 35 2167 plusmn 1147 NaturalMarch 0 62April 0 40
September 0 5B 30108 90435 April 3 52 1354 plusmn 618 Natural
November 0 77C 30214 9203 April 3 22 1126 plusmn 549 FarmD 29701 91206 April 0 22 FarmE 29888 91206 April 2 20 479 plusmn 662 FarmF Unknown February 0 20 Farm
Table 1 Sampling of natural and farmed crayfish Sites are (A) Tulane Universityrsquos F Edward Herbert Research Center inBelle Chase (natural) (B) Maurepas Wildlife Management Area (natural) (C) Lafayette (farm) (D) Morgan City (farm) and (E)Belle River (farm) and (F) unknown location in Louisiana (farm) Bd Batrachochytrium dendrobatidis See Fig 1 for sampling
locations in Louisiana USA
Aut
hor c
opy
Brannelly et al Chytrid fungal infection in crayfish
crayfish than in the amphibians sampled in a givenmonth (Fig 2) However since crayfish and amphi -bians were sampled on different dates and in differ-ent years and in some cases also from different sitescaution should be used in comparing prevalence esti-mates between these 2 taxa In this study we did notfind Bd-infected crayfish in September (05 Bd+95 CI = 0 to 435 prevalence) or November (077Bd+ 95 CI = 0 to 48 prevalence) of 2012 butMcMahon et al (2013) found Bd at 173 prevalencein Louisiana crayfish in September of 2011 Our fail-ure to detect Bd in the fall could be due to yearly vari-ation in disease prevalence or an artefact of our smallsample size early in the fall Although our study didnot support the idea that crayfish act as disease reser-voirs during the late summer or fall months we diddetect Bd in crayfish during the spring and thereforecannot rule out the idea that these animals may playan important role in disease dynamics Summer tem-peratures in Louisiana typically exceed the thermaltolerance of Bd and amphibians may clear theirinfections during this time It is possible that crayfishare also able to clear Bd infections during high sum-mer temperatures but more research is needed totest this prediction
Farmed and wild-caught crayfish Bd prevalenceswere similar to each other in the spring when farmedcrayfish were available (Fig 2) It remains unclearwhether the effects of Bd on crayfish health shouldbe an important consideration for farmers Bd infec-tion has been shown to cause mortality in laboratory-exposed crayfish (McMahon et al 2013) but itsimpact on farmed and natural crayfish populationsremains unstudied While we did not examine ani-mals for pathology we did find that farmed crayfishhad significantly lower Bd loads than wild-caughtanimals The different conditions these animals ex -perienced directly prior to sampling could also havecontributed to the difference in pathogen load Wild-caught animals were separated upon capture andkept alive only for transport between the field andthe laboratory while the farmed crayfish were keptalive communally in a refrigerated room for up to 3 dbetween capture and sampling However beforeconcluding that farmed animals have lower patho-gen loads than natural populations a sample of cray-fish directly from the farms analogous to our naturalpopulation sampling methods is needed While com-munal refrigerated conditions might have impactedour Bd load results we found that farmed animalsheld in analogous conditions to those destined fortrade were positive for Bd at a similar prevalence towild-caught animals Six percent of the farmed indi-
viduals left the farm and were sold for consumptionwhile infected with Bd
Minimizing disease transmission between wildlifeand farmed animals is important and this topic hasbeen extensively studied in domesticated animalsglobally Wild animals can carry disease and infectindividuals in farmed populations which is particu-larly concerning for livestock and farm owners (egMycobacterium bovis Delahay et al 2001) Farmedanimals can also introduce spread and maintaininfection in natural populations which can be de -trimental to vulnerable species living near farms(eg chickens introducing avian malaria into nativeHawaiian bird populations Warner 1968) Bothfarmed and natural crayfish populations in south-eastern Louisiana were Bd positive in the springwhich is the time when they commonly share habi-tats with amphibians Crayfish infected with Bd inclose proximity with amphibian populations mayplay a role in amphibian disease dynamics espe-cially if infected crayfish are transported to areas thatwere previously Bd free
Our study found that farmed crayfish were Bdpositive during the trading season If farmed cray-fish that are globally traded are infected with Bdthen even a low prevalence of infection could haveimplications for global amphibian conservation Theinternational crayfish market is large with 40 000 to60 000 t traded per annum (Holdich 1993) whichequates to approximately 08 to 12 billion indivi -duals The North American species Procambarusclarkii makes up 85 of all crayfish trade (Holdich1993) Most of these globally traded crayfish origi-nate from farms in Louisiana (Holdich 1993) Cray-fish are the most widely introduced freshwatertaxon globally (Helms et al 2013) likely becomingestablished after escaping from farms (Gherardi2006) as farms rarely exercise biosecurity measuresto ensure isolation from natural populations Addi-tionally P clarkii is a highly successful invasivespecies once it escapes its native range It can re -produce by par thenogenesis can survive in ephe -meral ponds by burrowing deep into mud banks intimes of drought (which may help Bd persist) andtends to escape from farms and travel quickly tolocal ponds where it then reproduces (Gherardi2006) There is already unequivocal evidence thatthe invasive P clarkii has driven amphi bian declinethrough predation rather than pathogen spread inmany regions of Europe (Cruz et al 2006ab Cruz2008 Ficetola et al 2012) Although there is a clearcorrelation between amphi bian de clines and inva-sion by P clarkii in Europe the potential relation-
233A
utho
r cop
y
Dis Aquat Org 112 229ndash235 2015
ship between amphibian declines crayfish invasionand disease has not been explored
Trade of diseased individuals whether it be amphi -bians or other aquatic organisms might explain thespread of the Bd pathogen globally (Fisher amp Garner2007 Garner et al 2009 Schloegel et al 2009 Kolbyet al 2014) While the origin of Bd is still unclear itappears that strains of Bd found in parts of Europeare most closely related to strains found in the east-ern USA (Rosenblum et al 2013) which could beexplained by the import of invasive North Americanspecies like Rana catesbeiana and P clarkii for con-sumer purposes Spain is one place where the role ofcrayfish as an important alternative host for Bd de -serves further study as this country has been experi-encing some of the greatest amphibian declines inEurope (Gherardi et al 2001 Garner et al 2006) Thedeclines have been attributed to both amphibian pre-dation by the invasive P clarkii as well as chytrid-iomycosis (Gherardi et al 2001 Garner et al 2006)P clarkii were introduced into Spain twice for thepurposes of farming for consumption from farms inLouisiana between 1973 and 1974 (Hasburgo-Lorena1986) Since then crayfish have spread into Portugaland other parts of Europe either through importationfrom Spain or migration out of farms It is possiblethat Bd was introduced into the area through im -ported crayfish and subsequently spread to naturalamphibian populations Although amphibian tradehas been implicated in the global spread of Bd(Fisher amp Garner 2007 Garner et al 2009 Schloegelet al 2009) even regions devoid of commercial amphi -bian importation have been exposed to this patho-gen For example Bd has recently been de tected forthe first time in amphibians coming from Madagas-car (Kolby 2014) where North American Procam-barus spp have been introduced into the wild andare now rapidly spreading (Jones et al 2009) Whilethe correlation between amphibian disease and cray-fish presence has not been studied in EuropeanMalagasy or other ecosystems our study suggeststhat farmed P clarkii can carry Bd and that createsthe risk of zoonotic spill-over if appropriate controlmeasures are not taken
Bd disease dynamics are challenging to understandand predict and when we consider non-amphi bianhosts the story becomes increasingly complex Thereare current biosecurity protocols enacted globally toreduce disease spread through amphibian trade andcontaminated field equipment (Green et al 2009Murray et al 2011) but little attention has beendirected towards the prevention of Bd spread by non-amphibian hosts In this study we found that both
natural and farmed crayfish populations carry Bdinfection in Louisiana and that infection is seasonalin wild-caught animals similar to the pattern of pre -valence seen in amphibian populations in the sameregion While prevalence in our study was low cray-fish species may still be an important part of the disease dynamic especially because they are mass-distributed globally We would argue that a 60Bd infection prevalence in a species that is as widelytraded as P clarkii represents a significant risk anddeserves to be considered in biosecurity measures
Acknowledgements We thank Matthew Robak for helprunning the qPCR analysis Matthew Chatfield for help inthe laboratory and the field and Jonathan Kolby for com-menting on the manuscript
LITERATURE CITED
Boyle DG Boyle DB Olsen V Morgan JAT Hyatt AD (2004)Rapid quantitative detection of chytridiomycosis (Batra-chochytrium dendrobatidis) in amphibian samples usingreal-time Taqman PCR assay Dis Aquat Org 60 141minus148
Brannelly LA Chatfield MWH Richards-Zawacki CL (2012)Field and laboratory studies of the susceptibility of thegreen treefrog (Hyla cinerea) to Batrachochytrium den-drobatidis infection PLoS ONE 7 e38473
Chatfield MWH Brannelly LA Robak MJ Freeborn L Lail-vaux SP Richards-Zawacki CL (2013) Fitness conse-quences of infection by Batrachochytrium dendrobatidisin northern leopard frogs (Lithobates pipiens) EcoHealth10 90minus98
Cruz J (2008) Collapse of the amphibian community of thePaul do Boquilobo Natural Reserve (central Portugal)after the arrival of the exotic American crayfish HerpetolJ 18 197minus204
Cruz MJ Pascoal S Tejedo M Rebelo R (2006a) Predationby an exotic crayfish Procambarus clarkii on natterjacktoad Bufo calamita embryos its role on the exclusion ofthis amphibian from its breeding ponds Copeia 2006 274minus280
Cruz MJ Rebelo R Crespo EG (2006b) Effects of an intro-duced crayfish Procambarus clarkii on the distributionof south-western Iberian amphibians in their breedinghabitats Ecography 29 329minus338
Delahay RJ Cheeseman CL Clifton-Hadley RS (2001)Wildlife disease reservoirs the epidemiology of Myco -bacterium bovis infection in the European badger (Melesmeles) and other British mammals Tuberculosis (Edinb)81 43minus49
Ficetola GF Siesa ME De Bernardi F Padoa-Schioppa E(2012) Complex impact of an invasive crayfish on fresh-water food webs Biodivers Conserv 21 2641minus2651
Fisher MC Garner TWJ (2007) The relationship betweenthe emergence of Batrachochytrium dendrobatidis theinternational trade in amphibians and introduced amphi -bian species Fungal Biol Rev 21 2minus9
Garmyn A Van Rooij P Pasmans F Hellebuyck T Van DenBroeck W Haesebrouck F Martel A (2012) Waterfowl potential environmental reservoirs of the chytrid fungusBatrachochytrium dendrobatidis PLoS ONE 7 e35038
234A
utho
r cop
y
Brannelly et al Chytrid fungal infection in crayfish 235
Garner TWJ Perkins MW Govindarajulu P Seglie DWalker S Cunningham AA Fisher MC (2006) The emer -ging amphibian pathogen Batrachochytrium dendroba-tidis globally infects introduced populations of the NorthAmerican bullfrog Rana catesbeiana Biol Lett 2 455minus459
Garner TWJ Stephen I Wombwell E Fisher MC (2009) Theamphibian trade bans or best practice EcoHealth 6 148minus151
Gherardi F (2006) Crayfish invading Europe the case studyof Procambarus clarkii Mar Freshw Behav Physiol 39 175minus191
Gherardi F Renai B Corti C (2001) Crayfish predation ontadpoles a comparison between a native (Austropotamo-bius pallipes) and an alien species (Procambarus clarkii)Bull Fr Peche Piscicult 361 659minus668
Green DE Gray MJ Miller DL (2009) Disease monitoringand biosecurity Forestry Wildlife and Fisheries Publica-tions and Other Works Knoxville TN http traceten-nesseeedu utk_ forepubs2
Hasburgo-Lorena A (1986) The status of Procambarusclarkii population in Spain Freshw Crayfish 6 131minus136
Helms B Loughman ZJ Brown BL Stoeckel J (2013) Recentadvances in crayfish biology ecology and conservationFreshw Sci 32 1273minus1275
Holdich DM (1993) A review of astaciculture freshwatercrayfish farming Aquat Living Resour 6 307minus317
Jones JPG Rasamy JR Harvey A Toon A and others (2009)The perfect invader a parthenogenic crayfish poses anew threat to Madagascarrsquos freshwater biodiversity BiolInvasions 11 1475minus1482
Kilburn VL Ibaacutentildeez R Green DM (2011) Reptiles as potentialvectors and hosts of the amphibian pathogen Batra-chochytrium dendrobatidis in Panama Dis Aquat Org97 127minus134
Kolby JE (2014) Presence of the amphibian chytrid fungusBatrachochytrium dendrobatidis in native amphibiansexported from Madagascar PLoS ONE 9 e89660
Kolby JE Smith KM Berger L Karesh WB Preston APessier AP Skerratt LF (2014) First evidence of amphib-ian chytrid fungus (Batrachochytrium dendrobatidis) andranavirus in Hong Kong amphibian trade PLoS ONE 9 e90750
Kriger KM Hero JM Ashton KJ (2006) Cost efficiency in thedetection of chytridiomycosis using PCR assay Dis AquatOrg 71 149minus154
McClain WR Romaire RP (2007) Procambarid crawfish lifehistory and biology Publication No 2403 Southern Re -gional Aquaculture Center Mississippi State UniversityStoneville MS
McMahon TA Brannelly LA Chatfield MWH Johnson PTJand others (2013) Chytrid fungus Batrachochytrium den-drobatidis has nonamphibian hosts and releases chemi-cals that cause pathology in the absence of infectionProc Natl Acad Sci USA 110 210minus215
Murray K Skerratt LF Marantelli G Berger L Hunter DMahony MJ Hines H (2011) Hygiene protocols for thecontrol of diseases in Australian frogs A report for theAustralian Government Department of SustainabilityEnvironment Water Population and Communities Avail-able at wwwenvironmentgovau resource hygiene-protocols-control-diseases-australian-frogs
Newcombe RG (1998) Two-sided confidence intervals forthe single proportion comparison of seven methods StatMed 17 857minus872
Parker JM Mikaelian I Hahn N Diggs HE (2002) Clinicaldiagnosis and treatment of epidermal chytridiomycosisin African clawed frogs (Xenopus tropicalis) Comp Med52 265minus268
Rosenblum EB James TY Zamudio KR Poorten TJ and oth-ers (2013) Complex history of the amphibian-killingchytrid fungus revealed with genome resequencingdata Proc Natl Acad Sci USA 110 9385minus9390
Schloegel LM Picco AM Kilpatrick M Davies AJ HyattAD Daszak P (2009) Magnitude of the US trade inamphibians and presence of Batrachochytrium dendro-batidis and ranavirus infection in imported North Ameri-can bullfrogs (Rana catesbeiana) Biol Conserv 142 1420minus1426
Schloegel LM Ferreira CM James TY Hipolito M and oth-ers (2010) The North American bullfrog as a reservoir forthe spread of Batrachochytrium dendrobatidis in BrazilAnim Conserv 13 53minus61
Shapard EJ Moss AS San Francisco MJ (2012) Batra-chochytrium dendrobatidis can infect and cause mortal-ity in the nematode Caenorhabditis elegans Myco-pathologia 173 121minus126
Skerratt LF Berger L Speare R Cashins S and others (2007)Spread of chytridiomycosis has caused the rapid globaldecline and extinction of frogs EcoHealth 4 125minus134
Stuart SN Chanson JS Cox NA Young BE Rodrigues ASLFischman DL Waller RW (2004) Status and trends ofamphibian declines and extinctions worldwide Science306 1783minus1786
Voyles J Young S Berger L Campbell C and others (2009)Pathogenesis of chytridiomycosis a cause of catastrophicamphibian declines Science 326 582minus585
Warner RE (1968) The role of introduced diseases in theextinction of the endemic Hawaiian avifauna Condor 70 101minus120
Editorial responsibility Louise Rollins-Smith Nashville Tennessee USA
Submitted June 16 2014 Accepted October 30 2014Proofs received from author(s) December 22 2014
Aut
hor c
opy
Brannelly et al Chytrid fungal infection in crayfish
crayfish than in the amphibians sampled in a givenmonth (Fig 2) However since crayfish and amphi -bians were sampled on different dates and in differ-ent years and in some cases also from different sitescaution should be used in comparing prevalence esti-mates between these 2 taxa In this study we did notfind Bd-infected crayfish in September (05 Bd+95 CI = 0 to 435 prevalence) or November (077Bd+ 95 CI = 0 to 48 prevalence) of 2012 butMcMahon et al (2013) found Bd at 173 prevalencein Louisiana crayfish in September of 2011 Our fail-ure to detect Bd in the fall could be due to yearly vari-ation in disease prevalence or an artefact of our smallsample size early in the fall Although our study didnot support the idea that crayfish act as disease reser-voirs during the late summer or fall months we diddetect Bd in crayfish during the spring and thereforecannot rule out the idea that these animals may playan important role in disease dynamics Summer tem-peratures in Louisiana typically exceed the thermaltolerance of Bd and amphibians may clear theirinfections during this time It is possible that crayfishare also able to clear Bd infections during high sum-mer temperatures but more research is needed totest this prediction
Farmed and wild-caught crayfish Bd prevalenceswere similar to each other in the spring when farmedcrayfish were available (Fig 2) It remains unclearwhether the effects of Bd on crayfish health shouldbe an important consideration for farmers Bd infec-tion has been shown to cause mortality in laboratory-exposed crayfish (McMahon et al 2013) but itsimpact on farmed and natural crayfish populationsremains unstudied While we did not examine ani-mals for pathology we did find that farmed crayfishhad significantly lower Bd loads than wild-caughtanimals The different conditions these animals ex -perienced directly prior to sampling could also havecontributed to the difference in pathogen load Wild-caught animals were separated upon capture andkept alive only for transport between the field andthe laboratory while the farmed crayfish were keptalive communally in a refrigerated room for up to 3 dbetween capture and sampling However beforeconcluding that farmed animals have lower patho-gen loads than natural populations a sample of cray-fish directly from the farms analogous to our naturalpopulation sampling methods is needed While com-munal refrigerated conditions might have impactedour Bd load results we found that farmed animalsheld in analogous conditions to those destined fortrade were positive for Bd at a similar prevalence towild-caught animals Six percent of the farmed indi-
viduals left the farm and were sold for consumptionwhile infected with Bd
Minimizing disease transmission between wildlifeand farmed animals is important and this topic hasbeen extensively studied in domesticated animalsglobally Wild animals can carry disease and infectindividuals in farmed populations which is particu-larly concerning for livestock and farm owners (egMycobacterium bovis Delahay et al 2001) Farmedanimals can also introduce spread and maintaininfection in natural populations which can be de -trimental to vulnerable species living near farms(eg chickens introducing avian malaria into nativeHawaiian bird populations Warner 1968) Bothfarmed and natural crayfish populations in south-eastern Louisiana were Bd positive in the springwhich is the time when they commonly share habi-tats with amphibians Crayfish infected with Bd inclose proximity with amphibian populations mayplay a role in amphibian disease dynamics espe-cially if infected crayfish are transported to areas thatwere previously Bd free
Our study found that farmed crayfish were Bdpositive during the trading season If farmed cray-fish that are globally traded are infected with Bdthen even a low prevalence of infection could haveimplications for global amphibian conservation Theinternational crayfish market is large with 40 000 to60 000 t traded per annum (Holdich 1993) whichequates to approximately 08 to 12 billion indivi -duals The North American species Procambarusclarkii makes up 85 of all crayfish trade (Holdich1993) Most of these globally traded crayfish origi-nate from farms in Louisiana (Holdich 1993) Cray-fish are the most widely introduced freshwatertaxon globally (Helms et al 2013) likely becomingestablished after escaping from farms (Gherardi2006) as farms rarely exercise biosecurity measuresto ensure isolation from natural populations Addi-tionally P clarkii is a highly successful invasivespecies once it escapes its native range It can re -produce by par thenogenesis can survive in ephe -meral ponds by burrowing deep into mud banks intimes of drought (which may help Bd persist) andtends to escape from farms and travel quickly tolocal ponds where it then reproduces (Gherardi2006) There is already unequivocal evidence thatthe invasive P clarkii has driven amphi bian declinethrough predation rather than pathogen spread inmany regions of Europe (Cruz et al 2006ab Cruz2008 Ficetola et al 2012) Although there is a clearcorrelation between amphi bian de clines and inva-sion by P clarkii in Europe the potential relation-
233A
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y
Dis Aquat Org 112 229ndash235 2015
ship between amphibian declines crayfish invasionand disease has not been explored
Trade of diseased individuals whether it be amphi -bians or other aquatic organisms might explain thespread of the Bd pathogen globally (Fisher amp Garner2007 Garner et al 2009 Schloegel et al 2009 Kolbyet al 2014) While the origin of Bd is still unclear itappears that strains of Bd found in parts of Europeare most closely related to strains found in the east-ern USA (Rosenblum et al 2013) which could beexplained by the import of invasive North Americanspecies like Rana catesbeiana and P clarkii for con-sumer purposes Spain is one place where the role ofcrayfish as an important alternative host for Bd de -serves further study as this country has been experi-encing some of the greatest amphibian declines inEurope (Gherardi et al 2001 Garner et al 2006) Thedeclines have been attributed to both amphibian pre-dation by the invasive P clarkii as well as chytrid-iomycosis (Gherardi et al 2001 Garner et al 2006)P clarkii were introduced into Spain twice for thepurposes of farming for consumption from farms inLouisiana between 1973 and 1974 (Hasburgo-Lorena1986) Since then crayfish have spread into Portugaland other parts of Europe either through importationfrom Spain or migration out of farms It is possiblethat Bd was introduced into the area through im -ported crayfish and subsequently spread to naturalamphibian populations Although amphibian tradehas been implicated in the global spread of Bd(Fisher amp Garner 2007 Garner et al 2009 Schloegelet al 2009) even regions devoid of commercial amphi -bian importation have been exposed to this patho-gen For example Bd has recently been de tected forthe first time in amphibians coming from Madagas-car (Kolby 2014) where North American Procam-barus spp have been introduced into the wild andare now rapidly spreading (Jones et al 2009) Whilethe correlation between amphibian disease and cray-fish presence has not been studied in EuropeanMalagasy or other ecosystems our study suggeststhat farmed P clarkii can carry Bd and that createsthe risk of zoonotic spill-over if appropriate controlmeasures are not taken
Bd disease dynamics are challenging to understandand predict and when we consider non-amphi bianhosts the story becomes increasingly complex Thereare current biosecurity protocols enacted globally toreduce disease spread through amphibian trade andcontaminated field equipment (Green et al 2009Murray et al 2011) but little attention has beendirected towards the prevention of Bd spread by non-amphibian hosts In this study we found that both
natural and farmed crayfish populations carry Bdinfection in Louisiana and that infection is seasonalin wild-caught animals similar to the pattern of pre -valence seen in amphibian populations in the sameregion While prevalence in our study was low cray-fish species may still be an important part of the disease dynamic especially because they are mass-distributed globally We would argue that a 60Bd infection prevalence in a species that is as widelytraded as P clarkii represents a significant risk anddeserves to be considered in biosecurity measures
Acknowledgements We thank Matthew Robak for helprunning the qPCR analysis Matthew Chatfield for help inthe laboratory and the field and Jonathan Kolby for com-menting on the manuscript
LITERATURE CITED
Boyle DG Boyle DB Olsen V Morgan JAT Hyatt AD (2004)Rapid quantitative detection of chytridiomycosis (Batra-chochytrium dendrobatidis) in amphibian samples usingreal-time Taqman PCR assay Dis Aquat Org 60 141minus148
Brannelly LA Chatfield MWH Richards-Zawacki CL (2012)Field and laboratory studies of the susceptibility of thegreen treefrog (Hyla cinerea) to Batrachochytrium den-drobatidis infection PLoS ONE 7 e38473
Chatfield MWH Brannelly LA Robak MJ Freeborn L Lail-vaux SP Richards-Zawacki CL (2013) Fitness conse-quences of infection by Batrachochytrium dendrobatidisin northern leopard frogs (Lithobates pipiens) EcoHealth10 90minus98
Cruz J (2008) Collapse of the amphibian community of thePaul do Boquilobo Natural Reserve (central Portugal)after the arrival of the exotic American crayfish HerpetolJ 18 197minus204
Cruz MJ Pascoal S Tejedo M Rebelo R (2006a) Predationby an exotic crayfish Procambarus clarkii on natterjacktoad Bufo calamita embryos its role on the exclusion ofthis amphibian from its breeding ponds Copeia 2006 274minus280
Cruz MJ Rebelo R Crespo EG (2006b) Effects of an intro-duced crayfish Procambarus clarkii on the distributionof south-western Iberian amphibians in their breedinghabitats Ecography 29 329minus338
Delahay RJ Cheeseman CL Clifton-Hadley RS (2001)Wildlife disease reservoirs the epidemiology of Myco -bacterium bovis infection in the European badger (Melesmeles) and other British mammals Tuberculosis (Edinb)81 43minus49
Ficetola GF Siesa ME De Bernardi F Padoa-Schioppa E(2012) Complex impact of an invasive crayfish on fresh-water food webs Biodivers Conserv 21 2641minus2651
Fisher MC Garner TWJ (2007) The relationship betweenthe emergence of Batrachochytrium dendrobatidis theinternational trade in amphibians and introduced amphi -bian species Fungal Biol Rev 21 2minus9
Garmyn A Van Rooij P Pasmans F Hellebuyck T Van DenBroeck W Haesebrouck F Martel A (2012) Waterfowl potential environmental reservoirs of the chytrid fungusBatrachochytrium dendrobatidis PLoS ONE 7 e35038
234A
utho
r cop
y
Brannelly et al Chytrid fungal infection in crayfish 235
Garner TWJ Perkins MW Govindarajulu P Seglie DWalker S Cunningham AA Fisher MC (2006) The emer -ging amphibian pathogen Batrachochytrium dendroba-tidis globally infects introduced populations of the NorthAmerican bullfrog Rana catesbeiana Biol Lett 2 455minus459
Garner TWJ Stephen I Wombwell E Fisher MC (2009) Theamphibian trade bans or best practice EcoHealth 6 148minus151
Gherardi F (2006) Crayfish invading Europe the case studyof Procambarus clarkii Mar Freshw Behav Physiol 39 175minus191
Gherardi F Renai B Corti C (2001) Crayfish predation ontadpoles a comparison between a native (Austropotamo-bius pallipes) and an alien species (Procambarus clarkii)Bull Fr Peche Piscicult 361 659minus668
Green DE Gray MJ Miller DL (2009) Disease monitoringand biosecurity Forestry Wildlife and Fisheries Publica-tions and Other Works Knoxville TN http traceten-nesseeedu utk_ forepubs2
Hasburgo-Lorena A (1986) The status of Procambarusclarkii population in Spain Freshw Crayfish 6 131minus136
Helms B Loughman ZJ Brown BL Stoeckel J (2013) Recentadvances in crayfish biology ecology and conservationFreshw Sci 32 1273minus1275
Holdich DM (1993) A review of astaciculture freshwatercrayfish farming Aquat Living Resour 6 307minus317
Jones JPG Rasamy JR Harvey A Toon A and others (2009)The perfect invader a parthenogenic crayfish poses anew threat to Madagascarrsquos freshwater biodiversity BiolInvasions 11 1475minus1482
Kilburn VL Ibaacutentildeez R Green DM (2011) Reptiles as potentialvectors and hosts of the amphibian pathogen Batra-chochytrium dendrobatidis in Panama Dis Aquat Org97 127minus134
Kolby JE (2014) Presence of the amphibian chytrid fungusBatrachochytrium dendrobatidis in native amphibiansexported from Madagascar PLoS ONE 9 e89660
Kolby JE Smith KM Berger L Karesh WB Preston APessier AP Skerratt LF (2014) First evidence of amphib-ian chytrid fungus (Batrachochytrium dendrobatidis) andranavirus in Hong Kong amphibian trade PLoS ONE 9 e90750
Kriger KM Hero JM Ashton KJ (2006) Cost efficiency in thedetection of chytridiomycosis using PCR assay Dis AquatOrg 71 149minus154
McClain WR Romaire RP (2007) Procambarid crawfish lifehistory and biology Publication No 2403 Southern Re -gional Aquaculture Center Mississippi State UniversityStoneville MS
McMahon TA Brannelly LA Chatfield MWH Johnson PTJand others (2013) Chytrid fungus Batrachochytrium den-drobatidis has nonamphibian hosts and releases chemi-cals that cause pathology in the absence of infectionProc Natl Acad Sci USA 110 210minus215
Murray K Skerratt LF Marantelli G Berger L Hunter DMahony MJ Hines H (2011) Hygiene protocols for thecontrol of diseases in Australian frogs A report for theAustralian Government Department of SustainabilityEnvironment Water Population and Communities Avail-able at wwwenvironmentgovau resource hygiene-protocols-control-diseases-australian-frogs
Newcombe RG (1998) Two-sided confidence intervals forthe single proportion comparison of seven methods StatMed 17 857minus872
Parker JM Mikaelian I Hahn N Diggs HE (2002) Clinicaldiagnosis and treatment of epidermal chytridiomycosisin African clawed frogs (Xenopus tropicalis) Comp Med52 265minus268
Rosenblum EB James TY Zamudio KR Poorten TJ and oth-ers (2013) Complex history of the amphibian-killingchytrid fungus revealed with genome resequencingdata Proc Natl Acad Sci USA 110 9385minus9390
Schloegel LM Picco AM Kilpatrick M Davies AJ HyattAD Daszak P (2009) Magnitude of the US trade inamphibians and presence of Batrachochytrium dendro-batidis and ranavirus infection in imported North Ameri-can bullfrogs (Rana catesbeiana) Biol Conserv 142 1420minus1426
Schloegel LM Ferreira CM James TY Hipolito M and oth-ers (2010) The North American bullfrog as a reservoir forthe spread of Batrachochytrium dendrobatidis in BrazilAnim Conserv 13 53minus61
Shapard EJ Moss AS San Francisco MJ (2012) Batra-chochytrium dendrobatidis can infect and cause mortal-ity in the nematode Caenorhabditis elegans Myco-pathologia 173 121minus126
Skerratt LF Berger L Speare R Cashins S and others (2007)Spread of chytridiomycosis has caused the rapid globaldecline and extinction of frogs EcoHealth 4 125minus134
Stuart SN Chanson JS Cox NA Young BE Rodrigues ASLFischman DL Waller RW (2004) Status and trends ofamphibian declines and extinctions worldwide Science306 1783minus1786
Voyles J Young S Berger L Campbell C and others (2009)Pathogenesis of chytridiomycosis a cause of catastrophicamphibian declines Science 326 582minus585
Warner RE (1968) The role of introduced diseases in theextinction of the endemic Hawaiian avifauna Condor 70 101minus120
Editorial responsibility Louise Rollins-Smith Nashville Tennessee USA
Submitted June 16 2014 Accepted October 30 2014Proofs received from author(s) December 22 2014
Aut
hor c
opy
Dis Aquat Org 112 229ndash235 2015
ship between amphibian declines crayfish invasionand disease has not been explored
Trade of diseased individuals whether it be amphi -bians or other aquatic organisms might explain thespread of the Bd pathogen globally (Fisher amp Garner2007 Garner et al 2009 Schloegel et al 2009 Kolbyet al 2014) While the origin of Bd is still unclear itappears that strains of Bd found in parts of Europeare most closely related to strains found in the east-ern USA (Rosenblum et al 2013) which could beexplained by the import of invasive North Americanspecies like Rana catesbeiana and P clarkii for con-sumer purposes Spain is one place where the role ofcrayfish as an important alternative host for Bd de -serves further study as this country has been experi-encing some of the greatest amphibian declines inEurope (Gherardi et al 2001 Garner et al 2006) Thedeclines have been attributed to both amphibian pre-dation by the invasive P clarkii as well as chytrid-iomycosis (Gherardi et al 2001 Garner et al 2006)P clarkii were introduced into Spain twice for thepurposes of farming for consumption from farms inLouisiana between 1973 and 1974 (Hasburgo-Lorena1986) Since then crayfish have spread into Portugaland other parts of Europe either through importationfrom Spain or migration out of farms It is possiblethat Bd was introduced into the area through im -ported crayfish and subsequently spread to naturalamphibian populations Although amphibian tradehas been implicated in the global spread of Bd(Fisher amp Garner 2007 Garner et al 2009 Schloegelet al 2009) even regions devoid of commercial amphi -bian importation have been exposed to this patho-gen For example Bd has recently been de tected forthe first time in amphibians coming from Madagas-car (Kolby 2014) where North American Procam-barus spp have been introduced into the wild andare now rapidly spreading (Jones et al 2009) Whilethe correlation between amphibian disease and cray-fish presence has not been studied in EuropeanMalagasy or other ecosystems our study suggeststhat farmed P clarkii can carry Bd and that createsthe risk of zoonotic spill-over if appropriate controlmeasures are not taken
Bd disease dynamics are challenging to understandand predict and when we consider non-amphi bianhosts the story becomes increasingly complex Thereare current biosecurity protocols enacted globally toreduce disease spread through amphibian trade andcontaminated field equipment (Green et al 2009Murray et al 2011) but little attention has beendirected towards the prevention of Bd spread by non-amphibian hosts In this study we found that both
natural and farmed crayfish populations carry Bdinfection in Louisiana and that infection is seasonalin wild-caught animals similar to the pattern of pre -valence seen in amphibian populations in the sameregion While prevalence in our study was low cray-fish species may still be an important part of the disease dynamic especially because they are mass-distributed globally We would argue that a 60Bd infection prevalence in a species that is as widelytraded as P clarkii represents a significant risk anddeserves to be considered in biosecurity measures
Acknowledgements We thank Matthew Robak for helprunning the qPCR analysis Matthew Chatfield for help inthe laboratory and the field and Jonathan Kolby for com-menting on the manuscript
LITERATURE CITED
Boyle DG Boyle DB Olsen V Morgan JAT Hyatt AD (2004)Rapid quantitative detection of chytridiomycosis (Batra-chochytrium dendrobatidis) in amphibian samples usingreal-time Taqman PCR assay Dis Aquat Org 60 141minus148
Brannelly LA Chatfield MWH Richards-Zawacki CL (2012)Field and laboratory studies of the susceptibility of thegreen treefrog (Hyla cinerea) to Batrachochytrium den-drobatidis infection PLoS ONE 7 e38473
Chatfield MWH Brannelly LA Robak MJ Freeborn L Lail-vaux SP Richards-Zawacki CL (2013) Fitness conse-quences of infection by Batrachochytrium dendrobatidisin northern leopard frogs (Lithobates pipiens) EcoHealth10 90minus98
Cruz J (2008) Collapse of the amphibian community of thePaul do Boquilobo Natural Reserve (central Portugal)after the arrival of the exotic American crayfish HerpetolJ 18 197minus204
Cruz MJ Pascoal S Tejedo M Rebelo R (2006a) Predationby an exotic crayfish Procambarus clarkii on natterjacktoad Bufo calamita embryos its role on the exclusion ofthis amphibian from its breeding ponds Copeia 2006 274minus280
Cruz MJ Rebelo R Crespo EG (2006b) Effects of an intro-duced crayfish Procambarus clarkii on the distributionof south-western Iberian amphibians in their breedinghabitats Ecography 29 329minus338
Delahay RJ Cheeseman CL Clifton-Hadley RS (2001)Wildlife disease reservoirs the epidemiology of Myco -bacterium bovis infection in the European badger (Melesmeles) and other British mammals Tuberculosis (Edinb)81 43minus49
Ficetola GF Siesa ME De Bernardi F Padoa-Schioppa E(2012) Complex impact of an invasive crayfish on fresh-water food webs Biodivers Conserv 21 2641minus2651
Fisher MC Garner TWJ (2007) The relationship betweenthe emergence of Batrachochytrium dendrobatidis theinternational trade in amphibians and introduced amphi -bian species Fungal Biol Rev 21 2minus9
Garmyn A Van Rooij P Pasmans F Hellebuyck T Van DenBroeck W Haesebrouck F Martel A (2012) Waterfowl potential environmental reservoirs of the chytrid fungusBatrachochytrium dendrobatidis PLoS ONE 7 e35038
234A
utho
r cop
y
Brannelly et al Chytrid fungal infection in crayfish 235
Garner TWJ Perkins MW Govindarajulu P Seglie DWalker S Cunningham AA Fisher MC (2006) The emer -ging amphibian pathogen Batrachochytrium dendroba-tidis globally infects introduced populations of the NorthAmerican bullfrog Rana catesbeiana Biol Lett 2 455minus459
Garner TWJ Stephen I Wombwell E Fisher MC (2009) Theamphibian trade bans or best practice EcoHealth 6 148minus151
Gherardi F (2006) Crayfish invading Europe the case studyof Procambarus clarkii Mar Freshw Behav Physiol 39 175minus191
Gherardi F Renai B Corti C (2001) Crayfish predation ontadpoles a comparison between a native (Austropotamo-bius pallipes) and an alien species (Procambarus clarkii)Bull Fr Peche Piscicult 361 659minus668
Green DE Gray MJ Miller DL (2009) Disease monitoringand biosecurity Forestry Wildlife and Fisheries Publica-tions and Other Works Knoxville TN http traceten-nesseeedu utk_ forepubs2
Hasburgo-Lorena A (1986) The status of Procambarusclarkii population in Spain Freshw Crayfish 6 131minus136
Helms B Loughman ZJ Brown BL Stoeckel J (2013) Recentadvances in crayfish biology ecology and conservationFreshw Sci 32 1273minus1275
Holdich DM (1993) A review of astaciculture freshwatercrayfish farming Aquat Living Resour 6 307minus317
Jones JPG Rasamy JR Harvey A Toon A and others (2009)The perfect invader a parthenogenic crayfish poses anew threat to Madagascarrsquos freshwater biodiversity BiolInvasions 11 1475minus1482
Kilburn VL Ibaacutentildeez R Green DM (2011) Reptiles as potentialvectors and hosts of the amphibian pathogen Batra-chochytrium dendrobatidis in Panama Dis Aquat Org97 127minus134
Kolby JE (2014) Presence of the amphibian chytrid fungusBatrachochytrium dendrobatidis in native amphibiansexported from Madagascar PLoS ONE 9 e89660
Kolby JE Smith KM Berger L Karesh WB Preston APessier AP Skerratt LF (2014) First evidence of amphib-ian chytrid fungus (Batrachochytrium dendrobatidis) andranavirus in Hong Kong amphibian trade PLoS ONE 9 e90750
Kriger KM Hero JM Ashton KJ (2006) Cost efficiency in thedetection of chytridiomycosis using PCR assay Dis AquatOrg 71 149minus154
McClain WR Romaire RP (2007) Procambarid crawfish lifehistory and biology Publication No 2403 Southern Re -gional Aquaculture Center Mississippi State UniversityStoneville MS
McMahon TA Brannelly LA Chatfield MWH Johnson PTJand others (2013) Chytrid fungus Batrachochytrium den-drobatidis has nonamphibian hosts and releases chemi-cals that cause pathology in the absence of infectionProc Natl Acad Sci USA 110 210minus215
Murray K Skerratt LF Marantelli G Berger L Hunter DMahony MJ Hines H (2011) Hygiene protocols for thecontrol of diseases in Australian frogs A report for theAustralian Government Department of SustainabilityEnvironment Water Population and Communities Avail-able at wwwenvironmentgovau resource hygiene-protocols-control-diseases-australian-frogs
Newcombe RG (1998) Two-sided confidence intervals forthe single proportion comparison of seven methods StatMed 17 857minus872
Parker JM Mikaelian I Hahn N Diggs HE (2002) Clinicaldiagnosis and treatment of epidermal chytridiomycosisin African clawed frogs (Xenopus tropicalis) Comp Med52 265minus268
Rosenblum EB James TY Zamudio KR Poorten TJ and oth-ers (2013) Complex history of the amphibian-killingchytrid fungus revealed with genome resequencingdata Proc Natl Acad Sci USA 110 9385minus9390
Schloegel LM Picco AM Kilpatrick M Davies AJ HyattAD Daszak P (2009) Magnitude of the US trade inamphibians and presence of Batrachochytrium dendro-batidis and ranavirus infection in imported North Ameri-can bullfrogs (Rana catesbeiana) Biol Conserv 142 1420minus1426
Schloegel LM Ferreira CM James TY Hipolito M and oth-ers (2010) The North American bullfrog as a reservoir forthe spread of Batrachochytrium dendrobatidis in BrazilAnim Conserv 13 53minus61
Shapard EJ Moss AS San Francisco MJ (2012) Batra-chochytrium dendrobatidis can infect and cause mortal-ity in the nematode Caenorhabditis elegans Myco-pathologia 173 121minus126
Skerratt LF Berger L Speare R Cashins S and others (2007)Spread of chytridiomycosis has caused the rapid globaldecline and extinction of frogs EcoHealth 4 125minus134
Stuart SN Chanson JS Cox NA Young BE Rodrigues ASLFischman DL Waller RW (2004) Status and trends ofamphibian declines and extinctions worldwide Science306 1783minus1786
Voyles J Young S Berger L Campbell C and others (2009)Pathogenesis of chytridiomycosis a cause of catastrophicamphibian declines Science 326 582minus585
Warner RE (1968) The role of introduced diseases in theextinction of the endemic Hawaiian avifauna Condor 70 101minus120
Editorial responsibility Louise Rollins-Smith Nashville Tennessee USA
Submitted June 16 2014 Accepted October 30 2014Proofs received from author(s) December 22 2014
Aut
hor c
opy
Brannelly et al Chytrid fungal infection in crayfish 235
Garner TWJ Perkins MW Govindarajulu P Seglie DWalker S Cunningham AA Fisher MC (2006) The emer -ging amphibian pathogen Batrachochytrium dendroba-tidis globally infects introduced populations of the NorthAmerican bullfrog Rana catesbeiana Biol Lett 2 455minus459
Garner TWJ Stephen I Wombwell E Fisher MC (2009) Theamphibian trade bans or best practice EcoHealth 6 148minus151
Gherardi F (2006) Crayfish invading Europe the case studyof Procambarus clarkii Mar Freshw Behav Physiol 39 175minus191
Gherardi F Renai B Corti C (2001) Crayfish predation ontadpoles a comparison between a native (Austropotamo-bius pallipes) and an alien species (Procambarus clarkii)Bull Fr Peche Piscicult 361 659minus668
Green DE Gray MJ Miller DL (2009) Disease monitoringand biosecurity Forestry Wildlife and Fisheries Publica-tions and Other Works Knoxville TN http traceten-nesseeedu utk_ forepubs2
Hasburgo-Lorena A (1986) The status of Procambarusclarkii population in Spain Freshw Crayfish 6 131minus136
Helms B Loughman ZJ Brown BL Stoeckel J (2013) Recentadvances in crayfish biology ecology and conservationFreshw Sci 32 1273minus1275
Holdich DM (1993) A review of astaciculture freshwatercrayfish farming Aquat Living Resour 6 307minus317
Jones JPG Rasamy JR Harvey A Toon A and others (2009)The perfect invader a parthenogenic crayfish poses anew threat to Madagascarrsquos freshwater biodiversity BiolInvasions 11 1475minus1482
Kilburn VL Ibaacutentildeez R Green DM (2011) Reptiles as potentialvectors and hosts of the amphibian pathogen Batra-chochytrium dendrobatidis in Panama Dis Aquat Org97 127minus134
Kolby JE (2014) Presence of the amphibian chytrid fungusBatrachochytrium dendrobatidis in native amphibiansexported from Madagascar PLoS ONE 9 e89660
Kolby JE Smith KM Berger L Karesh WB Preston APessier AP Skerratt LF (2014) First evidence of amphib-ian chytrid fungus (Batrachochytrium dendrobatidis) andranavirus in Hong Kong amphibian trade PLoS ONE 9 e90750
Kriger KM Hero JM Ashton KJ (2006) Cost efficiency in thedetection of chytridiomycosis using PCR assay Dis AquatOrg 71 149minus154
McClain WR Romaire RP (2007) Procambarid crawfish lifehistory and biology Publication No 2403 Southern Re -gional Aquaculture Center Mississippi State UniversityStoneville MS
McMahon TA Brannelly LA Chatfield MWH Johnson PTJand others (2013) Chytrid fungus Batrachochytrium den-drobatidis has nonamphibian hosts and releases chemi-cals that cause pathology in the absence of infectionProc Natl Acad Sci USA 110 210minus215
Murray K Skerratt LF Marantelli G Berger L Hunter DMahony MJ Hines H (2011) Hygiene protocols for thecontrol of diseases in Australian frogs A report for theAustralian Government Department of SustainabilityEnvironment Water Population and Communities Avail-able at wwwenvironmentgovau resource hygiene-protocols-control-diseases-australian-frogs
Newcombe RG (1998) Two-sided confidence intervals forthe single proportion comparison of seven methods StatMed 17 857minus872
Parker JM Mikaelian I Hahn N Diggs HE (2002) Clinicaldiagnosis and treatment of epidermal chytridiomycosisin African clawed frogs (Xenopus tropicalis) Comp Med52 265minus268
Rosenblum EB James TY Zamudio KR Poorten TJ and oth-ers (2013) Complex history of the amphibian-killingchytrid fungus revealed with genome resequencingdata Proc Natl Acad Sci USA 110 9385minus9390
Schloegel LM Picco AM Kilpatrick M Davies AJ HyattAD Daszak P (2009) Magnitude of the US trade inamphibians and presence of Batrachochytrium dendro-batidis and ranavirus infection in imported North Ameri-can bullfrogs (Rana catesbeiana) Biol Conserv 142 1420minus1426
Schloegel LM Ferreira CM James TY Hipolito M and oth-ers (2010) The North American bullfrog as a reservoir forthe spread of Batrachochytrium dendrobatidis in BrazilAnim Conserv 13 53minus61
Shapard EJ Moss AS San Francisco MJ (2012) Batra-chochytrium dendrobatidis can infect and cause mortal-ity in the nematode Caenorhabditis elegans Myco-pathologia 173 121minus126
Skerratt LF Berger L Speare R Cashins S and others (2007)Spread of chytridiomycosis has caused the rapid globaldecline and extinction of frogs EcoHealth 4 125minus134
Stuart SN Chanson JS Cox NA Young BE Rodrigues ASLFischman DL Waller RW (2004) Status and trends ofamphibian declines and extinctions worldwide Science306 1783minus1786
Voyles J Young S Berger L Campbell C and others (2009)Pathogenesis of chytridiomycosis a cause of catastrophicamphibian declines Science 326 582minus585
Warner RE (1968) The role of introduced diseases in theextinction of the endemic Hawaiian avifauna Condor 70 101minus120
Editorial responsibility Louise Rollins-Smith Nashville Tennessee USA
Submitted June 16 2014 Accepted October 30 2014Proofs received from author(s) December 22 2014
