This document summarizes a study on the surveillance of ranaviruses in farmed American bullfrogs and sympatric amphibian, fish, and reptile species in Brazil. The study found high prevalence of ranaviruses in bullfrog farms with poor management practices and whole lifecycle production. Ranaviruses were detected at low levels in sympatric species near infected farms but not in other areas without bullfrog introduction. While disease was reported in farmed bullfrog tadpoles, no disease or mortality was observed in sympatric species despite some testing positive for ranaviruses. Future work is needed to further characterize the ranaviruses detected and their potential virulence.

1. Surveillance onSurveillance on
RANAVIRUSESRANAVIRUSES
2nd. INTERNATIONAL SYMPOSIUM ON
RANAVIRUSES
Knoxville, TN – 27-28 July 2013
RANAVIRUSESRANAVIRUSES
inin
Farmed frogs and SympatricFarmed frogs and Sympatric
Amphibia, Fish & ReptilesAmphibia, Fish & Reptiles
Rolando Mazzoni & Adriano Q. Mesquita
CPA/EVZ/UFG
BRAZIL

2. BrazilBrazil
American bullfrogsAmerican bullfrogs
BACKGROUND
Virus isolation from sick tadpolesVirus isolation from sick tadpoles
(farmed)(farmed)

3. Presence ofPresence of
“FV3 like” viruses“FV3 like” viruses
BACKGROUND
“FV3 like” viruses“FV3 like” viruses

4. OLDER TADPOLES & FROGSOLDER TADPOLES & FROGS
BACKGROUND
10% Positive10% Positive (conventional PCR)(conventional PCR)
No virus associated to lessionsNo virus associated to lessions (TEM)(TEM)

5. Bullfrogs are TEVBullfrogs are TEV -- FV3 carriersFV3 carriers
BACKGROUND
WOLF et al., 1968; WILLIAMS et al., 2005; WHITTINGTON et al., 2010
PRESENCE # DISEASE !!!!

6. FV3 in American BullfrogsFV3 in American Bullfrogs
•• INTRODUCEDINTRODUCED
oror
•• SouthSouth--American VirusAmerican Virus
BACKGROUND
Frog farms shed virus to the environment?Frog farms shed virus to the environment?
Pathogenic to other aquatic organisms?Pathogenic to other aquatic organisms?

7. OBJECTIVESOBJECTIVES
Ranavirus surveillanceRanavirus surveillance::
•• Farmed American bullfrogsFarmed American bullfrogs
•• Surrounding areasSurrounding areas
•• Other areasOther areas•• Other areasOther areas
Disease reportsDisease reports

8. MATERIAL & METHODSMATERIAL & METHODS
Samples were obtained from Am. bullfrogsSamples were obtained from Am. bullfrogs
•• EggsEggs
•• TadpolesTadpoles
•• FrogsFrogs
Sympatric amphibian, fish & reptilesSympatric amphibian, fish & reptiles
•• Frogs (Frogs (Leptodactylus ocellatusLeptodactylus ocellatus;; TrachycephalusTrachycephalus
typhoniustyphonius;;
•• Toads (Toads (BufoBufo marinusmarinus))
•• Lizards (Lizards (PhyllopezusPhyllopezus pollicarispollicaris))
•• Fish (Fish (Oreochromis niloticusOreochromis niloticus))
•• Snakes (Snakes (BothropsBothrops jararacussujararacussu))

9. GOIAS
BRAZIL
SAO
PAULO
SAMPLED AREAS

10. GOIAS
FARMS LOCATION AND SAMPLING SITES
Farm 3
Farm 2

11. SAMPLED ORGANSSAMPLED ORGANS
•• LIVERLIVER
•• KIDNEYKIDNEY
•• SPLEENSPLEEN
MATERIAL & METHODS
•• SPLEENSPLEEN
•• BLOODBLOOD
•• GONADSGONADS
•• SKINSKIN
•• MUSCLEMUSCLE

12. METHODMETHOD
•• REAL TIME PCRREAL TIME PCR
MATERIAL & METHODS
•• REAL TIME PCRREAL TIME PCR
•• TARGET SEQUENCE = MCPTARGET SEQUENCE = MCP

13. Positions: 97689 to 97751
Frog virus 3, complete genome
GenBank: AY548484.1
Tan et al. (2004)
MATERIAL & METHODS
RCVBRRCVBR--F = GAGCGTCACCCTCTCATTCF = GAGCGTCACCCTCTCATTC
RCVBRRCVBR--R = GCGTCCAGGTATGCCGTGR = GCGTCCAGGTATGCCGTG
RCVBRRCVBR--S = CGACATCAGCGCCCAGTCS = CGACATCAGCGCCCAGTC
Tm = 61°C

14. RESULTSRESULTS
•• Positive percentage for RVs in farmed bullfrogsPositive percentage for RVs in farmed bullfrogs
Farm % positive
01 1501
(40 samples)
15
02
(45 samples)
35
03
(150 samples)
100

15. RESULTS
Positive percentage for RVs in sympatric speciesPositive percentage for RVs in sympatric species
from Farm 03from Farm 03
Bullfrogs
(150 samples)
Amphibian
(12 samples)
Reptiles
(10 samples)
Fish
(20 samples)
100 10 10 10

16. RESULTS
Amphibian Reptiles Fish
Results for RVs in sympatric species from regionsResults for RVs in sympatric species from regions
where bullfrogs were not introducedwhere bullfrogs were not introduced
Amphibian
(12 samples)
Reptiles
(8 samples)
Fish
(12 samples)
Negative

17. RESULTS
DISEASE REPORTS IN FARMED FROGSDISEASE REPORTS IN FARMED FROGS
Farm
01 Sporadic
outbreak
(once)(once)
02 No outbreak
03
100%
mortality
in
tadpoles

18. RESULTS
DISEASE REPORTS IN OTHER SPECIESDISEASE REPORTS IN OTHER SPECIES
NO MORTALITIES
OR
DISEASES
REGISTERED

19. CONCLUSIONS
Farmed FrogsFarmed Frogs
•• High to moderate presence of RVsHigh to moderate presence of RVs
Farms with High RV prevalenceFarms with High RV prevalenceFarms with High RV prevalenceFarms with High RV prevalence
•• Poor managementPoor management
•• Whole cycleWhole cycle
•• High densitiesHigh densities
•• No biosecurity measuresNo biosecurity measures

20. CONCLUSIONS
Sympatric speciesSympatric species
•• Low detectionLow detection
•• No disease reportedNo disease reported
Other areasOther areas = No detection= No detection

21. CONCLUSIONS
Ranavirus in Frog FarmsRanavirus in Frog Farms
•• IntroducedIntroduced withwith AmericanAmerican bullfrogsbullfrogs
•• ContaminatingContaminating sympatricsympatric speciesspecies
Sympatric speciesSympatric species
•• NoNo diseasedisease yetyet reportedreported•• NoNo diseasedisease yetyet reportedreported
ResistanceResistance//lowlow susceptibilitysusceptibility ??
Low virulence ?Low virulence ?
Higher sensitivity with RTHigher sensitivity with RT--PCRPCR

22. FUTURE
DIRECTIONS
Experimental ChallengesExperimental Challenges
SequencingSequencing
Compare RCVCompare RCV--BR with RCVBR with RCV--Z and FV3Z and FV3
•• Virulence genes id.Virulence genes id.

23. THANKS !THANKS !