Ranavirus is a double-stranded DNA virus associated with amphibian, fish and reptile die-offs worldwide. International trade of live animals farmed for human consumption, such as the American bullfrog (Lithobates catesbeianus), plays a key role in spreading the pathogen. In Brazil, ranavirus has only been reported in captive bullfrog farms. We found infected tadpoles of both native species and the American bullfrog in the wild, and a case of mass mortality of amphibians and fish potentially associated with ranavirus. Dead animals presented skin ulcerations, hemorrhages, and edemas. We also found an overall prevalence of 37% of the amphibian chytrid in the area, and two bullfrog tadpoles were co-infected with both pathogens. We suggest that the interaction between the two pathogens should be investigated to improve global conservation of ectothermic vertebrates.

Ranavirus is an emergent virus in the Iridoviridae family associated with die-offs of ectothermic vertebrates worldwide (Chinchar 2002; Gray and Chinchar 2015). Introduction of this pathogen into different regions may be due to the pet trade, initially ornamental fish (Jankovich et al. 2015), and currently with the international movement of live amphibians (Schloegel et al. 2009; Kolby et al. 2014) such as the North American Bullfrog (Lithobates catesbeianus). The bullfrog can be infected with ranavirus without clinical disease (Hoverman et al. 2011), thereby acting as a vector for viral dissemination (Mazzoni et al. 2009; Schloegel et al. 2009). Lithobates catesbeianus is one of the main species farmed for human consumption (Schlaepfer et al. 2005), and Brazil is a major producer (Warkentin et al. 2009). However, in the early 1990s, several farms were abandoned, resulting in bullfrogs escaping into natural environments (Both et al. 2011). Such escapes are a risk of transmission of ranavirus to naïve and endemic ectothermic vertebrate populations, which could be catastrophic (Berger et al. 1998; Price et al. 2014). Because ranavirus was linked to die-offs in bullfrog farms in Brazil (Galli et al. 2006; Mazzoni et al. 2009), we hypothesized that this virus would be found in natural habitats, especially in the south of the Atlantic Forest, where feral populations of bullfrogs are widespread (Both et al. 2011).

On 17 November 2017 in the state of Rio Grande do Sul, southern Brazil (28°15′44″S, 52°24′22″W), we found a pond with several dying and dead American bullfrog tadpoles and a few dead fish, all showing signs of ranaviral disease, such as skin ulcerations, hemorrhages, and edemas (Fox et al. 2006; Miller et al. 2015). We collected samples for disease screening for ranavirus and Batrachochytrium dendrobatidis.

We collected tadpoles in nets of various sizes in two ponds 12 km apart, both located in highly disturbed habitats (Fig. 1A, B). We used different nets between ponds, but not between animals. Pond A (580 m elevation) is a natural pond where we found adult bullfrogs and tadpoles of native species of the families Bufonidae and Hylidae (not identified to species level), and pond B (724 m elevation) is an artificial pond where we only found bullfrog tadpoles and a few unidentified fish. We collected a total of 18 tadpoles of native species in pond A, and four bullfrog tadpoles (two dead and two lethargic individuals) in pond B. We collected a smaller number of specimens in pond B, because most dead animals were either in a state of advanced decomposition or in very deep, difficult to access parts of the pond. Each bullfrog tadpole was placed in an individual plastic bag. Native species were placed in plastic bags containing four or five individuals. Live individuals were anaesthetized and euthanized according to Brazilian regulations (Conselho Nacional de Controle de Experimentação Animal 2016). To avoid cross-contamination, a new pair of gloves was used before handling each tadpole. Kidneys, livers, and spleens were removed from all animals with sterile scissors. Pools of organ samples were stored in individual cryotubes with 1 mL of DNAzol® Reagent (Invitrogen®, Thermo Fisher Scientific, São Paulo, Brazil) for later DNA extraction following product specifications. After extraction, we used a spectrophotometer (Take3 Plate, BioTek Instruments, Winooski, Vermont, USA) to determine the amount of nucleic acid in each sample and diluted the samples to a final concentration of 7.5 ng/μL. We analyzed the presence or absence of viral DNA with a TaqMan real-time quantitative (q)PCR assay (Allender et al. 2013) using the 7500 ABI Fast Real-Time PCR System (Applied Biosystems, Carlsbad, California, USA). The threshold was set manually to 0.168 ΔRn (Rn value of an experimental reaction minus the Rn value of the baseline signal) to be comparable to the standard curve. We used two positive templates and two no-template controls in the assay. Mean cycle threshold for positive templates was 37. Therefore, we set the cycle threshold cutoff of the assay to 37.5 (10 DNA viral copies/μL), and samples with more than 10 DNA copies were considered positive for ranavirus. Although amphibians did not show clinical signs of chytridiomycosis, B. dendrobatidis is prevalent in the southern Atlantic Forest (James et al. 2015; Jenkinson et al. 2016); therefore, we also tested for the presence of this pathogen in the mouthparts of tadpoles with a qPCR assay (Boyle et al. 2004).

Figure 1

Sampling locations, lesions, and dead animals in the municipality of Passo Fundo, state of Rio Grande do Sul, southern Brazil, where sampling of tadpoles occurred in November 2017. (A) A natural pond, (B) an artificial pond, (C) skin ulcerations and (D) hemorrhage in a North American bullfrog (Lithobates catesbeianus) tadpole, (E) dead unidentified fish, and (F) dead bullfrog tadpoles in the artificial pond.

Figure 1

Sampling locations, lesions, and dead animals in the municipality of Passo Fundo, state of Rio Grande do Sul, southern Brazil, where sampling of tadpoles occurred in November 2017. (A) A natural pond, (B) an artificial pond, (C) skin ulcerations and (D) hemorrhage in a North American bullfrog (Lithobates catesbeianus) tadpole, (E) dead unidentified fish, and (F) dead bullfrog tadpoles in the artificial pond.

Close modal

We did not observe bullfrog tadpoles in pond A, and all native specimens seemed healthy. Contrarily, we found more than 20 dead bullfrog tadpoles in pond B (Fig. 1E, F) and no native anuran species. All observed individuals in this pond had severe skin lesions (Fig. 1C, D), and were positive for ranavirus. The two live tadpoles collected in pond B presented low ranavirus infection loads (15 and 21 copies/μL). The qPCR of dead tadpoles revealed higher infection loads (188 and 1,914 copies/μL). In pond A, five samples from native tadpoles amplified for the presence of ranavirus, but with small numbers of viral copies (between 10 and 12 genomic DNA/μL). The chytrid fungus was detected in seven of 19 tadpoles, including the native and invasive species (Table 1). However, infection load was lower than that expected for chytridiomycosis (Vredenburg et al. 2010), and we discarded chytridiomycosis as the potential cause of death of anurans in the pond.

Table 1

Results for the TaqMan quantitative (q)PCR assay for ranavirus detection in tadpole samples collected from two ponds in the municipality of Passo Fundo, state of Rio Grande do Sul, southern Brazil. Cycle threshold (Ct) for positive templates was Ct=37. We considered positive for ranavirus samples at Ct≥37.5 (bold). The presence or absence of Batrachochytrium dendrobatidis is also given, and infection load as the number of zoospore genome equivalent (GE) detected in the qPCR assay is provided for positive samples.

Results for the TaqMan quantitative (q)PCR assay for ranavirus detection in tadpole samples collected from two ponds in the municipality of Passo Fundo, state of Rio Grande do Sul, southern Brazil. Cycle threshold (Ct) for positive templates was Ct=37. We considered positive for ranavirus samples at Ct≥37.5 (bold). The presence or absence of Batrachochytrium dendrobatidis is also given, and infection load as the number of zoospore genome equivalent (GE) detected in the qPCR assay is provided for positive samples.
Results for the TaqMan quantitative (q)PCR assay for ranavirus detection in tadpole samples collected from two ponds in the municipality of Passo Fundo, state of Rio Grande do Sul, southern Brazil. Cycle threshold (Ct) for positive templates was Ct=37. We considered positive for ranavirus samples at Ct≥37.5 (bold). The presence or absence of Batrachochytrium dendrobatidis is also given, and infection load as the number of zoospore genome equivalent (GE) detected in the qPCR assay is provided for positive samples.

Despite bullfrogs being tolerant to ranavirus (Hoverman et al. 2011), we detected high loads of ranavirus and apparent signs of disease, and most dead animals were in advanced stages of decomposition, which is characteristic of death by ranavirus (Brunner et al. 2015; Miller et al. 2015). Studies on susceptibility to ranavirus across life stages have shown that tadpoles often die of infection whereas adults might clear the virus (Lesbarrères et al. 2012), which could explain the mortality of tadpoles observed here. However, without histopathologic examination, we do not have evidence to attribute these deaths to this pathogen.

Although ranavirus is known to occur in natural populations in South America (Zupanovic et al. 1998b; Fox et al. 2006), it has not previously been described in wild populations, as well as in native amphibian species, in Brazil. Because the susceptibility of native amphibian hosts to ranavirus in Brazil has not been investigated, the low infection loads that we detected (<12 copies/μL) could either represent the beginning stage of infection (Brunner et al. 2005) or be related to host tolerance to this pathogen (Zupanovic et al. 1998a; Roy and Kirchner 2000).

Studies on ranaviruses in Brazil are incipient (Mazzoni et al. 2009; Neves et al. 2016), so we are not aware of how long ranavirus has been present in Brazil, its geographic range and distribution, which strains are present in the wild, and the consequences of infection to native hosts. More studies on ranavirus infection among native species in Brazil must be carried out to clarify these questions, particularly in the Atlantic Forest of Brazil, which is a biodiversity hotspot (Myers et al. 2000) with recognized historic amphibian declines associated with pandemic pathogens (Carvalho et al. 2017).

We thank K. H. Teixeira for permission to access the area. We also thank K. L. Gendreau, G. M. Rosa, and two anonymous reviewers for improving the article. Methods were approved by the ethics committee from the Universidade Estadual de Campinas (Comissão de Éticas no Uso de Animais, CEUA/UNICAMP 4728-1/2017). This study was funded by São Paulo Research Foundation (FAPESP 2012/08846-3, 2016/25358-3, 2016/03344-0, 2017/01718-3, and 2017/01917-6), Coordination for the Improvement of Higher Education Personnel (CAPES 001), and the National Council for Scientific and Technological Development (CNPq 300896/2016-6). The sampling permit was provided by Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio/SISBio 60211).

Allender
MC,
Bunick
D,
Mitchell
MA.
2013
.
Development and validation of TaqMan quantitative PCR for detection of frog virus 3-like virus in eastern box turtles (Terrapene carolina carolina)
.
J Virol Methods
188
:
121
125
.
Berger
L,
Speare
R,
Daszak
P,
Green
DE,
Cunningham
AA,
Goggin
CL,
Slocombe
R,
Ragan
MA,
Hyatt
AD,
McDonald
KR,
et al.
1998
.
Chytridiomycosis causes amphibian mortality associated with population declines in the rainforests of Australia and Central America
.
Proc Natl Acad Sci U S A
95
:
9031
9036
.
Both
C,
Lingnau
R,
Santos
A
Jr,
Madalozzo
B,
Lima
LP,
Grant
T.
2011
.
Widespread occurrence of the American bullfrog, Lithobates catesbeianus (Shaw 1802) (Anura: Ranidae), in Brazil
.
S Am J Herpetol
6
:
127
134
.
Boyle
DG,
Boyle
DB,
Olsen
V,
Morgan
JAT,
Hyatt
AD.
2004
.
Rapid quantitative detection of Chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay
.
Dis Aquat Org
60
:
141
148
.
Brunner
JL,
Richards
K,
Collins
JP.
2005
.
Dose and host characteristics influence virulence of Ranavirus infections
.
Oecologia
144
:
399
406
.
Brunner
JL,
Storfer
A,
Gray
MJ,
Hoverman
JT.
2015
.
Ranavirus ecology and evolution: From epidemiology to extinction
.
In
:
Ranaviruses: Lethal pathogens of ectothermic vertebrates
,
Gray
M,
Chinchar
V,
editors
.
Springer
,
Cham, Switzerland
,
pp
.
71
104
.
Carvalho
T,
Becker
CG,
Toledo
LF.
2017
.
Historical amphibian declines and extinctions in Brazil linked to chytridiomycosis
.
Proc Biol Sci
284
:
20162254
.
Chinchar
VG.
2002
.
Ranaviruses (family Iridoviridae): Emerging cold-blooded killers
.
Arch Virol
147
:
447
470
.
Conselho Nacional de Controle de Experimentação Animal
.
2016
.
Normativas do Concea - Lei, Decreto, Portarias, Resoluções Normativas e Orientações Técnicas
.
www.mctic.gov.br. Accessed November 2017
.
Fox
SF,
Greer
AL,
Torres-Cervantes
R,
Collins
JP.
2006
.
First case of ranavirus-associated morbidity and mortality in natural populations of the South American frog Atelognathus patagonicus
.
Dis Aquat Org
72
:
87
92
.
Galli
L,
Pereira
A,
Márquez
A,
Mazzoni
R.
2006
.
Ranavirus detection by PCR in cultured tadpoles (Rana catesbeiana Shaw, 1802) from South America
.
Aquaculture
257
:
78
82
.
Gray
MJ,
Chinchar
GV.
2015
.
Ranaviruses: Lethal pathogens of ectothermic vertebrates
.
Springer
,
Berlin, Germany
,
246
pp
.
Hoverman
JT,
Gray
MJ,
Haislip
NA,
Miller
DL.
2011
.
Phylogeny, life history, and ecology contribute to differences in amphibian susceptibility to ranaviruses
.
Ecohealth
8
:
301
319
.
James
TY,
Toledo
LF,
Rödder
D,
Leite
DS,
Belasen
A,
Betancourt-Román
CM,
Jenkinson
TS,
Soto-Azat
C,
Lambertini
C,
Longo
AV,
et al.
2015
.
Disentangling host, pathogen, and environmental determinants of a recently emerged wildlife disease: Lessons from the first 15 years of amphibian chytridiomycosis research
.
Ecol Evol
5
:
4079
4097
.
Jankovich
JK,
Qin
Q,
Zhang
VG,
Chinchar
VG.
2015
.
Ranavirus replication: Molecular, cellular, and immunological events
.
In
:
Ranaviruses: Lethal pathogens of ectothermic vertebrates
,
Gray
M,
Chinchar
V,
editors
.
Springer
,
Cham, Switzerland
,
pp
.
105
139
.
Jenkinson
TS,
Betancourt-Román
CM,
Lambertini
C,
Valencia-Aguilar
A,
Rodriguez
D,
Nunes-de-Almeida
CHL,
Ruggeri
J,
Belasen
A,
Leite
DS,
Zamudui
KR,
et al.
2016
.
Amphibian-killing chytrid in Brazil comprises both locally endemic and globally expanding populations
.
Mol Ecol
25
:
2978
2996
.
Kolby
JE,
Smith
KM,
Berger
L,
Karesh
WB,
Preston
A,
Pessier
AP,
Skerrat
LF.
2014
.
First evidence of amphibian chytrid fungus (Batrachochytrium dendrobatidis) and ranavirus in Hong Kong amphibian trade
.
PLoS One
9
:
e90750
.
Lesbarrères
D,
Balseiro
A,
Brunner
J,
Chinchar
VG,
Duffus
A,
Kerby
J,
Miller
DL,
Robert
J,
Schock
DM,
Waltzek
T,
et al.
2012
.
Ranavirus: Past, present and future
.
Biol Lett
8
:
481
483
.
Mazzoni
R,
de Mesquita
AJ,
Fleury
LFF,
de Brito
WMED,
Nunes
IA,
Robert
J,
Morales
H,
Coelho
ASG,
Barthasson
DL,
Galli
L,
et al.
2009
.
Mass mortality associated with a frog virus 3-like Ranavirus infection in farmed tadpoles Rana catesbeiana from Brazil
.
Dis Aquat Org
86
:
181
191
.
Miller
DL,
Pessier
AP,
Hick
P,
Whittington
RJ.
2015
.
Comparative pathology of ranaviruses and diagnostic techniques
.
In
:
Ranaviruses: Lethal pathogens of ectothermic vertebrates
,
Gray
M,
Chinchar
V,
editors
.
Springer
,
Cham, Switzerland
,
pp
.
171
208
.
Myers
N,
Mittermeier
RA,
Mittermeier
CG,
da Fonseca
GA,
Kent
J.
2000
.
Biodiversity hotspots for conservation priorities
.
Nature
403
:
853
858
.
Neves
PV,
Catroxo
MH,
Hupolito
M,
de Oliveira
CR,
Ferreira
CM.
2016
.
Use of polyclonal antibodies, electron microscopy and histopathology to detect iridovirus-like particles in bullfrogs
.
Bol Inst Pesca
42
:
523
531
.
Price
SJ,
Garner
TWJ,
Nichols
RA,
Balloux
F,
Ayres
C,
de Alba
AM,
Bosch
J.
2014
.
Collapse of amphibian communities due to an introduced ranavirus
.
Curr Biol
24
:
2586
2591
.
Roy
BA,
Kirchner
JW.
2000
.
Evolutionary dynamics of pathogen resistance and tolerance
.
Evolution
54
:
51
63
.
Schlaepfer
MA,
Hoover
C,
Dodd
CK
Jr.
2005
.
Challenges in evaluating the impact of the trade in amphibians and reptiles on wild populations
.
BioScience
55
:
256
264
.
Schloegel
LM,
Picco
AM,
Kilpatrick
AM,
Davies
AJ,
Hyatt
AD,
Daszak
P.
2009
.
Magnitude of the US trade in amphibians and presence of Batrachochytrium dendrobatidis and ranavirus infection in imported North American Bullfrogs (Rana catesbeiana)
.
Biol Conserv
142
:
1420
1426
.
Vredenburg
VT,
Knapp
RA,
Tunstall
TS,
Briggs
CJ.
2010
.
Dynamics of an emerging disease drive large-scale amphibian population extinctions
.
Proc Natl Acad Sci U S A
107
:
9689
9694
.
Warkentin
IG,
Bickford
D,
Sodhi
NS,
Bradshaw
CJ.
2009
.
Eating frogs to extinction
.
Conserv Biol
23
:
1056
1059
.
Zupanovic
Z,
Lopez
G,
Hyatt
AD,
Green
B,
Bartran
G,
Parkes
H,
Whittington
RJ,
Speare
R.
1998
a
.
Giant toads Bufo marinus in Australia and Venezuela have antibodies against ‘ranaviruses'
.
Dis Aquat Org
32
:
1
8
.
Zupanovic
Z,
Musso
C,
Lopez
G,
Louriero
CL,
Hyatt
AD,
Hengstberger
S,
Robinson
AJ.
1998
b
.
Isolation and characterization of iridoviruses from the giant toad Bufo marinus in Venezuela
.
Dis Aquat Org
33
:
1
9
.