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EVALUATION OF A NOVEL DETECTION METHOD FOR RANAVIRUS INWATER SAMPLES FROM PINE MOUNTAIN WILDLIFE MANAGEMENT
AREA, LETCHER COUNTY KENTUCKY
By:
Matthew R. Pettus
Thesis Approved:
Chair, Advisory Committee
Member, Advisory Committee
Member, Advisory Committee
Dean, Graduate School
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STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the requirements for aMaster's degree at Eastern Kentucky University, I agree that the Library shall
make it available to borrowers under rules of the Library. Brief quotations from this
thesis are allowable without special permission, provided that accurateacknowledgment of the source is made.
Permission for extensive quotation from or reproduction of this thesis may begranted by my major professor, or in his absence, by the Head of Interlibrary
Services when, in the opinion of either, the proposed use of the material is forscholarly purposes. Any copying or use of the material in this thesis for financial gain
shall not be allowed without my written permission.
Signature _____________________________________
Date _________________________________________
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EVALUATION OF A NOVEL DETECTION METHOD FOR RANAVIRUS INWATER SAMPLES FROM PINE MOUNTAIN WILDLIFE MANAGEMENT
AREA, LETCHER COUNTY KENTUCKY
By:
Matthew R. Pettus
Bachelors of ScienceElmhurst CollegeElmhurst Illinois
2006
Submitted to the Faculty of the Graduate School ofEastern Kentucky University
in partial fulfillment of the requirementsfor the degree of
MASTER OF SCIENCEApril, 2010
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DEDICATION
This thesis is dedicated to my parents, Robert and Debbie Pettus. Without their
help this thesis would never have been completed. To my father, you have
always been there to support me. I thank you from the bottom of my heart. To
my mother, without the lessons you taught me I would not be here. Little woman,
you will always be loved and missed. You both have made me who I am, thank
you.
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ACKNOWLEGDEMENTS
I would like to thank my advisor and graduate committee chair, Dr. Paul Cupp.
Your knowledge and resources have helped make this project work. I would like
to thank my co-committee chair Dr. Marcia Pierce for all the long hours and
countless times you have stepped in to remedy my research woes. Also, for all
your help in getting this thesis written and ready for review. Dr. Stephen Richter
thank you for the use of your lab, as well as always making me feel welcome
even though I wasnt part of your lab. Finally, Id like to say thank you to all of the
graduate students. This would have been an impossible road without your
camaraderie.
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ABSTRACT
Throughout the world amphibian populations have declined over the last
ten years. Many factors have been implicated, yet emerging diseases are
currently having the largest effects on amphibian populations. This research was
intended to develop a protocol using water samples to detect Ranavirus, a
recently emerging infectious agent, from environmental water samples at Pine
Mountain Wildlife Management Area in Letcher County, Kentucky. Ranaviruses
are dsDNA viruses that have been implicated in localized amphibian declines.
Possible reservoirs for Ranaviruses include adult amphibians, reptiles, and
fishes. Direct transmission has been well documented and indirect transmission
is highly possible. Centrifugal filters were used to concentrate water samples
from a volume of 15 ml down to 200 l. PCR was performed on the concentrated
water samples and PCR products were separated using 1% agarose gel
electrophoresis. Tissue samples from animals living in each pond were also
taken for comparison to the water samples. Total samples obtained included 38
water samples and 98 tissue samples. All of the samples tested negative for
Ranavirus. To determine the lowest concentration of virus detectable by this
novel system, double distilled water (ddH2O) and pond water was seeded with
Ranavirus at a known concentration. This system could detect 13.3 PFU/l in
ddH2O and 106.4 PFU/l in pond water. While there may have been several
factors involved in this result, it is most likely that during the sampling period
Ranaviruswas not present in the Pine Mountain Wildlife Management Area.
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TABLE OF CONTENTS
CHAPTER Page
INTRODUCTION.................................................................................................. 1
Ranavirus Characteristics.................................................................... 3
Viral Reservoirs ................................................................................... 6
Possible Amphibian Reservoirs ................................................. 6
Non-Amphibian Reservoirs........................................................ 7
Ranavirus Transmission ..................................................................... 7
Sampling............................................................................................. 9
II.METHODS....................................................................................................... 11
Field Sampling................................................................................... 11
Laboratory Data Collection ................................................................ 12
Lowest Detectable level of Virus.............................................. 12
DNA Extraction from Water and Tissue Samples .................... 13
Amplification of DNA................................................................ 14
III. RESULTS...................................................................................................... 15
Controls ............................................................................................. 15
Lowest Detectable Level of Virus....................................................... 15
Water Samples .................................................................................. 15
Tissue Samples ................................................................................. 16
IV. DISCUSSION................................................................................................ 17
LITERATURE CITED ......................................................................................... 24
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APPENDIX.. ................................................. 31
VITA ................................................................................................................... 38
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LIST OF TABLES
TABLE
1. Latitude and longitude of ponds with and
without recorded die-offs and parkingplaces close to the ponds.............................................................................. 33
2. The pond number, species collected, andthe collection number of the samples fromJuly 10, 2008 ................................................................................................ 34
3. The pond number, species collected, andthe collection number of the samples fromJuly 25, 2008 ................................................................................................ 35
4. The pond number, species collected, andthe collection number of the samples fromAugust 8, 2008 .............................................................................................. 36
5. The pond number, species collected, andthe collection number of the samples fromAugust 26, 2008 ............................................................................................ 36
6. The pond number, species collected, andthe collection number of the samples fromSeptember 11, 2008 ...................................................................................... 37
7. The pond number, species collected, andthe collection number of the samples fromSeptember 28, 2008 ....................................................................................... 37
8. The pond number, species collected, andthe collection number of the samples fromOctober 9, 2008.............................................................................................. 38
9. The pond number, species collected, andthe collection number of the samples fromOctober 23, 2008.......................................................................................... 38
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LIST OF FIGURES
FIGURE
1. Lowest detectable level of virus in water samples using
20l, 10l, 5l, and 2.5l of Ranavirusstock virus ........................................ 16
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Chapter I
Introduction
Amphibians are the most threatened vertebrate class, more so than
eitherbirds or mammals (Gascon et al. 2007). There are 1856 endangered or
threatened species of amphibians; approximately 32% of amphibians are
globally threatened, as compared to 12% of birds and 23% of mammals
(Gascon et al. 2007). At least 2468 amphibian species (43.2%) are
experiencing some form of population decrease, whereas
28 species (0.5%)
are increasing and only 1552 species (27.2%) are stable. Currently, 1661 or
29.1% of amphibian species have an unknown trend (Gascon et al. 2007).
In the last decade, global amphibian population declines have steadily
gained more attention (Blaustein and Wake 1990). Many causes have been
proposed for this decline including increases in UV radiation (Broomhall et al.
2000), introduced species (Adams et al. 1999), and emerging infectious
disease (Daszak et al. 2003; Collins and Storfer 2003; Wake and Vredenburg
2008). While each of these is important, emerging infectious disease (EID)
has been implicated in single and multiple population die offs and is of
increasing importance each year (Jancovich et al. 2003; Daszak et al. 1999;
Carey et al. 2003). Declines have been increasingly seen in pristine areas
with little disturbance (Lips et al. 2003). EIDs have been found in both
pristine and impacted areas, and as the presence of these diseases increase
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we will see far-reaching consequences in our amphibian populations (Lips et
al. 2003; Jancovich et al. 2003).
Emergent infectious diseases are defined as diseases that are
expanding their range, taking on new hosts, or are newly discovered (Daszak
et al. 1999). There are two main emerging diseases implicated in the decline
of amphibian populations: chytrid fungus and Iridovirusinfections.
Chytridiomycosis is a fungal disease caused by Batrachochytrium
dendrobatidis, which was first described in 1998 (Berger et al. 1998). This
fungus attacks keratinized tissue including several skin layers in adult
amphibians and the mouth parts of larval amphibians. Three hypotheses
have been proposed to explain the death of amphibians infected with chytrid
fungus: damage to the skin reduces the ability of amphibians to cutaneously
breathe and/or osmoregulate; there is a release of fungal toxins that are
absorbed; or a combination of these events occur (Berger et al. 1998, Pessier
et al. 1999). Often highly virulent pathogens are dependent on the host
density and as the pathogen suppresses the host population, transmission
stops. This allows the host population to rebound (Anderson and May 1986;
Dobson and May 1986). This does not appear to be the case with chytrid.
The amphibian larval stages have been shown to live on after the death of the
adults, implying that the larvae may be reservoir hosts (Daszak et al. 2000).
This may allow chytrid to remain in reduced amphibian populations. In
addition, chytrid can live as a saprophyte, or survive on decaying matter,
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which may explain the lack of recolonization after amphibian die offs (Lips
1998; Lips 1999).
The second group of pathogens implicated in amphibian declines is the
Iridoviruses. Unlike chytrid fungus, the Iridoviruses are not a single species.
They are a group of viruses that infect fish, salamanders, frogs, and reptiles
(Mao et al. 1999, Johnson et al. 2008, Gray et al. 2009, Ariel et al. 2009).
Those affecting amphibians are in the genus Ranavirus. Several ranaviruses
are common in amphibians and mortalities have been reported at all latitudes,
and in most major families of anurans and urodeles (Carey et al. 2003;
Daszak et al.2003).
RanavirusCharacteristics
Ranaviruses were first isolated by Granoff et al. (1965) from Lithobates
pipiens. Ranaviruses are in the family Iridoviridae (Eaton et al. 2007).
Iridoviridae contains 5 genera. The Iridoviruses and Chloridoviruses infect
invertebrates. The Ranaviruses, Megalocystiviruses, and Lymphocystiviruses
infect vertebrates (Chinchar et al. 2009).
Ranaviruses have a diameter of 160-200 nm and have a linear
double-stranded (ds) DNA genome (Fauquet et al. 2005, Williams et al.
2005). Their nucleoprotein core is made up of a single coiled filament 10 nm
wide. The capsid symmetry is skewed with a T=133 or 147. The unit
genome size is ~105 kbp with a G+C content of ~54% (Fauquet et al. 2005).
Ranaviruses have a distinctive icosahedral shape that is often visible in the
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cytoplasm of infected cells in electron microscope images (Chinchar and
Hyatt 2008). The genome encodes for 100 gene products with several genes
thought to increase virulence and help in evasion of the host immune
response (Chinchar 2002, Chinchar et al. 2009). The viral infection and
immune response can be so overwhelming that cellular death can occur
within hours of infection (Chinchar et al. 2003; Williams et al. 2005).
Ranaviruses are known to infect anurans, urodeles, reptiles, and bony
fish (Mao et al. 1997, Williams et al. 2005). Three species of Ranavirusare
known to infect amphibians. These are Frog Virus 3 (FV3), Bohle iridovirus
(BIV), and Ambystoma tigrinumvirus (ATV) (Chinchar et al. 2005). The major
capsid protein (MCP) of these viruses make up approximately half the virus
weight and is highly conserved (Hyatt et al. 2000).
The major capsid protein (MCP) is an area that has been studied
extensively (Mao et al. 1999). While Hyatt et al. (2000) found that the MCP is
highly conserved there is still variation amongst isolated viruses. The genetic
variation in the MCP of ATV shows geographic differences (Ridenhour and
Storfer 2008), while FV3 seems to be more genetically conserved over its
geographic range (Schock et al. 2008). Despite this variation, Mao e al.
(1997) found that primers created for FV3 were able to adhere to nine wild
strain iridoviruses and that those strains were more closely related to FV3
than other iridoviruses. To this day the MCP remains one of the most
commonly used genes in identifying ranaviruses.
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Viral Reservoirs
Possible Amphibian Reservoirs
Amphibians are a likely reservoir for the ranaviruses. Due to the
biphasic life cycle in amphibians it is possible that reservoirs are both aquatic
and terrestrial. In aquatic environments it is probable that species with larva
that mature in >1 season, have incomplete metamorphosis, and/or are highly
aquatic adults are the most likely reservoirs (Gray et al. 2009). Gray et al.
(2007) reported that 57% of overwintering tadpoles of Lithobates catesbeiana
were infected with Ranavirus. Many individuals of overwintering tadpoles do
not show physical signs of infection (Miller et al. 2009). This trend has also
been shown in Desmognathus quadramaculatus(Gray et al. 2009). Because
of this, both larval and adult amphibians may help Ranaviruspersist in the
environment (Duffus et al. 2008)
In situations where amphibian larvae do not over winter, adults may
remain as a reservoir for Ranavirus. Sub-lethal infections in tadpoles have
been shown to continue after metamorphosis (Ariel et al. 2009). Brunner et
al. (2004) was able to show that ATV-infected Ambystoma tigrinumcould
survive through metamorphosis and that 7% of returning salamanders the
following year were infected with ATV. In the late 1990s, Jancovich et al.
(1997) hypothesized that tadpoles and asymptomatic adults act as the
reservoir for Ranavirus. As more research has come to light, it appears that
pre- and post-metamorphic amphibians are likely to be the preeminent
reservoirs for Ranavirus.
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2006). Skin contact of one second between an infected animal and an
uninfected animal has been shown to cause disease (Brunner et al. 2007).
Many amphibians are cannibalistic (Alford 1999, Rosen 2007, Pizzatto and
Shine 2008). Cannibalism may be the consumption of any life stage including
eggs, which Ranavirusalso has been found to infect (Tweedell and Granoff
1968, Alford 1999). Studies have shown that predation on infected
individuals can led to infection (Harp and Petranka 2006, Brunner et al. 2007).
Those species that have cannibalistic phenotypes may exhibit a lower
occurrence of this phenotype due to poor survivorship (Pfennig et al. 1991).
Indirect transmission is infection caused by virus circulating through
the environment. This can be through the water or through soil. Salamander
larvae exposed to ATV-infected individuals, but without direct contact, have
become infected (Greer et al. 2008). Harp and Petranka (2006) were able to
induce infection by exposing Lithobates sylvaticusto sediment from an active
natural die-off. These studies suggest that Ranavirusthat is shed into the
surrounding environment can infect new hosts. For indirect transmission to
occur, Ranavirusmust be able to persist in the environment. However, this
aspect of Ranavirusecology is not well understood. Jancovich et al. (1997)
showed that ATV could persist for up to 2 weeks. However, this may be
contingent on some level of moisture being present. Brunner et al. (2007)
found that sediment that was inoculated with ATV and then dried for four days
did not cause infection, while soil inoculated and kept moist caused 87%
mortality in salamanders. The length of viability is unknown for ranaviruses,
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during or after die-offs cannot aid us in understanding transmission or sub-
lethal infections.
Recently, a non-lethal method of sampling has been developed. St.
Amour and Lesbarreres (2007) have been able to detect the Ranavirusin toe
clippings. This has allowed investigators to determine the presence or
absence of the Ranavirusin field samples and determine if lab raised animals
are free of Ranavirus. Since the results are a positive or negative for the
presence of Ranavirusand animals need to be caught, restrained, and toes
removed, a faster method is possible.
The purpose of my research is the detection of Ranavirusin Kentucky
ponds using a water based non-lethal system. I plan to use this method as a
quick and effective means to identify the presence of infection, and hopefully
provide a tool for continued research into indirect transmission of ranaviruses.
My novel detection method using water samples will be compared to tissue
samples processed using the St. Amour and Lesbarreres (2007) protocols.
Water samples will allow quick field sampling and reduce the overall cost of
sampling, while maintaining the speed and reliability of PCR-based detection
methods.
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Chapter II
Materials and Methods
Sampling was done at Pine Mountain Wildlife Management Area
(PMWMA). This management area is one of Kentuckys last contiguous and
least disrupted forests. It is comprised of 4,849 acres located in the far
southeast corner of Kentucky in Letcher and Harlan counties. In 2002, more
than 50 ponds were built by Kentucky Department of Fish and Wildlife as
wildlife watering holes. When these ponds were monitored in 2004 and 2007
for amphibian populations it was found that there were active die-offs at
several ponds.
For this study, ponds were selected from the 20 best amphibian ponds
at PMWMA (J. MacGregor, personal communication, August 30 2008). Of
these 20 ponds, 11 had no previous die-offs and the remaining 9 had
recorded die-offs suspected to be from Ranavirusbut that were never verified
(J. MacGregor, personal communication, May 16 2008). Prior to entering the
field, 5 ponds were randomly selected from each of these two groups. These
ten ponds became the sampled ponds.
Field Sampling
At each pond, water and tissue samples were taken. Water samples
were collected from July 10, 2008 to October 23, 2008 at two week intervals.
Samples were taken at approximately 75 mm under the surface of the pond at
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the edges where amphibian adults and larvae were often seen. Water
samples consisted of 45 ml of pond water in a 50 ml plastic test tube. These
water samples were immediately placed on ice for transport to the laboratory.
Animals were obtained using a dip net. Tissue samples were taken from
adults, larvae, and eggs. Tissue samples were obtained from adults by
removing the third phalanx at the second joint with a pair of suture scissors.
The toe was immediately placed in 1 ml tubes containing 100% ethanol and
placed on ice. Tissue samples from larvae were obtained by tail clipping.
Approximately 2 cm of the distal tail tip were cut using suture scissors. The
samples were placed in 1ml tubes containing 100% ethanol and placed on
ice. While the traditional means of sampling larvae is tissue homogenation,
tail clippings have been used in many amphibian orders to test for Ranavirus
(St. Amour and Lesberreres 2007). Eggs were sampled by excising 3 to 4
eggs from the egg mass. Eggs were placed directly in 1 ml tubes containing
100% ethanol and placed on ice for transport to the laboratory.
Processing Samples in the Laboratory
Minimum Ranavirus Detectable Concentrations
A consistent control volume was needed for each following experiment.
A volume of 20 l was chosen because it was a volume that consistently
produced PCR products that were detected and was easily visible on gel
electrophoresis. These controls were at a concentration of 106.4 plaque
forming units (PFU)/l.
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Double distilled water was used to produce viral dilutions, which were
then used to determine the lowest concentration detectable by the water
testing method. Stock Ranaviruswas obtained from ATCC (Manassas, VA)
at a concentration of 8 x 107 PFU/ml. Double distilled water was seeded with
20 l, 10 l, 5 l, 2.5 l, 1.25 l, 0.625 l, and 0.3125l to create a total
volume was 15 ml. Concentrations of Ranaviruswere 106.4 PFU/l, 53.2
PFU/l, 26.6 PFU/l, 13.3 PFU/l, 6.65 PFU/l, 3.325 PFU/l, and 1.662
PFU/l respectively. The 15 ml sample was then pipetted into an Amicon 15
filter unit (Millipore; Billerica, MA) with a pore size of 3 kDa. The Amicon Ultra
15 centrifugal filter units containing the water samples were centrifuged until
the volume was reduced to 200 l. Using the reduced volume of 200 l, a
DNA extraction was performed using a MiniElute Virus Spin Kit(Qiagen;
Valencia, CA).
DNA Extraction from Water and Tissue Samples
Pond water samples were vortexed to homogenize the contents. The
maximum volume of water an Amicon 15 can process is 15 ml. Therefore,
sub-samples of 15 ml were taken from each 45 ml sample. These were pre-
filtered using a 0.45 micron syringe filter (Corning; Corning NY) to remove
bacteria, detritus, and other debris. The filtrate was then placed in an Amicon
15 filtration unit. Each Amicon 15 was centrifuged until the volume was
reduced to 200 l. DNA extraction was done using the MiniElute Virus Spin
Kit(Qiagen; Valencia, CA).
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DNA extractions from tissue samples were done using a DNeasy
Blood and Tissuekit (Qiagen; Valencia, CA).
Amplification of DNA
Forward and reverse primers for the MCP developed by Mao et al.
(1996) were used to detect the presence of Ranavirusthrough PCR. PCR
conditions during amplification followed protocols described in Mao et al.
(1996). Reactions were incubated for 5 minutes at 94C followed by 28
cycles consisting of 94C for 1 minute, 45C for 2 minutes, 55C for 3 minutes,
and a single cycle of 55C for 5 minutes. Gel electrophoresis was performed
using 1% agarose gels (50 ml volume). Each gel was produced using 45ml
distilled water, 5 ml of 10X TAE buffer, and 0.5g agarose. Samples were
prepared for electrophoresis by adding a 10 l aliquot of each PCR product
sample to 6.7 l distilled water and 3.3 l orange buffer (Invitrogen; Carlsbad,
CA). Samples were vortexed and 10 l of each were pipetted in a single lane
on the gel. 5 l of a 100 bp ladder was placed in the first and last wells of
each gel. Gels were electrophoresed at 100 volts and stained with ethidium
bromide (0.02%) before examination using a UV light box.
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Chapter III
Results
Controls
Initial testing determined Ranaviruscould be recovered from ddH2O
samples at 53.2 PFU/l, 106.4PFU/l, 159.6PFU/l, and 266.0PFU/l. A
consistent control volume was needed for each following experiment. These
controls each contained 106.4 PFU/l.
Lowest Concentration of RanavirusDetected
The sample of 2.5l was the lowest volume visible on gel
electrophoresis (Figure 1). The 2.5l of Ranavirusin 15ml distilled water was
a concentration of 13.3 PFU/l.
To determine the lowest detectable concentration in pond water, pond
water samples were seeded to concentrations of 266 PFU/l, 106.4 PFU/l,
53.2 PFU/l, 26.6 PFU/l, and 13.3 PFU/l. The lowest concentration that
had PCR products detectable by electrophoresis was 106.4 PFU/l.
Water Samples
A total of 38 water samples were taken from 10 ponds at PMWMA.
Ranaviruswas not detected in any environmental sample at PMWMA.
Six randomly selected environment samples from PMWMA were seeded with
virus stock solution at 106.4 PFU/l to determine if there were PCR inhibitors
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present in the water samples. Of these samples, four were visible after gel
electrophoresis. PCR and gel electrophoresis were attempted twice more on
the samples that tested negative. These samples in subsequent tests
continued to test negative.
Figure 1. Lowest detectable level of virus in water samples using 20l, 10l,5l, 2.5l of Ranavirusstock virus (8 x 107 PFU/ml). L-DNA Ladder,E-Empty Lane
Tissue Samples
A total of 90 tissue samples were taken from the experimental
ponds at PMWMA. No evidence of Ranavirusinfection were found in any
tissue samples collected at PMWMA.
L E E E E 2.5l 5l 10l20l L
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Chapter IV
Discussion
I was able to create a novel protocol for the detection of Ranavirusin
both seeded ddH20 and seeded pond water that is non-destructive. This
method differs from other water sampling protocols for emerging infectious
diseases (EID) in that it uses lower water sample volumes (see Krishtein et al.
2007). The goal for this novel detection method was to be used to detect
Ranavirusin small ephemeral ponds where the volume of water can be very
low, amphibian larval densities can be high, and Ranavirusinfection is
common. However, we were unable to detect Ranavirusin any
environmental water samples, so more research needs to be conducted to
further develop this method for use in the field.
This is the first attempt to create a water based sampling method for
the detection of Ranavirus. However, water sampling has been used to
detect other EIDs. Kirshtein et al. (2007) proposed a sampling protocol for
the detection of Batrachochytrium dendrobatidisin water and sediment.
Using 0.2 m filters and sampling between 64 l and 50 ml, they were able to
detect as little as 0.06 zoospores, or 10 copies of DNA. However, the lowest
concentration detectable in 50 ml of water was 30 zoospores per liter. The
protocol proposed for Ranavirusdetection in this paper uses lower volumes of
water, samples were filtered after collection from the field, and isolates a
smaller causative agent. The use of smaller water volumes may have caused
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an increase in false negative results. Samples were collected in the field and
put on ice until they could be returned to the lab for processing. This reduced
the amount of equipment and time needed to sample. Ranaviruses are
between 160-200 nm in size; because of their small size they are difficult to
filter out of water. The Amicon 15 (Millipor; Billerica, MA) was selected
because it had the ability to retain Ranavirusabove the filter and was easily
used in the lab, however it is limited to a 15 ml sample size. In future studies
using similar protocols for water sampling, it may be necessary to filter
multiple sub-samples of 15 ml through a single filter to reduce the chance of
false negative results. My goal was to keep the field sampling quick and the
laboratory work simple and thereby keep the cost, both in time and money
low, however using this system I was unable to detect Ranavirusin any
environmental water samples.
For water sampling to work an EID must be able to persist in the
environment. While this is not well established for Ranavirus, Brunner et al.
(2007) and Jancovich et al. (1997) showed Ranavirusinfections could be
initiated through inoculated sediment. Harp and Petranka (2006) also
showed that Ranaviruscould be transmitted through water between
Lithobates sylvaticatadpoles without contact. The novel detection method
presented here also anecdotally supports that Ranaviruscan be detected in
water, though only in seeded samples.
Ranaviruswas detected in concentrations as low as 13.3 PFU per l in
ddH2O and 106.4PFU per l in pond water, or13.3 x 103 PFU ml-1 and 10.6 x
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10 4 PFU ml-1respectively. The concentration of Ranavirusthat is biologically
significant is not known. However, Rojas et al. (2005) used a laboratory study
to suggest that an environmentally relevant concentration of Ranavirusvirions
in water inhabited with infected salamanders may be between 103 to 104 PFU
ml-1. The concentrations detected by my novel detection method in pond
water falls just above the projected biologically significant concentrations of
Ranavirus. It may be possible to reduce the lowest detectable volume by
adjusting PCR parameters, and reducing the chance of inhibitors in the pond
water. Inhibitors in the water are a possible problem with this system.
When six randomly selected samples of pond water were seeded with
20 l ofRanavirusat 8 x 107 PFU/ml, then filtered and processed normally,
only four tested positive for Ranavirus. This could lead to a high level of false
negatives. If Ranaviruswas present in these samples it is possible that the
primers do not adhere well to the wild strain of Ranaviruspresent at PMWMA.
However, this is a highly conserved region. Mao et al. 1997 tested nine
Iridovirus wild strains and was able to detect each of them using these
primers. In subsequent tests, positive results were not consistent and the
lowest detectable level of Ranavirusfluctuated. This may indicate that there
are PCR inhibitors in the water samples. However, more field research is
necessary to determine if these projected concentrations are indeed
biologically significant and whether or not inhibitors in the water are affecting
the lowest concentration detectable in pond water samples.
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The prevalence of Ranavirusin known infected areas has been
studied. In China, Ranaviruswas found to infect 5.7% of adults and 42.5% of
larva of Rana dybowskii(Xu et al. 2010). Gray et al. (2007) found that
between 15% and 40% of Rana clamitanstadpoles were infected with
Ranavirusdepending on time of the year and whether there was cattle access
to the wetland. These high percentages of infected animals were not found in
sampling sites at PMWMA. There may be several explanations for these
results. First, it is possible that sublethally infected animals tested negative
due to reduced levels of Ranaviruswithin their tissues. Brunner et al. (2007)
found that Ambystoma tigrinumlarvae that survived with sub-lethal infections
of ATV did not always test positive. Seven of ten sublethally infected animals
tested positive only once out of three tests. However, only a small portion of
individuals were sub-lethally infected. While this could have reduced the total
number of animals that tested positive it is not likely to cause all animals
sampled to test negative. Ninety-eight animals were sampled in this study
with most being tadpoles. With others reporting high percentages of tadpoles
being infected (Gray et al. 2007, Xu et al. 2010) it is not probable that sub-
lethal infections were a factor in PMWMA negative persistence of Ranavirus.
A second possibility is that through multiple exposures to Ranavirus,
the population at PMWMA is gaining immunity to Ranavirus. Gantress (2006)
found that Ranaviruswas cleared from Xenopus laevismore quickly after a
second exposure, and Majji et al. (2006) found that exposure to one
Ranavirusgave partial immunity to other Ranaviruses. Die-offs have
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occurred over the last six years at PMWMA (J. MacGregor, personal
communication, August 30 2008) and may be reducing a portion of the
susceptible adults and increasing the number of metamorphs that have
cleared a Ranavirusinfection. It is also possible that breeding has shifted to
reduce the presence of genes that lead to high-susceptibility to Ranavirus.
Teacher et al. (2009) found that Ranavirushad imposed selection for
particular haplotypes and that some amphibians were adapting to the
presence of Ranavirus. While more research would be necessary to
determine if this was occurring at PMWMA, it is possible that susceptible
individuals are dying and being replaced by animals that are less susceptible.
It is also possible that Ranaviruswas not detected at PMWMA
because it was not present at the time of sampling. Gray et al. (2007) found
that there was a higher percentage of infected tadpoles in the fall and winter.
Sampling in this study was conducted from July to October. This time frame
was chosen based on observations made at PMWMA (J. MacGregor,
personal communication, May 16 2008). Die-offs were observed historically
in May at PMWMA. Since sampling was done after May there is a possibility
that the concentration of Ranavirusin the water was lower than it would have
been if sampling was done earlier in the year. At PMWMA die-offs in wood
frogs and green frogs were most common. Due to my later sampling it is
possible that wood frog die-offs had already occurred and the level of
Ranaviruspresent was reduced. In this study a large amount of green frogs
were captured and tested without any evidence of Ranavirusbeing present.
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In future work at PMWMA it will be necessary to sample as early as possible
to determine if wood frog die-offs occur and whether or not there is an
increase in Ranavirusin water samples during this time. However, it is
possible that winter and fall infections and die-offs occur also at this location.
The lack of Ranavirusat PMWMA may have been due to the
misdiagnosis of the causative agent behind the die-offs that have occurred
there. The diagnosis of the causative agent was based on a few physical
symptoms and the fact that Ranavirushad been found in elsewhere in
Kentucky. While only one pond in Kentucky has had confirmed amphibian
Ranavirusdie-offs, Ranavirushas been confirmed in Terrapene carolinain
Ohio County, Kentucky via oral swabs (J. MacGregor, personal
communication, May 12 2009).
A recently emerging disease exhibits similar symptoms to Ranavirus.
Perkinsus-like disease, or Dermomycoidessp. causes lethargy, bloat, and
hemorrhaging of the skin (Cook 2009). While these previous symptoms are
similar to both fungal and viral EIDs, Perkinsus-like disease also causes
erratic swimming, and Davis et al. (2007) found that they were able to hand
catch infected animals. These symptoms have not been reported in the die-
offs at PMWMA; however, it will be necessary to continue to sample at
PMWMA during an active outbreak to determine definitively the causative
agent of the die-offs.
Another possibility is that after several drought years Ranavirusno
longer persisted in PMWMA. Die-offs may be affected by rainfall (J.
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MacGregor, personal communication, August 23 2007). Kentucky had lower
than average rainfall in Letcher County in both 2006 and 2007 (Robert Watts,
unpublished data). When sampling was done in 2008, Letcher County had
average rainfall for the year (National Weather Service Forecast Office).
Brunner el at. (2007) found that sediment that had dried for four days could
not cause infection. During sampling in 2008, 7 of 10 ponds dried for some
period of time over the sampling period, despite an average amount of
rainfall. It is a possibility that due to the occurrence of drought over a two
year span that Ranavirusdid not persist at PMWMA.
While many questions about the findings of this study persist, I am
confident that I have laid the ground work for water sampling to detect
Ranavirus. In the future it will be necessary to further refine this sampling
method by optimizing the PCR reaction and eliminating the possibility of
inhibitors in the water samples. Consistent long term sampling of PMWMA
will help to determine a base line of Ranavirusactivity in the area and
increase the sample size of both water and tissue collected. A positive
Ranavirusdie-off needs to be sampled to determine if Ranaviruscan be
detected in actual environmental samples and what concentrations of
Ranavirusare actually biologically relevant. Despite the future work needed, I
believe that this study has begun to establish the ground work necessary to
found a quick, inexpensive, and non-destructive testing method using water
samples that will elucidate the ecology of Ranaviruses.
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APPENDIX
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Table 1. Latitude and longitude of ponds with and without previous die-offs
and parking places close to the ponds.
Pond #
Latitude andLongitude for
Parking
Latitude andLongitude of Pond
LocationsPrevious Die-Offs
Documented
52 N 57 04' 14.8'' N 57 04' 11.1'' N
W 82 49' 10.0'' W 82 49' 08.0''
26 N 37 02' 36.3'' N 37 02' 41.6'' N
W 82 52' 48.0'' W 82 52' 45.8''
23 N 37 02' 23.1'' N 37 02' 19.5'' N
W 82 53' 04.5'' W 82 53' 04.5''
18 N 37 01' 47.8'' N 37 01' 44.8'' N
W 82 54' 00.4'' W 82 53' 57.6''
13 N 37 01' 32.5'' N 37 01' 31.9'' N
W 82 54' 49.9'' W 82 53' 49.7''
47 N 37 03' 47.2'' N 37 03' 43.1'' Y
W 82 50' 21.7'' W 82 50' 20.4''
44 N 37 03' 42.6'' N 37 03' 39.6'' Y
W 82 50' 38.3'' W 82 50' 37.6''
40 N 37 03' 11.3'' N 37 03' 10.9'' Y
W 82 51' 43.3'' W 82 51' 45.0''
36 N 37 03' 04.6'' N 37 03' 02.8'' Y
W 82 51' 57.6'' W 82 51' 01.0''
27 N 37 02' 42.6'' N 37 02' 38.3'' Y
W 82 52' 34.3 W 82 52' 34.3
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Table 2. The pond number, species collected, and the collection number of
the samples from July 10, 2008.
Pond Number Species Sampled Collection Number
26 N. viridescens Tail (adult) 26-1R. clamitans tail clip (Tadpole) 26-2
R. clamitans tail clip (Tadpole) 26-3
N. viridescens Tail (adult) 26-4
N. viridescens Tail (adult) 26-5
N. viridescens Tail (adult) 26-6
13 Rana clamitans toe (Adult) 13-1
R. clamitans tail clip (Tadpole) 13-2
R. clamitans tail clip (Tadpole) 13-3
Rana clamitans toe (Adult) 13-4
36 R. clamitans tail clip (Tadpole) 36-1
N. viridescens Tail (adult) 36-2
R. clamitans tail clip (Tadpole) 36-3R. clamitans tail clip (Tadpole) 36-4
40 Rana clamitans toe (Sm Adult) 40-1
R. clamitans tail clip (Tadpole) 40-2
R. clamitans tail clip (Tadpole) 40-3
R. clamitans tail clip (Tadpole) 40-4
44 Rana clamitans toe (Adult) 44-2
Rana clamitans (Adult) 44-2
47 Dry
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Table 3. The pond number, species collected, and the collection number of
the samples from July 25, 2008.
Pond Number Species Sampled Collection Number
52 Ambystoma sp. (larva) 52b-1Ambystoma sp. (larva) 52b-2
Rana clamitans tail clip (Tadpole) 52b-3
Rana clamitans tail clip (Tadpole) 52b-4
26 Notophthalmus viridescens Tail 26b-1
Rana clamitans toe (Sm. Meta) 26b-2
Rana clamitans toe (Sm. Meta) 26b-3
Rana eggs 26b-4
23 Dry
18 Dry
13 Notophthalmus viridescens Tail 13b-1
Rana clamitans toe (Adult) 13b-2
Hyla sp. (Tadpole) 13b-327 Notophthalmus viridescens Tail 27b-1
Rana clamitans toe (Adult) 27b-2
36 Notophthalmus viridescens Tail 36b-1
Rana clamitans toe (Adult)*** 36b-2Rana clamitans tail clip(Tadpole)*** 36b-3
Rana clamitans tail clip (Meta)*** 36b-4Rana clamitans tail clip(Tadpole)*** 36b-5
40 Notophthalmus viridescens Tail 40b-1
Rana clamitans tail clip (Tadpole) 40b-2
Rana clamitans toe (Sm. Meta) 40b-3
Rana clamitans tail clip (Tadpole) 40b-444 Rana clamitans toe (Adult) 36-1
Notophthalmus viridescens Tail 36-2
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Table 4. The pond number, species collected, and the collection number of
the samples from August 8, 2008.
Pond Number Species Sampled Collection Number
52 Ambystoma sp. 52C-1Notophthalmus viridescens 52C-2
Rana clamitans 52C-3
26Emerging Tads- Ranaclamitans 26C-1
Notophthalmus viridescens 26C-2
Rana clamitans 26C-3
23 Rana clamitans 23C-1
13 Rana clamitans tadpoles 13C-1
Notophthalmus viridescens 13C-2
27 Rana clamitans 27C-1
Rana sylvatica 27C-2
36 Notophthalmus viridescens 36C-1
40 Rana clamitans tadpole 40C-1
Notophthalmus viridescens 40C-2
Rana clamitans tadpole 40C-3
Rana clamitans adult 40C-4
44 Notophthalmus viridescens 44C-1
47 Dry
Table 5. The pond number, species collected, and the collection number of
the samples from August 26, 2008.
Pond Number Species SampledCollectionNumber
52 Bufo sp. Tadpoles 52D-1
23 Dry
13 Notophthalmus viridescens 13D-1
Rana clamitans Tadpoles 13D-2
Rana clamitans Tadpoles 13D-3
36 Rana clamitans 36D-1
47 Dry
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Table 6. The pond number, species collected, and the collection number of
the samples from September 11, 2008.
Pond Number Species SampledCollectionNumber
13 Rana clamitans Tadpole 13E -1
Rana clamitans Tadpole 13E -2
27 Rana clamitans Adult 27E -1
36 Rana clamitans Tadpole 36E -1
Rana clamitans Tadpole 36E -2
47 Dry
Table 7. The pond number, species collected, and the collection number of
the samples from September 28, 2008.
Pond Number Species SampledCollectionNumber
52 Ambystoma sp. Larva 52F-1
Ambystoma sp. Larva 52F-2
26 Rana clamitans Tadpoles 26F-1
Rana clamitans Tadpoles 26F-2
Rana clamitans Tadpoles 26F-3
18 Dry
13 Rana clamitans Tadpoles 13F -1
Rana clamitans Tadpoles 13F -2Rana clamitans Tadpoles 13F -3
Rana clamitans Tadpoles 13F -4
27 Rana clamitans Adult 27F -1
44 Rana clamitans Adult 44F-1
47 Dry
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Table 8. The pond number, species collected, and the collection number of
the samples from October 9, 2008.
Pond Number Species SampledCollectionNumber
52 Ambystoma sp. 52F2-1
Ambystoma sp. 52F2-2
26 Dry
23 Dry
18 Dry
13 Rana clamitans Tadpoles 13F2 -1
Rana clamitans Tadpoles 13F2 -2
Rana clamitans Tadpoles 13F2 -3
27 Rana clamitans Adult 27F2 -1
47 Dry
Table 9. The pond number, species collected, and the collection number of
the samples from October 23, 2008.
Pond Number Species SampledCollectionNumber
13 Rana clamitans Tadpole 13G-1
47 Dry
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Vita
Mathew Robert Pettus was born on January 7, 1979 to Robert and
Debbie Pettus in St. Louis, Missouri. At age 3, he and his family moved to the
northwest suburbs of Chicago. He went to elementary school at Central
School, junior high school at Westfield Junior High School, and high school at
Lake Park High School. In 1997, Matthew graduated high school and began
a long college career. In 2006, Matthew graduated from Elmhurst College
with a B.S. in biology. Matthew began work on a masters degree at Eastern
Kentucky University later that year. In 2010, Matthew received his M.S.
degree in biology from Eastern Kentucky University.