FISH AND AMPHIBIANS AS POTENTIAL RESERVOIRS OF MYCOBACTERIUM
ULCERANS, THE CAUSATIVE AGENT OF BURULI ULCER DISEASE
By
Sarah Jane Willson

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Entomology
2011

ABSTRACT

FISH AND AMPHIBIANS AS POTENTIAL RESERVOIRS OF MYCOBACTERIUM
ULCERANS, THE CAUSATIVE AGENT OF BURULI ULCER DISEASE
By
Sarah Jane Willson

Buruli ulcer is a skin disease associated with exposure to certain tropical water bodies. Much
remains unknown about the reservoir and transmission of this disease. Previous studies have
suggested that fish may concentrate M. ulcerans in their gills and intestines and then serve as
passive reservoirs of the bacteria. The current study was designed to expand on earlier studies by
using a PCR-based assay targeting the enoyl reductase (ER) domain of the plasmid responsible
for mycolactone production to screen multiple species of fish and amphibians from multiple
water bodies where M. ulcerans has been previously detected. This was done to test the
hypothesis that fish and amphibians serve as natural reservoirs of M. ulcerans, with some taxa or
feeding guilds being more likely to harbor the pathogen than others. ANOVA and non-metric
multidimensional scaling showed no fish or amphibian species or feeding guild served as a
reliable indicator of the presence of ER-positive mycobacteria in a water body. However,
specimens from certain water bodies were observed to have higher ER-positivity rates than
others. M. ulcerans in an adult frog was found and confirmed with VNTR analysis. This is the
first reported finding of the bacterium in a wild adult amphibian and suggests that amphibians
may warrant further study.

DEDICATION

Non nobis Domine, sed Tuo Nomine da gloriam.

iii

ACKNOWLEDGEMENTS

I am thankful to my advisor, Dr. Richard Merritt, and committee members, Dr. Michael
Kaufman and Dr. M. Eric Benbow for advice and support throughout this project. I would like
to thank everyone in the Walker / Kaufman Lab at MSU, especially Betsy Brouhard, Marissa
Cann, Garrett Berry, Blane Doyon, Lora Anderson and Dr. Edward Walker, for sharing lab space
and equipment. Dr. Michael Kaufman, Dr. Suzanne Thiem, Dr. Heather Williamson, and David
Malakauskas all assisted with the molecular aspects of this project. The logistics and field work
for this project would not have been possible without the planning and support of Dr. Mollie
McIntosh, Dr. Ryan Kimbirauskas, Paul Hoppe, and Stephanie Miller. I also want to thank
collaborators from Noguchi Memorial Institute for Medical Research in Ghana, especially Dr.
Daniel Boakye, Dr. Charles Quaye and Yaw Gaisah.

iv

TABLE OF CONTENTS

LIST OF TABLES……………………………………………………………………………….vi
LIST OF FIGURES……………………………………………………………………………...vii
INTRODUCTION………………………………………………………………………………..1
METHODS……………………………………………………………………………………….5
RESULTS………………………………………………………………………………………..11
DISCUSSION…………………………………………………………………………………...14
CONCLUSION………………………………………………………………………………….20
APPENDIX……………………………………………………………………………………...21
LITERATURE CITED…………………………………………………………………………..29

v

LIST OF TABLES

Table 1: Taxa collected in 2008 survey of areas considered likely to be ER positive (P) or ER
negative (N) based on water filter data…………………………………………………………..22

Table 2: Specimens collected in 2009 to be screened for M. ulcerans. All samples were collected
from districts considered to be endemic for Buruli Ulcer Disease……………………………....25

vi

LIST OF FIGURES

Figure 1: NMS ordination plot with three dimensions represented on two axes of fish
communities using species presence or absence data from 2008 in relation to ER positivity.
Triangles represent ER positive communities and circles represent ER negative communities.
Ellipses represent 95% confidence intervals. Stress: 0.1568934. For interpretation of the
references to color in this and all other figures, the reader is referred to the electronic version of
this thesis ……………………………….......................................................................................26
Figure 2: NMS ordination plot with three dimensions represented on two axes of amphibian
communities using species presence or absence data from 2008 in relation to ER positivity.
Triangles represent ER positive communities and circles represent ER negative communities.
Ellipses represent 95% confidence intervals. Stress: 0.06834609…………………………….....27
Figure 3: ER positivity rates of fish and amphibians and combined data for specimens collected
in 2009. …………………………………………………………………………………………28

vii

Introduction

Mycobacterium ulcerans MacCallum is the causative agent of Buruli ulcer disease
(MacCallum et al. 1948). In humans, Buruli ulcer disease manifests first as a small nodule and
can progress to extensive skin ulcerations. This disease tends to occur in tropical and subtropical
areas and is especially prevalent in the West-African countries of Benin, Ivory Coast, and Ghana
(W.H.O. 2008). On a large scale, Buruli ulcer disease has been correlated with agricultural land
use (Wagner et al. 2008). Local outbreaks have been linked to exposure to certain water bodies
but the reservoir(s) and method of transmission of M. ulcerans have not been determined (Aiga
et al. 2004; Jacobsen and Padgett 2010; Merritt et al. 2010; Raghunathan et al. 2005; Sopoh et al.
2010).
M. ulcerans is believed to have originated from the closely related Mycobacterium
marinum Aronson, a common environmental bacterium (Stinear et al. 2000). Though M.
ulcerans is also traditionally considered to be an environmental pathogen, recent analyses show
that the genome of M. ulcerans is contracting, consistent with an evolutionary shift from a freeliving ancestor to use of the more stable environment provided by a specific host (Stinear et al.
2007). In addition, M. ulcerans has acquired a virulence plasmid, pMUM001, which allows M.
ulcerans to produce mycolactone. Mycolactone is a toxin that may play an important role in
allowing M. ulcerans to use specific hosts. For example, salivary glands of certain aquatic
insects can be colonized by wild-type M. ulcerans, but not by mutant mycolactone-negative M.
ulcerans (Marsollier et al. 2005). PMUM001 occurs only in M. ulcerans and a few closely
related mycolactone-producing mycobacteria recently discovered in fish (Rhodes et al. 2005;
Ranger et al. 2006) and frogs (Trott et al. 2004). Though currently given separate species names,

1

it has been argued these other mycolactone-producing mycobacteria are simply different strains
of M. ulcerans (Pidot et al. 2010).
Possible hosts of M. ulcerans include predaceous aquatic insects (Marsollier et al. 2002),
fish (Kotlowski 2004), snails (Marsollier et al. 2004), terrestrial mammals (Fyfe et al. 2010) and
mosquitoes (Johnson 2009). In aquatic environments M. ulcerans may be concentrated by small
filtering organisms, such as filter feeding insects, which are then preyed upon by larger
organisms, causing further concentration of the bacteria (Portaels et al. 1999; Merritt et al. 2005).
Marsollier et al. (2005) suggested that M. ulcerans may be transferred to humans through the bite
of aquatic Hemiptera. However, this is unlikely to be the primary method of transmission
(Benbow et al. 2008), nor have these insects been shown to be a vector of any pathogen
associated with animals in nature (Merritt et al. 2010). More recent experimentation by Mosi et
al. (2008) showed colonization by M. ulcerans of the exoskeleton of biting water bugs
(Hemiptera: Belostomatidae) that had fed on M. ulcerans infected mosquito larvae. This
indicates that insects may still play a role as a reservoir in the transmission cycle.
Previous studies have suggested that fish may concentrate M. ulcerans in their gills and
intestines and then serve as passive reservoirs of the bacteria (Portaels et al. 2001; Eddyani et al.
2004; Kotlowski 2004). In these studies, the gills and intestines of several fish were found to be
positive for IS2404, an insertion sequence that occurs 209 times on the M. ulcerans chromosome
and four times on pMUM001 (Stinear et al. 2007). This insertion sequence was previously
thought to be specific to M. ulcerans, but is now known to occur in other mycobacteria (Yip et al.
2007). The use of the gills and intestine in the above studies raises the question of whether fish
were actually colonized by M. ulcerans, or whether it was simply being trapped by their gills and
passing through their digestive tracts. Feeding trials with frogs have shown that mycobacteria

2

can survive passage through the digestive tract and be passed out through the feces (Mok et al.
1987). Testing of internal organs other than the digestive tract is needed to show whether wild
fish are actually becoming infected with M. ulcerans.
In addition, it is possible that M. ulcerans may be localized in organs other than the
intestine and gills. Experimental exposure of Danio rerio Hamilton to M. marinum and
Mycobacterium peregrinum Bojalil showed that these mycobacteria are primarily acquired
through the intestine, and then disseminate to other internal organs. Though mycobacteriosis in
fish can potentially involve any of the organs, it most commonly involves the kidney and liver
(Belas et al.1995). With fish artificially infected with M. marinum and M. peregrinum,
mycobacteria were subsequently recovered from the intestines, livers, and spleens of infected
fish, but never from the gills (Harriff et al. 2007). It is possible that testing only the gills and
intestines of fish may have resulted in an underestimation of rates of infection.
The discovery of mycolactone-producing mycobacteria in frogs has also raised the
question of whether or not M. ulcerans might also occur in frogs. Many tadpoles feed by
filtering or scraping small algal particles, and could conceivably acquire and concentrate M.
ulcerans during this process, as suggested by Merritt et al. (2005). Though historically
considered to be an extracellular mycobacterium, M. ulcerans is capable of growing inside
amphibian cells at 28ºC (Drancourt et al. 2002).
It is unclear if the fish tested in earlier experiments were actually positive for M. ulcerans
or if the positive IS2404 tests resulted from the presence of other mycobacteria. More specific
PCR methods have recently been developed. Enoyl reductase (ER) PCR targets the ER domain
of the pMUM001 plasmid (Williamson et al. 2008), which only occurs in mycolactone
producing mycobacteria. This target occurs in only four places on the plasmid, making ER-PCR

3

less sensitive but more specific than IS2404 PCR (Durnez et al. 2009). Variable number tandem
repeat (VNTR) DNA typing is also being used to distinguish between M. ulcerans and other
mycolactone-producing mycobacteria. VNTR loci occur in varying numbers, with some
occurring only once in the genome making this the least sensitive, though highly specific, PCR
detection method (Lavender et al. 2008). Additionally, positive assay results are dependent on
currently known VNTR profiles, and it is likely that additional profiles exist.
The current study was designed to expand on earlier studies by screening multiple species
of fish and amphibians from multiple water bodies where M. ulcerans has been previously
detected. This was done to test the hypothesis that fish and amphibians serve as natural
reservoirs of M. ulcerans, with some taxa or feeding guilds being more likely to harbor the
pathogen than others. As such, it was predicted that certain fish and amphibian taxa could be
used as indicator species for the presence of environmental conditions favorable to M. ulcerans.
In addition, because fish and amphibians are mobile (increasing the chance of contact with the
bacterium), and provide a stable environment for the bacterium, PCR testing of fish and
amphibians would allow for more consistent detection of M. ulcerans in a water body than other
environmental sampling methods such as water filters. To this end, a survey of fish and
amphibian taxa was conducted in M. ulcerans positive and M. ulcerans negative sites.
Specimens were collected from M. ulcerans endemic areas for subsequent PCR screening for
mycolactone-producing mycobacteria, and positivity rates were compared for internal organs
versus external swabs, between different taxa, and between feeding guilds.

4

Methods
Study Location
This study was carried out as part of a larger study on the ecology of M. ulcerans in West
Africa. In July 2008, samples were collected in Ghana, along a gradient from the Volta Region,
where no M. ulcerans or mycolactone-producing mycobacteria have been detected, to the
Greater Accra area, where M. ulcerans and other mycolactone-producing mycobacteria have
been detected at multiple locations (Williamson et al. 2008). A second sampling season occurred
August 2009, in the Ga West and Tema districts of Accra, Ghana, where M. ulcerans was
previously detected (Williamson et al. 2008).

Collection and Preservation of Specimens
Three sampling techniques were employed at each site to maximize the variety of taxa
captured. Three collapsible live bait traps (Promar model TR-501, Gardena, CA) were deployed
at each site and baited with commercial catfish dough bait (Berkley, Spirit Lake, IA), and the
traps were set as far apart as possible around the site. Traps were submerged for approximately
one hour at each site. In addition, a seine (Cabela’s Inc., Sidney, NE) was used when the sites
were wadeable and without abundant aquatic vegetation. A D-net (Bioquip Products, Rancho
Dominguez, CA) was used to sample marginal areas and within aquatic plant beds.
Approximately one hour of combined seining and D-net sampling was performed at each site.
When adult frogs were observed at a site, they were captured with D-nets. Up to 30 individuals
of each fish and amphibian taxon present at the site were captured, and excess individuals were
released. The one exception to these methods was Lake Weija (2009 Site 9), where live fish
were purchased from local fishermen. Fish and tadpoles were euthanized with CO 2 and adult

5

frogs were euthanized with benzocaine hydrochloride (IACUC AUF # 06/08-090-00). Buckets
used to hold captured fish and amphibians were cleaned and treated with RNase
awayTM (Molecular Bioproducts, Inc., San Diego, CA) between sites.
In addition to collecting fish and amphibians, 500 ml of water was also collected and
filtered at each site during season two, to test for the presence of M. ulcerans. This water was
pre-filtered through a 1.6 μm fiberglass filter (Whatman Inc., Kent, UK) to remove large
particles and then filtered through a 0.2 μm nitrocellulose filter (Whatman Inc., Kent, UK). If
the filter became clogged multiple filters were used. The filter apparatus was cleaned with
RNase AwayTM between each site. The filters were saturated with 95% ethanol for preservation
and placed in foil wrappers for transport to Michigan State University, where they were stored at
-20ºC until processing.
In 2008, all tadpoles were preserved in 10% formalin and all fish were preserved in 95%
ethanol, and all organisms from a single site were grouped together. For 2009, half of all
specimens (fish and tadpoles) were preserved in 10% formalin and half were preserved in 95%
ethanol. Specimens in ethanol were either preserved individually or pooled into groups of 3
depending on size.
Fish and amphibians were identified to the lowest taxonomic level possible. Fish were
identified to genus and in most cases species level using Dankwa et al. (1999). Amphibians were
identified to the lowest possible taxonomic level using Channing and Howell (1999) and Schiotz
(1999). In most cases this was the generic level. Many tadpoles cannot be identified to the
species level, and the tadpoles of numerous species are not known. In addition, tadpole
morphology changes during development, and tadpole keys are based on fully grown tadpoles
(Channing and Howell 1999). Specimens were dissected and the intestines and kidneys removed

6

and stored in 95% ethanol. Instruments were cleaned between specimens using 95% ethanol
TM

followed by application of RNase Away

.

DNA Extraction
Year 2009 samples preserved in 95% ethanol were screened for M. ulcerans DNA. All
DNA extraction and PCR procedures were performed in a hood, under sterile conditions.
Negative controls were used throughout the process to ensure sterility and assess possible
contamination. M. ulcerans cells preserved in 95% ethanol were used as a positive control to
ensure that procedures were successful. For the dissected intestine and kidney samples, ethanol
was poured off of each sample and reserved in a sterile container. The wet mass of the
remaining sample was recorded and the sample was then homogenized with the previously
reserved ethanol in a glass tissue grinder. For small samples (less than or equal to 20 mg wet
mass), the entire sample was used for DNA extraction. For large samples, homogenate
containing approximately 20 mg of tissue was used for DNA extraction. In order to remove the
ethanol from the samples before DNA extraction, samples were centrifuged and the ethanol was
removed. The remaining pellet was rinsed with TE, centrifuged, and the supernatant was
removed.
Extraction was first attempted using the one tube method designed for extraction of M.
ulcerans in aquatic insects, mollusks, and fish (Kotlowski et al. 2004). However, satisfactory
detection was not achieved with this method. DNA was then extracted using a method optimized
for environmental mycobacteria (Käser et al. 2009), with the exception of the final step, in that
DNA was re-suspended in 25 µl TE instead of 100 µl water. Extractions were performed in
groups of 18 samples, and each group included a negative and a positive control. Extracted DNA
7

from a subset of 27 intestine samples and 23 kidney samples was spiked with M. ulcerans DNA
prior to PCR analysis to determine the level of PCR inhibition. Due to the presence of inhibitors,
it was necessary to further purify the extracted intestinal DNA according to manufacturer’s
®

instructions using PowerClean DNA Clean-Up Kits (MO BIO Laboratories, Inc., Carlsbad,
CA), and eluting with 50 µl of the provided elution solution. Negative and positive controls
were also cleaned using this procedure. Because the clean-up of the intestine samples resulted in
more dilute DNA than that of the kidney samples, 8 µl of intestine sample DNA versus 4 µl
kidney sample DNA was used per 25 µl PCR mixture.

PCR and Gel Electrophoresis
PCR was performed targeting the enoyl reductase domain of the plasmid responsible for
mycolactone production. The 25 µl PCR cocktails contained 2 µl of each primer (10 µM) (F: 5’GAGATCGGTCCCGACGTCTAC-3’, R: 5’-GGCTTGACTCATGTCACGTAAG-3’), 12.5 µl
TM

FailSafe

2x PCR buffer (EPICENTRE Biotechnologies, Madison, WI), 0.5 µl Go Taq

polymerase enzyme (Promega, Madison, WI), and either 4 µl of template DNA and 4 µl PCR
water (for kidney samples), or 8 µl template DNA (for intestine samples). PCRs were run using
primers and cycling conditions described in Williamson et al. (2008). Samples were loaded
along with positive and negative controls onto 0.8% TBE agarose gels stained with Ethidium
Bromide and fragment sizes were compared to a 1 Kb DNA ladder (Invitrogen, Carlsbad, CA).
ER-positive samples were then sent to the University of Tennessee for VNTR analysis as
previously described (Williamson et al. 2008).
Serial dilutions were made from a stock sample of M. ulcerans cells preserved in 95%
7

ethanol. The stock solution contained approximately 10 CFUs/ml. Aggregates of bacteria were
8

broken apart by passing the bacterial suspension through a 25 gauge needle 10 times
(Williamson et al. 2008) and eight 1:10 serial dilutions were made. To determine whether
sensitivity was changed by the addition of fish tissue, the internal organs of a commercially
purchased goldfish were dissected and processed using the previously mentioned techniques. 180
µl of 0.1 mg/µl (wet mass) tissue homogenate was spiked with 20 µl of each of the previously
made dilutions before being subjected to the Käser (2009) extraction procedure and ER PCR.

Data Analyses
For data collected in 2008, a Jaccard dissimilarity matrix was constructed separately for
both fish and amphibian communities. Separate non-metric multidimensional scaling (NMS)
analyses were performed on fish and amphibian communities using species presence or absence
data to determine if these communities were related to ER positivity. A scree plot was used to
determine the optimal number of dimensions to characterize the data. Once the optimal number
of dimensions was determined, 1000 initial starts were used for subsequent analysis.
Additionally, an ANOSIM was performed using 10,000 permutations to determine if there were
differences between ER positive and ER negative communities and an indicator species analysis
was performed to determine if specific taxa could be used to predict ER positivity. The above
statistical tests were performed using R version 2.13.1 (http://R-project.org, R Foundation for
statistical Computing, Vienna, Austria), with the ecodist (Goslee and Urban 2007), labdsv
(Roberts 2010), ellipse (Murdoch and Chow 1996), BiodiversityR (Kindt and Coe 2005), and
vegan (Oksanen et al. 2011) packages. A site was considered to be ER positive if any ER
positive filters had ever been recovered from the site (Heather Williamson, unpublished data).
For 2009, a separate one way ANOVA was performed on arcsine square root transformed

9

percent positive values for fish and amphibians, with site and species as factors. For fish an
additional ANOVA was performed using feeding guild as a factor. One location (Djorse)
consisted of two different water types, a lotic stream next to a lentic pond. Due to the differing
habitats, the location was classified as two different sites (Sites 1 and 2) in the analyses. Site 6
(Mensah Bar) was not included in season 2 analyses due to consistently ambiguous ER results
for that site. Results were considered to be significant at α=0.05. For significant ANOVAs,
Tukey Kramer post-hoc multiple comparison procedures were performed. JMP® Statistical
Discovery Software, version 7.0 (www.jmpin.com, SAS Institute, Inc., Cary, NC) was used for
ANOVAs.

10

Results

A total of 624 fish, representing 13 genera and at least 17 species, and a total of 350
amphibians, representing 10 genera, were collected (Tables 1 and 2). A scree plot showed a three
dimensional solution in the NMS analysis. NMS analyses showed that ER positivity was not
associated with fish or amphibian community structure (Figures 1 & 2). ANOSIM verified these
results (fish: p-value = 0.089191; amphibian: p-value = 0.38766). Indicator species analysis
showed a significant association of fish species Barbus sublineatus with ER positivity (p-value =
0.038), however it had a low indicator value of 0.4333.
Of the 271 intestine samples that were subjected to DNA extraction, the resulting DNA
from 141 samples (52%) was visibly discolored and showed PCR inhibition. A sub-sample of 27
intestinal DNA extractions that were not visibly discolored was spiked with M. ulcerans DNA to
determine the level of inhibition. Of these, 63% showed signs of inhibition. Of the 23 kidney
samples randomly selected and spiked with M. ulcerans DNA, none showed evidence of
inhibition. Spin columns were used in an attempt to remove inhibitors from the discolored
kidney samples. While this did result in removal of most of the brown coloration from the
samples, the PCRs remained completely inhibited. The MO BIO cleanup kit removed both the
brown coloration and the PCR inhibition.
With the one-tube procedure, the lowest detection limit using ER PCR was
approximately 1,600 CFUs. With the Käser extraction procedure, the lowest detection limit was
0.32 CFUs. Addition of goldfish tissue homogenate did not decrease the detection limit, but
additional non-specific binding was observed.

11

Negative controls consistently showed lack of contamination and positive controls
consistently showed that the extraction and PCR were successful. Site 6 (Mensah Bar) was
excluded from statistical analyses due to ambiguous PCR results for that site. PCRs of
specimens from this site consistently produced a fragment which was slightly larger than the
expected ER fragment, but was so close in size that it was not possible to conclusively determine
whether the specimens were ER positive. Overall ER positivity rates were 54% (128 / 238). For
fish, ER positivity was 39% (25 / 65), tadpoles were 59% (96 / 162), and adult frogs were 64%
(7 / 11). Positivity rates for all specimens from a single site ranged from a low of 6% at site 1 to
74% at site 8 (Figure 3). ANOVA results showed ER positivity rates for fish were significantly
different among locations (p-value = 0.0054), with a Tukey test indicating that specimens from
sites 2, 3, and 4 were most highly positive. ER positivity rates were not significantly different
between fish species or among feeding groups. Amphibian positivity rates were not significantly
related to location, genus identification or life stage (adult versus tadpole). When fish and
amphibian data were combined, positivity was not significantly related to site or to group (fish
versus tadpole versus adult frog).
Of the 128 positive specimens, 118 were identified as positive based on DNA from the
intestine. Five specimens had positive results from both the intestine and the kidney, and 5 had
positive results only from the kidney.
Twenty-seven external surface swabs were analyzed, and 4 of these were ER positive. Of
the 27 total swabs, 14 came from fish or amphibians in which either the kidney or intestine was
ER positive. Of these 14, only one external swab was also positive. The other 3 ER positive
external swabs came from fish and amphibian specimens in which the kidney and intestine were
ER negative.

12

One 0.2 μm nitrocellulose filter was tested from 7 sites sampled in season 2. Only the
filter from site 6 (Mensah Bar) tested ER positive, despite ER-positive specimens being collected
from every site.
One hundred seventy ER positive samples were sent to the University of Tennessee for
VNTR analysis. Three of these were successfully VNTR typed. Two were determined to be
Mycobacterium liflandii, a mycolactone-producing mycobacteria pathogenic to frogs. These
were recovered from the intestine of a Hyperolius tadpole (Site 10), and the intestine of a
predatory cichlid, Hemichromis bimaculatus (Site 2). One specimen, an adult frog from the
genus Leptopelis, (site 3) was confirmed to be positive for M. ulcerans. This is the first known
finding of M. ulcerans in an adult amphibian.

13

Discussion

Site ER positivity was not statistically associated with specific fish or amphibian
communities and showed no association between species identification and ER positivity.
However, this sampling scheme was qualitative in nature, representing a single point in time. To
fully characterize fish and amphibian communities, more intensive sampling methodologies
would be required. According to these data, the presence or absence of certain fish and
amphibian species appears unlikely to be useful as an indicator for the presence of M. ulcerans in
a water body.
Species and functional groups did not influence positivity rates in fish, but with fish data,
certain sites were significantly linked to positivity. This may suggest that the environmental
distribution of M. ulcerans is a part of the ecology of highly focal waterbodies, but it may also be
an artifact related to small sample sizes in those locations. Fish appear likely to come into
contact with the bacterium regardless of feeding guild. Additionally, while it is not apparent that
fish act as replicative reservoirs for M. ulcerans, they do accumulate bacteria at levels detectable
by standard PCR, and offer a potential tool for environmental screening. Functional group
analyses were not performed for amphibians because little is known about the specific feeding
methods of tadpoles, and because all adult frogs are predators. However, neither genus nor site
was significantly linked to positive ER status among amphibians.
The overall fish ER positivity rate (39%) was higher in this study than IS2404 positivity
rates reported in previous studies (10% in Eddyani et al. 2009 and 20% in Kotlowski et al. 2004).
This was unexpected considering the much higher sensitivity of IS2404 PCR. Possible reasons
for the difference include annual and seasonal variation in positivity rates, increased sample sizes

14

in the current study, and differing extraction methods. For example, the one-tube extraction
method used by Kotlowski was attempted in the current study, but the Käser extraction method
was chosen because it resulted in detection limits that were over 5,000 times more sensitive. Site
was shown to be an important factor in the current study, so number and location of sites could
also explain the difference. Non-specific binding was observed, particularly in the intestine
samples, so it was possible that this could have produced some false positives in samples from
this study. Contamination is always a possibility, but every effort was taken to avoid
contamination, and negative controls consistently showed a lack of contamination. In addition,
samples were processed in batches that usually included both kidney and intestine samples. If
contamination had occurred it would have impacted the results of both, not just intestines. The
fact that almost all of the kidney samples were negative provides evidence that contamination
was not a factor. The possibility of PCR inhibition in previous studies must be considered,
especially given the high amount of inhibition initially observed in the present study.
For amphibians, it is important to consider the slow rate of development of M. ulcerans
and the transience of the aquatic tadpole stage. It has been shown that M. ulcerans can replicate
within amphibian cells (Drancourt et al. 2002). Timing of tadpole development is highly
variable, depending on species, temperature, food availability, and density. Some Hyperolius
species go through metamorphosis after 5-6 weeks, while some Kassina species can take up to
10 months (Channing and Howell 2006). M. ulcerans or other mycolactone-producing
mycobacteria acquired by tadpoles with very short aquatic stages would potentially have very
little time to replicate while the amphibian remained in the tadpole stage, and might not be
detectable until after the frog has gone through metamorphosis. For this reason, adult frogs may
be better targets than tadpoles for future studies. The single specimen in which M. ulcerans was

15

verified by VNTR typing was an adult frog. This is the first report of M. ulcerans in a terrestrial
amphibian, and may represent a potential aquatic-terrestrial linkage. While M. ulcerans has
traditionally been associated with aquatic habitats, it has recently been found in terrestrial
mammals in Australia (Fyfe et al. 2010).
The majority of positive ER results came from intestine samples for both fish and frogs.
External swabs had an overall ER positivity rate of only 15%, suggesting that while bacteria may
sometimes adhere to the external surface of an organism, ER positive intestine results were
actually due to bacteria in the intestine, and not due to external contamination of samples. The
very low rate of ER positive kidney samples suggests that bacteria were acquired during feeding
and were passing through the intestine without causing a systematic infection. If the bacteria
were acquired through injection, such as during attack by predators, it would likely be detected
in the kidney as well (Mosi 2009).
Water filters had lower overall positivity rates than fish and amphibians. Unfortunately,
only a single filter could be processed from each site where water filtrate was collected due to
time constraints, though multiple sites were tested. One of 7 filters (14%) tested ER positive.
That filter was from site 6 (Mensah Bar), a site that produced ambiguous ER results and was
therefore removed from the fish and amphibian analyses. The 14% positivity rate for filters was
consistent with the 15% positivity rate for external swabs. Both of these would likely represent
bacteria suspended in the water column. Water filters have been used as a way to detect M.
ulcerans in water bodies (Williamson et al. 2008, Vandelannoote et al. 2010). However, this
method could potentially be missing M. ulcerans-positive locations simply due to the small
quantity of water filtered. If M. ulcerans was unevenly distributed in the water body, water
samples could prove to be inconsistent. Use of fish or amphibians may be a more effective way

16

to determine whether M. ulcerans is present in a water body due to the animals’ mobility and
subsequently greater chance of bacterial contact. Additionally, a greater detection rate in fish
guts versus water samples in this study suggests some retention within fish intestines, but further
research is needed to determine the residence time of M. ulcerans within fish guts. Preliminary
study by Mosi (2009) found that M. ulcerans could be detected in the guts of 9% of artificially
infected belostomatids 30 days post infection and in 30% of guts 60 days post infection, though
these findings are difficult to interpret due to small sample sizes.
Despite finding relatively high ER positivity rates in fish and amphibians, very few
samples were successfully VNTR typed. There are two potential explanations for this. First,
there were likely very low proportions of M. ulcerans or other mycolactone-producing
mycobacterial DNA in the samples, and many of the samples yielded low overall DNA
quantities. DNA was further depleted by early extraction issues and repeated PCRs. Many of
the ER bands were very faint, consistent with small quantities of DNA. VNTR amplification is
less sensitive than ER PCR, so it is likely that DNA could be detected by ER amplification but
not by VNTR amplification. Lavender et al. (2008) reported low sensitivity for VNTR of
-1

environmental samples, with over 100 genomes µL DNA required for identification. Fyfe et al.
5

(2010) reported that at least 10 organisms/gram must be present for successful VNTR analysis.
Low quantities of DNA in fish are consistent with the conclusion of Mosi (2009) that M.
ulcerans does not replicate in fish. For fish, it appears that M. ulcerans and other mycolactoneproducing mycobacteria are likely encountered during feeding, but then pass through the
digestive tract. This could be further investigated by testing fecal material separately from
intestinal tissue.

17

A second possibility for low success of VNTR typing was false ER positive results. As
discussed above, it was unlikely that contamination was an issue, given the low rate of ER
positivity among kidney samples that were processed along with the intestine samples. However,
it was possible that non-specific binding may have caused false positives. Non-specific bands
were observed in many intestine samples, and some were close in size to the expected ER band
size. This could be dealt with in future studies by re-designing primers, varying PCR protocols
and reagent concentrations, trying different polymerases, or by using different PCR targets.
Verification through sequencing would also be an important tool for determination of positive
PCR identity
The relatively high levels of PCR inhibition experienced during the course of this study
reinforce the need to consider inhibition when dealing with environmental samples.
Inhibition was likely caused by some combination of polysaccharides, polyphenols, and/or
humic acids. Polysaccharides are especially problematic in plant materials. They co-precipitate
with DNA during ethanol precipitation, thereby remaining in the extracted DNA (Sharma et al.
2002). Polyphenols and humic acids are the most common inhibitors of environmental samples,
especially samples including soil particles, and lakes with colored water. They can cause brown
color in extracted DNA, and covalently bind to DNA, which blocks the action of DNA
polymerase (Wilson 1997).
Methods to remove these inhibitors include precipitation of polysaccharides with high
concentrations of NaCl and Sarcosyl, or with SDS and potassium acetate. Polyphenols and
humic acids can be removed with Hexadecyltrimethylammonium bromide (CTAB) based
extractions, Sephadex or other biogel columns, cesium chloride density centrifugation, gel
electrophoresis, or Polyvinylpolypyrrolidone (PVP) (Dong et al. 2006). Commercial purification

18

kits such as the MO BIO kit used in this study are also used. However, because the methods
vary in effectiveness, there is a need for a standardized extraction method for use in M. ulcerans
studies in order to be able to meaningfully compare results across studies.
Numerous extraction methods have been used to test for M. ulcerans (Boom et al., 1990,
Durnez et al. 2009, Fyfe et al. 2007, Käser 2009, Ross et al. 1997, Williamson et al. 2008). The
Käser (2009) method was chosen for this study specifically because it was a recently developed,
optimized combination of well-known methods, was developed with testing of environmental
samples in consideration, was relatively inexpensive, and was validated with insect samples.
However, despite working well on control fish tissue, the method did not remove environmental
inhibitors from actual field samples. Other studies have also found problems with inhibition.
Vandelannote et al. (2010) found inhibition in 50/148 environmental samples based on Ct values
when using the Modified Boom extraction method (Boom et al. 1990, Durnez et al. 2009).
While using a commercial kit extraction, Fyfe et al. (2007) detected inhibition in some soil
samples extracts with the use of Ct values. The samples were re-analyzed after 10-fold dilution.
While dilution is feasible in the presence of high amounts of target DNA, environmental samples
may contain very small amounts of M. ulcerans DNA, which may not be detectable after dilution.
It should also be noted that dilution fails to remove inhibitors that are bound to the DNA, and is
an unsatisfactory treatment of nucleic acids contaminated by numerous environmentally-derived
inhibitory compounds.

19

Conclusion

In this study M. ulcerans in an adult frog was found and confirmed with VNTR analysis.
This is the first reported finding of the bacterium in a wild adult amphibian and suggests that
amphibians may warrant further study. Amphibians cannot be ruled out as reservoirs due to the
transience of their aquatic stages and higher ER-positivity rates as compared to fish. No fish or
amphibian species or feeding guild served as an indicator of the presence of ER-positive
mycobacteria in a water body. However, certain waterbodies were observed to have higher ERpositivity rates than others. While not useful as indicator species, fish and amphibians may still
be useful for the detection of ER-positive mycobacteria. Future studies on the environmental
distribution of M. ulcerans and other mycolactone-producing mycobacteria would benefit from
the development of a standardized extraction method carefully tested to ensure removal of
inhibitors.

20

APPENDIX

21

Table 1: Taxa collected in 2008 survey of areas considered likely to be ER positive (P) or ER
negative (N) based on water filter data.
Location

Fish

Amphibians
Count

Count

Site 1 (P)
Otinibi

Barbus sublineatus
Cyprinodontidae
Sarotherodon sp.

19
1
1

Hyperolius sp.
undetermined

Site 2 (P)
Danfa

Barbus sublineatus
Hemichromis bimaculatus
Sarotherodon sp.

6
8
6

none

Site 3 (P)
Teiman

Hemichromis bimaculatus
Parachanna obscura
Sarotherodon sp.

3
1
21

none

Site 4 (P)
Afiaman

Brienomyrus brachyistius
Hemichromis bimaculatus
Hemichromis fasciatus
Sarotherodon sp.

1
1
1
2

none

Site 5 (P)
Kotoku

Sarotherodon sp.

5

Afrixalus sp.
Hyperolius sp.

11
6

Site 6 (P)
Nsakina

Protopterus annectens

1

Afrixalus sp.
Hyperolius sp.

13
1

Site 7 (N)

6
25

Leptopelis sp.

6

Pampamwie

Barbus parablabes
Barbus sublinatus

Site 8 (N)
Ata Kofi

Barbus parablabes
Barbus sublineatus

9
26

Afrixalus sp.
Amnirana sp.
Hyperolius sp.

5
6
9

Site 9 (N)
Titiaka

Barbus trispilos
Larval fish
Sarotherodon sp.

1
1
1

Amnirana sp.
Bufo sp.
Leptopelis sp.

3
2
4

Site 10 (P)
Wawaso

Barbus macrops
Barbus sublineatus

1
9

none

22

1
3

Table 1 (cont’d)
Site 11 (N)
Asato

Cyprinidae

3

Amnirana sp.

Site 12 (P)
Okaniase

Barbus sublineatus

9

none

Site 13 (N)
Adaklu

Barbus macrops
Barbus trispilos
Sarotherodon sp.

25
11
1

none

Site 14 (N)
Adaklu

Barbus trispilos
Fundulosoma thierryi

19
7

Afrixalus sp.
Hyperolius sp.

1
10

Site 15 (N)
Adaklu

Aphyosemion walkeri
Barbus macrops
Barbus parablabes
Sarotherdon sp.

10
7
27
4

Afrixalus sp.
Hyperolius sp.

5
1

Site 16 (P)
Agodeke

Aphyosemion petersii
Barbus parablabes
Barbus trispilos
Propterus annectens

15
4
26
1

none

none
Site 17 (P)
Abutia Kloe

31

Afrixalus sp.
Hemisus sp.
Hyperolius sp.
Kassina sp.

6
1
6
1
1
2

Site 18 (P)
Wayanu

Barbus parablabes
Barbus sublineatus
Larval fish
Syndontus sp.

2
11
1
2

Bufo sp.
Hyperolius sp.

Site 19 (N)

Barbus macrops
Brienomyrus brachyistius
Hemichromis fasciatus
Sarotherodon sp.

30
2
1
7

none

Barbus macrops
Hemichromis bimaculatus
Sarotherodon sp.

28
4
10

Hyperolius sp.

Laweh Kope

Site 20 (N)
Asutuare

Junction

23

4

Table 1 (cont’d)
Barbus macrops
Hemichromis fasciatus
Sarotherodon sp.

41
1
11

none

Cyprinodontidae
Hemichromis bimaculatus
Sarotherodon sp.

2
1
5

Hyperolius sp.

Site 23 (N)
Mensah

Fundulosoma thierryi

9

None

Site 24 (N)
Saduase

none

Site 25 (N)

Sarotherodon sp.

31

none

Total Fish:

524

Total Amphibians:

Site 21 (N)
Asebi

Site 22 (N)
Dedenya

Afrixalus sp.
Chiromantis sp.
Hyperolius sp.

22

1
1
15

Oyibi

24

178

Table 2: Specimens collected in 2009 to be screened for M. ulcerans. All samples were collected from
districts considered to be endemic for Buruli Ulcer Disease.
Location

Fish

Amphibians
Count

Count

Site 1
(Djorse
stream)

Barbus sublineatus
Hemichromis bimaculatus
Epiplatys dageti

1
4
2

Bufo sp.

10

Site 2
(Djorse
pond)

Hemichromis bimaculatus

1

Afrixalus sp.
Ranidae

12
4

Site 3
(Otuaplem)

Hemichromis bimaculatus
Clarias anguillaris

3
1

Ranidae
Leptopelis sp.
Afrixalus sp.
Hemisus sp.
Xenopus sp.

2
1
4
10
12

Site 4
(Sarpeiman)

Hemichromis bimaculatus
Unidentified larval fish

3
1

Bufo sp.
Afrixalus sp.
Hyperolius sp.

13
1
8

Site 5
(Seduase)

none

Afrixalus sp.

9

Site 6
(Mensah bar)

Barbus macrops
Sarotherodon sp.

15
19

none

Site 7
(Achiaman)

Sarotherodon sp.
Clarias anguillaris
Hemichromis bimaculatus

23
1
2

Hyperolius sp.
Ranidae

17
1

5

Afrixalus sp.
Hyperolius sp.
Unidentified tadpole
Ranidae

6
38
1
3

15

none

4

Afrixalus sp.
Hyperolius sp.

10
10

100

Total Amphibians:

172

Hemichromis bimaculatus
Site 8
(Kwashikuma
Marsh)

Site 9
(Lake Weija)

Sarotherodon sp.

Sarotherodon sp.
Site 10
(Kwashikuma
Pond)
Total fish:

25

Figure 1: NMS ordination plot with three dimensions represented on two axes of fish
communities using species presence or absence data from 2008 in relation to ER positivity.
Triangles represent ER positive communities and circles represent ER negative communities.
Ellipses represent 95% confidence intervals. Stress: 0.1568934. For interpretation of the
references to color in this and all other figures, the reader is referred to the electronic version of
this thesis.

26

Figure 2: NMS ordination plot with three dimensions represented on two axes of amphibian
communities using species presence or absence data from 2008 in relation to ER positivity.
Triangles represent ER positive communities and circles represent ER negative communities.
Ellipses represent 95% confidence intervals. Stress: 0.06834609.

27

Percent ER Positive

fish
amphibians
combined

100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
1

2

3

4

5
Site

7

8

9

10

Figure 3: ER positivity rates of fish and amphibians and combined data for specimens collected
in 2009.

28

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34