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Introduction
Many recent amphibian declines are associated
with the emergence of a pathogenic fungus,
Batrachochytrium dendrobatidis (Bd), that causes
the disease chytridiomycosis in amphibians (Berger
et al. 1998). This deadly disease has been linked to
the decline of over 200 amphibian species worldwide
(Skerratt et al. 2007), with the majority of these declines
observed in Australia and the New World (e.g., Fisher
et al. 2009). Recent studies have documented the
presence of Bd in a variety of genera in at least 13
countries in sub-Saharan Africa, mostly in southern and
eastern Africa (Blackburn et al. 2010). However, aside
from one possible Bd-caused decline in Tanzania (for
the toad Nectophrynoides asperginis: Weldon & du
Preez 2004; Channing et al. 2006), there have been no
reported declines in African amphibians associated with
Bd infection. Recent surveys of museum specimens
reveal that Bd has a long history of presence in sub-
Saharan Africa, beginning in at least the early 1930s
(Soto-Azat et al. 2010), bolstering previous suggestions
that this pathogen may be endemic to Africa (Weldon
et al. 2004). Weldon et al. (2004) further proposed that
the global trade of Xenopus laevis from South Africa
may have caused the spread of Bd from Africa to other
regions (Weldon et al. 2004).
The earliest potential record of Bd in Africa (1933)
derives from preserved museum specimens collected
in Cameroon (Soto-Azat et al. 2010), which hosts
one of Africa’s richest amphibian faunas. However,
there are few previously published surveys of Bd in
western and central Africa, including Cameroon and
neighboring Nigeria. Investigation of ve individuals
of Hoplobatrachus occipitalis collected in Nigeria
during 1971 did not detect Bd (Oullett et al. 2005).
Surveys in Okomu National Park in Nigeria between
2007 and 2008 found one individual of Chiromantis
rufescens with a conrmed Bd infection (Imasuen et
al. 2009). Bd has also been detected recently much
farther east in Central Africa (Greenbaum et al. 2008).
However, rather than view this record as suggestive of
Bd infection elsewhere in Central Africa, this record
of Bd from eastern Democratic Republic of Congo
might be best interpreted as indicative of the margins
of the Bd-positive region encompassing East African
countries such as Kenya (Kielgast et al. 2009), Tanzania
(Weldon and du Preez 2004; Channing et al. 2006), and
Uganda (Goldberg et al. 2007; Soto-Azat et al. 2010).
Additional information is sorely needed to determine
the prevalence and potential impact of Bd in central and
Herpetology Notes, volume 4: 083-086 (2011) (published online on 24 February 2011)
Survey of the chytrid fungus Batrachochytrium dendrobatidis from
montane and lowland frogs in eastern Nigeria
Natalie M. M. Reeder1,$, Tina L. Cheng1,$, Vance T. Vredenburg1, and David C. Blackburn2*
1 Department of Biology, San Francisco State University, San
Francisco, California, 94132, USA;
2 Division of Herpetology, Biodiversity Institute, University of
Kansas, Lawrence, Kansas 66045, USA;
e-mail david.c.blackburn@gmail.com
* Corresponding author
$ These authors contributed equally
Abstract. The chytrid fungus Batrachochytrium dendrobatidis (Bd) that causes the amphibian disease chytridiomycosis might
have originated in sub-Saharan Africa. Based on historical museum collections, it has been detected in frog specimens from
Central Africa (Cameroon) as early as 1933. Yet, to date, there are few surveys of Bd for recently collected specimens from Central
Africa. We present the results of a study undertaken to evaluate the presence and prevalence of Bd in eastern Nigeria at lowland
and montane sites in Gashaka-Gumti National Park and in or near Ngel Nyaki Forest Reserve, respectively. Using quantitative
real-time PCR, we detected three low-intensity cases of B. dendrobatiis infection each in a different genus of frog (Amietophrynus,
Astylosternus, and Petropedetes), including in an undescribed species, at the lowland site in Gashaka-Gumti National Park. While
it is promising that Bd was detected at only low intensities and at only one site, the extent to which Bd is distributed in this region
of high amphibian diversity in Africa requires further evaluation.
Keywords. Amphibian declines, Africa, chytridiomycosis, Gashaka-Gumti National Park, Mambilla Plateau, Ngel Nyaki
Forest Reserve.
Natalie M. M. Reeder et al.
84
western Africa as well as to help resolve whether Bd
may have originated in Africa. We conducted a survey
of the distribution and prevalence of Bd in eastern
Nigeria at lowland and montane sites on or near the
Mambilla Plateau.
Materials and Methods
During the course of short opportunistic surveys in April 2009,
chytrid screening was carried out (by DCB) at lowland and mon-
tane sites near or on, respectively, the Mambilla Plateau (Fig. 1;
Table 1). The lowland eld site is based near the Kwano eld
camp of the Gashaka Primate Project within Gashaka-Gumti Na-
tional Park (Adamawa State). At this site, most frogs were coll-
ected in close proximity to a stream, including in the leaf litter, in
the water of the streams, on sandy banks, or on rock faces within
the stream. Frogs were collected from a handful of montane loca-
lities all near the eld station of the Nigerian Montane Forest Pro-
ject adjacent to the Ngel-Nyaki Forest Reserve near Yelwa village
(Taraba State). These montane sites included forested streams in
the forest reserve as well as tree-lined riparian corridors, grass-
land streams, eucalyptus plantations, and farms outside of the re-
serve. Nearly all frogs were collected active in the grass and leaf
litter, though some, especially male Leptopelis nordequatorialis,
were collected calling from trees at night. All specimens screened
for chytrid are deposited in the herpetology collections of the Mu-
seum of Comparative Zoology (Harvard University).
Infection status was determined through a standardized swabbing
protocol (Hyatt et al. 2007). Using a synthetic cotton swab, each
individual was swabbed with 30 strokes (ve times between the
toes on each hindfoot, ve times on each thigh, and ve times on
each side of the ventral abdomen). Swabs were extracted using a
PrepMan extraction and run in duplicate by quantitative real-time
PCR (qPCR) as described by Boyle et al. (2004) and Hyatt et al
(2007). Each plate contained negative controls. Standard samples
of known Bd quantity served as positive controls on each plate;
standards were obtained from the Australian Animal Health La-
boratory (courtesy of A. Hyatt). Samples were considered posi-
tive for Bd if qPCR revealed a measurable quantity (Genomic
Equivalent, GE > 0) of Bd in either of the two replicates during
the 40 cycle thresholds (CT) of the run. False positives were eli-
minated by ensuring that amplication curves followed standard
exponential growth patterns (Hyatt et al. 2007). Infection levels
on individual amphibians were quantied as zoospore equivalents
obtained by multiplying genomic equivalents by 80 to correct for
dilution during extraction and PCR.
Results and Discussion
We tested 34 individuals from ve currently
recognized families and ten genera from the lowland
and montane sites (Table 1). The overall prevalence
of Bd for our survey was 8.8% (3/34), with 0% (0/18)
prevalence at the montane site, and 18.8% (3/16)
prevalence at the lowland site (Table 1). Positive
samples all produced low-level infections (less than
1 zoospore equivalents), and were detected in three
lowland species: Astylosternus sp., Amietophrynus sp.,
Petropedetes sp. nov. Specimens from these species
were all collected in or adjacent to a large stream
running past the Kwano eld camp within Gashaka
Gumti National Park.
Specimens of two of the species tested for Bd
comprise type specimens of species described only
recently (Artholeptis palava: Blackburn et al. 2010;
Phrynobatrachus danko: Blackburn 2010) Other
specimens represents an undescribed species of
Petropedetes (Blackburn unpublished data). Bd was not
detected in Artholeptis palava and Phrynobatrachus
Locality Coordinates Elevation Species (Family) No. Tested No. Positive
Kwano Camp,
Gashaka Gumti N.P.
07 19' 47.9'' N
011 35' 06.0'' E
~540 m Amietophrynus sp. (Bufonidae)
Astylosternus sp. (Arthroleptidae)
Cardioglossa leucomystax (Arthroleptidae)
Hylarana galamensis (Ranidae)
Hyperolius sp. (Hyperoliidae)
Petropedetes sp. nov. (Petropedetidae)
Phrynobatrachus cornutus (Ranidae)
Phrynobatrachus natalensis (Ranidae)
3
1
2
2
1
2
4
1
1
1
0
0
0
1
0
0
Ngel Nyaki F.R. 07 05' 53.3'' N
011 03' 19.6'' E
~1460 m Phrynobatrachus steindachneri (Ranidae) 2 0
Kurmi Danko F.R. 07 06' 54.6'' N
011 01' 35.8'' E
~1500 m Phrynobatrachus danko (Ranidae) 3 0
Ngel Nyaki F.R. 07 05' 10.0'' N
011 03' 59.9'' E
~1550 m Arthroleptis palava (Arthroleptidae)
Astylosternus sp. (Arthroleptidae)
Leptodactylodon cf. bicolor (Arthroleptidae)
1
1
2
0
0
0
Yelwa 07 05' 17.0'' N
011 04' 41.1'' E
~1550 m Amietophrynus cf. villiersi (Bufonidae)
Leptopelis nordequatorialis (Arthroleptidae)
4
1
0
0
Yelwa 07 05' 18.1'' N
011 05' 06.6'' E
~1580 m Arthroleptis palava (Arthroleptidae) 1 0
Riparian forest,
near Ngel Nyaki F.R.
07 05' 00.5'' N
011 03' 51.0'' E
~1630 m Leptopelis nordequatorialis (Arthroleptidae) 3 0
Table 1. Species screened for Bd in this study.
Survey of the chytrid fungus in eastern Nigeria 85
danko, both from montane sites, but was detected in one
specimen of the undescribed species of Petropedetes.
The data presented here expand our knowledge of
the geographic and taxonomic distribution of Bd in
Nigeria and Africa. Though Bd was present in three
frog specimens, we did not observe any sick or dying
frogs associated with Bd infection during our survey.
In addition, the animals that were positive have very
low zoospore equivalents or low infection intensities.
Previous eld studies showed that high infection
intensities lead to disease and eventually population
extinctions whereas frogs with low infection intensities
(at the levels we found in this study) show no signs of
disease and populations persist with Bd (Vredenburg et
al. 2010; Briggs et al. 2010). Our results showing low
levels of Bd infection intensities corresponds with the
observation that there are no reports of mortality events
associated with Bd infection anywhere in central or
western Africa. Unfortunately, it is impossible to know
the dynamics of this host-pathogen system in the areas
surveyed in this study because of the opportunistic
nature of the survey and the lack of previous Bd
surveys at these sites.
Further studies are needed to test the susceptibility or
resistance of Nigerian amphibians to Bd. At present,
it remains difcult to evaluate the potential threat that
Bd represents to these amphibian populations. Future
studies could also potentially benet from a better
understanding of the natural immune responses of
these species to Bd infection. In addition, the testing of
additional historical museum specimens from Nigeria
should be conducted to determine the prevalence of Bd
in Nigeria, at least over the past century. Finally, due
to our limited sample sizes and geographic sampling,
our understanding of the distribution and threat of Bd
in Nigeria remains incomplete. With our discovery of
several low-level Bd-positive individuals, we hope
that this will lead to more thorough, repeated, and
widespread surveys of Bd in Nigeria.
Acknowledgements. Field research in Nigeria was facilitated
through collaboration with Hazel Chapman (University of
Canterbury, New Zealand) and the Nigerian Montane Forest
Project as well as Volker Sommer (University College London,
United Kingdom) and the Gashaka Primate Project. The Vice
Chancelor of Gombe State University, A. Mahadi, facilitated
components of eld research. Funding for the eld component
of this research was provided by the Museum of Comparative
Zoology (Harvard University) to DCB, the lab work, conducted
at San Francisco State University, was funded by NSF grant EF-NSF grant EF-
0723563 as part of the joint NSF-NIH Ecology of Infectious
Disease program.
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Accepted by Zoltán T. Nagy
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