Marburgvirus Resurgence in Kitaka Mine Bat Population after Extermination Attempts, Uganda

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LETTERS
panel B). We performed C. burnetii–
specic qPCR on the ticks; 14 (88%)
were positive.
We genotyped C. burnetii–positive
DNA from the feces and from 6 of the
16 ticks by using multispacer sequence
typing as described (5). All samples
were identied as MST17, the unique
genotype circulating in Cayenne (5).
After obtaining the laboratory re-
sults, we conrmed that a local group in
charge of the collection and treatment
of injured animals usually released
rehabilitated 3-toed sloths into Tiger
Camp. Residents of Tiger Camp regu-
larly observed and came into contact
with the sloths, and ticks were frequent-
ly observed on the fur of the animals.
Furthermore, 3 Q fever patients from
Cayenne reported contact with sloths.
Feces from the sloth in this study
were highly infectious for C. burnetii.
Because sloths live in tall trees and can
shed this bacterium in their feces, human
contamination might occur through in-
halation of infectious aerosols from fe-
ces. The high prevalence of C. burnetii
infection in ticks also suggests possible
transmission through tick bites or from
aerosols of tick feces that have been de-
posited on the skin of animal hosts; such
feces can be extremely rich in bacteria
and highly infectious (10).
In this 2013 outbreak of Q fever,
epidemiologic studies led to the iden-
tication of 3-toed sloths as a putative
source of C. burnetii infection. Further
investigations are needed to conrm
the role of sloths as a reservoir for C.
burnetii in French Guiana and to im-
plement efcient measures to prevent
transmission to humans.
Acknowledgments
We thank the French Forces Medi-
cal Service for its support. We also thank
G. Hyvert, T. Lamour, M. Sophie, and D.
Blanchet for their excellent assistance dur-
ing eld work and A. Abeille, T. Ameur,
and C. Nappez for processing the samples.
Funding was provided by the Foun-
dation Méditerranée Infection.
Bernard Davoust,1
Jean-Lou Marié,1
Vincent Pommier de Santi,
Jean-Michel Berenger,
Sophie Edouard,
and Didier Raoult
Authorafliations:Aix-Marseille Université,
Marseille,France (B.Davoust,J.-L.Marié,
J.-M. Berenger, S. Edouard, D. Raoult);
Groupe de Travail en Épidémiologie
AnimaleduServicedeSantédesArmées,
Toulon, France (J.-L. Marié); Direction
Interarmées du Service de Santé en
Guyane, Cayenne, France (V. Pommier
de Santi); and Centre d’Épidémiologie et
de Santé Publique des Armées, Marseille
(V.PommierdeSanti).
DOI:http://dx.doi.org/10.3201/eid2010.140694
References
1. Grangier C, Debin M, Ardillon V,
Mahamat A, Fournier P, Simmonnet C,
et al. Epidemiologie de la èvre Q en
Guyanne, 1990–2006. Le Bulletin de
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2009;10:2–4. http://www.invs.sante.fr/
publications/bvs/antilles_guyane/2009/
bvs_ag_2009_10.pdf
2. Epelboin L, Chesnais C, Boullé C,
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fever pneumonia in French Guiana: preva-
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dx.doi.org/10.1086/322034
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Address for correspondence: Didier Raoult,
Unité de Recherche en Maladies Infectieuses et
Tropicales Emergentes (URMITE) CNRS UMR
7278 IRD 198 INSERM U1095 Aix-Marseille
Université, Faculté de Médecine, 27 bd Jean
Moulin, 13385 Marseille CEDEX 5, France;
email: didier.raoult@gmail.com
Marburgvirus
Resurgence in
Kitaka Mine
Bat Population
after Extermination
Attempts, Uganda
To the Editor: Marburg virus
(MARV) and Ravn virus (RAVV),
collectively called marburgviruses,
cause Marburg hemorrhagic fever
(MHF) in humans. In July 2007, 4 cas-
es of MHF (1 fatal) occurred in miners
at Kitaka Mine in southern Uganda.
Later, MHF occurred in 2 tourists who
visited Python Cave, ≈50 km from
Kitaka Mine. One of the tourists was
from the United States (December
 EmergingInfectiousDiseases•www.cdc.gov/eid•Vol.20,No.10,October2014 1761
1These authors contributed equally to this
article.

LETTERS
2007) and 1 was from the Netherlands
(July 2008); 1 case was fatal (1,2,3).
The cave and the mine each contained
40,000–100,000 Rousettus aegyptia-
cus bats (Egyptian fruit bats).
Longitudinal investigations of
the outbreaks at both locations were
initiated by the Viral Special Patho-
gens Branch of the Centers for Dis-
ease Control and Prevention (CDC,
Atlanta, GA, USA, and Entebbe,
Uganda) in collaboration with the
Uganda Wildlife Authority (UWA)
and the Uganda Virus Research In-
stitute (UVRI). During these stud-
ies, genetically diverse MARVs and
RAVVs were isolated directly from
bat tissues, and infection levels of the
2 viruses were found to increase in ju-
venile bats on a predictable bi-annual
basis (4,5). However, investigations at
Kitaka Mine were stopped when the
miners exterminated the bat colony by
restricting egress from the cave with
papyrus reed barriers and then entan-
gling the bats in shing nets draped
over the exits. The trapping continued
for weeks, and the entrances were then
sealed with sticks and plastic. These
depopulation efforts were documented
by researchers from UVRI, the CDC,
the National Institute of Communi-
cable Diseases (Sandringham, South
Africa), and UWA during site visits
to Kitaka Mine (online Technical Ap-
pendix Figure, http://wwwnc.cdc.gov/
EID/article/20/8/14-0696-Techapp1.
pdf). In August 2008, thousands of
dead bats were found piled in the for-
est, and by November 2008, there was
no evidence of bats living in the mine;
whether 100% extermination was
achieved is unknown. CDC, UVRI,
and UWA recommended against ex-
termination, believing that any results
would be temporary and that such ef-
forts could exacerbate the problem if
bat exclusion methods were not com-
plete and permanent (6,7).
In October 2012, the most recent
known marburgvirus outbreak was de-
tected in Ibanda, a town in southwest
Uganda. Ibanda is ≈20 km from the
Kitaka Mine and is the urban center
that serves smaller communities in
the Kitaka area. This MHF outbreak
was the largest in Ugandan history: 15
laboratory-conrmed cases occurred
(8). In November 2012, an ecologic
investigation of the greater Ibanda/
Kitaka area was initiated. The inves-
tigation included interviews with lo-
cal authorities to locate all known
R. aegyptiacus colonies in the area.
Although minor colonies of small in-
sectivorous bats were found, the only
identiable colony of R. aegyptiacus
bats was found inside the re-opened
Kitaka Mine, albeit at much reduced
size, perhaps 1%–5% of that found be-
fore depopulation efforts.
To determine whether the R. ae-
gyptiacus bats that had repopulated
Kitaka Mine were actively infected
with marburgviruses, we tested 400
bats by using previously described
methods (4,5). Viral RNA was extract-
ed from ≈100 mg of liver and spleen
tissue by using the MagMAX Total
Nucleic Acid Isolation Kit (Applied
Biosystems, Foster City, CA, USA)
according to the manufacturer’s rec-
ommended protocol. The Fisher ex-
act test was conducted by using IBM
SPSS Statistics, version 19.0 (IBM
Corp., Armonk, NY, USA).
Of the 400 R. aegyptiacus bats
collected, 53 (13.3%) were positive
for marburgvirus RNA by quan-
titative reverse transcription PCR
(32/233 [13.7%] adults and 21/167
[12.6%] juveniles; online Technical
Appendix Table); marburgvirus was
isolated from tissue samples from
9 of the 400 bats. The overall level
of active infection was signicantly
higher than that found in Kitaka Mine
during 2007–2008 (5.1%) (5) (Fisher
exact test, p<0.001) and in other stud-
ies in Uganda (Python Cave [2.5%])
and Gabon (4.8%) (4,9). The reason
for the increase is not clear, but it may
be related to the effects of the exter-
mination and subsequent repopula-
tion. Increases in disease prevalence
in wildlife populations after culling
are not unprecedented (6,7). We
speculate that after the depopulation
attempt, a pool of susceptible bats be-
came established over time and was
subjected to multiple marburgvirus
introductions, as evidenced by the
genetic diversity of viruses isolated
from the bats (Figure). A pool of sus-
ceptible bats would have led to higher
levels of active infection within the
colony, thereby increasing the poten-
tial for virus spillover into the human
population. A signicant sex and age
bias was not detected with respect to
active infection during the breeding
season (Fisher exact test, p>0.5 for
both), and overall, the presence of vi-
rus-specic IgG among the bats was
16.5%, a nding consistent with that
in previous studies (4,5).
Phylogenetic analysis of viral
RNA genome fragment sequences in
this study showed high marburgvirus
genetic diversity, including the pres-
ence of RAVVs and MARVs. Se-
quences for isolates from 3 bats were
nearly identical to those of the MARV
isolates obtained from patients in the
2012 Ibanda outbreak (8), suggesting
that bats from Kitaka Mine were a
likely source of the virus.
Acknowledgments
We thank UVRI, the Uganda Minis-
try of Health, and UWA for their assistance
during the outbreak investigation. We also
thank R. Swanepoel and S. Balinandi for
the photographs used in this publication.
Funding for this study was provided
by the United States Department of Health
and Human Services.
Brian R. Amman,
Luke Nyakarahuka,
Anita K. McElroy,
Kimberly A. Dodd,
Tara K. Sealy, Amy J. Schuh,
Trevor R. Shoemaker,
Stephen Balinandi,
Patrick Atimnedi, Winyi Kaboyo,
Stuart T. Nichol,
and Jonathan S. Towner
1762 EmergingInfectiousDiseases•www.cdc.gov/eid•Vol.20,No.10,October2014

LETTERS
Author afliations: Centers for Disease
Control and Prevention, Atlanta, Georgia,
USA (B.R. Amman, A.K. McElroy, K.A.
Dodd,T.K. Sealy,A.J.Schuh,S.T.Nichol,
J.S.Towner);UgandaVirusResearchInsti-
tute, Entebbe, Uganda (L. Nyakarahuka);
Emory University,Atlanta (A.K. McElroy);
University of California, Davis, California,
USA (K.A. Dodd); Centers for Disease
Control and Prevention, Entebbe (T.R.
Shoemaker,S.Balinandi);UgandaWildlife
Authority,Kampala, Uganda(P.Atimnedi);
and Uganda Ministry of Health, Kampala
(W.Kaboyo)
DOI:http://dx.doi.org/10.3201/eid2010.140696
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Figure. Phylogeny of concatenated
marburgvirus nucleoprotein (NP) and
viral protein 35 (VP35) gene fragments
as determined by using the maximum-
likelihoodmethod.SequencesfromtheNP
(289–372nt)andVP35(203–213nt)genes
were amplied and determined from viral
RNA and then sequenced as described
elsewhere(4).Sequencenamesinboldface
represent those generated from samples
collected from bats during the November
2012outbreakinvestigationatKitakaMine,
Uganda. Underlined sequence names
represent those generated from samples
obtained from marburgvirus-infected
persons in Kabale and Ibanda, Uganda,
in 2012. Multiple sequence alignments
weregenerated,andamaximum-likelihood
analysis was conducted on concatenated
NPand VP35(208–580nt) sequencesby
using the PhyML method in conjunction
with the GTR+I+G nucleotide substitution
model implemented in SeaView version
4.2.12(10).NPandVP35genesequences
determined from samples in this study (in
boldface) were submitted to GenBank
(accession nos. KJ747211–KJ747234
and KJ747235–KJ747253, respectively).
Bayesian posterior probabilities above
50 are shown at the nodes. Scale bar
indicates nucleotide substitutions per site.
Ang,Angola;DRC,DemocraticRepublicof
Congo; Gab, Gabon; Ger,Germany; Ken,
Kenya;Net, Netherlands;Rav,Ravnvirus;
Uga,Uganda;Zim,Zimbabwe.

LETTERS
1764 EmergingInfectiousDiseases•www.cdc.gov/eid•Vol.20,No.10,October2014
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et al. Seasonal pulses of Marburg virus
circulation in juvenile Rousettus aegyp-
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6. Donnelly CA, Woodroffe R, Cox DR,
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Address for correspondence: Jonathan S.
Towner, Centers for Disease Control and
Prevention, 1600 Clifton Rd NE, Mailstop G14,
Atlanta, GA 30329-4027, USA; email: jit8@
cdc.gov
Detection of
Measles Virus
Genotype B3, India
To the Editor: Molecular epide-
miologic investigations and virologic
surveillance contribute notably to the
control and prevention of measles
(1). Nearly half of measles-related
deaths worldwide occur in India,
yet virologic surveillance data are
incomplete for many regions of the
country (2,3). Previous studies have
documented the presence of measles
virus genotypes D4, D7, and D8 in
India, and genotypes D5, D9, D11,
H1, and G3 have been detected in
neighboring countries (3,4).
Kerala, India’s southernmost
state, has high measles vaccination
coverage compared with many other
states in the country; however, the dis-
ease is still endemic in the region. Two
districts, Thiruvananthapuram and
Malappuram, report the highest num-
bers of cases (5). Baseline data on cir-
culating measles virus genotypes are
needed for measles elimination, but
such data are not available for Kerala.
In this context, we performed a pilot
genetic analysis of the measles virus
strains circulating in Thiruvanantha-
puram, the capital of Kerala. We used
throat and nasopharyngeal swab and
serum samples from children admit-
ted to Sree Avittom Thirunal Hospital
during measles outbreaks occurring
March–August 2012.
We used the Vero/human-SLAM
cell line (http://www.phe-culture
collections.org.uk). for isolation of
measles virus from throat and naso-
pharyngeal swab samples. For sero-
logic conrmation of cases, we used
a commercial measles IgM ELISA
kit (IBL International GmbH, Ham-
burg, Germany). Virus genotyping
was based on the 450-nt coding se-
quence for the carboxyl terminus of
nucleoprotein (N) of measles virus,
as recommended by the World Health
Organization (3,6). We extracted
RNA from the samples using TRIzol
reagent (GIBCO-BRL, Grand Island,
NY, USA). We performed reverse
transcription PCR using a Super-
Script One-Step RT-PCR kit with a
Platinum Taq system (Invitrogen,
Carlsbad, CA, USA) and previously
described primers (3,6). Amplicons
were subjected to bidirectional se-
quencing using a BigDye Terminator
v3.1 cycle sequencing kit (Applied
Biosystems, Foster City, CA, USA).
We edited and aligned nucleotide se-
quences using Bio Edit 7.1.11 soft-
ware (7). Phylogenetic analysis was
performed by using the maximum-
likelihood method implemented in
the MEGA5 program (8) to compare
the determined N gene sequences
with the World Health Organization
reference sequences of the 24 known
measles genotypes.
PCR products could be amplied
from 16 of the 24 samples analyzed.
Ten samples provided high quality
sequence reads for the N gene coding
region, which were used for further
analysis. Clinical and demographic
data for these 10 cases, virus isolation
status, and GenBank accession num-
bers of the sequences are summarized
in the Table.
Phylogenetic analysis revealed
1 of the 10 measles virus strains to
be of genotype D8 (online Technical
Appendix Figure 1, http://wwwnc.
cdc.gov/EID/article/20/10/13-0742-
Techapp1.pdf), a genotype previ-
ously found to be circulating in
Kerala and in other regions of India
(3,6,9,10). The other 9 virus strains
were closely related to B3 genotype
reference strains, indicating circu-
lation of the B3 genotype in Kerala
(online Technical Appendix Figure
1). The nucleotide sequences of 7 of
the 9 strains were identical, indicat-
ing a single chain of transmission.
The remaining 2 samples showed
sequence divergence, indicating in-
dependent sources of infection. In a
phylogenetic analysis comparing the
Kerala B3 genotypes and a dataset of