Published Ahead of Print 19 November 2010.
2011, 193(3):670. DOI: 10.1128/JB.00750-10.
Abraham Aseffa, Stephen V. Gordon and Noel H. Smith
Rudovick Kazwala, Gunilla Källenius, R. Glyn Hewinson,
Jakob Zinsstag, Paul van Helden, Françoise Portaels,
Soolingen, Anita L. Michel, Berit Djønne, Alicia Aranaz,
Pacciarini, Simeon Cadmus, Moses Joloba, Dick van
Laura Boschiroli, Annélle Müller, Naima Sahraoui, Maria
Mucavele, Bongo Nare Richard Ngandolo, Judith Bruchfeld,
Rigouts, Rebuma Firdessa, Adelina Machado, Custodia
Beatrice Boniotti, Sabrina Rodriguez, Markus Hilty, Leen
Hailu, Benon Asiimwe, Kristin Kremer, James Dale, M.
Stefan Berg, M. Carmen Garcia-Pelayo, Borna Müller, Elena
Important in East Africa
African 2, a Clonal Complex of
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JOURNAL OF BACTERIOLOGY, Feb. 2011, p. 670–678
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 193, No. 3
African 2, a Clonal Complex of Mycobacterium bovis Epidemiologically
Important in East Africa?†
Stefan Berg,1M. Carmen Garcia-Pelayo,1Borna Mu ¨ller,2Elena Hailu,3Benon Asiimwe,4Kristin Kremer,5
James Dale,1M. Beatrice Boniotti,6Sabrina Rodriguez,7Markus Hilty,8Leen Rigouts,17Rebuma Firdessa,3
Adelina Machado,16Custodia Mucavele,16Bongo Nare Richard Ngandolo,12Judith Bruchfeld,10
Laura Boschiroli,6,9Anne ´lle Mu ¨ller,2Naima Sahraoui,14Maria Pacciarini,6Simeon Cadmus,11
Moses Joloba,4Dick van Soolingen,5Anita L. Michel,18Berit Djønne,15Alicia Aranaz,7
Jakob Zinsstag,20Paul van Helden,2Franc ¸oise Portaels,17Rudovick Kazwala,13
Gunilla Ka ¨llenius,19R. Glyn Hewinson,1Abraham Aseffa,3
Stephen V. Gordon,21and Noel H. Smith22*
VLA Weybridge, New Haw, Surrey KT15 3NB, United Kingdom1; Division of Molecular Biology and Human Genetics, Faculty of Health
Sciences, Stellenbosch University, P.O. Box 19063, Tygerberg 7505, South Africa2; Armauer Hansen Research Institute, P.O. Box 1005,
Addis Ababa, Ethiopia3; Department of Medical Microbiology, Makerere University Medical School, P.O. Box 7072, Kampala, Uganda4;
Tuberculosis Reference Laboratory, National Institute for Public Health and the Environment (RIVM), Centre for Infectious Disease
Control (CIb/LIS), P.O. Box 1, 3720 BA Bilthoven, Netherlands5; Reparto Genomica, Istituto Zooprofilattico Sperimentale della
Lombardia e dell’Emilia, Via Bianchi n. 9, 25124 Brescia, Italy6; Dept. de Sanidad Animal, Facultad de Veterinaria, and Centro
Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense, Avenida, Puerta de Hierro s/n, 28040 Madrid, Spain7; Institute
for Infectious Diseases, University of Bern, Friedbu ¨hlstrasse 51, CH-3010 Bern, Switzerland8; Agence Franc ¸aise de Se ´curite ´ Sanitaire des
Aliments, 23 Avenue du Ge ´ne ´ral-de-Gaulle, 94706 Maisons-Alfort Cedex, France9; Unit of Infectious Diseases, Department of Medicine,
Solna, Karolinska Institutet, Karolinska University Hospital, 17177 Stockholm, Sweden10; Department of Veterinary Public Health &
Preventive Medicine, University of Ibadan, Ibadan, Nigeria11; Laboratoire de Recherches Ve ´te ´rinaires et Zootechniques de Farcha,
BP 433, N?Djame ´na, Chad12; Sokoine University of Agriculture, Morogoro, Tanzania13; Universite ´ Saad Dahlab, Route de Soumaa,
BP 270, Blida, Algeria14; Department of Animal Health, National Veterinary Institute, BP 750 Sentrum, N-0106 Oslo, Norway15;
Facudade de Veterinaria, Universidade Eduardo Mondlane, CP 257 Maputo, Mozambique16; Department of Microbiology, Institute of
Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium17; Faculty of Veterinary Science, University of Pretoria, Private Bag
X04, and ARC-Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort 0110, South Africa18; Department of Clinical Science
and Education, Karolinska Institutet, So ¨dersjukhuset, 11883 Stockholm, Sweden19; Swiss Tropical and Public Health Institute,
Socinstrasse 57, 4002 Basel, Switzerland20; College of Life Sciences and UCD Conway Institute, University College Dublin, Dublin 4,
Ireland21; and VLA Weybridge, New Haw, Surrey KT15 3NB, United Kingdom, and Centre for the Study of Evolution, University of
Sussex, Brighton BN1 9QL, United Kingdom22
Received 28 June 2010/Accepted 13 November 2010
We have identified a clonal complex of Mycobacterium bovis isolated at high frequency from cattle in Uganda,
Burundi, Tanzania, and Ethiopia. We have named this related group of M. bovis strains the African 2 (Af2)
clonal complex of M. bovis. Af2 strains are defined by a specific chromosomal deletion (RDAf2) and can be
identified by the absence of spacers 3 to 7 in their spoligotype patterns. Deletion analysis of M. bovis isolates
from Algeria, Mali, Chad, Nigeria, Cameroon, South Africa, and Mozambique did not identify any strains of
the Af2 clonal complex, suggesting that this clonal complex of M. bovis is localized in East Africa. The specific
spoligotype pattern of the Af2 clonal complex was rarely identified among isolates from outside Africa, and the
few isolates that were found and tested were intact at the RDAf2 locus. We conclude that the Af2 clonal complex
is localized to cattle in East Africa. We found that strains of the Af2 clonal complex of M. bovis have, in general,
four or more copies of the insertion sequence IS6110, in contrast to the majority of M. bovis strains isolated
from cattle, which are thought to carry only one or a few copies.
Bovine tuberculosis (TB), caused by Mycobacterium bovis, is
mainly a disease of cattle, but it is also a zoonosis infecting
humans. Bovine TB has been eradicated in Australia and many
European countries; however, it is still believed to be common
among cattle throughout the rest of the world. On the African
continent, information on the prevalence of bovine TB is
scarce and control programs are in place in only a few coun-
tries (6, 16). However, a number of reports suggest that the
disease is widely spread over the African continent and highly
prevalent in several countries (8, 21, 38, 42, 51), with infection
present mainly in cattle but also in wildlife (39).
M. bovis is one of seven species constituting the Mycobacte-
rium tuberculosis complex, which includes M. tuberculosis, one
of the most devastating bacterial pathogens of humans. There
is little or no exchange of chromosomal DNA between cells
from the M. tuberculosis complex, making this group of bacte-
ria highly clonal (14, 30, 53–54). In a strictly clonal population,
any mutation present in an ancestral strain will be present in all
* Corresponding author. Mailing address: VLA Weybridge, New
Haw, Surrey KT15 3NB, United Kingdom. Phone: 44 1273 873502.
Fax: 44 1932 357260. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jb
?Published ahead of print on 19 November 2010.
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its descendants and can be used to identify clonal complexes. A
series of deletions (regions of difference [RD]) within the M.
tuberculosis complex have been used to identify phylogenetic
relationships between members of the M. tuberculosis complex
(11), and for M. tuberculosis, different lineages and sublineages
have also been characterized by specific deletions (25, 60). In
a similar manner, we are exploring the relationships between
lineages of M. bovis that dominate in different geographical
regions around the world.
Spoligotyping, a PCR and hybridization technique, is the
most common genotyping technique for strains of the M. tu-
berculosis complex and assays polymorphism in 43 unique
spacer sequences found in the direct repeat (DR) region (36,
61). Each spoligotype pattern from strains of the animal-
adapted lineage of the M. tuberculosis complex is given a
unique identifier by www.Mbovis.org. Several studies of the
DR region in closely related strains of M. tuberculosis have
concluded that the evolutionary trend of this region is primar-
ily by loss of single or multiple contiguous spacers (23, 29, 63);
duplication of direct variable repeat (DVR) units or point
mutations in spacer sequences were found to be rare. Although
the absence of specific spacers, or groups of spacers, in a
spoligotype pattern can be indicative of a closely related group
of strains (clonal complex), spacers are frequently lost inde-
pendently in different lineages (homoplasy). Furthermore, the
interpretation of specific spacer loss, such as that of spacers 3
to 7 in the strains described in this article, can be ambiguous if
adjacent spacers in the spoligotype pattern are also deleted.
Recently a clonal complex of M. bovis, called African 1 (Af1)
(41), that is highly prevalent in several countries of west-cen-
tral Africa has been identified. In this article, we identify a
second M. bovis clonal complex common in East Africa and
name this group of strains the African 2 (Af2) clonal complex
of M. bovis.
MATERIALS AND METHODS
Bacterial strains. The majority of all M. bovis strains analyzed in this study
were isolated from cattle and are described in more detail in the supplemental
material. One hundred twenty strains were collected from six abattoirs in Ethi-
opia during 2006 to 2008 (8); nine strains were collected at an abattoir in
Kampala from cattle originating from seven districts in Uganda (5); ten strains
were collected from three sites in or close to the capital Bujumbura in Burundi
(48); and fourteen strains were collected from cattle at a Morogoro slaughter-
house in Tanzania (41). Additional population samples of M. bovis isolated from
cattle from South Africa (n ? 22) (40]), Chad (n ? 5) (35]), Mali (n ? 20) (42),
Cameroon (n ? 3), Nigeria (n ? 5), Mozambique (n ? 14), Algeria (n ? 17)
(51), Italy (n ? 93) (10), and Spain (n ? 20) (49]) were analyzed (see the
supplemental material). Also, two strains of M. bovis from humans, isolated in
Uganda and Sweden, were further investigated for this study.
All isolates were characterized by spoligotyping, and the majority were also
deletion typed for regions RD4 and RDAf2. Selected M. bovis isolates were
subjected to variable-number tandem repeat (VNTR) typing (24) and RDAf1
deletion typing (41) (see the supplemental material). Isolates of M. tuberculosis
H37Rv and M. bovis AF2122/97 were used as controls.
Spoligotyping, VNTR typing, and microarray analysis. Strains were spoligo-
typed according to the method of Kamerbeek et al. (36) with minor modifications
(12), and the exact tandem repeat (ETR) loci ETR-A to ETR-F were VNTR
typed as previously described (12, 24). The VNTR types are displayed as a series
of six integers representing the deduced number of repeats present at each locus.
All VNTR typing was performed at the VLA, Weybridge, United Kingdom.
For microarray analysis, two isolates (no. BTB0691 and BTB1091; see the
supplemental material) were selected from the Ethiopian M. bovis collection.
Both isolates lacked spacers 3 to 7 in their spoligotype pattern. Approximately 1
to 2 ?g genomic DNA was purified, and deletions were identified by microarray
analysis using previously published methods (26). Deletions found in regions
associated with repetitive elements and insertion sequences, which are known to
be prone to deletion events, were disregarded in this study.
Deletion typing. The identification of a strain as M. bovis was on the basis of
spoligotype signature (56) and growth characteristics; many of the isolates from
Uganda, Burundi, Tanzania, and Ethiopia were confirmed as M. bovis by deletion
typing of the RD4 region (11). The status of the RDAf2 region (deleted or
intact) was assessed by multiplex PCR with a set of three primers (primer set
Af2): two primers targeting the flanking regions of RDAf2 (RDAf2_Fw, 5?-AC
TGGACCGGCAACGACCTGG, RDAf2_Rev, 5?-CGGGTGACCGTGAACT
GCGAC) and one primer hybridizing with the internal region of RDAf2
(RDAf2_IntRev, 5?-CGGATCGCGGTGATCGTCGA). A PCR product of 458
bp (RDAf2 intact) or 707 bp (RDAf2 deleted) was identified by agarose gel
electrophoresis. Each PCR mixture contained 1 ?l of supernatant of heat-killed
mycobacterial cells, a final concentration of 1? HotStartTaq master mix
(Qiagen), 1 ?M primers RDAf2_FW, RDAf2_Rev, and RDAf2_IntRev, and
sterile distilled water to a final volume of 20 ?l. Thermal cycling was performed
with an initial denaturation step of 15 min at 96°C, 35 cycles of 30 s at 96°C, 30 s
at 55°C, and 1 min at 72°C, followed by a final elongation step of 10 min at 72°C.
PCR products were separated on a 1% agarose gel. Isolates subjected to RDAf1
typing were examined according to a previously described PCR protocol (41).
IS6110 RFLP typing. Genomic DNA was purified from selected M. bovis
strains (66), and approximately 2 ?g DNA was used for IS6110 restriction
fragment length polymorphism (RFLP) analysis according to the internationally
standardized protocol (62). In short, DNA was digested with the restriction
endonuclease PvuII, separated by agarose gel electrophoresis, and transferred to
a nylon membrane by Southern blotting. The membrane was hybridized with a
probe targeting the right-hand site of the IS6110 element (62, 65) and subse-
quently with a 36-bp oligonucleotide targeting the direct repeat region (65). The
probes were labeled using the enhanced chemiluminescence detection system
(ECL; Amersham). The IS6110 RFLP patterns were analyzed by using the
BioNumerics software program (Applied Maths, Sint Martens-Latum, Belgium),
and the dendrogram was prepared by using the Dice coefficient and unweighted-
pair group method using average linkages (UPGMA).
Nucleotide sequence accession numbers. The RDAf2 deletion junctions of 10
strains of the Af2 clonal complex from five countries were sequenced using
standard methods. The isolate name, country of origin, and GenBank accession
numbers for the sequences surrounding the RDAf2 deletion junctions are as
follows: BTB0890, Ethiopia, GU004183; BTB1067, Ethiopia, GU004182;
BTB1474, Ethiopia, GU004184; JN03, Uganda, GU004178; JN58, Uganda,
GU004179; SEA199701128, Somalia, GU004185; 940130, Burundi, GU004186;
940439, Burundi, GU004187; 11, Tanzania, GU004180; and B3, Tanzania,
GU004181 (see the supplemental material).
Isolates with spacers 3 to 7 absent. An extensive slaughter-
house study in Ethiopia of 58 M. bovis strains isolated from six
abattoirs dispersed throughout the country showed that many
isolates lacked spacers 3 to 7 in their spoligotype pattern, in
addition to the absence of spacers 9, 16, and 39 to 43 (8). We
supplemented this sample with an additional 62 isolates from
the same abattoirs and found that over 75% (n ? 91; total ?
120) of these Ethiopian isolates had spoligotype patterns that
were missing spacers 3 to 7 (Table 1).
Furthermore, three separate spoligotype surveys of bovine
TB in Ethiopian cattle from Addis Ababa and central/southern
Ethiopia showed similar results: over 80% of strains had spac-
ers 3 to 7 deleted (2, 9, 59).
From Uganda, which is situated close to Ethiopia, it was
recently shown that six of nine M. bovis isolates from cattle
originating from seven districts in both the northwest and
southern parts of the country also had spacers 3 to 7 missing in
their spoligotype pattern (5). The absence of spacers 3 to 7 in
Ugandan isolates was supported by a further spoligotype sur-
vey of 19 M. bovis isolates sampled from cattle from similar
regions of the country (44).
To further identify the clonal complexes of bovine TB in
VOL. 193, 2011 M. BOVIS AFRICAN 2 CLONAL COMPLEX671
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East Africa, we spoligotyped M. bovis strains previously col-
lected from cattle in Burundi and Tanzania (41, 48). All iso-
lates from Burundi (originating at the city of Bujumbura and
nearby in the western parts of the country; n ? 10) and 10 out
of 14 isolates from cattle sampled at a Morogoro slaughter-
house in south-central Tanzania had spacers 3 to 7 missing in
their spoligotype patterns (Table 1).
In total, 117 out of 153 (76%) of M. bovis isolates from these
four east African countries had spoligotype patterns missing
spacers 3 to 7, and we concluded that, in a manner similar to
that of the African 1 clonal complex in west-central Africa
(41), these isolates may represent a clonal complex of bovine
TB descended from an ancestral cell in which spacers 3 to 7
had been deleted. The commonest spoligotype pattern in this
data set was SB0133 (Table 2), which is similar to that of M.
bovis BCG (SB0120, which lacks spacers 3, 9, 16, and 39 to 43),
with the additional loss of spacers 4 to 7. Spoligotype pat-
tern SB0133 was found at high frequency in the Tanzanian
and Ethiopian samples and in a low number in Uganda and
was absent from the small sample of strains from Burundi
Identification of a specific deletion. To identify a phyloge-
netically informative deletion for the east African M. bovis
strains lacking spacers 3 to 7, two Ethiopian isolates of spoligo-
type SB0133, which represents the most complete spoligotype
pattern lacking spacers 3 to 7, were tested by microarray anal-
ysis using an M. tuberculosis-M. bovis composite amplicon mi-
croarray. This analysis showed that these two strains were
deleted for all regions that are commonly missing in strains of
M. bovis, including RD4 (11). Region RDAf1, which is deleted
TABLE 1. Spoligotype patterns of M. bovis strains isolated in four east African countriesa
(%) of strains?
aThe presumed ancestral spoligotype pattern of the Af2 clonal complex is shown in bold.
bInternational names for these spoligotype patterns were assigned by Mbovis.org (http://www.Mbovis.org).
cThe spoligotype pattern is shown as a series of 43 ones and zeros, corresponding to spacers 1 to 43 in spoligotyping, with 1 representing hybridization to the spacer
and 0 representing the absence of hybridization.
TABLE 2. Definition and summary of characteristics of the Af2 clonal complex of M. bovis
Definition................................................................................................Presence of deletion RDAf2 (14.1 kb between Mb0599 and Mb0610)
Spoligotype marker................................................................................Absence of spacers 3 to 7
Spoligotype signaturea...........................................................................1100000101111110111111111111111111111100000 (SB0133)
Distribution.............................................................................................At high frequency in East Africa (Uganda, Burundi, Tanzania, and Ethiopia)
IS6110 copy no.......................................................................................4 or more copies (infrequently less than 4)
aThe spoligotype signature represents the assumed spoligotype pattern in the progenitor strain of this clonal complex and is shown as a series of 43 ones and zeros
corresponding to spacers 1 to 43 in spoligotyping, with “1” representing hybridization to the spacer and “0” representing absence of hybridization. The international
name for this spoligotype pattern was assigned by Mbovis.org (http://www.mbovis.org).
672BERG ET AL.J. BACTERIOL.
on February 15, 2013 by guest
in members of the African 1 clonal complex of M. bovis,
present at high frequency in several countries in west-central
Africa, was intact, showing that these strains were not mem-
bers of the African 1 clonal complex (41), as was a region
called RDEu1, which is associated with isolates from cattle
originating in Great Britain (unpublished data). However, we
identified a unique region of chromosomal DNA of approxi-
mately 14 kb that was deleted in both Ethiopian isolates. The
endpoints of this deletion were characterized by nucleotide
sequencing and compared to the whole genome sequence of
M. bovis AF2122/97 (27). We determined that 14,094 bp were
deleted, and to our knowledge, this deletion has not previously
been described. We named this deletion Region of Difference
African 2 (RDAf2).
The RDAf2 deletion removed the entire coding sequences
of 10 genes from Mb0600c to Mb0609 and parts of Mb0599
and Mb0610 (corresponding to the genes Rv0585c to Rv0593
and parts of Rv0584 and Rv0594 in M. tuberculosis H37Rv).
The regions surrounding the RDAf2 deletion junction in the
genome of the sequenced strain M. bovis AF2122/97 showed
no evidence of the common M. tuberculosis complex insertion
sequences or repetitive DNA and were not GC rich. No sig-
nificant inverted or direct repeats could be identified at either
side of the deletion junctions in the RDAf2 region of the
AF2122/97 chromosome sequence. We therefore concluded
that this region of the chromosome is not prone to generate
homoplastic deletions and hence the RDAf2 deletion could be
a suitable phylogenetic marker to identify strains of a clonal
complex of M. bovis strains which descended from an ancestral
cell in which RDAf2 was deleted in addition to spacers 3 to 7
in the spoligotype pattern.
Distribution of RDAf2 among cattle isolates in East Africa.
To rapidly identify strains with the RDAf2 deletion, we devel-
oped a simple PCR method using two primers targeting both
flanking regions of RDAf2 and one primer targeting an
RDAf2 internal sequence. All 120 M. bovis isolates from cattle
from Ethiopia were tested by this deletion assay, and 91 of
these were deleted for RDAf2. Furthermore, we tested sam-
ples of M. bovis collected from cattle from Uganda (5), Bu-
rundi (48), and Tanzania (41) for the status of the RDAf2
region. The RDAf2 region was deleted in 6 out of 9 isolates
from Uganda, in all isolates sampled from Burundi (n ? 10),
and in 10 of the 14 isolates from Tanzania (Table 1). All strains
in these samples and throughout this article that were deleted
for RDAf2 were also missing spacers 3 to 7 in their spoligotype
To provide supportive evidence that the RDAf2 deletion
was identical by descent, we sequenced across the RDAf2
deletion junction in nine M. bovis isolates of different spoligo-
types (at least two from each of the four east African coun-
tries). The RDAf2 deletion endpoints were the same in all nine
strains, suggesting that in these strains the RDAf2 deletion is
identical by descent.
We concluded that a clonal complex of M. bovis strains,
defined by the deletion RDAf2 and marked by the loss of
spoligotype spacers 3 to 7, was present at high frequency in
Uganda, Burundi, Tanzania, and Ethiopia (Table 2). We
named this M. bovis clonal complex African 2.
Af2 in west-central Africa. The M. bovis Af1 clonal complex
has previously been identified at high frequency in samples of
strains from Mali, Nigeria, Chad, and Cameroon (41). In a
manner similar to that of Af2, described here, all isolates of the
Af1 clonal complex have a specific deletion, RDAf1, and have
spacer 30 missing in their spoligotype. To determine the phy-
logenetic relationship between Af1 and Af2 strains, a selection
of isolates which represent the population of Af1 strains
present in each of these west-central African countries were
deletion typed for the RDAf2 deletion. All Af1 strains from
Mali (n ? 13), Nigeria (n ? 5), Chad (n ? 5), and Cameroon
(n ? 3) were intact for RDAf2 (see the supplemental mate-
rial). Reciprocal deletion analysis showed that strains of Af2
are not deleted for RDAf1 (n ? 27), and we concluded that
Af1 and Af2 were phylogenetically distinct. We also performed
RDAf2 deletion typing of seven strains from Mali of spoligo-
types SB0134 and SB0991; these patterns represent non-Af1
isolates found in that country (42); these strains were intact at
the RDAf2 and RDAf1 loci (41). We concluded, based on the
dominance of the Af1 clonal complex in west-central Africa
(53), that the Af2 clonal complex was rare or absent from Mali,
Nigeria, Chad, and Cameroon.
Af2 elsewhere in Africa. Spoligotype surveys of M. bovis from
Algeria, Zambia, and South Africa suggest that strains with
spacers 3 to 7 deleted are absent or present at low frequency in
these countries (40, 43, 47, 51). Furthermore, a spoligotype
survey of over 100 strains from Madagascar did not disclose
any patterns with spacers 3 to 7 missing (47). To determine the
prevalence of Af2 in other African countries, we RDAf2
deletion typed samples representing previously published
spoligotype surveys of M. bovis from Algeria (n ? 17) (51) and
South Africa (n ? 20) (40), as well as an unpublished set of 14
strains from Mozambique; all were intact for the RDAf2 re-
From these spoligotype and deletion surveys of M. bovis in
African nations, we concluded that strains of the Af2 clonal
complex were present at high frequency in some east African
countries (Uganda, Burundi, Tanzania, and Ethiopia) but were
rare or absent in Algeria, Mali, Nigeria, Chad, Cameroon,
Zambia, South Africa, Mozambique, and Madagascar. This
observation echoes the localization of the Af1 clonal complex
to west-central Africa (41) (Fig. 1).
Global distribution of Af2. In unpublished data, we have
shown that a globally distributed clonal complex of M. bovis
called European 1 (Eu1), which is defined by a specific deletion
called RDEu1 and the loss of spoligotype spacer 11 (spacers 4
to 7 are usually present), is present at high frequency in many
parts of the world. RDAf2 deletion analysis of a sample of 21
Eu1 strains from Great Britain (see the supplemental material)
showed that they were intact at the RDAf2 locus, and recip-
rocal deletion analysis showed that a sample of RDAf2 strains
was intact at the RDEu1 locus. We concluded that strains of
the Eu1 clonal complex are not members of the Af2 clonal
complex and vice versa. The phylogenetic independence of Af2
and Eu1 implies that Af2 strains are rare or absent on the
British Isles, most of the New World, Australia, and New
Zealand, where the Eu1 clonal complex is virtually at fixation
In mainland Europe, where Eu1 is rare, we inspected pre-
viously published spoligotype surveys of cattle isolates of M.
bovis from France, Italy, Spain, Belgium, and Portugal for
isolates showing the spoligotype signature of Af2 strains: the
VOL. 193, 2011 M. BOVIS AFRICAN 2 CLONAL COMPLEX673
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loss of spacers 3 to 7. We identified small numbers of isolates
with the Af2 spoligotype signature from France (?2%), Italy
(?2%), and Spain (?1%) (3, 10, 31, 49, 52). Sixteen (2%) of
747 M. bovis isolates from northern Italy had either spoligotype
SB0133 (the most common Af2 spoligotype pattern in East
Africa) or SB1584. RDAf2 deletion typing showed that none of
the Italian strains were deleted for this region. Furthermore, in
a large survey of 5,585 M. bovis isolates from Spanish cattle, 20
isolates were, by spoligotype pattern, possible members of the
Af2 clonal complex (49). However, none of these Spanish
strains were deleted for RDAf2 (see the supplemental mate-
rial). We concluded that strains of the Af2 clonal complex were
rare or absent in cattle outside East Africa. In this respect, Af2
again mimics the Af1 clonal complex, which has not been
found in cattle outside west-central Africa (41).
Human isolates of Af2. The prevalence of bovine TB in
humans is unknown in most African countries, and M. bovis is
isolated only occasionally from humans. However, previously
published studies from Uganda identified three human M.
bovis isolates with spoligotype patterns identical to those of the
commonest Af2 strains found in that country (44–45). One of
these strains was deletion typed for RDAf2, and we confirmed
the region to be deleted. A previously unpublished M. bovis
strain, collected in Sweden from a patient born in Somalia, had
a spoligotype identical to the commonest spoligotype found in
cattle in the neighboring country Ethiopia (SB1176) and was
also deleted for RDAf2, with deletion boundaries identical to
those observed in strains isolated from cattle (see the supple-
IS6110 copy number. It has frequently been suggested that
M. bovis isolates from cattle have only one, or a few, copies of
the insertion element IS6110 (4, 7, 15, 17–18, 50, 64). However,
cattle isolates of M. bovis from Burundi and Uganda have been
shown to have multiple copies of IS6110 (5, 48). All 10 isolates
from Burundi that we have characterized as Af2 had four or
more IS6110 copies, and five of the Af2 isolates from Uganda
had six or more copies of IS6110. In contrast, two strains from
Uganda, previously shown to have only one copy of IS6110 (5),
were found to be intact at RDAf2 and therefore were not
members of the Af2 clonal complex.
To further explore the IS6110 copy number in Af2 strains,
we subjected four isolates of the Af2 clonal complex from
Ethiopia to IS6110 RFLP typing. Three of the Af2 isolates
contained between four and seven IS6110 copies, while a single
Af2 strain from Ethiopia had only two copies of IS6110 (Fig.
2). We also IS6110 RFLP typed six strains that were intact at
the RDAf2 locus (not members of the Af2 clonal complex)
from Ethiopia, Mali, and Chad; four of these isolates had only
one copy of IS6110; however, two strains, from Ethiopia and
Mali, had two and three copies, respectively (Fig. 2). In total,
including previously published IS6110 RFLP data (5, 48) on
strains identified here by deletion typing as Af2, 20 of 21 Af2
strains had four or more copies of IS6110.
We have identified an epidemiologically important clonal
complex of M. bovis which is found at high frequency in
Uganda, Burundi, Tanzania, and Ethiopia and have named this
clonal complex African 2 (Af2). The Af2 clonal complex is
epidemiologically important because it is commonly recovered
from cattle in these four countries, but we do not yet know how
phylogenetically distinct this clonal complex is from other
clonal complexes of M. bovis. Members of the Af2 clonal com-
plex are defined by a 14.1-kb deletion of chromosomal DNA
which we have named Region of Difference Af2 (RDAf2).
Sequencing of the RDAf2 region in nine isolates from the four
countries has shown that the deletion boundaries are identical;
in the absence of repetitive elements, or other features, flank-
ing the RDAf2 deletion that can promote homoplastic dele-
tions and the apparent strict clonality of M. bovis, we conclude
that this deletion is identical by descent in strains from each of
these four countries. That is, RDAf2 was deleted in the most
recent common ancestor of this clonal complex, and this region
is therefore deleted in all of its descendants. A definition and
summary of the Af2 clonal complex are shown in Table 2.
Strains of the Af2 clonal complex can be identified by the
loss of spacers 3 to 7 in the DR locus; however, this charac-
teristic is not necessarily specific. It is theoretically possible for
strains with the RDAf2 deletion to have these spacers present,
although we have not yet identified such an isolate. Further-
more, because the loss of spoligotype spacers can be subject to
homoplasy (54, 67), strains that are not members of the Af2
clonal complex (RDAf2 region intact) may also lack spacers 3
to 7; for example, two Nigerian strains of the African 1 clonal
complex (deleted for RDAf1) (41) had also lost spacers 3 to 7,
FIG. 1. Localization of the M. bovis Af1 and Af2 clonal complexes
in Africa. (A) The four west-central African countries where Af1
strains were found to be dominant are shown in yellow, and the four
east African countries where Af2 strains are highly prevalent are
shown in green. Isolates of the Af1 clonal complex are very rare or not
present in countries with the Af2 complex and vice versa. Countries
where no Af1 or Af2 strains have been identified are labeled in gray,
and countries where no isolates were studied are white. (B) Cattle
distribution on the African continent (gray shaded area). (Reprinted
from reference 32 with permission from the publisher.).
674BERG ET AL.J. BACTERIOL.
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as well as the signature loss of spacer 30, and were shown to be
intact for RDAf2.
Af2 in Africa. We showed by reciprocal deletion analysis that
strains of the Af2 clonal complex are not members of the
African 1 (Af1) clonal complex, which is virtually fixed in
Nigeria, Chad, and Cameroon and represents 65% of the iso-
lates from Mali (41). Isolates from Mali that were not members
of the dominant Af1 clonal complex have been given the pre-
liminary name African 5 (Af5) based on the common loss of
spacers 3 to 5 in their spoligotype pattern. RDAf2 deletion
analysis of Af5 strains from Mali showed they were not mem-
bers of the Af2 clonal complex, and we conclude that Af2 is
rare or absent in all four of these west-central African coun-
We have also subjected strains from Algeria, South Africa,
and Mozambique to RDAf2 deletion typing, and although the
number of strains sampled in each of these countries was small,
this supported our conclusion that the Af2 clonal complex
is not uniformly distributed throughout Africa. In general,
spoligotype surveys showed that strains with spacers 3 to 7
deleted are rare in these African nations, reinforcing the sug-
gestion of localization of Af2 to cattle in East Africa (Fig. 1).
Af2 in the rest of the world. An inspection of previously
published spoligotype surveys from countries throughout the
world did not identify any country with more than 2% of strains
with spacers 3 to 7 missing in their spoligotype patterns. In
unpublished data, we have observed that another clonal com-
plex of M. bovis, European 1, is virtually fixed in the British
Isles, most of the New World, Australia, and New Zealand and
therefore the Af2 clonal complex is rare or absent from these
countries. Furthermore, large spoligotype surveys of strains
from mainland Europe (10, 22, 49) and Iran (58) and RDAf2
deletion analysis of the few strains with the Af2 spoligotype
signature from Italy and Spain did not identify any Af2 strains
in cattle outside Africa.
We conclude that among the countries sampled, strains of
the Af2 clonal complex were common in cattle only in the four
east African nations; in this respect, Af2 resembles the Af1
clonal complex, which is apparently confined to cattle in west-
central Africa (41). Strains of the Af2 clonal complex represent
over 70% of the isolates from Uganda, Burundi, Tanzania, and
Ethiopia and have not been isolated from cattle elsewhere in
Localization of Af2 genotypes. If we assume that spoligotype
spacers are lost and never regained, then all the Af2 spoligo-
type patterns described here can be derived from an ancestral
spoligotype pattern equivalent to that of the vaccine strain
BCG (SB0120, missing spacers 3, 9, 16, and 39 to 43), with the
additional deletion of spacers 4 to 7 (SB0133). Although the
number of strains sampled here from Uganda, Burundi, and
Tanzania is small, the population structure of Af2 in these
countries showed remarkable differences; the most common
Af2 spoligotype pattern in each of the four east African coun-
tries surveyed was different. Spoligotype patterns SB0133 and
SB0303 (a single spacer 13 loss derivative of spoligotype pat-
tern SB0133) were the only Af2 patterns found in more than
one country in our data set. However, the frequency of strains
with these two patterns varied remarkably between countries.
SB0133 was most common in our sample from Tanzania (64%)
but was at much lower frequencies in the three other east
African nations (from 0 to 14%), and spoligotype pattern
SB0303 was common in Burundi (50%) but was only found in
a single isolate of the 120 strains from Ethiopia. This observa-
tion contrasts with the spoligotype distribution of the Af1
clonal complex, for which a single ancestral-type spoligotype
pattern was dominant in three of four west-central African
To further investigate the national differences between Af2
clones, we six-locus VNTR typed a sample of eight strains with
spoligotype pattern SB0133 from Tanzania and nine strains
with the same spoligotype pattern from Ethiopia (Table 3).
The Tanzanian strains were all of the same genotype (spoligo-
type plus VNTR type); however, this genotype was not found
among the nine SB0133 strains from Ethiopia. Finally, a single
isolate of spoligotype SB0303 from Ethiopia differed from the
genotypes found in five strains with that spoligotype from Bu-
rundi (Table 3).
Both the spoligotype surveys and the genotype comparisons
FIG. 2. IS6110 RFLP patterns, IS6110 copy numbers, RDAf2 deletion types, and spoligotypes of 12 M. bovis isolates from Africa and of the
reference M. bovis BCG strain P3. The arrow marks a ?1.9-kb restriction fragment commonly representing an IS6110 copy found in the DR region
of M. bovis strains.
VOL. 193, 2011M. BOVIS AFRICAN 2 CLONAL COMPLEX675
on February 15, 2013 by guest
suggest that the population of Af2 strains in each of these four
east African countries is unique. That is, for any isolate of Af2,
it should be possible, with reasonable accuracy, to determine
from its genotype its country of origin. This conclusion rein-
forces a continuing theme of national localization of M. bovis
genotypes initially described for Af1 strains in west-central
Africa (41). However, the genotype data presented here for
Af2 and, to a lesser extent, the spoligotype data must be in-
terpreted with care. Apart from Ethiopia, the sample sizes of
strains from the other three countries were very small and,
more significantly, isolated in only a few localized areas. Intra-
country geographical localization of M. bovis genotypes, as is
commonly seen in the United Kingdom (54) and as was dis-
cussed in a previous study (41), could confound the observa-
tion of national localization of the Af2 genotypes presented
Human isolates of Af2. Support for geographical localization
of Af2 genotypes comes from M. bovis strains isolated from
humans. In Uganda, several human isolates were shown to
have the same spoligotype pattern as those found in local cattle
(5, 45). Furthermore, a Somali immigrant was diagnosed with
abdominal TB shortly after arrival in Sweden, and the infection
was confirmed as bovine TB (unpublished data). This M. bovis
isolate was deleted for RDAf2 and had a genotype identical to
those found at high frequency in Ethiopia (SB1176; 5 2 5 4* 3
3.1) (Table 3). We do not know where this patient contracted
this disease, but it is possible that the original source of infec-
tion was cattle in Somalia. This epidemiological link to Somalia
implies that the Af2 clonal complex may be more widely dis-
tributed across the Horn of Africa than the present study
shows. However, it is possible that the Somali patient migrated
via Ethiopia and was infected during transit.
Evolution of the Af2 clonal complex. The simplest explana-
tion for the observed distribution and population structure of
the Af2 clonal complex throughout these east African coun-
tries is that this clonal complex of M. bovis spread between
these four countries. The progenitor strain, originating in one
place, would have had spoligotype pattern SB0133 (spacers 3
to 7 missing) and carried the RDAf2 deletion; all Af2 spoligo-
type patterns described here can be generated from SB0133 by
spacer loss. The country-specific population structures could
have evolved by drift during the spread of the Af2 clonal
complex between countries in a series of founder events or
subsequently as the population expanded in each country. This
explanation is similar to that used to explain the distribution of
Af1 strains throughout west-central African nations (41), but
with one important difference. The Af1 clonal complex of M.
bovis was fixed in three of the four west-central African coun-
tries in which it was sampled, and therefore it was suggested
that the Af1 clonal complex was transmitted through cattle
naive to bovine TB. The spoligotype surveys of strains from
East Africa (Uganda, Burundi, Tanzania, and Ethiopia)
showed that in three of these countries non-Af2 strains of M.
bovis are present at a reasonably high frequency (between 5
and 33%). There is no obvious relationship between the
spoligotype patterns of non-Af2 strains identified in Uganda,
Tanzania, and Ethiopia, and we do not know the temporal
relationship between the origin of these non-Af2 strains and
the Af2 clonal complex; the non-Af2 strains may have been
present before the introduction of the Af2 clonal complex or
may have been introduced recently from neighboring countries
that have not yet been surveyed for bovine TB. This question
may be resolved as more countries in Africa are surveyed for
genotypes of bovine TB.
Whether the progenitor of the Af2 clonal complex evolved
in Africa or evolved elsewhere and was subsequently imported
to Africa is unknown, although this may be resolved when the
phylogenetic relationship of this clonal complex to strains from
other countries is determined by whole-genome sequencing.
The RDAf2 deletion. The large RDAf2 deletion (14.1 kb)
affects 12 open reading frames on the M. bovis chromosome, of
which 9 belong to an operon, mce2. M. tuberculosis has four
homologous mce operons, mce1 to mce4 (14), whereas all M.
bovis strains are missing the entire mce3 operon due to a
deletion, RD7, which was lost early in the lineage of animal-
adapted strains that leads from the recent common ancestor
with M. tuberculosis to M. bovis (55). All members of the Af2
clonal complex have, in addition, lost the mce2 operon as a
consequence of the RDAf2 deletion.
Each mce operon includes two yrbE and six mce genes, which
show homology to ABC transporter permeases and substrate-
binding proteins, respectively (13), and are believed to be in-
volved in transport across the cell envelope. The mce4 operon
in M. tuberculosis has been identified as a cholesterol import
system (46), while the functions of the other three mce operons
are still unclear. However, several studies have shown that M.
tuberculosis isolates deleted for mce2 are attenuated in mice (1,
28, 37). It remains to be seen if strains of the M. bovis Af2
clonal complex, with a naturally deleted mce2 operon, would
also be attenuated in mice. However, isolates of the Af2 clonal
complex collected from cattle for this study were, in general,
isolated from typical tubercle lesions, suggesting that RDAf2
isolates are capable of causing typical tuberculosis-like pathol-
ogy in cattle. Further work is needed to assess any differences
in pathogenicity between strains of the Af2 clonal complex and
those of other clonal complexes of M. bovis.
IS6110 copy number. It has been suggested that M. bovis
from cattle has only one or two copies of the transposable
element IS6110 (4, 7, 15, 17–18, 39–40, 50, 64). Most other
species (or ecotypes ) of the M. tuberculosis complex have
multiple copies of IS6110, with the exception of some M. tu-
berculosis lineages; for example, strains of the TbD1 intact or
TABLE 3. Countries of isolation, spoligotypes, VNTR types, and
frequencies of M. bovis strains of the Af2 clonal complex with
spoligotype patterns that were found in multiple countries
Country Spoligotype ETR-VNTRa
Frequency, no. of
4 2 5 4* 2 3.1
3 2 5 4* 2 3.1
5 2 4 3* 3 3.1
5 2 4 3* 3 2.1
3 2 5 4* 3 3.1
5 2 4 3* 3 3.1
2 2 5 4* 3 3.1
2 2 5 6* 3 3.1
5 2 5 4* 3 3.1
5 2 5 4* 3 3.1
aAllele call for the ETR-A to -F loci (12).
bStrain isolated in Sweden.
676 BERG ET AL.J. BACTERIOL.
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“ancestral” lineage are frequently found to have low copy
numbers of IS6110 (20, 57). We here show that the Af2 clonal
complex of M. bovis is, in general, multicopy (four or more) for
IS6110. However, this is not a reliable characteristic of Af2
strains; isolate BTB1087 in this study was deleted for RDAf2
and contained only two copies of IS6110.
We suspect that the association between M. bovis and a low
copy number of IS6110 has developed because of the global
distribution of an M. bovis clonal complex which has, in gen-
eral, only one (or rarely two) copies of IS6110 (the European
1 clonal complex; unpublished data) and is present in many
developed nations with the technology to carry out IS6110
Strains of almost all species within the M. tuberculosis com-
plex carry an IS6110 element in the direct repeat (DR) region,
which is a hot spot integration region for insertion elements
(34). For M. bovis strains, this IS6110 copy in the DR region is
usually visualized as a ?1.9-kb PvuII restriction fragment in
IS6110 RFLP analysis (15, 19, 64). It has previously been
suggested that small variations in the size of this fragment
correlate with variation in the number of spoligotype spacers in
the DR region that are flanking the IS6110 DNA (64). We
observed in our RFLP analysis that the single copy of IS6110
in the two Ethiopian strains, BTB0893 and BTB0915 (both
intact for RDAf2), was integrated in a much larger restriction
fragment of ?4.2 kb. We suggest that this much larger frag-
ment may be explained by the loss of the PvuII restriction site
that is commonly found in spacer 36 (63); spacer 36 is absent
from the spoligotype pattern of strains BTB0893 and
BTB0915. Using a DR probe in RFLP analysis, we verified that
a single IS6110 copy was located in the DR region in those two
strains and in the other 10 isolates examined. We concluded
that all M. bovis strains investigated by RFLP typing in this
study carried an IS6110 copy in the DR region.
Conclusions. We have identified a second clonal complex of
M. bovis in Africa, found at high frequency in east African
cattle and with a distribution that does not overlap with the
previously identified west-central African clonal complex, Af-
rican 1. The geographical localization of the Af2 clonal com-
plex to these four east African countries, and perhaps to some
additional neighboring countries not yet surveyed, may have
been governed by geographical features that affect cattle den-
sity, trading, and movement in this part of Africa. For example,
Fig. 1B shows the cattle distribution in Africa (32), and it is
interesting to note the limited links between regions of high
cattle density in East Africa, where Af2 is prevalent, and re-
gions in west-central Africa, where Af1 dominates. The uneven
distribution of cattle in Africa may have contributed to local-
ized dominance of clonal complexes of M. bovis in different
regions of Africa.
However, these two clonal complexes, Af1 and Af2, may
represent groups of strains with different selective advantages
or behaviors, and comparing and contrasting the phenotypic
differences between these distinct divisions within M. bovis may
elucidate the molecular mechanisms of these differences and
identify the selective forces operating on both bovine TB and
its cattle host. For example, Bos taurus (European cattle) is
common in West Africa, where the African 1 clonal complex of
M. bovis dominates, whereas Bos indicus (Zebu; Asian cattle)
is common in East Africa, where African 2 dominates (32–33).
It will require further work to determine if the African 1 and
African 2 clonal complexes are specifically adapted to these
two different types of cattle or if the relationship merely rep-
resents demographic happenstance.
On a more practical level, the results presented here show
that the development of simple genotype schemes for M. bovis
within these east African countries is worthwhile and will aid
eradication schemes by identifying strains imported from
neighboring countries (41). Furthermore, now that the African
1 and African 2 clonal complexes have been identified, it is a
simple matter to sequence chromosomes of representative iso-
lates and gather a rich harvest of specific molecular polymor-
phisms to use in local epidemiological analysis. Comparative
genome sequence analysis will also resolve the phylogenetic
status of these clonal complexes and may show that the ma-
jority of bovine TB found in Africa originated elsewhere and
has been imported to the continent relatively recently. This, in
turn, will develop our understanding of the historical and
phylogeographical basis of bovine TB in Africa and inform our
understanding of the disease in Europe and throughout the
We thank A. Mulder at RIVM and M. Okker and K. Gover at the
VLA for excellent technical assistance.
This work was funded by the Wellcome Trust Livestock for Life and
Animal Health in the Developing World initiatives, the Swiss National
Science Foundation, the Swedish Heart-Lung Foundation, the Swedish
Research Council, the Swedish International Development Cooperation
Agency, the Damien Foundation (Belgium), the South African Medical
Research Council and National Research Foundation, the MacArthur
Foundation/University of Ibadan, the European Union Seventh Frame-
work Program (Integrated Control of Neglected Zoonoses), and the
Department of Environment, Food and Rural Affairs, United Kingdom.
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African 2, a Clonal Complex of Mycobacterium bovis
Epidemiologically Important in East Africa
Stefan Berg, M. Carmen Garcia-Pelayo, Borna Müller, Elena Hailu, Benon Asiimwe, Kristin Kremer, James Dale, M. Beatrice Boniotti,
Sabrina Rodriguez, Markus Hilty, Leen Rigouts, Rebuma Firdessa, Adelina Machado, Custodia Mucavele,
Bongo Nare Richard Ngandolo, Judith Bruchfeld, Laura Boschiroli, Annélle Müller, Naima Sahraoui, Maria Pacciarini, Simeon Cadmus,
Moses Joloba, Dick van Soolingen, Anita L. Michel, Berit Djønne, Alicia Aranaz, Jakob Zinsstag, Paul van Helden, Françoise Portaels,
Rudovick Kazwala, Gunilla Källenius, R. Glyn Hewinson, Abraham Aseffa, Stephen V. Gordon, and Noel H. Smith
VLA Weybridge, New Haw, Surrey KT15 3NB, United Kingdom; Division of Molecular Biology and Human Genetics, Faculty of Health Sciences, Stellenbosch University, P.O.
Box 19063, Tygerberg 7505, South Africa; Armauer Hansen Research Institute, P.O. Box 1005, Addis Ababa, Ethiopia; Department of Medical Microbiology, Makerere
University Medical School, P.O. Box 7072, Kampala, Uganda; Tuberculosis Reference Laboratory, National Institute for Public Health and the Environment (RIVM), Centre
for Infectious Disease Control (CIb/LIS), P.O. Box 1, 3720 BA Bilthoven, Netherlands; Reparto Genomica, Istituto Zooprofilattico Sperimentale della Lombardia e dell=Emilia,
Via Bianchi n. 9, 25124 Brescia, Italy; Dept. de Sanidad Animal, Facultad de Veterinaria, and Centro Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense,
Avenida, Puerta de Hierro s/n, 28040 Madrid, Spain; Institute for Infectious Diseases, University of Bern, Friedbühlstrasse 51, CH-3010 Bern, Switzerland; Agence Française
de Sécurité Sanitaire des Aliments, 23 Avenue du Général-de-Gaulle, 94706 Maisons-Alfort Cedex, France; Unit of Infectious Diseases, Department of Medicine, Solna,
Karolinska Institutet, Karolinska University Hospital, 17177 Stockholm, Sweden; Department of Veterinary Public Health & Preventive Medicine, University of Ibadan,
Ibadan, Nigeria; Laboratoire de Recherches Vétérinaires et Zootechniques de Farcha, BP 433, N’Djaména, Chad; Sokoine University of Agriculture, Morogoro, Tanzania;
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Weybridge, New Haw, Surrey KT15 3NB, United Kingdom, and Centre for the Study of Evolution, University of Sussex, Brighton BN1 9QL, United Kingdom
Volume 193, no. 3, p. 670–678, 2011. Page 671, column 2: Lines 7 through 14 should read as follows: “The status of the RDAf2 region
of RDAf2 (RDAf2_Fw, 5=-ACCGCCCTGTCCTATGTGAG, and RDAf2_Rev, 5=-TGACGGTTGCCTTTCTTGAC) and one primer
intact) or 711 bp (RDAf2 deleted) was identified by agarose gel electrophoresis.”
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