JOURNAL OF CLINICAL MICROBIOLOGY, June 2011, p. 2222–2229
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 6
Amplified Fragment Length Polymorphism Reveals Specific Epigenetic
Distinctions between Mycobacterium avium Subspecies
paratuberculosis Isolates of Various Isolation Types?
B. O’Shea,1* S. Khare,1P. Klein,2A. Roussel,3L. G. Adams,1T. A. Ficht,1and A. C. Rice-Ficht4
Department of Veterinary Pathobiology,1Department of Horticulture,2and Department of Large Animal Clinical Sciences,3
Texas A&M University, and Department of Molecular and Cellular Medicine, Texas A&M University Health Science Center,4
College Station, Texas
Received 3 June 2010/Returned for modification 2 September 2010/Accepted 28 March 2011
Amplified fragment length polymorphism (AFLP) was employed as a genetic analysis tool for the study of the
genetic relatedness of Mycobacterium avium subsp. paratuberculosis isolates harvested from bovine fecal sam-
ples and from bovine or human tissues. This analysis revealed genetic differences between these two isolate
types that were confirmed through cluster analysis. Dendrogram analysis separated these two isolate types
based on the isolation scheme (tissue-associated versus fecal M. avium subsp. paratuberculosis isolates).
Further sequence analysis of unique genetic regions from each isolation type revealed no genetic sequence
differences. However, Clustal DNA alignments identified AFLP restriction enzyme sites that were undigested
in the tissue-associated isolates. AFLP analysis also disclosed that the same AFLP restriction sites were
digested in all of the fecal isolates. Sequence analysis further revealed a consensus sequence upstream of the
undigested restriction sites for possible methyltransferase recognition in the tissue-associated M. avium subsp.
Mycobacterium avium subspecies paratuberculosis is the eti-
ologic agent of a chronic granulomatous enteritis of ruminants
known as Johne’s disease (12). M. avium subsp. paratuberculo-
sis has also been suspected to be involved in the chronic in-
flammatory bowel disorder in humans known as Crohn’s dis-
ease (5, 10, 27). Although prevalent mostly in the bovine host
species, this organism has been found to be an infectious agent
of numerous mammals and birds alike (6, 9). M. avium subsp.
paratuberculosis infections are transmitted through the fecal-
oral route by contaminated feed sources, and human cases are
suspected to be due to contaminated milk (28).
Many reports have examined the genetic differences and
strain diversity among M. avium subsp. paratuberculosis isolates
(1, 8, 29–31, 33, 35). These studies report that genetic differ-
ences exist between isolates from differing host species, loca-
tions, and strain types and that these genetic differences can
range from single-nucleotide polymorphisms (SNP) to large
genomic rearrangements. Previous data have confirmed that
amplified fragment length polymorphism (AFLP) analysis is
capable of detecting large polymorphic differences between
isolates (29) and can also detect genetic differences due to
SNPs at the AFLP restriction sites. Furthermore, the highly
sensitive AFLP technique can distinguish between isolates with
epigenetic differences at the AFLP restriction sites if the re-
striction site differences are due to methylation differences
(36). Although this information does not allow for the identi-
fication of the methylated DNA base responsible for the poly-
morphism, it serves as a starting point for future epigenetic
studies involving M. avium subsp. paratuberculosis isolates.
The importance of this information has been demonstrated
in multiple previous studies in which DNA methylation has
been found to play a key role in the differential regulation of
the corresponding gene products and to be able to influence
the virulence of the bacterial organism (2, 4, 7, 14, 16–18,
In this study, AFLP profiles of M. avium subsp. paratuber-
culosis isolates from tissue-associated (isolation from infected
tissue) and fecal (isolation from bovine fecal samples) sources
were analyzed for the distinction of isolate-specific banding
patterns. Bioinformatics tools were used to identify host-spe-
cific AFLP regions for genetic/epigenetic differences.
MATERIALS AND METHODS
Bacterial isolates. Four human M. avium subsp. paratuberculosis isolates were
obtained from the American Type Culture Collection (ATCC 49164, ATCC
43544, ATCC 43545, and ATCC 43015). Six fecal isolates from Texas Brahman
cattle with clinical cases were obtained from Michael Collins (University of
Wisconsin School of Veterinary Medicine [UWVM]): isolates T12, T136, T139,
T140, T141, and T143. Two bovine M. avium subsp. paratuberculosis isolates were
obtained from the ATCC: ATCC 19698 and ATCC 19851. All Texas Brahman
cattle isolates and ATCC 19698 were recovered from fecal material (fecal iso-
lates), while all human isolates were recovered from tissue (tissue associated),
and ATCC 19851 was isolated from the head of an infected cow (tissue associ-
ated). All isolates were identified as M. avium subsp. paratuberculosis by culture
requirements, colony morphology, and the presence of IS900 by PCR as de-
scribed previously (29).
Amplified fragment length polymorphism. AFLP analysis was performed with
the PstI and MseI restriction enzymes as described previously (29) on all isolates
except for isolate ATCC 19698, which was used in duplicate as an internal
control. In this case, an ATCC 19698 bacterial broth culture was divided into two
separate equal aliquots, and the entire AFLP procedure was performed sepa-
rately on each aliquot.
Cluster analysis. The BioNumerics computer analysis program, version 4.0
(Applied Maths, Austin, TX), was used for cluster analysis of AFLP-generated
* Corresponding author. Mailing address: Molecular and Cellular
Medicine, 440 Reynolds Medical Building, Texas A&M University
Health Science Center, College Station, TX 77843-1114. Phone: (979)
862-7474. Fax: (603) 646-2622. E-mail: Brian.J.Oshea@Dartmouth
?Published ahead of print on 6 April 2011.
data. The Dice method of band scoring was used to create a composite data set
from 4 independent AFLP primer sets in order to develop a summary dendro-
PCR confirmation of host-specific regions. AFLP-generated regions unique to
one isolation type were analyzed by agarose gel electrophoresis with ethidium
bromide staining. Regions were excised, purified using the Qiagen (Valencia,
CA) gel purification kit, and subsequently cloned into chemically competent
Escherichia coli cells using the TOPO 2.1 vector according to the manufacturer’s
protocols (Invitrogen, Carlsbad, CA). Plasmid DNAs of positive transformants
were purified with Qiagen’s Miniprep kit according to the manufacturer’s pro-
tocols and were subjected to sequence analysis (Lone Star Labs, Houston, TX).
DNA sequence analysis of M. avium subsp. paratuberculosis regions consisted of
queries in BLAST searches for sequence alignments against known genomes and
for possible sequence homology to known organisms. Sequences were also
aligned to the known genomic sequence of M. avium subsp. paratuberculosis K10,
and PCR primers external to these regions were derived. PCR primers were
designed to amplify regions flanking AFLP-derived regions and AFLP restriction
sites for evidence of single-nucleotide polymorphisms (SNP). The PCR condi-
tions used to amplify M. avium subsp. paratuberculosis regions with external
primers were as follows: a 20-?l reaction mixture consisting of 100 ng each
primer, 10 ?l FailSafe PreMix G (Epicenter), 1 U Taq DNA polymerase (Epi-
center Technologies, Madison, WI), and 7.6 ?l DNA eluted from FTA storage
cards (Whatman Inc., Clifton, NJ) according to the manufacturer’s protocols.
Thermocycler protocols were as follows: initial denaturation at 94°C for 5 min,
followed by 45 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min. A final
elongation was carried out for 10 min at 72°C. Amplicons were viewed on 2%
(wt/vol) SeaKem LE agarose gels with ethidium bromide staining.
Data mining of host-specific regions. PCR products corresponding to isola-
tion-specific regions were used as queries in BLAST searches of the NCBI
database and for alignment to the known M. avium subsp. paratuberculosis K10
genomic sequence. Regions of interest were also aligned to each other using
MacVector data analysis software in order to locate evidence of consensus
sequences used for possible recognition sites for DNA methyltransferases.
AFLP analysis of experimentally infected bovines. Male Holstein calves, 3 to
4 weeks old and weighing 45 to 55 kg, were fed milk replacer twice daily and
water ad libitum. The calves were clinically healthy before the experiment. All
experiments were performed under a protocol approved by the Texas A&M
University Institutional Animal Use and Care Committee. Calves were fasted for
24 h prior to the nonsurvival surgery; then they were anesthetized and main-
tained analgesic for the course of the 12-h experiment. In brief, for the prepa-
ration and sampling of ligated loops of the ileum and jejunum, anesthesia was
induced with propofol (Abbott Laboratories, Chicago, IL), followed by place-
ment of an endotracheal tube and maintenance with isoflurane (Abbott Labo-
ratories, Chicago, IL) for the duration of the experiment. The abdominal wall
was clipped and prepared aseptically with chlorhexidine and isopropanol prior to
opening. Sterile drapes were used as a barrier. The abdominal wall was opened,
and the entire length of the Peyer’s patch of the distal jejunum and ileum was
exteriorized. The lumen of the ileum and the distal jejunum proximal to the
Peyer’s patch were flushed with saline to remove any remaining intestinal digest
and were manually propelled into the cecum. Six- to 10-cm-long loops of the
distal jejunum and ileum were ligated with umbilical tape, leaving a 1- to 2-cm-
long area for the interloop. Control loops were injected with 3.0 ml of sterile
phosphate-buffered saline (PBS), and infected loops were injected with 3.0 ml of
sterile PBS containing 3 ? 109CFU of M. avium subsp. paratuberculosis ATCC
19698. The loops were placed back into the abdominal cavity, and the incision
was temporarily closed with Backhaus towel clamps. Intravenous (i.v.) sterile
2.5% dextrose and 0.45% normal physiological saline were used to maintain the
circulating blood volume at 5 ml/kg of body weight/h. At 0.5, 1, 2, 4, 8, and 12 h
after bacterial inoculation, loops were excised. Samples for bacteriologic culture
and DNA extraction were collected as described below. Electrocautery was used
to control hemorrhage after the excision of the loops. Throughout the experi-
mental procedure, the calves were monitored for vital signs (blood pressure,
heart rate, hydration status, anesthesia depth, and temperature). The calves were
euthanized with a rapid overdose (a single bolus at 60 mg/lb i.v.) of pentobarbital
sodium after the final 12-h loops were excised.
A 6-mm-diameter biopsy punch was used to collect two tissue samples from
Peyer’s patches for bacteriology. Intestinal tissue samples were washed three
times in PBS, weighed, homogenized in PBS, serially diluted, and plated onto
Herrold’s egg yolk medium containing amphotericin B, nalidixic acid, and van-
comycin (ANV) (Becton Dickinson and Company, Sparks, MD) for incubation
at 37°C. The cultures were observed visually weekly for any contamination, and
the final CFU counts were recorded on week 16 (19).
The M. avium subsp. paratuberculosis colonies were selected and grown in 7H9
broth containing Mycobactin J, oleic acid-albumin-dextrose-catalase (OADC),
and glycerol. DNA was prepared from this bacterial suspension for AFLP studies
of host-passaged bacteria.
AFLP analysis of bovine and human isolates. To determine
the polymorphic differences between isolates of differing iso-
lation types, AFLP analysis was performed on 4 human and 1
bovine tissue-associated M. avium subsp. paratuberculosis iso-
late from the ATCC, and the results were compared to those
of AFLP analysis of 6 fecal isolates from Texas Brahman cattle.
Duplications of ATCC 19698 were used for the normalization
of the AFLP protocol. AFLP primers and adapters were used
as described previously (29). By comparing these AFLP data,
four regions common only to M. avium subsp. paratuberculosis
isolates of tissue-associated origin (Fig. 1) were identified.
These regions corresponded to particular primer combinations
as follows: region 1, primers P-CG and M-AC (330 bp); region
2, primers P-GC and M-AC (220 bp); region 3, primers P-GG
and M-AC (240 bp); region 4, primers P-GG and M-AG (365
bp). Along with these tissue-associated isolate-specific regions,
two regions that exist only in isolates harvested from fecal
material were identified (Fig. 1). These regions correspond to
particular primer combinations as follows: region 5, primers
P-GC and M-AC (620 bp); region 6, primers P-GG and M-AT
(660 bp). The AFLP data suggested a genetic separation of M.
avium subsp. paratuberculosis isolates based on colonization in
tissue or shedding in the feces.
Cluster analysis. The overall genetic divergence of M. avium
subsp. paratuberculosis fecal and tissue-associated isolates was
determined by cluster analysis using BioNumerics data anal-
ysis software, version 4.0, employing the Dice method with
the unweighted-pair group method with arithmetic means
(UPGMA). A composite data set analyzing 4 independent
AFLP primer sets for each isolate was used to create a den-
drogram for the 6 fecal isolates, 5 tissue-associated isolates,
and 2 internal controls (ATCC 19698) (Fig. 2). These data
revealed a very tight (97% similarity) clustering of the ATCC
19698 internal-control isolates, confirming the very high repro-
ducibility of the AFLP technique. The cluster analysis also
revealed clustering of the fecal isolates, as well as clustering of
the tissue-associated isolates. These data also provided evi-
dence of multiple strains of M. avium subsp. paratuberculosis
within a single bovine herd. Isolates 139, 140, 141, and 143
were all isolated from individual cows in herd 32 from Texas.
The cluster analysis revealed a high (89%) similarity of isolates
140 and 141, but isolates 139 and 143 had only 74% similarity
to each other and 77% similarity to the other herd 32 isolates,
suggesting the possibility of 3 distinct M. avium subsp. paratu-
berculosis isolates within herd 32. The tissue-associated isolates
clustered together despite their different origins (human or
PCR confirmation of isolation-specific regions. Isolate-spe-
cific AFLP bands (regions 1 to 6) were sequenced as described
in Materials and Methods. A primer set was designed for the
specific region, and PCR analysis was performed, resulting in
amplicons for all samples. In contrast to the procedure used in
a previous report (29), primer pairs flanking the AFLP regions
were created in order to sequence the entire region, including
VOL. 49, 2011AFLP FOR M. AVIUM SUBSP. PARATUBERCULOSIS ISOLATES 2223
the restriction sites and flanking regions. Analysis of the se-
quence of each of these products revealed 100% sequence
homology to the M. avium subsp. paratuberculosis strain K10
sequence. While this evidence was inconsistent with the differ-
ences observed by AFLP analysis, the possibilities of SNPs at
the AFLP restriction sites and the genomic divergence of the
isolates used in this study from the sequenced K10 genome
were then investigated.
PCR results for numerous regions present only in tissue-
associated isolates and regions present only in fecal isolates
yielded identical amplicons for all isolates tested, regardless of
origin. These data suggested that there was no sequence vari-
ation event in any of the regions tested. These corresponding
PCR products were investigated using SNP analysis at AFLP
restriction sites that could lead to different banding patterns on
AFLP gels. Sequence analysis of all regions tested revealed no
SNPs at either PstI or MseI restriction sites. These data sug-
gested an unknown epigenetic trait as the cause of the AFLP
differences observed between tissue-associated and fecal M.
avium subsp. paratuberculosis isolates.
Data mining of isolation type-specific regions. After the
possibilities that insertions/deletions and single-nucleotide
polymorphisms at restriction sites could account for AFLP
differences between tissue-associated and feces-derived M.
avium subsp. paratuberculosis isolates were eliminated, the se-
quences of these regions were analyzed. Upon further exami-
nation, two AFLP tissue-associated isolate-specific regions, re-
gion 1 (fructose-6-phosphate amidotransferase) and region 3
(a hypothetical protein), revealed a PstI restriction site internal
to the AFLP-derived region. Sequence analysis of amplicons
FIG. 1. Composite AFLP gel comparison of fecal and tissue-associated M. avium subsp. paratuberculosis isolates. M, molecular weight markers.
Isolates in each panel, from left to right, are as follows: fecal isolates T12, T136, T139, T140, T141, and T143; ATCC 19698 (internal control);
tissue-associated isolates 19851, 43015, 43544, 43545, and 49164; duplicate internal control (ATCC 19698). (A) AFLP analysis with primers P-CG
and M-AC. The arrow points to region 1 (330 bp), found only in tissue-associated isolates. (B) AFLP analysis with primers P-GC and M-AC. The
top arrow points to region 5 (620 bp), present in fecal isolates only; the lower arrow points to region 2 (220 bp), present in tissue-associated isolates
only. (C) AFLP analysis with primers P-GG and M-AC. The arrow points to region 3 (240 bp), present in tissue-associated isolates only. (D) AFLP
analysis with primers P-GG and M-AG. The arrow points to region 4 (365 bp), present in tissue-associated isolates only. (E) AFLP analysis with
primers P-GG and M-AT. The arrow points to region 6 (660 bp), present in fecal isolates only.
2224 O’SHEA ET AL.J. CLIN. MICROBIOL.
obtained from fecal isolates with these primer sets (for region
1, forward primer 5?-GCCCAGATCACCGCCAACTAC-3?
and reverse 5?-CGCATACTTCCTCCCGAACG-3? were used
to amplify a 744-bp product; for region 3, forward primer
5?-GCAACGATTGTCCCAAACCC-3? and reverse primer 5?-
ACAGCACCGACGACGCATTC-3? were used to amplify a
474-bp product) revealed the same internal PstI sites. To de-
termine if these internal PstI sites were digested in the fecal
isolates during the AFLP process, AFLP primer sets were
designed to amplify a smaller, truncated region in the fecal
isolates, as indicated in Fig. 3.
AFLP amplification of M. avium subsp. paratuberculosis fe-
cal and tissue-associated isolates with internally derived AFLP
PstI primers with 3? diadenine ends yielded amplicons of an
appropriate size from all fecal isolates, while appropriate am-
plicons were absent from all tissue-associated isolates (Fig. 4).
Corresponding amplicons from fecal isolates were extracted
from gels and were subjected to sequencing as described
above. Sequence analysis confirmed 100% homology with
AFLP region 1 of tissue-associated origin. These data con-
firmed the presence of an internal PstI restriction site in all
fecal and tissue-associated isolates of M. avium subsp. paratu-
berculosis tested, but this restriction site was digested by the
AFLP process only in isolates of fecal origin. An explanation
for this occurrence could be a difference in the methylation
status of the PstI site between the fecal and tissue-associated
groups. These methylation events can inhibit proper PstI di-
gestion, yielding AFLP banding pattern differences.
The internal PstI sites of regions 1 and 3 were investigated in
detail in order to identify potential methylation sites that might
act as inhibitors of restriction of the internal PstI sites in
tissue-associated isolates. By aligning the internal PstI sites of
regions 1 and 3, a consensus sequence 22 bp upstream of the
internal PstI sites, common to both regions, was identified (Fig.
5). The positioning of consensus residues relative to the resis-
tant PstI site suggested that these residues could serve as a
binding site for a putative methylase.
AFLP analysis of experimentally infected bovines. To deter-
mine if host selective pressures could alter AFLP banding
patterns, bovine ileal loop analysis was performed. ATCC
19698 was isolated from bovine ileal loops at 30 min and at 1,
2, 4, 8, and 12 h. DNA from these isolates was analyzed by
AFLP, and the results were compared with those for the
ATCC 19698 inoculum (Fig. 6). The AFLP banding patterns
demonstrated clear differences between the inoculum and the
tissue-associated isolates obtained at all time points. These
banding pattern differences further supported our hypothesis
of a host pressure-induced epigenetic trait, unique to tissue-
associated M. avium subsp. paratuberculosis, which inhibits di-
gestion by PstI, causing AFLP banding pattern differences.
This was evident in that the inoculum isolate had a banding
pattern different from those of the isolates recovered at later
FIG. 2. Cluster analysis of AFLP data for fecal and tissue-associated M. avium subsp. paratuberculosis isolates. A comparative dendrogram of
fecal and tissue-associated isolates is shown with corresponding percentages of similarity. The isolate number, subspecies designation, host species,
origin of the sample, herd, cow identification number (ID), and isolation type are given on the right. N/A, not applicable.
FIG. 3. Graphical representation of the internal PstI site of region 1. Sequence analysis of region 1 revealed an internal PstI site that was not
digested in tissue-associated isolates on AFLP gels. New AFLP primers were designed with corresponding 3? diadenine ends for amplification from
fecal M. avium subsp. paratuberculosis (MAP) isolates.
VOL. 49, 2011AFLP FOR M. AVIUM SUBSP. PARATUBERCULOSIS ISOLATES 2225
time points (Fig. 6A to C, arrows). Moreover, this finding was
further supported by the fact that the tissue-associated isolates
from all time points, each representing an independent DNA
isolation, had banding patterns very similar to each other.
These banding pattern differences were not likely caused by
DNA rearrangement, due to the very short period within the
host cell (30 min). All of the time points fall far short of the
24-h M. avium subsp. paratuberculosis generation time.
In this study, M. avium subsp. paratuberculosis isolates that
were isolated from tissue (tissue associated) and fecal samples
were differentiated by the use of AFLP. Distinguishing genetic
variations were evident upon examination of AFLP-amplified
DNA and were unique to isolates of specific origins (tissue
associated or fecal) (Fig. 1). These genetic variations were not
confined to one isolation type of M. avium subsp. paratubercu-
losis; on the contrary, isolation-specific regions were found to
be present in fecal and tissue-associated isolates alike. These
data confirmed previously published results suggesting a dif-
ferentiation among M. avium subsp. paratuberculosis isolates
from different hosts (3, 13).
Since verification of the AFLP data suggested a genetic
separation of M. avium subsp. paratuberculosis tissue-associ-
ated and fecal isolates, cluster analysis was performed. This
analysis confirmed the AFLP observations that the banding
patterns of fecal and tissue-associated isolates were genetically
distinct. The cluster analysis demonstrated the reproducibility
of AFLP by the clustering of the duplicate sample of ATCC
19698 to a similarity of 97%. It is also noteworthy that the
ATCC 19698 isolate itself fell in the middle of the fecal cluster
of isolates (Fig. 2). This was anticipated, because ATCC 19698
was a fecal isolate of M. avium subsp. paratuberculosis. Another
important observation from the cluster analysis was the
uniqueness of three different bovine M. avium subsp. paratu-
berculosis strains from herd 32. Cluster analysis revealed that
isolates 140 and 141 clustered closely together, while isolates
139 and 143 diverged from one another, showing only 74%
similarity to each other and 77% similarity to the other herd 32
isolates (Fig. 2). Taken together, these data suggested that
herd 32 was infected with 3 strains of M. avium subsp. paratu-
berculosis. This observation correlates with the findings of Mo ¨-
bius et al., in which a number of genetically distinct M. avium
subsp. paratuberculosis isolates were recovered from single
herds in Germany (25). Dendrogram analysis further revealed
a clustering of the human M. avium subsp. paratuberculosis
isolates as a less similar group of isolates than the bovine
isolates. These data were reasonable in view of the fact that the
human isolates were recovered from regions all over the world
over a period of decades, in contrast with the bovine isolates,
which, with the exception of ATCC 19698, were isolated re-
cently exclusively from herds in Texas. These data validated
previous work in which multiplex PCR of IS900 integration loci
(MPIL) revealed that human M. avium subsp. paratuberculosis
isolates exhibit greater genetic diversity than bovine isolates
AFLP analysis not only revealed the genetic diversity of M.
avium subsp. paratuberculosis isolates from fecal and tissue-
associated samples but also identified 6 genetic regions specific
to one isolation type or the other. In this study, the genetic
basis for the isolation-specific variations was investigated. Se-
quence analysis, PCR analysis, and SNP analysis of the tissue-
associated isolate-specific regions revealed sequence homology
to the M. avium subsp. paratuberculosis K10 isolate and no
discernible difference from any fecal isolate. However, upon
examination of regions 1 and 3, an internal PstI restriction site
refractory to restriction during the AFLP process was appar-
ent. Regions 1 and 3 corresponded to AFLP bands unique to
tissue-associated isolates. Their apparent uniqueness was not,
however, based on any differences in the DNA sequence.
Therefore, we hypothesized that an epigenetic event such as
DNA methylation at this internal PstI site might be a reason
FIG. 4. Methylation-specific AFLP gel. A primer set consisting of
P-AA and M-AC was used. Isolates are as follows, from left to right:
molecular weight marker, fecal isolates (T12, T136, T139, T140, T141,
and T143), ATCC 19698, tissue-associated isolates (19851, 43015,
43544, 43545, and 49164), and ATCC 19698. The arrow indicates a
truncated AFLP region present in all fecal isolates and absent from all
tissue-associated isolates, corresponding to the 3? end of region 1.
2226 O’SHEA ET AL.J. CLIN. MICROBIOL.
for the unique AFLP banding patterns. An AFLP primer with
a 3? diadenine end was created for the amplification of the
truncated 3? region of region 1 in fecal isolates (Fig. 3). These
AFLP data revealed the presence of an amplicon correspond-
ing in size to this truncated region in the fecal isolates that was
absent from the tissue-associated isolates, suggesting the pos-
sibility of a tissue-associated isolate-specific methylation event
of the internal PstI site of region 1, inhibiting AFLP digestion
of tissue-associated isolate DNA. The idea that mycobacteria
contain methylated DNA that will inhibit restriction enzymatic
activity is not new. Although many studies have proven the
existence of DNA methylation in mycobacteria, no study has
proven where these methylation events take place or revealed
the particular methyltransferases required for these events
Support for the hypothesis of tissue-associated isolate-specific
methylation of the internal PstI site in region 1 was found in the
presence of a consensus sequence 22 bp upstream of both internal
PstI sites in regions 1 and 3. Although the methyltransferase has not
yet been identified, a putative enzyme recognition sequence, 5?-GC
AGnnGnnnGnnnnnnnnnnnnnnCTGCAG-3?, was observed. The
methylation site is not uncommon. For example, the restriction en-
zyme BsgI of Bacillus sphaericus recognizes a site 14 bp from the
restriction site (5?-GTGCAGnnnnnnnnnnnnnnnn?-3?). Addition-
ally, the restriction enzyme EcoP15I of E. coli P15 binds 25 bp
upstream of the restriction site (5?-CAGCAGnnnnnnnnn
nnnnnnnnnnnnnnnn?nn-3?). In the case of BsgI, as with the M.
constitute a perfect palindrome. These examples demonstrate the
possibility of a tissue-associated isolate-specific methylase as the ex-
banding patterns of M. avium subsp. paratuberculosis tissue-associ-
ated and feces-derived isolates. Although the evidence of DNA
methylation at these sites is somewhat circumstantial, investigations
into the nature of DNA methylation and the specific methyltrans-
ferase involved are currently under way. The preliminary results of
this research suggest a lack of 5-methyl-cytosine at these sites based
on sodium bisulfite modification analysis. This is not surprising in
view of previous reports of certain mycobacterial isolates lacking
the predominant methylated DNA base in mycobacteria (34). Cur-
these locations are under way.
It has been well established that DNA modifications can
lead to the survival of bacteria and/or modifications in the
virulence of bacteria (14, 17, 21, 23, 24, 32, 34). Although
confirming evidence for a role of PstI methylation in the sur-
vival of tissue-associated isolates or the virulence of M. avium
subsp. paratuberculosis has yet to be shown, the evidence for
such an effect is accumulating. DNA methylation has been
proven to play a role in the differences in transcription and
therefore gene product levels in bacteria (11, 14, 17, 21). These
data, coupled with the discovery of a consensus sequence that
could be a recognition sequence for a DNA methyltransferase,
support the contention that the methylation profiles of M.
avium subsp. paratuberculosis tissue-associated and feces-de-
rived isolates are unique and that these methylation patterns
may influence the transcription levels of the methylated genes.
This contention is also supported by the bovine ileal loop
AFLP analysis, which revealed differences in banding patterns
between tissue-associated isolates and the original inoculum. A
possible explanation for our detection of these differences
could be that the tissue-associated bacteria bearing the epige-
netic modification were not detectable in the original inoculum
but were selected during infection. An alternate explanation
could be the presence of a tissue-associated isolate-specific
methylation trait that is displayed only after the host cell in-
ternalizes the bacterium. Once the bacterium is internalized,
the site is methylated, inhibiting enzymatic restriction of PstI
and yielding the genetic differences observed by AFLP analysis
in the M. avium subsp. paratuberculosis tissue-associated iso-
lates. The latter explanation posits a host-dependent epige-
netic trait, which would therefore be reversible. It is our opin-
ion that the former is more likely the correct explanation. This
is further supported by the fact that the epigenetic patterns are
stable after the ileal loop subculturing, suggesting a state in-
dependent of host pressures. These data support the proposed
interpretation that the epigenetic trait common to M. avium
subsp. paratuberculosis tissue-associated isolates inhibits PstI
FIG. 5. Alignment of sequences of regions 1 and 3, with internal PstI sites aligned at position 42. Region 3 has an AFLP-digested PstI site at
position 3. Region 1 has an AFLP-digested PstI site at position 15. The consensus sequence shows evidence of a 4-bp stretch common to both
regions, starting at position 17. The internal AFLP PstI primer with a 3? diadenine end is located at position 48 of region 1.
VOL. 49, 2011AFLP FOR M. AVIUM SUBSP. PARATUBERCULOSIS ISOLATES2227
digestion, and thus, the data support the presence of an epi-
genetic trait expressed solely in M. avium subsp. paratubercu-
losis tissue-associated bacteria.
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