Proceedings of the Seventh International Colloquium on Paratuberculosis

False positive Mycobacterium avium subsp. paratuberculosis IS900 PCR and its diagnostic
implications.

Bölske G, Englund S, Johansson K-E and Königsson MH

Department of Bacteriology, National Veterinary Institute, SE-751 89 Uppsala, Sweden

Corresponding author: Bölske G. Phone +46 18 67 40 00. Fax +46 18 30 91 62.

E-mail goran.bolske@sva.se

Keywords: IS900, PCR, specificity.

SUMMARY

The insertion sequence IS900 has been considered specific for Mycobacterium avium subsp.
paratuberculosis (Map) and therefore has been used as the target gene for diagnostic PCR of Map.
From a healthy dairy cow in the Swedish Paratuberculosis Control Program, we have isolated and
characterised a mycobacterium harbouring one copy of a sequence with 94 % identity to IS900 at the
nucleic acid level. The isolate was shown to be related to Mycobacterium cookii, as assessed by 16S
rRNA sequencing. Strong amplifications were obtained with several PCR primers described for
detection of IS900. This finding shows the need of alternative PCR systems based on other genes than
IS900 to confirm the presence of Map.

INTRODUCTION or its gene and the phylogeny of mycobacteria

Paratuberculosis, caused by
Mycobacterium avium subsp. paratuberculosis
(Map), is rare or absent in Sweden. The Swedish
Paratuberculosis control program and most of the
Swedish surveillances to monitor freedom from
disease are based on culture, either from faeces
or from lymph nodes and intestine from the
slaughterhouses. During the culture procedure,
suspected colonies are picked and identified as
Map by PCR.
The PCR methods generally used for
identification and detection of Map are based on
IS900, an insertion sequence considered specific
for Map (5, 16, 17, 22). The insertion sequence
IS900 is a 1451 bp segment that lacks inverted
terminal repeats and does not generate direct
repeats in target DNA (10). It belongs to the
same family of insertion sequences as IS901,
IS902, and IS1110, which have been described in
M. avium subsp. avium (Maa), M. avium subsp.
silvaticum (Mas), and Maa respectively.
PCR based on IS900 has been used to
identify the presence of Map in milk, faecal
specimens, and human intestinal tissue without
primary culture or in cases when Map has not
been possible to cultivate or isolate (11). The
IS900 PCR also is used in some control programs
for paratuberculosis as the sole method to
identify the presence of Map.
A definite method for identification of
mycobacteria is sequence analysis of 16S rRNA
based on this method has been extensively
studied (20). Sequence analysis of the 16S rRNA
gene has been particularly useful for
identification of mycobacteria because of the
difficulties with the conventional typing methods
for these bacteria (15).
We have isolated a mycobacterium
positive for IS900 by PCR, which by further
investigation proved not to be Map. The
phenotypic features of the isolated organism were
investigated and partial sequencing of the IS900-
like fragment was performed. The nucleotide
sequence of the 16S rRNA gene was determined
and the phylogenetic relationship to other
mycobacteria was established. Amplification
with primers targeting mycobacterial genes other
than IS900 was investigated as complementary
tests to confirm or exclude the presence of Map.
MATERIALS AND METHODS
A single colony, suspected to be Map,
was obtained from a faecal culture of a healthy
dairy cow in the Swedish Paratuberculosis
Control program. The isolate (strain 2333),
detected on modified Löwenstein-Jensen medium
with mycobactin (14), was picked for
microscopic examination of acid-fast rods after
Ziehl-Neelsen staining, PCR for IS900 with
primers p36/p11 (5) and subculture on media
with and without mycobactin. Strain 2333 has

261
 Proceedings of the Seventh International Colloquium on Paratuberculosis
been deposited at the Culture collection of
Institute Pasteur and given no. CIP 107487.
Map, strain ATCC19698, was cultured
on modified Löwenstein-Jensen medium with
mycobactin at 37 ºC for 8 weeks. Strain 2333
was cultured on modified Löwenstein-Jensen
medium at 37 ºC for 7 weeks and Mycobacterium
cookii ATCC49103 at 30 ºC for 6 weeks.
Genomic DNA was purified from the cultured
mycobacteria as described previously (6).
Growth of strain 2333 and M. cookii was
studied on modified Löwenstein-Jensen medium at
30 ºC, 37 ºC and 45 ºC for up to 10 weeks.
Pigmentation with and without light exposure was
studied after 2 and 4 weeks incubation of the
cultures. Well-developed colonies of strain 2333
were subjected to the Accuprobe “M. avium
complex culture identification test” (M. avium
subspp.- and M. intracellulare-specific, GenProbe,
San Diego, CA, USA).
Strain 2333 and strain ATCC 19698 were
subjected to restriction fragment length
polymorphism analysis (RFLP). Preparation of
genomic DNA and digestion by restriction
endonucleases BstE and Pst were performed
according to Pavlík et al. (18). The resulting
restriction fragments were separated in 0.8 % w/v
agarose by pulsed field electrophoresis (Chef-DR®
Biorad, Hercules, CA, USA) at 5.3 V/cm with a
linear ramping from 0.3 s to 10.0 s for 10 h, and
transferred to Hybond-N+ membrane by Southern
blotting. The labelling and detection system Gene
ImagesTM Alk Phos DirectTM (Amersham Pharmacia
Biotech, Uppsala, Sweden) was used in accordance
with the instructions of the manufacturer. The 413
bp PCR product obtained by amplification of Map
DNA with primer p90 and p91 (Table 1), was
labelled with alkaline phosphatase and used as a
probe. Over night hybridisation and primary
washing were performed at 55 ºC.
Table 1.PCR primers used in this study.
Primer pair Specificity
p36 / p11 IS900
p90 / p91 IS900
150 / 921 IS900
s204 / s749 IS900
s347 / s535 IS900
MP3/ MP4 IS900
mpf/ mpr IS900
Mav17A / Mav17B Genus
f57a/ f57b f57
myc 3/ myc 1 us-p34
593/sva-001a 16S rRNA
a. The sequence for primer sva-001 was as follows: 5’-RSPb-ACC TTG TTA CGA CTT CGT CCC AAT C-3’
b. RSP is reversed universal sequencing handle with the following sequence 5’- CAC AGG AAA CAG CTA TGA CC-3’
262
Strain 2333 was investigated by PCR
with primers specific for IS900 (1,5,16,17,22).
PCR was also performed with primers specific
for the f57 gene, and the p34 gene (2), and with
the genus-specific primers 17A/17B (9). All PCR
primer pairs used in the study are listed in Table
1. Amplifications were performed in 50 µl and
were undertaken in a PTC-200 Thermo Cycler
(MJ Research, Waltham, MA, USA) (7). The
PCR products were analysed by electrophoresis
in 2 % agarose gels in 1X TBE-buffer, stained in
ethidium bromide and visualised by UV-light
transillumination. The Real-Time PCR with
primers mpf/mpr was undertaken in a Rotor-
Gene 2000 (Corbett Research, Sydney,
Australia).
Primers p90 and MP4 were used to
amplify a 1043 bp region of the IS900-like
sequence of strain 2333 under the PCR
conditions mentioned for primer p90/p91 above.
The 16S rRNA gene was amplified with primer
593 (F) and sva-001(R) complementary to the
universal region U1 and U8, respectively. The
resulting PCR products were sequenced by using
the Thermo sequenase cycle sequencing kit and
fluorescently labelled IS900 primers and 16S
rRNA primers (12), respectively. The IS900-like
sequence and the 16S rRNA sequence of strain
2333 have been deposited in the GenBank under
the accession number AF455252 and AY065649,
respectively.
Pre-aligned mycobacterial 16S rRNA
sequences were retrieved from the Ribosomal
Database Project (RDP, URL:
http://rdp.cme.msu.edu/html/) or from GenBank.
The sequence of Mycobacterium sp. strain 2333
were then manually aligned with the RDP
sequences. The final alignment comprised 1350
nucleotide positions (7). Phylogenetic
Amplicon size Source
278 Moss et al. (17)
413 Millar et al. (16)
229 Vary et al. (22)
563 Englund et al. (5)
210 Englund et al. (5)
314 Bauerfeind et al. (1)
115 Willemsen et al.(23)
317 Fries et al. (9)
439 Coetsier et al. (2)
257 Coetsier et al. (2)
~1500 Johansson et al. (12)
 Proceedings of the Seventh International Colloquium on Paratuberculosis
calculations were performed by using the
neighbour-joining method, implemented in the
phylogenetic program package PHYLIP (13).
RESULTS
The primary growth of the isolate
appeared as a yellow colony on one slope of
mycobactin-containing Löwenstein-Jensen
medium on the final reading at 4 months. The
colony analysed by PCR with primers
p36/p11, was IS900 positive and the isolate
was, therefore, first identified as Map. After
Ziehl-Neelsen staining of the colony, acid-fast
rods could be seen. Most rods were
comparatively long and slender and some were
slightly bent. This appearance is not typical
for Map. At subculture, growth appeared after
4 weeks and the strain grew equally well on
media with and without mycobactin. These
findings indicated the isolate was not Map.
Further, the Accuprobe M. avium complex test
was negative, providing another indication
that the isolate was not Map.
On subculture, strain 2333 had a
strong yellow to orange pigmentation that was
scotochromogenic. Growth appeared more
vigorous at 37 °C than at 30 °C. No growth
was observed at 45 °C. Visible colonies
appeared after approximately 3 weeks. Strain
2333 differed from Map in growth rate,
pigmentation of colonies, and microscopic
morphology. It was similar to M. cookii,
except for apparently the temperature
optimum; strain 2333 grew best at 37 °C while
M. cookii grew best at 30 °C.
Amplification of genomic DNA of strain
2333 with the IS900 specific primers p90/p91,
p36/p11, 150/921, s204/s749, s347/s535,
MP3/MP4 and mpf/mpr, yielded one single PCR
product, with the respective primer pairs. The
resulting amplicons were of the same sizes as the
corresponding PCR products from amplified
Map DNA. The result indicated strain 2333 to
harbour the IS900 sequence and would,
therefore, if only based on the PCR results, be
judged as an Map isolate. Amplification with
primers for the f57 sequence, which to our
knowledge is specific for Map (2) did not yield
any amplicons with DNA from strain 2333 but
produced a 439 bp PCR product with Map DNA.
Likewise, amplification with primer myc3 and
myc1, targeting the us-p34 sequence in the M.
tuberculosis complex and the M. avium complex
(2) yielded a 257 bp product with Map control
DNA but not with DNA from strain 2333. The
results of PCR based on the f57 sequence and the
us-p34 sequence showed strain 2333 not to be
Map, any subspecies of M. avium, or M.
263
intracellulare. With the genus-specific primers
17A/17B [9] a weak banding pattern was
obtained for strain 2333 whereas one distinct
band was observed for Map. The result showed
genetic differences between the strains and gave
further evidence for the strain 2333 not to be
Map.
The number of IS900-like fragments in
strain 2333 was determined by digestion with
BstEII and PstI followed by Southern blotting
with an IS900 specific probe. One single band
was obtained with BstEII and PstI, respectively,
confirming the presence of a single IS900-like
fragment. The control DNA of Map showed the
banding pattern described by Pavlík et al. (19)
verifying the specificity of the probe and the
stringency of the hybridisation conditions used.
Sequence data of the amplified IS900-
like sequence were generated from the nucleotide
positions corresponding to 28 to 1071 of the
IS900 nucleotide sequence (GenBank accession
number X16293). There were 58 nucleotide
differences between the IS900 sequences of Map
and the IS900-like sequence of strain 2333,
giving an identity at the nucleic acid level of
94.4%. Within the first 450 bases, where most
PCR primers are positioned, only 6 bases
differed from IS900. One mismatch was found in
the sequence for primer p91 and MP3,
respectively, and two mismatches were found in
the s749 primer sequence. None of these
mismatches prevented amplification.
In a previous study of IS900-like
sequences in mycobacteria (3), the resulting
IS900 PCR products were subjected to restriction
endonuclease analysis to identify false positive
results. The restriction enzyme HaeΙΙΙ was used
to examine the PCR product obtained by the
truncated version of primers 150/921, and AlwΙ,
and MseΙ were used to assess the PCR product
generated by the primers p90/p91. DNA of strain
2333 amplified with the original primers 150/921
and p90/p91 yielded PCR products where the
restriction sites for HaeΙΙΙ, AlwΙ, and MseΙ, were
identical to the restrictions sites in amplified
DNA of Map. For that reason, restriction
endonuclease analysis could not be used to solve
the problem with false positives. Sequence
analysis of the PCR product remains the definite
method to confirm the identity of the IS900
sequence.
A preliminary phylogenetic analysis of
Mycobacterium sp. strain 2333 and 70
representative mycobacterial species and strains
retrieved from RDP showed in which
phylogenetic cluster this strain grouped. A
number of species were selected from this group
together with a few from the list obtained by
similarity searches with the 16S rRNA sequence
 Proceedings of the Seventh International Colloquium on Paratuberculosis

of strain 2333 in
GenBank. In the
final phylogenetic
analysis (Figure
1) it was found
that strain 2333
grouped with M.
cookii and
Mycobacterium
sp. strain IMVS
B76676 (Figure
1). The similarity
values for strain
2333 to M. cookii
and
Mycobacterium
sp. strain IMVS
B76676 were Figure 1. Evolutionary tree based on 16S rRNA sequences showing the phylogenetic relations of Mycobacterium sp. strain
98.3 % and 98.8 2333 to other mycobacteria. Nocardia asteroides was used as outgroup. The bootstrap percentage values from 1000 re-
samplings of the data set are given at each node. The length of the scale bar corresponds to 1 substitution per 100 nucleotide
%, respectively. positions. Strain designations are given for taxa, which have not been assigned species status.
The similarity
value to Map was (GenProb, San Diego, Ca), would have been
only 96.4%. better. The best methods for such a confirmation
A long or extended helix 18 is should be PCR systems directed against other
characteristic for slow-growing mycobacteria and Map specific genes. These methods, however,
strain 2333 possessed an extended helix 18. The have to be evaluated further regarding their
sequence of the hypervariable region B (nucleotide specificity and general occurrence in Map.
position 433-503 according to Escherichia coli
An alternative to detect one specific
numbering), which comprises helix 18, is very
gene by PCR is the use of PCR primers targeting
informative for mycobacteria (15) and it was found
general DNA sequences, which yields species-
to be unique for strain 2333. In total, there were 51
specific fingerprints. We have been able
nucleotide differences in the 16S rRNA genes of
successfully to distinguish Map from other
Map and strain 2333.
mycobacteria by using a novel fingerprinting
In a recent study (3) it was shown that a
system based on primers targeting the
number of field isolates of mycobacteria possess
enterobacterial repetitive intergenic consensus
IS900-like sequences. These isolates had
(ERIC) element and IS900 (4,8). This system
significant 16S rRNA sequence similarities with
offers a complement to IS900 PCR for
M. intracellulare, M. scrofulaceum, and
identification of Map.
Mycobacerium sp. strain IWGMT 90236. As can
Our finding of an IS900-like sequence
be seen from Fig. 1, strain 2333 is not very
in a Mycobacterium sp. unrelated to Map has of
closely related to these taxa and the sequence
course consequences for the reliability of a
similarity was 96.9% to all of them. Thus,
positive IS900 PCR, performed directly on
phylogenetic data clearly show that strain 2333
clinical specimens for detection of Map. It
should not be classified as Map and this strain
creates difficulties in cases where the microbe
probably represents a new mycobacterial species.
cannot be isolated for confirmation. It then could
be confirmed by another specific PCR method,
DISCUSSION targeting another gene in Map. The f57 gene,
which we have tested here and on some further
The identification of colonies as Map strains (8), seems promising. Another possible
with IS900 PCR has to be confirmed when it gene for a confirming PCR could be the ISMav2
affects the diagnosis of new cases or new (21), which however has to be evaluated further.
outbreaks of paratuberculosis. This is primarily Another way of confirmation could be to
performed with the classical phenotypic methods sequence the PCR product and see if there are
as described in this case report. However, by any nucleotide differences compared to IS900.
using molecular methods, a faster confirmation As there are very few differences in this case
can be achieved. We used a genetic probe compared to IS900 and for another IS900-like
specific for the M. avium complex, but a probe sequence there may not be any differences in the
with narrower specificity, M. avium species sequence between the primer pairs.

264
 Proceedings of the Seventh International Colloquium on Paratuberculosis
CONCLUSIONS
An identification of Map based on
IS900 PCR for diagnosis of new cases or
outbreaks should always be confirmed either by
isolation of Map or by a PCR method targeting
another gene in Map. The same should of course
also hold true for surveys with IS900 PCR
directly on diverse specimens, (like human
intestine or milk).
Some previous results with positive
PCR for IS900 from different types of specimens
might be questioned. Some of the positive results
might have been generated from IS900-like
sequences instead of true IS900 genes.
ACKNOWLEDGEMENT
We thank Anna-Lena Andersson for
technical assistance. This study was financially
supported by grants from the Swedish Council
for Forestry and Agricultural
Research/FORMAS. This study also forms part
of the EU research collaboration FAIR6-CT98-
4373 "Concerted Action for the Setting up of an
European Veterinary Network on Diagnosis,
Epidemiology and Research of Mycobacterial
Diseases" .
REFERENCES
1. Bauerfeind R, Benazzi S, Weiss R, Schliesser
T, Willems H and Baljer G. 1996. Molecular
characterization of Mycobacterium
paratuberculosis isolates from sheep, goats, and
cattle by hybridization with a DNA probe to
insertion element IS900. J. Clin. Microbiol. 34,
1617-1621.
2. Coetsier C, Vannuffel P, Blondeel N, Denef JF,
Cocito C and Gala J.L. 2000. Duplex PCR for
differential identification of Mycobacterium
bovis, M. avium, and M. avium subsp.
paratuberculosis in formalin- fixed paraffin-
embedded tissues from cattle. J Clin Microbiol.
38, 3048-54.
3. Cousins DV, Whittington R, Marsh I, Master
A, Evans RJ and Kluver P. 1999. Mycobacteria
distinct from Mycobacterium avium subsp.
paratuberculosis isolated from faeces of
ruminants posessIS900-like sequences detectable
by IS900 polymerase chain reaction: implications
for diagnosis. Mol. Cell. Probes 14, 431-442.
4. Englund S. 2002. Molecular Biology Techniques
as a Tool for detection and Characterization of
Mycobacterium avium subsp. paratuberculosis.
Ph.D. thesis. Swedish University of Agricultural
Sciences, Uppsala. ISBN 91-576-6366-1.
5. Englund S., Ballagi-Pordány A, Bölske G and
Johansson KE. 1999. Single PCR and nested
265
PCR with a mimic molecule for detection of
Mycobacterium avium subsp. paratuberculosis.
Diagn. Microbiol. Infect. Dis. 33, 163-171.
6. Englund S, Bölske G, Ballagi-Pordány A and
Johansson KE. 2001. Detection of
Mycobacterium avium subsp. paratuberculosis in
tissue samples by single, fluorescent and nested
PCR based on the IS900 gene. Vet Microbiol. 81,
257-71.
7. Englund S, Bölske G and Johansson KE. 2002.
An IS900-like sequence found in a
Mycobacterium sp, other that Mycobacterium
avium subsp. paratuberculosis. FEMS Microbiol.
Lett. 209, 261-265.
8. Englund S, Heldtander Königsson M and
Bölske G. 2002. Alternative PCR for
Mycobacterium paratuberculosis. In:
Proceedings of the Seventh International
Colloquium on Paratuberculosis. Abstract no. 38.
Bilbao, Spain.
9. Fries JWU, Patel RJ, Piessens WF and Wirth
DF. 1990. Genus- and species-specific DNA
probes to identify mycobacteria using the
polymerase chain reaction. Mol. Cell. Probes 4,
87-105.
10. Green EP, Tizard MLV, Moss MT, Thompson
J, Winterbourne DJ, McFadden JJ and
Hermon-Taylor J. 1989. Sequence and
characteristics of IS900, an insertion element
identified in human Crohn' s disease isolate of
Mycobacterium paratuberculosis. Nucleic Acids
Res. 17, 9063-9073.
11. Hermon-Taylor J, Bull TJ, Sheridan JM,
Cheng J, Stellakis ML and Sumar N. 2000.
Causation of Crohn' s disease by Mycobacterium
avium subspecies paratuberculosis. Can J
Gastroenterol. 14, 521-39.
12. Johansson KE, Heldtander MU and Pettersson
B. 1998. Characterization of mycoplasmas by
PCR and sequence analysis with universal 16S
rDNA primers. Methods Mol Biol. 104, 145-65.
13. Johansson KE, Tully JG, Bolske G and
Pettersson B. 1999. Mycoplasma cavipharyngis
and Mycoplasma fastidiosum, the closest relatives
to Eperythrozoon spp. and Haemobartonella spp.
FEMS Microbiol Lett. 174, 321-6.
14. Jørgensen JB. 1982. An improved medium for
culture of Mycobacterium paratuberculosis from
bovine faeces. Acta vet. scand. 23, 352-335.
15. Kirschner P and Bottger EC. 1998. Species
identification of mycobacteria using rDNA
sequencing. Methods Mol Biol. 101, 349-61.
16. Millar DS, Withey SJ, Tizard MLV, Ford JG
and Hermon-Taylor J. 1995. Solid-phase
hybridization capture of low-abundance target
DNA sequences: application to the polymerase
chain reaction detection of Mycobacterium
paratuberculosis and Mycobacterium avium
subsp. silvaticum. Anal. Biochem. 226, 325-330.
17. Moss MT, Green EP, Tizard ML, Malik ZP
and Hermon-Taylor J. 1991. Specific detection
of Mycobacterium paratuberculosis by DNA
 Proceedings of the Seventh International Colloquium on Paratuberculosis

hybridization with a fragment of the insertion ISMav2, a novel insertion sequence-like element
element IS900. Gut. 32, 395-398. from Mycobacterium avium subspecies
18. Pavlík I, Bejcková M, Rozsypalová Z and paratuberculosis. FEMS Microbiol Lett. 196, 31-
Kosková S. 1995. Characterization by restriction 7.
endonuclease analysis and DNA hybridization 22. Vary PH, Andersen PR, Green E, Herman-
using IS900 of bovine, ovine, caprine and human Taylor J and McFadden JJ. 1990. Use of highly
dependent strains of Mycobacterium specific DNA probes and the polymerase chain
paratuberculosis isolated in various localities. reaction to detect Mycobacterium
Vet. Microbiol. 45, 311-318. paratuberculosis in Johne' s disease. J. Clin.
19. Pavlik I, Horvathova A, Dvorska L, Bartl J, Microbiol. 28, 933-937.
Svastova P, du Maine R and Rychlik I. 1999. 23. Willemsen PTJ, Bakker D, Damman M, Eger
Standardisation of restriction fragment length A and van Zijderveld FG. 1999. An errror in the
polymorphism analysis for Mycobacterium avium reported IS900 nucleotide sequence affects the
subsp. paratuberculosis. J Microbiol Methods. proposed expression of ORF2 (Hed) and the
38, 155-167. detection of M. a paratuberculosis using the
20. Pitulle C, Dorsch M, Kazda J, Wolters J and polymerase chain reaction. In: Proceedings of the
Stackebrandt E. 1992. Phylogeny of rapidly Sixth International Colloquium on
growing members of the genus Mycobacterium. Paratuberculosis. pp 466-471. Melbourne,
Int. J. Syst. Bacteriol. 42, 337-343. Australia.
21. Strommenger B, Stevenson K and Gerlach GF.
2001. Isolation and diagnostic potential of

266