Note: Descriptions are shown in the official language in which they were submitted.
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Title: Detection of Mycobacterium avium subspecies.
The invention relates to the field of microbiology,
more specifically to the field of detection and
identification of pathogenic micro-organisms, more
specifically to the detection and identification of
Mycobacterium avium subspecies paratuberculosis, an agent
which causes Johne's disease in many ruminants.
Mycobacteria are aerobic, Gram-positive, acid-fast
rod-shaped bacteria (0.2-0.6 x 1.0-10 um). The genus as a
whole is characterised by long-chain mycolic acids in the
cell-wall. Large amounts of lipids in the cell-wall result in
remarkable resistance to de-staining of stained cells, which
forms the basis of the Ziehl-Neelsen staining used to
identify Mycobacteria. In general, Mycobacteria are slow-
growing organisms. Mycobacterium avium subspecies
paratuberculosis is ranked as an extremely slow growing
organism. Many attempts have been made to improve the
cultivation conditions. Further possibilities to
significantly improve the growth rate of the bacterium in
vitro are virtually absent.
The taxonomic position of Mycobacterium avium
subspecies paratuberculosis is defined on 16S ribosomal RNA
sequence data (1). The bacterium is a member of the
Mycobacterium avium complex (MAC). This complex is composed
of Mycobacterium avium, represented by the three subspecies
avium, silvaticum and paratuberculosis, and of Mycobacterium
intracellulare (2).
Mycobacterium av.i.um subspecies paratuberculosis is,
among others, characterised from they other bacteria in this
complex by its requirement for mycobactin in the growth
medium (3).
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Paratuberculosis or Johne's disease, is a world-wide
occuring disease caused by the bacterium Mycobacterium avium
subspecies paratuberculosis. An estimated 30-400 of the
cattle farms in The Netherlands is affected by this disease
and approximately 20 of all cows in The Netherlands is
infected with this bacterium. The total damage as a result of
the,disease (loss of milk production., mortality and
restrictions in trade) is estimated at 45 million Dutch
guilders per annum with a damage of 50 to 300 guilders per
cow per annum on the affected farms. At this moment, infected
animals generally are, once detected, culled or removed from
the farm and generally destroyed.
Johne's disease is thought to be related to the
inflammatory bowel syndrome in humans, also known as Crohn's
disease. If the causal agent of John.e's disaese is indeed
also a casual of inflammatory bowel syndrome in humans, the
disease may well be placed on the list of animal to human
transmissible diseases or zoonoses.
In the mid-seventies a campaign was launched to
eradicate the disease. This campaign was not successful
mainly due to limited diagnostic possibilities. New attempts
are initiated in which the efforts a.re directed towards
identification, certification 'and canalisation of
paratuberculosis-unsuspected farms, and detection and
isolation of affected cattle and eradication of
paratuberculosis on infected farms. In the latter programme
both management measures and diagnosis play an important
role. A solid diagnostic test for the detection of
Mycobacterium avium subspecies parat:uberculosis will play a
pivotal role in such a campaign.
The detection of cattle infected with Mycobacterium
avium subspecies paratuberculosis is hampered by two
important factors: i) The clinical symptoms of illness are
first detectable after an incubation period of four to five
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years. Due to the slow progression of the infection and slow
development of an immune response, 'testing before the second
year in the life of a cow is therefore in general not even
considered useful. ii) The bacterium itself propagates
extremely slow as a result of which the only currently
reliable test to identify the bacterium - a conventional
cultivation of faecal samples - requires up to 6 months.
Furthermore, testing only once does not suffice. A minimal
sampling rate of two times per year per farm is necessary to
ensure effectiveness of the eradication program. In current
methods, 4 separate cultures per faecal sample are monitored
for growth over a period of up to half a year. This results
in significant accumulation of cultures. Large-scale
cultivation testing programs are therefore hampered by
problems of capacity. Moreover, culi~ivation methods are
frustrated by problems of cantaminai~ion with fungi and/or
Bacillus species. The Netherlands a:Lone harbours 30.000
cattle farms with a total of approx:Lmately 800.000 cows. If
every cow in The Netherlands were to be tested by cultivation
methods several millions of faecal aample cultures would have
to be stored for up to half a year <~t 37° C.
Recent years have seen various attempts to develop
alternative arid more rapid methods of detection.
Unfortunately, present alternative methods are considered
unsuitable for large scale testing programs. Most
contemporary diagnostic tests are s:imply not sensitive enough
to reliably detect Mycobacterium av_ium subspecies
paratuberculosis in faecal samples, also serological methods,
albeit suitable for large scale screening programmes are nor
sensitive enough.
Currently, there are at leant 8 different diagnostic
tests for the detection/demonstration of infection of
Mycobacterium av.ium subspecies paratuberculosis. Three of
these are direct methods for the dei~ection of Mycobacterium
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avium subspecies paratuberculosis cells. The other five are
tests that demonstrate the presence of an immune response by
the host-animal. In general, direct methods of detection are
considered superior with respect to reliability.
The three tests used for direct detection of the
bacterium are i) conventional cultivation (4), ii) BACTEC
(radiometrical or fluorimetrical) cultivation (5) and iii)
DNA probe testing (PCR) (6,7,8). The detection limit for
conventional cultivation is approximately 100 bacteria per
gram of faeces. As a result, conventional cultivation is
still one of the most sensitive methods of detection. The
BACTEC system for the detection of Mycobacterium avium
subspecies paratuberculosis is a revision of the BACTEC
system for the detection of M. tuberculosis in humans (Becton
Dickinson Diagnostics) that is used in general hospitals. For
paratuberculosis testing, the BACTEC growth medium is
supplemented with specific components to allow growth of
Mycobacterium avium subspecies paratubercu.Iosis in the
cultivation tubes. The method is more rapid (7 weeks) and
mare sensitive than conventional cultivation, but is also
much more expensive. In order to apply this method on a large
scale (1.000 samples/day) approximately 300 BACTEC incubators
are required. This makes BACTEC cultivation, be it
radiometric or fluorometric, not feasible.
The 5 tests that are used to detect an immune
response against the bacterium Myb. avium subsp.
paratuberculosis are i) the complement fixation (CF) test,
ii) agar gel immunodiffusion (AGID) test (specific for
sheep), iii) ELISA test on blood-serum (USDA-licensed), iv)
ELISA an milk, v) gamma interferon test (USDA-licensed).
Immunological tests (among which are the serological
and cellular immune-mediated assays) for the detection of
paratuberculosis are generally not considered satisfactory
with respect to sensitivity and specificity, especially in
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the case of diagnosis in sub-clinically infected animals.
Often in serological tests, false-positive results are found
due to cross-reactivity with similar antigens in other
mycobacteria or related organisms. False-negative reactions
5 occur in the case of so-called "serological non-responders";
animals in the terminal stage of the disease are often
anergic. Also, antibody responses develop very slowly. Before
three years of age, a very limited percentage of infected
animals develop a humoral immune response, and some do not
develop an immune response at all.
The Idexx Laboratories ELISA test on blood serum
exhibits a specificity (percentage animals that is free of
infection and tests negative) of 99o and a relative
sensitivity (percentage of M. paratubercu.Iosis-infected
animals that tests positive) of 45% in "sub-clinically"
infected animals (in this case cultivation results are taken
as 1000). In the clinical stage of infection the relative
sensitivity increases to approximately 850. Only animals of
age 20 months and older pan be tested reliably for
paratuberculosis with this test. In younger animals the test
is too insensitive unless the animals show clinical signs of
infection. The interpretation of the test results is
quantitative (increase in optical density in the ELISA} and
is proportional to the antibody density in the animal's
blood. High scores are strongly indicative of infection and
possible shedding of bacteria in faeces and milk. A
confirmation test (re-testing) must occur within 6 to 12
weeks. The test can, however, not be applied to animals that
have received vaccination against Johne's disease as this
results in false-positive test results. The system is
suitable for large scale testing but cannot be considered as
an indisputable evidence of infection.
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The detection of gamma interferon is a cellular
immune response assay. The method for determination of
interferon release by white blood-cells as a result of
Johnin-PPD stimulation is availablE: as a diagnostic kit
licensed by the US Department of Agrriculture (USDA). Since
the release of interferon precedes the formation of
antibodies during pathogenesis of paratuberculosis, infection
can be detected at an earlier stage compared to other
immunological tests. However, the sampled blood that is
treated with heparin to inhibit blood coagulation must be
handled with care and tested within. 12 hours after
collection. Therefore, the interferon-assay is only performed
on appointment and careful co-ordination between veterinary
practitioner and testing laboratory is required. As a result,
Z5 the test is not suited for large-scale testing.
The use of nucleic acid probe-tests for the detection
of Myb avium subsp. paratuberculosis has, albeit at first
sight a possible alternative for conventional cultivation,
proven to be practically'quite unfeasible. For one, this is
due to the extreme nucleic acid homologies that exist between
Myb aviurn subsp. paratuberculosis a.nd other Myb avium
subspecies, which are commonly found in samples such as
feacal samples of ruminants (McFadd.en, J.J., et al. 1987b. J.
Gen. Microbiol. 133:211-213; Saxega.ard, F., and I. Baess.
1988. Acta Pathol. Microbiol. Immun.ol. Stand. 96:37-42;
Yoshimura, H.H., and D.Y. Graham. 19$8. J. Clin. Microbiol.
26:1309-1312). For another reason, this is due to the low
effective level of detectable nucleic acid due both to the
extreme low cell densities of the target organism, the
presence of a large excess of non-target organisms, as well
as inhibition of enzymatic nucleic acid amplification
reactions by inhibitory substances in the faecal matrix (Van
der Giessen, J.W.B., et al. 1992. J'. Clin. Microbiol.
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30(5):1216-1219; Millar, D.S., et a.1.1995. Anal. Biochem.
226(2):325-330).
In general, nucleic acid-based tests can be directed
towards {genomic) DNA or its RNA transcript. Two problems
with using genomic DNA as target in. DNA amplification methods
for diagnostic purpose in general a.re the low copy number of
target molecules per cell and the relative unpredictability
of natural sequence variability within the total population
of target organisms. More or less related organisms may
suddenly show to possess very similar or homologous DNA
sequences (Kunze, Z.M., et al. 1991. Mol. Microbiol.
5(9):2265-2272; Moss, M.T., et al. 1992. J. Gen. Microbiol.
138:139-145). Such unpredictability limits the reliability
through the specificity. 'The specificity of a nucleic acid
based testing.system is determined by the presence of the
specific nucleic acid target sequence for hybridisation or
the proper identification of the target sites by the probes
or primers. The specificity of the test is therefore
determined by the genetic variation. in the target sequences
over the total population of the bacterium to be detected.
Knowledge about this variation is therefore important.
Mycobacteria are difficult to differentiate on the
basis of their genetic makeup due to the presence of
exceptional sequence conservation among species (Frothingham,
R., et al. 1994. J: Clin. Microbiol. 32(7):1639-1643). A
number of methodologies have been developed to accomplish the
distinction between the various species and subspecies.
A DNA probe test for the detection of Myb. avium
subsp. paratuberculosis is in one ease based on the presence
of an insertion sequence in the gen.ome of Myb. avium subsp.
paratuberculosis thought to be specific for this subspecies,
the so-called IS900 sequence (McFadlden, J.J., et al. 1987a.
Mol. Microbiol. 1:283-291; Vary, P.H., et al. 1990. J. Clin.
Microbiol. 28(5):933-937). For the detection of this
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paratubercu~osis insertion element, which, when present, is
present in 15-20 copies on the genome, a PCR reaction is
applied. However, the IS900 sequence is not present in all
strains of Myb. avium subsp. paratu,berculosis due to the
variation of genomic DNA as describ~ad above. Several studies
report on the absence of IS900 sequences in between 3 and 20%
of the clinical isolates of Mycobacterium paratuberculosis
obtained from animals with classical Johne's disease
(Thoresen, O.F., and I. Olsaker. 1994. Vet. Microbiol.
40:293-303; Bauerfeind, R., et al. 1996. J.Clin. Microbiol.
34(7):1617-1621). Consequently, a ts~st based on IS900 testing
may bear the risk of false-negative results, thereby missing
cows that should have been culled o:r destroyed. In 1992, Moss
et al. demonstrated the presence of IS900-like elements
(IS902) in the "wood pigeon strain" Mycobacterium avium
subsp. silvaticum (Moss, M.T., et a:L. 1992. J. Gen.
Microbiol. 138:139-145) which is closely related to
Mycobacterium avium subsp. paratube:~culosis. Other reports
have confirmed the presence of such homologous genetic
elements in Mycobacterium avium subspecies other than
paratuberculosis (Kunze, Z:M., et a:L. 1991. Mol. Microbiol.
5(9):2265-2272; Kunimoto, D., et al.. 1994. Am. Soc.
Microbiol. 9:182: Roiz, M.P., et al. 1995. J. Clin.
Microbiol. 33:2389-1391). More recently, while studying the
etiology of the lung disease sarcoidosis, E1-Zaatari et al.
discovered a bacterium belonging to the Mycobacterium avium
complex that failed to hybridise wii~h a silvaticum-specific
probe but was shown to contain the IS900 sequence, or a
closely related sequence, as inferrESd from IS900 PCR (E1-
Zaatari, F.A.K., et al. 1997. Scand,. J. Infect. Dis. 29:202-
204). According to test result for i~he IS900 sequences, the
bacterium should be identified as M~~cobacterium avium subsp.
paratuberculosis. The possible socio-economic consequences of
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such findings, however, demand careful interpretation and the
authors are accurate when accepting the possibilities of
having detected a IS900 homologue or an organism of the avium
complex not formerly known to possess the IS900 sequence.
Clearly, results of the IS900 test are, although extremely
useful in many cases, not unequivocally interpretable. In
fact, careful selection of PCR primers is essential to
discriminate between IS900 (Vary, P.H., et al. 1990. J. Clin.
Microbiol. 28(5):933-937), IS901 (Kunze, Z.M., et al. 1991.
Mol. Microbiol: 5(9):2265-2272) and IS902 (Moss, M.T., et al.
1992. J. Gen. Microbiol. 138:139-145) insertion elements and
IS900 hybridisation probes should b~e employed at utmost
stringency to evade the possibilities of erroneously
detecting homologous, but dissimilar genetic elements.
Consequently, a test based on IS900 testing may also bear the
risk of false-positive results, thereby marking cows as
infected that are perfectly healthy.
A further great limitation of this IS900 test is that
the PCR reaction itself '~.s inhibited very strongly by
substances that are naturally prese:rlt in faeces (Van der
Giessen, J.W.B., et al. 1992. J. Cl.in. Microbioi. 30(5):1216-
1219). This inhibitory effect limits the use of PCR for the
detection of paratuberculos.is in faeces based on the genomic
DNA targets, such as the IS900 sequence. To circumvent this
problem, dilution (1Ox) of the faeces sample prior to IS900
PCR testing can be applied. This will, to a limited extend
reduce PCR inhibition (Widjojoatmodjo, M.N., et al. 1992. J
Clin. Microbiol. 30:3195-3199 Vare:la, P., et al. 1994. J.
Clin. Microbiol. 32:1240-1248). However, it also greatly
reduces the sensitivity of the test. In recent years many
improvements to the original protocol for PCR detection of
the IS900 sequence have been made. ~~nong these are so-called
"hot start" PCR protocols to inacti~crate possible inhibitors,
as well as protocols based on bead beating with zirconium
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beads in combination with direct zirconium adsorption and
elution (Van der Giessen, J.W.B. 1993. Academic thesis,
Utrecht University, The Netherlands). Despite these
improvements, the PCR reaction for the direct detection of
5 Myb. avium subsp. paratuberculosis in faeces is still not as
sensitive as cultivation methods. Typically, only 10-30% of
the samples that are considered positive by cultivation are
diagnosed positive when direct IS900 PCR is used (Van der
Giessen, J.W.B., et al. 1992. J. Clin. Microbiol. 30(5):1216-
10 1219). The combined application of BACTEC pre-cultivation and
subsequent culture confirmation by IS900 PCR can reach
sensitivities comparable to conventional cultivation, and
such methods are frequently used (Evans, K.D., et al. 1992.
J. Clin. Microbiol. 30:2427-2431; Sockett, D.C., et al. 1992.
Can. J. Vet. Res. 56(2):148-153; Cousins, D.V., et al. 1995.
Aust. Vet. J. 72(12):458-462; Whittington, R.J., et al. 1998.
J. Clin. Microbiol.'36(3):701-707). Although this will reduce
the time in which paratuberculosis can be diagnosed to about
1 week, it will not circumvent the problem of the culture
logistics in large scale testing programs described earlier.
To further improve the sensitivity of direct IS900 testing,
samples may be "purified" by using immunomagnetic beads that
can specifically "pull" the Myb. av.i.um (subsp.
paratuberculosis) cells (Grant, I.R., et al. 1998. Appl.
Environ. Microbiol. 64(9):3153-3158) or DNA (Millar, D.S., et
a1.1995. Anal. Biochem. 226(2):325-330) from the sample that
is under investigation. Such methods have been developed for
paratuberculosis and the procedures for this can be
automated.
The eminent lack of accurate, rapid and reliable
tests for differentiation of the various Mycobacterium avi.um
complex bacteria and particularly paratuberculosis has
resulted in the development of a series of alternative DNA
probe tests. Among these are the one by Poupart et al.
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(Poupart, P., et al. 1993. J. Clin. Microbiol. 31(6):1601-
1005) in which a paratuberculosis-specific sequence is used
that was identified by screening a ~genomic library of
paratuberculosis in a transcription vector. Such a unique
sequence may be used as an RNA probe. Gormley et al.
(Gormley, E., et al. 1997. FEMS Microbiol. Lett. 147(1):63-
68) identified the use of restriction fragment length
polymorphisms in the PAN promotor region of pathogenic
mycobacteria as a valuable tool in differentiation of
paratuberculosis. More recently, Ellingson et al. (Ellingson,
J.L., et al. 1998. Mol. Cell. Probes 12(3):133-142) developed
a so-called di-oligonucleotide hybridization (dOH) assay for
the simultaneous detection of a Mycobacterium genus-specific
recA gene sequence and a paratuberculosis-specific 30 by hspX
sequence. All of these alternative tests are based on genomic
targets. An important drawback of these genomic methods,
however, is related to the low copy number at which these
genes or gene sequences occur in the cell. This limits the
sensitivity of such tests.
To facilitate large scale screening testing for
Johne's disease with the purpose of eradication several
conditions must be met. The test system must be specific for
Mycobacterium avium subspecies paratuberculosis and must
therefore be able to make a distinction between this
subspecies and the other subspecies of the Mycobacterium
avium complex that do not cause Johne's disease. Secondly,
the specificity of the test (no false-positives are wanted
due to the fact that positive cows are all destroyed) must be
close to a 100% (relative to conventional cultivation).
Thirdly, the sensitivity (no false-negatives) of the system
must approach that of conventional cultivation methods,
otherwise an eradication programme iaill not be successful. A
sensitivity of 100 cells per gram o:f faeces must at least be
reached. Although the IS900 probe test exhibits such
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sensitivities under artificial circumstances when tested on
M. paratuberculosis-spiked culture-negative DNA samples (Vary
et al. 1990. J. Clin. Microbiol. 28:933-937), in direct
detection applications in crude bovine manure detection
limits lower than 10,000-100,000 cells per gram of faeces are
rarely met (Vary et al. 1990. J. Clin. Microbiol. 28:933-937;
Van der Giessen et al. 1992. J. Clin. Microbiol. 30: 1226-
1219). Fourthly, the throughput--rate of the process must be
one working day (the total analysis does not necessarily be
completed within one day; a certain accumulation of samples
can be met). Fifthly, the assay must allow automation.
Finally, the system must reach capacities of approximately
1,000 analysis results per day.
Further conditions must be 3net in the case of high-
throughput screening with respect to reliability of test
results. Every method of analysis and specifically high-
throughput screening requires an analysis of reliability. On
the one hand, the reliability is determined by the chance of
acquiring false-negative'v'test results. This chance is
expressed as sensitivity. On the other hand, chances exist on
acquiring false-positive test resultst this chance is
expressed in the specificity. Although DNA target
amplification technologies such as such as PCR (Mullis et al.
1987. US patent 4,683,195), TMA (Enns, R.K. 1987. Gen-Probe,
Inc., San Diego, Calif.; Jonas, V., et al. 1993. J. Clin.
Microbiol. 31(9):2410-2416) or NASB.A (Compton, J. 1991.
Nature (London) 350;91-92), are extremely sensitive, a
negative test result does not necessarily preclude the
possibilities of isolating Myb. avium subsp. paratuberculosis
from the sample material. The test result is influenced by
method of sampling, and transport conditions, variability in
the sampling process, laboratory procedural errors, sample
miss-identification and, most importantly transcriptional
errors. Furthermore, these target amplification methods do
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not present the possibilities to verify the test result. Such
problems could be circumvented by using the technology of
fluorescence in situ hybridization (FISH) in which whole
bacterial cells are fluorescently stained with sequence-
s specific nucleic acid probes. FISH has as a unique advantage
that cells are individually visible. However, the direct
detection of very low numbers of intact cells of Myb. avium
subsp. paratuberculosis in faecal samples is hampered by the
high background population ~ ( 10~°-101 per gram) of other
faecal micro-organisms.
In conclusion, immunologica.l tests do not present
indisputable evidence of infection with the bacterium. The
sole proof is formed by demonstrating the presence of the
bacterium Myb. avium subsp. paratuberculosis in milk, blood
or faeces or other relevant samples. For large-scale
analysis, cultivation methods are unsuited. The only options
for large scale screening lie in the application of
diagnostic methods, which rely on the demonstration of
genetic material of the bacterium hlyb. avium subsp.
paratubercuZosis. However, the diagnosis of the disease,
i.e., the reliable demonstration of bacterial nucleic acid,
is among other things, hampered by the enormous similarity
between this subspecies and for example Mycobacterium av.ium
subspecies avium. Mycobacterium avium subspecies avzum is
prevalent as a normal commensal bacterium in birds, and is
relatively widespread. The currently available most sensitive
nucleic acid test is the demonstration of the presence of a
specific insertion element, IS900, present in the genome of
the subspecies paratuberculosis. However, the sensitivity of
this test is too low to allow direct detection of the
bacterium in faecal samples or other natural habitats of the
bacterium that contain substances inhibitory to enzymatic
nucleic acid amplification reactions. Furthermore, as
explained above, the IS900 sequence is sometimes absent in
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the genome of Mycobacterium avium subspecies
paratuberculosis, thereby creating the possibility of a
false-negative diagnosis and the IS900 sequence can sometimes
be found in the genome of other Mycobacterium avium
subspecies, thereby creating the risk of a false-positive
diagnosis.
The invention provides a method by which a rapid and
sensitive procedure for the detection and identification of
Mycobacterium avium subspecies paratubercuLosis in biological
and natural samples is achieved, whereby Mycobacterium avium
subspecies paratuberculosis can be discriminated from other
Mycobacterium avium subspecies, such as subspecies avium. The
invention provides a nucleic acid probe or primer allowing
detecting nucleic acid derived from Mycobacterium avium
whereby nucleic acid derived from N!ycobacterium avium
subspecies paratuberculosis can be discriminated from nucleic
acid derived from other Mycobacterium avium subspecies. In
particular, the invention provides a nucleic acid probe or
primer allowing detecting nucleic acid derived from
Mycobacterium avium whereby Mycobacterium avium subspecies
paratuberculosis can be discriminated from other
Mycobacterium avzum subspecies, said nucleic acid comprising
a stable and conserved mutation specific for Mycobacterium
avium subspecies paratuberculosis.
The invention provides a method for specifically
detecting nucleic acid derived from a causal agent of Johne's
disease in a sample whereby nucleic acid from Mycobacterium
avium subspecies paratuberculosis is discriminated from
nucleic acid from other Mycobacterium avium subspecies
comprising detecting in said nucleic acid a mutation
specifically conserved for Mycobacterium avium subspecies
paratuberculosis. Said mutation is specifically conserved
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throughout Nlyco~acterium avium subspecies paratuberculosis,
as opposed to the IS900 insertion sequence, which is
sometimes missing or is found in other subspecies.
In a preferred embodiment, the invention provides a
5 method wherein said nucleic acid is derived from 23S
ribosomal RNA, for example wherein said ribosomal RNA
comprises a nucleic acid as shown in figure 1, or wherein
said conserved mutation is located at position 754, 1363 or
3093 as shown in figure 2. The invention provides three
10 conserved mutations that are present in the functional
paratuberculosis 23S rRNA gene sequence. These can be used as
diagnostic targets to distinguish between the
paratuberculosis and avium subspecies. As such they are part
of the present invention in which they are referred to as
15 mutations 754, 1363 and 3093 (figure 2).
Of all genes in the genome, the ribosomal RNA genes
belong to the most stable - or least variant. Due to their
role as key elements in protein synthesis, ribosomal RNA's,
and consequently the gems encoding' them, are highly
conserved both in structure and seguence (Pace, N.R., et al.
1985. ASM News 51:4-12). The ribosomal RNA genes comprise a
mosaic of variable regions, which allow for discrimination
between lower taxa, alternated by ~:equences that are well
conserved, thus allowing for differentiation between higher
order taxa (Woese, C.R. 1987. Microbiol. Rev. 51:221-271;
Olsen, G.J., et al. 1986. Ann. Rev. Microbiol. 40:337-365).
Due to the high copy number of mature ribosomal RNA
molecules, as a part of the cellular ribosomes, and their
predictable, evolutionary regulated, sequence variability,
ribosomal RNA's have become a well established tool in the
identification of bacteria. However, among all bacteria, the
mycobacteria possess exceptional sE:quence conservation among
species (Frothingham, R., et al. 1994. J. Clin. Microbiol.
32(7):1639-1643). And the ribosomal RNA's do not represent
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feasible targets to differentiate between subspecies. Van der
Giessen et al. (Van der Giessen, J.W.B., et al. 1992. J. Med.
Microbiol. 36:255-263) attempted to developed a PCR test for
Myb. avium subsp. paratuberculosis based on the 16S ribosomal
RNA gene. Although several sequence differences between the
various paratuberculosis strains and avium strains were
found, the sequence comparisons between the 16S rRNA gene of
paratuberculos.is and its most closely related subspecies
avium did not reveal a single stable base in difference that
could be used as a distinction between paratuberculosis and
avium. The 16S ribosomal RNA test thus detected both
paratuberculosis and avium together and exhibited a
sensitivity that was between one and two orders of magnitude
lower than the IS900 test (Van der Giessen, J.W.B., et al.
1992. J. Clin. Microbiol. 30(5):1216-1219). This may be due
to the difference in copy number between the ribosomal RNA
operon (max 1-3 copies per genome) and the IS900 sequence
(15-20 copies). Although the presence of strain-specific
sequence differences, based on the comparison of gene
sequences from two separate isolatea (Van Der Giessen et al.
1994. Microbiology 140:1103-1108), may sometimes mistakenly
be interpreted as to yield diagnostically valuable
differentiation criteria for closely related species, they do
not provide reliable subspecies-level sequence information
and may not be used as such.
In a follow-up study, Van der Giessen et al.
performed a sequence comparison of the 23S rRNA genes of one
strain of paratuberculosis and one strain of avium (Van der
Giessen, J.W.B., et al. 1994. Microbiology (UK) 140:1103-
1108). The presence of 9 base differences between the two
sequences (3000 nucleotides each) was reported and the
possibilities to differentiate the two strains on the bases
of these differences was proposed. Also, Stone et al. (Stone,
B.B., et al. 1995. Int. J. System Eiacteriol. 45:811-819)
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performed a partial sequence analysis (226 bases) of the 23S
rRNA of a large number of different Mycobacteria, among which
were one avium and one paratuberculosis strain. Their results
indicated the presence of even more sequence differences
between the 23S rRNA gene sequence of their avium and
paratuberculosis strains. Among the 226 nucleotides for which
the sequence was determined, no less than 4 differences were
reported between the two strains. However, both the study of
Stone et al. and Van der Giessen et al. could not demonstrate
the presence of stable and conserved discriminatory
signatures or mutations in the paratuberculos.is 23S rRNA gene
compared to the av.ium gene that could be used to
differentiate paratuberculosis from avium.
The natural genetic variability of bacterial cells of
the same species but of different clonal origin within a
consortium or from geographically separated populations is
not well understood. Frequently, lack of knowledge in this
domain results in genetic tests that produce a prodigality of
false negative results d~.e to the fact that many field
isolates do not contain the presumed distinctive sequence
characteristics.
Development of a diagnostic test for paratuberculosis
on the basis of the diagnostic targets proposed by Van der
Giessen et al. would have resulted in a diagnostic test
system with a high rate of false negative test results due to
the proposition of target- or signature sequences that are
not conserved in paratuberculosis strains of different clonal
origin. No less than six of the nine mutations suggested by
Van der Giessen et al. can not be used to make a distinction
between avium and paratuberculosis cells since these
mutations are not conserved. These six mutations do not occur
in the type strain of paratuberculosis (ATCC 19698}. These
mutations are sequence positions in the 23S rRNA gene of the
investigated strains that represent; random mutations or
natural sequence variability in so far that these mutations
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do not exhibit evolutionary or phylogenetic significance and
for that reason they can not be used for distinctive testing.
More such mutations can be found by inter-strain sequence
comparison. However, they serve no diagnostic purpose. Such
mutations mask the subspecies-specific mutations that are
significant to subspecies-specific diagnostics of
paratuberculosis.
The present invention reveals three point mutations
as potential subspecies-specific target-sites that are
conserved in all of the 25 investigated reference strains and
field isolates of paratubercu~osis. Two of these mutations,
situated at positions 754 and 1363, are specifically proposed
for combined use in a diagnostic test for the subspecies
paratuberculosis as..these targets are situated in relatively
close proximity of one another.
Van der Giessen et al. suggested the presence of a
total of 9 mutations or 'mismatches' between the 23S rRNA
gene sequences of avium and paratuberculosis. They identified
two additional differences between the obtained sequences but
these were positioned im~tediately next to one another in a
non-transcribed spacer region and can therefore not be used
for sensitive detection as they are non transcribed a.n
functional ribosomal RNA. The conserved status of these
'mismatches' remains presently unknown. The nine 'mismatches'
in the 23S rRNA gene that were identified by Van der Giessen
et al. were proposed as potential diagnostic targets. The
present invention however reveals that only three conserved
mutations are present in the functional paratuberculosis 23S
rRNA gene sequence. These can be used as diagnostic targets
to distinguish between the paratube:rculosis and avium
subspecies. As such they are part of the present invention in
which they, are referred to as mutations 754, 1363 and 3093
(figure 2). The six remaining targeas suggested by Van der
Giessen et al. are not conserved (positions 1746,1747, 1843,
2718, 2810, and 3126 respectively i.n figure 2). A final
mutation identified through the present study is displayed in
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figure 2 as position 3188. This mut:ation is part of the
internal transcribed spacer region between the 23S and 5S
rRNA genes and can not be used for sensitive detection. Its
conserved status was recently conf~'_rmed by Scheibl and
Gerlach (Vet. Microbiol. (1997) 57:;151-158).
In all, over 60 difference~> between an individual
avium and an individual paratuberculosis strain can be
identified in the alignment present:ed in figure 2, of which
only three can be used for. subspecies-specific diagnostics
and are part of the present invention. This illustrates that
the present invention reveals parat:uberculosis targets not
identified before.
Differences in the ribosomal RNA genes are in general
not considered feasible to serve a:~ a basis for
differentiation between closely re~_ated species or
subspecies. In order to distinguish different species of the
genus Mycobacterium, the internal transcribed spacer (ITS)
region between the 16S and 23S rRNA genes that is not
transcribed into functional RNA ha:> recently gained more
attention (Frothingham. fit, et al. 1993. J. Bacteriol.
175(10):2818-2825; Glennon, M., et al. 1994. Tuber. Lung Dis.
75(5):353-360; Frothingham, R, and K.H. Wilsan. 1994. J.
Infect. Dis. 169(2):305-312.; Ji, Y.E., et al. 1994.
Microbiology. 140(Pt7):1763-1773). Due to the absence of
evolutionary consequences of mutations in the ITS region,
mutations occur more frequently in this non-transcribed
spacer than in the functional rRNA genes themselves. One
problem with such mutations is therefore that they are riot
fixed during evolution and that thE:y may even differ between
strains of the same species. For da_fferentiation of
subspecies, however, even ITS region sequences may sometimes
contain insufficient differences (E3ourque, S.N., et al. 1995.
Appl. Environ. Microbiol. 61(4):16'.3-1626). Isolates of
Mycobacterium leprae have also been found to contain
identical sequences in the ITS region (De Wit, M.Y.L., and
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P.R. Klatser. 1994. Microbiology 140:1983-1987). Recently,
Scheibl and Gerlach (Scheibl, P. and G.F. Gerlach. 1997. Vet.
Microbiol. 57(2-3):151-158) demonsi=rated that despite its
high potential mutation rate, the =CTS region of all
5 Mycobacterium avium subsp, paratuberculosis strains
investigated exhibited one common k>ase difference compared to
other Mycobacterium avium subspeciE;s. Two problems exist with
trying to use this difference as a differentiation criteria
between avium and paratuberculosis. For one the ITS region is
10 not transcribed into functional RNP, as a result of which the
copy number of this target remains too low to generate
improvement of the sensitivity of detection when compared to
available tests such as the IS900 probe test. But more
importantly, and unlike in ribosomal RNA's, base mutations in
15 the ITS region encounter no evolutionary pressure and
mutations can occur quite random and frequent. ITS region
mutations are therefore less stable that rRNA gene mutations.
This limits the use of ITS sequence's to strain level
differentiation of Myc. avium complex bacteria (Frothingham.
20 R, et al. 1993. J. Bacteriol. 175(10):2818-2825; Frothingham,
R, and K.H. Wilson. 1994. J. Tnfect. Dis. 169(2):305-312).
The present invention reveals the presence of three
stable and conserved mutations in the 23S rRNA of
Mycobacterium avium subspecies paratuberculosis which allow
the differentiation of this bacterium from other
Mycobacterium avium complex bacteria, while at the same time
providing for a high copy number target in the form of
functionally transcribed RNA.
For example, and further explained in the
experimental part, the invention provides a nucleic acid
probe or primer wherein said ribosomal RNA comprises a
nucleic acid sequence as shown in figure 1. Herein are
identified at least 4 unique mutations in the 23S rRNA gene
of subspecies paratuberculosis, of which three are
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transcribed into the functional ribosomal RNA of this
bacterium. The ribosomal RNA is present in some 1,000 to
10,000 copies in growing cells and constitute a far more
powerful target for direct enzymatic nucleic amplification
than the IS900 sequence.
The present invention reveals three point mutations
as potential subspecies-specific target-sites that are
conserved in reference strains and field isolates of
paratu~berculoszs. Two of these mutations, situated at
positions 754 and 1363, are specifically proposed for
combined use in a diagnostic test for the subspecies
paratuberculosis as these targets are situated in relatively
close proximity of one another. The present invention shows
that these three mutations are conserved and present in the
functional paratuberculosis 23S rRNA gene sequence. These can
be used as diagnostic targets to distinguish between the
paratuberculosis and avium subspecies. As such they are part
of the present invention in which they are referred to as
mutations 754, 1363 and 3093 (figure 2). A final mutation
identified through the present study is displayed in figure 2
as position 3188. This mutation is part of the internal
transcribed spacer region between the 235 and 5S rRNA genes
and can not be used for sensitive detection. Its conserved
status was recently confirmed by Scheibl and Gerlach (Vet.
Microbiol. (1997) 57:151-158). In all, over 60 differences
between an individual avium and an individual
paratuberculosis strain are identified in the alignment
presented in figure 2, of which only three can be used for
subspecies-specific diagnostics.
Nucleic acid detection generally utilises specific
hybridisation of a probe or primer to the nucleic acid to be
detected. Such nucleic detection in general is known in the
art and can far example be achieved with classical
hybridisation techniques, such as Northern or Southern
Blotting on nucleic acid derived directly from a sample or on
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nucleic acid derived via an amplification process, for
example via recombinant DNA techniques wherein said nucleic
acid is first amplified, be it indirectly in a host cell, for
example in a bacterium such as E, coli using a suitable
plasmid system or directly in a suitable amplification system
such as PCR or NASBA. Nucleic acid to be detected can also be
sequenced directly from the starting material, but more often
from nucleic acid that has first been amplified as described
above.
A probe or primer according to the invention
comprises a DNA, RNA or PNA oligonucleotide sequence specific
for a Mycobacterium avium subspecies with which said species
can be detected in a nucleic acid detection assay. Primers,
as defined herein, are understood to be unlabelled
oligonucleotides, which are selected to start (prime)
sequencing or amplification techniques. By selecting a primer
for a specific match, or, alternatively, for a specific
mismatch, for example for a nucleotide sequence having a.
specific mutation, deletion, insertion or other
discriminatory feature, said sequencing or amplification
techniques can be employed to specifically detect a nucleic
acid that may or may not be present. in a sample, and
discriminate it from other, related, nucleic acid sequence
that may be present.
Probes, as defined herein, are understood to be
labelled oligonucleotides, which are selected to detect and
label the desired nucleic acid, i.e. to report its presence.
Labelling is achieved with reporter molecules, which are
widely known in the art, such as radioactive labels, enzymes,
particles such as gold or silver particles,.chromophores,
fluorochromes, excitation or quencher molecules, and other
reporter molecules known in the art:. Probes are in general
used to detect a nucleic acid by hybridisation to said
nucleic acid, be it by hybridisation to a nucleic acid
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present in a sample, for example by in situ hybridisation
such as by FISH (fluorescent in situ hybridisation, or by
hybridisation to an amplified fragment of said nucleic acid
(also known as an amplicon). PCR or NASBA derived amplicons,
for example, can be detected by probing with such a probe,
for example by blotting techniques, or by more sophisticated
techniques such as Taqman or FRET technology.
The invention provides a nucleic acid probe or primer
allowing detecting nucleic acid derived from any
Mycobacterium avium, such as derived from its various
subspecies avium, silvaticum or paratuberculosis, whereby
nucleic acid derived from Mycobacterium avium subspecies
paratuberculosis can be discriminated from nucleic acid
derived from other Mycobacterium avium subspecies, however,
in a preferred embodiment, the invention provides a nucleic
acid probe or primer according to the invention for detecting
a causal agent of Johne's disease in a sample, preferably in
a sample derived from a ruminant, ~riost preferably from a cow.
In a most preferred emboeiiment, the: invention provides a
nucleic acid probe or primer allowing detecting nucleic acid
derived from Mycobacterium avium derived from a faecal, blood
or milk sample. Herewith the invention provides methods and
means to detect Mycobacterium aviunt in samples obtained from
animals that may or may not be suspected of having Johne's
disease, allowing discriminating between the various
Mycobacterium avium subspecies, and positively identifying
those animals for example infected with subspecies
paratuberculosis.
n general, both RT-PCR and NASBA are sensitive
methods for detection of bacterial ribosomal RNA's. As a
result, cells of pure cultures susx>ended in common buffers,
such as PBS, can be detected at sensitivities of only a few
cells per milliliter or gram (see figure 8 of the present
invention). However, nucleic acid extracts obtained from
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crude manure of fecal samples exert very strong inhibitory
effects on enzymatic amplification of specific nucleic acid
sequences both by (RT-)PCR and NASBA (see figure 6 of the
present invention). The reason for this is not fully
understood by the art. The result of this, however, is a very
low sensitivity of diagnostic assays in fecal samples, due to
the necessity of extensive sample dilution.
Selective recovery of the target cells from the
matrix, i.e. by immunomagnetic capture, is one way to
circumvent this problem as demonstrated by Grant et al. for
detection of M. paratuberculosis in milk (Appl. Environ.
Microbiol. 64, 1998, pp. 3153-3158) and by Widjojoatmodjo et
al. for detection of salmonella in fecal samples (J Clin
Microbiol 30, 1992, pp. 3195-3199). Another way is to attempt
excluding co-extraction of inhibitory compounds, such as
bile-acids, by cellulose adsorption purification of extracted
RNA's as demonstrated by Wilde et a.l. for detection of viral
RNA's from fecal samples (J. Clin. Microbiol. 28, 1990, pp.
1300-1307).
Yet another way, and one that presents an embodiment
of the present invention, is to usE: selective lysis and pre-
extraction procedures of non-target: materials while retaining
the target cells or the target nucleic acids in the sample.
Due to their extraordinary cellular: composition, cells of
Mycobacterium species are very resistant to lysis, at least
more resistant than most other bacterial organisms. Specific
procedures, separately known to thE: art to severely disturb
cellular integrity, such as repeated freeze-thawing or bead
beating with small beads, such as glass beads, are effective
ways to lyse most or all cells inc=Luding those of M.
paratuberculosis. In a preferred embodiment of the invention
its is provided that certain treatments that result in at
least partial lysis of mast fecal bacterial cells do not
effectively or only little lyse ce:Lls of M. paratuberculosis
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or other Mycobacteria. In a preferred embodiment, the
invention thus provides selective lysis of non-mycobacterial
organisms or matter to allow better' detection of a
mycobacterial organism. Therefor, the resistance to lysis of
M. paratuberculosis can be used to increase assay
sensitivity. These treatments may include, but are not
limited to, TRI-reagent processing of samples (see below) or
NaOH exposure. Further examples for the use of such selective
lysis treatment and their effect on. assay sensitivity are
given below. It is an embodiment of the present invention
that such selective or partial lysis treatments can be used
to reduce the presence of non-target cells, non-target
nucleic acids or compounds otherwise inhibitory to or
interfering with the desired specific enzymatic nucleic acid
amplification prior to extraction and purification of target
nucleic acids, thereby increasing the sensitivity of
diagnostic systems for detection of specific micro-organisms
at very low concentrations in complex matrices, such as M.
paratuberculosis in fecal samples, milk, sputum or blood.
In a preferred embodiment, the invention provides a
nucleic acid probe or primer derived from ribosomal RNA, such
as 23S ribosomal RNA. In the case of ribosomal RNA 1,000 to
10,000 copies are normally present in growing cells. This
copy.number is much higher than that of other genomic targets
that are generally present in 1 to 20 copies. The unique
advantage of RNA target amplification by, e.g., reverse
transcriptase PCR (RT-PCR) or NASBA. is therefore that these
methods are superior in sensitivity than the DNA
amplification methods for detection of bacterial cells, e.g.,
by PCR. However, also for hybridisation techniques, such as
in situ hybridisation, this high copy number is advantageous,
since it again allows superior sensitivity.
The invention provides a method for detecting nucleic
acid derived from Mycobacterium avium in a sample comprising
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using at least one nucleic acid prok>e or primer according to
the invention. An assay or method a:> provided by the
invention is for example based on the detection of one or
more specific point-mutations in the 23S ribosomal RNA of the
bacterium. The high copy number of ribosomal RNA's relative
to other genomic targets provides for a very high sensitivity
of the assay, thereby allowing for performance of the assay
in "difficult" matrices , such as faecal, sputum, blood or
milk samples. The invention provides a method or assay to
detect the bacterium in milk, faeces>, soil, feed, and any
other habitat in which the bacterium can be found. The speed
and ease with which the assay can bc~ performed relative to
conventional methods enables one to use it for routine
analyses of a large number of samples. Samples can comprise
those taken from individuals, such as cows, however, it is
also feasible to test bulk or tank milk samples, pooled
faecal or pooled blood samples with a method provided by the
invention.
Herewith, the indention provides a method for
detecting a causal agent of Johne's disease. Especially, a
method or assay as provided by the invention can be used to
discriminate between Mycobacterium avium subspecies
paratuberculosis and its closest relative Mycobacterium avium
subspecies avium.
The detection of Myb. avium subsp. paratuberculos.is
cells in faeces is greatly simplified by a method or assay
according to the invention because whole bacterial cells need
for example not be detected but DNA or RNA liberated from the
cells in the sample is analysed for the presence of specific
target or signature sequences. In order to develop a
functional test for the detection and identification of Myb.
avium subsp. paratuberculosis cells in faeces, the test must
be able to make a distinction between the three subspecies in
the Mycobacterium avium complex. This test has now been
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provided by the invention. A method as provided by the
invention has sufficiently high sensitivity to test faecal
samples. Said method can be performed using nucleic acid
amplification techniques, or hybridisation techniques, such
as situ hybridisation, for example as described herein.
Furthermore, the invention provides a diagnostic kit
comprising at least nucleic acid probe or primer according to
the invention, and optionally other means, such as buffers
and other reagents or instructions, to detect Mycobacterium
avium, preferably subspecies paratuberculosis, the causal
agent of Johne's disease.
The invention provides method for detecting a
ruminant infected with a causal agent of Johne's disease
comprising obtaining a sample from said cow and testing said
sample for the presence of nucleic acid using at least one
nucleic probe or primer, or a method or diagnostic kit
according to the invention. Herewith, the invention provides
a method for eradicating Johne's disease from a herd of
ruminants comprising using a method according to the
invention and further comprising culling or removing a
ruminant infected with a causal agent of Johne's disease
ruminant from said herd, after which said ruminant, for
example an infected cow, may be destroyed. Such a testing and
remove system, generally called a control or eradication
programme, is best performed under strict supervision of or
even prescribed by (veterinary) governmental authorities, but
may also very well be achieved in a voluntary effort by
,combined farmers or others involved. in the agricultural
community. In summary, the invention provides use of a
nucleic acid probe or primer allowing detecting nucleic acid
derived from Mycobacterium avium wruereby nucleic acid derived
from Mycobacterium avium subspecie~~ paratuberculosis can be
discriminated from nucleic acid derived from other
Mycobacterium avium subspecies. Such use is for example to
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detect a causal agent of Johne's di~;ease in faecal samples,
for example by using a nucleic acid detection method as
provided by the invention. Such use, as provided by the
invention, for example allows governmental authorities and
individual farmers, veterinarians, and concerned agricultural
organisations to eradicate Johne's disease from a herd of
ruminants, more specifically of cow:> infected with
Mycobacterium avium subspecies parat:uberculos.is.
The invention is further explained in the
experimental part of the description without limiting the
invention thereto.
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Experimental part
Detection of paratuberculosis-specific gene sequences
by PCR.
Source and identity of bacteria used in this study.
The source and identity as well as the condition of
the pure cultures from which nucleic acids were extracted is
presented in table 1, in which DSMZ is the Deutsche Sammlung
von Mikro-organismen and Zellculturen, ATCC is the American
Type Culture Collection.
fNA extraction and 23S rDNA sequencing
Total nucleic acids were isolated from colonies grown
on Lowenstein-Jensen medium or dire~~tly from lyophilized cell
pellets as obtained from culture collections. Methods used
were as described elsewhere (Aznar et al., 1994. Int. J.
System. Bacteriol. 44:330-337). The 23S rRNA genes were
amplified by using primes directed towards conserved regions
and the genes were sequenced commercially. The 23S rRNA genes
were aligned and checked for the presence of Myb. avium
subsp. paratuberculosis-specific sequences. Part of the
alignment is displayed in Fig. 1.
The paratuberculosis sequen<:e is characterised by the
presence of 3 single point-mutations as compared to the avium
arid silvaticum sequences. These three point-mutations
comprise of a transition of C in av:ium and silvaticum to a T
in the paratuberculosis gene. These point mutations can be
detected by using the polymerase ch<~in reaction such as
described now.
PCR detection of point-mutations.
PCR was performed on a Eppendorf Mastercycler
gradient with a temperature gradieni~ from 55 to 68 degrees
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centigrade. The PCR reaction mixture (100 ul) consisted of
the following components: 10 ul of ~_Ox reaction buffer; 0.3
~.l of Taq DNA polymerase (Promega carp.); 2 ~l of both
forward and reverse primer @ 200 ~ZM;~ 1 ~Zl of DNA template @
5 25 ng/ul; 4 pl of dNTP's and 80,7 ul of H20. Thermal cycles
consisted of a denaturing step at 95°C for 5 min, followed by
cycles of 95°C, gradient temp 55--68°C and 72 °C for 1,
2,
and 3 min respectively. PCR products were checked by standard
agarose gel-electrophoresis in 0.8o agarose gels and stained
10 with ethidium bromide.
A series of primers was tested to determine the
optimal site of the "mismatch" position within the probe
sequence relative to the avium sequence (as displayed in
15 table 2). When used in a PCR reaction several of these
primers were found to result in reliable discrimination
between subspecies avium and subspecies paratuberculoszs (see
figure 2).
This invention fbrms the key element of the
20 diagnostic procedure which comprises at least one of the
following steps:
1. Sampling of faeces
2. Transport and storage of the sample in RNA-
stabilizing solution.
25 3. Selective lysis of non-mycobacterial matter.
4. Liberation of the desired nucleic acid by lysis
of the bacterial cells.
5. Amplification of the :bacterium-specific
sequences by a method known to the art
30 6. Detection of the bacterium-specific sequences by
nucleic acid hybridization methods
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Further detailed description.
I
Example 1: Cell lysis, ribonucleic acid extraction
and RNA amplification from pure culiures of Mycobacterium
avium subspecies paratuberculosis.
Sample preparation. Pure cu7_tures of M.
paratuberculosis strain GDR were grown on Lowenstein-Jensen
gradient agars. A loop of cells was collected from the agar
surface and resuspended into glycerol (20o v/v} PBS. The
cell-suspensions were stored at -20"C until their use in
spiking experiments to determine then sensitivity of the
various detection assays. Frozen ce:Ll-suspensions were
rapidly thawed, washed twice in water and resuspended into
100 ul of water. A volume of 10 ul was fixed in 0.4o formalin
for subsequent determination of cel:1 number. For cell
counting, Sybr Green (Molecular Probes, Leiden, The .
Netherlands) was used as a DNA counter-stain. Briefly, a IO
ul volume of the washed and fixed cell suspension was added
to 1.0 ml of PBS. To this diluted suspension, Sybr Green was
added to yield a final concentration of 1/10,000-th of the
original manufacturer-stock. Staining occurred at 37°C for 10
min. After staining, the entire suspension was drawn over a
0,2 um pore-size black polycarbonate membrane filter
(Millipore, Etten-Leur, The Netherlands). The filters were
mounted on a microscope slide and cell counts were determined
using an Olympus BX-60 epifluorescence microscope (Paes
Nederland BV, Zoeterwoude, The Netherlands). A minimum of 15
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fields-of-view per sample were counted or until the total
number of counted cells was 300.
RNA extraction and quantification.
RNA was extracted from purE: culture cell suspensions
by using three basic methods. Modi:~ications were made to the
procedures in case cell lysis of Nf. paratuberculos.is was
incomplete. These modifications comprised the inclusion of
freeze/thawing cycles or bead-beating procedures. Basic
procedures were as directed by the manufacturers of the RNA
extraction systems. The following methods were tested for RNA
extraction from M, paratuberculosi,s cells: i) RNA extraction.
according to the silica-adsorption principle described by
Boom et al. (J. Clin. Microbiol. 28, 1990, pp. 495-503) which
was purchased commercially as part of the NucliSens Basic Kit
system for NASBA diagnostics (Orga:non Teknika, Boxtel, The
Netherlands), ii} RNA extraction according to the acid-
phenol/guanidinium principle described by Chomczynski and
Sacchi (Anal Biochem 162; 1987, pp. 156-159) which was
purchased commercially as TRI-reagent (Sigma Chemical Comp.,
Zwijndrecht, The Netherlands) and iii) a solid phase RNA
extraction with the RNeasy Plant kit purchased from Qiagen
(Hilden, Germany), which includes a protoplast disrupting
shredder filter. Extracts were dissolved in DEPC-treated
water containing the RNase inhibitor RNasin (Promega, Leiden,
The Netherlands) to prevent degradation.
Quantification of extracted nucleic acids was
performed by using the fluorochrome RiboGreen (Molecular
Probes, Leiden, The Netherlands) as an nucleic acid stain as
directed by the manufacturer. Fluorescence readings were made
on a Fluoroskan Ascent FL (Labsystems Oy, Helsinki, Finland)
with excitation filter at 485 nm and emission filter at 530
nm.
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The efficiency of RNA extraction from M.
paratuberculosis cells and the quality of the extract for
nucleic acid amplification purpose was further evaluated
empirically by NASBA and RT-PCR.
The yield of cellular RNA obtained from 1 x 10~5
cells of M. paratuberculosis strain GDR by various methods as
well as NASBA results with such extracts as determined
fluorimetrically with RiboGreen as a nucleic acid stain and
by NASBA ECL detection is presented hereunder.
Method pg of RNA extracted NASBA ECL counts
NucliSens 4,790 638,625
TRI 739 46,586
TRI + bead beating n.d. 725,521
RNeasy Plant 6,476 n.d.
NASBA amplification of 23S rRNA
NASBA was performed by using the commercial NucliSens
Basic Kit system for NASBA diagnostics (Organon Teknika).
Reaction conditions for NASBA amplification of RNA were as
directed by the manufacturer. Detection of M.
paratuberculosis-specific 23S-rRNA fragments was performed by
ECL detection using a biotin labeled probe. Measurements were
performed on a NucliSens reader (Organon Teknika).
NASBA primers and probes specific for M.
paratuberculosis were designed based on multiple alignments
of the complete 23S rRNA gene sequences from 5 M.
paratuberculosis and 5 M. avium strains, together with
sequences from M. .intracellulare and M. silvat.icum.
Alignments were constructed by using the Multiple Sequence
alignment tool from the BCM Search Launcher
(www.hgse.bcm.tmc.edu/ SearchLauncher/). Confirmation of the
existence of conserved M. paratuberculosis-specific sequences
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in the 23S rRNA gene was further evaluated by partial
sequencing and by point-mutation PCFt (primers with wild-type
position at 3'-end) from 12 additional paratubercuZosis
strains and 3 additional avium stra~_ns. Various NASBA primer
sets specific for M, paratuberculos_~s were designed and their
performance was tested empirically. The optimized primers and
probes are presented in figure 6. Optimized results were
obtained with one particular primer set (see figure 7).
RT-PCR amplification of 23S rRNA.
Primers specific for M. paratuberculosis were
designed based on multiple alignments of the complete 23S
rRNA gene sequences from 5 M. paratuberculosis and 5 M. avium.
strains, together with sequences from M. intrace11u1are and
M. s.ilvaticum. Alignments were constructed by using the
Multiple Sequence alignment tool from the BCM Search Launcher
(www.hgsc.bcm.tmc.edu/ SearchLauncher/). Confirmation of the
existence of conserved M. paratubercu.Zosis-specific sequences
in the 23S rRNA gene was further evaluated by partial
sequencing and by point-mutation PCR (primers with wild-type
position at 3'-end) from 12 additional paratuberculosi.s
strains and 3 additional avium strains. The primers used are
presented in figure 6. A total of 41 thermal cycles was
applied.
Sensitivity of the various amplification reactions
was determined by using standardized cell-suspensions of M.
paratuberculosis in 20% glycerol. M. paratubexculosis-
specific 23S-rRNA from TRI-reagent extracted and CF11
purified total RNA was transcribed into cDNA with AMV Reverse
Transcriptase (Promega Inc., Leiden, The Netherlands) at 50°C
for 90 min. The reaction condition:> were as directed by the
manufacturer. cDNA sequences were then amplified by PCR.
Reaction products were checked for correct length on 1.50
agarose gels using ethidium bromide staining. The agarose
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gels were blotted onto hybond N+ membranes (Amersham
Pharmacia) using a vacuum manifold and standard procedures
(Sambrook J, Fritch EF and Maniatis T. Molecular cloning: A
laboratory manual. 2nd ed. Cold Spring Harbor Labl. Press,
5 New York, 1989). DNA was crosslinked by UV irradiation using
a UV crosslinker (Amersham Inc., 's Hertogenbosch, The
Netherlands) at 700 Joules/cm2. Blots were stored dry until
use. For confirmation of PCR reaction product identity, blots
were hybridized with a fluorescein labeled probe (see figure
10 6) and stained by NBT-BCIP chromogenic staining and anti-
FITC-AP Fab fragments (Roche Diagno:>tics, Almere, The
Netherlands).
Assay sensitivity.
15 By using NASBA of 23S rRNA targets, cells of M.
paratuberculosis in pure culture cE:ll suspensions could
effectively be detected at numbers as law as 10 cells per ml
of sample (the lowest cell number t:ested), provided that
cells were disrupted by l5ead beating (see figure 8). Similar
20 detection sensitivities were obtained by R'~-PCR (see figure
9) .
Example 2: NASBA amplificai~ion and detection of
Mycobacterium paratuberculosis-specific RNA in total RNA
25 extracts from bovine fecal samples.
Sample preparation. Fresh cow manure samples were
collected from the rectum of individual animals from disease-
free farms. The manure was diluted 1:1 with PBS. After
30 thorough vortexing, samples were centrifuged at low speed
(100 x g) and aliquots of the supernatant representing 0,5
gram of the crude manure bacterial fractions were stored at -
20°C until use.
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Total RNA extraction from M'. paratuberculosis-free
bovine manure. For NASBA amplification, RNA was extracted by
using the commercial NucliSens Basic Kit (Organon Teknika)
based on silica adsorption. Briefly, to a cell pellet
obtained from 0,5 grams of a crude manure bacterial cell
fraction a volume of 10,0 ml of Lysis Buffer (NucliSens Basic
Kit) was added. Extraction of the RNA occurred with silica
(Basic Kit) as directed by the manufacturer. Further
purification was achieved by boiling of the extract (10 min,
100°C). Following extractian, the P;NA was even further
purified by the method of Wilde et al. (J. Clin. Microbiol.
28, 1990, pp. 1300-1307) using the cellulose fiber compound
CF-11 (Whatman International Ltd., Clifton NJ, USA). Briefly,
isopropanol precipitated total RNA was washed with 75%
ethanol and dried for 5 min at room temperature. The pellet
was resuspended in a 100 pl volume of DEPC-treated water and
heated to 55°C for 10 min. Upon cooling, extracts were
diluted 1:1 with 2 x STE containing 66% ethanol and an amount
of 30 mg of CF-11 was added. Samples were placed on a
rotating mixer for 45 min at room temperature. After
centrifugation and removal of the supernatant, the pellet was
washed 3 times with 1 x STE containing 20% ethanol. The RNA
was eluted in a 20 ul volume of 10--50o formamide in DEPC
water. The eluate was stored at -2t)°C until use.
RNA extraction from pure cultures of M.
paratubercu.Iosis.
RNA was extracted from pure culture cell suspensions
by using the NucliSens Basic Kit system for NASBA diagnostics
(Organon Teknika).
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NASBA amplification and detention. Non-purified total
RNA extracts from crude bovine manure were mixed with RNA
extracts obtained from pure cultures of M. paratuberculosis
in order to investigate the inhibiting effect of fecal matrix
compounds on the amplification reaction. Reaction conditions
for NASBA amplification were as described in example 1.
Various dilutions of non-purified crude manure RNA extract
were mixed with RNA extracted from 1,000,000 cells of M.
paratuberculosis strain GDR: Non-purified crude manure
extract showed a severe inhibitory effect on NASBA
amplification of M. paratuberculosis RNA (see figure 10).
This effect could be reduced by administering very low
amounts of bovine fecal matrix extract in the amplification
reaction or by purification of RNA extract as described.
Example 3: NASBA, amplification and detection of
Mycobacterium paratuberculosis cells in bovine fecal samples.
Sample preparation. Sample preparation was as
described in example 2.
Total RNA extraction from bovine manure spiked with
M. paratuberculosis cells. For NASBA amplification, RNA was
extracted by using the commercial TRI-reagent (Sigma).
Briefly, to a cell pellet obtained from 500, 50 and 5 mg of a
crude manure bacterial cell fracticsn, various amounts of M.
paratuberculosis cells ranging from 10,000 to 100 cells were
added. The pellets were resuspendecl in a 1 ml volume of 4%
NaOH. The partial lysate was centrifuged at 10,000 x g and
the remaining cell pellet was resu~~pended in 1 ml of TRI-
reagent. The TRI-reagent suspended cell fraction was
transferred to a mini bead beater vial, which contained 0,5
ml of 0,1 um sized glass beads (Biospec, Bartlesville OK,
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USA). Cells were lysed by bead beating in a mini beat beater
(Biospec) for 3 x 60 sec and cooled on ice. RNA was extracted
as advised by reagent the manufacturer. The RNA extract was
further purified by CF-11 adsorption as described in example
2.
NASBA amplification and detection. M.
paratuberculosis-specific 23S rRNA's in the extracts were
measured by NASBA amplification andl detection as described in
example 1. Sensitivity of the assay was between 100 and 1,000
cells per gram of feces (see figure: 11).
Example 4: Detection of Myc;obactexium
paxatubexculosis in spiked bovine fecal samples by reverse
transcription PCR (RT-PCR) and reverse transcription nested
PCR (RT-nested-PCR) .
Sample preparation. Sample preparation was as
described in example 2.
RNA extraction from bovine manure. Total RNA was
extracted from 0,5 grams of the crude manure bacterial
fraction described in example 3.
RT-PCR and RT-nested-PCR annplification.
RT-PCR procedures were as described in example 1. For
RT-nested-PCR, a 2 ul volume of PCI~ reaction product was
added to a second PCR reaction containing the nested
primerset (see figure 7). An additional 41 thermal cycles
were applied.
Following CF-11 purification and RT-PCR amplification
of extracted rRNA's, a detection limit of between 100 and
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1,000 cells of M. paratubercu~os.is per gram of manure was
achieved. However, using the nested PCR approach a further
improvement of sensitivity was achif~ved. As little as 10
cells per gram of crude manure could successfully be detected
with the RT-nested-PCR procedure (sE=e figure 12).
When nucleic acid amplificat=ion is applied on a large
scale, effective programs for decontamination or the
prevention of cross-contamination mast be installed. The
large amounts of amplicon that are generated in the rooms in
IO which the actual amplification step takes place must be
contained. Physical separation must be realised between
activities of reagent preparation, .sample handling and
amplification & detection. Another .approach is the
containment of the source of contamination: the amplification
reaction itself. This can be achieved by applying closed-
system amplification and detection :by which the reaction
vessel is never opened again. Alternatively, and possible
when applying PCR, or DNA amplification is the incorporation
of uracil-DNA glycosylase' in the system. This enzyme
specifically degradates uracyl containing sequences that are
incorporated in the amplicon prior to the initiation of new
amplification reactions (Ferre et al., 1996 In: A Laboratory
guide to RIdA. Isolation, analysis and synthesis. P.A. Krieg,
ed. pp. 175-221. Wiley-Liss, New York)
The sensitivity of the system is primarily determined
by the amount of detectable targets. Such targets can then be
amplified by PCR or NASBA. Other methods of amplification are
so-called signal amplification methods of which branched DNA
(bDNA) and LCR (ligase chain reaction) are examples.
Amplification targets can comprise RNA or DNA. In the case of
ribosomal RNA 1,000 to 10,000 copies are normally present in
growing cells. This copy number is much higher than that of
genomic targets that are generally present in 1 to 20 copies.
The unique advantage of RNA target amplification by, e.g.,
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reverse transcriptase PCR (RT-PCR) or NASBA is therefore that
these methods are superior in sensitivity than the DNA
amplification methods for detection of bacterial cells, e.g.,
by PCR.
5 Not only the actual assay (DNA amplification method)
or amplicon detection parts are important in the method of
detection of M. paratuberculosis, also the data analysis and
specifically the sample pre-treatment are critical processes
to be assigned. Sample preparation relates to the liberation
10 of DNA or RNA from M, paratuberculosis cells present in
sample.
RNA targets are present at much higher frequencies
then are DNA targets. Sensitivity is thus improved by using
RNA as the starting material for nucleic acid amplification
15 based detection methods. As a result, RT-PCR, NASBA, or TMA
technologies are preferred. The availability of unique RNA
targets allows the combination of :such amplification
technologies with RMNA targeted fluorescence in situ
hybridization (FISH) tech~iology. Strong fluorescent signals
20 are required for the detection of Myb. avium subsp.
paratuberculosis cells in faecal samples by FISH. It may be
expected that the slowly growing cE:lls contain very few
ribosomes. Signal amplification protocols such as proposed in
EP 97.20.2618 can be used to improve the signal in FISH
25 procedures. The use of FISH in the detection of M.
paratuberculosis in faecal samples may require the selective
enrichment of M. paratuberculosis cells from the
autochthonous background population (e.g., by cultivation or
immunomagnetic capture). Rapid mica=oscopic confirmation of
30 samples that have tested positive by nucleic acid
amplification methods can be realised through FISH. The high
reliability and confirmation possibilities of FISH and the
high sensitivity and speed.of the nucleic acid amplification
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41
technologies makes parallel development of both FISH and
NASBA or RT-PCR extremely valuable.
For the extraction of RNA from pure cultures of
bacteria or from relatively simple matrices, high-throughput
solid phase extraction procedures a:re commercially available.
An important aspect of the RNA extraction reaction is the
purity of the final extract. The faecal matrix is known to
contain substances that exert significant inhibitory action
on the enzymes of nucleic acid amplification technologies
that cannot be easily removed from the extract. Bilirubin and
bile-salts are known to inhibit the PCR reaction at
concentrations as low as 10 to 50 milligrams per millilitre
(Widjojoatmodjo et al., 1992. J Clin Microbiol 30:3195-3199).
Several procedures have been developed to improve nucleic
acid amplification from the fecal matrix. Among these are
100-fold (Varela et al., 1994. J Clin Micrabiol 32:1246-1248)
to 500 fold (Widjojoatmodjo et al., 1992. J Clin Microbiol
30:3195-3199) dilutions of the fecal samples to reduce
inhibition. Alternatively, ion-exchange column purification
(Kato et al., 1993. J Infect Dis 16'7:455-458) or
cetyltrimethylammonium bromide treatment of the extract
(Jiang et al., 1992. J Clin Microbiol 30:2529-2534) can be
used to reduce the amount of inhibiting compounds. Also
glass-matrix precipitation (Stacy-Phipps et al.,1995. J Clin
Microbial 33:1054-1059) or alternative resins or the use of
chaotropic compounds in the extraction (Shieh et al., 1995. J
Viral Methods 54:51-66) can improve: enzymatic nucleic acid
amplification. The reliability of accurately discriminating
between point mutations in amplification reactions can
substantially be increased by using competitor primers or,
preferably, PCR clamping with PNA (drum, H., et al. 1993:
Nucleic Acids Res. 21:5332-5336).
Possibilities for detection of the produced amplicon
are infinite. Technologies such as the chemiluminescence
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based hybridization protection (HP) assay with acridinium-
esters (AE) (Gen-Probe), fluorescence based technologies such
as the use of molecular beacons (Tyagi and Kramer. 1996.
Nature Biotechnol. 14:303-308), Taq-man procedures (Perkin
Elmer), the FRET principle (Roche Diagnostics) or the use of
intercalating dyes can be used to detect the amplicons in
various ways that all have their own specificities. Sandwich
hybridization assays in combination with magnetic bead
capture formats can also be applied..
Apart from the fact that an RNA-based approach
results in a much higher amount of initial target or
amplification-template this approach has another unique
advantage: contamination problems a.re significantly reduced
since RNA amplicons are much more labile than DNA amplicons
and will naturally deteriorate.
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Legends to the figures.
Figure i
Comparative sequence alignment at the four sites in
the 23S rRNA gene region that contain mutations specific for
Mycobacterium avium subsp. paratub~srculosis.
The short names represent the following strains:
paratGDR, Mycobacterium avium subspecies paratuberculosis
strain GDR; par44135, Mycobacterium avium subspecies
paratuberculos.is strain DSM 44135; parbovE5, Mycobacterium
avium subspecies paratuberculosis strain Spbov E5; paratGie,
Mycobacterium avium subspecies paratubercuiosis strain J2A;
par19698, Mycobacterium avium subspecies paratuberculosis
strain ATCC 19698 (Type strain); avi44157, Mycobacterium
avium subspecies avium strain DSM 44157; avi25291,
Mycobacterium avium subspecies aviu.m strain strain ATCC 25291
(Type strain); avi43216, Mycobacterium av.ium subspecies avium
strain DSM 43216: avi44158, Mycobacterium avium subspecies
avium strain DSM 44158; MYCAVIUM, Mycobacterium avium
subspecies avium strain strain 23435; int13950, Mycobacterium
intracellulare strain ATTC 13950 (Type strain); si144175,
Mycobacterium avium subspecies silvaticum strain DSM 44175
(Type strain); 23SEcoli, Escherichia co~i 23S rRNA gene
sequence Genbank J01695.
The first 5 species represent well characterized
strains of Mycobacterium avium subsp. paratuberculos.is. The
next 5 strains represent well characterized strains of
Mycobacterium avium subsp. avium. The four mutations that
form part of this invention are numbered 754, 1363, 3093 and
3188. The 3188 mutation is positioned in the ITS region
between the 23S and 5S rRNA genes. The boxed regions
represent the thymine residue in the rRNA operon of
Mycobacterium avium subsp. paratubercu.Iosis that form the
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basis of subspecies-specific detection methods as embodied in
the present invention.
Figure 2
Sequence alignment of the complete 23S rRNA gene of
selected Mycobacterium species. The data presented in this
alignment show that only positions numbered 754, 1363, 3093
and 3188 harbour sequence identities unique to the subspecies
paratubercu.Iosis of the Mycobacterium avium complex. The
species abbreviations are as in figure 1.
Figure 3.
Comparative sequence alignment of a 656 basepair
1S fragment of the 23S rRNA gene of selected Mycobacterium
species that overlaps with mutation 1363 in figs. 1 and 2.
The first 19 sequences represent the fragment of well
characterized strains as well as field isolates of
(presumably) Mycobacterium avium subsp. paratuberculosis.
The next 8 sequences represent the fragment of well
characterized strains as well as field isolates of
(presumably) Mycobacterium avium subsp. avium. This figure
shows that the mutation is also present in field isolates of
Mycobacterium avium subsp. paratubercu.Losis. The presence of
mutation 1363 for paratuberculosis is visible on position 150
of this alignment.
Figure 4
Temperature gradient PCR performed with the two
point-rnutat.ion primers that anneal specif~.cally with the
Mycobacterium avium subsp. paratube~rculosis 23S rRNA gene at
the position of mutations 754 and 1363. The figure represents
an agarose gel of PCR products obtained after amplification
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of a portion of the 23S rRNA genes from DNA from
Mycobacterium avium subspecies paratuberculosis strain GDR
(all upper slots of the gel) and Mycobacterium avium
subspecies avium strain 97-513 (all lower slots of the gel).
5 The primer pair consisted of primer 20F and primer 22R (as
described in table 2). A series of 10 different annealing
temperatures was applied by using the eppendorf Master cycler
gradient apparatus. From left to right over the gel, PCR
products from reactions with increasing annealing
10 temperatures were applied. At an annealing temperature of
66.5 °C, no PCR product is generated from Mycobacterium avium
subspecies avium, whereas a PCR product with this primer set
can still be obtained even at annealing temperatures of 68.9
~C
Figure 5
ParatubercuLosis-specific F~CR performed with the two
23S rRNA point-mutation primers used in the experiment of
fig. 4. The figure represents agarose gels of PCR products
obtained after amplification of a portion of the 23S rRNA
genes from DNA isolated from a large number of well
characterized strains as well as field isolates of
(presumably) Mycobacterium avium :>ubsp. paratuberculosis
(top) and of (presumably) MycobactE~rium avium subsp. avium
(bottom). An annealing temperature of 68 °C was used. No
product of the specific length (approximately 600 bases) was
obtained from DNA isolated from Mycobacterium avium subsp.
avium strains.
In the upper gel, the products of paratGl4, paratG53,
paratG63, parat390, parat437, parai~442, parat444, parat434,
parat421, parat412, parat423, para1~.424, parat415, and
paratG32 were obtained from PCR reactions with DNA from field
isolates of Mycobacterium avium si.zbsp. paratuberculosis. The
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other paratuberculosis strains are coded as described in the
legend to figure 1. The codes of the strains used in the
lower gel were as described in the legend to figure 1. The
codes avi97613 and avi97675 represent field isolates of
Mycobacterium av.ium subsp. avium.
Figure 6. Probes and primers used for the enzymatic
amplification of 23S ribosomal RNA sequences of M.
paratuberculosis by NASBA or RT-(nested)-PCR.
Figure 7. Effect of primer design on NASBA
amplification of 23S rRNA sequences from M. paratuberculosis.
Primerset A was chosen for further studies. By using the
optimized primer set, a detection sentitivity of Less than 10
cells per ml of buffer could be attained (see text).
Figure 8. Effect of cell lysis on detection of M.
paratubercu.2osis cells by NASBA. By using forced cell
disruption or selective ~.ysis much higher detection
sensitivities could be attained. TF;I-reagent itself could be
used as a selective lysis environment.
Figure 9. Southern blots from RT-PCR amplification of
RNA extracted from pure cultures of: M. paratuberculosis
strain GDR by TRI reagent with (above) and without (below)
additional bead beating procedures. Detection by RT-PCR is as
sensitive as NASBA (i.e., less thars 10 cells per ml).
Figure I0. Effect of matrix: inhibition on the
enzymatic amplification of 23S ribosomal RNA sequences of M.
paratuberculosis by NASBA. A total amount of RNA extracted
from 1,000,000 cells of M. paratubE~rculosis strain GDR was
added to crude manure RNA extract (see text).
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Figure 11. NASBA amplification and detection of M.
para,tuberculosis cells in bovine manure. Non-target cell
lysis was achieved by 4% NaOH treatment. Assay sensitivity
was further improved by CF-11 adsorption (see text).
Figure 12. Southern blot from RT-nested-PCR
amplification of M. paratuberculosi~~ cells in bovine manure.
As little as 10 cells per gram of manure could be detected by
the nested PCR approach. P.C., posit:ive control.
,r
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References
1. Thorel, M.F., et al. 1990. Numerical taxonomy of
mycobactin-dependent-mycobacteria, Emended description of
Mycobacterium avium, and description of Mycobacterium avium
subsp. avium subsp. nov., Mycobacterium avium subsp.
paratuberculosis subsp. nav. and Mycobacterium avium subsp.
si.tvaticum subsp. nov. Int. J. System. Bacteriol. 40:254-260.
2. Good, R.C. 1992. The genus Mycobacterium - Medical. In:
The Prokaryotes: a handbook on the biology of bacteria:
ecophysiology, isolation, identification, applications. 2na
ed. A. Balows et al., editors. Vol 2. pp. 1243. Springer
Verlag, New York.
3. Goodfellow, M. and L.G. Wayne. 1982, Taxonomy and
nomenclature, p. 471-521. In: C. Rai=ledge, and J. Stanford
(ed.), The biology of the mycobacteria, vol. 1. Physiology,
identification and classification. Academic Press, London.
4. Kent, P.T., and G.P. Kubica. 1985. Public health
mycobacteriology: a guide for the lESVel III laboratory. U.S.
Department of Health and Human Services, Centers for Disease
Control, Atlanta.
5. Siddiqi, S.H. 1988. BACTEC TB system: product and
procedure manual. Becton Dickinson Diagnostic Instrument
Systems, Towson. MD.
6. McFadden, J.J., et al. 1987a. The use of DNA probes
identifying restriction-fragment-length-polymorphisms to
examine the Mycobacterium avium complex. Mol. Microbiol.
1:283-291.
7. Vary, P.H., et al. 1990. Use of highly specific DNA
probes and the polymerase chain reaction to detect
Mycobacterium paratubercu.Losis in Johne's disease. J. Clin.
Microbiol. 28(5):933-937.
CA 02353580 2001-06-O1
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49
8. Moss, M.T., et al. 1991. Specific detection of
Mycobacterium paratuberculosis by DNA hybridisation with a
fragment of the insertion element IS900. Gut 32(9):395-398.
9. Benedictus, G. 1985. Some aspecas of bovine
paratuberculosis. Academic thesis, Utrecht University, The
Netherlands.
10. Wayne, Z.G., et al. 1993. Serovar determination and
molecular taxonomic correlation in Mycobacterium avium,
Mycobacterium intrace1.1u1are, and Mycobacterium scrofulaceum:
a cooperative study on the internat_Lonal working group on
mycobacterial taxonomy. Int. J. Syst=em. Bacteriol. 43(3):482-
489.
11. McFadden, J.J., et al. 1987b. Determination of genome
size and DNA homology between unclassified mycobacterium
species isolated from patients with Crohn's disease and other
mycobacteria. J. Gen. Microbiol. 133:211-213.
12. Saxegaard, F., and T. Baess. 1988. Relationship between
Mycobacterium avium, Mycobacterium paratuberculosis, and
'wood pigeon mycabacteri~..' Acta Pai~hol. Microbiol. Immunol.
Scand. 96:37-42.
13. Yoshimura, H.H., and D.Y. Graham. 1988. Nucleic acid
hybridization studies of mycobactin~-dependent mycobacteria.
J. Clin. Microbiol. 26:1309-1312.
14. Van der Giessen, J.GJ.B., et al.. 1992. Evaluation of the
abilities of three diagnostic tests based on the polymerise
chain reaction to detect Mycobacterium paratuberculosis in
cattle: application in a control program. J. Clin. Microbiol.
30(5):1216-1219.
25. Millar, D.S., et al. 1995. Solid-phase hybridization
capture of low-abundance target DNA sequences: application to
the polymerise chain reaction detection of Mycobacterium
paratuberculosis and Mycobacterium ~avium subsp. silvaticum.
Anal. Biochem. 226(2):325-330.
CA 02353580 2001-06-O1
WO 00/34517 PCT/NL99/00741
16. Kunze, Z.M., et al. 1991. IS90:L, a new member of a
widespread class of atypical insertion sequences, is
associated with pathogenicity in Mycobacterium avium. Mol.
Microbiol. 5(9):2265-2272.
5 17. Moss, M.T., et al. 1992. IS902,, an insertion element in
the chronic enteritidis-causing Mycobacterium avium subsp.
silvaticum. J. Gen. Microbiol. 138:139-145.
18. Frothingham, R., et al. 1994. l~xtensive DNA sequence
conservation throughout the Mycobacterium tuberculosis
10 complex. J. Clin. Microbiol. 32(7):1639-1643.
19. Thoresen, O.F. and I. Olsaker. 1994. Distribution and
hybridization patterns of the insertion element IS900 in
clinical isolates of Mycobacterium ,paratuberculbs.is. Vet.
Microbiol. 40:293-303.
I5 20. Bauerfeind, R., et al. 1996. Molecular characterization
of Mycobacterium paratubercu3osis isolates from sheep, goats,
and cattle by hybridization with a DNA probe to insertion
element IS900. J.Clin. Microbiol. 34(7):1617-1621.
21. Kunimoto, D., et al: 1994. Sequence homologous to IS900
20 present in some M. avium (abstract). Am. Soc. Microbiol.
9:182.
22. Roiz, .M.P., et al. 1995. Use of restriction fragment
length polymorphism as a genetic marker for typing
Mycobacterium avium strains. J. Clin. Microbiol. 33:1389-
25 1391.
23. E1-Zaatari, F.A.K., et al. 1997: Detection of
Mycobacterium avium complex in cerebrospinal fluid of a
sarcoid patient by specific polymerase chain reaction. Stand.
J. Infect. Dis. 29:202-204.
30 24. Widjojoatmodjo; M.N., et al. 1992. The magnetic immuno
polymerase chain reaction assay for direct detection of
salmonellae in fecal samples. J Clin Microbiol 30:3195-3199.
25. Varela, P., et al. 1994. Direct detection of Vibri.o
choZerae in stool samples. J Clin Mficrobiol 32:1246-1248
CA 02353580 2001-06-O1
WO 00/34517 PCTINL99/00741
51
26. Van der Giessen, J.W.B. 1993. A malecular approach to
the diagnosis and control of bovine paratuberculosis.
Academic thesis, Utrecht University, The Netherlands.
27. Evans, K.D., et al. 1992. Identification of
Mycobacterium tuberculosis and Mycobacterium avium - M.
intracellulare directly from primary BACTEC cultures by using
acridinium ester labeled DNA probes.. J. Clin. Microbiol.
30:2427-2431.
28. Sockett, D.C., et a1. 1992. Evaluation of conventional
and radiometric fecal culture and a. commercial DNA probe for
diagnosis of Mycobacterium paratubE~rcu.Iosis infections in
cattle. Can. J. Vet. Res. 56(2):148-153.
29. Cousins, D.V., et al. 1995. Use of BACTEC radiometric
culture method and polymerase chain reaction for the rapid
screening of faeces and tissues for Mycobacterium
paratuberculosis. Aust. Vet. J. 72(12):458-462
30. Whittington, R.J., et al. 1998. Rapid detection of
Mycobacterium paratuberculosis in clinical samples from
ruminants and in spiked ~nvironment;al samples by modified
BACTEC 12B radiometric culture and direct confirmation by
IS900 PCR. J. Clin. Microbiol. 36(3):702-707.
31. Grant, I.R., et al. 1998. Isolation of Mycobacterium
paratubercu.tosis from milk by immunomagnetic separation.
Appl. Environ. Microbiol. 64 (9) :31Ci3-3158.
32. Poupart, P., et al. 1993. Preparation of a specific RNA
probe for detection of Mycobacterium paratuberculosis and
diagnosis of Johne's disease. J. C7_in. Microbiol. 31(6):1601-
1605.
33. Gormley, E., et al. 1997. Identification and
differentiation of mycobacteria us~_ng the PAN promotor
sequence from Mycobacterium paratuberculos.is as a DNA probe.
FEMS Microbiol. Lett. 14711):63-68..
34. Ellingson, J.L., et al. 1998. Identification of a gene
unique to Mycobacterium avium subspecies paratuberculosis and
CA 02353580 2001-06-O1
WO 00/34517 PCT/NL99/00741
52 ,
application to diagnosis of paratube:rculosis. Mol. Cell.
Probes 12(3):133-142.
35. Van der Giessen, J.W.B., et al. 1992. Amplification of
16S ribosomal RNA sequences to detects Mycobacterium
paratuberculasis. J. Med. Microbiol. 36:255-263.
36. Van der Giessen, J.W.B., et a1. 1.994. Comparison of the
23S ribosomal RNA genes and the spacer region between the 16S
en 23S rRNA genes of the closely re7_ated Mycobacterium avium
and Mycobacterium paratuberculosis and the fast-growing
Mycobacterium phlei. Microbiology (LIK) 140:1103-1108.
37. Stone, B.B., et al. 1995. Comparison of Mycobacterium
23S rRNA sequences by high-temperature reverse transcription
and PCR. Int. J. System Bacteriol. 45:811-819.
38. Pace, N.R., et al. 1985. Analy~:ing natural microbial
populations by rRNA sequences. ASM News 51:4-12.
39. Woese, C.R. 1987. Bacterial evolution. Microbiol. Rev.
51:221-271.
40. Olsen, G.J., et al. 19.86. Microbial ecology and
evolution: a ribosomal RIBA approach,. Ann. Rev. Microbiol.
40:337-365.
41. Frothingham. R, et al. 1993. SE:quence-based
differentiation of strains in the Mycobacterium avium
complex. J. Bacteriol. 175(10):2818--2825.
42. Glennon, M., et al. 1994. The ribosomal intergenic
spacer region: a target for the PCR based diagnosis of
tuberculosis. Tuber. Zung Dis. 75(5;?:353-360.
43. Frothingham, R and K.H. Wilson.. 1994. Molecular
phylogeny of the Mycobacterium avium complex demonstrates
clinically meaningful divisions. J. Infect. Dis. 169(2):305-
312.
44. Ji, Y.E., et al. 1994. Nucleotide sequences of the
spacer-1, spacer-2 and trailer regions of the rrn operons and
secondary structures of precursor 2:35 rRNAs and precursor 5S
CA 02353580 2001-06-O1
WO 00/34517 PCTINL99/00741
53 '
rRNAs of slow-growing mycobacteria. Microbiology.
140(Pt7):1763-1773.
45. Bourque, S.N., et al. 1995. Comparative analysis of the
16S to 23S ribosomal intergenic spacer sequences of Bacillus
thuringiensis strains and subspecies and of closely related
species. Appl. Environ. Microbiol. E~1(4):1623-1626.
46. De Wit, M.Y.L. and P.R,. Klatser. 1994. Mycobacterium
leprae isolates from different sources have identical
sequences of the spacer region between the 16S and 23S
ribosomal RNA genes. Microbiology 140:1983-1987
47. Scheibl, P. and G.F. Gerlach. 1997. Differentiation of
Mycobacterium paratuberculosis isolates by rDNA-spacer
analysis and random amplified polymorphic DNA patterns. Vet.
Microbiol. 57 (2-3) :151-158.
~.5 48. Mullis et al. 1987. Process for amplifying, detecting,
and/or-cloning nucleic acid sequences. US patent 4,683,195.
49.Enns, R.K. 1987. Clinical studies summary report: the Gen-
Probe~ rapid diagnostic system for i~he Mycobacterium avium
complex. Gen-Probe, Inc.; San Diego,, Calif.
50. Jonas, V., et al. 1993. Detection and identification of
Mycobacterium tuberculosis directly from sputum sediments by
amplification of rRNA. J. Clin. Mic:robiol. 31(9):2410-2416.
51. Compton, J. 1991. Nucleic acid sequence-based
amplification. Nature (London) 350:!al-92.
52. drum, H., et al. 1993. Single base pair mutation
analysis by PNA directed PCR clamping. Nucleic Acids Res.
21:5332-5336.
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Table 1. Organisms used in this study
Mycobacterium avium subspecies paraauberculosis strain GDR
Mycobacterium avium subspecies para;tuberculosis strain DSM
44135
Mycobacterium avium subspecies paraauberculosis strain Spbov
E5
Mycobacterium avium subspecies paravtuberculosis strain ATCC
19698 (Type strain)
Mycobacterium avium subspecies avium strain 23435
Mycobacterium avium subspecies avium strain DSM 44158
Mycobacterium avium subspecies avium strain DSM 43216
Mycobacterium avium subspecies avium strain DSM 44157
Mycobacterium avium subspecies avium strain ATCC 25291 (Type
l5 strain)
Mycobacterium avium subspecies avium strain 97-613
Mycobacterium avium subspecies silvaticum strain DSM 44175
(Type strain)
Mycobacterium intracellu~are strain ATTC 13950 (Type strain)
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TablE: 2
Primers for point-mutation PCR. The site of the "mutation" is
indicated. Primers with code "F" are forward primers, those
5 with code "R" are reverse primers.
Code sequence (5'-> 3') Td (C)
20F CTGAATAGGGCGCATCCC 60
19F TGAATAGGGCGCATCCC~T 58
10 1F GAATAGGGCGCATCCCTT 58
3F AATAGGGCGCATCCGTTT 56
5F ATAGGGCGCATCCCTTTG 58
7F TAGGGCGCATCCCTTTGG 60
9F AGGGCGCATCCCTTTGGG 62
15 11F GGGCGCATCCCTTTGGGG 64
13F GGCGCATCCCTTTGGGGT 62
22R CTCCCTCCACCACCG 54
21R TCCCTCCACCACC T 52
15R CCCTCCACCACCG'~:aC 54
20 16R CCTCCACCACCGTCA 52
17R CTCCACCACCC-TCAC 52
18R CACCACC TCACCCG 54
22Ra CACCCTCCACCACC 54
2lRa ACGCTGCACCACCC~T 52
