A robust method for bacterial lysis and DNA purification to be used with real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in milk.
ABSTRACT A possible mode of transmission for the ruminant pathogen Mycobacterium avium subsp. paratuberculosis (MAP) from cattle to humans is via milk and dairy products. Although controversially, MAP has been suggested as the causative agent of Crohn's disease and its presence in consumers' milk might be of concern. A method to detect MAP in milk with real-time PCR was developed for screening of bulk tank milk. Pellet and cream fractions of milk were pooled and subjected to enzymatic digestion and mechanical disruption and the DNA was extracted by automated magnetic bead separation. The analytical sensitivity was assessed to 100 organisms per ml milk (corresponding to 1-10 CFU per ml) for samples of 10 ml. The method was applied in a study of 56 dairy herds to compare PCR of farm bulk tank milk to culture of environmental faecal samples for detection of MAP in the herds. In this study, 68% of the herds were positive by environmental culture, while 30% were positive by milk PCR. Results indicate that although MAP may be shed into milk or transferred to milk by faecal contamination, it will probably occur in low numbers in the bulk tank milk due to dilution as well as general milking hygiene measures. The concentration of MAP can therefore be assumed to often fall below the detection limit. Thus, PCR detection of MAP in milk would be more useful for control of MAP presence in milk, in order to avoid transfer to humans, than for herd prevalence testing. It could also be of value in assessing human exposure to MAP via milk consumption. Quantification results also suggest that the level of MAP in the bulk tank milk of the studied Danish dairy herds was low, despite environmental isolation of MAP from the herds.
- SourceAvailable from: Monika Dzieciol[Show abstract] [Hide abstract]
ABSTRACT: Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of paratuberculosis (Johne's disease) in ruminants and is suggested to be one of the etiologic factors in Crohn's disease in humans. Contaminated milk might expose humans to that pathogen. The aim of the present study was to develop a novel real-time PCR assay providing the additional possibility to detect viable Mycobacterium avium subsp. paratuberculosis (MAP) based on the MAP-specific Mptb52.16 target. The design included an internal amplification control to identify false negative results. Inclusivity and exclusivity tested on 10 MAP strains, 22 non-MAP mycobacteria, and 16 raw milk microflora strains achieved 100%. The detection limit in artificially contaminated raw milk was 2.42 × 101 MAP cells/ml milk. In a survey of naturally contaminated samples obtained from dairy herds with a known history of paratuberculosis, 47.8% pre-milk and 51.9% main milk samples tested positive. Real-time PCR-derived MAP-specific bacterial cell equivalents (bce) ranged from 1 × 100 to 5.1 × 102 bce/51 ml; the majority of samples had less than one bce per ml milk. Expression of the chosen target was detected in artificially contaminated raw milk as well as inoculated Dubos broth, thus confirming the real-time PCR assay's potential to detect viable MAP cells. Concentrating the DNA of a large sample volume in combination with the newly developed real-time PCR assay permitted quantification of low levels of MAP cells in raw milk and pasteurized milk. The selected target - Mptb52.16 - is promising with regard to the detection of viable MAP. Future studies integrating quantitative DNA- and RNA-based data might provide important information for risk assessment concerning the presence of MAP in raw milk and pasteurized milk.BMC Research Notes 10/2010; 3:251.
- Revista medica de Chile 06/2011; 139(6):794-801. · 0.36 Impact Factor
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ABSTRACT: Molecular diagnostic tests are widely implemented in animal and human health microbiology. Efficient nucleic acid extraction methods are essential in diagnostic laboratories and automation is a valuable tool for those with high throughput activity. Nucleic acid extraction protocols present variable efficiency depending on the composition of the specimen and the chemical-physical characteristics of the target pathogen. In the present study, we compared the DNA extraction performances of four automated methods (kits I, M, P, Q) adapted on a Hamilton Robotics “Microlab Starlet” extraction unit, and one manual method (kit R). Ten-fold dilutions of Mycobacterium avium subsp. paratuberculosis (MAP) were used to contaminate bovine central nervous system (CNS) and lung-spleen-liver pools (LSL). In consideration of its chemical-physical characteristics, MAP was selected as a template for pathogen DNA detection. Analytical performance and repeatability were assessed through downstream real-time PCR amplification, hands-on time (HOT), total turnaround time (TAT) and costs of all kits. MAP was detected differently depending on extraction kit and type of matrix analysed. Kits M and I showed the highest analytical performance on CNS (1MAP/ml) and LSL (10MAP/ml), respectively. Besides analytical results, kits I and M displayed high repeatability, the same HOT, very similar TAT, and were inexpensive. In conclusions, different standardized automated systems have been established with high throughput, sensitivity and repeatability for CNS and LSL. Our results also demonstrated that is necessary to assess the effectiveness of extraction kits in matrices not previously tested to avoid the risk of unreliable diagnostic outcomes. KeywordsAutomated–DNA extraction–Evaluation–IS900– Mycobacterium avium subsp. paratuberculosis –Real-time PCRWorld Journal of Microbiology and Biotechnology 01/2011; 27(1):31-37. · 1.26 Impact Factor
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A robust method for bacterial lysis and DNA purification to be used with real-time
PCR for detection of Mycobacterium avium subsp. paratuberculosis in milk
David Herthneka, Søren Saxmose Nielsenb, Ann Lindberga, Göran Bölskea,⁎
aNational Veterinary Institute (SVA), SE-751 89 Uppsala, Sweden
bDepartment of Large Animal Sciences, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark
a b s t r a c ta r t i c l ei n f o
Received 13 March 2008
Received in revised form 10 July 2008
Accepted 17 July 2008
Available online 20 July 2008
Mycobacterium avium subsp.
A possible mode of transmission for the ruminant pathogen Mycobacterium avium subsp. paratuberculosis
(MAP) from cattle to humans is via milk and dairy products. Although controversially, MAP has been
suggested as the causative agent of Crohn's disease and its presence in consumers' milk might be of concern.
A method to detect MAP in milk with real-time PCR was developed for screening of bulk tank milk.
Pellet and cream fractions of milk were pooled and subjected to enzymatic digestion and mechanical
disruption and the DNA was extracted by automated magnetic bead separation. The analytical sensitivity was
assessed to 100 organisms per ml milk (corresponding to 1–10 CFU per ml) for samples of 10 ml.
The method was applied in a study of 56 dairy herds to compare PCR of farm bulk tank milk to culture of
environmental faecal samples for detection of MAP in the herds. In this study, 68% of the herds were positive
by environmental culture, while 30% were positive by milk PCR.
Results indicate that although MAP may be shed into milk or transferred to milk by faecal contamination, it
will probably occur in low numbers in the bulk tank milk due to dilution as well as general milking hygiene
measures. The concentration of MAP can therefore be assumed to often fall below the detection limit. Thus,
PCR detection of MAP in milk would be more useful for control of MAP presence in milk, in order to avoid
transfer to humans, than for herd prevalence testing. It could also be of value in assessing human exposure to
MAP via milk consumption. Quantification results also suggest that the level of MAP in the bulk tank milk of
the studied Danish dairy herds was low, despite environmental isolation of MAP from the herds.
© 2008 Elsevier B.V. All rights reserved.
Mycobacterium avium subsp. paratuberculosis (MAP) is the causa-
tive agent of paratuberculosis or Johne's disease, a wasting disease of
ruminants with worldwide occurrence. This chronic disease has avery
long incubationperiod; in cattle usually varying from twoto tenyears.
Manifestations of the diseasearereducedmilkproduction, weightloss
and in later stages emaciation and diarrhea. The infection sites are
mainly the intestine and mesenteric lymph nodes, but intracellular
MAP in macrophages may circulate to other body sites. MAP has been
found in low numbers in many body locations, such as lymph nodes,
spleen and liver (Larsen et al., 1981; Wu et al., 2007) as well as in
semen (Ayele et al., 2004; Larsen and Kopecky, 1970), milk and milk
products (Ayele et al., 2005; Corti and Stephan, 2002; Grant et al.,
1998; O'Reilly et al., 2004; Stephan et al., 2007; Sweeney et al., 1992;
Taylor et al., 1981). During the progression of the disease, the animal
starts shedding MAP in the faeces. At first, usually intermittently but
in later stages, constantly and in high numbers, possibly more than
108colony forming units CFU/g faeces (Chiodini et al., 1984).
Dalziel (1913) was the first to suggest a link between MAP and
then, the etiology of the disease has been disputed. While some
2000; Uzoigwe et al., 2007), the majority of studies only suggest an
association between the disease and the organism, while considering
the causative potential of the latter unclear (Behr and Schurr, 2006;
Grant, 2005; Sartor, 2005; Shanahan and O'Mahony, 2005). However,
the fact that it has been found in infected cows' milk is one reason to be
concerned about the status of MAP as a possible zoonotic pathogen.
MAP has been shown to be able to survive experimental
pasteurization to some extent (Chiodini and Hermon-Taylor, 1993;
Grant et al., 1996; Grant et al., 2002b; Grant et al., 2005), although it
has been suggested that flawed modeling of the commercial
pasteurization process could allow a residual of the bacteria to
survive, while proper pasteurization does not (Rademakeret al., 2007;
Stabel et al.,1997). Yet reports claim to have isolated or found signs of
viable MAP in retail milk (Anon., 2000; Ayele et al., 2005; Ellingson
et al., 2005; Grant et al., 2002a; Millar et al., 1996). Several
explanations for the different results and conclusions in the above
and other related studies, such as strain dependence, varying degrees
Journal of Microbiological Methods 75 (2008) 335–340
⁎ Corresponding author Tel.: +46 18 674266.
E-mail address: email@example.com (G. Bölske).
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of cell clumping and difficulties of MAP culture, were suggested by
Klijn et al. (2001).
The most likely source of MAP in milk is considered to be faecally
(Stabel, 2000; Streeter et al., 1995; Sweeney et al., 1992). Information
about the amount of MAP in milk is sparse, however clinically ill cows
have been reported to typically excrete less than 100 CFU/ml in milk
(Giese and Ahrens, 2000) while milk from asymptomatic animals may
contain 2–8 CFU/50 ml (Sweeney et al., 1992), or even less. Due to the
fastidiousness of MAP, its tendency to aggregate and problems involved
with culture of MAP from milk, these figures are likely to be
underestimated (Grantand Rowe,2001).Theamountof MAPinfaecally
contaminated milk is hard to predict, since it depends on the amountof
2007). Considering the potentially very high concentration of MAP in
faeces, contaminated milk could contain much more MAP than the
above cited 100 CFU/ml. However, after mixing the contaminated milk
withmilk from non-infectedcows,MAPinthebulktankmilkis likelyto
be diluted to very low levels. Due to the random nature of faecal
contamination, the MAP status of the tank milk of a farm is likely to
change over time.
It may be useful to monitor the presence of MAP in bulk tank milk
from dairy herds in order to determine the level of MAP in milk
delivered for processing of consumers' milk. Detecting MAP in the
tankmilk mightalso be a methodforherd testing for paratuberculosis.
Due to the difficulties of faecal culture and the prolonged time it takes
to receive the final culture results, a sensitive PCR could be an
attractive alternative. Assessment of the prevalence and quantity of
MAP in tank milk can provide quantitative information regarding the
magnitude of human exposure via this route.
proper lysis of the cells, preferably achieved by mechanical disruption
(Hermon-Taylor et al., 2000; Herthnek, 2006; Lanigan et al., 2004;
Odumeru et al., 2001). This is critical for a high sensitivity, which is
necessary since concentrations of MAP in milk tend to be very low.
Efficient removal of PCR inhibitors, such as Ca2+ions in the case of milk,
and a way to monitor the presence of inhibition are equally important.
In this study we develop an extraction procedure for PCR on milk.
The procedure, followed by real-time PCR, is applied to bulk tank milk
from herds with and without paratuberculosis infection in order to
compare it to environmental fecal culture for herd screening. It was
also used for measurement of MAP contamination level in farm milk
intended for human consumption.
2. Materials and methods
2.1. Preparation of spiked milk
The MAP type strain (ATCC 19698) was cultured for eight weeks on
Löwenstein-Jensen medium, supplemented with mycobactin (4 mg/l,
Allied Monitor, Fayette, MO, USA). The colonies of one tube were
harvested and the bacteria were dispersed, washed free of excessive
free DNA and quantified in a microscope as previously described
(Herthnek et al., 2006). The suspension of MAP was then serially
diluted and added to milk from a Swedish herd that never tested
positive for paratuberculosis to obtain spiked milk samples with final
concentrations of 1–104organisms/ml. The samples were frozen and
later used for DNA extraction and real-time PCR as described below,
both for assessment of analytical sensitivity, quantification of MAP in
the clinical samples and for testing various parameters during the
development of the extraction method.
2.2. Herds and sampling
The study population included 56 dairy herds delivering milk to an
organic milk collection center located in central Jutland, Denmark. A
total of 65 herds delivered milk to this center, but six herds chose not
toparticipate andfrom three herds,bulkmilk samplesforPCR analysis
could not be obtained.
The median herd size for the period 1 October 2005 to 30
September 2006 was 92 cows (range: 12–296) and the average annual
milk yield during the same time was 7009 kg/cow (range: 4337–
9964). Herds were predominantly classified as Danish Jersey (50%)
andDanishHolstein (39%). Remainingherds wereeitherofDanish Red
breed or crossbreeds.
From each herd, environmental samples of mainly faeces were
collected one to three times, according to the following scheme: in
April 2006, all herds were visited, and environmental samples were
collected from six locations in the cows' areas. If all six samples were
negative or overgrown by contaminating flora, the herds were visited
again in September 2006. If all samples were negative on the second
visit, they were visited again in December 2006. Six samples were
collected on each sample date. Environmental samples were collected
from the near environment of the cows. In herds with loose housing
systems, three samples were collected from the site where the cows
enter the milking facility and three samples were taken at different
sites in the cow barn where all cows had to pass by, for example at
drinking troughs or feeding passages. In herds with tie stalls, six
samples were collected at different places behind the cows. By the
nature of the sampling method, all samples represented a pool of
faeces from several cows. Culture of environmental samples was
performed on Herrold's Egg Yolk Medium, as described previously
(Nielsen et al., 2004).
Milk for PCR testing was sampled from the bulk milk cooling tank
on one to three occasions from the 56 herds; all in all, 143 samples
were collected. Seven herds were sampled only once, and 13 herds
were sampled only twice, while the remaining herds were tested
three times. Eight samples contained an insufficient volume of ~5 ml,
instead of 10 ml, but were still included for analysis.
A small number of milk samples (n=8) from Swedish farms that
have acquired A status in the Swedish control program (Sternberg
et al., 2002) could be obtained and tested as negative controls. Most
farms in the program did not produce milk, hence the low number of
2.3. Extraction of DNA from milk samples
Frozen milk was thawed overnight at 8 °C. A volume of 10 ml was
transferred to tubes and centrifuged at 3000 g for 30 min at 10 °C. The
solid cream fraction of each tube was transferred with a loop to plastic
jars with lids and the whey fractions were discarded. The remaining
pellets, possibly containing precipitated casein, were incubated at
37 °C for 30–60 min (depending on the amount of precipitation) and
centrifuged at 3000 g for 15 min at room temperature. The newcream
fractions were either discarded (if a large amount of cream was
already harvested) or added to the plastic jars. If the milk was not too
acidic, the casein had been dissolved and could be discarded. The
openings of each tube were briefly touched against soft paper tissue,
leaving the pellets with as little liquid as possible. A mixture of 1000 µl
lysis buffer (2 mM EDTA, 400 mM NaCl, 10 mM Tris–HCl pH 8.0, 0.6%
SDS) and 2 µl of proteinase K (10 µg/µl, Sigma-Aldrich, St. Louis, MO,
USA) was used to dissolve the cream fraction in each jar. The dissolved
cream was transferred to the 10 ml-tubes and pooled with the
remaining pellets. The mixture was transferred to bead beating tubes
(2.0 ml, BioSpec Products, Inc., Bartlesville, OK, USA) containing 600 µl
of beads (0.1 mm zirconia/silica beads, BioSpec Products, Inc.), taking
care not to overfill them.
The tubes were incubated at 56 °C for 1 h, shaken in a cell disrupter
(MiniBeadbeater-8™, BioSpec Products) at 3200 rpm for 1 min and
centrifuged at 10,000 g for 1 min. Avoiding the cream fraction, 400 µl
of the liquid phase was transferred to sample tubes from the EZ1 DNA
Tissue Kit (Qiagen, Hilden, Germany). With the exception of the
D. Herthnek et al. / Journal of Microbiological Methods 75 (2008) 335–340
Author's personal copy
increased sample volume, the instructions for this kit was followed
and the samples were automatically processed in the BioRobot® EZ1
workstation (Qiagen), set at the bacteria protocol, an elution volume
of 50 µl and a sample volume of 200 µl.
During the period of testing, some improvements were made to the
method. The amounts of lysis buffer and beads used for bead beating
were increased, as was the volume of lysate added to the robot. The
template volume used for PCR was also changed from 2 µl to 5 µl.
Before completion of method development, a comparison of DNA
yield from pooled cream and pellet with that of only pellet was
performed on milk spiked with 1000 MAP/ml. Milk samples and PCR
tubes were in duplicates.
2.4. Real-time PCR
The primers and probes used for primary detection were those of
the previously described MP system on IS900, developed by PTJ
Willemsen (Herthnek and Bölske, 2006; Herthnek et al., 2006). The
PCR mixture for each reaction comprised of 8.625 µl or 5.625 µl of H2O
(Sigma-Aldrich) depending on the volume of template used, 2.5 µl of
10× PCR-buffer II (Applied Biosystems, Foster City, CA, USA), 5.0 µl of
MgCl2(25 mM, Applied Biosystems), 2.0 µl of GeneAmp® dNTP with
dUTP (2.5 mM each of dATP, dCTP, dGTP and 5.0 mM of dUTP, Applied
Biosystems), 0.75 µl of forward primer (10 µM), 0.75 µl of reverse
primer (10 µM), 0.5 µl of MAP-specific probe (10 µM), 0.5 µl of mimic-
specific probe (10 µM), 0.125 µl of AmpliTaq Gold® (5 U/µl, Applied
Biosystems) and 0.25 µl of AmpErase® (Uracil N-glycosylase, 1 U/µl,
Applied Biosystems). To this mixture, 2 or 5 µl of template DNA and
2 µl of the internal control plasmid pWIC9 (Herthnek et al., 2006) was
added, for at total volume of 25 µl. This was always performed in
duplicate. The concentration of pWIC9 was first calibrated against low
concentrations of MAP DNA to make sure that its reaction would not
compete with the target reaction. The real-time PCR reaction was
performed on a Rotor-Gene 3000™ (Corbett Research, Mortlake,
Australia) with the same program and analysis methods as previously
described (Herthnek et al., 2006).
2.5. Absolute quantification of MAP in milk with real-time PCR
DNA from duplicates of spiked milk samples for each MAP
concentration level was extracted as described above and run in
duplicates in the real-time PCR. From the resulting Ct-values (Cycle
threshold; number of PCR cycles needed for the fluorescence to reach
a certain threshold), a standard curve for MAP content in milk was
produced. Ct-values from positive milk samples in other runs were
adjusted according to the difference of the Ct-values of the positive
controls in the current run and those in the run with spiked samples.
MAP content in the tested milk was quantified using these adjusted
Ct-values with the standard curve.The calculated concentrations were
given in MAP/ml, not CFU/ml, since real-time PCR disregards the state
of viability of MAP.
2.6. Contamination precautions and confirmation
A number of steps were taken to avoid false positives in the
evaluation of the method. All laboratory work, except for the loading
of the EZ1 robot, was carried out in laminarair flowcabinets. Standard
procedures, such as the use of separate rooms for extraction, PCR
mixture preparation and PCR amplification, and the use of PCR
negative controls, were followed. PCR tubes were never re-opened
after amplification. In addition, the carry-over prevention kit with
GeneAmp® dNTP with uracil and AmpErase® Uracil N-glycosylase
(UNG) (Applied Biosystems) was used to remove any carry-over DNA
that, in spite of our careful handling of the samples, would
accidentally have leaked from tubes in previous runs. Positive controls
were added after all other tubes had been closed.
When faced with a MAP positive result by milk PCR, extensive
work was carried out both to confirm the identity of the MAP by
alternative PCR systems and to confirm the source of MAP to the milk
sample, by preparing and testing another 10 ml of the same milk. The
preferred alternative PCR system to confirm the primary PCR
detection was the previously described DH3 system on the MAP-
specific gene F57, but when considered not sensitive enough for very
low MAP concentrations, due to the single copy number of F57, the
DH1 system on the IS900 was used. DH1 targets a different part of the
IS900, that evades presently known cross-reacting strains. (Herthnek
and Bölske, 2006).
The analytical sensitivity of the new milk PCR method was
assessed to 100 organisms/ml for samples of 10 ml, although MAP
could also be detected in roughly three of four samples spiked with 10
organisms/ml. Note that 100 organisms of MAP correspond to less
than 5 CFU (Herthnek et al., 2006).
In the comparison of DNA extraction from milk with and without
cream fraction, the DNA from the pellet extraction yielded the Ct-
values 34.7, 35.0, 34.6 and one negative, while DNA from the pooled
pellet and cream yielded the Ct-values 33.7, 33.1, 32.7 and 32.8. On
average, this was 1.8 cycles earlier thanwhen the creamwas discarded
(P=0.044), when not counting the one negative.
Of the 143 bulk milk samples from the 56 herds, 19 samples from
17 herds (30%) were PCR positive by the primary MP real-time PCR
system. Results of environmental culture, which were positive in 38
herds (68%), were used as reference. Of the 89 milk samples from
these positiveherds,18samples (20%)weremilk PCRpositive.On herd
level,16 (42%) of the 38 culture positive herds were positive with milk
PCR, as seen in Table 1.
Ct-values yielded by the positive milk samples were always quite
high (34.2–40.9 cycles). By absolute quantification of the MAP content
in the milk, it appeared that MAP concentrations in the bulk milk
Comparison of the number of herds positive for MAP, using bulk milk PCR and
Milk PCREnvironmental CultureTotal
Fig.1. Estimated MAP content in positive milk, quantified by real-time PCR with spiked
milk as reference. When a sample was positive in only one replicate, it is here indicated
by one single data point. When there were several positive reactions, concentrations
calculated from the different Ct-values are in the span between the lower and higher
D. Herthnek et al. / Journal of Microbiological Methods 75 (2008) 335–340
Author's personal copy
samples rarelyexceeded 100 organisms/ml (Fig.1), which was alsothe
assessed detection limit. Forconcentrations under this limit, a positive
reaction with only two PCR replicates cannot be expected. This
observation was supported by the fact that positive PCR reactions
were often difficult to reproduce and in some cases, positive samples
could not be confirmed at all, due to lack of material to run further
replicates (Table 2).
The proportion of culture positive environmental samples out of
the 6–18 environmental samples cultured (and not overgrown) was
positivelycorrelated withthe proportionof positivemilk samples,and
also with the degree to which the positive milk samples could be
confirmed. In the group of culture positive herds where half or less of
the environmental culture samples were positive, 21% were PCR
positive by tank milk PCR. In the group where more than half of the
environmental culture samples were positive, 54% were positive by
milk PCR. Onlyamong these, the positive PCR could be confirmed both
on alternative PCR and by preparation of a new sample. Among farms
with all environmental culture samples positive (six out of six), 67%
were milk PCR positive.
The negative control milk samples from Swedish farms were
negative for MAP.
The choice of milk volume used in the protocol was based on the
assumption that the likelihood of detection of a scarce pathogen, and
thus the sensitivity of the test, will increase with increased sample
volume. We also acknowledged the fact that a larger volume is harder
to handle. The pellet and cream of 10 ml milk could readily be
dissolved and transferred to a 2 ml tube, while still having a
reasonably low viscosity. Although pretreatment with immunomag-
netic separation (IMS) (Grant et al.,1998; Khare et al., 2004; Metzger-
Boddien et al., 2006; O'Reilly et al., 2004) and peptide-mediated
capture (Stratmann et al., 2006; Stratmann et al., 2002) have
previously been applied for concentration of MAP and separation of
MAP from PCR inhibitors in milk, our approach was instead to capture
the nucleic acids of MAP after processing of the complete sample,
including the cream. As long as PCR-inhibitory substances are
successfully removed, this should reduce the risk of losing MAP that
do not attach to the magnetic beads used in the formerly mentioned
Previous studies have shown that MAP, with its fatty cell wall, may
segregate to both the pellet and the cream fraction upon centrifuga-
tion and that these fractions should be pooled to achieve the highest
yield of MAP (Gao et al., 2007; Gao et al., 2005; Millar et al., 1996;
Singh et al., 2007). Ourlimited experimenton spiked milk agreed with
this hypothesis. It should, however, be noted that MAP may behave
differently in milk of different fat content and in different storage
conditions. More replicates and different kinds of milk could have
been included in a more thorough comparison, if there would be
doubts that discardingof thecreamindeed decreases theyield of MAP.
Depending on the age and probably the protein content of milk
(and potentially other factors), centrifugation may cause a large
protein precipitate to pellet. If the milk had curdled, this precipitate
could be difficultorevenimpossible to dissolve, which is why the milk
needed to be collected and frozen as fresh as possible. If the milk was
relatively fresh, however, a precipitate could still form, but this was
generally softer and would readily be dissolved by incubation at 37 °C
for 30–60 min after the cream and whey fraction had been removed.
This stepmadethemethodmore robustandtolerant todifferent kinds
To reduce the amount of manual processing, we utilized the
automated extraction robot EZ1 for purification of nucleic acids.
According to the protocol of the EZ1 DNA Tissue Kit, the robot should
be loaded with 200 µl material. However, loading it with 400 µl
material originating from spiked milk (1000 MAP/ml) did not overfill
the tubes during the extraction and the extracted DNA produced a
real-time PCR signal one cycle earlier, indicating an anticipated
twofold increase in MAP yield. An approach for MAP DNA extraction
with automation resembling ours was applied previously and
independently by Bosshard (2006), but excluding the cream of the
Although the bead beating process should have disrupted the
bacteria to expose the MAP DNA, it is plausible that some of the DNA
will still linger in the fat phase of the lysate, despite the hydrophilicity
of nucleic acids. By centrifuging and transferring only the liquid phase
to the robot, this DNAwould then be lost. However, it was shown that,
if the lysate was transferred to the robot without separation, fat
droplets would stick to the magnetic beads and thereby acting as
vehicles for PCR inhibitory substances, which is why centrifugation
after bead beating was the preferred method.
The somewhat laborious spiking method, described earlier
(Herthnek et al., 2006), was used in order to avoid overestimation of
the analytical sensitivity, which may happen if the difference between
actual bacterial cell number and CFU is not acknowledged, or if the
presence of free MAP DNA in a colony is not taken into account. Free
DNA that forms in MAP colonies may, if not removed, lead to wrong
conclusions regarding the efficiency of the lysis method used. Since
the method of detection does not rely on the viability of the cells,
while quantification by culture indeed does, the use of CFU when
assessing analytical sensitivity of molecular diagnostic methods is not
recommended, especially when working with MAP, which is particu-
larly fastidious. The difficulty to determine the number of CFU of MAP
due to the lack of standardization of MAP culture in general has
previously been acknowledged (Grant and Rowe, 2001; Klijn et al.,
When comparing the percentage of infected herds where MAP was
detected with the respective methods, environmental culture and
milk PCR, it should be considered that while one to three samples
from one herd were taken for milk PCR testing, up to 18 samples were
taken for culture.
A rigorous cross contamination control, as described above, was
necessary since the expected weak signals yielded by milk samples
containing small amounts of MAP will be impossible to discriminate
from low grade cross contaminations. We believe that we have thus
reduced the risk of cross contamination to a minimum. Although one
cannot exclude the possibility that a positive sample that could not be
confirmed by testing DNA from a new preparation was actually a false
positive, it is very unlikely. The only source for cross contamination
concentrations of MAP, such contamination would, when introduced in
another sample, probably be diluted far below the limit of possible
detection. However, due to the reported occurrence of IS900 positive
non-MAP organisms in milk (Taddei et al., 2005), the identity of MAP
should, preferably, be confirmed by one of our previously developed
alternative PCR systems (Herthnek and Bölske, 2006) when a milk
sample becomes positive by our primary system on the IS900 gene.
Except for using this milk PCR method on milk for the purpose of
controlling the milk for consumers, there were hopes that milk
screening would be an easy way to test prevalence of MAP in herds.
That would require that MAP would be shed or contaminated into milk
Number of positive milk samples, possible to confirm on secondary backup sample and
identity confirmed by an alternative PCR system, respectively
aDNA from primary or secondary preparation, positive with IS900 DH1 or F57 DH3.
D. Herthnek et al. / Journal of Microbiological Methods 75 (2008) 335–340
Author's personal copy
to a sufficient degree, to fall within the detection limit even when it is
diluted with MAP-free milk from other cows. Apparently, in most
studied herds with paratuberculosis, the concentrations of MAP in the
tank milk were too low to be readily detected, and when the samples
were positive, the concentrations were usually lower than 100 MAP/ml.
Therefore, although the analytical sensitivity is high, the diagnostic
sensitivity remains low. There may be herds in other countries with
other clinical and management patterns, where a larger proportion of
the cows in a herd shed MAP in their milk and/or contaminate the milk
more due to less successful cleansing of the teats and udders, with
resulting higher concentrations of MAP in the tank milk. This should,
however, not be assumed to be the case. Thus, in our opinion, bulk tank
milk PCR result suggests that the herd is positive. Instead, milk PCR will
be more useful for control of MAP presence in consumers' milk. When
milk is tested together with spiked milk samples of known MAP
and together with data of MAP survival after pasteurization, the risk of
presence of viable MAP can be assessed. Rademaker et al. (2007)
reviewed several pasteurization studies and their respective reductions
of MAP viability in a table (4–7 log reduction) and Klijn et al. (2001)
investigated the causes of the different levels of reduction. Without
speculating about the potential hazard that some surviving MAP would
with similar disease status and hygienic standard as the Danish farms
tested in this study would be unlikely to contain any viable MAP if
pasteurized with commercial methods.
This study was supported by grants from the Swedish Farmers'
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