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TITLE: Combining culture techniques for Bartonella: The best of both worlds 1
Authors: Tarah Lynch, Jennifer Iverson, Michael Kosoy* 2
3
Affiliation: Centers for Disease Control and Prevention, Division of Vector Borne Diseases, Fort 4
Collins, Colorado, USA 5
Running title: Culture of Bartonella 6
Key words: Bacteria, Bartonella, culture, isolation, B. quintana, B. henselae, B. elizabethae, B. 7
tamiae 8
9
10
11
12
*Corresponding author: Dr. Michael Kosoy 13
Centers for Disease Control and Prevention, Division of Vector Borne Diseases 14
3150 Rampart Road, Fort Collins, CO, USA 80521 15
Ph: 970-266-3522 16
E: [email protected] 17
18
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.02403-10 JCM Accepts, published online ahead of print on 2 February 2011
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ABSTRACT 19
In this study we compared some common Bartonella culturing methodologies using four diverse 20
species causing human illnesses. Based on a review of the literature, we focused on three major 21
inconsistencies between protocols: base media, cell co-culture and temperature. Our data showed 22
that B. tamiae demonstrated temperature-dependent growth limitations between common 23
culturing conditions only 2oC apart. Additionally, growth of B. quintana was significantly 24
enhanced by the presence of mammalian cell co-culture within mammalian culture conditions, 25
however when the media was modified to incorporate insect culture-based media, co-culturing 26
with mammalian cells was no longer needed. In this study, we were able to overcome these 27
temperature and cell dependent limitations and accommodate all of the strains tested by 28
combining mammalian culture-based media with insect culture-based media. 29
INTRODUCTION 30
Bartonella is a genus of gram negative, facultative intracellular bacteria that have been detected 31
in a plethora of insect and mammalian hosts (5, 8, 14, 19, 23, 26). Numerous Bartonella species 32
have been associated with clinical illnesses ranging from mild skin lesions to more severe 33
manifestations including persistent fevers, neurological symptoms and endocarditis (6, 10). 34
These bacteria can be fastidious under current laboratory conditions and attempts to isolate pure 35
culture from biological specimens are often unsuccessful despite positive molecular detection (1, 36
7). 37
The majority of culturing methods for Bartonella species found in the literature focus on growth 38
requirements for the two species commonly associated with human infection, B. henselae and B. 39
quintana (3, 9, 22, 28, 36). Through a review of the literature on Bartonella isolation methods 40
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from biological samples, we found many differences in the culturing protocols. To summarize, 41
most laboratories use co-cultivation with mammalian cells or an axenic, insect culture-based 42
media or plating onto blood supplemented agar (15, 25). The mammalian cell co-culture and 43
insect-based media are usually used as an enrichment step before plating on agar. Of particular 44
interest is the variability between the cell culture systems as this method is traditionally 45
considered to be most successful for the initial isolation of Bartonella species (23, 24, 28, 32). 46
We focused our comparison on the protocols of three prominent laboratories that commonly 47
culture Bartonella spp and found the major differences to be localized to three main variables (i) 48
media base (RPMI, M199 or DMEM), (ii) mammalian cell line (bovine endothelial, human 49
endothelial, primate epithelial) and (iii) culturing temperature (35oC, 37
oC). 50
In 2005, a cell-free, liquid medium called Bartonella alpha proteobacteria growth medium 51
(BAPGM) was developed to detect Bartonella in veterinary and human blood samples (29). It is 52
a modified formulation of liquid media designed to support insect cells. This medium required 53
sheep blood supplementation, however upon discovering Candidatus Bartonella melophagi in 54
commercial sheep blood (4), some researchers now increase the amount of the blood sample 55
being tested instead of using commercial blood (25). More recently, Reiss et al. (33) reported 56
the benefits of another modified insect cell-based media (Schneider’s) that did not require whole 57
blood supplementation. This study demonstrated the ability to support three species of 58
Bartonella (B. henselae, B. quintana and B. vinsonii). Although this medium was not used to 59
isolate from biological samples in this study, we chose to use this formulation in contrast to the 60
BAPGM because whole blood additive was not necessary. 61
Our laboratory has a particular interest in the possible association of Bartonella species with 62
febrile illnesses and culture-negative infective endocarditis cases due to ongoing projects in 63
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many countries. We selected four representative species of Bartonella that have been associated 64
with human disease and are also phylogenetically diverse. B. henselae and B. quintana were 65
extensively studied as pathogens accounting for the majority of Bartonella infections; both have 66
been detected in cases of endocarditis (11, 18, 31). B. elizabethae was selected as it was 67
originally isolated from a case of human infective endocarditis in North America (12). Lastly, B. 68
tamiae was recently isolated from human patients in Thailand during the study on febrile 69
illnesses (25). 70
The main goals of this study were to compare mammalian cell culture based methodologies with 71
the cell-free, insect culture media-based protocols to determine the best conditions to culture 72
diverse Bartonella species that may be of clinical importance. Based on the review of current 73
protocols in the literature, we focused on three questions: (i) is there an optimal media to 74
accommodate diverse species of Bartonella? (ii) is the presence of mammalian cells necessary 75
for culturing? (iii) is there a difference between the commonly used culturing temperatures of 76
35oC and 37
oC? 77
MATERIALS AND METHODS 78
Bacterial isolates: The following strains were used in the current study, B. henselae 79
(ATCC49793), B. elizabethae (ATCC 49927), B. quintana (ATCC 51694) and B. tamiae (ATCC 80
BAA-1343). All strains were used at the lowest known laboratory passage available, estimated 81
between 4-8 passages. 82
Cell culture and growth curves: Growth curves were compared between five media variations 83
at two temperatures for a total of 10 test conditions (Summarized in Table 1). Briefly, the M10 84
(mammalian cell culture media) and MS10 (mammalian and insect-based combination) were 85
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analyzed with both the absence and presence of VeroE6 cells (primate kidney epithelial, ATCC 86
CRL-1586). The S10 (Schneider’s media) is a modification developed elsewhere from insect cell 87
culture-based media (33). The S10 alone was not able to support VeroE6 cells therefore this 88
media was only tested without cells. 89
Conditions involving the presence of VeroE6 cells were prepared by standard methods (16) 24 90
hours prior to inoculation. Briefly, VeroE6 cells were seeded into 24-well plates at 1x105 91
cells/ml in M10 media, which consists of M199 media (Sigma-Aldrich, St. Louis, MO) 92
supplemented with 10% fetal bovine serum (Invitrogen, Baltimore, MD), 10mM sodium 93
pyruvate (Invitrogen) and 10mM HEPES (Invitrogen). After 24 hours, the media was removed 94
and the cells rinsed with sterile phosphate buffered saline (PBS) before being replaced with the 95
appropriate pre-inoculated test condition (described below). 96
Media were inoculated similarly for both cell-free and VeroE6 cell conditions. Frozen stocks of 97
each bacterial isolate were diluted between 1.8x103 – 5.5x10
4 into 40 ml of each test condition 98
(see Table 1). The inoculated media was mixed thoroughly by pipetting before being aliquoted 99
into 24-well plates and placed at either 35oC with 5% CO2 or 37
oC with 5% CO2. Each 100
inoculated medium was aliquoted into triplicate wells per time point so that each well was only 101
sampled once. This protocol has been used previously (9) as a method to count total bacterial 102
growth (intra- and extracellular) when comparing conditions using cell culture. At every time 103
point the bottom of each well was scraped to remove the adherent VeroE6 cells and the media 104
mixed by pipetting before removing a 200µl sample. This plate was then discarded. The samples 105
were serially diluted in sterile PBS and dilutions then spotted in triplicate onto 10% rabbit blood 106
supplemented BHI agar plates and quantified (colony forming units/ml) based on a method 107
described previously (30). The initial inoculated media was quantified as time point 0, followed 108
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by days 1 (24 hr post inoculation), 3, 5, 7 and 9. Each experiment was conducted in triplicate and 109
repeated at least twice. The growth curve data are presented as medians + range. 110
Statistical analyses: The data were analyzed within each strain between conditions at the time 111
course endpoint of 9 days or at the peak of growth using the Kruskal-Wallis or Wilcoxon rank 112
sum tests with χ2 approximations. The day 9 endpoint was used for the purpose of comparing 113
media and cell presence as this was most biologically relevant to Bartonella culturing protocols, 114
which usually require incubation from 7 to 28 days (3, 23, 25). The peak growth of each strain 115
was compared between temperatures as it was the most logical point to show temperature 116
dependent differences as there was such a discrepancy in growth at day 9 as growth either 117
plateau or dramatically declined. Statistical analyses were conducted using JMP Statistical 118
software (SAS Institute, Cary, NC) with results considered significant if P < 0.05. 119
Immunofluorescence assay for autoagglutination: It has been observed previously that 120
autoagglutination is a phenotype corresponding to expression of trimeric autotransporter 121
adhesins in B. henselae and B. quintana, which are crucial for host cell adherence (20, 37). 122
Highly passaged laboratory strains have been shown to lose this phenotype (2). In the current 123
study, we examined this phenotype to firstly confirm that the strains being tested were able to 124
express the TAA proteins and secondly to qualitatively compare changes in this phenotype under 125
different environmental conditions (mammalian cell presence, temperature or media 126
formulation). Our protocol is a modification developed elsewhere by Riess et al (34). 127
Briefly, the bacteria was prepared by growing bacterial isolates in M10, MS10, or S10 media at 128
either 35°C with 5% CO2 or 37°C with 5% CO2 in 24-well plates. The bacteria were used 129
undiluted for all strains except B. elizabethae, which was diluted 1:2 in sterile PBS in order to 130
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maintain consistent bacterial numbers between strains. After 36 hours, 2µl of culture taken from 131
the bottom of each well was pipetted onto sterilized 10mm Gold Seal fluorescent antibody slides 132
(Daigger, Portsmouth, NH) and fixed for 10 minutes at room temperature with 4% 133
paraformaldehyde. Slides were washed 3 times with PBS containing 0.1% Triton (TX-PBS), 134
rinsed with distilled water, and air dried. Nonspecific binding was blocked using 20µl of 3% 135
BSA (Jackson ImmunoResearch, West Grove, PA) diluted in TX-PBS. After 30 minutes at 136
room temperature, excess blocking buffer was removed using a pipet and 20µl of mouse immune 137
serum (ProSci Inc., Poway, CA) diluted 1:32 in sterile PBS was added. After washing 3 times 138
with TX-PBS, 20µl fluorescein isothiocyanate (FITC)-labeled, goat anti-mouse IgG conjugate 139
(Kirkegaard & Perry Laboratories, Gaithersburg, MD) diluted 1:150 in sterile PBS was added. 140
Slides were incubated in a moist chamber for 30 minutes at room temperature, washed 2 times 141
with PBS, rinsed with distilled water, and air dried. Slides were then read using a Zeiss 142
Axioscope2 Plus camera and fluorescence microscope (Thornwood, NY) with 40x objectives 143
and 10x oculars. Images were processed using Adobe Photoshop CS (Adobe, San Jose, CA). 144
RESULTS 145
Figure 1 plots the growth curves for each Bartonella species tested in five conditions at 35oC and 146
at 37oC. The first two are the mammalian cell conditions with and without VeroE6 cells (M10-V 147
and M10 respectively), the axenic Schneider’s insect cell-based media (S10) and the last two are 148
a combination of the mammalian and insect media with and without VeroE6 cells (MS10-V and 149
MS10 respectively). 150
Visual inspection of the growth curves shows that within our period of observation, most strains 151
reach a peak of growth and then viability either plateau or decline. The only exception to this 152
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trend is B. quintana in M10 media, where the peak was minimal and the viability did not vary 153
much over the 9 day time course. Due to the logarithmic scale used to display the data, there are 154
some statistical differences that may not be evident without the further analysis. 155
At 9 days post inoculation, the colony forming unit (CFU) counts were consistently similar 156
between cultures grown in MS10 and S10 media (χ2
< 2.877, P>0.089) with two exceptions; 157
when B. tamiae and B. quintana were grown at 37oC, cultures grown in MS10 yielded 158
significantly higher CFU counts than those grown in S10 (χ2 > 8.366, P< 0.004). Additionally, in 159
each pair-wise comparison and at both 35oC and 37
oC, both MS10 and S10 consistently yielded 160
significantly higher CFU counts than M10 or M10-V (χ2 > 8.308, P < 0.004). 161
To determine if addition of mammalian cells (specifically in this study,VeroE6 cells) affected the 162
Bartonella viability by 9 days post inoculation, we compared CFU counts within media with and 163
without cells (M10-V with M10 and MS10-V with MS10). No significant differences were found 164
between endpoint viability for any of the strains tested, with the exception of B. quintana that 165
yielded conflicting results. At both 35oC and 37
oC, within the combination media conditions 166
(MS10/MS10-V) the CFU counts were significantly lower when cells were present (χ2 > 8.308, 167
P< 0.004). However, when growth in the mammalian conditions (M10/M10-V) the CFU counts 168
of B. quintana were significantly higher when cells were present (χ2 > 8.308, P< 0.004). 169
Using the rationale that the growth curves either plateau or decline after reaching the peak, we 170
compared the affects of temperature on peak CFU values. For visual comparison, there is a 171
horizontal line drawn that represents the peak growth at 35oC within each strain to compare the 172
peak at 37oC (Figure 1). The growth peak was significantly higher at 35
oC compared to 37
oC for 173
all strains in MS10 and S10 media (χ2 > 8.337, P< 0.004) with the exception of B. elizabethae 174
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which yielded no significant difference in peak growth between temperatures. We restricted our 175
analysis to these two media, as the mammalian conditions (M10/M10-V) growth peaks were 176
consistently lower than the S10 and MS10/MS10-V conditions. 177
The autoagglutination phenotype was examined for two main reasons. Firstly, to test if the 178
strains used in our experiments were able to express the trimeric autotransporter adhesins (TAA) 179
associated with this phenotype (37) as it has been shown to be lost after multiple passages in 180
vitro (2). And secondly, the assay was utilized to investigate temperature or media dependent 181
changes to this phenotype under our current test conditions. 182
Results of the agglutination assay are summarized in figure 2. Agglutination of B. tamiae was 183
noted at M10 and MS10 media, however in the S10 media, no agglutination was seen. This 184
change appeared to be media dependent only as results were similar between both temperatures. 185
Agglutination of B. henselae also appeared to be media dependent and temperature independent. 186
In M10 media, B. henselae cultures produced very large aggregations. This could explain the 187
large range in viability measured during the growth curves of B. henselae in the M10 and M10-V 188
conditions (figure 1B). In the MS10 and S10 media, B. henselae still agglutinated, however the 189
clumps were smaller in comparison. With respect to B. elizabethae, agglutination was seen in all 190
media and at both temperatures. There did not appear to be a difference in size or quantity of 191
aggregates in any condition which agrees with the similarities seen between conditions from the 192
quantitative growth curve data of B. elizabethae. Conversely, B. quintana did not agglutinate in 193
any of the conditions tested. 194
DISCUSSION 195
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The data presented in this study showed that there are media and temperature dependent growth 196
differences in vitro between diverse isolates of the Bartonella genus. We compared three 197
culturing techniques, the first using mammalian culture conditions with and without the presence 198
of VeroE6 cells (M10-V, M10 respectively), the second using insect culture-based media (S10) 199
and thirdly, a combination of the mammalian and insect based media with and without VeroE6 200
cells (MS10-V, MS10 respectively). 201
Our data showed that the combination media, MS10 and MS10-V were able to support all four 202
Bartonella spp. well at both 35oC and 37
oC. These growth curves were comparable to the S10 203
media in all cases except B. tamiae at 37oC, where viability of the bacteria in the S10 media 204
declined significantly lower than the MS10 and MS10-V conditions by the end of the time 205
course at day 9. 206
In the MS10 and MS10-V conditions, our data suggest that the presence of VeroE6 cells did not 207
significantly affect growth or sustainability of the bacterial cultures suggesting that cell culture 208
may not be necessary for culture of these bacterial species. However, due to the high bacterial 209
loads that were reached in these growth curves, it is difficult to interpret this to be true as 210
monolayers had lifted off between 5-7 days in most cases (data not shown). B. quintana was the 211
only strain for which the presence of cell culture under mammalian conditions (M10-V) was a 212
significant benefit compared to the same cell-free media (M10). These results agree with 213
previous work suggesting that under mammalian conditions, cell co-culture is important for B. 214
quintana growth (3, 23). Recent developments, however have shown that cell culture is 215
unnecessary using insect cell-based media (29, 33). In the near future, our laboratory will be 216
comparing the media conditions described in this study on biological samples to more accurately 217
test if cell co-culture is necessary upon initial isolation of Bartonella species that have not been 218
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adapted to laboratory conditions. For this reason, we see the ability of the MS10 media to sustain 219
mammalian cell culture as a benefit in the possibility that co-culture might be necessary. Our 220
laboratory also tested the combination, MS10 media with other cell lines and was able to 221
adequately support Huh-7 (Human hepatocyte) and THP-1 (Human monocyte) cell lines 222
(microscopic observations, data not shown). 223
VeroE6 cells were chosen for this study as there is evidence in the literature that both B. henselae 224
and B. quintana are able to adhere to different cell lines in vitro - including monocytes, 225
endothelial and epithelial cells from various mammalian hosts (2, 13, 21, 35). Additionally, 226
VeroE6 were used to successfully isolate B. tamiae for the first time (25). We acknowledge that 227
endothelial cells are typically chosen for culturing Bartonella species however they may not be 228
necessary when the media composition is changed as shown in the present work, with the 229
combination or replacement with insect cell-based media. As vector-borne bacteria, Bartonella 230
species are required to survive in various environments. Clinically, Bartonella species have been 231
shown to cause a variety of clinical manifestations such as bacteremia, endocarditis, bacillary 232
angiomatosis and peliosis where the bacteria interact with multiple cell types within the same 233
host (6, 10). Endothelial cells such as HUVECs provide an excellent in vitro model to study 234
pathogenesis as there is evidence that endothelial cells are invaded in vivo (8, 14) however, for 235
the purpose of a culturing protocol to isolate Bartonella species from biological material 236
endothelial cells may not be optimal as many of the cell lines require a higher passaging 237
frequency and extra growth factors which increases the cost substantially. 238
The bacterial strains for these experiments were used at the lowest passage possible as B. 239
henselae has been shown to undergo ‘phase variation’ after multiple laboratory passages and 240
show decreased autoagglutination associated with decreased cell adherence (2, 27). 241
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Immunofluorescence was used to assess the presence of the autoagglutination phenotype and 242
also to examine if there were changes associated with temperature or media differences in our 243
test conditions (summarized in figure 2). B. quintana was the only strain that did not show 244
agglutination in any condition. We still however, observed media and cell dependent differences 245
in growth (fig 1) which are similar to those found in the literature (22). Specifically, that co-246
culture with mammalian cells under mammalian conditions (M10-V) were required for growth, 247
however when insect cell-based media is incorporated (S10 or MS10), B. quintana were able to 248
grow in cell-free media. Autoagglutination is associated with expression of the TAA proteins 249
(27, 37). Although media dependent differences were seen with B. tamiae and B. henselae, it is 250
unclear whether the agglutination phenotype correlates to growth or viability of the culture. It 251
would be interesting to further investigate if there are changes in transcriptional regulation or 252
loss of genes associated with host adaptability or pathogenicity under these varying culture 253
conditions and also over a broader range in temperature to include lower temperatures more 254
relevant to a vector host. 255
There is a discrepancy in the literature concerning temperature for culturing Bartonella species. 256
The majority of studies that co-culture Bartonella species with mammalian cells use 37oC in 257
contrast to axenic insect culture-based media and agar which are cultured at 35oC (15, 17, 33). 258
Although a 2oC difference may seem minor or trivial, it was interesting to see that there were 259
statistically significant differences in peak growth between the two temperatures for three of the 260
four strains tested (B. elizabethae was the exception showing no significant difference in peak 261
growth between the two temperatures). Additionally, the drastic divergence in growth between 262
these temperatures for B. tamiae was surprising as this strain was isolated from a human febrile 263
patient. We are very interested to continue this investigation as the temperature dependent 264
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growth requirements may provide new insight into the life cycle and biological niche of this 265
microorganism. This information is important to know because these two temperatures are very 266
commonly used in a clinical laboratory setting and may sometimes be used interchangeably. 267
Additionally, because of the minor difference in temperature, incubator settings should be 268
calibrated properly as the temperatures can fluctuate and may impact the growth of some 269
Bartonella species. 270
The goals of this study were to compare key components of common Bartonella culturing 271
methodologies, as well as combine them to investigate the best conditions to culture diverse 272
Bartonella species of clinical importance. We have shown that by combining the mammalian and 273
insect cell-based media (MS10) we were able to achieve peak growth comparable to the insect 274
culture-based media alone (S10) at 35oC but at 37
oC the MS10 media performed significantly 275
better for both B. tamiae and B. quintana. Our data suggest that mammalian cell co-culture is not 276
necessary for growth of laboratory adapted Bartonella strains; however, the combination media 277
is still able to support mammalian cell culture which may remain an important variable during 278
initial isolation of Bartonella from biological samples. Our data also show that B. tamiae has 279
temperature dependent growth requirements that are not adequately met with the mammalian or 280
insect media alone at 37oC, regardless of mammalian cell co-culture. 281
This is the first study to our knowledge that has compared these methodologies using diverse 282
Bartonella spp. These findings are important for clinical microbiologists, as Bartonella species 283
are fastidious organisms and a diverse genus which are a growing concern as emerging or re-284
emerging pathogens. 285
286
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Table 1. Media formulations with and without VeroE6 cells (repeated for both 35oC and 37
oC) 287
Abbreviation Media VeroE6 cells Supplementation pH Reference
M10-V Present
M10
M199a
-
10% fetal bovine serum,
1mM sodium pyruvate,
10mM HEPES,
1ug/ml of Amphotericin Bb
7.6 (11)
S10 Schneider’s - 10% fetal bovine serum,
5% sucrose
1ug/ml of Amphotericin Bb
7.1 (23)
MS10-V Present
MS10
40% M199a
40% Schneider’s -
10% fetal bovine serum,
1mM sodium pyruvate,
10mM HEPES,
1ug/ml of Amphotericin Bb
7.4 This study
288 a
( with Earle’s salts and L-glutamine) bAmphotericin B is recommended when culturing biological specimens in our laboratory 289
FIGURE LEGENDS 290
Figure 1. Growth curves of four Bartonella species : B. tamiae, B. henselae, B. elizabethae and B. 291
quintana measured at two temperatues, 35oC and 37
oC. There are 5 conditions tested for each strain at 292
both temperatures. = M10, cell free media, = M10 + VeroE6 cells, = MS10, = MS10 + 293
VeroE6 cells and = S10, cell free media. 294
Figure 2. Differences in autoagglutination between temperature and media after 36 hours. Representative 295
immunofluorescent micrographs are shown of each bacterial strain grown in axenic medias (M10, MS10 296
and S10) at both 35oC and 37
oC. Images were taken with a 40X objective before the contrast was adjusted 297
and the images cropped using Adobe Photoshop. 298
299
ACKNOWLEDGEMENTS 300
Thank you to the ASM/CCID Postdoctoral Fellowship Program for funding, Dr. N. Zeidner for 301
the use of his IFA microscope, Dr. R. Eisen for her guidance and expertise with the statistical 302
analysis and also to Dr. R. Gilmore for his scientific advice and review of this manuscript. 303
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304
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