1 high prevalence and species diversity of helicobacter detected in
TRANSCRIPT
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High prevalence and species diversity of Helicobacter detected in wild house mice 1
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Wasimuddin,1,* Dagmar Čížková,1 Josef Bryja,1 Jana Albrechtová,1 Heidi C. Hauffe,2 3
and Jaroslav Piálek1 4
5
1Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, 603 65 Brno, 6
Czech Republic; 7
2 Department of Biodiversity and Molecular Ecology, Research and Innovation Centre, 8
Fondazione E. Mach, Via E. Mach 1, 38010 S. Michele all’Adige (TN), Italy 9
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*Corresponding author. Mailing address: Department of Population Biology, Institute of 11
Vertebrate Biology AS CR, 675 02 Studenec 122, Czech Republic. Fax: (420) 568-423-121. 12
E-mail: [email protected] 13
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Abstract 15
PCR diagnostics detected 100% prevalence of Helicobacter in 425 wild house mice (Mus 16
musculus) from across Central Europe. Of seven species identified, the five most frequent 17
were H. rodentium (78%), H. typhlonius (53%), H. hepaticus (41%), H. bilis (30%) and H. 18
muridarum (1%). Double infections were more common (42%) than single (30%) and triple 19
(21%) infections. Wild house mice could be considered potential reservoirs of Helicobacter 20
for both humans and other vertebrates. 21
22
The members of the genus Helicobacter comprise more than 30 species (9). Although a few 23
have been thoroughly studied for their impact on human and animal health (4, 5, 15, 23), as 24
Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01989-12 AEM Accepts, published online ahead of print on 7 September 2012
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well as on research (6), in most cases, zoonotic potential and mode of transmission remain 25
obscure (18), especially for intestinal helicobacters (22). In the case of rodents, most studies 26
have been conducted with laboratory mice, and studies of wild populations across large 27
geographical areas are virtually unknown. Therefore, screening of wild commensal rodents 28
like the house mouse, Mus musculus, known to host at least 11 intestinal helicobacters (3, 21, 29
22), for the occurrence and prevalence of Helicobacter species is considered crucial for 30
gaining insights into their ecology and epidemiology (8). 31
In this study, 425 individuals were trapped at 91 sites scattered within a 6,500 km2 32
rectangular area stretching from north-eastern Germany to western Czech Republic (Fig. 1, 33
Table S1). This area is occupied by two house mouse subspecies, M. m. domesticus and M. 34
m. musculus (see 10, 11 for their distribution). Specifically, we asked what is (i) the 35
prevalence and (ii) the diversity of Helicobacter spp. in natural populations of the house 36
mouse? We also asked (iii) is co-infection with several Helicobacter spp. common in wild 37
mice? 38
House mice were live-trapped inside and adjacent to houses and farms from 2008-39
2010 and dissected in a field laboratory. Extreme caution was taken to avoid cross-infections 40
and cross-contaminations (e.g. mice were housed singly until anesthetized; sterile dissection 41
a maximum of 24 hours after a mouse was trapped). The colon including faeces of each 42
mouse was put into a sterile 30 ml screw-cap microtube containing 70% EtOH and stored at 43
room temperature. DNA was isolated from fecal samples under sterile conditions at the 44
Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic 45
as described elsewhere (17). 46
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Using Helicobacter genus-specific primers (C97; 5'-GCT ATG ACG GGT ATC C 47
and C98; 5'-GAT TTT ACC CCT ACA CCA) part of 16S rRNA gene was amplified (14, 19) 48
for each mouse sample. Escherichia coli was used as a negative and H. equorum as a positive 49
control (provided by Faculty of Veterinary Medicine, University of Veterinary and 50
Pharmaceutical Sciences, Brno, Czech Republic) (1). Therefore, there is 100% prevalence of 51
Helicobacter infection in wild house mice in our study area. The same level of Helicobacter 52
prevalence was reported for 84 samples of three wild rodent species in Sweden; however, 53
house mice were absent in that study (12). Lower prevalence is reported in surveys of 54
laboratory mice (Table 1); however, this might reflect the eradication efforts adopted in most 55
rodent vivaria against these bacteria, improvement in nutrition or lack of continuous 56
reinfection. 57
Multiplex species-specific PCR (6) was used to screen for 5 Helicobacter species (H. 58
hepaticus, H. bilis, H. rodentium, and H. typhlonius and H. muridarum), and 401 samples 59
were positive for at least one of these species. PCR products of the 16S rRNA from two mice 60
each hosting only H. hepaticus, H. bilis, H. rodentium, or H. typhlonius were sequenced 61
(primers C97 and C98, see sequencing method below) and found to be identical with 62
previously published GenBank sequences (accession number are given in Fig. 2). No house 63
mouse carrying only H. muridarum in single infection was found; consequently, the PCR 64
product for this species could not be sequenced. The prevalence of individual Helicobacter 65
species in mouse samples was unequal, ranging from 78% for H. rodentium to 1% for H. 66
muridarum (Table 1). H. rodentium was the exclusive helicobacter in 25% of 401 positive 67
samples, whereas, H. typhlonius, H. hepaticus and H. bilis were present as a single species 68
only in 14%, 6%, and 2% of 401 Helicobacter-positive mice, respectively. None of the 69
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Helicobacter species showed a specific pattern of spatial distribution, instead the four most 70
common Helicobacter species were found in mice scattered across whole study area (Fig. 1). 71
This results suggest that co-infection of Helicobacter species is common in wild 72
house mouse individuals across our large study area. Among the 401 Helicobacter-positive 73
faeces samples, 279 were positive for more than one species (median was 2 Helicobacter 74
species per mouse). Double infections were detected in 42% of mice, single infection in 30%, 75
and co-infection with three or four Helicobacter species was detected in 21% and 6% of 76
mice, respectively. The most frequently observed helicobacter communities in double 77
infections were H. rodentium/H. typhlonius and H. rodentium/H. hepaticus, each reaching 78
15% from the total sample of 401 mice, whereas in triple infections the highest frequency 79
was observed for H. rodentium/H. typhlonius/H. bilis (9% of mice). This is in contrast to 80
previous reports showing that in laboratory mice, infections with single Helicobacter species 81
are most commonly observed (16, 20). Further screening should confirm whether multiple 82
infections are the prevailing pattern in wild house mice. 83
To identify the Helicobacter species in the remaining 24 samples with a negative 84
species-specific PCR, products from positive genus-specific PCR (422 bp long fragments of 85
16S rRNA gene) (19) were sequenced using BigDye Terminator v3.1 Cycle sequencing Kit 86
and ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, Carlsbad, CA). The sequences 87
were edited in Seqscape v2.5 (Applied Biosystems), cut to 364 bp, aligned using the same 88
software and searched using BLAST in GenBank (accessed at 89
http://blast.ncbi.nlm.nih.gov/Blast.cgi). In 13 samples the presence of ambiguous bases 90
suggested co-infection by two or more different Helicobacter species, and these samples 91
were excluded from further analyses. For the remaining 11 samples, a DNA-barcoding 92
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approach was used and their position on a neighbour-joining (NJ) tree was used to infer 93
species (Fig. 2). The 16S rRNA sequences were added to GenBank: three samples appeared 94
to represent H. typhlonius (accession number JX198315), and one sample H. hepaticus 95
(accession number JX198316); four were identical to H. mastomyrinus (accession number 96
JX198314) and two clustered with H. ganmani (accession number JX198318) (all showed 97
≥99% identity in BLAST searches). The species status of the Helicobacter from mouse 98
sample SK1050 (accession number JX198317) was not resolved (Fig. 2). 99
To conclude, there is a wide spectrum of Helicobacter species in wild house mice in 100
Central Europe. The diversity of Helicobacter species found in this sample of wild mice is 101
striking: we found representatives of 7 out of 11 recognized Helicobacter species previously 102
identified in Mus musculus (3, 21, 22), plus one unknown species, in an area of only 6,500 103
km2. Although other studies have also reported a high prevalence of helicobacters in house 104
mice (Table 1), this study showed that all mice from all 91 sites were infected with at least 105
one Helicobacter species in our study area which included part of two countries (Germany 106
and Czech Republic) and two different house mouse subspecies. Collectively, the results 107
indicate that Helicobacter spp. could be common bacteria infecting the majority of wild 108
house mice, but this needs to be confirmed in other geographical areas. The high prevalence 109
of H. bilis, H. hepaticus and H. typhlonius (Table 1) are intriguing given their ability to cause 110
disease (including inflammatory bowel disease) in some strains of laboratory mice (8, 22), 111
and should be studied further for their impact on house mouse ecology. Given that several of 112
the Helicobacter species detected in these samples can also infect humans (H. bilis, H 113
hepaticus) (6, 15, 22), and domestic animals such as dogs (H. bilis) (22), and as house mice 114
are mainly commensal (living in human habitations), further studies should be carefully 115
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conducted to confirm whether wild house mice could serve as a vector of Helicobacter 116
infection (15). Furthermore, since several species harbored by wild mice are known to 117
confound or potentially confound studies in laboratory mice by causing disease and are 118
difficult to eradicate (e.g. H. hepaticus, H. bilis, H. bilis + H. rodentium, H. typhlonius, 22), 119
care should be taken to avoid contact of breeding colonies of house mice with their wild 120
counterparts. Our results also indicate that wild mice could be source of novel intestinal 121
helicobacters, and we urge further screening of wild populations. 122
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ACKNOWLEDGEMENTS 124
We thank many of our colleagues for collecting mouse samples. We are grateful to Bohumil 125
Sak and Martin Kváč, Institute of Parasitology, Biology Centre of the Academy of Sciences 126
of the Czech Republic, České Budějovice, Czech Republic for providing DNA samples and 127
to Barbora Bezděková and Ján Futas, Faculty of Veterinary Medicine, University of 128
Veterinary and Pharmaceutical Sciences, Brno, Czech Republic for providing H. equorum 129
DNA. Ludovít Ďureje constructed maps of Helicobacter distribution. Karolína Sobeková and 130
Tomáš Albrecht helped in initial phases of this project. Oldřich Tomášek provided valuable 131
comments on an earlier version of the manuscript. This work was supported by the Czech 132
Science Foundation (project 206/08/0640) and the Fondazione Edmund Mach (HCH). 133
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TABLE 1. Helicobacter prevalence in laboratory and wild mice estimated in this and previous 209
studies. Records retrieved from literature are directly comparable to ours since they present 210
data based only on PCR amplification methods. 211
n.a. – not analyzed 212 213
214
215
216
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218
219
220
221
Helicobacter species
Infection in wild house mice (this study) %
Infection in mice in close vicinity of
laboratory facilities %
Infection in laboratory
mice %
Reference
H. bilis 30 n.a. 1 7
H. hepaticus 41 59 11-59 7, 16, 20
H. muridarum 1 n.a. 0 7
H. rodentium 78 10 6-17 7, 16, 20
H. typhlonius 53 29 26 16, 20
Other H. spp. 6 8 11 7, 16
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Figure caption 222 223 FIG. 1. Distribution of H. bilis (A), H. hepaticus (B), H. rodentium (C), and H. typhlonius (D) 224
in mouse populations across the Czech-German boundary. The middle panel indicates the 225
position of the study area in central Europe. Black symbols display populations with the 226
presence and open symbols with the absence of individual bacteria species. Numbers 227
correspond to localities listed in Table S1. 228
229 230 231 232 FIG. 2. Identification of Helicobacter samples that provided positive PCR with genus-specific 233
primers, but negative with species-specific primers. Neighbor-joining tree based on 16S 234
rDNA partial sequences was calculated in Mega v. 4 using K2-P distances among sequences. 235
Wolinella succinogenes and Campylobacter jejuni were used as outgroups. Samples 236
sequenced in this study are indicated by the host identification codes (starting with either SU 237
or SK). * indicate 11 Helicobacter species that have been previously recorded in house mice. 238
GenBank accession numbers are shown in brackets. Numbers above branches indicate the 239
bootstrap values (only those > 75 are shown). 240
241
Supplementary Table S1. 242
List of 91 sampling sites of the house mouse in Germany and the Czech Republic with sample 243
size per site and percentage of mice PCR-positive for each of five Helicobacter species. 244
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