R E S E A R C H A R T I C L E
Ying Bai
1,2
, Michael Y. Kosoy
2
, Jack F. Cully
3,4
, Thiagarajan Bala
3
, Chris Ray
1
& Sharon K. Collinge
1,5
1
Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, CO, USA;
2
Division of Vector-Borne Infectious Diseases, National
Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, CO, USA;
3
Division of Biology, Kansas State University,
Manhattan, KS, USA;
4
Kansas Cooperative Fish and Wildlife Unit, US Geological Survey, KS, USA; and
5
Environmental Studies Program, University of
Colorado, Boulder, CO, USA
Correspondence: Ying Bai, PO Box 2087,
Fort Collins, CO 80522, USA. Tel.: 1 970 266
3555; fax: 1 970 221 6435; e-mail: bby5@cdc.gov
Received 24 January 2007; revised 24 May
2007; accepted 25 May 2007.
First published online August 2007.
DOI:10.1111/j.1574-6941.2007.00364.x
Editor: Julian Marchesi
Keywords
Bartonella ; disease ecology; grasshopper mouse; Onychomys leucogaster ; jump.
Abstract
Rodent-associated Bartonella species are generally host-specific parasites in North
America. Here evidence that Bartonella species can ‘jump’ between host species is presented. Northern grasshopper mice and other rodents were trapped in the western USA. A study of Bartonella infection in grasshopper mice demonstrated a high prevalence that varied from 25% to 90% by location.
Bartonella infection was detected in other rodent species with a high prevalence as well. Sequence analyses of gltA identified 29 Bartonella variants in rodents, 10 of which were obtained from grasshopper mice. Among these 10, only six variants were specific to grasshopper mice, whereas four were identical to variants specific to deer mice or 13-lined ground squirrels. Fourteen of 90 sequenced isolates obtained from grasshopper mice were strains found more commonly in other rodent species and were apparently acquired from these animals. The ecological behavior of grasshopper mice may explain the occurrence of Bartonella strains in occasional hosts. The observed rate at which Bartonella jumps from a donor host species to the grasshopper mouse was directly proportional to a metric of donor host density and to the prevalence of Bartonella in the donor host, and inversely proportional to the same parameters for the grasshopper mouse.
The genus Bartonella includes a variety of genetically related
Gram-negative bacteria that parasitize vertebrate erythrocytes (Anderson & Neuman, 1997). Among them, the wellknown human pathogens are Bartonella bacilliformis , the agent of Carrion’s disease (Karem et al ., 2000), Bartonella quintana , the agent of trench fever (Bass et al ., 1997), and
Bartonella henselae , the agent of cat scratch disease (Margileth, 1993). Recently, Bartonella species have also been isolated from rodents, cats, dogs, and other domesticated and wild animals (Kordick et al ., 1996; Droz et al ., 1999;
Dehio et al ., 2001; Gundi et al ., 2004; Maillard et al ., 2004), leading to a surge in the number of descriptions of novel members of this genus. However, the public health importance of many of these isolates remains undefined.
Interest in rodents as reservoir hosts of Bartonella led to numerous studies that found a high level of genetic diversity of Bartonella among a variety of rodent species around the world (Birtles et al ., 1994; Kosoy et al ., 1997; Ying et al .,
2002; Castle et al ., 2004; Jardine et al ., 2005). The identity of some strains isolated from rodents and humans suggested that rodents might serve as reservoir hosts of certain
Bartonella species that cause human illness (Daly et al .,
1993; Ellis et al ., 1999; Welch et al ., 1999; Kosoy et al .,
2003). The close association between rodents and humans throughout the world makes the study of rodent-borne
Bartonella essential for determining the extent to which rodents may serve as a source of human infections. Although the transmission mechanisms by which individual rodents acquire Bartonella infections are not fully understood, arthropod vectors such as fleas have been implicated as potential vectors (Breitschwerdt & Kordick, 2000; Stevenson et al ., 2003) and experimental studies have demonstrated the role of fleas in the transmission of bartonellae among rodents (Bown et al ., 2004). Other transmission mechanisms might also exist, such as vertical transmission (Kosoy et al .,
1998).
Journal compilation c 2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original US government works
FEMS Microbiol Ecol 61 (2007) 438–448

Acquisition of nonspecific Bartonella by grasshopper mice 439
Evidence for cospeciation of Bartonella with natural hosts varies among studies and by geographic region. There is a strong association between specific Bartonella strains and certain rodent hosts: Rattus rats in Southern China (Ying et al ., 2002); Bandicota rats in Thailand (Castle et al ., 2004);
Richardson’s ground squirrels ( Spermophilus richardsonii ) and other rodent species in Canada (Jardine et al ., 2005); cotton rats ( Sigmodon hispidus ) and Peromyscus mice in the southeastern United States (Kosoy et al ., 1997); and ground squirrels and chipmunks in the western states (Kosoy et al .,
2003). However, observations in Europe have indicated that one single species of Bartonella frequently infects several different rodent species at a given site (Birtles et al ., 2001;
Holmberg et al ., 2003).
Although the absence of cospeciation has been observed, no study has quantified the distribution of strains of a
Bartonella species among different rodent species, to infer whether strains are host-specific or nonspecific. There is a possibility that generally host-specific strains may occasionally invade atypical hosts; in the terminology of Woolhouse et al . (2005), a pathogen lineage may appear to ‘jump’ from one host lineage to another. Such jumps not only fulfill interspecific transmission but might also be important for the maintenance of Bartonella in nature or the spread of
Bartonella among rodents.
Several rodent species co-occur in the prairie ecosystems in the western United States, including prairie dogs ( Cynomys spp.), deermice ( Peromyscus spp.), ground squirrels
( Spermophilus spp.), voles ( Microtus spp.), and grasshopper mice ( Onychomys spp.). Northern grasshopper mice
( Onychomys leucogaster ) occur chiefly in dry, sandy grasslands in the central and southwestern United States and northern Mexico. As the name implies, the diet of these mice consists largely of grasshoppers and other insects, supplemented by small mammals and a variety of plants. Bailey &
Sperry (1929) reported that animal matter, including other mice, makes up nearly 89% of the natural food; plant material comprises only 11%, a fact that has led to the grasshopper mouse being referred to as a ‘predatory’ species.
They are also reputed to usurp the burrows of other small mammals rather than take the time to construct their own
(Thomas, 1988). These special behaviors of grasshopper mice might increase the chance of contracting infections from other rodents directly or via ectoparasites and may facilitate their role in spreading infection among species.
Understanding the ecology of bacterial transmission among natural hosts is crucial for determining how and when these bacteria may infect humans.
In this report, Bartonella infections in the northern grasshopper mouse and several associated rodent species were studied. The objectives were to study the prevalence and diversity of Bartonella variants in this mouse and associated species by culturing and characterizing the cultures using genetic analyses, and to provide further evidence for (or against) cospeciation of Bartonella with its hosts by comparing the genetic diversity of Bartonella among the studied host species.
Study sites and trapping
Small mammals were trapped in four ‘grassland’ (graminoid-dominated) sites in the United States: (1) Badlands
National Park, located in southwestern South Dakota
(43 1 45
0
0
00
N, 102 1 30
0
0
00
W); (2) Cimarron National Grassland, located in the southwest corner of Kansas (37 1 7
0
27
00
N,
101 1 47
0
24
00
W); (3) Comanche National Grassland, located in southeastern Colorado (37
1
20 0 12 00 N, 103
1
4 0 07 00 W); and
(4) Thunder Basin National Grassland, located in northeastern Wyoming (43
1
41 0 48 00 N, 104
1
59 0 39 00 W).
Small mammals were captured in May–June and July–
September, 2003 at each site, using 7 7-trap grids with
20 m between traps. Eight grids were trapped at Badlands, and 12 grids were trapped at each of the other three National
Grasslands. Within each grid, 49 large Sherman live-traps
(H.B. Sherman Traps Inc., FL) were prebaited with oatmeal and set for three consecutive nights. Each day, trapped animals were removed and traps were rebaited and reset.
Data and blood collection
Rodents were anesthetized with a mixture of isofluorane
(Halocarbon Laboratories, River Edge, NJ) and oxygen using a vaporizer (SurgiVet, Waukesha, WI) before being subject to physical measurements and identified to species and sex. Each animal was scored for the absence or presence and number of fleas. Animals were bled from the retroorbital plexus, marked with a uniquely numbered ear tag for small animals (1005-1, National Bank & Tag Company,
Newport, KY), and released at the site of capture after recovery from anesthesia. The research methods were approved by the Institutional Animal Care and Use Committee of Kansas State University. Animals recaptured within the same trapping session were not reprocessed. Blood samples were placed in liquid nitrogen in the field and then transferred to a 70
1
C freezer before testing.
Culturing
Isolation of Bartonella followed the methods in Kosoy et al .
(1997). In brief, whole blood was diluted 1 : 4 in brain heart infusion (BHI) medium (BBL, Becton Dickinson Microbiology System), supplemented with 5% amphotericin B that has no effect on Bartonella growth but reduced the likelihood of fungal contaminants that can overgrow fastidious and slow-growing Bartonella colonies and then plated
FEMS Microbiol Ecol 61 (2007) 438–448 Journal compilation c 2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original US government works

440 Y. Bai et al .
on heart infusion agar containing 5% rabbit blood. Agar plates were incubated at 35
1
C for up to 4 weeks in an aerobic atmosphere of 5% carbon dioxide. Bacterial colonies were tentatively identified as Bartonella based on colony morphology and microscopic examination of Gram-stained bacteria, after subculturing to confirm purity. All morphologically distinct colony types from each sample were identified individually. Cultures were harvested in 5 mL of
BHI medium plus 10% glycerol.
Verification of Bartonella by PCR
Crude DNA extracts were obtained from isolates by heating a heavy suspension of the organisms at 95 1 C for 10 min.
Bartonella isolates were verified by PCR amplification of a region in the citrate synthase ( gltA ) gene that is specific for
Bartonella (Norman et al ., 1995). PCR amplifications were performed in a 50 m L reaction mixture containing 5 m L 10X
PCR buffer, 5 pmol of each primer, 200 m M each dNTP
(Invitrogen, Cergy-Pontoise, France) 2.5 U Taq DNA polymerase (EuroblueTaq, Eurobio, Les Ulis, France), and 2.5
m L
DNA. Two oligonucleotides, BhCS781.p (5 0 -GGGGAC-
CAGCTCATGGTGG-3 0 ) and BhCS1137.n
(5
CAAAAAGAACAGTAAACA-3
0 -AATG-
0
), were used as PCR primers to generate a 379-bp amplicon of the Bartonella gltA (Norman et al ., 1995). Positive and negative controls were included in each PCR run to evaluate the presence of appropriately sized amplicons and contamination, respectively. Each PCR was carried out in a PTC 200 Peltier thermal cycler (MJ Research Inc., MA) using the following program parameters: a 3-min denaturation at 95 1 C, followed by 35 cycles of 1-min denaturation at 95
1
C, 1-min annealing at 56
1
C, and 1-min elongation at 72
1
C. Amplification was completed by holding the reaction mixture at
72
1
C for 10 min. PCR products were analyzed for the presence of amplicons of the correct size by electrophoresis in 1.5% agarose gel with ethidium bromide staining.
Sequencing and analysis of DNA
Amplicons of the appropriate size were purified using the
QIAquick PCR Purification Kit (Qiagen, Maryland) and sequenced in both directions using the CEQquick start Kit
(Beckman, CA). Sequencing reactions were carried out in a
PTC 200 Peltier Thermal cycler using the same primers for
PCR assay at a concentration of 1–2 m M. Cycle parameters for the sequencing reactions were 45 cycles at 96
1
C for 20 s,
50
1
C for 20 s, and 60
1
C for 4 min.
Sequences were analyzed using
LASERGENE
(DNASTAR,
Madison, WI) sequence analysis software to determine the consensus of sequences for the amplified region of the glt A.
The
CLUSTAL V program within
MEGALIGN
(DNASTAR) was used to align and compare homologous Bartonella glt A sequences from the present study and from the GenBank database. Novel sequences from the current study were submitted to GenBank and assigned unique accession numbers. The resulting alignment was analyzed using neighbor joining and bootstrap programs available in the
PAUP software for parsimony analysis (Center for Biodiversity, Illinois Natural History Survey, Champaign, IL).
Definitions
In order to analyze ecological and epidemiological factors influencing the rate at which Bartonella jumps between species, ‘donor’ and ‘recipient’ species were identified.
Donor species were those in which a specific variant was typically found, and recipient species were those in which the same variant was rarely found. In this study, the grasshopper mouse was the only recipient identified for several variants that were typically found in other, donor species.
The Bartonella ‘jump rate’ is defined as the fraction of grasshopper mice that were infected with a variant that was otherwise found only in a donor species, among all Bartonella -positive grasshopper mice at a given site.
Statistical analysis
The relationship between jump rate and the proportional
‘contribution’ of donor hosts to the rodent community was examined. The contribution of a particular host species to the community was defined as the proportion of captures consisting of that host. The relationship between jump rate and proportional contribution of grasshopper mice to rodent community was also examined. Finally, the relationship between jump rate and overall Bartonella prevalence
(including all variants) in donor hosts, and in the grasshopper mouse was examined. These analyses were performed using generalized linear models constructed via the
‘genmod’ procedure in
SAS
(
STATISTICAL ANALYSIS SYSTEM
, version 9.1).
Because jump rate is confined to an interval (0–1), it cannot be modeled as a continuous response variable via linear regression. Instead, Poisson regression was used to model the jump rate as ln ð E ð V Þ = e Þ ¼ b
0
þ b
1 x
1
þ b
2 x
2
þ . . .
þ b n x n
; or ln ð E ð V ÞÞ ¼ ln ð e Þ þ b
0
þ b
1 x
1
þ b
2 x
2
þ . . .
þ b n x n
; where v is the number of apparent jumps observed in grasshopper mice in a given community (modeled as a
Poisson-distributed random integer with expected value
E( V )), e is the number of grasshopper mice examined for
Bartonella within that community, and the vector b represents the intercept ( b
0
) and regression coefficients associated with predictor variables x i
. The number of predictors that could be considered within any one model was limited by the small sample size ( n = 8, given four study sites and two
Journal compilation c 2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original US government works
FEMS Microbiol Ecol 61 (2007) 438–448

Acquisition of nonspecific Bartonella by grasshopper mice 441 potential donor species per site). Therefore, an information criterion (AIC c
; Burnham & Anderson, 2002) was used to compare support for several models based on one or two of the potential predictor variables. This approach also allowed to rank the relative support for individual predictors across all models considered, using Akaike weights (Burnham &
Anderson, 2002). The predictors considered included study site, donor species, relative (numerical) contribution of the donor species to the rodent community at the study site
(C
D
), relative contribution of the grasshopper mouse to the same community (C
G
), prevalence of Bartonella infection in the donor species at the study site (P
D
), and prevalence of
Bartonella infection in the grasshopper mouse at the same site (P
G
).
contributed 53.5%, 27.5%, and 7.6% of all captured small mammals, respectively (Table 1). Other rodent species are listed in Table 1. The northern grasshopper mouse was the dominant species in Cimarron (49.4%) and Comanche
(42.2%), whereas the deer mouse was dominant in Badlands
(76.4%) and Thunder Basin (77.6%). High numbers of 13lined ground squirrels were also found in Comanche
( n = 21; 15.6%) and Thunder Basin ( n = 24; 7.0%). The rodent community in Cimarron was the most diverse, with
10 rodent species.
Small mammal captures
881 small mammals were captured belonging to 14 species of 12 genera within five families of rodents during
May–September 2003 from the four study sites (Table 1).
The composition of rodent species varied by location, from five to 10 species per site. The deer mouse ( Peromyscus maniculatus , n = 471), northern grasshopper mouse
( n = 242), and 13-lined ground squirrel ( Spermophilus tridecemlineatus , n = 67) were captured at each site and
Prevalence and distribution of infection in rodents
Bartonella
Bartonella infections were found in 356 (52.4%) of 680 tested rodents. The infected rodents belonged to seven rodent species. Northern grasshopper mice had the highest prevalence (73.5%); deer mice and 13-lined ground squirrels had prevalences of 44.4% and 43.1%, respectively (Table 2).
In Badlands, the overall prevalence of Bartonella was
43.1%. The highest prevalence was in the deer mouse
(47.3%), which was higher than that in the grasshopper mouse (25.0%). In Thunder Basin, the overall prevalence was 52.3%. There, the prevalence of Bartonella was higher in
13-lined ground squirrels (58.3%) and in deer mice (55.3%) than in grasshopper mice (35.1%). The highest overall prevalence occurred at Cimarron, where an extremely high
Table 1.
Captured rodents and contribution of each species to the rodent community at four study sites, May–September 2003
Badlands, SD Cimarron, KS Comanche, CO Thunder Basin, WY Total
Species
PEFL
PELE
PEMA
REME
REMO
SPTR
THTA
Total
CHHI
DIOR
LECU
MIOC
MUMU
NEMI
ONLE
(
Captured n )
10
0
0
25
0
0
113
0
0
10
0
162
4
0
0
C w
(%)
2.5
0
0
6.2
0
0
15.4
0
0
69.8
0
100
0
0
6.2
(
Captured n )
17
21
0
3
119
1
0
0
5
12
4
7
52
0
241
C
(%)
0.4
0
1.2
49.4
1.7
2.9
21.6
0
100
0
2
5
7.1
8.7
0
(
Captured n )
0
57
1
1
10
3
0
1
1
21
0
0
40
0
135
C
(%)
0.7
0.7
0
42.2
0
0
29.6
0.7
0.7
15.6
0
100
7.4
2.2
0
(
Captured n )
0
41
1
0
0
0
266
1
343
0
8
2
0
0
24
C
(%)
0
2.3
0.6
0
12
0.2
0
0
0
77.6
0
0
7
0.3
100
Captured
( n )
31
32
2
13
1
3
242
4
7
471
1
6
67
1
881
C
(%)
3.5
3.6
0.2
1.5
0.1
0.3
27.5
0.4
0.8
53.5
0.1
0.7
7.6
0.1
100
Rodent species codes: CHHI, Chaetodipus hispidus ; DIOR, Dipodomys ordii ; LECU, Lemmiscus curtatus ; MIOC, Microtus ochrogaster ; MUMU, Mus musculus ; NEMI, Neotoma micropus ; ONLE, Onychomys leucogaster ; PEFL, Perognathus flavus ; PELE, Peromyscus leucopus ; PEMA, Peromyscus maniculatus ; REME, Reithrodontomys megalotis ; REMO, Reithrodontomys montanus ; SPTR, Spermophilus tridecemlineatus ; THAT, Thomomys talpoides .
w
C, relative numerical contribution to the rodent community (number of captured individuals of the species divided by total number of captured individuals).
FEMS Microbiol Ecol 61 (2007) 438–448 Journal compilation c 2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original US government works

442 Y. Bai et al .
prevalence in grasshopper mice (90.4%) and relatively low prevalence in deer mice (34.9%) were found. In contrast, the lowest overall prevalence, 41.4%, was at Comanche, where the prevalence was 81.5% in grasshopper mice and no infection was found in deer mice (Table 2).
Genetic diversity of Bartonella isolates in rodents
The gltA sequences of Bartonella cultures from the study sites exhibited considerable heterogeneity. A total of 198 sequences were obtained from seven rodent species and assigned to 29 genotypic variants (Table 3). Phylogenetic analysis showed that the 29 distinct Bartonella variants clustered into several clades with different levels of divergence. For some clades, host-specificity of Bartonella variants was observed. Specifically, nine Bartonella variants identified among 63 sequences of Bartonella isolates obtained from deer mice belonged to one clade. This clade also includes sequences of Bartonella isolates obtained from white-footed mice in this study, as well as hundreds of Bartonella isolates obtained from Peromyscus species (Fig. 1) in some other studies (Kosoy et al .,
1997, 2003; Jardine et al ., 2005). Similarly, two Bartonella variants identified among 22 sequences of Bartonella isolates from 13-lined ground squirrels were clustered into one clade along with numerous Bartonella isolates obtained from other ground squirrel species (Kosoy et al ., 2003), suggesting that this clade is specific for the genus of Spermophilus .
Genotypic variants of Bartonella in grasshopper mice
Based on partial gltA sequences, a total of 10 genotypic variants of Bartonella were found among 90 sequences of
Bartonella isolates in grasshopper mice from the four study sites. Representatives of each variant were submitted to GenBank and assigned a unique accession number
(Table 4). Three variants (DQ357609, DQ357610, and
DQ357611) were present only at Cimarron and
Comanche; two variants (DQ357612 and DQ357613) were present at all sites except Badlands; one variant
(DQ357614) was present only at Comanche; one variant
(EF028157) was present only at Badlands and Thunder
Basin; two variants (EF028158 and EF028159) were present only at Badlands; and one (EF028160) was present only at
Thunder Basin (Table 4).
Sequence analyses demonstrated that the 10 variants associated with grasshopper mice clustered into four phylogenetic groups (Fig. 2). Sequence divergence was
3.1–11.6% between groups. The first group, containing five variants from 57 isolates that were obtained from
Cimarron (39), Comanche (17), and Thunder Basin
(1) was specific to grasshopper mice. Sequence similarity was 98.9–99.7% within this group. The second group, containing 19 identical isolates obtained from Cimarron
(15), Comanche (3), and Thunder Basin (1), was also specific to grasshopper mice. The third group, containing three variants of 13 isolates obtained from Badlands (5) and
Table 2.
Prevalence (%) of Bartonella infection in rodents from four sites, May–September 2003
Badlands, SD Cimarron, KS Comanche, CO Thunder Basin, WY
Species
CHHI
DIOR
LECU
MIOC
MUMU
NEMI
ONLE
PEFL
PELE
PEMA
REME
REMO
SPTR
THTA
Total
(
Tested n )
0
91
0
0
20
0
0
130
0
8
2
0
0
9
0
Positive
(%)
0
0
0
5 (55.6)
0
0
5 (25)
0
0
43 (47.3)
0
0
3 (37.5)
0
56 (43.1)
(
Tested n )
2
115
2
7
43
0
12
19
0
3
11
0
214
0
0
Positive
(%)
0
0
0
8 (42.1)
0
1
104 (90.4)
0
4 (57.1)
15 (34.9)
0
0
5 (45.5)
0
137 (64)
(
Tested n )
0
34
1
0
54
0
1
15
0
116
7
2
0
1
1
Positive
(%)
0
0
0
1 (50)
0
0
44 (81.5)
0
0
0
0
0
3 (20)
0
48 (41.4)
(
Tested n )
0
152
0
0
37
0
0
24
1
220
0
5
1
0
0
Positive
(%)
0
0
0
4 (80)
0
0
13 (35.1)
0
0
84 (55.3)
0
0
14 (58.3)
0
115 (52.3)
(
Total
Tested n )
10
1
2
226
2
7
320
1
21
26
1
4
58
1
680
Positive
(%)
0
13 (50)
0
5 (50)
0
1 (50)
166 (73.5)
0
4 (57.1)
142 (44.4)
0
0
25 (43.1)
0
356 (52.4)
Rodent species codes: CHHI, Chaetodipus hispidus ; DIOR, Dipodomys ordii ; LECU, Lemmiscus curtatus ; MIOC, Microtus ochrogaster ; MUMU, Mus musculus ; NEMI, Neotoma micropus ; ONLE, Onychomys leucogaster ; PEFL, Perognathus flavus ; PELE, Peromyscus leucopus ; PEMA, Peromyscus maniculatus ; REME, Reithrodontomys megalotis ; REMO, Reithrodontomys montanus ; SPTR, Spermophilus tridecemlineatus ; THAT, Thomomys talpoides .
Journal compilation c 2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original US government works
FEMS Microbiol Ecol 61 (2007) 438–448

Acquisition of nonspecific Bartonella by grasshopper mice 443
Thunder Basin (8), with similarities of 98.9–99.7%, belongs to the clade that is ‘specific’ to Peromyscus . The last group, containing just one isolate obtained from Thunder Basin, belongs to the clade that is ‘specific’ to Spermophilus .
the only isolate obtained from Thunder Basin was identical to the Bartonella isolate found in a 13-lined ground squirrel from Thunder Basin (EF028160 and AY584570, respectively;
Fig. 1). A total of 14 apparent jumps were observed: 13 presumably occurred from deer mice to grasshopper mice at
Badlands (5) and Thunder Basin (8), and one presumably occurred from a 13-lined ground squirrel to a grasshopper mouse in Thunder Basin (1).
Jumps of Bartonella from other rodent species to grasshopper mice
In many cases, the sequences of Bartonella isolates obtained from grasshopper mice were identical to the sequences obtained from other rodent species. In group 3, mentioned above and in Fig. 2, one variant identified in grasshopper mice from Thunder Basin (eight isolates) and Badlands
(three isolates) was identical to Bartonella variants identified in deer mice from Thunder Basin (GenBank accession numbers EF028157 and AY064535, respectively; Fig. 1).
The other two variants in group 3, consisting of one isolate each, were obtained from Badlands and are identical to the sequences of Bartonella isolates obtained from deer mice captured in the same area (EF028159 and AY064534,
EF028158 and AF489536, respectively; Fig. 1). In group 4,
Table 3.
Distribution of genetic variants of Bartonella among rodents within study sites
Rodent species # Isolates # Sequences # Variants
Dipodomys ordii
Microtus ochrogaster
Neotoma micropus
Onychomys leucogaster
Peromyscus leucopus
Peromyscus maniculatus
Spermophilus tridecemlineatus
Total
13
5
1
175
4
142
25
365
13
5
1
90
4
63
22
198
1
10
4
2
1
9
2
29
Peromyscus clade
Pm12377wy
Pm12510sd
OL12062wy EF028157 BL(3) TB(8)
Pm11808wy AY064535
Pm12489sd
OL12645sd EF028159 BL(1)
Pm12498sd AY064534
OL12490sd EF028158 BL(1)
Pm12508sd AF489536
PL12637ks
Pm11622co
Pm11861ks
Pm11341wy
OL11384ks
OL12651co
OL11387ks
OL11424ks
OL11321ks
Nm11542ks
OL11799wy EF028160 TB(1)
St11800wy AY584570
St12072wy
Mo12494sd
Mo12511sd
OL11408ks
Do11528ks
Do11922ks
Do11538ks
Do11483wy
Spermophilus clade
Identical isolates
Identical isolates
Identical isolates
Identical isolates
5.9
4
Nucleotide Substitutions (x100)
2 0
Fig. 1.
Phylogenetic relationships between Bartonella variants obtained from the grasshopper mouse and other rodent species within the four study sites, based on the analysis of a 338-bp sequence in the citrate synthase gene ( gltA ). Numbers in parentheses indicate the number of isolates obtained from grasshopper mice at the given site that are identical to the representative strain for each variant with an assigned GenBank accession number. Do,
Dipodomys ordii ; Mo, Microtus ochrogaster ; Nm, Neotoma micropus ; PL, Peromyscus leucopus ; Pm, Peromyscus maniculatus ; OL, Onychomys leucogaster ; St, Spermophilus tridecemlineatus ; BL, Badlands; CI, Cimarron; CO, Comanche; TB, Thunder Basin.
FEMS Microbiol Ecol 61 (2007) 438–448 Journal compilation c 2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original US government works

444
Table 4.
Distribution of genetic variants of Bartonella in northern grasshopper mouse in study sites
Representative strain GenBank Acc No.
Cimarron Comanche Badlands
OL11522ks
OL11523ks
OL11524ks
OL11554ks
OL11531ks
OL12651co
OL12062wy
OL12490sd
OL12645sd
OL11799wy
Total
DQ357609
DQ357610
DQ357611
DQ357612
DQ357613
DQ357614
EF028157
EF028158
EF028159
EF028160
6
15
6
5
22
0
0
0
54
0
0
11
2
0
2
3
1
1
0
20
0
0
0
0
0
0
0
0
3
0
5
1
1
Thunder Basin
0
1
0
0
1
0
8
1
11
0
0
Y. Bai et al .
Total
8
19
7
6
34
2
11
1
90
1
1
Population parameters and jump rate of
Bartonella
The apparent jump rate for Bartonella was predicted well by parameters related to the relative density of donor and recipient species, and the relative prevalence of Bartonella infection in donor and recipient species (Fig. 3). Fourteen log-linear models of jump rate were considered based on various combinations, products, and ratios of the parameters in Table 5. The effects of study site could not be considered within this modeling framework, because models based on this four-level categorical variable required the estimation of a relatively large number of parameters, leading to difficulties with model convergence. However, no evidence was found for an effect of site on the jump rate, based on a nonparametric
ANOVA
( P = 0.47). Of the 14 loglinear models, three were relatively well supported by the data. In addition to the best model (shown in Fig. 3), models with similar support were based on either the contribution of the donor species to the local rodent community ( C
D
, a metric of density) or the relative contributions of the donor species and the grasshopper mouse ( C
D
/ C
G
). Metrics of support for these two models, relative to the model in Fig. 3, were D AIC c
= 2.10 and 2.12, respectively. Burnham & Anderson (2002) suggest that D AIC c values less than two indicate models with support similar to the best model, and only models with D AIC c values greater than seven can be disregarded in further analyses. The relative support for each individual predictor, across all 14 models, is shown in
Table 5.
Bartonella infection is widely distributed in a variety of rodent species around the world, and infection levels are commonly quite high (Birtles et al ., 1994; Kosoy et al ., 1997;
Ying et al ., 2002; Holmberg et al ., 2003). The present study corroborates this pattern. The overall prevalence of Bartonella infection in these grassland rodent communities of the western US was 52.4%, with the highest infection rate found in northern grasshopper mice (90%).
The present results identified a high level of sequence divergence among Bartonella variants within populations of the northern grasshopper mouse. Among 10 gltA variants identified in grasshopper mice, six variants were circulating only in grasshopper mice. These two groups contained most of the isolates cultured from grasshopper mice, and may presently be considered specific to the grasshopper mouse.
At the same time, this study provided evidence suggesting frequent jumps of Bartonella from some rodent species to the northern grasshopper mouse. Four Bartonella variants from grasshopper mice were identical to variants obtained from deer mice or 13-lined ground squirrels. The frequency of these variants in grasshopper mice was significantly associated with variables related to the potential for Bartonella to jump from other hosts in the community, including the relative density and disease prevalence in putative donor and recipient hosts. Thus, it appears likely that these variants of Bartonella were acquired as a result of Bartonella jumping from donor species to the grasshopper mouse. It could be demonstrated that these four variants were typical of deer mice or 13-lined ground squirrels because there have been numerous isolates from the two said species that are identical to them, respectively.
The fact that 11 of 14 putative jumps belonged to one variant of Bartonella suggests that this variant may be more successful at making the jump between species. The authors would like to call attention to the epidemiological and evolutionary processes that may be at play here. Among the many possibilities, one may be seeing evidence of the evolution of a more general strain of Bartonella , one that is now spreading among grasshopper mice without the need for repeated spillover from deer mice.
Bartonella jumps between host species have rarely been observed in rodents in North America and Asia. Jardine et al . (2006) reported the occurrence of a jump of a
Journal compilation c 2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original US government works
FEMS Microbiol Ecol 61 (2007) 438–448

Acquisition of nonspecific Bartonella by grasshopper mice 445
51
97
67
85
86
98
79
64
50
70
69
OL12490sd EF028158 BL (1)
B.v. arupensis AF214557
OL12645sd EF028159 BL (1)
OL 12062wy EF028157 BL (3) TB (8)
OL12651co DQ357614 CO (2)
OL11522ks DA357609 CI (6) CO (1)
OL11524ks DQ357611 CI (6) CO (2)
OL11531ks DQ357613 CI (22) CO (11) TB (1)
OL11523ks DQ357610 CI (5) CO (1)
B.v.berkoffii
DQ334266
B.vinsonii
U28074
B.taylorii
AF 191502
OL11799wy EF028160 TB (1)
B.washoensis
AF050108
Group 4
B.chomelii
AY254308
B.clarridgeiae
U84386
B.elizabethae
U28072
B.tribocorum
AJ005494
B.grahamii
DQ334256
Group 3
OL11554ks DQ357612 CI (15) CO (3) TB (1)
B.quintana U28073
B.henselae
L38987
B.koehlerae
AF176091
B.doshiae
AF207827
B.bacilliformis
U28076
Group 1
Group 2
Fig. 2.
Phylogenetic relationships between Bartonella variants obtained from grasshopper mice and reference Bartonella species generated from bootstrapped datasets using parsimony analysis of a 338-bp sequence in the citrate synthase gene ( gltA ). Numbers in parentheses indicate frequency of isolates obtained from grasshopper mice at the given site that are identical to the representative strain for each variant. OL, Onychomys leucogaster ; BL,
Badlands; CI, Cimarron; CO, Comanche; TB, Thunder Basin.
Bartonella strain typically associated with Spermophilus tridecemlineatus into Spermophilus richardsonii ; Ying et al .
(2002) reported that a single Rattus norvegicus acquired
Bartonella from Apodemus mice found in the same area of
China; and Castle et al . (2004) reported a single jump of the
Bandicota indica strain into R. rattus in Thailand. In contrast, the present study suggests a relatively high jump rate among grasshopper mice compared with other rodent species in the western United States. This high jump rate might be explained by species-specific behaviors that increase the chance of contracting infections from other rodents. Firstly, grasshopper mice could pick up fleas that usually parasitize other rodent species when usurping the burrows of other small mammals. If bartonellae are transmitted among rodents by fleas, shared parasites could explain why Bartonella strains can be shared between grasshopper mice and other rodents. Studies of Bartonella in fleas might help to identify the source of infection in grasshopper mice. Some flea species, such as Aetheca wagneri, Orchopeas leucopus , and Pleochaetis exilis, could be commonly shared between grasshopper mice and deer mice (Thomas, 1988).
Secondly, as a predator, grasshopper mice prey on small mice of other species, perhaps obtaining infection directly. If
Bartonella can be transmitted by mechanisms other than fleas, one should not ignore the possibility that Bartonella may jump from other rodent species to grasshopper mice during direct interactions associated with predation or attempted predation.
Although grasshopper mice may commonly acquire Bartonella strains from other rodent species, such jumps do not always occur, as demonstrated by the fact that grasshopper mice were not infected with bartonellae associated with other rodents at Cimarron and Comanche. The present results suggest that the ratio of potential donor to recipient
FEMS Microbiol Ecol 61 (2007) 438–448 Journal compilation c 2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original US government works

446 Y. Bai et al .
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
− 0.05
− 0.05
0.00
0.05
0.10
0.15
0.20
0.25
C P / C P
0.30
0.35
0.40
0.45
Fig. 3.
Observed values (open symbols) and predicted values (filled squares) of the putative jump rate or the fraction of infected grasshopper mice carrying strains of Bartonella that were otherwise found only in
‘donor’ species, such as deer mice (circles, n = 4) and 13-lined ground squirrels (diamonds, n = 4). Predicted values are based on the relative numerical contribution ( sensu Table 1) of the donor species ( C
D
) and the grasshopper mouse ( C
G
) to the rodent community, and on the relative prevalence of Bartonella infection in the donor species ( P
D
) and the grasshopper mouse ( P
G
). This model is based on a log-linear, Poisson regression of the observed number of jumps per site vs. the predictor values, using the (log-transformed) number of grasshopper mice examined for Bartonella at each site as the exposure level (offset) for each data point. The observed jump rate is directly related to C
D and P
D
, and inversely related to C
G and P
G
, as would be expected if these nonspecific strains found in grasshopper mice were acquired from donor species. See Table 5 for the relative support for each predictor across all models considered.
hosts, and the ratio of Bartonella prevalence in donor and recipient hosts, may control the jump rate. In a situation when donor hosts are more abundant, one would expect more successful transfer of infections from donor hosts to recipient hosts by increasing contact between the two hosts, and thus the jump rate is increased. A low level of prevalence of infections in the recipient host could benefit donor hosts by increasing the number of recipients (more infection-free animals) susceptible to the donor strain, and thus increases jumps. Jumps of Bartonella were not observed from rodent species other than deer mice and 13-lined ground squirrels to grasshopper mice. Partially, this can be explained as a result of the low contribution of other rodent species to these communities. Nevertheless, even if the proportion of the donor host is high, jumps still may not occur if the prevalence of Bartonella in the donor host is low. Other factors, such as ecological conditions and ectoparasite activity, may also affect the jump rate.
Recently, increased attention has been focused on the role of Bartonella as a threat to human health (Anderson &
Neuman, 1997; Breitschwerdt & Kordick, 2000; Comer et al .,
2001). Many recently described Bartonella species have been isolated from humans and linked to rodent reservoir:
Bartonella grahamii , first detected in bank voles ( Clethrionomys glareolus ) in the UK (Birtles et al ., 1995), was subse-
Table 5.
Predictors of the rate at which grasshopper mice may acquire
Bartonella from donor species
Predictor
C
D
C
G
P
D
P
G
Donor
Akaike weight
0.99
0.78
0.61
0.59
0.02
Effect
Positive, direct
Negative, inverse
Positive, direct
Negative, inverse
Positive (PEMA), negative (SPTR)
Predictor codes: C
D
, numerical contribution of donor species to the rodent community ( sensu Table 1); C
G
, contribution of the grasshopper mouse; P
D
, prevalence of Bartonella infection in the donor species; P
G
, prevalence in the grasshopper mouse; Donor, effect of donor species
(deer mouse or 13-lined ground squirrel) on the acquisition rate.
quently detected in a human patient with neuroretinitis
(Kerkhoff et al ., 1999); rats ( Rattus norvegicus ) are the reservoir host for Bartonella elizabethae, which has been detected in human patients with endocarditis (Ellis et al .,
1999); Bartonella vinsonii ssp.
arupensis was found in whitefooted mice ( Peromyscus leucopus ) and in a human patient with fever and bacteremia (Welch et al ., 1999); Iralu et al .
(2006) found serological evidence that rodent-associated
Bartonella was associated with febrile illness in humans in
New Mexico; and Bartonella washoensis , first found in a human patient with cardiac disease, was subsequently detected in a California ground squirrel ( Spermophilus beecheyi ) (Kosoy et al ., 2003). Although grasshopper mice are not particularly associated with people and are less likely to transmit bartonellae directly to humans, they may spread infections among different rodent species and serve as a reservoir due to a common acquisition of Bartonella from other rodent species, such as deer mice and 13-lined ground squirrels, which have both been closely linked to other agents of human diseases. Describing and understanding the ecology of zoonotic pathogens in their natural hosts is of epidemiologic interest and warrants further investigation for predicting and preventing disease occurrence in humans.
This research was supported by grants from the NSF/NIH joint program in Ecology of Infectious Diseases (DEB-
0224328), the National Center for Environmental Research
(NCER) STAR program of the US-EPA (R-82909101-0), and the USGS Species at Risk program. The authors acknowledge USFWS, Badlands National Parks, Thunder Basin
National Grasslands, Cimarron National Grassland, Comanche National Grassland, and Chihuahua National
Grasslands for their support. The authors thank all the field workers who helped in trapping rodents and collecting blood samples.
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Published by Blackwell Publishing Ltd. No claim to original US government works
FEMS Microbiol Ecol 61 (2007) 438–448

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