Experimental evidence of host speci®city ofBartonella infection in rodents

M.Y. Kosoy*, E.K. Saito, D. Green, E.L. Marston, D.C. Jones,J.E. Childs

Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease

Control and Prevention, Public Health Service, US Department of Health and Human Services, Atlanta,

GA, USA

Abstract

A large number of Bartonella species and genetic variants were compared for their abilityto cause bacteremia in di�erent rodent species: the cotton rat (Sigmodon hispidus ), white-footed mouse (Peromyscus leucopus ), BALB/c mouse and Wistar rat. Experimental data

supported ®eld observations that host speci®city can occur among certain Bartonella speciesand rodent species. Bacteremia could only be readily produced in cotton rats or white-footed mice if the strains used for inoculation were originally obtained from the same

species or from a phylogenetically close species. A few Bartonella colonies could beobserved in the blood of some BALB/c mice by 7 days after inoculation, but no evidence ofthe persistence of the infection was found. Host speci®city suggests the possibility of a longco-speciation of Bartonella species with their rodent hosts. Host±parasite relationships

measured by the duration and level of bacteremia and the minimal infectious dose mayserve as additional criteria for classi®cation of Bartonella isolates obtained from naturalenvironments. Published by Elsevier Science Ltd.

Keywords: Animal model; Bacteremia; Bartonella; Co-evolution; Experimental infection; Host±parasite

speci®city; Peromyscus; Rodent; Sigmodon hispidus

0147-9571/00/$ - see front matter Published by Elsevier Science Ltd.

PII: S0147 -9571 (99)00075 -2

Comparative Immunology, Microbiology

& Infectious Diseases 23 (2000) 221±238

www.elsevier.com/locate/cimid

* Corresponding author. Present address: CDC/DVBID, P.O. Box 2087, Foothills Campus, Fort Col-

lins, CO 80525, USA.

E-mail address: mck3@cdc.gov (M.Y. Kosoy).

Re sumeÂ

Nous avons inocule plusieurs espeÁ ces et ge notypes de bartonelles aÁ quatre espeÁ ces

di�e rentes de rongeurs (rat du coton, Sigmodon hispidus; souris aÁ pattes blanches,Peromyscus leucopus; souris BALB/c et rats Wistar), a®n de de terminer lesquelles, parmi cessouches, sont capables d'induire une bacte rie mie chez chacun de ces rongeurs. Les donne esexpe rimentales con®rment les observations de terrain qui indiquent qu'il existe un degre de

spe ci®cite certain entre hoà tes et bacte ries. Une bacte rie mie n'a pu eà tre produite que chez S.hispidus et P. leucopus inocule s avec une souche bacte rienne isole e, aÁ l'origine, de la meà meespeÁ ce ou d'une espeÁ ce proche de rongeur. De rares colonies de bartonelles ont e te isole es

du sang de souris BALB/c. Dans ce cas, cependant, la pe riode bacte rie mique ne semblaitpas persister au delaÁ du septieÁ me jour post-inoculation. Ces observations suggeÁ rent qu'il yait eu co-spe ciation entre espeÁ ces de rongeurs et espeÁ ces, ou ge notypes, de bartonelles. La

comparaison des dure es et niveaux de bacte rie mie, ainsi que des doses minimalesinfectieuses chez di�e rents couples hoà tes±parasites, pourrait fournir des criteÁ ressupple mentaires pour la classi®cation d'isolats de bartonelles. Published by Elsevier ScienceLtd.

Mots-cleÂ: ModeÁ le animal; Bacte rie mie; Bartonella; Co-e volution; Infection expe rimentale; Spe ci®citeÂ

hoà te±parasite; Peromyscus leucopus; Rongeur; Sigmodon hispidus

1. Introduction

Interest in bacteria of the genus Bartonella has been increasing with therecognition of several novel or re-emerging diseases caused by these pathogens.Examples include bacillary angiomatosis, occurring in immunocompromizedpersons caused by Bartonella henselae [1±3], and the description of a new urbantransmission cycle for B. quintana causing endocarditis among homeless alcoholicsin the United States and Europe [4,5].

In addition to new diseases, there have been numerous descriptions of novelmembers of this genus with isolates being obtained from rodents [6±9], cats [10±12], and dogs [13]. The human public health importance for many of these isolatesremains unde®ned, although some human pathogens, such as B. elizabethae [14],now have been linked to a rodent reservoir such as rats of the genus Rattus [7,15].Some new Bartonella isolates have been given species names, while others remainwith interim designations until further characterization. The application of limitedgenetic sequencing methods has permitted tentative identi®cation of newgenogroups of Bartonella as probable new species. However, the molecular typingof these organisms should be coupled with studies aimed at de®ning the biologicalproperties of these newly described bacteria. Phenotypic characteristics that couldin¯uence the appropriate taxonomic placement of Bartonella, such as host speciesspeci®city, have been largely inferred from ®eld investigations.

Data on the host speci®city of Bartonella isolates obtained from specimens

M.Y. Kosoy et al. / Comp. Immun. Microbiol. Infect. Dis. 23 (2000) 221±238222

collected from animals sampled under natural ®eld conditions remaincontroversial (Fig. 1). Numerous species of bacteria infecting erythrocytes in awide range of animals were originally described under the genus Grahamella,which has now been uni®ed with the genus Bartonella [16]. Historically, thesebacteria were considered to be host speci®c [17]. However, Birtles and Harrison [6]were unable to ®nd evidence of single host±single bacterium speci®city amongsamples obtained from 37 small mammals captured in the United Kingdom, andconcluded that Grahamella spp/Bartonella spp are not host speci®c.

The prevalence, geographic distribution and diversity of Bartonella isolates froma variety of rodents sampled from the major biotic communities of thesoutheastern United States have been described by Kosoy et al. [8]. This studyde®ned several new genotypes of Bartonella, based on limited sequence analysis ofthe gltA gene, and presented evidence that some host speci®city in Bartonellaeinfection occurs. The relationship between bacteria and host was not easilydeduced and appeared to vary among di�erent genera of rodents (Fig. 1). Cottonrats, Sigmodon hispidus, harbored multiple genotypes (designated A, B, and C) ofBartonella; at least one of these was shared by a second species of sympatricrodent (the rice rat, Oryzomys palustris, and Bartonella genotype A). However,multiple species of mice in the genus Peromyscus sampled from di�erent localesharbored an identical unique genotype (designated D) of Bartonella or closelyrelated variants of this genotype. Similarly, rats of the genus Rattus from Northand South America and Europe carry bacteria identical, or in some cases closelyrelated, to B. elizabethae [7,15].

Until recently, studies of the host±parasite interaction of Bartonella species werelimited because of the paucity of suitable experimental animal hosts. Most studiesof bacterial pathogenesis had been conducted in domestic cats inoculated with B.

Fig. 1. Three proposed models of Bartonella (former Grahamella or Rochalimaea ) host-speci®c

relationships with rodents species based on ®eld observations: I Ð pattern with simple host speci®city

as ``one animal species Ð one bacterial species'' [17]; II Ð no evidence of host speci®city between

rodent and bacterial species [6]; and III Ð complex character of host speci®city when some closely

related rodent species may share one bacterial species, and several bacterial species may co-exist in one

animal species [8]. The black dot is a rodent species, and the white dot is a bacterial species.

M.Y. Kosoy et al. / Comp. Immun. Microbiol. Infect. Dis. 23 (2000) 221±238 223

henselae [18±21]. Cats inoculated with B. henselae developed a persistentbacteremia for 1±8 months. Cats inoculated with B. quintana did not developbacteremia [19].

In a series of experiments, using three Bartonella species recovered fromnaturally infected cotton rats, high levels of bacteremia were produced in alllaboratory-raised cotton rats inoculated with either high or low doses of thechallenge species [22]. Bacteremia persisted for 35±77 days. After the initialbacteremia was cleared, subsequent challenge with a heterologous speciesproduced a detectable bacteremia in each animal inoculated, while eight of nineanimals challenged with the homologous Bartonella species remained non-bacteremic.

The objectives of the present study were to, (1) investigate the ability ofdi�erent Bartonella species and their variants to produce bacteremia in fourrodent species (cotton rat Ð Sigmodon hispidus, white-footed mouse ÐPeromyscus leucopus, BALB/c mouse Ð Mus musculus, and Wistar rat Ð Rattusnorvegicus ), (2) determine the minimal infectious dose (ID50) of the di�erentBartonella species and their variants required to establish bacteremia, and (3) usethe observed di�erences to develop additional criteria for the phenotypicclassi®cation of Bartonella di�erentiated largely by limited genotypic analyses.

2. Materials and methods

2.1. Bartonellae

Four well-characterized Bartonella species (B. henselae, B. quintana, B. vinsonii,and B. elizabethae ) and 10 genetically distinct Bartonella isolates obtained fromwild captured rodents from Colorado, Georgia and North Carolina were used forthe experimental study (Fig. 2). The isolates obtained from rodents included avariety of unique genetic variants that clustered into four distinct genotypes: A, B,C, and D [8]. The degree of similarity between any one of these genotypes, basedon comparison of a 338-bp sequence of the citrate synthase gene ( gltA ), iscomparable to the degree of similarity between the well-characterized species, B.elizabethae, B. henselae, B. quintana, and B. vinsonii [7]. Genetic di�erencesbetween the well-de®ned clusters of related strains, antigenic typing [23],phenotypic appearance of colonies [23], and di�erential growth characteristics insusceptible animals [22] indicate that these genotypes behave as unique biologicalspecies of the genus Bartonella. In this manuscript we will refer to the genotypesA, B, C, and D as species A, B, C, and D. Below the level of species we use term``genovar'' to refer to the genetically related but distinguishable Bartonella thatclustered in genetic and phenotypic analyses within species A, B, C, or D. Theterm ``strain'' will be used to refer to a unique isolate of Bartonella. The gltAsequences of the Bartonellae used in this study have the following GenBankaccession number: B. elizabethae - Z70009, B. henselae - L38987, B. quintana -Z70014, B. vinsonii - Z70015, genovar A1 (type strain Sh6397ga) - U84372,

M.Y. Kosoy et al. / Comp. Immun. Microbiol. Infect. Dis. 23 (2000) 221±238224

genovar A2 (type strain Sh6700ga) - U84373, genovar A3 (type strain Op6398ga) -U84374, genovar B1 (type strain Sh6396ga) - U84375, genovar B6 (type strainNm15586co) - AF110312, genovar C1 (type strain Sh6541ga) - U84377, genovarD1 (type strain PL7238nc) - U84379, genovar D2 (type strain Pm7772nc) -U84380, genovar D7 (type strain Pg8555ga) - U84385, and genovar D8 (typestrain Nm15594co) - AF128838.

All bacteria were cultivated on heart infusion agar plates supplemented with 5%rabbit blood (BBL, Becton Dickinson Microbiology System, Cockeysville, MD) at328C in an aerobic atmosphere with 5% carbon dioxide. Each isolate was grownon an agar plate until con¯uent. Cultures were harvested and suspended inphosphate-bu�ered saline, pH 7.6 (PBS). Serial 10-fold dilutions in brain heartinfusion medium were plated on agar to calculate inoculum dose by colony-forming units (CFU).

Fig. 2. A phylogenetic analysis and the level of similarity for the 13 Bartonella strains used in the study

based on a 338-bp sequence of the citrate synthase ( gltA ) gene. �A sequence homology at level of

0.9970 corresponds to only one nucleotide di�erence between compared pair of the cultures.

M.Y. Kosoy et al. / Comp. Immun. Microbiol. Infect. Dis. 23 (2000) 221±238 225

2.2. Rodents

Cotton rats were purchased from Virion System Inc. (Rockville, MD), white-footed mice from the Peromyscus Stock Center, University of South Carolina(Columbia, SC), and Wistar rats and BALB/c mice from Harlan Sprague±Dawley(Indianapolis, IN). In total, 88 cotton rats, 74 white-footed mice, 47 BALB/c miceand 33 Wistar rats were used in the study. The ages of the animals at the time ofinoculation ranged from 4 to 6 weeks old. The animals were housed separately incages in a speci®c-pathogen-free environment until experimental study and weregiven food and water ad libidum. All animals were negative for Bartonellainfection, based on plating of blood on agar and serological screening prior toexperimental work.

2.3. Infection of rodents

An inoculum was prepared by diluting known concentrations of bacteria insterile PBS. As described previously, animals were inoculated with 1.0 ml of thebacterial suspension of each Bartonella culture (three rodents per culture anddose) via a combination of intraperitoneal (0.5 ml) and subcutaneous routes(0.5 ml) using a 22-G needle [22]. Three rodents of each species received only1.0 ml of diluent as a negative control. Wistar and cotton rats were euthanizedwith CO2 gas, and BALB/c and white-footed mice were sacri®ced by anestheticoverdose (Iso¯urane). All protocols were reviewed and approved by the Centersfor Disease Control and Prevention Animal Care and Use Committee.

2.4. Blood sampling

Animals were anesthetized by intramuscular administration of a 1:10 mixture ofxylazine (Rompun, The Butler Company, Norcross, GA) and ketaminehydrochloride (Fort Dodge Laboratories Inc., Fort Dodge, IA). Blood samples(0.3±0.5 ml) were collected from animals at weekly intervals by retro-orbitalbleeding using sterile, heparinized, capillary tubes. Blood samples were stored atÿ708C until use.

2.5. Determination and quanti®cation of bacteremia

Bacteremia was determined by direct plating of blood collected from animals onheart infusion agar plates supplemented with 5% rabbit blood (BBL). Cultureswere examined daily for bacterial growth and CFU were assessed at 7±8 days.Blood was initially diluted 1:4 in brain heart infusion medium for primary plating,and serial four-fold dilutions of the original sample were applied when growth wastoo dense for initial CFU quanti®cation.

M.Y. Kosoy et al. / Comp. Immun. Microbiol. Infect. Dis. 23 (2000) 221±238226

2.6. Polymerase chain reaction (PCR) analysis

Cultures used for inoculations, as well as cultures of organisms recovered fromrodents during the experiments, were con®rmed as being of the original Bartonellatype by PCR ampli®cation of extracted DNA using gltA gene-speci®c primers,followed by sequence analysis, as previously described [8].

3. Results

3.1. Cotton rats Ð Bartonella

In total, 39 cotton rats were challenged with a high dose (107 CFU) of 13strains of Bartonella. All 9 of the cotton rats inoculated with genovars A1, B1,and C1 obtained from naturally infected cotton rats became bacteremic (Table 1).Of the remaining 30 cotton rats, bacteremia was only detected in the three animalsinoculated with strain Op15907ga; a strain of Bartonella identical by gltA geneanalysis to all other organisms of genovar A1, species A (Fig. 2). This strain wasone of ®ve obtained from rice rats, O. palustris, captured at a site dominated bycotton rats and where 98.4% (n=539) of the Bartonella-positive animals obtainedwere cotton rats. The total number of strains identical to Op15907ga obtainedfrom cotton rats from this site was 339 [23].

Inoculation of cotton rats with B. vinsonii, B. henselae, B. quintana, B.elizabethae, strain Op6398ga, and strain Nm15586co, did not result in detectablebacteremia within the 4 week observation period (Table 1). Strain Op6398ga,which is similar to Op15907ga by gltA gene analysis (Fig. 2) but belongs togenovar A3, was one of three genetically identical Bartonella strains obtained fromrice rats captured on McQueens Island, Georgia. Strain Nm15586co, obtainedfrom the Mexican woodrat, Neotoma mexicana, is genetically closest to species B(B1: Sh6396ga) from cotton rats and to B. vinsonii (Fig. 2). None of the strainsfrom group D infected the cotton rats.

The ID50 of Bartonella species A, B, and C, was established in cotton rats(Table 2), and ranged from <10 CFU (Sh6541ga, genovar C1, species C) toapproximately 103 CFU (Sh6540ga and Op15907ga, genovar A1, species A)(Table 2). Both strains of genovar A1 had comparable ID50s; however, theduration of bacteremia in cotton rats receiving the rice rat strain (Op15907ga) wasshorter compared to that of Sh6540ga (Table 2). Only 2/6 cotton rats receiving theisolate derived from rice rats remained bacteremic at 4 weeks compared to 7/8cotton rats receiving the homologous isolate (P=0.09, Fisher's exact test).

3.2. White-footed mice Ð Bartonella

In total, 36 white-footed mice, Peromyscus leucopus, were challenged with ahigh dose (107 CFU) of 12 strains of Bartonella. The 12 mice inoculated with thefour strains of species D Bartonella became bacteremic; each strain originated

M.Y. Kosoy et al. / Comp. Immun. Microbiol. Infect. Dis. 23 (2000) 221±238 227

Table

1

Experim

entalstudyofcottonrats,Sigmodonhispidus,challenged

with13strainsofBartonella

Bartonella

species

(genovar)

Strain

name

Strain

source:

anim

alspecies,countryorstate

Number

ofbacterem

ic/testedrats

per

weekafter

inoculationa

1week

2week

3week

4week

A(A

1)

Sh6540ga

Sigmodonhispidus,Georgia

3/3

3/3

3/3

3/3

A(A

1)

Op15907ga

Oryzomyspalustris,Georgia

3/3

3/3

2/3

0/3

B(B1)

Sh6396ga

Sigmodonhispidus,Georgia

3/3

3/3

3/3

3/3

C(C

1)

Sh6541ga

Sigmodonhispidus,Georgia

3/3

3/3

3/3

3/3

A(A

3)

Op6398ga

Oryzomyspalustris,Georgia

0/3

0/3

0/3

0/3

B(B6)

Nm15586co

Neotomamexicana,Colorado

0/3

0/3

0/3

0/3

D(D

1)

Pl7238nc

Peromyscusleucopus,NorthCarolina

0/3

0/3

0/3

0/3

D(D

2)

Pm7067nc

Peromyscusmaniculatus,NorthCarolina

0/3

0/3

0/3

0/3

D(D

8)

Nm15594co

Neotomamexicana,Colorado

0/3

0/3

0/3

0/3

B.vinsonii

Baker

Microtuspennsylvanicus,Canada

0/3

0/3

0/3

0/3

B.henselae

Houston-1

Human,Texas

0/3

0/3

0/3

0/3

B.quintana

OK90-268

Human,Oklahoma

0/3

0/3

0/3

0/3

B.elizabethae

F9251

Human,Massachusetts

0/3

0/3

0/3

0/3

aThreeBartonella-negativerats

per

each

strain

wereinoculatedwith107CFU

ofeach

inoculum.Bloodsampleswerecollectedweekly

beginningthe®rst

weekafter

inoculationduring4weekperiodofobservation.

M.Y. Kosoy et al. / Comp. Immun. Microbiol. Infect. Dis. 23 (2000) 221±238228

Table

2

Bacterem

iain

cottonrats,Sigmodonhispidus,challenged

withfourstrainsofBartonella

during4-w

eekperiodofobservation

Bartonella

species:genovar

(strain)

Inoculum

dose

(CFU)

Number

ofratsa

Number

ofbacterem

icrats

andlevel

ofbacterem

iabyweekafter

inoculationb

1week

2week

3week

4week

nMeanCFU

nMeanCFU

nMeanCFU

nMeanCFU

Sp.A:A1(Sh6540ga)

107

33

2.7�105

32.3�105

31.3�104

32.7�103

103

55

3.8�105

52.2�105

54.1�105

41.2�103

102

30

00

00

00

0

10

30

00

00

00

0

Sp.A:A1(O

p15907ga)

107

33

7.3�104

31.7�104

26.0�103

00

103

33

2.0�105

31.0�105

34.7�104

24.7�103

102

30

00

00

00

0

Sp.B:B1(Sh6396ga)

107

33

1.3�103

31.3�103

31.0�103

34.8�102

103

55

1.1�104

51.0�104

58.0�103

54.8�103

102

30

02

6.7�103

31.5�103

25.6�103

10

30

00

00

00

Sp.C:C1(Sh6541ga)

107

33

1.1�104

33.6�104

31.7�104

34.1�103

103

50

05

8.9�104

55.5�104

53.1�104

102

30

03

1.7�105

36.7�105

35.3�104

10

50

03

6.8�104

51.8�105

51.3�105

13

00

00

16.7�103

16.7�103

aThreeor®veBartonella-negativerats

per

each

strain

anddose

wereinoculated.

bLevel

ofbacterem

iameasuredbymeanCFU

per

1.0

mlofblood.

M.Y. Kosoy et al. / Comp. Immun. Microbiol. Infect. Dis. 23 (2000) 221±238 229

Table

3

Experim

entalstudyofwhite-footedmice,

Peromyscusleucopus,challenged

with12strainsofBartonella

Bartonella

species

(genovar)

Strain

name

Strain

source:

anim

alspecies,countryorstate

Number

ofbacterem

ic/testedmiceper

weekafter

inoculationa

1week

2week

3week

4week

D(D

1)

Pl7238nc

Peromyscusleucopus,NorthCarolina

3/3

3/3

3/3

3/3

D(D

2)

Pm7067nc

Peromyscusmaniculatus,NorthCarolina

3/3

3/3

3/3

3/3

D(D

2)

Pm15590co

Peromyscusmaniculatus,Colorado

3/3

3/3

3/3

3/3

D(D

7)

Pg8555ga

Peromyscusgossypinus,Georgia

1/3

3/3

3/3

3/3

D(D

8)

Nm15594co

Neotomamexicana,Colorado

0/3

0/3

0/3

0/3

A(A

1)

Sh6540ga

Sigmodonhispidus,Georgia

0/3

0/3

0/3

0/3

B(B1)

Sh6396ga

Sigmodonhispidus,Georgia

0/3

0/3

0/3

0/3

C(C

1)

Sh6541ga

Sigmodonhispidus,Georgia

0/3

0/3

0/3

0/3

B.vinsonii

Baker

Microtuspennsylvanicus,Canada

0/3

0/3

0/3

0/3

B.henselae

Houston-1

Human,Texas

0/3

0/3

0/3

0/3

B.quintana

OK90-268

Human,Oklahoma

0/3

0/3

0/3

0/3

B.elizabethae

F9251

Human,Massachusetts

0/3

0/3

0/3

0/3

aThreeBartonella-negativemiceper

each

isolate

wereinoculatedwith107CFU

ofeach

inoculum,andsamplesofbloodwerecollectedweekly

beginning

the®rstweekafter

inoculationduring4-w

eekperiodofobservation.

M.Y. Kosoy et al. / Comp. Immun. Microbiol. Infect. Dis. 23 (2000) 221±238230

from a wild mouse of the same genus (two strains from deer mice, P. maniculatus;one strain each from a white-footed mouse, P. leucopus, and a cotton mouse, P.gossypinus ) (Table 3). Two genetically identical strains of genovar D2 (Pm7067ncand Pm15590co) originated from P. maniculatus captured from North Carolinaand Colorado. None of the other Bartonella species and genovars tested causedbacteremia in Peromyscus mice during the 4 week observation period, includingNm15594co, obtained from the Mexican woodrat, which is genetically very similarto species D Bartonella isolated from Peromyscus (Fig. 2).

The ID50 of three Bartonella genovars belonging to species D was established inwhite-footed mice (Table 4), and ranged from approximately 104 CFU (Pl7238nc,genovar D1) to approximately 107 CFU (Pm15590co, genovar D2). Both ID50sand peak levels of bacteremia were higher than that ever observed in cotton rats.The isolate Pl7238nc (genovar D1) caused the highest level of bacteremia observedin this study (peak level > 1,000,000 CFU/ml). The minimum dose of inoculumrequired to produce bacteremia in laboratory-reared white-footed mice was higherwhen the initial isolate was a species other than P. leucopus (Table 4); however,only a single strain from P. leucopus was investigated here.

3.3. Laboratory mice and rats Ð Bartonella

In total, 43 BALB/c mice were challenged with a high dose of 107 CFU and alow dose of 103 CFU of nine strains of Bartonella. When 1:4 dilutions of bloodcollected from these mice 1 week p.i. were plated on agar, a few Bartonella-likecolonies were observed on 4 of the 43 plates. Counts ranged from one to ®vecolonies per plate and were obtained from all three mice inoculated with the highdose of strain Sh6540ga (species A, genovar A1) and from one of three miceinoculated with the high dose of isolate Sh6396ga (species B, genovar B1). Thebacterial colonies cultured from the blood of the BALB/c mice appeared smooth,circular, and non-adherent to the surface of the agar, a similar morphologicdescription of colonies of all Bartonella strains obtained from blood of cotton ratsand white-footed mice. Microscopic examination of smears revealed gram-negative, rod-shaped bacteria resembling Bartonella, but these were unusuallymonomorphic with elongated bodies. However, DNA extracted from theseorganisms, ampli®ed by PCR, and analyzed for the gltA gene sequence,demonstrated that the isolates were genetically identical to the Bartonella used forinoculation. When additional passages of these four cultures were made, themorphology of the organisms reverted to the pleomorphic rods typical of mostBartonella species. Other than the low recovery of bacteria from these few animalsduring the ®rst week p.i., no other BALB/c mice became bacteremic during the 4-week period of observation.

No bacteremia was detected from blood obtained from any of the 30 Wistarrats inoculated with high doses of 107 CFU of 10 Bartonella (Fig. 3).

M.Y. Kosoy et al. / Comp. Immun. Microbiol. Infect. Dis. 23 (2000) 221±238 231

Table

4

Bacterem

iain

white-footedmice,

Peromyscusleucopus,challenged

withfourstrainsofBartonella

obtained

from

threespeciesofgenusPeromyscus

Bartonella

species:genovar

(strain)

Inoculum

dose

(CFU)

Number

ofratsa

Number

ofbacterem

icrats

andlevel

ofbacterem

iabbyweekafter

inoculation

1week

2week

3week

4week

nMeanCFU

nMeanCFU

nMeanCFU

nMeanCFU

Sp.D:D1(Pl7238nc)

107

33

1.8�106

34.2�105

33.0�105

31.3�105

106

33

1.1�106

36.3�105

34.7�105

7.5�104

105

33

2.6�106

35.0�105

33.9�105

34.9�104

104

32

6.7�105

21.0�105

21.0�105

26.7�103

103

50

00

00

00

0

Sp.D:D2(Pm7067nc)

107

33

7.0�105

39.0�105

38.7�105

31.5�105

106

33

1.2�106

35.3�105

35.3�105

33.6�105

105

31

9.3�104

16.7�103

12.0�102

10

104

30

00

00

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Sp.D:D7(Pg8555ga)

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aThreeor®veBartonella-negativerats

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M.Y. Kosoy et al. / Comp. Immun. Microbiol. Infect. Dis. 23 (2000) 221±238232

4. Discussion

This is the ®rst report to compare a large number of Bartonella species andgenetic variants for their ability to cause bacteremia in di�erent rodent hosts,including rodent species associated with Bartonella maintenance in naturalenvironments. This study provides experimental data that support ®eldobservations that host speci®city can occur among certain Bartonella-rodent dyads[8,23]. Bacteremia could only be readily produced in cotton rats or white-footedmice when the strain used for inoculation was originally obtained from the samespecies or from congenic rodents (Fig. 3). A single exception to this observationoccurred when a Bartonella isolate, genetically identical by partial gltA genesequencing, obtained from a rice rat trapped among many infected cotton rats,caused bacteremia in cotton rats.

Recent investigations of rodent populations in regions of Europe, Africa, andNorth America have revealed a number of novel species or unnamed varieties ofBartonella. Attempts to provide universal and simple rules of a one-to-oneassociation between bacterial isolates and the rodent species infected quickly failed

Fig. 3. Culture results from blood samples from four species of rodents used as experimental hosts for

infection with 15 strains of Bartonella: 1 Ð evidence of prolonged high level bacteremia; 2 Ð no

evidence of bacteremia; 3 Ð low level of bacteremia one week after infection, possibly representing

residual bacteria from the original inoculum.

M.Y. Kosoy et al. / Comp. Immun. Microbiol. Infect. Dis. 23 (2000) 221±238 233

[6,8]. The data generated from molecular analyses of individual isolatescontradicted conclusions about strict and simple host speci®city among theseintra-erythrocytic organisms, which had been mostly described under genusGrahamella [17]. Based on the observation that a single Bartonella species could beisolated from several rodent species, Birtles et al. [6] suggested that if speciesspeci®city existed it might be associated with an arthropod vector and bacteriarather than with the bacteria and the vertebrate host.

However, although the hypothesis of ``one rodent species ± one bacterialspecies'' could not be unequivocally supported, some evidence of host speci®citywas observed in a large study involving hundreds of rodents and their associatedBartonella [8,23]. While three distinct Bartonella genotypes (species) wereassociated with cotton rats, none of them was found commonly in other rodentspecies (although they did occur in some sympatric species), and none of them hasbeen found among mice of the genus Peromyscus. On the other hand, severalPeromyscus species from widely separated regions (southeastern and westernUnited States) were found to share the same Bartonella species (D) and genovars[23]. The only other rodent that shared the D species was the golden mouse,Ochrotomys nuttalli, which is phylogenetically very close to the genus Peromyscus(Fig. 4). Moreover, host speci®city or some biological resistance to speciescrossover, may explain the pattern observed in nature where the most prevalentBartonella species typically infects most individuals of the numerically dominantspecies in the rodent community, yet does not attain high prevalence in other

Fig. 4. Phylogenetic relationships between some North American Sigmodontine rodents (fragment

modi®ed from Engel et al. [24]).

M.Y. Kosoy et al. / Comp. Immun. Microbiol. Infect. Dis. 23 (2000) 221±238234

sympatric rodent species. For example, in an area where cotton rats constitutednearly 80% of the rodent community, based on trapping success, and where theoverall prevalence of Bartonella infection in cotton rats was 79% (n = 539), onlyone of 80 white-footed mice from the same locality was infected [23]. However,the prevalence of Bartonella species D may reach 70% in white-footed mice inlocations where it is the dominant rodent species [8].

In a previous study, we experimentally produced Bartonella infections in cottonrats when one of the three naturally occurring Bartonella species (A, B, or C) wasused as an inoculum [22]. That study demonstrated that the cotton rat can serveas an experimental host to investigate the pathogenesis of Bartonella infection.The present study adds the laboratory-bred white-footed mouse to the list ofmammals available for in vivo study of Bartonella infections.

The present study also indicates limitations to the use of the classical laboratoryanimals for investigating experimental Bartonella infections when the bacteria usedoriginate from geographically di�erent sources or from phylogenetically distantanimals. No bacteremia was detected in any rodent, other than the transientinfections in a few BALB/c mice, when inoculums originated from heterologousrodents of the genera Microtus and Neotoma, or from a non-rodent source (Tables1 and 3, Fig. 3). One culture obtained from a rice rat successfully causedbacteremia in cotton rats (Fig. 3). This genovar was obtained from a localitywhere cotton rats were the dominant species in the rodent community, and thegreat majority of the isolates of this serovar from this location came from cottonrats. The isolation of this serovar from a rice rat is further evidence that inter-species transmission of Bartonella occurs among rodents and that the bacteriumcan maintain its virulence for the original host after passage in an additionalspecies. Such species crossover of infection might be expected in this situation, asthe phylogenetic relationship between Oryzomys and Sigmodon species [24] placesthe rice rat taxonomically closer to the cotton rat than to any other NorthAmerican rodent species (Fig. 4).

Although based on limited observations, it appears that the ID50 of Bartonellaspecies D introduced in P. leucopus was lower for isolates that originated from thehomologous species (0104 CFU) compared with genetically related isolates fromother Peromyscus species (106 CFU for strains from P. maniculatus from NorthCarolina and 107 CFU for strains from P. maniculatus from Colorado and P.gossypinus from Georgia). Clearly, the importance of phylogenetic relatedness ofrodent hosts, or presumably any group of taxonomically linked vertebrate species,in in¯uencing susceptibility to Bartonella infection is an area of major interest anddeserving additional detailed study.

None of a large set of the cultures resulted in visible bacteremia in traditionallaboratory animals: mice and rats. Although a few colonies of speci®c Bartonellaspecies could be observed in blood of some BALB/c mice 7 days after inoculationwith a high dose of the bacteria, no signs of persistence of the infection in themice were found more than 1 week after infection. Similarly, a limited number ofBartonella were identi®ed in the blood of BALB/c mice a short period afterinoculation with a very high dose of B. henselae by Karem et al. [25]. Karem et al.

M.Y. Kosoy et al. / Comp. Immun. Microbiol. Infect. Dis. 23 (2000) 221±238 235

could culture Bartonella 24 h after inoculation, but no live organisms were foundfrom the same animals by 48 h after infection. It is likely that the recovery ofBartonella after such a short period of the time is representative of residualorganisms from the original inoculum. These data suggest that the results of ®eldinvestigations on Bartonella species distribution in animal communities should beinterpreted cautiously because of the possibility of low-level or short-durationbacteremia in animals.

Several of our observations also indicate that genetic identity based on limitedsequence analysis of the gltA gene does not indicate phenotyic homogeneityamong Bartonella strains. In cotton rats, the level of bacteremia and the ID50 ofthe species A Bartonella originally isolated from a rice rat were similar to thosecaused by a genetically indistinguishable (by citrate synthase gene sequencing)isolate originally obtained from a cotton rat (Table 2). However, the duration ofthe bacteremia caused by the rice rat isolate in cotton rats was 3±4 weeks, muchshorter than that observed in the homologous dyad (Tables 1 and 2) [22].Similarly, morphological changes were apparent in the few isolates of Bartonellaobtained from BALB/c mice during their ®rst week p.i., yet based on limitedgenetic analysis, the recovered isolates were ``identical'' to the inoculatedgenotypes. Finally, our attempts to establish B. elizabethae infection in Wistar ratswere unsuccessful, yet two independent researchers have reported identifyingbacteria identical to B. elizabethae from Rattus norvegicus, on the basis of partialsequencing of the gltA gene [7,15]. In addition, another investigator has been ableto produce short-lived bacteremia in laboratory rats after injection with B.elizabethae and B. tribocorum; the latter species was isolated from the blood ofwild rats [9].

Although we can only speculate on why our laboratory rats failed to developbacteremia following inoculation with a Bartonella species linked to this rodentunder natural circumstances, several factors could have played a role. The strainof B. elizabethae used in our experiments was isolated from a human withendocarditis [14] and was not derived from a rodent. However, by partialsequencing this human isolate appeared identical to strains coming from wild-captured Rattus. The Wistar rat was selected for study because this rat wasestablished directly from crosses of captured wild rats and is presumably ``close''to the wild genotype [26]. Multiple B. elizabethae-related strains isolated fromwild-caught Rattus species and other Bartonella strains obtained from Muridaespecies (Old World rats and mice) may be considered as prospective candidatesfor establishing infections into a variety of laboratory animals.

Observation of the colony morphology of Bartonella cultured from the blood ofthe experimentally infected rodents revealed some di�erences compared to culturesof the same bacterial species, but obtained from wild-captured rodents. Only thesmooth phenotype of colonies of Bartonella obtained from cotton rats, white-footed mice, and BALB/c mice during our experimental study was observed.Direct plating of blood collected from wild-captured rodents demonstrated thatcolonies of Bartonella from 12.5% of cotton rats and 27.5% of Peromyscus miceappeared with the typical rough phenotype with slightly irregular shape and

M.Y. Kosoy et al. / Comp. Immun. Microbiol. Infect. Dis. 23 (2000) 221±238236

adherence to the agar [8]. It is possible that a passage of the Bartonella culturesthrough a susceptible host organism resulted in some phenotypic switching in theinfecting bacteria with the predominance of the smooth colony morphology.

In summary, although there were some indications of host speci®city at the levelof rodent species-Bartonella genovar, the main di�erence in susceptibility toinfection was observed above the taxonomic level of rodent genus or even abovethe phylogenetically close genera (e.g., Sigmodon and Oryzomys; Peromyscus andOchrotomys ). Evidence of host speci®city suggests the possibility of a long-termco-evolution or co-speciation of Bartonella with their rodent hosts. Data on host±parasite relationships measured by the minimal infectious dose and the durationand level of bacteremia may serve as additional criteria for biological classi®cationof Bartonella isolates obtained from natural environments.

References

[1] Koehler JE, Quinn FD, Berger TG, LeBoit PE, Tappero JW. Isolation of Rochalimaea species

from cutaneous and osseous lesions of bacillary angiomatosis. The New England Journal of

Medicine 1992;327:1625±31.

[2] Koehler JE, Sanchez MA, Garrido CS, Whitfeld MJ, Chen FM, Berger TG, Rodriguez-Barradas

MC, LeBoit PE, Tappero JW. Molecular epidemiology of Bartonella infections in patients with

bacillary angomatosis-peliosis. The New England Journal of Medicine 1997;337:1876±83.

[3] Regnery RL, Anderson BE, Clarridge JE, Rodrigues-Barradas M, Jones DC, Carr JH.

Characterization of a novel Rochalimaea species, R. henselae sp. Nov., isolated from blood of a

febrile, human immunode®ciency virus-positive patient. Journal of Clinical Microbiology

1992;30:265±74.

[4] Drancourt MJ, Mainardi J, Brouqui R, Vandenesch F, Carta A, Lehnert F, Etienne J, Goldstein

F, Acar J, Raoult D. Bartonella (Rochalimaea ) quintana endocarditis in three homeless men. The

New England Journal of Medicine 1995;332:419±23.

[5] Spach DH, Kanter AS, Dougherty MJ, Larson AM, Coyle MB, Brenner DJ, Swaminathan B,

Matar GM, Welch DF, Root RK, Stamm WE. Bartonella (Rochalimaea ) quintana bacteremia in

inner-city patients with chronic alcoholism. The New England Journal of Medicine 1995;332:424±8.

[6] Birtles RJ, Harrison TG, Molyneux DH. Grahamella in small woodland mammals in the U.K.:

isolation, prevalence and host speci®city. Annals of Tropical Medicine and Parasitology

1994;88:317±27.

[7] Birtles RJ, Raoult D. Comparison of partial citrate synthase gene ( gltA ) sequences for

phylogenetic analysis of Bartonella species. International Journal of Systematic Bacteriology

1996;46:891±7.

[8] Kosoy MY, Regnery RL, Tzianabos T, Marston EL, Jones DC, Green D, Maupin GO, Olson JG,

Childs J. Distribution, diversity, and host speci®city of Bartonella in rodents from the southeastern

United States. The American Journal of Tropical Medicine & Hygiene 1997;57:578±88.

[9] Heller R, Riegel P, Hansmann Y, Delacour G, Bermond D, Dehio C, Lamarque F, Monteil H,

Chomel B, Piemont Y. Bartonella tribocorum sp. nov., a new Bartonella species isolated from the

blood of wild rats. International Journal of Systematic Bacteriology 1998;48:1333±9.

[10] Groves MG, Harrington KS. Rochalimaea henselae infections: newly recognized zoonoses

transmitted by domestic cats. Journal of American Veterinary and Medical Association

1994;204:267±71.

[11] Chomel BB, Abbott RC, Kasten RW, Floyd-Hawkins KA, Kass PH, Glaser CA, Pedersen NC,

Koehler JE. Bartonella henselae prevalence in domestic cats in California: risk factors and

M.Y. Kosoy et al. / Comp. Immun. Microbiol. Infect. Dis. 23 (2000) 221±238 237

association between bacteremia and antibody titers. Journal of Clinical Microbiology

1995;33:2445±50.

[12] Heller R, Artois M, Xemar V, De Briel D, Gehin H, Jaulhac B, Monteil H, Piemont Y.

Prevalence of Bartonella henselae and Bartonella clarridgaiae in stray cats. Journal of Clinical

Microbiology 1997;35:1327±31.

[13] Kordick DL, Swaminathan B, Greene GE, Wilson KH, Whitney AM, O'Connor S, Hollis DG,

Matar GM, Steigerwalt AG, Malcolm GB, Hayes PS, Had®eld TL, Breitschwerdt EB, Brenner

DJ. Bartonella vinsonii subsp. Berkho�i subsp. Nov., isolated from dogs; Bartonella vinsonii subsp.

Vinsonii; and emended description of Bartonella vinsonii. International Journal of Systematic

Bacteriology 1996;46:704±9.

[14] Daly JS, Worthington MG, Brenner DJ, Moss WC, Hollis DG, weyant RS, Steigerwalt AG,

Weaver RE, Daneshvar MI, O'Connor SP. Rochalimae elizabethae sp. nov. isolated from a patient

with endocarditis. Journal of Clinical Microbiology 1993;31:872±81.

[15] Ellis BA, Regnery RL, Beati L, Marston E, Ksiazek TG, Rood M, Glass GG, Jones D, Childs JE,

Rats of the genus Rattus are reservoir hosts for pathogenic Bartonella: an Old World origin for a

New World disease? The Journal of Infectious Diseases 1999;180:220±4.

[16] Birtles RJ, Harrison TG, Saunders NA, Molyneux DH. Proposal to unify the genera Grahamella

and Bartonella, with description of Bartonella talpae comb. nov., Bartonella peromysci comb. nov.,

and three new species, Bartonella grahamii sp. nov., Bartonella taylorii sp. nov., and Bartonella

doshiae sp. nov. International Journal of Systematic Bacteriology 1995;45:1±8.

[17] Tyzzer EE. A comparative study of Grahamella, Haemobartonella and Eperythrozoa in small

mammals. Proceeding of the American Philosophical Society 1942;85:359±98.

[18] Greene CE, McDermott M, Jameson PH, Atkins CL, Marks AM. Bartonella henselae infection in

cats: evaluation during primary infection, treatment, and rechallenge infection. Journal of Clinical

Microbiology 1996;34:1682±5.

[19] Regnery RL, Rooney JA, Johnson AM, Nesby SL, Manzewitsch R, Beaver K, Olson JG.

Experimentally induced Bartonella henselae infections followed by challenge exposure and

antimicrobial therapy in cats. American Journal of Veterinary Research 1996;57:1714±20.

[20] Abbott RC, Chomel BB, Kasten RW, Floyd-Hawkins KA, Kikuchi Y, Koehler JE, Pedersen NC.

Experimental and natural infection with Bartonella henselae in domestic cats. Comparative

Immunology, Microbiology & Infectious Diseases 1997;20:41±51.

[21] Guptill L, Slater L, Wu CC, Lin TL, Glickman LT, Welch DF, HogenEsch H. Experimental

infection of young speci®c pathogen-free cats with Bartonella henselae. The Journal of Infectious

Diseases 1997;176:206±16.

[22] Kosoy MY, Regnery RL, Kosaya OI, Childs JE. Experimental infection of cotton rats with three

naturally occurring Bartonella species. Journal of Wildlife Diseases 1999;35:275±84.

[23] Kosoy MY. Unpublished data.

[24] Engel SR, Hogan KM, Taylor JF, Davis SK. Molecular systematics and paleobiogeography of the

South American Sigmodontine rodents. Molecular Biology and Evolution 1998;15:35±49.

[25] Karem KL, Dubois KA, McGill SL, Regnery RL. Characterization of Bartonella henselae speci®c

immunity in BALB/c mice, Immunology (in press).

[26] Altman PL, Katz DD. Mouse and rat. In: Altman PL, Katz DD, editors. Inbred and genetically

de®ned strains of laboratory animals. Bethesda, Maryland: Federation of American Societies for

Experimental Biology, 1979.

M.Y. Kosoy et al. / Comp. Immun. Microbiol. Infect. Dis. 23 (2000) 221±238238