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Veterinary Immunology and Immunopathology 116 (2007) 104–108

Short communication

Bartonella henselae infection in splenectomized domestic cats

previously infected with hemotropic Mycoplasma species

Jane E. Sykes a, Jennifer B. Henn b, Rickie W. Kasten b, Claire Allen c,Bruno B. Chomel b,*

a Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616, USAb Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, USA

c Companion Animal Health Center, School of Veterinary Medicine, University of California, Davis, CA 95616, USA

Received 25 September 2006; received in revised form 31 October 2006; accepted 18 December 2006

Abstract

Cat scratch disease is caused by Bartonella henselae and the domestic cat represents its main reservoir. In immunocompromised

patients, infection with B. henselae is characterized by more severe clinical forms than in non-immunocompromised individuals.

The objective of the present study was to investigate the characteristics of B. henselae (Houston-I strain) infection in four

splenectomized and three non-splenectomized cats, five of which were chronically infected with ‘Candidatus Mycoplasma

haemominutum’. No major clinical signs were observed in either group of cats. Cats in both splenectomized and non-

splenectomized groups became bacteremic within a week post-inoculation. Although bacteremia was on average 10 days longer

in the splenectomized cats, that difference was not statistically significant (P = 0.72). In both groups, the level of bacteremia peaked

within the same time frame; however, the level of bacteremia was about 10-fold higher in the splenectomized cats (P = 0.007). Such

a difference could be associated with a reduced immune response to the infection, especially a reduced ability to phagocytize

Bartonella organisms in the splenectomized cats. Concurrent infection with ‘Candidatus M. haemominutum’ did not appear to alter

the course of infection.

# 2007 Elsevier B.V. All rights reserved.

Keywords: Bacteremia; Bartonellosis; Hemoplasma; Splenectomy

1. Introduction

Bartonella spp. are vector-borne bacteria mainly

infecting erythocytes and endothelial cells of their hosts

and considered emerging pathogens in humans and

animals (Boulouis et al., 2005). In humans, cat scratch

disease is caused by Bartonella henselae and the

domestic cat represents its main reservoir (Chomel

et al., 2006). In immunocompromised patients, disease

* Corresponding author. Tel.: +1 530 752 8112;

fax: +1 530 752 2377.

E-mail address: bbchomel@ucdavis.edu (B.B. Chomel).

0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetimm.2006.12.004

associated with B. henselae tends to be more severe than

in immunocompetent individuals. Bacillary angioma-

tosis, a systemic vasculoproliferative disease, has been

mainly reported in AIDS patients (Spach and Koehler,

1998), and a few cases of bacillary angiomatosis and

peliosis hepatis have been diagnosed in recipients of

organ transplants (Ahsan et al., 1998; Juskevicius and

Vnencak-Jones, 2004). Bartonella-induced prolonged

fever and bacteremia has also been reported in

immunocompromised individuals, including a splenec-

tomized patient (Henriquez et al., 2004), suggesting that

the spleen may play an important role in the clearance of

Bartonella spp. In addition to filtration of antigen and

antibody complexes and bacteria, the spleen captures

J.E. Sykes et al. / Veterinary Immunology and Immunopathology 116 (2007) 104–108 105

and presents antigen to T cells and is a key organ for

generating B cell-mediated humoral immunity (Mrusek

et al., 2005; Brown, 1992). The lack of such functions

when the spleen is removed explains the increased

susceptibility to bacterial infections (Sullivan et al.,

1978). Interestingly, no natural case of bacillary

angiomatosis has ever been reported in domestic cats,

despite the fact that both feline immunodeficiency virus

(FIV) and B. henselae infections are quite common in

stray cats (Chomel et al., 2006; Pedersen et al., 1989).

The effect of splenectomy on Bartonella infection in cats

has not been previously investigated. Similarly, ‘Candi-

datus Mycoplasma haemominutum’ (Mhm) and Myco-

plasma haemofelis are common epierythrocytic

hemotropic mycoplasmas (hemoplasmas) that may infect

cats subclinically or be associated with hemolytic anemia

(Foley and Pedersen, 2001). Subclinical infection with

Mhm has been detected in approximately 15–20% of cats

(Jensen et al., 2001; Tasker et al., 2003; Lappin et al.,

2006), and hemoplasmas have the potential to share the

same mode of transmission as Bartonella (Woods et al.,

2005). In a study of community-source cats with outdoor

exposure or known flea exposure, DNA for a hemo-

plasma or Bartonella was detected in 21 and 23% of the

cats, respectively (Hackett et al., 2006). It was therefore

of interest to investigate the characteristics of B. henselae

infection in splenectomized cats compared to non-

splenectomized cats in animals previously infected with

hemoplasma species.

2. Materials and methods

Seven 6–12-month-old, specific pathogen-free (SPF)

cats were obtained from the Feline Nutrition Labora-

tory, University of California, Davis. Five cats (cats 157,

218, 147S, 158S and 181S) were female intact cats and

two cats (cats 146 and 189S) were male neutered. Prior

to enrollment, four of these cats had been splenecto-

mized (147S, 158S, 181S, and 189S) and subsequently

inoculated intraperitoneally with either Mycoplasma

haemofelis (Mhf) and ‘Candidatus M. haemominutum’

(Mhm) (cat 146), ‘Candidatus M. haemominutum’

(Mhm; cats 147S, 181S, 189S, and 218), or ‘Candidatus

Mycoplasma haematoparvum’ (Mhp, cats 157 and

158S). Serial PCR assays over a 6-week period

suggested successful infection in cats inoculated with

Mhf or Mhm; Mhp was never detected. The cats were

enrolled in the present study after verification that they

were all Bartonella culture and antibody negative. Cats

were also treated for 3 weeks with doxycycline (10 mg/

kg PO q24h), in an attempt to eliminate the hemoplasma

infection. Each cat was inoculated in three to four

different sites with 0.5 mL of a B. henselae Houston I

(ATCC # 49882) inoculum of 4 � 108 colony forming

units (CFU)/mL. Physical examination was performed

on all cats daily for the two first weeks and at least

weekly thereafter. Body temperature was recorded daily

during the first 2 weeks of the experiment. The cats were

housed in two different rooms (one each for splenecto-

mized and non-splenectomized cats) in a controlled

environment (temperature controlled, ectoparasite-free,

with restricted access). A blood sample (3 mL) was

collected from the jugular vein for B. henselae serology

and culture twice weekly during the first 3 weeks post-

inoculation (PI), weekly for the next 3 weeks and every

other week for the five following months. The project

was approved by the University of California Davis

Animal Use and Care Committee (Protocol # 10850).

Of the 3 mL of blood collected, 2 mL was placed into

EDTA tubes for Bartonella isolation and 1 mL of blood

was placed into serum separation tubes for serological

tests. The EDTA tubes were frozen at�70 8C and plated

a few days later onto 5% rabbit blood agar plates, as

previously described (Chomel et al., 1995). The plates

were incubated at 35 8C with 5% CO2 for 4 weeks and

were examined two to three times a week for bacterial

growth. The isolated strains were confirmed to be B.

henselae by PCR/RLFP of the citrate synthase gene

using HhaI and TaqI endonucleases (Chomel et al.,

1999). The number of colonies was counted and

calculated as colony forming units (CFL/mL).

Specific antibodies against B. henselae purified

antigens (outer membrane proteins, OMPs) were

detected using an ELISA, as previously described

(Henn et al., 2005), except that plates were sensitized

only with OMP antigens of B. henselae type I (Houston

I), serum samples were diluted at 1:100 in ELISA Tris–

milk buffer, and the conjugate used was an anti-cat IgG

phosphatase-labeled conjugate diluted at 1:4000.

To determine each cat’s hemoplasma infection

status, DNA was extracted from 200 mL aliquots of

EDTA blood collected on days 3, 27, 41, 62, and 103

after inoculation with B. henselae using a commercially

available kit (QIAmp blood and tissue kit, Qiagen Inc.,

Chatsworth, CA, USA), according to the instructions of

the manufacturer. DNA was eluted with 200 mL of

elution buffer. Conventional PCR was performed as

previously described, which results in amplification of a

193 base pair segment of the 16S rRNA gene of Mhm or

Mhp and a 170 base pair segment of the 16S rRNA gene

of Mhf (Jensen et al., 2001; Sykes et al., 2004). Positive

and negative controls were included routinely with

every run. PCR products were identified following

electrophoresis in 2.4% agarose gels.

J.E. Sykes et al. / Veterinary Immunology and Immunopathology 116 (2007) 104–108106

To compare mean levels of Bartonella bacteremia

between splenectomized and non-splenectomized cats,

an equal variance t-test was performed using MINITAB

statistical software Release 12 (MINITAB Inc., State

College, PA, USA). A nonparametric Mann–Whitney

rank sum test was performed to compare duration of

bacteremia, time to peak bacteremia, and ELISA OD

values for the splenectomized and non-splenectomized

groups of cats using MINITAB software.

3. Results and discussion

None of the cats showed any specific clinical signs

after Bartonella inoculation. On days 6 and 7 PI, two of

the three non-splenectomized cats had mild elevation in

rectal temperature (39.8 and 40 8C) that lasted for less

than 48 h, as documented previously in cats experi-

mentally infected with B. henselae (Guptill et al., 1997).

Fever has not previously been noted following infection

with the Houston I strain (Yamamoto et al., 2002).

Results of PCR analysis for hemoplasmas showed that

cats 146, 147S, 181S, 189S and 218 remained infected

with hemoplasmas throughout the study, and all cats

tested positive only for Mhm based on the size of the

PCR product. Cats 157 and 158S remained negative

using PCR.

By day 6 PI, all seven cats were bacteremic with B.

henselae. No significant differences in time to reach the

bacteremia peak were observed between the splenecto-

mized cats and the non-splenectomized control group

(P = 1.00) (Table 1). Bacteremia peaked between days 10

and 20 PI in the three non-splenectomized cats and

between days 13 and 20 in the four splenectomized cats

(Table 1). The mean level of bacteremia among the four

splenectomized cats was 3.9 � 105 CFU/mL (range:

Table 1

Mean level of bacteremia, duration of bacteremia and time for bacteremia to

inoculated with B. henselae Houston I strain

Catsa Mean level of bacteremia

(range) (CFU/mL)

Non-splenectomized (n = 3)

146 1.20 � 104 (1–2.60 � 104)

157 2.66 � 105 (1–1.00 � 106)

218 1.08 � 104 (1–3.40 � 104)

Splenectomized (n = 4)

147S 6.50 � 105 (8–1.60 � 106)

158S 6.76 � 105 (72–1.60 � 106)

181S 2.46 � 105 (40–5.00 � 105)

189S 9.12 � 104 (12–2.40 � 105)

a All cats except cats 157 and 158S were concurrently infected with

conventional PCR.

2.0 CFU/mL–9.6 � 105 CFU/mL), which was signifi-

cantly higher (P = 0.007) than in the three non-

splenectomized cats (mean: 8.9 � 104, range 1.0 CFU/

mL–3.5 � 105 CFU/mL). Bacteremia lasted between 70

and 111 days (median 89.5 days) in the splenectomized

group and between 73 and 85 days (median 82 days) in

the control group. This difference was not statistically

significant (P = 0.72). None of the cats developed any

relapse of bacteremia (defined as a positive blood culture

after two consecutive negative blood cultures) during the

rest of the study period. Such results have been noted in

non-splenectomized cats in previous studies (Regnery

et al., 1996; Yamamoto et al., 2002). The difference in

magnitude of bacteremia was most likely the result of a

reduced immune response to the infection, especially a

reduced ability to phagocytize Bartonella organisms in

the splenectomized cats. Few other studies of infection in

splenectomized cats have previously been conducted. A

higher level of parasitemia was observed in splenecto-

mized cats infected experimentally with Cytauxzoon felis

(Cowell et al., 1988).

Bartonella-specific IgG antibodies against OMPs

from B. henselae Houston I were detected using ELISA

in all seven cats, with a normalized optical density (OD)

increasing regularly by day 10 PI and reaching an OD

above 0.2 between days 27 and 34 PI. The normalized

OD stayed above that threshold until the end of the

study. No difference in the level of antibody response

(normalized OD) could be observed between the two

groups, which both had similar curves (Fig. 1). By

comparison, splenectomy was associated with an

increased antibody response in cats infected with C.

felis (Cowell et al., 1988). Studies of the humoral

response to vaccination in humans and rodents

following splenectomy have generally shown similar

peak in non-splenectomized and splenectomized cats experimentally

Duration of bacteremia

(days)

Time to peak bacteremia

(days)

82 20

73 17

85 10

111 20

97 17

82 17

70 13

‘Candidatus Mycoplasma haemominutum’ based on the results of

J.E. Sykes et al. / Veterinary Immunology and Immunopathology 116 (2007) 104–108 107

Fig. 1. Mean ELISA normalized optical density in splenectomized

(four cats) and non-splenectomized cats (three cats) inoculated with

Bartonella henselae Houston I.

or reduced antibody responses when compared with

non-splenectomized individuals (Clayer et al., 1992;

Aaberge et al., 1984; Brydak et al., 2004).

Previous infection of these cats with different

hemotropic Mycoplasma species did not appear to

significantly modify the course of the infection, which

was similar to that in previous studies of B. henselae

Houston I in SPF cats (Regnery et al., 1996; Yamamoto

et al., 2002). Two of the cats were not successfully

infected with hemoplasmas, and the course of B.

henselae infection in these cats was similar to that in the

cats that were infected with hemoplasmas, although of

interest, the non-splenectomized, uninfected cat had

higher magnitude of bacteremia than the non-splenec-

tomized, infected cats (repeated measures ANOVA,

P = 0.004). The failure of doxycycline therapy to

eliminate infection with Mhm is similar to the results of

a previous study showing failure of marbofloxacin to

eliminate infection (Tasker et al., 2006).

Limitations of this study included the small number

of cats used and the relatively short duration of the

study. Given a greater number of cats and a several year

study period, it is possible that disease expression and/

or relapse of bacteremia may have occurred in

splenectomized cats. The cats were co-infected with

Mhm, and sufficient numbers of uninfected controls

were not available for comparison. Nevertheless, this

study showed that over a 7-month period, splenectomy

does not appear to be associated with disease expression

in cats infected with B. henselae, despite a 10-fold

increase in the level of bacteremia. Cats appear to

maintain their ability to produce antibody against

blood-borne pathogens such as Bartonella despite the

absence of the important functions of the spleen relating

to antigen collection and presentation. Concurrent

infection of these cats with ‘Candidatus M. haemomi-

nutum’ did not appear to significantly modify the course

of B. henselae infection.

Acknowledgments

The present study was supported by a grant from the

Center for Companion Animal Health (George and

Phyllis Miller Feline Research Fund), School of

Veterinary Medicine, University of California, Davis.

The authors would like to thank LeAnn Lindsay, Sandy

Paff and Jennifer Strasser for their technical assistance.

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