# Bartonella spp. DNA associated with biting flies from California

Crystal Y. Chung, Rickie W. Kasten, Sandra M. Paff, Brian A. van Horn, Muriel Vayssier Taussat, Henri-Jean Boulouis, Bruno B. Chomel

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Crystal Y. Chung, Rickie W. Kasten, Sandra M. Paff, Brian A. van Horn, Muriel Vayssier Taussat, et al.. Bartonella spp. DNA associated with biting flies from California. Emerging Infectious Diseases, 2004, 10 (7), pp.1311-1313. hal-02677178

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Distributed under a Creative Commons Attribution 4.0 International License
*Bartonella* spp.
DNA Associated
with Biting Flies
from California

Crystal Y. Chung,* Rickie W. Kasten,*
Sandra M. Paff,* Brian A. Van Horn,*
Muriel Vayssier-Taussat,† Henri-Jean Boulouis,†
and Bruno B. Chomel*

*Bartonella* DNA was investigated in 104 horn flies (*Haematobia* spp.), 60 stable flies (*Stomoxys* spp.), 11 deer flies (*Chrysops* spp.), and 11 horse flies (*Tabanus* spp.) collected on cattle in California. Partial sequencing indicated *B. bovis* DNA in the horn fly pool and *B. henselae* type M DNA in one stable fly.

Bartonella spp. are vector-borne bacteria associated with numerous emerging infections in humans and animals (1). Four Bartonella species have been isolated from wild and domestic ruminants. B. schoenbuchensis and B. capreoli were recovered from wild roe deer (Capreolus capreolus) (2,3) in Europe, whereas B. bovis (formerly B. weissii) was recovered from domestic cattle in the United States and Europe (3–5). Strains similar to B. bovis and B. capreoli were also isolated from mule deer (Odocoileus hemionus) and elk (Cervus elaphus) from California (3,4). Recently, B. chomelii was recovered from bacteremic cows in France (6). A high prevalence of infection with various Bartonella species has been reported in domestic and wild ruminants in North America and Europe (2–4). Of the herds investigated in California, 95% of beef cattle and 17% of dairy cattle were bacteremic for B. bovis and 90% of the mule deer were bacteremic for Bartonella spp. (4). The main vector of these ruminant-infecting Bartonella spp. has not been identified.

The role of ticks as potential vectors for *Bartonella* in
cattle was investigated (7,8). In Europe, >70% of 121
*Ixodes ricinus* ticks collected from roe deer had 16S rRNA
gene sequences for *Bartonella* or other closely related
species (7). In California, *Bartonella* DNA was detected in
approximately 19% of 151 questing adult *I. pacificus* ticks
(8), but the direct role of ticks in *Bartonella* transmission
among ruminants has never been established. In a search
for an efficient *Bartonella* vector, which could explain
such high prevalence of infection in wild and domestic

ruminants, we tested biting flies for *Bartonella* spp. DNA
to establish the potential role of biting flies as vectors of
*Bartonella* in cattle.

The Study

Flies were collected by hand, with a bug net, at various
locations on the University of California campus, mainly
the dairy barn, beef barn, and feedlot, from early July to
mid-August 2003. Flies were identified on the basis of
morphologic characteristics visually or under binocular
lenses for the smaller flies by an experienced entomolo-
gist. Of the 370 biting flies collected, 104 (62%) of the
horn flies (*Haematobia* spp.), 60 (33%) of the stable flies
(*Stomoxys* spp.), 11 (92%) of the deer flies (*Chrysops*
spp.), and 10 (91%) of the horse flies (*Tabanus* spp.) were
tested for *Bartonella* DNA. The stable flies were collected
from the dairy and the feedlot barns. The horn flies, deer
flies, and horse flies were collected from the beef barn.

Before DNA extraction, the flies were placed in a sterile
1.5-mL microtube, washed with 70% ethanol, and rinsed
with sterile water. Because of size differences among the
flies, 2-3 horn flies were grouped together in a single
microtube, while each stable fly was placed in an individ-
ual vial. The abdomen of deer flies and horse flies was first
removed and then placed in individual vials. DNA extrac-
tion was performed by using the DNeasy Tissue Kit
(Qiagen, Valencia, CA) according to the manufacturer's
instructions, with some minor adjustments. The amount of
reagents for the deer and horse flies were doubled, and the
flies were incubated in a waterbath overnight at 55°C.

*Bartonella* DNA was detected by polymerase chain
reaction (PCR) using primers for the citrate synthase (gltA)
gene, as previously published (9). Undiluted DNA extract-
ed from the flies was used as the DNA template. As a pos-
itive control, a low concentration of *B. henselae* was added
to a separate set of the same DNA template. A negative
control was made by using sterile water instead of the
DNA template. Using gel electrophoresis, we analyzed
PCR products for the appearance of an ≈380-bp fragment.
Any evidence of a 380-bp fragment was further analyzed
by restriction fragment length polymorphism (RFLP) pro-
cedures, by using *TaqI* (Promega Corp., Madison, WI),
*HhaI*, *AciI*, and *MseI* endonucleases (New England
Biolabs, Beverly, MA), and DNA sequence analysis
(Davis Sequencing, Davis, CA).

Four of the 60 stable flies and one pool (2 flies) of the
45 horn fly pools showed a 380-bp fragment. PCR/RFLP
analysis confirmed *Bartonella* DNA in one of the four sta-
ble flies and in the horn fly pool. However, for the three
other stable flies, the PCR/RFLP profiles did not match
any known *Bartonella* digestion profile. The sequence
obtained from the horn fly pool (*Haematobia* spp.) collect-
ed in the beef cattle barn was identical to that for *B. bovis*

<footnote>*University of California, Davis, California, USA; and †Unité Mixte de Recherche, Ecole Nationale Veterinaire d'Alfort, Maisons-Alfort, France</footnote>
(Figure 1). The sequence obtained from a stable fly (Stomoxys spp.) collected in the dairy cattle barn was identical to that for *B. henselae* type M (Marseille) (Figure 2). The highlighted area indicates the divergence between *B. henselae* type H (Houston I) and *B. henselae* type M, as previously described (10).

<figure><img src="image_2.png" /><figcaption>Figure 1. Alignment of BhCS.781p/BhCS.1137n *gltA* gene ampli-
cons for 306 bp of *Bartonella bovis* (GenBank accession no.
af293394), a *B. bovis* isolate (cow 2226) from a Californian cow
and the horn fly pool (fly-HO17).</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>Figure 2. Alignment of BhCS.781p/BhCS.1137n *gltA* gene ampli-
cons for 328 bp of *Bartonella henselae* type H (GenBank acces-
sion no. baoglt), *B. henselae* type M (isolate ucd-U4) from a
California cat and the stable fly DNA extract (fly-SO13). The high-
lighted region indicates base pair difference.</figcaption></figure>

**Conclusions**

This identification of *Bartonella* DNA is the first asso-
ciated with horn and stable flies and the first identification
of *B. henselae* from a biting fly. It is also the first report of
identification of *Bartonella* DNA from flies from North
America. This finding demonstrates, as for ticks, that
*Bartonella* DNA is present in various biting insects. We
found a very low percentage of *Bartonella* DNA-positive
flies, in contrast to the very high prevalence (57 [88%] of
65 observed in *Hippoboscidae* adult flies (*Lipoptena cervi*
and *Hippobosca equina*) collected from domestic cattle
and wild roe deer in France (H.J. Boulouis, pers. comm.).
This low prevalence may be related to the fact that differ-
ent fly species were tested but more likely could be asso-
ciated with a low level of *Bartonella* bacteremia in our
herds. In a previous study, only 17% of cows in a dairy
herd were bacteremic (4), and prevalence was even lower
in another dairy herd from Tulare, in the central valley of
California (B.B. Chomel et al., unpub. data). A follow-up
for this study would be to collect blood from herds at the
University of California, Davis, and establish the status of
*Bartonella* bacteremia. Future research should include col-
lecting flies in different locations and herds in which high
levels of bacteremia were previously detected. Inhibitory
factors were unlikely to be associated with such a low
prevalence because spiked controls were systematically
detected.

Identification of *B. henselae* DNA in a stable fly indi-
cates the wide range of blood-sucking arthropods that can
harbor this human pathogen. The partial *gltA* sequence was
identical to that for *B. henselae* type Marseille, the most
common type found in cats and humans in California (11).
Fleas have been shown to be an efficient vector of *B.
henselae* (12–14). More recently, *B. henselae* DNA was
identified in adult questing *I. pacificus* ticks from
California and from *I. ricinus* ticks collected on humans in
Italy (8,15). The role of ticks as potential vectors of *B.
henselae* in humans has also been suggested (16–18).
Since *Bartonella* are likely to be present in biting flies,
investigating the potential of biting flies as either mechan-
ical or biologic vectors of *Bartonella* in cattle and possibly
humans should be pursued.

**Acknowledgments**

We thank Robin Houston for helping identify flies.

Ms. Chung's summer fellowship was funded by the Center
for Comparative Medicine, University of California, Davis,
through a training grant from the National Institutes of Health.

Ms. Chung is a second-year student at the School of
Veterinary Medicine, University of California, Davis. This study
was performed as her NIH summer fellowship through the Center
for Comparative Medicine at University of California, Davis.
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Address for correspondence: Bruno B. Chomel, Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, USA; fax: 530-752-2377; email: bbchomel@ucdavis.edu

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