Role of Hippoboscidae Flies as Potential Vectors of Bartonella spp. Infecting Wild and Domestic Ruminants

Abstract

The putative role of biting flies in Bartonella transmission among ruminants was investigated. Amplification of the Bartonella citrate synthase gene from 83 Hippoboscidae was detected in 94% of 48 adult Lipoptena cervi flies, 71% of 17 adult Hippobosca equina flies, 100% of 20 adult Melophagus ovinus flies, and 100% of 10 M. ovinus pupae. Our findings suggest that Hippoboscidae play a role in the transmission of Bartonella among ruminants. The vertical transmission of Bartonella in M. ovinus and the presence of Bartonella DNA in all samples suggest a symbiotic association between Bartonella and M. ovinus.

Bartonella spp. are intracellular small gram-negative bacteria transmitted by blood-sucking arthropods and considered to be emerging pathogens in humans and animals (1, 8, 9, 22). In recent years, these organisms have been identified in a wide range of wild and domestic mammals (4, 6, 7, 11), some of which have been associated with zoonoses. Recently, four new Bartonella species have been isolated from ruminants: B. schoenbuchensis and B. capreoli were recovered from wild roe deer (Capreolus capreolus) (3, 10), whereas B. bovis (3) and B. chomelii (20) were recovered from domestic cattle. There are no pathological outcomes associated with Bartonella infection in ruminants (5, 7).

Arthropod vectors involved in the transmission of Bartonella spp. among ruminants are still unknown. As blood-sucking ectoparasites of ruminants, flies of the family Hippoboscidae are good candidates for the transmission of Bartonella. Among Hippoboscidae, Lipoptena, Hippobosca, and Melophagus are the three main genera which parasitize mammals (14, 18). The deer ked (Lipoptena cervi), the predominant Lipoptena species in Europe, parasitizes cervids (15), whereas the louse fly (Hippobosca equina) parasitizes cows and horses and the sheep ked (Melophagus ovinus) is a permanent ectoparasite of sheep (Ovis aries) (18).

The aim of this study was to determine if Hippoboscidae could be putative vectors of Bartonella spp. in ruminants. We investigated whether Bartonella DNA could be detected in adult and pupal stages of Hippoboscidae collected from ruminants (domestic cattle and roe deer) known to be naturally infected with Bartonella spp. We extended the study to Hippoboscidae collected from sheep and horses for which no evidence of Bartonella infection had ever been demonstrated.

Collection and identification of Hippoboscidae.

Eighty-three Hippoboscidae flies of different species, stages, and genders were collected and taxonomically identified under a binocular lens (18). Samples were obtained from the hosts or from our parasitology collection (Table 1). Each sample was stored in absolute ethanol.

TABLE 1.

Taxonomic identification, origin, and repartition of the 83 samples of Hippoboscidae tested, and results of specific PCR amplification of their Bartonella citrate synthase (gltA) genes

Species of Hippoboscidae	Evolutive stage	No. of flies	Host (no. of hosts)	Origin (yr of sampling)	No. of samples positive by PCR for Bartonella gltA gene/ total no. of samples (%)
Lipoptena cervi	Adult	10	Wild roe deer (1)	Midi Pyrénéces (2000)	8/10	= 45/48 (94)
14	Wild roe deer (unknown)	Unknown (1994)	13/14
24	Wild roe deer (1)	Bourgogne (2003)	24/14
	Pupa	3	Wild roe deer (unknown)	Unknown (1994)	0/3	= 0/3 (0)
Hippobosca equina	Adult	2	Cow (1)	Ile de France (2001)	2/2	= 12/12 (100)
10	Cow (3)	Rhone Alpes (2002)	10/10
	Adult	5	Horse (1)	Centre (2003)	0/5	= 0/5 (0)
	Pupa	1	Horse (1)	Centre (2003)	0/1	= 0/1 (0)
Melophagus ovinus	Adult	6	Sheep (1)	Normandie (2003)	6/6	= 20/20 (100)
4	Sheep (1)	Ile de France (2002)	1/1
2	Sheep (1)	Bourgogne (2003)	1/1
8	Sheep (2)	Roumanie (2004)	8/8
	Pupa	3	Sheep (unknown)	Unknown	3/3	= 10/10 (100)
2	Sheep (2)	Normandie (2003)	2/2
1	Sheep (1)	Bourgogne (2003)	1/1
4	Sheep (2)	Roumanie (2004)	4/4

DNA extraction and PCR.

Each fly and pupa were washed three times in sterile water baths and once in a 70% ethanol bath and then dried. DNA was then extracted after the flies were crushed with a bead beater as previously described (16).

A 380-bp fragment of the citrate synthase (gltA) gene of the genus Bartonella was amplified in fly DNA extracts by PCR, using specific primers (BhCs781 and BhCs1137) as described previously (20, 26). All reactions were performed at least twice.

PCR-RFLP.

PCR-restriction fragment length polymorphism (PCR-RFLP) was performed with 10 μl of the PCR product of the gltA gene obtained from 63 of 83 extracts of Hippoboscidae (45 L. cervi, 12 H. equina, and 6 M. ovinus extracts) by using restriction endonucleases TaqI, MseI, and DdeI (New England Biolabs, Beverly, Mass.) as previously described (17, 21). Patterns were compared to the PCR-RFLP profiles of six ruminant-related species or strains, i.e., B. capreoli (CIP 106691 and CCUG 43827) (3), B. schoenbuchensis strain R1 (DSM 13525 and NCTC 13165) (10), B. bovis strain R3, (CIP 106692 and CCUG 43828) (3), B. chomelii (A828^T and CIP 107869^T) (20), and A971, isolated from a Formosan Sika deer (Cervus nippon taiouanus) in a nature park in France (21).

Sequencing and sequence analysis.

Ten amplified fragments (seven obtained from L. cervi, one obtained from H. equina, and two obtained from M. ovinus) were sequenced by GenomeExpress (Meylan, France). Another fragment, obtained from an L. cervi parasite for which a mixed profile was found by PCR-RFLP analysis, was first cloned in a pCR2-1 TOPO TA cloning vector according to the manufacturer's instructions (Invitrogen, Paisley, United Kingdom). Recombinant plasmid DNA was isolated from the selected white positive colonies with a Wizard Plus miniprep DNA purification system (Promega, Madison, Wis.).

Sequences were compared with known sequences listed in the GenBank nucleotide sequence databases by using the BLAST search option of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov).

Detection of Bartonella DNA in flies of Hippoboscidae by specific PCR amplification of the citrate synthase gene (gltA).

Among the 83 Hippoboscidae, 51 flies were identified as L. cervi (48 adults and three pupae), 18 were identified as H. equina (17 adults and one pupa), and 14 were identified as M. ovinus (eight adults and six pupae) (Table 1).

The presence of the specific amplified fragment was detected from 45 (94%) L. cervi adults, from 20 (100%) M. ovinus adults, and from 12 (71%) H. equina adults. All H. equina flies collected on cows (n = 11) were positive, whereas none of those collected on horses (n = 6) resulted in a PCR product DNA.

All 10 M. ovinus pupae resulted in a PCR product, whereas none of the 4 L. cervi and H. equina pupae displayed the expected amplified fragment.

Identification of Bartonella DNA present in flies of Hippoboscidae.

PCR-RFLP analysis of gltA amplified from the 63 samples of flies of Hippoboscidae were performed and compared to the profiles of the ruminant-infecting Bartonella strains (Table 2). Using TaqI, MseI and DdeI, we identified four different profiles for known ruminant strains. Profile 1 was found in B. schoenbuchensis R1 and in 30 samples comprising 26 L. cervi and 4 M. ovinus samples. Four sequences obtained from L. cervi extracts showed 100% sequence similarity with the gltA gene of B. schoenbuchensis strain R1.

TABLE 2.

PCR-RFLP profiles, distribution, and sequence identification by partial sequence analysis of the gltA genes of ruminant-infecting Bartonella-reference strains obtained from different species of Hippoboscidae

PCR-RFLP profile type (associated strain[s])	Fragment size(s) (bp) after digestion witha:			Species of Hippoboscidae flies showing the profile type	No. of samples	No. of sequenced fragments	Closest Bartonella species (% DNA identity based on a fragment of the gltA gene)
	TaqI	DdeI	MseI
1 (B. schoenbuchensis R1)	ND	300, U	180, U	L. cervi	26	4	B. schoenbuchensis R1 (100)
				M. ovinus			No sequence available
				Adult	1
				Pupa	3
2 (B. chomelii, B. schoenbuchensis	ND	ND	180, U	L. cervi	4	1	B. schoenbuchensis R3 (100)
R3, and A971)				H. equina	12	1	B. chomelii (100)
				M. ovinus			A971 cervid strain (99)
				Adult	1	1
				Pupa	1	1
3 (B. bovis)	150, 250	ND	150, 220	None
4 (B. capreoli)	ND	100, 250	120, 180	None
New (no strain associated)	ND	ND	200, 180	L. cervi	6	2	A971 cervid strain (95)
Mixed (profiles 1 and 2)	ND	ND, 300, U	180, U	L. cervi	7	1
						Clone 1	B. schoenbuchensis R1 (100)
						Clone 2	B. chomelii (100)

^aND, sample was not digested; U, fragment was of undetectable size.

Profile 2 was found for B. schoenbuchensis R3 strain, B. chomelii, and strain A971, and profile 2 was also found in 16 samples (2 L. cervi, 2 M. ovinus, and 12 H. equina samples). Sequence analysis of the gltA gene fragment obtained from two M. ovinus samples presenting this PCR-RFLP profile showed 99% similarity with the A971 sequence, whereas the sequence obtained from L. cervi showed 100% similarity with the B. schoenbuchensis strain R3 sequence. The sequence obtained from one H. equina extract had 100% similarity with the B. chomelii gltA gene sequence.

Profiles 3 and 4 were found for B. bovis and B. capreoli, respectively, but for none of the fly samples.

Six L. cervi samples had a new RFLP profile different from those of the reference strains. Two fragments with this new RFLP profile showed 95% similarity with the A971 gltA sequence, 94% similarity with those of a B. schoenbuchensis-related strain (GenBank accession number AJ564635), and from 92 to 93% similarity to other ruminant-related Bartonella species (B. capreoli and B. chomelii).

A mixed profile between profiles 1 and 2 was obtained for seven samples (L. cervi). For this PCR-RFLP mixed profile, sequencing of cloned fragments allowed the distinction between two different sequences displaying 100% DNA similarity with B. schoenbuchensis and B. chomelii.

The present study is the first report of the presence of Bartonella in ruminant blood-sucking flies. Up to 84% of the flies of Hippoboscidae carried ruminant-related Bartonella DNA, which strongly suggests that Hippoboscidae might play a role in the transmission of Bartonella among ruminants.

PCR-RFLP profiles using the gltA gene were consistently similar to those of other ruminant-related Bartonella spp., as confirmed by sequence analysis. Among L. cervi samples, we observed different PCR-RFLP profiles. Most of the profiles and sequences obtained were related to B. schoenbuchensis. One sequence corresponding to an unrecognized PCR-RFLP profile shared less than 96% sequence similarity with those of the validated species or strains and therefore could not be identified by its gltA sequence (19). This finding was corroborated by the fact that undescribed Bartonella strains remain to be identified in ruminants (7, 21). H. equina flies presented Bartonella DNA related to the B. chomelii sequence (20). Flies of Hippoboscidae are common blood-sucking parasites of ruminants (18) and are already known for their vectorial role in the life cycles of pathogens such as Trypanosoma melophagium for M. ovinus and sheep (2, 23). Ruminants have the potential to become bacteremic with Bartonella, and we found that most of the flies feeding on ruminants were infected with Bartonella at a relatively high level (using quantitative PCR [data not shown]). Furthermore, a preliminary study conducted in our laboratory indicated that immunoglobulin G raised against ruminant-related Bartonella species could be detected in horses despite the fact that we did not culture Bartonella from horse blood. This fact suggests that Bartonella is transmitted from bovine to equine by a common vector and that Hippoboscidae are the most likely candidates. We propose that flies of the family Hippoboscidae may be vectors for Bartonella, as has been shown for horse-biting flies and in cases of equine infectious anemia (13) and for fleas transmitting B. henselae (12). Investigations to identify the localization of Bartonella within the flies are necessary to determine the exact role of Hippoboscidae in Bartonella transmission.

We extended the study to Hippoboscidae flies feeding on horses and sheep, which are animals for which no evidence of Bartonella infection has been reported. We cultured blood samples of more than 100 horses, 150 sheep, and 84 big horn sheep (Ovis canadensis), but we did not isolate Bartonella (reference 7 and our unpublished data). None of the H. equina organisms collected from horses presented Bartonella DNA, suggesting that the Bartonella DNA-positive louse flies likely acquired Bartonella after blood meals on infected cows.

Surprisingly, Bartonella DNA was present in all of the M. ovinus samples, even at the pupal stage. As M. ovinus is wingless, its transmission from one sheep to another occurs by contacts between ewes and lambs (18). The presence of Bartonella DNA in M. ovinus, along with the absence of Bartonella sp. isolation from sheep, might be explained by a symbiotic association between Bartonella and the ectoparasite without transmission to ruminants. The fact that 100% of the tested pupae were Bartonella DNA positive strengthens this hypothesis. Previous studies of tick endosymbionts showed that symbionts were closely related to bacterial pathogens transmitted by ticks and had lost the ability to infect vertebrates (24). Moreover, a 16S DNA sequence from Wolbachia melophagi (GenBank accession number X89110), which has been described as an endosymbiont of M. ovinus (25, 27), is closer to a B. schoenbuchensis 16S DNA (99% similarity) than to other Wolbachia 16S rDNA sequences (between 80 and 83.5% similarity with other insect endosymbiont Wolbachia species sequences), suggesting that this sequence is more closely related to Bartonella than to Wolbachia. As no Bartonella DNA was detected in L. cervi and H. equina pupae, the endosymbiontic nature of Bartonella is thus limited to M. ovinus.

The present study reports the first demonstration of the presence of Bartonella in ruminant blood-sucking flies and should lead to further investigations of Bartonella sp. transmission. Finally, the possible role of Bartonella sp. as a symbiont will change the way we consider Bartonella biology.