Guidelines for the Isolation,Molecular Detection,and Characterization of Bartonella Species

Abstract

Bartonellae are fastidious, facultative, intracellular vector-borne bacteria distributed among mammalian reservoirs worldwide. The pathogenic potential of many Bartonella spp. has increased the interest in these bacteria and advanced their research. Isolation of Bartonella spp. is laborious using classical bacteriological methods and requires specific conditions and prolonged incubation periods. In contrast, molecular methods for detection of Bartonella DNA are considered as more practical and sensitive than the former. Among the molecular methods, the use of real-time PCR assays for primary screening of Bartonella spp., followed by several molecular confirmatory assays, using either conventional or real-time PCR, is recommended. Although primary isolation of Bartonella is a laborious task, we encourage its application to all PCR-positive samples as this is the most reliable proof for the presence of live bacteria. Moreover, a successful trial will enable a broader molecular characterization and speciation of isolated colonies. The present guideline gathers and summarizes recommendations, including advantages and limitations of isolation and molecular detection of Bartonella from mammalian and arthropod samples.

Introduction

B artonella organisms are vector-borne, gram-negative, facultative intracellular bacteria, which establish long-lasting intraerythrocytic infections in adapted reservoirs (Boulouis et al. 2005, Chomel et al. 2009). They are distributed worldwide and have been isolated from several mammalian species, including humans and domestic and wild animals (Vayssier-Taussat et al. 2009, Kosoy 2010). Due to their hemotropic lifestyle, bartonellae are typically transmitted by blood-sucking arthropods within mammalian reservoir communities. Sand flies, lice, fleas, biting flies (e.g., Hippoboscidae, Muscidae), and ticks are among the arthropods associated with Bartonella transmission and/or infection (Cotté et al. 2008, Chomel et al. 2009, Tsai et al. 2011). Special attention has been given to this bacterial genus due to the pathogenicity exhibited by many of its species, including the two anthropogenic bartonellae (Bartonella bacilliformis and Bartonella quintana) and the zoonotic species (e.g., Bartonella henselae, Bartonella grahamii, Bartonella elizabethae, Bartonella koehlerae and Bartonella rochalimae) (Daly et al. 1993, Avidor et al. 2004, Eremeeva et al. 2007, Lydy et al. 2008, Irshad and Gordon 2009, Chomel and Kasten 2010, Ramdial et al. 2012, Oksi et al. 2013).

The diagnosis of Bartonella spp. infection has been considered a challenging task due to the difficulty to isolate these bacteria in vitro, requiring specific conditions that will be discussed below. These characteristics limited not only the detection of infected animals but also the identification of the species involved since they are inert to most classical biochemical assays (Regnery et al. 1992, Clarridge et al. 1995, Bermond et al. 2000). Thus, molecular detection assays (PCR) were rapidly adopted to improve their sensitivity and facilitate the identification. Moreover, reservoirs and vectors are commonly infected with more than one Bartonella species or variants, thus the detection of a particular Bartonella genotype may represent just a portion of the infection repertoire, most probably the dominant genotype (Gurfield et al. 1997, Abbot et al. 2007, Gutiérrez et al. 2014b). Yet, the sole application of molecular methods can represent an obstacle for accurate identification and description of novel Bartonella spp. Therefore, the expertise of both molecular and culture isolation methods is essential and complementary in the diagnosis of Bartonella spp.

Accumulative experience in Bartonella research has led to significant improvements of molecular and culture isolation practices, which have provided a more exhaustive determination of Bartonella among animal samples. The present guidelines intend to gather and summarize practical recommendations for both techniques applied on mammalian and arthropod samples. Special attention is given to the handling of samples collected from the wild.

Sample Collection and Processing

A proper collection of a sample from an animal is the first essential step for successful detection of bartonellae. Due to their hemotropism, blood is the ideal sample source for Bartonella diagnosis from reservoir animals (Kosoy et al. 1999, Schulein et al. 2001). However, it should be noted that bacteremia can be cyclic (Morick et al. 2013) and therefore low blood bacterial levels, below the detection limit of the applied assay, can represent false-negative results. If possible, several tests over time should be performed to overcome this potential limitation. Other tissue samples such as the spleen and liver could be obtained at necropsy or clinical autopsies (Guptill et al. 1997, Maruyama et al. 2004, Angelakis et al. 2009, Morick et al. 2009). When blood is not available such as in the case of carcasses, the spleen is probably the most pertinent internal organ for isolation and detection of Bartonella spp. since it has been demonstrated experimentally that bartonellae are retained and filtered in this organ (Deng et al. 2012). In incidental hosts, Bartonella species are unable to produce durable bacteremia (Vayssier-Taussat et al. 2009), in such cases, the screening of tissues (e.g., spleen, liver, lymph nodes, and skin lesions) is recommended. Recruited samples should be transported and kept at 4°C until their subsequent analysis. When samples are not processed in the day of collection, they should be kept frozen (−20°C or −80°C) until use.

When isolation of Bartonella organisms is intended, it is extremely important to minimize contamination by skin-associated bacteria and other environmental contaminants due to the slow-growing characteristic of bartonellae. Shaving and disinfection of the mammalian's skin with ethanol 70% before sample collection is required when the sample is collected through venipuncture. For small mammalians, such as rodents, other bleeding procedures include retro-orbital bleeding, tail bleeding, and cardiac puncture (Hui et al. 2007). However, most of these techniques require anesthetics and need to be performed by trained practitioners. Blood samples need to be collected in sterile anticoagulated tubes. EDTA tubes have been widely used as the preferred collection tubes and have shown to maintain the viability of Bartonella spp. for a long term (Brenner et al. 1997). Importantly, all sampling procedures, collection techniques, frequency, and the maximum volume of sample allowed for a particular animal species must be approved by the institutional corresponding animal care authorities.

Arthropod specimens can be collected from the mammalian hosts by inspecting carefully the mammalian's fur, using a hairbrush or toothbrush and clean tweezers. If possible, all specimens observed on the host should be collected (or at least 10–20 when present). If the goal is DNA detection only, the ectoparasites can be collected into tubes containing sterile 70% ethanol and transported or stored at room temperature (with the lid covered to prevent ethanol volatilization). When bacterial isolation is intended, the arthropod should be kept alive until further processing in the laboratory. For this purpose, the arthropods can be collected in a flask/tube with a small portion of vegetation (e.g., pieces of grass) or wet paper to maintain high humidity during transportation.

Isolation of Bartonella Organisms

Bartonellae are fastidious bacteria characterized by slow-growing rates. Isolation of Bartonella species from natural reservoir hosts is relatively easy, while more difficult from incidental hosts. They require blood-enriched media under humid and increased carbon dioxide (CO₂) atmospheres (5%). The incubation periods for primary isolations of Bartonella vary across species and animal host source. Visible Bartonella colonies from primary isolations can be obtained as soon as 3–5 days, but usually require longer periods of up to 5–6 weeks (Maurin et al. 1994, Brenner et al. 1997, Kosoy et al. 1997, Breitschwerdt et al. 2001). Subculturing of isolated colonies usually requires shorter incubation periods, ranging from 4 to 10 days (fully grown colonies) at their optimal temperatures (between 27°C and 37°C, depending on the Bartonella species), and 5% CO₂ and high humid atmosphere.

Samples can be directly plated on the appropriate agar media, but certain pretreatments have shown to enhance Bartonella isolation. When blood samples are used, lysis of the erythrocytes by lysis-centrifugation or freezing–thawing techniques has been shown to promote the release of intraerythrocytic bartonellae (Welch et al. 1992, Brenner et al. 1997, Heller et al. 1997). Additionally, infected cells can be concentrated by centrifugation to increase the chances of isolation (Chomel et al. 1996). Moreover, to reduce the overgrowth and impact of coinfecting bacteria (common in wild animals), blood and/or tissue samples can be diluted and homogenized (1:2–1:16) in liquid media, such as brain heart infusion (BHI) broth, before the seeding of a sample in an agar medium. This liquid solution can be supplemented with 5% amphotericin to reduce potential fungal contamination (Kosoy et al. 1997, Bai et al. 2011).

For isolation of Bartonella species from arthropod samples, it is recommended to pretreat the specimens before cultivation in agar media. Decontamination of superficial bacteria from the arthropod with 70% ethanol or ethanol–iodine solutions, followed by sterile water or phosphate-buffered saline (PBS) washes (incubations of 5–10 min), results in reduction of contaminants and does not prevent the isolation of Bartonella (Koehler et al. 1994, Flexman et al. 1995, La Scola et al. 2001, Dehio et al. 2004, Kernif et al. 2014). Then, the arthropod can be homogenized in a liquid medium (e.g., Luria-Bertani, BHI broths) and subsequently plated on agar as described below.

A variety of agar media have been used for the isolation of bartonellae. Columbia, Brucella, BHI, and Trypticase soy-based agars, all supplemented with 5% blood (usually rabbit or sheep), and chocolate agar are the most common solid media used for Bartonella isolation (Koehler et al. 1992, Regnery et al. 1992, Schwartzman et al. 1993, Kosoy et al. 1997, Ellis et al. 1999, Dehio et al. 2004). In a pioneering study, Koehler et al. (1992) compared the use of different media for the isolation of Bartonella spp. from human samples. The authors highlighted the effectiveness of chocolate agar, which promoted its use in following studies. Notably, a previous report described the contamination of sheep blood, with Bartonella, used as a supplement for culture medium (Bemis and Kania 2007). Therefore, it is crucial to check the sterility of blood used for preparation of each new batch of media. For this aim, incubation of noninoculated control plates (negative controls) for at least 6 weeks, at optimal conditions, is recommended. Due to the extended incubation periods, it is crucial to prevent overdrying of the agar and contaminations by sealing the plates, using semipermeable membranes such as commercial shrink seals.

The phenotype of Bartonella colonies varies according to the Bartonella species. A particular species may present different colony morphologies during isolation. Moreover, variation according to the level of passage in agar plates is commonly observed. Primary colonies can be deeply invaginated or raised, cauliflower-like or circular, gray or creamy, smooth or rough-firm, adherent or nonadherent, and/or embedded in the surface of the agar (Regnery et al. 1992, Kosoy et al. 1997). The most evident phenotypic characteristic that may assist in the selection of potential Bartonella colonies is their slow-growing rate during incubation. Accordingly, once Bartonella colonies appear in the agar plate, they usually continue to grow slowly and produce small size changes over several days.

Various liquid media have been described for the isolation and culture of Bartonella species. Commercial broth media supplemented with hemin or histidine–hematin were first reported to support B. henselae and B. quintana growth (Schwartzman et al. 1993, Wong et al. 1995, Chenoweth et al. 2004). Later, the use of media bases for the maintenance of insect cell cultures has provided the most promising broths for Bartonella cultures. The Bartonella-Alpha-Proteobacteria growth medium (BAPGM) (Maggi et al. 2005) and Schneider's insect-based liquid medium supplemented with fetal calve serum and sucrose (Riess et al. 2008) have demonstrated their capability to serve as proper media for isolation of several Bartonella species. A combination of liquid culture, followed by isolation on blood-based agar media, has also been shown to be successful for the primary isolation of Bartonella spp. from human and dog samples (Breitschwerdt et al. 2007, Duncan et al. 2007). However, since these media are not selective for Bartonella spp. only, overgrowth of coinfecting bacteria can limit their use for primary isolations.

Molecular Detection of Bartonella spp. DNA

DNA extraction from animal samples

When choosing the DNA extraction method, two major aspects need to be considered: the presence of high concentrations of PCR inhibitors in the animal blood and tissue samples (Al-Soud and Radstrom 2001) and the efficacy of the DNA extraction method. To overcome the former, many commercial blood and tissue-based kits include PCR inhibitor neutralizers. The ability to amplify DNA from the extracted sample needs to be evaluated and confirmed when a new method is chosen, for instance, by targeting a host-associated locus (Roux and Raoult 1999, Halos et al. 2004, Morick et al. 2011). Second, the Bartonella loads in blood samples from animal reservoirs may be very low, representing a small proportion of infected erythrocytes, less than 5% (Harms and Dehio 2012). In such cases, mechanical and biochemical lysis combined methods and/or prolonged incubations in the buffer lysis (Roux and Raoult 1999) can enhance the recovery of the Bartonella DNA from the blood samples. Additionally, a pre-enrichment culture step of the Bartonella in liquid medium before DNA extraction has also shown to enhance the Bartonella DNA detection from clinical samples (Duncan et al. 2007, Bai et al. 2010).

For arthropod samples, a superficial cleaning step before DNA extraction is critical to remove potential biological contaminants and remnants of the ethanol used for storage and transportation. Washes usually include an immersion in fresh sterile ethanol 70% for 5–10 min, followed by two to three immersions of sterile water or PBS. After the washing steps, the arthropod can be processed for DNA extraction using tissue-based DNA extraction protocols (e.g., commercial kits, phenol–chloroform protocols), using single individual arthropods or pools (2–20 individuals) depending on the size of the specimens. The use of arthropod pools increases the chances of detection of Bartonella DNA; however, it prevents the capability to determine coinfections with several Bartonella spp. in single arthropods and restricts prevalence determination. For small specimens (e.g., fleas, lice, and mites), maceration of the whole arthropod with a sterile pestle in a sterile tube containing small volume of PBS (∼50–100 μL) is recommended to avoid DNA losses. For larger arthropods, such as hard ticks, mincing the specimens into small pieces, by a bead beater or a sterile scalpel, and separating the exoskeleton by centrifugation, facilitate the mechanical destruction of the internal arthropod tissues and reduce the saturation of silica columns with excess exoskeleton (Halos et al. 2004, Harrus et al. 2011). To increase the efficiency of DNA extraction from the arthropod tissues, long incubation (over 2 h) in the buffer lysis is usually needed. In hard ticks, the presence of low quantities of the targeted DNA can be overcome by DNA preamplification protocols before the specific PCR (Michelet et al. 2014) or by using nested PCR assays (Cotté et al. 2008).

Amplification of Bartonella DNA

Amplification of Bartonella DNA from animal and human samples by PCR-based techniques has been extensively assessed. Conventional PCR assays were initially described for direct detection of Bartonella DNA from samples of clinical cases (Relman et al. 1990, Anderson et al. 1994, Birtles et al. 2000). However, when conventional PCR is used for the detection of Bartonella spp. in subclinical reservoirs, the sensitivity of the assays is usually limited. This is attributed to the low Bartonella loads in wild animals, which may result in false-negative results. Therefore, the use of more sensitive techniques, such as nested and real-time PCR assays, has improved the sensitivity of Bartonella diagnosis (Rampersad et al. 2005, Diaz et al. 2012, Gutiérrez et al. 2013). The disadvantage of the latter assay is the small size of the amplicons, limiting its specificity. Thus, confirmatory real-time PCR assays targeting additional loci or the combination with conventional PCR assays for larger amplicons are recommended.

Many conserved and housekeeping loci have been developed as targets for the characterization and molecular detection of Bartonella organisms (see Table 1 for recommended targets). Among them, the citrate synthase gene (gltA) and the RNA polymerase β-subunit gene (rpoB) are the most commonly used targets for the identification of Bartonella spp. due to their potent discriminatory power (La Scola et al. 2003), their relative stability as housekeeping genes, and their extensive GenBank database. However, homologous recombination events have been reported within the gltA, potentially leading to some species misidentification (e.g., in rodent-associated bartonellae) (Paziewska et al. 2011, Buffet et al. 2013). Recommendations for the identification of Bartonella variants are included in the Experts’ Advice section below.

Table 1.

Author-Recommended PCR Targets and Primers Required for the Detection and Characterization of Bartonella spp.

Targets recommended for molecular characterization of isolates
Bartonella locus		Primers	Approximate size (bp)	Assay type	Reference
Citrate synthase	gltA	443F and 1210R; Bhcs.781p and Bhcs.1137n	340–370	Conventional PCR	Norman et al. (1995); Birtles and Raoult (1996)
β subunit of the RNA polymerase	rpoB	1400F and 2300R	825	Conventional PCR	Renesto et al. (2001)
16S-23S Internal transcribed spacer	ITS	321s and 983as	621–704	Conventional PCR	Maggi et al. (2005)
16S ribosomal RNA gene	16S	p24E and p12B	280	Conventional PCR	Bergmans et al. (1995)
Riboflavin synthase	ribC	Barton-1 and Barton-2	588	Conventional PCR	Johnson et al. (2003)
NADH dehydrogenase gamma subunit	nuoG	F and R	346	Conventional PCR/real-time PCR	Colborn et al. (2010)
Cell division protein	ftsZ	Bfp1 and Bfp2	900	Conventional PCR	Zeaiter et al. (2002)
60 kDa Heat shock protein	groEL	Hs233 and Hs1630	720	Conventional PCR	Marston et al. (1999)

Targets recommended for direct molecular detection (from blood/tissue/arthropod)
mtRNA	ssrA	ssrAF and ssrAR	350	Real-time PCR	Diaz et al. (2012)
16S-23S Internal transcribed spacer	ITS	321s and H493as	200	Real-time PCR	Maggi et al. (2005); conditions for real-time PCR described in Gutiérrez et al. (2013)
β subunit of the RNA polymerase	rpoB	600F and 800R	200	Real-time PCR	Morick et al. (2009)

The sensitivity to detect positive samples varies considerably among the described PCR assays. Notably, many of the described primers in the literature were developed for the amplification and characterization of Bartonella isolates (from colonies). Consequently, once applied for direct detection of Bartonella DNA, cross-reaction with the host gDNA and/or with coinfecting microorganisms has been reported (Maggi and Breitschwerdt 2005, Colborn et al. 2010, Gutiérrez et al. 2013). Therefore, nonspecific amplification can mislead the diagnosis of Bartonella if the amplicons obtained are not sequenced or characterized. Although sequencing of positive amplicons is preferable, other techniques such as high-resolution melting (HRM) analysis can assist in rapid discrimination of Bartonella DNA amplicons according to their melt profiles (Morick et al. 2009, Gutiérrez et al. 2013).

Coinfection of hosts with more than one Bartonella species or genotype is a well-known phenomenon (Gurfield et al. 1997, Abbot et al. 2007, Chan and Kosoy 2010, Gutiérrez et al. 2014b). Taking into consideration that PCR-based assays are biased toward the most predominant species, in samples containing several species or genotypes, the molecular detection of a particular species does not rule out the presence of other coinfecting Bartonella spp. It has been observed that amplification of additional loci can lead to the detection of distinctly related Bartonella spp. DNA, suggesting coinfection (Gutiérrez et al. 2013, 2014a). The latter phenomenon can occur due to different primer-annealing sensitivities among the primers used that may favor detection of one of the species over the other (Whiley et al. 2008). Another possibility is that the diverse DNA sequences are originated from a single recombinant strain, as has been described earlier in rodent-associated bartonellae (Harrus et al. 2009, Paziewska et al. 2011, Buffet et al. 2013). Although both scenarios have been demonstrated, the former seems to be a more common event. Ideally, to characterize coinfecting Bartonella genotypes by molecular methods, multiple amplicons of a single-locus target (e.g., gltA) can be sequenced, using cloning libraries (in Escherichia coli vectors) or 454-pyrosequencing platforms (Abbot et al. 2007, Gutiérrez et al. 2014b). Furthermore, in cases where the Bartonella spp. involved in a particular animal population have been well characterized and seem homogeneous worldwide (e.g., feline-associated and bovine-associated bartonellae), species-specific primers may be developed to test the potential coinfecting spp. in previously screened positive samples (Bereswill et al. 1999, Rolain et al. 2003, Cherry et al. 2009). The development of species-specific primers is currently facilitated with the availability of the complete and/or partial genomes of many Bartonella spp.

In wild animals, such as carnivores, rodents, bats, and their associated ectoparasites, the detection of yet uncharacterized Bartonella spp. and genotypes is a common phenomenon (Diniz et al. 2009, Inoue et al. 2009, Morick et al. 2010, Bai et al. 2012). To prevent the erroneous relationship of loci from different genotypes (during coinfection) with one uncharacterized strain, independent description of the amplicons is recommended. Only further characterization of isolated colonies will eventually guarantee accurate identification.

The advantages and limitations of the use of molecular detection assays for Bartonella diagnosis have led to the following recommendations: (1) real-time PCR should be chosen over conventional PCR assays when Bartonella infection is assessed among animal hosts; (2) characterization (sequencing) of the amplicons obtained is indispensable; and (3) additional loci preferably of longer length should be targeted to confirm positive samples, detect false-negative samples, and to potentially detect coinfecting Bartonella spp. Furthermore, when a new screening of samples is planned, evaluation of the sensitivity and specificity of different primers to the intended samples is recommended. Ideally, the assay should be standardized so as to quantify the detection limit of the assay selected.

Molecular Detection Versus Culture Isolation

The culture isolation of a Bartonella species from an infected animal will always be the preferred direct method for the diagnosis and characterization of the species involved. However, despite the improvements in culturing methods, these assays are still laborious, time-consuming, and their sensitivity to detect Bartonella-positive samples from wild animals is considerably low. In contrast, molecular diagnostic methods offer a more rapid, specific, and sensitive tool to determine the Bartonella infections. Studies that have compared both methods have demonstrated the higher sensitivity of real-time PCR assays over isolation in more than twofold of the positive samples (Mietze et al. 2011, Diaz et al. 2012, Gutiérrez et al. 2013). Additionally, different Bartonella spp./strains present different growing rates in culture, significantly biasing the infection description in coinfected animals, using this sole methodology. Thus, applying the appropriate PCR targets, followed by a characterization of the obtained DNA sequences, molecular methods can represent more sensitive and discriminatory assays for the detection of Bartonella within animal populations. Moreover, we encourage the attempt of culture isolation of DNA-positive samples since isolated colonies enable a broader molecular characterization and speciation of the infecting bacteria. Figure 1 illustrates the recommended work-chart in the detection of Bartonella in animal samples.

FIG. 1.

Recommended work-chart for detection of Bartonella in animal samples. Those should be prepared for DNA extraction, followed by molecular screening for Bartonella DNA, preferably by real-time PCR assay. Positive amplicons need to be characterized, preferably by sequencing. Then, Bartonella-positive samples should be screened for additional loci. Blood and/or tissue samples confirmed to be PCR positive can be assessed for Bartonella isolation. Pretreatment of the samples before seeding in agar plates is recommended.

Experts’ Advice on the Identification of Bartonella

The high genetic diversity of Bartonella species makes the identification of Bartonella isolates or uncultured organisms a challenging task. La Scola et al. (2003) proposed a criterion based on the sequencing of 327-bp gltA and 825-bp rpoB fragments. Accordingly, if an isolate showed similarities lower than 96.0% and 95.4% for the latter genes (of the validated species), respectively, it was proposed to be considered a new species. However, using these housekeeping loci and additional ones (e.g., ITS, ribC, groEL), many isolates or uncultured bartonellae sequences have shown to represent variants of the validated Bartonella spp. (with sequence similarities between 95% and 99%), as well as more distinctly related variants (similarities below 95%), and/or cases of mixed origin species (i.e., isolates carrying two or more loci closely related to different validated Bartonella spp.). Moreover, due to the frequent coinfection of hosts with more than one Bartonella species, the identification of Bartonella needs to be carefully addressed. Thus, in the case of isolates, we recommend to identify all Bartonella isolates with at least three to five loci and use only well-isolated colonies (i.e., at least two repassages in agar plates from single colonies). Moreover, phylogenetic analysis using concatenated sequences is recommended to support the differentiation and diagnosis of newly isolated variants (Harrus et al. 2009, Buffet et al. 2013). In addition, we recommend the isolation of several colonies (three to five colonies) from the primary isolation plates to facilitate the detection of potential coinfections. When direct molecular detection (i.e., uncultured organisms) is performed for the identification of the host-infecting Bartonella, screening of at least two to three loci is highly recommended. The latter will allow a more robust confirmation of positive samples and potentially identify coinfecting bartonellae. With the constant decrease in costs of whole-genome sequencing (WGS) techniques, future characterization of new Bartonella species and variants will require the use of WGS.

Conclusions

As Bartonella bacteria emerge constantly and their importance in public health increases, rapid diagnostic tools are required. We suggest the use of real-time PCR assays for the initial screening of Bartonella spp., followed by several molecular confirmatory assays targeting several loci (preferably of longer fragments than those amplified by the real-time assay). In addition, we encourage the use of culture isolation as it denotes the presence of live bacteria. Moreover, the isolated colonies enable a broader molecular characterization and accurate speciation of the bacteria.

Footnotes

Acknowledgments

This study was supported by TD1303 COST Action entitled, European Network for Neglected Vectors and Vector-Borne Infections (EurNegVec), and by the Israel Science Foundation (grant number 30/11 to Shimon Harrus).

Author Disclosure Statement

No competing financial interests exist.

References

Abbot

, Aviles

, Eller

, Durden

. Mixed infections, cryptic diversity, and vector-borne pathogens: evidence from Polygenis fleas and Bartonella species. Appl Environ Microbiol, 2007; 73:6045–6052.

Al-Soud

, Radstrom

. Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol, 2001; 39:485–493.

Anderson

, Sims

, Regnery

, Robinson

, et al. Detection of Rochalimaea henselae DNA in specimens from cat-scratch disease patients by PCR. J Clin Microbiol, 1994; 32:942–948.

Angelakis

, Khamphoukeo

, Grice

, Newton

, et al. Molecular detection of Bartonella species in rodents from the Lao PDR. Clin Microbiol Infect, 2009; 15 Suppl 2:95–97.

Avidor

, Graidy

, Efrat

, Leibowitz

, et al. Bartonella koehlerae, a new cat-associated agent of culture-negative human endocarditis. J Clin Microbiol, 2004; 42:3462–3468.

Bai

, Calisher

, Kosoy

, Root

, et al. Persistent infection or successive reinfection of deer mice with Bartonella vinsonii subsp. arupensis . Appl Environ Microbiol, 2011; 77:1728–1731.

Bai

, Kosoy

, Boonmar

, Sawatwong

, et al. Enrichment culture and molecular identification of diverse Bartonella species in stray dogs. Vet Microbiol, 2010; 146:314–319.

Bai

, Recuenco

, Gilbert

, Osikowicz

, et al. Prevalence and diversity of Bartonella spp. in bats in Peru. Am J Trop Med Hyg, 2012; 87:518–523.

Bemis

, Kania

. Isolation of Bartonella sp. from sheep blood. Emerg Infect Dis, 2007; 13:1565–1567.

10.

Bereswill

, Hinkelmann

, Kist

, Sander

. Molecular analysis of riboflavin synthesis genes in Bartonella henselae and use of the ribC gene for differentiation of Bartonella species by PCR. J Clin Microbiol, 1999; 37:3159–3166.

11.

Bergmans

, Groothedde

, Schellekens

, van Embden

, et al. Etiology of cat scratch disease: comparison of polymerase chain reaction detection of Bartonella (formerly Rochalimaea) and Afipia felis DNA with serology and skin tests. J Infect Dis, 1995; 171:916–923.

12.

Bermond

, Heller

, Barrat

, Delacour

, et al. Bartonella birtlesii sp. nov., isolated from small mammals (Apodemus spp.). Int J Syst Evol Microbiol, 2000; 50 Pt 6:1973–1979.

13.

Birtles

, Hazel

, Bown

, Raoult

, et al. Subtyping of uncultured bartonellae using sequence comparison of 16 S/23 S rRNA intergenic spacer regions amplified directly from infected blood. Mol Cell Probes, 2000; 14:79–87.

14.

Birtles

, Raoult

. Comparison of partial citrate synthase gene (gltA) sequences for phylogenetic analysis of Bartonella species. Int J Syst Bacteriol, 1996; 46:891–897.

15.

Boulouis

H-J

, Chang

C-C

, Henn

, Kasten

, et al. Factors associated with the rapid emergence of zoonotic Bartonella infections. Vet Res, 2005; 36:383–410.

16.

Breitschwerdt

, Maggi

, Duncan

, Nicholson

, et al. Bartonella species in blood of immunocompetent persons with animal and arthropod contact. Emerg Infect Dis, 2007; 13:938–941.

17.

Breitschwerdt

, Sontakke

, Cannedy

, Hancock

, et al. Infection with Bartonella weissii and detection of Nanobacterium antigens in a North Carolina beef herd. J Clin Microbiol, 2001; 39:879–882.

18.

Brenner

, Rooney

, Manzewitsch

, Regnery

. Isolation of Bartonella (Rochalimaea) henselae: effects of methods of blood collection and handling. J Clin Microbiol, 1997; 35:544–547.

19.

Buffet

J-P

, Pisanu

, Brisse

, Roussel

, et al. Deciphering Bartonella diversity, recombination, and host specificity in a rodent community. PLoS One, 2013; 8:e68956.

20.

Chan

K-S

, Kosoy

. Analysis of multi-strain Bartonella pathogens in natural host population—do they behave as species or minor genetic variants?. Epidemics, 2010; 2:165–172.

21.

Chenoweth

, Somerville

, Krause

, O'Reilly

, et al. Growth characteristics of Bartonella henselae in a novel liquid medium: primary isolation, growth-phase-dependent phage induction, and metabolic studies. Appl Environ Microbiol, 2004; 70:656–663.

22.

Cherry

, Maggi

, Cannedy

, Breitschwerdt

. PCR detection of Bartonella bovis and Bartonella henselae in the blood of beef cattle. Vet Microbiol, 2009; 135:308–312.

23.

Chomel

, Boulouis

H-J

, Breitschwerdt

, Kasten

, et al. Ecological fitness and strategies of adaptation of Bartonella species to their hosts and vectors. Vet Res, 2009; 40:29.

24.

Chomel

, Kasten

. Bartonellosis, an increasingly recognized zoonosis. J Appl Microbiol, 2010; 109:743–750.

25.

Chomel

, Kasten

, Floyd-Hawkins

, Chi

, et al. Experimental transmission of Bartonella henselae by the cat flea. J Clin Microbiol, 1996; 34:1952–1956.

26.

Clarridge

3rd , Raich

, Pirwani

, Simon

, et al. Strategy to detect and identify Bartonella species in routine clinical laboratory yields Bartonella henselae from human immunodeficiency virus-positive patient and unique Bartonella strain from his cat. J Clin Microbiol, 1995; 33:2107–2113.

27.

Colborn

, Kosoy

, Motin

, Telepnev

, et al. Improved detection of Bartonella DNA in mammalian hosts and arthropod vectors by real-time PCR using the NADH dehydrogenase gamma subunit (nuoG). J Clin Microbiol, 2010; 48:4630–4633.

28.

Cotté

, Bonnet

, Le Rhun

, Le Naour

, et al. Transmission of Bartonella henselae by Ixodes ricinus . Emerg Infect Dis, 2008; 14:1074–1080.

29.

Daly

, Worthington

, Brenner

, Moss

, et al. Rochalimaea elizabethae sp. nov. isolated from a patient with endocarditis. J Clin Microbiol, 1993; 31:872–881.

30.

Dehio

, Sauder

, Hiestand

. Isolation of Bartonella schoenbuchensis from Lipoptena cervi, a blood-sucking arthropod causing deer ked dermatitis. J Clin Microbiol, 2004; 42:5320–5323.

31.

Deng

, Le Rhun

, Lecuelle

, Le Naour

, et al. Role of the spleen in Bartonella spp. infection. FEMS Immunol Med Microbiol, 2012; 64:143–145.

32.

Diaz

, Bai

, Malania

, Winchell

, et al. Development of a novel genus-specific real-time PCR assay for detection and differentiation of Bartonella species and genotypes. J Clin Microbiol, 2012; 50:1645–1649.

33.

Diniz

, Billeter

, Otranto

, De Caprariis

, et al. Molecular documentation of Bartonella infection in dogs in Greece and Italy. J Clin Microbiol, 2009; 47:1565–1567.

34.

Duncan

, Maggi

, Breitschwerdt

. A combined approach for the enhanced detection and isolation of Bartonella species in dog blood samples: pre-enrichment liquid culture followed by PCR and subculture onto agar plates. J Microbiol Methods, 2007; 69:273–281.

35.

Ellis

, Regnery

, Beati

, Bacellar

, et al. Rats of the genus Rattus are reservoir hosts for pathogenic Bartonella species: an Old World origin for a New World disease?. J Infect Dis, 1999; 180:220–224.

36.

Eremeeva

, Gerns

, Lydy

, Goo

, et al. Bacteremia, fever, and splenomegaly caused by a newly recognized Bartonella species. N Engl J Med, 2007; 356:2381–2387.

37.

Flexman

, Lavis

, Kay

, Watson

, et al. Bartonella henselae is a causative agent of cat scratch disease in Australia. J Infect, 1995; 31:241–245.

38.

Guptill

, Slater

, Wu

, Lin

, et al. Experimental infection of young specific pathogen-free cats with Bartonella henselae . J Infect Dis, 1997; 176:206–216.

39.

Gurfield

, Boulouis

, Chomel

, Heller

, et al. Coinfection with Bartonella clarridgeiae and Bartonella henselae and with different Bartonella henselae strains in domestic cats. J Clin Microbiol, 1997; 35:2120–2123.

40.

Gutiérrez

, Cohen

, Morick

, Mumcuoglu

, et al. Identification of different Bartonella species in the cattle tail louse (Haematopinus quadripertusus) and in cattle blood. Appl Environ Microbiol, 2014a; 80:5477–5483.

41.

Gutiérrez

, Morick

, Cohen

, Hawlena

, et al. The effect of ecological and temporal factors on the composition of Bartonella infection in rodents and their fleas. ISME J, 2014b; 8:1598–1608.

42.

Gutiérrez

, Morick

, Gross

, Winkler

, et al. Bartonellae in domestic and stray cats from Israel: comparison of bacterial cultures and high-resolution melt real-time PCR as diagnostic methods. Vector Borne Zoonotic Dis, 2013; 13:857–864.

43.

Halos

, Jamal

, Vial

, Maillard

, et al. Determination of an efficient and reliable method for DNA extraction from ticks. Vet Res, 2004; 35:709–713.

44.

Harms

, Dehio

. Intruders below the radar: molecular pathogenesis of Bartonella spp. Clin Microbiol Rev, 2012; 25:42–78.

45.

Harrus

, Bar-Gal

, Golan

, Elazari-Volcani

, et al. Isolation and genetic characterization of a Bartonella strain closely related to Bartonella tribocorum and Bartonella elizabethae in Israeli commensal rats. Am J Trop Med Hyg, 2009; 81:55–58.

46.

Harrus

, Perlman-Avrahami

, Mumcuoglu

, Morick

, et al. Molecular detection of Rickettsia massiliae, Rickettsia sibirica mongolitimonae and Rickettsia conorii israelensis in ticks from Israel. Clin Microbiol Infect, 2011; 17:176–180.

47.

Heller

, Artois

, Xemar

, De Briel

, et al. Prevalence of Bartonella henselae and Bartonella clarridgeiae in stray cats. J Clin Microbiol, 1997; 35:1327–1331.

48.

Hui

, Huang

, Ebbert

, Bina

, et al. Pharmacokinetic comparisons of tail-bleeding with cannula- or retro-orbital bleeding techniques in rats using six marketed drugs. J Pharmacol Toxicol Methods, 2007; 56:256–264.

49.

Inoue

, Maruyama

, Kabeya

, Hagiya

, et al. Exotic small mammals as potential reservoirs of zoonotic Bartonella spp. Emerg Infect Dis, 2009; 15:526–532.

50.

Irshad

, Gordon

. Bartonella henselae neuroretinitis in a 15-year-old girl with chronic myelogenous leukemia. J AAPOS, 2009; 13:602–604.

51.

Johnson

, Ayers

, McClure

, Richardson

, et al. Detection and identification of Bartonella species pathogenic for humans by PCR amplification targeting the riboflavin synthase gene (ribC). J Clin Microbiol, 2003; 41:1069–1072.

52.

Kernif

, Leulmi

, Socolovschi

, Berenger

, et al. Acquisition and excretion of Bartonella quintana by the cat flea, Ctenocephalides felis felis . Mol Ecol, 2014; 23:1204–1212.

53.

Koehler

, Glaser

, Tappero

. Rochalimaea henselae infection. A new zoonosis with the domestic cat as reservoir. JAMA, 1994; 271:531–535.

54.

Koehler

, Quinn

, Berger

, LeBoit

, et al. Isolation of Rochalimaea species from cutaneous and osseous lesions of bacillary angiomatosis. N Engl J Med, 1992; 327:1625–1631.

55.

Kosoy

. Ecological associations between bacteria of the genus Bartonella and mammals. Biol Bullet, 2010; 37:716–724.

56.

Kosoy

, Regnery

, Kosaya

, Childs

. Experimental infection of cotton rats with three naturally occurring Bartonella species. J Wildl Dis, 1999; 35:275–284.

57.

Kosoy

, Regnery

, Tzianabos

, Marston

, et al. Distribution, diversity, and host specificity of Bartonella in rodents from the Southeastern United States. Am J Trop Med Hyg, 1997; 57:578–588.

58.

La Scola

, Fournier

, Brouqui

, Raoult

. Detection and culture of Bartonella quintana, Serratia marcescens, and Acinetobacter spp. from decontaminated human body lice. J Clin Microbiol, 2001; 39:1707–1709.

59.

La Scola

, Zeaiter

, Khamis

, Raoult

. Gene-sequence-based criteria for species definition in bacteriology: the Bartonella paradigm. Trends Microbiol, 2003; 11:318–321.

60.

Lydy

, Eremeeva

, Asnis

, Paddock

, et al. Isolation and characterization of Bartonella bacilliformis from an expatriate Ecuadorian. J Clin Microbiol, 2008; 46:627–637.

61.

Maggi

, Breitschwerdt

. Potential limitations of the 16S-23S rRNA intergenic region for molecular detection of Bartonella species. J Clin Microbiol, 2005; 43:1171–1176.

62.

Maggi

, Duncan

, Breitschwerdt

. Novel chemically modified liquid medium that will support the growth of seven Bartonella species. J Clin Microbiol, 2005; 43:2651–2655.

63.

Marston

, Sumner

, Regnery

. Evaluation of intraspecies genetic variation within the 60 kDa heat-shock protein gene (groEL) of Bartonella species. Int J Syst Bacteriol, 1999; 49 Pt 3:1015–1023.

64.

Maruyama

, Izumikawa

, Miyashita

, Kabeya

, et al. First isolation of Bartonella henselae type I from a cat-scratch disease patient in Japan and its molecular analysis. Microbiol Immunol, 2004; 48:103–109.

65.

Maurin

, Roux

, Stein

, Ferrier

, et al. Isolation and characterization by immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blot, restriction fragment length polymorphism-PCR, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis of Rochalimaea quintana from a patient with bacillary angiomatosis. J Clin Microbiol, 1994; 32:1166–1171.

66.

Michelet

, Delannoy

, Devillers

, Umhang

, et al. High-throughput screening of tick-borne pathogens in Europe. Front Cell Infect Microbiol, 2014; 4:103.

67.

Mietze

, Morick

, Kohler

, Harrus

, et al. Combined MLST and AFLP typing of Bartonella henselae isolated from cats reveals new sequence types and suggests clonal evolution. Vet Microbiol, 2011; 148:238–245.

68.

Morick

, Baneth

, Avidor

, Kosoy

, et al. Detection of Bartonella spp. in wild rodents in Israel using HRM real-time PCR. Vet Microbiol, 2009; 139:293–297.

69.

Morick

, Krasnov

, Khokhlova

, Gottlieb

, et al. Investigation of Bartonella acquisition and transmission in Xenopsylla ramesis fleas (Siphonaptera: Pulicidae). Mol Ecol, 2011; 20:2864–2870.

70.

Morick

, Krasnov

, Khokhlova

, Gottlieb

, et al. Transmission dynamics of Bartonella sp. strain OE 1-1 in Sundevall's jirds (Meriones crassus). Appl Environ Microbiol, 2013; 79:1258–1264.

71.

Morick

, Krasnov

, Khokhlova

, Shenbrot

, et al. Bartonella genotypes in fleas (Insecta: Siphonaptera) collected from rodents in the Negev desert, Israel. Appl Environ Microbiol, 2010; 76:6864–6869.

72.

Norman

, Regnery

, Jameson

, Greene

, et al. Differentiation of Bartonella-like isolates at the species level by PCR-restriction fragment length polymorphism in the citrate synthase gene. J Clin Microbiol, 1995; 33:1797–1803.

73.

Oksi

, Rantala

, Kilpinen

, Silvennoinen

, et al. Cat scratch disease caused by Bartonella grahamii in an immunocompromised patient. J Clin Microbiol, 2013; 51:2781–2784.

74.

Paziewska

, Harris

, Zwolinska

, Bajer

, et al. Recombination within and between species of the alpha proteobacterium Bartonella infecting rodents. Microb Ecol, 2011; 61:134–145.

75.

Ramdial

, Sing

, Ramburan

, Dlova

, et al. Bartonella quintana-induced vulval bacillary angiomatosis. Int J Gynecol Pathol, 2012; 31:390–394.

76.

Rampersad

, Watkins

, Samlal

, Deonanan

, et al. A nested-PCR with an Internal Amplification Control for the detection and differentiation of Bartonella henselae and B. clarridgeiae: an examination of cats in Trinidad. BMC Infect Dis, 2005; 5:63.

77.

Regnery

, Anderson

, Clarridge

3rd , Rodriguez-Barradas

, et al. Characterization of a novel Rochalimaea species, R. henselae sp. nov., isolated from blood of a febrile, human immunodeficiency virus-positive patient. J Clin Microbiol, 1992; 30:265–274.

78.

Relman

, Loutit

, Schmidt

, Falkow

, et al. The agent of bacillary angiomatosis. An approach to the identification of uncultured pathogens. N Engl J Med, 1990; 323:1573–1580.

79.

Renesto

, Gouvernet

, Drancourt

, Roux

, et al. Use of rpoB gene analysis for detection and identification of Bartonella species. J Clin Microbiol, 2001; 39:430–437.

80.

Riess

, Dietrich

, Schmidt

, Kaiser

, et al. Analysis of a novel insect cell culture medium-based growth medium for Bartonella species. Appl Environ Microbiol, 2008; 74:5224–5227.

81.

Rolain

, Franc

, Davoust

, Raoult

. Molecular detection of Bartonella quintana, B. koehlerae, B. henselae, B. clarridgeiae, Rickettsia felis, and Wolbachia pipientis in cat fleas, France. Emerg Infect Dis, 2003; 9:338–342.

82.

Roux

, Raoult

. Body lice as tools for diagnosis and surveillance of reemerging diseases. J Clin Microbiol, 1999; 37:596–599.

83.

Schulein

, Seubert

, Gille

, Lanz

, et al. Invasion and persistent intracellular colonization of erythrocytes. A unique parasitic strategy of the emerging pathogen Bartonella . J Exp Med, 2001; 193:1077–1086.

84.

Schwartzman

, Nesbit

, Baron

. Development and evaluation of a blood-free medium for determining growth curves and optimizing growth of Rochalimaea henselae . J Clin Microbiol, 1993; 31:1882–1885.

85.

Tsai

, Chang

, Chuang

, Chomel

. Bartonella species and their ectoparasites: selective host adaptation or strain selection between the vector and the mammalian host?. Comp Immunol Microbiol Infect Dis, 2011; 34:299–314.

86.

Vayssier-Taussat

, Le Rhun

, Bonnet

, Cotte

. Insights in Bartonella host specificity. Ann N Y Acad Sci, 2009; 1166:127–132.

87.

Welch

, Pickett

, Slater

, Steigerwalt

, et al. Rochalimaea henselae sp. nov., a cause of septicemia, bacillary angiomatosis, and parenchymal bacillary peliosis. J Clin Microbiol, 1992; 30:275–280.

88.

Whiley

, Lambert

, Bialasiewicz

, Goire

, et al. False-negative results in nucleic acid amplification tests-do we need to routinely use two genetic targets in all assays to overcome problems caused by sequence variation?. Crit Rev Microbiol, 2008; 34:71–76.

89.

Wong

, Thornton

, Kennedy

, Dolan

. A chemically defined liquid medium that supports primary isolation of Rochalimaea (Bartonella) henselae from blood and tissue specimens. J Clin Microbiol, 1995; 33:742–744.

90.

Zeaiter

, Liang

, Raoult

. Genetic classification and differentiation of Bartonella species based on comparison of partial ftsZ gene sequences. J Clin Microbiol, 2002; 40:3641–3647.