Novel Chemically Modified Liquid Medium That Will Support the Growth of Seven Bartonella Species

Abstract

Bacteria of the genus Bartonella, a member of the Alphaproteobacteria, are fastidious, gram-negative, aerobic bacilli that comprise numerous species, subspecies, and subtypes. In human and veterinary medicine, species isolation remains a vital component of the diagnostic and therapeutic management of Bartonella infection. We describe a novel, chemically modified, insect-based liquid culture medium that supports the growth of at least seven Bartonella species. This medium will also support cocultures consisting of different Bartonella species, and it facilitated the primary isolation of Bartonella henselae from blood and aqueous fluid of naturally infected cats. This liquid growth medium may provide an advantage over conventional direct blood agar plating for the diagnostic confirmation of bartonellosis.Due to their zoonotic potential, their vector transmission, which includes sandflies, lice, fleas, and ticks, and their frequent adaptation to a mammalian reservoir host, Bartonella species are considered among the newest and most significant emerging pathogens (1,3,8,12,27,29,39,45). These bacteria are highly adapted to a mammalian reservoir host; further, these organisms have been shown to cause a long-lasting intraerythrocytic bacteremia in both humans and animals (11,14,22,24,25,37). Bartonella species are also the causative agents of Carrion's disease (Oroya fever and verruga peruana) (Bartonella bacilliformis) (6), trench fever (B. quintana) (11,40,43), endocarditis (B. elizabethae, B. henselae, B. vinsonii subsp. berkhoffii, B. washoensis, B. clarridgeae) (3, 7, 9, 16, 17, 21, 40, 44), bacillary angiomatosis in immunocompromised patients (B. quintana, B. henselae) (14,30,48), neuroretinitis (B. grahamii) (28), and cat scratch disease (B. henselae, B. clarridgeiae) (5,31,42).Because Bartonella species frequently induce persistent intravascular infections, it has been difficult to attribute chronic disease causation to infection in humans and companion animals; much of this difficulty may be related to the few and often very subtle clinical abnormalities that are reported by a patient or observed in a sick animal. Confirming disease causation is especially difficult in retrospective or prospective animal studies in which Bartonella bacteremia can be detected in overtly healthy, natural reservoir hosts-a paradigm in opposition to Koch's postulates for disease causation (12, 23). Nevertheless, an increasingly diverse spectrum of Bartonella-associated infections have been recognized in people and in dogs due to the development of new approaches to improving serologic and molecular diagnostic testing methods, which prove to be, in most instances, more sensitive than conventional culture methods for the isolation of Bartonella species (13,15,26,30,33,38,41,46,49). Primary isolation of Bartonella species following lysis centrifugation, or freezing of a blood sample, followed by application to a blood agar plate, is the most widely used method for the microbiological diagnosis of bartonellosis. Isolation of Bartonel...

Canine bartonellosis: serological and molecular prevalence in Brazil and evidence of co-infection withBartonella henselaeandBartonella vinsoniisubsp.berkhoffii

“…Two hundred microliters of blood from Bartonella ITS region-PCR positive dogs and/or IFA seroreactive dogs were inoculated into liquid Bartonella alpha-Proteobacteria growth medium (BAPGM) [35] and incubated at 35 • C in 5% CO 2 , water-saturated atmosphere. After a 14-day incubation period, 1 mL from liquid culture was sub-inoculated onto BAPGM/blood-agar plates and incubated for 14 days.…”

Bartonellaspecies detection in captive, stranded and free-ranging cetaceans

“…Molecular detection, cloning and bacterial isolation from blood and tissues were performed using previously-described methods [13,[26][27][28][29]. EDTAanticoagulated blood samples (n = 95 animals) and tissues (n = 15 freshly dead animals) were analyzed.…”

“…Tissue and ectoparasite samples were screened only by direct extraction PCR. For blood samples only, a pre-enrichment culture was established from the original sample using liquid BAPGM [29]; after a seven day incubation period a sample was removed for molecular screening using conventional and real-time PCR (n = 50 animals). Finally, a BAPGM blood agar plate was subinoculated using the liquid pre-enrichment blood culture, maintained for at least three weeks, at which time colony growth was again tested by conventional and real-time PCR (n = 50 animals).…”

“…Because conventional microbiological techniques lack sensitivity for Bartonella spp., bartonellosis is usually diagnosed by PCR amplification or serology [3,6,26,29]. Recently, the development of a more sensitive isolation approach, using Bartonella Alpha-Proteobacteria growth medium (BAPGM) followed by real time PCR has facilitated the molecular detection or isolation of Bartonella spp.…”

“…Recently, the development of a more sensitive isolation approach, using Bartonella Alpha-Proteobacteria growth medium (BAPGM) followed by real time PCR has facilitated the molecular detection or isolation of Bartonella spp. from the blood of sick and healthy animals [8,13,29]. These improved techniques led to the first recognition of bloodborne Bartonella spp.…”