A Bartonella vinsonii berkhoffii typing scheme based upon 16S–23S ITS and Pap31 sequences from dog, coyote, gray fox, and human isolates

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Abstract

Since the isolation of Bartonella vinsonii subspecies berkhoffii from a dog with endocarditis in 1993, this organism has emerged as an important pathogen in dogs and as an emerging pathogen in people. Current evidence indicates that coyotes, dogs and gray foxes potentially serve as reservoir hosts. Based upon sequence differences within the 16S–23S ITS region and Pap31 gene, we propose a classification scheme that divides B. vinsonii subsp. berkhoffii isolates into four distinct types. Two conserved sequences, of 37 and 18 bp, respectively, are differentially present within the ITS region of each of the four B. vinsonii subsp. berkhoffii types. To date, B. vinsonii berkhoffii types I, II, and III have been identified in the US, type III in Europe and type IV in Canada. Based upon the proposed genotyping scheme, the geographic distribution of B. vinsonii berkhoffii types needs to be more thoroughly delineated in future molecular epidemiological studies involving Bartonella infection in coyotes, dogs, gray foxes, human beings and potentially other animals or in arthropod vectors. Strain typing may help to better define the reservoir potential, carriership patterns, modes of transmission, and geographic distribution for each B. vinsonii berkhoffii type.

Introduction

Bacteria of the genus Bartonella (alpha subdivision of the class Proteobacteria [α-proteobacteria]) are fastidious, gram-negative, aerobic bacilli with more than 20 described species or subspecies [1], [2], [3], [4], [5]. Because of their zoonotic potential; transmission by a wide range of arthropod vectors, including sand flies, lice, fleas, and potentially ticks; and their ability to persistently infect mammalian reservoir hosts, bacteria of the genus Bartonella are considered emerging pathogens [6], [7], [8], [9], [10], [11]. There are three Bartonella vinsonii subspecies: Bartonella vinsonii vinsonii (type strain Baker, ATCC VR-152), Bartonella vinsonii berkhoffii (type strain 93-CO1, ATCC 51672) and Bartonella vinsonii arupensis (type strain OK 94-513, ATCC 700727) [12]. Bartonella vinsonii vinsonii was isolated from a vole in Canada by Baker in 1946 [13], B. vinsonii berkhoffii was isolated from a dog with endocarditis in our laboratory in 1993 [14], [15] and B. vinsonii arupensis was isolated from Peromyscus leucopus from Minnesota and Wisconsin in 1998 and from a cattle rancher from Wyoming in 1994 [12], [16].

Bartonella vinsonii berkhoffii, which was originally isolated from the blood of a dog with intermittent epistaxis and endocarditis, has emerged as an important canine pathogen. The organism has been associated with a wide variety of clinical manifestations including polyarthritis, cutaneous vasculitis, endocarditis, myocarditis, epistaxis, and granulomatous inflammatory disease [8], [14], [15], [17], [18], [19], [20], [21]. In 1998, Kordick et al. described persistent B. vinsonii berkhoffii infection in a sub-clinically infected dog during a 14-month testing period [20]. When this isolate (designated A [20] and Winnie in this manuscript) and two other B. vinsonii berkhoffii isolates obtained from subclinically infected dogs (designated B and C [20]) were subjected to restriction fragment length polymorphism (PCR-RFLP) using HaeIII, the restriction pattern differed from the 93-CO1 isolate obtained from the dog with endocarditis. Since, the isolation of the original B. vinsonii berkhoffii ATCC type strain, we have identified four distinct B. vinsonii berkhoffii types by amplification and sequencing of cultured organisms or PCR amplicons derived from blood or tissue samples obtained from healthy or sick dogs (this study, [15], [19], [20]), from coyotes (Canis latrans) (this study, [10]), from gray foxes (Urocyon cinereoargenteus, this study), from a dog with mitral and aortic valve endocarditis that had not traveled outside of Saskatchewan, Canada (this study) and from human blood samples (this study).

Based upon sequence differences within the 16S–23S ITS region and the bacteriophage associated heme-binding protein Pap31 gene, we propose a genotyping scheme that divides B. vinsonii subsp. berkhoffii isolates into four distinct types. Strain typing should help to better define the reservoir potential, carriership patterns, modes of transmission, and geographic distribution for each B. vinsonii berkhoffii type. In addition, we propose, that B. vinsonii berkhoffii strain 93-CO1 (ATCC 51672), isolated in 1993 at North Carolina State University College of Veterinary Medicine be retrospectively designated as B. vinsonii berkhoffii type I and that strain G7464 isolated from a healthy Greyhound blood donor at the University of Georgia College of Veterinary Medicine (dog B in Ref. [20]), based only on PCR-RFLP of the 16S–23S ITS region) and the isolate designated Winnie in this manuscript (isolate A in Ref. [20]) be retrospectively designated as a type II strains. Type III and type IV stains will be described for the first time as a component of this study.

Section snippets

Strains sources

The sources of B. vinsonii berkhoffii isolates or DNA are summarized in Table 1. A total of 27 strains, including six dog isolates, three gray fox isolates, nine coyote isolates, DNA extracted from six infected aortic valve samples obtained from dogs with endocarditis, and DNA extracted from three human blood samples were sequenced and aligned (Table 1). In addition, the sequence from a previously described human with B. vinsonii berkhoffii endocarditis, deposited in GenBank as accession number

Typing scheme, ITS amplification and sequence alignment

After alignment of 28 sequences from isolates and tissue samples, four clusters could be easily differentiated (Fig. 1, Fig. 2). The proposed genotyping scheme, strains and sources used in this study and the GenBank sequence submission access numbers are summarized in Table 1 for the ITS and Pap31 gene sequences. The 16S–23S ITS region sequences for B. vinsonii berkhoffii type I, type II, type III and type IV, some of the isolates reported in this study, were deposited in the GenBank database

Discussion and conclusions

Bartonella vinsonii berkhoffii sequences derived from the 16S to 23S sRNA intergenic spacer region and from the Pap31 gene amplified from coyote, dog and gray fox and human strains identified differences that allow for a clear division of this subspecies into four distinct strain types. The differences in the16S–23S ITS region are especially important, because there are two specific insertion regions that are variably expressed among the four types. The 37 bp insertion located at position 154 is

Acknowledgements

We thank Dr Susan Taylor from the Western College of Veterinary Medicine, University of Saskatchewan, for providing heart valve tissue from the dog with Bartonella vinsonii berkhoffii type IV infection. We also wish to thank Tonya Lee for editorial assistance and Rick Kasten for technical assistance.

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