Coyotes (Canis latrans) as the reservoir for a human pathogenic Bartonella sp.: molecular epidemiology of Bartonella vinsonii subsp. berkhoffii infection in coyotes from central coastal California

Abstract

Bartonella vinsonii subsp. berkhoffii was originally isolated from a dog suffering infectious endocarditis and was recently identified as a zoonotic agent causing human endocarditis. Following the coyote bite of a child who developed clinical signs compatible with Bartonella infection in Santa Clara County, Calif., this epidemiological study was conducted. Among 109 coyotes (Canis latrans) from central coastal California, 31 animals (28%) were found to be bacteremic with B. vinsonii subsp. berkhoffii and 83 animals (76%) had B. vinsonii subsp. berkhoffii antibodies. These findings suggest these animals could be the wildlife reservoir of B. vinsonii subsp. berkhoffii. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the gltA and 16S rRNA genes for these 31 isolates yielded similar profiles that were identical to those of B. vinsonii subsp. berkhoffii. Partial sequencing of the gltA and 16S rRNA genes, respectively, indicated 99.5 and 100% homology between the coyote isolate and B. vinsonii subsp. berkhoffii (ATCC 51672). PCR-RFLP analysis of the 16S-23S intergenic spacer region showed the existence of two different strain profiles, as has been reported in dogs. Six (19%) of 31 Bartonella bacteremic coyotes exhibited the strain profile that was identified in the type strain of a canine endocarditis case (B. vinsonii subsp. berkhoffii ATCC 51672). The other 25 bacteremic coyotes were infected with a strain that was similar to the strains isolated from healthy dogs. Based on whole bacterial genome analysis by pulsed-field gel electrophoresis (PFGE) with SmaI restriction endonuclease, there was more diversity in fingerprints for the coyote isolates, which had at least 10 major variants compared to the two variants described for domestic dog isolates from the eastern United States. By PFGE analysis, three Bartonella bacteremic coyotes were infected by a strain identical to the one isolated from three healthy dog carriers. Further studies are necessary to elucidate the mode of transmission of B. vinsonii subsp. berkhoffii, especially to identify potential vectors, and to determine how humans become infected.

FIG. 1

PCR (lanes 2 to 5) and PCR-RFLP (lanes 6 to 9, TaqI digestion; lanes 10 to 13, HhaI digestion) analyses of the gltA gene of coyote isolates with the set of primers suggested by Regnery et al. (40). Lanes 1 and 14, standard 100-bp molecular ladder; lanes 2, 6, and 10, coyote isolates; lanes 3, 7, and 11, B. vinsonii subsp. berkhoffii ATCC 51672; lanes 4, 8, and 12, B. vinsonii ATCC VR152; lanes 5, 9 and 13, B. henselae (strain U-4, University of California, Davis).

FIG. 2

PCR-RFLP analysis (lanes 2 to 11, TaqI digestion; lanes 12 to 21, MseI digestion) of the gltA gene of coyote isolates with the set of primers suggested by Norman et al. (37). Lanes 1 and 22, standard 100-bp molecular ladder; lanes 2 to 9 and 12 to 19, coyote isolates; lanes 10 and 20, B. vinsonii subsp. berkhoffii ATCC 51672; lanes 11 and 21, B. henselae (strain U-4; University of California, Davis).

FIG. 3

PCR-RFLP analysis of the 16S rRNA gene of coyote isolates with DdeI (A) or MnlI (B) restriction endonuclease. Lanes 1 to 12, coyote isolates; lane 13, B. vinsonii subsp. berkhoffii ATCC 51672; lane 14, 100-bp molecular ladder.

FIG. 4

PCR-RFLP analysis of the 16S-23S ITS region of coyote isolates with HaeIII restriction endonuclease. Lanes 1 and 15, standard 100-bp molecular ladder; lanes 2 to 10 and 13, coyote isolates (type II); lanes 11 and 12, coyote isolates (type I); lane 14, B. vinsonii subsp. berkhoffii ATCC 51672 (type I).

FIG. 5

(A) Fingerprints of B. vinsonii subsp. berkhoffii isolates by PFGE with SmaI digestion. Lane M, molecular size markers; lanes 2 to 11, B. vinsonii subsp. berkhoffii patterns I to X of the coyote isolates; lane 12, ATCC strain 51672 (pattern XI); lanes 13 to 15, isolates from three healthy dogs (pattern II). (B) Dendrogram of the fingerprints as determined by unweighted-pair group method using average linkage clustering. NC95-C02, NC95-C03, and NC95-C04 are B. vinsonii subsp. berkhoffii isolates from three healthy dogs. ID, identification.