Richard N. Brown

Structure of the Expression Site Reveals Global Diversity in MSP2 (P44) Variants in Anaplasma phagocytophilum

ABSTRACT Anaplasma phagocytophilum, a recently reclassified bacteria in the order Rickettsiales, infects many different animal species and causes an emerging tick-borne disease of humans. The genome contains a large number of related genes and gene fragments encoding partial or apparently full-length outer membrane protein MSP2 (P44). Previous data using strains isolated from humans in the United States suggest that antigenic diversity results from RecF-mediated conversion of a single MSP2 (P44) expression site by partially homologous donor sequences. However, whether similar mechanisms operate in naturally infected animal species and the extent of global diversity in MSP2 (P44) are unknown. We analyzed the structure and diversity of the MSP2 (P44) expression site in strains derived from the United States and Europe and from infections of different animal species, including wildlife reservoirs. The results show that a syntenic expression site is present in all strains of A. phagocytophilum investigated. This genomic locus contained diverse MSP2 (P44) variants in all infected animals sampled, and variants also differed at different time points during infection. Although similar variants were found among different populations of U.S. origin, there was little sequence identity between U.S. strain variants (including genomic copies from a completely sequenced U.S. strain) and expression site variants infecting sheep and dogs in Norway and Sweden. Finally, the possibility that combinatorial mechanisms can generate additional diversity beyond the basic donor sequence repertoire is supported by the observation of shared sequence blocks throughout the MSP2 (P44) hypervariable region in reservoir hosts. These data suggest similar genetic mechanisms for A. phagocytophilum variation in all hosts but worldwide diversity of the MSP2 (P44) outer membrane protein.

Gray Foxes (Urocyon cinereoargenteus) as a Potential Reservoir of a Bartonella clarridgeiae-Like Bacterium and Domestic Dogs as Part of a Sentinel System for Surveillance of Zoonotic Arthropod-Borne Pathogens in Northern California

ABSTRACT Two species of Bartonella, a novel Bartonella clarridgeiae-like bacterium and B. vinsonii subsp. berkhoffii, were isolated from rural dogs and gray foxes in northern California. A novel B. clarridgeiae-like species was isolated from 3 (1.7%) of 182 dogs and 22 (42%) of 53 gray foxes, while B. vinsonii subsp. berkhoffii was isolated from 1 dog (0.5%) and 5 gray foxes (9.4%). PCR and DNA sequence analyses of the citrate synthase (gltA) gene and the 16S-23S intergenic spacer region suggested that strains infecting dogs and gray foxes were identical. Fifty-four dogs (29%) and 48 gray foxes (89%) had reciprocal titers of antibodies against Bartonella spp. of ≥64. The high prevalence of bacteremia and seroreactivity to Bartonella spp. in gray foxes suggests that they may act as a reservoir species for the B. clarridgeiae-like species in this region. Domestic dogs were also tested for other arthropod-borne infectious agents. Fifty-one dogs (28%) were positive for Dirofilaria immitis antigen, seventy-four (40%) were seroreactive to Anaplasma phagocytophilum, and five (2.7%) were seropositive for Yersinia pestis. Fourteen dogs (7.6%) were PCR positive for A. phagocytophilum. Polytomous logistic regression models were used to assess the association of Bartonella antibody titer categories with potential risk factors and the presence of other vector-borne agents in domestic dogs. Older dogs were more likely to be seroreactive to Bartonella spp. There was no association between the exposure of dogs to Bartonella and the exposure of dogs to A. phagocytophilum in this study.

Infective Endocarditis in a Dog and the Phylogenetic Relationship of the Associated “Bartonella rochalimae” Strain with Isolates from Dogs, Gray Foxes, and a Human

The first case of canine endocarditis caused by “Bartonella rochalimae” is reported. By PCR-restriction fragment length polymorphism, sequence, and phylogenetic analyses, Bartonella isolates from a dog with endocarditis, 22 gray foxes, and three dogs, described as B. clarridgeiae like, were confirmed to belong to the new species “B. rochalimae,” suggesting canids as the natural reservoir.

PREVALENCES OF ZOONOTIC BACTERIA AMONG SEABIRDS IN REHABILITATION CENTERS ALONG THE PACIFIC COAST OF CALIFORNIA AND WASHINGTON, USA

Many seabirds are rehabilitated annually by wildlife rehabilitation centers along the Pacific Coast, USA. Although various strains of zoonotic bacteria have been isolated from seabirds, risks to rehabilitators at these centers have not been well documented. From November 2001 through January 2003, we determined the prevalence of detectable enteric fauna by isolation and characterization of Gram-negative bacteria from cloacal swabs taken from 26 common murres (Uria aalge), 49 gulls (Larus spp.), and 14 other seabirds treated by rehabilitators in California and Washington (USA). At least 25 bacterial species were identified, including multiple strains of Escherichia coli, as well as Enterobacter cloacae, Citrobacter freundii, and Klebsiella pneumoniae. Antibiotic resistance was found in 13 of 19 bacterial isolates tested, including E. coli, K. pneumoniae, Acinetobacter baumanii, and Pseudomonas aeruginosa. Potential transfer of these bacteria poses a risk to wildlife rehabilitators and to seabirds in these centers, as well as to free-ranging birds.

Distinct ecologically relevant strains of Anaplasma phagocytophilum.

To the Editor: Anaplasma phagocytophilum was defined to include Ehrlichia phagocytophila, E. equi, and the agent of human granulocytic ehrlichiosis. Nevertheless, we and others have found phenotypic and genetic differences from diverse regions and hosts and conclude preliminarily that ecologically separate strains might exist that should be distinguished. Two precedents include ruminant strains of A. phagocytophilum in Europe and the Ap-Variant 1 from ruminants and ticks of North America and Europe. In Europe, A. phagocytophilum infects livestock, rodents, and humans, with some species such as European cattle showing severe disease and high antibody prevalence. In contrast, cattle infection is rare in the United States, despite being common in other species. Experimental infection of cattle with California equine–origin strain MRK failed to induce disease or marked rickettsemia (1). Thus, even though European strains have ruminant tropism, an equine strain does not. Ap-Variant 1 is found in ticks and deer in North America. This strain is distinctive in the 16S rRNA, major surface protein 4 (msp4), msp2, and ankA genes (2). Deer, goats, and tick-derived cell lines can be infected with Ap-Variant 1, but rodents cannot (3). Our recent data examining A. phagocytophilum in western North America show at least 2 phenotypes: strains originating from sciurids (chipmunks and tree squirrels) and strains from woodrats (the previously postulated reservoir). In a survey of 2,121 small mammals in areas of California with enzootic Ap-Variant 1, seroprevalence was highest in tree squirrels (71%), woodrats (50%), and chipmunks (up to 28%), and PCR prevalence was highest in tree squirrels (16%) and chipmunks (34%) (4). We showed that chipmunks were competent reservoirs for A. phagocytophilum through exposure in the field, successful inoculation with strain MRK, and transmission through Ixodes pacificus to mice. However, discrepancy in the phenotype of strains originating from woodrats and chipmunks is substantial when these strains are inoculated into horses. One chipmunk strain can infect both rodents and horses (important laboratory animal models for human infection), whereas woodrat strains show restricted rodent-only tropism. A naturally infected redwood chipmunk was trapped in Mendocino County, California, exsanguinated, and documented to be positive for A. phagocytophilum by using real-time PCR, with a cycle threshold (Ct) of 31.31. An adult horse was negative for infection and exposure by PCR and immunofluorescence assay, premedicated with flunixin meglumine and diphenhydramine, and inoculated with 1.5 mL of infected chipmunk whole blood in EDTA. The mare was monitored daily for 16 days, including blood smear, serologic testing, and PCR, and assessment of behavior and attitude, rectal temperature, and legs for edema, swelling, or pain. She became ill 12 days postinoculation, with a body temperature of 40.0oC, lethargy, depression, and inappetance. Blood smears showed A. phagocytophilum morulae in neutrophils, and she was PCR positive on day 13 (Ct 37). Thus, infection from this chipmunk strain was indistinguishable from that induced when human-origin A. phagocytophilum was inoculated into this horse. The mare recovered after treatment. In contrast, woodrat strains show rodent-host tropism but are not infectious to horses. We attempted to infect 3 horses with A. phagocytophilum from naturally infected, PCR-positive woodrats from Hoopa Valley, Humboldt County (1 pool of 4, 1 single) and Henry Cowell State Park, Santa Cruz County (N = 1); both sites are enzootic for A. phagocytophilum. Woodrats were bled into tubes containing EDTA, and blood was kept cool and screened that day by real-time PCR and serologic testing. The PCR-positive samples were divided into rodent and horse inocula. The horses were negative for infection and exposure using PCR and immunofluorescence assay, premedicated as described above, and then inoculated with 6 mL of A. phagocytophilum–infected woodrat blood. These horses never became infected on the basis of clinical signs, serology, blood smears, and PCR. Each horse was reinoculated 1–2 months later with 1.5 mL of an equine-tropic strain (MRK or chipmunk) to verify susceptibility to infection. All 3 became ill within 12–13 days postinoculation, with substantial increase in body temperature (>39.4o C), lethargy, depression, and inappetance. Blood smears showed A. phagocytophilum morulae in neutrophils, and the animals were PCR positive and seroconverted. We considered the woodrat inocula unlikely to be noninfectious because aliquots of the same samples produced rickettsemia according to PCR in C3H/HeJ mice and uninfected woodrats. All 3 horses recovered after treatment. Although some woodrat A. phagocytophilum strains are genetically similar to human and equine strains, others differ from sciurid, human, horse, and dog strains, with conserved blocks within the msp2 gene (5). A phylogenetic tree based on the omp1n gene clusters a sciurid strain with local horses, distinct from northern California woodrats (Appendix Figure). Some woodrat strains have rodent-only tropism; Ap-Variant 1 does not infect rodents. Strains from sciurids and white-footed mice infect multiple laboratory animals and perhaps humans as well. Thus, epidemiologic studies evaluating human risk need to incorporate these distinctions and further ecologic and molecular genetic studies are necessary. With increasing reports of dissimilar genotypes of A. phagocytophilum from multiple regions of the world, defining distinct phenotypes and using nomenclature that appropriately clarifies the distinctions are important.

Anaplasma phagocytophilum Infection in Small Mammal Hosts of Ixodes Ticks, Western United States

A total of 2,121 small mammals in California were assessed for Anaplasma phagocytophilum from 2006 through 2008. Odds ratios were >1 for 4 sciurids species and dusky-footed woodrats. High seroprevalence was observed in northern sites. Ten tick species were identified. Heavily infested rodent species included meadow voles, woodrats, deer mice, and redwood chipmunks.

Zoonotic Bartonella Species in Fleas Collected on Gray Foxes (Urocyon cinereoargenteus)

Bartonella spp. are fastidious, gram-negative, rod-shaped bacteria and are usually vector-borne. However, the vector has not been definitively identified for many recently described species. In northern California, gray foxes (Urocyon cinereoargenteus) are infected with two zoonotic Bartonella species, B. rochalimae and B. vinsonii subsp. berkhoffii. Fleas (range 1-8 fleas per fox) were collected from 22 (41.5%) of 54 gray foxes from urban and backcountry zones near Hoopa, California. The flea species were determined, and DNA was individually extracted to establish the Bartonella species harbored by these fleas. Of the 108 fleas collected, 99 (92%) were identified as Pulex simulans. Overall, 39% (42/108) of the fleas were polymerase chain reaction (PCR)-positive for Bartonella, with B. rochalimae and B. vinsonii subsp. berkhoffii identified in 34 (81%) and 8 (19%) of the PCR-positive fleas, respectively. There was no difference between the prevalence of Bartonella spp. in P. simulans for the urban and backcountry zones. Fourteen (64%) of the 22 foxes were Bartonella bacteremic at one or more of the capture dates. In 10 instances, both the foxes and the fleas collected from them at the same blood collection were Bartonella-positive. B. rochalimae was the predominant species identified in both foxes and fleas. The competency of Pulex fleas as a vector of B. rochalimae has not been confirmed and will need to be demonstrated experimentally. Pulex spp. fleas readily feed on humans and may represent a source of human exposure to zoonotic species of Bartonella.

Spatial analysis of the exposure of dogs in rural north-coastal California to vectorborne pathogens

Between 0 and 50 per cent of the dogs in eight rural villages in far northern California with a high risk of tickborne diseases were seropositive for Anaplasma phagocytophilum and Bartonella vinsonii subspecies berkhoffii, and between 0 and 10 per cent were seropositive for Borrelia burgdorferi. The odds ratio for the co-exposure of individual dogs to B vinsonii berkhoffii and A phagocytophilum was 18·2. None of the diseases was associated with the sex of the dogs, whether they slept out of doors, or whether tick-preventive measures were taken. When the villages were assessed for landscape risk factors, a particularly high seroprevalence for B vinsonii berkhoffii and A phagocytophilum was observed in a village at a relatively high altitude and greater distance from the Pacific coast, and montane hardwood conifer woodland was most associated with a high seroprevalence for these two pathogens.

Prevalence and burden of two rickettsial phylotypes (G021 and G022) in Ixodes pacificus from California by real-time quantitative PCR

The western black-legged tick, Ixodes pacificus Cooley and Kohls, commonly bites humans in the far western U.S. In addition to transmitting Lyme borreliosis and anaplasmosis, it is a host of nonpathogenic bacteria as well as some of unknown pathogenicity. In this study, we report the detection, prevalence, and burden of 2 rickettsial phylotypes with unknown pathogenicity in I. pacificus ticks from 6 California counties using real-time quantitative PCR with phylotype-specific primers and probes. Prevalence of rickettsial phylotypes G021 and G022 from 247 I. pacificus ticks was 100% and 2.0%, respectively. The median burden of phylotype G021 was 7.3 per tick cell, whereas the burden of phylotype G022 was 0.8 per tick cell. The burden of phylotype G021 significantly differed between collection sites and between vegetation habitats. Ticks collected from the coastal sage scrub habitat of southern California had a lower burden of phylotype G021 when compared to central California oak woodland, northern California oak woodland, and mixed evergreen and ponderosa pine-oak habitats of northern California. No significant correlation was found between the burden of the phylotype G021 in the presence and absence of the phylotype G022 in I. pacificus, suggesting that the presence of these Rickettsia species do not interfere with each other in I. pacificus.

NATURAL AND EXPERIMENTAL BORRELIA BURGDORFERI INFECTIONS IN WOODRATS AND DEER MICE FROM CALIFORNIA

Sequestration of spirochetes and concurrent histopathologic lesions were evaluated in tissues of Borrelia burgdorferi-infected dusky-footed woodrats (Neotoma fuscipes) and deer mice (Peromyscus maniculatus). Rodents were either wild-caught and naturally infected or were experimentally infected by tick bite, by intramuscular (i.m.) injection of cultured spirochetes, or by i.m. injection of tick suspensions. Samples of host tissues, including skin, blood, ear, brain, eye, heart, lung, liver, spleen, kidney, and urinary bladder, were removed from up to 21 woodrats and four deer mice and cultured in BSK II medium. Borreliae-positive cultures of ear punch biopsies were obtained from 10 of 11 woodrats and from all of four deer mice. Additionally, positive cultures were obtained from three of 36 skin biopsies of woodrats, and from one of 36 cultures of woodrat blood. In contrast, spirochetes were not observed in 505 cultures of internal organs or whole blood. Samples of tissues from seven naturally infected woodrats, four experimentally infected woodrats, and nine experimentally infected deer mice also were examined for histopathologic lesions. Nonsuppurative cellular infiltrates were recognized in samples from most tissue types from woodrats, but few lesions were observed in tissues from deer mice. Recognized lesions in woodrats that were consistent with infections of Lyme borreliosis in other species included synovitis, myositis, and myocarditis. Such lesions were more common in woodrats than in deer mice. Inflammatory lesions, especially synovitis, were more common in woodrats with long-term infections than in woodrats with relatively short-term infections. No clinical signs of disease were observed in either species of rodent.