The emerging tick-borne pathogen Anaplasma phagocytophilum infects humans, domestic animals, and wildlife throughout the Holarctic. In the western U.S., the ecology of A. phagocytophilum is particularly complex, with multiple pathogen strains, tick vectors, and reservoir hosts. A recent phylogenetic analysis of A. phagocytophilum strains isolated from various small mammal hosts in California documented distinct clustering of woodrat strains separate from sciurid (chipmunk and squirrel) strains. Here, we identified strains of A. phagocytophilum in various Ixodes tick species in California and related these genotypes to those found among reservoir and clinical hosts from the same areas. The sequences from all of the nidicolous (nest-dwelling) Ixodes ticks grouped within a clade that also contained all of the woodrat-origin A. phagocytophilum strains. Two of the I. pacificus sequences were also grouped within this woodrat clade, while the remaining five belonged to a less genetically diverse clade that included several sciurid-origin strains as well as a dog, a horse, and a human strain. By comparing A. phagocytophilum strains from multiple sources concurrently, we were able to gain a clearer picture of how A. phagocytophilum strains in the western U.S. are partitioned, which hosts and vectors are most likely to be infected with a particular strain, and which tick species and reservoir hosts pose the greatest health risk to humans and domestic animals.
Anaplasma phagocytophilum; Ixodes spp.; Nidicolous ticks; Ixodes pacificus; ank gene; Phylogeny
During 2012–2013 in California, USA, 3 wild golden eagles were found with severe skin disease; 2 died. The cause was a rare mite, most closely related to Knemidocoptes derooi mites. Cautionary monitoring of eagle populations, habitats, and diseases is warranted.
ectoparasite; electron microscopy; golden eagle; Knemidocoptes sp.; mange; raptor; skin disease; wild; parasites
The Companion Animal Parasite Council hosted a meeting to identify quantifiable factors that can influence the prevalence of tick-borne disease agents among dogs in North America. This report summarizes the approach used and the factors identified for further analysis with mathematical models of canine exposure to tick-borne pathogens.
Anaplasma; Ehrlichia; Borrelia burgdorferi; Tick-borne infections; Prevalence map factors; Ticks; Ixodidae; Prostriata; Metastriata
Microtus californicus scirpensis is an endangered, isolated subspecies of California vole. It requires water pools and riparian bulrush (Schoenoplectus americanus) and occupies some of the rarest habitat of any North American mammal. The minimally vegetated, extremely arid desert surrounding the pools is essentially uninhabitable for Ixodes species ticks. We describe an enzootic cycle of Borrelia carolinensis in Ixodes minor ticks at a site 3500 km distant from the region in which I. minor is known to occur in Tecopa Host Springs, Inyo County, eastern Mojave Desert, California. Voles were live-trapped, and ticks and blood samples queried by PCR and DNA sequencing for identification and determination of the presence of Borrelia spp. Between 2011–2013, we found 21 Ixodes minor ticks (prevalence 4–8%) on Amargosa voles and Reithrodontomys megalotis. DNA sequencing of 16S rRNA from ticks yielded 99% identity to I. minor. There was 92% identity with I. minor in the calreticulin gene fragment. Three ticks (23.1%), 15 (24%) voles, three (27%) house mice, and one (7%) harvest mice were PCR positive for Borrelia spp. Sequencing of the 5S-23S intergenic spacer region and flagellin gene assigned Amargosa vole Borrelia strains to B. carolinensis. Ixodes minor, first described in 1902 from a single Guatemalan record, reportedly occurs only in the southeast American on small mammals and birds. The source of this tick in the Mojave Desert and time scale for introduction is not known but likely via migratory birds. Borrelia strains in the Amargosa ecosystem most closely resemble B. carolinensis. B. carolinensis occurs in a rodent-I. minor enzootic cycle in the southeast U.S. although its epidemiological significance for people or rodents is unknown. The presence of a tick and Borrelia spp. only known from southeast U.S. in this extremely isolated habitat on the other side of the continent is of serious concern because it suggests that the animals in the ecosystem could be vulnerable to further incursions of pathogens and parasites.
16S rRNA; Amargosa vole; Borrelia burgdorferi sensu lato; calreticulin; Microtus californicus scirpensis; Migration
Anaplasma phagocytophilum is an obligately intracellular, tick-transmitted, bacterial pathogen of humans and other animals. In order to evade host immunity during the course of infection, A. phagocytophilum utilizes gene conversion to shuffle approximately 100 functional pseudogenes into a single expression cassette of the msp2(p44) gene, which encodes the major surface antigen, major surface protein 2 (Msp2). The role and extent of msp2(p44) recombination in a reservoir host for A. phagocytophilum have not been evaluated. In the current study, we explored patterns of recombination and expression site variability of the msp2(p44) gene in three chronically infected woodrats, a reservoir for the disease in the Western USA. All three woodrats developed persistent infection of at least 6 months duration; two of them maintained active infection for at least 8 months. In total, we detected the emergence of 60 unique msp2(p44) expression site variants with no common temporal patterns of expression site recombination among the three A. phagocytophilum populations. Both the strength of infection (i.e. pathogen load) and the genetic diversity of pseudogenes detected at the msp2(p44) expression site fluctuated periodically during the course of infection. An analysis of the genomic pseudogene exhaustion rate showed that the repertoire of pseudogenes available to the A. phagocytophilum population could in theory become depleted within a year. However, the apparent emergence of variant pseudogenes suggests that the pathogen could potentially evade host immunity indefinitely. Our findings suggest a tightly co-evolved relationship between A. phagocytophilum and woodrats in which the pathogen perpetually evades host immunity yet causes no detectable disease.
•Mange causes significant morbidity and mortality in western gray squirrels.•N. centrifera was identified on affected squirrels in southern California.•Burrowing of mites in the skin causes severe dermatitis.•Sequences for the ITS-2 region of N. centrifera were submitted to GenBank.
Notoedric mange, caused by the contagious, burrowing mite Notoedres centrifera, has been associated with several large-scale population declines of western gray squirrels (Sciurus griseus) and has been a significant obstacle to population recovery in Washington State where the species is listed as threatened. In 2009, residents and wildlife rehabilitators in the isolated San Bernardino Mountains of southern California reported a dramatic die-off of western gray squirrels, in what had been a previously dense and robust population. Individuals were observed suffering from abnormal neurologic behaviors (ataxia and obtundation) and severe skin disease. Full necropsy of five squirrels from the epidemic showed that all had moderate to severe infestation with mange mites and severe dermatitis characterized by hyperkeratosis, acanthosis, intralesional mites, intracorneal pustules and superficial bacteria. Mites from affected squirrels were evaluated by light and electron microscopy and identified as N. centrifera based on morphologic criteria. Additionally, the internal transcribed spacer-2 region of the mite was cloned, sequenced and accessioned in GenBank. The cause for the abnormal neurologic behavior was not confirmed on post-mortem examination. However, we hypothesize that mange can cause incoordination and obtundation as a result of malnutrition and dehydration, and intense pruritis may induce abnormal or erratic behavior that could be mistaken for neurologic signs. While we have characterized the severe impact this disease can have on individual animals, more work is needed to understand the impact on squirrel populations, particularly in view of the anecdotal reports of dramatic population declines that may take decades to recover.
Dermatitis; Internal transcribed spacer; ITS-2; Notoedres centrifera; Sciurid
Anaplasma phagocytophilum is an emerging tick-borne pathogen that infects humans, domestic animals and wildlife throughout the Holarctic. In the far-western United States, multiple rodent species have been implicated as natural reservoirs for A. phagocytophilum. However, the presence of multiple A. phagocytophilum strains has made it difficult to determine which reservoir hosts pose the greatest risk to humans and domestic animals. Here we characterized three genetic markers (23S–5S rRNA intergenic spacer, ank and groESL) from 73 real-time TaqMan PCR-positive A. phagocytophilum strains infecting multiple rodent and reptile species, as well as a dog and a horse, from California. Bayesian and maximum-likelihood phylogenetic analyses of all three genetic markers consistently identified two major clades, one of which consisted of A. phagocytophilum strains infecting woodrats and the other consisting of strains infecting sciurids (chipmunks and squirrels) as well as the dog and horse strains. In addition, analysis of the 23S–5S rRNA spacer region identified two unique and highly dissimilar clades of A. phagocytophilum strains infecting several lizard species. Our findings indicate that multiple unique strains of A. phagocytophilum with distinct host tropisms exist in California. Future epidemiological studies evaluating human and domestic animal risk should incorporate these distinctions.
Anaplasma phagocytophilum is an obligately intracellular tick-transmitted bacterial pathogen of humans and other animals. During the course of infection, A. phagocytophilum utilizes gene conversion to shuffle ∼100 functional pseudogenes into a single expression cassette of the msp2(p44) gene, which codes for the major surface antigen and major surface protein 2 (MSP2). The role and extent of msp2(p44) recombination, particularly in hosts that only experience acute infections, is not clear. In the present study, we explored patterns of recombination and expression of the msp2(p44) gene of A. phagocytophilum in a serially infected mouse model. Even though the bacterium was passed rapidly among mice, minimizing the opportunities for the host to develop adaptive immunity, we detected the emergence of 34 unique msp2(p44) expression cassette variants. The expression of msp2(p44) pseudogenes did not follow a consistent pattern among different groups of mice, although some pseudogenes were expressed more frequently than others. In addition, among 263 expressed pseudogenes, 3 mosaic sequences each consisting of 2 different pseudogenes were identified. Population genetic analysis showed that genetic diversity and subpopulation differentiation tended to increase over time until stationarity was reached but that the variance that was observed in allele (expressed pseudogene) frequency could occur by drift alone only if a high variance in bacterial reproduction could be assumed. These findings suggest that evolutionary forces influencing antigen variation in A. phagocytophilum may comprise random genetic drift as well as some innate but apparently nonpurifying selection prior to the strong frequency-dependent selection that occurs cyclically after hosts develop strong adaptive immunity.
Anaplasma phagocytophilum; msp2(p44); antigen variation; recombination; drift; selection
The redwood chipmunk contributes to the maintenance of tick-borne diseases in northern California. The range of redwood chipmunks overlaps that of western black-legged ticks and tick-borne disease, including granulocytic anaplasmosis and Lyme borreliosis. Chipmunks have high Anaplasma phagocytophilum PCR- and seroprevalence, are infested with a diversity of Ixodes spp. ticks, and are reservoir competent for Borrelia burgdorferi. We hypothesized that chipmunks could maintain tick-borne disease on the forest floor while also potentially bridging infection to arboreal sciurids as well. We used radio-telemetry to evaluate chipmunk movement and use of trees, characterized burrows, described prevalence of tick-borne disease, and identified ticks on these chipmunks. A total of 192 chipmunks from Hendy Woods, Mendocino County, California, USA, was evaluated between November 2005 and April 2009. The mean density was 2.26–5.8 chipmunks/ha. The longest detected life span was 3 years. Female weights ranged from 80–120 g and males from 80–180 g. The A. phagocytophilum and Borrelia spp. seroprevalence was 21.4% and 24.7%, respectively, and PCR prevalence for these pathogens was 10.6% and 0%, respectively. Ixodes spp. ticks included I. angustus, I. ochotonae, I. pacificus, and I. spinipalpis. The mean infestation level was 0.92 ticks/chipmunk. Based on telemetry of 11 chipmunks, the greatest distance traveled ranged from 0.14–0.63 km for females and 0.1–1.26 km for males. Areas occupied by chipmunks ranged from 0.005–0.24 km2 for females and 0.006–0.73 km2 for males. On 3 occasions, chipmunks were found in trees. Burrows were identified under a moss-covered redwood log, deep under a live redwood tree, under a Douglas fir log, in a clump of huckleberry, in a root collection from an overturned Douglas fir tree, and in a cluster of exposed huckleberry roots. The biology of the redwood chipmunk has multiple features that allow it to be an important reservoir host for tick-borne disease in northwestern California.
Anaplasma phagocytophilum; Borrelia burgdorferi sensu lato; Relapsing fever; Borrelia spp; Reservoir; Rodent
Ixodes spp. tick-borne zoonotic diseases are present across the Holarctic in humans, domestic animals, and wildlife. Small mammals are reservoirs for the rickettsial pathogen Anaplasma phagocytophilum and tick vectors may include catholic-feeding bridge vectors as well as host-specialist or nidicolous ticks. Far western North American communities in which A. phagocytophilum is maintained are complex ecologically, with multiple reservoir host and tick species, multiple strains of the bacterial pathogen A. phagocytophilum and differences in dynamics of hosts and vectors across heterogeneous landscapes. We evaluated sites in northern California in order to identify primarily nidicolous ticks and the hosts they infest. A total of 667 ticks was found in 11 study sites, including 288 on flags and 379 attached to small mammals. Larvae were over-represented among attached ticks and adults on flags. The most abundant species was I. pacificus. Two-hundred fourteen nidicolous ticks were found, most abundantly I. angustus and I. spinipalpis. All adult I. ochotonae, I. auritulus, I. angustus, I. jellisoni, and I. woodi were female, while the male:female ratio of I. spinipalpis was 1.2:1 and 1:1 for I. pacificus. The greatest number of ticks was obtained from Tamias ochrogenys, Peromyscus spp., and Neotoma fuscipes. Of 234 small mammal individuals that were infested with Ixodes spp., only 81 (34.6%) were infested with I. pacificus. The remaining infested small mammals hosted nidicolous tick species. Eight ticks were PCR-positive, including 6 I. pacificus (one adult, one larva, and 6 nymphs), and 2 adult I. ochotonae and high PCR prevalences of 18% and 9% were detected in woodrats and chipmunks, respectively. Nymphal I. angustus ticks were active year-long with a possible increase in August while larval activity was only observed in December and spring months and adults only during spring and fall. Overall, we show high tick species richness and year-round high levels of infestation on rodents by several different nidicolous ticks in areas where A. phagocytophilum is enzootic, including on reported reservoir species.
Anaplasma phagocytophilum; Granulocytic anaplasmosis; Ixodes angustus; Ixodes ochotonae; Ixodes pacificus
Anthropogenic environmental change is often implicated in the emergence of new zoonoses from wildlife; however, there is little mechanistic understanding of these causal links. Here, we examine the transmission dynamics of an emerging zoonotic paramyxovirus, Hendra virus (HeV), in its endemic host, Australian Pteropus bats (fruit bats or flying foxes). HeV is a biosecurity level 4 (BSL-4) pathogen, with a high case-fatality rate in humans and horses. With models parametrized from field and laboratory data, we explore a set of probable contributory mechanisms that explain the spatial and temporal pattern of HeV emergence; including urban habituation and decreased migration—two widely observed changes in flying fox ecology that result from anthropogenic transformation of bat habitat in Australia. Urban habituation increases the number of flying foxes in contact with human and domestic animal populations, and our models suggest that, in addition, decreased bat migratory behaviour could lead to a decline in population immunity, giving rise to more intense outbreaks after local viral reintroduction. Ten of the 14 known HeV outbreaks occurred near urbanized or sedentary flying fox populations, supporting these predictions. We also demonstrate that by incorporating waning maternal immunity into our models, the peak modelled prevalence coincides with the peak annual spill-over hazard for HeV. These results provide the first detailed mechanistic framework for understanding the sporadic temporal pattern of HeV emergence, and of the urban/peri-urban distribution of HeV outbreaks in horses and people.
Hendra virus; Pteropus; flying fox; bat virus; connectivity; metapopulation disease model
Granulocytic anaplasmosis (GA) is an emerging tick-transmitted disease that persists in rodent- Ixodes ricinus-complex tick cycles across the Holarctic. Although the putative reservoir for anaplasmosis in the western United States is the dusky-footed woodrat (Neotoma fuscipes), this rodent was not shown reservoir-competent because of failure of infection from woodrats to other animals via ticks. Redwood chipmunks are common in habitats where Anaplasma phagocytophilum is common, have high PCR- and seroprevalence, and are infested with a diversity of Ixodes spp. ticks. Experimental infection of seven wild-caught A. phagocytophilum-negative redwood chipmunks induced persistent periods of recurrent rickettsemia during the persistent phase of infection. Of three animals for which xenodiagnosis was attempted, all successfully infected pools of I. pacificus larvae during the primary rickettsemia. We show that chipmunks are reservoir-competent for GA and may be important for maintaining infection in nature.
disease ecology; Ixodes; sciurid; tick-borne disease
Anaplasma phagocytophilum is a tick-transmitted bacterial pathogen of humans and other animals, and is an obligate intracellular parasite. Throughout the course of infection, hosts acquire temporary resistance to granulocytic anaplasmosis as they develop immunity specific for the major antigen, major surface protein 2 (Msp2). However, the bacterium then utilizes a novel recombination mechanism shuffling functional pseudogenes sequentially into an expression cassette with conserved 5′ and 3′ ends, bypassing host immunity. Approximately 100 pseudogenes are present in the only fully sequenced human-origin HZ genome, representing the possibility for almost unlimited antigenic diversity. In the present study, we identified a select group of 20% of the A. phagocytophilum HZ msp2 pseudogenes that have matched preferentially to human, canine, and equine expression cassettes. Pseudogenes cluster predominantly in one spatial run limited to a single genomic island in less than 50% of the genome but phylogenetically related pseudogenes are neither necessarily located in close proximity on the genome nor share similar percent identity with expression cassettes. Pseudogenes near the expression cassette (and the origin) are more likely to be expressed than those farther away. Taken together, these findings suggest that there may be natural selection pressure to retain pseudogenes in one cluster near the putative origin of replication, even though global recombination shuffles pseudogenes around the genome, separating pseudogenes that share genetic origins as well as those with similar identities.
We analyzed the structure of the expression site encoding the immunoprotective protein MSP2/P44 from multiple Anaplasma phagocytophilum strains in the United States. The sequence of p44ESup1 had diverged in Ap-variant 1 strains infecting ruminants. In contrast, no differences were detected between A. phagocytophilum strains infecting humans and domestic dogs.
Anaplasma; strain diversity; msp2/p44; OMPs; rickettsia; zoonoses; USA; dispatch
Zoonoses; Anaplasma phagocytophilum; livestock; horses; woodrats; humans; letter
Hendra virus (HeV) is a lethal paramyxovirus which emerged in humans in 1994. Poor understanding of HeV dynamics in Pteropus spp. (flying fox or fruit bat) reservoir hosts has limited our ability to determine factors driving its emergence. We initiated a longitudinal field study of HeV in little red flying foxes (LRFF; Pteropus scapulatus) and examined individual and population risk factors for infection, to determine probable modes of intraspecific transmission. We also investigated whether seasonal changes in host behaviour, physiology and demography affect host–pathogen dynamics. Data showed that pregnant and lactating females had significantly higher risk of infection, which may explain previously observed temporal associations between HeV outbreaks and flying fox birthing periods. Age-specific seroprevalence curves generated from field data imply that HeV is transmitted horizontally via faeces, urine or saliva. Rapidly declining seroprevalence between two field seasons suggests that immunity wanes faster in LRFF than in other flying fox species, and highlights the potentially critical role of this species in interspecific viral persistence. The highest seroprevalence was observed when animals showed evidence of nutritional stress, suggesting that environmental processes that alter flying fox food sources, such as habitat loss and climate change, may increase HeV infection and transmission. These insights into the ecology of HeV in flying fox populations suggest causal links between anthropogenic environmental change and HeV emergence.
Hendra virus; Henipavirus; Pteropus scapulatus; zoonotic emerging infectious diseases; flying fox
Bartonella rochalimae was first isolated from the blood of a human who traveled to Peru and was exposed to multiple insect bites. Foxes and dogs are likely natural reservoirs for this bacterium. We report the results of experimental inoculation of two dogs, five cats and six guinea pigs with the only human isolate of this new Bartonella species. Both dogs became bacteremic for 5–7 weeks, with a peak of 103–104 colony forming units (CFU)/mL blood. Three cats had low bacteremia levels (< 200 CFU/mL) of 6–8 weeks’ duration. One cat that remained seronegative had two bacterial colonies isolated at a single culture time point. A fifth cat never became bacteremic, but seroconverted. None of the guinea pigs became bacteremic, but five seroconverted. These results suggest that dogs could be a reservoir of this strain of B. rochalimae, in contrast to cats and guinea pigs.
Bartonella rochalimae; dogs; cats; guinea pigs; zoonosis
A total of 1,618 ticks [420 individual (adults) and pooled (larvae and nymphs) samples], 369 rodents (Apodemus agrarius, Rattus norvegicus, Tscherskia triton, Mus musculus, and Myodes regulus), and 34 shrews (Crocidura lasiura) that were collected in northern Gyeonggi-do near the Demilitarized Zone (DMZ) of Korea during 2004-2005, were assayed by PCR for selected zoonotic pathogens. From a total of 420 individual and pooled tick DNA samples, Anaplasma (A.) phagocytophilum (16), A. platys (16), Ehrlichia (E.) chaffeensis (63), Borrelia burgdorferi (16), and Rickettsia spp. (198) were detected using species-specific PCR assays. Out of 403 spleens from rodents and shrews, A. phagocytophilum (20), A. platys (34), E. chaffeensis (127), and Bartonella spp. (24) were detected with species-specific PCR assays. These results suggest that fevers of unknown causes in humans and animals in Korea should be evaluated for infections by these vector-borne microbial pathogens.
Bartonella; Borrelia; Rickettsia; rodents; Crocidura lasiura; tick-borne pathogens
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.
anaplasmosis; disease ecology; reservoir; dispatch
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.
There are only two reports in the literature demonstrating the presence of Campylobacter spp. in marine mammals. One report describes the isolation of a new species, Campylobacter insulaenigrae sp. nov., from three harbor seals (Phoca vitulina) and a harbor porpoise (Phocoena phocoena) in Scotland, and the other describes the isolation of Campylobacter jejuni, Campylobacter lari, and an unknown Campylobacter species from northern elephant seals (Mirounga angustirostris) in California. In this study, 72 presumptive C. lari and unknown Campylobacter species strains were characterized using standard phenotypic methods, 16S rRNA PCR, and multilocus sequence typing (MLST). Phenotypic characterization of these isolates showed them to be variable in their ability to grow either at 42°C or on agar containing 1% glycine and in their sensitivity to nalidixic acid and cephalothin. Based on both 16S rRNA PCR and MLST, all but 1 of the 72 isolates were C. insulaenigrae, with one isolate being similar to but distinct from both Campylobacter upsaliensis and Campylobacter helveticus. Phylogenetic analysis identified two C. insulaenigrae clades: the primary clade, containing exclusively California strains, and a secondary clade, containing some California strains and all of the original Scottish strains. This study demonstrates the inability of phenotypic characterization to correctly identify all Campylobacter species and emphasizes the importance of molecular characterization via 16S rRNA sequence analysis or MLST for the identification of Campylobacter isolates from marine mammals.
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.
In order to investigate the prevalence of tick-borne infectious agents among ticks, ticks comprising five species from two genera (Hemaphysalis spp. and Ixodes spp.) were screened using molecular techniques. Ticks (3,135) were collected from small wild-caught mammals or by dragging/flagging in the Republic of Korea (ROK) and were pooled into a total of 1,638 samples (1 to 27 ticks per pool). From the 1,638 tick samples, species-specific fragments of Anaplasma phagocytophilum (1 sample), Anaplasma platys (52 samples), Ehrlichia chaffeensis (29 samples), Ehrlichia ewingii (2 samples), Ehrlichia canis (18 samples), and Rickettsia rickettsii (28 samples) were amplified by PCR assay. Twenty-one pooled and individual tick samples had mixed infections of two (15 samples) or three (6 samples) pathogens. In addition, 424 spleen samples from small captured mammals (389 rodents, 33 insectivores, and 2 weasels) were screened for selected zoonotic pathogens. Species-specific DNA fragments of A. phagocytophilum (110 samples), A. platys (68 samples), E. chaffeensis (8 samples), E. ewingii (26 samples), E. canis (51 samples), and Rickettsia sp. (22 samples) were amplified by PCR assay. One hundred thirty small mammals had single infections, while 4, 14, and 21 striped field mice (Apodemus agrarius) had mixed infections of four, three, and two pathogens, respectively. Phylogenetic analysis based on nucleotide sequence comparison also revealed that Korean strains of E. chaffeensis clustered closely with those from China and the United States, while the Rickettsia (rOmpA) sequences clustered within a clade together with a Chinese strain. These results suggest that these agents should be considered in differential diagnosis while examining cases of acute febrile illnesses in humans as well as animals in the ROK.
Eschars can be used for genetic characterization of O. tsutsugamushi during the convalescent phase.
To verify the value of eschars for the diagnosis of scrub typhus and to characterize genotypes of Orientia tsutsugamushi in patients, we examined eschars and blood specimens of 7 patients from Shandong Province, People's Republic of China, for O. tsutsugamushi by polymerase chain reaction targeting the Sta56 gene. All 7 eschars and acute-phase blood samples were positive, while no specific DNA amplicons were obtained from the 7 convalescent-phase blood samples collected after antimicrobial drug therapy. The findings indicate that patients' eschars can be used for detection and genetic characterization of O. tsutsugamushi during the convalescent phase.
Scrub typhus; eschar; Orientia tsutsugamushi
We report the first documented case of endocarditis associated with Bartonella clarridgeiae in any species. B. clarridgeiae was identified as a possible etiological agent of human cat scratch disease. Infective vegetative valvular aortic endocarditis was diagnosed in a 2.5-year-old male neutered boxer. Historically, the dog had been diagnosed with a systolic murmur at 16 months of age and underwent balloon valvuloplasty for severe valvular aortic stenosis. Six months later, the dog was brought to a veterinary hospital with an acute third-degree atrioventricular block and was diagnosed with infective endocarditis. The dog died of cardiopulmonary arrest prior to pacemaker implantation. Necropsy confirmed severe aortic vegetative endocarditis. Blood culture grew a fastidious, gram-negative organism 8 days after being plated. Phenotypic and genotypic characterization of the isolate, including partial sequencing of the citrate synthase (gltA) and 16S rRNA genes indicated that this organism was B. clarridgeiae. DNA extraction from the deformed aortic valve and the healthy pulmonic valve revealed the presence of B. clarridgeiae DNA only from the diseased valve. No Borrelia burgdorferi or Ehrlichia sp. DNA could be identified. Using indirect immunofluorescence tests, the dog was seropositive for B. clarridgeiae and had antibodies against Ehrlichia phagocytophila but not against Ehrlichia canis, Ehrlichia ewingii, B. burgdorferi, or Coxiella burnetii.