Natural and experimental infection of white-tailed deer (Odocoileus virginianus) from the United States with an Ehrlichia sp. closely related to Ehrlichia ruminantium

An Ehrlichia sp. (Panola Mountain [PM] Ehrlichia sp.) closely related to Ehrlichia ruminantium was recently detected in a domestic goat experimentally infested with lone star ticks (LSTs, Amblyomma americanum) collected from Georgia, USA. The infected goat exhibited pyrexia and mild clinical pathologic abnormalities consistent with ehrlichiosis. At least two other Ehrlichia species (Ehrlichia chaffeensis and Ehrlichia ewingii) are maintained in nature by a cycle involving LSTs as the primary vector and white-tailed deer (Odocoileus virginanus) as a known or suspected reservoir. To investigate the possibility that white-tailed deer are potential hosts of the PM Ehrlichia sp., whole blood samples collected from 87 wild deer from 2000 to 2002 were screened with a species-specific nested PCR assay targeting the citrate synthase gene. In addition, two laboratory-raised white-tailed deer fawns were each infested with 120 wild-caught LST adults from Missouri, USA, and blood samples were periodically collected and tested for the PM Ehrlichia sp. Of 87 deer tested from 20 locations in the southeastern United States, three (3%) deer from Arkansas, North Carolina, and Virginia were positive for the PM Ehrlichia sp. Wild-caught ticks transmitted the PM Ehrlichia sp. to one of two deer fawns, and colony-reared nymphal LSTs acquired the organism from the deer, maintained it transstadially as they molted to adults, and transmitted the PM Ehrlichia sp. to two na?ve fawns. These findings indicate that white-tailed deer are naturally and experimentally susceptible to infection with an Ehrlichia sp. closely related to E. ruminantium and are able to serve as a source of infection to LSTs.     

Characterization of Ehrlichia species from Ixodes ovatus ticks at the foot of Mt. Fuji, Japan

A total of 390 adult ticks (288 Ixodes ovatus and 102 I. persulcatus ) collected at the foot of Mt. Fuji and two near cities in Shizuoka prefecture, Japan, were examined for Ehrlichia infection by isolation with laboratory mice from whole tick tissues. Ehrlichial DNAs were detected from the spleens of mice inoculated with tissues from I. ovatus, but not I. persulcatus. The prevalence of ehrlichiae in the ticks was estimated to be ca. 3%. The 16S rDNA analysis revealed that the sequences of 8 ehrlichial isolates (termed "Shizuoka" isolates) obtained were identical, and they were very similar, but not identical, to those of two Ehrlichia species strain variants recently isolated in Japan, followed by Ehrlichia chaffeensis in the US. Analysis of parts of the omp-1 multigene family specific for monocytic ehrlichiosis agents showed that the Shizuoka isolates were distinct from other ehrlichial organisms. The Shizuoka isolates caused death in immunocompetent laboratory mice, suggesting that they are highly pathogenic in mice. The data show that the Shizuoka isolates are likely to be a new strain variant of Ehrlichia species in Japan. Further characterization and surveillance will be required in Japan due to the presence of these human ehrlichiosis agent-like organisms.     

Presence of a novel Ehrlichia sp. in Ixodes granulatus found in Okinawa, Japan

Ehrlichia -specific DNA fragments of Ehrlichia omp-1 and groEL genes were found in two I. granulatus ticks which had been collected from wild small mammals in a subtropical zone in Japan. The DNA sequences of groEL and 16SrDNA of the suspected Ehrlichia were clustered into a group of E. chaffeensis , E. muris , and Ehrlichia sp. HF565 found in I. ovatus, but were distinctly different. Therefore the Ehrlichia strain was designated as a novel Ehrlichia sp. 360. The Ehrlichia sp. 360 was detected in I. granulatus but not in any other ticks. This suggests that I. granulatus is a probable vector of Ehrlichia sp. 360 in Japan.     

Granulocytic Ehrlichia infection in Ixodid ticks and mammals in woodlands and uplands of the U.K.

Abstract .The prevalence of infection with Ehrlichiae of the Ehrlichia phagocytophila genogroup (the granulocytic Ehrlichiae), in questing Ixodes ricinus ticks of U.K. upland and woodland habitats, was investigated by PCR. The prevalence of infection in the three feeding stages of I. ricinus indicated that granulocytic Ehrlichiae are transmitted transstadially with no, or inefficient, transovarial transmission. The presence of infected ticks in both habitats indicates that endemic cycles of granulocytic Ehrlichia (GE) infection are maintained by both domesticated sheep and by wild reservoirs, and coexist with endemic cycles of Borrelia burgdorferi infection. Moreover, demonstration, for the first time, of GE infection in engorged Ixodes trianguliceps ticks and blood collected from wild rodents, suggests that European wild rodents are competent reservoirs. GE infection prevalence in nymphal and adult I. ricinus was significantly greater in uplands than woodlands, which is consistent with ticks of all three feeding stages feeding on reservoir-competent sheep in uplands. In one woodland studied, pheasants are important hosts for nymphal I. ricinus but are incompetent or inefficient reservoirs, so reducing GE infection prevalence in I. ricinus ticks in this habitat. 16S rRNA sequences of GE from ticks of these U.K. habitats, showed a high degree of homology with those of granulocytic Ehrlichiae isolated from humans, but also showed some evidence of genetic diversity of granulocytic ehrlichiae in the U.K. The implications of these findings, for the taxonomy of granulocytic ehrlichiae and the potential for human infections to occur in the U.K., is discussed.     

Ehrlichia ruminantium: genomic and evolutionary features

Ehrlichia ruminantium is the causative agent of heartwater, an important tick-borne disease of livestock in Africa and the Caribbean that threatens the American mainland. The genome sequences of three strains of E. ruminantium have recently been published, revealing the presence of specific features related to genomic plasticity. E. ruminantium strains have traces of active genomic modifications, such as high substitution rates, truncated genes and the presence of pseudogenes and many tandem repeats. The most specific feature is the presence in all Ehrlichia of independent long-period tandem repeats, which are associated with expansion or contraction of intergenic regions.     

Ehrlichia ruminantium infection (heartwater) in wild animals

Several wild animal species have been implicated as hosts of Ehrlichia ruminantium (formerly Cowdria ruminantium), the rickettsial agent causing heartwater, a fatal disease of domestic ruminants in sub-Saharan Africa and eastern Caribbean. However, evidence for infection in most wild species is inconclusive because of inadequate diagnostic techniques. Infection has been proven only in 12 African ruminants, three non-African ruminants and two African rodents. A subclinical carrier state occurs in eight of the African ruminant species. Further studies on E. ruminantium infection in wild animal species are needed in order to determine the host range of this pathogen accurately. The host range of Ehrlichia ruminantium in wildlife is reviewed here and the role played by these species in the epidemiology and spread of heartwater is discussed.     

Novel genetic variants of Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma centrale, and a novel Ehrlichia sp. in wild deer and ticks on two major islands in Japan

Wild deer are one of the important natural reservoir hosts of several species of Ehrlichia and Anaplasma that cause human ehrlichiosis or anaplasmosis in the United States and Europe. The primary aim of the present study was to determine whether and what species of Ehrlichia and Anaplasma naturally infect deer in Japan. Blood samples obtained from wild deer on two major Japanese islands, Hokkaido and Honshu, were tested for the presence of Ehrlichia and Anaplasma by PCR assays and sequencing of the 16S rRNA genes, major outer membrane protein p44 genes, and groESL. DNA representing four species and two genera of Ehrlichia and Anaplasma was identified in 33 of 126 wild deer (26%). DNA sequence analysis revealed novel strains of Anaplasma phagocytophilum, a novel Ehrlichia sp., Anaplasma centrale, and Anaplasma bovis in the blood samples from deer. None of these have been found previously in deer. The new Ehrlichia sp., A. bovis, and A. centrale were also detected in Hemaphysalis longicornis ticks from Honshu Island. These results suggest that enzootic cycles of Ehrlichia and Anaplasma species distinct from those found in the United States or Europe have been established in wild deer and ticks in Japan.     

Evidence to show that an agent that cross-reacts serologically with Cowdria ruminantium in Zimbabwe is transmitted by ticks

The serological diagnosis of heartwater based on reactions to the immunodominant Cowdria ruminantium major antigen protein-1 (MAP-1) is impaired by the detection of false-positive reactions. In this study, the prevalence of false-positive reactions on seven heartwater-free farms in Zimbabwe was determined to be 8-94% by immunoblotting against C. ruminantium antigens. The highest prevalence of false-positives on Spring Valley Farm correlated with the presence of Rhipicephalus evertsi evertsi ticks. The other tick species found on these seven farms were Hyalomma truncatum and Hyalomma marginatum rufipes. Rhipicephalus evertsi evertsi ticks collected from Spring Valley Farm and fed on seronegative sheep caused seroconversion in one of two sheep. This sheep developed a mild febrile reaction and C. ruminantium MAP-1 antigen reactive antibodies 3 weeks after the ticks started feeding. Polymerase chain reactions (PCRs), conducted using C. ruminantium-specific primers on ticks collected from the seven farms and on some of the R. e. evertsi ticks that had caused seroconversion in one sheep, were negative. However, some of these ticks gave positive PCRs with DNA primers which amplify a 350 bp DNA fragment of the 16s rRNA gene from all ehrlichial agents indicating the presence of infection with one or more Ehrlichia species. Although attempts to isolate the cross-reacting agent from the sheep were unsuccessful, this study demonstrates that false-positive reactions with the MAP-1 C. ruminantium antigen are associated with agents transmitted by ticks.     

Tissue Distribution of the Ehrlichia muris-Like Agent in a Tick Vector

Human pathogens transmitted by ticks undergo complex life cycles alternating between the arthropod vector and a mammalian host. While the latter has been investigated to a greater extent, examination of the biological interactions between microbes and the ticks that carry them presents an equally important opportunity for disruption of the disease cycle. In this study, we used in situ hybridization to demonstrate infection by the Ehrlichia muris-like organism, a newly recognized human pathogen, of Ixodes scapularis ticks, a primary vector for several important human disease agents. This allowed us to assess whole sectioned ticks for the patterns of tissue invasion, and demonstrate generalized dissemination of ehrlichiae in a variety of cell types and organs within ticks infected naturally via blood feeding. Electron microscopy was used to confirm these results. Here we describe a strong ehrlichial affinity for epithelial cells, neuronal cells of the synganglion, salivary glands, and male accessory glands.     

Comparison of efficacy of American and African Amblyomma ticks as vectors of heartwater (Cowdria ruminantium) infection by molecular analyses and transmission trials

The ability of Amblyomma americanum, Amblyomma cajennense, Amblyomma maculatum, and Amblyomma variegatum to acquire and transmit Cowdria ruminantium infection was investigated. Uninfected nymphs were fed on clinically reacting C. ruminantium-infected sheep and then analyzed for infection by specific DNA detection assays and by tick transmission trials. By polymerase chain reaction (PCR), the mean infection prevalence of A. maculatum ticks (50.7%) was similar to that of A. variegatum, Elevage strain (43.5%; P = 0.83) and Petit Bourg strain (45.9%; P = 0.26) ticks. Though Amblyomma hebraeum were not tested by PCR, by DNA probe their infection prevalence was 94%. In contrast, A. americanum and A. cajennense ticks demonstrated very low susceptibility to C. ruminantium, and the prevalence of infection by PCR was approximately 1%. The higher susceptibility of A. maculatum and A. variegatum to C. ruminantium correlated with superior vector efficiency, depicted by similar prepatent periods and severity of disease transmissions to sheep. Amblyomma americanum and A. cajennense failed to transmit infection, confirming that low susceptibility to C. ruminantium correlates with the poor vector status of these species. These results highlight the importance of A. maculatum as a potential vector that is likely to play a major role in the establishment and maintenance of heartwater, if the disease were to be introduced to the U.S.A., Central, and South America.     

Highly prevalent Coxiella sp. bacterium in the tick vector Amblyomma americanum

Laboratory-reared and field-collected Amblyomma americanum ticks were hosts of a Coxiella sp. and a Rickettsia sp. While the Coxiella sp. was detected in 50 of 50 field-collected ticks, the Rickettsia sp. was absent from 32% of ticks. The Coxiella sp. showed evidence of a reduced genome and may be an obligate endosymbiont.     

Phylogenetic position of Cowdria ruminantium (Rickettsiales) determined by analysis of amplified 16S ribosomal DNA sequences.

The 16S ribosomal DNA sequence of Cowdria ruminantium, the causative agent of heartwater disease in ruminants, was determined. An analysis of this sequence showed that C. ruminantium forms a tight phylogenetic cluster with the canine pathogen Ehrlichia canis and the human pathogen Ehrlichia chaffeensis. Although a close relationship between the genus Cowdria and several members of the tribe Ehrlichieae has been suspected previously, the tight phylogenetic cluster with E. canis and E. chaffeensis is surprising in view of known differences in host preference and target cells.     

Longitudinal analysis of tick densities and Borrelia, Anaplasma, and Ehrlichia infections of Ixodes ricinus ticks in different habitat areas in The Netherlands

From 2000 to 2004, ticks were collected by dragging a blanket in four habitat areas in The Netherlands: dunes, heather, forest, and a city park. Tick densities were calculated, and infection with Borrelia burgdorferi and Anaplasma and Ehrlichia species was investigated by reverse line blot analysis. The lowest tick density was observed in the heather area (1 to 8/100 m2). In the oak forest and city park, the tick densities ranged from 26 to 45/100 m2. The highest tick density was found in the dune area (139 to 551/100 m2). The infection rates varied significantly for the four study areas and years, ranging from 0.8 to 11. 5% for Borrelia spp. and 1 to 16% for Ehrlichia or Anaplasma (Ehrlichia/Anaplasma) spp. Borrelia infection rates were highest in the dunes, followed by the forest, the city park, and heather area. In contrast, Ehrlichia/Anaplasma was found most often in the forest and less often in the city park. The following Borrelia species were found: Borrelia sensu lato strains not identified to the species level (2.5%), B. afzelii (2.5%), B. valaisiana (0.9%), B. burgdorferi sensu stricto (0.13%), and B. garinii (0.13%). For Ehrlichia/Anaplasma species, Ehrlichia and Anaplasma spp. not identified to the species level (2.5%), Anaplasma schotti variant (3.5%), Anaplasma phagocytophilum variant (0.3%), and Ehrlichia canis (0.19%) were found. E. canis is reported for the first time in ticks in The Netherlands in this study. Borrelia lusitaniae, Ehrlichia chaffeensis, and the human granylocytic anaplasmosis agent were not detected. About 1.6% of the ticks were infected with both Borrelia and Ehrlichia/Anaplasma, which was higher than the frequency predicted from the individual infection rates, suggesting hosts with multiple infections or a possible selective advantage of coinfection.     

Transmission of Ehrlichia chaffeensis from lone star ticks (Amblyomma americanum) to white-tailed deer (Odocoileus virginianus)

Amblyomma americanum is an aggressive ixodid tick that has been implicated as a vector for several bacterial agents. Among these is Ehrlichia chaffeensis, which causes human monocytic (or monocytotropic) ehrlichiosis. In this study, experimental tick transmission of E. chaffeensis from infected lone star ticks to deer was revisited, and the question of whether it would be possible to re-isolate the organism from deer was asked, because this had not been done previously. Here, we were able to transmit a wild strain of E. chaffeensis from acquisition-fed lone star ticks to white-tailed deer. Ehrlichia chaffeensis was re-isolated from one white-tailed deer on multiple days during the infection and from another deer on one day during the infection. Peak rickettsemias for E. chaffeensis-infected deer were 17 DPI with acquisition-fed ticks and 14 DPI with needle-inoculated deer. This study supports the role of the lone star tick and white-tailed deer as vector and reservoir host for E. chaffeensis, demonstrating culture re-isolation of E. chaffeensis in deer infected by experimental tick transmission for the first time.     

Population-based evaluation of the Ehrlichia ruminantium MAP 1B indirect ELISA

The indirect MAP 1B ELISA based on the recombinant MAP 1B fragment of the immunodominant MAP I protein of Ehrlichia ruminantium is considered to be the most sensitive and specific assay for the serodiagnosis of heartwater. In this study, we evaluated its reliability in detecting exposure to E. ruminantium in field populations of domestic ruminants in Zimbabwe. Cattle and goat herds in endemically stable areas with high infection pressure and an expected close to 100% prevalence of E. ruminantium exposure were sampled. Bovine sera (858) and caprine sera (706) collected at seven locations representative of the two main production systems (communal lands and large scale commercial farms) in the two main agroecological zones of Zimbabwe (highveld and lowveld) were analysed. The prevalence of MAP 1B-specific antibodies in goats was similar and high, ranging from 67 to 100%, at all except one site (43%). Age-specific differences in goats (1, 2, 3, 4, 5 years) were not observed. In contrast, MAP 1B seroprevalence in cattle was significantly lower (P < 0.001), ranging from 46 to 61% in the lowveld communal area and from 24 to 33% in the remaining areas (P < 0.001). Age-specific differences in seroprevalence (1, 2, 3, 4, 5-7 + years) were similarly not evident in cattle (P < 0.15). Hence, the indirect MAP 1B ELISA may be an unreliable indicator of past exposure to heartwater in field-infected cattle in Zimbabwe. Although the reasons for this low response in field cattle are not fully understood, this study illustrates the need for field validation of the performance of new diagnostic tests prior to their use for epidemiological purposes.     

Susceptibility of Amblyomma americanum to natural and experimental infections with Theileria cervi

One hundred fifty Amblyomma americanum were examined between March and September 1986 from Cookson Hills Wildlife Refuge in eastern Oklahoma (USA). Of these ticks, 11% (17 of 150) were infected with Theileria cervi. Field-collected nymphal ticks had an 8% (3 of 37) prevalence of infection averaging 1.0 infected acini/nymph. Female ticks had a 16% prevalence of infection averaging 1.6 infected acini/female; T. cervi was not observed in salivary glands of field-collected male ticks. When laboratory reared A. americanum nymphs were allowed to feed on experimental white-tailed deer (Odocoileus virginianus) with varying T. cervi parasitemias (less than 1, 2, 6 and greater than 20%), only ticks which fed on deer with parasitemias greater than 1% became infected. Although prevalence and intensity of infection varied in the infected ticks, there was no significant difference in prevalence of infection between males and females. However, females did acquire significantly greater intensities than males. The data from these studies confirm that T. cervi overwinters in A. americanum and suggests that the prevalence, intensity and abundance of infection of T. cervi in ticks is influenced by the parasitemia of the deer host. Furthermore, fawns may play a more important role in the epidemiology of T. cervi transmission than do adult deer because of the coordination between tick activity patterns and deer fawning.     

Ehrlichia chaffeensis in archived tissues of a white-tailed deer.

White-tailed deer (Odocoileus virginianus) play an integral role in the natural history of Ehrlichia chaffeensis, the causative agent of human monocytic ehrlichiosis (HME). Paraffinized tissues from a white-tailed deer submitted as a diagnostic case to the Southeastern Cooperative Wildlife Disease Study (Athens, Georgia, USA) in October of 198.5 and originally described as infected with an unidentified rickettsial organisim were re-examined by specific nested polymerase chain reaction (PCR) for evidence of infection with Ehrlichia spp. Ehrlichia chaffeensis was identified from the bone marrow and inguinal lymph node of this deer based on amplification of a characteristic sequence-confirmed 16S rDNA fragment from these tissues. Parallel PCR tests on the same samples were negative for 16S rDNA fragments of the agent of human granulocytic ehrlichiosis (HGE) and for an Ehrlichia-like organism widely distributed in white-tailed deer populations. This report describes detection of E. chaffeensis in archived tissue from a deer collected before the index case of human monocytic ehrlichiosis was established.     

我国东北地区蜱中检出蜱传埃立克体DNA

应用16S rRNA基因构建的特异性引物进行半套式PCR,检测东北地区蜱标本中埃立克体DNA,并对扩增产物进行克隆和序列测定,与GenBank中已知序列进行同源性比较。结果从全沟硬蜱和森林革蜱中均扩增出了查菲埃立克体DNA,扩增片段与美国分离株(GenBankM73222)相对应片段完全一致,同源性为100%;另外从全沟硬蜱中扩增出了粒细胞埃立克体DNA,扩增片段与美国分离株(GenBankU02521)相对应片段相差2个碱基,同源性为99.7%。表明我国东北边境地区有埃立克体病的病原体存在,该地区可能存在埃立克体病的自然疫源地。