Tick-Borne Fever in Lambs of Different Ages

Four groups of lambs aged 1 week, 4 weeks, 1/2 year and 1 year old respectively were inoculated with Ehrlichia phagocytophila infected blood. Clinical signs, temperature reactions, haematological changes and parasitaemia were more moderate in lambs inoculated with E. phagocytophila at the age of 1 week than those recorded in the older animals. The clinical response to tick-borne fever (TBF) appears to be more severe with increasing age of the lambs. The lymphocyte reactivity to mitogens was reduced in the TBF infected lambs, and was most pronounced in lambs in the 3 older age groups.     

Experimental infection of dusky-footed wood rats (Neotoma fuscipes) with Ehrlichia phagocytophila sensu lato

Dusky-footed wood rats, Neotoma fuscipes, have been implicated in the natural maintenance of Ehrlichia phagocytophila sensu lato, the agent of human granulocytic ehrlichiosis (HGE), in northern California based on high seroprevalence and amplification of E. phagocytophila s.l. DNA from wood rat blood. In order to further assess granulocytic ehrlichiosis in wood rats, we examined wild-caught wood rats for infection and then performed experimental intra-peritoneal infections with E. phagocytophila s.l. in horse or wood rat blood, and tested animals for 120 days by polymerase chain reaction (PCR) and serology. Of 15 wood rats collected from northern California, three were antibody and PCR-positive for E. phagocytophila s.l. at the time of capture. The naturally infected wood rats remained PCR-positive for a mean of 52 days (+/- 7 SD). Experimental i.p. passage of E. phagocytophila s.l. in wood rat blood was successful in three of four wood rats and the mean duration of PCR-positivity was 75 days (+/- 21.2 SD). Experimental infection with E. phagocytophila s.l. in horse blood succeeded in all four of the recipients and the mean duration of PCR-positivity of 81 days (+/- 17.5 SD). No infected individual appeared to be ill based on feeding behavior, activity, and hydration status. These data confirm that wood rats are susceptible to E. phagocytophila s.l., may develop prolonged infection without clinical ehrlichiosis, and may play a role in maintaining E. phagocytophila s.l. in nature.     

Granulocytic ehrlichiosis in a roe deer calf in Norway

A case of granulocytic ehrlichiosis is described in a roe deer (Capreolus capreolus) calf from Norway. The calf was heavily infested with Ixodes ricinus and died from Escherichia coli septicemia. Granulocytic Ehrlichia sp. was detected by polymerase chain reaction (PCR) from several organs and sequence determination identified a variant of human granulocytic ehrlichiosis (HGE) agent. This is the first report of a possible clinical granulocytic Ehrlichia sp. infection in a roe deer.     

Ehrlichiosis in a moose calf in Norway

A case of granulocytic ehrlichiosis in a moose calf (Alces alces) in Norway is described. The animal was heavily infested with ticks (Ixodes ricinus), and died from a Klebsiella pneumoniae septicemia. Examination of blood smears from the calf revealed cytoplasmic inclusions (morulae) typical of infection with Ehrlichia phagocytophila in the granulocytes. Ehrlichia sp. was detected by polymerase chain reaction (PCR) in blood from the calf, and in the ticks. Sequence determination identified it as E. phagocytophila. This is the first report of ehrlichiosis in moose.     

Detection of natural foci of babesiosis and granulocytic ehrlichiosis in Russia

The use of microscopy, infection of golden hamsters and the polymerase chain reaction made it possible to find out that about 30% of common red-backed voles (Clethrionomys glareolus), inhabiting the taiga forests of the southern part of the Western Urals (the Chusovskoi district of the Perm region), were infected with Babesia microti and simultaneously (a third of them) with Ehrlichia (Cytoecetes) phagocytophila, the causative agent of granulocytic ehrlichiosis. The sequencing of 18S rDNA of strain "Mys", isolated in Russia, revealed its identity to American B. microti strain GI, pathogenic for humans. The main vector supporting the circulation of B. microti in the natural foci in the region where these investigations were conducted was, seemingly, the tick Ixodes trianguliceps, Thus, for the first time the data proving the presence of reservoir hosts infected with B. microti and granulocytic E. phagocytophila, pathogenic for humans, in Russia were presented.     

Ehrlichia ewingii sp. nov., the etiologic agent of canine granulocytic ehrlichiosis.

The 16S rRNA gene was amplified, cloned, and sequenced from the blood of two dogs that were experimentally infected with the etiologic agent of canine granulocytic ehrlichiosis. The 16S rRNA sequence was found to be unique when it was compared with the sequences of other members of the genus Ehrlichia. The most closely related species were Ehrlichia canis (98.0% related) and the human ehrlichiosis agent (Ehrlichia chaffeensis) (98.1% related); all other species in the genus were found to be phylogenetically much more distant. Our results, coupled with previous serologic data, provide conclusive evidence that the canine granulocytic ehrlichiosis agent is a new species of the genus Ehrlichia that is related to, but is distinct from, E. canis and all other members of the genus. We propose the name Ehrlichia ewingii sp. nov.; the Stillwater strain is the type strain.     

Molecular and microscopical evidence of Ehrlichia spp. and Borrelia burgdorferi sensu lato in patients,animals and ticks in the Czech Republic

We report moderately severe cases of human ehrlichiosis and a lethal one caused by human granulocytic Ehrlichia, the HGE agent, closely related to Ehrlichia phagocytophila and Ehrlichia equi. Their vector is the Ixodes ricinus tick, which also transmits Borrelia burgorferi sensu lato in central, west and east regions of the Czech Republic. The diagnosis was established by PCR with sequence analysis of the genes encoding 16S rRNA of Ehrlichia and with reverse hybridization by using enzyme linked immunosorbent assay with different covalently coupled probes to the activated plate.Ten out of 47 patients and 10 huntsmen were PCR positive and 7 of them seroconverted to the HGE. Coinfection of Ehrlichia phagocytophila with Borrelia burgdorferi sensu lato was detected in 3 patients. Ehrlichia spp., the HGE agent, was isolated and propagated only from one patient in the HL-60 promyelocytic cell line. The maintenance of Ehrlichia in culture and in patients was assayed also by immunocytological staining and electron microscopy. Sequence or hybridization analysis of PCR results in different wild mammals and birds showed significant sources of Ehrlichia fagocytophila in nature. Three variants of E. phagocytophila in wild roe deer and boars, as well as for the first time in birds, have been described. Cultures from the blood of horses, and from the spleen and kidney specimens of roes and boars, PCR positive for Ehrlichia spp., displayed a disappearing level of the pathogen or contamination with other bacteria.     

Risk factors in habitats of the tick Ixodes ricinus influencing human exposure to Ehrlichia phagocytophila bacteria

Ixodes ricinus L. (Acari: Ixodida) were sampled during 1996-99 in southern Scotland, on vegetation using cloth drags, on humans by removal from clothing and on roe deer (Capreolus capreolus L.) by searching legs of culled deer. Developmental microclimate was recorded by automatic recorders and questing microclimate by portable instruments during tick collections. Ticks and deer were examined for infection with Ehrlichia phagocytophila bacteria (Rickettsiales) using microscopy and polymerase chain reaction. This pathogen causes tick-borne fever of sheep in Europe and human granulocytic ehrlichiosis in North America, but in Europe human clinical ehrlichiosis due to E. phagocytophila has not been recorded despite serological evidence of exposure. Among three types of habitat, coniferous woodland was most infested with questing ticks (560 ticks/km of drag; mean numbers collected on long trousers: 24.3 larvae, 13.5 nymphs and 0.8 adult ticks/km walked), deciduous woodland had slightly lower infestation (426 ticks/km drag) and upland sheep pasture had much lower infestation (220 ticks/km drag). Of the three main vegetation types, bracken was least infested (360 ticks/km drag), ericas most (430 ticks/km drag) and grassland had intermediate infestation density (413 ticks/km drag). Questing and developmental microclimates were poor predictors of exposure within these habitats, except lower infestation of pastures was attributed to greater illumination there. Collectors who walked a total of 300 km through all habitats (taking 360 h in all seasons), wearing cotton trousers hanging outside rubber boots, were bitten by only four nymphs and 11 larvae of I. ricinus (but no adult ticks). There was a negative correlation between densities of deer and ticks collected, although presence of deer remains a major indicator of exposure. The proportion of infected ticks was fairly uniform at four sites studied. Overall prevalence of E. phagocytophila in I. ricinus was 3.3% in nymphs (40/1203) but only approximately 1.5% in adults of both sexes (although males do not bite). It was estimated that nymphs of I. ricinus gave 4.4% probability of one infected bite/person/year (for occupational exposure during this research) due to presence in all seasons and habitats, their human biting rate of 0.011 nymphs/h or 0.013 nymphs/km and widespread infection with E. phagocytophila. The frequency distribution of intensity of infection in ticks was approximately normal (mean 98 morulae/nymph infected), thus there is a high risk of receiving a high dose from any one infected tick bite.     

Serologic and molecular evidence of Ehrlichia spp. in coyotes in California

In order to determine the role of coyotes in the epidemiology of granulocytic and monocytic ehrlichial agents in California (USA), we tested 149 serum samples for antibodies against Ehrlichia equi, E. risticii, and E. canis, using an indirect immunofluorescent antibody test. Polymerase chain reaction (PCR) assay was used to survey for the presence of members of the E. phagocytophila genogroup, E. risticii and E. canis in blood samples of 95 coyotes. Sixty-eight (46%) samples were seropositive for E. equi, two (1%) for E. risticii and none of the samples had antibodies reactive to E. canis. Two and one coyote were positive for E. risticii and members of the E. phagocytophila genogroup by PCR assay, respectively. In contrast, the 95 samples were negative for E. canis by PCR. Ninety-five percent of the 68 E. equi seropositive coyotes and the one coyote PCR positive for members of the E. phagocytophila genogroup originated from a coastal area. However, the two E. risticii seropositive coyotes and the two coyotes PCR positive for E. risticii were from northern California. Sequence analysis of the three amplified PCR products revealed the agent to be similar in two coyotes to the sequences of E. risticii from horses originating from northern California and identical in one coyote to the agent of human granulocytic ehrlichiosis and E. equi from California. Thus, coyotes are exposed to granulocytic ehrlichiae and E. risticii and may play a role in the epidemiology of these ehrlichial agents in California.     

Molecular cloning and characterization of a cDNA encoding cellobiose dehydrogenase from the wood-rotting fungus Grifola frondosa

A total of 305 Ixodes ricinus ticks (243 nymphs and 62 adults) were collected from three different regions of Thuringia in Middle Germany which are known to be endemic for Borrelia burgdorferi. Our aim was to investigate the carrier rate of ticks for granulocytic Ehrlichia species. The presence of ehrlichial 16S ribosomal DNA was investigated by polymerase chain reaction. Using primers specific for the Ehrlichia phagocytophila group PCR fragments of 151 bp and 943 bp, respectively, were produced in positive samples. Adult ticks showed a significantly higher infection rate (4/62; 6.5%) compared to nymphs (3/243; 1.2%). Prevalence rates varied between 0 and 3.8% regarding the different areas under investigation. The nucleotide sequences showed high similarity (between 97.5% and 99% identity) to the known sequences of the three E. phagocytophila group members HGE agent, E. phagocytophila and Ehrlichia equi. The sequence data did not allow a final classification to a particular member of this group.     

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.     

Serological Investigation of Granulocytic Ehrlichia Infection in Sheep in Norway

Serum samples of 749 sheep from 75 sheep flocks in Norway, i.e. 361 lambs (6 to 7 months old) and 388 adults (>1.5 year), were analysed for antibodies to Ehrlichia equi. Ten animals from each flock were examined. Seropositive animals were found along the coast of southern Norway from Vestfold to S?r-Tr?ndelag (as far north as 63°38'N). Seropositive sheep were not found in southeast, east or northern Norway. Thirty-two flocks were seropositive, although tick-borne fever had only been diagnosed earlier in half of these. In 78% of the seropositive flocks, more than 80% of the sheep were seropositive. A total of 35.7 % and 36.3 % of lambs and adults were found seropositive, respectively. However, the overall seroprevalence among animals that had been grazing on Ixodes pastures were 0.80 for the lambs and 0.84 for the adults. Mean antibody titres (± SD) (log₁₀) in seropositive lambs and adults were 2.59 (± 0.449) and 2.70 (± 0.481), respectively. No significant differences in either seroprevalence or mean antibody titre between sheep of different ages were obtained in this study. Based on antibodies 94% of sheep flocks on Ixodes pastures were infected with a granulocytic Ehrlichia infection. The association between seropositive flocks and Ixodes infested pasture shows a very high degree of agreement (p < 0.00001). The present study indicates that granulocytic Ehrlichia infection in sheep is underdiagnosed in Norway.     

The Effect of Two Different Oxytetracycline Treatments in Experimental Ehrlichia phagocytophila Infected Lambs

The effect of 2 different oxytetracycline treatments in acute E. phagocytophila infected lambs was investigated. Twenty 5-month-old lambs of the Dala and Rygja breeds were used. Ten lambs were inoculated intravenously with a stabilate of an ovine E. phagocytophila strain. On the third day of fever, 4 lambs were given long-acting oxytetracycline (Terramycin prolongatum vet^?, Pfizer) (20 mg/kg) intramuscularly and another 4 lambs were given short-acting oxytetracycline (Terramycin vet^?, Pfizer) (10 mg/kg) intravenously for 5 consecutive days. The lambs were examined for the presence of Ehrlichia infection by blood smear evaluation, polymerase chain reaction (PCR) and antibody titre against E. equi. One month after the last antibiotic treatment, 250 ml citrate blood from each of these lambs were inoculated into each of 10 susceptible lambs, which were observed during the following 6 weeks. The results indicate that oxytetracycline given in the acute stage of the infection may effectively teminate the development of fever, rickettsemia and weight reduction in E. phagocytophila infected lambs. No difference was observed between the 2 treatment groups. However, at least 3 of 8 antibiotic treated lambs (37.5%) were still infected with granulocytic Ehrlichia 3 months after treatment.     

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.     

The mouse as a model for investigation of human granulocytic ehrlichiosis: current knowledge and future directions

The use of laboratory mice to investigate correlates of infectious disease, including infection kinetics, cellular alterations, cytokine profiles, and immune response in the context of an intact host has expanded exponentially in the last decade. A marked increase in the availability of transgenic mice and research tools developed specifically for the mouse parallels and enhances this research. Human granulocytic ehrlichiosis (HGE) is an emerging, zoonotic disease caused by tick-borne bacteria. The HGE agent (Anaplasma phagocytophila) is one of two recognized pathogens to cause human granulocytic ehrlichiosis (HGE). The mouse model of HGE complements in vitro tissue culture studies, limited in vivo large animal studies, and ex vivo studies of human and ruminant neutrophils, and promises new avenues to approach mechanisms of disease. In the overview reported here, we focus principally on current research into HGE pathogenesis using the mouse model. Included is a discussion of current changes in ehrlichial classification and nomenclature, a review of ehrlichial biology and ecology, and highlights of clinical disease in animals and people.     

Molecular survey of Anaplasma phagocytophilum and Ehrlichia canis in red foxes (Vulpes vulpes) from central Italy

During the 2007-2008 hunting season, 150 spleen samples were collected from free-ranging red foxes (Vulpes vulpes) in central Italy. The specimens were tested by two nested PCR assays to detect DNA of Anaplasma phagocytophilum, etiologic agent of granulocytic ehrlichiosis of animals and humans, and DNA of Ehrlichia canis, which causes the monocytic ehrlichiosis in canids. None of the foxes were PCR-positive for E. canis; 25 (16.6%) were positive for A. phagocytophilum. No specific gross alterations were detected at necropsy, and no histopathologic lesions found on PCR-positive spleen samples.     

Granulocytic ehrlichiosis and tick infestation in mountain lions in California

Forty-seven mountain lions (Puma concolor) collected year-round in 1996 to 1998 from the Sierra Nevada foothills, the northern coast ranges, and in Monterey County (California, USA) were examined for infestation with Ixodes pacificus and Dermacentor variabilis ticks. Ticks were found predominantly in winter and spring. The seroprevalence of granulocytic ehrlichiae (GE) antibodies (Ehrlichia equi or the agent of human granulocytic ehrlichiosis) was 17% and the PCR-prevalence of DNA characteristic of GE in blood was 16%. There were eight polymerase chain reaction (PCR)-positive but seronegative mountain lions, one that was PCR-positive and seropositive, and eight that were PCR-negative and seropositive. Nineteen percent of engorged tick pools from mountain lions were PCR-positive. Because mountain lions inhabit tick-infested habitat and are frequently bitten by I. pacificus, surveillance for GE antibodies and DNA in mountain lions and other vertebrate hosts may be useful as indicators for geographical regions in which humans are at risk of GE infection.     

On molecular taxonomy: what is in a name?

Gene sequences of small portions of the genome are often used for premature detailed taxonomic changes, neglecting polyphasic taxonomy, which should also consider phenotypical characteristics. Three examples are given: (i) Recently, members of the genera Eperythrozoon and Haemobartonella have been moved, correctly so, from the Rickettsiales to the Mycoplasmatales, but were assigned to the genus Mycoplasma, mostly on the basis of 16S rRNA sequence analysis. Not only is the 16S rRNA sequence similarity between 'classical' Mycoplasma and these species of Eperythrozoon and Haemobartonella less than that between some other well-recognised bacterial genera, but their biological differences amply justify their classification in different genera of the Mycoplasmatales. Furthermore, the move creates considerable confusion, as it necessitates new names for some species, with more confusion likely to come when the 16S rRNA sequences of the type species of Eperythrozoon, a name which has priority over Mycoplasma, will be analysed. (ii) In the Rickettsiales, members of the genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolhbachia are so closely related phylogenetically on the basis of 16S rRNA sequences, and for some also of groESL operon sequences, that they have recently been fused, correctly so, into one family, the Anaplasmataceae, while the tribes Ehrlichieae and Wolbachieae have been abolished. Sequence diversity within the 'classical' genus Ehrlichia has led to classifying E. phagocytophila (including E. equi and the agent of human granulocytic ehrlichiosis), E. platys and E. bovis in the genus Anaplasma, while others have been retained in Ehrlichia, which also includes Cowdria ruminantium. E. sennetsu and E. risticii have been transferred to the genus Neorickettsia. 16S rRNA and GroEL sequences of 'classical' Anaplasma and some members of 'classical' Ehrlichia do show a close relationship, but differences in citrate synthase gene sequences, the GC content of this gene, and sequences of the gene encoding the beta-subunit of RNA polymerase, not to speak of the phenotypical differences, do not justify the fusion into one genus. Because of the phylogenetical diversity in Ehrlichia it is recommended that a new genus name be created for the E. phagocytophila genogroup (and E. platys and E. bovis). (iii) One of the conclusions of studies on the phylogeny of ticks of the subfamilies Rhipicephalinae and Hyalomminae, based on nucleotide sequences from 12S rRNA, cytochrome c oxidase I, the internal transcribed spacer 2, 18S rRNA, as well as morphological characters, is that Boophilus should be considered as a subgenus of Rhipicephalus. While Boophilus and Rhipicephalus are undoubtedly close, the obviously important morphological and biological differences between the genera Rhipicephalus and Boophilus are thus overruled by similarities in the sequences of a number of genes and this leads to considerable confusion. Polyphasic taxonomy amply justifies maintaining Boophilus as a separate genus, phylogenetically near to Rhipicephalus. This note is a plea for a cautious and balanced approach to taxonomy, taking into account molecular genotypical information, as far as is possible from different genes, as well as phenotypical characteristics.     

Infections of granulocytic ehrlichiae and Borrelia burgdorferi in white-tailed deer in Connecticut

Serum or whole blood samples, obtained from 141 white-tailed deer (Odocoileus virginianus) in Connecticut (USA) during 1980, 1991, and 1996, were analyzed to detect past or current infections of Ehrlichia phagocytophila genogroup organisms and Borrelia burgdorferi. When the BDS or NCH-1 strains of granulocytic ehrlichiae were used separately in indirect fluorescent antibody (IFA) staining methods, antibody positivity rates varied from 25 to 64% in 1991 and 1996, respectively. All 50 sera tested from 1980 collections were negative. Although percentages of sera with B. burgdorferi antibodies, as detected by an enzyme-linked immunosorbent assay, also differed (23 to 53%), there were coexisting antibodies to both bacteria in 20 (49%) of 41 sera. In tests on specificity, 19 deer sera with ehrlichial antibodies also were tested by IFA staining procedures for Anaplasma marginale antibodies; one serum with a titer of 1:5,120 to ehrlichial antigen reacted to A. marginale antigen at a serum dilution of 1:320. In parallel analyses of 69 sera, results of Western blot analyses for ehrlichial infections in deer were concordant (72% agreement) with those of IFA staining methods containing ehrlichial antigen. All positive immunoblots showed bands to peptides of the NCH-1 strain of the human granulocytic ehrlichiosis (HGE) agent having molecular masses of about 44, 105, or 110 kDa. In polymerase chain reaction (PCR) studies of blood samples from 63 deer, 11 (18%) specimens were positive for 16S ribosomal DNA of an Ehrlichia phagocytophila genogroup organism, whereas 23 (37%) samples were positive for the DNA of the 44 kDa gene of the HGE agent. White-tailed deer are exposed to different tick-borne bacteria in areas where Ixodes scapularis ticks are abundant and may, in some instances, have had concurrent infections.