Human Granulocytic Ehrlichiosis Agent Research Articles

Correction for Zhi et al., "Cloning and Expression of the 44-Kilodalton Major Outer Membrane Protein Gene of the Human Granulocytic Ehrlichiosis Agent and Application of the Recombinant Protein to Serodiagnosis".

Correction for Zhi et al., "Cloning and Expression of the 44-Kilodalton Major Outer Membrane Protein Gene of the Human Granulocytic Ehrlichiosis Agent and Application of the Recombinant Protein to Serodiagnosis".

A lifelong study of a pack Rhodesian ridgeback dogs reveals subclinical and clinical tick-borne Anaplasma phagocytophilum infections with possible reinfection or persistence

BackgroundVarious tick-borne infections often occur without specific clinical signs and are therefore notoriously hard to diagnose separately in veterinary practice. Longitudinal studies over multiple tick seasons performing clinical, serological and molecular investigations in parallel, may elucidate the relationship between infection and disease. In this regard, six related Rhodesian Ridgeback dogs living as a pack became subject of lifetime studies due to ongoing tick infestations and recurring clinical problems. Blood samples for diagnostic tests were obtained throughout the years 2000 to 2009.MethodsData collected from clinical observations, hemograms, serology and detection of Anaplasma phagocytophilum, either by microscopy or by DNA amplification and typing, were placed in a time line. This dataset essentially presents as a prospective study enabling the association of the Anaplasma infections with occurring disease.ResultsAll six dogs were infected, and two of them developed particular clinical symptoms that could be associated with Anaplasma infections over time. More specifically, episodes of general malaise with fever and purpura with thrombocytopenia and bacterial inclusions in granulocytes, were found concurrently with Anaplasma DNA and specific antibodies in peripheral blood samples. DNA from A. phagocytophilum variant 4 (of 16S rRNA) was found in multiple and sequential samples. DNA-sequences from variant 1 and the human granulocytic ehrlichiosis (HGE) agent were also detected.ConclusionsIn this study two lifelong cases of canine anaplasmosis (CGA) are presented. The data show that dogs can be naturally infected concurrently with A. phagocytophilum variant 1, variant 4 and the HGE agent. The ongoing presence of specific antibodies and Anaplasma DNA in one dog indicates one year of persisting infection. Treatment with doxycycline during recurring clinical episodes in the other dog resulted in transient clinical improvement and subsequent disappearance of specific antibodies and DNA suggesting that re-infection occurred.

Isolation and Short-Term Persistence of Ehrlichia ewingii in Cell Culture.

Ehrlichia ewingii is the causative agent of human and canine granulocytic ehrlichiosis. Since its discovery in 1970, little work has been done to characterize the pathogen or study the transmission dynamics due to the inability to grow the agent in vitro. The aim of this study was to assess the suitability of multiple cell lines and media formulations for propagation of E. ewingii in cell culture. In this study, we present an overview of attempts to isolate E. ewingii from the buffy coat of goats naturally infected by Amblyomma americanum ticks, as well as a methodology for maintaining the pathogen for up to 16 weeks in culture. The most promising results were seen with HL-60 cells differentiated by the addition of 1.5% DMSO to the media and supplemented with 8 mM l-glutamine. Cultures were passaged multiple times, and fluorescence and morulae were observed by indirect fluorescent antibody test and Diff-Quik staining. It is our hope that this information will provide a foundation for future attempts to propagate and maintain E. ewingii in cell culture.

The genus Anaplasma: new challenges after reclassification.

Summary The genus Anaplasmais one of four distinct genera in the family Anaplasmataceae, which are obligate intracellular pathogens vectored by ticks and found exclusively within parasitophorous vacuoles in the host cell cytoplasm. The 2001 reclassification of the order Rickettsiales expanded the genus Anaplasma, which previously contained pathogens that were host specific for ruminants (A. marginale, A. centrale and A. bovis), by adding A. phagocytophilum, a unification of three organisms previously classified as Ehrlichia (E. equi, E. phagocytophila and the unnamed agent of human granulocytic ehrlichiosis). Also included in the genus Anaplasma were A. bovis (formerly E. bovis), A. platys (formerly E. platys) and Aegyptianella pullorum. Despite the genomic relatedness of the regrouped organisms, many aspects of their biology are diverse, including their host specificity, host cell preferences, major surface proteins (MSPs) and tick vectors. This review focuses on the two most important pathogens: A. marginale, which causes bovine anaplasmosis, and A. phagocytophilum, the aetiologic agent of tick-borne fever in sheep and human granulocytic anaplasmosis, an emerging tick-borne disease of humans. For both pathogens, strain diversity is much greater than previously recognised. While MSPs were found to be useful in phylogenetic studies and strain identification, highly conserved MSPs were found to affect the specificity of serologic tests. Comparison of these two important pathogens highlights the challenges and insight derived from reclassification and molecular analysis, both of which have implications for the development and evaluation of diagnosis and control strategies.

The infection of reintroduced ruminants - Bison bonasus and Alces alces - with Anaplasma phagocytophilum in northern Poland.

The north-eastern part of Poland is considered an area of high risk for infection with tick-borne diseases, including with human granulocytic ehrlichiosis (HGE) agents. The etiological agent of HGE is Anaplasma phagocytophilum. As the animal reservoir for A. phagocytophilum in the environment serve the species from Cervidae and Bovidae families. European bison (Bison bonasus) and elk (Alces alces) are the big ruminant species, reintroduced to the forests of Middle Europe after many decades of absence. In the foci of zoonotic diseases they are able to play a role as natural reservoir to pathogens, however, their status as protected animals means their study has been rare and fragmentary. The studies of B. bonasus were conducted in Białowieża Primeval Forest and A. alces in Biebrza National Park. PCR amplifications were performed using primers amplifing the end of the groES gene, the intergenic spacer and approximately two-thirds of the groEL gene in the first round, and primers that span a 395-bp region of the groEL gene were used in the second round. The positive results were obtained in B. bonasus and A. alces, the prevalence of infection was 66.7 and 20.0%, respectively. Randomly selected samples were sequenced, sequences were compared with GenBank entries using Blast N2.2.13 and determined as A. phagocytophilum. The results presented herein are the first record of the presence of Anaplasma phagocytophilum in A. alces, and at the same time confirm the previous observations regarding the infection of B. bonasus with A. phagocytophilum.

Detection of Vector-Borne Agents in Lone Star Ticks, <I>Amblyomma americanum</I> (Acari: Ixodidae), From Mississippi

In this study, we evaluated Amblyomma americanum (lone star tick) in Mississippi for the presence of Ehrlichia chaffeensis, causative agent of human monocytic ehrlichiosis; Ehrlichia ewingii, causative agent of human and canine granulocytic ehrlichiosis; Borrelia lonestari, putative agent of southern tick-associated rash illness; Francisella tularensis, the agent of tularemia; and Rickettsia spp., particularly R. amblyommii, a suspected pathogen. We collected adult A. americanum from four regions of Mississippi: Northeast, Northwest, Southeast, and East. Of the ticks collected, 192 were dissected and DNA was extracted for nested polymerase chain reaction (PCR) assays to detect the above bacteria. In all, 3% of tick extracts had evidence of Borrelia sp., 4% for E. chaffeensis, 6% for E. ewingii, and 44% for a Rickettsia species. As determined by sequencing, most Rickettsia spp. were R. amblyommii. In addition, extracts from 42 pools (total of 950) of larval A. americanum collected in Southwest Mississippi were tested for the presence of E. chaffeensis and Rickettsia species. Of these extracts from pools, nine of 37 (24%) were PCR positive for a Rickettsia sp., most often, R. amblyommii; none had evidence of E. chaffeensis, supporting the ability of lone star ticks to transovarially transmit R. amblyommii, but not E. chaffeensis. This study demonstrates E. chaffeensis, E. ewingii, "B. lonestari", and R. amblyommii in A. americanum by PCR for the first time in Mississippi. Understanding the prevalence and epidemiology of these agents in Mississippi should increase awareness of tick-borne disease in the medical community.

The natural history of Anaplasma phagocytophilum

Anaplasma phagocytophilum is the recently designated name replacing three species of granulocytic bacteria, Ehrlichia phagocytophila, Ehrlichia equi and the agent of human granulocytic ehrlichiosis, after the recent reorganization of the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales.Tick-borne fever (TBF), which is caused by the prototype of A. phagocytophilum, was first described in 1932 in Scotland. A similar disease caused by a related granulocytic agent was first described in horses in the USA in 1969; this was followed by the description of two distinct granulocytic agents causing similar diseases in dogs in the USA in 1971 and 1982. Until the discovery of human granulocytic anaplasmosis (HGA) in the USA in 1994, these organisms were thought to be distinct species of bacteria infecting specific domestic animals and free-living reservoirs. It is now widely accepted that the agents affecting different animal hosts are variants of the same Gram-negative obligatory intracellular bacterium, which is transmitted by hard ticks belonging to the Ixodes persulcatus complex. One of its fascinating features is that it infects and actively grows in neutrophils by employing an array of mechanisms to subvert their bactericidal activity. It is also able to survive within an apparently immune host by employing a complex mechanism of antigenic variation. Ruminants with TBF and humans with HGA develop severe febrile reaction, bacteraemia and leukopenia due to neutropenia, lymphocytopenia and thrombocytopenia within a week of exposure to a tick bite. Because of the severe haematological disorders lasting for several days and other adverse effects on the host's immune functions, infected animals and humans are more susceptible to other infections.

Distinct Ecologically Relevant Strains ofAnaplasma phagocytophilum

Distinct Ecologically Relevant Strains of<i>Anaplasma phagocytophilum</i>

Death of a horse infected experimentally with Anaplasma phagocytophilum

A 19-year-old horse that was one of a group of six horses infected experimentally with Anaplasma phagocytophilum for a study of the pathogenesis of equine granulocytic ehrlichiosis died suddenly two...

Evaluation of an Improved PCR Diagnostic Assay for Human Granulocytic Ehrlichiosis.

Background: Human granulocytic ehrlichiosis (HGE) is a tick-borne disease caused by an agent similar to Ehrlichia equi. The etiologic agent has only recently been cultivated, and clinical and laboratory findings are nonspecific. Severity and fatal outcome are associated with delays in diagnosis and therapy. Methods and Results: The sensitivity of the polymerase chain reaction (PCR) technique for HGE was enhanced at least 250-fold for identification of the 16S ribosomal RNA gene of the HGE agent in blood by shortening cycle segments, using 35 rounds of amplification, employing eLONGase polymerase, and by using SYBR Green I for detection of amplified products. From 17 patients with suspected HGE, acute-phase blood was tested by PCR and for E. equi antibodies in acute and convalescent sera. The HGE PCR detected ehrlichial DNA in 1 ng of genomic leukocyte DNA and in as little as 0.6 infected leukocyte per microliter of blood. Of the 17 patients with suspected HGE, 7 (41%) were serologically confirmed, and six of these seven had positive PCRs (86% sensitivity). Ten seronegative patients had negative PCRs, and no patient had a positive PCR and negative serology (100% specificity). Conclusions: The polymerase chain reaction for the HGE agent 16S rRNA gene is a sensitive and specific method for the early confirmation of HGE at a time when therapeutic decisions are required.

Detection and Identification of Ehrlichia spp. in Ticks Collected in Tunisia and Morocco

A broad-range 16S rRNA gene PCR assay followed by partial sequencing of the 16S rRNA gene was used for the detection of members of the family Anaplasmataceae in ticks in North Africa. A total of 418 questing Ixodes ricinus ticks collected in Tunisia and Morocco, as well as 188 Rhipicephalus ticks from dogs and 52 Hyalomma ticks from bovines in Tunisia, were included in this study. Of 324 adult I. ricinus ticks, 16.3% were positive for Ehrlichia spp., whereas only 3.4 and 2.8% of nymphs and larvae, respectively, were positive. A large heterogeneity was observed in the nucleotide sequences. Partial sequences identical to that of the agent of human granulocytic ehrlichiosis (HGE) were detected in I. ricinus and Hyalomma detritum, whereas partial sequences identical to that of Anaplasma platys were detected in Rhipicephalus sanguineus. However, variants of Anaplasma, provisionally designated Anaplasma-like, were predominant in the I. ricinus tick population in Maghreb. Otherwise, two variants of the genus Ehrlichia were detected in I. ricinus and H. detritum. Surprisingly, a variant of Wolbachia pipientis was evidenced from I. ricinus in Morocco. These results emphasized the potential risk of tick bites for human and animal populations in North Africa.

Sequence Analysis of the msp4 Gene of Anaplasma phagocytophilum Strains

The causative agent of human granulocytic ehrlichiosis was recently reclassified as Anaplasma phagocytophilum, unifying previously described bacteria that cause disease in humans, horses, dogs, and ruminants. For the characterization of genetic heterogeneity in this species, the homologue of Anaplasma marginale major surface protein 4 gene (msp4) was identified, and the coding region was PCR amplified and sequenced from a variety of sources, including 50 samples from the United States, Germany, Poland, Norway, Italy, and Switzerland and 4 samples of A. phagocytophilum-like organisms obtained from white-tailed deer in the United States. Sequence variation between strains of A. phagocytophilum (90 to 100% identity at the nucleotide level and 92 to 100% similarity at the protein level) was higher than in A. marginale. Phylogenetic analyses of msp4 sequences did not provide phylogeographic information but did differentiate strains of A. phagocytophilum obtained from ruminants from those obtained from humans, dogs, and horses. The sequence analysis of the recently discovered A. phagocytophilum msp2 gene corroborated these results. The results reported here suggest that although A. phagocytophilum-like organisms from white-tailed deer may be closely related to A. phagocytophilum, they could be more diverse. These results suggest that A. phagocytophilum strains from ruminants could share some common characteristics, including reservoirs and pathogenicity, which may be different from strains that infect humans.

Tick- and flea-borne rickettsial emerging zoonoses

Between 1984 and 2004, nine more species or subspecies of spotted fever rickettsiae were identified as emerging agents of tick-borne rickettsioses throughout the world. Six of these species had first been isolated from ticks and later found to be pathogenic to humans. The most recent example is Rickettsia parkeri, recognized as a human pathogen more than 60 years after its initial isolation from ticks. A new spotted fever rickettsia, R. felis was also found to be associated with fleas and to be a human pathogen. Similarly, bacteria within the family Anaplasmataceae have been considered to be of veterinary importance only, yet three species have been implicated in human diseases in recent years, including Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis, Anaplasma phagocytophilum, the agent of human anaplasmosis (formerly known as "human granulocytic ehrlichiosis agent", E. equi and E. phagocytophila), and finally Ehrlichia ewingii, which causes granulocytic ehrlichiosis in humans. We present here an overview of the various tick- and flea-borne rickettsial zoonoses described in the last 20 years, focusing on the ecological, epidemiological and clinical aspects.

Acute Clinical, Hematologic, Serologic, and Polymerase Chain Reaction Findings in Horses Experimentally Infected with a European Strain of Anaplasma phagocytophilum

Six horses were experimentally infected by administration of horse blood containing a Swedish strain of Anaplasma phagocytophilum. The polymerase chain reaction (PCR) signal was consistently detected 2-3 days before appearance of clinical signs and persisted 4-9 days beyond abatement of clinical signs, whereas diagnostic inclusion bodies were 1st noted on average 2.6 +/- 1.5 (SD) days after onset of fever. Clinical signs and hematologic changes were largely indistinguishable from those previously reported for diseases caused by A phagocytophilum (formerly Ehrlichia equi--"Californian agent") and the human-derived human granulocytic ehrlichiosis agent. Horses 1st demonstrated antibody response 12-16 days after inoculation, 2 cases of which were still febrile, and serotiters rapidly peaked within 3-7 days of clinical illness. One horse died during the acute stage of disease, but initial clinical signs and hematologic changes were similar to those of other infected horses. This report shows that, despite minor genetic differences, a European equine-derived strain of A. phagocytophilum may be similar in pathogenicity to the Californian agent. The PCR used holds promise to widen the diagnostic window and would also be diagnostic during the initial days of clinical disease when inclusions in neutrophils in blood smears are not yet apparent.

Anaplasma phagocytophilumModulates gp91phoxGene Expression through Altered Interferon Regulatory Factor 1 and PU.1 Levels and Binding of CCAAT Displacement Protein

Infection of neutrophil precursors with Anaplasma phagocytophilum, the causative agent of human granulocytic ehrlichiosis, results in downregulation of the gp91(phox) gene, a key component of NADPH oxidase. We now show that repression of gp91(phox) gene transcription is associated with reduced expression of interferon regulatory factor 1 (IRF-1) and PU.1 in nuclear extracts of A. phagocytophilum-infected cells. Loss of PU.1 and IRF-1 correlated with increased binding of the repressor, CCAAT displacement protein (CDP), to the promoter of the gp91(phox) gene. Reduced protein expression of IRF-1 was observed with or without gamma interferon (IFN-gamma) stimulation, and the defect in IFN-gamma signaling was associated with diminished binding of phosphorylated Stat1 to the Stat1 binding element of the IRF-1 promoter. The diminished levels of activator proteins and enhanced binding of CDP account for the transcriptional inhibition of the gp91(phox) gene during A. phagocytophilum infection, providing evidence of the first molecular mechanism that a pathogen uses to alter the regulation of genes that contribute to an effective respiratory burst.

Seroepidemiology of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in wild mice captured in Northern Turkey

An expedition across the Asian part of the Black Sea coast and national parks of Northern Turkey was organized in the summer of 2001 to investigate the presence of Borrelia burgdorferi sensu lato (s.l.), Lyme borreliosis agent, and Anaplasma phagocytophilum, human granulocytic ehrlichiosis, agent, in wild mice. A total of 65 Apodemus flavicollis, Apodemus sylvaticus, Microtus epiroticus, Crocidura suaveolens and Mus macedonicus, were captured. Two out of 22 Apodemus sylvaticus specimens were seropositive for B. afzelii by enzyme-linked immunosorbent assay as confirmed by Western blotting, however cultures of skin and bladder samples from all small mammals in Barbour-Stoenner-Kelly's medium-II remained negative for B. burgdorferi s.l. All sera tested were negative for Anaplasma phagocytophilum by indirect immunofluorescent assay. The prevalence of B. burgdorferi s.l. and Anaplasma phagocytophilum is low in wild mice of the Asian part of Northern Turkey.

In vivo and in vitro studies on Anaplasma phagocytophilum infection of the myeloid cells of a patient with chronic myelogenous leukaemia and human granulocytic ehrlichiosis

Aims: The occurrence of human granulocytic ehrlichiosis (HGE) in a patient with chronic myelogenous leukaemia (CML) provided an opportunity to study whether Anaplasma phagocytophilum, the aetiological agent of HGE, infects...

Human granulocytic ehrlichiosis in Europe.

Human granulocytic ehrlichiosis (HGE) is a tick-borne zoonosis caused by Anaplasma phagocytophilum. A MEDLINE literature search revealed that the European story of HGE began in 1995 with an article on the presence of serum antibodies to A. phagocytophilum. At present, there is seroepidemiological information indicating the presence of infection with HGE agent(s) for several European countries; seroprevalence rates range from zero or very low to up to 28%. The proportion of seropositive persons increases with age and is higher in persons exposed to ticks. Knowledge of the causative agent of HGE and of animal reservoirs in Europe is limited. Ixodes ricinus is a recognized vector of A. phagocytophilum in Europe. Prevalence of the agent in questing I. ricinus is usually higher in adult ticks than in nymphs and ranges from zero or very low to > 30%. Pronounced differences between countries and marked variability by localities were established. Up to March 2003 about 65 human patients (all but one were adults) with confirmed HGE and several patients fulfilling criteria for probable HGE had been reported. The majority of them came from Central Europe (Slovenia) and Scandinavia (Sweden) but there are individual reports from several other European countries. The patients presented with an acute febrile illness that as a rule occurred after a tick bite; the majority had leukopenia and/or thrombocytopenia, elevated concentration of C-reactive protein and mild abnormalities of liver function test results. A small number of patients does not permit reliable conclusions on the clinical features of European HGE; however, there is an impression that at least in central Europe (but maybe not in Scandinavia) the disease is, from the clinical angle, only mild to moderately severe and (most likely) self-limited. The relatively high proportion of the population with HGE serum antibodies and the presence of A. phagocytophilum (like) agent(s) in ticks, small mammals and deer as found in several European countries are discordant with the rather low number of patients with proven HGE. The discordance may indicate inadequate awareness among European physicians, limited recording and reporting of the disease, and/or the presence of and the infection of humans with nonpathogenic A. phagocytophilum (like) strains present in ticks. Additional studies are needed to better define the biological and public health significance of HGE in Europe.

Detection of Anaplasma phagocytophilum in animals by real-time polymerase chain reaction.

The aim of this study was to detect Anaplasma phagocytophilum in wild and domesticated animals and to identify the phylogenetic relationships of different strains of this bacterium. We adapted six published conventional methods targeting 16S fragments for real-time polymerase chain reaction. Initial screening of samples from 419 animals found 37 Anaplasma positives, later confirmed with several different primers and a TaqMan probe. We also performed DNA quantification and melting curve analysis. The nucleic acid of Anaplasma sp. was detected in a higher percentage of cases in members of the deer family, hares, bank voles and mice (12.5 approximately 15%) than in foxes, boars, cows, and horses (around 4 approximately 6%). We also performed blood analysis of cows, horses, mice, and ticks removed from animals, evaluating the presence of antibodies against granulocytic Anaplasma sp. Finally, we subjected 11 randomly selected PCR amplified products to direct sequencing and we constructed the corresponding phylogenetic tree with respect to the Ehrlichia equi sequence, homologous to the human granulocytic ehrlichiosis agent. Mutual identity of the sequencing ranged from 99% to 100%.

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.