Anaplasma phagocytophilum Hijacks Flotillin and NPC1 Complex To Acquire Intracellular Cholesterol for Proliferation, Which Can Be Inhibited with Ezetimibe.

The intracellular cholesterol transport protein Niemann-Pick type C1 (NPC1) and lipid-raft protein flotillin (FLOT) are required for cholesterol uptake by the obligatory intracellular bacterium Anaplasma phagocytophilum and for infection, and each protein localizes to membrane-bound inclusions containing replicating bacteria. Here, we found striking localization of FLOT2 in NPC1-lined vesicles and a physical interaction between FLOT2 and NPC1. This interaction was cholesterol dependent, as a CRAC (cholesterol recognition/interaction amino acid cholesterol-binding) domain mutant of FLOT2 did not interact with NPC1, and the cholesterol-sequestering agent methyl-ß-cyclodextrin reduced the interaction. The stomatin-prohibitin-flotillin-HflC/K domain of FLOT2, FLOT21-183, was sufficient for the unique FLOT2 localization and interaction with NPC1. NPC1, FLOT2, and FLOT21-183 trafficked to the lumen of Anaplasma inclusions. A loss-of-function mutant, NPC1P691S (mutation in the sterol-sensing domain), did not colocalize or interact with FLOT2 or with Anaplasma inclusions and inhibited infection. Ezetimibe is a drug that blocks cholesterol absorption in the small intestine by inhibiting plasma membrane Niemann-Pick C1-like 1 interaction with FLOTs. Ezetimibe blocked the interaction between NPC1 and FLOT2 and inhibited Anaplasma infection. Ezetimibe did not directly inhibit Anaplasma proliferation but inhibited host membrane lipid and cholesterol traffic to the bacteria in the inclusion. These data suggest that Anaplasma hijacks NPC1 vesicles containing cholesterol bound to FLOT2 to deliver cholesterol into Anaplasma inclusions to assimilate cholesterol for its proliferation. These results provide insights into mechanisms of intracellular cholesterol transport and a potential approach to inhibit Anaplasma infection by blocking cholesterol delivery into the lumen of bacterial inclusions. IMPORTANCE Cholesterol influences membrane fluidity and forms membrane microdomains called lipid rafts that serve as organizing centers for the assembly of signaling molecules. Flotillin (FLOT) is a cholesterol-binding lipid-raft protein. The cholesterol-binding membrane glycoprotein Niemann-Pick type C1 (NPC1) is critical for managing cellular cholesterol level and its intracellular transport, and mutation of the gene encoding NPC1 causes the fatal cholesterol storage disease, Niemann-Pick disease, type C. Both FLOT and NPC1 are trafficked to inclusions created by the cholesterol-dependent bacterium Anaplasma phagocytophilum and required for cholesterol uptake by this bacterium for replication. Our novel findings that FLOT2 interacts physically with NPC1 and resides inside both bacterial inclusions and NPC1-containing vesicles underscore the important role for FLOT2 in infection, the intracellular transport of cholesterol in NPC1 vesicles, and cholesterol homeostasis. Both NPC1-FLOT2 interaction and A. phagocytophilum infection can be inhibited by ezetimibe, suggesting possible pharmacological intervention of intracellular cholesterol hijacking by Anaplasma.

Functional inhibition or genetic deletion of acid sphingomyelinase bacteriostatically inhibits Anaplasma phagocytophilum infection in vivo.

Anaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis. It poorly infects mice deficient in acid sphingomyelinase (ASM), a lysosomal enzyme critical for cholesterol efflux, and wild-type mice treated with desipramine that functionally inhibits ASM. Whether inhibition or genetic deletion of ASM is bacteriostatic or bactericidal for A. phagocytophilum and desipramine's ability to lower pathogen burden requires a competent immune system were unknown. Anaplasma phagocytophilum-infected severe combined immunodeficiency disorder (SCID) mice were administered desipramine or PBS, followed by the transfer of blood to naïve wild-type mice. Next, infected wild-type mice were given desipramine or PBS followed by transfer of blood to naïve SCID mice. Finally, wild-type or ASM-deficient mice were infected and blood transferred to naïve SCID mice. The percentage of infected neutrophils was significantly reduced in all desipramine-treated or ASM-deficient mice and in all recipients of blood from these mice. Infection was markedly lower in ASM-deficient and desipramine-treated wild-type mice versus desipramine-treated SCID mice. Yet, infection was never ablated. Thus, ASM activity contributes to optimal A. phagocytophilum infection in vivo, pharmacologic inhibition or genetic deletion of ASM impairs infection in a bacteriostatic and reversible manner and A. phagocytophilum is capable of co-opting ASM-independent lipid sources.

Molecular diagnosis of autochthonous human anaplasmosis in Austria - an infectious diseases case report.

BACKGROUND: The diagnosis of human anaplasmosis remains elusive and is probably often missed. This case report highlights the efficacy of molecular diagnostic techniques. CASE PRESENTATION: We would like to report the case of a 74-year-old man who was admitted to hospital because of a high fever, marked chills, transient diplopic images and vertigo, 6 weeks after multiple tick bites. The laboratory results showed mild anemia, marked thrombocytopenia and leukopenia and a moderately elevated C-reactive protein. The initial serology seemed to indicate an active infection with Borrelia spp., and Anaplasma phagocytophilum was detected in peripheral blood by polymerase chain reaction (PCR) and subsequent sequencing. The patient received intravenous ceftriaxone for 14 days and oral doxycycline for 4 weeks and made a fast and complete recovery. CONCLUSIONS: While human anaplasmosis has been reported very rarely in Austria, it should be considered as a differential diagnosis in febrile patients with low leukocyte and platelet counts with elevated levels of C-reactive protein after exposure to tick bites. Molecular detection of A. phagocytophilum is the technique of choice allowing rapid and reliable diagnosis.

A review on the eco-epidemiology and clinical management of human granulocytic anaplasmosis and its agent in Europe.

Anaplasma phagocytophilum is the agent of tick-borne fever, equine, canine and human granulocytic anaplasmosis. The common route of A. phagocytophilum transmission is through a tick bite, the main vector in Europe being Ixodes ricinus. Despite the apparently ubiquitous presence of the pathogen A. phagocytophilum in ticks and various wild and domestic animals from Europe, up to date published clinical cases of human granulocytic anaplasmosis (HGA) remain rare compared to the worldwide status. It is unclear if this reflects the epidemiological dynamics of the human infection in Europe or if the disease is underdiagnosed or underreported. Epidemiologic studies in Europe have suggested an increased occupational risk of infection for forestry workers, hunters, veterinarians, and farmers with a tick-bite history and living in endemic areas. Although the overall genetic diversity of A. phagocytophilum in Europe is higher than in the USA, the strains responsible for the human infections are related on both continents. However, the study of the genetic variability and assessment of the difference of pathogenicity and infectivity between strains to various hosts has been insufficiently explored to date. Most of the European HGA cases presented as a mild infection, common clinical signs being pyrexia, headache, myalgia and arthralgia. The diagnosis of HGA in the USA was recommended to be based on clinical signs and the patient's history and later confirmed using specialized laboratory tests. However, in Europe since the majority of cases are presenting as mild infection, laboratory tests may be performed before the treatment in order to avoid antibiotic overuse. The drug of choice for HGA is doxycycline and because of potential for serious complication the treatment should be instituted on clinical suspicion alone.

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

BACKGROUND: Various 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. METHODS: Data 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. RESULTS: All 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. CONCLUSIONS: In 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.

Doxycycline treatment efficacy in dogs with naturally occurring Anaplasma phagocytophilum infection.

OBJECTIVES: To evaluate doxycycline treatment efficacy and post-treatment pathogen persistence in dogs naturally infected with Anaplasma phagocytophilum in endemic regions of the USA. MATERIALS AND METHODS: Symptomatic dogs in four US states (MN, WI, CT and CA) were evaluated before treatment with doxycycline and approximately 30 and 60 days post-treatment. Clinicopathological parameters, co-exposures and A. phagocytophilum DNA in whole blood and lymph node samples were compared between A. phagocytophilum infected and uninfected dogs. RESULTS: In total, 42 dogs fulfilled the inclusion criteria, with 16 dogs (38%) blood PCR-positive and 26 dogs (62%) blood PCR-negative for A. phagocytophilum. At initial evaluation, the proportion of clinicopathological abnormalities was similar between A. phagocytophilum infected and uninfected dogs, although thrombocytopenia and lymphopenia were statistically more prevalent among A. phagocytophilum infected dogs. Treatment with doxycycline resulted in resolution of all clinical abnormalities in infected dogs; four dogs had persistent haematological abnormalities, including mild leukopenia, eosinopenia and lymphopenia. All 16 infected dogs became blood PCR-negative approximately 30 and 60 days after treatment onset. Additionally, 13/13 (100%) lymph node specimens tested post-treatment were PCR-negative. Select clinicopathological abnormalities persisted in uninfected dogs after treatment. CLINICAL SIGNIFICANCE: The results of this study support the efficacy of doxycycline therapy for clinical treatment of dogs naturally infected with A. phagocytophilum in the USA. This study did not find clinical, haematological or microbiological indicators that supported the persistence of A. phagocytophilum infection in naturally infected dogs following treatment with doxycycline for 28 days.

Ixodes scapularis Tick Cells Control Anaplasma phagocytophilum Infection by Increasing the Synthesis of Phosphoenolpyruvate from Tyrosine.

The obligate intracellular pathogen, Anaplasma phagocytophilum, is the causative agent of life-threatening diseases in humans and animals. A. phagocytophilum is an emerging tick-borne pathogen in the United States, Europe, Africa and Asia, with increasing numbers of infected people and animals every year. It is increasingly recognized that intracellular pathogens modify host cell metabolic pathways to increase infection and transmission in both vertebrate and invertebrate hosts. Recent reports have shown that amino acids are central to the host-pathogen metabolic interaction. In this study, a genome-wide search for components of amino acid metabolic pathways was performed in Ixodes scapularis, the main tick vector of A. phagocytophilum in the United States, for which the genome was recently published. The enzymes involved in the synthesis and degradation pathways of the twenty amino acids were identified. Then, the available transcriptomics and proteomics data was used to characterize the mRNA and protein levels of I. scapularis amino acid metabolic pathway components in response to A. phagocytophilum infection of tick tissues and ISE6 tick cells. Our analysis was focused on the interplay between carbohydrate and amino acid metabolism during A. phagocytophilum infection in ISE6 cells. The results showed that tick cells increase the synthesis of phosphoenolpyruvate (PEP) from tyrosine to control A. phagocytophilum infection. Metabolic pathway analysis suggested that this is achieved by (i) increasing the transcript and protein levels of mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), (ii) shunting tyrosine into the tricarboxylic acid (TCA) cycle to increase fumarate and oxaloacetate which will be converted into PEP by PEPCK-M, and (iii) blocking all the pathways that use PEP downstream gluconeogenesis (i.e., de novo serine synthesis pathway (SSP), glyceroneogenesis and gluconeogenesis). While sequestering host PEP may be critical for this bacterium because it cannot actively carry out glycolysis to produce PEP, excess of this metabolite may be toxic for A. phagocytophilum. The present work provides a more comprehensive view of the major amino acid metabolic pathways involved in the response to pathogen infection in ticks, and provides the basis for further studies to develop novel strategies for the control of granulocytic anaplasmosis.

Prophylactic treatment with flumethrin, a pyrethroid (Bayticol(®), Bayer), against Anaplasma phagocytophilum infection in lambs.

BACKGROUND: [corrected] Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila) causes the disease tick-borne fever (TBF) in domestic ruminants and has for decades been one of the main scourges for the sheep industry in the coastal areas of Norway. Current control strategies are based on reduction of tick infestation by chemical acaricides. METHODS: In the present study, we investigated if frequent pour-on applications of pyrethroids would reduce tick infestion rate and seroprevalence of A. phagocytophilum infection in sheep. Forty lambs, one month old, of the Norwegian White Sheep breed were used. The lambs belonged to the experimental sheep flock at the Department of Production Animal Clinical Sciences. None of the lambs had been on I. ricinus infested pasture before turnout (day 0). All lambs were twins and twenty lambs were treated with a pour-on pyrethroid (Bayticol(®), Bayer A/S, DK-2300) with a dose of 5 ml on days 0, 14, 28, 42, 56, 70, 84, 98, 112 and 128. Twenty lambs were untreated controls. The lambs were collected every fourteen days on pasture for treatment. In addition, the lambs were examined for ticks, blood sampled, weighed, and rectal temperature was recorded. RESULTS AND CONCLUSION: A significant reduction in tick infestion rate was detected on treated lambs. However, the present results indicate that frequent acaricide treatment does not reduce the seroprevalence to A. phagocytophilum on tick-infested pasture.

Subversion of NPC1 pathway of cholesterol transport by Anaplasma phagocytophilum.

Intracellular cholesterol amounts, distribution and traffic are tightly regulated to maintain the healthy eukaryotic cell function. However, how intracellular pathogens that require cholesterol, interact with the host cholesterol homeostasis and traffic is not well understood. Anaplasma phagocytophilum is an obligatory intracellular and cholesterol-robbing bacterium, which causes human granulocytic anaplasmosis. Here we found that a subset of cholesterol-binding membrane protein, Niemann-Pick type C1 (NPC1)-bearing vesicles devoid of lysosomal markers were upregulated in HL-60 cells infected with A. phagocytophilum, and trafficked to live bacterial inclusions. The NPC1 localization to A. phagocytophilum inclusions was abolished by low-density lipoprotein (LDL)-derived cholesterol traffic inhibitor U18666A. Studies using NPC1 siRNA and the cell line with cholesterol traffic defect demonstrated that the NPC1 function is required for bacterial cholesterol acquisition and infection. Furthermore, trans-Golgi network-specific soluble N-ethylmaleimide-sensitive factor attachment protein receptors, vesicle-associated membrane protein (VAMP4) and syntaxin 16, which are associated with NPC1 and LDL-derived cholesterol vesicular transport were recruited to A. phagocytophilum inclusions, and VAMP4 was required for bacteria infection. Taken together, A. phagocytophilum is the first example of a pathogen that subverts the NPC1 pathway of intracellular cholesterol transport and homeostasis for bacterial inclusion membrane biogenesis and cholesterol capture.

Elimination of Borrelia burgdorferi and Anaplasma phagocytophilum in rodent reservoirs and Ixodes scapularis ticks using a doxycycline hyclate-laden bait.

A field trial was conducted in a Lyme disease-endemic area of New Jersey to determine the efficacy of a doxycyline hyclate rodent bait to prophylactically protect and cure small-mammal reservoirs and reduce infection rates in questing Ixodes scapularis ticks for Borrelia burgdorferi and Anaplasma phagocytophilum. The doxycycline-laden bait was formulated at a concentration of 500 mg/kg and delivered during the immature tick feeding season in rodent-targeted bait boxes. The percentage of infected small mammals recovered from treated areas after 2 years of treatment was reduced by 86.9% for B. burgdorferi and 74% for A. phagocytophilum. Infection rates in questing nymphal ticks for both B. burgdorferi and A. phagocytophilum were reduced by 94.3% and 92%, respectively. Results from this study indicate that doxycycline-impregnated bait is an effective means of reducing infection rates for B. burgdorferi and A. phagocytophilum in both rodent reservoirs and questing I. scapularis ticks.

The clinical characteristics and outcomes of patients with human granulocytic anaplasmosis in China.

OBJECTIVES: The incidence of human granulocytic anaplasmosis (HGA), a tick-borne disease caused by the obligate intracellular bacterium Anaplasma phagocytophilum, has increased across the world. However, information on HGA is lacking in China. The purpose of this study was to investigate the clinical features and outcomes of HGA patients in China. METHODS: A total of 83 patients with HGA from the provinces of Hubei and Henan in China, who were admitted to Union Hospital between March 2009 and September 2010, were included in this study. We investigated the epidemiology, clinical features, laboratory markers, and therapeutic effects in these patients. We also analyzed life-threatening complications such as systemic inflammatory response syndrome (SIRS)/multiple organ dysfunction syndrome (MODS) following HGA and assessed the risk factors for a poor clinical outcome. RESULTS: In our study, an HGA outbreak peak was observed for the months May to August. The highest age-specific incidence occurred among the group of patients aged 50-59 years. With regard to patient occupation and pathological origin, we found that 73 of the 83 patients with HGA had a peasant occupation. With respect to symptoms, 45 patients had no complications and 38 patients diagnosed with HGA met SIRS criteria, of whom 25 rapidly developed MODS. The mortality for the entire cohort was 26.5%. The factors predictive of patients developing MODS and an adverse outcome were advanced age, disturbance of consciousness, highly elevated lactate dehydrogenase, creatinine, and aspartate aminotransferase levels, and the presence of SIRS. Moreover, MODS was found to be an independent predictor of death. CONCLUSIONS: In China, HGA patients had severe clinical symptoms and high rates of complications and mortality. These findings may provide useful information so that physicians will be on the alert for severe complications after a diagnosis of HGA; they will also be useful for optimizing supportive care for HGA-related critical illness. Prompt treatment and close monitoring of severe complications such as SIRS and MODS are of great importance in saving patient lives.

The prenylation inhibitor manumycin A reduces the viability of Anaplasma phagocytophilum.

Anaplasma phagocytophilum is an obligately intracellular bacterium and is the causative agent of human granulocytic anaplasmosis (HGA), an emerging and major tick-borne disease in the USA and other parts of the world. This study showed that the prenylation inhibitor manumycin A effectively blocked A. phagocytophilum infection in host cells (HL-60 or RF/6A cells). A. phagocytophilum infection activated extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase in host cells, and manumycin A treatment reduced ERK activation in A. phagocytophilum-infected host cells. As ERK activation is required for A. phagocytophilum infection, we examined whether manumycin A inhibited the bacteria directly or through host ERK signalling. Treatment of A. phagocytophilum alone with manumycin A significantly reduced the bacterial infectivity of host cells and bacterial viability in the absence of host cells, whereas pre-treatment of host cells did not inhibit bacterial infection in host cells. The inhibitory effect of manumycin A on A. phagocytophilum infection in host cells was achieved even at a concentration 100 times lower than that required for effective inhibition of mammalian cell signalling. These results suggested that manumycin A directly inactivates the bacterium, resulting in reduced infection and ERK1/2 activation. Thus, the manumycin group of drugs may have a therapeutic potential for HGA.

In-vitro studies on the susceptibility of ovine strains of Anaplasma phagocytophilum to antimicrobial agents and to immune serum.

Five strains of Anaplasma phagocytophilum, the causative agent of tick-borne fever of sheep and cattle and human granulocytic anaplasmosis, were tested in vitro for their susceptibility to nine antibiotics using the continuous tick cell line ISE6. Minimum inhibitory concentrations (MICs) were evaluated by comparing the percentage of infection of Giemsa-stained antibiotic-treated Anaplasma phagocytophilum-infected cells with that of untreated controls after 6 days of culture. The minimum bactericidal concentrations (MBCs) were evaluated after washing infected cells with antibiotic-free medium and further incubation of 6 days before comparing the percentage of infection of Giemsa-stained antibiotic-treated and untreated cells and by comparing the number of copies of the p44 gene by real-time polymerase chain reaction using p44-specific primers. The tick cell culture system was also used to assay the possible neutralizing effects of immune serum on cell-free bacteria in vitro. The neutralizing effects of immune serum were evaluated by comparing the number of copies of the p44 gene in samples inoculated with cell-free bacteria after 1h incubation with two-fold dilutions of immune sera obtained 21 days after infection with those inoculated with cell-free bacteria after incubation for 1h with two-fold dilutions of sera obtained before infection. Doxycycline, rifampin and ciprofloxacin were the most effective compounds, with MICs of 0.125 microg/ml, 0.5 mg/ml and 1 microg/ml, respectively. There was total resistance to penicillin, ampicillin, ceftriaxone and streptomycin and only very limited susceptibility to gentamycin and chloramphenicol. Both doxycycline and rifampin were also bactericidal at the same concentrations. Exposure of bacteria to immune ovine sera resulted in significant reductions of the number of copies of p44 gene.

A sustained-release formulation of doxycycline hyclate (Atridox) prevents simultaneous infection of Anaplasma phagocytophilum and Borrelia burgdorferi transmitted by tick bite.

Current prophylaxis for infected tick bites consists of personal protective measures directed towards ticks. This study compared the efficacy of a single oral dose of doxycycline with that of a single injection of sustained-release doxycycline in a model of Lyme borreliosis and Anaplasma phagocytophilum infection. Dosages of doxycycline were equilibrated based on previously determined peak plasma levels in mice [oral, 2.4 microg (ml plasma)(-1); sustained release, 1.9 microg (ml plasma)(-1)] determined 8 h after inoculation. In challenge experiments where five Borrelia burgdorferi-infected and five A. phagocytophilum-infected nymphs were used per mouse, only 20 and 30 % of mice were protected from B. burgdorferi and A. phagocytophilum infection, respectively, using oral doxycycline. In contrast, 100 % of mice receiving sustained-release doxycycline were protected from A. phagocytophilum infection, as indicated by real-time PCR of blood samples, quantitative PCR and culture isolation of spleen samples, and protected against B. burgdorferi infection as demonstrated by culture of ear, heart and bladder. Although 15-40 copies of A. phagocytophilum could be amplified from the spleens of mice treated with sustained-release doxycycline, no viable A. phagocytophilum from these spleens could be cultured in HL-60 cells. In contrast, 7/10 mice receiving oral doxycycline were PCR- and culture-positive for A. phagocytophilum, with copy numbers ranging from 800 to 10 000 within the spleen, as determined by quantitative PCR. Other correlates with A. phagocytophilum infection included a significant difference in spleen mass (mean of 110 mg for sustained-release treatment versus a mean of 230 mg for oral treatment) and the number of splenic lymphoid nodules (mean of 8 for sustained-release treatment versus mean of 12.5 for oral doxycycline) as determined by histopathology. These studies indicate that a single injection of a sustained-release formulation antibiotic may offer a viable prophylactic treatment option for multiple infectious agents in patients presenting with tick bites.

Human granulocytic anaplasmosis during pregnancy: case series and literature review.

We describe the clinical and laboratory manifestations and pregnancy outcomes of 6 women who received a diagnosis of human granulocytic ehrlichiosis during pregnancy. Human granulocytic ehrlichiosis did not seem to present in a fulminant fashion, and all treated patients had excellent responses to rifampin or doxycycline therapy. Perinatal transmission was documented in 1 neonate, who responded well to treatment. There do not appear to be any long-term adverse sequelae in children born from these pregnancies (mean follow-up duration, 21 months).

Dissociation between inhibition and killing by levofloxacin in human granulocytic anaplasmosis.

Human granulocytic anaplasmosis (HGA) is a potentially fatal tick-borne infection caused by Anaplasma phagocytophilum. Treatment options are limited for this entity, with doxycycline being the drug of choice. Certain fluoroquinolones such as levofloxacin are active against A. phagocytophilum in vitro. We report a hospitalized patient with HGA who improved coincident with a 13-day course of levofloxacin therapy, but clinically and microbiologically relapsed 15 days after completion of treatment. Relapse of infection after levofloxacin therapy was reproduced in a severe combined immune-deficient (SCID) mouse infection model. Quinolone therapy should not be considered curative of HGA.

Intra-leukocyte expression of two-component systems in Ehrlichia chaffeensis and Anaplasma phagocytophilum and effects of the histidine kinase inhibitor closantel.

The two-component system (TCS) composed of a pair of a sensor histidine kinase and a response regulator, allows bacteria to sense signals and respond to changes in their environment through specific gene activation or repression. The present study examined TCS in the obligatory intracellular bacteria Ehrlichia chaffeensis and Anaplasma phagocytophilum, that cause human monocytic ehrlichiosis (HME) and human granulocytic anaplasmosis (HGA) respectively. The genomes of E. chaffeensis and A. phagocytophilum were each predicted to encode three pairs of TCSs. All six genes encoding three histidine kinases and three response regulators were expressed in both E. chaffeensis and A. phagocytophilum cultured in human leukocytes. Pretreatment of host cell-free E. chaffeensis or A. phagocytophilum with closantel, an inhibitor of histidine kinases, completely blocked the infection of host cells. Treatment of infected cells 1 day post infection with closantel cleared infection in dose-dependent manner. All six genes in E. chaffeensis were cloned, recombinant proteins were expressed, and polyclonal antibodies were produced. Double immunofluorescence labelling and Western blot analysis revealed that all six proteins were expressed in cell culture. Autokinase activities of the three recombinant histidine kinases from E. chaffeensis were inhibited by closantel in vitro. A number of E. chaffeensis genes, including the six TCS genes, were downregulated within 5-60 min post closantel treatment. These results suggest that these TCSs play an essential role in infection and survival of E. chaffeensis and A. phagocytophilum in human leukocytes.

Prophylactic use of sustained-release doxycycline blocks tick-transmitted infection by Anaplasma phagocytophilum in a murine model.

A sustained-release formulation of doxycycline hyclate was tested for its ability to block Anaplasma phagocytophilum infection in mice. Mice treated with sustained-release doxycycline showed no splenomegaly and their blood samples were negative by PCR on days 7, 14, and 21. Control mice treated with either oral doxycycline or water had significant splenomegaly and were PCR positive at multiple time points. The sustained-release doxycycline formulation was shown to be efficacious for preventing tick-transmitted A. phagocytophilum infection in a mouse model.

Evaluation of antibiotic susceptibilities of Ehrlichia canis, Ehrlichia chaffeensis, and Anaplasma phagocytophilum by real-time PCR.

We determined MICs of antibiotics against Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia canis by real-time quantitative PCR. The doubling times of the organisms were established: 19 h for E. chaffeensis, 26 h for A. phagocytophilum, and 28 h for E. canis. In comparison to the reference method for determining sensitivities, which uses Diff-Quick staining, our PCR assay was very sensitive and specific. We confirmed that doxycycline and rifampin are highly active against these bacteria and found variable susceptibilities to fluoroquinolones; A. phagocytophilum was susceptible, but E. canis and E. chaffeensis were only partly susceptible. Beta-lactam compounds, cotrimoxazole, macrolide compounds, and telithromycin showed no activity against any of the three organisms. Thiamphenicol was found to be more active than chloramphenicol. For the first time, we showed that these three species have numerous point mutations in their 23S RNA genes, with those at positions 754, 2057, 2058, 2059, and 2611 (Escherichia coli numbering) known to confer resistance to macrolide compounds in other bacteria. The role of each of these mutations in resistance to these drugs should be investigated in the future. Our study confirms previous reports that quantitative PCR is a reliable method for determining antibiotic susceptibility; therefore, it might be useful for screening new drugs.

New real-time PCR-based method for in vitro susceptibility testing of Anaplasma phagocytophilum against antimicrobial agents.

Up to now, only a few isolates of Anaplasma phagocytophilum have been tested for their susceptibility against a small number of antimicrobial agents. In addition, as with other fastidious or intracellular bacteria, the test methods are laborious and neither minimal inhibitory concentration (MIC) definitions, nor the test conditions and the inocula are standardised to date. A new 16S-rDNA-based real-time PCR assay has been developed and used under standardised conditions to analyse the activity of seven antimicrobial agents against two A. phagocytophilum isolates. After 72 h incubation, MICs were determined by software-assisted calculation of bacterial growth in samples and controls from semi-quantitative PCR results. In our study, the rank order of potency on a mg/l basis for the antimicrobial agents with enhanced in vitro activity against A. phagocytophilum was moxifloxacin (MIC: < or = 0.03 mg/l) > doxycycline (MIC: < or = 0.125 mg/l) > ciprofloxacin (MIC: 0.125 mg/l). Gentamicin, ampicillin, azithromycin and cethromycin showed no activity against the isolates tested in this investigation. Our new 16S-rDNA-PCR-based microdilution test system was shown to be sensitive, reproducible and reliable. The assay is capable of testing larger numbers of isolates and antimicrobial agents under standardised and very precise test conditions and may therefore offer a competent technical solution of the difficulties known to be associated with in vitro testing of other bacterial pathogens that grow intracellularly, such as chlamydia or rickettsia.