Liver Is a Generative Site for the B Cell Response to Ehrlichia muris.

The B cell response to Ehrlichia muris is dominated by plasmablasts (PBs), with few-if any-germinal centers (GCs), yet it generates protective immunoglobulin M (IgM) memory B cells (MBCs) that express the transcription factor T-bet and harbor V-region mutations. Because Ehrlichia prominently infects the liver, we investigated the nature of liver B cell response and that of the spleen. B cells within infected livers proliferated and underwent somatic hypermutation (SHM). Vh-region sequencing revealed trafficking of clones between the spleen and liver and often subsequent local clonal expansion and intraparenchymal localization of T-bet+ MBCs. T-bet+ MBCs expressed MBC subset markers CD80 and PD-L2. Many T-bet+ MBCs lacked CD11b or CD11c expression but had marginal zone (MZ) B cell phenotypes and colonized the splenic MZ, revealing T-bet+ MBC plasticity. Hence, liver and spleen are generative sites of B cell responses, and they include V-region mutation and result in liver MBC localization.

Evasion of host antioxidative response via disruption of NRF2 signaling in fatal Ehrlichia-induced liver injury.

Ehrlichia is Gram negative obligate intracellular bacterium that cause human monocytotropic ehrlichiosis (HME). HME is characterized by acute liver damage and inflammation that may progress to fatal toxic shock. We previously showed that fatal ehrlichiosis is due to deleterious activation of inflammasome pathways, which causes excessive inflammation and liver injury. Mammalian cells have developed mechanisms to control oxidative stress via regulation of nuclear factor erythroid 2 related 2 (NRF2) signaling. However, the contribution of NRF2 signaling to Ehrlichia-induced inflammasome activation and liver damage remains elusive. In this study, we investigated the contribution of NRF2 signaling in hepatocytes (HCs) to the pathogenesis of Ehrlichia-induced liver injury following infection with virulent Ixodes ovatus Ehrlichia (IOE, AKA E. japonica). Employing murine model of fatal ehrlichiosis, we found that virulent IOE inhibited NRF2 signaling in liver tissue of infected mice and in HCs as evidenced by downregulation of NRF2 expression, and downstream target GPX4, as well as decreased NRF2 nuclear translocation, a key step in NRF2 activation. This was associated with activation of non-canonical inflammasomes pathway marked by activation of caspase 11, accumulation of reactive oxygen species (ROS), mitochondrial dysfunction, and endoplasmic reticulum (ER) stress. Mechanistically, treatment of IOE-infected HCs with the antioxidant 3H-1,2-Dithiole-3-Thione (D3T), that induces NRF2 activation, attenuated oxidative stress and caspase 11 activation, as well as restored cell viability. Importantly, treatment of IOE-infected mice with D3T resulted in attenuated liver pathology, decreased inflammation, enhanced bacterial clearance, prolonged survival, and resistance to fatal ehrlichiosis. Our study reveals, for the first time, that targeting anti-oxidative signaling pathway is a key approach in the treatment of severe and potential Ehrlichia-induced acute liver injury and sepsis.

Changes in the Seroprevalence of Tick-Borne Rickettsia and Ehrlichia Among Soldiers-Fort Liberty, North Carolina, 1991-2019.

We obtained samples from the Department of Defense Serum Repository from soldiers who were stationed at Fort Liberty, North Carolina, between 1991 and 2019 to assess temporal trends in tick-borne rickettsiosis and ehrlichiosis. Serological evidence of infection was common, with nearly 1 in 5 (18.9%) demonstrating antibodies. We observed significant decreases in Rickettsia seroprevalence (adjusted odds ratio [aOR], 0.42 [95% CI, .27-.65], P = .0001) while over the same period Ehrlichia seroprevalence, albeit less common, nearly doubled (aOR, 3.61 [95% CI, 1.10-13.99], P = .048). The increase in Ehrlichia seroprevalence likely reflects increased transmission resulting from the expanding geographic range of the lone star tick.

Ehrlichia SLiM ligand mimetic activates Hedgehog signaling to engage a BCL-2 anti-apoptotic cellular program.

Ehrlichia chaffeensis (E. chaffeensis) has evolved eukaryotic ligand mimicry to repurpose multiple cellular signaling pathways for immune evasion. In this investigation, we demonstrate that TRP120 has a novel repetitive short linear motif (SLiM) that activates the evolutionarily conserved Hedgehog (Hh) signaling pathway to inhibit apoptosis. In silico analysis revealed that TRP120 has sequence and functional similarity with Hh ligands and a candidate Hh ligand SLiM was identified. siRNA knockdown of Hh signaling and transcriptional components significantly reduced infection. Co-immunoprecipitation and surface plasmon resonance demonstrated that rTRP120-TR interacted directly with Hh receptor Patched-2 (PTCH2). E. chaffeensis infection resulted in early upregulation of Hh transcription factor GLI-1 and regulation of Hh target genes. Moreover, soluble recombinant TRP120 (rTRP120) activated Hh and induced gene expression consistent with the eukaryotic Hh ligand. The TRP120-Hh-SLiM (NPEVLIKD) induced nuclear translocation of GLI-1 in THP-1 cells and primary human monocytes and induced a rapid and expansive activation of Hh pathway target genes. Furthermore, Hh activation was blocked by an α-TRP120-Hh-SLiM antibody. TRP120-Hh-SLiM significantly increased levels of Hh target, anti-apoptotic protein B-cell lymphoma 2 (BCL-2), and siRNA knockdown of BCL-2 dramatically inhibited infection. Blocking Hh signaling with the inhibitor Vismodegib, induced a pro-apoptotic cellular program defined by decreased mitochondria membrane potential, significant reductions in BCL-2, activation of caspase 3 and 9, and increased apoptotic cells. This study reveals a novel E. chaffeensis SLiM ligand mimetic that activates Hh signaling to maintain E. chaffeensis infection by engaging a BCL-2 anti-apoptotic cellular program.

Dissecting the impact of Anaplasma phagocytophilum infection on functional networks and community stability of the tick microbiome.

CONTEXT: Pathogens can manipulate microbial interactions to ensure survival, potentially altering the functional patterns and microbiome assembly. The present study investigates how Anaplasma phagocytophilum infection affects the functional diversity, composition, and assembly of the Ixodes scapularis microbiome, with a focus on high central pathways-those characterized by elevated values in centrality metrics such as eigenvector, betweenness, and degree measures, in the microbial community. METHODS: Using previously published data from nymphs' gut V4 region's amplicons of bacterial 16S rRNA, we predicted the functional diversity and composition in control and A. phagocytophilum-infected ticks and inferred co-occurrence networks of taxa and ubiquitous pathways in each condition to associate the high central pathways to the microbial community assembly. RESULTS: Although no differences were observed concerning pathways richness and diversity, there was a significant impact on taxa and functional assembly when ubiquitous pathways in each condition were filtered. Moreover, a notable shift was observed in the microbiome's high central functions. Specifically, pathways related to the degradation of nucleosides and nucleotides emerged as the most central functions in response to A. phagocytophilum infection. This finding suggests a reconfiguration of functional relationships within the microbial community, potentially influenced by the pathogen's limited metabolic capacity. This limitation implies that the tick microbiome may provide additional metabolic resources to support the pathogen's functional needs. CONCLUSIONS: Understanding the metabolic interactions within the tick microbiome can enhance our knowledge of pathogen colonization mechanisms and uncover new disease control and prevention strategies. For example, certain pathways that were more abundant or highly central during infection may represent potential targets for microbiota-based vaccines.

Neurological manifestations of ehrlichiosis among a cohort of patients: prevalence and clinical symptoms.

BACKGROUND: Ehrlichiosis is a potentially fatal tick-borne disease that can progress to involve the central nervous system (CNS) (i.e., neuro-ehrlichiosis), particularly in cases where diagnosis and treatment are delayed. Despite a six-fold national increase in the incidence of ehrlichiosis over the past 20 years, recent data on the prevalence and manifestations of neuro-ehrlichiosis are lacking. METHODS: We conducted a retrospective chart review of all patients tested for ehrlichiosis at University of North Carolina Health facilities between 2018 and 2021 and identified patients who met epidemiological criteria for ehrlichiosis as established by the Council of State and Territorial Epidemiologists and employed by the Centers for Disease Control and Prevention. We estimated the prevalence of neurological symptoms and described the spectrum of neurological manifestations in acute ehrlichiosis, documenting select patient cases in more detail in a case series. RESULTS: Out of 55 patients with confirmed or probable ehrlichiosis, five patients (9.1%) had neurologic symptoms, which is notably lower than previous estimates. Neurological presentations were highly variable and included confusion, amnesia, seizures, focal neurological deficits mimicking ischemic vascular events, and an isolated cranial nerve palsy, though all patients had unremarkable neuroimaging at time of presentation. All but one patient had risk factors for severe ehrlichiosis (i.e., older age, immunosuppression). CONCLUSIONS: Neuro-ehrlichiosis may lack unifying patterns in clinical presentation that would otherwise aid in diagnosis. Clinicians should maintain a high index of suspicion for neuro-ehrlichiosis in patients with acute febrile illness, diverse neurological symptoms, and negative neuroimaging in lone star tick endemic regions.

Molecular diagnosis and characterization of Anaplasma marginale and Ehrlichia ruminantium infecting beef cattle of Maputo Province, Mozambique.

BACKGROUND: Members of the Anaplasmataceae family, such as the Anaplasma and Ehrlichia species, cause economic losses and public health risks. However, the exact economic impact has not been comprehensively assessed in Mozambique due to limited data available on its basic epidemiology. Therefore, we investigated the molecular occurrence and identity of Anaplasma and Ehrlichia spp. infecting beef cattle in Maputo province, Mozambique. METHODS: A total of 200 whole blood samples were collected from apparently healthy beef cattle. Whole blood DNA was extracted and tested for presence of Anaplasma spp. and Ehrlichia ruminantium DNA through amplification of the 16S rRNA and map1 genes. Positive samples to Anaplasma spp. were subject to PCR assay targeting the A. marginale-msp5 gene. Amplicons obtained were purified, sequenced and subject to phylogenetic analyses. RESULTS: Anaplasma spp., A. marginale and E. ruminantium were detected in 153 (76.5%), 142 (71%) and 19 (9.5%) of all the samples analyzed, respectively. On this same sample group, 19 (9.5%) were co-infected with A. marginale and E. ruminantium. The 16S rRNA sequences of Anaplasma spp. obtained were phylogenetically related to A. marginale, A. centrale and A. platys. Phylogenetic analysis revealed that A. marginale-msp5 nucleotide sequences were grouped with sequences from Asia, Africa and Latin America, whereas E. ruminantium-map1 DNA nucleotide sequences were positioned in multiple clusters. CONCLUSION: Cattle in Maputo Province are reservoirs for multiple Anaplasma species. A high positivity rate of infection by A. marginale was observed, as well as high genetic diversity of E. ruminantium. Furthermore, five new genotypes of E. ruminantium-map1 were identified.

Surveillance of tick-borne pathogens in domestic dogs from Chad, Africa.

BACKGROUND: Tick-borne pathogens are understudied among domestic animals in sub-Saharan Africa but represent significant threats to the health of domestic animals and humans. Specifically, additional data are needed on tick-borne pathogens in Chad, Africa. Surveillance was conducted among domestic dogs in Chad for selected tick-borne pathogens to measure (1) the prevalence of antibodies against Anaplasma spp., Borrelia burgdorferi, and Ehrlichia spp.; (2) the prevalence of infections caused by Hepatozoon spp., Ehrlichia canis, Anaplasma platys, and Babesia spp.; and (3) associations of pathogens with demographic, spatial, and temporal factors. Blood samples were collected from domestic dogs at three time points (May 2019, November 2019, June 2020) across 23 villages in southern Chad. RESULTS: Of the 428 dogs tested with the IDEXX SNAP 4Dx test in May 2019, 86% (n = 370, 95% CI = 83-90%) were positive for antibodies to Ehrlichia spp., 21% (n = 88, 95% CI = 17-25%) were positive for antibodies to Anaplasma spp., and 0.7% (n = 3, 95% CI = 0.1-2%) were positive for antibodies to Borrelia burgdorferi. Four different pathogens were detected via PCR. Hepatozoon spp. were most commonly detected (67.2-93.4%, depending on the time point of sampling), followed by E. canis (7.0-27.8%), A. platys (10.1-22.0%), and Babesia vogeli (0.4-1.9%). Dogs were coinfected with up to three pathogens at a single time point, and coinfections were most common in May 2019 compared to November 2019 and May 2020. CONCLUSIONS: Overall, this study provides new data about the epidemiology of tick-borne pathogens in domestic dogs in Chad, with potential implications for dog and human health.

An intracellular nanobody targeting T4SS effector inhibits Ehrlichia infection.

Infection with obligatory intracellular bacteria is difficult to treat, as intracellular targets and delivery methods of therapeutics are not well known. Ehrlichia translocated factor-1 (Etf-1), a type IV secretion system (T4SS) effector, is a primary virulence factor for an obligatory intracellular bacterium, Ehrlichia chaffeensis In this study, we developed Etf-1-specific nanobodies (Nbs) by immunizing a llama to determine if intracellular Nbs block Etf-1 functions and Ehrlichia infection. Of 24 distinct anti-Etf-1 Nbs, NbD7 blocked mitochondrial localization of Etf-1-GFP in cotransfected cells. NbD7 and control Nb (NbD3) bound to different regions of Etf-1. Size-exclusion chromatography showed that the NbD7 and Etf-1 complex was more stable than the NbD3 and Etf-1 complex. Intracellular expression of NbD7 inhibited three activities of Etf-1 and E. chaffeensis: up-regulation of mitochondrial manganese superoxide dismutase, reduction of intracellular reactive oxygen species, and inhibition of cellular apoptosis. Consequently, intracellular NbD7 inhibited Ehrlichia infection, whereas NbD3 did not. To safely and effectively deliver Nbs into the host cell cytoplasm, NbD7 was conjugated to cyclized cell-permeable peptide 12 (CPP12-NbD7). CPP12-NbD7 effectively entered mammalian cells and abrogated the blockade of cellular apoptosis caused by E. chaffeensis and inhibited infection by E. chaffeensis in cell culture and in a severe combined-immunodeficiency mouse model. Our results demonstrate the development of an Nb that interferes with T4SS effector functions and intracellular pathogen infection, along with an intracellular delivery method for this Nb. This strategy should overcome current barriers to advance mechanistic research and develop therapies complementary or alternative to the current broad-spectrum antibiotic.

Iron robbery by intracellular pathogen via bacterial effector-induced ferritinophagy.

Iron is essential for survival and proliferation of Ehrlichia chaffeensis, an obligatory intracellular bacterium that causes an emerging zoonosis, human monocytic ehrlichiosis. However, how Ehrlichia acquires iron in the host cells is poorly understood. Here, we found that native and recombinant (cloned into the Ehrlichia genome) Ehrlichia translocated factor-3 (Etf-3), a previously predicted effector of the Ehrlichia type IV secretion system (T4SS), is secreted into the host cell cytoplasm. Secreted Etf-3 directly bound ferritin light chain with high affinity and induced ferritinophagy by recruiting NCOA4, a cargo receptor that mediates ferritinophagy, a selective form of autophagy, and LC3, an autophagosome biogenesis protein. Etf-3-induced ferritinophagy caused ferritin degradation and significantly increased the labile cellular iron pool, which feeds Ehrlichia Indeed, an increase in cellular ferritin by ferric ammonium citrate or overexpression of Etf-3 or NCOA4 enhanced Ehrlichia proliferation, whereas knockdown of Etf-3 in Ehrlichia via transfection with a plasmid encoding an Etf-3 antisense peptide nucleic acid inhibited Ehrlichia proliferation. Excessive ferritinophagy induces the generation of toxic reactive oxygen species (ROS), which could presumably kill both Ehrlichia and host cells. However, during Ehrlichia proliferation, we observed concomitant up-regulation of Ehrlichia Fe-superoxide dismutase, which is an integral component of Ehrlichia T4SS operon, and increased mitochondrial Mn-superoxide dismutase by cosecreted T4SS effector Etf-1. Consequently, despite enhanced ferritinophagy, cellular ROS levels were reduced in Ehrlichia-infected cells compared with uninfected cells. Thus, Ehrlichia safely robs host cell iron sequestered in ferritin. Etf-3 is a unique example of a bacterial protein that induces ferritinophagy to facilitate pathogen iron capture.

Host Cells Upregulate Phosphate Transporter PIT1 to Inhibit Ehrlichia chaffeensis Intracellular Growth.

Ehrlichia chaffeensis infects and proliferates inside monocytes or macrophages and causes human monocytic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. After internalization, E. chaffeensis resides in specialized membrane-bound inclusions, E. chaffeensis-containing vesicles (ECVs), to evade from host cell innate immune responses and obtain nutrients. However, mechanisms exploited by host cells to inhibit E. chaffeensis growth in ECVs are still largely unknown. Here we demonstrate that host cells recognize E. chaffeensis Ech_1067, a penicillin-binding protein, and then upregulate the expression of PIT1, which is a phosphate transporter and transports phosphate from ECVs to the cytosol to inhibit bacterial growth. We found that host cells upregulate the PIT1 expression upon E. chaffeensis infection using transcriptome sequencing, qRT-PCR and Western blotting, and PIT1 is localized on the ECV membrane in infected THP-1 cells using confocal microscopy. Silence of PIT1 using shRNA enhances E. chaffeensis intracellular growth. Finally, we found that E. chaffeensis Ech_1067 induces the upregulation of PIT1 expression through the MyD88-NF-κB pathway using recombinant protein for stimulation and siRNA for silence. Our findings deepen the understanding of the innate immune responses of host cells to inhibit bacterial intracellular growth and facilitate the development of new therapeutics for HME.

Significance of Anaplasma phagocytophilum, Babesia caballi, and Theileria equi as etiologic agents in horses with clinical manifestations from the metropolitan area of Rio De Janeiro, Brazil.

Equine Piroplasmosis (EP) and Equine Granulocytic Anaplasmosis (EGA) are diseases that affect horses, transmitted by ixodid ticks, causing a nonspecific febrile syndrome. Equine Piroplasmosis is endemic in Brazil, and most horses are in enzootic stability. Serological and molecular studies carried out on horses in Brazil have shown the presence of Anaplasma phagocytophilum, however, the clinical relevance of this infection has not yet been established. The present study aims to evaluate the importance of Babesia caballi, Theileria equi, and A. phagocytophilum as etiological agents in horses with clinical manifestations suggestive of these diseases in the metropolitan mesoregion of Rio de Janeiro. A total of 45 animals with clinical signs were submitted to DNA extraction followed by qPCR test. Anaplasma phagocytophilum, Neorickettsia risticii and Theileria haneyi were not found in any of the horses with clinical signs, however 62.2% were infected with at least one agent of EP. Theileria equi was the most frequent etiologic agent (35.5%), followed by coinfection (15.5%) and B. caballi (11.2%). These results suggest that A. phagocytophilum has minor clinical importance in the region, while EP is frequently found in symptomatic horses, representing an important differential diagnosis in suspected cases.

DETECTION OF BABESIA CF. ODOCOILEI, BABESIA CAPREOLI, AND ANAPLASMA PHAGOCYTOPHILUM IN CERVIDS OF THE SCOTTISH HIGHLANDS, UNITED KINGDOM.

Outbreaks of suspected tick-borne disease (redwater fever) have been reported in captive deer of the Scottish Highlands. In this pilot study, polymerase chain reaction and amplicon sequencing were used to detect tick-borne pathogens in opportunistically collected blood and spleen samples from 63 (healthy, n = 44; diseased, n = 19) cervids, and 45 questing and feeding ticks (Ixodes ricinus) from the outbreak sites in 2021-2022. Potentially pathogenic Babesia species were detected in deer but not identified in ticks, Anaplasma phagocytophilum was detected in both deer and ticks, and Borrelia afzelii was detected in ticks but not in deer. Sequencing confirmed Babesia capreoli and Babesia cf. odocoilei parasitemia in clinically healthy red deer (Cervus elaphus), B. capreoli parasitemia in clinically healthy domestic reindeer (Rangifer tarandus tarandus), and two cases of B. cf. odocoilei-associated hemolytic anemia in white-lipped deer (Cervus albirostris), of which one was fatal despite imidocarb treatment. White-lipped deer appear to be highly susceptible to babesiosis caused by B. cf. odocoilei. This investigation highlights the importance of disease surveillance, including molecular diagnostics, for the detection of emerging tick-borne pathogens in managed populations of cervids.

Ehrlichia Notch signaling induction promotes XIAP stability and inhibits apoptosis.

Ehrlichia chaffeensis has evolved multiple strategies to evade innate defenses of the mononuclear phagocyte. Recently, we reported the E. chaffeensis tandem repeat protein (TRP)120 effector functions as a Notch ligand mimetic and a ubiquitin ligase that degrades the nuclear tumor suppressor, F-box and WD repeat domain-containing 7, a negative regulator of Notch. The Notch intracellular domain (NICD) is known to inhibit apoptosis primarily by interacting with X-linked inhibitor of apoptosis protein (XIAP) to prevent degradation. In this study, we determined that E. chaffeensis activation of Notch signaling increases XIAP levels, thereby inhibiting apoptosis through both the intrinsic and executioner pathways. Increased NICD and XIAP levels were detected during E. chaffeensis infection and after TRP120 Notch ligand mimetic peptide treatment. Conversely, XIAP levels were reduced in the presence of Notch inhibitor DAPT. Cytoplasmic and nuclear colocalization of NICD and XIAP was observed during infection and a direct interaction was confirmed by co-immunoprecipitation. Procaspase levels increased temporally during infection, consistent with increased XIAP levels; however, knockdown (KD) of XIAP during infection significantly increased apoptosis and Caspase-3, -7, and -9 levels. Furthermore, treatment with SM-164, a second mitochondrial activator of caspases (Smac/DIABLO) antagonist, resulted in decreased procaspase levels and increased caspase activation, induced apoptosis, and significantly decreased infection. In addition, RNAi KD of XIAP also decreased infection and significantly increased apoptosis. Moreover, ectopic expression of TRP120 HECT Ub ligase catalytically defective mutant in HeLa cells decreased NICD and XIAP levels and increased caspase activation compared to HeLa cells with functional HECT Ub ligase catalytic activity (TRP120-WT). This investigation reveals a mechanism whereby E. chaffeensis modulates Notch signaling to stabilize XIAP and inhibit apoptosis.

Neoehrlichiosis in Symptomatic Immunocompetent Child, South Africa.

We describe a case of neoehrlichiosis in an immunocompetent child with acute febrile illness in South Africa. Neoehrlichiosis was diagnosed by PCR on 16S rDNA from bone marrow aspirate. Phylogenetic analysis indicated an organism closely related to Candidatus Neoehrlichia. Clinicians should be aware of possible ehrlichiosis even in immunocompetent patients.

Canine ehrlichiosis seropositivity and associated factors in Kenya and Tanzania: a retrospective study.

Canine ehrlichiosis is an important tick-borne disease caused by bacteria in the Ehrlichia genus with species such as E. canis, E. ewingii and E. chaffeensis resulting in a severe dog illness. This study determined the occurrence of canine ehrlichiosis antibodies and its associated factors in Kenya and Tanzania. This was a retrospective study that evaluated laboratory records of 400 samples from Kenya and Tanzania submitted to Pathologists Lancet Kenya for the IDEXX SNAP 4Dx™ Plus test between the years 2016 and 2021. Records of all samples submitted to the Pathologists Lancet Kenya veterinary laboratory for the diagnostic tests were retrieved, examined, and compiled. Descriptive statistics and univariable and multivariable logistic regression were considered during analysis. The overall proportion of samples that tested positive for canine ehrlichiosis was 23% (92/400). Samples from Kenya accounted for 61% (245/400) of samples, and the percent positive was 31% (29/245). The samples from Tanzania accounted for 39% (155/400), and the percent positive was 69% (63/155). In the final model, the odds of a sample testing positive was 1.7 times for those submitted from July to December compared with those submitted from January to June. Blood samples of dogs from Tanzania had 5.31 times the odds of testing positive on the SNAP test when compared with those from Kenya. This study reports high percent positive in samples originating from Tanzania and those received during the year's second half.

Host membrane lipids are trafficked to membranes of intravacuolar bacterium Ehrlichia chaffeensis.

Ehrlichia chaffeensis, a cholesterol-rich and cholesterol-dependent obligate intracellular bacterium, partially lacks genes for glycerophospholipid biosynthesis. We found here that E. chaffeensis is dependent on host glycerolipid biosynthesis, as an inhibitor of host long-chain acyl CoA synthetases, key enzymes for glycerolipid biosynthesis, significantly reduced bacterial proliferation. E. chaffeensis cannot synthesize phosphatidylcholine or cholesterol but encodes enzymes for phosphatidylethanolamine (PE) biosynthesis; however, exogenous NBD-phosphatidylcholine, Bodipy-PE, and TopFluor-cholesterol were rapidly trafficked to ehrlichiae in infected cells. DiI (3,3'-dioctadecylindocarbocyanine)-prelabeled host-cell membranes were unidirectionally trafficked to Ehrlichia inclusion and bacterial membranes, but DiI-prelabeled Ehrlichia membranes were not trafficked to host-cell membranes. The trafficking of host-cell membranes to Ehrlichia inclusions was dependent on both host endocytic and autophagic pathways, and bacterial protein synthesis, as the respective inhibitors blocked both infection and trafficking of DiI-labeled host membranes to Ehrlichia In addition, DiI-labeled host-cell membranes were trafficked to autophagosomes induced by the E. chaffeensis type IV secretion system effector Etf-1, which traffic to and fuse with Ehrlichia inclusions. Cryosections of infected cells revealed numerous membranous vesicles inside inclusions, as well as multivesicular bodies docked on the inclusion surface, both of which were immunogold-labeled by a GFP-tagged 2×FYVE protein that binds to phosphatidylinositol 3-phosphate. Focused ion-beam scanning electron microscopy of infected cells validated numerous membranous structures inside bacteria-containing inclusions. Our results support the notion that Ehrlichia inclusions are amphisomes formed through fusion of early endosomes, multivesicular bodies, and early autophagosomes induced by Etf-1, and they provide host-cell glycerophospholipids and cholesterol that are necessary for bacterial proliferation.

Human case of Anaplasma phagocytophilum infection in Eastern Taiwan.

Human granulocytic anaplasmosis (HGA) is a tick-borne infection caused by the bacterium Anaplasma phagocytophilum. In this study, we report an indigenous case of clinically diagnosed HGA. The patient was a 41-year-old man who experienced a tick bite and later developed fever, chills, myalgia, malaise, thrombocytopenia, leukocytosis with a left shift, elevated hepatic transaminase levels, and splenomegaly upon admission to the hospital. Immunofluorescence assays detected seroconversion against A. phagocytophilum, whereas tests for spotted fever group rickettsiae, murine typhus, scrub typhus, Q fever, and ehrlichiosis were negative. ELISA and Western blot analysis using recombinant MSP2 protein confirmed the exposure to A. phagocytophilum. Oral doxycycline and intravenous ceftriaxone were prescribed, and the patient made a full recovery. Our findings indicate the presence of HGA on the main island of Taiwan. Precautions against tick bites should be taken when engaging in outdoor activities, and HGA should be considered by physicians in the differential diagnosis.

Donor-derived Ehrlichiosis: 2 Clusters Following Solid Organ Transplantation.

Ehrlichiosis has been infrequently described as transmissible through organ transplantation. Two donor-derived clusters of ehrlichiosis are described here. During the summer of 2020, 2 cases of ehrlichiosis were reported to the Organ Procurement and Transplantation Network (OPTN) and the Centers for Disease Control and Prevention (CDC) for investigation. Additional transplant centers were contacted to investigate similar illness in other recipients and samples were sent to the CDC. Two kidney recipients from a common donor developed fatal ehrlichiosis-induced hemophagocytic lymphocytic histiocytosis. Two kidney recipients and a liver recipient from another common donor developed ehrlichiosis. All 3 were successfully treated. Clinicians should consider donor-derived ehrlichiosis when evaluating recipients with fever early after transplantation after more common causes are ruled out, especially if the donor has epidemiological risk factors for infection. Suspected cases should be reported to the organ procurement organization and the OPTN for further investigation by public health authorities.

Serologic Evidence of Human Exposure to Ehrlichiosis Agents in Japan.

In retrospective analyses, we report 3 febrile patients in Japan who had seroconversion to antibodies against Ehrlichia chaffeensis antigens detected by using an immunofluorescence and Western blot. Our results provide evidence of autochthonous human ehrlichiosis cases and indicate ehrlichiosis should be considered a potential cause of febrile illness in Japan.