Non-Canonical Inflammasome Pathway: The Role of Cell Death and Inflammation in Ehrlichiosis.

Activating inflammatory caspases and releasing pro-inflammatory mediators are two essential functions of inflammasomes which are triggered in response to pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). The canonical inflammasome pathway involves the activation of inflammasome and its downstream pathway via the adaptor ASC protein, which causes caspase 1 activation and, eventually, the cleavage of pro-IL-1b and pro-IL-18. The non-canonical inflammasome pathway is induced upon detecting cytosolic lipopolysaccharide (LPS) by NLRP3 inflammasome in Gram-negative bacteria. The activation of NLRP3 triggers the cleavage of murine caspase 11 (human caspase 4 or caspase 5), which results in the formation of pores (via gasdermin) to cause pyroptosis. Ehrlichia is an obligately intracellular bacterium which is responsible for causing human monocytic ehrlichiosis (HME), a potentially lethal disease similar to toxic shock syndrome and septic shock syndrome. Several studies have indicated that canonical and non-canonical inflammasome activation is a crucial pathogenic mechanism that induces dysregulated inflammation and host cellular death in the pathophysiology of HME. Mechanistically, the activation of canonical and non-canonical inflammasome pathways affected by virulent Ehrlichia infection is due to a block in autophagy. This review aims to explore the significance of non-canonical inflammasomes in ehrlichiosis, and how the pathways involving caspases (with the exception of caspase 1) contribute to the pathophysiology of severe and fatal ehrlichiosis. Improving our understanding of the non-canonical inflammatory pathway that cause cell death and inflammation in ehrlichiosis will help the advancement of innovative therapeutic, preventative, and diagnostic approaches to the treatment of ehrlichiosis.

Glomerulotubular pathology in dogs with subclinical ehrlichiosis.

Subclinical stage of ehrlichiosis is characterized by absence of clinical or laboratory alterations; however, it could lead to silent glomerular/tubular changes and contribute significantly to renal failure in humans and animals. The aim of this study was to evaluate glomerular and tubular alterations in dogs with subclinical ehrlichiosis. We evaluated renal biopsies of 14 bitches with subclinical ehrlichiosis and 11 control dogs. Samples were obtained from the left kidney, and the tissue obtained was divided for light microscopy, immunofluorescence, and transmission electron microscopy. Abnormalities were identified by light microscopy in 92.9% of dogs with ehrlichiosis, but not in any of the dogs of the control group. Mesangial cell proliferation and synechiae (46.1%) were the most common findings, but focal segmental glomerulosclerosis and ischemic glomeruli (38.4%), focal glomerular mesangial matrix expansion (30.7%), mild to moderate interstitial fibrosis and tubular atrophy (23%), and glomerular basement membrane spikes (23%) were also frequent in dogs with ehrlichiosis. All animals with ehrlichiosis exhibited positive immunofluorescence staining for immunoglobulins. Transmission electron microscopy from dogs with ehrlichiosis revealed slight changes such as sparse surface projections and basement membrane double contour. The subclinical phase of ehrlichiosis poses a higher risk of development of kidney damage due to the deposition of immune complexes.

Impact of tick-borne Anaplasma phagocytophilum infections in calves of moose (Alces alces) in southern Norway.

The Gram-negative, obligate intracellular tick-transmitted pathogen Anaplasma phagocytophilum can cause acute febrile diseases in humans and domestic animals. The expansion of the tick Ixodes ricinus (Linnaeus, 1758) in northern Europe due to climate change is of serious concern for animal and human health. The aim of the present study was to investigate the impact of A. phagocytophilum infection in moose Alces alces (Linnaeus) calves by evaluating the carcass weights of infected and non-infected animals and examining animal tissues samples for co-infections with either species of Babesia Starcovici, 1893 or bacteria of the genus Bartonella. The carcasses of 68 free-ranging moose calves were weighed by hunters during the hunting seasons from 2014 to 2017 in two regions in southern Norway and spleen samples were collected. Anaplasma phagocytophilum was detected in moose sampled from locations infected with ticks with a prevalence of 82% (n = 46). The carcass weights of A. phagocytophilum-infected calves (n = 46) and non-infected (n = 22) calves were compared. Although the average weight of infected calves (45.6 kg) was lower than that of non-infected calves (46.5 kg), the difference was not statistically significant. Three different variants of the bacterium 16S rRNA gene were identified. The average weight of animals infected with variant I was 49.9 kg, whereas that of animals infected with variant III was 42.0 kg, but the difference was not statistically significant (p = 0.077). Co-infections of A. phagocytophilum with Bartonella spp. or with Babesia spp. were found in 20 and two calves, respectively. A triple infection was found in two calves. Sequence analysis of the 18S rRNA gene of Babesia-positive samples revealed the presence of Babesia cf. odocoilei (Emerson et Wright, 1970). Strains of Bartonella closely related to Bartonella bovis (Bermond, Boulouis, Heller, Laere, Monteil, Chomel, Sander, Dehio et Piemont, 2002) were identified based on phylogenetic analysis of the gltA and rpoB genes. The loss of body mass in moose calves in the tick-infected site was probably influenced by multiple factors.

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.

Frequency of co-seropositivities for certain pathogens and their relationship with clinical and histopathological changes and parasite load in dogs infected with Leishmania infantum.

In canine leishmaniosis caused by the protozoan Leishmania infantum, little is known about how co-infections with or co-seropositivities for other pathogens can influence aggravation of this disease. Therefore, the objectives of this study were to evaluate the frequency of co-infections with or co-seropositivities for certain pathogens in dogs seropositive for L. infantum and their relationship with clinical signs, histological changes and L. infantum load. Sixty-six L. infantum-seropositive dogs were submitted to clinical examination, collection of blood and bone marrow, culling, and necropsy. Antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato, Ehrlichia spp. and Toxoplasma gondii and Dirofilaria immitis antigens were investigated in serum. Samples from different tissues were submitted to histopathology and immunohistochemistry for the detection of Leishmania spp. and T. gondii. Quantitative real-time PCR was used to assess the L. infantum load in spleen samples. For detection of Coxiella burnetii, conventional PCR and nested PCR were performed using bone marrow samples. All 66 dogs tested positive for L. infantum by qPCR and/or culture. Fifty dogs (76%) were co-seropositive for at least one pathogen: T. gondii (59%), Ehrlichia spp., (41%), and Anaplasma spp. (18%). Clinical signs were observed in 15 (94%) dogs monoinfected with L. infantum and in 45 (90%) dogs co-seropositive for certain pathogens. The L. infantum load in spleen and skin did not differ significantly between monoinfected and co-seropositive dogs. The number of inflammatory cells was higher in the spleen, lung and mammary gland of co-seropositive dogs and in the mitral valve of monoinfected dogs. These results suggest that dogs infected with L. infantum and co-seropositive for certain pathogens are common in the region studied. However, co-seropositivities for certain pathogens did not aggravate clinical signs or L. infantum load, although they were associated with a more intense inflammatory reaction in some organs.

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.

mTORC1-mediated polarization of M1 macrophages and their accumulation in the liver correlate with immunopathology in fatal ehrlichiosis.

A polarized macrophage response into inflammatory (M1) or regenerative/anti-inflammatory (M2) phenotypes is critical in host response to multiple intracellular bacterial infections. Ehrlichia is an obligate Gram-negative intracellular bacterium that causes human monocytic ehrlichiosis (HME): a febrile illness that may progress to fatal sepsis with multi-organ failure. We have shown that liver injury and Ehrlichia-induced sepsis occur due to dysregulated inflammation. Here, we investigated the contribution of macrophages to Ehrlichia-induced sepsis using murine models of mild and fatal ehrlichiosis. Lethally-infected mice showed accumulation of M1 macrophages (iNOS-positive) in the liver. In contrast, non-lethally infected mice showed polarization of M2 macrophages and their accumulation in peritoneum, but not in the liver. Predominance of M1 macrophages in lethally-infected mice was associated with expansion of IL-17-producing T, NK, and NKT cells. Consistent with the in vivo data, infection of bone marrow-derived macrophages (BMM) with lethal Ehrlichia polarized M0 macrophages into M1 phenotype under an mTORC1-dependent manner, while infection with non-lethal Ehrlichia polarized these cells into M2 types. This work highlights that mTORC1-mediated polarization of macrophages towards M1 phenotype may contribute to induction of pathogenic immune responses during fatal ehrlichiosis. Targeting mTORC1 pathway may provide a novel aproach for treatment of HME.

Emerging Roles of Autophagy and Inflammasome in Ehrlichiosis.

Human monocytic ehrlichiosis (HME) is a potentially life-threatening tick-borne rickettsial disease (TBRD) caused by the obligate intracellular Gram-negative bacteria, Ehrlichia. Fatal HME presents with acute ailments of sepsis and toxic shock-like symptoms that can evolve to multi-organ failure and death. Early clinical and laboratory diagnosis of HME are problematic due to non-specific flu-like symptoms and limitations in the current diagnostic testing. Several studies in murine models showed that cell-mediated immunity acts as a "double-edged sword" in fatal ehrlichiosis. Protective components are mainly formed by CD4 Th1 and NKT cells, in contrast to deleterious effects originated from neutrophils and TNF-α-producing CD8 T cells. Recent research has highlighted the central role of the inflammasome and autophagy as part of innate immune responses also leading to protective or pathogenic scenarios. Recognition of pathogen-associated molecular patterns (PAMPS) or damage-associated molecular patterns (DAMPS) triggers the assembly of the inflammasome complex that leads to multiple outcomes. Recognition of PAMPs or DAMPs by such complexes can result in activation of caspase-1 and -11, secretion of the pro-inflammatory cytokines IL-1ß and IL-18 culminating into dysregulated inflammation, and inflammatory cell death known as pyroptosis. The precise functions of inflammasomes and autophagy remain unexplored in infections with obligate intracellular rickettsial pathogens, such as Ehrlichia. In this review, we discuss the intracellular innate immune surveillance in ehrlichiosis involving the regulation of inflammasome and autophagy, and how this response influences the innate and adaptive immune responses against Ehrlichia. Understanding such mechanisms would pave the way in research for novel diagnostic, preventative and therapeutic approaches against Ehrlichia and other rickettsial diseases.

Molecular identification of Ehrlichia ewingii in a polyarthritic Texas dog.

An 8-year-old, neutered male, Golden Retriever presented for bilateral carpal joint effusion. A complete blood count revealed mild leukopenia and marked thrombocytopenia. Samples were sent to the Texas A&M Veterinary Medical Diagnostic Laboratory for blood smear review and serologic testing for tick-borne diseases. Numerous morulae were observed within neutrophils, and antibodies against Ehrlichia canis were detected at a 1:512 dilution via the indirect fluorescent antibody (IFA) test. As neutrophilic morulae are morphologically indistinguishable between Ehrlichia ewingii and Anaplasma phagocytophilum, and genus-wide cross-reactivity is possible with serologic testing, additional molecular testing was performed. Quantitative real-time polymerase chain reaction (qPCR) followed by conventional PCR and Sanger sequencing were performed on serum identified with E ewingii as the sole disease-causing agent. Three months after diagnosis and treatment, no morulae were found, molecular testing for E ewingii detected no DNA, and convalescent IFA testing demonstrated a continued detection of antibodies for E canis at a 1:512 dilution. To the authors' knowledge, this is the first reported case of E ewingii confirmed with molecular diagnostics in a Texas dog. The zoonotic transmission potential of E ewingii should be noted as Texas supports competent tick vectors, and dogs represent effective sentinels for human ehrlichiosis. This report also highlights the utility of molecular diagnostics when serologic and microscopic evaluations are not sufficient in providing the species-level identity of a causative agent.

Subversion of RAB5-regulated autophagy by the intracellular pathogen Ehrlichia chaffeensis.

Intracellular pathogens often exploit RAB functions to establish a safe haven in which to survive and proliferate. Ehrlichia chaffeensis, an obligatory intracellular bacterium, resides in specialized membrane-bound inclusions that have early endosome-like characteristics, e.g., resident RAB5 GTPase and RAB5 effectors, including VPS34 (the catalytic subunit of class III phosphatidylinositol 3-kinase), but the inclusions lack late endosomal or lysosomal markers. Within inclusions, Ehrlichia obtains host-derived nutrients by inducing RAB5-regulated autophagy using Ehrlichia translocated factor-1 deployed by its type IV secretion system. This manipulation of RAB5 by a bacterial molecule offers a simple strategy for Ehrlichia to avoid destruction in lysosomes and obtain nutrients, membrane components, and a homeostatic intra-host-cell environment in which to grow.

Type I IFNs drive hematopoietic stem and progenitor cell collapse via impaired proliferation and increased RIPK1-dependent cell death during shock-like ehrlichial infection.

Type I interferons (IFNα/ß) regulate diverse aspects of host defense, but their impact on hematopoietic stem and progenitor cells (HSC/HSPCs) during infection remains unclear. Hematologic impairment can occur in severe infections, thus we sought to investigate the impact of type I IFNs on hematopoiesis in a tick-borne infection with a virulent ehrlichial pathogen that causes shock-like disease. During infection, IFNα/ß induced severe bone marrow (BM) loss, blunted infection-induced emergency myelopoiesis, and reduced phenotypic HSPCs and HSCs. In the absence of type I IFN signaling, BM and splenic hematopoiesis were increased, and HSCs derived from Ifnar1-deficient mice were functionally superior in competitive BM transplants. Type I IFNs impaired hematopoiesis during infection by both limiting HSC/HSPC proliferation and increasing HSPC death. Using mixed BM chimeras we determined that type I IFNs restricted proliferation indirectly, whereas HSPC death occurred via direct IFNαR -mediated signaling. IFNαR-dependent signals resulted in reduced caspase 8 expression and activity, and reduced cleavage of RIPK1 and RIPK3, relative to Ifnar1-deficient mice. RIPK1 antagonism with Necrostatin-1s rescued HSPC and HSC numbers during infection. Early antibiotic treatment is required for mouse survival, however antibiotic-treated survivors had severely reduced HSPCs and HSCs. Combination therapy with antibiotics and Necrostatin-1s improved HSPC and HSC numbers in surviving mice, compared to antibiotic treatment alone. We reveal two mechanisms whereby type I IFNs drive hematopoietic collapse during severe infection: direct sensitization of HSPCs to undergo cell death and enhanced HSC quiescence. Our studies reveal a strategy to ameliorate the type I IFN-dependent loss of HSCs and HSPCs during infection, which may be relevant to other infections wherein type I IFNs cause hematopoietic dysfunction.

Epidemiology, genetic variants and clinical course of natural infections with Anaplasma phagocytophilum in a dairy cattle herd.

BACKGROUND: Anaplasma phagocytophilum is an obligate intracellular, tick-transmitted bacterium that causes granulocytic anaplasmosis in humans and several mammalian species including domestic ruminants where it is called tick-borne fever (TBF). Different genetic variants exist but their impact with regard to putative differences in host associations and pathogenicity are not yet completely understood. METHODS: Natural infections with A. phagocytophilum in a dairy cattle herd in Germany were investigated over one pasture season by using serology, haematology, blood chemistry and polymerase chain reaction (PCR). Sequence analysis of partial 16S rRNA, groEL, msp2 and msp4 genes of A. phagocytophilum was carried out in order to trace possible genetic variants and their relations between cattle, roe deer (Capreolus capreolus) and ticks (Ixodes ricinus) in this area. RESULTS: In total 533 samples from 58 cattle, 310 ticks, three roe deer and one wild boar were examined. Our results show (i) typical clinical symptoms of TBF in first-time infected heifers, such as high fever, reduced milk yield, lower limb oedema and typical haematological and biochemical findings such as severe leukopenia, erythropenia, neutropenia, lymphocytopenia, monocytopenia, a significant increase in creatinine and bilirubin and a significant decrease in serum albumin, γ-GT, GLDH, magnesium and calcium; (ii) a high overall prevalence of A. phagocytophilum infections in this herd as 78.9% (15/19) of the naïve heifers were real-time PCR-positive and 75.9% (44/58) of the entire herd seroconverted; and (iii) a high level of sequence variation in the analysed genes with five variants of the 16S rRNA gene, two variants of the groEL gene, three variants of the msp2 gene and four variants in the msp4 gene with certain combinations of these variants. CONCLUSIONS: In cattle particular combinations of the genetic variants of A. phagocytophilum occurred, whereas three roe deer showed different variants altogether. This is indicative for a sympatric circulation of variants in this small geographical region (< 1 km2). Both re- and superinfections with A. phagocytophilum were observed in five cattle showing that infection does not result in sterile immunity. For prevention of clinical cases we suggest pasturing of young, not pregnant heifers to reduce economical losses.

Canine Monocytic Ehrlichiosis among working dogs of organised kennels in India: A comprehensive analyses of clinico-pathology, serological and molecular epidemiological approach.

Canine Monocytic Ehrlichiosis (CME) is a serious tick-borne rickettsial disease affecting canine populations globally. Besides few reports from stray and pet dogs from localised geographical regions (cities/towns/small states), a comprehensive study on prevalence of Ehrlichia canis (E. canis) among working dogs from different geo-climatic zones of India was pertinently lacking. Study of CME among these dog populations was thus carried out, encompassing clinical aspects and different diagnostic methodologies viz., microscopy, serology and molecular biology. During the two-year study period, clinical specimens from 225 cases suspected of canine ehrlichiosis were examined for clinical pathology and presence of the haemoparasites. Overall prevalence of ehrlichiosis by microscopic examination, commercial dot-ELISA kit and nested PCR assay was estimated to be 1.3%, 19.1% and 5.8%, respectively, which were found to be statistically significant by McNemar Chi squared test (p<0.05). It was also observed that possibly due to widespread use of doxycycline therapy in field, CME presently does not remain a potential threat which it uses to pose earlier. However, concurrent infections of E. canis and Babesia gibsoni were found to be mostly fatal. Keeping in view of high number of apparently healthy dogs (24) out of total positive cases (46) observed during the study, it is recommended that prevalence studies on CME should also involve screening of apparently healthy dogs. Phylogenetic analysis carried on partial sequencing of 16S rRNA of E. canis strains revealed that all of the Indian strains clustered in a single clade with other E. canis species from India and rest of the world. Molecular divergence was observed among the sequences of Brazilian and American isolates which were also included in the present study. These findings have thus opened a new paradigm for planning of pragmatic control strategies against CME.

First report of Ehrlichia infection in goats, China.

Ehrlichiosis is an emerging infectious disease of domestic animals which is transmitted by ticks. This disease has been reported earlier in most parts of China in dogs, cattle and humans, but there is no published data regarding this disease in goats. The present study provided the evidence of Ehrlichia infection in goats in Wuhan, China on the basis of clinical signs, gross lesions, serum-biochemical, histopathological and PCR. Twenty four goats were presented to the veterinary hospital of Huazhong Agricultural University during July, 2016. The goats were diagnosed for Ehrlichia in monocytic and granulocytic forms by blood smear examination. Further confirmation was done by PCR examination, while histopathological examination revealed degeneration and inflammation in different tissues. The biochemical criterion and blood samples analysis showed significant (P < 0.05) changes. The present study reported that goats are naturally exposed to Ehrlichia infection. To the best of our knowledge, this is the first clinical report of Ehrlichia infection in goats infested with infected ticks.

Implication, clinical and biological impact of vector-borne haemopathogens in anaemic dogs in France: a prospective study.

OBJECTIVES: To prospectively describe the clinical and biological impact of vector-borne haemopathogens in anaemic dogs in France and occurrence of haemolysis. MATERIALS AND METHODS: This prospective descriptive cohort study includes 134 client-owned dogs that were anaemic on admission at the Veterinary Teaching Hospital of VetAgro Sup, Lyon, France. They underwent comprehensive screening with PCR to detect a panel of vector-borne haemopathogens, SNAP Leishmania and SNAP 4Dx Plus (IDEXX). RESULTS: Vector-borne haemopathogen-associated anaemia accounted for 17·2% (23/134) of anaemic cases. PCR for Babesia species, Mycoplasma species, Anaplasma platys or Ehrlichia canis and positive serology for Leishmania species, occurred in 11/23, 10/23, 2/23, 1/23 and 1/23 cases, respectively. Two dogs had positive PCR for both Babesia and Mycoplasma species. All dogs infected with Mycoplasma species alone had neoplastic disease. Vector-borne haemopathogens were the second most common cause of haemolysis (7/23, 30·4%). In the multivariable regression model, males were overrepresented ( OR : 2·82, P=0·03) and haemolysis ( OR : 3·31, P=0·01) was more frequent in dogs with vector-born haemopathogen-associated anaemia. CLINICAL SIGNIFICANCE: Vector-borne haemopathogens are a common cause of anaemia in this geographical region.

Experimental evaluation of Peromyscus leucopus as a reservoir host of the Ehrlichia muris-like agent.

BACKGROUND: The Ehrlichia muris-like agent (EMLA) is a newly recognized human pathogen in the North Central United States. Although blacklegged ticks (Ixodes scapularis) have been identified as capable vectors, wild reservoirs have not yet been established for EMLA. As key hosts for I. scapularis, white-footed mice (Peromyscus leucopus) are important reservoirs for various tick-borne pathogens, and potentially, for EMLA. The objective of this study was to evaluate reservoir competence in P. leucopus using a natural vector. RESULTS: Mice acquired EMLA infection from feeding ticks and were able to transmit infection to naïve ticks. Transmission between simultaneously feeding tick life stages was also demonstrated. Infections in mice were acute and severe, with systemic dissemination. Limited host survival and clearance of infection among survivors resulted in a narrow interval where EMLA could be acquired by feeding ticks. CONCLUSIONS: Peromyscus leucopus is a competent reservoir of EMLA and likely to play a role in its enzootic transmission cycle. The duration and severity of EMLA infection in these hosts suggests that tick phenology is a critical factor determining the geographic distribution of EMLA in North America.

Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase.

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.

Ehrlichiosis Induced Aseptic Meningitis and Second-Degree Heart Block in an Adolescent Male.

We present a previously healthy 16-year-old male with ehrlichiosis-induced aseptic meningitis and subsequent second-degree heart block.