Cutaneous Plasmacytosis Associated with Ehrlichiosis in an African-American Patient

Cutaneous plasmacytosis is a rare disease that presents clinically with multiple red-brown papules and plaques with minimal to no epidermal change. Histopathologic findings include a perivascular dermal infiltration of polyclonal plasma cells. The etiology of cutaneous plasmacytosis is unknown, but hypothesized to be due to persistent or repeated antigenic stimulation. Ehrlichia represents a family of obligate intracellular bacteria that have been associated with the development of plasma cell dyscrasias in the veterinary literature. We present a case of a 67-year-old male patient with the development of progressively worsening cutaneous plasmacytosis following prolonged hospitalization secondary to ehrlichiosis sepsis. The patient initially presented with isolated cutaneous involvement and normal laboratory findings that eventually progressed to include multiple laboratory abnormalities, including anemia, hyperproteinemia, and elevated serum creatinine. Further diagnostic workup was declined by the patient despite evidence of progression to systemic plasmacytosis or multiple myeloma.     

Development of a Quantitative PCR Assay for Differentiating the Agent of Heartwater Disease,Ehrlichia ruminantium,from the Panola Mountain Ehrlichia

Panola Mountain Ehrlichia (PME) is an emerging Ehrlichia sp. reported in ten US states. Based on the sequence homology of all known genes, PME is closely related to Ehrlichia ruminantium (ER), the causative agent of heartwater. Heartwater is an economically important tick‐borne disease of cattle, sheep and goats responsible for stock losses in sub‐Saharan Africa. Unfortunately, ER was imported to the Caribbean islands in the 19th century, and the presence of this foreign animal disease in the Caribbean poses a threat to the US mainland. If introduced, a heartwater outbreak would cause massive losses of naïve livestock. The serologic assay of choice to diagnose heartwater is cross‐reactive with Ehrlichia spp., including PME, as we demonstrate here, which would confound disease surveillance in the event of a heartwater outbreak. The purpose of this study was to develop a diagnostic assay capable of rapidly distinguishing between these pathogens. Using synthetic MAP‐1B peptides for ER and PME, we tested the cross‐reactivity of this assay using sera from infected livestock. The MAP‐1B ELISA cannot distinguish between animals infected with PME and ER. Therefore, a dual‐plex Taqman^™ qPCR assay targeting the groEL gene of PME and ER was developed and validated. Primers were designed that are conserved among all known strains of ER, allowing for the amplification of strains from the Caribbean and Africa. The assay is highly sensitive (10 copies of DNA) and specific. This assay distinguishes between infection with PME and ER and will be a valuable tool in the event of heartwater outbreak on the US mainland, or for epidemiological studies involving either disease‐causing organism.     

Absence of Myelofibrosis in Dogs with Myelosuppression Induced by Ehrlichia canis Infection

Bone marrow (BM) pathology was assessed in 10 dogs with Ehrlichia canis-induced aplastic pancytopenia. BM core biopsy sections were stained with haematoxylin and eosin and with haematoxylin/van Gieson and Gordon and Sweets' reticulin stain for the detection of collagen and reticulin fibres, respectively. Iron stores were assessed by Perls' Prussian blue staining. There was no significant deposition of collagen or reticulin in any sample, but in seven dogs the BM was depleted of haemosiderin. These findings suggest that myelofibrosis does not play a significant role in the development of BM failure in canine monocytic ehrlichiosis and that iron deficiency may exacerbate the anaemia in the myelosuppressive phase of the disease.     

Antigenic protein modifications in Ehrlichia

To develop effective vaccination strategies against Ehrlichia , we have previously reported developing an animal model of cross-protection in which C57BL/6 mice primed with E. muris were resistant to lethal infection with Ixodes ovatus ehrlichia (IOE). Polyclonal antibody produced in mice after priming with E. muris and later injected with IOE-detected antigenic proteins in E. muris and IOE cell lysates. Cross-reaction of antigenic proteins was observed when we probed both the E. muris and IOE cell lysates with IOE and E. muris -specific polyclonal antibody. Analysis of the total proteins of E. muris and IOE by two dimensional electrophoresis showed that both E. muris and IOE have the same antigenic proteins. Finally, studies on post-translational protein modifications using a novel technique, Eastern blotting, showed that E. muris proteins are more lipoylated and glycosylated than those of IOE.     

PCR检测蜱中查菲埃立克体DNA及其序列分析

应用从查菲埃立克体１６ＳｒＲＮＡ基因序列高变区构建的特异引物，进行ＰＣＲ，检测蜱标本中病原体ＤＮＡ。结果从云南采集的龟形花蜱、从福建采集的越原血蜱和卵形硬蜱扩增出３８９ｂｐ的特异ＤＮＡ片段。收集龟形花蜱和越原血蜱的特异ＰＣＲ产物，通过与Ｔ载体连接，进行克隆和序列测定。其ＤＮＡ序列与美国查菲埃立克体分离株对应位置相差一个核苷酸，与其它种埃立克体的同源性为８０．７％～９６．１％。这是首次在我国发现埃立克体存在的病原线索，表明在我国南方可能存在人单核细胞埃立克体感染的自然疫源地     

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.

Ehrlichia spp., rickettsial obligatory intracellular bacteria cause tick-borne infectious diseases, which are greatly rising in worldwide prevalence (1, 2). Ehrlichia chaffeensis causes human monocytic ehrlichiosis, a severe flu-like illness accompanied by hematologic abnormalities and hepatitis, which can be fatal (2 to 5% mortality) (3–6). In humans, E. chaffeensis infects monocytes and macrophages and concocts unique membrane-bound compartments (inclusions). The inclusions have early endosome-like characteristics, including the presence of transferrin (Tf), transferrin receptor (TfR), and vacuolar-type H⁺-ATPase as well as the small GTPase RAB5 and its effectors, but the inclusions lack late endosomal or lysosomal markers or NADPH oxidase (7–9). Within the inclusions, Ehrlichia acquires all nutrients, including iron, for its reproduction to yield numerous mature infectious forms.Iron serves as a cofactor in many processes of bacteria and eukaryotes, including electron transfer, energy metabolism, oxygen transport, oxygen sensing, and DNA synthesis and repair (10). Ehrlichia is an obligate aerobe and is absolutely dependent on host iron for ATP synthesis via the electron transport chain, because its glycolytic pathway is incomplete and it lacks ATP−ADP translocase, unlike Rickettsia and Chlamydia (11). E. chaffeensis lacks the siderophore biosynthesis pathway and Fe³⁺ uptake regulator (11). Nonetheless, Ehrlichia acquires iron from the host cell labile cellular iron (LCI) pool, and pretreating human monocytes with the membrane-permeable iron chelator deferoxamine blocks E. chaffeensis infection (12). Ehrlichia enhances host cell iron uptake via up-regulating TfR messenger RNA (mRNA) (13) and acquires iron from the holoTf, as E. chaffeensis endosomes intersect with TfR-recycling endosomes and are slightly acidic—enough to release iron from holoTf (7). In fact, treatment of macrophages with interferon-γ down-regulates TfR mRNA and almost completely inhibits Ehrlichia infection, and addition of holoTf abrogates this inhibition (12). However, TfR mRNA levels return to basal level after 24 h postinfection when bacterial exponential growth begins (13), and treatment with interferon-γ can no longer inhibit infection at this point (12), suggesting that alternative or additional iron acquisition mechanisms exist to support exponential intracellular growth of Ehrlichia.The bacterial type IV secretion system (T4SS) translocates bacterial proteins and nucleoprotein complexes (called “effectors,” as they bring about responses) from bacteria to eukaryotic cells (14). Rickettsial organisms including E. chaffeensis have T4SS, sometimes referred to as T4aSS, similar to the virB/virD system of Agrobacterium tumefaciens (15, 16). The dot/icm system of Legionella pneumophila, sometimes referred to as T4bSS, secretes ∼300 effectors with redundant functions; hence, each effector can be knocked out, but the mutant lacks a phenotype (17). In contrast, the total number of T4aSS effectors is much lower [for example, fewer than six effectors exist in A. tumefaciens (15)], but each effector has a crucial role in infection/disease. To date, only a handful of T4SS effectors have been identified for rickettsial organisms, and even fewer have been functionally characterized (18–21). VirD4 is a well-established coupling protein involved in escorting translocated DNA and proteins in A. tumefaciens (15). By a bacterial two-hybrid screen using E. chaffeensis VirD4 as bait, we previously identified three Ehrlichia proteins that directly bind to Ehrlichia VirD4: ECH0825 (Ehrlichial translocated factor-1, Etf-1), ECH0261 (Etf-2), and ECH0767 (Etf-3) (22). All three were formerly annotated as hypothetical proteins, as they lack sequence similarity to previously known proteins or protein domains or motifs. Etf-1 has key roles in Ehrlichia infection of human cells by blocking host cell apoptosis and inducing RAB5-regulated autophagy for nutrient (amino acids) acquisition (9, 22, 23). Etf-2 is a unique RABGAP5 structural mimic that lacks GTPase-activating protein activity (24). Etf-2 directly binds RAB5-GTP on the Ehrlichia inclusion membrane and impedes the fusion of Ehrlichia-containing early endosomes with lysosomes (24). Whether Etf-3 is secreted or has any biological function is unknown. In the present study, we discovered that Etf-3 is a true T4SS effector that is secreted, binds tightly to ferritin, and induces ferritinophagy to provide free Fe²⁺ for intracellular Ehrlichia.     

内蒙古中西部草原蜱媒病原体多样性及基因型分析

目的了解内蒙古中西部草原蜱类的群落结构、携带病原体多样性及基因型。方法于2016-2019年春夏季,在内蒙古中西部草原,采用动物体表搜集法采集蜱标本,进行蜱种鉴定。解剖摘取蜱的唾液腺并提取基因组DNA,以斑点热立克次体柠檬酸合成酶A (gltA)、疏螺旋体以鞭毛蛋白B (flaB)、埃立克体属以外膜蛋白质1 (omp1)、无形体属主要表面蛋白2 (msp2)为靶基因进行PCR扩增初筛。立克次体gltA初筛阳性样品经限制性片段长度多态性(RFLP)分类,再根据蜱种和地区每类选20~30个代表性样品进行gltA、立克次体外膜蛋白A (rOmpA)基因测序。扩增序列测序后用BLAST、 Clustal W和MEGA 7.0软件进行同源性分析,以邻接法构建系统进化树。结果共采集成蜱3 822只,经形态学特征和特异性18S r RNA基因分型法鉴定,隶属于2属3种,分别为草原革蜱、亚东璃眼蜱和边缘璃眼蜱,其中草原革蜱占55.7%(2 129/3 822)、亚东璃眼蜱占30.0%(1 147/3 822),为该地区的优势蜱种。PCR检测结果显示,立克次体gltA基因阳性蜱1 899只,阳性率为49.7%(1 899/3 822), gltA基因阳性样品根据RFLP结果分为两类,两类样品的gltA基因序列均为581 bp,与R. raoultii (DQ365804)或R. aeschlimanni (KT873466)的同源性为100%;两类样品的rOmpA基因均长367 bp,与R. raoultii (AH015610)或R. aeschlimanni (U83466)的同源性为100%,与gltA基因的结果相符。3 822只蜱中,R. raoultii和R. aeschlimanni的阳性率分别为37.2%(1 422/3 822)和12.5%(477/3 822),其中草原革蜱中分别为58.5%(1 245/2 129)和11.1%(477/2 129);亚东璃眼蜱中分别为15.4%(177/1 147)和0;边缘璃眼蜱中分别为0和44.0%(240/546)。疏螺旋体flaB基因阳性蜱28只,阳性率为0.7%(28/3 822),其中草原革蜱中为0.8%(16/2 129),亚东璃眼蜱为1.0%(12/1 147)。共获得疏螺旋体flaB基因序列10条,与莱姆病主要病原体B. garinii (AB035602)和B. afzelii PKo (NC008277)的同源性分别为90.6%~100%和95.6%~100%。亚东璃眼蜱中B. garinii和B. afzelii的阳性率分别为0.9%(10/1 147)和0.2%(2/1 147),草原革蜱中均为0.4%(8/2 129)。3 822只蜱中omp1基因阳性1只,TA克隆后获得8个氨基酸序列相同的克隆和3个氨基酸序列存在差异的克隆,11个克隆的氨基酸序列与E. muris的同源性最高,但仅为65%~69%。3 822只蜱中均未检出无形体属菌群。系统进化树分析结果显示,3种蜱感染的立克次体均与R. raoultii和R. aeschlimanni聚在一簇。在获得的10条疏螺旋体菌群flaB基因序列中,源于草原革蜱和亚东璃眼蜱的1条序列与B. garinii聚在一簇,草原革蜱的另1条序列与B. afzelii聚在一簇,其余8条序列与B. garinii和B. afzelii的flaB基因序列处在不同的分支。草原革蜱感染的埃立克体属菌与目前已知的埃立克体属菌群关系较远,形成独自的聚类。结论内蒙古中西部草原存在草原革蜱、亚东璃眼蜱和边缘璃眼蜱,蜱类中广泛存在斑点热立克次体和莱姆病螺旋体的感染,是R. raoultii、R. aeschlimanni、 B. garinii和B. afzelii潜在的自然疫源地。有必要加强该地区蜱媒传染病的预防控制工作。