Anaplasmataceae: Dichotomous Autophagic Interplay for Infection.

Autophagy is a vital conserved degradative process that maintains cellular homeostasis by recycling or eliminating dysfunctional cellular organelles and proteins. More recently, autophagy has become a well-recognized host defense mechanism against intracellular pathogens through a process known as xenophagy. On the host-microbe battlefield many intracellular bacterial pathogens have developed the ability to subvert xenophagy to establish infection. Obligately intracellular bacterial pathogens of the Anaplasmataceae family, including Ehrlichia chaffeensis, Anaplasma phaogocytophilium and Orientia tsutsugamushi have developed a dichotomous strategy to exploit the host autophagic pathway to obtain nutrients while escaping lysosomal destruction for intracellular survival within the host cell. In this review, the recent findings regarding how these master manipulators engage and inhibit autophagy for infection are explored. Future investigation to understand mechanisms used by Anaplasmataceae to exploit autophagy may advance novel antimicrobial therapies and provide new insights into how intracellular microbes exploit autophagy to survive.

Epidemiology and genotyping of Anaplasma marginale and co-infection with piroplasms and other Anaplasmataceae in cattle and buffaloes from Egypt.

BACKGROUND: Anaplasma marginale is an obligate intracellular bacterium and the main cause of bovine anaplasmosis in tropical and subtropical regions. In Egypt, data regarding the prevalence of A. marginale in ruminant hosts and of the circulating genotypes is lacking. This study therefore aimed to (i) investigate the presence, epidemiology and genotypes of A. marginale in cattle and buffaloes in Egypt, (ii) to evaluate suitable diagnostic tools and (iii) to identify co-infections of A. marginale with other selected tick-borne pathogens. METHODS: Blood samples were collected from 394 animals (309 cattle and 85 buffaloes) from three different areas in Egypt. For the detection of A. marginale infection, several tests were compared for their sensitivity and specificity: blood smear analysis, enzyme-linked immunosorbent assay (ELISA), PCR, real-time PCR and reverse line blot (RLB) assay. Co-infections with A. marginale, piroplasms and other Anaplasmataceae were surveyed by RLB while A. marginale genotypes were identified by amplifying and sequencing the partial msp1α gene. RESULTS: Anaplasma marginale DNA was amplified by qPCR in 68.3% of cattle and 29.4% of buffaloes. RLB showed infection with A. marginale in 50.2% of cattle and 42.5% of buffaloes. Blood smear analysis detected this agent in 16.2% of cattle and 2.4% of buffaloes. ELISA showed specific antibodies against A. marginale in 54.9% of cattle. Anaplasma marginale was associated, in cattle and buffaloes, with several tick-borne pathogens (Theileria annulata, Babesia bovis, Babesia bigemina, Babesia occultans and Anaplasma platys). A significant difference of A. marginale infection level was noticed in cattle, where animals between 3-5-years-old had a higher prevalence (79.2%) compared to those older than 5 years (36.4%) and younger than 3 years (59.7%) and one year (64.5%), respectively (P = 0.002281). Microsatellite analysis identified 15 different genotypes. CONCLUSIONS: The epidemiological findings revealed high prevalence of A. marginale in cattle and buffaloes in all the investigated areas. The circulation of diverse genotypes was observed, most of these A. marginale genotypes being specific for Egypt. The qPCR assay was confirmed to be the most sensitive tool for detection of A. marginale in cattle and buffaloes even in the carrier state, highlighting the importance of using suitable diagnostic tests.

Genetic variability of Anaplasmataceae circulating in small mammals and ticks in an Ixodes persulcatus/Ixodes trianguliceps sympatric area in Russian Siberia.

A total of 705 rodents from Myodes, Microtus, and Apodemus genera, 396 adult questing Ixodes persulcatus, and 115 Ixodes larvae and nymphs taken from rodents (and then molted under laboratory conditions to nymphs and adults) were collected in 2013-2018 in Omsk province, Russian Siberia, and examined for the presence of Anaplasmataceae. DNA of Anaplasma phagocytophilum was detected in 29.5 % rodents, 3.8 % questing I. persulcatus, two molted adult I. persulcatus, and one molted adult Ixodes trianguliceps. Ehrlichia muris DNA was found in specimens from 12.1 % rodents, 3.0 % questing I. persulcatus, 14 % molted adult I. persulcatus, and one molted adult I. trianguliceps. Neoehrlichia mikurensis DNA was found in 0.6 % blood samples. It was suggested that in the studied area A. phagocytophilum and E. muris are mainly transmitted to small rodents by I. trianguliceps and I. persulcatus, respectively. Based on groEL gene sequence analysis, three phylogenetic clusters of A. phagocytophilum (clusters 4, 5, 6, according to Jaarsma et al., 2019) were identified. Most of genotyped A. phagocytophilum isolates obtained from rodents (87.6 %) and a single isolate found in a molted adult I. trianguliceps belonged to cluster 5. Cluster 6 contained 11.8 % genotyped specimens from rodents, and one questing and two molted adult I. persulcatus, while cluster 4 included specimens from 93 % genotyped questing I. persulcatus and one vole. The finding of A. phagocytophilum from clusters 5 and 6 in voles from the same sampling area indicated that clusters 5 and 6 segregate according to the tick-carriers, but not the geography. Most of the genotyped specimens of E. muris and N. mikurensis corresponded to typical genotypes detected in Asian Russia previously. In addition, new genetic variants of E. muris and N. mikurensis, which significantly differed from other known isolates and formed separate branches on phylogenetic trees, were identified in several voles.

Prevalence of Anaplasmataceae and Filariidae species in unowned and military dogs in New Caledonia.

Dogs are competent reservoir hosts of several zoonotic agents, including Filariidae nematodes and Anaplasmataceae family bacteria. The latter family unites human and veterinary pathogens (Anaplasma, Ehrlichia and Neorickettsia bacteria) with Wolbachia, some of which are obligatory endosymbionts of pathogenic filarial nematodes. The epidemiology of Anaplasmataceae and Filariidae species infecting dogs living in kennels in New Caledonia was studied. 64 EDTA blood samples were screened for the presence of Anaplasmataceae and filarial nematodes. Molecular study was conducted using primers and probe targeting the of 23S rRNA long fragment of Anaplasmataceae species. Next, all blood sample was screened for the presence of Filariidae species targeting the primers and probe targeting the COI gene, as well as primers targeting the COI and 5S rRNA genes of all filarial worms. Anaplasma platys was identified in 8/64 (12.5, 95% confidence interval [CI]: 4.4-20.6%) and Wolbachia endosymbiont of Dirofilaria immitis in 8/64 (12.5%, CI: 4.4-20.6%). Filariidae species investigation was performed and showed that 11/64 (17.2%, CI: 7.9-26.4%) dogs were infected with D. immitis, whereas, 2/64 (3.1%, CI: 0.0-7.3%) were infected with Acanthocheilonema reconditum. Finally, we checked the occurrence of co-infection between Anaplasmataceae and Filariidae species. Co-occurrence with Wolbachia endosymbiont of D. immitis was observed in seven dogs, one dog was co-infected with A. platys and A. reconditum and another was co-infected with Wolbachia endosymbiont of D. immitis and A. reconditum. These results are the first report of Anaplasmataceae and Filariidae occurring in dogs in New Caledonia.

A new case of the enigmatic Candidatus Neoehrlichia sp. (FU98) in a fox from the Czech Republic.

This study reports a new case of Candidatus Neoehrlichia sp. (FU98) infection in a fox from the Czech Republic, and provides confirmatory evidence on the occurrence of this newly identified sequence type. However, further studies are needed to investigate the distribution, host range and possible vector(s) for this bacterium, as well as its impact on animals and humans.

Natural Anaplasmataceae infection in Rhipicephalus bursa ticks collected from sheep in the French Basque Country.

Rhipicephalus bursa is one of 79 species of the genus Rhipicephalus in the family of Ixodidae. In this study, we investigated Anaplasmataceae bacteria associated with R. bursa collected after an epizootic outbreak of ovine anaplasmosis. 76 adult ticks, (60 male and 16 female ticks), were removed from sheep in two farms and all identified as R. bursa, all females were partially engorged. We found that 50% of the ticks were positive in the initial Anaplasmataceae qPCR screening. Bacterial species was identified by analyzing the sequences of amplicons of 23S rRNA, groEL and rpoB genes. 22.4% of ticks contained DNA of Anaplasma phagocytophilum and 7.9% the DNA of Anaplasma ovis. Based on 23S rRNA and groEL genes analysis, we found that 19.7% of ticks contained a potentially new species of Ehrlichia. We propose the status of Candidatus for this uncultured species and we provisionally name it Candidatus Ehrlichia urmitei. No Wolbachia were identified. These results show that R. bursa can be a carrier of Anaplasmataceae bacteria.

Emerging tick-borne infections in mainland China: an increasing public health threat.

Since the beginning of the 1980s, 33 emerging tick-borne agents have been identified in mainland China, including eight species of spotted fever group rickettsiae, seven species in the family Anaplasmataceae, six genospecies in the complex Borrelia burgdorferi sensu lato, 11 species of Babesia, and the virus causing severe fever with thrombocytopenia syndrome. In this Review we have mapped the geographical distributions of human cases of infection. 15 of the 33 emerging tick-borne agents have been reported to cause human disease, and their clinical characteristics have been described. The non-specific clinical manifestations caused by tick-borne pathogens present a major diagnostic challenge and most physicians are unfamiliar with the many tick-borne diseases that present with non-specific symptoms in the early stages of the illness. Advances in and application of modern molecular techniques should help with identification of emerging tick-borne pathogens and improve laboratory diagnosis of human infections. We expect that more novel tick-borne infections in ticks and animals will be identified and additional emerging tick-borne diseases in human beings will be discovered.

Survey of Ixodes pacificus Ticks in California Reveals a Diversity of Microorganisms and a Novel and Widespread Anaplasmataceae Species.

Ixodes pacificus ticks can harbor a wide range of human and animal pathogens. To survey the prevalence of tick-borne known and putative pathogens, we tested 982 individual adult and nymphal I. pacificus ticks collected throughout California between 2007 and 2009 using a broad-range PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) assay designed to detect a wide range of tick-borne microorganisms. Overall, 1.4% of the ticks were found to be infected with Borrelia burgdorferi, 2.0% were infected with Borrelia miyamotoi and 0.3% were infected with Anaplasma phagocytophilum. In addition, 3.0% were infected with Babesia odocoilei. About 1.2% of the ticks were co-infected with more than one pathogen or putative pathogen. In addition, we identified a novel Anaplasmataceae species that we characterized by sequencing of its 16S rRNA, groEL, gltA, and rpoB genes. Sequence analysis indicated that this organism is phylogenetically distinct from known Anaplasma species with its closest genetic near neighbors coming from Asia. The prevalence of this novel Anaplasmataceae species was as high as 21% at one site, and it was detected in 4.9% of ticks tested statewide. Based upon this genetic characterization we propose that this organism be called 'Candidatus Cryptoplasma californiense'. Knowledge of this novel microbe will provide awareness for the community about the breadth of the I. pacificus microbiome, the concept that this bacterium could be more widely spread; and an opportunity to explore whether this bacterium also contributes to human or animal disease burden.

Co-infection with 'Candidatus Neoehrlichia mikurensis' and Borrelia afzelii in an Ixodes ricinus tick that has bitten a human in Romania.

Despite the vast importance of ticks as disease vectors, the infectious agents transmitted by ticks are still incompletely known in many areas. Here, we report for the first time the detection of the bacterium 'Candidatus Neoehrlichia mikurensis' in Romania, in an Ixodes ricinus tick obtained from a human. Furthermore, the tick also had a co-infection with Borrelia afzelii. 'Candidatus Neoehrlichia mikurensis' is one of the most recent discoveries of a tick-borne agent, and has been found in human patients in several European countries as well as in China.

Understanding Anaplasmataceae pathogenesis using "Omics" approaches.

This paper examines how "Omics" approaches improve our understanding of Anaplasmataceae pathogenesis, through a global and integrative strategy to identify genes and proteins involved in biochemical pathways key for pathogen-host-vector interactions. The Anaplasmataceae family comprises obligate intracellular bacteria mainly transmitted by arthropods. These bacteria are responsible for major human and animal endemic and emerging infectious diseases with important economic and public health impacts. In order to improve disease control strategies, it is essential to better understand their pathogenesis. Our work focused on four Anaplasmataceae, which cause important animal, human and zoonotic diseases: Anaplasma marginale, A. phagocytophilum, Ehrlichia chaffeensis, and E. ruminantium. Wolbachia spp. an endosymbiont of arthropods was also included in this review as a model of a non-pathogenic Anaplasmataceae. A gap analysis on "Omics" approaches on Anaplasmataceae was performed, which highlighted a lack of studies on the genes and proteins involved in the infection of hosts and vectors. Furthermore, most of the studies have been done on the pathogen itself, mainly on infectious free-living forms and rarely on intracellular forms. In order to perform a transcriptomic analysis of the intracellular stage of development, researchers developed methods to enrich bacterial transcripts from infected cells. These methods are described in this paper. Bacterial genes encoding outer membrane proteins, post-translational modifications, eukaryotic repeated motif proteins, proteins involved in osmotic and oxidative stress and hypothetical proteins have been identified to play a key role in Anaplasmataceae pathogenesis. Further investigations on the function of these outer membrane proteins and hypothetical proteins will be essential to confirm their role in the pathogenesis. Our work underlines the need for further studies in this domain and on host and vector responses to infection.

Ixodes ricinus abundance and its infection with the tick-borne pathogens in urban and suburban areas of Eastern Slovakia.

BACKGROUND: Raising abundance of ticks and tick-borne diseases in Europe is the result of multiple factors including climate changes and human activities. Herein, we investigated the presence and seasonal activity of Ixodes ricinus ticks from 10 urban and suburban sites in two different geographical areas of southeastern and northeastern Slovakia during 2008-2010. Our aim was to study the abundance of ticks in correlation with the environmental factors and their infection with Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Neoehrlichia mikurensis. METHODS: Questing I. ricinus ticks were collected from ten urban and suburban sites in Eastern Slovakia. A total of 670 ticks were further analysed for the presence of B. burgdorferi s.l., A. phagocytophilum and N. mikurensis by molecular methods. Tick site and environmental relations were analysed using General Linear Models (LM). The differences between the number of Lyme borreliosis cases between the Kosice and Bardejov regions during a ten-year period were tested by Wilcoxon matched pairs test. RESULTS: In total, 2921 (1913 nymphs, 1008 adults) I. ricinus ticks were collected from 10 study sites during the main questing season. Tick activity and relative abundance differed between locations and months. Temperature and humidity were the main factors affecting the tick abundance and questing activity. Out of 670 examined ticks, 10.15% were infected with spirochetes from B. burgdorferi s.l. complex (represented by B. afzelii, B. garinii, B.valaisiana and B. burgdorferi s.s.), 2.69% with the A. phagocytophilum and 2.39% with N. mikurensis. The number of Lyme borreliosis cases per 100,000 inhabitants in the Bardejov region was significantly higher than in the Kosice region. CONCLUSIONS: Our data indicate that the risk of infection with tick-borne pathogens in Eastern Slovakia is common since 15.2% of ticks were infected at least with one of the tested microorganisms. Even though the abundance of ticks was affected by the microclimatic conditions and the prevalence of pathogens differed between the habitats, the infection risk for humans is also affected by human activities leading to an increased contact with infected ticks.

Characterization of 'Candidatus Neoehrlichia lotoris' (family Anaplasmataceae) from raccoons (Procyon lotor).

Recently, a novel ehrlichial organism was isolated from a raccoon (Procyon lotor) and the isolate (RAC413) was infectious to two naïve raccoons but not laboratory mice, rats or rabbits. In this study, amplification and sequencing of four gene targets (16S rRNA gene, groESL, gltA and rpoB) confirmed that the novel ehrlichial organism was a member of the family Anaplasmataceae and was most closely related to, but distinct from, 'Candidatus Neoehrlichia mikurensis' TK4456(R) and IS58. RAC413 shared the highest sequence similarity with members of the genus Ehrlichia (94.2-95.1, 80.9-83.1, 67.9-71.9 and 39.9-40.7 % similarity for the 16S rRNA gene, groESL, gltA and rpoB, respectively). No sequence variation in three sequences (16S rRNA gene, groESL and gltA) was observed between the RAC413 isolate and five additional sequences amplified from blood of naturally infected raccoons from several geographically isolated populations in the south-eastern USA. Serum samples from four experimentally infected raccoons did not react to Ehrlichia canis, Ehrlichia chaffeensis, Anaplasma marginale or Anaplasma phagocytophilum antigens in an immunofluorescence assay or an Ehrlichia ewingii peptide in an ELISA format. On the basis of the distinctive molecular and serological characteristics and apparent host specificity of this ehrlichial organism, it is proposed that this organism be designated 'Candidatus Neoehrlichia lotoris' (reference strain RAC413).

Raccoons (Procyon lotor), but not rodents, are natural and experimental hosts for an ehrlichial organism related to "Candidatus Neoehrlichia mikurensis".

"Candidatus Neoehrlichia mikurensis" has been reported from a variety of rodent and Ixodes tick species in Europe and Asia. Recently, an ehrlichial organism closely related to "Candidatus Neoehrlichia mikurensis" was cultured from a raccoon (Procyon lotor) from Georgia, USA. To determine prevalence and distribution, we conducted a molecular survey of free-ranging raccoons (n=197) from 10 populations in 3 states and found that infections were common in tick-infested populations (50-94%). In an effort to determine the host range of this organism, 10 species of rodents (n=137) trapped in 3 areas where positive raccoons had been detected were tested; all were negative. In addition, captive bred raccoons and several common laboratory animals (mice, rats, and rabbits) were inoculated with the raccoon ehrlichial isolate (strain RAC413). Raccoons became infected with the culture isolate but all other hosts were refractory to infection. The 16S rRNA gene sequence (1379bp) of the RAC413 isolate was most similar (98.4-98.8%) to members of the "Candidatus Neoehrlichia mikurensis" group and phylogenetic analysis confirmed this organism was related to, but distinct from, "Candidatus Neoehrlichia mikurensis". Based on the molecular and natural history uniqueness of this organism from raccoons, we propose that this represents a novel species in the "Candidatus Neoehrlichia" group of ehrlichial organisms.

Mechanisms to create a safe haven by members of the family Anaplasmataceae.

Members of the family Anaplasmataceae are obligatory intracellular bacteria with unique host cell specificities. Depending on each bacterial species, granulocytes, platelets, endothelial cells, monocytes, macrophages, red blood cells, and cells of invertebrates are specifically infected. This unique host cell specificity has been the major hurdle to overcome in order to cultivate this group of bacteria. Because these bacteria cannot survive outside host cells, once released from a host cell, they need to rapidly induce signals for their own internalization into another host cell unique to each species. How these bacteria enter and continue to survive and replicate within the host milieu, then exit the host cell is largely unknown. Recently, however, unique strategies employed by some of these bacteria for successful parasitism of mammalian leukocytes have begun to be uncovered. When these bacteria interact with host cells, signals are transduced both inside the host cells and inside the bacteria. These signals disable the alarm system, as well as microbicidal mechanisms, of the leukocytes and condition the host cells to accept these intruders to share space and nutrient resources. Signals transduced inside the bacteria allow them to finely tune their metabolism and physiology in the new host cell environment and to disguise themselves as "insiders" so that their sojourn does not upset the host cell physiology until they have sufficiently multiplied. This paper discusses our recent findings on these topics.

Infection exclusion of the rickettsial pathogen anaplasma marginale in the tick vector Dermacentor variabilis.

Anaplasma marginale is a tick-borne, rickettsial cattle pathogen that is endemic in several areas of the United States. Recent studies (J. de la Fuente, J. C. Garcia-Garcia, E. F. Blouin, J. T. Saliki, and K. M. Kocan, Clin. Diagn. Lab. Immunol. 9:658-668, 2002) demonstrated that infection of cultured tick cells and bovine erythrocytes with one genotype of A. marginale excluded infection with other genotypes, a phenomenon referred to as infection exclusion. The present study was undertaken to confirm the phenomenon of infection exclusion of A. marginale genotypes in a tick vector, Dermacentor variabilis. Only one genotype of A. marginale (Virginia isolate) was detected by PCR in ticks that fed first on a calf infected with a Virginia isolate and second on a calf infected with an Oklahoma isolate. These studies demonstrate that infection exclusion of A. marginale genotypes also occurs in naturally infected ticks, as well as in cattle and cultured tick cells, and results in establishment of only one genotype per tick.

Ehrlichia muris sp. nov., identified on the basis of 16S rRNA base sequences and serological, morphological, and biological characteristics.

The 16S rRNA gene of a new infectious agent, strain AS145T (T = type strain), which was isolated from a wild mouse in Japan, was amplified by using the PCR. The amplimers were directly sequenced by dideoxynucleotide methods with Taq DNA polymerase. Sequence comparisons with other members of the tribe Ehrlichieae and related species revealed that the infectious agent isolated from the mouse is a new species of the genus Ehrlichia that is most closely related to Ehrlichia chaffeensis (level of sequence similarity, 97.9%), an agent of human ehrlichiosis in the United States. This result was consistent with the results of an immunoblot analysis performed with immune sera against different ehrlichiosis agents. On the basis of these findings and other morphological, biological, and serological characteristics of the organism, we propose that ehrlichiae with these properties belong to a new species, Ehrlichia muris.

Ultrastructural aspects of intraerythrocytic development of a Venezuelan strain of Anaplasma marginale.

Blood samples from splenectomized calves infected with a Venezuelan strain of Anaplasma marginale were studied by electron microscope. A. marginale appears to invade erythrocytes by the invagination of the host cell plasma membrane. The parasite reproduces by binary or multiple fission, changing from a rounded to an irregularly polyhedral form during and after division. The mature Anaplasma, within the host red cell modifies the erythrocytes cytoplasma and external membrane producing pores through which the parasite can escape without lysing the host erythrocyte.