The Obligate Intracellular Bacterial Pathogen Anaplasma phagocytophilum Exploits Host Cell Multivesicular Body Biogenesis for Proliferation and Dissemination.

Anaplasma phagocytophilum is the etiologic agent of the emerging infection, granulocytic anaplasmosis. This obligate intracellular bacterium lives in a host cell-derived vacuole that receives membrane traffic from multiple organelles to fuel its proliferation and from which it must ultimately exit to disseminate infection. Understanding of these essential pathogenic mechanisms has remained poor. Multivesicular bodies (MVBs) are late endosomal compartments that receive biomolecules from other organelles and encapsulate them into intralumenal vesicles (ILVs) using endosomal sorting complexes required for transport (ESCRT) machinery and ESCRT-independent machinery. Association of the ESCRT-independent protein, ALIX, directs MVBs to the plasma membrane where they release ILVs as exosomes. We report that the A. phagocytophilum vacuole (ApV) is acidified and enriched in lysobisphosphatidic acid, a lipid that is abundant in MVBs. ESCRT-0 and ESCRT-III components along with ALIX localize to the ApV membrane. siRNA-mediated inactivation of ESCRT-0 and ALIX together impairs A. phagocytophilum proliferation and infectious progeny production. RNA silencing of ESCRT-III, which regulates ILV scission, pronouncedly reduces ILV formation in ApVs and halts infection by arresting bacterial growth. Rab27a and its effector Munc13-4, which drive MVB trafficking to the plasma membrane and subsequent exosome release, localize to the ApV. Treatment with Nexinhib20, a small molecule inhibitor that specifically targets Rab27a to block MVB exocytosis, abrogates A. phagocytophilum infectious progeny release. Thus, A. phagocytophilum exploits MVB biogenesis and exosome release to benefit each major stage of its intracellular infection cycle: intravacuolar growth, conversion to the infectious form, and exit from the host cell. IMPORTANCE Anaplasma phagocytophilum causes granulocytic anaplasmosis, a globally emerging zoonosis that can be severe, even fatal, and for which antibiotic treatment options are limited. A. phagocytophilum lives in an endosomal-like compartment that interfaces with multiple organelles and from which it must ultimately exit to spread within the host. How the bacterium accomplishes these tasks is poorly understood. Multivesicular bodies (MVBs) are intermediates in the endolysosomal pathway that package biomolecular cargo from other organelles as intralumenal vesicles for release at the plasma membrane as exosomes. We discovered that A. phagocytophilum exploits MVB biogenesis and trafficking to benefit all aspects of its intracellular infection cycle: proliferation, conversion to its infectious form, and release of infectious progeny. The ability of a small molecule inhibitor of MVB exocytosis to impede A. phagocytophilum dissemination indicates the potential of this pathway as a novel host-directed therapeutic target for granulocytic anaplasmosis.

The Unfolded-Protein Response Triggers the Arthropod Immune Deficiency Pathway.

The insect immune deficiency (IMD) pathway is a defense mechanism that senses and responds to Gram-negative bacteria. Ticks lack genes encoding upstream components that initiate the IMD pathway. Despite this deficiency, core signaling molecules are present and functionally restrict tick-borne pathogens. The molecular events preceding activation remain undefined. Here, we show that the unfolded-protein response (UPR) initiates the IMD network. The endoplasmic reticulum (ER) stress receptor IRE1α is phosphorylated in response to tick-borne bacteria but does not splice the mRNA encoding XBP1. Instead, through protein modeling and reciprocal pulldowns, we show that Ixodes IRE1α complexes with TRAF2. Disrupting IRE1α-TRAF2 signaling blocks IMD pathway activation and diminishes the production of reactive oxygen species. Through in vitro, in vivo, and ex vivo techniques, we demonstrate that the UPR-IMD pathway circuitry limits the Lyme disease-causing spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale (anaplasmosis). Altogether, our study uncovers a novel linkage between the UPR and the IMD pathway in arthropods. IMPORTANCE The ability of an arthropod to harbor and transmit pathogens is termed "vector competency." Many factors influence vector competency, including how arthropod immune processes respond to the microbe. Divergences in innate immunity between arthropods are increasingly being reported. For instance, although ticks lack genes encoding key upstream molecules of the immune deficiency (IMD) pathway, it is still functional and restricts causative agents of Lyme disease (Borrelia burgdorferi) and anaplasmosis (Anaplasma phagocytophilum). How the IMD pathway is activated in ticks without classically defined pathway initiators is not known. Here, we found that a cellular stress response network, the unfolded-protein response (UPR), functions upstream to induce the IMD pathway and restrict transmissible pathogens. Collectively, this explains how the IMD pathway can be activated in the absence of canonical pathway initiators. Given that the UPR is highly conserved, UPR-initiated immunity may be a fundamental principle impacting vector competency across arthropods.

Citizen Science Provides an Efficient Method for Broad-Scale Tick-Borne Pathogen Surveillance of Ixodes pacificus and Ixodes scapularis across the United States.

Tick-borne diseases have expanded over the last 2 decades as a result of shifts in tick and pathogen distributions. These shifts have significantly increased the need for accurate portrayal of real-time pathogen distributions and prevalence in hopes of stemming increases in human morbidity. Traditionally, pathogen distribution and prevalence have been monitored through case reports or scientific collections of ticks or reservoir hosts, both of which have challenges that impact the extent, availability, and accuracy of these data. Citizen science tick collections and testing campaigns supplement these data and provide timely estimates of pathogen prevalence and distributions to help characterize and understand tick-borne disease threats to communities. We utilized our national citizen science tick collection and testing program to describe the distribution and prevalence of four Ixodes-borne pathogens, Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Anaplasma phagocytophilum, and Babesia microti, across the continental United States. IMPORTANCE In the 21st century, zoonotic pathogens continue to emerge, while previously discovered pathogens continue to have changes within their distribution and prevalence. Monitoring these pathogens is resource intensive, requiring both field and laboratory support; thus, data sets are often limited within their spatial and temporal extents. Citizen science collections provide a method to harness the general public to collect samples, enabling real-time monitoring of pathogen distribution and prevalence.

Duration of tick attachment necessary for transmission of Anaplasma phagocytophilum by Ixodes scapularis (Acari: Ixodidae) nymphs.

This study assessed the duration of tick attachment necessary for a successful transmission of Anaplasma phagocytophilum by an infected I. scapularis nymph. Individual nymphs were placed upon BALB/c mice and allowed to feed for predetermined time intervals of 4 to 72 h. Ticks removed from mice at predetermined intervals were tested by PCR for verification of infection and evaluation of the bacterial load. The success of pathogen transmission to mice was assessed by blood-PCR at 7, 14 and 21 days postinfestation, and IFA at 21 days postinfestation. Anaplasma phagocytophilum infection was documented in 10-30 % of mice, from which ticks were removed within the first 20 h of feeding. However, transmission success was ≥70% if ticks remained attached for 36 h or longer. Notably, none of the PCR-positive mice that were exposed to infected ticks for 4 to 8 h and only half of PCR-positive mice exposed for 24 h developed antibodies within 3 weeks postinfestation. On the other hand, all mice with detectable bacteremia after being infested for 36 h seroconverted. This suggests that although some of the ticks removed prior to 24 h of attachment succeed in injecting a small amount of A. phagocytophilum, this amount is insufficient for stimulating humoral immunity and perhaps for establishing disseminated infection in BALB/c mice. Although A. phagocytophilum may be present in salivary glands of unfed I. scapularis nymphs, the amount of A. phagocytophilum initially contained in saliva appears insufficient to cause sustainable infection in a host. Replication and, maybe, reactivation of the agent for 12-24 h in a feeding tick is required before a mouse can be consistently infected.

Environmental determinants of Anaplasma phagocytophilum infection in cattle using a kernel density function.

The study of vector-borne zoonotic diseases often relies on partial data, because of the constraints associated with observing various elements of the transmission cycle: the pathogen, the vector, the host - wild or domestic. Each angle comes with its own practical challenges, leading to data reflecting poorly either on spatial or temporal dynamics, or both. In this study, we investigated the effect of landscape on the presence of bovine ehrlichiosis infection in Walloon cattle. This disease is transmitted to cattle through the bite of a tick infected by the bacterium Anaplasma phagocytophilum. The first case of bovine ehrlichiosis in the southern region of Belgium (Wallonia) was detected in 2005 and the high seroprevalence found in herds suggests that the disease is endemic. The presence of antibodies of A. phagocytophilum in one cow selected in each of 1445 herds in 2010 and 2011 was detected using indirect immunofluorescence. Samples were geolocated at the farm. However, the precise location of infection remains uncertain. To account for the data sparsity, we elaborated a spatial index for the intensity of the presence of seropositive animals, based on a non-parametric kernel density estimation. We examined this index with the landscape surrounding the pastures, using multiple regressions. Landscape factors were selected using a conceptual framework based on the ecological resources needed for the transmission cycle of A. phagocytophilum. Results suggest that our spatial index adequately reflected infection presence in cattle in Wallonia, which was highest in central regions, corresponding to more forested and fragmented landscapes. We noticed that the presence of large hosts, wild or domestic, as well as the composition and configuration of the landscape of the pasture, influenced the capacity of the pasture to support the presence of bovine ehrlichiosis in Walloon herds. This is consistent with the ecology of A. phagocytophilum and current knowledge about risk factors of tick-borne diseases in cattle at the regional scale. The nature of the kernel density index, based on uncertainties over the location of cases positive to A. phagocytophilum, reflected the infectiousness profile at the landscape and not at the pasture level. Results also highlighted that the effects of some environmental variables remain, even when considering the different agro-geographic regions of Wallonia, which present contrasted landscapes and different levels of intensity of A. phagocytophilum infection. The kernel density index is a useful tool to help veterinary practitioner to quickly target areas where A. phagocytophilum infection is likely.

A Comparative Spatial and Climate Analysis of Human Granulocytic Anaplasmosis and Human Babesiosis in New York State (2013-2018).

Human granulocytic anaplasmosis (HGA) and human babesiosis are tick-borne diseases spread by the blacklegged tick (Ixodes scapularis Say, Acari: Ixodidae) and are the result of infection with Anaplasma phagocytophilum and Babesia microti, respectively. In New York State (NYS), incidence rates of these diseases increased concordantly until around 2013, when rates of HGA began to increase more rapidly than human babesiosis, and the spatial extent of the diseases diverged. Surveillance data of tick-borne pathogens (2007 to 2018) and reported human cases of HGA (n = 4,297) and human babesiosis (n = 2,986) (2013-2018) from the New York State Department of Health (NYSDOH) showed a positive association between the presence/temporal emergence of each pathogen and rates of disease in surrounding areas. Incidence rates of HGA were higher than human babesiosis among White and non-Hispanic/non-Latino individuals, as well as all age and sex groups. Human babesiosis exhibited higher rates among non-White individuals. Climate, weather, and landscape data were used to build a spatially weighted zero-inflated negative binomial (ZINB) model to examine and compare associations between the environment and rates of HGA and human babesiosis. HGA and human babesiosis ZINB models indicated similar associations with forest cover, forest land cover change, and winter minimum temperature; and differing associations with elevation, urban land cover change, and winter precipitation. These results indicate that tick-borne disease ecology varies between pathogens spread by I. scapularis.

Incompetence of the Asian Longhorned Tick (Acari: Ixodidae) in Transmitting the Agent of Human Granulocytic Anaplasmosis in the United States.

The Asian longhorned tick, Haemaphysalis longicornis Neumann (Acari: Ixodidae), was recently introduced into the United States and is now established in at least 15 states. Considering its ability for parthenogenetic propagation and propensity for creating high-density populations, there is concern that this tick may become involved in transmission cycles of endemic tick-borne human pathogens. Human granulocytic anaplasmosis (HGA) caused by Anaplasma phagocytophilum is one of the more common tick-borne diseases in the United States, especially in the northeastern and midwestern states. There is considerable geographical overlap between HGA cases and the currently known distribution of H. longicornis, which creates a potential for this tick to encounter A. phagocytophilum while feeding on naturally infected vertebrate hosts. Therefore, we evaluated the ability of H. longicornis to acquire and transmit the agent of HGA under laboratory conditions and compared it to the vector competence of I. scapularis. Haemaphysalis longicornis nymphs acquired the pathogen with the bloodmeal while feeding on infected domestic goats, but transstadial transmission was inefficient and PCR-positive adult ticks were unable to transmit the pathogen to naïve goats. Results of this study indicate that the Asian longhorned tick is not likely to play a significant role in the epidemiology of HGA in the United States.

Protein and DNA Biosynthesis Demonstrated in Host Cell-Free Phagosomes Containing Anaplasma phagocytophilum or Ehrlichia chaffeensis in Axenic Media.

Rickettsiae belong to the Anaplasmataceae family, which includes mostly tick-transmitted pathogens causing human, canine, and ruminant diseases. Biochemical characterization of the pathogens remains a major challenge because of their obligate parasitism. We investigated the use of an axenic medium for growth of two important pathogens-Anaplasma phagocytophilum and Ehrlichia chaffeensis-in host cell-free phagosomes. We recently reported that the axenic medium promotes protein and DNA biosynthesis in host cell-free replicating form of E. chaffeensis, although the bacterial replication is limited. We now tested the hypothesis that growth on axenic medium can be improved if host cell-free rickettsia-containing phagosomes are used. Purification of phagosomes from A. phagocytophilum- and E. chaffeensis-infected host cells was accomplished by density gradient centrifugation combined with magnet-assisted cell sorting. Protein and DNA synthesis was observed for both organisms in cell-free phagosomes with glucose-6-phosphate and/or ATP. The levels of protein and DNA synthesis were the highest for a medium pH of 7. The data demonstrate bacterial DNA and protein synthesis for the first time in host cell-free phagosomes for two rickettsial pathogens. The host cell support-free axenic growth of obligate pathogenic rickettsiae will be critical in advancing research goals in many important tick-borne diseases impacting human and animal health.

Red and fallow deer determine the density of Ixodes ricinus nymphs containing Anaplasma phagocytophilum.

BACKGROUND: The density of Ixodes ricinus nymphs infected with Anaplasma phagocytophilum is one of the parameters that determines the risk for humans and domesticated animals to contract anaplasmosis. For this, I. ricinus larvae need to take a bloodmeal from free-ranging ungulates, which are competent hosts for A. phagocytophilum. METHODS: Here, we compared the contribution of four free-ranging ungulate species, red deer (Cervus elaphus), fallow deer (Dama dama), roe deer (Capreolus capreolus), and wild boar (Sus scrofa), to A. phagocytophilum infections in nymphs. We used a combination of camera and live trapping to quantify the relative availability of vertebrate hosts to questing ticks in 19 Dutch forest sites. Additionally, we collected questing I. ricinus nymphs and tested these for the presence of A. phagocytophilum. Furthermore, we explored two potential mechanisms that could explain differences between species: (i) differences in larval burden, which we based on data from published studies, and (ii) differences in associations with other, non-competent hosts. RESULTS: Principal component analysis indicated that the density of A. phagocytophilum-infected nymphs (DIN) was higher in forest sites with high availability of red and fallow deer, and to a lesser degree roe deer. Initial results suggest that these differences are not a result of differences in larval burden, but rather differences in associations with other species or other ecological factors. CONCLUSIONS: These results indicate that the risk for contracting anaplasmosis in The Netherlands is likely highest in the few areas where red and fallow deer are present. Future studies are needed to explore the mechanisms behind this association.

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

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

An efficient microinjection method to generate human anaplasmosis agent Anaplasma phagocytophilum-infected ticks.

Ticks are important vectors that transmit several pathogens including human anaplasmosis agent, Anaplasma phagocytophilum. This bacterium is an obligate intracellular rickettsial pathogen. An infected reservoir animal host is often required for maintenance of this bacterial colony and as a source for blood to perform needle inoculations in naïve animals for tick feeding studies. In this study, we report an efficient microinjection method to generate A. phagocytophilum-infected ticks in laboratory conditions. The dense-core (DC) form of A. phagocytophilum was isolated from in vitro cultures and injected into the anal pore of unfed uninfected Ixodes scapularis nymphal ticks. These ticks successfully transmitted A. phagocytophilum to the murine host. The bacterial loads were detected in murine blood, spleen, and liver tissues. In addition, larval ticks successfully acquired A. phagocytophilum from mice that were previously infected by feeding with DC-microinjected nymphal ticks. Transstadial transmission of A. phagocytophilum from larvae to nymphal stage was also evident in these ticks. Taken together, our study provides a timely, rapid, and an efficient method not only to generate A. phagocytophilum-infected ticks but also provides a tool to understand acquisition and transmission dynamics of this bacterium and perhaps other rickettsial pathogens from medically important vectors.

Epidemiological study on Anaplasma phagocytophilum in cattle: Molecular prevalence and risk factors assessment in different ecological zones in Iran.

Anaplasma phagocytophilum is a tick-borne pathogen affecting humans and domestic animals worldwide. This study aimed to determine the molecular epidemiology and its associated risk factors of A. phagocytophilum infection in cattle in four ecological zones of Iran. A multi-stage stratified random sampling method was utilized during 2017-2018. A total of 1851 blood samples from 320 cattle farms were collected and examined using specific nested polymerase chain reaction (nPCR) based on the 16S rRNA gene. The overall prevalence of A. phagocytophilum was 15.5% (286/1851) by using nPCR. All four zones were A. phagocytophilum positive, the presence of A. phagocytophilum DNA was detected in eight out of nine tested provinces. Univariable analysis of risk factors indicated that climate, altitude, longitude, latitude, season, farm-type, feeding method, hygiene of the farm, tick infestation, use of acaricides by the farmer, distance from other farms, contact with wild animals, race, sex, and milk yield were significant determinants (P < 0.05) for A. phagocytophilum infection. The multivariable analysis determined that longitude, latitude, season, feeding method, and hygiene of the farm remained as significant risk factors for A. phagocytophilum infection (P < 0.05). Specific (SaTScan) cluster analysis identified two high risks and four low risks statistically significant clusters for A. phagocytophilum infection amongst the study areas (P < 0.001). Phylogenetic analysis indicated that A. phagocytophilum 16S rRNA isolates were 96-99% identical to sequences deposited in the GenBank. To the best of our knowledge, this is the first comprehensive molecular study on the epidemiology and risk factors analysis of A. phagocytophilum infection in cattle in different climatic zones of Iran. Further investigations are necessary to be performed regarding the tick vectors, reservoir animals, and the zoonotic potential of the A. phagocytophilum in the endemic region of Iran.

Binding of Host Cell Surface Protein Disulfide Isomerase by Anaplasma phagocytophilum Asp14 Enables Pathogen Infection.

Diverse intracellular pathogens rely on eukaryotic cell surface disulfide reductases to invade host cells. Pharmacologic inhibition of these enzymes is cytotoxic, making it impractical for treatment. Identifying and mechanistically dissecting microbial proteins that co-opt surface reductases could reveal novel targets for disrupting this common infection strategy. Anaplasma phagocytophilum invades neutrophils by an incompletely defined mechanism to cause the potentially fatal disease granulocytic anaplasmosis. The bacterium's adhesin, Asp14, contributes to invasion by virtue of its C terminus engaging an unknown receptor. Yeast-two hybrid analysis identified protein disulfide isomerase (PDI) as an Asp14 binding partner. Coimmunoprecipitation confirmed the interaction and validated it to be Asp14 C terminus dependent. PDI knockdown and antibody-mediated inhibition of PDI reductase activity impaired A. phagocytophilum infection of but not binding to host cells. Infection during PDI inhibition was rescued when the bacterial but not host cell surface disulfide bonds were chemically reduced with tris(2-carboxyethyl)phosphine-HCl (TCEP). TCEP also restored bacterial infectivity in the presence of an Asp14 C terminus blocking antibody that otherwise inhibits infection. A. phagocytophilum failed to productively infect myeloid-specific-PDI conditional-knockout mice, marking the first demonstration of in vivo microbial dependency on PDI for infection. Mutational analyses identified the Asp14 C-terminal residues that are critical for binding PDI. Thus, Asp14 binds and brings PDI proximal to A. phagocytophilum surface disulfide bonds that it reduces, which enables cellular and in vivo infection.IMPORTANCEAnaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis, an emerging potentially fatal disease and the second-most common tick-borne illness in the United States. Treatment options are limited, and no vaccine exists. Due to the bacterium's obligatory intracellular lifestyle, A. phagocytophilum survival and pathogenesis are predicated on its ability to enter host cells. Understanding its invasion mechanism will yield new targets for preventing bacterial entry and, hence, disease. We report a novel entry pathway in which the A. phagocytophilum outer membrane protein Asp14 binds host cell surface protein disulfide isomerase via specific C-terminal residues to promote reduction of bacterial surface disulfide bonds, which is critical for cellular invasion and productive infection in vivo Targeting the Asp14 C terminus could be used to prevent/treat granulocytic anaplasmosis. Our findings have broad implications, as a thematically similar approach could be applied to block infection by other intracellular microbes that exploit cell surface reductases.

Prevalence of Infection and Co-Infection and Presence of Rickettsial Endosymbionts in Ixodes Scapularis (Acari: Ixodidae) in Connecticut, USA.

Ixodes scapularis is currently known to transmit 7 pathogens responsible for Lyme disease, anaplasmosis, babesiosis, tick-borne relapsing fever, ehrlichiosis, and Powassan encephalitis. Ixodes scapularis can also be colonized by endosymbiotic bacteria including those in the genus of Rickettsia. We screened 459 I. scapularis ticks submitted to the Connecticut Agricultural Experiment Station Tick Testing Laboratory with the objectives to (1) examine differences in infection prevalence of Borrelia burgdorferi, Anaplasma phagocytophilum, Babesia microti, and Borrelia miyamotoi, (2) evaluate whether prevalence of co-infections occur at the same frequency that would be expected based on single infection, and (3) determine the presence of rickettsial endosymbionts in I. scapularis. The prevalence of infection in I. scapularis was highest with Bo. burgdorferi sensu lato (nymph = 45.8%; female = 47.0%), followed by A. phagocytophilum (nymph = 4.0%; female = 6.9%), Ba. microti (nymph = 5.7%; female = 4.7%), and Bo. miyamotoi (nymph = 0%; female = 7.3%). We also identified rickettsial endosymbionts in 93.3% of I. scapularis. Nymphs were significantly more likely to be infected with Bo. burgdorferi if they were infected with Ba. microti, whereas adult females were significantly more likely to be infected with Bo. burgdorferi if they were infected with A. phagocytophilum. Our study suggests that the infection prevalence of Bo. burgdorferi is not independent of other co-circulating pathogens and that there is a substantially higher infection of Bo. miyamotoi in I. scapularis females compared with nymphs in this study. High prevalence of infection and co-infection with multiple pathogens in I. scapularis highlights the public health consequences in Connecticut, a state endemic for Lyme and other tick-borne diseases.

Prevention of transmission of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum by Ixodes spp. ticks to dogs treated with the Seresto® collar (imidacloprid 10% + flumethrin 4.5%).

The capability of imidacloprid 10% + flumethrin 4.5% (Seresto®) collars to prevent transmission of Borrelia burgdorferi sensu lato (Bbsl) and Anaplasma phagocytophilum (Ap) by naturally infected ticks was evaluated in two studies with 44 dogs. In each study, one group served as non-treated control, whereas the other groups were treated with the Seresto® collar. All dogs were exposed to naturally Bbsl- and Ap-infected hard ticks (Ixodes ricinus, Ixodes scapularis). In study 1, tick infestation was performed on study day (SD) 63 (2 months post-treatment [p.t.]); in study 2, it was performed on SD 32 (one month p.t.) respectively SD 219 (seven months p.t.). In situ tick counts were performed 2 days after infestation. Tick counts and removals followed 6 (study 1) or 5 days (study 2) later. Blood sampling was performed for the detection of specific Bbsl and Ap antibodies and, in study 1, for the documentation of Ap DNA by PCR. Skin biopsies were examined for Bbsl by PCR and culture (only study 1). The efficacy against Ixodes spp. was 100% at all time points. In study 1, two of six non-treated dogs became infected with Bbsl, and four of six tested positive for Ap; none of the treated dogs tested positive for Bbsl or Ap. In study 2, ten of ten non-treated dogs became infected with Bbsl and Ap; none of the treated dogs tested positive for Bbsl or Ap; 100% acaricidal efficacy was shown in both studies. Transmission of Bbsl and Ap was successfully blocked for up to 7 months.

Simultaneous infection of cattle with different Anaplasma phagocytophilum variants.

Anaplasma phagocytophilum is a tick-transmitted Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It causes tick-borne fever in cattle and sheep. We report here the case of a 5-year-old cow from Germany with clinically overt granulocytic anaplasmosis presenting with fever, lower limb oedema and drop in milk-yield. The herd encompassed 10 animals, 8 other animals showed subclinical infection. The strains from the 9 A. phagocytophilum positive cows were molecularly characterized using ankA gene-based and multilocus sequence typing (MLST). Seven of 9 (78%) animals were infected simultaneously with different ankA variants belonging to ankA clusters I and IV. MLST analysis also revealed the presence of multiple strain types. This could be due to co-transmission or superinfection. Hosts harboring diverse A. phagocytophilum strains might enable the emergence of new ankA variants and/or MLST sequence types via bacterial recombination.

Anaplasma phagocytophilum modifies tick cell microRNA expression and upregulates isc-mir-79 to facilitate infection by targeting the Roundabout protein 2 pathway.

The microRNAs (miRNAs) are a class of small noncoding RNAs that have important regulatory roles in multicellular organisms including innate and adaptive immune pathways to control bacterial, parasite and viral infections, and pathogens could modify host miRNA profile to facilitate infection and multiplication. Therefore, understanding the function of host miRNAs in response to pathogen infection is relevant to characterize host-pathogen molecular interactions and to provide new targets for effective new interventions for the control infectious diseases. The objective of this study was to characterize the dynamics and functional significance of the miRNA response of the tick vector Ixodes scapularis in response to Anaplasma phagocytophilum infection, the causative agent of human and animal granulocytic anaplasmosis. To address this objective, the composition of tick miRNAs, functional annotation, and expression profiling was characterized using high throughout RNA sequencing in uninfected and A. phagocytophilum-infected I. scapularis ISE6 tick cells, a model for tick hemocytes involved in pathogen infection. The results provided new evidences on the role of tick miRNA during pathogen infection, and showed that A. phagocytophilum modifies I. scapularis tick cell miRNA profile and upregulates isc-mir-79 to facilitate infection by targeting the Roundabout protein 2 (Robo2) pathway. Furthermore, these results suggested new targets for interventions to control pathogen infection in ticks.

Ixodes scapularis Src tyrosine kinase facilitates Anaplasma phagocytophilum survival in its arthropod vector.

Anaplasma phagocytophilum, the agent of human anaplasmosis, is an obligate intracellular bacterium that uses multiple survival strategies to persist in Ixodes scapularis ticks. Our previous study showed that A. phagocytophilum efficiently induced the tyrosine phosphorylation of several Ixodes proteins that includes extended phosphorylation of actin at tyrosine residue Y178. In order to identify the tyrosine kinase responsible for the A. phagocytophilum induced tyrosine phosphorylation of proteins, we combed the I. scapularis genome and identified a non-receptor Src tyrosine kinase ortholog. I. scapularis Src kinase showed high degree of amino acid sequence conservation with Dsrc from Drosophila melanogaster. We noted that at different developmental stages of I. scapularis ticks, larvae expressed significantly higher levels of src transcripts in comparison to the other stages. We found that A. phagocytophilum significantly reduced Src levels in unfed nymphs and in nymphs while blood feeding (48 h during feeding) in comparison to the levels noted to relative uninfected controls. However, A. phagocytophilum increased Src levels in fully engorged larvae and nymphs (48 h post feeding) and in vitro tick cells in comparison to the relative uninfected controls. Inhibition of Src kinase expression and activity by treatment with src-dsRNA or Src-inhibitor, respectively, significantly reduced A. phagocytophilum loads in ticks and tick cells. Overall, our study provides evidence for the important role of I. scapularis Src kinase in facilitating A. phagocytophilum colonization and survival in the arthropod vector.

Tick-Pathogen Interactions: The Metabolic Perspective.

The first tick genome published in 2016 provided an invaluable tool for studying the molecular basis of tick-pathogen interactions. Metabolism is a key element in host-pathogen interactions. However, our knowledge of tick-pathogen metabolic interactions is very limited. Recently, a systems biology approach, using omics datasets, has revealed that tick-borne pathogen infection induces transcriptional reprograming affecting several metabolic pathways in ticks, facilitating infection, multiplication, and transmission. Results suggest that the response of tick cells to tick-borne pathogens is associated with tolerance to infection. Here we review our current understanding of the modulation of tick metabolism by tick-borne pathogens, with a focus on the model intracellular bacterium Anaplasma phagocytophilum.

Three genetically distinct clades of Anaplasma phagocytophilum in Ixodes scapularis.

Human granulocytic anaplasmosis (HGA) is an emerging disease in Canada because of range expansion by the arthropod vector, Ixodes scapularis. These ticks carry the Ap-ha variant of Anaplasma phagocytophilum (Ap-ha), which has been implicated in causing HGA, and the Ap-variant 1, which is not associated with human infection. We report the detection of 13 genotypes of the ankyrin (ankA) gene among 76 infected blacklegged ticks. Haplotype network and phylogenetic analyses revealed that the ankA genotypes corresponding to the Ap-ha variant did not form a monophyletic assemblage. They formed two distinct clades (Clades I and III), one of which was genetically more similar in nucleotide and amino acid sequences to genotypes of Ap-variant 1 that comprised Clade II. Additional work is needed to explore the evolutionary history of A. phagocytophilum in North America, and to determine if there are differences in pathogenicity or clinical symptoms associated with the two divergent groups of the Ap-ha variant given the significant differences in ankA amino acid sequence.