scholarly journals Dissecting Flavivirus Biology in Salivary Gland Cultures from Fed and Unfed Ixodes scapularis (Black-Legged Tick)

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. e02628-18 ◽  
Author(s):  
Jeffrey M. Grabowski ◽  
Olof R. Nilsson ◽  
Elizabeth R. Fischer ◽  
Dan Long ◽  
Danielle K. Offerdahl ◽  
...  
Keyword(s):  
Salivary Glands ◽  
Cellular Localization ◽  
Ixodes Scapularis ◽  
Blood Feeding ◽  
The United States ◽  
Embryonic Cells ◽  
Content Type ◽  
Virus Like Particles ◽  
Powassan Virus ◽  
Tick Salivary Glands

ABSTRACT The Ixodes scapularis tick transmits a number of pathogens, including tick-borne flaviviruses (TBFVs). In the United States, confirmed human infections with the Powassan virus (POWV) TBFV have a fatality rate of ∼10% and are increasing in incidence. Tick salivary glands (SGs) serve as an organ barrier to TBFV transmission, and little is known regarding the location of TBFV infection in SGs from fed ticks. Previous studies showed I. scapularis vanin (VNN) involved with TBFV infection of I. scapularis ISE6 embryonic cells, suggesting a potential role for this gene. The overall goal of this study was to use SG cultures to compare data on TBFV biology in SGs from fully engorged, replete (fed) ticks and from unfed ticks. TBFV multiplication was higher in SGs from fed ticks than in those from unfed ticks. Virus-like particles were observed only in granular acini of SGs from unfed ticks. The location of TBFV infection of SGs from fed ticks was observed in cells lining lobular ducts and trachea but not observed in acini. Transcript knockdown of VNN decreased POWV multiplication in infected SG cultures from both fed and unfed ticks. This work was the first to identify localization of TBFV multiplication in SG cultures from a fed tick and a tick transcript important for POWV multiplication in the tick SG, an organ critical for TBFV transmission. This research exemplifies the use of SG cultures in deciphering TBFV biology in the tick and as a translational tool for screening and identifying potential tick genes as potential countermeasure targets. IMPORTANCE Tick-borne flaviviruses (TBFVs) are responsible for more than 15,000 human disease cases each year, and Powassan virus lineage 2 (POWV-L2) deer tick virus has been a reemerging threat in North America over the past 20 years. Rapid transmission of TBFVs in particular emphasizes the importance of preventing tick bites, the difficulty in developing countermeasures to prevent transmission, and the importance of understanding TBFV infection in tick salivary glands (SGs). Tick blood feeding is responsible for phenomenal physiological changes and is associated with changes in TBFV multiplication within the tick and in SGs. Using SG cultures from Ixodes scapularis female ticks, the primary aims of this study were to identify cellular localization of virus-like particles in acini of infected SGs from fed and unfed ticks, localization of TBFV infection in infected SGs from fed ticks, and a tick transcript (with associated metabolic function) involved in POWV-L2 infection in SG cultures.

scholarly journals An Ixodes scapularis protein required for survival of Anaplasma phagocytophilum in tick salivary glands

2006 ◽  
Vol 203 (6) ◽  
pp. 1507-1517 ◽  
Author(s):  
Bindu Sukumaran ◽  
Sukanya Narasimhan ◽  
John F. Anderson ◽  
Kathleen DePonte ◽  
Nancy Marcantonio ◽  
...  
Keyword(s):  
Gene Expression ◽  
United States ◽  
Salivary Glands ◽  
Anaplasma Phagocytophilum ◽  
Ixodes Scapularis ◽  
The United States ◽  
Salivary Protein ◽  
Specific Requirement ◽  
Obligate Intracellular ◽  
Tick Salivary Glands

Anaplasma phagocytophilum is the agent of human anaplasmosis, the second most common tick-borne illness in the United States. This pathogen, which is closely related to obligate intracellular organisms in the genera Rickettsia, Ehrlichia, and Anaplasma, persists in ticks and mammalian hosts; however, the mechanisms for survival in the arthropod are not known. We now show that A. phagocytophilum induces expression of the Ixodes scapularis salp16 gene in the arthropod salivary glands during vector engorgement. RNA interference–mediated silencing of salp16 gene expression interfered with the survival of A. phagocytophilum that entered ticks fed on A. phagocytophilum–infected mice. A. phagocytophilum migrated normally from A. phagocytophilum–infected mice to the gut of engorging salp16-deficient ticks, but up to 90% of the bacteria that entered the ticks were not able to successfully infect I. scapularis salivary glands. These data demonstrate the specific requirement of a pathogen for a tick salivary protein to persist within the arthropod and provide a paradigm for understanding how Rickettsia-like pathogens are maintained within vectors.

scholarly journals Flavivirus Infection of Ixodes scapularis (Black-Legged Tick) Ex Vivo Organotypic Cultures and Applications for Disease Control

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Jeffrey M. Grabowski ◽  
Konstantin A. Tsetsarkin ◽  
Dan Long ◽  
Dana P. Scott ◽  
Rebecca Rosenke ◽  
...  
Keyword(s):  
Organ Culture ◽  
Salivary Glands ◽  
Vaccine Development ◽  
Ex Vivo ◽  
Ixodes Scapularis ◽  
The United States ◽  
Hemorrhagic Disease ◽  
Organ Cultures ◽  
Powassan Virus ◽  
Langat Virus

ABSTRACT Ixodes scapularis ticks transmit many infectious agents that cause disease, including tick-borne flaviviruses (TBFVs). TBFV infections cause thousands of human encephalitis cases worldwide annually. In the United States, human TBFV infections with Powassan virus (POWV) are increasing and have a fatality rate of 10 to 30%. Additionally, Langat virus (LGTV) is a TBFV of low neurovirulence and is used as a model TBFV. TBFV replication and dissemination within I. scapularis organs are poorly characterized, and a deeper understanding of virus biology in this vector may inform effective countermeasures to reduce TBFV transmission. Here, we describe short-term, I. scapularis organ culture models of TBFV infection. Ex vivo organs were metabolically active for 9 to 10 days and were permissive to LGTV and POWV replication. Imaging and videography demonstrated replication and spread of green fluorescent protein-expressing LGTV in the organs. Immunohistochemical staining confirmed LGTV envelope and POWV protein synthesis within the infected organs. LGTV- and POWV-infected organs produced infectious LGTV and POWV; thus, the ex vivo cultures were suitable for study of virus replication in individual organs. LGTV- and POWV-infected midgut and salivary glands were subjected to double-stranded RNA (dsRNA) transfection with dsRNA to the LGTV 3′ untranslated region (UTR), which reduced infectious LGTV and POWV replication, providing a proof-of-concept use of RNA interference in I. scapularis organ cultures to study the effects on TBFV replication. The results contribute important information on TBFV localization within ex vivo I. scapularis organs and provide a significant translational tool for evaluating recombinant, live vaccine candidates and potential tick transcripts and proteins for possible therapeutic use and vaccine development to reduce TBFV transmission. IMPORTANCE Tick-borne flavivirus (TBFV) infections cause neurological and/or hemorrhagic disease in humans worldwide. There are currently no licensed therapeutics or vaccines against Powassan virus (POWV), the only TBFV known to circulate in North America. Evaluating tick vector targets for antitick vaccines directed at reducing TBFV infection within the arthropod vector is a critical step in identifying efficient approaches to controlling TBFV transmission. This study characterized infection of female Ixodes scapularis tick organ cultures of midgut, salivary glands, and synganglion with the low-neurovirulence Langat virus (LGTV) and the more pathogenic POWV. Cell types of specific organs were susceptible to TBFV infection, and a difference in LGTV and POWV replication was noted in TBFV-infected organs. This tick organ culture model of TBFV infection will be useful for various applications, such as screening of tick endogenous dsRNA corresponding to potential control targets within midgut and salivary glands to confirm restriction of TBFV infection.

scholarly journals Water absorption through salivary gland type I acini in the blacklegged tick, Ixodes scapularis

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3984 ◽  
Author(s):  
Donghun Kim ◽  
Paulina Maldonado-Ruiz ◽  
Ludek Zurek ◽  
Yoonseong Park
Keyword(s):  
Water Absorption ◽  
Salivary Glands ◽  
Ixodes Scapularis ◽  
Blood Feeding ◽  
Type I ◽  
Atpase Inhibitor ◽  
Excess Water ◽  
Long Duration ◽  
Tick Salivary Glands ◽  
Gland Type

Tick salivary glands play critical roles in maintaining water balance for survival, as they eliminate excess water and ions during blood feeding on hosts. In the long duration of fasting in the off-host period, ticks secrete hygroscopic saliva into the mouth cavity to uptake atmospheric water vapor. Type I acini of tick salivary glands are speculated to be involved in secretion of hygroscopic saliva based on ultrastructure studies. However, we recently proposed that type I acini play a role in resorption of water/ions from the primary saliva produced by other salivary acini (i.e., types II and III) during the tick blood feeding phase. In this study, we tested the function of type I acini in unfed female Ixodes scapularis. The route of ingested water was tracked after forced feeding of water with fluorescent dye rhodamine123. We found that type-I acini of the salivary glands, but not type II and III, are responsible for water uptake. In addition, the ingestion of water through the midgut was also observed. Injection or feeding of ouabain, a Na/K-ATPase inhibitor, suppressed water absorption in type I acini. When I. scapularis was offered a droplet of water, ticks rarely imbibed water directly (5%), while some approached the water droplet to use the high humidity formed in the vicinity of the droplet (23%). We conclude that during both on- and off-host stages, type I acini in salivary glands of female Ixodes scapularis absorb water and ions.

scholarly journals Spotted Fever GroupRickettsiaInfection and Transmission Dynamics inAmblyomma maculatum

2019 ◽  
Vol 87 (4) ◽  
Author(s):  
Chanakan Suwanbongkot ◽  
Ingeborg M. Langohr ◽  
Emma K. Harris ◽  
Wellesley Dittmar ◽  
Rebecca C. Christofferson ◽  
...  
Keyword(s):  
Salivary Glands ◽  
Spotted Fever ◽  
Attachment Site ◽  
Spotted Fever Group ◽  
Tick Feeding ◽  
Tick Saliva ◽  
Content Type ◽  
Feeding Site ◽  
Vertebrate Hosts ◽  
Tick Salivary Glands

ABSTRACTTick vectors are capable of transmitting several rickettsial species to vertebrate hosts, resulting in various levels of disease. Studies have demonstrated the transmissibility of both rickettsial pathogens and novelRickettsiaspecies or strains with unknown pathogenicity to vertebrate hosts during tick blood meal acquisition; however, the quantitative nature of transmission remains unknown. We tested the hypothesis that if infection severity is a function of the rickettsial load delivered during tick transmission, then a more virulent spotted fever group (SFG)Rickettsiaspecies is transmitted at higher levels during tick feeding. UsingAmblyomma maculatumcohorts infected withRickettsia parkerior “CandidatusRickettsia andeanae,” a quantitative PCR (qPCR) assay was employed to quantify rickettsiae in tick salivary glands and saliva, as well as in the vertebrate hosts at the tick attachment site over the duration of tick feeding. Significantly greater numbers ofR. parkerithan of “Ca. Rickettsia andeanae” rickettsiae were present in tick saliva and salivary glands and in the vertebrate hosts at the feeding site during tick feeding. Microscopy demonstrated the presence of both rickettsial species in tick salivary glands, and immunohistochemical analysis of the attachment site identified localizedR. parkeri, but not “Ca. Rickettsia andeanae,” in the vertebrate host. Lesions were also distinct and more severe in vertebrate hosts exposed toR. parkerithan in those exposed to “Ca. Rickettsia andeanae.” The specific factors that contribute to the generation of a sustained rickettsial infection and subsequent disease have yet to be elucidated, but the results of this study suggest that the rickettsial load in ticks and during transmission may be an important element.

scholarly journals Variation in the Microbiota of Ixodes Ticks with Regard to Geography, Species, and Sex

2015 ◽  
Vol 81 (18) ◽  
pp. 6200-6209 ◽  
Author(s):  
Will Van Treuren ◽  
Loganathan Ponnusamy ◽  
R. Jory Brinkerhoff ◽  
Antonio Gonzalez ◽  
Christian M. Parobek ◽  
...  
Keyword(s):  
New York ◽  
North Carolina ◽  
South Carolina ◽  
Lyme Disease ◽  
Ixodes Scapularis ◽  
Illumina Miseq ◽  
The United States ◽  
Upper Midwest ◽  
Content Type ◽  
Ixodes Ticks

ABSTRACTIxodes scapularisis the principal vector of Lyme disease on the East Coast and in the upper Midwest regions of the United States, yet the tick is also present in the Southeast, where Lyme disease is absent or rare. A closely related species,I. affinis, also carries the pathogen in the South but does not seem to transmit it to humans. In order to better understand the geographic diversity of the tick, we analyzed the microbiota of 104 adultI. scapularisand 13 adultI. affinisticks captured in 19 locations in South Carolina, North Carolina, Virginia, Connecticut, and New York. Initially, ticks from 4 sites were analyzed by 454 pyrosequencing. Subsequently, ticks from these sites plus 15 others were analyzed by sequencing with an Illumina MiSeq machine. By both analyses, the microbiomes of female ticks were significantly less diverse than those of male ticks. The dissimilarity between tick microbiomes increased with distance between sites, and the state in which a tick was collected could be inferred from its microbiota. The genusRickettsiawas prominent in all locations.Borreliawas also present in most locations and was present at especially high levels in one site in western Virginia. In contrast, members of the familyEnterobacteriaceaewere very common in North CarolinaI. scapularisticks but uncommon inI. scapularisticks from other sites and in North CarolinaI. affinisticks. These data suggest substantial variations in theIxodesmicrobiota in association with geography, species, and sex.

scholarly journals Polymicrobial Nature of Tick-Borne Diseases

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Santiago Sanchez-Vicente ◽  
Teresa Tagliafierro ◽  
James L. Coleman ◽  
Jorge L. Benach ◽  
Rafal Tokarz
Keyword(s):  
Climate Change ◽  
Anaplasma Phagocytophilum ◽  
Ixodes Scapularis ◽  
The Elderly ◽  
Amblyomma Americanum ◽  
Babesia Microti ◽  
Borrelia Miyamotoi ◽  
Content Type ◽  
Powassan Virus ◽  
Polymicrobial Infections

ABSTRACT Tick-borne diseases have doubled in the last 12 years, and their geographic distribution has spread as well. The clinical spectrum of tick-borne diseases can range from asymptomatic to fatal infections, with a disproportionate incidence in children and the elderly. In the last few years, new agents have been discovered, and genetic changes have helped in the spread of pathogens and ticks. Polymicrobial infections, mostly in Ixodes scapularis, can complicate diagnostics and augment disease severity. Amblyomma americanum ticks have expanded their range, resulting in a dynamic and complex situation, possibly fueled by climate change. To document these changes, using molecular biology strategies for pathogen detection, an assessment of 12 microbes (9 pathogens and 3 symbionts) in three species of ticks was done in Suffolk County, New York. At least one agent was detected in 63% of I. scapularis ticks. Borrelia burgdorferi was the most prevalent pathogen (57% in adults; 27% in nymphs), followed by Babesia microti (14% in adults; 15% in nymphs), Anaplasma phagocytophilum (14% in adults; 2% in nymphs), Borrelia miyamotoi (3% in adults), and Powassan virus (2% in adults). Polymicrobial infections were detected in 22% of I. scapularis ticks, with coinfections of B. burgdorferi and B. microti (9%) and of B. burgdorferi and A. phagocytophilum (7%). Three Ehrlichia species were detected in 4% of A. americanum ticks. The rickettsiae constituted the largest prokaryotic biomass of all the ticks tested and included Rickettsia amblyommatis, Rickettsia buchneri, and Rickettsia montanensis. The high rates of polymicrobial infection in ticks present an opportunity to study the biological interrelationships of pathogens and their vectors. IMPORTANCE Tick-borne diseases have increased in prevalence in the United States and abroad. The reasons for these increases are multifactorial, but climate change is likely to be a major factor. One of the main features of the increase is the geographic expansion of tick vectors, notably Amblyomma americanum, which has brought new pathogens to new areas. The clinical spectrum of tick-borne diseases can range from asymptomatic to fatal infections, with a disproportionate incidence in children and the elderly. In addition, new pathogens that are cotransmitted by Ixodes scapularis have been discovered and have led to difficult diagnoses and to disease severity. Of these, Borrelia burgdorferi, the agent of Lyme disease, continues to be the most frequently transmitted pathogen. However, Babesia microti, Borrelia miyamotoi (another spirochete), Anaplasma phagocytophilum, and Powassan virus are frequent cotransmitted agents. Polymicrobial infection has important consequences for the diagnosis and management of tick-borne diseases.

scholarly journals Tick Saliva Enhances Powassan Virus Transmission to the Host, Influencing Its Dissemination and the Course of Disease

2015 ◽  
Vol 89 (15) ◽  
pp. 7852-7860 ◽  
Author(s):  
Meghan E. Hermance ◽  
Saravanan Thangamani
Keyword(s):  
Low Dose ◽  
Expression Profiles ◽  
The United States ◽  
High Dose ◽  
Tick Saliva ◽  
Study Objective ◽  
Content Type ◽  
Virus Dose ◽  
Powassan Virus ◽  
Pharmacologically Active

ABSTRACTPowassan virus(POWV) is an encephalitic tick-borne flavivirus which can result in serious neuroinvasive disease with up to a 10% case fatality rate. The study objective was to determine whether the salivary gland extract (SGE) fromIxodes scapularisticks facilitates the transmission and dissemination of POWV in a process known as saliva-activated transmission. Groups of BALB/c mice were footpad inoculated with either a high dose of POWV with and without SGE or a low dose of POWV with and without SGE. Mice from each group were sacrificed daily. Organ viral loads and gene expression profiles were evaluated by quantitative real-time PCR. Both groups of mice infected with high-dose POWV showed severe neurological signs of disease preceding death. The presence of SGE did not affect POWV transmission or disease outcome for mice infected with the high dose of POWV. Neuroinvasion, paralysis, and death occurred for all mice infected with the low dose of POWV plus SGE; however, for mice infected with the low dose of POWV in the absence of SGE, there were no clinical signs of infection and no mice succumbed to disease. Although this group displayed low-level viremias, all mice were completely healthy, and it was the only group in which POWV was cleared from the lymph nodes. We conclude that saliva-activated transmission occurs in mice infected with a low dose of POWV. Our study is the first to demonstrate virus dose-dependent saliva-activated transmission, warranting further investigation of the specific salivary factors responsible for enhancing POWV transmission.IMPORTANCEPowassan virus(POWV) is a tick-borne flavivirus that continues to emerge in the United States, as is evident by the surge in number and expanding geographic range of confirmed cases in the past decade. This neuroinvasive virus is transmitted to humans by infected tick bites. Successful tick feeding is facilitated by a collection of pharmacologically active factors in tick saliva. In a process known as saliva-activated transmission, tick bioactive salivary molecules are thought to modulate the host environment, making it more favorable for the transmission and establishment of a pathogen. This phenomenon has been demonstrated for several tick-borne pathogens; however, a systematic investigation of the role of tick saliva on dissemination and pathogenesis of a tick-borne viral disease has never been attempted before. This study will fill that gap by systematically examining whether the presence of tick saliva contributes to the transmission and dissemination of POWV in mice.

scholarly journals Complex Population Structure of Borrelia burgdorferi in Southeastern and South Central Canada as Revealed by Phylogeographic Analysis

2014 ◽  
Vol 81 (4) ◽  
pp. 1309-1318 ◽  
Author(s):  
S. Mechai ◽  
G. Margos ◽  
E. J. Feil ◽  
L. R. Lindsay ◽  
N. H. Ogden
Keyword(s):  
United States ◽  
Borrelia Burgdorferi ◽  
Ixodes Scapularis ◽  
The United States ◽  
Sensu Stricto ◽  
Content Type ◽  
South Central ◽  
Central Canada ◽  
Complex Population ◽  
Reservoir Hosts

ABSTRACTLyme disease, caused by the bacteriumBorrelia burgdorferisensu stricto, is an emerging zoonotic disease in Canada and is vectored by the blacklegged tick,Ixodes scapularis. Here we used Bayesian analyses of sequence types (STs), determined by multilocus sequence typing (MLST), to investigate the phylogeography ofB. burgdorferipopulations in southern Canada and the United States by analyzing MLST data from 564B. burgdorferi-positive samples collected during surveillance. A total of 107 Canadian samples from field sites were characterized as part of this study, and these data were combined with existing MLST data for samples from the United States and Canada. Only 17% of STs were common between both countries, while 49% occurred only in the United States, and 34% occurred only in Canada. However, STs in southeastern Ontario and southwestern Quebec were typically identical to those in the northeastern United States, suggesting a recent introduction into this region from the United States. In contrast, STs in other locations in Canada (the Maritimes; Long Point, Ontario; and southeastern Manitoba) were frequently unique to those locations but were putative descendants of STs previously found in the United States. The picture in Canada is consistent with relatively recent introductions from multiple refugial populations in the United States. These data thus point to a geographic pattern of populations ofB. burgdorferiin North America that may be more complex than simply comprising northeastern, midwestern, and Californian groups. We speculate that this reflects the complex ecology and spatial distribution of key reservoir hosts.

scholarly journals Imaging of Borrelia turicatae Producing the Green Fluorescent Protein Reveals Persistent Colonization of the Ornithodoros turicata Midgut and Salivary Glands from Nymphal Acquisition through Transmission

2016 ◽  
Vol 83 (5) ◽  
Author(s):  
Aparna Krishnavajhala ◽  
Hannah K. Wilder ◽  
William K. Boyle ◽  
Ashish Damania ◽  
Justin A. Thornton ◽  
...  
Keyword(s):  
Life Cycle ◽  
Green Fluorescent Protein ◽  
Salivary Glands ◽  
Fluorescent Protein ◽  
Blood Feeding ◽  
Relapsing Fever ◽  
Tick Feeding ◽  
Content Type ◽  
Green Fluorescent ◽  
Infectious Cycle

ABSTRACT Relapsing fever (RF) spirochetes colonize and are transmitted to mammals primarily by Ornithodoros ticks, and little is known regarding the pathogen's life cycle in the vector. To further understand vector colonization and transmission of RF spirochetes, Borrelia turicatae expressing a green fluorescent protein (GFP) marker (B. turicatae-gfp) was generated. The transformants were evaluated during the tick-mammal infectious cycle, from the third nymphal instar to adult stage. B. turicatae-gfp remained viable for at least 18 months in starved fourth-stage nymphal ticks, and the studies indicated that spirochete populations persistently colonized the tick midgut and salivary glands. Our generation of B. turicatae-gfp also revealed that within the salivary glands, spirochetes are localized in the ducts and lumen of acini, and after tick feeding, the tissues remained populated with spirochetes. The B. turicatae-gfp generated in this study is an important tool to further understand and define the mechanisms of vector colonization and transmission. IMPORTANCE In order to interrupt the infectious cycle of tick-borne relapsing fever spirochetes, it is important to enhance our understanding of vector colonization and transmission. Toward this, we generated a strain of Borrelia turicatae that constitutively produced the green fluorescent protein, and we evaluated fluorescing spirochetes during the entire infectious cycle. We determined that the midgut and salivary glands of Ornithodoros turicata ticks maintain the pathogens throughout the vector's life cycle and remain colonized with the spirochetes for at least 18 months. We also determined that the tick's salivary glands were not depleted after a transmission blood feeding. These findings set the framework to further understand the mechanisms of midgut and salivary gland colonization.

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