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The transfection of plasmids into cell lines for the transient expression of exogenous proteins is a fundamental method for characterizing their functions, cellular localization and interactions. Currently, only a few reports on tick transfection systems and expression plasmids specifically constructed for tick cell lines have been published. In this study, the transcriptome of the tick cell line IDE8 was analyzed to screen for highly-expressed genes. The upstream sequences of these genes were selected as possible tick-derived promoters, and their promoter activity was evaluated using a luciferase assay. Four IDE8-derived sequences with promoter activity were identified, and the promoter activities of three common mammalian promoters, CMV, PGK and CAG, were studied and compared in the IDE8 and IRE/CTVM19 tick cell lines. In the two tick cell lines, the efficiency of the CAG promoter was considerably higher than that of CMV, PGK and the four newly-identified tick promoters. Additionally, time course experiments revealed that the protein expression driven by mammalian promoters reached peak levels on day 3, while the protein expression driven by our constructed tick-derived promoters reached peak levels on day 2 in tick cells. By comparing the transfection efficiency of three transfection reagents with different mechanisms in tick cell lines, we identified Effectene (with Enhancer, Qiagen) as the most effective reagent for tick cells. The findings of this study suggested that there are differences between tick and mammalian cell lines in their response to the transfection system. These findings will contribute to future studies on topics including tick protein function, tick genetic modification and tick-host-pathogen interactions.

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The blacklegged tick, Ixodes scapularis, a species of significant importance to human and animal health, harbors an endosymbiont Rickettsia buchneri sensu stricto. The symbiont is largely restricted to the ovaries, but all life stages can harbor various quantities or lack R. buchneri entirely. The endosymbiont is cultivable in cell lines isolated from embryos of Ixodes ticks. Rickettsia buchneri most readily grows and is maintained in the cell line IRE11 from the European tick, Ixodes ricinus. The line was characterized by light and electron microscopy and used to analyze the growth dynamics of wildtype and GFPuv-expressing R. buchneri. qPCR indicated that the genome copy doubling time in IRE11 was >7 days. Measurements of fluorescence using a plate reader indicated that the amount of green fluorescent protein doubled every 11 days. Two 23S rRNA probes were tested via RNA FISH on rickettsiae grown in vitro and adapted to evaluate the tissue tropism of R. buchneri in field-collected female I. scapularis. We observed strong positive signals of R. buchneri in the ovaries and surrounding the nucleus of the developing oocytes. Tissue tropism in I. scapularis and in vitro growth dynamics strengthen the contemporary understanding of R. buchneri as a transovarially transmitted, non-pathogenic endosymbiont.

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Although tick-borne infectious diseases threaten human and animal health worldwide, with constantly increasing incidence, little knowledge is available regarding vector-pathogen interactions and pathogen transmission. In vivo laboratory study of these subjects using live, intact ticks is expensive, labor-intensive, and challenging from the points of view of biosafety and ethics. Several in vitro models have been developed, including over 70 continuous cell lines derived from multiple tick species and a variety of tick organ culture systems, facilitating many research activities. However, some limitations have to be considered in the translation of the results from the in vitro environment to the in vivo situation of live, intact ticks, and vertebrate hosts. In this review, we describe the available in vitro models and selected results from their application to the study of tick-borne viruses, bacteria, and protozoa, where possible comparing these results to studies in live, intact ticks. Finally, we highlight the strengths and weaknesses of in vitro tick culture models and their essential role in tick-borne pathogen research.

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Members of the family Anaplasmataceae are obligate intracellular bacteria that replicate within membrane bound vacuoles in the cytoplasm of cells in vertebrate and invertebrate hosts. This study reports a putative new Anaplasma species in gopher tortoises in Florida. Two Florida gopher tortoises (Gopherus polyphemus) presented at the University of Florida Veterinary Hospital with anemia and intracytoplasmic vacuoles filled with bacteria within erythrocytes. The bacteria within these parasitophorous vacuoles were morphologically similar to Anaplasma marginale. We inoculated ISE6 cells with blood from one tortoise and isolated bacterial colonies consistent with A. marginale. Molecular characterization targeting Anaplasmataceae 16S rRNA sequences indicated that the clinical isolate, named here provisionally as "Candidatus Anaplasma testudinis", grouped within the genus Anaplasma on a separate clade, most closely related to the A. marginale, Anaplasma ovis and Anaplasma centrale group. We next screened archived red blood cells from 38 wild gopher tortoises with documented clinical anemia. Fourteen of the 38 wild tortoises, representing 5 of 11 geographical locations were PCR-positive for Anaplasmataceae spp. Sequencing analysis revealed 16S rRNA sequence identical to "Ca. A. testudinis". The clinical presentation of significant anemia associated with "Ca. A. testudinis" in a threatened species could have conservation implications. Importantly, the availability of a clinical isolate will aid further studies to develop diagnostic tests and to investigate potential tick vectors and infectivity for other wildlife and domestic animal species.

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Tick-borne pathogen co-infections are common in nature. Co-infecting pathogens interact with each other and the tick microbiome, which influences individual pathogen fitness, and ultimately shapes virulence, infectivity, and transmission. In this review, we discuss how tick-borne pathogens are an ideal framework to study the evolutionary dynamics of co-infections. We highlight the importance of inter-species and intra-species interactions in vector-borne pathogen ecology and evolution. We also propose experimental evolution in tick cell lines as a method to directly test the impact of co-infections on pathogen evolution. Experimental evolution can simulate in real-time the long periods of time involved in within-vector pathogen interactions in nature, a major practical obstacle to cracking the influence of co-infections on pathogen evolution and ecology.

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Candidatus (Ca.) Neoehrlichia mikurensis is the cause of neoehrlichiosis, an emerging tick-borne infectious disease characterized by fever and vascular events. The bacterium belongs to the Anaplasmataceae, a family of obligate intracellular pathogens, but has not previously been cultivated, and it is uncertain which cell types it infects. The goals of this study were to cultivate Ca. N. mikurensis in cell lines and to identify possible target cells for human infection. Blood components derived from infected patients were inoculated into cell lines of both tick and human origin. Bacterial growth in the cell cultures was monitored by real-time PCR and imaging flow cytometry. Ca. N. mikurensis was successfully propagated from the blood of immunocompromised neoehrlichiosis patients in two Ixodes spp. tick cell lines following incubation periods of 7-20 weeks. Human primary endothelial cells derived from skin microvasculature as well as pulmonary artery were also susceptible to infection with tick cell-derived bacteria. Finally, Ca. N. mikurensis was visualized within circulating endothelial cells of two neoehrlichiosis patients. To conclude, we report the first successful isolation and propagation of Ca. N. mikurensis from clinical isolates and identify human vascular endothelial cells as a target of infection.

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Tick-borne encephalitis virus (TBEV) is the causative agent of tick-borne encephalitis (TBE), a vector-borne zoonotic neuroinfection. For successful circulation in natural foci the virus has to survive in the vector for a long period of time. Information about the effect of long-term infection of ticks on properties of the viral population is of great importance. In recent years, changes in the eco-epidemiology of TBEV due to changes in distribution of ixodid ticks have been observed. These changes in TBEV-endemic areas could result in a shift of the main tick vector species, which in turn may lead to changes in properties of the virus. In the present study we evaluated the selective pressure on the TBEV population during persistent infection of various species of ticks and tick cell lines. TBEV effectively replicated and formed persistent infection in ticks and tick cell lines of the vector species (Ixodes spp.), potential vectors (Dermacentor spp.) and non-vector ticks (Hyalomma spp.). During TBEV persistence in Ixodes and Dermacentor ticks, properties of the viral population remained virtually unchanged. In contrast, persistent TBEV infection of tick cell lines from both vector and non-vector ticks favoured selection of viral variants with low neuroinvasiveness for laboratory mice and substitutions in the E protein that increased local positive charge of the virion. Thus, selective pressure on viral population may differ in ticks and tick cell lines during persistent infection. Nevertheless, virus variants with properties of the original strain adapted to mouse CNS were not eliminated from the viral population during long-term persistence of TBEV in ticks and tick cell lines.

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Canine monocytic ehrlichiosis is caused by Ehrlichia canis, a small gram-negative coccoid bacterium that infects circulating monocytes. The disease is transmitted by the brown dog tick Rhipicephalus sanguineus s.l. and is acknowledged as an important infectious disease of dogs and other members of the family Canidae worldwide. E. canis is routinely cultured in vitro in the canine monocyte-macrophage cell line DH82 and in non-vector Ixodes scapularis tick cell lines, but not in cells derived from its natural vector. Here we report infection and limited propagation of E. canis in the tick cell line RSE8 derived from the vector R. sanguineus s.l., and successful propagation through six passages in a cell line derived from the experimental vector Dermacentor variabilis. In addition, using bacteria semi-purified from I. scapularis cells we attempted to infect a panel of cell lines derived from non-vector species of the tick genera Amblyomma, Dermacentor, Hyalomma, Ixodes and Rhipicephalus with E. canis and, for comparison, the closely-related Ehrlichia ruminantium, causative agent of heartwater in ruminants. Amblyomma and non-vector Dermacentor spp. cell lines appeared refractory to infection with E. canis but supported growth of E. ruminantium, while some, but not all, cell lines derived from Hyalomma, Ixodes and Rhipicephalus spp. ticks supported growth of both pathogens. We also illustrated and compared the ultrastructural morphology of E. canis in DH82, RSE8 and I. scapularis IDE8 cells. This study confirms that E. canis, like E. ruminantium, is able to grow not only in cell lines derived from natural and experimental tick vectors but also in a wide range of other cell lines derived from tick species not known to transmit this pathogen.

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This study aimed to describe the association of Borrelia burgdorferi s.s. with ixodid tick cell lines by flow cytometry and fluorescence and confocal microscopy. Spirochetes were stained with a fluorescent membrane marker (PKH67 or PKH26), inoculated into 8 different tick cell lines and incubated at 30°C for 24 h. PKH efficiently stained B. burgdorferi without affecting bacterial viability or motility. Among the tick cell lines tested, the Rhipicephalus appendiculatus cell line RA243 achieved the highest percentage of association/internalization, with both high (90%) and low (10%) concentrations of BSK-H medium in tick cell culture medium. Treatment with cytochalasin D dramatically reduced the average percentage of cells with internalized spirochetes, which passed through a dramatic morphological change during their internalization by the host cell as observed in time-lapse photography. Almost all of the fluorescent bacteria were seen to be inside the tick cells. PKH labeling of borreliae proved to be a reliable and valuable tool to analyze the association of spirochetes with host cells by flow cytometry, confocal and fluorescence microscopy.

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Anaplasma phagocytophilum is an intracellular rickettsial pathogen transmitted by Ixodes spp. ticks, which causes granulocytic anaplasmosis in humans, horses and dogs and tick-borne fever (TBF) in ruminants. In the United States, human granulocytic anaplasmosis (HGA) is highly prevalent while TBF has not been reported. However, in Europe the situation is the opposite, with high prevalence for TBF in sheep and low prevalence of HGA. The origin of these differences has not been identified and our hypothesis is that different A. phagocytophilum isolates impact differently on tick vector capacity through inhibition of apoptosis to establish infection of the tick vector. In this study we used three different isolates of A. phagocytophilum of human, canine and ovine origin to infect the Ixodes ricinus-derived cell line IRE/CTVM20 and the Ixodes scapularis-derived cell line ISE6 in order to characterize the effect of infection on the level of tick cell apoptosis. Inhibition of apoptosis was observed by flow cytometry as early as 24h post-infection for both tick cell lines and all three isolates of A. phagocytophilum, suggesting that pathogen infection inhibits apoptotic pathways to facilitate infection independently of the origin of the A. phagocytophilum isolate and tick vector species. However, infection with A. phagocytophilum isolates inhibited the intrinsic apoptosis pathway at different levels in I. scapularis and I. ricinus cells. These results suggested an impact of vector-pathogen co-evolution on the adaptation of A. phagocytophilum isolates to grow in tick cells as each isolate grew better in the tick cell line derived from its natural vector species. These results increase our understanding of the mechanisms of A. phagocytophilum infection and multiplication and suggest that multiple mechanisms may affect disease prevalence in different geographical regions.

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Lumpy skin disease (LSD) is of substantial economic importance for the cattle industry in Africa and the Near and Middle East. Several insect species are thought to transmit the disease mechanically. Recent transmission studies have demonstrated the first evidence for a role of hard (ixodid) ticks as vectors of lumpy skin disease virus (LSDV). The aim of this study was to attempt in vitro growth of the virus in Rhipicephalus spp. tick cell lines and investigate in vivo the presence of the virus in ticks collected from cattle during LSD outbreaks in Egypt and South Africa. No evidence was obtained for replication of LSDV in tick cell lines although the virus was remarkably stable, remaining viable for 35 days at 28°C in tick cell cultures, in growth medium used for tick cells and in phosphate buffered saline. Viral DNA was detected in two-thirds of the 56 field ticks, making this the first report of the presence of potentially virulent LSDV in ticks collected from naturally infected animals.

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BACKGROUND: Although Alkhumra haemorrhagic fever virus (AHFV) has been isolated from ticks, epidemiological data suggest that it is transmitted from livestock to humans by direct contact with animals or by mosquito bites, but not by ticks. This study was carried out to assess the ability of the virus to replicate in tick cells in vitro. METHODS: AHFV was inoculated into cell lines derived from the hard ticks Hyalomma anatolicum (HAE/CTVM9) and Rhipicephalus appendiculatus (RAE/CTVM1) and the soft tick Ornithodoros moubata (OME/CTVM24). Inoculated cells were directly examined every week for 4 weeks by real-time reverse transcription PCR and by IFAT using polyclonal antibodies. RESULTS: AHFV RNA was detected in all three inoculated tick cell lines throughout the 4-week observation period at levels up to almost twice that of the inoculum, but none of them exhibited a cytopathic effect. AHFV antigen could be detected in all three cell lines by IFAT. Titration of tick cell culture suspension in LLC-MK2 cells yielded AHFV titres of 10(6.6) 50% tissue culture infective dose (TCID50)/ml for OME/CTVM24 and 10(5.5) TCID50/ml for RAE/CTVM1 cells after 4 weeks of culturing; no viable virus was detected in HAE/CTVM9 cells. CONCLUSION: This is the first description of propagation of AHFV in tick cells.

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