Development of a polyclonal competitive enzyme-linked immunosorbent assay for detection of antibodies to Ehrlichia ruminantium

A polyclonal competitive enzyme-linked immunosorbent assay (PC-ELISA) is described for detection of antibodies to Ehrlichia (Cowdria) ruminantium by using a soluble extract of endothelial cell culture-derived E. ruminantium as the antigen and biotin-labeled polyclonal goat immunoglobulins as the competitor. For goats, the diagnostic sensitivity and specificity were both 100% with a cutoff of 80% inhibition (80 PI), with detection of antibodies for 550 days postinfection. For cattle, diagnostic sensitivity and specificity were 86 and 100%, respectively, with a cutoff of 50 PI and 79 and 100% with a cutoff of 70 PI. Cross-reactions with high-titer experimental or field antisera to other Ehrlichia and Anaplasma species were observed at up to 68 PI in cattle and up to 85 PI in sheep, and therefore to exclude these cross-reactions, cutoffs of 70 PI for bovine serology and 85 PI for small-ruminant serology were selected. Application of the PC-ELISA to bovine field sera from South Africa gave a higher proportion of positive results than application of the murine macrophage immunofluorescent antibody test or indirect ELISA, suggesting a better sensitivity for detection of recovered cattle, and results with bovine field sera from Malawi were consistent with the observed endemic state of heartwater and the level of tick control practiced at the sample sites. Reproducibility was high, with average standard deviations intraplate of 1.2 PI and interplate of 0.6 PI. The test format is simple, and the test is economical to perform and has a level of sensitivity for detection of low-titer positive bovine sera that may prove to be of value in epidemiological studies on heartwater

Growth of Cowdria ruminantium, the causative agent of heartwater, in a tick cell line

The tick-borne rickettsia Cowdria ruminantium has been propagated continuously for over 500 days in the Ixodes scapularis tick cell line IDE8 by using the Gardel isolate from bovine endothelial cells as an inoculum. Infection of the tick cells was confirmed by PCR, karyotyping, electron microscopy, and reinfection of bovine cells

Identification of a novel ß-adrenergic octopamine receptor-like gene (ßAOR-like) and increased ATP-binding cassette B10 (ABCB10) expression in a Rhipicephalus microplus cell line derived from acaricide-resistant ticks

Background: The cattle tick Rhipicephalus (Boophilus) microplus is an economically important parasite of livestock. Effective control of ticks using acaricides is threatened by the emergence of resistance to many existing compounds. Several continuous R. microplus cell lines have been established and provide an under-utilised resource for studies into acaricide targets and potential genetic mutations associated with resistance. As a first step to genetic studies using these resources, this study aimed to determine the presence or absence of two genes and their transcripts that have been linked with acaricide function in cattle ticks: β-adrenergic octopamine receptor (βAOR, associated with amitraz resistance) and ATP-binding cassette B10 (ABCB10, associated with macrocyclic lactone resistance) in six R. microplus cell lines, five other Rhipicephalus spp. cell lines and three cell lines representing other tick genera (Amblyomma variegatum, Ixodes ricinus and Hyalomma anatolicum). Methods: End-point polymerase chain reaction (PCR) was used for detection of the βAOR gene and transcripts in DNA and RNA extracted from the tick cell lines, followed by capillary sequencing of amplicons. Quantitative real-time PCR (qPCR) was performed to determine the levels of expression of ABCB10. Results: βAOR gene expression was detected in all Rhipicephalus spp. cell lines. We observed a second amplicon of approximately 220 bp for the βAOR gene in the R. microplus cell line BME/CTVM6, derived from acaricide-resistant ticks. Sequencing of this transcript variant identified a 36 bp insertion in the βAOR gene, leading to a 12-amino acid insertion (LLKTLALVTIIS) in the first transmembrane domain of the protein. In addition, nine synonymous SNPs were also discovered in R. appendiculatus, R. evertsi and R. sanguineus cell lines. Some of these SNPs appear to be unique to each species, providing potential tools for differentiating the tick species. The BME/CTVM6 cell line had significantly higher ABCB10 (P = 0.002) expression than the other R. micropluscell lines. Conclusions: The present study has identified a new βAOR gene and demonstrated a higher ABCB10 expression level in the BME/CTVM6 cell line, indicating that tick cell lines provide a useful experimental tool for acaricide resistance studies and further elucidation of tick genetics

Cold storage and cryopreservation of tick cell lines

<p>Abstract</p> <p>Background</p> <p>Tick cell lines are now available from fifteen ixodid and argasid species of medical and veterinary importance. However, some tick cell lines can be difficult to cryopreserve, and improved protocols for short- and long-term low temperature storage will greatly enhance their use as tools in tick and tick-borne pathogen research. In the present study, different protocols were evaluated for cold storage and cryopreservation of tick cell lines derived from <it>Rhipicephalus </it>(<it>Boophilus) decoloratus</it>, <it>Rhipicephalus </it>(<it>Boophilus) microplus, Ixodes ricinus </it>and <it>Ixodes scapularis</it>. For short-term cold storage, cells were kept under refrigeration at 6°C for 15, 30 and 45 days. For cryopreservation in liquid nitrogen, use of a sucrose-phosphate-glutamate freezing buffer (SPG) as cryoprotectant was compared with dimethylsulfoxide (DMSO) supplemented with sucrose. Cell viability was determined by the trypan blue exclusion test and cell morphology was evaluated in Giemsa-stained cytocentrifuge smears.</p> <p>Results</p> <p>Cold storage at 6°C for up to 30 days was successful in preserving <it>R</it>. (<it>B.) microplus</it>, <it>R</it>. (<it>B.) decoloratus, I. ricinus </it>and <it>I. scapularis </it>cell lines; lines from the latter three species could be easily re-cultivated after 45 days under refrigeration. While cell lines from all four tick species cryopreserved with 6% DMSO were successfully resuscitated, the <it>R</it>. (<it>B</it>.) <it>decoloratus </it>cells did not survive freezing in SPG and of the other three species, only the <it>R</it>. (<it>B</it>.) <it>microplus </it>cells resumed growth during the observation period.</p> <p>Conclusions</p> <p>This constitutes the first report on successful short-term refrigeration of cells derived from <it>R</it>. (<it>B.) decoloratus</it>, <it>R</it>. (<it>B.) microplus</it>, and <it>I. ricinus</it>, and use of SPG as an alternative to DMSO for cryopreservation, thus making an important contribution to more reliable and convenient tick cell culture maintenance.</p

The role of Cytochrome P450s towards the control of ticks and other arthropods

Introduction Ticks most notably Rhipicephalus (Boophilus) microplus are becoming increasingly resistant to acaricides .This resistance is often broken down into the 3 main mechanisms of detoxification involving 3 groups of enzymes; Cytochrome P450s (CYPs), Glutathione-S-transferases (GST) or Esterases (EST or carboxylesterases) (Foil et al., 2004) Material and Methods Acaricide resistance research to date, has focused on identifying which acaricides are becoming redundant in terms of effectiveness to kill the arthropod. Often, this research uses mortality based experimental procedures (Li et al., 2003; Miller et al., 2005), and investigations into the molecular/enzymatic basis of resistance have focused on GSTs and ESTs rather than CYPs. Another method used in our laboratory investigating the role of CYPs in resistance is employing tick cell cultures as a model system developed in partnership with the Roslin Institute (Bell-Sakyi et al., 2007). This system provides an ideal way to monitor CYP expression before, during and after various treatments such as acaricides. In Anopheles gambiae, expression levels of CYPs, GSTs and ESTs were monitored using a microarray following infection of the mosquito by Plasmodium (Felix et al., 2010). A similar micro array approach could be employed with ticks and mites as more data becomes available. Results Our group is monitoring the expression levels of different CYPs in tick cell lines as well as looking to identify novel CYPs and work on phylogenetic links for those genes between different arthropod groups. We are also investigating polymorphisms between different tick cell lines and different arthropod species. Discussion and Conclusions Among the arthropods, research strongly suggests that the CYP6 (Hemingway et al., 2004) and CYP9families are most highly associated with xenobiotic resistance and a lot of research has been carried out looking at these CYP families in various insect species. In mosquito species some resistance to pesticides is due to their CYP enzymes evolving to detoxify these chemicals at increasing concentrations (Nikou et al., 2003

Identification and validation of internal reference genes for real-time quantitative polymerase chain reaction-based studies in Hyalomma anatolicum ticks

Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne viral disease caused by the orthonairovirus CCHF virus (CCHFV). Ticks of the genus Hyalomma are the viral reservoir and they represent the main vector transmitting the virus to their hosts during blood feeding. However, how CCHFV replicates in its natural arthropod host cells and the nature of virus/host interactions are still largely unknown. With the aim of developing tools for use in this field, we identified and validated expression of four candidate endogenous control tick genes in a Hyalomma anatolicum-derived cell line. These genes will be useful for normalization of viral/cellular transcripts in infection/expression studies or as internal controls in molecular epidemiology surveys of pathogens transmitted by Hyalomma ticks

Defensins from the tick Ixodes scapularis are effective against phytopathogenic fungi and the human bacterial pathogen Listeria grayi

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: Ixodes scapularis is the most common tick species in North America and a vector of important pathogens that cause diseases in humans and animals including Lyme disease, anaplasmosis and babesiosis. Tick defensins have been identified as a new source of antimicrobial agents with putative medical applications due to their wide-ranging antimicrobial activities. Two multigene families of defensins were previously reported in I. scapularis. The objective of the present study was to characterise the potential antimicrobial activity of two defensins from I. scapularis with emphasis on human pathogenic bacterial strains and important phytopathogenic fungi. [Methods]: Scapularisin-3 and Scapularisin-6 mature peptides were chemically synthesised. In vitro antimicrobial assays were performed to test the activity of these two defensins against species of different bacterial genera including Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis, and Listeria spp. as well as Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa along with two plant-pathogenic fungi from the genus Fusarium. In addition, the tissue-specific expression patterns of Scapularisin-3 and Scapularisin-6 in I. scapularis midgut, salivary glands and embryo-derived cell lines were determined using PCR. Finally, tertiary structures of the two defensins were predicted and structural analyses were conducted. [Results]: Scapularisin-6 efficiently killed L. grayi, and both Scapularisin-3 and Scapularisin-6 caused strong inhibition (IC value: ∼1 μM) of the germination of plant-pathogenic fungi Fusarium culmorum and Fusarium graminearum. Scapularisin-6 gene expression was observed in I. scapularis salivary glands and midgut. However, Scapularisin-3 gene expression was only detected in the salivary glands. Transcripts from the two defensins were not found in the I. scapularis tick cell lines ISE6 and ISE18. [Conclusion]: Our results have two main implications. Firstly, the anti-Listeria and antifungal activities of Scapularisin-3 and Scapularisin-6 suggest that these peptides may be useful for (i) treatment of antibiotic-resistant L. grayi in humans and (ii) plant protection. Secondly, the antimicrobial properties of the two defensins described in this study may pave the way for further studies regarding pathogen invasion and innate immunity in I. scapularis.Miray Tonk is a Marie Curie Early Stage Researcher supported by the POSTICK ITN (Post-graduate training network for capacity building to control ticks and tick-borne diseases) within the FP7- PEOPLE – ITN programme (EU Grant No. 238511). This project was partially supported by the Grant Agency of the Czech Republic (GACR P302/11/1901) and with institutional support RVO: 60077344 from Biology Centre, Institute of Parasitology as well as grant ANTIGONE (EU-7FP; 278976). James J. Valdés was sponsored by project CZ.1.07/2.3.00/30.0032, co-financed by the European Social Fund and the state budget of the Czech Republic. Radek Šíma was supported by the Grant 13-12816P (GA CR). Mohammad Rahnamaeian and Andreas Vilcinskas acknowledge the Ministry for Science and Art of the State of Hesse (Germany) for funding the LOEWE Center of Insect Biotechnology and Bioresources. Zdeněk Franta was supported by Alexander von Humboldt Research Fellowship for Postdoctoral Researchers.Peer Reviewe

Uukuniemi Virus as a Tick-Borne Virus Model.

International audienceIn the last decade, novel tick-borne pathogenic phleboviruses in the family Bunyaviridae, all closely related to Uukuniemi virus (UUKV), have emerged on different continents. To reproduce the tick-mammal switch in vitro, we first established a reverse genetics system to rescue UUKV with a genome close to that of the authentic virus isolated from the Ixodes ricinus tick reservoir. The IRE/CTVM19 and IRE/CTVM20 cell lines, both derived from I. ricinus, were susceptible to the virus rescued from plasmid DNAs and supported production of the virus over many weeks, indicating that infection was persistent. The glycoprotein GC was mainly highly mannosylated on tick cell-derived viral progeny. The second envelope viral protein, GN, carried mostly N-glycans not recognized by the classical glycosidases peptide-N-glycosidase F (PNGase F) and endoglycosidase H (Endo H). Treatment with β-mercaptoethanol did not impact the apparent molecular weight of GN On viruses originating from mammalian BHK-21 cells, GN glycosylations were exclusively sensitive to PNGase F, and the electrophoretic mobility of the protein was substantially slower after the reduction of disulfide bonds. Furthermore, the amount of viral nucleoprotein per focus forming unit differed markedly whether viruses were produced in tick or BHK-21 cells, suggesting a higher infectivity for tick cell-derived viruses. Together, our results indicate that UUKV particles derived from vector tick cells have glycosylation and structural specificities that may influence the initial infection in mammalian hosts. This study also highlights the importance of working with viruses originating from arthropod vector cells in investigations of the cell biology of arbovirus transmission and entry into mammalian hosts. Tick-borne phleboviruses represent a growing threat to humans globally. Although ticks are important vectors of infectious emerging diseases, previous studies have mainly involved virus stocks produced in mammalian cells. This limitation tends to minimize the importance of host alternation in virus transmission to humans and initial infection at the molecular level. With this study, we have developed an in vitro tick cell-based model that allows production of the tick-borne Uukuniemi virus to high titers. Using this system, we found that virions derived from tick cells have specific structural properties and N-glycans that may enhance virus infectivity for mammalian cells. By shedding light on molecular aspects of tick-derived viral particles, our data illustrate the importance of considering the host switch in studying early virus-mammalian receptor/cell interactions. The information gained here lays the basis for future research on not only tick-borne phleboviruses but also all viruses and other pathogens transmitted by ticks