Field-collected Ixodes Scapularis Research Articles

Microbiota of field-collected Ixodes scapularis and Dermacentor variabilis from eastern and southern Ontario, Canada

The microbiota of hard ticks has been an area of growing interest due to the potential role that the broader microbial community may play in pathogen carriage and transmission. In the last two decades, Ontario, Canada has experienced rapid changes in the risk of tick-borne disease, primarily due to the range expansion of Ixodes scapularis. Another human-biter, Dermacentor variabilis, is a longstanding resident of the province, but currently poses minimal risk of pathogen transmission. To examine the microbiota of these two species, we collected adult and nymphal I. scapularis and D. variabilis from the eastern and southern regions of the province via tick dragging, and conducted next generation sequencing of 19 samples (composed of 45 ticks) via Illumina MiSeq. A total of 1400469 sequences were detected (median 69118/sample; range 23350–155227). The most abundant families of bacteria were unclassified Clostridiales and Ruminococcaceae for both I. scapularis and D. variabilis. No significant differences in the relative abundances of any phylum, class, order, family or genus were detected between locations (east vs south), sex, life stage or tick species. There were no differences in community membership or structure based on unifrac and AMOVA analyses. Female and male ticks had lower microbial diversity when compared to nymphs, based on the Simpson’s index and Shannon evenness index. The findings of our study differ from previous studies of these tick species conducted in other geographic areas, and highlight the potential role geography and related ecological factors may have in shaping the tick microbiota.

Detection of Borrelia burgdorferi, Anaplasma phagocytophilum and Babesia microti, with two different multiplex PCR assays

We have developed 2 real-time multiplex PCR assays for detection of Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti. The efficiency and sensitivity of each multiplex PCR assay was evaluated using field-collected Ixodes scapularis ticks that were positive for each of the pathogens, cloned plasmids harboring each of the PCR targets, and laboratory I. scapularis infected with B. burgdorferi B31. There was no difference in efficiency or sensitivity when comparing the multiplex PCR with the individual PCR reactions. If the 2 multiplex PCR assays are used in the same analysis, field-collected ticks that only harbor B. miyamotoi can also be identified. The multiplex assays are fast and cost-effective methods for screening and detecting pathogens in ticks, when compared to single-target PCR.

Identifying the last supper: utility of the DNA barcode library for bloodmeal identification in ticks

Ticks are among the most important vectors of disease in the Northern Hemisphere, and a better understanding of their feeding behaviour and life cycle is critical to the management and control of tick-borne zoonoses. DNA-based tools for the identification of residual bloodmeals in hematophagous arthropods have proven useful in the investigation of patterns of host use in nature. Using a blind test approach, we challenged the utility of the DNA barcode library for the identification of vertebrate bloodmeals in engorged, field-collected Ixodes scapularis. Universal vertebrate primers for the COI barcode region successfully amplified DNA from the host bloodmeal and only rarely amplified tick DNA. Of the 61 field-collected ticks, conclusive genus- and species-level identification was possible for 72% of the specimens. In all but two cases, barcode-based identification of the bloodmeal was consistent with the morphological identification of the vertebrate host the ticks were collected from. Possible explanations for mismatches or ambiguities are presented. This study validates the utility of the DNA barcode library as a valuable and reliable resource for the identification of unknown bloodmeals in arthropod vectors of disease. Future directions aimed at the refinement of these techniques to gain additional information and to improve the amplification success of digested vertebrate DNA in tick bloodmeals are discussed.

Rapid identification of vector-borne flaviviruses by mass spectrometry

Flaviviruses are a highly diverse group of RNA viruses classified within the genus Flavivirus, family Flaviviridae. Most flaviviruses are arthropod-borne, requiring a mosquito or tick vector. Several flaviviruses are highly pathogenic to humans; however, their high genetic diversity and immunological relatedness makes them extremely challenging to diagnose. In this study, we developed and evaluated a broad-range Flavivirus assay designed to detect both tick- and mosquito-borne flaviviruses by using RT-PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) on the Ibis T5000 platform. The assay was evaluated with a panel of 13 different flaviviruses. All samples were correctly identified to the species level. To determine the limit of detection for the mosquito-borne primer sets, serial dilutions of RNA from West Nile virus (WNV) were assayed and could be detected down to an equivalent viral titer of 0.2 plaque-forming units/mL. Analysis of flaviviruses in their natural biological background included testing Aedes aegypti mosquitoes that were laboratory-infected with dengue-1 virus. The assay accurately identified the virus within infected mosquitoes, and we determined the average viral genome per mosquito to be 2.0 × 10 6. Using human blood, serum, and urine spiked with WNV and mouse blood and brain tissues from Karshi virus-infected mice, we showed that these clinical matrices did not inhibit the detection of these viruses. Finally, we used the assay to test field-collected Ixodes scapularis ticks collected from sites in New York and Connecticut. We found 16/322 (5% infection rate) ticks positive for deer tick virus, a subtype of Powassan virus. In summary, we developed a single high-throughput Flavivirus assay that could detect multiple tick- and mosquito-borne flaviviruses and thus provides a new analytical tool for their medical diagnosis and epidemiological surveillance.

Reservoir Competency of Goats for Anaplasma phagocytophilum

The susceptibility of goats to infection by Anaplasma phagocytophilum (A. phagocytophilum) strains Ap-Variant 1 and Ap-ha was assessed by infestation of goats with field-collected Ixodes scapularis (I. scapularis) ticks. Both strains were infectious in the goat model. These results demonstrate that goats can be used in a laboratory setting to propagate A. phagocytophilum. Transmission from an infected goat to a naïve goat by I. scapularis tick feeding was used to demonstrate that goats are reservoir-competent for the Ap-Variant 1 strain.

Three Multiplex Assays for Detection of <I>Borrelia burgdorferi</I> sensu lato and <I>Borrelia miyamotoi</I> sensu lato in Field-Collected <I>Ixodes</I> Nymphs in North America

Two hundred fifty New Jersey field-collected Ixodes scapularis Say ticks and 17 Colorado Ixodes spinipalpis Hadwen & Nuttall ticks were tested using three separate multiplex real-time polymerase chain reaction (PCR) assays. One assay targets the rrs-rrlA IGS region of Borrelia spp. to detect Borrelia burgdorferi sensu lato (s.l.) and Borrelia miyamotoi s.l. The second assay targets the ospA region of B. burgdorferi s.l. to detect B. burgdorferi sensu stricto (s.s.), Borrelia bissettii, and Borrelia andersonii. The final assay targets the glpQ region of B. miyamotoi s.l. to differentiate B. miyamotoi LB-2001 and Borrelia lonestari. A testing scheme combining these tests yielded 18% of tested I. scapularis ticks surveyed from New Jersey positive for B. burgdorferi s.s., 3.2% I. scapularis ticks positive for B. miyamotoi LB-2001, and 41.2% I. spinipalpis ticks positive for B. bissettii surveyed from Colorado.

Three Multiplex Assays for Detection ofBorrelia burgdorferisensu lato andBorrelia miyamotoisensu lato in Field-CollectedIxodesNymphs in North America

Two hundred fifty New Jersey field-collected Ixodes scapularis Say ticks and 17 Colorado Ixodes spinipalpis Hadwen & Nuttall ticks were tested using three separate multiplex real-time polymerase chain reaction (PCR) assays. One assay targets the rrs-rrlA IGS region of Borrelia spp. to detect Borrelia burgdorferi sensu lato (s.l.) and Borrelia miyamotoi s.l. The second assay targets the ospA region of B. burgdorferi s.l. to detect B. burgdorferi sensu stricto (s.s.), Borrelia bissettii, and Borrelia andersonii. The final assay targets the glpQ region of B. miyamotoi s.l. to differentiate B. miyamotoi LB-2001 and Borrelia lonestari. A testing scheme combining these tests yielded 18% of tested I. scapularis ticks surveyed from New Jersey positive for B. burgdorferi s.s., 3.2% I. scapularis ticks positive for B. miyamotoi LB-2001, and 41.2% I. spinipalpis ticks positive for B. bissettii surveyed from Colorado.

Multiplex real-time PCR for detection of anaplasma phagocytophilum and Borrelia burgdorferi.

A multiplex real-time PCR assay was developed for the simultaneous detection of Anaplasma phagocytophilum and Borrelia burgdorferi. The assay was tested on various Anaplasma, Borrelia, Erhlichia, and Rickettsia species, as well as on Bartonella henselae and Escherichia coli, and the assay was found to be highly specific for A. phagocytophilum and the Borrelia species tested (B. burgdorferi, B. parkeri, B. andersonii, and B. bissettii). The analytical sensitivity of the assay is comparable to that of previously described nested PCR assays (A. phagocytophilum, 16S rRNA; B. burgdorferi, fla gene), amplifying the equivalent of one-eighth of an A. phagocytophilum-infected cell and 50 borrelia spirochetes. The dynamic range of the assay for both A. phagocytophilum and B. burgdorferi was >/=4 logs of magnitude. Purified DNA from A. phagocytophilum and B. burgdorferi was spiked into DNA extracted from uninfected ticks and from negative control mouse and human bloods, and these background DNAs were shown to have no significant effect on sensitivity or specificity of the assay. The assay was tested on field-collected Ixodes scapularis ticks and shown to have 100% concordance compared to previously described non-probe-based PCR assays. To our knowledge, this is the first report of a real-time multiplex PCR assay that can be used for the simultaneous and rapid screening of samples for A. phagocytophilum and Borrelia species, two of the most common tick-borne infectious agents in the United States.

Protection against tick-transmitted Lyme disease in dogs vaccinated with a multiantigenic vaccine

In an effort to develop a safe and effective vaccine for the prevention of Lyme borreliosis that addresses concerns raised over currently available vaccines, dogs were vaccinated twice with a multiantigenic preparation of Borrelia burgdorferi, strain N40, on days 0 and 20 of the experiment. About 70 and 154 days after the first immunization, dogs were challenged by exposing them to field-collected Ixodes scapularis ticks harboring B. burgdorferi. Vaccinated dogs were completely protected from infection by all criteria utilized to assess infection, developed high-titer anti- B. burgdorferi serum antibodies and growth inhibitory activity which persisted for over 200 days, and did not demonstrate any untoward consequence of vaccination. Serum absorption experiments revealed that borreliacidal and most likely protective antibodies in dogs receiving the multiantigenic preparation were not only elicited against the OspA antigen, but were also produced against additional yet to be determined targets on B. burgdorferi organisms. These data demonstrate that a multiantigenic vaccine is effective in preventing Lyme disease transmitted via the natural vector.

Molecular typing of Borrelia burgdorferi sensu lato by PCR-restriction fragment length polymorphism analysis.

The etiologic agent of Lyme borreliosis, Borrelia burgdorferi sensu lato, has been isolated from many biologic sources in North America and Eurasia, and isolates have been divided into three distinct genospecies (B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii). In order to explore the possible association of genospecies with disease manifestation, 60 isolates of B. burgdorferi sensu lato were subjected to 5S rDNA-linked restriction fragment length polymorphism (RFLP) analysis. The results confirmed earlier studies which indicated that virtually all North American isolates are B. burgdorferi sensu stricto, whereas Eurasian strains fall into all three genospecies. Thirty-five isolates were further characterized by PCR amplification of a region of the 16S-23S rDNA spacer and HinfI digestion of the products. This method resulted in the subdivision of B. burgdorferi sensu stricto into two distinct PCR-RFLP types. In contrast, B. garinii isolates all displayed an identical pattern. Additionally, a number of previously unclassified North American isolates (25015, DN127, 19857, 24330) showed distinctively different PCR-RFLP patterns. The application of this method for the typing of uncultured B. burgdorferi directly in biologic samples was demonstrated by analysis of several field-collected Ixodes scapularis tick specimens. The described PCR-RFLP technique should allow for the direct and rapid molecular typing of B. burgdorferi-containing samples and facilitate studies of the relationship between spirochete genotype and clinical disease.