Ixodes scapularis

We investigated the design of potential immunogenic peptides of the gSG6 protein using bio-informatic tools. The strategy was i) to identify potential immunogenic epitopes predicted by algorithms; and ii) to research the specificity of An. gambiae gSG6 peptide sequence compared to the genome/ Expressed Sequence Tag (EST) libraries of other organisms.
This analysis was based on the amino acid sequence of mature An. gambiae gSG6 (“UniProtKB/TrEMBL:Q9BIH5” and “gi:13537666”, [23] (link)).
The identification of putative linear B-cell epitopes of An. gambiae gSG6 was performed by computerized predictions of antigenicity based on physico-chemical properties of the amino-acid sequences with the BcePred database [24] and with the FIMM database [25] (link). We also identified the MHC class 2 binding regions using the ProPred-2 online service [26] (link).
Sequence alignments were done with the Tblastn program in Vectorbase database [27] (link) which enabled comparing a sequence of gSG6 peptides with known genomes or EST libraries of Aedes aegypti, Ixodes scapularis, Culex pipiens, Pediculus humanus, Glossina morsitans, Rhodnius prolixus, Lutzomia longipalpis and Phlebotomus papatasi. Concomitantly, we investigated sequence alignments with the Blast program to compare the gSG6 peptides sequence with all non-redundant GenBank CDS database [28] (link).
Peptides were synthesized and purified (>80%) with Genosys (Sigma-Genosys, Cambridge, UK) with an added N-terminal biotin. All peptides were shipped lyophilized and they were resuspended in 0.22 µm filtered milliQ water and stored in aliquots at −80°C.

Auanema rhodensis n. sp. (strain SB347) was originally isolated from blood-engorged deer ticks (Ixodes scapularis) that were used as bait for nematodes. The ticks were placed in the upper layer of the soil in Kingston (University of Rhode Island), R.I., United States, in September 2001 by E. Zhioua (W. Sudhaus, pers. comm.). Subsequently, a laboratory culture of A. rhodensis SB347 was established by W. Sudhaus^{14 (link)}. A. freiburgensis n. sp. (strain SB372) was isolated from a dung pile in Freiburg, Germany, in August 2003 by W. Sudhaus. Both strains have been kept in the laboratory on NGM plates seeded with Escherichia coli OP50, as is standard for C. elegans²⁶ and preserved cryogenically (e.g. at the NYU Rhabditid Collection).
Both species produce males and females, and hermaphrodites after passage through the dauer stage^{19 (link)}. The genders were collected separately as follows. In A. rhodensis, most female embryos are produced by their mother within the first 15 hours of adulthood^{19 (link), 22 (link)}. To obtain females, dauer juveniles were placed individually on a small agar plate seeded with OP50 and cultured at 20 °C until adulthood. After these hermaphrodites oviposited 25 eggs or fewer, they were removed. The F1 generation developed into adult females. To obtain hermaphrodites, dauer juveniles were transferred from old cultures onto seeded NGM plates and collected with the Baermann funnel technique after they reached adulthood. Males were hand-picked from 3- to 7-day-old cultures. For A. freiburgensis, females were obtained by letting hermaphrodites self-fertilize on individual plates. Most self-progeny under these conditions are either female or male. Hermaphrodites were obtained by isolating dauer juveniles from crowded plates and letting them develop into adults.

The primary objective of the study described herein was to evaluate the efficacy of a fipronil deer feed against I. scapularis and A. americanum ticks parasitizing white-tailed deer under pen conditions. The vector-host association and treatment concept are presented in Fig. 1. Ixodes scapularis was selected because it is a vector of seven human pathogens, with the most notable being those causing Lyme disease [31 (link), 32 (link)]. Lyme disease is the most common vector-borne disease in the USA, occurring most frequently in the Northeast and Midwest of the USA, and is estimated to account for approximately 500,000 human cases per year [32 (link)–34 (link)]. Amblyomma americanum was selected because it is suspected to vector five or more disease agents transmissible to humans [35 (link)], and is also linked with southern tick-associated rash illness (STARI) [36 (link)] and red meat allergy [37 (link), 38 ].Fig. 1

Vector-host association (a) and impact of fipronil deer feed consumption by white-tailed deer on reproductive female ticks (b). a Adult females attach to white-tailed deer and blood feed for approximately 6–11 days. Fully engorged females drop off of the host and begin the reproductive process. Females then oviposit and produce thousands of eggs. b Adult female ticks blood-feeding on white-tailed deer expire and are prevented from feeding to engorgement and detaching, subsequently preventing them from successfully ovipositing and reducing the reproductive rate

Ticks were acquired from the Oklahoma State Tick Rearing Facility (OSU) (Stillwater, OK, USA). Equal numbers of each sex and species (I. scapularis and A. americanum) were obtained. For each lot of I. scapularis and A. americanum and prior to shipment to the study site, OSU screened a subsample of ticks (n = 10) for pathogens using standardized PCR assays. Ixodes scapularis were screened for B. burgdorferi and Anaplasma phagocytophilum. Amblyomma americanum were screened for the presence of Ehrlichia chaffeensis, Francisella tularensis and Rickettsia rickettsii. All PCR-screened ticks were negative for the above pathogens. Once ticks arrived at the study site, they were housed in an industry-standard desiccator with the relative humidity maintained at > 90% until enclosed in a feeding capsule for attachment to deer.
The feeding capsules utilized in this study were specifically designed for holding blood-feeding I. scapularis and A. americanum. Feeding capsules allow for the containment and localization of ticks and aid in facilitating blood-feeding [40 (link)]. The traditional stockinet sleeve method for feeding ticks on cattle [41 (link)–43 ] was determined to be inadequate for white-tailed deer. We instead developed a feeding capsule for deer application, which was in part based upon feeding capsules for ticks (referred to hereafter as tick feeding capsules) previously designed for tick-feeding on rabbits and sheep [44 ]. To make each capsule, sheets of ethylene–vinyl acetate foam were cut into three square pieces. Each square had a different outside area, allowing for flexibility (base, approx. 12 × 12 cm; middle, approx. 9 × 9 cm; top, approx. 7 × 7 cm), and had a combined depth of approximately 18 mm. The center of each square was cut away, creating an opening. The inner surface areas of the base and middle piece openings were each approximately 7 × 7 cm; the top piece had a smaller opening (approx. 1.5 × 1.5 cm) through which the ticks were to be inserted, which decreased the probability that ticks would escape through the top of the capsule (Additional file 3: Figure S2).
Deer were anesthetized using an intramuscular injection of telazol and xylazine at dosages of approximately 3 mg/kg and approximately 2.5 mg/kg, respectively. Once fully anesthetized, deer were weighed to the nearest 0.1 kg using a certified balance. Prior to blood collection and capsule attachment, large patches of fur on the neck were trimmed using electric horse clippers (Wahl®; Wahl Clipper Corp., Sterling, IL, USA). Prior to capsule attachment, 10 ml of blood was collected from the jugular vein of each deer using a 20-gauge needle. The blood from each individual deer was immediately placed into a vacutainer containing EDTA and was centrifuged for 10 min at 7000 revolutions/min. The plasma was transferred to 1.5-ml centrifuge tubes, which were then stored at − 20 °C until analysis.
Two identical tick feeding capsules were attached to opposing sides of the neck of each deer using a liberal amount of fabric glue (Tear Mender, St. Louis, MO, USA). Each capsule was held firmly in place for > 3 min to allow it to adhere to the skin and fur. For each deer, 20 I. scapularis mating pairs were placed within one capsule, and 20 A. americanum mating pairs were placed within the second capsule. Prior to tick attachment, 20 ticks (all same species and sex) were placed into a modified 5-ml syringe. Ticks were chilled in ice for approximately 5–10 min to slow movement. The 20 mating pairs were then carefully plunged into the capsules and a fine mesh lid was applied and reinforced with duct tape. Representative photos and video of the tick attachment process are presented in Fig. 2 and Additional file 4: Video S1, respectively. The capsules were further secured to deer by wrapping the neck with a veterinary bandage (3 M Company, St. Paul, MN, USA).Fig. 2

Tick capsule attachment and tick attachment. a Female ticks being plunged into capsule, b plunger being removed prior to mesh lid being secured, c completed, secured capsule being checked to ensure all corners are adhered to the neck, d closeup of completed capsule containing 20 Ixodes scapularis mating pairs

After completion of capsule and tick attachment, deer were given tolazine via intramuscular injection at a dose of 4 mg/kg to reverse the effects of the anesthetic. Deer were then housed in individual pens, observed closely until they were mobile and moving normally and monitored routinely for the remainder of the day.

The study took place in Dutchess County, New York, which has experienced high incidence rates of Lyme disease and other tick-borne diseases since the 1990s (Eisen et al, 2016 (link)). We selected two tick control interventions that had been demonstrated in small-scale studies to be effective in reducing population size of blacklegged ticks and that were considered safe for people, pets, and the environment (Dolan et al, 2004 (link); Schulze et al, 2017 (link); Schulze et al, 2007 (link); Williams et al, 2018 (link)).
One intervention was the deployment of TCS bait boxes, which attract small mammals to a food source inside an enclosed device and apply the tick-killing chemical fipronil to these mammals. Fipronil is lethal to ticks but harmless to mammals (Dolan et al, 2004 (link)). The other intervention was the biopesticide Met52, which consists of spores of the F52 strain of the fungus Metarhizium brunneum. Met52 solution is mixed with water and sprayed on the ground and low-lying vegetation where ticks dwell. By killing ticks attached to small mammals, TCS bait boxes are expected to affect the abundance of host-seeking (questing) ticks the following year, whereas Met52 targets host-seeking ticks, with impacts expected within days to weeks after deployment.
As previously described in detail (Keesing et al, 2022 (link)), we selected 24 residential neighborhoods that had reported high incidence of tick-borne diseases in Dutchess County in recent prior years. Neighborhoods consisted of ∼100 adjacent 1- and 2-family residences at moderate to high density, including their respective yards. Overall average property size was 0.19 ha. After a year of exhaustive efforts to recruit eligible households in each neighborhood to participate in the study, we enrolled a mean of 34% of properties in each neighborhood (range 24–44%).
Each of the neighborhoods was randomly assigned to one of four treatment categories, with six neighborhoods assigned to each category: (1) active TCS bait boxes and active Met52; (2) active TCS bait boxes and placebo Met52; (3) placebo TCS bait boxes and active Met52; and (4) placebo TCS bait boxes and placebo Met52. All participating properties in each neighborhood received the same treatment category. Placebo TCS bait boxes were identical to active bait boxes except that they contained no fipronil. Placebo Met52 consisted of water only. Participating households agreed not to deploy broadcast acaricides independent of our study throughout its duration.
Both products were used according to label instructions. TCS bait boxes, covered with galvanized steel shrouds (active and placebo), were deployed twice annually, in spring and mid-summer, at an average rate of 5.9 boxes per property (38/ha), at least 10 meters apart, preferentially in sites frequented by small mammals. Active Met52 was sprayed by truck-mounted high-pressure sprayers (GNC Industries, Inc.) at a concentration of 2.22 L per 378.5 L of water. Placebo Met52 (water only) was sprayed using the same truck-mounted sprayers at the same rate of 4 L of spray per 93 m² at a pressure of 1.2–1.4 MPa. Spraying of active and placebo Met52 occurred twice each year, immediately before (April—early May) and during (late May–late June) the peak activity period for nymphal blacklegged ticks in this region (Ostfeld et al, 2018 (link)). Further details are provided in the supplementary online appendix of Keesing et al (2022 (link)) (https://wwwnc.cdc.gov/eid/article/28/5/21-1146_article).
The study design was double-masked ( = “double-blind”), in that neither the members of participating households nor the team of researchers collecting data were aware of the treatment category of any of the neighborhoods. All data collection, entry, and compilation were conducted with the treatment categories remaining masked.