Hyperemia

A. gambiae Keele strain mosquitoes were maintained on a 10% sugar solution in laboratory culture at 27°C and 70% humidity with a 12 hrs light/dark cycle according to standard rearing procedures [49] . A single cohort of adult female mosquitoes were collected immediately after eclosion, and either maintained under normal, non-sterile insectary conditions or placed into a sterile environment. Following, adult female mosquitoes were daily given fresh filtered sterilized 10% sucrose solution containing 15 µg gentamicin sulphate (Sigma) and 10 units/10 µg of penicillin-streptomycin (Invitrogen) per ml, respectively. Each cohort of mosquitoes was simultaneously membrane-fed freshly washed human erythrocytes resuspended to 40% haematocrit using human serum. As far as possible, every care was taken to maintain the sterility of the blood and membrane-feeding apparatus used to feed the mosquitoes, in order to prevent the antibiotic-treated mosquitoes acquiring bacterial infection during the process of membrane-feeding. The mosquitoes were starved for 8 hrs before feeding to encourage engorgement, and sugar solution was replaced once blood feeding had finished. At 24 hrs after blood feeding, 20 mosquitoes from each replicate of each cohort was collected and dissected on ice. RNA was extracted from dissected tissues at the assayed time points using the RNeasy kit (Qiagen). The quantification of RNA concentrations was performed using a Spectrophotometer (Eppendorf).

The methodology for measuring endothelial function and vascular reactivity using DTM has been previously described [21 (link)–25 (link)]. All DTM tests were performed using a VENDYS® 6000 Portable System (Endothelix, Houston, TX), a PC-based system that fully automates the cuff reactive hyperemia protocol. The general test setup and a sample VENDYS test report are shown in Figure 1. During subject preparation, blood pressure cuffs were placed on both of the subject's upper arms, and VENDYS skin temperature sensors were affixed to both of the subject's index fingers. The software-driven DTM test began with an automated measurement of blood pressure and heart rate obtained from the left arm cuff. Following a 5-minute period of patient and temperature stabilization, a 5-minute cuff occlusion (cuff inflated to 30 mmHg above systolic BP) of the right arm was performed. During the cuff occlusion period, fingertip temperature in the right hand decreased because of the absence of warm circulating blood. When the cuff was released after the 5-minute occlusion, hyperemic blood flow to the forearm and hand was restored, and this resulted in a “temperature rebound” in the fingertip that is directly related to the subject's hyperemic blood flow response, endothelial function, and vascular reactivity [21 (link), 22 (link)]. Using the recorded fingertip temperatures, the ambient temperature of the testing room, the observed slope of temperature decline, and a multivariate bioheat formula, the VENDYS software calculated and plotted a zero reactivity curve (ZRC). The ZRC served as an internal control and showed the expected temperature rebound curve, if zero vascular reactivity was present and the other variables remained the same. In other words, the ZRC is the expected temperature curve, if no vasodilatation and subsequent reactive hyperemia had occurred [21 (link)]. Vascular reactivity index (VRI) was determined by taking the maximum difference between the observed temperature rebound curve and the ZRC during the reactive hyperemia period. VRI ranged from 0.0 to 3.5 and was classified as being indicative of poor (0.0 to <1.0), intermediate (1.0 to <2.0), or good (≥2.0) vascular reactivity.
The VENDYS DTM Test Registry includes age, sex, blood pressure, heart rate, VRI, and fingertip temperature measurements recorded during DTM tests. The Registry does not include other health related information. All DTM tests were performed in ambulatory care clinical settings. This study includes a total of 6,084 patients from 18 clinics that volunteered to submit their data to the Registry. The number of each type of medical practice is as follows: cardiology = 9, general/family practice = 4, antiaging = 3, and internal medicine = 2.
Statistical analyses were performed using MATLAB (The MathWorks, Inc., Natick, MA). Variable data were expressed as mean ± SD. VRI scores in men and women were compared using unpaired Student's t-test. Comparisons of categorical data (e.g., proportion of subjects with good VRI in men versus women) were performed using Fisher's exact test. Pairwise correlations were examined using Pearson's correlation coefficient, and correlations between VRI and multiple patient characteristics (i.e., age, sex, blood pressure, and heart rate) were evaluated using multiple linear regression analysis. p value < 0.05 was considered significant. When performing statistical comparisons, tests with missing data were excluded from the comparison. “Cold Finger Flag” was defined as the condition in which the right finger temperature at start of cuff occlusion (time 300 s) is ≤27°C. Previous DTM testing had shown that right finger t300 temperatures < 27°C often resulted in technically poor results. “Sympathetic Response Flag” was defined as the condition in which left finger temperature continuously declines (>0.5°C temperature drop over a 5-minute time period) after right arm-cuff occlusion. When evaluating VRI, tests that exhibited “Cold Finger Flag” (n = 353) or “Sympathetic Response Flag” (n = 294) were excluded from the analyses. In addition to monitoring temperature at the index finger of the right arm, we studied temperature changes at the index finger of the left (nonoccluded) arm and observed interesting signals that are currently under further investigations and not included in the results below.

A total of 4938 I. scapularis ticks collected in passive surveillance in 2012 (excluding the 68 used in validation) were tested using the developed testing protocol (Figure 
1). The screening 23S and confirmatory ospA real-time PCR were used to assess B. burgdorferi infection as described above. The screening 23S PCR is a multiplex assay that also detects the presence of A. phagocytophilum DNA using primers specific for the msp2 gene (ApMSP2f and ApMSP2r)
[14 (link)]. Confirmation of infection with A. phagocytophilum is achieved by an in-house real-time PCR assay targeting 16S rRNA. The Borrelia miyamotoi-specific IGS real-time PCR was used to detect B. miyamotoi infections that were subsequently confirmed by B. miyamotoi glpQ real-time PCR. All real-time PCR assays were conducted using the conditions described above for B. miyamotoi IGS. Tick extracts that were positive for Borrelia spp. infection in the screening 23S real-time PCR, but negative for B. burgdorferi in the ospA real-time PCR, and negative in the B. miyamotoi IGS assay, were tested by 16S-23S IGS nPCR with the aim of sequencing products to identify other infecting Borrelia species.
Associations of infections and co-infections in ticks with province of origin, level of engorgement of the tick, host of origin, and tick instar were investigated by logistic regression in STATA version 11.0 for Windows (STATACorp, College Station, TX, USA). The most parsimonious multivariable model was created by backwards and forwards elimination and substitution of variables. Logistic regression models were used to investigate whether or not there were significant associations between infections of ticks with different pathogens. The level of significance throughout was P < 0.05.

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

The post-attachment period spanned the initial 8 days following tick attachment (day 0 to day 8). During this time, deer were housed individually in pens (Additional file 5: Figure S3) and checked daily to ensure adequate health and wellbeing and to ensure that the capsules remained firmly attached. At day 6 and day 8 post-attachment, the deer were anesthetized in the previously described manner, and the capsules were opened to monitor the condition of the ticks. These time points were selected because I. scapularis was the primary species of concern and reportedly takes approximately 6–11 days to reach engorgement and detach [45 ]. A minimum of 48 h was required between sedations for each deer because of PSU IACUC policy forbidding sedation of animals on consecutive days.
Ticks were easily visible by the naked eye. The inside of each capsule was carefully scanned for attached and detached ticks. The numbers of total ticks recovered and their attachment status (attached, detached), feeding status (flat, partially engorged, fully engorged) and condition (alive, dead) were recorded. Any dead ticks (attached, detached) were removed from deer. At the conclusion of tick observations on day 8 post-attachment, capsules were completely removed, and any live or dead ticks were manually removed from the deer.
Fully engorged, live detached female ticks were collected, weighed to the nearest 0.0001 g using an analytical balance (Mettler-Toledo, LLC, Columbus, OH, USA), and maintained individually in vials. Engorged females were maintained in a desiccator (> 90% relative humidity) and were allowed approximately 14–28 days to complete oviposition [45 ]. After oviposition was completed, females were removed, and egg masses weighed to the nearest 0.0001 g. Egg masses were monitored for the emergence of larvae, with eggs embryonating within approximately 35–50 days [45 ]. Egg masses were monitored for approximately 2–3 weeks to estimate the proportion of hatched eggs.