Signal and Noise: Proton Pump Inhibitors and the Risk of Dementia?

The association between proton pump inhibitor (PPI) use and dementia remains controversial. This cohort study re-examines this issue, addressing shortcomings identified in previous publications using a population-based and a high-dimension propensity-score matched cohort to follow patients for up to 22 years. Cox regression models using baseline characteristics, a lag period, and time-varying variables were used to examine the risk of dementia by cumulative PPI exposure. High-dose PPI users (> 180 days of use) had significantly higher risk of dementia in crude Cox models. After adjustment for medical diagnoses and prescription drug use, these associations disappeared. Among high-dose users starting PPI therapy between 46 and 55 years old, the unadjusted hazard ratio (HR) was 1.55 (95% confidence interval (CI) 1.14, 2.10); the adjusted hazard ratio (aHR) was 1.10 (95% CI 0.80, 1.51). For high-dose users starting therapy between 56 and 65 years, HR = 1.22 (95% CI1.03, 1.44); aHR = 0.99 (95% CI 0.83, 1.17). High-dose users between the ages of 66 and 75 years had no association with the risk of dementia. The use of lag models or time-varying parameters similarly found some association with dementia in crude, but not multivariable Cox models. Although high-dose PPI users were more likely to develop dementia, they were more likely to be diagnosed with dementia risk factors, such as diabetes and cardiovascular disease, which are risk factors for dementia. Controlling for these conditions using multivariable models or a propensity-score matched cohort eliminated this association.

Time to benefit and the long-term persistence of new users of oral bisphosphonates.

INTRODUCTION: This study aimed to examine long-term persistence in new users of oral bisphosphonates in a population-wide cohort in Manitoba, Canada. MATERIALS AND METHODS: A longitudinal observational study was conducted using administrative health data characterizing long-term bisphosphonate persistence in those who started treatment between 1997 and 2018. Treatment discontinuation was evaluated using Kaplan-Meier methods. Cox regression was used to examine associations between discontinuation and osteoporosis diagnosis, previous fractures, and age. A sub-analysis of users with FRAX scores examined the relationship between 10-year fracture risk estimations and discontinuation. RESULTS: Of 42,249 new bisphosphonate users, median age was 71 years, with 88.6% being female. Median duration of bisphosphonate use was 0.95 years (IQR 0.25, 3.9 years). Overall, 47.9% of incident users persisted up to 1 year, 25.0% persisted up to 3 years, and 14.1% up to 5 years. Presence of an indication for bisphosphonate use was associated with decreased discontinuation risk. Persistence generally increased with age. Having a BMD test performed was a predictor of lower discontinuation. The strongest predictor was having an osteoporosis diagnosis [HR for discontinuation = 0.68 (95% CI 0.66, 0.70)]. In users with FRAX scores (n = 14,114), moderate-risk [HR = 0.86 (95% CI 0.77, 0.96)] and high-risk users [HR = 0.77 (95% CI 0.69, 0.85)] were less likely to discontinue compared to lower-risk users. CONCLUSIONS: A rapid decline in bisphosphonate persistence was shown. Almost half of users would not be expected to achieve clinically relevant benefits with a persistence of less than 1 year. Allowing informed choice in high-risk patients may be the best way to focus on those likely to benefit and persist with treatment.

Opioid prescribing by dentists in Manitoba, Canada: A longitudinal analysis.

BACKGROUND: Overuse of opioids has become a health crisis in the United States and Canada. Opioid prescribing practices have subsequently come under scrutiny, with limited study regarding prescribing patterns of dentists. METHODS: A longitudinal analysis was conducted on all adult patients to whom an opioid was dispensed from 2014 through 2017 in Manitoba, Canada. Rates of dental opioid prescribing, milligram morphine equivalents (MMEs) per prescription, and guideline adherence were determined. Additional analyses evaluated the contribution of dentist prescribing to first opioid use and opioid use 90 days before and after first dental opioid prescription. RESULTS: Dentist prescriptions accounted for 3.8% of all opioid prescriptions and 0.58% of total MMEs. Codeine with acetaminophen combinations were the primary opioid prescribed (97.4%), followed by tramadol/acetaminophen (1.7%) and oxycodone with acetaminophen (0.7%). Overall, 30 or less tablets were supplied in 96.1% of prescriptions. Prescriptions were written for 5 or less days in 89.1% of cases and for 7 or less days in 95.2% of cases. In the 90-day before-after analysis, 87.8% of patients received only 1 prescription from their dentist, with an additional 9.8% receiving only 2 prescriptions. Dentists were responsible for 20.6% of first opioid prescriptions, with 5.6% written for 50 or more MMEs per day. CONCLUSIONS: Compared with available guidelines, prescribed quantities were mostly appropriate, suggesting that the overall contribution of dentists to opioid overuse is limited. PRACTICAL IMPLICATIONS: Dental opioid stewardship can be encouraged through an enhanced regulatory monitoring program with local review to guide efforts to further improve opioid prescribing. Continued efforts are warranted to prescribe smaller quantities and for greater avoidance of opioid use for dental pain.

Patterns, Policy and Appropriateness: A 12-Year Utilization Review of Blood Glucose Test Strip Use in Insulin Users.

OBJECTIVES: Considerable attention has been paid to the rising costs of the use of blood glucose test strips (BGTS). Insulin users have generally been treated as a single homogeneous group, resulting in policies that cap usage (8.2 strips/day) in provincial drug insurance programs. The objective of this study was to conduct a utilization review of BGTS by insulin users and to evaluate use patterns against current insulin use patterns and BGTS policy. METHODS: BGTS usage was examined in a cohort of insulin users with type 1 and type 2 diabetes over a 12-year period (2001 to 2013) using the population-based administrative data in Manitoba, Canada. RESULTS: Total BGTS strip use increased by 121%, from $4.3 to $9.5 million. However, the number of insulin users also increased by 115%. Use has been stable at 1.5 strips per day per person since 2004 by insulin users with type 2 diabetes but has risen from 1.9 to 3.0 strips per day per person in those with type 1 diabetes. Mean daily test strip use was below the number of daily tests recommended for patients using insulin as per the current Canadian guidelines, with 11% and 15% of insulin users with type 1 and type 2 diabetes not claiming any BGTS use and a further 15% (type 1) and 28% (type 2) using fewer than 1 strip per day. CONCLUSIONS: BGTS use per insulin user has been stable for most of the past decade, and the vast majority of use falls well below provincial insurance caps. The amount of low-level testing (0 to <1 strip/day) suggests that greater attention should be directed to ensuring a safe level of testing by all insulin users.

Cost of shingles: population based burden of disease analysis of herpes zoster and postherpetic neuralgia.

BACKGROUND: Around 30% of the population will experience herpes zoster (HZ), 10% of whom develop postherpetic neuralgia (PHN). Together, these illnesses produce a significant economic burden to the healthcare system. METHODS: Administrative healthcare data collected over the period of April 1st 1997 to March 31st 2014 were analyzed to determine the healthcare system burden of HZ using direct medical costs. Episodes of HZ were identified using international classification of disease (ICD) codes. Trends in age-adjusted (AA) HZ-rates were analyzed by piecewise-regression. Total annual and per-episode costs were determined for drug treatment, medical care, and hospitalizations within each year. RESULTS: The incidence of HZ increased by 49.5% from 1997/98 to 2013/14. Piecewise-regression of AA-rates revealed a steady AA-rate of 4.7 episodes/1000 person-years (PY) from 1997/98 to a breakpoint in 2008/09, after which rates began to increase reaching 5.7 episodes/1000 PY in 2013/14. Drug costs rose significantly (p <0.03) from $89.77/episode (95% CI: $82.96, $96.59) to $127.34/episode (95% CI: $117.24, $137.44). Medical costs increased (p <0.0001) from $57.98/episode (95% CI; $55.26, $60.70) to $78.84/episode (95% CI; $74.08, $83.61). Hospitalization rates declined from 3.10% in 1997/98 to 1.36% in 2011/12, resulting in cost dropping from $397/episode (95% CI; $284, $511) to $195/episode (95% CI; $129, $260). Total annual costs of HZ and PHN were $1,997,183 in 2011/12, 4.7% lower than the 1997/98 costs of $2,095,633. CONCLUSION: A significant increase in annual number of HZ cases was observed, driven largely by demographic factors. A 21% increase in the AA-incidence reveals changes in HZ rates beyond those expected by population shifts. The large increase in incidence of HZ, with rising per episode medical and prescription costs were offset by dramatic drops in hospitalization rates, the net effect of which has been to hold the total costs relatively constant. However, the decrease in hospitalization rates slowed over the last half of the study, settling at 1.3% in the last 4 study years. The likely future of HZ burden is one of rising costs, primarily driven by the demographic shifts of an increasing and aging population.

Price of pain: population-based cohort burden of disease analysis of medication cost of herpes zoster and postherpetic neuralgia.

BACKGROUND: Pain is a main symptom of herpes zoster (HZ), and postherpetic neuralgia (PHN) is a frequent complication occurring in 5% to 15% of cases, causing moderate to severe neuropathic pain. A population-based observational study was conducted to evaluate the treatment patterns and economic burden of prescription drug treatment of HZ and PHN pain in the province of Manitoba (Canada) over a period of 15 years. METHODS: Administrative health care data, including medical and hospital separation records, were examined to identify episodes of HZ using International Classification of Diseases-9/10 codes between April 1, 1997 and March 31, 2014. Episodes of PHN were identified using medical and prescription claims. Incident use of analgesic, antidepressant, or anticonvulsant drugs was used to determine prescription pain costs. RESULTS: The age-adjusted incidence of HZ increased from 4.7 episodes/1,000 person-years in 1997/98 to 5.7/1,000 person-years in 2013/14. PHN occurred in 9.2% of HZ cases, a rate that did not change over the study period (P=0.57). The annual cost to treat HZ pain rose by 174% from 1997/98, reaching CAD $332,981 in 2011/12, 82.8% (95% confidence interval [CI] 81.2%, 84.3%) of which was related to PHN. The per episode cost of HZ rose by 111% from $31.59 (95% CI $25.35, $37.84) to $66.81 (95% CI $56.84, $76.78) and by 94% for PHN from $292 (95% CI $225, $358) to $566 (95% CI $478, $655). These increases were driven by increasing use of anticonvulsants, primarily gabapentin, which accounted for 57% of the increase in cost. CONCLUSION: There has been an increase in the incidence of HZ and PHN and in the average cost associated with the prescription treatment of their resultant neuropathic pain. The primary driver of the increased episodic cost is the increased use of gabapentin. These changes have resulted in a substantial increase in the economic burden associated with HZ and PHN.

The changing landscape of antiviral treatment of herpes zoster: a 17-year population-based cohort study.

BACKGROUND: Herpes zoster (HZ) is a common viral disease that produces a painful vesicular rash. Early use of antiviral medications is recommended, as it reduces pain and speeds healing. A population-based observational study was conducted to evaluate the changing burden of HZ in the province of Manitoba (Canada) over a period of 17 years. METHODS: Administrative health care data including medical and hospital records were examined, and International Classification of Diseases, Ninth Revision, Clinical Modification and International Classification of Diseases, Tenth Revision, Clinical Modification codes were used to identify episodes of HZ between April 1, 1997 and March 31, 2014 in persons aged 20 or over. Annual age-adjusted incidence and hospitalization rates were calculated. Prescription records of HZ-diagnosed persons for acyclovir, valacyclovir, and famciclovir were used to calculate the rates and costs of antiviral treatment. RESULTS: There were 73,893 identified cases of HZ and 1,245 HZ-related hospitalizations between 1997 and 2013. Of these episodes, 42,270 (57.2%) were treated with antiviral medications at a total cost of $4,708,065 (CAD). The age-adjusted incidence of HZ rose from 4.67/1,000 person years in 1997/1998 to 5.67/1,000 person years in 2013/2014, a 21.9% increase. Antiviral treatment rates increased from 41.7% to 66.2% of all diagnosed episodes. Mean treatment costs per episode dropped from $127.29 in 1997/1998 to $56.06 in 2013/2014, primarily due to the introduction of generic antiviral medications. The total cost of antiviral treatment peaked in 2005/2006 at $329,935 and dropped steadily thereafter to $223,973 in 2013/2014. HZ-related hospitalization rates decreased from 3.1% to 0.9%. CONCLUSION: While both the incidence of HZ and the rates of antiviral treatment have risen substantially, the economic burden from antiviral treatment has been decreasing since a peak in 2005/2006 and was only 3.2% higher in 2013/2014 than in 1997/1998. This drop in cost is attributed to the introduction of generic antiviral drugs.

The safety of meperidine prescribing in older adults: A longitudinal population-based study.

BACKGROUND: Meperidine (pethidine) is an opioid analgesic that offers little advantage relative to other opioids and several disadvantages including limited potency, short duration of action, and the production of a neurotoxic metabolite (normeperidine) with a long half-life. Older adults are more sensitive to meperidine's side effects and may have diminished renal function which leads to the accumulation of normeperidine. The Institute for Safe Medication Practices has suggested avoiding meperidine in older adults, limiting its dose (≤600 mg/day) and duration of use (≤48 h). The objective of this study was to determine the level of meperidine use in older adults and assess the dosage and duration of meperidine with reference to these safety recommendations. METHODS: A longitudinal study using administrative healthcare data was conducted to examine meperidine utilization and levels of high dose and extended duration prescribing among persons ≥65 years of age between April 1, 2001, and March 31, 2014 in Manitoba, Canada. The number of meperidine prescriptions, users, duration of treatment, defined daily doses (DDD) dispensed and number of prescribers were determined over the study period. RESULTS: In the Manitoba older adult population there was a marked decline in meperidine users and prescriptions from 2001 to 2014. There was an average use of 26.4 (95 % CI 24.0-28.8) DDDs of meperidine per user per year. While only 3.7 % of the prescriptions exceeded the 600 mg maximum daily dose, 96.7 % of prescriptions exceeded the recommended 2 days of therapy. For the remaining users of meperidine, the amount of meperidine used per person rose from 18.98 to 56.14 DDDs/user/year over the study period. The number of prescribers of meperidine declined throughout the study, but low DDD prescribers declined more quickly than high DDD prescribers. CONCLUSIONS: While meperidine use has declined, the remaining use appears to be decreasing in safety, with more meperidine prescribed per user. This seems to be driven by the continued prescribing by a small number of high DDD prescribers. Targeted educational initiatives directed at this small group of prescribers may be helpful. Alternatively removing meperidine from medication insurance schemes may provide additional incentive to avoid meperidine in older adults.

Safety of fentanyl initiation according to past opioid exposure among patients newly prescribed fentanyl patches.

BACKGROUND: Although a convenient opioid delivery system, transdermal fentanyl patches have caused several deaths and resulted in safety warnings reminding prescribers that fentanyl patches should be prescribed only for patients who have adequate prior exposure to opioids. We conducted a longitudinal analysis of the safety of fentanyl initiation by examining past opioid exposure among patients newly prescribed fentanyl patches. METHODS: We identified all patients in the province of Manitoba who were newly prescribed fentanyl patches between Apr. 1, 2001, and Mar. 31, 2013. We converted all prior opioid use to oral morphine equivalents and determined the average daily dose in the 7-30 days before initial fentanyl patch use. Fentanyl initiation was considered unsafe if the patient's pre-fentanyl opioid exposure was below the recommended level. RESULTS: We identified 11 063 patients who began using fentanyl patches during the study period. Overall, fentanyl initiation was deemed unsafe in 74.1% of cases because the patient's prior opioid exposure was inadequate. Women and patients 65 years of age and older were more likely than men and younger patients, respectively, to have inadequate prior opioid exposure (p < 0.001 for each comparison). The proportion of patients who had unsafe prescriptions for fentanyl patches decreased significantly over the study period, from 87.0% in 2001 to 50.0% in 2012 (p < 0.001). INTERPRETATION: The safety of fentanyl initiation improved over the study period, but still half of fentanyl patch prescriptions were written for patients with inadequate prior opioid exposure. Review of prior opioid exposure may be a simple but important way to improve the safe use of fentanyl patches.

The effectiveness and limitations of regulatory warnings for the safe prescribing of citalopram.

BACKGROUND: Citalopram is the most commonly prescribed antidepressant in Canada. Concerns have been raised about its cardiac safety, and a dose-dependent prolongation of the QT interval has been documented. Drug interactions involving concomitant use of other medications that prolong the QT interval or increase citalopram levels by interfering with its metabolism increase the cardiac risk. Regulatory bodies (Health Canada and the US Food and Drug Administration) issued warnings and required labeling changes in 2011/2012, suggesting maximum citalopram doses (<40 mg for those <65 years; <20 mg for those ≥65 years) and avoiding drug interactions that increase cardiac risk. The purpose of this study is to assess the impact of these warnings on citalopram prescribing practices. METHODS: A quasi-experimental interrupted time series analysis was conducted using all citalopram prescribing data from the population of Manitoba, Canada from 1999 to 2014. This allowed for the examination of high-dose prescribing (above regulatory warning levels) and the number of interacting medications per citalopram prescription. RESULTS: There was a dramatic decline in the prescribing of high doses in both age groups, with a 64.8% decline in those <65 years and 33.6% in those ≥65 years. Segmented regression models indicated significant breakpoints in the third quarter of 2011 for both age groups (P<0.0001), corresponding to the time the regulatory warnings were issued. There appeared to be no impact of the warnings on the prescribing of interacting medications. The number of interacting medications actually increased in the postwarning period (<65, 0.78-0.81 interactions per citalopram prescription; ≥65, 0.93-0.94, P<0.001). CONCLUSION: Regulatory changes appear to have produced an important reduction in the high-dose prescribing of citalopram. In contrast to this relatively simple dosage change, there was no indication that the more complex issue of resolving drug-drug interactions was impacted by regulatory warnings.

Voluntary warnings and the limits of good prescribing behavior: the case for de-adoption of meperidine.

BACKGROUND: Meperidine (pethidine) offers little to no therapeutic advantage over other opioids, may be more prone to abuse, and produces a neurotoxic metabolite with a long half-life. The Institute for Safe Medication Practices (ISMP) issued warnings in 2004 and 2005 suggesting that meperidine be avoided, and when used, it should be in limited doses (<600 mg/24 h) and for a limited duration (<48 hours). Hospitals have responded to these warnings, but much less is known about meperidine prescribing in the community setting. This study examined the potential impact of ISMP warnings on the prescribing of meperidine using time series analysis. METHODS: A population-based longitudinal cross-sectional study was conducted to examine oral meperidine utilization among persons 16 years of age and older in Manitoba, Canada, between April 1, 2001 and March 31, 2014. Amounts of meperidine were expressed using defined daily doses (DDDs), the equivalent of 400 mg of meperidine per day. The number of meperidine prescriptions and users per quarter were determined and analyzed using regression analysis. RESULTS: There were 49,063 prescriptions for 442,641 DDDs of meperidine dispensed to 9,374 distinct users. The number of DDDs of meperidine per 1,000 persons peaked in the second quarter of 2003 at 11.75, and then dropped to a low of 5.36 by 2014. This represented a marked decline in the numbers of users and prescriptions over the study period. The piecewise regression model revealed a significant breakpoint in the last quarter of 2004 (F (3, 48)=337.00, P<0.0001). In contrast to these findings, among the remaining users, there was an increase in the amount of meperidine per prescription (increase of 0.34 DDDs/prescription/year; F(1, 50)=434, P<0.0001, R (2)=0.89) and the amount of meperidine per user (increase of 1.17 DDDs/user/year; F(1, 50)=653.5, P<0.0001, R (2)=0.93). CONCLUSION: Following the ISMP warnings, meperidine use dramatically declined. Unfortunately, the remaining users of meperidine are using more meperidine and receiving more meperidine in each prescription. This pattern of results suggests that there may be limits to voluntary safety warnings. Policy action such as removal of medication insurance coverage may represent a logical next step to reverse or de-adopt meperidine and further enhance patient safety.

Salivary gland degeneration and vitellogenesis in the ixodid tick Amblyomma hebraeum: Surpassing a critical weight is the prerequisite and detachment from the host is the trigger.

The normal engorged body weight of female ixodid ticks (Acari: Ixodidae) is about 100x the unfed weight. Virgin female Amblyomma hebraeum normally do not feed beyond 10x the unfed weight. However, about 10-20% of a population of virgins will feed to perhaps 20x the unfed weight, but not much beyond that. In A. hebraeum, when females surpass about 10x the unfed weight, the following changes in physiology occur if they are removed from the host: (a) they will not reattach if given the opportunity, (b) their salivary glands (SGs) will undergo autolysis within 4 days if they are mated or 8 days if they are virgin, and (c) egg maturation and oviposition will occur in due course. Mated or virgin female ticks removed from the host below about 10x the unfed weight do not experience the latter changes (Kaufman, W.R., Lomas, L., 1996. 'Male Factors' in ticks: their role in feeding and egg development. Invertebrate Reproduction and Development 30, 191-198). In 1984 we named this transitional weight, the 'critical weight' (CW). Its absolute value is probably a species-specific characteristic (Kaufman, W.R., 2007. Gluttony and sex in female ixodid ticks: how do they compare to other blood-sucking arthropods? Journal of Insect Physiology 53, 264-273). Although mated females tend to engorge within a day of surpassing the CW, virgin females surpassing the CW can remain attached to the host for at least several weeks more. It is not known whether the physiological changes in the SGs and ovaries listed above occur in those large virgins that remain attached, although we suppose that this would be maladaptive. Instead, we hypothesize in this study that surpassing the CW is only a prerequisite for inducing these changes, and that detachment is the actual trigger. We support our hypothesis by demonstrating that large virgins, remaining attached to a host for 8 days, did not undergo SG degeneration nor complete egg maturation during the attachment period. Those changes occurred only within 8 days following detachment. So some type of sensory information associated with attachment to the host, and still undefined, inhibits expression of the physiological changes hitherto associated merely with surpassing the CW.

Effect of 20-hydroxyecdysone and haemolymph on oogenesis in the ixodid tick Amblyomma hebraeum.

Earlier work from our laboratory indicated that injection of 20-hydroxyecdysone (20E) into non-vitellogenic female Amblyomma hebraeum ticks stimulates the synthesis of vitellogenin (Vg), but not its uptake into oocytes [Friesen, K., Kaufman, W.R., 2004. Effects of 20-hydroxyecdysone and other hormones on egg development, and identification of a vitellin-binding protein in the ovary of the tick, Amblyomma hebraeum. Journal of Insect Physiology 50, 519-529]. In contrast, Thompson et al. [Thompson, D.M., Khalil, S.M.S., Jeffers, L.A., Ananthapadmanaban, U., Sonenshine, D.E., Mitchell, R.D., Osgood, C.J., Apperson, C.S., Roe, M.R., 2005. In vivo role of 20-hydroxyecdysone in the regulation of the vitellogenin mRNA and egg development in the American dog tick, Dermacentor variabilis (Say). Journal of Insect Physiology 51, 1105-1116] demonstrated that injection of 20E into virgin female Dermacentor variabilis ticks stimulated both vitellogenesis and Vg uptake into oocytes. In addition to the species difference in the two studies there were substantially different methods for injecting 20E. In our earlier work we injected small partially fed ticks after removing them from the host. Thompson et al. injected the females while they remained attached to the host. So in this study we repeated our earlier experiments on A. hebraeum using on-host injection. We also injected 20E into off-host ticks with or without haemolymph collected from engorged ticks (days 2-10 post-engorgement), or from large partially fed mated ticks in the rapid phase of engorgement, to see whether we might detect a 'vitellogenin uptake factor' (VUF) in haemolymph. Off-host injection of 20E (0.45microg/g body weight (bw)) did not induce ovary development beyond that of vehicle-injected controls. But ticks in this study, receiving 20E plus haemolymph from engorged ticks, showed a significant increase in ovary weight beyond that of 20E alone (1.31+/-0.05% bw; 34 for 20E plus haemolymph and 1.03+/-0.05% bw; 25 for 20E alone). However, in normal engorged A. hebraeum, the ovary exceeds 7% bw at the onset of oviposition. As in our earlier work, in this study 20E stimulated Vg-synthesis (3.9+/-0.5mgVt-equivalents/ml) beyond that occurring in vehicle-injected ticks (0.76+/-0.14mgVt-equivalents/ml), and there was a further increase in ticks injected with 20E plus haemolymph from engorged ticks (8.9+/-1.0mgVt-equivalents/ml). On-host injection of 20E alone (6microg20E/g bw) did not produce a statistically significant increase in oocyte length over that of vehicle-injected controls, whereas on-host injection of 20E plus engorged haemolymph resulted in significantly larger oocytes (261+/-57microm) compared to vehicle-injected controls (132+/-11microm), compared to 20E alone (131+/-12microm), or haemolymph alone (124+/-24microm). There was a marked stimulation of Vg-synthesis by 31microg20E/g bw (6.0+/-1.5mgVt-equivalents/ml) compared to vehicle-injected controls (1.02+/-33mgVt-equivalents/ml). Vt accumulation by ovaries was significantly greater in ticks treated with haemolymph (12+/-3microgVt/mg ovary) or 20E plus haemolymph (56+/-26microgVt/mg ovary) compared to vehicle-injected controls (5.1+/-1.5microgVt/mg ovary). There was also a significant effect of 6microg20E/g bw plus engorged haemolymph on ovary weight (1.74+/-0.29% bw) compared to vehicle-injected ticks (0.95+/-0.10% bw), but not compared to ticks injected with 20E alone (1.25+/-0.19% bw). We conclude that at least some of the differences observed between the two laboratories relate to the species difference, and that there is some evidence that the engorged haemolymph of A. hebraeum contains a VUF.

Effects of 20-hydroxyecdysone and other hormones on egg development, and identification of a vitellin-binding protein in the ovary of the tick, Amblyomma hebraeum.

Partially fed adult female Amblyomma hebraeum ticks were injected with 20-hydroxyecdysone (20E; up to 43 microg/g body weight (bw)), juvenile hormone III (JH III; up to 100 microg/g bw), bovine insulin (up to 2000 mU/g bw), or triiodothyronine (up to 200 ng/g bw) in an attempt to stimulate vitellogenesis. Of these, only 20E stimulated synthesis and release of vitellogenin (Vg). Immunoblot analysis revealed that Vg-synthesis occurred in the fat body. However, consistent with earlier observations suggesting that a distinct signal may be required for Vg-uptake, there was no significant Vg-uptake by oocytes of partially fed, 20E-treated ticks. Because Vg-uptake commonly occurs via receptor-mediated endocytosis (i.e., a specific Vg-receptor), we attempted to identify a vitellin (Vt)-binding protein in ovaries of engorged female ticks. A single 86 kDa Vt-binding protein was identified, even under reducing conditions (2-mercaptoethanol), by a ligand-blotting technique. Sodium salt of suramin (5 mM) inhibited binding of Vt to the 86 kDa protein. However, this protein was also detected in ovaries from small partially fed ticks (50-100 mg), suggesting that the inability of 20E to stimulate Vg-uptake in partially fed ticks may not have been due to the absence of a Vg-receptor.

Quantification of vitellogenesis and its control by 20-hydroxyecdysone in the ixodid tick, Amblyomma hebraeum.

Ovaries of the ixodid tick, Amblyomma hebraeum Koch, grew rapidly after engorgment as a result of yolk uptake. At 26 degrees C, oviposition began by day 10 post-engorgement, plateaued on days 16-18, and ended by day 38. Vitellin (Vt) was partially purified from ovaries of day 10 engorged ticks by gel filtration and ion exchange chromatography. This Vt comprises seven major and several minor polypeptides. Two polypeptides (211 and 148 kD) from haemolymph of engorged female ticks corresponded to minor polypeptides of similar molecular weight in the ovary. The haemolymph titre of the 211 and 148 kD polypeptides increased up to the onset of oviposition. These polypeptides were absent in males and non-vitellogenic females (day 0 engorged or day 10 partially-fed females), and were thus designated as vitellogenin (Vg). Antibodies raised against haemolymph Vg211 and 148 recognized these polypeptides in partially purified Vt, as well as six of the seven major polypeptides. Using these antibodies we developed an indirect, competitive ELISA to quantify Vg. Rise in haemolymph Vg-concentration lagged slightly behind the rise in haemolymph ecdysteroid (ES)-concentration, and Vg-synthesis was stimulated by injections of 20E into non-vitellogenic females. These observations indicate that an ES is the vitellogenic hormone in A. hebraeum.