Epidemiology and Genetic Diversity of Grapevine Leafroll-Associated Viruses in British Columbia.

Grapevine leafroll disease (GLD) is a complex associated with one or more virus species belonging to the family Closteroviridae. The majority of viruses in this complex are vectored by one or more species of mealybugs (Pseudococcidae) and/or scale insects (Coccidae). Grape-growing regions of British Columbia (BC), including Okanagan, Similkameen, and Fraser valleys and Kamloops (BC central interior), Vancouver, and Gulf islands, were surveyed during the 2014 and 2015 growing seasons for the presence of four major grapevine leafroll-associated viruses, including Grapevine leafroll-associated virus 1 (GLRaV-1), GLRaV-2, GLRaV-3, and GLRaV-4. In total, 3,056 composite five-vine samples were collected from 153 Vitis vinifera and three interspecific hybrid vineyard blocks. The results showed GLRaV-3 to be the most widespread, occurring in 16.7% of the composite samples, followed by GLRaV-4 (3.9%), GLRaV-1 (3.8%), and GLRaV-2 (3.0%). Mixed infections of two or more GLRaVs were found in 4.1% of the total samples. The relative incidence of GLRaVs differed among regions and vineyard blocks of a different age. Characterization of partial CO1 region from a total of 241 insect specimens revealed the presence of Pseudococcus maritimus, Parthenolecanium corni, and other Pulvinaria sp. in BC vineyards. Spatial patterns of GLRaV-3 infected grapevines in three vineyard blocks from three different regions in the Okanagan Valley showed variable degrees of increase in disease spread ranging from 0 to 19.4% over three growing seasons. Regional differences in the relative incidence and spread of GLD underline the need for region-based management programs for BC vineyards.

Aphid Transmission of the Ontario Isolate of Plum Pox Virus.

Utilization of timed virus acquisition access probes in studies of plum pox virus (PPV) transmission by aphids demonstrated that endemic species transmitted the virus readily from plum, Prunus domestica (L.) Batsch; peach, P. persica (L.); or dwarf flowering almond, P. glandulosa Thunberg., to peach seedlings. The green peach aphid, Myzus persicae (Sulzer), was shown to be the most efficient vector. Acquisition of virus by green peach aphids from infected peach leaves resulted in 18-28% infected peach seedlings, while aphids previously fed on infected leaves of plum transferred virus to 36% of peach seedlings. Although the spirea aphid, Aphis spiraecola (Patch), was a less efficient vector than M. persicae it is perhaps more important for the spread of PPV due to its greater abundance and occurrence earlier in the season when peach trees are thought to be more susceptible to infection. Virus transmission rates varied depending on the virus source and healthy test plant species. In contrast to many previous studies, aphid inoculation of the experimental host Nicotiana benthamiana Domin occurred at a low rate, never exceeding 4%. Acquisition of PPV by M. persicae from infected peach fruit was greatly reduced compared with acquisition from leaves. The results of this research indicate that the Ontario isolate of PPV-D is readily transmissible by aphids to peach and natural spread of the virus needs to be considered in future management or eradication programs.

Are fluoride levels in drinking water associated with hypothyroidism prevalence in England? A large observational study of GP practice data and fluoride levels in drinking water.

BACKGROUND: While previous research has suggested that there is an association between fluoride ingestion and the incidence of hypothyroidism, few population level studies have been undertaken. In England, approximately 10% of the population live in areas with community fluoridation schemes and hypothyroidism prevalence can be assessed from general practice data. This observational study examines the association between levels of fluoride in water supplies with practice level hypothyroidism prevalence. METHODS: We used a cross-sectional study design using secondary data to develop binary logistic regression models of predictive factors for hypothyroidism prevalence at practice level using 2012 data on fluoride levels in drinking water, 2012/2013 Quality and Outcomes Framework (QOF) diagnosed hypothyroidism prevalence data, 2013 General Practitioner registered patient numbers and 2012 practice level Index of Multiple Deprivation scores. FINDINGS: We found that higher levels of fluoride in drinking water provide a useful contribution for predicting prevalence of hypothyroidism. We found that practices located in the West Midlands (a wholly fluoridated area) are nearly twice as likely to report high hypothyroidism prevalence in comparison to Greater Manchester (non-fluoridated area). INTERPRETATION: In many areas of the world, hypothyroidism is a major health concern and in addition to other factors-such as iodine deficiency-fluoride exposure should be considered as a contributing factor. The findings of the study raise particular concerns about the validity of community fluoridation as a safe public health measure.

Insecticide susceptibility of three species of cutworm (Lepidoptera: Noctuidae) pests of grapes.

Climbing cutworms in the genus Abagrotis are economically important pests of grapes in the Okanagan Valley of British Columbia (BC). Grapes are recently introduced into many areas of the region, and the association between crop and pest is new and still evolving. This has led to limited information being available on pest management strategies, including the evaluation of chemical controls compatible with local production practices. Few insecticides are currently registered in Canada for cutworm control on grapes, and our study was initiated to provide information on the efficacy of potential control materials. We were also interested in the relative susceptibilities of the three most common cutworm species attacking grape buds in BC--Abagrotis orbis (Grote), Abagrotis reedi Buckett, and Abagrotis nefascia (Smith). Dose-response bioassays with nine insecticides were conducted on neonate larvae using Bok Choy leaf disks, and on fourth-instar larvae using diet incorporation. There were considerable differences in the toxicity of insecticides within species for neonates and fourth instars. For some materials, the relative toxicity to neonates and fourth instars were not correlated. Response to insecticides among the three species showed variation as well, and correct identification of the species complex present in individual locations is important in choosing the best available control material.

The effect of exercise on behavioral and psychological symptoms of dementia: towards a research agenda.

BACKGROUND: Behavioral and psychological symptoms of dementia (BPSD) are common and are core symptoms of the condition. They cause considerable distress to the person with dementia and their carers and predict early institutionalization and death. Historically, these symptoms have been managed with anxiolytic and antipsychotic medication. Although potentially effective, such medication has been used too widely and is associated with serious adverse side-effects and increased mortality. Consequently, there is a need to evaluate non-pharmacological therapies for behavioral and psychological symptoms in this population. One such therapy is physical activity, which has widespread health benefits. The aim of this review is to summarize the current findings of the efficacy of physical activity on BPSD. METHOD: Published articles were identified using electronic and manual searches. Rather than systematically aggregating data, this review adopted a rapid critical interpretive approach to synthesize the literature. RESULTS: Exercise appears to be beneficial in reducing some BPSD, especially depressed mood, agitation, and wandering, and may also improve night-time sleep. Evidence of the efficacy of exercise on improving other symptoms such as anxiety, apathy, and repetitive behaviors is currently weak or lacking. CONCLUSION: The beneficial effect of exercise type, its duration, and frequency is unclear although some studies suggest that walking for at least 30 minutes, several times a week, may enhance outcome. The methodological shortcomings of current work in this area are substantial. The research and clinical implications of current findings are discussed.

Host plant suitability and feeding preferences of the grapevine pest Abagrotis orbis (Lepidoptera: Noctuidae).

Thirteen plant species were tested for their suitability as hosts for Abagrotis orbis (Grote), a climbing cutworm pest of grapevines in British Columbia. Choice tests were also conducted to investigate larval feeding preferences for the Brassicaceae species joi choi, Brassica rapa variety. Chinensis L., spring draba; Draba verna L.; and shepherd's purse, Capsella bursa-pastoris (L.) Medik; compared with postdormant buds of grape, Vitis vinifera L. (Vitaceae), and leaves of nine other plant species from several families. Results showed that tah tsai, Brassica rapa L. variety rosularis (M. Tsen & S. H. Lee) Hanelt (Brassicaceae), is a superior host for A. orbis based on shorter time to adult eclosion, heavier pupae, and higher rates of survival. Later-instar larvae died when fed draba, whereas those reared on shepherd's purse did not survive beyond the third instar. White clover, Trifolium repens L. (Fabaceae), and grape leaves were unsuitable hosts throughout development. Fifth-instar A. orbis preferred plants of the Brassicaceae family, dandelion, Taraxacum officinale Weber (Asteraceae), and strawberry, Fragaria sp. L. (Rosaceae), compared with postdormant grape buds. The results of this study suggest that the winter annual mustards draba and shepherd's purse that often grow abundantly in vine rows might help reduce climbing cutworm damage to the buds of grapevines.

Relationship of insecticide tolerance to esterase enzyme activity in Aphis pomi and Aphis spiraecola (Hemiptera: Aphididae).

Green apple aphid, Aphis pomi De Geer, and Aphis spiraecola Patch (both Hemiptera: Aphididae), are sympatric aphid species that are pests of apples (Malus spp.) and other crops. A. spiraecola has been shown to be significantly more tolerant to several insecticides compared with A. pomi. To establish the mechanisms contributing to this difference in insecticide response, clones of both species were collected from British Columbia, Canada, and Washington state. Dose-response bioassays were conducted to determine relative tolerances to the insecticides pirimicarb, dimethoate, and imidacloprid; these results have been reported previously. Samples of adult aphids from each clone were assayed for the activity of esterase enzymes often involved in the detoxification of insecticides. A. spiraecola had higher esterase activity compared with A. pomi; this was apparent for two model substrates, alpha-naphthyl acetate (alpha-NA) and alpha-naphthyl butyrate (alpha-NB). Aphid clones of both species collected from Washington had higher esterase activity than clones collected from British Columbia. Clones from both species and locations hydrolyzed alpha-NA to a greater extent than alpha-NB. Esterase activity measured with both substrates was significantly positively correlated with the relative response to pirimicarb and dimethoate; a significant positive correlation also was found for hydrolysis of alpha-NB and imidacloprid. The apparent involvement of esterases in the differential response of A. pomi and A. spiraecola to insecticides indicated that the choice of control chemicals for A. spiraecola should not involve chemistries that are metabolized predominantly by esterases.

First Report of Aster Yellow Phytoplasmas ('Candidatus Phytoplasma asteris') in Canadian Grapevines.

In North America, elm yellows, aster yellows (AY), and X-disease phytoplasmas have been detected in American grapevines (1), and recently, Bois noir was detected in Canadian vineyards from British Columbia (BC) and Ontario (ON) (2). Typical symptoms of grapevine yellows (GY) include leaf rolling and chlorosis, uneven or total lack of lignification of canes, flower abortion or berry withering, and stunting. In 2006 and 2007, independent surveys were conducted by the Canadian Food Inspection Agency (CFIA) and Agriculture and Agri-Food Canada (AAFC) to detect phytoplasmas in Canadian vineyards containing different cultivars in BC, ON, Québec (QC), Nova Scotia, New Brunswick, and Prince Edward Island. The CFIA collected and tested 651 fresh leaf samples from recently imported grapevines and older grapevines in the same or neighboring blocks displaying symptoms typical of those associated with disease caused by phytoplasmas. Many vineyards were surveyed only once. AAFC collected and tested 3,485 samples from symptomatic and asymptomatic grapevines from established vineyards in ON, BC, and QC. The same vineyards were sampled in ON and BC both years; QC vineyards were only sampled in 2007. AAFC-collected leaf samples were freeze dried and stored at -20°C before processing. CFIA samples were tested by a modified real-time PCR assay and TaqMan probe targeting the 16S ribosomal RNA gene that detects a wide range of known phytoplasmas (2). Positive samples were confirmed by conventional PCR using the phytoplasma-specific primers P1/P7 (3) and the resulting ~1,800-bp fragment was cloned and sequenced as previously described (2). DNA extracted by AAFC was amplified by nested PCR technology with universal phytoplasma specific primer pairs P1/P6 and R16R2/R16F2 (3) and the resulting 1,200-bp fragment was cloned and sequenced. Two plants, one located in ON in 2006 and the other in BC in 2007, were found to be infected with an AY-like phytoplasma by the CFIA. The phytoplasmas detected in both infected plants had a 99.9% nt sequence identity with AY phytoplasma sequences from GenBank (Accession Nos. AF222063 and AY665676, respectively), with the BC isolate also showing 100% identity to a strain of AY, ash witches'-broom phytoplasma (GenBank Accession No. AY566302). AAFC detected phytoplasma DNA in both years in a total of 17 symptomatic plants and 21 asymptomatic plants from different vine varieties in ON, BC, and QC. Positive samples were found to have a 99.0% nt sequence identity to AY subgroup 16SrI-A (GenBank Accession No. AY180956). Sequences were exchanged for confirmation of phytoplasma identity and were deposited in Genbank under Accession Nos. FJ659844 and FJ824597. Phytoplasma strains were identified for all plants in which phytoplasmas were detected. Results show that AY is present in vineyards in the provinces of ON, BC, and QC. To our knowledge, this is the first report of AY being detected in grapevines in Canada. References: (1) E. Boudon-Padieu. Bull. O I V, 79:299, 2003. (2) M. Rott et al. Plant Dis. 91:1682, 2007. (3) E. Tanne et al. Phytopathology 91:741, 2001.

Influence of mixtures of kaolin particle film and synthetic insecticides on mortality of larval obliquebanded leafrollers (Lepidoptera: Tortricidae) from resistant and susceptible populations.

Experiments were conducted to determine potential interactions between kaolin particle film and three insecticides on neonate larvae of the obliquebanded leafroller, Choristoneura rosaceana (Harris) (Lepidoptera: Tortricidae). Kaolin did not significantly affect the toxicity of azinphosmethyl or indoxacarb to an insecticide-susceptible population when applied simultaneously with either insecticide in a 7-d leaf disk bioassay. Methoxyfenozide was slightly more toxic to the same leafroller population when coapplied with kaolin. When these bioassays were repeated on a multiresistant laboratory strain of C. rosaceana, mixtures of kaolin with either azinphosmethyl or indoxacarb were significantly more toxic than the insecticides alone, 3.1- and 7.7-fold more toxic for azinphosmethyl:kaolin and indoxacarb:kaolin, respectively. Mixtures of kaolin and methoxyfenozide did not differ in toxicity to the resistant leafroller population from the toxicity of methoxyfenozide alone. Kaolin alone had no effect on leafroller mortality over the 7-d duration of the bioassay. Although the toxicities of mixtures of kaolin with azinphosmethyl or indoxacarb are only moderately higher than those of the insecticides alone, they may be high enough to provide control of leafroller populations that have become difficult to manage due to the development of insecticide resistance.

Susceptibilities of apple aphid and spirea aphid collected from apple in the Pacific Northwest to selected insecticides.

Laboratory bioassays using leaf disks of apple dipped in test solutions of insecticides demonstrated that the apple aphid, Aphis pomi De Geer, and the spirea aphid, Aphis spiraecola Patch, differed significantly in susceptibility to a number of insecticides registered for control of aphids on apple (Malus spp.). Compared with A. pomi, A. spiraecola was approximately four- and three-fold less susceptible to pirimicarb and lambda-cyhalothrin, respectively, whereas there was little difference in response to dimethoate. Pymetrozine is thought to act on aphids primarily as a feeding inhibitor. Exposure of aphids to this material generated data that fit the probit model for only half the tested clones. However, the LC50 value for one clone of A. spiraecola was nearly 1,000 times higher than the value for one clone of A. pomi. Although the results from these trials did not indicate that either species had developed significant levels of resistance to the test materials, differences in LC50 levels of > 10-fold suggest insecticide tolerances and the possibility of control failures in the future. The demonstrated differences in susceptibility to insecticides between these two morphologically similar species also should be considered during the evaluation of new products for use on apple.

Baseline susceptibilities to imidacloprid for green apple aphid and spirea aphid (Homoptera: Aphididae) collected from apple in the Pacific northwest.

Susceptibilities to the neonicotinyl insecticide imidacloprid were determined for clones of apple aphid, Aphis pomi De Geer, and spirea aphid, Aphis spiraecola Patch, collected from conventional and organic apple orchards and from crab apple and wild apple in Washington state and British Columbia over a period of 6 yr. For aphids collected during 1996--1998, adults were dipped in test solutions by using the Food and Agriculture Organization protocol, and third instars and adults were reared on treated apple leaf disks. During the final 3 yr of study, bioassays involved only third instars on treated leaf material. Tests showed that A. spiraecola was significantly more tolerant to imidacloprid compared with A. pomi. Depending on the bioassay method and aphid developmental stage, average LC50 values for A. spiraecola were 4.4 -5.7 times higher than those for A. pomi established under the same test conditions. Clones of both species from Washington were marginally more tolerant to imidacloprid than clones from British Columbia, but the differences were generally not significant. Average measures of susceptibility for clones from organic orchards or unsprayed trees also did not differ from those for clones from conventional orchards, and there was no evidence for increasing LC50 values over the 6 yr of study. Differences in susceptibility to insecticides between these two anatomically similar species should be considered during the testing of new products for use on apple.

Nicotiana occidentalis: A New Herbaceous Host for Blueberry scorch virus.

Blueberry scorch virus(BlScV) is a carlavirus that causes a serious disease of blueberries (Vaccinium corymbosum L.) in North America (2). In aphid-transmission studies of BlScV using blueberry as host and test species, we found the rate of transmission to be low, and a lengthy incubation period was required before BlScV could be detected. For sequencing studies, RNA extraction from blueberry using standard methods was unreliable and inefficient. These problems prompted a search for alternate hosts. Of 12 herbaceous hosts screened for BlScV transmission using the blueberry aphid, Ericaphis fimbriata Richards, with mechanical transmission, only Nicotiana occidentalis (Wheeler) became infected. After 3 to 4 weeks, infection of N. occidentalis with BlScV resulted in mild symptoms that included pronounced leaf twisting and swollen leaf veins. Infection with BlScV was confirmed using a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) kit (Agdia Inc., Elkhart, IN), polyclonal antibodies to BlScV from the antiserum collection at the Pacific Agri-Food Research Center, Summerland, British Columbia, Canada, and reverse transcription-polymerase chain reaction (RT-PCR). Forward (5'-NTAAACACTCCCGAATATAC-3') and reverse (5'-CAGATTGCTTATCCGGCTTC-3') primers were designed with the published sequence of BlScV isolate NJ-02 (GenBank Accession No. NC003499). An amplicon of the expected size was generated and sequenced. BLAST analysis indicated that the nucleotide sequence of the amplified fragment was 87% identical to the corresponding sequence in NJ-02 (1). N. occidentalis was readily infected with BlScV following aphid or mechanical inoculations from blueberry. With E. fimbriata as the aphid vector, the transmission rate from blueberry to N. occidentalis was approximately 26%, compared with 70% for mechanical inoculations. Mechanical transfer of BlScV between infected N. occidentalisplants resulted in a 100% transmission rate. Recently, with N. occidentalis, we have completely sequenced two strains of BlScV from British Columbia, Canada and identified several aphid vector species. The identification of N. occidentalis as an herbaceous host of BlScV greatly facilitates future studies on the virus. References: (1) T. D. Cavileer et al. J. Gen. Virol. 75: 711, 1994. (2) R. R. Martin and P. R. Bristow. Phytopathology 78:1636, 1988.

Chemical residues and bioactivity of tebufenozide applied to apple foliage.

Tebufenozide, an insect growth regulator that acts as an ecdysone agonist, was applied at recommended label rate to apple trees in August 1997 and May 1998. Foliar residues were determined by high-performance liquid chromatography from samples collected 2, 24, 48 and 96 h post-spray, and at weekly intervals thereafter for 9 weeks in 1997 and 11 weeks in 1998. Foliage sampled at the same times was used in bioassays to determine residual toxicity to first-instar obliquebanded leafrollers, Choristoneura rosaceana (Harris). Residue decay followed first-order kinetics in both years, with residual half-life of 36.3 days in 1997 and 7.2 days in 1998. Estimates of the time needed to reduce bioactivity to 50% in bioassays were 18.7 days in 1997 and 36.3 days in 1998. The accuracy of equations describing decay of tebufenozide residues and bioactivity over time was not improved by using degree-day accumulations as the independent variable.

Susceptibility of leafrollers (Lepidoptera: Tortricidae) from organic and conventional orchards to azinphosmethyl, spinosad, and Bacillus thuringiensis.

Populations of obliquebanded leafroller, Choristoneura rosaceana Harris, and three-lined leafroller, Pandemis limitata Robinson, were obtained from seven sites in the Okanagan and Similkameen Valleys of British Columbia and assayed for their responses to three insecticides using a leaf disk bioassay. Lethal concentration ratios (LCRs) were calculated for all populations compared with a susceptible laboratory colony of C. rosaceana; significant variation was detected in response to all three insecticides. LCRs were 0.86-15.52 for azinphosmethyl, 0.38-2.37 for spinosad (Success), and 0.58-4.89 for Bacillus thuringiensis (Foray). Correlation analysis indicated no cross-resistance among the three insecticides. Leafroller populations obtained from apple orchards managed with organic production practices were more susceptible to azinphosmethyl than leafrollers obtained from conventionally managed sites. Conversely, the highest levels of tolerance to B. thuringiensis were observed in the populations from organic sites, possibly reflecting usage patterns; B. thuringiensis is one of the few insecticides allowed under organic production guidelines. All populations were highly susceptible to spinosad, which may be a useful tool for resistance management programs if used judiciously.

Comparison of bioassay techniques for determining baseline susceptibilities to imidacloprid for green apple aphid (Homoptera: Aphididae).

The susceptibility of a clone of green apple aphid, Aphis pomi (De Geer), to the neonicotinyl insecticide imidacloprid was determined by direct and indirect bioassay techniques. Aphid numbers were assessed on potted apple seedlings treated with various concentrations of imidacloprid, adults were dipped in test solutions as per the Food and Agriculture Organization protocol, or nymphs and adults were reared on treated apple leaf disks. Effective concentrations required to kill half of the test population (EC50) varied depending on the bioassay technique, ranging from as low as 0.064 ppm for first instars reared for 3 d on treated leaf disks to 1.79 ppm for adult apterae dipped in solutions of imidacloprid and held for 24 h on clean leaf disks. When imidacloprid was directly applied to aphids, mortality continued to increase over 3 d, but the difference was not statistically significant between day 2 (1.36 ppm) and day 3 (1.19 ppm). Toxicity of neonicotinyls to aphids is expressed rather slowly and primarily after oral ingestion. The effect of imidacloprid on reproduction of green apple aphid was also assessed for adult apterae reared on treated leaf disks. Contrary to previous reports, our results demonstrated that imidacloprid does not have a direct negative effect on the reproductive physiology of this species. Negative effects can mostly be attributed to the antifeedant activity of this compound and the protracted time to death. The results of this study contribute to a better understanding of the most suitable techniques for assessing aphid mortality after exposure to these new insecticides and provides a baseline susceptibility to imidacloprid for green apple aphid.

Resistance and cross-resistance to four insecticides in populations of obliquebanded leafroller (Lepidoptera: Tortricidae).

Populations of obliquebanded leafroller, Choristoneura rosaceana (Harris), were collected from organic and conventionally managed orchards located in the Okanagan and Similkameen Valleys of British Columbia Neonate F1 progeny were assayed for resistance to azinphosmethyl, tebufenozide, methoxyfenozide, and indoxacarb using a leaf disk bioassay. Significant differences in resistance levels among populations were observed for all four insecticides. Insects collected from organic sites were more susceptible to all insecticides than were insects collected from conventional sites. Resistance to the benzoylhydrazine insect growth regulators tebufenozide and methoxyfenozide was highly correlated with resistance to azinphosmethyl across populations, indicating cross-resistance between these compounds. The highest levels of resistance were observed with indoxacarb, but resistance levels to indoxacarb did not correlate with those for azinphosmethyl. Dose-response regression lines for tebufenozide were parallel across populations, suggesting that the resistance mechanism(s) were quantitatively, but not qualitatively, different. Cross-resistance between azinphosmethyl and benzoylhydrazine insecticides indicates that a resistance management strategy for obliquebanded leafroller involving the rotation of these materials is not likely to be successful.

Emergency medicine and public health: new steps in old directions.

Emergency medicine and public health have opportunities to interact in at least 4 areas: surveillance of diseases, injuries, and health risks; monitoring health care access; delivering clinical preventive services; and developing policies to protect and improve the public's health. Recent, cross-cutting initiatives and innovations in these 4 areas follow pathways first explored more than a generation ago and provide an important impetus for future work. An analysis of recent contributions also points to various obstacles and challenges that must be addressed to take full advantage of existing and rapidly developing ties between emergency medicine and public health. The connections between these 2 fields will continue to create important partnership opportunities and the strong possibility of achieving new benefits for patients, the public, and the professionals who serve them.

TaqMan-based detection of Trichomonas vaginalis DNA from female genital specimens.

A double-labeled fluorescent probe was designed and evaluated for detecting Trichomonas vaginalis DNA in a 5' nuclease (TaqMan) assay. The T. vaginalis-specific probe contains a 5'-fluorescein (5'-FAM) and a 3'-rhodamine (TAMRA) derivative. Female genital secretions were collected on Amplicor (Roche Molecular, Indianapolis, Ind.) swabs and by a transport system used for Chlamydia trachomatis and/or Neisseria gonorrhoeae DNA detection by PCR. Five hundred fifty-two female genital specimens, of which 248 (45%) were vaginal specimens and 304 (55%) were introital, were tested for both T. vaginalis DNA and viable microorganisms using the 5' nuclease assay and broth culture, respectively. Of these, 304 of 552 (55%) were also evaluated by direct microscopic examination for the characteristic motile organism. After resolving discrepancies, the comparisons produced an analytical sensitivity and specificity for the TaqMan-based PCR assay of 97.8 and 97.4%, respectively. As a result, DeltaRQ values (differences in fluorescence due to probe hybridization and resulting 5'-FAM cleavage from the specific PCR product) of > or =2.0 and < or =1.5 were established for T. vaginalis-positive and -negative cutoffs, respectively. DeltaRQ values between 1.5 and 2.0 were considered indeterminate. Overall findings revealed a high level of agreement between PCR and culture for detecting T. vaginalis. Potential benefits of the 5' nuclease assay include a greater sensitivity compared to direct microscopic examination and the ease of testing large numbers of clinical specimens in a significantly shorter turnaround time compared to culture.

Type A allatostatins from Drosophila melanogaster and Diplotera puncata activate two Drosophila allatostatin receptors, DAR-1 and DAR-2, expressed in CHO cells.

The type-A allatostatins A (AST-A) are a group of insect peptides with a common C-terminal motif Y/FXFGL-NH(2). The existence of at least four putative type A Drosophila melanogaster ASTs (called type A drostatins or DST-As) has been predicted from the sequence of a recently cloned DST-A preprohormone [C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 273, 126-1131]. SRPYSFGL-NH(2), (DST-3A), the only DST isolated from Drosophila so far, activated the first cloned DST-A GPCR (DAR-1) [N. Birgül et al. (1999) EMBO J. 18, 5892-5900]. A newly cloned orphan Dm GPCR, which shares 47% overall and 60% transmembrane region sequence identity with DAR-1, was classified as a second putative Dm DST-A receptor (DAR-2) [C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 273, 571-577]. Although activation of DAR-2 by DSTs has been postulated, no experimental evidence for that has been presented to date. In this study, we expressed both DAR-1 and DAR-2 in CHO cells and used a GTPgammaS and a Ca(2+) mobilization assay for pharmacological evaluation of the receptors. Synthetically prepared DST-As, as well as selected Diplotera punctata (cockroach) ASTs, activated DAR-1 and DAR-2 in both functional assays indicating ligand redundancy and cross species activity. Cell pretreatment with pertussis toxin led to some differences in the nature and magnitude of signaling pathways at the DAR-1 and DAR-2 receptors, suggesting possible differential coupling to cellular effector system(s) and distinct biological functions of each receptor in vivo.