Quantitative Risk Assessments of Hepatitis A Virus and Hepatitis E Virus from Raw Oyster Consumption.

A quantitative risk assessment of hepatitis A virus (HAV) and hepatitis E virus (HEV) from raw oyster consumption from farm and retail was evaluated over three seasons. This risk assessment comprises four steps: hazard identification, dose-response assessment, exposure assessment, and risk characterization. We used probabilistic models for prevalence, concentration, and oyster consumption. HEV dose-response (DR) model based on HEV dosing in chimpanzees and used to perform a dose-response assessment of HEV was proposed. Both HAV and HEV were simultaneously enumerated by real-time PCR to determine viral doses. The probabilistic prevalences of HAV and HEV were in the ranges of 8-20% and 8-40%, respectively. The best-fit DR model was the beta-Poisson with alpha and N50 equal to 216.9 and 3.03 × 107 , respectively. After running the Monte Carlo simulation, the annual cases of foodborne hepatitis A and hepatitis E from raw oyster consumption from farms were 9,264-17,526 and 1-604, respectively, while those at retail were 7,694-14,591 and 1-204, respectively. This study suggested that consuming farm oysters poses a significantly higher risk of hepatitis A than hepatitis E. The best-fit DR model for HEV developed in this study could determine risks of hepatitis E from raw oyster consumption in Thailand.

Wolbachia supergroups A and B in natural populations of medically important filth flies (diptera: muscidae, calliphoridae, and sarcophagidae) in Thailand.

Filth flies, belonging to suborder Brachycera (Family; Muscidae, Calliphoridae and Sarcophagidae), are a major cause of nuisance and able to transmit pathogens to humans and animals. These insects are distributed worldwide and their populations are increasing especially in sub-tropical and tropical areas. One strategy for controlling insects employs Wolbachia, which is a group of maternally inherited intracellular bacteria, found in many insect species. The bacteria can cause reproductive abnormalities in their hosts, such as cytoplasmic incompatibility, feminization, parthenogenesis, and male lethality. In this study we determined Wolbachia endosymbionts in natural population of medically important flies (42 females and 9 males) from several geographic regions of Thailand. Wolbachia supergroups A or B were detected in 7 of female flies using PCR specific for wsp. Sequence analysis of wsp showed variations between and within the Wolbachia supergroup. Phylogenetics demonstrated that wsp is able to diverge between Wolbachia supergroups A and B. These data should be useful in future Wolbachia-based programs of fly control.

Comparative field efficacy of newly developed formulations of larvicides against Aedes aegypti (L.) (Diptera: Culicidae).

Aedes aegypti (L.) is known as vector of dengue and chikungunya fever. Larvicides are used to control this vector. We evaluated the efficacy of newly developed formulations of larvicides to control Ae. aegypti under field conditions for 24 weeks post single application. Mosdop P and Mosdop TB containing diflubenzuron (2% and 40 mg/tablet, respectively) as the active ingredient, were applied at a dosage of 0.1 mg a.i./1 and Mosquit TB10, Mosquit TB100 and Temecal containing temephos (1%, 10% and 1%, respectively) as the active ingredient were applied at a dosage of 1 mg active ingredent (a.i.) to 200 liter water storage jars. Two water regimens were used in the jars: in one regimen the jar was kept full of water all the time and in the other regimen a full jar had half the volume removed and refilled weekly. The larvicidal efficacy was reported as the level of inhibition of emergence (IE%) calculated based on the pupal skins in the jars versus the original number of larvae added. Mosdop P, Mosdop TB, Mosquit TB10, Mosquit TB100 and Temecal showed complete larvicidal efficacy (100% IE) in the constantly full jars for 16, 17, 14, 20 and 13 weeks posttreatment, respectively; in the jars where half the volum of water was replaced weekly, the larvicides had complete larvicidal efficacy (100% IE) for 19, 20, 17, 24 and 15 weeks post-treatment, respectively. The five larvicide regimens evaluated in this study are effective for controlling Ae. aegypti larvae.

Oxyresveratrol Attenuates Inflammation in Human Keratinocyte via Regulating NF-kB Signaling and Ameliorates Eczematous Lesion in DNCB-Induced Dermatitis Mice.

Oxyresveratrol (ORV) is one of the novel antioxidants having been extensively studied in recent years. One of the main sources of ORV is Artocarpus lakoocha, which has been used in traditional medicine in Thailand for decades. However, the role of ORV in skin inflammation has not been clearly demonstrated. Therefore, we investigated the anti-inflammatory effects of ORV on dermatitis model. The effect of ORV was examined on human immortalized and primary skin cells exposed to bacterial components including peptidoglycan (PGN) and lipopolysaccharide (LPS) and 2,4-Dinitrochlorobenzene (DNCB)-induced dermatitis mouse model. PGN and LPS were used to induce inflammation on immortalized keratinocytes (HaCaT) and human epidermal keratinocytes (HEKa). We then performed MTT assay, Annexin V and PI assay, cell cycle analysis, real-time PCR, ELISA and Western blot in these in vitro models. H&E staining, immunohistochemistry (IHC) staining with CD3, CD4 and CD8 markers were used to evaluate the effects of ORV in in vivo model of skin inflammation using BALB/c mice. Pretreatment of HaCaT and HEKa cells with ORV inhibited pro-inflammatory cytokine production through inhibition of NF-κB pathway. In DNCB-induced dermatitis mouse model, ORV treatment reduced lesion severity, and skin thickness and numbers of CD3, CD4 and CD8 T cells in the sensitized skin of mice. In conclusion, it has been demonstrated that ORV treatment can ameliorate inflammation in the in vitro models of skin inflammation and in vivo models of dermatitis, suggesting a therapeutic potential of ORV for treatment of skin diseases particularly eczema.

Insecticide resistance in bedbugs in Thailand and laboratory evaluation of insecticides for the control of Cimex hemipterus and Cimex lectularius (Hemiptera: Cimicidae).

Bedbugs are found in many countries around the world, and in some regions they are resistant to numerous insecticides. This study surveyed bedbugs in Thailand and determined their resistance to insecticides. The surveys were carried out in six provinces that attract large numbers of foreign tourists: Bangkok, Chonburi, Chiang Mai, Ubon Ratchathani, Phuket, and Krabi. Bedbugs were collected from hotels and colonized in the laboratory to evaluate their resistance to insecticides. Cimex hemipterus (F.) was found in some hotels in Bangkok, Chonburi, Phuket, and Krabi, whereas Cimex lectularius L. was found only in hotels in Chiang Mai. No bedbugs were found in Ubon Ratchathani. The colonized bedbugs showed resistance to groups of insecticides, including organochlorines (dichlorodiphenyl trichloroethane, dieldrin), carbamates (bendiocarb, propoxur), organophosphates (malathion, fenitrothion), and pyrethroids (cyfluthrin, deltamethrin, permethrin, lambda-cyhalothrin, etofenprox) in tests using World Health Organization insecticide-impregnated papers. The new insecticides imidacloprid (neonicotinoid group), chlorfenapyr (pyrrole group), and fipronil (phenylpyrazole group) were effective against the bedbugs; however, organophosphate (diazinon), carbamates (fenobucarb, propoxur), and pyrethroids (bifenthrin, cypermethrin, esfenvalerate, etofenprox) were ineffective. Aerosols containing various pyrethroid insecticides with two to four different active ingredients were effective against the bedbugs. The results obtained from this study suggested that both species of bedbugs in Thailand have developed marked resistance to various groups of insecticides, especially those in the pyrethroid group, which are the most common insecticides used for pest control. Therefore, an integrated pest management should be implemented for managing bedbugs in Thailand.

Competitive suppression between chikungunya and dengue virus in Aedes albopictus c6/36 cell line.

Aedes albopictus C6/36 cell line was used to evaluate dengue virus serotype-3 (DENV-3) and chikungunya virus (CHIKV) co-infection. Virus infection was determined using a one-step duplex reverse transcriptase polymerase chain reaction (D- RT-PCR). D-RT-PCR was positive for both viruses when equal multiplicity of infection (MOI) was utilized. Co-infection with different titers between a higher CHIKV titer (MOI = 1.0) than DENV-3 titer (MOI = 0.1) showed similar results with that of equal titer. However, co-infection with a lower CHIKV titer (MOI = 0.1) than DENV-3 titer (MOI = 1.0) revealed positive results for only dengue virus infection, suggesting DENV competitive suppression of CHIKV. This competitive suppression occurred in mixed-infection samples but not in individually double infection (super infection) samples which produced both dengue and chikungunya virus progenies. Both virus replications depend on the amount of virus titer rather than serial infection. These findings have provided information regarding pathogen-pathogen interaction in host cell, which could be used to predict outbreaks, and to develop virus detection and vector control.

Detection of Changuinola virus (Reoviridae: Orbivirus) in field-caught sand flies in southern Thailand.

BACKGROUND: Phlebotomine sand flies are vectors for several pathogenic bacteria, parasites and viruses that have significant impacts on public health. Sand fly-associated viruses that cause diseases in humans and animals have recently received more attention. This study aimed to detect pathogenic viruses belonging to the Orbivirus genus, Phlebovirus genus, Flavivirus genus and family Rhabdoviridae in several field-caught sand fly species in southern Thailand. METHODS: Sand flies were collected in southern Thailand using CDC light traps. Each sample was processed individually for virus screening using RT-PCR and sequencing. RESULTS: Seven out of 60 sand fly samples (two samples of Idiophlebotomus spp., three of Phlebotomus papatasi and two of Sergentomyia khawi) were positive for the Orbivirus genus, which is closely related to Changuinola virus (CGLV). Phlebovirus genus, Flavivirus genus and family Rhabdoviridae were negative in all samples. CONCLUSIONS: CGLV causes Changuinola virus disease or Changuinola fever, a febrile illness in Central and South America. The virus has never been reported in Thailand. This study is the first report of the detection of CGLV in sand flies from Thailand. An extensive study of sand flies from other regions of the country and the associations between sand flies, viruses and vertebrate hosts in Thailand should be undertaken.

Zika virus isolation, propagation, and quantification using multiple methods.

Zika virus (ZIKV) was isolated from the archival urine, serum, and autopsy specimens by intrathoracic inoculation of Toxorhynchitis splendens and followed by three blind sub-passaging in C6/36 mosquito cells. The virus isolates were identified using an immunofluorescence assay and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). This study analyzed 11 ZIKV isolates. One isolate (0.6%) was obtained from 171 urine samples, eight (8.7%) from 92 serum samples and two from tissues of an abortive fetus. After propagation in C6/36 cells, ZIKV was titrated by plaque and focus forming unit (FFU) assays in Vero cell monolayers, and viral genomes were determined via real-time and digital RT-PCR. Plaque and FFU assay quantitations were comparable, with the amount of infectious viruses averaging 106-107 PFU or FFU/ml. Real-time RT-PCR semi-quantified the viral genome numbers, with Ct values varying from 12 to 14. Digital RT-PCR, which precisely determines the numbers of the viral genomes, consistently averaged 10-100 times higher than the number of infectious units. There was good correlation between the results of these titration methods. Therefore, the selection of a method should be based on the objectives of each research studies.

Identification of blood meal of field caught Aedes aegypti (L.) by multiplex PCR.

Laboratory bred female Aedes aegypti (L.) was used to determine sensitivity of multiplex PCR for detecting human blood meal. Human blood DNA was detected in live fully fed mosquitoes until 3 days after blood feeding, and for 4 weeks when stored at -20 degrees C. Among 890 field caught female mosquito samples examined for vertebrate DNA by multiplex PCR, results were positive for human, pig, dog, cow and mixture of 2 host DNA at 86.1, 3.4, 2.1, 1.0 and 3.6%, respectively, while 3.9% of the samples were negative. Blood feeding pattern must be considered when mosquito control strategies become employed.

Field evaluation in Thailand of spinosad, a larvicide derived from Saccharopolyspora spinosa (Actinomycetales) against Aedes aegypti (L.) larvae.

Two formulations of spinosad, direct application tablet (DT) and 0.5% granules (GR), at 3 dosages (0.25, 0.5 and 1.0 mg/l) in 200-liter earthen jars were evaluated against the larvae of Aedes aegypti. Two water regimens were used in the jars: jar full all the time and a full jar in which half the volume of the water was removed and replaced at each assessment interval. All treatments and controls were replicated 4 times and challenged with cohorts of 25 third-instar larvae of Ae. aegypti at weekly intervals during the study. The number of pupal skins (indicating successful emergence of adults) in the treated and control regimens were counted 7 days post-addition and they were used to calculate inhibition of emergence (% IE) based on the original number of larvae used. The DT formulation at the highest concentration (1.0 mg/l) yielded 79-100% IE for 34 days in the full jars, efficacy declining beyond this period. However, the longevity of this dosage was much longer with 90-100% IE for 62 days post-treatment in the water exchange regimen. The target and manufacturer-recommended concentration of 0.5 mg/l of DT gave good control (92-100% IE) for 20 days, declining below 92% IE thereafter in full jars. This dose also yielded good control with IE of 97-100% for 27 days in the water exchange regimen. The 0.5% GR formulation at all 3 dosages showed higher efficacy and greater longevity in the jars than the DT. In the full jars, all 3 dosages produced IE of 76-100% for 55 days post-treatment. In the water exchange regimen, the efficacy and longevity were increased by about one week, up to 62 days post-treatment. It is clear that the DT formulation can be used effectively against Ae. aegypti larvae at a target dose of 0.5 mg/l in 200-liter jars. This dose can be increased to 1.0 mg/l if slightly longer residual activity is desired. In containers where water is consumed and more water added, the longevity of efficacy will be longer for the DT than in jars which remain full all the time. GR (0.5%) gave longer control than DT. GR (0.5%) floated on the surface and produced scum and an oily film, features not desirable in stored water.

Intramolecular integration assay validates integrase phi C31 and R4 potential in a variety of insect cells.

Phage phi C31 and R4 integrases are site-specific and unidirectional serine recombinases. We have analyzed the ability of these integrases to mediate intramolecular integration between their attB and attP sites in 7 important insect cell lines as a means of predicting their relative mobility in the corresponding insect species. Both integrases exhibit significantly higher frequencies in Drosophila S2 cells than in the other insect cell lines examined, but do work well in all of the species tested. Our results, coupled with previous results of the activity of phi C31 integrase in D. melanogaster and Aedes aegypti, suggest the family of serine catalyzed integrases will be useful site-specific integration tools for functional genome analysis and genetic engineering in a wide range of insect species.

Outbreak of chikungunya fever in Thailand and virus detection in field population of vector mosquitoes, Aedes aegypti (L.) and Aedes albopictus Skuse (Diptera: Culicidae).

We investigated chikungunya fever outbreak in the southern part of Thailand. Human plasma specimens obtained from suspected patients and adult wild-caught mosquitoes were detected for chikungunya virus employing reverse transcriptase-polymerase chain reaction technique. Chikungunya virus was detected in about half of the blood specimens whereas a range of 5.5 to 100% relative infection rate was found in both sexes of the vector mosquitoes, Aedes aegypti (L.) and Ae. albopictus Skuse. The infection rate in Ae. albopictus was higher than in Ae. aegypti, with relative infection rate in male of both species being higher than in female. The appearance of chikungunya virus in adult male mosquitoes of both species reveals a role of transovarial transmission of the virus in field population of the mosquito vectors. These findings have provided further understanding of the relationship among mosquito vectors, chikungunya virus and epidemiology of chikungunya fever in Thailand.

The Prevalence of Bartonella Bacteria in Cattle Lice Collected from Three Provinces of Thailand.

Cattle lice are obligatory blood-sucking parasites, which is the cause of animal health problems worldwide. Recently, several studies have revealed that pathogenic bacteria could be found in cattle lice, and it can act as a potential vector for transmitting louse-borne diseases. However, the cattle lice and their pathogenic bacteria in Thailand have never been evaluated. In the present study, we aim to determine the presence of bacterial pathogens in cattle lice collected from three localities of Thailand. Total genomic DNA was extracted from 109 cattle louse samples and the Polymerase Chain Reaction (PCR) of 18S rRNA was developed to identify the cattle louse. Moreover, PCR was used for screening Bartonella spp., Acinetobacter spp., and Rickettsia spp. in cattle louse samples. The positive PCR products were cloned and sequenced. The phylogenetic tree based on the partial 18S rRNA sequences demonstrated that cattle lice species in this study are classified into two groups according to reference sequences; Haematopinus quadripertusus and Haematopinus spp. closely related to H. tuberculatus. The pathogen detection revealed that Bartonella spp. DNA of gltA and rpoB were detected in 25 of 109 samples (22.93%) both egg and adult stages, whereas Acinetobacter spp. and Rickettsia spp. were not detected in all cattle lice DNA samples. The gltA and rpoB sequences showed that the Bartonella spp. DNA was found in both H. quadripertusus and Haematopinus spp. closely related to H. tuberculatus. This study is the first report of the Bartonella spp. detected in cattle lice from Thailand. The finding obtained from this study could be used to determine whether the cattle lice can serve as a potential vector to transmit these pathogenic bacteria among cattle and may affect animal to human health.

Vertical transmission of Zika virus in Culex quinquefasciatus Say and Aedes aegypti (L.) mosquitoes.

Several mosquito species have been described as vectors for the Zika virus (ZIKV), such as those in the Aedes, Anopheles, Mansonia and Culex genera. Our previous survey studies were found the ZIKV RNA positive in both male, female and larvae of Culex quinquefasciatus Say and Aedes aegypti (L.) mosquitoes collected from active ZIKV infected patients' homes in Thailand. Therefore, the aims of this study were to investigate whether ZIKV could be vertically transmitted in Cx. quinquefasciatus, Ae. aegypti and Ae. albopictus. Laboratory and field colonies of these mosquito species were maintained and artificially fed with ZIKV in human blood. Fully engorged mosquitoes (F0) were selected and reared for the vertical transmission study. The subsequent mosquito generations were fed with human blood without the virus. ZIKV in the mosquitoes was detected by hemi-nested RT-PCR and sequencing. C6/36 cells were used to isolate ZIKV from samples that tested positive by hemi-nested RT-PCR. Moreover, ZIKV was identified by immunocytochemical staining 7 days after infection in several organs of infected F0 females, including the salivary glands, midguts, yoke granules and facet cells of the eye. The localization of the ZIKV antigen was identified by the presence of the specific antibody in the salivary glands, midguts, yoke granules and facet cells. ZIKV was detected in female and male Cx. quinquefasciatus until the F6 and F2 generations, respectively. The isolated virus showed cytopathic effects in C6/36 cells by 5 days postinfection. The results suggested that the vertical transmission of ZIKV occurs in Cx. quinquefasciatus in the laboratory. However, we were able to detect the presence of ZIKV in Ae. aegypti in only the F1 generation in both male and female mosquitoes, and Ae. albopictus mosquitoes were not able to vertically transmit the virus at all. Data obtained from this study could be valuable for developing a better understanding of the role of Cx. quinquefasciatus as a potential vector for ZIKV transmission in Thailand and may be useful in creating more effective mosquito vector control strategies in the future.

Synergistic Toxicity of Plant Essential Oils Combined with Pyrethroid Insecticides against Blow Flies and the House Fly.

Blow flies (Diptera: Calliphoridae) and the house fly (Diptera: Muscidae) are filth flies of medical importance, and control of their population is needed. As insecticide applications have resulted in fly resistance, and the exploration of plant essential oils (EOs) has increased against filth flies, this study assessed the combination of EOs with pyrethoids to enhance toxic efficacy. The EOs of five effective plants were screened initially against the house fly (Musca domestica L.). Their chemical constituent was performed using gas chromatography-mass spectrometry (GC-MS) analysis. The main components of Boesenbergia rotunda (Zingiberaceae) rhizome, Curcuma longa (Zingiberaceae) rhizome, Citrus hystrix (Rutaceae) fruit peel, Ocimum gratissimum (Lamiaceae) seed, and Zanthoxylum limonella (Rutaceae) fruit were δ-3-caren (35.25%), ß-turmerone (51.68%), ß-pinene (26.56%), p-cumic aldehyde (58.21%), and dipentene (60.22%), respectively. The screening test revealed that the three most effective plant EOs were from B. rotunda, C. longa and O. gratissimum, which were selected for the combination with two pyrethroid insecticides (permethrin and deltamethrin), in order to enhance their synergistic efficacy against the blow flies, Chrysomya megacephala Fabricius, Chrysomya rufifacies Macquart, and Lucilia cuprina Wiedemann, and the house fly. Synergistic action was presented in almost all of the flies tested with permenthrin/deltamethrin/EOs mixtures. It was interesting that the combination of deltamethrin with three EOs showed a synergistic effect on all of the tested flies. However, an antagonistic effect was observed in C. megacephala and M. domestica treated with permethrin-B. rotunda and C. megacephala treated with permethrin-O. gratissimum. The LD50 of insecticides decreased when combined with plant EOs. This alternative strategy will be helpful in developing a formula for effective fly control management.

Larvicidal efficacy of new formulations of temephos in non-woven sachets against larvae of Aedes aegypti (L.) (Diptera: Culicidae) in water-storage containers.

Three new formulations of temephos (LAVIFOS SG 1%, MOSQ SG 1% and AZAI-SS ZG 1%) were evaluated for larvicidal efficacy against larvae of Aedes aegypti (L.) in water-storage jars under field-simulated conditions. LAVIFOS SG 1% and MOSQ SG 1% are sand granule formulations, whereas AZAI-SS ZG 1% is zeolite granule formulation. Each formulation contained 1% temephos as an active ingredient. Each formulation was packed in a non-woven sachet at quantity of 20 g per sachet and placed in a 200-liter glazed clay jar to obtain a dosage of 1 mg/l (one sachet per jar). Each treatment and control (jar without larvicide) was replicated four times. A concurrent set of treatments and controls were carried out in parallel, but the water in each treated and control jars was removed and refilled weekly. All jars (treatment and control) were challenged weekly by adding 25 third-instar larvae per jar and assessment was made of larval mortality by counting pupal skins one week after the addition of larvae. The three formulations provided complete larvicidal efficacy (100%) for at least 24 weeks post-treatment (the length of this study). In the jars where all the water was removed and refilled weekly, LAVIFOS SG 1%, and MOSQ SG 1% provided complete larvicidal efficacy for at least 24 weeks post-treatment, whereas AZAI-SS ZG 1% showed complete larvicidal efficacy for 16 weeks post-treatment. AZAI-SS ZG 1% still demonstrated a high degree of larvicidal activity (93-99%) from 17 to 24 weeks post-treatment. The present study reveals an excellent residual efficacy of the three new formulations of temephos against larvae of Aedes aegypti in water-storage jars lasting for at least 16 to 24 weeks post-treatment. These new formulations will make the control of DHF vectors in Thailand more cost effective as they are removable and retrievable sachets that can be reused after cleaning the water-storage containers.

Simulated field evaluation of the efficacy of two formulations of diflubenzuron, a chitin synthesis inhibitor against larvae of Aedes aegypti (L.) (Diptera: Culicidae) in water-storage containers.

Tablet (40 mg a.i./tablet) and granular (2% a.i.) formulations of diflubenzuron, a chitin synthesis inhibitor, insect growth regulator, were evaluated for larvicidal efficacy against the larvae of Aedes aegypti (L.) in water-storage containers under field conditions in Thailand. Each formulation was applied to 200-1 clay jars at 5 different dosages (0.02, 0.05, 0.1, 0.5 and 1 mg/l a.i.). The jars were covered with solid celocrete sheets and placed in the shade under a roof. Another experiment was also carried out using 3 different dosages (0.1, 0.5 and 1 mg/l) where half the water in each treated jar and the control was removed and refilled weekly. Each treatment was replicated four times. The treatments were challenged by adding 25 3rd instar larvae/jar weekly. Assessments were made of each treatment through emergence inhibition (%EI) by removing and counting pupal skins one week after larval addition. Using these assessment techniques, a high degree of larvicidal efficacy (96-100%EI) was achieved with 4 dosages (0.05, 0.1, 0.5 and 1 mg/l) of both (tablet and granular) formulations for a period of 23 weeks post-treatment. The efficacy of the lowest dosage (0.02 mg/l) of tablet and granular formulations lasted for 21 and 22 weeks post-treatment, respectively. Under the conditions of water removal and weekly refilling, a high degree of larvicidal efficacy (96-100%El) at the 3 dosages was obtained with the tablet formulation 18 to 21 weeks post-treatment, whereas the efficacy of the granular formulation persisted 15 to 23 weeks post-treatment depending on the dosage. This study clearly demonstrates a high level of residual activity with both formulations of diflubenzuron against larvae of Ae. aegypti in water-storage containers. Considering environmental factors and water-use conditions, it is likely that dosages of 0.05 to 0.1 mg a.i./l are effective dosages providing long-lasting control for 3 to 4 months in the field.

Field evaluation of novaluron, a chitin synthesis inhibitor larvicide, against mosquito larvae in polluted water in urban areas of Bangkok, Thailand.

Novaluron, an insect growth regulator, a benzoylphenyl urea insecticide, was evaluated in the field against the larvae of polluted-water mosquitoes. The study was carried out in highly polluted sites infested with populations of mosquito larvae, mostly Culex quinquefasciatus Say, in low-income communities in urban areas of Bangkok, Thailand. An EC10 formulation was premixed in water and applied by pressurized spray tank to plots ranging from 180 to 1,000 m2 at the rate of 0.1 ml EC 10/m2 (equal to 10 mg a.i./m2) of the breeding sites. Assessments were made by sampling mosquito larvae and pupae to determine the trends of immature populations before treatment and weekly after treatment. Reduction of the populations in percents were then computed by comparing counts of immature mosquitoes (larvae and pupae) to the pretreatment counts at each particular site. It was found that the immature populations of mosquitoes in the treated areas were dramatically suppressed and remained at extremely low levels for 3-7 weeks after the treatment depending on the prevailing conditions of each experimental site. No negative impact on fishes or aquatic plants in the treated areas were detected during the study period and three months after the experiment was discontinued. Novaluron is an effective agent to control immature populations of polluted-water mosquitoes, especially Cx. quinquefasciatus in habitats in urban areas. This IGR larvicide may play an important role in vector control programs in terms of effectiveness, environmental friendliness and strategies for insecticide-resistance management in vector mosquitoes.

Repellent activity of essential oils against cockroaches (Dictyoptera: Blattidae, Blattellidae, and Blaberidae) in Thailand.

Seven commercial essential oils extracted from the plant species Boesenbergia rotunda (L.) Mansf., Citrus hystrix DC., Curcuma longa L., Litsea cubeba (Lour.) Pers., Piper nigrum L., Psidium guajava L. and Zingiber officinale Roscoe, and naphthalene as a control, were evaluated for repellent activity against the three cockroach species Periplaneta americana (L.), Blattella germanica (L.) and Neostylopyga rhombifolia (Stoll) under laboratory conditions. The essential oil derived from Citrus hystrix showed the best repellency over other candidate essential oils and naphthalene. The essential oil of Citrus hystrix exhibited complete repellency (100%) against P. americana and B. germanica, and also showed the highest repellency (among the essential oils tested) of about 87.5% against N. rhombifolia under laboratory conditions. In the field, Citrus hystrix essential oil formulated as a 20% active ingredient in ethanol and some additives provided satisfactory repellency of up to 86% reduction in cockroaches, mostly P. americana and N. rhombifolia with a residual effect lasting a week after treatment. Citrus hystrix essential oil has good potential for being used as a cockroach repellent. Further improvements in efficacy and residual activity may be realized with appropriate formulations.

Detection of an Unknown Trypanosoma DNA in a Phlebotomus stantoni (Diptera: Psychodidae) Collected From Southern Thailand and Records of New Sand Flies With Reinstatement of Sergentomyia hivernus Raynal & Gaschen, 1935 (Diptera: Psychodidae).

Although female sand flies are best known as the vectors of Leishmania parasites and viruses, several previous reports have demonstrated that these insects can also act as vectors for the trypanosomes of bats, lizards, and snakes. In this report, we created an inventory of Phlebotomine sand flies from southern Thailand. A novel trypanosome was found in a specimen of Phlebotomus stantoni, and two sand fly species newly recorded in the country, Sergentomyia khawi and Sergentomyia hivernus, were described. PCR primer pairs specific for the internal transcribed spacer 1 (ITS1) and the small subunit ribosomal DNA (SSU rDNA) gene of trypanosomatids were used to demonstrate the presence of the parasite in the sand fly. In addition, the Cytochrome b (CytB) gene was used to identify the sand fly species. Among the 45 samples of the sand fly that were collected, seven samples were Ph. stantoni sand flies and a single sample was positive for Trypanosoma sp. through PCR analysis. This study represents the first detection of Trypanosoma sp. in a sand fly from Thailand. The ITS1 and SSU rDNA sequences indicated that this specimen is suspected to be a novel Trypanosoma species. Further studies of this suspected new Trypanosoma species, including its vertebrate hosts and pathogenic potential, are therefore necessary.