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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.

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.

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.

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.

Aedes aegypti (L.) and Ae. albopictus (Skuse) are accepted as primary vectors of mosquito-borne diseases, such as Dengue Haemorrhagic Fever (DHF), chikungunya fever and Zika fever.

Sound waves are physical energy caused by vibrating material. The energy of the vibrator is transferred to particles of the medium that are exposed to sound.

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 &delta, 3-caren (35.25%), &beta, turmerone (51.68%), &beta, 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.

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.

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.

Tabanus rubidus is one of the most medically and veterinary important haematophagous insects. We demonstrated the profile of the Internal Transcribed Spacer 2 (ITS2) of ribosomal DNA in T. rubidus collected from various regions of Thailand and Myanmar. The ITS2 sequences in this study is high in A+T content (80.1%) and ranged from 589 to 622 bp. The highest intra and interspecific divergence among samples from same region was 1.2% and 11.2%, respectively. The sequences from this study were grouped by the same collecting region into individual clade using the Neighbor Joining with Kimura 2-Parameter tree. The data obtained in this study is useful for accurate species identification using ITS2 sequence which is the first step to provide the biological data for better integrated vector management programs for surra disease.

BACKGROUND: Bed bugs [both Cimex hemipterus (F.) and Cimex lectularius L.] are highly resistant to pyrethroids worldwide. An important resistance mechanism known as ‘knockdown resistance’ (kdr) is caused by genetic point mutations on the voltage-gated sodium channel (VGSC) gene. Previous studies have identified two point mutations (V419L and L925I) on the VGSC gene in C. lectularius that are responsible for kdr-type resistance. However, the kdr mutations in C. hemipterus have not been investigated. RESULTS: Four novel mutations, L899V (leucine to valine), M918I (methionine to isoleucine), D953G (asparticacid to glycine) and L1014F (leucine to phenylalanine), were identified in the domain II region of the C. hemipterus VGSC gene. This region has been widelyinvestigatedforthestudyofkdr-typeresistancetopyrethroidsinotherinsectpests.TheV419LandL925Ikdr mutations as previously identified in C.lectulariuswere not detected in C.hemipterus. CONCLUSION:M918IandL1014Fareconsideredtobeprobablekdrmutationsandmayplayessentialrolesinkdr-typeresistance to pyrethroids in C. hemipterus. Further studies are under way in the authors’ laboratory to determine the non-kdr-type resistance mechanisms in C.hemipterus. ©2 014 Society of Chemical Industry

Trikatu is composed of dried fruits of Piper nigrum L and Piper retrofractum Vahl, and dried rhizomes of Zingiber officinale R. Although this preparation has been used to relieve pruritis, pain, and inflammation for a long time, there is no clinical evidence to confirm its efficacy and safety. Therefore, we performed a double-blind, within person-randomized controlled study of 30 healthy volunteers to determine efficacy and safety of topical Trikatu on mosquito bite reactions.All subjects were bitten by Aedes aegypti laboratory mosquitoes on their forearms and they were randomly assigned arms to apply either Trikatu or reference product on the mosquito bite papule. The main outcome was the difference of papule size reduction at 30 min, measured by a caliper, between the Trikatu and reference arms. Pruritis, redness, pain, and patient satisfaction were assessed at 15, 30, 60, 180, and 360 min as secondary outcomes.There were no significant differences between treatment and reference arms on any outcome at any time of measurement.Trikatu did not show additional effects for relieving mosquito bite reaction as compared with the reference product containing camphor, menthol, and eucalyptus. For further study, it is very important to consider a proper selection of subjects, comparator product, and concentration of extract when Trikatu preparation is investigated.

SUMMARYInfection with dengue, the most prevalent mosquito-borne virus, manifests as dengue fever (DF) or the more fatal dengue haemorrhagic fever (DHF). DHF occurs mainly when an individual who has acquired antibodies to one serotype is inoculated with another serotype. It was reported that mosquito control may have increased the incidence of DF and DHF due to age-dependency in manifesting these illnesses or an immunological mechanism. Tetravalent dengue vaccine is currently being tested in clinical trials. However, seroconversions to all four serotypes were achieved only after three doses. Therefore, vaccines may predispose vaccinees to the risk of developing DHF in future infections. This study employed an individual-based computer simulation, to emulate mosquito control and vaccination, incorporating seroconversion rates reported from actual clinical trials. It was found that mosquito control alone would have increased incidence of DF and DHF in areas of high mosquito density. A vaccination programme with very high coverage, even with a vaccine of suboptimal seroconversion rates, attenuated possible surges in the incidence of DF and DHF which would have been caused by insufficient reduction in mosquito abundance. DHF cases attributable to vaccine-derived enhancement were fewer than DHF cases prevented by a vaccine with considerably high (although not perfect) seroconversion rates. These predictions may justify vaccination programmes, at least in areas of high mosquito abundance. In such areas, mosquito control programmes should be conducted only after the vaccination programme with a high coverage has been initiated.

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.

Background The re-emergence of chikungunya (CHIK) fever in Thailand has been caused by a novel lineage of chikungunya virus (CHIKV) termed the Indian Ocean Lineage (IOL). The Aedes albopictus mosquito is thought to be a primary vector of CHIK fever in Thailand, whereas Ae. aegypti acts as a secondary vector of the virus. The vertical transmission is believed to be a primary means to maintain CHIKV in nature and may be associated with an increased risk of outbreak. Therefore, the goal of this study was to analyze the potential of these two Thai mosquito species to transmit the virus vertically and to determine the number of successive mosquito generations for the virus transmission. Methods Two-hundred-and-fifty female Ae. aegypti and Ae. albopictus mosquitoes were artificially fed a mixture of human blood and CHIKV IOL. Mosquito larvae and adults were sampled and screened for CHIKV by one-step qRT-PCR. LLC-MK2 cell line was used to isolate CHIKV in the mosquitoes each generation. The virus isolate was identified by immunocytochemical staining and was confirmed by sequencing. Both mosquito species fed on human blood without CHIKV and uninfected LLC-MK2 cells were used as controls. Results Aedes aegypti and Ae. albopictus mosquitoes were able to transmit CHIKV vertically to F5 and F6 progenies, respectively. The virus isolated from the two mosquito species caused cytopathic effect in LLC-MK2 cells by 2 days post-infection and immunocytochemical staining showed the reaction between CHIKV IOL antigen and specific monoclonal antibody in the infected cells. DNA sequence confirmed the virus transmitted vertically as CHIKV IOL with E1-A226V mutation. No CHIKV infection was observed in both mosquito species and LLC-MK2 cells from control groups. Conclusions The study demonstrated that Ae. aegypti and Ae. albopictus mosquitoes from Thailand are capable of transmitting CHIKV IOL vertically in the laboratory. Our results showed that Ae. albopictus is more susceptible and has a greater ability to transmit the virus vertically than Ae. aegypti. This knowledge would be useful for risk assessments of the maintenance of CHIKV in nature, which is crucial for disease surveillance, vector control and the prevention of potential CHIKV epidemics.

SUMMARYDengue haemorrhagic fever (DHF) is caused by dengue virus transmitted by Aedes mosquitoes; mean age of patients varies temporally and geographically. Variability in age of patients may be due to differences in transmission intensity or demographic structure. To compare these two hypotheses, the mean age of DHF patients from 90 districts in northern Thailand (1994–1996, 2002–2004) was regressed against (i) Aedes abundance or (ii) demographic variables (birthrate, average age) of the district. We also developed software to quantify direction and strength of geographical gradients of these variables. We found that, after adjusting for socioeconomics, climate, spatial autocorrelation, the mean age of patients was correlated only with Aedes abundance. The geographical gradient of mean age of patients originated from entomological, climate, and socioeconomic gradients. Vector abundance was a stronger determinant of mean age of patients than demographic variables, in northern Thailand.

SUMMARYExpansion of dengue has been attributed to urbanization. To test this concept, we examined dengue transmission intensities in Thailand. We used the inverse of mean age of dengue haemorrhagic fever (DHF) cases as a surrogate of dengue transmission intensity (or force of infection). The transmission intensity in Bangkok decreased rapidly since the mid-1990s, to levels that are currently lower than in other regions. Regression analysis revealed that transmission intensity is highest in the Northeastern rural region, mainly due to scarcity of private water wells. Private wells reduce the need for household water containers, the major breeding sites for vectors. Cumulatively, these results show that urbanization is not necessarily associated with intense dengue transmission in Thailand. Paradoxically, the DHF incidence in Bangkok has surpassed other regions despite declines in transmission intensity. This finding implies the existence of endemic stability (i.e. low incidence of a clinical illness in spite of high transmission intensity).