Exsheathment and midgut invasion of nocturnally subperiodic Brugia malayi microfilariae in a refractory vector, Aedes aegypti (Thailand strain).

Exsheathment and midgut invasion of nocturnally subperiodic Brugia malayi microfilariae were analyzed using light and scanning electron microscopy in a refractory vector, Aedes aegypti (Thailand strain). Results showed that exsheathed microfilariae represented only approximately 1% of the total microfilaria midguts dissected at 5-min post-infected blood meal (PIBM). The percentage of exsheathed microfilariae found in midguts progressively increased to about 20, 60, 80, 90, and 100% at 1-, 2-5-, 6-12-, 18-36-, and 48-h PIBM, respectively. Importantly, all the microfilariae penetrating the mosquito midguts were exsheathed. Midgut invasion by the exsheathed microfilariae was observed between 2- and 48-h PIBM. SEM analysis revealed sheathed microfilariae surrounded by small particles and maceration of the microfilarial sheath in the midguts, suggesting that the midguts of the refractory mosquitoes might have protein(s) and/or enzyme(s) and/or factor(s) that induce and/or accelerate exsheathment. The microfilariae penetrated the internal face of the peritrophic matrix (PM) by their anterior part and then the midgut epithelium, before entering the hemocoel suggesting that PM was not a barrier against the microfilariae migrating towards the midgut. Melanized microfilariae were discovered in the hemocoel examined at 96-h PIBM suggesting that the refractory mosquitoes used melanization reactions against this parasite. This study provided evidence that A. aegypti (Thailand strain) has refractory mechanisms against B. malayi in both midgut and hemocoel.

Cytogenetic, crossing and molecular evidence of two cytological forms of Anopheles argyropus and three cytological forms of Anopheles pursati (Diptera: Culicidae) in Thailand.

Nine and 11 isolines of Anopheles argyropus and Anopheles pursati, respectively, were established from individual females collected from cow-baited traps, and the characteristics of metaphase chromosomes were investigated in their F1-progenies. As determined by the different amounts of extra heterochromatin on sex chromosomes, 2 types of X (X1, X2) and Y (Y1,Y2), and 2 types of X (X1, X2) and 3 types of Y (Y1, Y2, Y3) chromosomes were obtained from An. argyropus and An. pursati, respectively. These types of sex chromosomes comprised 2 [Forms A (X1, Y1) and B (X1, X2, Y2)] and 3 [Forms A (X1, X2, Y1), B (X1, X2, Y2) and C (X2, Y3)] karyotypic forms of An. argyropus and An. pursati, respectively. All karyotypic forms acquired from An. pursati are new one that were discovered in this study, of which Forms A, B and C were found generally in Chiang Mai Province, while only 1 isoline of Form B was obtained in Ratchaburi Province. Form A was recovered from An. argyropus only in Ubon Ratchathani Province, whereas Form B from that species was found commonly in both Ubon Rathchathani and Nakhon Si Thammarat Provinces. Crossing experiments among the 2 and 3 isolines representing 2 and 3 karyotypic forms of An. argyropus and An. pursati, respectively, indicated genetic compatibility in yielding viable progenies and synaptic salivary gland polytene chromosomes through F2-generations. The conspecific natures of these karyotypic forms in both species were further supported by very low intraspecific sequence variations (average genetic distance: An. argyropus = 0.003-0.007, An. pursati = 0-0.005) of ribosomal DNA (ITS2) and mitochondrial DNA (COI and COII).

Morphological and protein analyses of adult female salivary glands of Anopheles barbirostris species A1 (Diptera: Culicidae).

Morphology and protein profiles of female salivary glands of Anopheles barbirostris species A1 were analyzed. Female glands consisted of a distinctive tri-lobed structure connected to a main salivary canal, a single medial and two lateral lobes with proximal and distal portions. Cellular architecture was similar among the lobes, with secretory material appearing as large masses. Cells of the proximal-lateral lobes contained secretory masses with a finely filamentous aspect. In the distal-lateral lobes, cells had a dense secretory product with mottled pattern. Cells of the medial lobe had secretory masses which were uniformly stained and highly electron dense. Following emergence, the glands accumulated secretory material rapidly and developed completely within three days. Degenerative changes including loss of stored secretion and increase of cytoplasmic vacuolation and concentric lamellar structures were observed from day 16 post emergence that correlated with total amount of the salivary gland proteins determined during development. SDS-PAGE, nanoLC-MS, and glycoprotein analysis revealed at least eleven major protein bands, of which each morphological region contained different major proteins. Two glycoproteins, apyrase/5'-nucleotidase and D7, were identified. These results form a basis for further studies on details of cytopathological changes of malarial infected glands and roles of the proteins in disease transmission.

Cytogenetic, crossing and molecular evidence of two cytological forms of Anopheles argyropus and three cytological forms of Anopheles pursati (Diptera: Culicidae) in Thailand

Nine and 11 isolines of Anopheles argyropus and Anopheles pursati, respectively, were established from individual females collected from cow-baited traps, and the characteristics of metaphase chromosomes were investigated in their F1-progenies. As determined by the different amounts of extra heterochromatin on sex chromosomes, 2 types of X (X1, X2) and Y (Y1,Y2), and 2 types of X (X1, X2) and 3 types of Y (Y1, Y2, Y3) chromosomes were obtained from An. argyropus and An. pursati, respectively. These types of sex chromosomes comprised 2 [Forms A (X1, Y1) and B (X1, X2, Y2)] and 3 [Forms A (X1, X2, Y1), B (X1, X2, Y2) and C (X2, Y3)] karyotypic forms of An. argyropus and An. pursati, respectively. All karyotypic forms acquired from An. pursati are new one that were discovered in this study, of which Forms A, B and C were found generally in Chiang Mai Province, while only 1 isoline of Form B was obtained in Ratchaburi Province. Form A was recovered from An. argyropus only in Ubon Ratchathani Province, whereas Form B from that species was found commonly in both Ubon Rathchathani and Nakhon Si Thammarat Provinces. Crossing experiments among the 2 and 3 isolines representing 2 and 3 karyotypic forms of An. argyropus and An. pursati, respectively, indicated genetic compatibility in yielding viable progenies and synaptic salivary gland polytene chromosomes through F2-generations. The conspecific natures of these karyotypic forms in both species were further supported by very low intraspecific sequence variations (average genetic distance: An. argyropus = 0.003-0.007, An. pursati = 0-0.005) of ribosomal DNA (ITS2) and mitochondrial DNA (COI and COII).

Metaphase karyotypes of Anopheles paraliae (Diptera:Culicidae) in Thailand and evidence to support five cytological races.

Sixteen isoline colonies of Anopheles paraliae were established from wild-caught females collected from cow-baited traps at 4 locations in Thailand. They showed 3 types of X (X1, X2, X3) and 5 types of Y (Y1, Y2, Y3, Y4, Y5) chromosomes based on the number and amount of major block(s) of heterochromatin present in the heterochromatic arm, and were designated as Forms A (X3, Y1), B (X1, X2, X3, Y2), C (X3, Y3), D (X1, X2, X3, Y4) and E (X3, Y5). Form A was found in Songkhla Province, Form B was obtained in Ratchaburi, Nakhon Si Thammarat and Songkhla Provinces, Form C was acquired in Chanthaburi Province, Form D was recovered in Ratchaburi and Songkhla Provinces, and Form E was encountered in Ratchaburi Province. Hybridization experiments among the 7 isoline colonies, which represented the 5 karyotypic forms of An. paraliae, revealed genetic compatibility in providing viable progenies and synaptic salivary gland polytene chromosomes through F2-generations, and thus suggest the conspecific nature of these karyotypic forms. These results were supported by the very low intraspecific sequence divergence (mean genetic distance = 0.000-0.002) of the nucleotide sequences in ribosomal DNA (ITS2) and mitochondrial DNA (COI and COII) of the 5 forms.

Relative developmental and reproductive fitness associated with F1534C homozygous knockdown resistant gene in Aedes aegypti from Thailand.

The effect of permethrin resistance, conferred by a homozygous mutation (F1534C) in the voltage-gated sodium channel protein, upon the reproductive fitness of Aedes aegypti (PMD-R strain) from Thailand was evaluated by comparing with a pyrethroid-susceptible sub colony (PMD strain). The parameters evaluated included larval development time, pupation success, adult emergence, egg production and hatchability, mating ability, female wing length and adult longevity. Larval development times were similar with very low mortality of larvae, pupae and emerging adults among either strain. However, PMD produced significantly fewer females than PMD-R. The mean numbers of eggs laid by PMD (54.2±15.9) and PMD-R (54.6±14.5) strains were not significantly different but the hatchability of PMD eggs (53.7%) was lower than PMD-R eggs (71.2%). The mean wing length of PMD females (2.85±0.15 mm) was longer than PMD-R females (2.74±0.09 mm). The insemination rates for both strains were 100%. The longevity of both strains was mostly not significantly different, over 90% of both sexes surviving at day 30. Our results suggest that the presence of the homozygous F1534C mutation does not lead to fitness reductions. This is in accordance with the high frequency of this allele found among wild populations of Ae. aegypti in many countries. These results also suggest that the removal of pyrethroid insecticide selection pressure may not lead to a regression of 1534C alleles in pyrethroid resistant Ae. aegypti.

Geographic distribution and genetic compatibility among six karyotypic forms of Anopheles peditaeniatus (Diptera: Culicidae) in Thailand.

Fifty-three isolines of Anopheles peditaeniatus were established from individual wild-caught females collected from cow-baited traps in 17 provinces of Thailand. Three types of X (X1, X2, X3) and 6 types of Y (Y1,Y2, Y3, Y4, Y5, Y6) chromosomes were determined based on different amounts of major block(s) of heterochromatin. These sex chromosomes comprised 6 karyotypic forms designated as Forms A (X3, Y1), B (X1, X2, X3, Y2), C (X3, Y3), D (X1, X2, X3, Y4), E (X1, X2, X3,Y5) and F (X2, X3, Y6). Form F is a new metaphase karyotype discovered in this study and is commonly found in all regions. Form A was found only in Lampang province, whereas Form E is widespread throughout the country. Forms B, C and D were obtained from the northern, northeastern, western and southern regions. Crossing experiments among the 11 isoline colonies representing the 6 karyotypic forms of An. peditaeniatus indicated genetic compatibility yielding viable progenies and complete synapsis of salivary gland polytene chromosomes through to the F2-generations. The results suggested the conspecific nature of these karyotypic forms which were further supported by very low intraspecific variation (genetic distance = 0.000-0.003) of nucleotide sequences in ribosomal DNA (ITS2) and mitochondrial DNA (COI and COII).

Simulium (Nevermannia) chomthongense, a new species of black fly (Diptera: Simuliidae) from Chiang Mai, Thailand.

Simulium (Nevermannia) chomthongense sp. nov. is described from female, male, pupal and larval specimens collected from Doi Inthanon National Park and Doi Phahompok National Park, Chiang Mai, Thailand. This new species, first reported as S. (Eusimulium) sp. A, and later regarded as S. (N.) caudisclerum Takaoka & Davies, described from peninsular Malaysia, is distinguished from S. (N.) caudisclerum in the male by the number of enlarged upper-eye facets and the relative size of the hind basitarsus against the hind tibia and femur, and in the pupa by the relative length of the stalks of paired filaments against the common basal stalk and the color of the dorsal surface of abdominal segments 1- 3 (or 4). Taxonomic and molecular notes are provided to separate this new species from four other known species of the vernum species-group, which share an accessory sclerite on the larval abdomen, a rare characteristic in this species-group.

The impact of a national program to eliminate lymphatic filariasis in selected Myanmar immigrant communities in Bangkok and Ranong Province, Thailand.

Some immigrants from Myanmar to Thailand have brought Wuchereria bancrofti infections with them, causing a community health problem for Thai citizens. The seroprevalence of bancroftian filariasis was detected in 438 and 512 Myanmar immigrants residing in Bangkok and Ranong Provinces, respectively, along with 81 Thai citizens living in Bangkok. The immunochromatograpy card test was positive in 5 Myanmar immigrants living in Bangkok and 1 living in Ranong for a prevalence of 0.63%. Antifilarial IgG4 antibodies were found in 21 Myanmar immigrants living in Bangkok and 14 living in Ranong for a prevalence of 3.68%. None of the samples from Thai citizens were positive with either test. These prevalence rates are lower than those observed between 2001 and 2005. The Thai mass drug administration program to eliminate lymphatic filariasis among Myanmar immigrants appears to be a successful public health strategy.

Multiplex assay to identify Korean vectors of malaria.

Following the recent emergence of malaria in South Korea, vector control has been an important task. For this, vector identification is very important. Earlier, two PCR-based assays have been described. But, poor species resolution and their ability to include only 4-5 species limit their use. Thus, it has now become important to revise the assay identifying these members. In this study, a new assay based on internal transcribed spacer 2 and 28S of ribosomal DNA has been described. The assay successfully identified all the Korean malaria vector mosquitoes. Therefore, it is an indispensable tool to study ecology, abundance and biology of these species.

Field evaluation of G10, a celery (Apium graveolens)-based topical repellent, against mosquitoes (Diptera: Culicidae) in Chiang Mai province, northern Thailand.

The potential of G10, a celery (Apium graveolens)-based topical product, as a repellent against natural mosquito populations was evaluated in comparison to commercial (Insect Block 28) and standard (25% DEET) repellents in Chiang Mai province, northern Thailand. These repellent products afforded encouragingly excellent personal protection against a broad range of mosquito species belonging to various genera, including Aedes, Anopheles, Armigeres, Culex, and Mansonia. No mosquito bite was observed on the volunteers treated with G10 and Insect Block 28 throughout the field study, whereas two species, i.e., six A. barbirostris and two A. subalbatus, came to bite or land on 25% DEET-treated volunteers. Thus, it can be concluded that while G10 and Insect Block 28 exhibited similarly powerful repellent activities with complete (100%) protection, 25% DEET was effective in minimizing bites with 99.68% protection. G10 formula was also studied for physical properties and biological stability after being kept under two conditions; a heating and cooling cycle, and varying temperature and time storage. Most samples of stored G10 not only demonstrated a similarity in appearance and physical properties, but also provided comparable repellency to that of the fresh preparation. These findings encourage commercial development of G10 formula as an alternative to conventional synthetic repellents.

Celery-based topical repellents as a potential natural alternative for personal protection against mosquitoes.

Celery-based products were investigated for chemical composition, skin irritation, and mosquito repellency in comparison to commercial repellents and the standard chemical, N,N-diethyl-3-methylbenzamide (DEET), with a goal to develop a natural alternative to synthetic repellents for protection against mosquitoes. Chemical identification by gas chromatography coupled with mass spectrometry discovered that the major constituents of Apium graveolens hexane extract (AHE) were 3-n-butyl-tetrahydrophthalide (92.48%), followed by 5.10% beta-selinene and 0.68% gamma-selinene. Evaluation of skin irritation in 27 human volunteers revealed no irritant potential from 25% ethanolic AHE solution. Laboratory investigated repellent against female Aedes aegypti mosquitoes demonstrated that G10 formula, the best AHE-developed product, provided remarkable repellency with a median protection time of 4.5 h (4.5-5 h), which was greater than that of ethanolic DEET solution (25% DEET, 3.5 h) and comparable to that of the best commercial repellent, Insect Block 28 (28.5% DEET, 4.5 h). According to significantly promising results, including highly effective repellency and no potential skin irritation or other side effects, the G10 formula is a worthwhile product that has the promise of being developed for commercialized registration. This developed AHE product could be an acceptable and affordable alternative to conventional synthetic chemicals in preventing mosquito bites, and in turn, helping to interrupt mosquito-borne disease transmission.

Seasonal and diurnal biting activities and zoonotic filarial infections of two Simulium species (Diptera: Simuliidae) in northern Thailand.

Seasonal and daily biting activity patterns, and natural filarial infections of adult black flies attracted to human bait were investigated at Ban Pang Faen, a rural area in Chiang Mai Province in northern Thailand. Collections were carried out twice a month from 0600 to 18-00 hours from January 2005 to February 2006. Among ten Simulium species collected, S. nodosum and S. asakoae were predominant occupying 57.3% and 37.2% of the total 16,553 females, respectively. These two predominant species showed different patterns in seasonal abundance: majority of S. nodosum (86.7%) were collected in hot season (from mid February to mid May), while most of S. asakoae (74.5%) were collected in rainy season (from mid May to mid October). For the daily biting activity, S. nodosum had two patterns: the main one was unimodal with a peak from 17-00 to 18-00, and the other was bimodal and had the major peak from 1600 to 18-00 and the minor one from 07-00 to 09-00. The pattern of S. asakoae was mostly unimodal with a peak from 06-00 to 10-00. The filarial larvae found in S. nodosum and S. asakoae were morphologically different from each other. The short and thick infective larvae found in S. asakoae differed from all known filarial larvae; it is suggested that they might be a bird parasite, Splendidofilariinae or Lemdaninae. The infection of the mammophilic S. nodosum with large Onchocerca type infective larvae was confirmed in this area. Natural filarial infections were found in each month (except December) in either S. nodosum or S. asakoae or in both. Monthly infection rates with all stages of larvae were 0.6-5.0% for S. nodosum, and 1.0-4.0% for S. asakoae. It is suggested that people in this village are exposed to the risk of infection with zoonotic filariae throughout the year.

Repellent activity of selected essential oils against Aedes aegypti.

Essential oils extracted from ten plant species were screened for repellency against Aedes aegypti mosquitoes. Three oils; Zanthoxylum piperitum, Anethum graveolens and Kaempferia galanga, exerted protection against A. aegypti, with median complete-protection times of 1, 0.5 and 0.25 h, respectively. The protection times were increased significantly by incorporating 10% vanillin. The highest potential was established from Z. piperitum oil +10% vanillin (2.5 h, range=1-2.5 h). Mixtures from pairs of the effective oils possessed slight repellency that ranged from 0-0.5 h. None of the oil combinations repelled A. aegypti for longer than their constituent oil alone. With vanillin added, however, each oil mixture provided improved protection, which was approximately equal to oil on its own. GC/MS analysis revealed that the main component of Z. piperitum fruit oil was limonene (37.99%), with minor amounts of sabinene (13.30%) and beta-myrcene (7.17%). Repellent testing of stored samples of Z. piperitum fruit oil against A. aegypti demonstrated that repellent activity of those kept at -20 degrees C or 4 degrees C was present for a period of at least 3 months. Therefore, the essential oil of Z. piperitum fruit may prove useful in the development of mosquito repellents as an effective personal protection measure against mosquito bites.

Aromatic plant-derived essential oil: an alternative larvicide for mosquito control.

Five aromatic plants, Carum carvi (caraway), Apium graveolens (celery), Foeniculum vulgare (fennel), Zanthoxylum limonella (mullilam) and Curcuma zedoaria (zedoary) were selected for investigating larvicidal potential against mosquito vectors. Two laboratory-reared mosquito species, Anopheles dirus, the major malaria vector in Thailand, and Aedes aegypti, the main vector of dengue and dengue hemorrhagic fever in urban areas, were used. All of the volatile oils exerted significant larvicidal activity against the two mosquito species after 24-h exposure. Essential oil from mullilam was the most effective against the larvae of A. aegypti, while A. dirus larvae showed the highest susceptibility to zedoary oil.

Effectiveness of Zanthoxylum piperitum-derived essential oil as an alternative repellent under laboratory and field applications.

Recently, there were considerable efforts made to promote the use of environmentally friendly and biodegradable natural insecticides and repellents, particularly from botanical sources. In this study, Zanthoxylum piperitum-derived essential oil isolated by steam distillation was investigated and compared to the standard synthetic repellent, N,N-diethyl-3-methylbenzamide (DEET), for repellency against mosquitoes under laboratory and field conditions. The oil of Z. piperitum alone and also with 5% vanillin added repelled laboratory-reared female Aedes aegypti with the median protection times of 1.5 and 2.5 h, respectively. These repellency times were proven to be significantly lower than those of DEET-based products: 25% DEET and 25% DEET + 5% vanillin demonstrating 3.5 and 5.5 h, respectively. However, when applied under field conditions, Z. piperitum oil + 5% vanillin was found to provide better protection against a wide range of natural mosquito populations than 25% DEET + 5% vanillin. While the product of DEET was effective in reducing bites with 99.7% protection, the protective effect of Z. piperitum oil product appeared complete (100%). It had a protective effect against Aedes gardnerii, Anopheles barbirostris, Armigeres subalbatus, Culex tritaeniorhynchus, Culex gelidus, Culex vishnui group, and Mansonia uniformis. The better repellency against a wide range of field mosquitoes derived from Z. piperitum oil products suggested an advantage of efficacy by providing a broad spectrum of activity. Therefore, Z. piperitum could have potential for use in the development of combined repellents as a natural active ingredient, synergist, or additive to conventional synthetic chemicals, particularly in situations when DEET is ineffective and impractical.

Diagnostic value of IgG isotype responses against Brugia malayi antifilarial antibodies in the clinical spectrum of brugian filariasis.

To study the diagnostic significance of antifilarial IgG subclasses in the clinical spectrum of brugian filariasis, IgG1, IgG3 and IgG4 antifilarial antibodies were determined in an exposed population comprising 74 asymptomatic amicrofilaraemics, 30 microfilaraemics, 20 lymphangitis and 16 elephantiasis patients resident in Narathiwart province, an area endemic for Brugia malayi lymphatic filariasis in southern Thailand. The dominant isotype of antifilarial antibody was IgG4. A significantly higher percentage of individuals were positive for IgG1 in the microfilaraemic and lymphangitis groups compared with the elephantiasis and endemic normal patients, while a significantly higher positive rate of IgG3 was found in those with lymphangitis. The possible role of these isotypes for diagnostic purposes and the pattern of antibody response in various clinically manifesting groups are discussed.

Evaluation of the colchicine-like activity of Gloriosa superba-extracted fractions for mosquito (Diptera: Culicidae) cytogenetic study.

Four fractions of Gloriosa superba L., i.e., hexane fraction, dichloromethane fraction 1, dichloromethane fraction 2, and methanol fraction, were investigated for colchicine-like activity using a mosquito cytogenetic assay. The results revealed that the latter three fractions yielded promisingly high colchicine-like activity, whereas the hexane fraction yielded very low activity compared with 1% colchicine in a 0.85% sodium chloride solution. The metaphase rates and average number of metaphase chromosomes per positive brain ganglion (range) of Aedes aegypti L. larvae after incubation with 0.25-2% solutions of dichloromethane fraction 1, dichloromethane fraction 2, 0.5-2% solutions of methanol fraction, and 1% colchicine solution were 90-100% and 7 (2-19) to 22 (7-47); 90-100% and 4 (1-11) to 30 (4-73); 95-100% and 11 (1-28) to 17 (2-62); and 100% and 6 (2-11), respectively. The temperature stability tests of the three promising fractions were performed by heating 0.5% working solution at 121 degrees C for 15 min and preparing 0.5% working solution from stock frozen at -20 degrees C for 10 mo. These fractions also yielded satisfactory outcomes of metaphase rates and an average number of metaphase chromosomes per positive brain ganglia compared with 1% colchicine solution.

Mosquito repellency of the seeds of celery (Apium graveolens L.).

When the mosquito repellencies of four fractions of Apium graveolens seeds (one hexane, two dichloromethane and one methanolic) were investigated in the laboratory, all four were found to offer human volunteers some protection against female, adult Aedes aegypti. The hexane fraction, however, was found to exhibit the highest repellency in the laboratory, with median effective doses (ED50) and ED95 of 0.41 and 2.93 mg/cm2 skin, respectively. Only this fraction, which was also found to provide protection against mosquito bites for 3.5 h when applied, in the laboratory, at a concentration of 250 mg/ml, was then investigated for its repellency in the field and its stability. In storage, it was found to retain its repellency for at least 2 months, although significant reductions in its repellency were observed (in terms of shortened complete-protection times) after 3 months, whatever the temperature of storage (-20 degrees C, 4 degrees C, or room temperature). When applied to the skin of volunteers under field conditions, the hexane fraction showed strong repellent activity against a wide range of mosquito species belonging to various genera (Ae. gardnerii, Ae. lineatopennis, Armigeres subalbatus, Culex tritaeniorhynchus, Cx. vishnui group, Cx. quinquefasciatus and Mansonia uniformis). It appeared not to cause dermal irritation or any other adverse effect, either during 6 months of use or in the following 3 months of follow-up. Mosquito repellents based on extracts of Ap. graveolens seeds could be developed commercially, as an effective personal-protection measure against mosquito bites and the diseases caused by mosquito-borne pathogens.

Toxicity of crude rhizome extract of Kaempferia galanga L. (Proh Hom).

The ethanolic rhizome extract of Kaempferia galanga L. (Zingiberaceae) was studied by conventional pharmacological methods including the Hippocratic screening test, and acute and subacute toxicities in rats. The hexane fraction was tested for dermal irritation in rabbits. The ethanolic extract, when tested by the Hippocratic screening test, demonstrated signs that indicated CNS depression such as a decrease in motor activity and respiratory rate, and a loss of screen grip and analgesia. In the acute toxicity test, oral administration of 5 g/kg of Kaempferia galanga produced neither mortality nor significant differences in the body and organ weights between controls and treated animals. Moreover, both gross abnormalities and histopathological changes were not comparatively detectable between all controls and treated animals of both sexes. In subacute toxicity studies, no mortality was observed when varying doses of 25, 50 or 100 mg/kg of ethanolic Kaempferia galanga extract were administered orally per day for a period of 28 days. There were no significant differences in the body and organ weights between controls and treated animals of both sexes. Hematological analysis showed no differences in any of the parameters examined (WBC count, platelet, hematocrit and hemoglobin estimation) in either the control or treated groups of both sexes. However, the differential leukocyte counts showed a slight but significant decrease of lymphocyte count in the 50 and 100 mg/kg male rat groups. In the blood chemistry analysis, no significant change occurred in the blood chemistry parameters, including glucose, creatinine, blood urea nitrogen (BUN), aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (Alk-P), total protein and albumin of both sexes. Pathologically, neither gross abnormalities nor histopathological changes were observed. No sign of irritation was observed during the dermal irritation test of the hexane fraction of Kaempferia galanga.