Nematocidal effect of Piper retrofractum Vahl on morphology and ultrastructure of Strongyloides stercoralis third-stage infective larvae.

In a previous research work aimed at discovering natural helminthicides as alternatives to conventional synthetic drugs, Piper retrofractum fruit hexane extract (PHE) has been shown to possess promising nematocidal activity against the third-stage infective larvae of Strongyloides stercoralis. Thus, this study was designed to evaluate the chemical composition and the impact of PHE on symptom and structural alterations of S. stercoralis. Chemical analysis of PHE by gas chromatography-mass spectrometry demonstrated 26 different compounds, constituting 100% of the total composition. The main components were 4-acetylphenyl (4-benzoylphenoxy) acetate (14.86%) and octyl methoxycinnamate (12.72%). Nematocidal bioassays revealed promising potential of PHE against S. stercoralis larvae, with an LC50 value of 0.059 mg/ml, while the reference drug ivermectin exerted higher efficacy, with an LC50 value of 0.020 µg/ml. Behavioural observations under light microscopy revealed that PHE-treated S. stercoralis larvae moved slowly, became paralysed and eventually died during 24 h of incubation. The dead larvae appeared under light microscope as straight worms with unknown vacuoles of different sizes inside their internal bodies. Morphological alterations of the PHE-treated S. stercoralis larvae, such as straight bodies with swollen cuticle, faded transverse annulations and faded longitudinal striations, as well as shallow and smooth lateral longitudinal grooves, were seen clearly under scanning electron microscopy. Ultrastructural changes in the treated larvae, such as protruded lateral longitudinal grooves, loose muscle with vacuolation, dissociation between the hypodermis and cuticle and marked intracellular disorganization with vacuolation, were detected under transmission electron microscopy. The results of this study provide evidence that PHE is toxic against S. stercoralis and also a potential new alternative for anti-Strongyloides chemotherapy.

Angelica sinensis (Umbelliferae) with proven repellent properties against Aedes aegypti, the primary dengue fever vector in Thailand.

Botanical resources with great diversity in medicinal and aromatic plants are a rich and reliable source for finding insect repellents of plant origin, which are widely popular among today's consumers. Although some herbal-based repellents have been proven comparable to or even better than synthetics, commercially available natural repellents generally tend to be expensive, with short-lived effectiveness. This critical flaw leads to ongoing research for new and effective repellents, which provide longer protection against vector and nuisance-biting insects, while remaining safe, user friendly, and reasonably priced. This study aimed to evaluate the repellent activity of plant-derived products against the primary dengue vector, Aedes aegypti, by following the human bait technique of World Health Organization guidelines. Preliminary laboratory screening tests for repellency of 33 plant species clearly demonstrated Angelica sinensis as the most effective repellent from each kind of extracted product, with its essential oil and ethanolic extract having median complete protection times of 7.0 h (6.0-7.5) and 2.5 h (2.0-2.5), respectively. Due to its low yield (0.02 %), pungent smell, and little cause of irritation, A. sinensis essential oil did not qualify as a candidate for further repellent assessment. However, subsequent extractions of A. sinensis with different organic solvents of increasing polarity provided four extractants with varying degrees of repellency against A. aegypti. The hexane extract of A. sinensis provided excellent repellency, with a median complete protection time of 7.5 h (6.5-8.5), which was longer than that of ethanol (2.5, 2.0-2.5 h), acetone (1.75, 0.5-2.5 h), and methanol extracts (0.5, 0-1.0 h). By being the most effective product, A. sinensis hexane extract gave significant protection comparable to that of its essential oil and the standard synthetic repellent, N,N-diethyl-3-methylbenzamide (DEET: 6.25, 5.0-6.5 h). Qualitative gas chromatography/mass spectrometry analysis demonstrated the presence of phthalides and phthalates, including 3-N-butylphthalide, butylidenephthalide, ligustilide, and di-iso-octyl phthalate, as the principal constituents in A. sinensis products. The success of A. sinensis products, particularly that of hexane extract, has proved their potential as bioactive candidates in the next step for developing and producing alternative natural repellents with commercial aspirations.

First report of canine cutaneous myiasis caused by Chrysomya bezziana Villeneuve (Diptera: Calliphoridae) in Thailand.

Although myiasis caused by the blow fly, Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), has been reported in Thailand, all of the cases were human. This study described three cutaneous myiasis cases caused by C. bezziana in dogs in Chiang Mai province, northern Thailand. The removal of fly larvae together with specific treatment to cure all cases in this study was represented.

Hemocyte classification of three mosquito vectors: Aedes togoi, Anopheles lesteri and Culex quinquefasciatus.

Insect blood cells or hemocytes play an important role in the defense against parasites and other pathogenic organisms. However, the hemocyte types of three mosquito vectors, Aedes togoi, Anopheles lesteri and Culex quinquefasiatus are not well known. Therefore, the aim of this study was to characterize the hemocytes of these three mosquito species based on morphology using light microscopy. The abdominal cutting and perfusion method was used in this study as it took the fewest steps, provided the largest number of hemocytes and yielded less contamination with fat body cells. Hemocyte typing, based on morphology, revealed three types of hemocytes (prohemocytes, oenocytoids and granulocytes) that were contained in the hemolymph of all three mosquito species. This study demonstrated that the use of distinct morphology with light microscopy provided sufficient criteria to characterize and differentiate mosquito hemocytes. This technique will be useful in terms of cost saving and for new researchers who begin to study in this field.

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.

Antimosquito property of Petroselinum crispum (Umbellifereae) against the pyrethroid resistant and susceptible strains of Aedes aegypti (Diptera: Culicidae).

The increasing and widespread resistance to conventional synthetic insecticides in vector populations has underscored the urgent need to establish alternatives in the mosquito management system. This study was carried out with the aim to investigate the antimosquito property, larvicidal and adulticidal potential, of plant products against both the pyrethroid-susceptible and resistant strains of Aedes aegypti. Seventeen plant products, including essential oils and ethanolic extracts, were obtained by steam distillation and extraction with 95 % ethanol, respectively. Their larvicidal activity was screened, using World Health Organization (WHO) procedures against A. aegypti, Muang Chiang Mai-susceptible (MCM-S) strain. The most effective product was a candidate for investigating larvicidal and adulticidal potential against three laboratory strains of A. aegypti, comprising MCM-S, Pang Mai Dang-resistant (PMD-R), and Upakut-resistant (UPK-R). Potential toxicity of the plant candidate was compared with that of synthetic temephos, permethrin, and deltamethrin. Chemical constituents of the most effective plant product also were analyzed by gas chromatography-mass spectrometry (GC-MS). Results obtained from the preliminary screening revealed the varying larvicidal efficacy of plant-derived products against MCM-S A. aegypti, with mortality ranging from 0 to 100 %. The larvicidal activity of seven effective plant products was found to be dose dependent, with the highest efficacy established from Petroselinum crispum fruit oil, followed by oils of Foeniculum vulgare, Myristica fragrans, Limnophila aromatica, Piper sarmentosum, Curcuma longa, and M. fragrans ethanolic extract (LC50 values of 43.22, 44.84, 47.42, 47.94, 49.19, 65.51, and 75.45 ppm, respectively). Essential oil of P. crispum was then investigated further and proved to be a promising larvicide and adulticide against all strains of A. aegypti. The pyrethroid-resistant strains of both PMD-R and UPK-R A. aegypti showed significant resistance to temephos, permethrin, and deltamethrin in either the larval or adult stage. Interestingly, high susceptibility to P. crispum oil was observed in the larvae and adults of MCM-S, which are pyrethroid-susceptible A. aegypti, and comparable to those of the pyrethroid-resistant strains, PMD-R and UPK-R. GC-MS analysis of P. crispum oil demonstrated that 19 compounds, accounting for 98.25 % of the whole oil, were identified, with the main constituents being thymol (42.41 %), p-cymene (27.71 %), and γ-terpinene (20.98 %). In conclusion, the profound larvicidal and adulticidal potential of P. crispum oil promises to form a new larvicide and adulticide against either the pyrethroid-susceptible or resistant strain of A. aegypti. Consequently, P. crispum oil and its constituents can be used or incorporated with other chemicals/measures in integrated mosquito management for controlling A. aegypti, particularly in localities with high levels of pyrethroid and organophosphate resistance.

Short Communication: First report of canine cutaneous myiasis caused by Chrysomya bezziana Villeneuve (Diptera: Calliphoridae) in Thailand

@#Although myiasis caused by the blow fly, Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), has been reported in Thailand, all of the cases were human. This study described three cutaneous myiasis cases caused by C. bezziana in dogs in Chiang Mai province, northern Thailand. The removal of fly larvae together with specific treatment to cure all cases in this study was represented.

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.

Potassium permanganate staining for differentiation the surface morphology of Opisthorchis viverrini, Haplorchis taichui and Phaneropsolus bonnei eggs.

Potassium permanganate staining method was developed for differentiation Opisthorchis viverrini, Haplorchis taichui and Phaneropsolus bonnei eggs. The surfaces of O. viverrini, H. taichui and P. bonnei eggs stained permanently and temporarily were similar in appearance even the staining procedures were varied both in concentration and time. Determined under light microscope set at 400x, all of these eggs were oval-shaped, operculated at one pole and indistinct small knob at posterior end. O. viverrini eggs showed the distinct musk-melon-like prominent ridges on the surface. Haplorchis taichui eggs had a light striae pattern while P. bonnei eggs had a smooth egg shell. Length of these trematode eggs were significant different (chi2 test, p < 0.05). Mean +/- SD of O. viverrini, H. taichui and P. bonnei eggs were 26.34 +/- 1.65 microm, 29.03 +/- 1.48 microm and 23.00 +/- 1.49 microm, respectively. Regarding of their width, the mean +/- SD of O. viverrini, H. taichui and P. bonnei eggs were 15.54 +/- 0.69 microm. 14.94 +/- 0.91 microm and 12.25 +/- 1.02 microm, respectively. The means of width of O. viverrini and H. taichui eggs were not significantly different (chi2 test, p > 0.05), however, they were significantly different from those of P. bonnei (chi2 test, p < 0.05). Temporary staining using 1% w/v concentration and only 1 minute of time is useful in the mass fecal examination survey for the prevalence and intensity of truly Opisthorchis infection.

Intensity of trematode metacercariae in cyprinoid fish in Chiang Mal Province, northern Thailand.

The intensity of infection of trematode metacercariae was determined in five species of cyprinoid fish collected from Mae Ngud reservoir, Chiang Mai Province. These species were Thynnichthys thynnoides, Puntioplites proctozysron, Hampala macrolepidota, Puntius leiacanthus and Puntius gonionotus. T. thynnoides contained the highest number (83.0%) of metacercariae, whereas P. gonionotus had the fewest (0.19%). The caudal fin was the area of highest infection of metacercariae (49.39%), with the anal fin having the fewest (5.93%). Most metacercariae was isolated from Haplorchis taichui (63.27%), with an intensity variation of 0.3-165.2 metacercariae/fish.

In vitro excystation of Haplorchis taichui (Trematoda: Heterophyidae).

The effects of trypsin, bile extract, temperature and acid-based condition for the in vitro excystation of Haplorchis taichui metacercariae were studied. At 37 degrees C, approximately half the number of metacercariae excysted when exposed to 1% trypsin for 15 minutes with no more excystation found beyond this time. Increasing trypsin concentration seemed to reduce the excystation rate while bile extract was, however, unlikely to be an absolute requirement. A temperature of 37 degrees-41 degrees C yielded a similar excystation result in combination with 1% trypsin; however, less excystation occurred at a lower temperature of 35 degrees C. The acid-based environment of pH 8 gave the best excystation result in association with 1% trypsin at a temperature of 39 degrees C. Higher and lower basicity produced a smaller excystation rate. An environmental condition of 1% trypsin at pH 8 and a temperature of between 37 degrees-41 degrees C was recommended for the in vitro excystation of H. taichui metacercariae. The relatively broad temperature and pH range condition for the excystation of H. taichui corresponded with various definitive hosts that were infected naturally by this fluke.

In vitro development of Haplorchis taichui (Trematoda: Heterophyidae).

Newly excysted metacercariae of Haplorchis taichui were cultured in a candle jar set at 37 degrees C. Both monophasic culture media [0.85% NaCl, RPMI 1640, RPMI 1640+10% fetal calf serum (FCS)] and diphasic culture media [RPMI 1640 + egg yolk agar, RPMI 1640 + 5%, 10% or 15% blood in blood agar (BA), RMPI 1640 + 5%, 10% and 15% FCS with 5% blood in BA] were used in vitro. Parasites survived for only 1 day in 0.85% NaCl without any development. In RPMI 1640 with egg yolk agar and RMPI 1640 + 5%, 10% FCS, the parasite survived for 3-5 days. In contrast, worms survived for 12-14 days in RPMI 1640 with blood agar without any change in result in a different concentration of blood in BA. The ovary and testes were observed after 3 days incubation in this media. Nevertheless, only 1 parasite in RPMI 1640 with 15% blood in BA had vitellaria and eggs at day 6. RPMI 1640 with blood agar can be used as short-term maintenance for the in vitro culture of H. taichui. However, further studies are needed.

Development of Haplorchis taichui (Trematoda: Heterophyidae) in Mus musculus mice.

The development of Haplorchis taichui was studied in sixteen mice, Mus musculus. Metacercarial cysts of H. taichui were obtained from the freshwater fish, Thynnichthys thynnoides, collected in Chiang Mai Province, north Thailand. Approximately 200 active metacercariae were orally introduced into each mouse. Two mice were randomly sacrificed and necropsied daily from day 2-9 postinfection (pi). Two peaks of increment in the length and width of worms were found at day 3 and 7 pi. H. taichui was rapid in maturation, similar to other minute intestinal flukes. Rudimentary sex organs were found at day 2 pi. Spermatozoa in a seminal receptacle, vitellaria and eggs were seen as early as 3 days pi. The number of eggs increased daily to approximately 50-60 eggs thereafter. Mice can serve as a suitable experimental definitive host for harvesting adult H. taichui, especially in 1-week pi.

Screening for larvicidal activity of ten carminative plants.

Ten species of plants, reported to possess carminative property, were screened for larvicidal potential against Culex quinquefasciatus by exposing early 4th instar larvae to a series of concentrations of the ethanolic extracts of the plants. Mortality counts were made after 24 hours exposure. Probit analysis using computerized Harvard Programming (Hg1, 2) was employed to determine the LC50, LC95 and LC99 values in order to compare the larvicidal potency of the ten plants. Marked larvicidal effects were seen with Kaempferia galanga, Illicium vernum and Spilanthes acmella having LC50 values of 50.54, 54.11 and 61.43 ppm, respectively.

Surface ultrastructure of excysted metacercariae of Haplorchis taichui (Trematoda: Heterophyidae).

The morphology of newly excysted juvenile Haplorchis taichui was studied using a light and a scanning electron microscope (SEM). The reproductive organs were well developed. The whole body surface was covered with numerous transverse rows of scale-like spines, which had 3-11 points at the tip. The spines on the dorsal were similar with those on the ventral surfaces in shape, size and number of points. The spines in anterior were digitated into 10-11 points, then 8-9 points and 7 points. The presence of 3 points in each spine was observed in the area adjacent to the excretory pore. Two types of sensory papillae existed throughout the body: type I, ciliated knob-like swellings and type II, round swellings of the tegument. The rapid maturation of H. taichui results from the development of both internal and external organs during the newly excysted stage.

Autogenous Aedes togoi sub-colony (Chanthaburi, Thailand strain), an efficient laboratory vector in study of filariasis.

Comparative filarial susceptibility and biology between stock colony and selectively autogenous Aedes. togoi sub-colony were carried out to determine the laboratorial vector-capacity and viability of autogenous sub-colony. The results of susceptibility revealed that the selectively autogenous Ae. togoi sub-colony yielded higher susceptibility than the stock colony, ie Dirofilaria immitis: susceptibility rates=80.00% [Exp1(F8)] and 76.19% [Exp2(F17)] (autogenous sub-colony), 53.33% (Exp1) and 71.43% (Exp2) (stock colony); Brugia malayi: susceptibility rates=83.33% [Exp1(F17)] and 84.38% [Exp2(F19)] (autogenous sub-colony), 81.25% (Exp1) and 75.00% (Exp2) (stock colony), but not at the level of statistically significant differences except the Exp1 of D. immitis, which was significant difference. In addition, the average No. L3 per infected mosquito in the selective autogenous sub-colony (D. immitis: Exp1=3.37, Exp2=3.19; B. mallayi: Exp1=8.80, Exp2=3.37) was also higher than in stock colony (D. immitis: Exp1=2.44, Exp2=2.73; B. malayi: Exp1=7.85, Exp2=3.02), but not at the level of statistically significant difference. The results of comparisons on some biological aspects demonstrated that most of the cases they have similar biology except the average egg deposition per gravid female of stock colony (130.17+/-43.33) was significantly more than selectively autogenous sub-colony(F9) (94.33+/-13.69), egg length x width 575.62+/-18.06 microm x 186.15+/-9.35 microm of stock colony was significantly larger than selectively autogenous sub-colony(F9) 560.49+/-18.96 microm x 177.99+/-8.40 microm, and mean longevity of adult female of stock colony [41.60(6-61)] was significantly longer than selectively autogenous sub-colony(F9) [35.00(5-39)]. The selectively autogenous sub-colony was established and twenty-two successive generations have been colonized.

Larvicidal, adulticidal and repellent effects of Kaempferia galanga.

Four fractions of Kaempferia galanga (hexane fraction, dichloromethane fraction 1, dichloromethane fraction 2 and methanolic fraction) were tested for larvicidal activity toward fourth instar Culex quinquefasciatus. The hexane fraction was found to exhibit the highest larvicidal effect with the LC50 of 42.33 ppm. Testing for adulticidal activity, the hexane fraction did not show any promising adulticidal effect. However, it caused a knockdown effect which might be useful as a repellent. It was then tested for repellent activity in human volunteers both in laboratory and field studies. In a laboratory study, the hexane fraction possessed repellency against Aedes aegypti (ED50 value of 30.73 microg/cm2), and provided biting protection for 3 hours. In a field study, it could protect against certain mosquitos, ie, Armigeres subalbatus, Anopheles barbirostris, An. aconitus, Mansonia uniformis, Cx. quinquefasciatus, Cx. gelidus, Cx. tritaeniorhynchus and Ae. aegypti. The hexane fraction did not cause dermal irritation when applied on human skin.

Prevalence of trematode metacercariae in cyprinoid fish of Ban Pao district, Chiang Mai Province, northern Thailand.

The prevalence of trematode metacercariae in natural cyprinoid fish was carried out in Ban Pao district, Chiang Mai Province, northern Thailand. Fish were collected from both man-made water reservoirs and natural sources during July 1996-May 1997. Nine species of cyprinoid fish harbored the metacercariae of Opisthorchis viverrini and small intestinal flukes of the family Heterophyidae, ie Haplorchis taichui, H. pumilio and Centrocestus spp. Metacercariae of H. taichui were found approximately 384 times more than O. viverrini. Among the fish collected, Puntius leiacanthus contained the highest number of metacercariae of H. taichui (182 metacercariae/fish) whereas P. orphoides contained the highest number of metacercariae of O. viverrini (1.4 metacercariae/fish). Metacercariae of H. taichui found in this area were year-round, with the highest and lowest incidence rates being in winter and rainy season, respectively. The difference between H. taichui and O. viverrini with respect to their prevalences are discussed.

Movability of advanced third-stage larva of Gnathostoma spinigerum exposed to albendazole sulphoxide in vitro.

Movability of advanced third-stage larvae of Gnathostoma spinigerum exposed to albendazole sulphoxide (AlbSO), the active metabolite of albendazole, was determined in vitro. Larvae in control groups moved actively with the whole body for all 21 days of the study period. In larvae treated with AlbSO 1 microg/ml, the movement was significantly reduced after 11 days exposed to the drug and to be only a part of body on the 15th-21st days. In larvae treated with AlbSO 2 microg/ml, the movement was initiated in decreasing after 9th days and to be only a part of body on the 12th-17th days. Finally, worms were immobile but not dead on the 20th-21st days. Although there was no larvae died at 21st days exposed to AlbSO in both concentrations; but all worms were sluggish and may die later. These lethargic worms may not be able to migrate in patients and leading to cure. Albendazole may not be benefit for acute symptom clearance; however, it can prevent the recurrent migratory swelling after the treatment of 21 day-course.