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.

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.

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.

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.

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.

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.

Scanning electron microscopic observations on advanced third-stage larva of Gnathostoma spinigerum after in vitro exposure to albendazole sulphoxide.

Gnathostomiasis is the parasitic disease caused by the migration of an advanced third-stage larva of Gnathostoma spinigerum. To date, albendazole is claimed to be the effective drug in preventing the reoccurrence of migratory swelling in patients. After being exposed to 1 and 2 micrograms/ml albendazole sulphoxide (AlbSO) in vitro, the parasites moved deteriorately, however, no dead larva was found even exposed to these concentrations for 21 consecutive days. The topographical alterations after 21 days of albendazole sulphoxide exposure are described using a scanning electron microscope. The marked changes in surface morphology were observed in both neck and body regions. The tegumental surface on the neck region was swollen and covered with fuzzy materials, whereas, the spines on the posterior region of the body were dislodged. These changes would probably lead to reduction of intermittent cutaneous migratory swelling in human gnathostomiasis patients.

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.

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.

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.