Observations of the third instar larva and puparium of Chrysomya bezziana (Diptera: Calliphoridae).

Observations on the ultrastructure of the third instar larva and puparium of the Old World screw-worm fly, Chrysomya bezziana, are presented utilizing both light microscopy (LM) and scanning electron microscopy (SEM). Results of this study indicate that the shape of the intersegmental spines between the pro- and mesothorax markedly differ from other blow fly species (Chrysomya megacephala, Chrysomya rufifacies, Chrysomya nigripes, Lucilia cuprina) in being broad-based with sharp recurved tips. Other characters such as the posterior spiracles, number of papillae on the anterior spiracles, oral grooves, and posterior spiracular hairs also differ. The strong and robust mouthhooks may explain the ability of larvae to penetrate deeply into human tissues. Perforated sieve plates covered with antler-like projections were observed within the anterior spiracles of the puparium of C. bezziana. The posterior spiracular discs each bear three spiracular slits with approximately 2-microm wide openings that were viewed either open or closed by a membrane underneath. This study expands our knowledge of the fine details of the external morphology of both the third instar larva and puparium of C. bezziana, which is an obligatory myiasis-producing species in many regions. A key to differentiate the third instar of C. bezziana from other blow flies in Thailand is given.

Surface ultrastructure of the puparia of the blow fly, Lucilia cuprina (Diptera: Calliphoridae), and flesh fly, Liosarcophaga dux (Diptera: Sarcophagidae).

Surface ultrastructure of the puparia of the blow fly, Lucilia cuprina, and flesh fly, Liosarcophaga dux, are presented utilizing scanning electron microscopy (SEM). Emphasis was focused on characteristic features of the puparia that could be used for differentiation from other forensically important fly species. The puparium of L. cuprina typically measures 6.2+/-0.2 mm in length and 2.3+/-0.1 mm in width. Each anterior spiracle contains a single row of 5-7 papillae. The intersegmental spines between the prothorax and mesothorax are triangular with constricted tips. A clustered bubble membrane, comprising about 30 mammillate structures, is positioned dorsolaterally on each side of the first abdominal segment in young puparia but is replaced by short, tubular respiratory horns in aged puparia. The posterior end of the puparium is broadly truncate and bears a pair of medially positioned posterior spiracles that each contains three straight spiracular slits. The puparium of L. dux is larger in comparison to L. cuprina and typically measures 9.9+/-0.3 mm in length and 3.8+/-0.2 mm in width. An anterior spiracle of this species contains 14-17 papillae. The intersegmental spines between the prothorax and mesothorax are broad and triangular. A convoluted structure of unknown function was observed at the dorsolateral edge of segments 5-11. In L. dux, the caudal segment of the puparium is slightly tapered and abruptly truncated and contains a pair of posterior spiracles that are located within a deep concavity in the terminal end. Each posterior spiracular disc appears D-shaped, with a pronounced medial projection and three vertically oriented long, narrow spiracular slits. The anatomical features presented herein may be useful for identification of fly puparia of these two species in future forensic entomological investigations.

The first documented forensic entomology case in Thailand.

The forensic entomology case described herein is the first such case documented in Thailand. A mummified corpse of a 32-yr-old man was discovered in a forested habitat, with the larvae of six species of flies (Diptera) found in association with the corpse at the time of its discovery, i.e., those of Hydrotaea (=Ophyra) spinigera Stein (family Muscidae), Piophila casei (L.) (family Piophilidae), Megaselia scalaris (Loew) (family Phoridae), Sagus sp. (family Stratiomyidae), and larvae of two unidenitified flesh fly species (family Sarcophagidae). The presence and age of the larval specimens of P. casei, M. scalaris, and H. spinigera gave entomological evidence that the postmortem interval for the corpse was 3-6 mo. This report also documets some of the forensically important fly species that occur in Thailand.

Scanning electron microscopy of the third-instar Piophila casei (Diptera: Piophilidae), a fly species of forensic importance.

The morphology of third-instar Piophila casei (L.) is described by means of scanning electron microscopy. Features of the anterior cephalic region and creeping welts that are used in larval skipping and creeping, respectively, are highlighted. Morphological features classically used for larval identification are also illustrated.

Experimental infection of Gnathostoma spinigerum larvae in prawns and tadpoles.

Naturally captured Lanchester's freshwater prawns (Macrobrachium lanchesteri) and farm-bred Rana regulosa tadpoles were assessed for their capability of being the first or second intermediate hosts of Gnathostoma spinigerum. Seventy specimens from each animal group were randomized into a control group and investigated for larvae of G. spinigerum by pressing them between two pieces of glass and examination under stereomicroscope. No Gnathostoma larvae were found in the entire control group. Another 120 specimens of each animal were used in two treatment groups; 60 being exposed to the first-stage larvae, G. spinigerum, and 60 exposed to cyclops containing the third-stage larvae for 7 days. No larvae of G. spinigerum were found in the prawns of both treatment groups that resembled tadpoles exposed to the first-stage larvae. In contrast, 18.3% of tadpoles, which were exposed to cyclops containing third-stage larvae, were infected. Lanchester's freshwater prawns cannot serve as intermediate host of G. spinigerum, while R. regulosa can serve as the second intermediate host.

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.

Prevalence of Opisthorchis viverrini infection among villagers harboring Opisthorchis-like eggs.

The precise occurrence of Opisthochis viverrini infection in humans, who were positive for Opisthorchis-like eggs in a stool examination, was determined using the potassium permanganate staining method. In the 68 specimens examined, there were more individual O. viverrini eggs (38.24%) than singular Haplorchis taichui eggs (29.41%). One-fourth of the total specimens contained a mixed infection of O. viverrini and H. taichi eggs. The median ratio of O. viverrini: H. taichui eggs in mixed infection cases was 2.29 (min = 1, max = 17.5). It is suggested that chemotherapy with praziquentel treatment should be given to patients who are positive for O. viverrini-like eggs.

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.

Chrysomya rufifacies (Macquart) as a forensically-important fly species in Thailand: a case report.

The first documented use of the larvae of the hairy maggot blow fly, Chrysomya rufifacies, for estimating the postmortem interval (PMI) of a decomposing human corpse in Thailand is reported. A honeycomb-like wound observed on one leg of the corpse was infested with numerous third-stage C. rufifacies larvae. Based on the pupae and larvae present in accordance with the ambient temperature previously recorded, six days postmortem was estimated for a corpse at the time of its discovery and investigation. Since adult C. rufifacies specimens have been collected in many parts of both urban and mountainous areas in Thailand, more biological information about this blowfly species is needed to increase the accuracy of forensic investigations where the fly is present.

Surface ultrastructure of the third-instar larvae of Hydrotaea spinigera Stein (Diptera: Muscidae), a fly species of forensic importance.

The surface ultrastructure of third-instar larvae of Hydrotaea (= Ophyra) spinigera Stein was observed with scanning electron microscopy. The number of papillae in each anterior spiracle ranges from 5-7. Abdominal segments 4-12 possess ventral welts. Each posterior spiracular disc bears 3 nearly straight slits, with the lower and middle slits closer together than the middle and upper slits. Although the larvae of H. spinigera are generally similar to two other closely-related species in Thailand, Hydrotaea (= Ophyra) leucostoma (Wiedemann) and Hydrotaea (= Ophyra) chalcogaster (Wiedemann), some of the morphological features can help in differentiating these species, thus allowing more accurate identification of species of fly larvae in forensic investigation.

Lack of efficacy of quinine and artemether against advanced third-stage larvae of Gnathostoma spinigerum in vitro.

The efficacy of quinine and artemether--the effective blood schizontocide in malarial treatment--has been in vitro tested with the advanced third-stage larvae of Gnathostoma spinigerum. All larvae were collected from freshwater eel (Fluta alba) and exposed to the culture medium, each containing either quinine dihydrochloride or artemether at a final concentration of 20 microg/ml and 0.5 microg/ml, respectively for 21 consecutive days. Larval motility was assessed daily and the topographical changes were assessed using scanning electron microscope after 21-days of drug exposure. All worms moved actively for 21 days of study period and no change in surface ultrastructure was observed. Quinine and artemether at these concentrations have no effect on movement and topographical changes on the advanced third-stage larvae of this parasite.

Successful eradication of Ascaris lumbricoides and hookworm infection after three repeated doses of albendazole.

Three repeated orally doses of albendazole 400 mg in 6 weekly intervals were evaluated in Thai hill-tribe students who had at least one kind of soil-transmitted helminths (i.e. Ascaris lumbricoides, hookworm and Trichuris trichiura). Stool examination and parasite egg count were performed using Beaver's standard direct smear method and Kato-Katz's cellophane thick smear method prior to treatment and then 1 month after the first, second and third dose of drug administrations. A single dose of albendazole was very effective against A. lumbricoides and hookworm infections, with cure rates of 98.68 per cent and 92.16 per cent, respectively. The second and third dosages eradicated A. lumbricoides and hookworm infections, respectively. Conversely, the first to third cure rates for T. trichiura infection were relatively low, being 37.76-58.16 per cent. Three repeated doses of albendazole proved to be beneficial in eradication of A. lumbricoides and hookworm infections, and decreased the prevalence of T. trichiura infected cases. For eradication of T. trichiura infection, further regimen and period of drug administration is required.

Musca domestica as a mechanical carrier of bacteria in Chiang Mai, north Thailand.

The common house fly, Musca domestica L., was assessed for its potential as a mechanical carrier of bacteria in urban areas of Chiang Mai province, north Thailand. Sixty-one specimens (61.0%) were found to carry bacteria, with each harboring 1-5 bacteria. No significant difference between the sex of flies for carrying bacteria was found. A total of 21 bacteria was isolated, of which the most common was coagulase negative staphylococci (n = 38), followed by Viridans streptococci (n = 9). The highest bacterial load for all bacteria isolated was 10(3)-10(4) (67%), followed by > 10(4) (26%) colonies per fly. It is recommended that fly control management measures, including sanitation improvement, should be implemented.

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.

Mechanical carrier of bacterial enteric pathogens by Chrysomya megacephala (Diptera: Calliphoridae) in Chiang Mai, Thailand.

Chrysomya megacephala was studied regarding its mechanically bacterial carrier in urban areas of Chiang Mai, northern Thailand. Fifty-six adult flies were randomly collected using sweep insect net during April-May, 1999 from 3 fresh food markets and examined for bacteriological isolation. Among them, 49 flies (87.5%) were bacterial carriers. The total 22 bacterial species and 8 groups were isolated. Three species previously reported as the bacterial enteric pathogens causing diarrheal disease were isolated from 5 flies, ie Aeromonas hydrophila, Edwardsiella tarda and Vibrio cholerae non-01, with their prevalence rates in flies being 3.579, 1.79% and 3.57%, respectively. Five possible bacteria enteric pathogens, ie Aermononas sobria, Citrobacter freundii, Escherichia coli, Providencia alcalifaciens and Pseudomonas aeruginosa, were isolated from 21 flies with the prevalence rates in flies being 5.34%, 3.57%, 26.79%, 7.14% and 1.79%, respectively. The bacterial load isolated from all 3 pathogenic species was entirely found more than 10 colony per fly, indicating the high chance for disease transmission via this fly species. C. megacephala may play the possible and/or important role of bacterial enteric pathogens transmission, thereby promoting the public health personnel for sanitation improvement in fresh food markets and fly control management in these particular areas.

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.