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.

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.

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.

Experimental transmission of subperiodic Wuchereria bancrofti to the leaf monkey (Presbytis melalophos), and its periodicity.

Infective larvae of nocturnally subperiodic Wuchereria bancrofti were obtained from laboratory-raised Aedes togoi mosquitoes which had fed on an infected human volunteer from Kanchanaburi, Thailand, and 471 of them were inoculated subcutaneously into the inguinal regions of an immunosuppressed Presbytis melalophos. Microfilaremia was first detected 287 days after inoculation, and 77 days later, at 364 days of infection, the number of microfilariae determined at 2-hour intervals reached a maximum of 619/20 mm3 at 4 hours past midnight, and the daytime levels were 11-35/20 mm3; the corresponding levels in the human volunteer were 121 and 13-32/20 mm3. Thus the nocturnal periodicity was relatively greater in the monkey than it was in the human host. Eight days after the periodicity study the monkey died and 77 adult worms were recovered from the popliteal (4), inguinal (58), iliac (6) and para-aortic (9) lymph nodes and 1 each from the ovary and liver. Of 56 females, only half were gravid.

Scanning electron microscopy of eggs of Mansonia uniformis, Ma. indiana, Ma. annulifera, and Ma. annulata (Diptera: Culicidae).

The eggs of Mansonia uniformis, Ma. indiana, Ma. annulifera, and Ma. annulata are described with the aid of scanning electron micrographs. Comparisons of egg structure among the four species showed that the eggs differed discernibly with respect to the outer chorionic tubercles on the anterior tube, the spines surrounding the raised central micropyle within the anterior cup, and the outer chorionic tubercles in the posterior region. Attempts to produce a key for identification of these eggs under scanning electron microscopy are discussed.

Scanning electron microscopy of eggs of Mansonia bonneae (Diptera: Culicidae).

The egg of Mansonia bonneae (Edwards) is described by means of scanning electron microscopy (SEM) and compared with other sympatric species reported recently in previous studies. Ma. bonneae differed from Ma. uniformis (Theobald), Ma. indiana (Edwards), Ma. annulifera (Theobald), and Ma. annulata (Leicester) in that it has six variably sized dog-tooth spines surrounding the raised central micropyle, and only a single nipplelike stud at the anterior one-third of the egg. A key for identification of the eggs of the five sympatric Mansonia species under SEM is presented.

Applicability of presence-absence and sequential sampling for ovitrap surveillance of Aedes (Diptera: Culicidae) in Chiang Mai, northern Thailand.

Applicability of presence or absence sampling for ovitrap surveillance of Aedes aegypti (L.) and Aedes albopictus (Skuse) was examined for data collected in Chiang Mai, northern Thailand. Distribution of eggs per trap was contagious but did not fit the negative binomial distribution with a common k. The relationship between the mean number of eggs per trap and the proportion of positive traps was described using Gerrard & Chiang's model, which does not assume particular distribution patterns. Using this relation, the mean number of eggs per trap with confidence limits can be estimated without egg counts. Potential usefulness of presence or absence sequential sampling for decision making (when to initiate vector control for prevention of dengue hemorrhagic fever) was shown.

Scanning electron microscopy of the egg of Mansonia dives (Diptera: Culicidae).

The egg of Mansonia dives (Schiner) is described by means of scanning electron micrographs and compared with other species described in previous studies. The egg of Ma. dives differs from those of Ma. uniformis (Theobald), Ma. indiana (Edwards), Ma. annulifera (Theobald), Ma. annulata (Leicester), and Ma. bonneae (Edwards) in that it has a tuberculated anterior cup. A key for identification of the eggs of the six Mansonia species, based on electron microscopy, is presented.

Scanning electron microscopy of Aedes lineatopennis (Diptera: Culicidae) eggs.

The eggs of Aedes lineatopennis (Ludlow) are described by means of a scanning electron microscopy. The Ae. lineatopennis egg has a rather boat-shaped, fragmented micropylar collar, and membrane-like wall enclosing many tubercles of exochorionic sculpture.

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.

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.

Screening of ten plant species for metaphase chromosome preparation in adult mosquitoes (Diptera: Culicidae) using an inoculation technique.

The screening of 10 plant species (Aloe barbadensis Mill., Asparagus officinalis L., As. plumosus Bak., As. racemosus Willd., As. sprengeri Regel, Codyline fruticosa Goppert, Dracaena loureiri Gagnep., Gloriosa superba L., Hemerocallis flava L., and Sansevieria cylindrica Bojer) for colchicine-like substance(s) using a mosquito cytogenetic assay revealed that a 1% solution of dried Gl. superba rhizome extracted in 0.85% sodium chloride solution could be used instead of a 1% colchicine in Hanks' balanced salt solution. The metaphase rates and average number of metaphase chromosomes per positive mosquito of Aedes aegypti (L.) after intrathoracic inoculation with 1% Gl. superba-extracted solution were 100% and 29.80 in females, and 90% and 25.78 in males, whereas the inoculation with 1% colchicine solution yielded 100 and 90% metaphase rates, and 20.90 and 12.22 average number of metaphase chromosomes per positive mosquito in females and males, respectively. The application of Gl. superba-extracted solution for metaphase chromosome preparation in other mosquito genera and species [e.g., Culex quinquefasciatus Say, Toxorhynchites splendens (Wiedemann), and Anopheles vagus (Döenitz)] also has yielded the satisfactory results.

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.

Scale pattern variations of Aedes aegypti in Chiang Mai, northern Thailand.

Abdominal tergal scale pattern variations of Aedes aegypti were studied in Chiang Mai, northern Thailand for specimens reared from eggs laid in ovitraps set monthly both indoors and outdoors in urban and rural areas. The populations predominantly consisted of the dark type form, regardless of macrohabitats (urban vs. rural), microhabitats (indoor vs. outdoor) or seasons (dry vs. rainy). Among 6,003 specimens, no individuals had pale-scales continuing from the tergal base to its apex on the third tergum and posteriorly.

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.

Observations on the D/V ratio of male genitalia of Culex pipiens complex in Amphur Muang Chiang Mai, Northern Thailand.

Mosquitoes belonging to the Culex pipiens complex were collected from various locations in Amphur Muang Chiang Mai, Northern Thailand, and identified by using D/V ratios of male genitalia. Most of the mosquitoes were Cx. pipiens quinquefasciatus, having D/V ratios of 0.46 +/- 0.09. Among these mosquitoes, 1.23% (6/490) had D/V ratios between 0.8-0.9 These forms were considered as Cx. p. quinquefasciatus which showed extreme variations from the generally acceptable criterion.

Experiments in crossing two strains of Anopheles barbirostris van der Wulp 1884 (Diptera: Culicidae) in Thailand.

Crossing experiments between two strains of Anopheles (Anopheles) barbirostris from Chumphon and Chon Buri provinces in Thailand were done by induced copulation in order to determine the genetic relationship. On comparison of the F1 hybrids and those of their parent species as the control, there was a difference in the number of eggs laid, hatchability and viability. The low viability of the F1 hybrids with high larval and pupal mortalities, producing only a few F hybrids, and the fact that F1 hybrids' salivary chromosome showed asynapsis suggest there exists reproductive isolations between both strains. The data presented suggest that these two strains exhibit possible presence of a species complex in An. barbirostris.

Paragonimiasis in Mae Hong Son Province northern Thailand: case report.

Two cases of paragonimiasis have been reported from Srisangval Hospital, Mae Hong Son Province, Thailand. These two patients (Thai-Karen) resided in Nam-Piang-Din, Mae Hong Son. The preliminary survey for epidemiological data in Nam-Piang-Din, Mae Hong Son revealed that most of Thai-Karen villagers had the habit of eating raw crabs and fresh water prawns. A survey for intermediate and reservoir hosts are still in progress and this area can be considered as an additional area for paragonimiasis in Thailand.