Dengue viruses pose a considerable global public health problem with an estimated 100 million cases of illness every year. This illustrates the need for rapid and reliable diagnostic methods for proper patient management and disease control. Currently, laboratory diagnosis depends on serology or virus isolation, with both methods having certain drawbacks. Alternatively, reverse transcription and polymerase chain reaction (RT-PCR) offers the potential for the rapid, highly sensitive and specific detection of dengue viruses. Since we occasionally encounter the problem of insufficient amounts of patient serum for the direct detection of dengue viruses, a method was developed for the extraction of viral RNA after biological amplification in mosquito larvae. Using this method, 15 of 19 clinical samples tested were correctly identified using RT-PCR.
A new bufonid amphibian, belonging to a new monotypic genus, is described from the Andaman Islands, in the Bay of Bengal, Republic of India, based on unique external morphological and skeletal characters which are compared with those of known Oriental and other relevant bufonid genera. Blythophryne gen. n. is distinguished from other bufonid genera by its small adult size (mean SVL 24.02 mm), the presence of six presacral vertebrae, an absence of coccygeal expansions, presence of an elongated pair of parotoid glands, expanded discs at digit tips and phytotelmonous tadpoles that lack oral denticles. The taxonomic and phylogenetic position of the new taxon (that we named as Blythophryne beryet gen. et sp. n.) was ascertained by comparing its 12S and 16S partial genes with those of Oriental and other relevant bufonid lineages. Resulting molecular phylogeny supports the erection of a novel monotypic genus for this lineage from the Andaman Islands of India.
Two laboratory trials were conducted to determine the effect of the addition of spores (conidia) of the nematophagous fungus, Arthrobotrys oligospora, on the development of the ruminant parasite, Strongyloides papillosus, in cultures of bovine faeces. Both studies showed that at a concentration of 2000 conidia/g faeces virtually eliminated infective larvae (> 99% reduction), following 14 days incubation under ideal conditions (25 degrees C and saturated humidity) for free-living development of this parasite species. In one trial, a high level of control was also observed at a 10-fold decrease in conidia concentration (200 spores/g faeces). This work has demonstrated, in principle, that A. oligospora could provide a practical biological control agent against S. papillosus infecting intensively raised young ruminants in the humid tropics/subtropics.
Control of nematode parasites of small ruminants in a wet, tropical environment using the nematophagous fungus, Duddingtonia flagrans, was assessed in this study. Two methods of fungal delivery were tested, namely as a daily feed supplement, or incorporated into feed blocks. Initially, pen trials were conducted with individually penned groups of sheep and goats at dose rates of 125,000 spores and 250,000 spores/kg live weight per day. At the lower dose rate this reduction was between 80 and 90% compared with the pre-treatment levels. At the higher dose rate, there was virtually complete suppression (>99% reduction) of larval recovery. Trials using the fungal feed blocks, showed that when animals were individually penned, they consumed only small amounts of the block (particularly goats), hence little effect on larval recovery in faecal cultures was observed. Grouping animals according to species and dose rate induced satisfactory block consumption and subsequent high levels of larval reduction in faecal cultures. These larval reductions were mirrored by the presence of fungus in faecal cultures. This work was followed by a small paddock trial, whereby three groups of sheep were fed either a feed supplement without fungal spores, supplement with spores, or offered fungal blocks. The dose rate of spores in the latter two groups was 500,000 spores/kg live weight per day. Egg counts were significantly reduced in the two fungal groups, compared with the control group and the latter required two salvage anthelmintic treatments to prevent mortality due to haemonchosis. Pasture larval numbers on the two fungal group plots were also much lower than on the control plot.
An estimated 13 million people in the Oriental Region have brugian filariasis. The filarial parasites that cause this disease exist in periodic and sub-periodic forms and are transmitted by four genera of mosquito: Anopheles, Mansonia and, less frequently, Coquillettidia and Ochlerotatus. In most endemic countries, control of the disease has been entirely based on chemotherapy, although house-spraying and use of insecticide-treated bednets can be quite effective against the vectors of nocturnally periodic Brugia malayi and B. timori. The vector-control methods that may be applied against the Mansonia mosquitoes that transmit the parasites causing sub-periodic brugian filariasis are reviewed here. Most of the conventional methods for controlling the immature, aquatic stages of mosquitoes have proved unsatisfactory against Mansonia. The reason is that, unlike the those of other genera, the larvae and pupae of Mansonia spp. are relatively immobile and obtain air not at the water surface but from the underwater roots, stems and leaves of floating plants to which the larvae and pupae attach. Removal of host plants can be very effective in reducing Mansonia productivity, whereas large-scale use of herbicides is restricted by the potential adverse effects on the ecosystem. Environmental management in water-development projects remains the best option.
An Aedes survey using various larval survey methods was conducted in 12 urban housing areas and 29 vacant lands in Sibu town proper. Aedes albopictus larvae were found in all areas surveyed while Aedes aegypti larvae were present in 10 localities and 4 vacant lands. There were no significant difference in the house index, breteau and larval density index of these two Aedes (Stegomyia) species from the survey areas. The proportion of containers positive with Ae. aegypti and Ae. albopictus in area outside the house compound and near the house fencing were 3.2 times higher than outdoor compound. The indoor/outdoor breeding ratio for Ae. aegypti alone is 1.6:1. The most preferred breeding habitats outdoor were plastic cups and used tires while indoor habitats were ant traps and flower vases. In the vacant lands, the average number of larvae per containers was significantly higher than in houses and over 51% of the containers inspected were positive. Shared breeding between Ae. aegypti and Ae. albopictus larvae accounted for 9% in house surveys and 4.5% in vacant land survey. The use of various methods in Aedes larval survey may provide essential information in the study of vector epidemiology in dengue and dengue hemorrhagic fever transmission.
Successful colonization of Mansonia dives, the principal vector of subperiodic Brugia malayi was established in a field insectary. Mean egg clusters laid on Eichhornia crassipes, Pistia stratiotes, Homalomena cordata and polystyrofoam strips were 12.0, 10.4, 9.5 and 13.7 respectively. However, the mean number of first instar larvae hatched from each egg cluster laid by females on the three plant substrates (range 51.1 to 58.6) was higher than that laid on the polystyrofoam strips (41.8). There were no significant differences in the success pupation and adult emergence rates among the three host plants used as attachment substrates. Adult emergence occurred at a mean of 10.8 days. The first adult emergence was observed at the 25th day after hatching and continued till the 50th day. The 50% mortality rates for the adults were estimated as 8 days for the males and 14 days for the females. The mean gonotrophic cycle ranged from 3.8 to 4.3 days with a mean of 4.04 days. 63.6% of Ma. dives females oviposited in a medium of rat dung and water. The mean incubation period of eggs ranged from 5.2 to 6.5 days with a mean of 5.7 days. The biology of Ma. dives and Ma. bonneae is briefly compared.
Studies on larval population densities and adult emergence rates of the Brugian filariasis vectors Mansonia bonneae Edwards and Ma. dives Schiner were conducted in freshwater swamp forest bordering the Sadong River, Serian District, Sarawak, East Malaysia, during 1984-85. Three species of aquatic host-plants in the Family Araceae were identified as supporting immature stages of the Ma. bonneae/dives complex. Proportions of positive plants were 4.7%, 6.5% and 3.4% with 6.4 +/- 2.6, 7.3 +/- 2.8 and 10.1 +/- 1.1 larvae per positive plant, respectively, for the plant species Homalomena cordata Schott, H. rostrata Griffiths and Hydrostemma motleyi (Hook. f.) Mabberley. These data indicate no significant preferences between the three types of host-plant. Detailed monitoring of the host-plant H. cordata revealed no significant monthly fluctuations in larval density per plant nor the proportion of positive plants. 11.6% of larvae were Ma. dives and 88.4% were Ma. bonneae. Mean daily yields of Ma. bonneae/dives adults per square metre of H. cordata vegetated water surface were 0.45 males plus 0.57 females during the wet season (December-February) compared with 0.2 males plus 0.31 females during the dry season (June-August). Thus output of adults per plant was approximately halved, and suitable breeding areas were further reduced, during the dry season. By extrapolation from these rates, a crude mean estimate for productivity of Ma. bonneae/dives females is 1.6 million per hectare per annum in swamp forest habitats vegetated with any of the host-plants studied.
This study focuses on the larvicidal, oviposition, and ovicidal effects of a crude extract of Artemisia annua against Aedes aegypti, Anopheles sinensis, and Culex quinquefasciatus. Dried cells of Artemisia annua from cell suspension cultures were extracted using hexane. The extract showed moderate larvicidal effects against mosquitoes. At 24-h post treatment, the LC50 values for Anopheles sinensis, Aedes aegypti, and Culex quinquefasciatus were recorded as 244.55, 276.14, and 374.99 ppm, respectively. The percentage mortality of larvae was directly proportional to the tested concentration. Anopheles sinensis was found to be the most susceptible species, whereas Culex quinquefasciatus was the most tolerant to the Artemisia annua extract. The results indicated that the Artemisia annua extract showed concentration-dependent oviposition deterrent activity and had a strong deterrent effect. At 500 ppm, the percentage effective repellency was more than 85% compared with the control group for all the species, with oviposition activity index values of -0.94, -0.95, and -0.78 for Aedes aegypti, Anopheles sinensis, and Culex quinquefasciatus, respectively. In the ovicidal assay, the percentage hatchability of eggs after treatment with 500 ppm of Artemisia annua extract was significantly lower than the control, with values of 48.84 ± 4.08, 38.42 ± 3.67, and 79.35 ± 2.09% for Aedes aegypti, Anopheles sinensis, and Culex quinquefasciatus, respectively. Artemisia annua was found to be more effective against Aedes aegypti and Anopheles sinensis compared with Culex quinquefasciatus. This study indicated that crude extract of A. annua could be a potential alternative for use in vector management programs.
The objective of this study was to elucidate the association of various risk factors with dengue cases reported in Lundu district, Sarawak, by analyzing the interaction between environmental, entomological, socio-demographic factors. Besides conventional entomological, serological and house surveys, this study also used GIS technology to generate geographic and environmental data on Aedes albopictus and dengue transmission. Seven villages were chosen based on the high number of dengue cases reported. A total of 551 households were surveyed. An overall description of the socio-demographic background and basic facilities was presented together with entomological and geographical profiles. For serological and ovitrap studies, systematic random sampling was used. Serological tests indicated that 23.7% of the 215 samples had a history of dengue, either recent or previous infections. Two samples (0.9%) were confirmed by IgM ELISA and 49 samples (22.8%) had IgG responses. A total of 32,838 Aedes albopictus eggs were collected in 56 days of trapping. Cluster sampling was also done to determine whether any of the risk factors (entomological or geographical) were influenced by geographical location. These clusters were defined as border villages with East Kalimantan and roadside villages along Lundu/Biawas trunk road. The data collected were analyzed using SPSS version 10.01. Descriptive analysis using frequency, means, and median were used. To determine the association between variables and dengue cases reported, and to describe the differences between the two clusters of villages, two-sample t-test, and Pearson's Chi-Square were used. Accurate maps were produced with overlay and density function, which facilitates the map visualization and report generating phases. This study also highlights the use of differential Global Positioning System in mapping sites of 1m accuracy. Analysis of the data revealed there are significant differences in clusters of villages attributable to container density, house density, distance of the house from the main road, and number of Ae. albopictus eggs from ovitraps set indoor, outdoor and in dumping sites (Person's Chi-Square = 6.111, df = 1, p < 0.01). Further analysis using t-test showed that house density, container density, indoor mosquitoes egg count, outdoor mosquitoes egg count, and dumping sites mosquitoes egg count were higher at the roadside villages compared to border villages. A number of potential risk factors including those generated from GIS were investigated. None of the factors investigated in this study were associated with the dengue cases reported.
Larvae of Aedes albopictus obtained from dengue endemic areas in Selangor, Malaysia were evaluated for their susceptibility to operational dosage of temephos (1 mg/L). Larval bioassays were carried out in accordance to modified WHO standard methods. Biochemical microassay of enzymes in Ae. albopictus was conducted to detect the emergence of insecticide resistance and to define the mechanisms involved in temephos resistance. The 50% mortality lethal time (LT50) for Ae. albopictus tested against temephos ranged between 58.65 to 112.50 minutes, with resistance ratio ranging from 0.75 - 1.45. This study addressed the fluctuation of time-related susceptibility status of Ae. albopictus towards insecticide. Significant difference on the weekly enzyme levels of non-specific esterases, mixed function oxidases and glutathione S-transferases was detected (p ≤ 0.05). No significant correlation was found between temephos resistance and enzyme activity (p > 0.05). Only glutathione S-transferases displayed high level of activity, indicating that Ae. albopictus may be resistant to other groups of insecticide. The insensitive acetylcholinesterase was detected in some field collected Ae. albopictus populations, indicating the possibility of emergence of carbamate or other organophosphate resistance in the field populations. Continuous resistance monitoring should be conducted regularly to confirm the efficacy of insecticides for dengue control.
A study on insect succession of monkey carcass in a forested area in Ulu Gombak, Selangor, Malaysia was conducted from 9 May to 18 June 2007. The third instar of the housefly, Musca domestica (Linnaeus) (Diptera: Muscidae) were only found on dry stage of a decomposed (Day-33) monkey carcass (Macaca fascicularis Raffles). This observation revealed that M. domestica maggots were found together with other muscid fly maggots, Hydrotaea (=Ophyra) spinigera (Stein) (Diptera: Muscidae) on dry stage of a carcass. However, the role of M. domestica on forensic entomological study remains unknown. This study recorded the first finding of M. domestica maggots on primate carcass in Malaysia.
The bioefficacy of nine commercial formulations of temephos against Aedes aegypti, Aedes albopictus and Culex quinquefasciatus larvae were evaluated in the laboratory. WHO larval bioassay with operational dosage of temephos at 1 mg/L was performed. The larval mortality was recorded every 5 minutes until complete mortality was achieved. All formulations of temephos exhibited various toxicity level against Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus. Generally, larvae of Cx. quinquefasciatus was susceptible to all formulations of temephos, followed by Ae. aegypti and Ae. albopictus.
The inhibitory activity of diflubenzuron, a chitin synthesis inhibitor, on the ecdysis of Aedes sp. larvae was evaluated in earthen jars and automobile tires. Two formulations of diflubenzuron were used in this study: Dimilin(R) WP (wettable powder), 25% and Dimilin GR (granular), 2%. The equivalent rate of 25 g/ha, 50 g/ha and 100 g/ha active ingredients for both WP and GR formulations were used in this study. Generally, at the higher dosage of 100 g/ha, both formulations were more effective against Aedes mosquitoes. On the whole, the WP formulation appeared to perform better than the GR formulation in terms of residual activity.
Larvae obtained from Taman Samudera (Gombak, Selangor), Kampung Banjar (Gombak, Selangor), Taman Lembah Maju (Cheras, Kuala Lumpur) and Kampung Baru (City centre, Kuala Lumpur) were bioassayed with diagnostic dosage (0.012 mg/L) and operational dosage (1 mg/L) of temephos. All strains of Aedes aegypti and Aedes albopictus showed percentage mortality in the range of 16.00 to 59.05 and 6.4 to 59.50 respectively, after 24 hours. LT50 values for the 6 strains of Ae. aegypti and Ae. albopictus were between 41.25 to 54.42 minutes and 52.67 to 141.76 minutes respectively, and the resistance ratio for both Aedes species were in the range of 0.68 to 1.82 when tested with operational dosage, 1 mg/L temephos. These results indicate that Aedes mosquitoes have developed some degree of resistance. However, complete mortality for all strains were achieved after 24 hours when tested against 1 mg/L temephos.
Larvae of Aedes aegypti and Aedes albopictus obtained from 6 consecutive ovitrap surveillance (OS) in Taman Samudera and Kg. Banjar were evaluated for their susceptibility to temephos. Larval bioassays were carried out in accordance with WHO standard methods, with diagnostic dosage (0.012 mg/L) and operational dosage (1 mg/L) of temephos respectively. Aedes aegypti and Ae. albopictus obtained from six OS in Taman Samudera showed resistance to diagnostic dosage of temephos with percentage mortality between 5.3 to 72.0 and 9.3 to 56.0, respectively, while Ae. aegypti and Ae. albopictus obtained from Kg. Banjar showed resistance to temephos with percentage mortality between 16.0 to 72.0 and 0 to 50.6, respectively. Only two strains of Ae. aegypti from Kg. Banjar were susceptible to temephos with 93.3% (OS 2) and 100% (OS 3) mortality. The 50% mortality at lethal time (LT50) for all strains of Ae. aegypti and Ae. albopictus tested against operational dosage of temephos showed range between 36.07 to 75.69 minutes and 58.65 to 112.50 minutes, respectively, and complete mortality was achieved after 24 hours. Our results indicated that there is weekly variations of the resistance status for Ae. aegypti and Ae. albopictus. Aedes susceptibility to temephos is changing from time to time in these two study sites. It is essential to continue monitoring the resistance of this vector to insecticides in order to ensure the efficiency of program aimed at vector control and protection of human health.
The bioefficacy of a commercial formulation of temephos, Creek against Aedes aegypti larvae was studied in the laboratory. Earthen jars were filled with 10 L tap water each. One g of temephos (Creek) sand granule formulation was added into each earthen jar as recommended by the manufacturer. The final test concentration of Creek was 1 mg a.i./L. One earthen jar was filled with 10 L tap water and served as a test control (untreated). Thirty late 3(rd) or early 4(th) instar of lab-bred Ae. aegypti larvae were added into each earthen jar. Mortality of the larvae was recorded after 24 hours and percent mortality was calculated. Test was repeated every week. The results showed that complete larval mortality was achieved after 24 hours. The residual effect lasted 15 weeks (105 days), indicating that Creek is effective at the dosage recommended by the manufacturer which is 1 mg a.i./L.
Toxicity tests carried out on the larvae of A. aegypti and C. quinquefasciatus showed the former to be more tolerant of all insecticides tested, the order of toxicity being temephos greater than DDT greater than DDVP greater than malathion greater than lindane greater than carbaryl; also the second instar larvae of A. aegypti were more susceptible than fourth instar larvae. Enzyme kinetic studies on the total non-specific esterases and CarEs of adults and larvae of both species showed the Km values for total esterases of adult A. aegypti to be 0.333 mM vs 0.233 mM for C. quinquefasciatus; for adult CarEs it was 0.250 mM vs 0.220 mM. For total larval esterases of A. aegypti it was 0.112 mM vs 0.175 mM for C. quinquefasciatus: and for larval CarES it was 0.159 mM vs 0.213 mM respectively. Although some correlation between in vivo toxicity (LD50 values) and in vitro esterase inhibition (I50 values) between species could be discerned, overall correlation could not be established.
This study was conducted to evaluate the effectiveness of a floor gully come with grating to prevent the oviposition of Aedes aegypti in the floor trap. In order to conduct the test, two containers were placed into a mosquito cage (30 cm × 30 cm × 30 cm). Both containers were filled with declorinated seasoned tap water and covered with floor gully c/w grating and normal floor gully, respectively. A total of 50 gravid Ae. aegypti females were then released into the cage and left for a week. All the eggs obtained from the test were allowed to remain inside the containers for the eggs to hatch. The number of hatched larvae was counted and recorded. Five replicates were conducted concurrently. There was a significant difference of Ae. aegypti larvae obtained between container with floor gully c/w grating and normal floor gully (p < 0.05). A total of 96.41% reduction of Ae. aegypti larvae was obtained in the container with floor gully c/w grating compared with the normal floor gully, indicating that the floor gully c/w grating used in this study was able to prevent oviposition of Ae. aegypti in holding water.
The pathogenicity of Bacillus sphaericus strain 1593 was tested against laboratory-reared larvae of four local species of mosquitoes of public health importance in Malaysia; Aedes aegypti, Anopheles balabacensis, Mansonia uniformis and Culex quinquefasciatus. The bacteria was shake-cultured at 28 +/- 1 degrees C for three days, using Glucose-Yeast Extract Salts medium. After which, the spores and vegetative cells were harvested and stored at 4 degrees C before use. Conditions for bioassays were mean temperature of 25 +/- 1 degrees C and relative humidity 65 +/- 5.0. Twenty third-instar larvae of each species were assayed in 90 ml of diluted spore solution. Each concentration and a control were replicated three times for each bioassay. Larval mortalities at 24 hours and 48 hours were taken and analyzed through Probit Analysis using a computer (IBM 370). LC50 values after 48 hours of exposure showed an increasing order of larval susceptibility as follows: Ae. aegypti (417.70 x 10(4)), An. balabacensis (45.84 x 10(4)), Ma. uniformis (18.23 x 10(4)) and Cx. quinquefasciatus (4.14 x 10(4) spores/ml). With the ability to kill 90% of the Cx. quinquefasciatus larvae tested with just a concentration of 10(5) spores/ml, B. sphaericus (strain 1593) has shown good potential as a biocontrol agent for this species of mosquito.