International Postgraduate Research Conference (IPRC)
Permanent URI for this communityhttp://repository.kln.ac.lk/handle/123456789/155
Browse
5 results
Search Results
Item Optimization of Irradiation Dose for Sterilization of Aedes aegypti (Linnaeus) Mosquitoes for Application of Sterile Insect Technique (SIT) Program in Sri Lanka(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Ranathunge, T.; Adikari, D.; Harishchandra, J.; Gunawardene, N.S.; Hapugoda, M.There is an increasing demand for exploration of the potential of applying sterile insect techniques (SIT) in area-wide integrated vector management (AW-IVM) in many countries. Sterility of male insects can be accomplished with ionizing irradiation and SIT focus on release of sufficient sterile male mosquitoes to induce sterility in the wild females which over time causes decline of the target mosquito population. Therefore, current study was focused on determining the effects of different doses of radiation on survival, flight ability and reproductive capacity of local strains of Aedes aegypti (Linnaeus) for SIT application in Sri Lanka. Adults Ae. aegypti were maintained under standard laboratory conditions at the Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka. Male pupae and female pupae were separated using a Fay and Morlan glass plate technique. A total of 100 male Ae. aegypti pupae were exposed to each different irradiation doses (40, 50, 60, 70 and 80 Gy) using Co60 source of gamma rays. Effects of irradiation on pupal mortality, flight ability, fertility and adult male survival were monitored under laboratory conditions. One-way analysis of variance (ANOVA) was used to investigate the significance in the variations among observed factors. Kaplan-Meier survival analysis was conducted to estimate the survival functions of irradiated males. The survival in relation to different radiation doses were compared using ANOVA followed by Tukey’s pairwise comparison. The survival of irradiated pupae was invariably greater than 90% in control- and in test groups and they did not differ significantly (P > 0.05). Irradiation had no significant adverse effects on the flight ability (capacity to fly out of a test device) of male mosquitoes, which consistently exceeded 90%. The fertility of female mated with irradiated male was significantly reduced in Ae. aegypti at all doses and zero fertility was observed at 70 and 80 Gy. Ae. aegypti, fertility in irradiated males mated with female was less than 1% at 50 and 60 Gy. The male mean survival time was reduced by irradiation in a dose-dependent manner. However, the mean survival time in control and sterilizing doses of 40, 50, 60 and 70 did not differ significantly (P > 0.05). Therefore, 50 Gy dose will be used as the optimal radiation dose Ae. aegypti population for future evaluations of SIT-based control. The results of the present study will be applied to studies of male sexual competitiveness and for stepwise evaluations of the SIT for suppression of Ae. aegypti population in Sri Lanka.Item Assessment of Real Time Polymerase Chain Reaction (PCR) Assay for the Early Diagnosis of Leptospirosis in Humans(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Uduwawala, U.M.H.U.; Manamperi, A.; Gunaratna, G.P.S.; Karunanayake, L.; Chandani, W.L.; Hapugoda, M.Leptospirosis is the most widespread zoonotic disease worldwide having a great impact on health issues in developing countries. It is caused by a pathogenic spirochete of the genus Leptospira where humans become infected through contact with the urine of infected animals. It is often exceptionally under-recognized as the clinical manifestation mimics variety of similar disease conditions that occur in the same environmental and climatologic conditions which accentuate the importance of laboratory diagnosis of leptospirosis. At present, no hospital based facilities are available for acute confirmation of the disease. The existing practice is retrospective confirmation with serological diagnosis. Therefore, the establishment of acute phase diagnosis will help in monitoring the disease, determining when hospital admission is required and reduce case fatalities. The objective of this study was to establish and evaluate a molecular-based assay to provide laboratory confirmation of leptospirosis at the acute phase of the infection (1-5 days of fever). Patients fulfilling clinical criteria stipulated by the accepted case definition were selected for the study and patients who failed to show evidence of sero conversion were considered as true negatives. A real time Polymerase Chain Reaction (PCR) assay with targeting a 203 bp fragment in the secY gene which is conserved among pathogenic serovars of Leptospira was established using a reference DNA sample (L.interrogans serovar Icterohaemorrhagiae strain RGA). Analytical sensitivity and the analytical specificity of the assay were calculated. The accuracy of the real time PCR was determined by a panel of acute blood samples collected from laboratory confirmed leptospirosis patients (n=35) and non-leptospirosis (n=44) patients based on Microscopic Agglutination Test (MAT) and/or IgM immunochromatography. Patients who failed to give positive test results either with MAT or IgM immunochromatography were considered as true negatives. Analytical sensitivity was approximately 314 genome equivalents per reaction and analytical specificity showed no amplification of Leptospira saprophytic sp. and other micro-organisms. The assay could effectively detect Leptospira DNA from clinically diagnosed leptospirosis suspected patients with 60.0% (21/35) diagnostic sensitivity and 77.27% (34/44) diagnostic specificity. This may be attributed to some samples failing laboratory confirmation despite their collection based on clinical suspicion. Therefore, real time PCR established can be used for rapid and definitive diagnosis of leptospirosis during the acute phase of infectionItem Isolation of a Potential Microbial Agent for Controlling Dengue Vector Mosquitoes in Sri Lanka(19th Conference on Postgraduate Research, International Postgraduate Research Conference 2018, Faculty of Graduate Studies,University of Kelaniya, Sri Lanka, 2018) Induwara, R.; Fernando, M.; Ranathunge, T.; Parakrama, G.; Hapugoda, M.Controlling dengue vector mosquito is the most appropriate controlling method for dengue in the absence of an effective drug or a vaccine for dengue viruses. Vector control can be performed using variety of approaches such as environmental management, chemical and biological control etc.; which have been used in recent vector control activities. Due to the development of resistant varieties against chemical insecticides, the present study attempted to identify larvicidal activity of bacteria collected from the natural environment, as an ideal environmental friendly and effective strategy for controlling Aedesaegypti (Linnaeus), a dengue vector mosquito species. Zero dengue infection was reported in Pudumurippu area in the District of Kilinochchi, Northern Province of Sri Lanka in a preliminary survey. Spore forming microbes were isolated through spread plate technique using water and sediment samples collected from a reservoir in Pudumurippu. Larvicidal activity of each isolate was tested by Ae. aegypti third instar Larvae (L3) in vitro. The highest larvicidal activity was observed in an isolated bacterium from a reservoir water sample under laboratory and field conditions. This bacterium was presumptively identified and subjected to 16s-rRNA sequence analysis. Larvicidal activity of this bacterium was compared with a currently used Bacillus thuringiensisisraelensis (Bti). As well as optimum physiological characteristic features of isolated strain was determined by growing the bacteria strain on nutrient agar supplemented with different NaCl concentrations and different pH values. Isolated bacterium was confirmed as a new strain of Bacillus cereus (SL001; MG827268). This bacterial strain showed the highest larvicidal activity at 5% (1×105 CFU/ml), with mean cumulative mortality rate 92±4.1% and 84.2 ±5.3% at 48 hours’ post challenged under laboratory and field conditions respectively. When compared with Bti, this novel strain showed significantly higher (p < 0.05) larvicidal activity. B. cereus (SL001) displayed high growth rate while tolerating wide range of salinity (0-30gL-1) and pH (6-10). Based on the findings, B. cereus (SL001) with the highest larvicide efficiencies could be an ideal candidate for biological controlling of Ae. aegypti dengue vector mosquitoes in Sri Lanka. Further analysis of this bacterium is on going at present.Item Laboratory Evaluation of the Bio-control Efficacy of Selected Copepods on Dengue Vectors of Sri Lanka(19th Conference on Postgraduate Research, International Postgraduate Research Conference 2018, Faculty of Graduate Studies,University of Kelaniya, Sri Lanka, 2018) Udayanga, N.W.B.A.L.; Ranathunge, R.M.T.; Iqbal, M.C.M.; Abeyewickreme, W.; Hapugoda, M.Among variety of alternatives for management of dengue vectors, biological control remains as a promising approach, due to its high efficacy, sustainability and low impacts on human and ecosystem health. Copepods are considered asa leading predator of mosquito larvae including Aedes vectors. However, the predatory potential of different copepods on bio-control of dengue vectors has been less studied in Sri Lanka. Therefore, the current study aimed to evaluate the predatory success of five locally abundant copepods on both AedesaegyptiandAe. albopictus. Copepod collections were made from water bodies and rock pools located in the Kandy and Gampaha districts by using a plankton net. After morphological identification, single gravid copepods of different species were used to establish copepod cultures under standard laboratory conditions. Five adult copepods of each species were transferred into containers and groups of 200 first instar Ae. Aegypti larvae were introduced in to each container, separately. The number of surviving larvae in each container was recorded at 3 hour intervals up to 24 hours. Five replicates were conducted for each copepod species. Same experimental design was followed for Ae. albopictus. General Linear Modelling technique (GLM) followed by Tukey’s pair-wise comparison was used to make statistical inferences on the significance of average larvalconsumption rates by studied copepod species. SPSS (version 23) was used for the statistical analysis. Five species of copepods, namely Cyclops languides, C. varicans, C. vernalis, Mesocyclopleuckarti and M. scrassus were considered during the study. Average predation rates of 34.9±1.80 and 33.5±1.06 for Ae. aegypti and Ae. albopictus, respectively, were indicated by M. leuckartias the highest predation rates. Meanwhile, M. scrassus showed the second highest predation rates for both Aedes vectors. On the other hand, the lowest predatory efficacy was shown by C. languides with 10.6+1.60 and 8.4+1.10 for Ae. aegypti and Ae. albopictus, respectively. The average consumption rates of different copepod species varied significantly (p<0.05 at 5% level of significance). The type of Aedes species had a significant influence on the predatory efficacy of studied copepods (p< 0.05) under laboratory conditions. In conclusion, M. leuckartiand M. scrassus that reported the highest predation efficacies on both Aedes larvae could be suggested as potential bio-control agents for dengue vector management approaches in Sri Lanka after semi field and field settings.Item Sterile Insect Technique for Controlling Aedes albopictus (Skuse); Sex Separation and Mating Competitiveness(19th Conference on Postgraduate Research, International Postgraduate Research Conference 2018, Faculty of Graduate Studies,University of Kelaniya, Sri Lanka, 2018) Harishchandra, J.; Hapugoda, M.; Premaratne, R.G.; Abeyewickreme, W.Sterile Insect Technique (SIT) introduced by Edward F. Knipling in 1950s has been shown as an effective and sustainable genetic approach to control populations of selected pest insects in area wide integrated pest management programmes. Successfulness of this approach depends on the availability of an efficient sexing method and production of sterile males competitive enough to compete with wild males. In the context of developing SIT for the control of dengue vector Aedes albopictus (Skuse) in Sri Lanka, a method for separation of male pupae was tested and mating competitiveness of irradiated males against wild males was evaluated under laboratory conditions. Male pupae of the F42 generation of Ae. albopictus (Gampaha strain) were separated from female pupae using mechanical sieving method. A set of three stainless steel sieves of mesh sizes 1.4, 1.25 and 1.12mm were placed one on the other in descending order of size towards the bottom, forming a column immersed in a water jar. All pupae were poured on to the top sieve and shaken gently for seven minutes. Pupae were collected from each sieve and the male and female counts were taken after emergence of adults. Male and female pupae used for the competitiveness assessment were separated by the above sieving method. Male pupae (F42, age 28-40hours) were exposed to Gamma radiation (Gamma 220, Atomic Energy of Canada Ltd., Co60) at a dose of 50Gy by placing 300 male pupae in each plastic vial with 10ml of water. Dosimetry was performed using Frickey method. Wild fertile males and females reared from field-collected eggs from Gampaha (F1) were used to compete and cross with irradiated males respectively in different ratios. Two days after the emergence, the adult mosquitoes were caged (Bug Dorm 30X30X30cm) with ratios of irradiated males: wild males: wild virgin females 1:1:1, 3:1:1 and 5:1:1 (females, n = 100). Fertile control was set with wild males and wild virgin females 1:1. and an irradiated control was set with irradiated males with wild virgin females 1:1. Mosquitoes were allowed to mate for 3 days with access to 10% sugar solution. Then the mosquitoes were fed with cattle blood and engorged females were isolated in oviposition tubes. After 4 days, females were returned into cages and the filter papers containing eggs were allowed to dry. Eggs were hatched after one week and the hatching rates were compared. From the pupae retained in the sieve of mesh size 1.12mm, 98.3% were males. The mean hatch rates of fertile and irradiated controls and with ratios of irradiated males: wild fertile males: wild virgin females 1:1:1, 3:1:1, 5:1:1 were 80.24%±1.63SEM, 2.42%±0.57SEM, 56.74%±5.79SEM, 37.33%±4.76SEM and 34.23%±4.73SEM respectively. Fried Competitive Index (C) of irradiated males compared to controls was 0.43. Results show sieving method can be used for separating male pupae for irradiation with 98% accuracy. Irradiated males were less than 50% competitive as wild males under laboratory conditions