Medicine
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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty
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Item Diversity of Wolbachia infections in Sri Lankan mosquitoes with a new record of Wolbachia supergroup B infecting Aedes aegypti vector populations(Nature Publishing Group, 2024) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S; Abeyewickreme, W.; Chandrasena, T.G.A.N.; Thayanukul, P.; Kittayapong, P.Wolbachia bacteria are common endosymbionts of insects and have recently been applied for controlling arboviral vectors, especially Aedes aegypti mosquito populations. However, several medically important mosquito species in Sri Lanka were present with limited information for the Wolbachia infection status. Therefore, the screening of Wolbachia in indigenous mosquitoes is required prior to a successful application of Wolbachia-based vector control strategy. In this study, screening of 78 mosquito species collected from various parts of the country revealed that 13 species were positive for Wolbachia infection, giving ~ 17% infection frequency of Wolbachia among the Sri Lankan mosquitoes. Twelve Wolbachia-positive mosquito species were selected for downstream Wolbachia strain genotyping using Multi Locus Sequencing Type (MLST), wsp gene, and 16S rRNA gene-based approaches. Results showed that these Wolbachia strains clustered together with the present Wolbachia phylogeny of world mosquito populations with some variations. Almost 90% of the mosquito populations were infected with supergroup B while the remaining were infected with supergroup A. A new record of Wolbachia supergroup B infection in Ae. aegypti, the main vectors of dengue, was highlighted. This finding was further confirmed by real-time qPCR, revealing Wolbachia density variations between Ae. aegypti and Ae. albopictus (p = 0.001), and between males and females (p < 0.05). The evidence of natural Wolbachia infections in Ae. aegypti populations in Sri Lanka is an extremely rare incident that has the potential to be used for arboviral vector control.Item The Diversity of midgut bacteria among wild-caught Phlebotomus argentipes (Psychodidae: Phlebotominae), the Vector of leishmaniasis in Sri Lanka(Hindawi Pub. Co., 2020) Gunathilaka, N.; Perera, H.; Wijerathna, T.; Rodrigo, W.; Wijegunawardana, N.D.A.D.ABSTRACT: Phlebotomus argentipes is the main suspected vector for leishmaniasis in Sri Lanka. Investigations on the presence of aerobic bacteria in the gut of sand flies which evidence a potential approach to control leishmaniasis transmission through a paratransgenic strategy are still not available for the local sand fly populations. Field-caught unfed female sand flies collected from three selected Medical Officer of Health (MOH) areas (Polpithigama, Maho, and Galgamuwa) in Kurunegala District, Sri Lanka from August to December 2018 were used. Prokaryotic 16S ribosomal RNA partial gene was amplified and sequenced. Morphological identification revealed the presence of only one sand fly species, P. argentipes (n = 1,969). A total of 20 organisms belonging to two phyla (Proteobactericea and Furmicutes) were detected within the gut microbial community of the studied sand fly specimens. This study documents the first-ever observation of Rhizobium sp. in the midgut of P. argentipes. The presence of Bacillus megaterium, which is considered as a nonpathogenic bacterium with potential use for paratransgenic manipulation of P. argentipes suggest that it may be used as a delivery vehicle to block the vectorial transmission of Leishmania parasites. In addition, Serratia marcescens may be used as a potential candidate to block the parasite development in sand fly vectors since it has evidenced antileishmanial activities in previous investigations. Hence, further studies are required to gain full insight into the potential use of this bacterium in the control of Leishmania parasites through paratransgenesis.Item Aedes aegypti lines for combined sterile insect technique and incompatible insect technique applications: the importance of host genomic background(Blackwell Publishing Ltd, 2020) Carvalho, D.O.; Torres-Monzon, J.A.; Koskinioti, P.; Wijegunawardana, N.D.A.D.; Liang, X.; Pillwax, G.; Xi, Z.; Bourtzis, K.ABSTRACT: Aedes aegypti L. (Diptera: Culicidae), being the primary vector of pathogenic arboviruses, is a target for the development of novel genetic approaches to complement current conventional vector control strategies such as the combined sterile insect and incompatible insect technique (SIT/IIT). A transinfected line of Ae. aegypti carrying the wAlbB Wolbachia strain (WB2) was introgressed into two genomic backgrounds, Brazil and Mexico, producing two new Ae. aegypti strains (WB2-BRA and WB2-MEX). These strains were evaluated with respect to several life-history traits such as fecundity, fertility, longevity, pupa size, pupation curve, and male mating competitiveness, as well as their response to irradiation. Our results show that the impact of Wolbachia infection depends on the genomic background and that the Brazilian one had no significant effect, whereas the Mexican one negatively affected fertility, longevity, and pupal size. Interestingly, Wolbachia-infected Ae. aegypti lines required a lower irradiation dose to achieve complete female sterility than the uninfected ones. The present findings are discussed given the potential use of Wolbachia-infected Ae. aegypti lines in combined SIT/IIT population suppression programs.Item Optimization of Aedes albopictus rearing procedures for combined sterile insect techniques (SIT) and Wolbachia-based laboratory studies in Sri Lanka(Springer Science and Business Media Deutschland GmbH, 2020) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Abeyewickreme, W.; Chandrasena, T.G.A.N.; Dassanayake, R.S.; Manamperi, A.The establishment of a laboratory colony is an essential first step for conduction of laboratory studies on the biology and control of Aedes albopictus mosquitoes. Therefore, with the objective of generating high quality mosquitoes as research material for utilization in ongoing vector control studies and to disseminate authenticated, high-quality Ae. albopictus mosquito rearing information to the research community, maintenance of an Ae. albopictus mosquito colony was initiated at the Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka. A self-mating colony was established from eggs of the F1 progeny of individuals collected as free-living larvae in Narahenpita (Western Province), Sri Lanka. The mean temperature of 28 °C (± 2 °C) and relative humidity of 80% (± 5%) was constantly maintained inside the insectary. Lighting was provided by fluorescent lights, regulated with 12:12 h continuous dark and light period. Pest insects were controlled manually. Mosquitoes were maintained on bovine blood provided via an artificial membrane feeding system and a continuous supply of 10% sugar supplements. Larvae were maintained in deoxygenate water and fed with International Atomic Energy Agency (IAEA) recommended diet of tuna meal, bovine liver powder, and brewery yeast in a ratio of 37.5:27:10.5 in 1 L. Data on fecundity, fertility, larvae death, pupation, adult emergence, adult mosquito longevity were recorded. Adhering to bio-safety, all discarded materials were boiled thoroughly and incinerated if required. This report on the establishment and maintenance of a laboratory colony of Ae. albopictus will be of value for identifying the critical requirements essential under artificial conditions.Item Evaluation of the effects of Aedes vector indices and climatic factors on dengueincidence in Gampaha District, Sri Lanka(Hindawi Publishing Corporation, 2019) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Chandrasena, T.G.A.N.; Dassanayake, R.S.; Udayanga, N.W.B.A.L.; Abeyewickreme, W.Constant monitoring of Aedes vector indices such as Aedes mosquito abundance and ovitrap data is important for the control of dengue epidemics. Therefore, the current study attempted to evaluate the effect of larval and climatic factors on the incidence of dengue outbreaks in the Gampaha district. Based on the distribution of previously reported dengue cases, 34 households in Narangodapaluwa PHI area, Ragama, Sri Lanka, were selected randomly, and entomological surveillance was done fortnightly using adult mosquito catches and larval surveillance techniques for a period of two years. Further, weekly ovitrap surveillance was conducted for one year, by maintaining four ovitraps in a single house, two indoors and two outdoors at ground and at a height of 1.5-2 m. Based on the findings, larval indices, namely, Breteau index (BI), House index (HI), and Container index (CI), were calculated, along with the Ovitrap index (OI). The study area was positive for Ae. albopictus with an adult capturing range of 1~15/34 households. BI initially remained < 3%, which subsequently decreased up to 0. No significant difference in OI was found between the ovitraps placed at ground level and at a height of 1.5-2m (p>0.05), 95% level of confidence. The OI varied from 56.9% to 94.7% during the study period of 12 months, indicating two peaks at the monsoons. Statistics of one-way ANOVA revealed a significant difference in the monthly OI during the study period (p≤0.001) with two peaks representing the monsoonal rainfall patterns. Pearson's correlation analysis revealed that the association between dengue cases and larval indices (BI, CI, HI, and OI) and meteorological parameters was not significant (p<0.05). Migration of mosquitoes and patients could be considered as possible factors affecting the absence of a significant relationship.Item Male isolation of dengue vector Aedes mosquitoes for sterile insect technique by spiking blood with ivermectin(Sri Lanka Association for the Advancement of Science, 2015) Abeyewickreme, W.; Wijegunawardana, N.D.A.D.; Ranathunge, R.M.T.B.There is an increasing demand for exploration of the potential for applying the Sterile Insect Technique (SIT) in area-wide integrated vector management (AW-IVM) in many countries. However, because female mosquitoes, unlike male mosquitoes, can transmit disease, means to eliminate them from the mass production process are a critical pre-requisite. In addition, the efficiency of the SIT programme could be increased by not releasing female mosquitoes due to the fact that sterile males can then only focus on wild females to achieve mating. Therefore, mosquito SIT programmes success will depend on exclusive release of sterile males, which is impossible on a large scale without efficient sex separation methods. Many different sex separations for different stages of mosquitoes are currently being attempted to successfully establish a sexing mechanism for the above purpose. Among them, the mostly attempted methods broadly include genetic sexing methods, molecular methods, mechanical methods and behavioral methods. For all blood feeding mosquitoes, sex separation could occur at the adult stage by spiking blood with insecticides (malathion, dieldrin) or other mosquito toxins (ivermectin, spinosad), as behavioral tools. In this study, a veterinary preparation of ivermectin (Ivotec,1% w/v) which is used to treat dogs infested with the filarial worm Dirofilaria repens in Sri Lanka was used as a tool for sex separation of adult Aedes albopictus and Aedes aegypti mosquitoes by feeding on spiked blood. In an initial attempt by spiking blood with 5 parts per million (5 ppm) ivermectin, 40% of the blood–fed females could survive beyond 24 hours post feeding. However, when the dosage of ivermectin was increased to 7 ppm the survival rate decreased below 40%. Since there had been high mortality of both females and males even in the control cages in which mosquitoes were blood-fed without ivermectin it was suggested that the age of the mosquitoes used may be a factor for high mortality of mosquitoes in the control experiment. When the experiment was repeated with 7 ppm ivermectin in blood using 4-5 day old mosquitoes more than 65% mortality could be observed within 12 hours post feeding in the experimental cage with a significant difference in mortality compared to the controlItem Maternal transformation of Wolbachia isolated from infected mosquito hosts to Aedes aegypti using micro-injection based procedure: an approach towards integrated dengue vector control(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Chandrasena, T.G.A.N.; Dassanayake, R.S.; Ruanareerate, T.; Kittayapong, P.; Abeyewickreme, W.BACKGROUND: The maternal transformation of Wolbachia (Wb) to Aedes mosquitoes is an approach that has been widely used in Aedes mosquito population control strategies in the integrated dengue vector control efforts. This study details usage of such approach in the maternal transformation of Aedes aegypti using the Wb isolated from infected mosquito hosts using micro-injection based procedure for the first time in Sri Lanka. METHODS: Twenty five ovaries of each adult female Aedes albopictus and Culex quinquifaciatus mosquitoes (14-18 days old) were dissected and Wb was isolated and suspended in 20 μl of 1X Phosphate Buffered Saline (PBS). The isolated samples were kept on ice to maximize the bacteria viability. The exudate was then used in the trans-infection of Ae. aegypti mosquitoes. Infection of Wb into the mosquito Ae. aegypti was carried out by maternal/adult microinjection. To create stable trans-infected lines, the regions near the 3rd leg emerged from the thorax of Ae. aegypti were microinjected to incorporate Wb into the developing germ line and then to facilitate the transmission of Wb to offspring. The volume used for injection based on the swelling of the abdomen, which was monitored throughout the experiment while maintaining the injection pressure, time and number of injecting times. Virgin female Aedes mosquitoes survived after microinjection was fed with 1 ml of 10% sugar solution containing 10,000 units Penicillin/Streptomycin per 100 ml. Survival rate was monitored at 1, 3 and 24 hour/s following microinjection procedure. Survived female mosquitoes resulted from injected line (generation 0 [G0]) was placed in vials with three uninfected male mosquitoes to establish iso-female lines. After egg laying 3 times, G0 females were sacrificed and total DNA was extracted, and PCR was carried out (wsp primers 81F and 691R) using DNA extracted from Ae. aegypti to detect the presence of Wb DNA and its density in the latter mosquito using dilution series of extracted DNA. To select for a stable infection, only offspring from females that came positive for Wb by PCR screening was used as parental stocks. For the samples positive by wsp PCR, strain identification was carried out by performing PCR with 4 different primer sets; wPip, wAlbA, wAlbB and wsp specific for Wb. Females that tested negative for Wb were discarded along with their progeny. The resulting triple infected line will be monitored periodically by PCR to confirm infection status. RESULTS: Wolbachia-triple infected line of Ae. aegypti mosquito was created by using isolated wAlbA+wAlbB+wPip Wolbachia strains. Infection status of newly developed line was monitored by PCR up to G2 and will be continued to G8. Experimental work will be started from G5 to evaluate the use of newly develop Ae. aegypti mosquito line for Wb-based approach under laboratory condition. CONCLUSION: Direct maternal micro-injection technique was used to create Wb infected mosquito line of Ae aegypti, which could be applied in Wb-based dengue vector control activities in Sri Lanka once the line is established.Item Knowledge and practices of dengue control and their effect on breeding sites and vector populations(Sri Lanka Medical Association, 2010) Pinidiyapathirage, M.J.; Gunethilake, M.A.G.N.; Wijegunawardana, N.D.A.D.OBJECTIVE: To describe the association between knowledge and practices of dengue control with the observed dengue breeding sites and vector populations in a community. METHODS: A cross-sectional survey was conducted among households in Negombo area in July 2009 during the height of the dengue epidemic in the country. Knowledge and practices in this community were measured using two methodologies. The first was a questionnaire, to be answered by the household respondent; the second, a mosquito larval survey, to identify potential and actual mosquito breeding sites in the corresponding household. The use of the two instruments allowed comparison of respondent knowledge and reported practice with the actual condition of the compound. RESULTS: All respondents (n=120) have heard of dengue, 89% knew at least one symptom of dengue and 93% knew that a mosquito was responsible for transmission. 60% correctly stated the preferred biting times of the vector and 80% knew that discarded tyres, bottles and plastic containers with water collections were the potential vector breeding sites. On inspection, 104 (87%) households had at least one potential breeding source. However, Aedes aegypti/albopictus larvae/pupae were observed only in water samples collected from 3 (2.5%) households. Knowledge about dengue symptoms, methods of prevention or level of education had no association with the potential breeding sites found. Low income was significantly associated with having at least one potential breeding source in the household. CONCLUSION: Although the level of knowledge about dengue was high in this community, we found only httle evidence that this knowledge was put into practice.Item Impact of repeated annual Diethylcarbamazine-Albendazole mass treatment on transmission of Wuchereria bancrofti in the Gampaha district.(Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, 2007) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Abeyewickreme, W.Entomological and parasitological surveys are critical for the baseline evaluation of impact of repeated annual. Diethylcarbamazine-Albendazole mass treatment on the reduction of microfilaria density and level transmission of Wuchereria bancrofti in Culex quinquefaciatus. The objective of this study was to assess the current situation, following the mass drug administration programme in the Gampaha district, with regard to lymphatic filariasis, using entomological and parasitological data. A pilot survey was carried out using parasitological, 'clinical and entomological indicators in 21 sites in 7 Medical Officer of Health areas of Gampaha district to assess the current filariasis situation. The localities were selected from. the Medical Officer of Health areas based on previous data obtained from the regional Anti Filariasis Campaign office. Results indicate that 76.19% (16/21) sites were infested with mosquitoes positive for Wuchereria bancroft and the positivity of 1.44% (31/2157) was observed among the mosquitoes caught from households in the selected sites. The microfilariae wasa determined to be 15.5 per positive mosquito. The parasitological result was indicated 0.017 % prevalence of lymphatic fllariasis in the selected population. Data recorded by the Anti Filariasis Campaign Gampaha in 1994, suggested that significant decrease of infective rate, positivity of mosquito and microfilaria density, which are respectively 90%, 3.05% and 23. Study confirms that active transmission of Wuchereria bancrofti is currently taking place in the Gampaha district, despite, the mass drug administration Programme been implemented since 2002. This study highlights the urgent requirement of a proper screening programme combined with anti filarial treatment and vector control programme to minimize filarial morbidity and interrupt filarial transmission within the country.Item Ovi-trap based surveillance for Aedes in dengue endemic Gampaha District, Sri Lanka during rainy season(Faculty of Tropical Medicine, Mahidol University, 2008) Hapangama, H.A.D.C.; Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Benjamin, S.; Abeyewickreme, W.Dengue is endemic in Gampaha District, Sri Lanka. Aedes aegypti and Aedes albopictus have been incriminated as vectors in urban and suburban environments. An ovitrap-based weekly surveillance was carried out to determine the distribution and abundance of Dengue vectors, in six dengue endemic areas; Galudupita, Gongitota, Wanawasala, Kerangapokuna, Mabole and Welikadamulla in the Gampaha District during May and June 2008 in both indoors and outdoors. Galudupita and Gongitota gave the highest ovitrap indices, both indoors and outdoors as 97% and 73% respectively. Of the total positive ovitraps(1320), positivity for Ae. albopictus, Ae. aegypti and both were 63.5%, 32.9% and 3.6% respectively. Highest mean number of Ae. aegypti and Ae. albopictus larvae per ovitrap in both indoor and outdoor ovitraps were observed Galudupita and Gongitota respectively. However, statistical analysis showed that ovitrap index of Ae. aegypti in six study was not significantly different from each other (P > 0.05). Outdoor mean number of Ae. aegypti larvae per ovitrap in Galudupita, was significantly higher than other sites (F=3.587, P=0.012). Indoor Ae. albopictus ovitrap index of Gongitota was significantly higher than of Welikadamula (F=2.59, P=0.046) while outdoor Ae. albopictus ovitrap index of Gongitota and Wanawasala was significantly higher than of Welikadamula F=3.19 P=.0.02). Results irnplicate that Ae. albopictus prefers more vegetation as observed in Gongitota and Wanawasala. In highly populated and urbanized Galudupita, area with less vegetation, Ae. albopictus population was less than Ae. aegypti even outdoors. Study implicates that Ae. albopictus is the predominant indoor and outdoor breeder during rainy season.