Graduate Studies

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Aedes albopictus the “underrated” Asian Tiger
(University of Kelaniya, 2010) Jayasooriya, D.H.S.W.; Gunawardene, Y.I.N.S.; Manamperi, A.; de Silva, H.J.; Abeyewickreme, W.
Introduction The mosquito Aedes aegypti was thought to be the main vector responsible for virtually all dengue epidemics; while Aedes albopictus was considered a vector in which the virus is maintained but does not cause epidemics. Objective The study was conducted covering three endemic districts in Sri Lanka to determine the role of genus Aedes during dengue transmission. Methods and Material Mosquitoes were collected within a 350m radius from the location of the positive patients. Heads and abdomens of 63 pools were tested for DENV RNA with and RT-PCR-LH-(P32) assays Results Discussion Ae. albopictus was present in majority of the locations in all districts surveyed. Ae. albopictus was found in 13/17 (76.47%), 24/25 (96%)and 19/22 (86.36%) sites in Colombo, Gampaha and Kurunegala respectively. The RT-PCR-LH-(P32) assays indicated that 5/25 (20%) sites in Gampaha, 2/17 (11.76%) in Colombo and 6/22 (27.27%) in Kurunegala were positive for DENV. In Gampaha and Colombo there were 3 and 1 of DEN-2 positive pools respectively, while there were 2 and 1 of DEN-3 positive pools respectively. A higher number of positive pools (4/1or 21.05%) for DEN-1 and 1/1(5.26 %) for DEN-4 were found in Kurunegala. In Kurunegala one pool was positive for both DEN-2 and DEN-4 indicating the circulation of multiple serotypes within close proximity. Moreover one of the three DEN-2 positive pools in Gampaha consisting of only male Ae. albopictus mosquitoes is supportive of the belief of vertical transmission of DENV. In a DEN-4 positive location in Kurunegala HI was found to be10%, BI= 1and CI= 5.88 %while anotherDEN-2 positive site in Wattala showed HI of 5.55%and a BI of 5.55 suggesting active transmission. The abundance of Ae. albopictus in all districts and the findings indicating that100% of the positive pools were made of Ae. albopictus in this study highlights the importance of Ae. albopictus in the transmission dynamics dengue. The ability of Ae. albopictus to be infected with low viremia and the degree to which it permits replication within the mosquito itself could have an impact on the transmission and these verity of the disease. Co-circulation of two or more serotypes in a single pool or in different pools of mosquitoes within the same district is suggestive of hyper endemic transmission dengue in the three districts. The greater susceptibility of Ae. albopictus to infection by DENV is said to lead to greater virus adaptation. Sri Lanka as a whole would be at serious risks for multiple outbreaks in future. Our results indicate that Ae. albopictus is more efficient in dengue transmission than previously thought. The results shed light on the efficiency of Ae. albopictus as a vector in transmitting DENV in the absence or low abundance of Ae. aegypti in Sri Lanka. The present study suggests that Ae. albopictus sp is underrated in terms of transmission potential during peak transmission periods of dengue in Sri Lanka. Key words: RT-PCR-LH-(P32) RT-PCR-Liquid Hybridization with P32 radio isotope, HI-House hold Index, BI- Breteau Index, CI-Container Index,DENV-Dengue Virus Authors wish to acknowledge the financial assistance rendered by the NSF Sri Lanka (GrantNo:SIDA/2006/BT/02)and the IAEA (Grant NoTC SRL 6/028).
Application of a Real Time Polymerase Chain Reaction (PCR) for Detection of Pathogenic Leptospira in Clinical Samples
(University of Kelaniya, 2012) Denipitiya, D.T.H.; Jiffrey, A.M.; Abeyewickreme, W.; Wellawaththge, C.; Hapugoda, M.D.
Leptospirosis, is a zoonotic disease with worldwide distribution, caused by pathogenic species of the genus Leptospira. It has the greatest impact on health in developing countries where it is often grossly under-recognized. Clinical features are similar to a range of other infectious diseases that occur in the same environmental and climatologic conditions. Therefore, laboratory confirmation is essential for proper management of leptospirosis patients. Molecular assays offer definitive laboratory confirmation of leptospirosis at the early phase of infection (1-5 days of fever) within a few hours. The objective of this study was to establish and evaluate potential use of a real time- PCR assay for early, definitive laboratory confirmation of leptospirosis patients. A SYBR green-based real time PCR assay targeting a 203 bp fragment on the secY gene which is conserved among pathogenic serovars of Leptospira was established using a reference DNA sample (Leptospira interrogans strain RGA). Analytical specificity of the assay was tested with the DNA from pathogenic and non-pathogenic Leptospira spp. and five other micro organisms. Analytical sensitivity of the assay was tested using serial dilutions of the reference sample. A panel of acute blood samples (n=150) collected during early phase of infection (1-5 days of fever) from leptospirosis suspected patients was used for evaluation of real time PCR vs qualitative PCR. The results show, real time PCR assay with high analytical specificity (100%) was established and the assay shows 100 times higher sensitivity over qualitative PCR assay (1.3 pg/ml). Real time PCR and qualitative PCR could diagnose current leptospirosis infection in 37.3% (56/150) and 19.3% (29/150) suspected patients respectively. These results indicate high sensitivity of real time PCR over qualitative PCR for diagnosis of leptospirosis patients. In conclusion, this study shows that real time PCR has the potential to facilitate rapid and sensitive diagnosis of acute leptospirosis during early phase of infection.
Assessment of artificial blood feeding methods and efficacy of different blood meal sources in mass rearing of Aedes aegypti for innovative vector controlling strategies
(Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2016) Gunathilaka, P.A.D.H.N.; Ranathunga, R.M.T.B.; Udayanga, N.W.B.A.L.; Abeyewickreme, W.
Background Mass rearing of vector mosquitoes of dengue is a crucial requirement for implementation of innovative vector controlling strategies such as Sterile Insect Technique (SIT), Incompatible Insect Technique (IIT) and transgenic mosquitoes. Selection of the artificial membrane-feeding technique and blood meal source have been recognized as key considerations in mass rearing of vectors. Methodology Three artificial membrane feeding techniques (Glass plate method, Metal plate and Hemotek membrane feeding method) and three blood sources (human, cattle and chicken) were evaluated based on feeding rates, fecundity and hatching rates of Ae. aegypti, under laboratory conditions. One-way ANOVA, cluster analysis, analysis of variance (ANOSIM) and principal coordinates (PCO) analysis, were used to investigate the significance in the variations among blood feeding. Results Feeding rates of Ae. aegypti significantly differed among the membrane feeding techniques as suggested by one-way ANOVA (p <0.05). The metal plate method was identified as the most efficient and cost effective feeding technique. Blood feeding rate of Ae. aegypti was higher with human blood followed by cattle and chicken blood, respectively. However, no significant difference was observed among mosquitoes fed with cattle blood and human blood, in terms of fecundity, oviposition rate and fertility. Conclusions Metal plate feeding technique could be recommended as the most effective membrane feeding technique for mass rearing of Ae. aegypti, due to its high feeding rate and cost effectiveness. Since, obtaining human blood for insectary colony maintenance is problematic due certain ethical issues, cattle blood could be recommended for mass rearing Ae. aegypti.
Assessment of possible social and entomological risk factors affecting transmission of dengue in the District of Gampaha
(Faculty of Graduate Studies, University of Kelaniya, 2015) Withanage, G.P.W.K.; Abeyewickreme, W.; Hapugoda, M.D.
Dengue is the most important mosquito-borne viral infection transmitted to humans in Sri Lanka causing more than 30,000 cases annually. The objective of the study was to identify possible social and entomological risk factors affecting transmission of dengue in Gampaha district where the second highest number of dengue cases recorded during last ten years. Four Medical Officer of Health (MOH) areas were selected based on annual number of dengue cases greater than 250 for last ten years. One GramaNiladhari (GN) division was selected from each MOH area as a study area where the highest number of dengue incidents reported. In each study area, a cluster of 150 households was selected and household and entomological surveys were conducted. The selected areas were Eriyawatiya (Kelaniya MOH), Welikadamulla (Wattala MOH), Akbar town (Mahara MOH), and 3-Kurana (Negombo MOH) GN divisions. There were 2577 population in 600 households in the study areas and 53.5% were females. Average size of a homestead was 17 perches and most of the households (98%-588/600) were individual house. Main vegetations in the homesteads were grass and bushes (97%-583/600) and potential breeding places of dengue vector mosquitoes were observed in 96.1% (577/600) households. Main dengue vector mosquito present in the captured Aedes adult mosquitoes was Aedes albopictusis (92.9%-183/197). Most prominent breeding places were discarded bottles and tins (4.0%-15/371), plant axils (9.7%-36/371), plastic containers (26.4%-98/371), and roof gutters (4.3%-16/371) in the observed premises and 9.2% (34/371) were Aedes larvae positive. Most frequent mosquito preventive measure was bed-nets (30.3%-182/600), but mosquito coils (30.8%-185/600) and vaporizers (17.6%- 106/600) were frequently used. Participants have adequate knowledge about the disease, but they neglect preventive measures and highly depend on government vector control programs. Unplanned households, crowded conditions, poor waste management systems, and negligence to pursue preventive measures may be possible risk factors and therefore, frequent encouragement is needed to control dengue transmission.
Blood-feeding patterns of Anopheles mosquitoes in malaria-endemic areas of Sri Lanka
(University of Kelaniya, 2012) Gunathilaka, P.A.D.H.N.; Fernando, M.A.S.T.; Hapugoda, M.D.; Wijeyerathne, P.; Wickremasinghe, A.R.; Abeyewickreme, W.
Background: Studies on host preference patterns in blood-feeding of anopheline mosquitoes are crucial for incriminating them as malaria vectors. However, little information is available on the host preferences of Anopheles mosquitoes in Sri Lanka. Therefore, the objective of the present study was to determine the hematophagic tendency of the anophelines. Methods: Adult Anopheles mosquitoes were collected using Cattle Baited Trap Collection (CBTC), Cattle Baited Net Collection (CBNC), Window Trap Collection (WTC), and Hand Collection (HC) from selected sentinel sites in Mannnar (3) and Trincomalee (5) Districts during June 2011- June 2012. Each blood fed mosquito was processed in to 9 cm whatman filter papers within 24 hours after blood meal has taken. DNA was extracted using the dried blood meal protocol of the QIAmp DNA mini kit. A multiplexed, Real Time Polymerase Reaction (RT- PCR) assay targeting 8 animals was developed for two panels (Panel 1: Bovine, cat, pig, monkey: Panel 2: Human, rat, dog, chicken) to identify the host meal of Anopheles. Human Blood Index (HBI), Forage Ratio (FR) and Host Feeding Index (HFI) were calculated. Results: A total of 216 field caught freshly engorged females mosquitoes belonging to 12 Anopheles species was analyzed. The host preference of anophelines observed in this study was bovine (86.17%), human (1.84%), cat (0.46%) and pig (0.46%). Only 6.91 % was positive for both human and bovine. In addition 5.0 % of the total samples tested were unknown. The overall HBI and HFI in the present study were low indicating the humans were not the preferred host for the tested anopheline species. Nevertheless, a small proportion engorged An. aconitus (0.37), An. culicifacies (0.27), An. barbirostris (0.2), An. annularis (0.125) and An. subpictus (0.12), An. peditaeniatus (0.08), An. pseudojamesi (0.04) and An. barbumbrosus (0.04) contained human blood, The FRs for human were <1.0 for most of the anophelines, except An. aconitus (1.04). Conclusion: The presence of human blood, in mosquito species indicates the possibility of them transmitting malaria. Hence, further studies on vector competence are needed to determine the role of each of the above anopheline species currently as efficient vectors of malaria.
Breeding of Anopheles culicifacies in different waterbodies in the district of Trincomalee
(University of Kelaniya, 2012) Gunathilaka, P.A.D.H.N.; Fernando, M.A.S.T.; Hapugoda, M.D.; Wijeyerathne, P.; Wickremasinghe, A.R.; Abeyewickreme, W.
Introduction: Anopheles culicifacies (Diptera: Culicidae), the major vector of malaria in Sri Lanka is known to breed in clean and clear water. This study was focused to understand the larval habitats of the major malaria vector with the eco system changes in the Trincomalee district of the Eastern Province. Method: Potential larval habitats for Anopheles mosquitoes were surveyed on a monthly basis for 17 months (January 2011 –June 2012) in 4 different selected sampling sites (Murthankulam, Kommnaimottai, Paranamadawachchiya and Kokmotawewa). Collected larvae were identified using standard taxonomic keys. The species Distribution (C) and Density (D) were calculated. Results: A total of 2996 larval specimens representing 13 Anopheles species were reported from 16 different breeding habitats namely, waste water (n= 635), built well (n= 1229), earth well (n=149), agricultural well (n=9), rain water collection (n=89), animal hoof print (n=17), burrow pit (n=256), rock pool (n=10), canal (n=15), irrigation canal (n=27), lake margin (n=27), tank margin (n=448), pond margin (n=15), marshy land (n=13), paddy field (n=15) and slow moving water (n=42). An. culicifacies was observed as the most predominant species throughout the survey. According to Density criterion, An. culicifacies (44.0%), An. subpictus (19.2%), An. barbirostris (13.2%), An. peditaeniatus (10.28%) and An. nigerrimus (8.7%) were within the dominant class; (D > 5%). Two species (An. vagus, An. pallidus) were in the subdominant class (1< D <5%). Only An. annularis, An. varuna, An. barbumbrosus, An. pseudojamesi, An. jamesii and An. tessellatus were the satellite species (D < 1%). An. nigerrimus, An. subpictus and An. peditaeniatus can be regarded as constant according to distribution (C= 80.1-100%). Only An. vagus was the most frequently reported (C= 60.1 – 80%) species. All other Anopheles including An. culicifacies were observed as infrequent species (C= 20.1 – 40%) and no species was identified as sporadic appearance (C= 0 – 20%). Most productive breeding site for An. culicifacies were drains covered with waste water (Density= 81.57%) in remote areas. Interpretation & conclusion: These results indicate that An. culicifacies has adapted to breed in a wide range of water bodies including waste water collections although they are considered to breed in clean and clear water. The survival of the major vector mosquito in widespread water bodies could be responsible for the increase in the incidence of malaria in the future.
Characterization of a Dengue Positive Population in Colombo Municipal Council (CMC) Medical Officer of Health (MOH), Colombo
(Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2016) Lakmal, M.G.K.; Udayanga, N.W.B.A.L.; Gunathilaka, P.A.D.H.N.; Fernando, S.D.; Karunathilaka, K.; Abeyewickreme, W.
Dengue that has caused 29,777 suspected infections in 2015 has emerged the most challenging health issue faced in Sri Lanka. Approximately 51% of dengue infections are reported from the Western Province. The susceptibility of a population to dengue is influenced by a combination of socio-economic, environmental and epidemiological factors. The current study was conducted to characterize a population of dengue patients residing in a high dengue risk area in terms of their socio-economic appearance. An interviewer administrated questionnaire was used to collect information on the socio-economic aspects of a group of 140 randomly selected dengue positive patients residing in Colombo Municipal Council (CMC) Medical Officer of Health (MOH) area. Out of 140, a majority (63.6%, n=89) were females, while the age groups of 31-40 and 21-30 years were recognized as the most susceptible age groups for dengue. High proportion of the patients (79.3%, n=111) were having an educational level below secondary school (O/L) with a total family income below 20,000 LKR (80.7%, n=113). All the homesteads were smaller (<5 perch) in size, with small roads for access (77.1%, n=108) and/or a footpath (22.9%, n=32) as the access route. Majority of the households (69.3%, n=97) were temporary with moderate conditions (51.4%, n=72), that were mainly utilized for both residential and commercial purposes (63.5%, n=89) at once. In a majority of households’ solid waste was disposed within 7 days (62.1%) via the Municipal Council or by keeping near the main roads (37.9%). Source separation of waste, home gardening and composting by the study population was minimal (n=137, 97.9%). Pipe water remained as the only water source while approximately 27.9% (n=39) households were lacking sanitary facilities. Use of mosquito coils and bed-nets were the most preferred mosquito biting prevention methods. Low educational level, income, poor living conditions and limitations in solid waste disposal methods were recognized as potential risk factors for high infection rates of dengue in this population. Special focus should be given to these key areas by the relevant vector controlling authorities in order to facilitate controlling of dengue outbreaks within the CMC area.
Characterization of Anopheline larval habitats and species composition of aquatic macro-invertebrates in Trincomalee District, Sri Lanka
(Faculty of Graduate Studies, University of Kelaniya, 2015) Ranathunga, R.M.T.B.; Gunathilaka, P.A.D.H.N.; Kannangara, D.N.; Abeyewickreme, W.; Hapugoda, M.D.
Malaria control methods that aim to reduce adult vector populations by targeting their aquatic immature stages. A better fundamental understanding of the biology and ecology of these essential stages could contribute to the implementation of current control methods and to the development of novel strategies. Objective of this study was to examine breeding habitat diversity and analysis of richness, diversity and geographical distribution of Anopheline larvae and the species composition of aquatic macro-invertebrates in their oviposition sites in Trincomalee District. Fifteen major permanent breeding places in five possible malaria sensitive sites (Gomarankadawala, Ichchallampaththu, Mollipothana, Padavisiripura and Thoppur) in Trincomalee District were selected. Anopheles larvae and macro-invertebrates were collected using standard methods for 16 months (April, 2013-July, 2014) and they were identified microscopically. The Shannon diversity index (H') was used to characterize species diversity at the five study sites by its abundance and evenness of the species present. ANOVA were used to analyze the correlation between macro-invertebrates and mosquito larval abundance. In total, 4478 including 11 species of Anopheles larvae were identified. An. subpictus, An. nigerrimus and An. peditaeniatus (71%) were the most abundant and widely-distributed species. Anopheline larval diversity was highest in Mollipothana (H‘=1.986). Whereas in Gomarankadawala, Ichchallampaththu and Thoppur where H‘=1.721, H‘= 0.857 and H‘=0.762 respectively. In total, 28 species of aquatic macro-invertibrates were identified and highest diversity was recorded in Mollipothana, Gomarankadawala and Ichchallampaththu (H‘=3.14-2.56). This deduces that the species richness and diversity of Anopheles mosquitoes and macro-invertebrates are higher in these areas. The presence of permanent breeding places may be the factors for this phenomenon. This study represents the first systematic update to the distribution of macro-invertebrates associated with Anopheles mosquito oviposition sites in Trincomalee District. Knowledge generated on the ecology of Anopheles mosquitoes will help to eliminate malaria vectors in the country.
Climatic factors affecting density of Anopheles vector mosquitoes in Ampara District, Sri Lanka
(University of Kelaniya, 2014) Kannangara, D.N.; Ranathunge, R.M.T.B.; Abeyewickreme, W.; Hapugoda, M.D.; Subasinghe, S.M.C.U.P.
Background: Apart of many vector-borne diseases malaria played a major role during past decades in Sri Lanka. Controlling strategies had effectively addressed this issue so that there were no malaria patients recently. However it has been observed that abundance of vector mosquitoes in districts like Ampara is high, which signifies a potential of spreading of malaria in the area in future. Identification of the relationship between the climatic factors and vector density could be a cost effective way in controlling the mosquito instead of costly strategies currently followed. This study attempts to identify the relationship exists between climatic factors and the vector density in Ampara District.
Comparison of recombinant protein and cell lysate antigens for detection of anti-chikungunya (CHIK) IgM antibody
(University of Kelaniya, 2011) Athapaththu, A.M.M.; Abeyewickreme, W.; Hapugoda, M.; Khanna, N.; Inouve, S.; Tun, M.M.N.; Gunasena, S.
Chikungunya (CHIK) virus specific antigen which has high specificity and low cross reactivity with other related diseases is required for laboratory confirmation. The objective of this study is to compare two antigens for detection of anti-CHIK antibody. In this study, two antigens (viral cell lysate and recombinant protein) were evaluated for detection of anti-CHIK antibody by using IgM ELISA. A novel recombinant protein antigen was designed based on envelope domain, a critical antigenic region of the major structural protein. This protein was expressed in Escherichia coli and resultant protein was affinity purified and 10mg with >95% of purity per liter of culture was obtained. Cell lysate antigen was prepared using a crude culture fluid. Two antigens were evaluated separately using a panel of well characterized serum samples obtained from the Dept. of Virology (WHO Reference Centre for Viral Reference and Research), Institute of Tropical Medicine, Nagasaki University. A total of 64 serum samples confirmed as positives and 22 confirmed as negatives were used to evaluate the antigens. Specificity and sensitivity of the recombinant protein antigen was 48% and 90% respectively. Specificity and sensitivity of the viral lysate antigen was 17% and 100% respectively. Viral lysate antigens can cause biohazard risk, high production cost and cross reactivity with other organisms of the same genus/family. Recombinant protein antigen which shows high specificity and sensitivity used in this study is important to overcome problems associated with viral lysate antigen. Testing of a large number of samples is needed to reconfirm this finding. Acknowledgment: Financial assistance and technical co-operation by International Center for Genetic Engineering and Biotechnology (ICGEB CRP SRL 08/02), National Science Foundation (NSF/RG/2009/BT/01) and International Atomic Energy Authority (IAEA/SRL/5/042) is acknowledged.
Density of Aedes aegypti and Aedes Albopictus in some dengue endemic areas
(University of Kelaniya, 2000) Hapugoda, G.P.G.M.D.; de Silva, N.R.; Abeyewickreme, W.; Rajamanthri, R.
Detection of pathogenic Leptospira species in rat blood samples by molecular-based assays
(University of Kelaniya, 2013) Denipitiya, D.T.H.; Chandrasekharan, N.V.; Abeyewickreme, W.; Hapugoda, M.D.
Background: Leptospirosis is a worldwide zoonotic infection, caused by pathogenic species of the genus Leptospira. It was traditionally known as ‘rat fever’ in Sri Lanka, because rodents, especially rats, are considered to be the most important reservoirs or maintenance hosts of Leptospira. In 2012, the highest numbers of cases were reported in the District of Gampaha. The objective of this study is to detect pathogenic Leptospira species in rat blood samples by molecular based assays. Method: Rats (n=38) were trapped in a high risk area (Mirigama) in the District of Gampaha, from May 2012 to February 2013 by using live traps. Each rat was anesthetized by using diethyl ether and 2-3 ml sample of blood was collected from each rat. Blood samples collected from all rats were tested by molecular- based assays and a serological assay. Qualitative Polymerase Chain Reaction (PCR), real time PCR and Loop Mediated Isothermal Amplification (LAMP) were used as molecular-based assays which targetted conserved gene regions among pathogenic serovars of Leptospira species. Microscopic Agglutination Test (MAT), the Gold Standard assay for detection of anti Leptospira antibody was used as a serological assay. Results and Discussion: Of the 38 rat blood samples, molecular-based assays confirmed Leptospira infection in 5% (2/38), 16% (6/38) and 11% (4/38) by qualitative PCR, real time PCR and LAMP assay respectively. None of the samples was positive by MAT. After first infection, some Leptospira species live in the host animal as commensal bacteria. Therefore, host does not stimulate antibody production further and that may be below the detection level of the antibody by MAT. Conclusions: Results of molecular based assays showed that Leptospira are circulating among the rats tested in this study, although at the time of collection, their antibody levels were too low to detect by MAT, which had the lowest detection limit of 1:800.
Determination of appropriate positioning of the ovitraps for dengue mosquito surveillance
(Faculty of Graduate Studies, University of Kelaniya, 2015) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Chandrasena, T.G.A.N.; Dassanayake, R.S.; Gunathilaka, P.A.D.H.N.; Abeyewickreme, W.
Three months ovitrap survey was conducted to assess the suitable position in placing the ovitraps for dengue vector mosquito surveillance and this study was initiated due to loss of valuable data from our previous studies as a result of physical damage of the ground kept ovitarps. Thirty four households in the Ragama Medical Officer of Health area in Gampaha District were selected to conduct the ovitrap survey during the period of May to July, 2015 to select the most appropriate positioning of the ovitrap. The conventional black plastic ovitraps (3.2x 2.7 cm) were used in this purpose to collect aquatic stages of Aedes mosquitoes while placing plywood paddle (4 x 0.5 cm) over the upper rim of each coded ovitrap. A total of 136 ovitraps were used in the study site providing four ovitraps (2 each indoor & outdoor) for each house while one of the ovitraps of indoor and outdoor being hung and other being kept on the ground. In positioning ovitraps, the outdoor ones were kept 3m away from the house while leaving indoor ovitraps in the living room in close proximity to racks/hanging clothes or partially shaded places. Following collection of samples at each week, ovitraps were washed thoroughly, refilled with new water and a new paddle, and corresponding data were recorded and analyzed. These analyses revealed that number of larvae and the number of Aedes mosquito eggs present in the two different ovitrap positions (Ground kept vs Hung) were not significantly different; in spite of significant difference (P=0.001) between the outside and inside placements. Further, significantly higher values were observed for both number of mosquito eggs and larvae present in each ovitrap kept outside (60 and 13 respectively) than those placed inside (32 and 3 respectively). Furthermore, slightly higher values were observed for hung ovitraps (49 and 9 respectively) than ones kept on the ground (43 and 7 respectively). Finally, ovitrap placed above the ground level was selected in continuing the routine ovitrap survey, as there was considerable reduction of mechanical damage to the latter thus facilitating continuous data collection.
Effects of socio-economic and socio-cultural risk factors on low malaria transmission in Mannar and Trincomalee Districts of Sri Lanka
(University of Kelaniya, 2014) Gunathilaka, P.A.D.H.N.; Abeyewickreme, W.; Ranathunge, R.M.T.B.; Hapugoda, M.D.; Wickremasinghe, A.R.
The socio-economic and socio-cultural practices of the people differ between communities and play an important role in malaria control operations. The present study was conducted to assess the influence of household socio-economic and socio-cultural status on reduction of malaria transmission in the Districts of Mannar and Trincomalee, which are endemic regions for malaria in Sri Lanka.
Eliminating female Aedes mosquitoes by spiking blood meals with toxicants as a sex separation method in the context of the sterile insect technique and Incompatible Insect Technique
(Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2016) Ranathunga, R.M.T.B.; Gunathilaka, P.A.D.H.N.; Udayanga, N.W.B.A.L.; Wijegunawardena, N.D.A.; Abeyewickreme, W.
Ivermectin and spinosad have longevity reducing effects in several insect species, including disease transmitting mosquitoes after feeding blood contaminated with above toxicants on hosts. Therefore, this approach could play an enormous role in mosquito control operations by its use in the female elimination process during mass-rearing, enabling the release of only sterile males in the context of the sterile insect technique (SIT) and incompatible insect technique (IIT) with Wolbachia bacterium. Blood meals of cattle origin were spiked with diffeerenct concentrations (2, 4, 6, 8 and 10 ppm) of Ivermectin (Ivotec, 1% w/v) and Spinosad (Spinosyn, 12% w/v) and offered to a batch of adult Ae. aegypti (n = 300) and Ae. albopictus (n = 300) having 1:1 sex ratio in order to obtain an optimal dose for quick and total female elimination. After 24 hours all remaining females and males were aspirated out and transferred in to new cages separately. An additional blood meal with the initially fed concentrations of ivermectin and spinosad was provided after 24 hours, followed by a 48-hour observation of mortality. This experiment was repeated by feeding cattle blood containing a mixture of ivermectin and spinosad in 1:1 ratio from each concentration. Spiked blood with 8 ppm ivermectin and spinosad killed all the fed females of Ae aegypti and Ae. abopictus within 24 to 48 hours. It was observed that the number of fed females gradually increased, when decreasing the toxicity in combination of ivermectin and spinosad (1:1) in blood for both species. Male mortality did not show any significant differences during the study. Mixed feeding of blood with 8 ppm ivermectin and 8 ppm spinosad have shown as the viable treatment to eliminate female Ae aegypti and Ae. abopictus from laboratory colonies.
Emerging Spatio-Temporal Trends in Dengue Incidence in Poojapitiya Medical Officer of Health (MOH), Central province, Sri Lanka
(Faculty of Graduate Studies, University of Kelaniya, 2015) Udayanga, N.W.B.A.L.; Gunathilaka, P.A.D.H.N.; Iqbal, M.C.M.; Kusumawathie, P.H.D.; Abeyewickreme, W.
Dengue has emerged as one of the major threats to the health sector of many countries including Sri Lanka, due to the increasing number of reported dengue cases over time. Therefore, a statistical and geo-informatics based analysis of the spatio-temporal trends in dengue distribution was carried out to identify recent spatial and temporal trends in dengue distribution patterns, while assessing the susceptibility of the population to dengue infection in the Poojapitiya Medical Officer of Health (MOH) area. Monthly records of reported dengue cases from 2009 to 2013 of the Poojapitiya MOH area were acquired and spatial maps of the recorded dengue case distribution in each Grama Niladhari Division (GND) were developed by using Arc GIS (version 10.2). Normal Chi square test coupled with Paired-Chi square test in MINITAB (version 14.12.0) were used to investigate the impact of gender and age on the infection. Galhinna GND indicated the highest susceptibility to dengue throughout the study period, while the rest of the GNDs indicate less (37 GNDs) or null (29 GNDs) susceptibilities. As suggested by the results of the Paired-Chi square test [>Χ 2 (66, 0.95) = 85.951], the emergence of dengue outbreaks indicated a significantly declining trend in recorded dengue cases in all of the GNDs (regardless of the outbreak in 2012) during recent years. The Percentage Infected Male : Female Ratio (PIMFR) suggested that the, males had a relatively high susceptibility for dengue infection than females with 63.9 : 36.1 of average PIMFER (p=0.00<0.05 at 95% of significance). According to the Paired-Chi square test, the vulnerability of the age groups shifts significantly throughout the study period [>Χ 2 (7, 0.95) = 14.067], where the age groups of 11 - 20 and 21 – 30 indicated high vulnerability to dengue, while >61 age group remained as the least vulnerable group for dengue infection. The identification of the potential high risk GNDs with high susceptibility to the infection of dengue along with the vulnerable age groups in the community would assist the relevant vector controlling agencies to concentrate their efforts, ensuring the effective controlling of dengue outbreak occurrence within the of Poojapitiya MOH.
Evaluation the Oviposition Behaviour of Aedes Aegypti and Ae. Albopictus Mosquitoes; Main and Secondary Vector for Dengue Transmission in Sri Lanka under Laboratory Conditions
(Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2016) Gunathilaka, P.A.D.H.N.; Ranathunga, R.M.T.B.; Udayanga, N.W.B.A.L.; Abeyewickreme, W.
Studies on the oviposition behavior of Aedes albopictus and Ae. aegypti are critical for effective controlling of dengue vector breeding. However, less attention has been drawn on such aspects of vector biology and bionomics in Sri Lanka. Therefore, the objective of the current study was to evaluate the oviposition behavior of dengue vectors under laboratory conditions. Ovitraps with different sizes, colours, sources of water, sodium chloride (NaCl) concentration (0.2, 0.5, 1, 2 and 3%) and presence and absence of larvae were kept within separate cages of batches of 1000 Ae. albopictus and Ae. aegypti with (1:1, male: female ratio). Female mosquitoes were allowed to lay eggs and the number of eggs laid in different ovitraps were enumerated. ANOVA and cluster analysis were used to investigate the significance in the variations among oviposition behaviours. The mean number of eggs laid by both Ae. albopictus and Ae. aegypti increased with the increasing size of the ovitraps. Ae. albopictus indicated the highest mean number of eggs in 0.2% of NaCl than that of distilled water, while the egg laying preference reduced with the increasing salinity for both species. Drain water with low dissolved oxygen level (0.43 ± 0.12 mg/l) was the preferred water source for both species and indicated a significantly high oviposition rate in ovitraps with larvae. Black colour ovitraps attracted the majority of the gravid females, while white was least preferred. There were no significant variations among oviposition behaviors of Ae. albopictus and Ae. aegypti under laboratory conditions. Back coloured ovitraps were the preferred breeding place.
IGamma- ray irradiation as a method for sterilization of males of Aedes albopictus (skuse) for its control through Sterile Insect Technique (SIT)
(Faculty of Graduate Studies, University of Kelaniya, 2015) Harishchandra, J.; Abeyewickreme, W.; Hapugoda, M.D.; Premaratne, R.G.; Gilles, J.R.L.
Sterile Insect Technique (SIT) can be integrated with other conventional control methods to suppress population of Aedes albopictus, a vector of dengue in Sri Lanka. A study was conducted to determine the effective dose of gamma radiation for producing sterile males of Ae. albopictus mosquitoes. A batch of male pupae (n=32) aged 24-48 hours in F1 was irradiated using Gamma 220, (Co60) irradiator with 25, 30, 40, 50, 60 and 70Gy in duplicates. Adult emergence rate and male longevity were recorded. Virgin females from the same cohort (F1) were introduced into each cage for mating and fed blood starting 5 days after emergence. Then, females were isolated in individual tubes and hatching rate of individual egg batches was calculated after two weeks of egg maturation in hatching solution. Spermacheca of female mosquitoes were dissected and insemination rates were calculated after egg laying. Male mosquito pupae in F1 showed low mortality (0-3.12%) immediately after exposing to radiation (0-48 hours). After 14 days of the irradiation, survival rate of male mosquitoes in F1 were 82.7%, 79.7%, 56.3%, 73.4%, 76.6%, 76.1% and 56.3% when irradiated at 0Gy, 25Gy, 30Gy, 40Gy, 50Gy, 60Gy and 70Gy respectively. No significant difference was observed among survival of males in different doses except in 30 Gy and 70 Gy (Kaplan-Meier survival analysis, Log Rank test). Low survival rate at 30 Gy could be due to mosquito handling errors and need to be retested. Based on the hatching rate of the F2 progeny, males (F1) showed 89%, 93%, 97%, 99%, 99% and 100% sterility when irradiated at 25Gy, 30Gy, 40Gy, 50Gy, 60Gy and 70Gy respectively. Insemination rate of the irradiated males among different doses and controls were above 90% in F1 based on spermatheca dissection of bloodfed females (F1). 50 Gy is recommended as the most suitable radiation dose to produce 99% sterility in Ae. albopictus males which has 76% survival after 14 days of irradiation. This dose can be used to produce sterile males of Ae. albopictus for a SIT programme.
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.
Larvivorous Potential of Four Fish Species Against Dengue Vectors in The Kandy District of Sri Lanka
(Faculty of Graduate Studies, University of Kelaniya, 2015) Kusumawathie, P.H.D.; Jayasooriya, G.A.J.S.K.; Gunathilaka, P.A.D.H.N.; Hapugoda, M.D.; Abeyewickreme, W.
There is a growing concern on the effects of insecticides that are used for the control of mosquito vectors of human diseases. Introducing a self propagating predator into the vector breeding habitats may present continual biological control of vector population. In order to control dengue vectors, namely, Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) (Diptera: Culicidae), larval control is one of the major strategies. The objective of this study was to evaluate the larvivorous potential of fish species against dengue vectors. Four fish species, namely, Poecilia reticulata (guppy), Aplocheilus dayi (Udahandaya), Oreochromis niloticus (Tilapia) and Puntius bimaculatus were collected from natural breeding habitats. They were placed in fish stock cement tanks at the Regional Office of the Anti Malaria Campaign in Kandy till they were used for the study that was conducted from January – June 2015. In order to determine the larvivorous potential of the fish species, five fish of each species were placed in separate enamel trays containing de-chlorinated water and allowed to acclimatize for half an hour. Afterwards, a batch of 25 Ae. aegypti larvae was introduced to each tray and counted the number of larvae remaining after 5, 10, 30 minutes, 1 and 24 hours. When all 25 larvae are consumed, another batch of 25 larvae was introduced to those trays and this was continued till the study was completed. At the end of study period (24 hours), the number of larvae consumed per fish for each fish species was determined. This study was carried out in the laboratory with 6 replicates. The experiment was repeated for Ae. albopictus. Overall, Oreochromis niloticus, Poecilia reticulata, Aplocheilus dayi and Puntius bimaculatus consumed 97.6, 66.9, 49.3 and 47.5 Ae. aegypti and 98.4, 62.3, 51.2 and 45.4 Ae. albopictus larvae respectively. All four fish species showed larvivorous activity and Oreochromis niloticus showed the highest larvivorous potential followed by Poecilia reticulata. Since Ae. aegypti and Ae. albopictus breed in water storage containers such as cement tanks, barrels and ornamental ponds, the fish species that showed high larvivorous potential may be considered for dengue vector larval control in such containers. In order to achieve good community participation for the use of larvivorous fish for dengue vector control, effective health education is of utmost importance.