STUDIES OF FILARIASIS IN KEBAN AGUNG AND GUNUNG AGUNG VILLAGES IN SOUTH BENGKULU, SUMATERA, INDONESIA I11 :

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1 Bulletin Penetitian Kesehatan (Health Studies in Indonesia) Vol. XI1 No. I 1984 STUDIES OF FILARIASIS IN KEBAN AGUNG AND GUNUNG AGUNG VILLAGES IN SOUTH BENGKULU, SUMATERA, INDONESIA I11 : Natural and artificial infections of vector mosquito species * Sudomo, M.', Suwarto ' and Lim Boo ~ iat~ ABSTRACT Sejumlah nyamuk yang terdiri dari 4 species Mansonia spp dan nyamuk yang terdiri dun dua species Anopheles spp dari desa Keban Agung dun Gunung Agung telah dipenksa dengan jalan pembedahan untuk mengetahui adanya infeksi larva Brugia, selama periode 24 bulan. Infeksi larva Brugia pada nyamuk Mansonia spp lebih tinggi daripada pada nyamukanopheles spp. Pada semua species nyamuk Mansonia pemah ditemukan mengundung larva Brugia stadium 3 (stadium infektifl sedangkan pada Anopheles tidak pernah ditemukan larva Brugia stadium ini. Dalam percobmn penginfeksian dengan penderita filariasis (B. rnalayi) dengan empat species Mansonia dun lima species Anopheles terbukti bahwa semua species Mansonia rentan terhadap B. malayi sedangkan pada Anopheles hanya 2 species yang dqat d iinfeksi. INTRODU~ION MATERIALS AND METHODS Longitudinal studies were conducted on fila The study areas (Fig. 1) and the procedures of riasis vectors following the survey on mosquito the monthly routine nocturnal collections of fauna by Sudomo et al in two filariasis landing mosquitoes from indoors and outdoors endemic areas in South Bengkulu. Suzuki et al. have been documented by Sudomo et al. (1983). (1981) in their preliminary survey of filariasis Most of the Mansonia and Anopheles spp. collected monthly were individually dissected to vectors in the study areas, revealed several Mansonia spp. and some of the Anopheles spp. as po examine for filariasis infection. Larvae collected tential vectors of Brugia malayi. The studies were were mounted in glycerine and identified under further extended for a 24 month period to the compound microscope. The larvae were identified as Brugia sp. with the aid of the key by establish (1) seasonal variations of natural infection in mosquito vectors; (2) confirmation of Nelson (1961, and Rarnachandran (197). They potential vectors, and (3) experimental infections were reconfirmed by Dr. Mak Joon Wah of Malaysia and Mr. Purnomo of Indonesia. of various mosquito spp to assess the susceptibility of these mosquitoes to the parasite. Nine mosquito species collected by light traps at various periods from Keban Agung and Gunung * This study is supported by filariasis component of Agung villages were used for experimental the UNDP/World BankjWHO Special Programme studies. They were sorted out and divided into for Research and Training in ~iopical Diseases. two batches, one for the experiments, ad 1. Health Ecology Research Centre, National Institute another for the control. ~h~ mosquitoes were of Health Research and Development, Ministry of Health, P.. Box 226, Jakarta, Indonesia. fed on four microfilariae (MF) carriers with 96, 2. WHO/Vector Bioloav and Control Research Unit 84, g8 and 129 microfilariae per 2 mm3 blood. 11, P.. BOX 32, Jakarta, Indonesia. After feeding, they were held in cages measuring

2 NATURAL AND ARTIFICIAL INFECTIONS OF VECTOR MOSQUITO SPECIES Nipa palm Swampy areas Rice fields secondary forest (rubber) Field station Fig. 1. Map showing study areas in South Bengkulu, Sumatera, Indonesia. 45

3 SUDOMO, M. ET.AL. 3 cm3 and fed with sugar water only. The holding period for these mosquitoes was up to 13.5 days, and any mosquito which died during the holding period was dissected. Survivors were sacrified at the end of the period. The holding period was based on Suzuki et al. (1981) who showed that the Mf development to infective larvae in the mosquitoes were from days. Equal numbers of mosquitoes for each of the species were used as controls. These mosquitoes were kept in identical cages separately, and at the same time. The same procedures were used for the control mosquitoes as that of the experimental ones, except that these mosquitoes were fed with sugar water only. RESULTS A total of mosquitoes comprising four Mansonia spp. and two Anopheles spp. were examined from Keban Agung, and results are shown in Table 1. Among the four Mansonia spp.,ma. bonneae and Ma. dives \r/ found with a higher natural infection rate and wormload than Ma. uniformis or Ma. annulata. The natural infection rates of An. peditaeniatus and An. nigemmus was exceptionally low, but none of them were found harboring infective Brugia larvae. In Gunung Agung 5,629 mosquitoes of the same species as of Keban Agung were examined. Of the four Mansonia spp. the lowest natural infection rate was found in Ma. uniformis; Ma. bonneae had the highest wormload compared to all the other species. Like in Keban Agung infection rates of An. nigerrimus and An. peditaeniatus were very low, and no infective Brugia larvae was found. (Table 2). Nine mosquito species were used for experimental infections, and results are presented in Table 3. Of the four Mansonia spp. higher success of infection rates were shown in Ma. bonneae, Ma. dives and Ma. uniformis than in Ma. annulata. However, there was no marked difference shown in the development of Mf to infective larvae among the four Mansonia spp. Ma. uniformis was found with higher wormload of Brugia larvae than the other species, and the lowest was found in Ma. annulata. In the controls only one mosquito of Ma. bonneae and Ma. dives was found with one Brugia larvae each at 13.5 days. For Anopheles species only An. peditaeniatus and An. nigerrimus were found to take the infection, however development of Mf to 3 rd larvae was found to be very low as compared to Mapspnia species. In the control, one mosquita/2f An, peditaeniatus and An. nigerrimus which died at 6 days and 4 days respectively Table 1. Natural infection with filarial larvae of lahding mosquito species from indoors and outdoors collections combined in Keban Agung village at South Bengkulu, Sumatera. Mosquitoes species No. of mosquitoes examined % positive %with infective larvae Total number of larvae Mean no. larvae per infected mosquito 1 st & 2nd Ma. bonneae Ma. dives Ma. uniforrnis Ma. annulata An. peditaeniatus An. nigerrimus f f f f * 1.16* 3.38 f f f f 1.22 * Less than 1 infected mosquitoes. 46

4 NATURAL AND ARTIFICIAL INFECTIONS OF VECTOR MOSQUITO SPECIES Table 2. Natural infection with filarial larvae of landing mosquito species from indoors and outdoors collections combined in Gunung Agung village at South Bengkulu, Sumatera. Mosquito species No. of mosquitoes examined % positive %with infective larvae Total number of larvae Mean no. latvae per infected mosquito 1st & 2nd Stage Ma. bonneae Ma. dives Ma. uniformis Ma. annulata An. peditaeniaius An. nigerrimus k f ' 2. k * 1.6 * 3.36 k 1.78 ' * 1.6 k.8 * Less than 1 infected mosquitoes. Table 3. Percentage of development of Mf to infective larvae ( ) in various mosquito species 'from days in experimental infection and mean number of larvae per infected mosquito. I Mosquito species Ma. bonneae Ma. dives Ma. uniformis Ma. annulata An. peditaeniatus An. nigerrimus An. vagus An. philippinensis Coq. nigrosignata No.of mosq. tested % positive larvae 'I 2; Experimental infections YOdevelop ' TL:iF 1 To, lst& 2nd s s I Mean larvae per infected mosquito lst 2nd s k O O k k k f k k k.44.7 '.1 1 * NO. of masquitoes Died at 24 days n + Control Died at 58 days n lx 11 lx Sacrified at 13.5 days n " X 37 lx X n = Numberdissected; + = Number positive; *C = Brugia sp. x = Dirofilariaspp. was found with one Brugia larvae each, and of those that were sacrified at 13.5 days, three of the former and one of the latter were found with 7 and 2 Dirofilaria larvae. None of the An. vagus, An. philippinensis and Coq. nigrosignata used Were infected. In the control group, few Coq. nigrosignata which either died or sacrified were found with DiroJilaria larvae only. = Less than 1 individuals; For the purpose of analyses, the natural infection in all Mansonia spp. were considered together. Correlationcoefficient tests show that there was no correlation between monthly infection and infectivity rates in the four mosquito species with either density or rainfall. It is however significant that the estimated number of infective Mansonia spp. averaged at.3 per

5 SUDOMO. M. ET.AL. man hour (range.24 to.92) in Keban DISCUSSION Agung and.36 (range.22 to.11) in Gunung Agung (Fig. 2). The estimation of the The present studies on natural infection and infection and infectivity rates of the four Manso susceptibility of filariasis vectors at Keban and nia spp. revealed a high infective rates in almost Gunung Agung, Bengkulu show that four Manall months during the year particularly in Keban sonia spp. are good vectors of Brugia filariasis Agung and to a lesser degree in Gunung Agung. in the areas. The higher mean wormload of...* infection Keban Agung 17 Gunung Agung f 1981 ^ * 1982 Fig. 2. Estimated number of infected and infective Mansonia spp. per manhour

6 NATURAL AND ARTIFICIAL INFECTIONS OF VECTOR MOSQUITO SPECIES Brugia larvae in Ma. bonneae and Ma. dives found in natural infections was also reflected in experimental infections with these two species being higher than in Ma. annulata. The mean wormload in naturally infected Ma. uniformis was lower than Ma. bonneae and Ma. dives; however experimentally, Ma. uniformis was found to be highly susceptible. It is therefore concluded that among the four Mansonia species, Ma. bonneae, Ma. dives and Ma. uniformis are more efficient vectors than Ma. annulata. The present findings further support the preliminary observations by Suzuki et al. (1981) on these four species as the principal vectors of periodic B, malayi in the study areas. A very low rate of natural infection with brugian larvae was found in An. peditaeniatus and An, nigerrimus, but no larvae were detected in them. Experimentally, these mosquitoes were found to take the infection very well, but the percentage with development of Mf to infective larvae was negligibly low. Suzuki et al. (1981) examined 1517 An. hyrcanus group (=An. peditaeniatus and An. nigerrimus) but found none of them was naturally infected; however, experimentally they showed that the Mf could develop to larvae, 8% days onward after feeding on Mf carriers. It was most unfortunate that during these experimental infections with both Mansonia and Anopheles spp. there were no control experiments. Thus it is quite likely that the brugian larvae found in these two mosquitoes may be due to natural infection. As B. pahangi is prevalent among the domestic cats in the study area (Suzuki et al., 1981 ; Lim & Sudomo (in press) there is a likelyhood that the larvae found in their experiments could be B. pahangi. None of the An. vagus, An. philippinensis and Coq. nigrosignata.used in these experiments were found to take the infection. Based on the present studies, it is unlikely that Anopheles spp. may play a significant role in the B. malayi transmission in the study areas. In conclusion it was apparent from the experimental and natural infection results that the four Mansonia spp. are the principle vectors of B. malayi with Ma. bonneae and Ma. dives being more efficient vectors than Ma. uniformis and Ma. annulata. Considering the estimation of high infective numbers of these mosquitoes and the total number of bites per man as received over a period, the role of Mansonia spp. in the transmission of B. malayi on these two areas cannot be over emphasized. Although the infective larvae of different Bmgia spp. are morphologically indistinguishable, experimental evidence obtained, corroborates this view. SUMMARY A total of 13,133 and 5,629 mosquitoes comprising of four Mansonia spp. and two Anopheles spp. was examined for natural infection with Bmgia larvae in both Keban Agung and Gunung Agung villages, Sumatera for a 24 month period. The natural infection rates of Mansonia spp. were significantly higher than the Anopheles spp. All the Mansonia spp. were found with infective Brugia larvae as compared to none found in the Anopheles spp. Experimental infections with four Mansonia spp. and five Anopheles spp. to microfilariae carriers show that all the four Mansonia spp. were susceptible while only two of the five Anopheles spp. took the infections. The natural and experimental infections of all the four Mansonia spp. were found to be naturally efficient and successful experimental vectors of B. malayi. It is, thus concluded that the Mansonia spp. are the principle vectors of B. malayi transmission in both the study areas. ACKNOWLEDGEMENTS The authors are grateful to the field staff, National Institute of Health Research and Development (NIHED) Jakarta, and also to Dr. E. Oswari, Director of Health Services, Bengkulu and Dr. Abu Hanifah, Chief, CDC/Bengkulu, for their supports. Thanks are also due to Dr. N, G. Gratz, Director, VBC, Geneva, Dr. C. P. Pant, Chief, ECV, Geneva for their criticisms, and Dr. Y. H. Bang, Project Leader, VBCRU 2 Jakarta for his support. They are also grateful to Dr. P. K. Rajagopalan, Vector Control Research Centre, Pondi

7 SUDOMO, M. ET. AL. cherry, India, for his advice; Dr. Mak Joon Wah, Filariasis Division, Institute for Medical Research, Kuala Lumpur, Malaysia and to Mr. Pur nomo, Staff of the Navy Medical Research Unit, Jakarta, Indonesia for their confirmation of the Bmgia larvae in the vectors. REFERENCES I;im Boo Liat and M. Sudomo (in manuscript). Studies of filariasis in Keban Agung and Gunung Agung villages in South Bengkulu, Sumatera V: Animal fiiariasis. Nelson, G.A. (196). The identification of filarial larvae in their vectors. Indian J. Mariology, 14 : Ramachandran, C.P. (197). A guide to methods and technique in filariasis investigation. Institu te for Medical Research Bulletin, Kuala Lumpur No. 15 : 39 pp. Sudomo, M., Suwarto and Lim Boo Liat (1983). Studies of Filariasis in Keban Agung and Gunung Agung villages in South Bengkulu, Sumatera, Indonesia : I. The mosquito fauna with reference to seasonal studies of two Anopheles and Culex tntaeniorhynchus. Bull. Health Studies in Indonesia XI. 1 : 2535 Suzuki, T., M. Sudomo, Y.H. Bang and Lim Boo Liat (1981). Studies on malayan fiariasis in Bengkulu (Sumatera), Indonesia with special reference to vector confirmation. Southeast Asian J. Trop. Med. Pub. Hlth. 12 : 4754.