Malaria sub-project. Activities already done. Activities planned for next year. WP MAL 1 Landscape, biotopes, habitats (First 12 months)

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1 EDEN 12 first months activity report Malaria sub-project Partner :. No. 12. ITALY Roberto Romi Date : November 2005 EDEN Mal- ITALY 1

2 Malaria sub-project WP MAL 1 Landscape, biotopes, habitats (First 12 months) Activities already done The model region is the geographical area named "Maremma", a coastal plain in Central Italy, that extends along the Tyrrhenian Sea, from South Toscana Region (Grosseto) to North Lazio Region (Viterbo) and inland to the Apennine foothills, with an extension of over 5000 squared kilometres (Km 2 ). n Maremma, malaria was hyperendemic up to the early 1950 s, when in Italy the disease was eradicated. At that time, the main malaria vector was Anopheles labranchiae, a member of the An. maculipennis complex, still now largely abundant in this area. In this model region a smaller study area was selected, on the basis of our previous study on An. labranchiae distribution in Central Italy. The study area extends from mid Toscana Region, with the northern limit in Val di Merse (San Casciano, N E) Province of Siena, to high Lazio Region, southern limit the town of Tarquinia ( N E) Province of Viterbo, for about 200 kilometres length and a mean depth of 16 Km (from sea to the hills, up to a maximum of 300 metres in altitude). The whole surface of the area may be estimated around 3,300 Km 2 (most of which in Province of Grosseto). A large variety of biotopes and human activities are present. The main rural settlements are represented by farms with presence of domestic animals (cows, sheep, pigs, poultry). For the longitudinal study of the Anophelinae populations five restricted sites, representatives of the whole area, were selected. The five sites (see map in Annex 1), are represented by as many farms where potential larval breeding sites (rice-fields, irrigation canals, small permanent streams) were recognized. The five sites are: 1 - Tarquinia Campo Magliano (Province of Viterbo); 2- Tarquinia Valfragida (Viterbo); 3 - Ampio (Grosseto); 4 - Principina Terra (Grosseto); 5 San Casciano (Siena), (Annex 1, Fig1). The acquisition of more detailed data on environmental, climatic and social conditions changes, occurred from past endemic periods up to present, is still in progress. Activities planned for next year - To continue collections and elaboration of detailed data on landscape, biotopes and habitats of the study area. - A seasonal survey will be carried out in three natural humid reserves present in the study area (Oasis of Burano Lake, Uccellina Natural Park, and Feniglia Pinewood Reservation Province of Grosseto) in order to evaluate their contribution to the abundance of anopheline mosquitoes. - Selection of a second model area to collected data on potential malaria vectors other than An. labranchiae (in years 2-4). It will be part of the delta of the Po river (Rovigo and Venezia Provinces), another historical malarious zone, where An. sacharovi (disappeared since late 1960 s), was the main vector. However, other members of the complex (still present) An. atroparvus, An. maculipennis ss and An. messeae, were suspected to play a role as secondary malaria vectors. Many historical and recent data on Anopheline mosquitoes are available. 2

3 WP MAL 2 Vector bionomics and competence (First 12 months) Activities already done Historical data (generic up to late 1970 s, more detailed since 1980) on bionomics and distribution of local Anophelinae mosquitoes in the study area have been collected and their management is still in progress. For distribution and population dynamic studies of the mosquitoes two monthly collections of larvae and indoor resting adult were carried out from April to October (in total 14 surveys). In particular, from June to September, one of the two monthly surveys included also night catches with light and light/co2 ( carbon dioxide dry ice) CDC traps and catches on human bait. The traps (two per sites) were placed close to the animal shelters, and the human baits (two or three per sites) operated in an open space close to houses. Processing of the collected Anophelines (morphological and molecular identification, blood meal identification, female longevity evaluation) are in progress. However preliminary results are presented in Annex 1, at the end of this report. The historical data on vector competence of European anophelines were collected. Attempt of rearing a large number of An. labranchiae specimens at the laboratory was done, in order to try experimental transmissions in Nijmegen (Netherlands) planned for years 3 and/or 4. Activities planned for next year - Optimisation of field and laboratory procedures and data analysis. - Second season of longitudinal surveillance in the 5 stations of the study area. - To elaborate data on length of gonotrophic cycles, longevity, survival rate. - To initiate field work and to evaluate the anthropophily of supposed zoophilic species (An. atroparvus, An. messeae) in the delta Po river area (second model rea). WP MAL 3 Public health and human activities (First 10 months) Activities already done Bibliographical data on malaria historical situation and the impact of control campaigns on malaria incidence have been acquired but their elaboration is still in progress. Data on imported malaria in Italy since the 1960 s have been collected and elaborated. Data analysis about will be available as soon as possible. Recent data on imported malaria incidence and public health activities in the study area have been collected in collaboration of the Department of Infectious Diseases, Ministry of Health. Data collection of the extent of human movements in travelling abroad and the risk related to potentially infective reservoirs for An. labranchiae in the study area, is still on going (Tab. 6 Annex 1). Characterization of human population migrations from malaria endemic countries into the study area. Demographical data related to communities located in the study area are showed in Tab. 7 Annex 1. Activities planned for next year : 3

4 - To complete the analysis of data on imported cases, human distribution and public health activities, in order to determine potential transmission areas. WP MAL 5 Data management (First 12 months) Activities already done The distribution of potential malaria vectors over the study area, will be managed through a GIS framework (Mapinfo). At present the data base managed by the GIS is limited to the part of the study area (scale 1: , 1: and 1:5.000) belonging to Toscana region and Grosseto Province. Raster cartography of Lazio Region will be found and georeferenced according to the Italian system of coordinate (Gauss Boaga: fuse ovest). Activities planned for next year - To complete the GIS framework with the part of the study area belonging to Lazio region. - A dynamic statistic model of malaria risk in the study area, as a function of different variable factors, will also be set up in collaboration with the University of Rome La Sapienza, Faculty of Statistics. Name of scientists and institutions involved Istituto Superiore di Sanità, Roma: Department of Infectious, Parasitic and Immuno-mediated Diseases; Unit of Vector Borne Diseases and International Health: Roberto Romi, Daniela Boccolini, Marco Di Luca, Luciano Toma, Francesco Severini, Antonio Massa, Francesca Mancini Barbieri. Ministry of Health, Roma: Dipartment of Health Prevention, Infectious Diseases Office: Stefania D Amato. Unità Funzionale di Zoologia Ambientale, ASL 9, Grosseto: Regional Reference Center for Medical Entomology and Vector Control in Toscana: Angelo Tamburro, Maurizio Cocchi, Giorgio Pontuale. 4

5 Annex 1 Anopheline Surveillance in Maremma, Italy (Roberto Romi et al.) Introduction Field researches were carried out in Maremma, a physical region between Lazio and Toscana Regions, Central Italy. Mosquito collections were carried out in 5 sites (farms) located in 3 main areas (Fig.1): Tarquinia (sites 1and 2: Campo Magliano e Valfragida) Grosseto (sites 3 and 4: Ampio e Principina) Siena (site 5: S. Casciano) respectively representatives of the southern limit, middle and northern limit of the distribution area of the potential malaria vector An. labranchiae in Maremma. These areas present a large variety of biotopes and human activities. In particular, the sites of Grosseto Province include an area (site 4) where the vector density is the highest of the study region because of the presence of ricefields and the area (site 3) where an autochthonous introduced malaria case occurred in Results Fortnightly collections of larvae and indoor resting adult Anophelines were carried out from April to October 2005, for a total of 14 surveys. In June-September one of the two surveys included also night catches with light and light/co2 CDC trapping (two per sites) and catches on human bait. Anophelines, mainly belonging to the An. maculipennis complex, were identified by egg morphology and by molecular analysis: 1. Species collected Ten species were found, belonging to 6 different genus. Anopheles Aedes Ochlerotatus Culex Culiseta claviger albopictus caspius pipiens annulata labranchiae detritus longiareolata maculipennis s.s plumbeus A total of 4147 adult mosquitoes was collected, 3806 of which were Anophelines, most (3707 = 97%) belonging to the An. maculipennis complex. Mosquitoes were collected either by indoor resting places (animal shelters and close premises), human bait catches, CDC-light traps (with and without CO2). Details are shown in Tab.1 5

6 Val di Merse (Toscana Region) 5 Maremma (Toscana Region) Maremma (Lazio Region) Fig. 1 Map of the study area showing the physical region named Maremma. This region is composed by 3 main areas belonging to in two administrative regions of Central Italy, Lazio and Toscana. Figures represent the five surveillance sites (farms) located respectively in the administrative Provinces of Viterbo (1 e 2), Grosseto (3 e 4) and Siena (5). 6

7 Tab. 1. Adult mosquitoes collected in the study area by site. Figures are the sum of the specimens collected with the different methods previously described (April - October 2005). Site Anophelinae Culicinae TOT An mac sl An spp. 1) Campo Magliano ) Valfragida ) Ampio ) Principina ) S. Casciano TOT Until now, part of the Anophelines belonging to the maculipennis complex have been identified mainly by egg morphology; few specimens were identified by diagnostic PCR. Molecular identification is going on and will be completed during next months. A provisional rate of species identification versus the total number of An. maculipennis s.l. collected by site is shown in Fig. 2. Fig.2. Rate of species identification within the global number of An. maculipennis s.l. collected by site. A-B = sites 1 and 2 (Tarquinia). C-D = sites 3 and 4 (Grosseto); E = site 5 (S. Casciano, Siena). A SITE 1 Campo Magliano B SITE 2 Valfragida 23.4% 3.2% 46.7% 45.8% 73.4% 7.5% An lab An mac ss An mac sl An lab An mac ss An mac sl 7

8 C SITE 3 Ampio D SITE 4 Principina 23.5% 7.06% 0.03% 0.2% 76.3% An lab An mac ss An mac sl 92.91% An lab An mac ss An mac sl E SITE 5 S. Casciano 1,1% 39,1% 59,8% An lab An mac ss An mac sl 2. Seasonal dynamics For 7 months, regular daily collections in animal shelters and related premises were conducted in all sites, fortnightly. Night catches were carried out in three of the five sites one week/month, both on human baits and by CDC traps (June-September). For each night, two teams of two persons collected mosquitoes on themselves from sunset (h 8-9 p.m.) to h 1-2 a.m, and two CDC-light traps (1 without and 1 with CO2) were hung in different places/site from sunset to h 9 a.m. The seasonal dynamics of An. maculipennis s.l. populations of the study sites are reported in Fig.3: results show that populations increase progressively, even if irregularly, and reach a peak around August-September. 8

9 Fig.3. Seasonal dynamic of An. maculipennis s.l populations by site of collection. (April-October 2005). A-B = sites 1 and 2 (Tarquinia). C-D = sites 3 and 4; (Grosseto); E = site 5 (S. Casciano, Siena) A SITE 1 Campo Magliano 0 6/4 10/5 24/5 8/6 21/6 18/7 26/7 10/8 23/8 6/9 21/9 4/10 25/10 An mac complex B SITE 2 Valfragida 0 6/4 10/5 25/5 8/6 21/6 18/7 26/7 10/8 23/8 24/8 6/9 21/9 4/10 25/10 An mac complex C SITE 3 Ampio /4 11/5 24/5 8/6 21/6 18/7 26/7 9/8 24/8 8/9 21/9 5/10 25/10 An mac complex 9

10 D SITE 4 Principina 0 19/4 10/5 11/5 25/5 9/6 22/6 18/7 27/7 9/8 24/8 25/8 7/9 21/9 4/10 26/10 An mac complex E SITE 5 S. Casciano 0 18/4 12/5 26/5 8/6 22/6 12/7 22/7 17/8 31/8 6/9 27/9 An mac complex 3. CDC trap collections As previously assessed, since the s up to 1990 s, CDC light traps (without as well as with CO2) did not attract selectively anopheline mosquitoes in Maremma. As above reported, two CDC traps were operating besides the catches on human bait, one time per month in the same date and site. A total of 117 An. maculipennis s.l. was collected with CDC traps from June to September Human biting rate Human biting rate (HBR) was recorded during four nights from June to September in 4 of the 5 study sites, In site 1 and 2, two couples of two persons located at 2 different points, collected mosquitoes on their legs and arms. A single couple was operating in site 5 (S. Casciano). Anopheline collection was recorded by hour from sunset h. 8.9 p.m. to h 1-2 a.m. 10

11 Tab. 2, Total number of An. maculipennis s.l. females collected on human baits in 4 sites of the study area from June to September In all four sites, most of the specimens were collected during the first two hours of exposition (h 8-10 p.m.); latest collection at h. 1 a.m. More information will be provided by An. maculipennis complex specimens identification. site June July August September 2) Valfragida ) Ampio ) Principina 59* ) S. Casciano *corresponding about to 0.75, 2.7, 11.1 and 7.3 bites/man/nigh respectively. 5. Feeding preferences The source of the blood meal of most of the freshly engorged Anophelines collected during the study period will not investigated because they came from animal shelters. This analysis will be carried out only on a very few number of An. maculipennis s.l. specimens collected in resting sites (different kind of premises) other than shed. Blood meals will be analysed during the next months using an ELISA assay. 6. Resting places Potential resting places were explored in order to determine resting preferences of An. maculipennis s.l. populations and to collect blood fed and gravid females. Most of the Anophelines were collected into the sites reported below (Tab. 3). As attended, results show that members of the An. maculipennis complex were found abundantly into sheds or anthropic places. 11

12 Tab. 3. Different mosquito resting sites and total number of mosquitoes collected indoor in the five study sites, Maremma, Italy (April-October 2005). Study site 1) Campo Magliano 2) Valfragida 3 ) Ampio 4) Principina 5) S. Casciano TOT Resting site Animal shelters (cow stable, pigsty, poultry, sheep shelter) Anthropic places (garage, tools deposit, seed/fodder storage room) Animal shelters (cow stable, poultry, rabbit hutch) Anthropic places (garage, storage rooms, machine shop) Animal shelters (horse stables) Anthropic places (house porches, fodder storage room, machine shop, garage) Animal shelters (poultry, sheep shelter) Anthropic places (machine milking room, dairy workshop room) Animal shelters (pigsty, poultry) Anthropic places (tools deposit, machine shop) An. maculipennis s.l. 124 Anopheles spp Culicinae Total mosquitoes/site

13 7. Parity rate Because of the scarce results of the night catches on human bait, only data from site 4 (Principina) were suitable for parity rate assessment. Females of Anopheles maculipennis s.l. were dissected in order to determine the parity rate using Detinova method. Dissections were carried out on a total of 49 specimens collected from June to September Results show a low parity rate of An. maculipennis s.l. females in June, because the growth of the mosquito population at that time, that reaches a peak near the end of August with a value of Fig 4. Parity rate (and crude numbers of parous and nulliparous specimens) of An. maculipennis s.l. females collected when landing on human bait, in Principina, Grosseto (June-September 2005). Site 4 - Principina Parity rate N. specimens /6 27/7 25/8 7/9 Par. rate 0,29 0,20 0,75 0,66 PAR NULL DISSECT ,9 0,8 0,7 0,6 0,5 Parity rate 0,4 0,3 0,2 0, Breeding sites For each study site the potential breeding sites present near the five farms were recorded and mapped. These breeding sites were regularly investigated during the 14 surveys, from April to October. Data relating to the most productive larval breeding sites (located not more than 500 metres from the respective farm) are reported in Tab. 4. PCR identification of collected An. maculipennis complex larvae will provide additional information on the species composition. 9

14 Tab. 4. Total number of larvae/pupae collected in representative (the most productive) Anopheline breeding site for each study site. Data are the sum of 14 surveys, carried out from April to October Study site (breeding site) 1 Campo Magliano (irrigation canal) Anopheles spp (N. larvae/pupae) Presence of water (N. times/14)* Positivity for Anophelines (N. times/14) Mean number of larvae/dip Valfragida (irrigation canal) Ampio (land reclamation canal) Principina (a. rice field) (b. irrigation canal) S. Casciano (rice field) TOTAL *Number of times that the breeding sites was flooded out of 14 surveys 14

15 Tab 7. Demographic data of the 18 towns (community) included in the study area up to 300 metres of altitude (ISTAT, 2003) Community Siena Province Surface (Km2) Inhabitants Density (by Km2) Italians Newly recorded Strangers Newly recorded *Population growth/decrease San Casciano , Monticiano , Murlo , Radicondoli , Sovicille , Casole D Elsa , tot Grosseto Province Grosseto , Monte Argentario , Capalbio , Castiglione della P , Orbetello , Magliano In Toscana , Scansano , tot Viterbo Province Monte Romano , Canino , Montalto Di Castro , Tuscania , Tarquinia , tot TOTAL

16 Tab 6. Strangers regularly resident in the 18 towns of the study area, coming from continents where malaria is still endemic (ISTAT, 2000) REGION (PROVINCE) Community Africa Asia Central America Oceania TOSCANA (SIENA) San Casciano Bagni Monticiano Murlo Radicondoli Sovicille Casole D'Elsa tot TOSCANA (GROSSETO) Grosseto Monte Argentario Capalbio Castiglione Della Pescaia Orbetello Magliano In Toscana Scansano tot LAZIO (VITERBO) Monte Romano Canino Montalto Di Castro Tuscania Tarquinia tot TOTAL Grand Total