TBD+RBD

1. AGM 26-30/03/2012, Izmir, Turkey FONDAZIONE EDMUND MACH TBD+RBD Some practical issues The Italian way by Valentina Tagliapietra, Heidi C. Hauffe, Annapaola Rizzoli and all the team

2. Better understanding of rainbo diversity in Europe and adjacent regions Study those poorly known parameters/factors we need for better modelling ‘rainbo’ disease spread and dynamics Assess spatial distribution, prevalence and diversity of selected tick-borne pathogens in questing and feeding ticks Estimates of vector competence and vector capacity for some tick-borne diseases Understand transmission mechanisms between ticks and hosts AGM 26-30/03/2012, Izmir, Turkey FONDAZIONE EDMUND MACH TBD RBD

3. AGM 26-30/03/2012, Izmir, Turkey FONDAZIONE EDMUND MACH Joint TBD- Rainbo protocol List of the pathogens to be monitored TBEV: serological screening in rodents and virus detection in ticks; Borrelia spp: serological screening in rodents and PCR in tissues (ears) and in questing ticks; Anaplasma phagocytophilum: serological screening in rodents and PCR in tissues (blood, spleen) and in questing ticks; Cowpox virus: Serological screening in rodents but preserve lungs for PCR; Hantaviruses: Serological screening in rodents but preserve lungs for PCR; LCMV: Serological screening in rodents but preserve lungs for PCR; For ROBOs PCR sequencing can be done later if needed.

4. AGM 26-30/03/2012, Izmir, Turkey FONDAZIONE EDMUND MACH • Sampling protocol • Rodents collection and blood sampling • Trapping and handling of rodents as standard procedures adopted by each team. Be sure the method remains the same at each locality. • However the following standards are recommended. • Small mammals will be • Live trapped and euthanized after anaesthesia. • Individually stored in plastic bag. • Frozen as soon as possible. All dissection procedures, ticks (all stages) and other ecto-parasite counts, will be done later in lab to maximize the field work efficiency. • Blood sample two aliquota: 1 (min 75µl) in EDTA tube to allow DNA extraction and identification of Anaplasma spp; 1 without (min. 50 µl) to be centrifuged for Hantav, Cowpox, LCMV, TBEV, Anaplasma sp serological screening. RBC and sera samples are than later frozen at -80°C. If no blood can be obtained than put heart in PBS or blood clots from thoracic cavity during the dissection and refrozen.

5. AGM 26-30/03/2012, Izmir, Turkey FONDAZIONE EDMUND MACH Sampling protocol Study sites and trapping protocol • Select one study area with few kilometers in diameter (possibly where also TBE have been reported). • Suggested sampling pattern: the study area should be divided into four subareas, and each subarea will be trapped at each seasonal trapping. In each subarea there are 5 short lines 200-m long with 20 live traps. Line transects need not be strictly linear. If not possible at least one extended annual trapping in the best season. • Mixture of habitats to reach host diversity, but emphasis on forests habitats where the major reservoir species (both for TBDs and for Rainbo pathogens) are usually more abundant. • Seasons in local perspective (tick activity, rodent breeding season); number of seasonal trappings depends on local conditions and resources; • Live trapping over two nights (standard density index estimates based on this). In low densities trapping can/should be continued to obtain a minimum number of animals (30 individuals for common species, bank vole, yellow-necked mouse, wood mouse, for other species these numbers will not always be reached during low densities).

6. AGM 26-30/03/2012, Izmir, Turkey FONDAZIONE EDMUND MACH Sampling protocol Autopsy In case the animal is done immediately you can also place half of each organ in formaline (we use a formaline buffered at 10%) for histological studies.

7. AGM 26-30/03/2012, Izmir, Turkey FONDAZIONE EDMUND MACH Sampling protocol • Questing ticks collection • Ticks dragging contemporarily with rodent sampling. • 1 m2 white blankets with stop and ticks (nymphs and adults) collection every 5-10 m. Larvae counted and removed from the blanket at the end. • Drag along 3 transects located between the live trap lines and with the same length. • Ticks identification • Ticks storage at -80°C or RNA later at -20°C.

8. AGM 26-30/03/2012, Izmir, Turkey FONDAZIONE EDMUND MACH EDENext long term study site: CAVEDINE, Tn, Italy • 750 m a.s.l. • 4 trapping grids with 64 traps (8x8) each • Ugglan special live traps 2 • CMR method • live trapping • Data series: 2000 to present Activities scheduled in 2011-2012 • 10-11 rodent monitoring sessions (every four weeks) • 2 days/3 nights • standardized informations • samples/informations taken/recorded from every animal at every capture: • macro parasites (ticks, mites, fleas) • saliva sample • urine sample • blood sample • glycemia • faeces • tissue sample (just at first capture, tail tip and ear biopsy) • collection of beech seeds (5 times from July to October)

9. Study sites Trento

10. TYPE OF HABITAT Mixed beech forest with sparse understory and dense shurb cover

11. Early warning site (EWS): Laghi di Lamar, TN, Italy • 800 m a.s.l. • 5 linear transects with 20 traps at 10-m intervals • Ugglan special live traps 2 • Live trapping • Animals killed with isofluorane Activities scheduled in 2011-2012 • 4 seasonal sessions (May-June-August-October) • minimum 2 days/3 nights • standardized informations • informations collected from every animal at every capture: • saliva sample • blood sample • glycemia • faeces

12. Data collection -Long term monitoring site in Cavedine (Tn) -Sampling period: 2000-2008 -Data collected: Annual density of rodents (CMR methods) Pattern of tick infection on rodents Masting (qualitative data provided by the National forestry office) TBE incidence data at regional and national levels