TBD and RBD: some practical issue: the Italian way

TBD+RBD

Some practical issues

The Italian way

AGM 26-30/03/2012, Izmir, Turkey FONDAZIONE EDMUND MACH

by

Valentina Tagliapietra, Heidi C. Hauffe, Annapaola Rizzoli and all the team

RBD

• 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

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.

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.

Sampling 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

Sampling protocol

BRAIN FROZEN -80°C Ljungan

LIVER FROZEN -80°C

LUNG FROZEN -80°C LCMV

HEART FROZEN -80°C

SPLEEN FROZEN -80°C Borrelia, Anaplasma, Rickettsia,

Bartonella, Babesia, LCMV PANCREAS FROZEN -80°C

KIDNEY FROZEN -80°C LCMV

GUT (ALCOOL 70°)

FAECES FROM INTESTINE -80°C Ljungan HIND FOOT -80°C

TAIL ETHANOL

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.

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.

Sampling protocol

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)

Trento

TYPE OF HABITAT

Mixed beech forest with sparse understory and dense shurb cover

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

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