1. Genetic diversity of Anaplasma phagocytophilum and reservoir competence of wild life animals for tick-borne pathogens in northern Italy.  
Ivana Baráková1,2, Giovanna Carpi3, Fausta Rosso2, Michal Chvostač1, Valentina Tagliapietra2, Heidi C. Hauffe2, Annapaola Rizzoli2, Markéta Derdáková1
1 Institute of Zoology, SAS, Dúbravská cesta 9, Bratislava, Slovakia
2 Fondazione Edmund Mach, Via Edmund Mach, 1, San Michele All'adige Trento, Italy
3 Yale School of Public Health, 60 College Street, New Haven, CT 06520, USA

2. Vectors and reservoir host
Second most important vectors after mosquitoes
in Europe – among the most important vectors of the viral, bacterial and protozoan diseases
Ixodes ricinus
Epidemiologically- the most important vector tick in moderate regions of Europe
Pathogen
reservoir host
Borrelia
birds, rodents, lizards
Rickettsia spp.
rodents, roe deer
Babesia spp.
roe deer
Anaplasma phagocytophilum
domestic and wild ruminants, dogs, fox, bear, rodents

3. Ecology of A.phagocytophilum
In USA
In Europe
vector- Ixodes scapularis
2 different genetic variants
Ap-variant 1 – isolated from Ixodes scapularis ticks, free-living ungulates – reservoir hosts, NOT PATHOGENIC FOR HUMANS
Ap-ha variant – isolated from Ixodes scapularis tick, humans, sick dogs, horses, rodents, PATHOGENIC FOR HUMANS (Massung a kol. 2002; 2006)
most important vector- I. ricinus
minor vectors I. trianguliceps (Bown et al., 2009) a D. Marginatus (de la Fuente et al., 2005)
Higher variability of genotypes and not clear ecological associations less human cases than in US;
In England, rodents are reservoirs of distinct genotypes that are probably transmitted by I. trianguliceps ticks and not I. ricinus ticks

4. Are there two different enzootic cycles of AP circulating in northern Italy?

5. Main Goals Phylogenetic analysis
1. Observe genetic variation of Anaplasma phagocytophilum on molecular level (ticks from host and vegetation and blood samples from rodents)
- amplification of msp4 and groEL genes
Phylogenetic analysis

6. Main Goals 2. Reservoir competence
To obtain data on reservoir competence of various pathogens (Anaplasma, Borrelia spp., Babesia spp. and Rickettsia spp.) in larvae ticks from various host in Northern Italy

7. Material and methods Phylogenetic analysis Alignment – ClustalW
Phylogenetic trees – MEGA4
DNA extraction from ticks (Qiagen), DNA extraction from blood samples (Fermentas)
PCR detection of genes (tick samples):
16S Anaplasma
18S Babesia
23S, 5S Borrelia
17k Rickettsia
Confirmation by sequencing
RT-PCR detection (blood samples):
-Confirmation by sequencing
Questing ticks positive for Anaplasma
(Fausta Rosso)
Genetic variability of positive samples were analyzed on two loci>
msp4
groEL
Confirmation by sequencing

8. Collection sites in Northern Italy
VALLE DEI LAGHI:
Lamar
Cavedine
Pietramurata
Mt. Bondone
Covelo
Lasino
Calavino
Cadine
Lundo
San Giovanni

9. Prevalence of AP in Northern Italy in feeding, questing ticks and host
Host (n° of tick)
A.phagocytophilum
Deer (49)
14,3% (5-A, 2-L)
Rodents (49)
6,1% (3-N)
Sheep (13)
7,7% (1-A)
Birds (27)
11% (3-N)
Dogs (30)
3,3% (1-N)
Humans (115)
2,6% (3-N)
Questing ticks °
A.phagocytophilum
821
1,8%
Blood samples
A.phagocytophilum
Rodents (1295)
0,3%

10. Maximum Likelihood Tree of the MSP4 sequences
Anaplasma phagocytophilum roe deer Northen Italy
Anaplasma phagocytophilum Apodemus flavicollis Northen Italy
Anaplasma phagocytophilum Dog Northen Italy
Anaplasma phagocytophilum bird Northen Italy
Anaplasma phagocytophilum rodent Northen Italy
MSP4 gene
Clade 1
Anaplasma phagocytophilum Mufflon Northern Italy
Diffrent ecotype from birds from Italy
Clade 2
Samples from rodents (from blood)
100% identical sequence from I. trianguliceps
No I. ricinus feeding on rodents were found with this genotype

11. Phylogenetic tree of groEL sequences constructed by Maximum parsimony
Anaplasma phagocytophilum sheep Northen Italy
Anaplasma phagocytophilum roe deer Northen Italy
Anaplasma phagocytophilum Apodemus flavicollis Northen Italy
Anaplasma phagocytophilum mufflon Northen Italy
Anaplasma phagocytophilum bird Northen Italy
groEL gene
Clade 1
Diffrent ecotype from birds from Italy
Clade 2
Samples from rodents from Italy

12. Reservoir competence Pathogen birds (n=91) rodents (n=342)
wild ungulats (n=184)
Borrelia
B. garinii
23% (21)
B. valaisiana
8.8% (8)
B.burgdorferi
B. lusitaniae
7.7% (7)
B. afzelii
9.6% (33)
Rickettsia spp.
R. helvetica
5.5% (5)
2.6% (9)
8.2% (15)
R. slovaca
0.3% (1)
R. monacensis
2.3% (8)
2.2% (4)
Babesia spp.
B. EU1
0.5% (1)
B. capreoli
1% (1)
Anaplasma
A. phagocytophilum
4.3% (8)

13. Conclusions Ecology of AP
Phylogenetic analysis confirmed wide heterogeneity of A. phagocytophilum strains associated with different hosts
two main clades, one containing I. ricinus ticks as vectors and strains circulating in different large vatebrate hosts (deer, sheep, dog, humans) and other one containing strains found just in rodents which are 100% identical with the strains found in I. trianguliceps in UK and Slovakia
These results support the theory of two distinct enzootic cycles
Reservoir competance
No larvae I.ricinus ticks were found positive for AP, indicating that strains from rodents can’t be transmited by I.ricinus ticks
Wild ungulates may play role as reservoirs for Rickettsia spp.
We sugest not only wild ungulates but also rodents as reservoir for Babesia EU1

14. Thank you for your attention

15. Results Prevalence of pathogens in northern
Italy in feeding, questing ticks and host
Host (n° of tick)
Anaplasma
Babesia
Borrelia
Rickettsia
Deer (49)
14,3% (5-A, 2-L)
2% (1-N)
2% (1-A)
12,3% (3-A, 2-N, 1-L)
Rodent (49)
6,1% (3-N)
4% (2-N)
Sheep (13)
7,7% (1-A) 0%
Bird (27)
11% (3-N)
7,4% (2-N)
37% (10-N)
3,7% (1-N)
Dog (30)
3,3% (1-N)
Humans (115)
2,6% (3-N)
0,8%* (1-N)
9,6%* (2-A, 9-N)
7%* (2-A, 6-N)
Questing ticks °
Anaplasma°
821
1,8%
Blood
Anaplasma
Rodents (1295)
0,3%
° Screening of questing ticks was done by Fausta Rosso
* Positive samples not confirmed by sequencing yet