1. health and fitness
2. disease

Ref.no:
Emerging tick-borne diseases, their prevalence, reservoirs and genetic
variability in northern Italy.
1, 2 Ivana
Baráková, 2 Markéta Derdáková, 1 Fausta Rosso, 3 Giovanna Carpi, 2 Diana Zelinková, 2 Michal Chvostač,
1 Heidi C. Hauffe, 1 Annapaola Rizzoli
1Institute
of Zoology, Slovak Academy of Sciences, Dúbravská cesta 9, Bratislava, Slovakia
2 Fondazione Edmund Mach di San Michele all'Adige, Via E. Mach, 1 (TN), Italy
3 Yale School of Public Health, 60 College Street, New Haven, CT 06520, USA
Introduction
The developement of meta-communities risk model for tick borne infections require the
evaluation of several parameters including the prevalence of infection for various pathogens in
questing ticks, the identification of the competent reservoir hosts and the assessment of their
reservoir capacity. In the current study we determined the prevalence of five emerging
pathogens (Anaplasma phagocytophilum, Rickettsie spp., Babesia spp., Borrelia spp and
Candidatus N. mikurensis ) in questing I. ricinus ticks and determine their occurrence in ticks
detached from various hosts (wild ungulates, rodents, sheep, birds, dogs and humans) with the
aim to clarify their role as reservoirs for the various pathogens in natural foci of alpine region in
Italy. Finally we explored in detail the genetic variability of A. phagocytophilum and its ecological
associations with hosts and vectors in the area.
Figure A, B: Bayesian phylogenetic tree of msp4, groEl sequences of A.
phagocytophilum strains circulating in northern Italy. These phylogenetic
analyses shows two main clades, one containing sequences from different hosts
(deer, sheep, bird, dogs, humans) and other one containing sequences from
rodents. Sequences from rodents are 100% identical with sequence from I.
trianguliceps. None of the tested I. ricinus were found with this genotype feeding
on rodents.
groEL gene
Materials and methods
Ticks were immediately placed in to the 70% ethanol until DNA was extracted. The DNA from ticks was extracted using QIAamp
DNA Investigator kit (QIAxcel) or by alkaline hydrolysis method (Guy and Stanek, 1991). In order to verify that DNA was
successfully amplified from I. ricinus tick a ̴ 470 bp fragment of the 16S rRNA gene was obtained from each sample. A fragment of
the 16S rRNA gene (546 bp) of A. phagocytophilum was amplified by means of nested PCR, with primers previously reported
(Massung et al. 1998). Presence of Babesia sp. was detected by amplifying the partial 18S rRNA gene. Presence of Borrelia
burgdorferi s.l. was detected by amplification of 5S-23S rDNA intergenic spacer region. Rickettsia sp. was detected by amplifying
partially the 17K gene (480 bp). Genetic variability of A. phagocytophilum was identidied by amplification of msp4 and groEl gene.
Amplified DNA was visualized by BioAnalyzer, Agilent 2100 or by QIAxcel screengel. Positive PCR products were purified by
enzymatic Exosap-IT (USB Corporation) and sequenced in both directions with an ABI BigDye terminator kit (Applied Biosystems,
Monza, Italy) and analysed on an ABI prism 3130 automated sequencer.
Results
29.4%
3.7% 100% 0%
5.9%
0%
0%
0%
11.8% 100%
6. 5% 0%
6.25% 0%
0%
9.7%
0%
0%
0%
66.7% 33.3% 0%
0%
Rodents
27
0
8
0
4/31
35
6.5%
6.5% 50%
Birds
24
0
0
2
0/26
26
11.5%
7.7% 100% 0%
40%
50%
12.5% 25%
12.5% 3.8%
100%
0%
0%
0%
Sheep
13
0
0
0
10/3
13
10%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
10%
0%
Dog
12
15 0
0
7/20
27
5%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
Human
88
0
0
25/63
88
3.2%
0%
0%
0%
6.3%
25%
75%
0%
0%
3.2%
50%
0%
50%
0%
Questing
ticks
2133 0
38%
0.5%
0
0
0 339/179
4
2133
1,8%
50%
0%
R. slovaca
44
R. monacensis
17/27
R. raoultii
0
R. helvetica
0
Babesia spp.
0
Rickettsia spp.
ed
Borrelia luisitaniae
(A/N)*
Borrelia valaisiana
screen
Borrelia afzelii
stage
Borrelia garinii
ticks
B. burgdorferi s.l.
per
I. turdus
n. of
I. trianguliceps
N. ticks
I. hexagonus
I. ricinus
Tick host
group (n.
Individual
s sampled
per group)
Wild
44
ungulate
Babesia microti
Total
Babesia venatorum
A. phagocytophilum
Tab.1: Prevalence of various pathogens in nymphs and adult ticks detached from hosts and in questing ticks.
0,36% 100% 0% 21.6%
33.4%
21.5% 21.5%
3.9%
9%
61.3%
0%
N.mikurensis
R. slovaca
R. monacensis
R. helvetica
Rickettsia spp.
B. lusitaniae
msp4 gene
B. turdi
B. valaisiana
B. afzelii
screened
B. garinii
larvae ticks
B. burgdorferi s.l.
group)
Total n. of
B. capreoli
sampled per
B. venatorum
(n. Individuals
Babesia spp.
Tick host group
A. phagocytophilum
Tab.2: Prevalence of various pathogens in larvae ticks detached from wildlife hosts.
Wild ungulates
185
5.4% 0.5% 100%
0%
0%
0%
0%
0%
0%
0%
9.7%
66.7% 27.8% 5.6% 0%
Rodents
358
0%
0%
10%
3%
97%
0%
0%
0%
4.5%
56.2% 43.8% 0%
5.3%
Birds
87
4.6% 2.3% 50%
50%
44.8%
43.6%
2.6%
16%
15.4% 2.6%
5.7%
5.7%
0%
1.7% 100%
0%
0%
Conclusions
The capacity of various host to contribute to the epidemiological cycle of the studied pathogens is highly
variable. The highest prevalence of infection with A. phagocytophilum and Rickettsia spp. was detected in ticks
detached, either at adult and nymphal stage and larval stage, from wild ungulates. B. burgdorferi s.l. and Babesia
spp. were mostly recorded in ticks collected from birds, were also an unusual species of Borrelia (Borrelia turdi)
was detected for the first time in this area. The pathogen Candidatus N. mikurensis was recorded only in larvae
detached from rodents.
Phylogenetic analysis of two genetic loci in positive samples for A. phagocytophilum have shown that
genotypes in ticks from some hosts (deer, sheep, dogs, human) were distinct from genotypes found in blood
samples collected from rodents (Fig. A,B). Msp4 and GroEL sequences of A. phagocytophilum genotypes from
rodents were identical to the genotype found in I. trianguliceps in UK and Slovakia. Our results suggest that
rodents in Europe may be reservoirs of A. phagocytophilum strains which are different from the strains circulating
in the other hosts species examined. Our study from northern Italy support the evidence that in Europe the
circulation of various A. phagocytophilum variants are associated with different epidemiological cycles (Barakova
et al., 2014, Genetic and ecologic variability among Anaplasma phagocytophilum strains, northern Italy, PMID:
24855911.)
Acknowledgements: This study was partially funded by the European Commission under the FP7 program (EDENEXT, www.edenext.eu). We thanks to all
those persons kindly helping in field work and providing feedings ticks from various sources. VEGA 2/0055/11, the Slovak Research and Development Agency
under contract No. APVV–0267-10,