Genetic diversity of Anaplasma phagocytophilum and reservoir competence of wild life animals for tick-borne pathogens in northern Italy.

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Genetic diversity of Anaplasma phagocytophilum and reservoir competence of wild life animals for tick-borne

pathogens in northern Italy.

Ivana Baráková^1,2, Giovanna Carpi³, Fausta Rosso², Michal Chvostač¹, Valentina Tagliapietra², Heidi C. Hauffe², Annapaola Rizzoli², Markéta Derdáková¹

1 Institute of Zoology, SAS, Dúbravská cesta 9, 845 06 Bratislava, Slovakia

2 Fondazione Edmund Mach, Via Edmund Mach, 1, 38010 San Michele All'adige Trento, Italy

3 Yale School of Public Health, 60 College Street, New Haven, CT 06520, USA

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Vectors and reservoir host 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

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Ecology of A.phagocytophilum

 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)

 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

 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

In USA In Europe

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Are there two different enzootic cycles of AP

circulating in northern Italy?

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Main Goals

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

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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

Main Goals

2. Reservoir competence

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Material and methods

 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):

16S Anaplasma

-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

Phylogenetic analysis

• Alignment – ClustalW

• Phylogenetic trees – MEGA4

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Collection sites in Northern Italy

VALLE DEI LAGHI:

Lamar Cavedine Pietramurata Mt. Bondone Covelo

Lasino Calavino Cadine Lundo

San Giovanni

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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%

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MSP4 gene

Diffrent ecotype from birds from Italy

Samples from rodents (from blood) 100% identical sequence from I.

trianguliceps

No I. ricinus feeding on rodents were found with this genotype

Anaplasma phagocytophilum Mufflon Northern Italy IRH004711-MSP4

IRHO12211-MSP4 IRH015611-MSP4 IRH018611-MSP4

268D Ixodes ricinus Slovakia IRH004511-MSP4

IRH004611-MSP4

Ixodes ricinus NORWAY13.66 AP100 roe deer Spain APElsa

OS15apsheep Slovakia Ixodes ricinus 19MVF Slovakia 14OVF sheep Slovakia AP27FVF I.ricinus Slovakia

AP12MVFV I. ricinus Slovakia AP13MVFV I. ricinus Slovakia

APV1 Ap variant USA APHZ human USA APWIH

APMRK AP136

Anaplasma phagocytophilum Rothirsch3 IRH004811-MSP4

Anaplasma phagocytophilum Cairn EF442006 Anaplasma phagocytophilum Mufflon4 AY530195

Anaplasma phagocytophilum bovAll56 IRH017311-MSP4

Anaplasma phagocytophilum ZJ-China Anaplasma phagocytophilum roe

Anaplasma phagocytophilum 18MVF tick Slovakia HU23-MSP4

HU148-MSP4 HU119-MSP4 HD158-MSP4

Anaplasma phagocytophilum kfv 007 rodent England OS56 Anaplasma ovis Slovakia

Anaplasma marginale Stillwater 2 100

100

100 80 57

99

70 64

84 99 87

100 52

30 40

28

33

30 67

86

52 73

53 63

0.01

Anaplasma phagocytophilum roe deer Northen Italy

Anaplasma phagocytophilum Apodemus flavicollis Northen Italy Anaplasma phagocytophilum Dog Northen Italy

Anaplasma phagocytophilum roe deer Northen Italy Anaplasma phagocytophilum roe deer Northen Italy

Anaplasma phagocytophilum bird Northen Italy

Anaplasma phagocytophilum rodent Northen Italy Anaplasma phagocytophilum rodent Northen Italy Anaplasma phagocytophilum rodent Northen Italy Anaplasma phagocytophilum rodent Northen Italy

Maximum Likelihood Tree of the MSP4 sequences

Clade 1

Clade 2

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groEL gene

Anaplasma phagocytophilum Slovakia Ixodes ricinus male from vegetation Anaplasma phagocytophilum Ixodes ricinus questing Germany

Anaplasma phagocytophilum Ixodes ricinus Austria Italy181 Anaplasma phagocytophilum Ixodes ricinus Italy889 Anaplasma phagocytophilum Ixodes ricinus IRH002611-GROEL

IRH002611-GROEL(2) IRH004711-GROEL IRH004711-GROEL(2) IRH018611-GROEL

Anaplasma phagocytophilum tick-from wild boar Slovenia IRH012211-GROEL

IRH04511-GROEL IRH04511-GROEL(2)

California woodrat horse

Italy782 Anaplasma phagocytophilum Ixodes ricinus Anaplasma phagocytophilum dog-7414

Anaplasma phagocytophilum human Anaplasma phagocytophilum horse

Anaplasma phagocytophilum sheep IRH004811-GROEL

IRH004811-GROEL(2)

Italy737 Anaplasma phagocytophilum Ixodes ricinus IRH017411-GROEL

Anaplasma phagocytophilum Ixodes persulcatus Ixodes persulcatus Anaplasma phagocytophilum Sv-vole8 Myodes rutilus gi328900325 Anaplasma phagocytophilum Sorex araneus

Anaplasma phagocytophilum Swiss rodent EHR-NE1 Clethrionomys glareolus gi6409326 Anaplasma phagocytophilum Slovakia-Hylov rodent ear Myodes glareolus2

HU148-GROEL HU23-GROEL HU119-GROEL

Anaplasma platys RDC 100

100 92

100 100 100

100

100 100

73 100 100 100

71 100

70 100

100 100

100

20

Anaplasma phagocytophilum sheep Northen Italy

Anaplasma phagocytophilum Apodemus flavicollis Northen Italy Anaplasma phagocytophilum roe deer Northen Italy

Anaplasma phagocytophilum mufflon Northen Italy

Anaplasma phagocytophilum bird Northen Italy

Samples from rodents from Italy

Diffrent ecotype from birds from Italy

Phylogenetic tree of groEL sequences constructed by Maximum parsimony

Clade 1

Clade 2

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Reservoir competence

Pathogen birds (n=91) rodents (n=342) wild ungulats (n=184)

Borrelia

B. garinii 23% (21) 0 0

B. valaisiana 8.8% (8) 0 0

B.burgdorferi

B. lusitaniae 7.7% (7) 0 0

B. afzelii 0 9.6% (33) 0

Rickettsia spp. R. helvetica 5.5% (5) 2.6% (9) 8.2% (15)

R. slovaca 0 0.3% (1) 0

R. monacensis 0 2.3% (8) 2.2% (4)

Babesia spp. B. EU1 0 2.3% (8) 0.5% (1)

B. capreoli 1% (1) 0 0

Anaplasma A. phagocytophilum 0 0 4.3% (8)

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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

Conclusions

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Thank you

for your attention

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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) 4% (2-N) 4% (2-N)

Sheep (13) 7,7% (1-A) 0% 0% 0%

Bird (27) 11% (3-N) 7,4% (2-N) 37% (10-N) 3,7% (1-N)

Dog (30) 3,3% (1-N) 0% 0% 0%

Humans (115) 2,6% (3-N) 0,8%* (1-N) 9,6%* (2-A, 9-N) 7%* (2-A, 6-N)

Blood Anaplasma

Rodents (1295) 0,3%

° Screening of questing ticks was done by Fausta Rosso

* Positive samples not confirmed by sequencing yet

Questing ticks ° ^Anaplasma°

821 1,8%