Occurrence and identification of risk areas of Ixodes ricinus-borne pathogens: a cost-effectiveness analysis in north-eastern Italy

  • Gioia Capelli1Email author,

    Affiliated with

    • Silvia Ravagnan1,

      Affiliated with

      • Fabrizio Montarsi1,

        Affiliated with

        • Silvia Ciocchetta1,

          Affiliated with

          • Stefania Cazzin1,

            Affiliated with

            • Elena Porcellato1,

              Affiliated with

              • Amira Mustafa Babiker1,

                Affiliated with

                • Rudi Cassini2,

                  Affiliated with

                  • Annalisa Salviato1,

                    Affiliated with

                    • Giovanni Cattoli1 and

                      Affiliated with

                      • Domenico Otranto3

                        Affiliated with

                        Parasites & Vectors20125:61

                        DOI: 10.1186/1756-3305-5-61

                        Received: 3 January 2012

                        Accepted: 27 March 2012

                        Published: 27 March 2012

                        Abstract

                        Background

                        Ixodes ricinus, a competent vector of several pathogens, is the tick species most frequently reported to bite humans in Europe. The majority of human cases of Lyme borreliosis (LB) and tick-borne encephalitis (TBE) occur in the north-eastern region of Italy. The aims of this study were to detect the occurrence of endemic and emergent pathogens in north-eastern Italy using adult tick screening, and to identify areas at risk of pathogen transmission. Based on our results, different strategies for tick collection and pathogen screening and their relative costs were evaluated and discussed.

                        Methods

                        From 2006 to 2008 adult ticks were collected in 31 sites and molecularly screened for the detection of pathogens previously reported in the same area (i.e., LB agents, TBE virus, Anaplasma phagocytophilum, Rickettsia spp., Babesia spp., "Candidatus Neoehrlichia mikurensis"). Based on the results of this survey, three sampling strategies were evaluated a-posteriori, and the impact of each strategy on the final results and the overall cost reductions were analyzed. The strategies were as follows: tick collection throughout the year and testing of female ticks only (strategy A); collection from April to June and testing of all adult ticks (strategy B); collection from April to June and testing of female ticks only (strategy C).

                        Results

                        Eleven pathogens were detected in 77 out of 193 ticks collected in 14 sites. The most common microorganisms detected were Borrelia burgdorferi sensu lato (17.6%), Rickettsia helvetica (13.1%), and "Ca. N. mikurensis" (10.5%). Within the B. burgdorferi complex, four genotypes (i.e., B. valaisiana, B. garinii, B. afzelii, and B. burgdorferi sensu stricto) were found. Less prevalent pathogens included R. monacensis (3.7%), TBE virus (2.1%), A. phagocytophilum (1.5%), Bartonella spp. (1%), and Babesia EU1 (0.5%). Co-infections by more than one pathogen were diagnosed in 22% of infected ticks. The prevalences of infection assessed using the three alternative strategies were in accordance with the initial results, with 13, 11, and 10 out of 14 sites showing occurrence of at least one pathogen, respectively. The strategies A, B, and C proposed herein would allow to reduce the original costs of sampling and laboratory analyses by one third, half, and two thirds, respectively. Strategy B was demonstrated to represent the most cost-effective choice, offering a substantial reduction of costs, as well as reliable results.

                        Conclusions

                        Monitoring of tick-borne diseases is expensive, particularly in areas where several zoonotic pathogens co-occur. Cost-effectiveness studies can support the choice of the best monitoring strategy, which should take into account the ecology of the area under investigation, as well as the available budget.

                        Keywords

                        Ixodes ricinus tick-borne diseases surveillance economic evaluation Italy.

                        Background

                        Ticks are second only to mosquitoes as vectors of zoonotic pathogens and are recognized as the primary vectors of vector-borne diseases in temperate climates [1].

                        Ixodes ricinus (Acari: Ixodidae), also known as "wood", "sheep" or "castor-bean" tick, is the ixodid species most frequently reported to bite humans in Europe [2], and acts as a major vector of viral, bacterial, and protozoan agents, which infect many domesticated and wild animals, as well as humans [3]. For instance, this species can transmit the tick-borne encephalitis virus (TBEv), Borrelia burgdorferi sensu lato (s.l.), the aetiological agent of Lyme borreliosis (LB), as well as other pathogens, e.g. Rickettsia, Anaplasma and Babesia spp. [4]. The distribution of tick-transmitted pathogens (TTPs) is primarily dependent on tick density and the availability of animal reservoirs. I. ricinus acts as vector of several pathogens mostly because of its large host spectrum, being able to feed on more than 300 animal species [2].

                        In Italy, I. ricinus occurs throughout the peninsula and its populations reach the highest density in hilly and pre-alpine northern areas, characterized by a temperate climate, with cold winters, and cool and humid summers [5]. These areas represent the optimal I. ricinus biotope, consisting of microhabitats characterized by humidity above 85% and a well conserved biocenosis of wild animals (including small and large mammals, birds, and reptiles). The north-eastern region of Italy accounts for the majority of human cases of LB and TBE [6]; the first cases of Human Granulocytic Anaplasmosis (HGA) by Anaplasma phagocytophilum have also been reported in the same area [7, 8].

                        According to Heiman et al.[1], tick-borne diseases (TBDs) are also likely to become among the infectious threats, one of the main concerns for public health in Europe within the coming years; therefore, well planned, efficient, and cost-effective surveillance systems need to be implemented. The first step towards planning TBDs surveillance should consist in assessing the panel of pathogens occurring in a given area and their relative epidemiological importance, in relation to their prevalence in vectors and hosts and the severity of the diseases that they cause. Alongside burden of pathogens, information on vector density and dynamics also needs to be aquired. In order to assess the spatial and temporal distribution of I. ricinus and the environmental factors associated with its occurrence in north-eastern Italy, the Ministry of Health launched a three year-project (code RC-IZSVe 11/04), whose results have been published elsewhere [9, 10]. In the present study, adult ticks collected through the previous years were screened for all the pathogens known or suspected to occur in north-eastern Italy, including TBEv, LB agents, A. phagocytophilum, Rickettsia spp., Babesia spp. and the recently described bacterium "Candidatus Neoehrlichia mikurensis".

                        The aims of this study were to assess the suitability of adult tick screening for (i) detecting the occurrence of endemic and emergent pathogens in north-eastern Italy, and (ii) identifying areas at risk for pathogen transmission to animals and humans. Based on the results of this survey, different strategies for collection of ticks and pathogen screening, as well as their relative costs, were evaluated and discussed.

                        Over the past few years, central and local Governments have drastically reduced funds to the majority of institutions involved in monitoring vector-borne diseases. This will inevitably impact on ways of approaching research and surveillance actions in terms of sampling design, and data collection and analyses.

                        Methods

                        Study area

                        From 2006 to 2008, I. ricinus ticks were collected in an area of north-eastern Italy (45°30'52"N to 46°32'4"N and 11°9'52"E to 13°1'14"E) within the regions of Veneto and Friuli Venezia Giulia (FVG), including five provinces (i.e., Vicenza, Verona, Treviso, Pordenone, and Udine) (Figure 1). Sampling was carried out in the south-eastern slope of hilly and pre-alpine areas in habitats suitable for growth and survival of I. ricinus, characterized by heterogeneous deciduous woodland and mixed forest, and occurrence of domestic and/or wild animals. The altitudes of the sites investigated ranged between 120 and 1308 m above sea level (a.s.l.). All sites were close to human dwellings or easily accessible through footpaths.
                        http://static-content.springer.com/image/art%3A10.1186%2F1756-3305-5-61/MediaObjects/13071_2012_Article_541_Fig1_HTML.jpg
                        Figure 1

                        Map of north-eastern Italy showing the 31 sites in which adult ticks were found (yellow: sites negative for pathogens; red: sites positive for one or more pathogens; number of pathogens/site is also reported within each red symbol).

                        Tick sampling and identification

                        From 2006 to 2008 a permanent site for each province was monitored monthly, whereas another 50 sites were monitored on one occasion each month (herein after defined as temporary sites). Ticks were collected by dragging using a 1 m2 white flannel cloth, through 50 m transects, stopping every 2.5 m to prevent their detachment. Once collected, ticks were kept refrigerated at + 4°C, counted, grouped according to their developmental stage, and identified based on morphological features [11]. All adults collected throughout the three years at 31 sites (5 permanents and 26 temporary) were molecularly processed.

                        Biomolecular analyses for the identification of pathogens and sequencing

                        Nucleic acids were extracted from single adult ticks using All Prep DNA/RNA mini Kit (Qiagen, Inc., Valencia, CA), according to the manufacturer's instructions and then kept frozen at -80°C. Target genes, primers, and probes used for testing and the size of the PCR amplification products are listed in Table 1 and 2.
                        Table 1

                        Biomolecular method used for pathogen identification, target genes, primers, probes and references.

                        Species

                        method

                        gene

                        primers

                        Nucleotide sequence (5'- 3')

                        Amplicon

                        size (bp)c

                        Ref.

                        Ixodes

                        PCR

                        16S ribosomal RNA

                        F-16sIxodes

                        AAAAAAATACTCTAGGGATAACAGCGTAA

                        97

                        [12]

                        (extraction control)

                          

                        R-16sIxodes

                        ACCAAAAAAGAATCCTAATCCAACA

                          
                           

                        16s-Ixodes-Probe

                        TTTTGGATAGTTCATATAGATAAAATAGTTTGC GACCTCG

                          

                        B. burgdorferi s.l.

                        real time PCR (duplex)

                        23S-rRNA

                        Bb23Sf

                        CGAGTCTTAAAAGGGCGATTTAGT

                        75

                        [14]

                           

                        Bb23Sr

                        GCTTCAGCCTGGCCATAAATAG

                          
                           

                        Bb23Sp-FAM

                        AGATGTGGTAGACCCGAAGCCGAGTG

                          

                        A. phagocytophilum

                        real time PCR (duplex)

                        msp2

                        ApMSP2f

                        ATGGAAGGTAGTGTTGGTTATGGTATT

                        77

                        [14]

                           

                        ApMSP2r

                        TTGGTCTTGAAGCGCTCGTA

                          
                           

                        ApMSP2p-HEX

                        TGGTGCCAGGGTTGAGCTTGAGATTG

                          

                        B. burgdorferi s.l.

                        PCR

                        flagellin

                        FLA1

                        AGAGCAACTTACAGACGAAATTAAT

                        482

                        [16]

                           

                        FLA2

                        CAAGTCTATTTTGGAAAGCACCTAA

                          

                        A. phagocytophilum

                        PCR

                        msp2

                        msp2-3f

                        CCAGCGTTTAGCAAGATAAGAG

                        334

                        [15]

                           

                        msp2-3r

                        GMCCAGTAACAACATCATAAGC

                          

                        TBEv

                        rRT-PCR

                        3' non-coding region

                        F-TBE 1

                        GGGCGGTTCTTGTTCTCC

                        67

                        [12]

                           

                        R-TBE 1

                        ACACATCACCTCCTTGTCAGACT

                          
                           

                        TBE-Probe-WT

                        TGAGCCACCATCACCCAGACACA

                          

                        TBEv

                        nested PCR

                        non-structural protein NS5

                        FSM-1

                        GAGGCTGAACAACTGCACGA

                        357

                        [13]

                           

                        FSM-2

                        GAACACGTCCATTCCTGATCT

                          
                          

                        non-structural protein NS5

                        FSM-1i

                        ACGGAACGTGACAAGGCTAG

                        251

                         
                           

                        FSM-2i

                        GCTTGTTACCATCTTTGGAG

                          

                        Rickettsia spp.

                        PCR

                        citrate synthase

                        RpCS.877p

                        GGGGGCCTGCTCACGGCGG

                        381

                        [17]

                           

                        RpCS1258n

                        ATTGCAAAAAGTACAGTGAACA

                          

                        Cand. N. mikurensis

                        PCR

                        groEL

                        NM-128s

                        AACAGGTGAAACACTAGATAAGTCCAT

                        1024

                        [19]

                           

                        NM-1152as

                        TTCTACTTTGAACATTTGAAGAATTACTAT

                          

                        Babesia/Theileria

                        PCR

                        18S rRNA

                        RLB-F2

                        GACACAGGGAGGTAGTGACAA

                        400

                        [18]

                           

                        RLB-R2

                        CTAAGAATTTCACCTCTGACAGT

                          
                        Table 2

                        Primers and UPL used for genospecies identification of Borrelia burgdorferi s.l. in co-infected ticks using real time PCR assays

                        Genospecies

                        Target gene

                        5'→ 3'primer sequence

                        UPL number

                        Amplicon

                        size (bp) c

                        B. burgdorferi s.s.

                        OspAa

                        TCTTGAAGGAACTTTAACTGCTGA

                        TGAAACTTCCCCAGATTTTGA

                        #119

                        97

                        B. afzelii

                        OspA

                        GACTCCGCAGGTACCAATTT

                        AAAGCGTTTTTAAGTTCATCAAGTG

                        #98

                        71

                        B. garinii

                        Flab

                        TCTGCTATGATTATGCCACCA CCTTTGCCTAAGAATTGATTACCA

                        #2

                        74

                        B. valaisiana

                        Fla

                        CCAAATGCACATGTTGTCAAA

                        TTTGCAGGTTGCATTCCA

                        #132

                        78

                        aOspA: Outer surface protein A gene; bFla: flagellin gene; cbp: base pairs

                        To ensure the effectiveness of the nucleic acid extraction, a real time PCR targeting the 16S rRNA was applied [12].

                        A real time PCR was used for TBEV detection [12]. Positive results in real-time PCR were confirmed by a nested real time (RT)-PCR [13]. A multiplex RT-PCR was used for the simultaneous detection of A. phagocytophilum and B. burgdorferi s.l. [14].

                        All samples positive for A. phagocytophilum were confirmed by a specific PCR [15] and sequenced. To determine the genospecies of B. burgdorferi s.l., a conserved region of the flagellin gene was amplified by PCR for all the B. burgdorferi s.l. positive samples according to a protocol previously published [16], followed by genetic sequencing of the PCR products. Sequence electropherograms of B. burgdorferi s.l. were checked for quality and to reveal the presence of double nucleotide peaks. When double peaks were detected in both (i.e., for primers forward and reverse) high-quality sequence electropherograms and their location corresponded to the variable sites specific for a certain genospecies, a multiple infection was suspected. To confirm the presence of co-infections of B. burgdorferi genospecies, four RT-PCR assays were performed by using Universal Probe Library (UPL) (Roche, Mannheim, Germany), presynthesized, fluorescence-labelled locked nucleic acid (LNA) hydrolysis probes, to detect specifically B. burgdorferi s.s., B. afzelii, B. garinii and B. valaisiana. Primers and probes number (Table 2) were chosen by free online software (UPL Assay Design Center web service; https://​www.​roche-applied-science.​com) and the UPL probe from the Roche Universal Probe Library collection. Real time PCR was performed with a reaction mixture consisting of 2 μl of DNA, 5 μl of 2× Light Cycler 480 Probes Master (Roche, Mannheim, Germany), 300 nM of each Borrelia species primer set and 200 nM of each corresponding UPL probe with a thermal cycling profile consisting of an initial activation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 10 s and annealing/extension at 60°C for 30 s and a final cooling step at 40°C for 30 s. Fluorescence data were collected in the annealing/extension phase at 60°C.

                        Rickettsia spp., Babesia spp., and "Ca. N. mikurensis" were amplified with protocols described in the literature [1719] and the species identity determined by genetic sequencing.

                        RT-PCRs were carried out on a Rotor Gene 6000 real-Time PCR system (Corbett, Australia) and traditional PCRs on a GeneAmp®PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA).

                        All PCR products were sequenced using the Big Dye Terminator v 3.1 cycle sequencing kit (Applied Biosystem, Foster City, CA, USA). The products of the sequencing reactions were purified using PERFORMA DTR Ultra 96-Well kit (Edge BioSystems, Gaithersburg, MD, USA) and sequenced in a 16-capillary ABI PRISM 3130 × l Genetic Analyzer (Applied Biosystem, Foster City, CA, USA). Sequence data were assembled and edited with SeqScape software v 2.5 (Applied Biosystem, Foster City, CA, USA), aligned and compared with representative sequences available in GenBank.

                        Statistical analysis

                        Differences in the prevalence of pathogens in relation to tick gender, province/region of origin, and month/year of collection were tested by using χ2 or Fisher's exact test, when appropriate. The correlation between number of adults examined and number of pathogens recovered was tested by linear regression. The software used was SPSS (SPSS Inc., Chicago, IL) for windows, version 13.0.

                        Cost estimation

                        Costs of molecular procedures were calculated as described by Cattoli et al.[20], adjusted for DNA/RNA extraction used in this study. Travel costs (distance range from the sites, i.e., 62-218 km), included fuel, tolls and meals for staff involved in tick collections. Costs for staff were calculated based on the number of working days and on staff salary scales of Istituto Zooprofilattico Sperimentale delle Venezie, Italy (2011).

                        Results

                        Occurrence of pathogens and co-infections

                        During the 146 dragging collections performed throughout the three years, 193 adult ticks (i.e., 95 females and 97 males) were collected in 31 sites (range 1-47 ticks per site). At least one pathogen was detected in 77 (39.9%) ticks from 14 sites (45%). Overall, 11 pathogens were identified with variable prevalence (Table 3), with B. burgdorferi s.l. the most common (17.6%), followed by R. helvetica (13.1%) and "Ca. N. mikurensis" (10.5%). Four genotypes within the B. burgdorferi complex (i.e., B. valaisiana, B. garinii, B. afzelii, and B. burgdorferi sensu stricto) were identified. TBE virus, A. phagocytophilum, R. monacensis, and Babesia EU1 (proposed name B. venatorum) were detected more rarely (Table 3). GenBank accession numbers of the most representative sequences are reported in Table 3.
                        Table 3

                        Pathogens and their prevalence (P) detected in 193 adult Ixodes ricinus from 2006 to 2008 in north-eastern Italy, permanent and temporary sites positives and year of detection.

                        Pathogens [accession numbers]

                        pos.

                        ticks

                        P

                        perm.

                        sites

                        n = 5

                        temp.

                        sites

                        n = 26

                        year of detection

                             

                        2006

                        2007

                        2008

                             

                        n = 43

                        n = 83

                        n = 67

                        Borrelia burgdorferi s.l.

                        34

                        17.6%

                             

                           B. valaisiana [GU581273]

                        12

                        6.2%

                        2

                        3

                        x

                        x

                        x

                           B. afzelii [GU581269, GU581270]

                        10

                        5.2%

                        2

                        2

                        x

                        x

                        x

                           B. garinii [GU581274-GU581277]

                        8

                        4.1%

                        1

                        3

                        x

                        x

                        -

                           B. burgdorferi s.s. [GU581271, GU581272]

                        6

                        3.1%

                        2

                        3

                        x

                        x

                        x

                        Rickettsia helvetica* [JQ669952, JQ669953]

                        25

                        13.1%

                        4

                        5

                        x

                        x

                        x

                        Ca. Neoehrlichia mikurensis* [JQ669946]

                        20

                        10.5%

                        3

                        3

                        x

                        x

                        x

                        R. monacensis* [JQ669950, JQ669951]

                        7

                        3.7%

                        -

                        3

                        x

                        -

                        -

                        TBE flavivirus [JQ669945]

                        4

                        2.1%

                        1

                        -

                        -

                        x

                        -

                        Anaplasma phagocytophilum [JQ669947,

                        JQ669948, JQ669949]

                        3

                        1.5%

                        2

                        1

                        -

                        x

                        -

                        Bartonella spp.

                        2

                        1.0%

                        2

                        -

                        -

                        x

                        -

                        Babesia EU1 (B. venatorum)* [JQ669954]

                        1

                        0.5%

                        1

                        -

                        -

                        -

                        x

                        Total

                        77

                        39.9%

                        5

                        9

                           

                        GenBank accession numbers are also reported.

                        * adult tested 191

                        The overall pathogen infection rate was significantly higher in females than in male ticks (46.2% vs. 29.9%; p < 0.01); considering single species, this difference was significant (p < 0.05) only for B. burgdorferi s.l., B. garinii and R. helvetica. All pathogens were detected in the permanent sites examined, with the exception of R. monacensis which was only detected in temporary sites (Table 3). Whilst highly prevalent pathogens (i.e., LB agents, R. helvetica and "Ca. N. mikurensis") were detected in both permanent and temporary sites, those with low prevalence rates (e.g., TBEv, Bartonella spp., and Babesia EU1) were only detected in permanent sites (Table 3), most likely due to the high intensity of sampling. Out of 77 positive ticks, 60 (78%) harboured a single infection, 13 (17%) were co-infected by two pathogens, and 4 (5%) by three pathogens. Pathogen associations are reported in Table 4 which describes the co-infections detected in 13 female and in 4 male ticks (p < 0.05).
                        Table 4

                        Pathogen association in co-infected ticks

                        Co-infected ticks

                        Pathogen associations

                        double co-infection

                         

                        3

                        R. helvetica-B. garinii

                        3

                        R. helvetica-Ca. N. mikurensis

                        1

                        R. monacensis-B. afzelii

                        1

                        R. monacensis-Ca. N. mikurensis

                        1

                        R. monacensis-B. valaisiana

                        1

                        B. afzelii-Ca. N. mikurensis

                        1

                        B. garinii/B. valaisiana

                        1

                        B. garinii-Ca. N. mikurensis

                        1

                        TBE-B. burgdorferi s.s.

                        triple co-infection

                         

                        1

                        TBE-B. burgdorferi s.s.-B. afzelii

                        1

                        R. monacensis-B. afzelii-Ca. N. mikurensis

                        1

                        R. monacensis-B. burgdorferi s.s.-Ca. N. mikurensis

                        1

                        B. valaisiana-Babesia EU1-Ca. N. mikurensis

                        Pathogen spatial and temporal distribution

                        Pathogen prevalence and species diversity in spatial distribution were different in the five provinces monitored (Table 5), with the Northern provinces (i.e., Udine, Pordenone, and Treviso) displaying the highest adult tick density and composition of pathogen species (Figure 1). In particular, out of 11 TTPs, 8 and 10 were detected only in Treviso and Udine, respectively. The higher overall prevalence of TTPs in Pordenone was linked specifically to infections by B. afzelii, R. monacensis and "Ca. N. mikurensis". Despite the small number of adult ticks (Table 5) collected in the southern provinces (i.e., Verona and Vicenza), high prevalent pathogens (R. helvetica and B. valaisiana) were detected in the same areas. All pathogens except Babesia EU1 were detected in FVG region, whereas R. monacensis, B. afzelii, and TBEv were not detected in adult ticks in Veneto region.
                        Table 5

                        Pathogens prevalence according to province of origin (permanent and temporary sites all over the three years) and significant differences*

                         

                        Friuli Venezia Giulia region

                        Veneto region

                        provinces

                        Pordenone

                        n = 47

                        Udine

                        n = 60

                        Treviso

                        n = 64

                        Vicenza

                        n = 10

                        Verona

                        n = 12

                        pathogens

                        pos ticks

                        %

                        pos ticks

                        %

                        pos ticks

                        %

                        pos ticks

                        %

                        pos ticks

                        %

                        Lyme agents:

                        14

                        29.8a

                        8

                        13.3a

                        10

                        15.6

                        -

                        -

                        2

                        16.7

                           B. valaisiana

                        3

                        6.4

                        2

                        3.3

                        5

                        7.8

                        -

                        -

                        2

                        16.7

                           B. afzelii

                        8

                        17.0b

                        2

                        3.3b

                        -

                        -

                        -

                        -

                        -

                        -

                           B. garinii

                        2

                        4.3

                        3

                        5.0

                        3

                        4.7

                        -

                        -

                        -

                        -

                           B. burgdorferi s.s.

                        2

                        4.3

                        2

                        3.3

                        2

                        3.1

                        -

                        -

                        -

                        -

                        R. helvetica

                        6

                        13.0

                        6

                        10.0

                        10

                        15.6

                        1

                        10.0

                        2

                        16.7

                        Ca. N. mikurensis

                        9

                        19.6

                        5

                        8.3

                        6

                        9.4

                        -

                        -

                        -

                        -

                        R. monacensis

                        6

                        13.0c

                        1

                        1.7c

                        -

                        -

                        -

                        -

                        -

                        -

                        TBEv

                        -

                        -

                        4

                        6.7

                        -

                        -

                        -

                        -

                        -

                        -

                        A. phagocytophilum

                        -

                        -

                        2

                        3.3

                        1

                        1.6

                        -

                        -

                        -

                        -

                        Bartonella spp

                        -

                        -

                        1

                        1.7

                        1

                        1.6

                        -

                        -

                        -

                        -

                        Babesia EU1

                        -

                        -

                        -

                        -

                        1

                        1.6

                        -

                        -

                        -

                        -

                        Total

                        36

                        76.6 ABCd

                        28

                        46.7 Ae

                        29

                        45.3 B

                        1

                        10.0 Ce

                        4

                        33.3 d

                        * Equal letter corresponds to significant difference (lower case = p < 0.05; upper case = p < 0.01)

                        The number of pathogens identified ranged from one to seven per single site (Figure 1) and, in general, the number of ticks/site was positively correlated (R2 = 0.83) with the number of pathogens detected. Interestingly, up to six pathogens were detected in 13 adults ticks collected in a single temporary site of the Treviso province. Although ticks and pathogens could be found from February to December throughout the three years of sampling, the density of adult ticks peaked in May and June, with all the 11 TTPs detected from April to June.

                        Possible scenarios for tick sampling and pathogen screening

                        Based on the results of this study, three different tick collection scenarios were pictured, and the results obtained compared with those above. The strategies hypothesized were as follows: tick collection throughout the year and testing of female ticks only (strategy A); collection from April to June and testing of adult male and female ticks (strategy B); collection from April to June and testing of female ticks only (strategy C).

                        The results of the three alternative strategies are summarized in Table 6. The prevalence of TTPs assessed using these three protocols did not differ significantly from the results of the initial screening. The prevalence calculated at province level resulted in a pathogen scenario similar to that of the initial screening for strategy A and B, whereas the small number (n = 67) of ticks collected in strategy C led to very high prevalence confidence intervals (data not shown).
                        Table 6

                        Pathogen prevalence according to the initial screening (all adults) and different sampling strategies (A, B, C) and prevalence difference among each strategy compared to the initial screening (Δ)

                           

                        Strategy A

                        Strategy B

                        Strategy C

                        Pathogens

                        all adults

                        n = 193

                        female ticks

                        all year

                        n = 95

                        Δ

                        all ticks

                        April-June

                        n = 127

                        Δ

                        female ticks

                        April-June

                        n = 67

                        Δ

                         

                        pos

                        %

                        pos

                        %

                        %

                        pos

                        %

                        %

                        pos

                        %

                        %

                        B. burgdorferi s.l.

                        34

                        17.6

                        23

                        24.2

                        6.6

                        19

                        15.0

                        2.6

                        14

                        20.9

                        3.3

                           B. valaisiana

                        12

                        6.2

                        6

                        6.3

                        0.1

                        6

                        4.7

                        -1.5

                        4

                        6.0

                        -0.2

                           B. afzelii

                        10

                        5.2

                        7

                        7.4

                        2.2

                        4

                        3.1

                        -2.0

                        2

                        3.0

                        -2.2

                           B. garinii

                        8

                        4.1

                        7

                        7.4

                        3.2

                        7

                        5.5

                        1.4

                        6

                        9.0

                        4.1

                           B. burgdorferi s.s.

                        6

                        3.1

                        5

                        5.3

                        2.2

                        4

                        3.1

                        0.0

                        4

                        6.0

                        2.9

                        R. helvetica

                        25

                        13.1

                        18

                        19.1

                        6.1

                        20

                        15.7

                        2.7

                        14

                        21.2

                        8.1

                        Ca. N. mikurensis

                        20

                        10.5

                        8

                        8.5

                        -2.0

                        11

                        8.7

                        -1.8

                        5

                        7.6

                        -2.0

                        R. monacensis

                        7

                        3.7

                        3

                        3.2

                        -0.5

                        2

                        1.6

                        -2.1

                        1

                        1.5

                        -2.2

                        TBEv

                        4

                        2.1

                        4

                        4.2

                        2.1

                        4

                        3.1

                        1.1

                        4

                        6.0

                        3.9

                        A. phagocytophilum

                        3

                        1.6

                        3

                        3.2

                        1.6

                        3

                        2.4

                        0.8

                        3

                        4.5

                        2.9

                        Bartonella spp.

                        2

                        1.0

                        0

                        0.0

                        -1.0

                        2

                        1.6

                        0.5

                        0

                        0.0

                        -1.0

                        Babesia EU1

                        1

                        0.5

                        0

                        0.0

                        -0.5

                        1

                        0.8

                        0.3

                        0

                        0.0

                        -0.5

                        Total

                        77

                        39.9

                        44

                        46.3

                        6.4

                        50

                        39.4

                        -0.5

                        31

                        46.3

                        6.4

                        The occurrence of all the 11 pathogens was confirmed by strategy B, while strategies A and C did not allow detection of sporadic pathogens (i.e., Bartonella spp., Babesia EU1), which were exclusively harboured by male ticks in this study. Out of 14 sites where pathogens were detected in the initial screening, 13, 11, and 10 were positive for pathogens using strategy A, B and C, respectively. The decrease in the number of ticks screened resulted in a loss of pathogen species detected in each single site. In particular, strategies A, B, and C did not allow the detection of 1-2 pathogens in 7, 3 and 7 sites, respectively.

                        Estimated costs (i.e., laboratory, travel and staff expenses) for the three strategies proposed are illustrated in Table 7. Compared with the initial screening, the costs of alternative strategies A, B and C were reduced by approximately one third, half and two thirds, respectively. Pros and cons of each strategy are illustrated in Table 8.
                        Table 7

                        Estimated costs (€) of different tick sampling strategies and pathogen screening for a three year study

                         

                        All ticks

                        Strategy A

                        Strategy B

                        Strategy C

                         

                        n

                        n

                        n

                        n

                        DNA/RNA extraction (x2)

                        388

                        3706

                        190

                        2438

                        254

                        1824

                        134

                        1286

                        biomolecular analyses

                        1018

                        7010

                        510

                        3516

                        669

                        4608

                        359

                        1634

                        sequencing

                        101

                        1818

                        59

                        1062

                        81

                        1458

                        55

                        990

                        draggings (travel costs)

                        146

                        24000

                        146

                        24000

                        71

                        9000

                        71

                        9000

                        Staff

                                

                           1 grant (sampling)

                        96

                        7234

                        96

                        7234

                        36

                        2713

                        36

                        2713

                           1 entomologist

                        32

                        4874

                        16

                        2399

                        21

                        3207

                        11

                        1692

                           1 technician

                        64

                        7932

                        32

                        3905

                        42

                        5220

                        22

                        2754

                           1 biotechnologist

                        112

                        26507

                        56

                        13277

                        75

                        17758

                        41

                        9697

                        Total

                        (reduction of costs)

                         

                        83081

                         

                        57832

                        (30%)

                         

                        45788

                        (45%)

                         

                        29765

                        (64%)

                        Table 8

                        Pros and cons of strategies A, B, and C in terms of results and costs

                        Strategies description

                        PROS

                        CONS

                        Strategy A

                        (pathogen detection in female ticks collected all over the year)

                        Good general pathogen detection

                        in the area

                        Good identification of risk sites

                        Good pathogen prevalence

                        assessment

                        No detection of sporadic

                        pathogens

                        High loss of single pathogen

                        detections per site

                        Low reduction of general costs

                        (30%)

                        No reduction of travel costs

                        Strategy B

                        (pathogen detection in all ticks

                        collected in the period April-June)

                        Excellent pathogen detection in

                        the area

                        Excellent pathogen prevalence

                        assessment

                        Low loss of single pathogen

                        determination per site

                        High reduction of travel costs (62%)

                        Medium efficiency in identifying

                        risk sites

                        Low reduction of laboratory

                        costs (33%)

                         

                        Detection of sporadic pathogens

                         

                        Strategy C

                        (pathogen detection in female ticks collected in the period April-June)

                        Good general pathogen detection

                        in the area

                        High reduction of general and

                        specific costs (64%)

                        Low efficiency of pathogen

                        prevalence assessment at local level

                        Non detection of sporadic pathogens

                        High loss of pathogen detection

                        per site

                        Discussion

                        The collection of adult ticks over a three-year period combining the use of permanent and temporary sampling sites provided relevant information on the occurrence of pathogens in the area under investigation. Up to 11 pathogens were detected in about 40% of I. ricinus individuals sampled from north-eastern Italy, with one or more pathogens occurring in 14 collection sites. The pathogens detected in the present study had already been identified from 1989 to date in I. ricinus collected in the same area [3, 2132], with the exception of B. lusitaniae, which was detected once in nymphs [33], and B. divergens which was isolated from cattle only [34]. However, this study reports a comprehensive survey of TTPs occurring at one time in this area.

                        LB agents and Rickettsia species were the most prevalent pathogens in ticks and are therefore regarded as the most likely transmissible agents to animals and humans in this area. The study monitored and confirmed the occurrence of other emergent pathogens, such as A. phagocytophilum, and Babesia EU1. Interestingly, it also ascertained the presence and the distribution of "Ca. N. mikurensis" for the first time in Italy. The relevant prevalence of ticks positive to "Ca. N. mikurensis" (more than 10%) is of particular interest considering the role of this pathogen as the aetiological agent of human infections in Germany, Switzerland, and Sweden [3537] and in a dog in Germany [19]. Indeed, following the primary isolation from rats (Rattus norvegicus) and Ixodes ovatus ticks [38] in Japan, this bacterium has been included in the list of emerging pathogens in Europe [39]. TBEv and A. phagocytophilum were detected in a few sites of those monitored (Table 3). The low prevalence and the scattered distribution patterns recorded for these agents, which often occur in local foci of transmission [40, 41], complicates monitoring of tick vectors, calling for the use of other tools, such as serological methods and clinical case reports, for supporting surveillance strategies. Bartonella spp. was also detected in I. ricinus and, in spite of the increasing number of infections reported in ticks [42, 43], the role played by I. ricinus in the transmission of this pathogen to animals and humans is disputable. However, recent laboratory evidence showed that the transmission of Bartonella birtlesii by I. ricinus ticks may occurr in naive mice [44].

                        Twenty-seven percent of positive ticks displayed co-infections by two or even three pathogens. Co-infections have been frequently reported in Europe not only in questing ticks [4547, 43, 48], but also in ticks removed from humans [49], as well as domestic and wild animals [50, 51].

                        Co-infections in questing I. ricinus confirm the wide host range of this tick species and the role played by mammals, such as small rodents, or birds, as reservoirs of several pathogens simultaneously. The frequent finding of co-infections in adult ticks should stimulate an increased awareness of physicians and veterinarians of potential multiple infections in vertebrate hosts, leading to different or atypical clinical presentations [52].

                        The present study indicates that screening of adult ticks is a successful strategy to maximize the probability of pathogen detection. The rationale for monitoring adult ticks is that the pathogen rate of infection in adult questing ticks is usually higher than in nymphs, as a consequence of the transtadial transmission of agents accumulated during the blood meal on different hosts [52].

                        However, despite the fact that the original screening strategy was focussed on a relatively small number of adult ticks, this strategy had considerable costs (table 7). Hence, other sampling strategies were hypothesized a-posteriori, in order to evaluate their effeciency in terms of data collected and reduction of costs. Reducing the sampling time to three months (strategies B and C) instead of the whole year, decreased costs consistently (i.e., travel and staff costs), by reducing the draggings from the initial 146 to 71. Nonetheless, strategy C resulted in a loss of data, especially at local level (provinces and sites).

                        Specific screening of female ticks (strategies A and C) was justified by the higher pathogen rate of infection found in I. ricinus females compared to males. Nevertheless, the screening of females only resulted in the fact that sporadic pathogens were not detected.

                        Strategy B (processing of all adult ticks from April to June) was the most cost-effective choice, and represented the best compromise for both cost reduction and reliability of results (Table 8). Therefore, this strategy is recommended as basis for circulation studies of TTPs in this specific context. However, other areas characterized by different climate, tick dynamics, and pathogen prevalence may need modifications in terms of sample size and time of tick collection.

                        Conclusions

                        The actions that should be planned in a surveillance programme vary according to objectives (e.g., detection of major zoonotic pathogens only or emergent ones as well), ecological characteristics of pathogens to examine, estimation of costs, and budget availability. When dealing with a TBD, systematic tick collections should be undertaken in order to assess the size of the vector population and the pathogen infection rates. According to the European Center for Diseases Control [53] local, national and international health authorities should control the occurrence of a given vector-borne disease, e.g. endemic or non-endemic diseases.

                        This study indicates that, in the ecological landscape of north-eastern Italy, a complete picture of TTPs occurrence and of areas at risk of transmission can be drawn by systematic screening of adult ticks throughout a three-year time frame. These data can support decision makers to plan further surveillance activities. Nevertheless, tick collection and pathogen detection are expensive, especially in areas where several zoonotic TTPs coexist. Strategy B here proposed proved to fulfil the original aims of the study, being also cost-effective.

                        In addition, a thoughtful optimization of the diagnostic procedures could contribute to reduce costs, enabling a comprehensive, cost-effective, broad spectrum detection platform. Under the above circumstances, advanced biomolecular technologies, such as suspension array, reverse line blot hybridization, and novel sequencing technologies (e.g., pyrosequencing or next generation sequencing), have opened new perspectives towards maximizing results and reducing costs at the same time. The use of more sensitive approaches is likely to increase the number of pathogen species detected, as well as of co-infections diagnosed in a given area.

                        Declarations

                        Acknowledgements and Funding

                        This study was supported by the Italian Ministry of Health (project codes RC-IZSVe 11/04 and RC-IZSVe 07/07) and by the Veneto region.

                        Publication of the CVBD7 thematic series has been sponsored by Bayer Animal Health GmbH.

                        Authors wish to thank Vincenzo Lorusso and Cinzia Cantacessi for their helpful comments on the text.

                        Authors wish to thank too the colleagues who provided the positive controls for PCRs: Alessandra Torina (Rickettsia conorii) of the National Reference Centre for Anaplasma, Babesia, Rickettsia and Theileria (CRABaRT), Sicily, Italy; Bernard Carcy (Babesia divergens, strain Rouen 1987) of the Laboratoire de Biologie cellulare et moleculaire, Montpellier, France; Martin Pfeffer ("Candidatus Neoehrlichia mikurensis'", field strain from I. ricinus) of the Institut für Tierhygiene und Öffentliches Veterinärwesen, University of Leipzig, Germany; Maria Grazia Ciufolini (TBE virus, strain HYPR) of the Istituto Superiore di Sanità, Rome, Italy; Marco Martini (Anaplasma phagocytophilum, field strain from I. ricinus) of the Faculty of Veterinary Medicine, University of Padua, Italy; Alda Natale (European strain B. burgdorferi N34) of the Immunology Laboratory, Istituto Zooprofilattico Sperimentale delle Venezie.

                        Authors would like to also thank all the colleagues of Local Veterinary Units of Veneto and Friuli Venezia Giulia regions, who helped with the choice of sites and collection of ticks throughout the years.

                        Authors’ Affiliations

                        (1)
                        Istituto Zooprofilattico Sperimentale delle Venezie
                        (2)
                        Dipartimento di Scienze Sperimentali Veterinarie, Università degli Studi di Padova
                        (3)
                        Dipartimento di Sanità Pubblica e Zootecnia, Università degli Studi di Bari

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