The influence of on the phenology of the tick and the prevalence of the burgdorferi s.l. complex

Master Thesis Marijke Warnas 840425-929-070 Supervisor: Willem Takken June-November, 2005 Department of Entomology Wageningen UR

Summary

Cases of have been rising strongly over the last couple of years in the Netherlands. It is caused by the bacterial agents of the s.l. complex, which, in the Netherlands, are transmitted by the sheep tick, . Cattle can act as a host for the adult stage of ticks. Lately, environmental policies in the Netherlands have stimulated introduction of large cattle into natural areas to create more diverse landscapes. In this study we assessed our hypotheses that the introduction of cattle into a woodland area raises the tick populations and number of infected ticks in the area. Four plots (with and without cattle in oak and pine forest) were sampled weekly for questing ticks by blanket dragging. Captured ticks were analyzed for using the Reverse Line Blot (RLB) technique. A sample of ticks was also analyzed using the Restriction Fragment Length Polymorphism (RFLP) technique to check for differences in analysis accuracy between the two techniques. Rodents were caught in all four plots during a 1-week period. In this study we found that introduction of cattle does not have a positive effect on tick populations or infection in ticks. Actually, it seems the opposite is true. Significantly more ticks were found in the areas without cattle than in those were cattle could roam free. Overall, the number of caught ticks was low. 18.8% of the caught ticks were infected with the Borrelia burgdorferi s.l. complex. B. afzelii seems to be the predominant species of the complex in this area. No significant difference in detection efficiency was found between the RLB and RFLP techniques. The number of rodents caught in the area was very low; the highest number of rodents was caught in the pine and oak forests without cattle. It seems that cattle have a negative effect on the presence of rodents in the area. No direct relation between the number of rodents and the number of ticks can be observed in this study.

- 1 - Table of Contents

1. INTRODUCTION ...... - 3 - 1.1 LYME BORRELIOSIS ...... - 3 - 1.1.1 Borrelia burgdorferi s.l...... - 3 - 1.1.2 Human pathology ...... - 3 - 1.2 IXODES RICINUS BIOLOGY ...... - 5 - 1.3 LYME BORRELIOSIS, I. RICINUS AND INTRODUCTION OF CATTLE ...... - 6 - 1.5 AIM OF THIS STUDY AND RESEARCH QUESTIONS...... - 8 - 2. MATERIAL AND METHODS ...... - 9 - 2.1 STUDY SITE ...... - 9 - 2.2 CLIMATE RECORDING ...... - 9 - 2.3 COLLECTION OF TICKS ...... - 10 - 2.4 DNA EXTRACTION ...... - 11 - 2.5 POLYMERASE CHAIN REACTIONS ...... - 12 - 2.5.1 Polymerase Chain Reaction for RFLP ...... - 12 - 2.5.2 Polymerase Chain Reaction for RLB ...... - 14 - 2.6 RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) ...... - 14 - 2.7 REVERSE LINE BLOT (RLB)...... - 15 - 2.7.1 Preparation of the RLB membrane ...... - 16 - 2.7.2 Hybridization of the PCR product...... - 16 - 2.8 EXAMINATION OF HOSTS ...... - 18 - 2.8.1 Rodent trapping ...... - 18 - 2.8.2 Cattle inspection ...... - 18 - 2.9 DATA ANALYSIS ...... - 18 - 3 RESULTS ...... - 19 - 3.1 CLIMATE ...... - 19 - 3.2 I. RICINUS PHENOLOGY ...... - 19 - 3.3 B. BURGDORFERI S.L. ...... - 26 - 3.4 RLB VERSUS RFLP TECHNIQUE ...... - 27 - 3.5 HOST EXAMINATION ...... - 28 - 4 DISCUSSION ...... - 29 - 4.1 I. RICINUS PHENOLOGY ...... - 29 - 4.2 HOSTS ...... - 29 - 4.3 B. BURGDORFERI S.L. INFECTIONS ...... - 29 - 4.4 RLB VERSUS RFLP TECHNIQUE ...... - 30 - 4.5 CONCLUSIONS ...... - 30 - 5 ACKNOWLEDGEMENTS...... - 31 - 6 REFERENCES: ...... - 32 - APPENDIX 1: CLIMATE DATA ...... - 34 - APPENDIX 2: RAW TICK DATA ...... - 38 -

- 2 - 1. Introduction

1.1 Lyme borreliosis

1.1.1 Borrelia burgdorferi s.l.

Lyme borreliosis is a chronic human disease, occurring in the Northern hemisphere. Its symptoms include skin, nervous system and heart and joint manifestations. Although the disease rarely leads to death, it can severely impair the lives of untreated patients(Gray 1998). Lyme borreliosis is caused by spirochetes belonging to the Borrelia burgdorferi s.l. complex. The B. burgdorferi s.l. complex comprises at least three genospecies pathogenic to humans: B. afzelii, B. garinii and B. burgdorferi s.s.. Others such as B. lusitaniae (LeFleche and al. 1997) and B. valaisiana (Wang, van Dam et al. 1997) have not yet been shown to be harmful to humans. Although B. burgdorferi s.s. is the only to cause Lyme disease in North America, it seems to be comparatively rare in Europe. In Europe the genospecies B. afzelii, B. garinii and B. valaisiana are the most frequent. Even within Europe differences in genospecies frequency has been found. In the Netherlands, Slovakia and in Switzerland B. afzelii seems to be the predominant genospecies (Rijpkema and Bruinink 1996; Humair, Rais et al. 1999; Hanincova, Schafer et al. 2003) , in England and Ireland however B. afzelii seems to be rare or even absent and B. garinii and B. valaisiana are dominant (Kirstein, Rijpkema et al. 1997; Kurtenbach, Peacey et al. 1998). These geographical distributions could be caused by host-relations of the various genospecies; B. garinii and B. valaisiana seem to be associated predominantly with (Humair and al. 1995; Humair and Gern 1998; Humair, Rais et al. 1999) while B. afzelii and B. burgdorferi s.s. are transmitted mostly by rodents (Humair and al. 1998; Kurtenbach, Peacey et al. 1998)

1.1.2 Human pathology

In general Lyme borreliosis progresses in 3 stages. In the first stage is characterized by a localized infection characterized by a circular rash. This rash is known as (EM, figure 1), it starts as a red spot at the site of the bite of the infected tick. It then spreads out, often leaving a blank spot in the middle, forming white-and-red concentric circles. This is why erythema migrans is sometimes also referred to as a “target-shaped” rash. Not all patients suffering from Lyme borreliosis however develop erythema migrans, approximately 10% of patients never get the rash (Nadelman and Wormser 1998). Besides the EM numerous other symptoms have been reported, including: fatigue, muscle pain, joint pain, headache, fever and/or chills, and stiff neck.

- 3 -

Fig. 1: Erythema migrans or “target-shaped”rash, in a patient infected with Lyme disease (source: www.natuurkalender.nl)

In the second stage of the diseases, multiple symptoms can manifest. Firstly, the EM will fade away, followed by an onset of cardiac, neurological or arthritic symptoms. These symptoms can occur weeks after the clearing of the EM, but it’s also possible it will take several months before new symptoms are presented. The third stage consists of a persistent or chronic occurrence of a degenerative skin condition on the limbs (acrodermatitis chronica atrophicans) and the progression of the cardiac, arthritic or neurological complications, further symptoms include: paralysis, disturbed motor and/or speech control and fatigue (Nadelman and Wormser 1998). The variance in clinical manifestations may in part be explained by infections with the different pathogenic strains of B. burgdorferi s.l. According to van Dam et al. (Dam and al. 1993) B. garinii can be associated with neuroborreliosis, B. burgdorferi s.s. with arthritis and B. afzelii with the degenerative skin condition (acrodermatitis chronica atrophicans). However, there seems to be considerable overlap (Dam and al. 1993). It is unclear how the different strains could cause these different symptoms.

- 4 - 1.2 Ixodes ricinus biology

The members of the complex, of which I. ricinus is a part, have very flexible and therefore complex lifecycles. These ticks are capable of entering to overcome unfavorable conditions, both unfed ticks and those a developmental phase are capable of using this mechanism (Gray 1998). Tow kinds of diapause have been observed: 1) a behavioural diapause, where unfed ticks go into a state of quiescence to overcome unfavorable questing times or 2) developmental diapause, where engorged stages or eggs arrest development until a more favorable time. Due to this system, the I. ricinus life- cycle, while it can be finished within 2 years, may take up to 7 years to be completed. The diapause is the cause for the large variance in time needed for the different stages to complete in the following description of the ticks life-cycle. All tick stages quest on vegetation in their search for a suitable host. Recently hatched larvae can stay hiding in the lower scrubs where they hatched out of their eggs for up to 75 days. When they do come out of hiding they climb up to higher positions in order to find their first host to attach themselves to. Although larvae seem to prefer small rodents as their hosts, they will feed on larger such as birds, rabbits or even deer. After feeding on their host for up to 6 days, they dislodge themselves and again find shelter in the lower scrubs to develop into nymphs, this can take 24 to 426 days. Once in the nymphal stage ticks take 10 to 540 days to start host seeking behaviour. The nymphs can feed on a very wide range of hosts, from small rodents to large such as cattle and deer. When they have successfully attached themselves to a host they will feed for up to 7 days after which they once more hide in the lower vegetation where they will develop into adults. The transformation from to tick can take up to 810 days and requires multiple molting sessions. Adult male ticks rarely feed and never engorges. Adult female ticks can feed on a variety of animals but generally needs larger animals of at least the size of a hare (Lepus spp.) for her to be able to fully engorge. Mating usually occurs on the host, but can also take place in the vegetation (Gray 1987). Up to 27 days after feeding the female will lay from 500 to 3000 eggs. She will generally lay her eggs in the leaf litter or top bottom layer, the eggs can remain there for up to 400 days. Figure 2 shows a simplified overview of the I. ricinus life cycle. Host-seeking behaviour usually occurs in the late spring and early summer, and to a lesser extent in autumn, for all tick stages, avoiding the low temperatures of winter and the low relative humidity in summer. Considering ticks are most vulnerable to when questing for hosts, they normally show no active behaviour below temperatures of 7°C or when relative humidity is below 80% (Gray 1998; Randolph, Green et al. 2002), thereby showing a preference for climates with cool summers and moderate winters.

- 5 -

Fig. 2: I. ricinus life cycle. The relative size of the animals approximates their significane as hosts for the different tick stages in a typical woodland area. (Courtesy of J. Gray and B.Kaye)

1.3 Lyme borreliosis, I. ricinus and introduction of cattle

In Europe, the principle of the B. burgdorferi s.l. complex is the hard tick Ixodes ricinus. Ticks in all life-stages ingest the Borrelia species when feeding on an infected host, and can maintain the spirochetes in the midgut through its entire life. As soon as the tick starts its next feeding, the spirochetes penetrate the midgut wall and invade various tissues, most importantly the salivary glands, by which the spirochetes are injected into the host’s bloodstream during feeding (Fingerle and al. 2002). Considering the migration to the salivary glands during feeding, of spirochetes to the host should not be possible for at least two days after attachment, although regurgitation of the gut contents has been suggested as to explain earlier spirochete transmissions, none have been able to prove this hypothesis. Although transovarial (adult infected female to eggs) transmission has been shown, the frequency of larval infections is considered too low (usually less than 1%) to be of importance for Borrelia infection rates, it may however, play a significant role in the preservation of the spirochetes in nature (Gray 1998). I. ricinus is able to feed on a large number of hosts, of which 50 have been identified as reservoir hosts for B. burgdorferi s.l. (Gern, Estrada-Pena et al. 1998). Of these 50 animals 32 have been identified as reservoir hosts in Europe, all small or medium sized mammals and small birds. Although larger mammals, such as cattle or deer, do serve as hosts for the ticks they are apparently incapable to serve as reservoirs for B. burgdorferi s.l.. They do however serve as reproduction hosts for I. ricinus, thereby contributing to

- 6 - the population of the ticks. Also ticks can become infected by feeding on larger (non- infected) hosts through non-systemic horizontal transmission, when two or more ticks are feeding on the same area on the host. This way, larger mammals might be able to contribute to the spread of B. burgdorferi s.l. and thus, Lyme disease. Recently, Dutch governmental policies have increased the number of large mammals in natural areas, mostly in order to recover or maintain local . Medical data report that the number of tick bites and cases of Lyme disease doubled from 1994 to 2001 (Boon and Pelt 2003). Figure 1 shows the increase in reported cases of erythema migrans, the indicative rash of Lyme disease. It has been suggested that the large increase in tick bites and Lyme cases is due to the increased number of large grazers in our nature reserves, since the animals may increase the tick population by serving as reproduction hosts and increase B. burgdorferi s.l. transmission through co-feeding, since they can feed multiple ticks at a time (Mik, Pelt et al. 1997). Although some is known about influences of cattle on ticks and Borrelia, none are clear-cut and none focused specifically on the influence of cattle. Therefore, in this research we will compare oak (Quercus robur) and scotch pine (Pinus sylvestris) forests, both with and without cattle in tick abundance and population composition as well as prevalence of the different genospecies of the B. burgdorferi s.l. complex.

Fig. 3: Erythema migrans, per 100.000 people, in the Netherlands in 1994, 2001 en 2005 (source: RIVM 2006).

- 7 - 1.5 Aim of this study and research questions

Considering the increase of cases of Lyme disease and the potential seriousness of the disease it is imperative that the potential effect of the current Dutch nature conservation policy of introducing cattle is studied. In this study we will continue a study conducted earlier this year in the same area (Gassner 2005; Gassner, Verbaarschot et al. 2008). That study found significantly less ticks in plots with cattle, and no significant effect of cattle on Borrelia infections. 25,6% of nymphs and 33,3 of adults were found to be infected with B. burgdorferi s.l., which is a very high infection rate. In this study we will continue the research to determine whether these findings hold or change throughout the seasons. The previous study used a new technique to examine for infection with B. burgdorferi s.l., RFLP (Restriction Fragment Length Polymorphism) ,where previous studies used the RLB (Reverse Line Blot) technique. In this study we will also examine if the high infection rate found in the area can be explained by a higher sensitivity of the RFLP technique.

Concluding, the main research question posed in this study is:

Does introduction of cattle in a woodland area affect densities of I.ricinus populations and/or risk of infection with parasites of the B. burgdorferi s.l. complex?

Secondary research questions posed in this study are: What is the course of the abundance and population composition of I. ricinus over the season? Are there significant differences in abundance and population composition of I. ricinus between Oak and Scotch Pine forests? Are there significant differences in the infection rates of the different genospecies of the B. burgdorferi s.l. complex between Oak and Scotch Pine forests and/or between areas with and without cattle? Is there a significant difference in sensitivity for B. burgdorferi s.l. between the RLB and RFLP technique? Do mixed infections of the B. burgdorferi s.l. complex occur within I. ricinus?

- 8 - 2. Material and Methods

2.1 Study site

The study area in the forestry Oostereng at the Renkumse Heide was situated approximately 2 km north of Renkum in the Netherlands (52°00’27N, 5°45’20E). The area consists of part rough grassland pasture and part Oak (Quercus robur) and Scotch Pine (Pinus sylvestris) forest. Our study site consisted of approximately 5 km2, of which about 3 km2 is accessible to cattle (Bos taurus). The area accessible to the cattle contained both forest and pasture. The area available to cattle is bounded by a 1.5m high barbwire fence. Within this area 5 different study sites of 400m2 were defined: A: Oak forest closed to cattle B: Pine forest closed to cattle C: Oak forest open to cattle D: Pine forest open to cattle E: Grassland pasture Figure 3 shows the location of the different sample plots within the study site. The grassland pasture was not sampled in this study, since a previous study showed no ticks resided there (Gassner 2005).The grassland pasture and adjacent forest have been grazed by cattle since 2001. The group of approximately 10 cattle are present throughout the year. Numerous wildlife is present in the area such as roe deer, badgers, foxes, rabbits, hares and several other rodent species as well as several species of birds. All these animals are known to be potential hosts for Ixodes ricinus.

2.2 Climate recording

Three data loggers (type Gemini Tinytag Plus TPG 1500), recording temperature and relative humidity every minute, were placed in both Oak plots and one of the Pine forest plots (Plot D: accessible by cattle) every week (due to a lack of data loggers, no recordings were made in the Pine forest plot without cattle). To prevent damage by cattle or other animals, a metal cage was placed over the data loggers. The data loggers were hung from the cage at a distance of 5 cm from the ground. A small cover was hung over the data loggers to shelter them from rain (Figure 4). Every week new data loggers were placed and the data from the ones in the plots uploaded to a computer using Easyview software (version 5.5.1.1, 2002. Intab interface teknik AB, Sweden).

- 9 -

Fig 4: On the left: Location of study site within the Netherlands. On the right: Detail of study site. The red dotted line depicts the fence in which cattle is kept. White areas represent pasture, gray areas forest (from (Gassner 2005)

2.3 Collection of ticks

Ticks were collected on a weekly basis for 13 weeks during a period from June till October (week 26 till 38). Sampling was started at approximately 9.00 and ended around 13:00. Sampling was done by dragging a 1m2 cotton blanket, equipped with a tube and rope at the front and weights at the back to improve contact with the forest floor. An area of 200m2 was sampled as shown in figure 5. The ticks were collected from the blanket with forceps after every 25m2 subplot. Life stage of the ticks was determined upon capture. Larvae were pooled for every subplot to a maximum of 10. The ticks were stored by 25m2 subplot in eppendorf tubes with 70% ethanol. The tubes were stored at 4° C until DNA extraction Sampling was postponed to the next day in case of heavy wind or rain.

- 10 -

s c s c

50 50 m

25m

s c s c

2m Fig.5: Plot layout. S: Sampling strip. C: Control strip Dotted line and arrows indicate blanket dragging route. Stars mark where ticks were collected from the blanket

2.4 DNA extraction

DNA extraction was performed as described by Schouls et al., 1999 (Schouls, Van De Pol et al. 1999):

1. Individual ticks were placed on tissue paper using forceps and left to dry for 20 minutes. 2. Dried ticks were put in 0,5ml vials containing 100µl 0,7M laboratory grade Ammonium Hydroxide (stock 29%). 3. Ticks were submerged using sterile forceps. 4. Vials were closed and put in boiling water for 30 minutes. 5. The vials were then put on ice for 2 minutes, followed by centrifugation at 14.000 RPM for 20 seconds. 6. Afterwards the vials were opened and put inside a fume hood at approximately 95°C for a minimum of 20 minutes, allowing the ammonium to evaporate.

- 11 - 7. When the ammonium has evaporated (this was checked by smelling the vials for traces of ammonia), leaving about 70% of the original volume, the vials were closed and stored at -20°C until further analysis.

2.5 Polymerase Chain Reactions

Prior to PCR all reaction components were vortexed and spun down. Reaction components were kept on ice when possible during preparations. All PCR preparations were conducted in a flow cabinet using sterile filter and autoclaved tips and vials. The reaction mixes were made in bulk, vortexed and subsequently distributed over the appropriate number of vials, after which the DNA extract was added.

2.5.1 Polymerase Chain Reaction for RFLP

The PCR protocol was adopted and adjusted from Michel et al., 2003 (Michel, Wilske et al. 2004). This protocol amplifies part of the outer surface protein (Osp-A) gene and consist of a PCR followed by a nested PCR, in which the product of the first PCR serves as the template for the second, nested, PCR. See table 1 for details on the primers. With every PCR run a number of positive and negative controls were included to validate the PCR, these were: - Culture extracts of B. burgdorferi s.s., B. valaisiana, B.garinii and B. afzelii - 2 positive and 2 negative field samples - 1 H2O - 1 Ammonium Hydroxide (DNA extraction solution)

Table 1: PCR primers , DNA sequence and target region on Borrelia Osp-A genes.

Primer Amplification Sequence Position V1a Primary 5'-GGGAATAGGTCTAATATTAGC–3' 18–38 (forward) amplification V1b Primary 5'-GGGGATAGGTCTAATATTAGC–3' 18–38 (forward) amplification V3a Nested 5'-GCCTTAATAGCATGTAAGC–3' 37–55 (forward) amplification V3b Nested 5'-GCCTTAATAGCATGCAAGC–3' 37–55 (forward) amplification R1 Both 5'-CATAAATTCTCCTTATTTTAAAGC–3' 832–855 (reverse) amplifications R37 Both 5'-CCTTATTTTAAAGCGGC–3' 829–845 (reverse) amplifications

- 12 - The reaction mix for each sample, adding up to a volume of 25µl, contained:

o 12,5 µl HotstarTaq master mix kit (Qiagen) o 1 µl (20pmol) Primer V1a (Forward) o 1 µl (20pmol) Primer V1b (Forward) o 1 µl (20pmol) Primer R1 (Reverse) o 1 µl (20pmol) Primer R37 (Reverse) o 6 µl H2O (provided with Hotstar kit) o 2,5 µl DNA extract (when pipetting the extract the tick was crushed with the tip of the pipette, after which the extract was pipetted up and down to homogenize)

The primary amplification was performed under the following conditions: 1. Hot start for polymerase activation: 95°C for 15 minutes 2. 30 cycles of: Denaturing: 94°C for 15 min. Annealing: 48°C for 45 sec. Extension: 72°C for 45 sec. 3. Final Extension: 72°C for 7 minutes

The optimized nested amplification mix, adding up to a volume of 50µl contained:

o 5 µl Supertaq PCR buffer (Ht biotechnology Ltd.) o 2 µl (20pmol) primer V3a (Forward) o 2 µl (20pmol) primer V3b (Forward) o 2 µl (20pmol) Primer R1 (Reverse) o 2 µl (20pmol) Primer R37 (Reverse) o 1µl DNTP’s (0,2M in PCR mix) o 0, 2µl (1U) Supertaq polymerase (Ht biotechnology Ltd.) o 30,8µl H2O (autoclaved miliQ) o 5µl PCR product from primary PCR

The nested amplification was performed under the following conditions: 1. Hot start for polymerase activation: 95°C for 5 minutes 2. 30 cycles of: Denaturing: 94°C for 45 sec.. Annealing: 48°C for 45 sec. Extension: 72°C for 1 min. 3. Final Extension: 72°C for 7 minutes

Finished samples were stored at -20°C.Nested PCR product were visualized on a 1% ethidium bromide stained agarose gel, recorded using an UV light table and Kodak DC 120 camera and analyzed with 1D software (Kodak).

- 13 - 2.5.2 Polymerase Chain Reaction for RLB

The PCR protocol uses primers which target the intergenic spacer region between the 5S and 23S ribosomal RNA genes. If Borrelia DNA is present this leads to a 225 base-pair sized product. The primer attached to the 5S end is biotinylated. See table 2 for primer sequences. The reaction mix was made in bulk, vortexed and subsequently distributed over the appropriate number of vials, after which the DNA extract was added.

Table 2: PCR primers, DNA sequence Primer Sequence 23S 5’-TCA GGG TAC TTA GAT GGT TCA CTT-3’ 5S 5’-GAG TTC GCG GGA TAG GTT ATT-3’

The reaction mix for each sample, adding up to a volume of 50µl, contained:

o 25 µl HotstarTaq master mix kit (Qiagen) o 4 µl (10pmol) primer R 23S o 4 µl (10pmol) primer R 5S o 12 µl H2O (provided with Hotstar kit) o 5 µl DNA extract (when pipetting the extract the tick was crushed with the tip of the pipette, after which the extract was pipetted up and down to homogenize)

The amplification was performed under the following conditions: 1. Hot start for polymerase activation: 95°C for 15 min. 2. 10 cycles of: Denaturing: 94°C for 45 sec. Annealing 60°C for 30 sec. (minus 1°C each cycle) Extension 72°C for 30 sec. 3. 40 cycles of: Denaturing: 94°C for 1 min. Annealing 50°C for 30 sec. Extension 72°C for 30 sec.

Finished samples were stored at -20°C.

2.6 Restriction Fragment Length Polymorphism (RFLP)

The RFLP protocol was adopted from Michel et al. (Michel, Wilske et al. 2004). Each sample from the nested PCR that was found positive for B. burgdorferi s.l. was digested separately by using 5 different restriction enzymes in order to generate genospecies specific digestion products. This technique detects B. burgdorferi s.s., B. valaisiana, B.garinii and B. afzelii, these strains can also be detected by the Reverse Line Blot (RLB) technique.

- 14 - For each enzyme and each sample, the reaction mix contained: o 9,45 µl H2O (autoclaved miliQ) o 2 µl Buffer (table 3) o 0,5 µl BSA o 0.05 µl (0,5U) Enzyme (table 3) o 7 µl Borrelia positive nested PCR product

See table 3 for enzyme details and specific digestion temperatures. Digestions were run overnight. The 65°C incubation of Kpn21 was run in a PCR machine in order to prevent evaporation. These samples were thoroughly vortexed and resuspended with a pipette in order to dissolve the digestion products. Digestion products were loaded with 5 µl loading buffer on a 2% ethidium bromide agarose gel running at 60 V for 65 minutes. Banding patterns are recorded in accordance with the PCR product visualization.

Table 3: Restriction enzymes, their incubation temperatures and their predicted RFLP banding patterns. See Michel et al., 2003 (Michel, Wilske et al. 2004) for more strains of Borrelia. *No Osp-a type. ** Published name, promega catalog name: CSP45I. ***published name, promega catalog name: ACCIII Borrelia species OSP-a Predicted RFLP pattern (bp) type SspI SfuI** BglI Kpn21*** HindIII (37°C) (37°C) (37°C) (65°C) (37°C) B. burgdorferi s.s 1 534/264 798 798 429/369 654/144 B. afzelii 2 798 537/261 798 798 798 B. valaisiana * 801 801 801 801 465/336 B.garinii 3 801 801 758/43 429/372 801 B.garinii 4 798 798 556/242 798 798 B.garinii 5 798 798 798 549/195/54 654/144 B.garinii 6 801 801 801 429/252/120 657/144 B.garinii 7 801 801 758/43 429/195/177 585/144/72 B.garinii 7 801 801 758/43 428/372 657/144

2.7 Reverse Line Blot (RLB)

The RLB procedure for Borrelia was developed at the Research Laboratory for Infectious Diseases, National Institute of Public Health and the Environment (RIVM) and is used there as a standard procedure for detecting and genotyping pathogenic microorganisms. In this study 9 types of oligonucleotide probes are used that target the following genospecies: Borrelia burgdorferi s.l. (3 different probes), B. burgdorferi s.s, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, and B. ruskii. The probes are synthesized with a 5’ terminal amino group. This amino group binds covalently to an activated membrane, keeping the probe molecules in place when the membrane is deactivated.

- 15 - 2.7.1 Preparation of the RLB membrane

1. Oligonucleotides were diluted to the optimized concentrations (app. 4) ranging from approximately 85 nM to 665 nM (12.5-100 pmol/150 µl.) in 150 µl. 500 mM NaHCO3.( The oligonucleotides were stored at -20º C. ) 2. The Biodyne C membrane was cut to the appropriate size, extending about 0.7 cm on both sides of the support cushion. A number of small triangles (few mm.) were cut in at the rim of one of the sides to mark the membrane. (This was done with thoroughly washed hands, no powdered gloves. The membrane was only handled carefully at the outer rim.) 3. The membrane was then put in a rolling bottle with 10 ml. freshly prepared 16% EDAC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in demineralised water. The membrane was incubated for 10 min., rolling at room temperature. 4. The EDAC solution was removed and 100 ml. of demineralised water was put into the rolling bottle to rinse the membrane with gentle shaking. With tweezers, the membrane was then placed onto a support cushion, taking care that the triangles are at the lower right corner of the membrane seen from the experimenter. 5. Afterwards, the membrane and support cushion were placed neatly under the openings of a clean miniblotter system and the screws fastened hand-tight. Residual water was removed by aspiration with a vacuum source. 6. The slots of the miniblotter were filled with 150 µl. of the dilute oligonucleotide solution. The first and last slot were left empty. Care was taken to prevent air bubbles in the system. 7. The first and last slots were filled with diluted pen ink (1:100). The blot was incubated for at least 1 min. at room temperature. 8. The oligonucleotides and ink were removed by aspiration, in the same order as they were applied. 9. The membrane was removed from the miniblotter using tweezers and placed in a rolling bottle with 100-150 ml. 100 mM NaOH for maximum of 10 min. to inactivate the membrane. The NaOH was drained and the membrane flushed by gentle shaking with 100-150 ml. demineralised water. 10. The membrane was placed in a plastic container and washed for 5 min. at 60º C. in 100 ml. 2x SSPE/01.%SDS (app.4). 11. The membrane could now be used for the RLB. When storing the membrane it was washed in 100-150 ml. 20mM EDTA (ph 8) for 15 min. at room temperature. It was put in an inert plastic container with enough 20 mM EDTA to keep the membrane fully covered with liquid, the membrane was then stored at 4°C.

2.7.2 Hybridization of the PCR product

1) The following buffer solutions were made for treating 1 membrane (mixing procedures app. 4.) - 80 ml. 2xSSPE/0.1%SDS (room temperature). - 400 ml. 2xSSPE/0.5%SDS (200 ml at 51º C., 200 ml at 42º C.). - 200 ml. 2xSSPE (room temperature).

- 16 - Diluted buffers can be stored 2 days at maximum. 2) 10 µl of PCR product was added to 150µl 2xSSPE/0.1%SDS in 2 ml eppendorf vials. 3) The vials were placed 10 minutes in a boiling water bath or heath-block to denature the DNA. 4) The vials were then placed directly on ice. 5) While denaturing the PCR product, a membrane was incubated for 5 minutes in about 60 ml 2xSSPE/0.1%SDS at room temperature. 6) After denaturing, the membrane was placed on a support cushion in the miniblotter. In such a way that the slots are perpendicular to the line pattern of the oligonucleotides (90º anticlockwise). 7) Residual fluid was removed from the slots by aspiration and the slots are filled with the diluted PCR products. The first, last and empty slots were filled with 2xSSPE/0.1%SDS to prevent cross-flow. 8) The blot was incubated for 60 minutes at 41º C on a horizontal surface. 9) The PCR products were removed by aspiration and the membrane was placed with tweezers in a rolling bottle. 10) Afterwards, 100 ml of pre-heated 2xSSPE/0.5%SDS buffer was put to the membrane. The rolling bottle was rolled in a hybridization oven at 51º C for 10 minutes. 11) This was repeated with the second 100 ml with the same conditions. 12) Subsequently, the buffer was drained from the rolling bottle. Next, 10 ml of 1:4000 streptavidine-peroxidase in 2xSSPE/0.5%SDS at room temperature was added and the rolling bottle was rolled for 30 to 40 minutes at 42º C in the oven. 13) The streptavidine solution was removed and the membrane washed (in the same bottle) 2 times with 100 ml 2xSSPE/0.5%SDS at 42º C for 10 minutes. 14) The membrane was moved with tweezers to a plastic container and washed 2 times for 5 minutes with 100 ml 2xSSPE at room temperature on a platform shaker. 15) The membrane was then moved to another plastic container and incubated by gentle manual swirling for 1-2 min in 20 ml pre-mixed ECL detection liquid (10 ml. of liquid A, 10 ml. of liquid B). 16) Finally, the membrane was removed from the ECL liquid, letting it drip dry. After letting it drip the membrane was sealed in transparent plastic and subsequently placed in a photo casing and covered with photographic film in a dark room. 17) The film and membrane were left in the photo casing overnight. 18) The next day, the photographic film was removed from the photo casing in a dark room and placed in a developing machine.

- 17 - 2.8 Examination of hosts

2.8.1 Rodent trapping

Rodent trappings were conducted during a five-day period in September 2005 in all four tick capture plots. In each plot, 20 Longworth traps were placed in the control strips next to the tick sampling strips. Traps were pre-baited for two days prior to the actual trapping. Traps were baited with oatmeal, peanut butter and a mealworm. Bait was refreshed daily, or when eaten. The traps were inspected four times every day with 6 hour intervals (12 p.m., 6 a.m., 12 a.m. and 6p.m.) Captured animals were marked by clipping fur at unique places for identification if recaptured. For each captured the species and number, life stage and placement of ticks were recorded, after which it was released.

2.8.2 Cattle inspection

During our research there was one opportunity to inspect the cattle for infestation with ticks. Cattle was rounded up from the meadow, and inspected on the presence of ticks on neck, head and legs. Ticks were counted and collected.

2.9 Data analysis

Data was analyzed using SPSS 15.0 and Microsoft Excel 2003. The data was tested for normality using a Kolmogorov-Smirnov test. Not normally distributed data that could not be transformed was analyzed using the appropriate non-parametric (Wilcoxon, Mann- Whitney U or Kruskal-Wallis) test followed by a Scheffe post-hoc comparison test when necessary.

- 18 - 3 Results

3.1 Climate

95% confidence intervals were calculated for the differences between all plots . Upper limits of differences overall were very small (max 0,47), especially for saturation deficit (max 0,15), which is a good indicator for tick activity (Randolph et al., 2002). Upper limits represent a value that is not exceeded in 95% of cases. All upper limits can be found in Appendix 1. Because such small differences are of little biological consequence data from the data loggers were averaged. Figure 6 shows the daily mean, minimum and maximum temperatures during the study period. Figure 7 shows mean, minimum and maximum relative humidity. Figure 8 shows daily precipitation as measured on the Haarweg meteorological station. Ticks show no questing behaviour during rainy conditions. Temperatures did not drop below 5˚C during the study period. Mean relative humidity was almost constantly higher than 80%. During the beginning of the study period and a short period in July there were short hot and dry periods. Relative humidity was low and saturation deficit exceeded 4 mmHg. These conditions were shown to be unfavorable to questing ticks by Randolph et al. (2002). Saturation deficit is shown in figure 9. The dashed lines indicate physiological values that influence tick questing behaviour.

3.2 I. ricinus phenology

In total 511 I. ricinus ticks were caught during the 13-week study period. Of the ticks caught 351 were larvae, 157 were nymphs, 2 were adult males and 1 was an adult female. Figure 10 gives an overview of the phenology of larvae and nymphs during out study period. Using a Kolmogorov-Smirnov test, the dataset was examined for normality. The data was found to be not normally distributed and could not be transformed by either log or square root transformation. Either a Kruskal-Wallis test, followed by Scheffe post-hoc comparison or a Mann-Whitney U test was used to distinguish differences between the plots. The oak plot without cattle yielded significantly more larvae than any of the other plots (p=0,00). The oak plot without cattle also held more nymphs than any of the other plots (p=0,00), the pine plot without cattle held significantly more nymphs than the two plots with cattle (p=0,007 and p=0,04). Overall, plots without cattle had significantly more nymphs and larvae than plots with cattle (p=0,00). Oak plots had significantly more larvae (p=0,00) and nymphs (p=0,03) than pine plots. Figure 11 gives an overview of the distribution of larvae and nymphs between the plots. The phenology of all ticks combined is shown in figure 12.

- 19 -

esting ticks. esting

indicates the physiological threshold of 5ºC, below which conditions are unfavorable for the which threshold conditions physiological indicates qu are of unfavorable below 5ºC, Fig: 6: Average daily minimum, mean and maximum temperatures, recorded by data loggers on site. The dashed The line site. dashed data recorded loggers on temperatures,by maximum Fig: mean and Average daily minimum, 6:

- 20 -

h conditions are unfavorable for for unfavorable h conditions are

dashed line indicates the physiological threshold of 80%, below whic line the threshold 80%, physiological indicates dashed below of ticks. questing

Fig 7: Average daily minimum, mean and maximum relative humidity, recorded by data loggers on site. The The site. data recorded loggers Figon mean relative and by daily maximum humidity, minimum, 7: Average

- 21 - 04.10.2005

01.10.2005

28.09.2005

25.09.2005

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19.09.2005

16.09.2005

13.09.2005

10.09.2005

07.09.2005

04.09.2005

01.09.2005

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26.08.2005

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14.08.2005 Date

11.08.2005

08.08.2005

05.08.2005

Daily precipitation Daily 02.08.2005

30.07.2005

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18.07.2005

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09.07.2005

06.07.2005

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30.06.2005

27.06.2005

8: Daily precipitation, recorded at Haarweg meteorological station, Wageningen. meteorological Haarweg recorded at Daily precipitation, 8:

0,00

5,00

Fig.

10,00

15,00

20,00 25,00 Precipitation (mm) Precipitation

- 22 -

physiological threshold of 4 mmHg, below which conditions are favorable for questing for conditions ticks. are questing favorable threshold physiological ofbelow mmHg, which 4

Fig. 9: Average daily saturation deficit, as recorded by data loggers on site. The dashed line indicates the indicates recorded The on line by site. Fig.dashed data saturation loggers deficit, as Average daily 9:

- 23 -

A B

Fig. 10: I. ricinus phenology. A: phenology of I. ricinus larvae in the different study plots. B: phenology of I. ricinus nymphs in the different study plots

- 24 -

Larvae Nymphs

cattle with D: Pine,

ticks between the between ticks

cattle with C: Oak, I. ricinus I. ricinus Plot

cattle no B: Pine,

Distribution of ticks between plots between ticks of Distribution

different plots.

cattle no A: oak,

Fig. 11: Distribution of Fig.caught of Distribution 11: 0 300 200 100

ticks caught of #

04.10.2005

27.09.2005

Larvae Nymphs 20.09.2005

05.09.2005

31.08.2005

23.08.2005

17.08.2005 Date

10.08.2005

02.08.2005

18.07.2005

11.07.2005

04.07.2005

Phenology of ticks in all plots combined plots all in ticks of Phenology

Phenology of cumulative sum of ticks caught in all all Phenology caught cumulative ticks in sum of of plots.

27.06.2005 Fig.12: 0

80 60 40 20

100 Sum of caught ticks in all plots all in ticks caught of Sum - 25 -

3.3 B. burgdorferi s.l. infections

160 I. ricinus ticks (157 nymphs, 2 adult males and 1 adult female) were analyzed for infection with B. burgdorferi s.l. using the RLB technique. Of 160 samples, 30 were found positive for infection, yielding an infection percentage of 18,8%. All four species of the B. burgdorferi s.l. complex were found to be present at the study site. 11 positives were found to be B. afzelii (36.7%), 8 were found to be B. garinii (26,7%), 5 were B. valaisiana (16,7%) and another 5 were B. burgdorferi sensu stricto (16,7%). Two mixed infections were found, one combining B. burgdorferi s.s. and B. garinii, the other B. burgdorferi s.s. and B. valaisiana. Overall the oak plot without cattle had the most infected samples. No infections were found in the oak plot with cattle. Although in absolute numbers the plots with cattle had significantly fewer infected ticks than plots without cattle (p=0,00), this can be attributed to the higher number of ticks suitable for analysis that were found in the plots without cattle. No difference was found between oak and pine plots (p=0,828). Figure 13 depicts the distribution of infected ticks in the different plots. Although the pine plot with cattle has the highest infection rate (28,75%), compared to oak without cattle (15,05%) and pine without cattle (17,78%), this provides no significant result. Infection percentages per plot are given in Figure 14. Distribution of infections

B. Burgdorferi s.s 20 B. valaisiana B. garinii N=93 B. afzelli

15

10 N=45

ticks infected of #

5 N=14

N=8

0 A: Oak, no cattle B: Pine, no cattle C: Oak, with cattle D: Pine, with cattle Plot Fig. 13: Distribution of infections of the different species of B. burgdorferi s.l. in caught I. ricinus ticks. The number of ticks analyzed is given above each bar.

- 26 - Infection percentage per plot

30.00

N=4

25.00

20.00

N=9 15.00

N=15

10.00 Infection Percentage (%) Percentage Infection

5.00

0.00 A: oak, no cattle B: Pine, no cattle C: Oak, with cattle D: Pine, with cattle Plot Fig. 14: Percentage of I. ricinus ticks infected with B. burgdorferi s.l. per plot.

The absolute number of infected ticks per plot is given in each bar.

3.4 RLB versus RFLP technique

125 DNA-extracts from ticks collected in the field were examined for infection with both the RLB and RFLP method. RFLP found 33 positives, the RLB found 26. The RFLP method found 10 positives where the RLB found none, on the other hand the RLB found 3 positives where the RFLP did not detect infection. Using a Wilcoxon matched pairs test no significant difference was found between the techniques (p=0,651). In two instances the two techniques found two different species of B. burgdorferi s.l. for one sample. In both cases the RFLP technique detected B. valaisiana and the RLB found B. afzelii.

- 27 - 3.5 Host examination

During the 5-day rodent trapping all rodents caught were wood mice (Apodemus sylvaticus) except for one bank vole caught in the pine plot without cattle. This plot yielded the highest number of caught rodents. At least 3 wood mice in this plot were recaptured, seven managed to escape before identification or marking could take place. Two of these mice were infested with four and one larvae each. Four captures were made in the oak plot without cattle, one of which was a recaptured wood mouse. These 3 wood mice were infested with the most ticks, with one individual mouse infested with 8 larvae. Another mouse was infested with one larvae and one nymph, upon recapture the larvae was still present, but the nymph had dislodged. One wood mouse, without was caught in the oak plot with cattle. No rodents were captured in the pine forest plot with cattle. Table 4 gives an overview of rodents caught per plot and their infestation with ticks. The number of rodents caught was very low. Table 4: Number of rodents caught in the different plots with the total amount of larvae and nymphs found on the captured animals.

Plot Habitat type # captures # larvae # nymphs A Oak, no cattle 4 10 1 B Pine, no cattle 16 5 0 C Oak, with cattle 1 0 0 D Pine, with cattle 0 0 0

Upon the on opportunity to inspect the cattle for tick infestation, five of the seven present cattle were checked. Two nymphs were found on the cattle, both on the legs.

- 28 - 4 Discussion

4.1 I. ricinus phenology

During this study relatively few ticks were found on the study site. Higher numbers of ticks of all stages were caught on the same site during the spring of the same year. Low numbers of adults in this period have been reported before and are caused by most of the adults laying their eggs in spring, after which they die. New adults do not emerge until fall (Ostfeld, Cepeda et al. 1995). Most larvae were caught at the beginning of the study at the end of June. Nymph captures remained mostly constant throughout the study period. The low number of ticks can not be explained by the climactic data. Saturation deficit, which greatly influences tick questing behaviour, was found to be continuously low during our study period with exception of a dry period between 7 – 19 July. These are very favorable conditions; when saturation deficit drops below 4 mmHG, ticks are less vulnerable to desiccation and are therefore more likely to show questing behaviour (Perret, Guigoz et al. 2000; Randolph, Green et al. 2002). No discernible effect of saturation deficit on the tick population can be observed in our data.

4.2 Hosts

Very few rodents were caught in this experiment, especially compared to a similar experiment that was conducted in June of the same year. In that experiment 256 rodents were caught in the same time span, with almost half the traps used as in this study (Gassner 2005). In this research we caught a total of 21 rodents. There seems to be no clear reason for this dramatic drop in rodent population. The low number of nymphs (1) feeding on the wood mice is probably caused by nymphs questing higher on the vegetation and therefore attaching to large hosts. Wild rabbits (Oryctolagus cuniculus) were observed in the area on multiple occasions, and could serve as hosts for the nymph population. Because rodents are hosts for the earlier tick life stages, their populations can influence tick populations. Although the two plots with the most rodents also yielded the most ticks, the plot with the most ticks was not the one with the most captured rodents. In this research, no clear relation can be seen between the number of caught rodents and the number of ticks found in the plots. There is an indication that cattle had a negative influence on the presence of rodents, fewer rodents were caught in the plots with cattle. However, this could not be proven statistically due to the low number of rodents caught.

4.3 B. burgdorferi s.l. infections

This study found an infection percentage of 18,8%. Although this is a high infection rate, between April and June of the same year, a higher mean infection percentage of 26,7% was found in the same plots. Probably part of this difference can be explained by the fact that fewer adult ticks were caught during our study period, which was largely in summer, when adults are less active (Ostfeld, Cepeda et al. 1995). The infection rate in adult ticks is higher since they have had two blood meals and thus two opportunities to become infected (Gray 1998).

- 29 - No infected ticks were found in the oak plot with cattle. This plot seemed the plot to be most frequented by the cattle present, it being closest to the meadow where the cattle spent most of their time. Infection rates per plot did not correspond with those found in the previous study. The plot with cattle and pine had the highest rate in this study, but it had the lowest in the earlier one. We found no infections in the oak with cattle plot when before it had the highest infection rate. This could indicate a shift in infections over the seasons. However, in both studies numbers were too low to yield any significant results. Therefore no conclusions can be drawn on the subject, longer studies are necessary to gain sufficient numbers for stronger statistical analysis.

4.4 RLB versus RFLP technique

Although the RFLP method found more infections than the RLB method, this difference was not significant. Both techniques are an excellent tool in determining infections of the B. burgdorferi s.l. complex. In general the RFLP technique is the cheaper and faster strategy, although we had trouble keeping it running properly during this study. The RLB technique seems to be slightly more reliable, but is more cumbersome and expensive. The RFLP technique has the added advantage of being able to determine two different strains of B. valaisiana, six different Osp-A types of B. garinii, two strains of B. burgdorferi s.s., two strains of B. lusitaniae and a separate Borrelia strain: Borrelia A14s.

4.5 Conclusions

Our hypothesis, that introduction of cattle into a woodland area increases I.ricinus populations and B. burgdorferi s.l. infections in an area, is rejected in this study. The opposite effect was observed. Fewest ticks and infections were found in the plot where the cattle seemed to spent the most time when in the woodland area. Although cattle have the potential to act as reproduction hosts and thus increase the tick population, they do not act as such in our study area. Possibly by entering the forest they change it in such a way that it becomes unfavorable for juvenile tick stages and their hosts. In the oak plot near the meadow where the cattle was most present in the forest, a herb layer was absent. A herb layer protects ticks from desiccation and also provides shelter for the rodents that feed the ticks in their early life stages. If, by wandering in the forest, the cattle reduce the herb layer, their very presence diminishes their chances to encounter older tick stages to feed on them. At the one opportunity we had to inspect the cattle we found just two nymphs feeding on them. Inevitably some adult ticks will feed on the cattle present in the area, but their numbers are too small to increase the tick population.. Roe deer were found to be more prevalent in the areas without cattle (Wielenga 2006). If roe deer act as reproduction host for tick populations and the presence of cattle drives them away, tick populations will decrease if cattle is introduced in an area. A model created by Buskirk and Ostfeld (Vanbuskirk and Ostfeld 1995) indicated that density of ticks was more sensitive to the availability of hosts for juveniles than hosts for adults. When only small numbers of ticks are feeding on the cattle, the likelihood of ticks transmitting infections through co-feeding seems less likely. No effect in infection percentage between plots with and without cattle was found in our study. A recent study in France found that cattle limited the prevalence of the B. burgdorferi s.l. complex (Richter and Matuschka 2006). Potentially large mammals can reduce infection percentages because infected ticks can lose their infection during the course of feeding on them (Telford, Mather et al. 1988;

- 30 - Jaenson and Talleklint 1992; Matuschka, Heiler et al. 1993). Because apparently very few ticks are feeding on the cattle in our study site, no such effect can be seen in our data. Although no effect can be seen in infection percentages, introduction of cattle has a significant negative effect on tick population density. Introduction of large herbivores might therefore reduce the changes of people getting infected with Lyme disease in the area where they are introduced, although different effects may be found in different areas.

5 Acknowledgements

I would like to thank Willem Takken for his supervision and advice during my thesis. Special thanks go out to Patrick Verbaarschot for his great help during the labwork. Saskia Wielenga is thanked for her cooperation in the rodent trapping and Jan Wierenga for his help in collecting ticks from the cattle. I am grateful to the Meteorology and Air Quality section of Wageningen UR for providing me with the precipitation data. I would like to thank Fedor Gassner for his advice on this thesis. Furthermore, I would like to thank my family and friends who supported me in numerous ways during this thesis.

- 31 - 6 References:

Boon, S. d. and W. v. Pelt (2003). "Verdubbeling consulten voor tekenbeten en ziekte van Lyme." Infectieziekten bulletin 14(5): 162-163. Dam, A. P. and e. al. (1993). "Different genospecies of Borrelia burgdorferi are associated with distinct clinical manifestations of Lyme borreliosis." Clinical Infectious Diseases 17: 708-717. Fingerle and e. al. (2002). "Dynamics of dissemination and outer surface protein expression of different European Borrelia burgdorferi s.l. strains in artificially infected Ixodes ricinus nymphs." Journal of Clinical Microbiology 40(4): 1456-1463. Gassner, F. (2005). Dynamics of tick and Borrelia populations in a natural woodland, in which cattle is resident. Laboratory of Entomology. Wageningen, Wageningen University and Research Centre: 41. Gassner, F., P. Verbaarschot, R. C. Smallegange, J. Spitzen, S. E. Van Wieren and W. Takken (2008). "Variations in Ixodes ricinus density and Borrelia infections associated with cattle introduced into a woodland in The Netherlands." Appl Environ Microbiol 74(23): 7138-44. Gern, L., A. Estrada-Pena, F. Frandsen, J. S. Gray, T. G. Jaenson, F. Jongejan, O. Kahl, E. Korenberg, R. Mehl and P. A. Nuttall (1998). "European reservoir hosts of Borrelia burgdorferi sensu lato." Zentralbl Bakteriol 287(3): 196-204. Gray, J. S. (1987). "Mating and Behavioral Diapause in Ixodes-Ricinus L." Experimental & Applied Acarology 3(1): 61-71. Gray, J. S. (1998). "The ecology of ticks transmitting Lyme borreliosis." Experimental and Applied Acarology 22(5): 249-258. Hanincova, K., S. M. Schafer, S. Etti, H. S. Sewell, V. Taragelova, D. Ziak, M. Labuda and K. Kurtenbach (2003). "Association of with rodents in Europe." Parasitology 126: 11-20. Humair, P. F. and e. al. (1995). "Strain variation of Lyme disease spirochetes isolated from Ixodes ricinus ticks and rodents collected in two endemic areas in Switzerland." Journal of 32: 433-438. Humair, P. F. and e. al. (1998). "An avian reservoir (Turdus merula) of the Lyme borreliosis spirochetes." Zentralblatt fur Bakteriologie 287: 521-538. Humair, P. F. and L. Gern (1998). "Relationship between Borrelia burgdorferi s.l. species, red squirrels (Scurius vulgaris) and Ixodes ricinus in enzootic areas in Switzerland." Acta Tropica 69: 213-227. Humair, P. F., O. Rais and L. Gern (1999). "Transmission of Borrelia afzelii from Apodemus mice and Clethrionomys voles to Ixodes ricinus ticks: differential transmission pattern and overwintering maintenance." Parasitology 118 ( Pt 1): 33-42. Jaenson, T. G. and L. Talleklint (1992). "Incompetence of roe deer as reservoirs of the Lyme borreliosis spirochete." J Med Entomol 29(5): 813-7. Kirstein, F., S. Rijpkema, M. Molkenboer and J. S. Gray (1997). "The distribution and prevalence of B. burgdorferi genomospecies in Ixodes ricinus ticks in Ireland." Eur J Epidemiol 13(1): 67-72. Kurtenbach, K., M. Peacey, S. G. T. Rijpkema, A. N. Hoodless, P. A. Nuttall and S. E. Randolph (1998). "Differential transmission of the genospecies of Borrelia burgdorferi sensu lato by game birds and small rodents in England." Applied and Environmental Microbiology 64(4): 1169-1174.

- 32 - LeFleche and e. al. (1997). "Characterization of Borrelia lusitaniae sp. Nov. by 16S ribosomal DNA sequence analysis." International Journal of Systematical Bacteriology 47: 921-925. Matuschka, F. R., M. Heiler, H. Eiffert, P. Fischer, H. Lotter and A. Spielman (1993). "Diversionary Role of Hoofed Game in the Transmission of Lyme-Disease Spirochetes." American Journal of Tropical Medicine and Hygiene 48(5): 693-699. Michel, H., B. Wilske, G. Hettche, G. Gottner, C. Heimerl, U. Reischl, U. Schulte-Spechtel and V. Fingerle (2004). "An ospA-polymerase chain reaction/restriction fragment length polymorphism-based method for sensitive detection and reliable differentiation of all European Borrelia burgdorferi sensu lato species and OspA types." Med Microbiol Immunol 193(4): 219-26. Mik, E., W. v. Pelt, B. Docters van Leeuwen, A. Veen, J. F. v. d. Schellekes and M. W. Borgdorff (1997). "The geographical distribution of tick bites and erythema migrans in general practice in the Netherlands." International journal of Epidemiology 26: 251-457. Nadelman, R. B. and G. P. Wormser (1998). "Lyme borreliosis." Lancet 352(9127): 557-65. Ostfeld, R. S., O. M. Cepeda, K. R. Hazler and M. C. Miller (1995). "Ecology of Lyme-Disease - Habitat Associations of Ticks (Ixodes-Scapularis) in a Rural Landscape." Ecological Applications 5(2): 353-361. Perret, J. L., E. Guigoz, O. Rais and L. Gern (2000). "Influence of saturation deficit and temperature on Ixodes ricinus tick questing activity in a Lyme borreliosis-endemic area (Switzerland)." Parasitol Res 86(7): 554-7. Randolph, S. E., R. M. Green, A. N. Hoodless and M. F. Peacey (2002). "An empirical quantitative framework for the seasonal population dynamics of the tick Ixodes ricinus." Int J Parasitol 32(8): 979-89. Richter, D. and F. R. Matuschka (2006). "Modulatory effect of cattle on risk for Lyme disease." Emerging Infectious Diseases 12(12): 1919-1923. Rijpkema, S. and H. Bruinink (1996). "Detection of Borrelia burgdorferi sensu lato by PCR in questing Ixodes ricinus larvae from the Dutch North Sea island of Ameland." Experimental & Applied Acarology 20(7): 381-385. Schouls, L. M., I. Van De Pol, S. G. Rijpkema and C. S. Schot (1999). "Detection and identification of Ehrlichia, Borrelia burgdorferi sensu lato, and species in Dutch Ixodes ricinus ticks." J Clin Microbiol 37(7): 2215-22. Telford, S. R., T. N. Mather, S. I. Moore, M. L. Wilson and A. Spielman (1988). "Incompetence of Deer as Reservoirs of Borrelia-Burgdorferi." Annals of the New York Academy of Sciences 539: 429-430. Vanbuskirk, J. and R. S. Ostfeld (1995). "Controlling Lyme-Disease by Modifying the Density and Species Composition of Tick Hosts." Ecological Applications 5(4): 1133-1140. Wang, G., A. P. van Dam, A. Le Fleche, D. Postic, O. Peter, G. Baranton, R. de Boer, L. Spanjaard and J. Dankert (1997). "Genetic and phenotypic analysis of Borrelia valaisiana sp. nov. (Borrelia genomic groups VS116 and M19)." Int J Syst Bacteriol 47(4): 926-32. Wielenga, S. (2006). The effect of cattle presence and roe deer density on the number of questing ticks in oak forest, pine forest and a meadow. Wageningen, Wageningen University and Research Centre.

- 33 - Appendix 1: Climate Data

Upper limits of the 95% confidence interval for the differences between plots. In 95% of cases data did not exceed the given value.

Compared plots Temperature Relative Humidity Saturation Deficit (ºC) (%) (mmHG) A-C 0.37 0.47 0.11 C-D 0.13 0.27 0.15 D-A 0.25 0.24 0.04

- 34 -

30.08.2005

28.08.2005

26.08.2005

Temperature plot D plot Temperature

Temperature plot C plot Temperature Temperature plot A plot Temperature

24.08.2005

22.08.2005

20.08.2005

18.08.2005

16.08.2005

14.08.2005

12.08.2005

10.08.2005

08.08.2005

06.08.2005

04.08.2005

02.08.2005 Date 31.07.2005

Temperature 29.07.2005

27.07.2005

25.07.2005

23.07.2005

21.07.2005

19.07.2005

11.07.2005

09.07.2005

07.07.2005

05.07.2005

03.07.2005

01.07.2005 forticks. the which threshold conditions physiological indicates questing are of unfavorable below 5ºC,

29.06.2005 Average daily minimum, mean and maximum temperatures per plot, recorded by data loggers on site. The dashed line dashed recorded loggers The on per by site. plot, data temperatures and Average daily maximum minimum, mean 27.06.2005

- 35 -

10.00

12.50

15.00

17.50

20.00 22.50 Mean Temperature (C) Temperature Mean

ration deficit per plot, as recorded by data loggers on site. The dashed line indicates the indicates The line by site. dashed data recorded loggers plot, rationas on per deficit

for conditions ticks. are questing favorable threshold physiological ofbelow mmHg, which 4

Average Average daily satu

- 36 -

physiological threshold of 4 mmHg, below which conditions are favorable for questing for conditions ticks. are questing favorable threshold physiological ofbelow mmHg, which 4

Average daily saturation deficit per plot, as recorded by data loggers on site. The dashed line indicates the indicates The line by site. dashed data recorded loggers plot, as on Average daily per saturation deficit

- 37 - Appendix 2: Raw tick data

B=burgdorferi s.s. V=valaisiana G=garinii A=afzelii In the “infected” and “B”, “V”, “G”, “A” co1umns a 1 indicates a positive result.

Adults Adults Nr. Date Plot Larvae Nymphs Analyzed Infected B V G A (m) (f) Analysis 27.06.2005 A1a 0 0 0 0 0 0 . . . . 27.06.2005 A1b 5 2 0 0 368-369 2 0 . . . . 27.06.2005 A2a 2 3 0 0 370-372 3 0 . . . . 27.06.2005 A2b 8 3 0 0 373-375 3 0 . . . . 27.06.2005 A3a 11 2 0 0 376-377 2 1 . . 1 . 27.06.2005 A3b 14 1 0 0 378 1 0 . . . . 27.06.2005 A4a 11 1 0 0 379 1 0 . . . . 27.06.2005 A4b 25 4 0 0 380-383 4 0 . . . . 27.06.2005 B1a 0 1 0 0 384 1 1 . . . . 27.06.2005 B1b 0 1 0 0 385 1 0 . . . . 27.06.2005 B2a 0 0 0 0 0 0 . . . . 27.06.2005 B2b 1 2 0 0 386-387 2 0 . . . . 27.06.2005 B3a 2 1 0 0 388 1 0 . . . . 27.06.2005 B3b 1 1 0 0 389 1 0 . . . . 27.06.2005 B4a 1 0 0 0 0 0 . . . . 27.06.2005 B4b 0 0 0 0 0 0 . . . . 27.06.2005 C1a 0 0 0 0 0 0 . . . . 27.06.2005 C1b 0 0 0 0 0 0 . . . . 27.06.2005 C2a 0 0 0 0 0 0 . . . . 27.06.2005 C2b 0 0 0 0 0 0 . . . . 27.06.2005 C3a 0 0 0 0 0 0 . . . . 27.06.2005 C3b 2 0 0 0 0 0 . . . . 27.06.2005 C4a 6 0 0 0 0 0 . . . . 27.06.2005 C4b 2 0 0 0 0 0 . . . . 27.06.2005 D1a 1 0 0 0 0 0 . . . . 27.06.2005 D1b 0 0 0 0 0 0 . . . . 27.06.2005 D2a 0 1 0 0 390 1 0 . . . . 27.06.2005 D2b 0 0 0 0 0 0 . . . . 27.06.2005 D3a 0 0 0 0 0 0 . . . . 27.06.2005 D3b 0 1 0 0 391 1 0 . . . . 27.06.2005 D4a 3 0 0 0 0 0 . . . . 27.06.2005 D4b 0 1 0 0 392 1 0 . . . . 04.07.2005 A1a 2 0 0 0 0 0 . . . . 04.07.2005 A1b 4 1 0 0 393 1 0 . . . . 04.07.2005 A2a 1 0 0 0 0 0 . . . . 04.07.2005 A2b 3 1 0 0 394 1 0 . . . . 04.07.2005 A3a 10 0 0 0 0 0 . . . . 04.07.2005 A3b 17 0 0 0 0 0 . . . . 04.07.2005 A4a 9 4 0 0 395-398 4 0 . . . . 04.07.2005 A4b 6 0 0 0 0 0 . . . . 04.07.2005 B1a 2 2 0 0 399-400 2 1 . . 1 . 04.07.2005 B1b 0 0 0 0 0 0 . . . . 04.07.2005 B2a 1 1 0 0 401 1 0 . . . .

- 38 - Adults Adults Nr. Date Plot Larvae Nymphs Analyzed Infected B V G A (m) (f) Analysis 04.07.2005 B2b 0 1 0 0 402 1 0 . . . . 04.07.2005 B3a 1 2 0 0 403-404 2 1 . . 1 . 04.07.2005 B3b 0 0 0 0 0 0 . . . . 04.07.2005 B4a 1 1 0 0 405 1 0 . . . . 04.07.2005 B4b 1 0 0 0 0 0 . . . . 04.07.2005 C1a 0 0 0 0 0 0 . . . . 04.07.2005 C1b 0 0 0 0 0 0 . . . . 04.07.2005 C2a 0 0 0 0 0 0 . . . . 04.07.2005 C2b 0 0 0 0 0 0 . . . . 04.07.2005 C3a 0 0 0 0 0 0 . . . . 04.07.2005 C3b 0 0 0 0 0 0 . . . . 04.07.2005 C4a 0 0 0 0 0 0 . . . . 04.07.2005 C4b 0 0 0 0 0 0 . . . . 04.07.2005 D1a 0 0 0 0 0 0 . . . . 04.07.2005 D1b 0 1 0 0 406 1 0 . . . . 04.07.2005 D2a 0 1 0 0 407 1 0 . . . . 04.07.2005 D2b 0 1 0 0 408 1 1 1 . . . 04.07.2005 D3a 0 0 0 0 0 0 . . . . 04.07.2005 D3b 0 0 0 0 0 0 . . . . 04.07.2005 D4a 0 1 0 0 409 1 1 . . . 1 04.07.2005 D4b 0 0 0 0 0 0 . . . . 11.07.2005 A1a 2 0 0 0 0 0 . . . . 11.07.2005 A1b 0 3 0 0 410-412 3 1 . . . 1 11.07.2005 A2a 4 4 0 0 413-416 4 1 1 . . . 11.07.2005 A2b 1 0 0 0 0 0 . . . . 11.07.2005 A3a 1 1 0 0 417 1 1 . 1 . . 11.07.2005 A3b 2 0 0 0 0 0 . . . . 11.07.2005 A4a 3 0 0 0 0 0 . . . . 11.07.2005 A4b 3 0 0 0 0 0 . . . . 11.07.2005 B1a 0 0 0 0 0 0 . . . . 11.07.2005 B1b 0 0 0 0 0 0 . . . . 11.07.2005 B2a 0 0 0 0 0 0 . . . . 11.07.2005 B2b 0 0 0 0 0 0 . . . . 11.07.2005 B3a 1 0 0 0 0 0 . . . . 11.07.2005 B3b 0 0 0 0 0 0 . . . . 11.07.2005 B4a 0 0 0 0 0 0 . . . . 11.07.2005 B4b 0 0 0 0 0 0 . . . . 11.07.2005 C1a 0 0 0 0 0 0 . . . . 11.07.2005 C1b 0 0 0 0 0 0 . . . . 11.07.2005 C2a 0 0 0 0 0 0 . . . . 11.07.2005 C2b 0 0 0 0 0 0 . . . . 11.07.2005 C3a 0 0 0 0 0 0 . . . . 11.07.2005 C3b 0 0 0 0 0 0 . . . . 11.07.2005 C4a 0 0 0 0 0 0 . . . . 11.07.2005 C4b 0 0 0 0 0 0 . . . . 11.07.2005 D1a 0 0 0 0 0 0 . . . . 11.07.2005 D1b 0 0 0 0 0 0 . . . . 11.07.2005 D2a 0 0 0 0 0 0 . . . .

- 39 - Adults Adults Nr. Date Plot Larvae Nymphs Analyzed Infected B V G A (m) (f) Analysis 11.07.2005 D2b 1 0 0 0 0 0 . . . . 11.07.2005 D3a 0 0 0 0 0 0 . . . . 11.07.2005 D3b 0 0 0 0 0 0 . . . . 11.07.2005 D4a 0 0 0 0 0 0 . . . . 11.07.2005 D4b 0 0 0 0 0 0 . . . . 18.07.2005 A1a 0 0 0 0 0 0 . . . . 18.07.2005 A1b 1 0 0 0 0 0 . . . . 18.07.2005 A2a 2 2 0 0 418-419 2 1 1 1 . . 18.07.2005 A2b 1 2 0 0 420-421 2 0 . . . . 18.07.2005 A3a 4 1 0 1 422-423 2 1 . 1 . . 18.07.2005 A3b 1 1 0 0 424 1 0 . . . . 18.07.2005 A4a 10 0 0 0 0 0 . . . . 18.07.2005 A4b 9 1 0 0 425 1 0 . . . . 18.07.2005 B1a 0 1 0 0 426 1 0 . . . . 18.07.2005 B1b 0 0 0 0 0 0 . . . . 18.07.2005 B2a 0 1 0 0 427 1 0 . . . . 18.07.2005 B2b 0 0 0 0 0 0 . . . . 18.07.2005 B3a 0 1 0 0 428 1 0 . . . . 18.07.2005 B3b 0 0 0 0 0 0 . . . . 18.07.2005 B4a 0 0 0 0 0 0 . . . . 18.07.2005 B4b 0 0 1 0 429 1 1 . . . 1 18.07.2005 C1a 0 0 0 0 0 0 . . . . 18.07.2005 C1b 0 0 0 0 0 0 . . . . 18.07.2005 C2a 0 0 0 0 0 0 . . . . 18.07.2005 C2b 0 0 0 0 0 0 . . . . 18.07.2005 C3a 0 0 0 0 0 0 . . . . 18.07.2005 C3b 0 1 0 0 430 1 0 . . . . 18.07.2005 C4a 2 0 0 0 0 0 . . . . 18.07.2005 C4b 0 0 0 0 0 0 . . . . 18.07.2005 D1a 0 0 0 0 0 0 . . . . 18.07.2005 D1b 0 0 0 0 0 0 . . . . 18.07.2005 D2a 0 0 0 0 0 0 . . . . 18.07.2005 D2b 1 1 0 0 431 1 0 . . . . 18.07.2005 D3a 0 0 0 0 0 0 . . . . 18.07.2005 D3b 0 0 0 0 0 0 . . . . 18.07.2005 D4a 0 0 0 0 0 0 . . . . 18.07.2005 D4b 0 0 0 0 0 0 . . . . 02.08.2005 A1a 2 0 0 0 0 0 . . . . 02.08.2005 A1b 3 1 0 0 432 1 1 1 . 1 . 02.08.2005 A2a 1 0 0 0 0 0 . . . . 02.08.2005 A2b 1 0 0 0 0 0 . . . . 02.08.2005 A3a 2 0 0 0 0 0 . . . . 02.08.2005 A3b 1 3 0 0 433-435 3 1 1 . . 1 02.08.2005 A4a 0 2 0 0 436-437 2 0 . . . . 02.08.2005 A4b 0 0 0 0 0 0 . . . . 02.08.2005 B1a 0 1 0 0 438 1 1 . 1 . . 02.08.2005 B1b 1 0 0 0 0 0 . . . . 02.08.2005 B2a 1 0 0 0 0 0 . . . .

- 40 - Adults Adults Nr. Date Plot Larvae Nymphs Analyzed Infected B V G A (m) (f) Analysis 02.08.2005 B2b 0 1 0 0 439 1 0 . . . . 02.08.2005 B3a 0 1 0 0 440 1 1 . . . 1 02.08.2005 B3b 1 1 0 0 441 1 0 . . . . 02.08.2005 B4a 1 1 0 0 442 1 0 . . . . 02.08.2005 B4b 1 0 0 0 0 0 . . . . 02.08.2005 C1a 0 0 0 0 0 0 . . . . 02.08.2005 C1b 0 0 0 0 0 0 . . . . 02.08.2005 C2a 0 0 0 0 0 0 . . . . 02.08.2005 C2b 0 0 0 0 0 0 . . . . 02.08.2005 C3a 0 0 0 0 0 0 . . . . 02.08.2005 C3b 0 0 0 0 0 0 . . . . 02.08.2005 C4a 0 0 0 0 0 0 . . . . 02.08.2005 C4b 0 0 0 0 0 0 . . . . 02.08.2005 D1a 1 0 0 0 0 0 . . . . 02.08.2005 D1b 0 0 0 0 0 0 . . . . 02.08.2005 D2a 0 0 0 0 0 0 . . . . 02.08.2005 D2b 0 0 0 0 0 0 . . . . 02.08.2005 D3a 0 0 0 0 0 0 . . . . 02.08.2005 D3b 0 0 0 0 0 0 . . . . 02.08.2005 D4a 0 0 0 0 0 0 . . . . 02.08.2005 D4b 0 0 0 0 0 0 . . . . 10.08.2005 A1a 0 0 0 0 0 0 . . . . 10.08.2005 A1b 4 0 0 0 0 0 . . . . 10.08.2005 A2a 1 0 0 0 0 0 . . . . 10.08.2005 A2b 0 1 0 0 443 1 1 . 1 . . 10.08.2005 A3a 0 0 0 0 0 0 . . . . 10.08.2005 A3b 0 1 0 0 1 0 . . . . 10.08.2005 A4a 4 2 0 0 444-445 2 0 . . . . 10.08.2005 A4b 1 2 0 0 446-448 2 0 . . . . 10.08.2005 B1a 0 1 0 0 449 1 0 . . . . 10.08.2005 B1b 0 0 0 0 0 0 . . . . 10.08.2005 B2a 2 2 0 0 450-451 2 0 . . . . 10.08.2005 B2b 1 0 0 0 0 0 . . . . 10.08.2005 B3a 0 2 0 0 452-453 2 1 . . . 1 10.08.2005 B3b 1 1 0 0 454 1 0 . . . . 10.08.2005 B4a 1 0 0 0 0 0 . . . . 10.08.2005 B4b 0 0 0 0 0 0 . . . . 10.08.2005 C1a 0 0 0 0 0 0 . . . . 10.08.2005 C1b 0 0 0 0 0 0 . . . . 10.08.2005 C2a 0 0 0 0 0 0 . . . . 10.08.2005 C2b 0 0 0 0 0 0 . . . . 10.08.2005 C3a 0 0 0 0 0 0 . . . . 10.08.2005 C3b 0 0 0 0 0 0 . . . . 10.08.2005 C4a 0 0 0 0 0 0 . . . . 10.08.2005 C4b 0 0 0 0 0 0 . . . . 10.08.2005 D1a 0 1 0 0 455 1 0 . . . . 10.08.2005 D1b 0 1 0 0 456 1 1 . . . 1 10.08.2005 D2a 0 0 0 0 0 0 . . . .

- 41 - Adults Adults Nr. Date Plot Larvae Nymphs Analyzed Infected B V G A (m) (f) Analysis 10.08.2005 D2b 0 0 0 0 0 0 . . . . 10.08.2005 D3a 0 0 0 0 0 0 . . . . 10.08.2005 D3b 0 0 0 0 0 0 . . . . 10.08.2005 D4a 0 0 0 0 0 0 . . . . 10.08.2005 D4b 0 0 0 0 0 0 . . . . 17.08.2005 A1a 4 2 0 0 457-458 2 0 . . . . 17.08.2005 A1b 4 0 0 0 0 0 . . . . 17.08.2005 A2a 0 1 0 0 459 1 0 . . . . 17.08.2005 A2b 4 0 0 0 0 0 . . . . 17.08.2005 A3a 0 2 0 0 460-461 2 2 . . . 2 17.08.2005 A3b 7 0 0 0 0 0 . . . . 17.08.2005 A4a 2 0 0 0 0 0 . . . . 17.08.2005 A4b 2 1 0 0 462 1 0 . . . . 17.08.2005 B1a 0 2 0 0 463-464 2 0 . . . . 17.08.2005 B1b 1 0 0 0 0 0 . . . . 17.08.2005 B2a 1 2 0 0 465-466 2 1 . . 2 . 17.08.2005 B2b 0 0 0 0 0 0 . . . . 17.08.2005 B3a 0 0 0 0 0 0 . . . . 17.08.2005 B3b 2 1 0 0 497 1 0 . . . . 17.08.2005 B4a 1 0 0 0 0 0 . . . . 17.08.2005 B4b 2 1 0 0 468 1 0 . . . . 17.08.2005 C1a 0 0 0 0 0 0 . . . . 17.08.2005 C1b 0 0 0 0 0 0 . . . . 17.08.2005 C2a 0 0 0 0 0 0 . . . . 17.08.2005 C2b 0 0 0 0 0 0 . . . . 17.08.2005 C3a 0 0 0 0 0 0 . . . . 17.08.2005 C3b 0 0 0 0 0 0 . . . . 17.08.2005 C4a 0 0 0 0 0 0 . . . . 17.08.2005 C4b 0 0 0 0 0 0 . . . . 17.08.2005 D1a 1 0 0 0 0 0 . . . . 17.08.2005 D1b 1 1 0 0 469 1 0 . . . . 17.08.2005 D2a 0 0 0 0 0 0 . . . . 17.08.2005 D2b 0 0 0 0 0 0 . . . . 17.08.2005 D3a 1 1 0 0 470 1 1 . . . 1 17.08.2005 D3b 0 0 0 0 0 0 . . . . 17.08.2005 D4a 0 0 0 0 0 0 . . . . 17.08.2005 D4b 0 0 0 0 0 0 . . . . 23.08.2005 A1a 3 1 0 0 471 1 0 . . . . 23.08.2005 A1b 1 1 0 0 472 1 0 . . . . 23.08.2005 A2a 0 0 0 0 0 0 . . . . 23.08.2005 A2b 2 0 0 0 0 0 . . . . 23.08.2005 A3a 1 1 0 0 473 1 0 . . . . 23.08.2005 A3b 1 1 0 0 474 1 0 . . . . 23.08.2005 A4a 0 0 1 0 475 1 0 . . . . 23.08.2005 A4b 0 1 0 0 476 1 0 . . . . 23.08.2005 B1a 2 0 0 0 0 0 . . . . 23.08.2005 B1b 0 0 0 0 0 0 . . . . 23.08.2005 B2a 1 0 0 0 0 0 . . . .

- 42 - Adults Adults Nr. Date Plot Larvae Nymphs Analyzed Infected B V G A (m) (f) Analysis 23.08.2005 B2b 0 0 0 0 0 0 . . . . 23.08.2005 B3a 0 0 0 0 0 0 . . . . 23.08.2005 B3b 0 0 0 0 0 0 . . . . 23.08.2005 B4a 0 2 0 0 477-478 2 0 . . . . 23.08.2005 B4b 0 0 0 0 0 0 . . . . 23.08.2005 C1a 0 0 0 0 0 0 . . . . 23.08.2005 C1b 0 0 0 0 0 0 . . . . 23.08.2005 C2a 0 0 0 0 0 0 . . . . 23.08.2005 C2b 0 0 0 0 0 0 . . . . 23.08.2005 C3a 0 0 0 0 0 0 . . . . 23.08.2005 C3b 0 0 0 0 0 0 . . . . 23.08.2005 C4a 0 0 0 0 0 0 . . . . 23.08.2005 C4b 0 0 0 0 0 0 . . . . 23.08.2005 D1a 0 2 0 0 479-480 1 0 . . . . 23.08.2005 D1b 1 0 0 0 0 0 . . . . 23.08.2005 D2a 0 0 0 0 0 0 . . . . 23.08.2005 D2b 0 0 0 0 0 0 . . . . 23.08.2005 D3a 0 0 0 0 0 0 . . . . 23.08.2005 D3b 0 0 0 0 0 0 . . . . 23.08.2005 D4a 0 0 0 0 0 0 . . . . 23.08.2005 D4b 0 0 0 0 0 0 . . . . 31.08.2005 A1a 3 1 0 0 481 1 0 . . . . 31.08.2005 A1b 3 0 0 0 0 0 . . . . 31.08.2005 A2a 0 1 0 0 482 1 1 . . 1 . 31.08.2005 A2b 0 1 0 0 483 1 0 . . . . 31.08.2005 A3a 0 0 0 0 0 0 . . . . 31.08.2005 A3b 2 0 0 0 0 0 . . . . 31.08.2005 A4a 1 0 0 0 0 0 . . . . 31.08.2005 A4b 1 0 0 0 0 0 . . . . 31.08.2005 B1a 0 0 0 0 0 0 . . . . 31.08.2005 B1b 0 0 0 0 0 0 . . . . 31.08.2005 B2a 1 0 0 0 0 0 . . . . 31.08.2005 B2b 0 0 0 0 0 0 . . . . 31.08.2005 B3a 0 0 0 0 0 0 . . . . 31.08.2005 B3b 0 0 0 0 0 0 . . . . 31.08.2005 B4a 0 0 0 0 0 0 . . . . 31.08.2005 B4b 2 0 0 0 0 0 . . . . 31.08.2005 C1a 0 2 0 0 484-485 2 0 . . . . 31.08.2005 C1b 0 1 0 0 486 1 0 . . . . 31.08.2005 C2a 0 1 0 0 487 1 0 . . . . 31.08.2005 C2b 0 0 0 0 0 0 . . . . 31.08.2005 C3a 0 0 0 0 0 0 . . . . 31.08.2005 C3b 0 0 0 0 0 0 . . . . 31.08.2005 C4a 0 2 0 0 488-489 2 0 . . . . 31.08.2005 C4b 0 0 0 0 0 0 . . . . 31.08.2005 D1a 0 0 0 0 0 0 . . . . 31.08.2005 D1b 0 0 0 0 0 0 . . . . 31.08.2005 D2a 0 0 0 0 0 0 . . . .

- 43 - Adults Adults Nr. Date Plot Larvae Nymphs Analyzed Infected B V G A (m) (f) Analysis 31.08.2005 D2b 1 0 0 0 0 0 . . . . 31.08.2005 D3a 1 0 0 0 0 0 . . . . 31.08.2005 D3b 0 0 0 0 0 0 . . . . 31.08.2005 D4a 0 0 0 0 0 0 . . . . 31.08.2005 D4b 0 0 0 0 0 0 . . . . 05.09.2005 A1a 6 0 0 0 0 0 . . . . 05.09.2005 A1b 0 1 0 0 490 1 0 . . . . 05.09.2005 A2a 1 0 0 0 0 0 . . . . 05.09.2005 A2b 2 1 0 0 491 1 0 . . . . 05.09.2005 A3a 1 1 0 0 492 1 0 . . . . 05.09.2005 A3b 5 0 0 0 0 0 . . . . 05.09.2005 A4a 2 0 0 0 0 0 . . . . 05.09.2005 A4b 2 0 0 0 0 0 . . . . 05.09.2005 B1a 0 0 0 0 0 0 . . . . 05.09.2005 B1b 0 0 0 0 0 0 . . . . 05.09.2005 B2a 1 0 0 0 0 0 . . . . 05.09.2005 B2b 0 0 0 0 0 0 . . . . 05.09.2005 B3a 0 0 0 0 0 0 . . . . 05.09.2005 B3b 0 0 0 0 0 0 . . . . 05.09.2005 B4a 0 2 0 0 493-494 2 0 . . . . 05.09.2005 B4b 0 0 0 0 0 0 . . . . 05.09.2005 C1a 0 0 0 0 0 0 . . . . 05.09.2005 C1b 0 0 0 0 0 0 . . . . 05.09.2005 C2a 0 0 0 0 0 0 . . . . 05.09.2005 C2b 0 0 0 0 0 0 . . . . 05.09.2005 C3a 0 0 0 0 0 0 . . . . 05.09.2005 C3b 0 0 0 0 0 0 . . . . 05.09.2005 C4a 0 0 0 0 0 0 . . . . 05.09.2005 C4b 0 0 0 0 0 0 . . . . 05.09.2005 D1a 0 0 0 0 0 0 . . . . 05.09.2005 D1b 0 0 0 0 0 0 . . . . 05.09.2005 D2a 0 0 0 0 0 0 . . . . 05.09.2005 D2b 0 0 0 0 0 0 . . . . 05.09.2005 D3a 0 0 0 0 0 0 . . . . 05.09.2005 D3b 0 0 0 0 0 0 . . . . 05.09.2005 D4a 0 0 0 0 0 0 . . . . 05.09.2005 D4b 0 0 0 0 0 0 . . . . 20.09.2005 A1a 0 0 0 0 0 0 . . . . 20.09.2005 A1b 0 1 0 0 495 1 0 . . . . 20.09.2005 A2a 3 0 0 0 0 0 . . . . 20.09.2005 A2b 1 1 0 0 496 1 0 . . . . 20.09.2005 A3a 0 2 0 0 497-498 2 0 . . . . 20.09.2005 A3b 2 2 0 0 499-500 2 0 . . . . 20.09.2005 A4a 5 1 0 0 501 1 0 . . . . 20.09.2005 A4b 0 1 0 0 502 1 0 . . . . 20.09.2005 B1a 1 2 0 0 2 0 . . . . 20.09.2005 B1b 0 2 0 0 503-504 2 0 . . . . 20.09.2005 B2a 0 0 0 0 505-506 0 0 . . . .

- 44 - Adults Adults Nr. Date Plot Larvae Nymphs Analyzed Infected B V G A (m) (f) Analysis 20.09.2005 B2b 2 0 0 0 0 0 . . . . 20.09.2005 B3a 0 0 0 0 0 0 . . . . 20.09.2005 B3b 0 0 0 0 0 0 . . . . 20.09.2005 B4a 2 1 0 0 507 1 0 . . . . 20.09.2005 B4b 0 0 0 0 0 0 . . . . 20.09.2005 C1a 0 0 0 0 0 0 . . . . 20.09.2005 C1b 0 0 0 0 0 0 . . . . 20.09.2005 C2a 0 0 0 0 0 0 . . . . 20.09.2005 C2b 0 0 0 0 0 0 . . . . 20.09.2005 C3a 0 0 0 0 0 0 . . . . 20.09.2005 C3b 0 0 0 0 0 0 . . . . 20.09.2005 C4a 0 0 0 0 0 0 . . . . 20.09.2005 C4b 0 0 0 0 0 0 . . . . 20.09.2005 D1a 0 0 0 0 0 0 . . . . 20.09.2005 D1b 0 0 0 0 0 0 . . . . 20.09.2005 D2a 0 0 0 0 0 0 . . . . 20.09.2005 D2b 0 0 0 0 0 0 . . . . 20.09.2005 D3a 0 0 0 0 0 0 . . . . 20.09.2005 D3b 0 0 0 0 0 0 . . . . 20.09.2005 D4a 0 0 0 0 0 0 . . . . 20.09.2005 D4b 0 0 0 0 0 0 . . . . 27.09.2005 A1a 0 0 0 0 0 0 . . . . 27.09.2005 A1b 0 1 0 0 508 1 0 . . . . 27.09.2005 A2a 0 2 0 0 509-510 2 0 . . . . 27.09.2005 A2b 0 1 0 0 511 1 0 . . . . 27.09.2005 A3a 0 0 0 0 0 0 . . . . 27.09.2005 A3b 1 1 0 0 512 1 1 . . . 1 27.09.2005 A4a 0 0 0 0 0 0 . . . . 27.09.2005 A4b 0 1 0 0 513 1 0 . . . . 27.09.2005 B1a 0 0 0 0 0 0 . . . . 27.09.2005 B1b 0 0 0 0 0 0 . . . . 27.09.2005 B2a 0 0 0 0 0 0 . . . . 27.09.2005 B2b 0 0 0 0 0 0 . . . . 27.09.2005 B3a 0 0 0 0 0 0 . . . . 27.09.2005 B3b 0 0 0 0 0 0 . . . . 27.09.2005 B4a 0 0 0 0 0 0 . . . . 27.09.2005 B4b 0 0 0 0 0 0 . . . . 27.09.2005 C1a 0 0 0 0 0 0 . . . . 27.09.2005 C1b 0 0 0 0 0 0 . . . . 27.09.2005 C2a 0 0 0 0 0 0 . . . . 27.09.2005 C2b 0 0 0 0 0 0 . . . . 27.09.2005 C3a 0 0 0 0 0 0 . . . . 27.09.2005 C3b 0 0 0 0 0 0 . . . . 27.09.2005 C4a 0 0 0 0 0 0 . . . . 27.09.2005 C4b 0 0 0 0 0 0 . . . . 27.09.2005 D1a 0 0 0 0 0 0 . . . . 27.09.2005 D1b 0 0 0 0 0 0 . . . . 27.09.2005 D2a 0 0 0 0 0 0 . . . .

- 45 - Adults Adults Nr. Date Plot Larvae Nymphs Analyzed Infected B V G A (m) (f) Analysis 27.09.2005 D2b 0 0 0 0 0 0 . . . . 27.09.2005 D3a 0 0 0 0 0 0 . . . . 27.09.2005 D3b 0 0 0 0 0 0 . . . . 27.09.2005 D4a 0 0 0 0 0 0 . . . . 27.09.2005 D4b 0 0 0 0 0 0 . . . . 04.10.2005 A1a 1 1 0 0 514 1 0 . . . . 04.10.2005 A1b 1 6 0 0 515-520 6 1 . . 1 . 04.10.2005 A2a 0 1 0 0 521 1 0 . . . . 04.10.2005 A2b 3 1 0 0 522 1 0 . . . . 04.10.2005 A3a 5 1 0 0 523 1 0 . . . . 04.10.2005 A3b 5 1 0 0 524 1 0 . . . . 04.10.2005 A4a 0 0 0 0 0 0 . . . . 04.10.2005 A4b 0 0 0 0 0 0 . . . . 04.10.2005 B1a 0 0 0 0 0 0 . . . . 04.10.2005 B1b 0 1 0 0 525 1 0 . . . . 04.10.2005 B2a 2 0 0 0 0 0 . . . . 04.10.2005 B2b 1 1 0 0 526 1 0 . . . . 04.10.2005 B3a 1 0 0 0 0 0 . . . . 04.10.2005 B3b 0 0 0 0 0 0 . . . . 04.10.2005 B4a 1 0 0 0 0 0 . . . . 04.10.2005 B4b 0 0 0 0 0 0 . . . . 04.10.2005 C1a 0 1 0 0 527 1 0 . . . . 04.10.2005 C1b 0 0 0 0 0 0 . . . . 04.10.2005 C2a 0 0 0 0 0 0 . . . . 04.10.2005 C2b 0 0 0 0 0 0 . . . . 04.10.2005 C3a 0 0 0 0 0 0 . . . . 04.10.2005 C3b 0 0 0 0 0 0 . . . . 04.10.2005 C4a 0 0 0 0 0 0 . . . . 04.10.2005 C4b 0 0 0 0 0 0 . . . . 04.10.2005 D1a 0 0 0 0 0 0 . . . . 04.10.2005 D1b 0 0 0 0 0 0 . . . . 04.10.2005 D2a 0 0 0 0 0 0 . . . . 04.10.2005 D2b 0 0 0 0 0 0 . . . . 04.10.2005 D3a 0 0 0 0 0 0 . . . . 04.10.2005 D3b 0 0 0 0 0 0 . . . . 04.10.2005 D4a 0 0 0 0 0 0 . . . . 04.10.2005 D4b 1 0 0 0 0 0 . . . .

- 46 -