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Keywords:

  • Lyme borreliosis;
  • Ixodes ricinus ticks;
  • Borrelia burgdorferi sensu lato;
  • DEET;
  • repellents

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

The ever-increasing number of Lyme borreliosis patients led us to consider more effective procedures for disease prevention. The aim of our study was to monitor the annual activity and infectivity of Ixodes ricinus ticks in the Pisárky region, City of Brno, CR, and to test the responses of the locally-captured ticks to selected repellents. The result of regular one-hour-perweek monitoring in 2011 was the collection of ticks that directly reflected the highest number of Lyme disease patients (4,835) detected throughout the period of recording in the Czech Republic. The ticks were examined for spirochaetes by dark field microscopy. The positive samples were identified by PCR analysis, confirming that 76% of these were infected with Borrelia burgdorferi sensu lato. Ticks were most abundant in May and June, with August having the highest risk for spirochaetal infection. Tick activity was statistically correlated with temperature. The moving-object-bioassay was used to study repellent efficiency on the Ixodes ricinus nymphs captured in the above-mentioned suburban park. Five selected commercial repellents based on DEET (N, N-diethyl3methylbenzamide) showed statistically different effects on the non-repellent control group.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Tick-borne infections rank among the most frequent arthropod-borne diseases in many parts of the world, including Europe. We focused on Ixodes ricinus ticks, the main vectors of pathogens causing the most frequent arthropod-borne infections, including Lyme borreliosis (LB), or tick-borne encephalitis, in Europe (Jaenson et al. 2012). The tick-transmitted diseases, especially borreliosis, represent a growing health problem in the Czech Republic. The highest number of patients diagnosed with LB disease over the monitored years was registered in 2011 (EPIDAT – database of the National Institute of Public Health).

The spread and length of the development cycle of I. ricinus, the vector of LB, depends on environmental conditions and weather. As indicated by changing environmental factors during recent years, the tick life-cycle has been shortening and ticks, as well as their pathogens, have migrated to areas where they previously did not occur (Hubálek et al. 2002, Materna et al. 2005, Stünzner et al. 2006). This study also emphasized regular monitoring of the number and proportion of ticks infected with Borrelia burgdorferi sensu lato and their activity related to temperature. Pisárky Park in Brno appears to have optimal conditions for ticks, with approximately 65 ticks per h that have been captured. Awareness of the critical months for LB infection is among the most important considerations for LB prevention. The Pisárky location was chosen because it is a leisure area where people go for walks and relaxation. One way to keep nuisance arthropods away from people and animals and to avoid tick-borne infections is the use of chemical repellents.

DEET (N, N-diethyl3methylbenzamide) is a standard repellent that, together with citriodiol picaridine and IR3535, is among the four active substances recommended by the World Health Organization for long-lasting repellency against nearly all arthropods (Sorge 2009). The ‘moving-objectbioassay’ (MOB) (Dautel et al. 1999) is one of the possible approaches for repellency testing. It consists of a rotating heated drum that mimics body warmth, movements of a potential host or, for example, a specific host's smell. Truly efficient tick repellents must be effective despite the presence of attractants. Tested compounds are applied to a small exterior area of the drum, and as the drum rotates, an approaching tick can attach to the drum as if it was a passing host. Ticks that approach the drum on a rod (imitation of vegetation) are expected to be host-seeking. To a certain extent, MOB eliminates the practical, ethical, and economical disadvantages of testing on live hosts. Numerous modifications of this bioassay have been created, which also makes the assay suitable for testing of animal repellents. Two aims of this study were to show possibilities of prevention in the form of efficiency of the five repellents and to define the critical months for LB infection in the leisure area of a large city.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Locality

The urban park of Brno-Pisárky is located 2 km outside the center of the Moravian metropolis Brno (Žákovská et al. 2007). The altitude is 197–210 m above sea level and the location is situated towards the bottom of the Pisárky Valley. The predominant local vegetation is formed by a deciduous oak-hornbeam forest and characteristic representatives of herbs. The biotope also contains a wide range of small and middle-sized mammals, including hedgehogs (Erinaceus europaeus) and several species of squirrels (Sciurus vulgaris). The locality was chosen because human visitors come to the park for walks with pets and to spend their free time. Under such conditions, infected ticks could represent a real health problem in this area.

Tick collection

Ticks were collected at regular intervals (one h/week) by flagging, dragging a white flannel flag (1×1 m) over low vegetation. Two collectors alternated each week throughout the year. All collected ticks were placed into test tubes and stored alive at 5° C until they were examined. Air temperature was monitored during the collection. Midgut tissue was removed from each tick, suspended in a drop of saline, and examined by dark field microscopy (DFM) at 400×. Remains of the positive ticks and suspension from microscopic evaluation were transferred to a sterile Eppendorf vial and processed for PCR.

DNA isolation and PCR reaction

Procedures were followed according to Nejedlá et al. (2009). Borrelia-specific DNA samples were isolated from 50 homogenates using the DNA isolation kit QIAamp DNA Blood Mini Kit (Qiagen, Germany). DNA was eluted from QIAamp membrane in 40 μl of elution buffer. A volume of 4 μl of this prepared solution was used for amplification. The nested PCR assay was based on the specific flagellin sequence amplification (Picken et al. 1996) for detection of B. burgdorferi s.l. The 40 μl of PCR mixture contained 1× Hot- StarTaq Master Mix (Qiagen, Germany), 0.1 pmol of FL3 and FL5 primers, and 20 pmol of FL6 and FL7 primers, 100 μM of dUTP (Sigma, U.S.A.), 102 internal competitive standard (Genex CZ, Czech Republic), and 4 μl of template DNA received after standard DNA isolation. All PCR runs were performed on a thermocycler (PTC-200, MJ Research) with the following profile: an initial activation step at 96° C for 12 min, 30 cycles consisting of a denaturation step for 10 s at 96° C, an annealing step for 10 s at 68° C, an extension step for 40 s at 72° C, and an additional 45 cycles consisting of 10 s of denaturation at 96° C, 10 s of annealing at 54° C, and an extension step at 72° C for 30 s. The resulting products of amplification were separated on 2% agarose gel containing ethidium bromide (5 μg/1 ml) and visualized under UV illumination. If the sample contained DNA of B. burgdorferi s.l., a 276 bp long amplification product was visible. If the sample did not contain the DNA of B. burgdorferi s.l., only an amplification of the internal competitive standard (plasmid DNA with sequences corresponding to primers FL6 and FL7) product of 420 bp was detected, which served as a control for a successful run of each single reaction. No amplification product was detectable in the case of inhibition of PCR reaction. As a positive control for both PCR and DNA isolation, a reference strain provided by the microbiology laboratory was used.

Repellents tested

Five commercial products were randomly chosen for this study, all of which were based on DEET, while their additional active ingredients (permethrin, deltamethrin, pyrethroids) were present only in low percentages and were considered rather toxic than repellent.

Experiment 1: DEET 25 % + permethrin < 1 %; additives: buthyl acetate, petrol fraction, propane, butane (producer: Leroy, Czech Rep.)

Experiment 2: DEET 14 % + permethrin 0.2 %; additives: propane, butane, ethanol (Lybar, Czech Rep.)

Experiment 3: DEET 14 % + deltamethrin 0.01 %; additives: propane, butane, ethanol (Lybar, Czech Rep.)

Experiment 4: DEET 10 % + pyrethroids (extract from dalmatian chrysanthemum); additives: isobutane, propane, butane, ethanol (Aromatica, Czech Rep.)

Experiment 5: DEET 7 %; additives unknown (Hydra farmacosmetici, Poland).

Repellency testing

To examine the responses of the ticks towards repellents, the modified moving object bioassay was used (Dautel et al. 1999). This assay was chosen for practical as well as ethical reasons, as it offers advantages of host-associated stimuli and tick contact while avoiding live hosts, since field-collected ticks potentially transmit serious pathogens.

During our experiments, tested compounds were applied onto an exterior area (8×2 cm) of the heated drum (OD 9 cm, height 13 cm, temperature 36.5 ± 0.5º C, sprayed matt white, 24 turns min−1). As the drum rotated and the exterior site passed by, a tick could approach the drum on a flat iron stick (imitation of vegetation) and attach to the drum as if it was a passing host.

A total of 250 nymphs was processed according to the experimental procedure, representing the majority of the developmental stages detected within the monitored area and that visitors to the park are often attacked by. The ticks were first offered untreated filter paper only (control experiment). Ticks that successfully attached to the filter paper were considered active and host-seeking and were subsequently exposed to the experiments with repellents. Fifty active nymphs were tested per repellent.

The aim of this part of the study was to mimic a practical usage of the products. The dosage of repellents followed the instructions in the user manuals as if in a real situation and reflected the dispenser performance (heavy/light aerosol, rapid/slow action). The products were tested within a 0 – 10 min time interval after evaporation of the solvent.

In both control experiments and tests with repellents, each nymph was observed for a maximum of two min. The distance between the nymph and the experimenter during the test was at least 50 cm. The experiments were performed at room temperature and relative humidity, which varied according to the weather.

Statistical analyses

Data obtained from the individual experiments were added to a parametric and structured dataset. The software package STATISTICA 9, 10 (StatSoft CR) was used for statistical analyses. A χ2 test was used to evaluate results of the repellency testing and activity and infectivity of the individual stages of the ticks. Linear efficiency of the individual products according to the applied dose was evaluated by Spearman's correlation as well as dependency of the ticks’ activity on temperature. One–way ANOVA test was used for the study of activity and infectivity in the individual months during the year.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Tick collection

I. ricinus ticks were monitored at the same time for one h per week from March to November, 2011. The total number of ticks collected on 29 occasions was 598 individuals, out of which 50 were positive for the presence of spirochaetes (8.4%) by the DFM method. The DFM-positive samples were further investigated by PCR. The average number of one-h tick collecting was 23.1. Representation of the various developmental stages is random and significantly different statistically. Ticks were present each month of the year, from March to October, 2011. The number of collected individuals fluctuated and these changes were also related to temperature changes at the location (Figure 1). While May and June were both statistically different from the other months and critical for tick occurrence, two typical peaks in tick activity were recorded, the first in June and the second in August (Figure 2). The overall spirochaetal prevalence detected by the DFM method in the Pisárky location of Brno was 8.4% (50/598). Positive ticks appeared in the spring one month after the first collection, and a growing infectivity during the season was found (Figure 2). Highest numbers of positive ticks from all collected months were in August (13/98, 13.2%). Critical dates for individual collections of highest positive ticks were two days in July when the infectivity reached 17.9% (5/28) and 14.3% (3/21). If comparing infectivity to the individual months, then May, June, July, and August are statistically different from the others. No positive ticks were found in October and November (Figure 2).

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Figure 1. Tick activity and influence of temperature.

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Figure 2. Number of ticks and prevalence in each month in 2011.

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Temperature is recorded in Figure 1. According to statistical testing using Spearman's rank correlation at the significance level of 0.05%, we found a dependence of tick numbers on temperature (Figure 1). DFM-positive samples (50, 8.4 %) were identified by PCR analysis while 76% of these were confirmed to be B. burgdorferi s.l. (38, 6.4 %).

Repellency testing

Experiment 1: A formulation with 25% DEET and <1 % permethrin.

Out of the total of 50 tested nymphs, 49 (98%) were repelled.

Experiment 2: Experimental set (DEET 14% + permethrin 0.2%).

Out of the total of 50, 44 (88%) of the nymphs were repelled and did not attach to the repellent-treated area and 6 (12%) were not repelled.

Experiment 3: A formulation with DEET 14% + deltamethrin 0.01%.

Out of the total of 50, three (6%) of the nymphs attached to the drum and 47 (94%) were repelled.

Experiment 4: Mixture of DEET 10% + pyrethroids as active substances.

Out of the total 50, 39 (78%) of the ticks were repelled and 11 (22%) clung to the filter paper of the drum.

Experiment 5: The product with 7% DEET.

Out of the total 50, this product efficiently repelled 37 (74%) of the ticks, but 13 (26%) did attach.

Table 1 summarizes data on the amounts of the applied products and active ingredients. Our data indicate that all repellents showed significant repellency during the experiments compared to the control group (χ2 test, P<0.001; Figure 3). Statistical evaluation showed among the individual products are not significant (χ2 test, P>0.05). Furthermore, efficiency of the individual products according to the applied amounts of the active ingredient DEET did not show linear character (Spearman's correlation, rs=0.7, P=0.188); the repellent efficiency was not directly dependent on the amount of active ingredient DEET (Figure 4).

Table 1. Amounts and basic statistic parameters of the applied products/active ingredients. 1: DEET 25% + permethrin < 1%, 2: DEET 14% + permethrin 0.2%, 3: DEET 14% + deltamethrin 0.01 %, 4: DEET 10% + pyrethroids, 5: DEET 7%.
ProductControl(mg/cm2)12345
Mean amount of product03.455.313.725.813.56
Mean amount of DEET00.860.740.520.580.25
Median amount of product03.455.383.85.753.5
Min. concentration of product03.04.383.55.063.2
Max. concentration of product03.86.134.06.564.0
image

Figure 3. Repellency dependence on the amount of the active DEET ingredient.

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image

Figure 4. Repellency of the tested products dosed according to the individual instructions on the package as provided by the producers.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

We present the results of an annual observation of the activity of I. ricinus ticks and Borrelia infectivity in a park located in Pisárky, Brno, CR, during the year 2011. The selected number of collected nymphs was further tested against five chosen repellents while recording their individual efficiency.

In a previous study in 1996–2002, there were 162 B. burgdorferi s.l. positive samples from I. ricinus in this locality (Žákovská et al. 2008b). From these, a total of ten isolated strains (6.2%) were obtained, seven of which were identified as B. afzelii and three as B. garinii, using PCR-RFLP (Žákovská et al. 2008a). The presence of B. garinii suggests a role for rodents in the circulation of B. burgdorferi s.l. in this area, but no exact data confirm that. In the year 2011, a total of 598 I. ricinus ticks were collected in Pisárky between March and November, 2011. The statistical tests divided tick activity during the year into the following four statistically different groups: (1) March and September, (2) April, July, and August, (3) May and June, and (4) October. No ticks were found in November. In our long-term research performed in the same locality from 1996 to 2002, the annual activity could be divided into three statistically different periods. In the six-year study (1996–2002), the curve of seasonal abundance reached two main peaks, one in May and the other in August (Žákovská et al. 2007). Occurrence of ticks during our one-year study in 2011 also reached two peaks: one maximum in June (151) and the second peak in August (91). Our results were consistent with Černý et al. 1965, who described the maximum occurrence of ticks in May and June, with another less pronounced peak in autumn. These results correspond to the findings of other authors where two peaks of activity were also registered (Peťko et al. 1996, Siuda 1996).

Another part of our study examined the effect of climate on the abundance of I. ricinus. Dependence of tick occurrence on various climatic conditions had already been investigated and confirmed (Alekseev and Dubinina 2000, Materna et al. 2005). Ticks generally need a high humidity to compensate for any deficit in body water (Krober and Guerin 1999). During our collection intervals in Pisárky, the climate was relatively humid, while the mean value was 53.8%, and tick activity was directly proportional to the temperature. The mean temperature measured in this locality was 18.7° C. The temperature was measured on each day of collection, but several parameters could have been influenced by factors several days earlier. Weather fluctuation was reflected in the number of collected individuals, which ranged from one to 65, with a mean of 23.1). For example, 2011 was characteristic of a rapid onset of tick numbers in April (82/month) because of the hot days in early spring. The second consideration was a very dry autumn with low numbers of ticks in October. Danielova et al. 2006 stated that climate modifications, such as the higher temperature in spring and autumn, cause longer development of ticks and their possible shift to higher altitudes. Further records on weather dependency come from western Germany, where the presence of B. burgdorferi sensu lato in ticks (14%) has increased in the region of Siebengebirge over the last 15 years due to unknown changes, some of them perhaps related to climate changes (Kampen et al. 2004).

Out of our 598 samples, only the 50 DFM positive ones were examined by the simple PCR protocol described by Picken et al. 1996, which gave 38 positive results (6.4%). The DFM method revealed 50 samples positive for the presence of spirochaetes (8.4%). The DFM method is commonly used for the detection of spirochetes in ticks (Žákovská et al. 2008a, Aleksejev et al. 2001, Stünzner et al. 2006, Hubálek et al. 2002). A reason for finding fewer positive samples in PCR could be that our PCR sensitivity cannot detect a very low number of borreliae (from 0–10 per tick). Another reason is that some of the spirochaetes may not belong in the genus Borrelia. Similar results can be observed in other studies. For example, Aleksejev et al. 2001, reported that 64.8% of ticks that yielded a Borrelia PCR product out of 100% of DFM-positive ticks, while our experiment result was 76%. Differences in positive findings between the DFM and PCR methods are reported by Stünzner et al. 2006 and Stanczak et al. 1999 who tested 2,285 I. ricinus ticks by IFA and PCR. It is apparent that different detection techniques may produce divergent epidemiological data.

The average infectivity during a long-term study (1996–2002) in the same location was 5.8% (Žákovská et al. 2008b), which appears to be lower than the result of the 2011 study (8.4%). However, in the long-term study, the level of infection changed not only within one year but also over the years, starting at 2.1% (2000) and culminating at 13.2% in 1998, as also recorded by Petko et al. 1996 in their five-year observation in Forest Park in Košice (Slovakia). The study of Hubálek et al. 2002, carried out in an area of the South Moravia and Lower Austria, described much greater DFM infectivity (24.6%) in 1,517 individual ticks. One of the reasons for the higher number of positive ticks compared to our results could be better reservoir competence of the locality in the lower Thaya ecosystem. The prevalence of a Borrelia infection in ticks is one of the most essential components of the Lyme borreliosis risk assessment. To have meaningful information on when to avoid walking in parks or when to be most wary of ticks is one way of preventing tick-borne infections. Critical months for the occurrence of ticks in 2011 were May and June, but the month of August showed the highest risk of spirochaetal infection, suggesting that the highest risk of transmission of the tick-borne borreliae in Pisárky locality is in May, June, and August. Similar conclusions were also reported by Piesman et al. (1990).

Many in vivo and in vitro assays (Dremova and Smirnova 1970, Ndungu et al. 1999, Jaenson et al. 2006) have been developed to examine the response of ticks to potential repellents. Experiments in our study were carried out with the modified moving-object-bioassay (Dautel et al. 1999), which is a test system that provides the advantages of host-associated stimuli and is cheap, timesaving, and avoids ethical problems and animal suffering. However, it is possible for the results to be influenced by an investigator exhaling the carbon dioxide within the closed room.

Only the short-time repellency of nymphs was examined in this study. We focused on this developmental stage because it is predominant in the monitored area and therefore of high epidemiological importance. All tested products showed significant repellency against the field-collected nymphs, but the hypothesis about linear efficiency of the individual products according to the applied amounts of DEET had to be rejected at the 5% significance level. Such outcomes could be a result of many factors, such as air temperature, humidity and pressure, physiological/pathological state, individual history and age of the ticks and infection by pathogens, and can be also expected with real usage.

It is essential to note that the field-collected ticks were strongly repelled by all tested products, because as far as the authors know, many studies have been carried out on laboratory-reared, pathogen-free ticks, often on different tick species (Soares et al. 2010, Zhang et al. 2009). However, such results can be influenced by the “laboratory history” of the ticks (light-dark regime, feeding pattern, unnatural environment). We tested ticks that attacked a live host, as in real situations, collected shortly before the test directly from their natural habitat. Experiments with host-seeking ticks without previous influence of laboratory breeding, carried out on the apparatus providing host-associated stimuli (warmth, movement), can significantly contribute to an investigation of tick-host behavior and safe repellent testing. Another contribution of this study is that it analyzes the efficiency of currently available modern products.

All tested products showed significant repellency and protection. The number of Lyme borreliosis patients in the Czech Republic in 2011 (4,835) is the highest detected in the history of monitoring these patients in the CR, and even within a relatively small area such as Pisárky, up to 65 individual ticks can be caught during 1 h of flagging. To defend ourselves against both the high infectivity and invasion of ticks under their optimal environmental conditions, we must have knowledge and awareness of the critical times and make the application of appropriate insect repellents an essential part of our visits to these environments.

Acknowledgments

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

We thank Dr. Martin Vácha for helpful comments concerning the design of the bioassay and technical support. This study was supported by grant MUNI/C 0776/012. Authors declare that they have no conflict of interest regarding this article.

REFERENCES CITED

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED
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