BORELLIA BURGDORFERI INFECTION IN REMOVED TICKS AND ANTI-BORRELIA ANTIBODIES IN INFESTED PATIENTS ADMITTED TO THE PASTEUR INSTITUTE, NOVI SAD

Introduction . The primary objectives of this study were (1) to determine the presence of Borrelia burgdorferi infection in ticks removed from patients for the purpose of singling out sites with increased risk of Lyme borreliosis, and (2) to determine the presence of IgM and/or IgG antibodies against B. burgdorferi sensu lato (s. l.) complex in sera of patients who had ticks removed. Materials and Methods . From 108 ticks removed from patients, all were examined zoologically and a sub-sample of 91 ticks was tested using PCR analysis to determine the presence of DNA indicating B. burgdorferi infection. To detect anti- Borrelia IgM and/ or IgG antibodies in 61 patients bitten by ticks, we used line recombinant immunoblot test. Results and Conclusions. The most common tick identified was Ixodes ricinus . B. burgdorferi s. l. was present in 37 of 91 tested ticks (40.7%). Seroconversion against B. burgdorferi s. l. antigen was detected in 12 of 61 patients (19.7%). Most of the infected ticks were from the province of Vojvodina (11 municipalities), with the city of Novi Sad proving to be the site with the highest number of infected ticks, 6 in total.


INTRODUCTION
Lyme borreliosis is a multisystemic tick-borne disease caused by bacteria from the Borrelia burgdorferi sensu lato complex (B. burgdorferi s. l.). In Europe, the dominant role in transmitting and maintaining the causative agent of Lyme borreliosis belongs to the hard ticks from the genus Ixodes, primarily Ixodes ricinus (Jensen et al., 2000). The life cycle of this tick comprises three stages: larva, nymph and adult. According to available data in Serbia, a third of the I. ricinus population in the country is infected with B. burgdorferi (Potkonjak, 2018).
The B. burgdorferi s. l. complex contains around 18 species and two genomospecies. The total number of species is subject to change, as new isolates are continually being described on a genetic and antigenic basis. The species with the highest pathogenic potential for humans are B. afzelli, B. burgdorferi sensu stricto (s. s.), B. garinii and B. spielmanni. The species B. bissetti, B. valaisiana and B. lusitaniae are considered only mildly pathogenic for humans (Rudenko et al., 2011).
In Europe, the primary reservoirs of the B. burgdorferi s. l. complex are various species of mammals (hedgehogs, mice, voles, squirrels, dormice and rabbits), birds (blackbirds, pheasants, auks and snipes) and certain reptiles. Once infected via tick bite, small rodents become a permanent source of infection for other ticks, particularly larvae and nymphs (Mannelli et al., 2012).
Lyme borreliosis can be confirmed through direct and indirect methods. Directly, the presence of Borrelia can be demonstrated by cultivation of the spirochetes on specific growth media, using antigen detection via direct immunofluorescence on the intestinal contents of ticks (Ružić-Sabljić and Cerar, 2017). Polymerase chain reaction (PCR) can be used to detect the spirochete DNA in the ticks themselves, as well as in clinical material. Indirect methods for confirming Borrelia infection include PCR and serological tests for Borrelia-specific antibodies in the serum, cerebrospinal or synovial fluid. IgM and IgG antibodies can be detected via indirect immunofluorescent test (IFA), enzymic immunological tests (ELISA) or immunoblot tests (WB). The primary objectives of this study were to: (1) find potential geographical localities with increased risk for Lyme borreliosis by determining the presence of B. burgdorferi infection in ticks removed from patients, and; (2) determine the presence of IgM and/ or IgG antibodies against B. burgdorferi s. l. complex in sera of patients who had ticks removed.

Tick collection
A total of 108 individual ticks were removed and collected from the skin of patients who were infested with ticks while engaged in outdoor activities from March to October 2018. Ticks were individually stored in sterile plastic tubes and labeled until species determination. Information on date and location of attachment were collected.

Sera collection
In order to detect and identify B. burgdorferi s. l. complex antibodies via the line recombinant immunoblot assay (Mikrogen Diagnostik, Germany), a total of 61 blood samples were collected over the course of 2018 from patients from which a tick was removed and analyzed initially. The blood was sampled at least one month after the tick bite.

Species identification and developmental stage determination of ticks
Species and development stage identification of ticks was conducted in the Parasitology Laboratory of the Pasteur Institute immediately after tick collection. The ticks were examined under a stereomicroscope (ST-30-2LR Bino Stereo, magnification 20-40×). Identification was based on morphological characteristics, according to the key by Estrada Pena et al. (2004). After identification, ticks were individually placed in Eppendorf tubes with 70% ethanol until further analysis.

DNA extraction from tick samples
Ticks for molecular analysis were randomly chosen from the pool of 108 ticks to make sample size n=91. DNA was extracted from individual ticks via alkaline lysis with 50 mM NaOH. According to the protocol, prior to extracting the tick from the Eppendorf tube, the 70% ethanol was removed with a pipette and the tick was rinsed twice in phosphate-buffered saline (PBS), pH 7.4. The PBS was then removed and 50 mM of NaOH was added in a volume equal to the volume of the tick. The tick was macerated, vortexed and suspended via centrifuge at high rotation, and then heated in the NaOH in a thermoblock at 95°C. The DNA in the heat-treated sample was neutralized with 1M Tris-HCl (pH 7.0), vortexed and centrifuged. The extracted DNA was transferred to a new Eppendorf tube and its concentration was measured via fluorimeter, after which it was stored at -70°C until further use. The presence of B. burgdorferi s. l. DNA was confirmed for individual tick DNA samples. For PCR amplification of the 16S rRNA gene, a commercial kit (Norgen Biotek, Canada) for B. burgdorferi s. l. detection was used. Amplification of the selected 277 bp sequence was carried out in a Techne TC-PLUS Thermal Cycler in 40 cycles consisting of the following: denaturation at 94°C (15 seconds), hybridization at 60°C (30 seconds) and elongation at 72°C (45 seconds).

Line recombinant immunoblot assay (LRIT)
To determine the presence of B. burgdorferi s. l. complex IgM and/or IgG antibodies in human sera, commercial line recombinant immunoblot kits (recomLine Borrelia, Mikrogen Diagnostik, Germany) were used. These highly specific and sensitive tests use recombinant proteins as antigens in order to detect five species of the B. burgdorferi

Interpretation of LRIT
The LRIT result was obtained by allotting points to individual bands, as per the manufacturer's recommendations. The number of points remains the same regardless of reaction intensity (noted as +, ++ or +++). The number of points for OspC and p18 bands is calculated only once, even when more than one or all OspC or p18 bands react. Finally, based on the total points, a positive (>7 points), borderline (6 points) or negative (≤5 points) result is given. The evaluation of total points for specific antigens in the recomLine Borrelia test for reliable and fast result estimation is shown in Table 1.

Statistical analysis
To ascertain if there were statistically significant differences in representation of specific tick developmental stages, the Chi-square test was performed in Microsoft Excel 2013 v.15.0 included in Office 2013 package. The threshold for statistical significance in the differences observed was 0.05.

Tick species and developmental stage identification
All the examined ticks (n=108) belonged to the family Ixodidae, with the species I. ricinus accounting for 102 ticks. The remaining ticks belonged to three other species: Rhipicephalus sanguineus (3 individuals), Dermacentor marginatus (2) and Haemaphysalis punctata (1). Of the I. ricinus ticks, 51 were nymphs, 36 were adult females and 15 were larvae. Two adult females and one nymph were found in the R. sanguineus sample. Both D. marginatus individuals were nymphs, and the single H. punctata was an adult female.
The highest number of ticks originated from the Novi Sad (54) and Irig (11) municipalities. A smaller number of ticks were collected from Beočin (7) and Sremski Karlovci (5), and the remaining 31 ticks originated from other localities in Serbia and four other countries (Croatia, Austria, Germany and Bosnia and Herzegovina) ( Figure  1). The ticks from the Novi Sad municipality consisted of 7 larvae, 27 nymphs and 20 adult females, with the Chi-square test demonstrating a statistically significant difference in the representation of specific developmental stages (p=0.003) ( Figure 2).   Figure 3). With the exception of a single D. marginatus, DNA examination revealed all of the infected ticks were I. ricinus. Ticks infected with Borrelia originated from 12 municipalities in Serbia and one municipality in Bosnia and Herzegovina, and they belonged to different developmental stages. Of the 37 infected ticks, the majority were nymphs (20, or 54.1%). Twelve adult females (32.4%) and five larvae (13.5%) were also infected with Borrelia. The majority of Borrelia-positive ticks from Serbia originated from the northern part of the country, the autonomous province of Vojvodina (11 municipalities), and one municipality in western Serbia (Ljubovija) (Figure 3). In Vojvodina, Borrelia infections were particularly common in ticks from different sites in the Srem (Syrmia) region, especially around Mt. Fruška Gora (Bukovac, Popovica, Stražilovo, Banstol, Rakovac-Beočin, Ledinci, Irig-Vrdnik-Iriški venac, Ruma). In the Bačka region, Borrelia-positive O n L i n e F i r s t ticks were found in four municipalities (Novi Sad (19), Temerin (1), Vrbas (2) and Bački Petrovac (1)), with the urban center of Novi Sad containing the highest number of infected ticks, a total of 6. With regards to the developmental stage of the ticks, most of the patients were exposed to nymph bites (54). More nymphs than other developmental stages were collected from patients infected in the Novi Sad area. An explanation can be found in the overlap between the maximum activity of the nymphs, which are the most aggressive stage in the tick life cycle, and extended periods of human outdoor activity in springtime. Due to their small size, larvae and nymphs are difficult to spot. They feed for 2-4 and 4-6 days respectively, which leads to increased risk of Borrelia transmission, and then detach from the skin of their host (Banović et al., 2019b). The three Metastriata species are far less frequently found in Serbia than I. ricinus. The assemblage of ticks noted in the current study is typical of forest ecosystems of Mt. Fruška Gora, from where most of the studied ticks originated. These habitats possess diverse plant and animal communities that are under low anthropogenic pressure, providing favorable conditions for the maintenance of tick life cycles. However, some of the patients observed/treated for tick bites were from urban areas with high anthropogenic pressure. In habitats such as these, ticks can survive in favorable areas with adequate microclimatic conditions, such as parks, open grassy areas and other plant surfaces that are not regularly treated against ticks.  (Savić et al., 2010) and 21.13% (Potkonjak et al., 2014). In the Belgrade area and its adjacent municipalities, reported values of Borrelia prevalence in ticks range from 21.9% to 31.7% (Cakanac et al., 1993;Milutinovic et al., 2006). On the other hand, a different study  found that over 40% of I. ricinus ticks were infected with these bacteria, similar to the results of the current study. According to prior studies (Milutinovic et al., 2008;Radulović et al., 2010;Tomanović et al., 2010) (Banović et al., 2019a).
When interpreting the results of the current study, physical (climatic) and biotic factors of the study area must be considered. These include amount of seasonal rainfall, temperature and light regime and especially relative humidity, as well as use of chemical treatments and other anthropogenic influences, including maintenance of greenery in urban areas. All these factors can interact to affect tick abundance and pathogen prevalence. According to data from the Republic Hydrometeorological Institute of Serbia (Republic Hydrometeorological Service of Serbia, 2019), 2018 was the warmest year since meteorological records began, with the highest minimal spring and summer temperatures and moderate rain in Serbia as a whole. Higher quantities of rainfall were noted in west, southeast and central parts of Serbia. May and June experienced higher than average rainfall, and the rainiest month was July, which is not typical of rainfall patterns in Serbia, an area of temperate continental climate (Republic Hydrometeorological Service of Serbia, 2019). Such climatic conditions exerted a positive influence on the abundance and activity of nymphs and adult ticks during spring and summer of 2018. Consequently, more people than usual were exposed to tick bites in this period, as is consistent with the results of our research.
Numerous studies confirmed the presence of B. burgdorferi in other European countries and determined factors that influence the varying prevalence of this spirochete in ticks. These include the availability of adequate tick reservoirs, characteristics of the dynamics of the enzootic cycles, habitat type, elevation, geographic distribution of O n L i n e F i r s t principal vectors, and most importantly, geographic distribution of Borrelia species. According to Stünzner et al. (2006), tick infection prevalence is reduced with an increase in elevation. In the current study, most infected ticks originated from Mt. Fruška Gora and the Novi Sad municipal area, which are less than 539 m above sea level (this being the elevation of the highest peak on Fruška Gora, Crveni Čot). Golubić and Zember (2001) noted Borrelia prevalence (45%) similar to ours (41.9%) in Croatia, where geographic and climatic characteristics are similar to the Srem region in Vojvodina.
Nymphs were the developmental stage most commonly infected with Borrelia in the current study (54.1% of infected ticks). Nymphs are considered responsible for infecting humans with the causative agent of Lyme borreliosis in the majority of cases (Steere et al., 2016), as demonstrated in the current study. The higher risk of contracting the disease from nymphs than from other stages results from a combination of two factors: a higher Borrelia prevalence in nymphs than in larvae, and the nymphs spending more time feeding on the host (along with smaller size and lesser visibility) than the adults. However, in a similar study of ticks collected from vegetation in the Czech Republic (Hubálek et al., 2003), the authors noted a higher prevalence of Borrelia in male I. ricinus (26.1%) than in females (24.9%) or nymphs (16.8%), which contrasts with the results of the current study. A second group of Czech authors arrived at a similar conclusion after seven years of monitoring Borrelia prevalence in I. ricinus: prevalence was highest in males (10.3%), with lower prevalences in females (6.9%), nymphs (5.6%) and larvae (1.6%) (Žákovská et al., 2008).
Factors influencing infection risk in humans include the presence and abundance of ticks in different types of habitat, Borrelia prevalence in ticks, and extended periods of possible exposure to ticks. The line recombinant immunoblot assay, performed one month after exposure to tick bites, demonstrated that sera sampled during the early infection stage predominantly contained IgM antibodies, as opposed to sera sampled in the late infection stage where IgG is dominant. The early, or IgM stage of the disease, is characterized by reactions with OspC; in the late IgG stage, strong reactions occur with recombinant proteins (p100, VlsE, p58, p39 and p18). VlsE is an early marker of IgG response, but it is also found in late stages of Lyme borreliosis, along with p100 and/or p18 (Bušová et al., 2018) A study in Sweden and parts of Finland reported that 28% of ticks removed from patients were infected with Borrelia, and that 3.5% of patients achieved seroconversion (Wilhelmsson et al., 2016). Here, we report a higher prevalence of tick infection with Borrelia (40.6%) as well as more frequent seroconversion in our patients (19.7%) compared to reports from Sweden, Åland Islands and Neuchatel. Possible reasons for higher seroconversion in the current study should be further investigated and may lie in the patient demographic characteristics and in the duration of tick feeding on humans, since in Neuchatel, ticks were removed relatively quickly, within 36 h of attachment.

CONCLUSION
One hundred and eight specimens of ticks removed from the skin of patients at the Pasteur Institute in Novi Sad were identified as belonging to four species: I. ricinus (n=102), R. sanguineus (n=3), D. marginatus (n=2) and H. punctata (n=1).
The nymphs were the most common developmental stage found on patients in the Novi Sad area.
In the total sample of infected ticks, the largest number belonged to the nymph stage (20 ticks, or 54.1%).
The presence of IgM or IgG antibodies against B. burgdorferi s. l. was recorded in the sera of 12 of the 61 patients tested (19.7%).
Data from this study indicate the need for further research, which should be extended to other regions of Serbia. A wider population sample should be subjected to seroconversion studies, paying particular attention to individuals whose profession entails a risk of tick bites, due to prolonged time in the ticks' natural habitat. Constant monitoring of the principal reservoir and most important vector of Borrelia, the tick I. ricinus, as well as monitoring the factors that enable the completion of its natural life cycle, are significant measures in the prevention of Lyme borreliosis.
O n L i n e F i r s t