Ph.D. Thesis

Researches regarding the

interaction of tick (Ixodidae) –

Borrelia – receptive host;

genetic diversity of some

species from the genus Borrelia (SUMMARY OF Ph.D. THESIS)

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ABSTRACT

In the last decade, the vector-borne diseases transmitted by ticks have become more important. The global warming, the urbanization of the natural habitat of the ticks, the population growth of some vertebrate host species in the arthropod’s habitat areas, affects the biology of vectors and the transmission of pathogens, thus the annual incidence of vectore-borne disease is increasing (GRAY, 2009). The incidence of vector-borne diseases is increased in geographical areas with distributed pathogen vectors, infections being frequent in the main activity period of the tick vectors.

The spirochaetes of the genus Borrelia are the most widespread bacterial pathogens transmitted by ticks. The species of the genus Borrelia represents different pathogenicity in the vertebrate hosts and is preserved within a cyclic system which implies the presence of some tick species (PARTE, 2014). Phylogenetically, the Borrelia species, are divided in three groups: the causative agents of Lyme borreliosis (LB group), the causative agents of relapsing fever (RF group), respectively reptiles associated Borrelia species (REP group) whose pathogenicity in humans is unknown at the moment.

Lyme borreliosis is the most widespread vector-borne disease transmitted by ticks in Europe, North America and Asia, caused by the spirochaetes from the Borrelia burgdorferi sensu lato complex (MASUZAWA, 2004). Due to the multisystemic affection in humans, is also called the „illness with 1000 faces”, affecting the skin, the joints, the nervous system, the heart (CIUTĂ, 2012). Humans are considered terminal hosts because they cannot further transmit the pathogens, closing the infection cycle. At the moment, there are 20 LB causative agents with different geographic distribution. The etiologic agent of the zoonosis circulates in ticks, in the wild as well in domestic vertebrate hosts, affecting some animal species and humans.

In Europe, the most frequently reported species, B. miyamotoi, the causative agent of RF, it seems to be present in different tick species, rodents, micromammals, birds and human (WAGEMAKERS, 2015). Phylogenetically, B. miyamotoi, belongs to the RF group, but can produce similar symptoms with the causative agents of LB and RF. Studies show that this species can be found in each developmental stage of the ticks, in larvae, nymphs and adults, demonstrating the transovarial transmission of the bacteria (SCOLES, 2001).

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The REP group includes species isolated from ticks or from ticks parasitizing these vertebrates, and includes the bacteria, Borrelia turcica. The ecology and pathogenicity of the bacteria is incomplete elucidated. Phylogenetic data suggest that this bacteria clusters with the species from LB and RF group.

In the most of the cases the reservoir hosts are common animal species from the spontaneous fauna, maintaining and transmitting the pathogens to vectors, or via successive chain of vectors which in turn transmits the disease to the definitive host (ANDERSON, 1991). The reservoir hosts are species that satisfy some specific conditions, such as the capacity to keep the pathogen alive for long periods of time, the capacity to transmit the pathogen to a vector and the capacity to be a common host for this vector (GERN, 1998).

The role of the micromammals is considered to have a great epidemiologic importance, also micromammals are markers for evaluating the distribution of some tick species and due to their high occurrence rate in the forested habitats, they are an important group of reservoir hosts (HANINCOVÁ, 2003).

Due to the extensive media coverage in recent years regarding the infection risk with Borrelia spp. transmitted by tick bites, the amount of epidemiologic data in Romania increases annually. To date, the epdiemiologic data in Romania is limited (BRICIU, 2011; 2014), but are some serological studies on dogs, horses, (KISS, 2011), and molecular studies on hedgehogs (DUMITRACHE, 2012), mustelids (GHERMAN, 2012) and on ticks collected from vegetation (COIPAN, 2010; 2011; KALMÁR, 2013).

In this context, the present study has the following general objectives:

Determination the molecular prevalence and the diversity of some species from the genus Borrelia in questing Ixodes ricinus ticks from Romania and analysis of the spatial distribution of species involved in the infection;

Provision of epidemiological data regarding the presence of Borrelia spp. in the tissues of some animal species and in the body of the ticks parasitizing these animals.

Evaluation the possibility of transmission and determination of the molecular prevalence of Borrelia turcica in Hyalomma aegyptium ticks parasitizing tortoise species, Testudo graeca, from Romania.

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Characterization of the molecular structure and determination of the DNA conformation via Fourier transformed infrared spectroscopy (FT-IR) of the Borrelia turcica spirochaetes.

The present thesis contains 163 pages and is structured in two parts: the first part presents the current state of knowledge, divided into 5 chapters, and the second part, the personal contributions organized in 10 chapters.

The first part is structured into 5 chapters and summarizes information from the literature regarding the vectorial role of Ixodidae ticks, the principal vector-borne diseases transmitted by ticks, LB and RF, the biology of the Borrelia spp. and the competent reservoir hosts, as some data on the principal detection and identification methods of Borrelia spp.

Chapter 1 presents data on the biologic cycle of Ixodidae ticks and their role/vectorial competence. Chapter 2 details the clinical manifestations of the two vector-borne diseases caused by etiologic agents, LB and RF, diseases transmitted by ticks. Chapter 3 describes the general characteristics of the Borrelia spp. pathogen, detailing the morphological structure of this bacterium. Chapter 4 presents the reservoir hosts for Borrelia spp., also approaching the topic of interactions between tick – Borrelia – vertebrate hosts. The last chapter of the first part, Chapter 5, is devoted to the detection and identification methods of Borrelia spp. Within there are presented the principal molecular and spectroscopic detection and identification methods in the tick body and in the vertebrate hosts tissues.

The second part of the study is structured into 10 chapters, including the working hypothesis/objectives (Chapter 6) and the general methodologies (Chapter 7), followed by the own researches (Chapters 8 - 13), general conclusions and recommendations (Chapter 14), respectively the originality and the innovative contributions of the thesis (Chapter 15). The thesis ends with the references (397 titles).

The aim of Chapter 8 was to identify the genospecies of the B. burgdorferi s.l. complex, as well the analysis of the spatial distribution of the species implicated in the infection of the questing I. ricinus ticks from Romania.

The information regarding the infection of Borrelia spp. in questing and in engorged ticks attached to humans, are still limited in Romania. The first study, at national level, on the infection of questing I. ricinus ticks, was conducted by our team within a research project, IDEI-PCCE CNCSIS 84, 7/2010 (DUMITRACHE, 2012; KALMÁR, 2013). For identification of the B. burgdorferi s.l. genospecies involved in the infection, two identification

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methods were applied, including: RFLP and sequencing. Thus, from 172 B. burgdorferi s.l. infected I. ricinus ticks, 141 (82%, 95% CI: 75.4 - 87.4) had monospecific infections The most frequently detected species were B. afzelii (126/12.221; 1.03% or 73.25% from total infected ticks), followed by B. garinii (65/12.221; 0.53% or 37.79% from total infected ticks) and B. burgdorferi s.s. (12/12.221; 0.10% or 6.98% from total infected ticks). Co-infections were detected in 31 ticks (18%) in 14 localitis: B. afzelii + B. garinii, B. garinii + B. burgdorferi s.s. and one tick infected with B. afzelii + B. burgdorferi s.s. From the total of 65 infected I. ricinus with B. afzelii, 30 (46.2%, 95% CI: 33.7 - 59.0) were co-infections. The most widespread genospecies in Romania is B. afzelii, this species was detected in 46 locations, followed by B. garinii in 23 locations and B. burgdorferi s.s. in 12 locations.

The overall prevalence of infections with different Borrelia species identified in other European countries is coparabale with our data obtained in this study. The dominant genospecies in Romania are B. afzelii and B. garinii, B. afzelii seems to be a sympatric genospecies with B. garinii, founded excusively in hilly regions, and with B. burgdorferi s.s. with a limited distribution. Although, European studies emphasize that B. afzelii is the most common genospecies in Europe and in particularly in Romania too (COPIAN, 2011). In the present study, was evaluated for the first time, on national levels, the genetic diversity of different B. burgdorferi s.l. genospecies in I. ricinus ticks collected from vegetation, and was analyzed the spatial distribution of the identified species.

The aim of Chapter 9 was to determine the prevalence of infection with B. miyamotoi in questing I. ricinus ticks from Transylvania and the analysis of the spatial distribution of this species. From phylogenetically point of view B. miyamotoi belongs to the RF group, but produces the same symptoms and clinical manifestations as the species from both, RF and LB groups (KRAUSE, 2015). Borrelia miyamotoi is distributed in Europe, America, Russia, Japan (FONVILLE, 2014). Although the existence of this species was known since 1995, it received little attention until the first reports of human cases. However, there is no data on the infection with B. miyamotoi in questing I. ricinus ticks in Romania. To obtain an indication of the presence of B. miyamotoi, potentially pathogenic bacteria, in questing I. ricinus ticks, ticks were randomly selected and analyzed from fourteen locations of Transylvania. Thus, 468 questing I. ricinus ticks were analyzed for B. miyamotoi infection. The pathogen detection was performed by quantitative PCR targeting the flab gene (FONVILLE, 2014). From the total of 468 (males 26.92% females 63.25%, nymphs 9.83%) questing I. ricinus

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ticks included in this study, 7 ticks were infected with B. miyamotoi, thus, the overall prevalence was 1.5%. The infections were detected in 5 locations, and the local prevalence, in the tick infected localities, varies between 1.61% - 8.33%.

The infection prevalence with B. miyamotoi determined in other European countries are similar to the results obtained in this study. The geographic distribution of B. miyamotoi is limited, but it’s assumed that the distribution of this species is similar with the distribution of LB species (KRAUSE, 2015). The results suggest a mosaic spreading of B. miyamotoi in questing I. ricinus in the investigated region, being the first report in Romania.

The aim of Chapter 10 was to provide epidemiologic data regarding the presence of Borrelia spp. in micromammalian tissues originating from Romania and in ticks parasitizing this animal species. Micromammals are competent reservoir hosts for ticks and transmit pathogens to vectors which are transmitted further to the definitive host (BOWMANN, 2008). Micromammals, especially different rodent species are competent reservoirs for ticks (MIHALCA, 2012a; 2013). These hosts are considered markers for evaluation of the distribution of some tick species (MIHALCA, 2012b). Due to the fact that micromammals are competent hosts for different tick species (MIHALCA, 2012b), and due to the high parasitic level of these species, micromammals play an important role in vector-borne diseases transmitted by ticks.

In this context, 401 micromammals (11 species) were collected from 7 counties from Romania: 9 rodent species – mice (Apodemus agrarius, A. flavicollis, A. sylvaticus, A. uralensis, Micromys minutus, Microtus agrestis, M. arvalis, M. subterraneus, Myodes glareolus) and 2 shrews (Crocidura leucodon, C. suaveolens). From 393 animals (98%) heart and liver samples were collected, from 2 animals (0.50%) only the heart and from 6 (1.5%) only the liver. Regarding the infection with Borrelia spp. in I. ricinus ticks (n=301) attached to micromammals, none of the ticks were infected with these spirochaetes.

The overall prevalence of infection with Borrelia spp. in micromammalian tissues was 4.99%. The highest prevalence was registred in C. suaveolens (7/49; 1.45%), followed by A. agrarius (3/87; 0.75%) and M. arvalis (3/53; 0.75%), A. flavicollis (2/49; 0.50%) and M. glareolus (2/32; 0.50%), M. agrestis (1/2; 0.5%), M. minutus (1/11; 0.50%), C. leucodon (1/21; 0.25%). The infected tissues were the heart (13/395; 3.29%) as well the liver (11/399; 2.76%). The most frequently detected species from the total infected was B. afzelii (14/20; 70%), followed by B.

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garinii, B. burgdorferi s.s. and B. miyamotoi with a prevalence of 10% (2/20) in case of each species. The correlation of genospecies with the hosts, showed that B. afzelii was detected in case of 8 micromammal species (A. agrarius, A. flavicollis, C. leucodon, C. suaveolens M. minutus, M. agrestis, M. arvalis, M. glareolus), detected in ones in case of each listed species. Borrelia garinii, B. burgdorferi s.s. and B. miyamotoi were detected in case of two animal species with the following associations: B. garinii - A. agrarius, B. garinii - M. arvalis respectively B. burgdorferi s.s. - A. agrarius şi B. burgdorferi s.s. - M. arvalis. In case of Borrelia miyamotoi - A. flavicollis and M. glareolus associations, the infected tissue was the liver.

The present study is the first which evaluates the prevalence of infection with Borrelia spp. in the tissues collected from rodents and insectivores from Romania, in correlation with the ticks parasitizing these animals. At the same time, is the first study which reports the presence of Borrelia spp. in tissues of insectivores, C. leucodon, and respectively C. suaveolens. The reservoir role of the two insectivore species has not been evaluated until now.

The aim of Chapter 11 was to evaluate the transmission of infection with B. turcica in different developmental stages of H. aegyptium ticks and in T. graeca tortoises. The importance and the epidemiologic role of tortoises (reptiles) have increased, mainly due to the international pet trade of the animals originated from the wild (BURRIDGE, 2011). Various emerging and/or zoonotic pathogens have been isolated and characterized from reptiles or from their associated ticks parasitizing these vertebrates (PAŞTIU, 2012). Reptiles, imported from the wild are harboring various tick species that facilitates the introduction of non-native pathogens, thus poising a significant public health risk (BURRIDGE, 2000; 2011; 2003).

The study was conducted in July of 2013. In total 28 engorged ticks were collected after complete detachment from 6 tortoises (T. graeca). All the nymphs were maintained in laboratory conditions till their molting in adults. For obtaining the spirochaetes culture, the intestines and salivary glands were obtained from each unfed (molted nymphs) and from engorged adults. The presence of spirochaetes was detected by DFM/MCF and PCR using specific primers, designed in this study for detection of B. turcica targeting gyrB and glpQ genes (Figure 1).

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Fig. 1. The developmental stages of the H. aegyptium ticks, their infection with B.

turcica obtained after molting and culture, respectively after PCR analysis (KALMÁR, 2015a).

The present study is the first one which evaluates the possibilities of transmitting the infection with B. turcica in H. aegyptium ticks collected from T. graeca from Romania. The high prevalence of H. aegyptium on T. graeca (PAŞTIU, 2012), and the results obtained in this study, indicate the possibility of preserving of B. turcica in nature within a cyclic system. Thus, we suppose, the natural vertebrate hosts for B. turcica could be the micromammals (e.g. rodents, hedgehogs) which are preferred by the immature stages (larvae, nymphs) of the ticks. At the same time, the negative blood samples can be associated with low bacteremia. Considering that, the blood samples of tortoises present no infections with B. turcica, suggests that, the infection of nymphs might have occurred during a previous blood meal on another host; via transovarial transmission; or by co-feeding mechanism of ticks on another host. The obtained results confirm, for the first time, the transstadial transmission of B. turcica pathogen in H. aegyptium ticks. This transmission highlights the importance of supervision of tortoises regarding the vector-borne diseases.

The aim of Chapter 12, was to determine by PCR the prevalence of infections with B. turcica in H. aegyptium ticks engorged to Dobrogean tortoises, T. graeca from Romania. Thus, DNA extracted previously from H. aegyptium ticks attached to T. graeca, tortoises and captured in Tulcea and

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Constanța counties were used (PAŞTIU, 2012). From the total of 448 ticks, the overall prevalence of infection with B. turcica was 17.41%.

The epidemiologic studies regarding the infection of H. ageyptium ticks with B. turcica are limited. Borrelia turcica was studied for the first time by GÜNER et al. in 2003. These spirochaetes were isolated from H. aegyptium ticks attached to T. graeca originated from Turkey. The results of infection prevalence with B. turcica obtained in the present study, are comparable with the prevalence rate obtained by GÜNER et al. (2003). The infection risks are influenced by a series of factors. One of these factors is the climate change which influences the ecology and the biology of the vectors, of the pathogen and of the host. In epidemiologic studies, the population densities influence the overall prevalence of infection with a given species.

The aim of Chapter 13 was to characterize the molecular structure of B. turcica bacteria by Fourier transform infrared spectroscopy (FT-IR), a method which allows the identification of the molecular structure of a given cell of an organism, and the identification of some cellular components, different proteins, lipids and carbohydrates, providing at the same time information about the conformation of DNA and RNA (OUST, 2004). Due to the difference of the molecular structure of microorganisms, this technique allows the identification of different bacteria species on the basis of emitted wavelengths (D’SOUZA, 2009).

The FT-IR analysis was carried out on B. turcica culture and on its isolated DNA. The scarped culture and DNA from the surface of discs were homogenized with potassium bromide (KBr). The spectral domains in case of the culture were included between ~ 500 –3500 cm-1, and ~ 700 – 1700 cm-1 in case of the DNA isolated from B. turcica. The registered FT-IR spectra in case of B. turcica culture includes 21 absorption bands. The registered bands in this spectral region are characteristic to the functional groups from the structure of proteins, nucleic acids, phospholipids and carbohydrates. Regarding the FT-IR spectra in case of the isolated DNA from B. turcica, 27 peaks were registered, which are characteristic to the molecular structure of DNA. The registered spectral differences of the pathogenic species, B. afzelii, B. garinii, B. burgdorferi s.s., comparatively with B. turcica, investigated in the present study, may be caused by the differences in the cell composition of the spirochates. The proteins of the B. turcica bacteria struct, determined by FT-IR, have α-helix conformations, and the DNA has B shape

The study has been supported by the project IDEI-PCCE CNCSIS 84, 7/2010 “Fundamental and applied studies of eco-epidemiology, biology

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and molecular genetics of the Lyme disease vector”, EurNegVec COST TD1303, in collaboration with National Institute of Public Health and Envrionment, Ministry of Health, Welfare and Sport, and by the co-financed project POSDRU 159/1.5/S/136893.

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