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Background: Borrelia lusitaniae is a species within the complex Borrelia burgdorferi sensu lato, associated with lizards as reservoirs and Ixodes ricinus as its main vector. Borrelia lusitaniae is predominantly distributed in Central and Southeast Europe, and in countries of the Mediterranean basin, such as Portugal, Morocco, Tunisia, and Italy where this spirochete appears to infect vector ticks more frequently than other genospecies. Evolution of this zoonotic tick-borne microparasite is shaped by different environmental factors. Comprehensive phylogenetic analysis may give insight into how B. lusitaniae spreads to new geographic locations. Aim: We applied Bayesian statistical methods to B. lusitaniae multilocus sequence typing (MLST) data to study the migration routes of B. lusitaniae and its potential for further spread. Results: The discrete phylogeographic analysis placed origins of B. lusitaniae in Southeast Europe and identified at least two introductions of B. lusitaniae from Europe to North Africa. Estimated effective reproductive potential (Re), as a key indicator for a pathogen spread, suggested potential for further spread. Conclusion: The results of this study can provide beneficial information about the potential for further spread of B. lusitaniae in Europe and North Africa and estimation of necessity for the development of strategies to monitor and control Lyme borreliosis.

Kompleks Borrelia burgdorferi sensu lato trenutno obuhvata dvadest i jednu opisanu vrstu borelija a karakteriše ih značajna varijabilnost u pogledu geografske distribucije kao i specifičnosti na relaciji domaćini rezervoari- kompetentni vektor. Poznato je da samo određeni sojevi vrsta borelija izazivaju bolest kod ljudi. U Severnoj Americi, lajmsku boreliozu uzrokuje samo jedna vrsta borelija-Borrelia burgdorferi sensu stricto (s.s.), dok u Evropi infekciju kod ljudi može uzrokovati nekoliko različitih vrsta borelija-Borrelia afzelii, Borrelia garinii, Borrelia bavariensis, Borrelia spielmanii i Borrelia burgdorferi s.s. Vrste Borrelia valaisiana, Borrelia lusitaniae i Borrelia bissettii identifikovane su samo u pojedinačnim slučajevima i prepoznate su kao potencijalni patogeni. Različite vrste borelija povezane su sa različitim kliničkim manifestacijama lajmske borelioze; B. afzelii obično ostaje lokalizovana u koži, B. garinii i B. bavariensis se obično povezuju s poremećajima nervnog sistema, dok se B. burgdorferi s.s. često povezuje sa razvojem artritisa. Iako postoje registrovani slučajevi lajmske borelioze u Srbiji, ne postoje podaci o tome koje vrste borelija izazivaju određene kliničke manifestacije lajmske borelioze. Region Srbije karakteriše visoka rasprostranjenost i raznovrsnost vrsta borelija u krpeljima i njihovim domaćinima. Istraživanja prisustva borelija u krpeljima kao i mikrobiološka izolacija sojeva borelija iz krpelja u Srbiji, ukazala su na dominaciju vrste B. lusitaniae, slede vrste B. afzelii, B. bavariensis, B. garinii, B. valaisiana i B. burgdorferi s.s. Takođe, B. lusitaniae je detektovana u krpeljima sakupljenim sa guštera i u uzorcima slezine lisica, dok je B. valaisiana detektovana u krpeljima sakupljenim sa šakala i ptica. Ispitivanje in vitro osetljivosti lokalnih sojeva borelija (izolovanih iz krpelja vrste Ixodes ricinus sakupljenih sa vegetacije) na ljudski serum, pokazalo je da su sojevi B. valaisiana otporni na komplement, dok su, The twenty-one described Borrelia species with considerable variability in host and vector associations, and geographical distribution have been classified to the Borrelia burgdorferi sensu lato complex. Only a subset of Borrelia species is known to cause human illness. In North America, Lyme borreliosis (LB) is predominantly caused by the Borrelia burgdorferi sensu stricto (s.s.), while in Europe, the overall diversity of Borrelia species is higher and LB can be caused by Borrelia afzelii, Borrelia garinii, Borrelia bavariensis, Borrelia spielmanii, and Borrelia burgdorferi s.s., while Borrelia valaisiana, Borrelia lusitaniae and Borrelia bissettii have been identified in single cases only and are recognized as potential pathogens. Different Borrelia species are involved in distinct clinical manifestations of LB; B. afzelii usually remains localized in the skin, B. garinii and B. bavariensis are usually associated with nervous system disorders, while B. burgdorferi s.s. is commonly associated with development of the arthritis. Although there are registered cases of LB in Serbia, there is no data on which Borrelia species cause certain clinical manifestations of LB. Region of Serbia is characterized by high prevalence and diversity of Borrelia species in ticks and their vertebrate hosts. The studies on the presence and isolation of Borrelia from ticks from Serbia pointed to the domination of B. lusitaniae followed by B. afzelii, B. bavariensis, B. garinii, B. valaisiana and B. burgdorferi s.s. Also, B. lusitaniae was detected in ticks collected from lizards and in spleen samples collected from red foxes, while B. valaisiana was detected in ticks collected from golden jackals and birds. In vitro susceptibility of local Borrelia strains isolated from ticks to human complement, showed B. valaisiana strains resisted complement-mediated killing, while certain B. lusitaniae strains (in the presence of complement) were equally motile as the only existing one B. lusitaniae

Introduction: Zoonotic tick-borne diseases (TBDs) represent an increasing threat in urban areas, including city parks and green recreational areas. Borrelia miyamotoi is pathogenic to humans but there is still little information about its circulation in nature and potential local impact on human health, while tick-borne encephalitis virus (TBEV) is endemic in many European countries and sporadic autochthonous cases in humans have been reported in Serbia. The objective of our study was to determine the prevalence of two emerging zoonotic tick-borne pathogens (TBPs) − B. miyamotoi and TBEV in urban areas in the city of Belgrade (24 localities), four localities in Eastern Serbia, one in the province of the Vojvodina. Methods: At 29 localities, a total of 480 ticks were collected from March to June 2021, and identified to the species level by using morphological keys. DNA and RNA were extracted from individual tick samples, while for molecular detection, probe-based qPCR and nested PCR were applied.Results: Among 480 ticks, 445 were identified as Ixodes ricinus (213 males, 176 females, 56 nymphs), 30 as Dermacentor reticulatus (18 females, 12 males), three Dermacentor marginatus females, one Haemaphysalis concinna female, and one Haemaphysalis punctata male. Using probe-based qPCR, the prevalence of B. miyamotoi in ticks was 1.04% (DNA was detected in five I. ricinus, two females and two males collected from three Belgrade localities and one male from Eastern Serbia), while sequencing was successful in samples of two I. ricinus females collected from two Belgrade localities and one male from Eastern Serbia. TBEV RNA was not detected in any sample. Conclusions: This is the first report on the presence of the emerging zoonotic pathogen B. miyamotoi in I. ricinus ticks in urban green areas in Serbia, indicating the risk of B. miyamotoi disease. Risk areas within cities should be identified and knowledge regarding TBPs and TBDs among the general population in urban areas sh

Background The epidemiological status concerning many canine tick-borne diseases (TBDs) in Serbia is still insufficiently known. Objectives Our study aimed to investigate the presence of tick-borne pathogens of the family Anaplasmataceae and Hepatozoon spp., as a cause of illnesses accompanied by clinical signs that can occur in dogs with anaplasmosis, ehrlichiosis and hepatozoonosis. Methods Dogs are included in the study based on the presence of a minimum of three clinical and/or pathological findings that could be associated with anaplasmosis, ehrlichiosis and hepatozoonosis. During the study (April–October 2018), 11 dogs met the conditions to be included in the survey. Identification of the causative agent in the blood of diseased dogs was performed by conventional PCR followed by sequencing. Results The presence of the pathogens was confirmed in three animals (3/11, 27.3%). The presence of Ehrlichia canis was confirmed in 3-month-old female Rottweiler puppy, an 8-year old Miniature Schnauzer female was positive for Hepatozoon canis infection, while 4-year-old mixed breed male dog was co-infected with both mentioned pathogens. These are the first cases of autochthonous infection with E. canis and H. canis in dogs from Serbia confirmed by molecular methods. Conclusions The results of our study indicate the importance of molecular methods to establish a reliable diagnosis of TBDs. Also, the confirmed presence of causative agents of canine monocytic ehrlichiosis and hepatozoonosis in Serbia appeals to veterinary practitioners that it is necessary to exclude the presence of those diseases in suspicious dogs.

Tropical Diseases of the University Clinical Centre of Serbia with a clinical presentation of disseminated erythema migrans. The patient and the mother could not recall if there had been a tick bite. A sample of blood was taken, and antibiotic therapy with amoxicillin was started immediately. Human serum sample was checked for the presence of IgM antibody against Borrelia burgdorferi sensu lato by commercial ELISA test and the sample was positive for IgM. Blood was collected into the sterile K2EDTA tube, immediately transported to the Laboratory at the Institute for Medical Research, National Institute of the Republic of Serbia, University of Belgrade, and centrifuged twice at 2200 rpm for 17 min at room temperature. After centrifugation, one part of the serum was served for DNA extraction using ammonium hydroxide, ethanol, and sodium acetate while the sediment was inoculated into a 6 mL tube with Barbour-Stoenner-Kelly-H (BSK-H) medium under aseptic conditions and incubated as 33°C. After 16 days of incubation, viable, motile, and spiral-shaped microorganisms were observed in the initial BSK-H culture under dark-field microscopy, and incubation was prolonged for 29 days. DNA from the culture was extracted using centrifugation, dissolving the sediment in the water, and heating at 95°C for 10 minutes. After extracting DNA from the human serum and the culture, rrf-rrl rDNA intergenic spacer and flagellin gene (flaB) were amplified by conventional PCR, and sequencing of obtained PCR products was performed commercially (Macrogen, Amsterdam, the Netherlands). After analysis of the sequences obtained, Borrelia lusitaniae was confirmed in human serum and culture. This is the first isolate of B. lusitaniae from a human blood sample that confirms that B. lusitaniae can disseminate via the hematogenous route.

In the present study, the effectiveness of six antimicrobial agents have been tested against 24 borrelia strains isolated from Ixodes ricinus ticks (11 Borrelia lusitaniae, eight Borrelia afzelii, three Borrelia garinii and two Borrelia valaisiana) and one B. lusitaniae strain isolated from human skin. The minimum inhibitory concentration range of antimicrobial agents was as follows: amoxicillin, 0.125–2 mg/L; doxycycline, 0.125–1 mg/L, ceftriaxone, 0.016–0.063 mg/L; cefuroxime, 0.063–1 mg/L; azithromycin, 0.0017–0.11 mg/L; amikacin 32–512 mg/L. Potentially pathogenic B. lusitaniae and B. valaisiana species were more susceptible to amoxicillin and azithromycin than pathogenic B. afzelii and B. garinii (P < 0.05); B. garinii, B. lusitaniae and B. valaisiana were more susceptible to doxycycline than B. afzelii (P < 0.05) while all species showed same susceptibility to ceftriaxone and cefuroxime (P > 0.05). This study is the first report on in vitro susceptibility of isolates from Serbia to antimicrobial agents and the first report on susceptibility of larger number of isolates of potentially pathogenic species B. lusitaniae. We showed that antimicrobial agents in vitro inhibit growth of borrelia strains very effectively, indicating the potential of their equally beneficial use in the treatment of Lyme borreliosis.

Barbour-Stoenner-Kelly II (BSK-II) and BSK-H media were used for cultivation and isolation of fastidiousBorreliaspecies - the causative agents of Lyme borreliosis. Culture media have a limited shelf life and require adequate storage. Our goal was to assess how the growth ofBorreliawould be affected by prolonged storage of media and inadequate storage conditions (BSK-H stored at +4 degrees C for 2.5 years and BSK-II stored at -20 degrees C for 11 years). Growth of differentBorrelia afzelii,Borrelia garinii,Borrelia lusitaniaeandBorrelia valaisianastrains was assessed during 2 weeks of incubation at 33 degrees C. Monitored parameters included cell count per mL, morphology and motility. The results of this study have shown weaker growth of borrelia strains in BSK-H at +4 degrees C (median final cell number of 1.5 x 10(6)/mL) than in BSK-II at -20 degrees C (median final cell number of 7.75 x 10(6)/mL) and in fresh BSK-H media (median final cell number of 8.95 x 10(6)/mL). Duration of storage of media had no impact onBorreliamorphology and motility. Our results indicate that temperature of -20 degrees C is optimal for long-term storage of medium, BSK-II stored for 11 years provided effective support to growth ofBorreliaand may be employed for cultivation.

Background The epidemiological status concerning many canine tick-borne diseases (TBDs) in Serbia is still insufficiently known. Objectives Our study aimed to investigate the presence of tick-borne pathogens of the family Anaplasmataceae and Hepatozoon spp., as a cause of illnesses accompanied by clinical signs that can occur in dogs with anaplasmosis, ehrlichiosis and hepatozoonosis. Methods Dogs are included in the study based on the presence of a minimum of three clinical and/or pathological findings that could be associated with anaplasmosis, ehrlichiosis and hepatozoonosis. During the study (April–October 2018), 11 dogs met the conditions to be included in the survey. Identification of the causative agent in the blood of diseased dogs was performed by conventional PCR followed by sequencing. Results The presence of the pathogens was confirmed in three animals (3/11, 27.3%). The presence of Ehrlichia canis was confirmed in 3-month-old female Rottweiler puppy, an 8-year old Miniature Schnauzer female was positive for Hepatozoon canis infection, while 4-year-old mixed breed male dog was co-infected with both mentioned pathogens. These are the first cases of autochthonous infection with E. canis and H. canis in dogs from Serbia confirmed by molecular methods. Conclusions The results of our study indicate the importance of molecular methods to establish a reliable diagnosis of TBDs. Also, the confirmed presence of causative agents of canine monocytic ehrlichiosis and hepatozoonosis in Serbia appeals to veterinary practitioners that it is necessary to exclude the presence of those diseases in suspicious dogs.

Wild canids are recognized as important reservoir hosts for several vector-borne pathogens and are a good sentinel species for epidemiological surveillance. Identifying the reservoir hosts for particular zoonotic pathogens in a specific area enables the implementation of adequate measures in order to prevent the occurrence of zoonotic diseases in humans and domestic animals. The present review aims to summarize the role of wild canids in the epidemiology of tick-borne zoonoses in Serbia based on previously published papers and to discuss the available data from an epidemiological point of view. The results of previously published studies indicate that wild canids are potential reservoir hosts for several zoonotic tick-borne pathogens in Serbia, and hosts for several tick species which are proven competent vectors. Based on molecular studies conducted so far, the presence of Anaplasma phagocytophilum, was confirmed in jackals while the presence of Candidatus Neoehrlichia micurensis (FU98), Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia lusitaniae was proven by PCR in analyzed red foxes. There is a need for continuous epidemiological monitoring of diseases caused by pathogens previously confirmed in wild canids. Also, further research is needed to reveal the role of other wild and domestic animals in the epidemiology of tick-borne zoonoses in Serbia., Divlje kanide su prepoznate kao važni domaćini rezervoari za nekoliko vektorima prenosivih zoonotskih patogena i predstavljaju dobre sentinel vrste za epidemiološki nadzor. Identifikacija domaćina rezervoara za pojedine zoonotske patogene na određenom području omogućava primenu odgovarajućih mera kako bi se sprečila pojava zoonoza kod ljudi i domaćih životinja. Cilj ove studije bio je da se predstavi uloga divljih kanida u epidemiologiji zoonoza koje se prenose vektorima u Srbiji na temelju prethodno objavljenih radova i da se dostupni podaci prodiskutuju sa epidemiološkog stanovišta. Na osnovu dosadašnjih molekularnih istraživanja u Srbiji, divlje kanide su prepoznate kao potencijalni rezervoari za nekoliko krpeljima prenosivih zoonotskih patogena. Kod šakala je potvrđeno prisustvo Anaplasma phagocytophilum, dok je prisustvo Candidatus Neoehrlichia micurensis (FU98), Borrelia burgdorferi sensu stricto, Borrelia garinii i Borrelia lusitaniae molekularnim metodama potvrđeno kod lisica. Rezultati pokazuju da su divlje kanide potencijalni domaćini rezervoari i izvor zoonotskih patogena u Srbiji i da predstavljaju dobre domaćine za više vrsta krpelja koji su potvrđeni kompetentni vektori pojedinih patogena. Potrebno je kontinuirano epidemiološko praćenje bolesti uzrokovanih patogenima čije je prisustvo prethodno potvrđeno kod divljih kanida. Takođe, potrebna su dodatna istraživanja kako bi se otkrila uloga drugih divljih i domaćih životinja u epidemiologiji zoonoza koje se prenose vektorima u našoj zemlji.

Human activities such as deforestation, urbanization, and environmental pollution lead to a reduction in the spatial boundary between wild animals, domestic animals and humans. These activities increase the risk for the emergence of pathogens from the sylvatic cycle in the population of domestic animals and humans. Foxes are recognized as potential reservoirs for a number of bacterial pathogens of medical and public health concern. The aim of the present study was to investigate the prevalence and spatial distribution of bacterial tick-borne pathogens from the Anaplasmataceae family, Borrelia burgdorferi sensu lato (s.l.), Rickettsia spp., Coxiella burnetii, Francisella tularensis, Bartonella spp., in the red fox population from Serbia and to discuss the obtained results from the epidemiological point of view. Legally hunted red foxes (Vulpes vulpes) from 14 localities in Serbia were included in the study and spleen samples from 129 animals were tested with conventional PCR assays for the presence of bacterial tick-borne pathogens. DNA of Candidatus Neoehrlichia sp. (FU98), Borrelia burgdorferi sensu stricto, Borrelia lusitaniae, and Borrelia garinii was detected in 6 (4.7%), 1 (0.8%), 2 (1.6%) and 1 (0.8%) animals, respectively. Co-infection by Candidatus Neoehrlichia sp. (FU98) and B. garinii was detected in one animal. All samples were negative for other tested bacterial tick-borne pathogens. The results of the present study indicate the potential role of foxes in natural cycles of Candidatus Neoehrlichia sp. (FU98) and causative agents of Lyme borreliosis in the investigated areas. Further research is required to elucidate the role of foxes in the epidemiology of these and other tick-borne zoonotic pathogens in the Republic of Serbia.

Spirochetes from the Borrelia burgdorferi sensu lato (s.l.). (Spirochaetales: Spirochaetaceae) species complex, including the causative agents of Lyme borreliosis, have been isolated from ticks, vertebrate reservoirs and humans. Previous analyses based on direct molecular detection in ticks indicated a considerable diversity of B. burgdorferi s.l. complex in Serbia. The present study aimed (a) to isolate borrelia strains from Serbia; (b) to determine their genotypic characteristics; and (c) to establish a collection of viable B. burgdorferi s.l. strains for further biological, ecological and genetic studies. For the present study, 231 adult Ixodes ricinus (Ixodida: Ixodidae) ticks from 16 ecologically different localities in Serbia were individually processed to cultivate B. burgdorferi s.l. This led to the isolation of 36 strains. A hbb gene quantitative real-time polymerase chain reaction (PCR) based on melting temperature determination and ospA gene sequencing were used to genotype the isolated spirochetes. The species identified based on the hbb gene real-time PCR were: Borrelia lusitaniae (44.4%), Borrelia afzelii (36.1%), Borrelia garinii (13.9%) and Borrelia valaisiana (5.6%), whereas the ospA sequence analysis revealed the occurrence of Borrelia bavariensis. This is the first report of the isolation of B. lusitaniae, B. garinii, B. bavariensis and B. valaisiana strains in Serbia.

Tick-borne haematozoans cause severe diseases in domestic animals, and some of them have zoonotic potential. The results of previous studies in Europe point to the important role of foxes in natural endemic cycles of several tick-borne pathogens, including protozoa. The aim of the present research was to acquire information on the prevalence and distribution of tick-borne protozoan parasites among foxes in Serbia. Legally hunted foxes from 14 localities throughout Serbia were analysed. Spleen samples were collected from 129 animals and tested for the presence of Babesia spp. and Hepatozoon spp. by PCR. In total, 79/129 (61.2%) of the tested foxes were positive for H. canis, while the presence of two Babesia species was confirmed: B. vulpes (37/129, 28.7%) and B. canis (1/129, 0.8%). Co-infection with B. vulpes and H. canis was present in 26/129 (20.2%) foxes and one animal (1/129, 0.8%) was co-infected by B. canis and H. canis. The results of this study indicate the important role of foxes in the epizootiology of B. vulpes and H. canis in the Republic of Serbia and stress the need for further research to clarify all elements of the enzootic cycle of the detected pathogens, including other reservoirs, vectors, and transmission routes.

Lyme borreliosis is caused by the spirochete Borrelia burgdorferi sensu lato, a fastidious bacterium that replicates slowly and requires special conditions to grow in the laboratory. Borrelia isolation from clinical material is a golden standard for microbiological diagnosis of borrelial infection. Important factors that affect in vitro borrelia growth are temperature of incubation and number of borrelia cells in the sample. The aim of the study was to assess the influence of temperature on borrelia growth and survival by evaluation and comparison of growth of 31 different borrelia strains at five different temperatures and to determine the influence of different inoculums on borrelia growth at different temperatures. Borreliae were cultured in the MKP medium; the initial and final number of spirochetes was determined by dark field microscopy using Neubauer counting chamber. The growth of borrelia was defined as final number of cells/mL after three days of incubation. For all three Borrelia species, the best growth was found at 33°C, followed by 37, 28, and 23°C, while no growth was detected at 4°C (P<0.05). The growth of B. afzelii species was weaker in comparison to the other two species at 23, 28, 33 and 37°C (P<0.05), respectively. There was no statistically significant difference between the growth of B. garinii and B. burgdorferi sensu stricto at 28, 33, and 37°C (P>0.05), respectively. Inoculum had statistically significant influence on growth of all three Borrelia species at all tested temperatures except at 4°C.

Antimicrobial resistance is a natural biological phenomenon in their struggle for existence and represents a major health care issue, associated with high mortality and morbidity. The first cases of mass emergence of resistant strains were observed in the middle of the 20th century, and since then cases of resistance have been reported all over the world, and in the last two decades even more frequently are reported multiple bacterial resistance. Factors that contribute to the development of bacteria resistance are abuse in the use of antibacterial agents, in humans or livestock, and releases of antibacterial agents into the environment. Moreover, the development of new effective antibiotics is decreasing, contrary to increasing the overall effort for the synthesis of new ones. Identification and reporting of bacterial resistance, as well as monitoring of the use of antibiotics in the outpatient and inpatient setting, today is the obligation of all countries.

Lyme borreliosis is a multisystem disorder characterized by a wide spectrum of clinical manifestations. In Europe, the main aetiological agents are Borrelia afzelii, Borrelia garinii and B. burgdorferi sensu stricto, but Borrelia spielmanii, Borrelia valaisiana, Borrelia lusitaniae and Borrelia bissettii have also been reported as rare or potential causes of human disease. In North America, B. burgdorferi sensu stricto is the only human pathogen. The most common clinical manifestation of Lyme borreliosis is a skin lesion, erythema migrans, which may resolve spontaneously without antibiotic treatment. However, the pathogen can spread to other tissues and organs, causing more severe manifestations that can involve a skin, nervous system, joints, and/or heart, requiring antibiotic therapy. The most commonly used antibiotics in the treatment of Lyme borreliosis are: amoxicillin, ceftriaxone, cefuroxime, doxycycline and azithromycin., Lajmska borelioza je multisistemska bolest koju karakteriše širok spektar kliničkih manifestacija. U Evropi je primarno prouzrokovana vrstama B. afzelii, B. garinii i B. burgdorferi sensu stricto, ali Borrelia spielmanii, Borrelia valaisiana, Borrelia lusitaniae and Borrelia bissettii se takođe navode kao retki ili potencijalni uzročnici oboljenja kod ljudi. U Severnoj Americi, kao jedini humani patogen, dokazana je vrsta B. burgdorferi sensu stricto. Najčešća klinička manifestacija lajmske borelioze je kožna lezija, erythema migrans, koja može da prođe spontano, bez terapije antibioticima. Međutim, patogen se može širiti do drugih delova kože i organa i tako prouzrokovati teže manifestacije kako na koži, tako i na nervnom sistemu, zglobovima i/ili srcu, pri čemu je terapija antibioticima neophodna. Najčešće korišćeni antibiotici u terapiji lajmske borelioze su: amoksicilin, ceftriakson, cefuroksim, doksiciklin i azitromicin.

Lyme borreliosis is the most common vector-borne disease in the northern hemisphere. The agents of Lyme borreliosis are borrelia, bacteria of the family Spirochaetaceae, which are grouped in Borrelia burgdorferi sensu lato species complex. Borreliae are fastidious, slow-growing and biochemically inactive bacteria that need special attention and optimal conditions for cultivation. The isolation of Borrelia from clinical material and their cultivation is a time-consuming and demanding procedure. Cultivation lasts from 9 up to 12 weeks, which is much longer than is necessary to grow most other human bacterial pathogens. Although B. burgdorferi sensu lato is susceptible to a wide range of antimicrobial agents in vitro, up to now the susceptibility of individual Borrelia species to antibiotics is defined only partially.

Lajmska borelioza je multisistemska bolest koju karakteriše širok spektar kliničkih manifestacija. U Evropi je primarno prouzrokovana vrstom B. afzelii a ređe vrstama B. garinii i B. burgdorferi sensu stricto. Borelije mogu biti izolovane iz različitog kliničkog materijala kao što su koža, krv i cerebrospinalna tečnost (CST). Modifikovana Kelly-Pettenkofer-ova (MKP) podloga je jedna od najčešće korišćenih hranljivih podloga za izolaciju i kultivaciju borelija. Prvi cilj ove doktorske disertacije je bio proceniti da li pojedinačne vrste borelija (B. afzelii, B. garinii i B. burgdorferi sensu stricto) imaju sposobnost da rastu u MKP podlogama koje su prethodno bile čuvane u frižideru na +4 °C u vremenskom periodu od manje od jednog meseca do 12 meseci, i kako dugotrajno čuvanje podloga može uticati na rast i morfologiju borelija. Rast borelija je procenjen posle 5 dana inkubacije na 33 °C; procenjen je broj borelija/mL, morfologija i pokretljivost. Rezultati su pokazali, da je starost MKP podloga imala statistički značajan uticaj na rast B. afzelii (P=0,021) i B. garinii (P=0,004) ali ne i na rast B. burgdorferi sensu stricto (P=0,204), dok starost podloga nije imala uticaj na morfologiju borelija i njihovu pokretljivost. U ovom radu je pokazano, da hranljive podloge starije od jednog meseca pa sve do 12 meseci, mogu podržati rast borelija. Optimalna temperatura za rast borelija u in vitro uslovima, predstavljena je u širokom rasponu od 30 do 37 °C. Borelije obično rastu na temperaturi 30-34 °C, a ponekad i na 35- 37 °C. Drugi cilj ove doktorske disertacije je bio proceniti i uporediti rast trideset jednog različitog soja vrsta B. afzelii, B. garinii i B. burgdorferi sensu stricto na pet različitih temperatura (4, 23, 28, 33 i 37 ºC) kako bi se procenio uticaj temperature na rast i preživljavanje borelija i definisala optimalna temperatura za njhovu kultivaciju. Rast je definisan kao konačni broj borelija/mL posle 3 dana inkubacije. Sve tri vrste borelija najbolje su, Lyme borreliosis is a multisystem disorder characterized by a wide spectrum of clinical manifestations. In Europe, it is predominantly caused by B. afzelii and less frequently by B. garinii and B. burgdorferi sensu stricto. Borrelia can be isolated from different clinical material, such as skin, blood and cerebrospinal fluid (CSF). Modified Kelly-Pettenkofer (MKP) medium is one of several media used for isolation and cultivation of Borrelia. The first aim of this doctoral dissertation was to assess whether particular Borrelia species (B. afzelii, B. garinii and B. burgdorferi sensu stricto) have the ability to grow in MKP medium stored at +4 °C for periods for one moth up to one year, and how prolonged storage may influences borrelia growth and morphology. The growth of Borrelia was evaluated after 5 days of incubation at 33 °C: cell count per mL, morphology and motility were assessed. The results of the present study showed that the duration of storage of MKP medium had statistically significant influence on growth of B. afzelii (P=0.021) and B. garinii (P=0.004) but not on growth of B. burgdorferi sensu stricto (P=0.204), while duration of storage of the medium had no impact on Borrelia morphology and motility. The results of the study indicate that medium stored for more than one and up to 12 months support borrelia growth. The optimum temperature for growth of Borrelia in vitro was presented widely as 30-37 °C. Borreliae are usually grown at 30-34 °C but sometimes at 35 or 37 °C. A second aim of this doctoral dissertation was to evaluate and compare growth of thirty-one different Borrelia strains (B. afzelii, B. garinii, and B. burgdorferi sensu stricto) at five different temperatures (4, 23, 28, 33, and 37 ºC) in order to find the influence of temperature on borrelia growth and survivance and to define the optimal temperature for cultivation of Borrelia. For all three Borrelia species, the best growth (growth was defined as final number of cells/mL after three