The role of particular ticks developmental stages in the circulation of tick-borne pathogens in Central Europe. 5. Borreliaceae

The developmental cycles of all B. burgdorferi s.l. genospecies present typical, main pattern described in the 90thies. The simple scheme might be modified according to the biology of species and hosts preference. Central European genospecies of B. burgdorferi s.l. can be associated with four groups of hosts playing the role of animal reservoirs. The group 1 contains genospecies associated with rodents as primary animal reservoir ­ B. afzelii, B. garinii, B. burgdorferi sensu stricto, strains B. bavariensis (B. garinii OspA serotype 4). The group 2 involves B. valaisiana and most of B. garinii strains, associated with birds. The group 3 involves B. spielmanii, the reservoir hosts are Gliridae, and hedgehogs. The group 4 includes B. lusitaniae, the hosts are lizards. B. miyamotoi enzootic cycle seems to be similar to B. burgdorferi complex, however, differs by the transovarial transmission possibility. The divisions are not extreme; in the hosts group, infected with appropriate Borrelia genospecies, very often are found the specimens infected with other genospecies.

Genomic and phenotypic characterization of Borrelia afzelii BO23 and Borrelia garinii CIP 103362.

In recent years, the number of Lyme disease or borreliosis cases in Eurasia has been dramatically increasing. This tick-borne disease is caused by Borrelia burgdorferi sensu lato, which includes B. burgdorferi sensu stricto, the main species found in North America, and B. afzelii and B. garinii, which are primarily responsible for the disease in Eurasia. Currently, research on Lyme disease has focused mainly on B. burgdorferi while B. afzelii and B. garinii, which cause disease with distinctly different symptoms, are less studied. The purpose of this study is to evaluate B. afzelii BO23 and B. garinii CIP 103362 as model organisms to study Eurasian Lyme disease. To begin our analyses, we sequenced, annotated the chromosomes of both species and compared them to B. burgdorferi strain B31. We also assayed shuttle vector, pBSV2, for transformation efficacy and demonstrated that these strains can be cultured on solid media. In addition, we characterized how physicochemical parameters (e.g., oxygen, osmolarity, oxidative stress) affect both growth and motility of the bacteria. Finally, we describe each strain's antibiotic susceptibility and accessed their ability to infect mice. In conclusion, B. afzelii BO23 was more practical for in vitro and in vivo studies than B. garinii CIP 103362.

The abundance of the Lyme disease pathogen Borrelia afzelii declines over time in the tick vector Ixodes ricinus.

BACKGROUND: The population dynamics of vector-borne pathogens inside the arthropod vector can have important consequences for vector-to-host transmission. Tick-borne spirochete bacteria of the Borrelia burgdorferi (sensu lato) species complex cause Lyme borreliosis in humans and spend long periods of time (>12 months) in their Ixodes tick vectors. To date, few studies have investigated the dynamics of Borrelia spirochete populations in unfed Ixodes nymphal ticks. METHODS: Larval ticks from our laboratory colony of I. ricinus were experimentally infected with B. afzelii, and killed at 1 month and 4 months after the larva-to-nymph moult. The spirochete load was also compared between engorged larval ticks and unfed nymphs (from the same cohort) and between unfed nymphs and unfed adult ticks (from the same cohort). The spirochete load of B. afzelii in each tick was estimated using qPCR. RESULTS: The mean spirochete load in the 1-month-old nymphs (~14,000 spirochetes) was seven times higher than the 4-month-old nymphs (~2000 spirochetes). Thus, the nymphal spirochete load declined by 80% over a period of 3 months. An engorged larval tick acquired ~100 spirochetes, and this population was 20 times larger in a young, unfed nymph. The spirochete load also appeared to decline in adult ticks. Comparison between wild and laboratory populations found that lab ticks were more susceptible to acquiring B. afzelii. CONCLUSION: The spirochete load of B. afzelii declines dramatically over time in domesticated I. ricinus nymphs under laboratory conditions. Future studies should investigate whether temporal declines in spirochete load occur in wild Ixodes ticks under natural conditions and whether these declines influence the tick-to-host transmission of Borrelia.

Comparison of Growth of Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi Sensu Stricto at Five Different Temperatures.

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.

Co-infections and transmission dynamics in a tick-borne bacterium community exposed to songbirds.

We investigated the transmission dynamics of a community of tick-borne pathogenic bacteria in a common European songbird (Parus major). Tick-naïve birds were infested with three successive batches (spaced 5 days apart) of field-collected Ixodes ricinus nymphs, carrying the following tick-borne bacteria: Rickettsia helvetica (16.9%), Borrelia garinii (1.9%), Borrelia miyamotoi (1.6%), Anaplasma phagocytophilum (1.2%) and Candidatus Neoehrlichia mikurensis (0.4%). Fed ticks were screened for the pathogens after moulting to the next developmental phase. We found evidence for early transmission (within 2.75 days after exposure) of R. helvetica and B. garinii, and to a lesser extent of A. phagocytophilum based on the increased infection rates of ticks during the first infestation. The proportion of ticks infected with R. helvetica remained constant over the three infestations. In contrast, the infection rate of B. garinii in the ticks increased over the three infestations, indicating a more gradual development of host tissue infection. No interactions were found among the different bacterium species during transmission. Birds did not transmit or amplify the other bacterial species. We show that individual birds can transmit several pathogenic bacterium species at the same time using different mechanisms, and that the transmission facilitation by birds increases the frequency of co-infections in ticks.

First detection and molecular identification of Borrelia garinii spirochete from Ixodes ovatus tick ectoparasitized on stray cat in Taiwan.

Borrelia garinii spirochete was detected for the first time in Ixodes ovatus tick ectoparasitized on stray cat in Taiwan. The genetic identity of this detected spirochete was determined by analyzing the gene sequence amplified by genospecies-specific polymerase chain reaction assays based on the 5S-23S intergenic spacer amplicon (rrf-rrl) and outer surface protein A (ospA) genes of B. burgdorferi sensu lato. Phylogenetic relationships were analyzed by comparing the sequences of rrf-rrl and ospA genes obtained from 27 strains of Borrelia spirochetes representing six genospecies of Borrelia. Seven major clades can be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. Phylogenetic analysis based on rrf-rrl gene revealed that this detected spirochete (strain IO-TP-TW) was genetically affiliated to the same clade with a high homogeneous sequences (96.7 to 98.1% similarity) within the genospecies of B. garinii and can be discriminated from other genospecies of Borrelia spirochetes. Interspecies analysis based on the genetic distance values indicates a lower level (<0.022) of genetic divergence (GD) within the genospecies of B. garinii, and strain IO-TP-TW was genetically more distant ( >0.113) to the strains identified in I. ovatus collected from Japan and China. Intraspecies analysis also reveals a higher homogeneity (GD<0.005) between tick (strain IO-TP-TW) and human (strain Bg-PP-TW1) isolates of B. garinii in Taiwan. This study provides the first evidence of B. garinii isolated and identified in an I. ovatus tick in Asia, and the higher homogeneity of B. garinii between tick and human strain may imply the risk of human infection by I. ovatus bite.

Influence of MKP medium stored for prolonged periods on growth and morphology of Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi sensu stricto.

Modified Kelly-Pettenkofer (MKP) medium is one of the several media used for isolation and cultivation of Borrelia. The aim of the study 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 1 month up to 1 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 this 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), whereas 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 1 and up to 12 months supports Borrelia growth.

Ixodes ricinus ticks infected with the causative agent of Lyme disease, Borrelia burgdorferi sensu lato, have higher energy reserves.

Ticks use their energy reserves to maintain their water balance, search for hosts and transmit tick-borne pathogens. However, the influence of tick-borne pathogens on the energy reserves of the tick vector has not been well studied. The relationship between Borrelia burgdorferi sensu lato (s.l.) infection status and fat content in questing Ixodes ricinus nymphs was examined. Nymphs were sampled from the field. Their body mass and fat content were measured, and their Borrelia genospecies infection status (using reverse line blot analysis), and spirochete load (using quantitative PCR) were analysed. Of the 900 nymphs tested, 21.2% were infected with a variety of Borrelia genospecies. Borrelia-infected nymphs had 12.1% higher fat content than uninfected ticks after correcting for body size. For the subset of Borrelia-infected nymphs, no relationship was found between spirochete load and fat content and bioenergetics calculations suggest that Borrelia spirochetes consume a negligible fraction of the tick energy reserves. While the mechanism that causes the association between Borrelia infection and higher fat content in I. ricinus nymphs remains unknown, the present study complements our previous findings that Borrelia-infected nymphs had higher survival times under desiccating conditions and walked less within a humidity gradient.

Loss of plasmids of Borrelia burgdorferi sensu lato during prolonged in vitro cultivation.

In the present study we analyzed stability of plasmid content in 34 Borrelia strains of three different species (13 Borrelia afzelii, 10 Borrelia garinii and 11 Borrelia burgodorferi sensu stricto) using pulse field gel electrophoresis (PFGE). During long-term in vitro cultivation consisting of 50 passages, plasmid loss was established in 46% of B. afzelii, 40% of B. garinii and 36% of B. burgdorferi sensu stricto strains. Loss of plasmids occurred as early as between the 5th and 10th passage, affected only plasmids in the range 9-41 kb but not plasmids in the range 50-68 kb and manifested with the loss of one to up to three plasmids.

Growth, cysts and kinetics of Borrelia garinii (Spirochaetales: Spirochaetacea) in different culture media.

The aim of the present paper was to evaluate cyst formation and growth parameters of Borrelia garinii in a range of media differing in formulation and cost. A qualitative assessment of morphology and motility of B. garinii was conducted. All media were prepared aseptically and used in test tubes or Petri dishes. For each medium, the initial spirochete concentration was standardized to 10(3) spirochets/mL. The following culture media were suitable to grow B. garinii: Barbour-Stoenner-Kelly, brain heart infusion and PMR. Growth was minimal at six weeks post-inoculation and maximum spirochete density was observed between 9-12 weeks. Often, the cultures developed cysts of different sizes, isolated or in groups, with a spiraled portion of variable sizes, mainly in unfavorable culture media. Brazilian Lyme disease-like illness, also known as Baggio-Yoshinari syndrome (BYS), is a new and interesting emerging tick-borne disease, caused by Borrelia burgdorferi sensu lato spirochetes, only during its cystic forms. It has been assumed that the peculiar clinical and laboratory features of BYS are consequential to the absence of a human sucker Ixodes ricinus complex tick at risk areas in Brazil, supporting the concept that the borrelia phenotypic expression pattern is modified as it is transmitted through the host.

Growth, cysts and kinetics of Borrelia garinii (Spirochaetales: Spirochaetacea) in different culture media

The aim of the present paper was to evaluate cyst formation and growth parameters of Borrelia garinii in a range of media differing in formulation and cost. A qualitative assessment of morphology and motility of B. garinii was conducted. All media were prepared aseptically and used in test tubes or Petri dishes. For each medium, the initial spirochete concentration was standardized to 10³ spirochets/mL. The following culture media were suitable to grow B. garinii: Barbour-Stoenner-Kelly, brain heart infusion and PMR. Growth was minimal at six weeks post-inoculation and maximum spirochete density was observed between 9-12 weeks. Often, the cultures developed cysts of different sizes, isolated or in groups, with a spiraled portion of variable sizes, mainly in unfavorable culture media. Brazilian Lyme disease-like illness, also known as Baggio-Yoshinari syndrome (BYS), is a new and interesting emerging tick-borne disease, caused by Borrelia burgdorferi sensu lato spirochetes, only during its cystic forms. It has been assumed that the peculiar clinical and laboratory features of BYS are consequential to the absence of a human sucker Ixodes ricinus complex tick at risk areas in Brazil, supporting the concept that the borrelia phenotypic expression pattern is modified as it is transmitted through the host.

Study on Lyme borreliosis focus in the Lublin region (eastern Poland).

A suburban focus of Lyme borreliosis situated 11 km from the southern border of the city of Lublin (eastern Poland) was characterized. The focus covers an area of circa 100 km(2), surrounding 3 localities inhabited by circa 7,500 people engaged mostly in farming. It was demonstrated that on the area of focus the infection rate of Ixodes ricinus ticks with Borrelia burgdorferi, frequency of serological response of inhabitants to the antigen of Borrelia burgdorferi, and incidence of Lyme borreliosis were significantly (p<0.001) greater compared to the whole territory of Lublin province, and were respectively 13.1 % vs. 4.7 %, 33.0 % vs. 13.7 %, and 0.002 % vs. 0.00075 % .

Two ways of experimental infection of Ixodes ricinus ticks (Acari: Ixodidae) with spirochetes of Borrelia burgdorferi sensu lato complex.

A previously reported procedure for the introduction of Borrelia spirochetes into tick larvae by immersion in a suspension of spirochetes was tested on Ixodes ricinus (L.) ticks and three of the most medically important European Borrelia genomic species, B. burgdorferi sensu stricto, B. garinii and B. afzelii. The procedure was compared with "classical" infection of nymphs by feeding on infected mice. Both methods yielded comparable results (infection rate 44-65%) with the exception of B. afzelii, which produced better results using the immersion method (44%) compared with feeding on infected mice (16%). Nymphs infected by the immersion method at the larval stage were able to transmit the infection to naïve mice as shown by serology and PCR detection of spirochetal DNA in organs. The immersion method is faster than feeding on infected mice and provides more reproducible conditions for infection. It can be exploited for studies on both pathogen transmission and Borrelia-vector interactions.

The tick salivary protein Salp15 inhibits the killing of serum-sensitive Borrelia burgdorferi sensu lato isolates.

Borrelia burgdorferi, the agent of Lyme disease, is transmitted by ticks. During transmission from the tick to the host, spirochetes are delivered with tick saliva, which contains the salivary protein Salp15. Salp15 has been shown to protect spirochetes against B. burgdorferi-specific antibodies. We now show that Salp15 from both Ixodes ricinus and Ixodes scapularis protects serum-sensitive isolates of Borrelia burgdorferi sensu lato against complement-mediated killing. I. ricinus Salp15 showed strong protective effects compared to those of I. scapularis Salp15. Deposition of terminal C5b to C9 (one molecule each of C5b, C6, C7, and C8 and one or more molecules of C9) complement complexes, part of the membrane attack complex, on the surface of B. burgdorferi was inhibited in the presence of Salp15. In the presence of normal human serum, serum-sensitive Borrelia burgdorferi requires protection against complement-mediated killing, which is provided, at least in part, by the binding to the tick salivary protein Salp15.

Evaluation of a modified culture medium for Borrelia burgdorferi sensu lato

The aim of the present study was to assess the possible use of a modified medium, prepared in the laboratory using the constituents of Barbour-Stonner-Kelly (BSK) medium and medium 199 as base, for the culture of Borrelia strains, comparing the growth of individual strains in this medium and in the BSK-H medium, and the protein profile and antigenic characteristics of Borrelia proteins expressed in these media. A qualitative evaluation of growth of Borrelia species was made with acceptable results (morphology and motility), but during a quantitative evaluation using the three main genospecies of Borrelia, the better results were obtained with a B. burgdorferi sensu stricto strain. The modified medium did not enable the growth of a B. afzelii strain. The protein profile and antigenic characteristic of the expressed proteins in the modified medium were studied with satisfactory results. These results suggest the modified medium as an alternative for the cultivation of Borrelia strains, with some limitations, in poorly-resourced laboratories.

[Role of biological assays in the diagnosis of Lyme borreliosis presentations. What are the techniques and which are currently available?]. / Place des méthodes biologiques dans le diagnostic des différentes manifestations de la borréliose de Lyme. Quelles sont les techniques? Quelles sont celles disponibles actuellement?

The biological diagnosis of Borrelia burgdorferi sensu lato infection is usually made by antibody detection in patient sera. Thus, serological testing (Elisa, immunoblotting) is essential for a biological diagnosis. Specific antibody detection is usually done in serum and CSF of patients suspected of Lyme borreliosis. Laboratories must follow European recommendations to validate these assays in routine practice. Antibody detection lacks sensitivity in the early cutaneous phase of the infection. Therefore, serological testing is not recommended for the diagnosis of erythema migrans. The interpretation of serology must take into account the variability of Elisa sensitivity and specificity and the lack of standardization for Western-blotting in Europe. Besides these indirect diagnosis techniques, there is also direct detection of spirochetes by culture or by in vitro DNA amplification but these require adequate samples. These molecular tests must not be performed routinely, but only for specific clinical situations and in specialized laboratories only.

Complementation of a Borrelia afzelii OspC mutant highlights the crucial role of OspC for dissemination of Borrelia afzelii in Ixodes ricinus.

Alteration of the outer surface protein (Osp) composition--especially that of OspA and OspC--seems to be important for the adaptation of Borrelia burgdorferi sensu lato to its endothermic hosts (mammals) and poikilothermic vectors (ticks). OspA possibly mediates adherence to tick midgut cells thus enabling the borreliae to survive in the vector, while OspC is associated with borrelial invasion of the tick salivary glands and infection of the mammalian hosts. Here we describe the first successful transformation and complementation of a Borrelia afzelii ospC mutant with the wild-type ospC in trans. To test the influence of OspC on the dissemination behavior in ticks, unfed Ixodes ricinus nymphs were artificially infected by capillary feeding either with B. afzelii wild type, the B. afzelii ospC mutant or the ospC-complemented clone. Tick midguts and salivary glands were investigated after different time intervals for the presence of borreliae and for OspA and OspC by immunfluorescence staining with monoclonal antibodies. While the B. afzelii wild-type strain exhibiting abundant OspC on its surface disseminated to the salivary glands, the OspC-negative mutant was only present in the tick midguts. The ospC-complemented clone which constitutively expresses the wild-type ospC was again able to colonize the salivary glands. This finding demonstrates that OspC is crucial for dissemination of B. afzelii from the tick midgut to the salivary glands, a prerequisite for infection of the warm-blooded host. A summary of the detailed data presented here has already been given in Goettner et al. [2006. OspC of B. afzelii is crucial for dissemination in the vector as shown by transformation and complementation of a European OspC-deficient B. afzelii strain. Int. J. Med. Microbiol. 296S1(Suppl. 40), 122-124].

Canine borreliosis: a laboratory diagnostic trial.

The aim of this study was to investigate samples from dogs suggestive of active canine borreliosis (group A) by culture and PCR and the detection of antibodies against Borrelia burgdorferi sensu lato in order to confirm a presumptive clinical diagnosis of canine borreliosis by laboratory results. Criteria for such a diagnosis were: history of tick exposure, lameness, neurological signs, nephropathy, lethargy, anorexia, and fever. A total of 302 samples comprising EDTA blood, urine, synovial fluid, cerebrospinal fluid, and tissue (skin, synovial membrane, kidney) from 98 dogs (26 with arthritis, 46 with neurological signs, 21 with nephropathy, 5 with non-specific symptoms) were collected and examined. Moreover, 55 healthy dogs (group B) and 236 dogs with symptoms or injuries unlikely to be associated with borreliosis (group C) were included in this study. Blood serum samples collected from all individuals (n=389) were analysed by ELISA. Twenty-one (21%) out of 98 dogs from group A, 4 (7%) out of 55 from group B and 15 (6%) out of 236 dogs from group C were positive for antibodies against B. burgdorferi sensu lato. The seroprevalences between groups A, B and C differed significantly. None of the corresponding samples investigated by PCR and culture were positive for spirochetal DNA or viable spirochetes. Borrelia afzelii was grown from one EDTA-blood sample but the corresponding blood serum sample remained antibody-negative. Consequently, the etiologic role of B. afzelii in this case is unclear. In approximately 40% of the presumptive canine borreliosis cases, other lesions have been found to be responsible for clinical signs. This study affirms that a definitive diagnosis of canine borreliosis cannot be made by clinical symptoms and serology based on a single consultation. Moreover, this study clearly revealed that the diagnostic sensitivity is enhanced by a thorough consideration and exclusion of other diseases.

Evaluation of a modified culture medium for Borrelia burgdorferi sensu lato.

The aim of the present study was to assess the possible use of a modified medium, prepared in the laboratory using the constituents of Barbour-Stonner-Kelly (BSK) medium and medium 199 as base, for the culture of Borrelia strains, comparing the growth of individual strains in this medium and in the BSK-H medium, and the protein profile and antigenic characteristics of Borrelia proteins expressed in these media. A qualitative evaluation of growth of Borrelia species was made with acceptable results (morphology and motility), but during a quantitative evaluation using the three main genospecies of Borrelia, the better results were obtained with a B. burgdorferi sensu stricto strain. The modified medium did not enable the growth of a B. afzelii strain. The protein profile and antigenic characteristic of the expressed proteins in the modified medium were studied with satisfactory results. These results suggest the modified medium as an alternative for the cultivation of Borrelia strains, with some limitations, in poorly-resourced laboratories.

Enhanced culture of Borrelia garinii and Borrelia afzelii strains on a solid BSK-based medium in anaerobic conditions.

The growth of 29 human strains from the three main pathogenic species of Borrelia burgdorferi sensu lato on a solid BSK-based medium was compared in two culture atmospheres: 3% CO(2) air and anaerobiosis. All strains grew under anaerobic conditions, whereas only 13 strains were able to grow in aerobiosis with 3% CO(2) (P<0.001). In the latter condition, 75% of the B. burgdorferi sensu stricto strains grew versus 33% of the B. garinii and B. afzelii strains. These data suggest that, especially for B. garinii and B. afzelii species, anaerobic conditions enhance growth yield and speed of low-passage Borrelia strains.