First isolation of Borrelia lusitaniae DNA from green lizards (Lacerta viridis) and Ixodes ricinus ticks in the Czech Republic.

This study aimed to reveal whether green lizards (Lacerta viridis), common hosts of tick larvae and nymphs, might be involved in the transmission cycle of Borrelia burgdorferi sensu lato in the Czech Republic. Green lizards were sampled in two areas at the Tiché Údolí Nature Reserve (site A: 50.1482 N, 14.3669E; site B: 50.1476 N, 14.3745 E), Central Bohemian Region, Czech Republic. The skin biopsy specimens and attached ticks (if any) were collected from 52 captured lizards. Also, questing ticks from both areas were collected by flagging. The touchdown polymerase chain reaction and gel electrophoresis revealed Borrelia lusitaniae in three lizard tissue samples. Most lizards (19/30, 63%) had at least one Borrelia positive tick. Borrelia lusitaniae formed 92% (34/37) and 59% (17/29) of all borreliae detected in larvae and nymphs, respectively. Borrelia lusitaniae (6/10, 60%) was also the major pathogen in questing nymphs from site B. At site A, 13% (2/16) of questing nymphs were positive for B. lusitaniae. Based on our data, it can be assumed that B. lusitaniae is a common pathogen at lizard sites in the Czech Republic, and further research to prove this hypothesis is therefore highly recommended. As lizards often inhabit urban areas, the data presented may also contribute to raising awareness of the possible spread and risk of Borrelia infection.

Metamorphoses of Lyme disease spirochetes: phenomenon of Borrelia persisters.

The survival of spirochetes from the Borrelia burgdorferi (sensu lato) complex in a hostile environment is achieved by the regulation of differential gene expression in response to changes in temperature, salts, nutrient content, acidity fluctuation, multiple host or vector dependent factors, and leads to the formation of dormant subpopulations of cells. From the other side, alterations in the level of gene expression in response to antibiotic pressure leads to the establishment of a persisters subpopulation. Both subpopulations represent the cells in different physiological states. "Dormancy" and "persistence" do share some similarities, e.g. both represent cells with low metabolic activity that can exist for extended periods without replication, both constitute populations with different gene expression profiles and both differ significantly from replicating forms of spirochetes. Persisters are elusive, present in low numbers, morphologically heterogeneous, multi-drug-tolerant cells that can change with the environment. The definition of "persisters" substituted the originally-used term "survivors", referring to the small bacterial population of Staphylococcus that survived killing by penicillin. The phenomenon of persisters is present in almost all bacterial species; however, the reasons why Borrelia persisters form are poorly understood. Persisters can adopt varying sizes and shapes, changing from well-known forms to altered morphologies. They are capable of forming round bodies, L-form bacteria, microcolonies or biofilms-like aggregates, which remarkably change the response of Borrelia to hostile environments. Persisters remain viable despite aggressive antibiotic challenge and are able to reversibly convert into motile forms in a favorable growth environment. Persisters are present in significant numbers in biofilms, which has led to the explanation of biofilm tolerance to antibiotics. Considering that biofilms are associated with numerous chronic diseases through their resilient presence in the human body, it is not surprising that interest in persisting cells has consequently accelerated. Certain diseases caused by pathogenic bacteria (e.g. tuberculosis, syphilis or leprosy) are commonly chronic in nature and often recur despite antibiotic treatment. Three decades of basic and clinical research have not yet provided a definite answer to the question: is there a connection between persisting spirochetes and recurrence of Lyme disease in patients?

A serological study of antibodies to Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato in the sera of healthy individuals collected two decades apart.

Sera collected from healthy individuals from the general population in the Czech Republic during repeated cross-sectional surveys were analyzed. Samples collected in the same six districts in two time periods, 1978-1989 and 2001, were compared. The study subjects were divided into six age categories between 10 and 59 years. Overall, 434 samples from 1978-1989 and 270 samples from 2001 were screened for Anaplasma phagocytophilum (AP) and Borrelia burgdorferi sensu lato (BB). The anti-AP positivity rates were 13.1% and 11.5% in the first and second period, respectively, and did not differ significantly between the periods (P = 0.559). The anti-BB antibodies were detected in 33.9% and 14.8% of study subjects, respectively. The positivity rates were significantly lower in the second period (P<0.001). No considerable changes were observed in the sex distribution of positive findings between the two periods. The highest positivity rates of anti-AP antibodies were found in the 10-14 year age group: 16.0% in 1978-1989 and 16.7% in 2001. The age distribution of the anti-AP antibody positivity rates did not change substantially (P = 0.872). In 1978-1989, the lowest anti-BB antibody positivity rate (26.7%) was found in the 10-14 year age group, with a gradual increase with age to 41.1% in 50-59 year-olds. In 2001, the positivity rate in the 10-14 year age group was 26.2% and was not significantly different from that in the first period (P = 0.955). However, the positivity rates in the older age groups 15-59 years decreased significantly (P<0.001) and varied between 8.3% and 15.1%.

[Guideline for the diagnosis and treatment of Lyme borreliosis].

The national guideline aims to highlight the latest knowledge about clinical manifestations of the infection, to summarize the diagnostic algorithm and to recommend the appropriate antibiotic therapy with respect to evidence-based medicine. The recommendations are consistent with most European guidelines as well as those published by the IDSA. The guideline provides the most recent information on the epidemiology, etiology and pathogenesis of Lyme borreliosis, dermatological, neurological and musculoskeletal involvement, the appropriate diagnostic procedure and prevention. Some information is also provided about post-treatment Lyme disease syndrome. Recommended oral and intravenous antimicrobials are listed in a table showing the doses and duration of therapy. The guideline also mentions diagnostic methods to be avoided or whose results should be interpreted with caution. Although the guideline cannot account for all individual variations among patients, it may provide instructions to physicians in typical and frequent clinical situations.

Detection of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in questing ticks Ixodes ricinus from the Czech Republic.

The aim of the present study is to compare the prevalence of Borrelia burgdorferi sensu lato (s.l.) and Anaplasma phagocytophilum in questing ticks by using molecular methods from spring to autumn 2007. A total of 526 Ixodes ricinus ticks were collected from vegetation in three different sampling sites, representing an urban area (city park), a suburban area (village) and a natural montane habitat. The prevalence of B. burgdorferi s.l. was 17.3% (7.5% in males, 18.7% in females and 20.2% in nymphs), while 4.4% of ticks (13.1% of males, 3.7% of females and 1.6% of nymphs) tested positive for A. phagocytophilum. We found higher rates of Anaplasma infection in ticks from the urban area (8.6%) than from the suburban (0.8%) and natural (1.6%) habitats in the spring months. The prevalence of Borrelia infection in the urban park increased significantly from spring (14% in March) to autumn (50% in October). The Anaplasma positivity in the urban area in the autumn months (2.2%) was significantly lower than in the spring and summer months (9.6%). The prevalence of A. phagocytophilum was significantly higher in male ticks than in females and nymphs. For B. burgdorferi s.l., the inverse was true. We conclude that infection risks associated with the presence of Anaplasma and Borrelia in ticks in cities may be comparable to those in natural ecosystems or may be even higher. Our results indicate the need for the surveillance of tick-borne pathogens in urban areas.

Emergence and genetic variability of Anaplasma species in small ruminants and ticks from Central Europe.

Anaplasmoses are common tick-borne zoonotic bacterial diseases of livestock and free-living ungulates from the genus Anaplasma that are recently emerging in Central Europe. The main aim of this study was to analyze the prevalence and genetic variability of Anaplasma phagocytophilum and Anaplasma ovis in small ruminants and questing ticks from six different sites in Slovakia and the Czech Republic using the PCR of the msp4 gene followed by the sequence analysis. At two farms from southeastern Slovakia, 66.1% small ruminants were infected with A. ovis in contrast to one positive animal from both sites in northern Slovakia. It was represented by two different genotypes. A. phagocytophilum was present in all tested flocks with the infection prevalence ranging from 0.9% to 5.7%. None of the tested questing ticks carried A. ovis. A. phagocytophilum was detected in 1.1% and 7.8% of questing Ixodes ricinus ticks collected around the farms located in southeastern and northern Slovakia, respectively. A. phagocytophilum revealed higher intraspecific diversity than A. ovis.

Detection of Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato in dogs in the Czech Republic.

The aim of this study is to present molecular, serologic, and clinical findings for dogs that were naturally infected with Anaplasma phagocytophilum or Borrelia burgdorferi sensu lato (s. l.) in the Czech Republic. This data can provide information relevant to human infection. In total, blood samples from 296 dogs and 118 engorged ticks were examined. Samples were tested for A. phagocytophilum using polymerase chain reaction (PCR) amplification, nested PCR, and direct sequencing of the 16S rDNA, and for B. burgdorferi s. l. using PCR amplification of the 16S rDNA and restriction fragment length polymorphism analysis of the 5S-23S rDNA intergenic spacer. In addition, blood samples were screened for antibodies to these bacteria. Ten (3.4%) dogs were PCR-positive for A. phagocytophilum. Morulae of A. phagocytophilum in granulocytes were found in two of these dogs. Nine of the PCR-positive dogs had clinical signs related to anaplasmosis. Statistically significant differences in the PCR detection rates were found between breeds and between symptomatic and asymptomatic dogs. Infection with Borrelia garinii was detected by PCR in a dog with meningoencephalitis. DNA of A. phagocytophilum and B. burgdorferi s. l. (B. garinii or Borrelia afzelii) was detected in 8.5% and 6.8% of ticks, respectively. Immunoglobulin (Ig) G seropositivity to A. phagocytophilum was 26%. Significant differences were found with respect to breed and gender. IgM and IgG antibodies to B. burgdorferi s. l. were detected in 2.4% and 10.3% of dogs, respectively. Our findings suggest that the exposure to B. burgdorferi s. l. exists in dogs in the Czech Republic, and exposure to A. phagocytophilum is common.