Tick-borne pathogens Bartonella spp., Borrelia burgdorferi sensu lato, Coxiella burnetii and Rickettsia spp. may trigger endocarditis.

BACKGROUND: Infections caused by tick-borne pathogens such as Bartonella spp., Borrelia burgdorferi s.l., Coxiella burnetii, and Rickettsia spp. are capable of causing serious lesions of the mitral and aortic valves, leading to a need for valve replacement. OBJECTIVES: The aim of the study was to determine whether such cases are sporadic or frequent. An additional goal was to establish effective diagnostic methods to detect these infections. MATERIAL AND METHODS: The study involved 148 patients undergoing valve replacement. Blood samples were drawn for serological testing. Samples of the removed mitral and aortic valves were tested with polymerase chain reaction and immunohistochemical staining. RESULTS: Specific antibodies to Bartonella spp. were detected in 47 patients (31.7%) and in 1 of the healthy controls (1%) (p < 0.05). Antibodies to B. burgdorferi spirochetes were found in 18 of the patients (12.2%) and in 6 blood donors from the control group (5.8%) (p < 0.1). Antibodies to Rickettsia spp. were detected in 12 (8.1%) and to C. burnetii phase I and II antigens in the serum of 1 patient. All the participants in the control group were seronegative to C. burnetii and Rickettsia spp. antigens. Polymerase chain reaction (PCR) tests for detection of Bartonella spp., B. burgdorferi s.l., C. burnetii and Rickettsia spp. DNA in the valve samples were all negative. Inflammation foci with mononuclear lymphoid cells in the aortic and mitral valves were seen in sections stained with hematoxiline and eozine. In sections dyed using the indirect immunofluorescence method with hyperimmune sera, Bartonella spp. and Rickettsia spp. were found. CONCLUSIONS: The results obtained indicate that laboratory diagnostics for patients with heart disorders should be expanded to include tests detecting tick-borne zoonoses such as bartonelloses, Lyme borreliosis, rickettsioses and Q fever.

Prevalence of Borrelia Burgdorferi Sensu Lato in Ticks from the Ternopil Region in Ukraine.

INTRODUCTION: Lyme borreliosis/Lyme disease is caused by Borrelia burgdorferi and is one of the most common vector-borne diseases transmitted by ticks. MATERIAL AND METHODS: A total of 136 Ixodes ricinus ticks, collected in the Ternopil (Ukraine) region, including 126 adults (70 females and 56 males), and 10 nymphs were examined. The identification of the species and their developmental form was based on morphological characteristics. RESULTS: PCR with B5S-Bor and 23S-Bor primers resulted in Borrelia burgdorferi sensu lato DNA amplification among six ticks (4.4%). The detailed analysis based on the DNA sequencing showed the presence of DNA of Borrelia afzelii in four samples; the remaining two represented Borrelia burgdorferi sensu lato complex, although their genospecies were not determined. The research confirmed the dominance of Borrelia afzelii genospecies in the ticks from Ukraine. CONCLUSION: It seems reasonable to undertake similar research in ticks from other regions of Ukraine. Knowledge in this field can be useful for public health and planning the prevention of tick-borne diseases.

Francisella tularensis/Rickettsia spp. co-infections in patients with skin changes and lymphadenopathy.

INTRODUCTION: Tularemia and spotted fever group rickettsioses (SFG) can be transmitted by ticks and have a number of common clinical symptoms. Most characteristic are a maculopapular or vesicular rash or an eschar at the site of the tick or insect bite accompanied by painful lymph nodes. The aim of this study was to determine whether Rickettsia spp./Francisella tularensis mixed infections occurred in patients with similar symptoms who were diagnosed with either Rickettsia spp. or F. tularensis infection. MATERIAL AND METHODS: Thirty-six cases from 2011-2014, including 15 individuals with clinically and serologically recognized SFG and 21 with tularemia, were analyzed retrospectively using immunofluorescence for detection of Rickettsia spp. or ELISA for detection of F. tularensis. RESULTS: Of the 36 cases examined, specific high titers of antibodies to Rickettsia spp. were found in 1 (4.4%) patient with tularemia and specific high titers of antibodies to F. tularensis were detected in 1 (6.7%) patient with SFG. CONCLUSIONS: The results of our study show that in infections with fever, enlarged lymph nodes and skin lesions after tick and insect bites, laboratory testing of both diseases - SFG rickettsiosis and tularemia - should be implemented. Identification of F. tularensis and Rickettsia spp. mixed infections is crucial in order to administer appropriate antibiotics and to avoid treatment failure and relapse.

An outbreak of leptospirosis imported from Germany to Poland.

BACKGROUND: Leptospirosis is a zoonotic disease caused by spirochetes of the Leptospiraceae family. In both humans and animals the main route of infection is indirect contact - through water or other products contaminated with urine containing spirochetes. Infection most commonly occurs through ingestion of water or food contaminated with Leptospira spp. OBJECTIVES: The aim of the study was to characterize cases of leptospirosis imported to Poland from Germany in 2014 and to analyze methods that are helpful for making a diagnosis. MATERIAL AND METHODS: The 10 patients examined were reported as suspected leptospirosis cases on the basis of clinical symptoms and epidemiological investigations. They originated from different regions of Poland and had been working together at a strawberry plantation in the Cloppenburg district of Lower Saxony in Germany. Blood and urine samples were tested by PCR and serum samples by serology. All ELISA positive and negative cases were examined using a reference microscopic agglutination test (MAT). RESULTS: In the tested group, 6 individuals (60%) were seropositive according to the ELISA, and 2 of them were confirmed by the MAT. The PCR results for the blood and urine samples were negative. CONCLUSIONS: Using the ELISA in the diagnosis of leptospirosis allowed the disease to be identified much faster, differentiating classes of antibodies and recognizing levels of them that are too low to be detectable by the MAT.

Bactericidal Activity of Octenidine to Various Genospecies of Borrelia burgdorferi, Sensu Lato Spirochetes in Vitro and in Vivo.

The aim of our studies was to invent a reliable method for detection of the bactericidal activity of disinfectants against Borrelia burgdorferi in suspension (in vitro) and in cell line cultures (in vivo). In the suspension method, 0.01% octenidine at 20°C and 35°C was bactericidal to Borrelia afzeli; Borrelia garini, B. burgdorferi sensu stricto after 5 minutes treatment. Increase of the temperature to 35°C speed up the bactericidal effect to 1 minute. The bactericidal action of octenidine towards B. burgdorferi spirochetes growing in fibroblasts was less effective and needed a longer time to kill them than in the suspension.

Seroprevalence of Bartonella Species in Patients with Ocular Inflammation.

Bartonella species, vector-borne etiologic agents of many systemic or self-limited infections, are responsible for a widening spectrum of diseases in humans, including inflammatory conditions of the eye. The aim of this study was to determine whether there is any relationship between uveitis and the evidence of Bartonella spp. infection in the serum, ocular fluid, and cataract mass in patients with intraocular inflammation. Polymerase chain reaction (PCR)-based tests and DNA sequencing were performed on surgery-extracted specimens of intraocular fluid and lens mass of 33 patients. Sera from 51 patients and 101 control subjects were tested for the presence of specific antibodies against Bartonella spp. Neither IgM-class antibodies against Bartonella spp. nor Bartonella spp. DNA were detected. A specific IgG-class antibody was found in 33.3% of the patients with uveitis. The rate of positive Bartonella serology was higher among the uveitis patients than that in control subjects. This high rate may in part result from unrecognized indirect mechanisms rather than the immediate presence and multiplication of Bartonella spp. in the eyeball. Nonetheless we believe that screening for Bartonella spp. should become part of the diagnostic workup in uveitis.

Borrelia miyamotoi - new etiologic agent of neuroborreliosis?

Borrelia miyamotoi spirochetes discovered in Ixodes persulcatus ticks in Japan, in 1994 and documented in ticks and rodents in moderate climate zone of northern hemisphere. They belong to tick-borne relapsing fever group spirochetes. Borrelia miyamotoi is an etiologic agent of B.miyamotoi disease with acute febrile illness, including fever, headache, dizziness, fatigue, chills, and muscle and joint pain. Recurrence of fever has been observer in more than 10% patients. In some patients meningoencephalitis, encephalitis and cranial neuritis were observed. Laboratory recognition is based mainly upon PCR testing. Serological testing is limited due to inaccessibility of tests.

Analysis of nanomechanical properties of Borrelia burgdorferi spirochetes under the influence of lytic factors in an in vitro model using atomic force microscopy.

BACKGROUND: Atomic force microscopy (AFM) is an experimental technique which recently has been used in biology, microbiology, and medicine to investigate the topography of surfaces and in the evaluation of mechanical properties of cells. The aim of this study was to evaluate the influence of the complement system and specific anti-Borrelia antibodies in in vitro conditions on the modification of nanomechanical features of B. burgdorferi B31 cells. MATERIAL AND METHODS: In order to assess the influence of the complement system and anti-Borrelia antibodies on B. burgdorferi s.s. B31 spirochetes, the bacteria were incubated together with plasma of identified status. The samples were applied on the surface of mica disks. Young's modulus and adhesive forces were analyzed with a NanoScope V, MultiMode 8 AFM microscope (Bruker) by the PeakForce QNM technique in air using NanoScope Analysis 1.40 software (Bruker). RESULTS/CONCLUSION: The average value of flexibility of spirochetes' surface expressed by Young's modulus was 10185.32 MPa, whereas the adhesion force was 3.68 nN. AFM is a modern tool with a broad spectrum of observational and measurement abilities. Young's modulus and the adhesion force can be treated as parameters in the evaluation of intensity and changes which take place in pathogenic microorganisms under the influence of various lytic factors. The visualization of the changes in association with nanomechanical features provides a realistic portrayal of the lytic abilities of the elements of the innate and adaptive human immune system.

Bacterial tick-borne diseases caused by Bartonella spp., Borrelia burgdorferi sensu lato, Coxiella burnetii, and Rickettsia spp. among patients with cataract surgery.

BACKGROUND: Clinical data have shown that tick-borne diseases caused by Borrelia burgdorferi sensu lato, Bartonella spp., Coxiella burnetii, and Rickettsia spp. can affect the central nervous system, including the eye. The aim of this study was to establish a relationship between the incidence of cataract and evidence of bacterial infections transmitted by ticks. MATERIAL AND METHODS: Fluid with lenticular masses from inside of the eye and blood from 109 patients were tested by PCR and sequencing. Sera from patients and the control group were subjected to serological tests to search specific antibodies to the bacteria. RESULTS: Microbiological analysis revealed the presence of Bartonella sp. DNA in intraoperative specimens from the eye in 1.8% of patients. Serological studies have shown that infections caused by B. burgdorferi sensu lato and Bartonella sp. were detected in 34.8% and 4.6% of patients with cataract surgery, respectively. CONCLUSIONS: Presence of DNA of yet uncultured and undescribed species of Bartonella in eye liquid indicates past infection with this pathogen. Specific antibodies to B. burgdorferi sensu lato and Bartonella sp. are detected more frequently in patients with cataract compared to the control group. This could indicate a possible role of these organisms in the pathological processes within the eyeball, leading to changes in the lens. Further studies are needed to identify Bartonella species, as well as to recognize the infectious mechanisms involved in cataract development.

Q fever outbreaks in Poland during 2005-2011.

BACKGROUND: Q fever is a health problem affecting humans and animals worldwide. In Poland, previous studies have pointed to 2 sources of outbreaks of the disease: the importation of infected animals and their products, and natural domestic foci. In the last decade, 5 outbreaks have occurred in cattle farms in south Poland in Malopolskie, Podkarpackie, Opolskie, and Silesian provinces. The aim of this study was to characterize the Q fever foci in Poland. MATERIAL AND METHODS: A total of 279 individuals were included. Levels of serum IgM and IgG antibodies to phase I and II C. burnetii antigens were assayed by indirect immunofluorescence method. Bacterial DNA from all specimens were detected with PCR with primer pairs specific to the htpAB-associated repetitive element, and amplicons were sequenced. RESULTS: Infection was recognized in 67 individuals out of 279 tested in all foci. Twenty-five individuals presented clinical symptoms of acute Q fever. DNA of C. burnetii was found in 8 human blood samples obtained from 3 farm workers and 5 family members. CONCLUSIONS: The described outbreaks demonstrate that the main source of human infections in Poland is infected cattle.

Prevalence of rickettsioses in Poland in 2006-2012.

INTRODUCTION: Tick-borne diseases, such as rickettsioses, anaplasmosis, Lyme boreliosis and bartonellosis are often difficult to correctly diagnose. All these disease are present in Poland. OBJECTIVES: The aim of the study was to estimate a prevalence of Rickettsia spp. infections in humans in Poland in 2006 to 2012 based on the results made in the Laboratory of Rickettsiae, Chlamydiae and Spirochetes, NIPH-NIH in Warsaw. MATERIAL AND METHODS: The levels of Rickettsia spp. and Anaplasma phagocytophilum IgM and IgG antibodies were determined by indirect immunofluorescent assay (IFA). From 2006 to 2012, serum samples derived from 180 humans suspected for rickettsioses, including 84 patients suspected for the infections with typhus and spotted fever group (SFG) rickettsiae, and 96 patients suspected for anaplasmosis. RESULTS: Specific serum antibodies to the SFG rickettsiae have been detected in 5 persons (2.7%). Granulocytic anaplasmosis has been recognized in 9 patients (4.9%). While the reporting and registration of rickettsioses are obligatory in Poland less than 50% of detected cases are reported. CONCLUSIONS: Presented date indicate that in Poland rickettsioses are often unrecognized resulting in their underestimation. If research for rickettsiosis are made immediately after infection, antibodies will not be detect.

Characteristics of Bartonella spp. infections in Poland in the years 2009-2012 identified in the laboratory of National Institute of Public Health--National Institute of Hygiene.

INTRODUCTION: Various Bartonella species, (Gram-negative aerobic bacilli) are etiologic agents ofzoonotic diseases called bartonelloses, which manifest with different symptoms depending on the bacterial species, reservoir and vector. In Poland and Europe, the most common bacterial species of the genus Bartonella is Bartonella henselae. MATERIAL AND METHODS: [corrected] Serum samples derived from patients with clinical symptoms suggesting Bartonella spp. infection, sent in 2009-2012 to the Laboratory of Rickettsiae, Chlamydiae and Spirochaetes of National Institute of Public Health--National Institute of Hygiene in Warsaw were tested. Levels of specific IgM and IgG antibodies to B. henselae and B. quintana antigens were detected with indirect immunofluorescence method (IFA). RESULTS: Six hundred sixty three serum samples were examined from humans with clinical symptoms suggestive bartonellosis, in 2009-2012. Specific antibodies for B. henselae were detected in 435 patients (65.6%). IgM antibodies were found in 93 patients (21.4%) including 11 patients (2.5%) with IgM only. IgG antibodies were identified in 424 people (78.6%) of whom 342 had IgG antibodies only. The antibodies of both classes were detected in 82 people (18.9%). B. quintana infections were not found. The majority of samples for study of bartonellosis were submitted in the autumn. In patients with confirmed bartonellosis, the most common symptoms of disease were lymphadenopathy (86 people, 13%), fever (13 patients, 2%) and nodular changes in various organs (13 patients, 2%). CONCLUSIONS: Infections caused by Bartonella spp. in Poland should be monitored to acquire the information on the frequency and distribution of disease in the country and their clinical course.

Case report of African tick-bite fever from Poland.

A confirmed case of rickettsiosis acquired in South Africa and recognized in Poland was described. The patient fulfilled clinical criteria highly suggestive of African tick bite fever, such as eschars, regional lymphadenitis, cutaneous rash within 10 days after his return from sub-Saharan Africa. Infection with Rickettsia africae was confirmed by polymerase chain reaction and sequencing.

Q fever at the turn of the century.

Q fever is an infectious zoonotic disease characterized by sudden fever, headache, and atypical pneumonia, caused by Coxiella burneti--an obligatory intracellular parasite. Based on phylogenetic analysis of the genes sequences, the classification was changed and C. burnetii species was included to the gamma subgroup of the proteobacteria, Legionellales order and Coxiellaceae family. This analysis showed more than 99% sequence similarity of 16SrRNA gene among the strains isolated in different regions of the world. Q fever is a widespread in the world zoonosis. Its main reservoir in the rural environment are farm animals: cows, sheep, goats, and urban pets such as dogs, cats, rabbits. In acute infection these bacteria are detected in various internal organs such as lungs, liver, spleen, and in excretion in urine, faeces and milk. During childbirth, they occur in large number in the amniotic fluid and placenta. Recently, it has been found that free-living amoeba Acanthamoeba castellani may also be a reservoir of the pathogen. The intra-amoebal location of C. burnetii cells was observed.

Susceptibility of Polish Bartonella henselae strains.

Due to the fastidious nature of B. henselae and the limited number of available isolates worldwide, there are few data on its in vitro susceptibility to antibiotics. We determined the minimal inhibitory concentrations (MIC) of ten antimicrobial agents against 11 feline isolates of B. henselae by Etest method. The lowest MICs were obtained for rifampicin < or = 0.002 mg/L. MICs of all isolates were < 0.016 mg/L for ampicilin, amoxicillin/clavulanic acid, tetracycline and ranged from 0.016 to 0.032 mg/L for azithromycin. The MICs for two tested fluoroquinolones: ciprofloxacin and levofloxacin ranged from 0.016 to 0.125 mg/L. The highest MICs were obtained for gentamicin ranging from 0.025 to 2.0 mg/L. Sulphonamide resistance genes sul 1, sul 2, sul 3 were not found in any of the tested isolates. Etest methodology seems to be a reliable method for determination of B. henselae susceptibility, however standardization is strongly desired.

Spotted fever rickettsiosis caused by Rickettsia raoultii--case report.

We report the case of rickettsial eschar-associated spotted fever, most probable due to Rickettsia raoultii, an emerging pathogen, which was previously described in patients with tick-borne lymphadenopathy (TIBOLA), also called Dermacentor-borne necrosis erythema and lymphadenopathy (DEBONEL). The pathogenicity of R. raoultii is not well established. The survey of ticks from Poland (Ixodes ricinus and Dermacentor reticulatus) revealed that R. raoultii occur in all regions of Poland and predominate over other rickettsiae of spotted fever group--R. slovaca and R. helvetica. A 17-year-old otherwise healthy girl was admitted to Department of Zoonotic and Tropical Diseases because of fever, eschar and rash. Multiple disseminated small lesions were present on the skin of her head, trunk and limbs, also palms and soles, and mucosa of her lips. The majority of them had necrotic center slightly elevated with redness around, single ones had vesicular appearance. The lymph nodes on the right side of her neck were enlarged. Laboratory investigations revealed: leukopenia (with 22% of bands in differential), thromocytopenia, slightly elevated C-Reactive Protein, as well as procalcytonin. The quick improvement was observed with a treatment with ceftriaxone and doxycycline. Two weeks after the onset of disease, IgG serum antibodies titer of 128 reacting with R. rickettsii antigen only was detected. IFA tests with six SFG rickettsial species demonstrated the strongest reaction with R. raoultii group antigens in a titer of 64. The case we report, resembling boutonneuse fever, with leukopenia, thrombocytopenia and septic parameters indicates possible higher virulence of R. raoultii than it was previously observed.

Characterization of Polish feline B. henselae isolates by multiple-locus tandem repeat analysis and pulse-field gel electrophoresis.

Knowledge about molecular epidemiology of B. henselae is important for recognizing the geographical distribution of strains and identification of isolates virulent for humans. Eleven Polish feline B. henselae isolates were typed, using 2 different techniques: pulse-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA). PFGE analysis distinguished 6 different PFGE types, with subtypes within 3 of them, whereas 10 MLVA types were assigned. Global diversity index (D.I.) for MLVA equaled 0.93. For 7 isolates, the results of MLVA confirmed cluster assignments based on PFGE. Both PFGE and MLVA results were in accordance with epidemiological data. Although PFGE has been previously demonstrated to be a suitable method for the differentiation of B. henselae isolates/strains, our results show the superiority of MLVA over PFGE with respect to higher discriminatory power, distinguishing genotypes I and II isolates, easier analysis of results, and possibility to compare the numerical data obtained by different laboratories. With MLVA, 7 new profiles were observed, compared to previous results from around the world; whereas 3 known profiles were previously described mainly in European B. henselae isolates. Our results confirm that some VNTR profiles can be used as specific geographical markers.

Borrelia burgdorferi sensu lato as activators of the complement system in in vitro model.

INTRODUCTION: The following parameters were analyzed: C5a, which is significant in classical and alternative pathways of the complement system activation, and factor H, the major function of which is to regulate the alternative pathway. Factor H, in the case of Borrelia infection, is combined by CRASPs proteins of spirochetes, and thus prevents C3b molecules from contact with the pathogen, opsonisation and lysis of bacteria. MATERIAL AND METHODS: The experimental material in the model for the presented work consisted of whole blood of healthy persons (without the presence of antibodies anti-Borrelia) and persons with clinical symptoms of Lyme disease, which was stimulated with three genospecies of spirochetes recognized as pathogenic in Poland and Europe: B. afzelii, B. burgdorferi s.s. and B. garinii. RESULTS: The increase in the level of C5a in the experimental model after in vitro stimulation of whole blood with three genospecies Borrelia can be treated as an indicator of an effective activation of the complement's cascade. The increase in level C5a in the plasma relies on the genospecies and the strongest is for B. garinii. The decrease in the level of factor H, observed after the incubation of whole blood with three genospecies Borrelia, shows that this parameter was included in the spirochetes' mechanisms acting against factors of the innate immunity system of a host, which prevents lysis of bacteria via the alternative pathway. CONCLUSIONS: The results obtained on the basis of the in vitro model can be analysed from the aspect of spirochetes' real contact with a host's organism during the bite of infected ticks. Despite blocking of the alternative pathway, Borrelia initiate the activation cascade regardless of antibodies via the first contact of a host's organism with spirochetes, or in accordance with antibodies during the infection or subsequent contact with bacteria.

[Leptospira spp. and Coxiella burnetii infections occuring in Radomskie District in people of selected professional groups]. / Zakazenia Leptospira spp. i Coxiella burnetii wystepujace w powiecie radomskim u ludzi z wybranych grup zawodowych.

INTRODUCTION: Leptospirosis and Q fever are a zoonotic diseases with global occurring. OBJECTIVES: The aim of this study was to determine the prevalence of Leptospira spp. and C. burnetii in humans, who have contacts with infected animals or are exposed to an environment potentially contaminated with these bacteria. MATERIAL AND METHODS. Blood serum samples originating from 177 veterinarians and farmers and 134 garbage collectors (blood samples) were examined. Control group consisted 43 blood samples derived from blood donors. For the detection of specific IgM and IgG antibodies of Leptospira spp., enzyme-linked immunosorbent assay (ELISA) was used. Indirect immunofluorescence method (IFA) was used for detection of specific IgG C. burnetii antibodies. DNA of Leptospira spp. and C. burnetii was detected by PCR method with appropriate pairs of primers. RESULTS: Specific IgG C. burnetii antibodies of phase II were detected in sera of 4.4% of the farmers and veterinarians, and in 12% of garbage men. Antibodies in blood donors was not found. Antibodies of Leptospira spp. were present in the serum of 23.6% of farmers and veterinarians, 26.2% of garbage men and 14% of blood donors. C. burnetii DNA was detected in one sample derived from the veterinarian (1.1%). Leptospira spp. DNA was not detected in tested material. Blood samples from farmers, veterinarians and garbage collectors showed the higher prevalence of antibodies of Leptospira spp. and C. burnetii as compared to the control group (blood donors). CONCLUSIONS: Beside farmers and veterinarians, garbage collectors should be consider as high risk group of contracting leptospirosis and Q fever. Both leptospirosis as well as Q fever should be considered in the differential diagnosis in humans with animals and animals' material contact when they reveal flu-like symptoms.