[Serology of Lyme borreliosis and human granulocytic ehrlichiosis in 2005-2010]. / Sérologie lymeské borreliózy a humánní granulocytární ehrlichiózy v letech 2005-2010.

The subject of this study is serological screening of blood and CSF (cerebrospinal fluid) samples for the presence of borrelial and ehrlichial antibodies. A total of 165 patients suspected to be at risk of Lyme disease were tested. Indirect immunofluorescence and enzyme immunoassay were used as diagnostic methods. Ehrlichial antibodies were detected in 36 (21.8%) patients. Borrelial antibodies were found in 70 samples (42.4%). The widening range of tick-borne diseases brings about the need for more data on these zoonoses.

Phenotypic and genotypic analysis of Borrelia spp. isolated from Ixodes ricinus ticks by using electrophoretic chips and real-time polymerase chain reaction.

The genotype of Borrelia burgdorferi sensu lato was detected in 371 out of 1244 ticks. Borrelia determination was based on partial sequencing of the 16S rRNA gene and real-time polymerase chain reactions for identification and quantitation of ospA and recA genes. Different Borrelia spp. were identified; B. garinii in 40% ticks followed by B. afzelii (36.3%), B. burgdorferi sensu stricto (12.9%), B. valaisiana (3.5%), B. lusitaniae (0.8%), B. bissettii (0.5%) and B. miyamotoi-like (0.5%). Cultivation of 30 borrelia strains in BSK-H medium, among them B. valaisiana, B. bissettii-like and B. miyamotoi-like strains was unique in Czechia. Calibrated microfluidic-based quantification showed differences in the concentration of the nucleic acids and molar mass of the outer surface proteins of different Borrelia spp. with standard sensitivity and specificity and was helpful for their identification. The outer surface protein OspA was absent in B. miyamotoi-like and the OspB protein in B. valaisiana, B. lusitaniae and in three subtypes of B. garinii.

Significance of Borrelia infection in development of dilated cardiomypathy (a pilot study).

A heart involvement known as Lyme carditis (LC), a consequence of Lyme borreliosis (LB), is relatively rare in contrast to the involvement of skin, joints and nervous system; it accounts for < 4% of all these patients in European countries. However, the diagnosis of the disease belongs to the most difficult challenges. While various forms of AV blocks dominate in the USA as confirmed by the literature, there is a clear predominance of arrhythmias of various incidence in the Czech Republic. The authors of this article focused on the form belonging to the rarest manifestations of LC, namely dilated cardiomyopathy (DCMP). The goal was to elucidate the etiological participation of Borrelia infection in the development of DCMP, which has attracted controversial opinions so far. In total, 33 patients with DCMP were enrolled in the study, 23 males and 10 females, with mean age 57.7 years (range 24-76 years). ELISA NRLB KC 90 method was used in all blood samples for detection of Borrelia infection (BI), Western blot method was used for confirmation, followed by identification of DNA of pathogenic Borreliae using PCR method. Bioptic material was examined by electronmicroscopy with an attempt to detect Spirochaetae in myocardium. 16 patients were excluded from the study owing to the absence of signs of LB. The study group included 17 patients (3 females, 13 males) with mean age 58 years (range 43-76 years), in whom the presence of Bb was proved by identification of DNA of pathogenic Borreliae or by electronmicroscopic detection of Spirochetae in myocardial bioptic sample. The findings obtained during the study confirmed that BI very probably participated in the development of dilated cardiomyopathy. It may be concluded that most of cases were either unapparent forms of LB or insufficiently treated cutaneous forms of this disease.

[The information value of detection of borrelia antibodies in the healthy blood donors and in the population at risk]. / Informacní hodnota prukazu antiborreliových protilátek u zdravých dárcu krve a rizikové skupiny populace.

BACKGROUND: Geographic differences of particular species of ticks and variant strains of Borrelias impede the comparison of local and foreign experience. The authors tried to support some literary observations with their own results and designed a study aimed at determination of anti-Borrelia antibodies (AB) in the selected population samples. The goal of the study was to find and statistically evaluate: a) difference in distribution of values of AB in the sample of probands from the risk and non-risk environment (foresters in Sumava and Prague blood donors, respectively) by means of ELISA method, b) tick bite frequency in the risk and non-risk groups, c) to determine and statistically evaluate the prevalence of probands with increased levels of antiborrelial antibodies in the local study population by ELISA method and the prevalence of probands with non-normal values found by Western blot (WB) method. METHODS AND RESULTS: In the group of Prague blood donors (control group), in total 200 probands were examined; in the risk group, 71 probands from Sumava forest were examined. Blood specimens were examined in the National Reference Laboratory for Lyme Borreliosis in Prague. In all specimens, ELISA NRLB KC90 was used. Western blot assay was used for confirmation. CONCLUSIONS: On the basis of literature review from Europe and USA, it is possible to generalize the following, despite some equivocations: 1. regional differences are not significant, with the exception of high-risk territories or "occupational exposure"; 2. there is no significant difference between males and females; 3. considerable caution is necessary in comparing the control and risk groups; prerequisite for such comparison is defining the characteristics of both groups.

Lyme borreliosis infection as a cause of dilated cardiomyopathy.

The relatively low percent of patients affected with the cardiac form of Lyme borreliosis is difficult to diagnose, especially if the disease manifests itself in ways other than atrio-ventricular blockade. The advanced stage of Lyme carditis manifesting as dilated cardiomyopathy is a special case of this affliction. The authors of this report present clinical experience with an attempt to support the working hypothesis about involvement of Lyme borreliosis infection in the development of dilated cardiomyopathy. The patients were clinically examined thoroughly with special attention to the cardiovascular system. In addition to the basic clinical methods, the following procedures have been employed: dynamic Holter's electrocardiography, exercise ECG test, coronarography, and myocardial biopsy. From laboratory methods pertaining to the detection of Borrelia, ELISA method, Western blot, PCR, electron microscopy and histopathological analysis were used. In all three cases, clinical and laboratory findings provided the evidence of the borreliosis infection involvement in the development of dilated cardiomyopathy.

Lyme borreliosis--waiting for Lyme carditis? A long-term prospective study.

A long-term prospective study of patients with confirmed non-cardiac form of Lyme disease (n=221) over a mean follow-up period of 40.6 months is reported. The study revealed no case of Borrelia-related cardiac involvement developed after several years in patients who had received antibiotic therapy in the early period. Therefore, these patients do not need follow-up by a cardiologist.

Localization of Borrelia burgdorferi in murine Lyme borreliosis by electron microscopy.

Lyme borreliosis is a newly recognized systemic infection with protean clinical manifestations. Because the localization of the causative spirochete (Borrelia burgdorferi) in infected tissues is unknown, we used electron microscopy to find spirochetes in the hearts of chronically infected mice. There were three predominant locations for the spirochete in the hearts. In mice infected for one month or less, the spirochetes were mostly in or around blood vessels. They were either in the lumen or in the perivascular space. Mice infected for more than one month had B. burgdorferi in cardiac myocytes as well, often with clear spaces around them. The third area in which spirochetes were common was collagen fibers; the borreliae were wrapped around fibers with their long axis parallel to the fibers. The number of spirochetes was relatively low, but there was no appreciable decrease in numbers of spirochetes with increasing time postinfection. Inflammatory infiltrates were primarily in the endocardium and pericardium, but spirochetes were generally not in or near areas of inflammation. These data are consistent with previously published information that have identified the heart as a site of chronic infection and inflammation in the mouse. The studies extend our understanding of the behavior of the spirochete in vivo by identifying common locations of B. burgdorferi and by noting the disparity between infection and inflammation.

The influence of a magnetic field on manganese transport into rat brain.

The aim of this study was to detect the effect of a magnetic field on manganese transport into rat brains. An experimental group of Female Wistar rats was given 0.48 mg Mn2+ per kg body weight intratracheally twice a week for 3 months and simultaneously exposed to a magnetic field: B = 10 mT, f = 50 Hz for 1 hr. Rats in one control group of rats received the same dose of manganese as the experimental group but were not exposed to the magnetic field. Rats in a second control group had neither exposure to manganese nor exposure to the magnetic field. After the last dose, all rats were sacrificed and their brains and other tissues were analyzed for manganese content. The results indicated that the magnetic field had a positive effect on increasing the manganese content in the brains of rats in the experimental group relative to those of the control groups. Visual evoked potentials (VEP) measured at the end of the exposure periods on randomly selected experimental and control rats showed a shortened but not statistically significant latency of the P1 peak of VEP in rats that had been exposed to both factors but not in control rats.

Seronegative Lyme arthritis caused by Borrelia garinii.

A case of a female patient suffering from Lyme arthritis (LA) without elevated antibody levels to Borrelia burgdorferi sensu lato is reported. Seronegative Lyme arthritis was diagnosed based on the classic clinical manifestations and DNA-detected Borrelia garinii in blood and synovial fluid of the patient, after all other possible causes of the disease had been ruled out. The disease was resistant to the first treatment with antibacterial agents. Six months after the therapy, arthritis still persisted and DNA of Borrelia garinii was repeatedly detected in the synovial fluid and the tissue of the patient. At the same time, antigens or parts of spirochaetes were detected by electron microscopy in the synovial fluid, the tissue and the blood of the patient. The patient was then repeatedly treated by antibiotics and synovectomy has been performed.

Interaction of Borrelia burgdorferi sensu lato with Epstein-Barr virus in lymphoblastoid cells.

Since the possibility of interruption of latent EBV infection has been suggested by the induction of the lytic virus cycle with chemical substances, other viruses, and by immunosuppression, we hypothesized that the same effect might happen in B. burgdorferi sensu lato infection as happens in Lyme disease patients with positive serology for both agents. We have observed EBV replication in lymphoblastoid cells after superinfection with B. garinii and B. afzelii strains after 1 and 4 h of their interaction. We found that viral and borrelial antigens persisted in the lymphoblasts for 3 and 4 days. Morphological and functional transformation of both agents facilitate their transfer to daughter cells. Association with lymphoblasts and internalization of B. garinii by tube phagocytosis increased replication of viruses more successfully than B. afzelii and chemical inductors. Demonstration of such findings must be interpreted cautiously, but may prove a mixed borrelial and viral cause of severe neurological disease.

The effect of an antiphlogistic incorporated in liposomes on experimentally induced inflammation.

The antiphlogistic Ibuprofen incorporated in liposomes caused a decrease of the inflammatory edema induced by Carrageenan in the distal part of the rat's hind leg after both the intramuscular and percutaneous administration. The antiphlogistic effect of free Ibuprofen in the cream was weaker. Intramuscular administration of empty liposomes slowed down in the initial stages the development of inflammation and slightly diminished the size of edema.

Electron microscopic demonstration of the interaction of liposomes and cells in vitro.

We have investigated the interaction of liposomes with the continuous cell lines P388D1 and L-132 and mouse peritoneal macrophages. To distinguish the liposomes from other vesicular structures, we have used liposomes with incorporated protein G and gold. A heterogeneous mixture of multilamellar liposomes 30 nm up to 1000 nm in size has been employed. Samples were examined at different time intervals. We found differences in the uptake of liposomes with regard to size and rate. Cells of a P388D1 monolayer took up liposomes by endocytosis very early after addition of liposomes and the number of lysosomes in their cytoplasm increased. In L-132 cells, first a fusion occurred between liposomes and the cell cytoplasmic protrusions, in the cytoplasm of which the mitochondria had multiplied. Peritoneal macrophages phagocytosed mainly large multilamellar liposomes and the membranous system of Golgi apparatus was the most prominent structure in the cytoplasm. Phagocytosis in P388D1 and L-132 cells was noted sporadically as late as 24 h after addition of liposomes to the cells.

Electron microscopic demonstration of the penetration of liposomes through skin.

To verify the penetration of liposomes through skin, we have used liposomes with encapsulated protein G-gold conjugate in a gel vehicle. Skin samples were examined 2, 4, 24, 48, and 72 h after liposome application. Our findings show that the penetration of liposomes through skin depends mainly on their size. Liposomes up to 600 nm in diameter penetrate through skin rather easily, whereas liposomes 1000 nm and more in diameter remain interiorized in the stratum corneum. The main penetration of liposomes proceeds along the hair sheaths as indicated by larger amounts of free liposomes in the corium of guinea pigs.

Interactions of invasive and noninvasive strains of Neisseria meningitidis with monkey epithelial cells, mouse monocytes and human macrophages.

Adherence and phagocytosis of invasive and noninvasive Neisseria meningitidis strains was investigated using light, fluorescence and electron microscopy. Invasive strains were isolated from the cerebrospinal fluid and/or blood of the patients with invasive meningococcal disease and noninvasive strains from the nasopharynx and/or larynx of healthy carriers. Adherence/endocytosis was studied on monkey kidney cells (the LLC-MK2 cell line) and phagocytosis on mouse monocytes and human macrophages (the P388D1 and U-937 cell lines, respectively). Although invasive and noninvasive meningococci isolated in the same cluster showed identical genotype and phenotype markers, they were found to interact differently with epithelial cells as well as with monocytes/macrophages. Invasive isolates displayed higher adherence to the surface of LLC-MK2 cells compared to noninvasive ones. Phagocytosis by P388D1 cells of noninvasive strains was effective and the bacteria were damaged by cytolysis. In contrast, invasive bacteria frequently persisted in "coiling" vacuoles and in effect could destroy the host cell. This is the first demonstration of coiling phagocytosis induced by meningococci. Efficiency of phagocytosis by U-937 cells was significantly higher for the noninvasive than invasive strains. Different behaviour of invasive and noninvasive strains of N. meningitidis observed during 4 hours of interactions with epithelial cells and monocytes/macrophages reflects well the higher pathogenic potential of invasive bacteria.

Molecular and microscopical evidence of Ehrlichia spp. and Borrelia burgdorferi sensu lato in patients, animals and ticks in the Czech Republic.

We report moderately severe cases of human ehrlichiosis and a lethal one caused by human granulocytic Ehrlichia, the HGE agent, closely related to Ehrlichia phagocytophila and Ehrlichia equi. Their vector is the Ixodes ricinus tick, which also transmits Borrelia burgorferi sensu lato in central, west and east regions of the Czech Republic. The diagnosis was established by PCR with sequence analysis of the genes encoding 16S rRNA of Ehrlichia and with reverse hybridization by using enzyme linked immunosorbent assay with different covalently coupled probes to the activated plate. Ten out of 47 patients and 10 huntsmen were PCR positive and 7 of them seroconverted to the HGE. Coinfection of Ehrlichia phagocytophila with Borrelia burgdorferi sensu lato was detected in 3 patients. Ehrlichia spp., the HGE agent, was isolated and propagated only from one patient in the HL-60 promyelocytic cell line. The maintenance of Ehrlichia in culture and in patients was assayed also by immunocytological staining and electron microscopy. Sequence or hybridization analysis of PCR results in different wild mammals and birds showed significant sources of Ehrlichia fagocytophila in nature. Three variants of E. phagocytophila in wild roe deer and boars, as well as for the first time in birds, have been described. Cultures from the blood of horses, and from the spleen and kidney specimens of roes and boars, PCR positive for Ehrlichia spp., displayed a disappearing level of the pathogen or contamination with other bacteria.

Scanning electron microscopy of the surface of the adult Multiceps endothoracicus and a comparison of its larval and adult scoleces.

Scanning electron microscopy disclosed differences in various areas of the surface of the adult tapeworm, and in larval and adult scoleces of M. endothoracicus. Microtriches with apically crooked spikes were found in the circlet of hooks of the adult tapeworm, and in the area between the outer and inner sucker margins, i.e., in sites important for intestinal attachment. Dimorphic microtriches were present on the inner margin of the suckers and around the genital pore, while the only area to contain dimorphic microtriches in the larva was the surface of the rostellum. An illustration is given of the egg and its fixation to uterine branches.

The morphogenesis of microtriches in the tegument of Taenia hydatigena throughout its larval development.

An electron microscope study was made on the development of microtriches, basic cytoplasmic inclusions, rod-shaped bodies and lamellated bodies in the larva of T. hydatigena. In the initial stage of its development, the tegument contains rod-shaped bodies and spineless or branching microtriches. An electron-dense substance released from rod-shaped bodies constitutes the basic substance of the tegument. Lamellated, secretory bodies appear in the tegument at the time of a differentiation of the scolex anlage. We believe that contents of lamellated bodies are released to the external environment either by their fusion with surface channels or their secernment in microtriches of the scolex anlage. The released secretion cements microtriches of a less active bladder in order to produce a defence barrier against host cells. Microtriches which have completed their secernment in an environment containing the secretory substance, close up in a point composed of the electron dense substance released from rod-shaped bodies. At the stage of encystment, microtriches become detached from the cementing substance which then is deposited on the inner side of the cyst wall. Lamellated bodies vacuolate.

The fine structure of larvae of Taenia hydatigena aged seven and ten days.

Studies on the fine structure of larval T. hydatigena during their initial phase of development have shown that the structure of their differentiating tegument is dissimilar to that of the adult cestode. It differs from it both in the number and shape of organelles (microvilli, rod-shaped bodies, invaginations of the basal membrane, mitochondria) present at the early ontogenesis of the bladder. Typical of the initial phase of the production of rod-shaped bodies in sub-tegumental syncytial cells is their limited appearance in the distal cytoplasm and the character of microvilli lacking both an electron-dense layer below the plasma membrane and distal points. At 10 days, during the further differentiation of the perinuclear cytoplasm, the number of rod-shaped bodies and invaginations of the basal membrane in the tegument increases, and more vesicles and cisternae of the Golgi apparatus appear in the cells. The layer of circular muscles and fibrils has not yet started to differentiate in the subtegument. It is dependent for its differentiation on the secretory activity of cells with dilated cisternae of the endoplasmic reticulum. The scolex anlage and the zone of growth of the bladder differentiate from proliferating germ cells. Glycogen-depositing cells are present at this stage of development. Later these cells change into glycogen-containing structures.

Fine structure of cells and tissues of Cysticercus tenuicollis aged 13 and 16 days.

A study has been made on the fine structure of three postembryonic phases of C. tenuicollis. We observed the differentiation of the villus-like microtriches without a distinct point. A lysis of host cells was caused by a secretion released from vesicles and droplets in the microthrix border. We distinguished 5 types of little-specialized cells during the initial phase of bladder development. The smallest of these cells were persisting oncospheral germ cells. The differentiation of cells terminated with the differentiation of the excretory system in a 16 day-old larva.

The fine structure and histochemistry of bleb-like extrusions in the microthrix border of the larval Multiceps endothoracicus.

The study on the fine structure and the histochemistry of microtriches in the spiral canal and the scolex of a larva of the polycephalic cestode Multiceps endothoracicus suggested a defensive mechanism of a part of the parasite against invading cells of the parasite against invading cells of the tissue reaction of the host. Numerous vesicles and bleb-like extrusions were present in the transitory zone of the opening of the spiral canal and in the rostellar region of the scolex in close contact with deteriorating host cells. Both, the vesicles and bleb-like extrusions gave a positive reaction for SS and SH groups and displayed an enzymatic activity of acid phosphatase (AP) and nonspecific esterase (NE). Scanning and transmissive electron microscopy revealed the process of formation of bleb-like extrusions from the distal cytoplasm and showed that the appearance of the microtriches was being changed by an accumulation of products of secernation and secretion responsible for a lysis of the host cells.