Assessment of antibodies against surface and outer membrane proteins of Anaplasma phagocytophilum in Lyme borreliosis and tick-borne encephalitis paediatric patients.

To examine evidence of positive antibodies against immunogenic proteins of Anaplasma phagocytophilum in patients with other tick-borne infections and to diagnose possible co-infections, 412 serum specimens were tested by immunoblotting using three specific Anaplasma antigens: surface proteins p44 and Asp62 and outer membrane protein A (OmpA). In total, 284 serum samples from children with Lyme borreliosis and 12 serum samples from children with tick-borne encephalitis were tested. Sera from patients with viral aseptic meningitis (n = 47) and from blood donors (n = 69) were used as controls. Among all serum specimens from patients with tick-borne infections submitted for this study, six samples (2·0%) showed positive IgM reactions and seven samples (2·4%) were IgG positive for A. phagocytophilum by immunoblot. Borderline reactivity was found in 30 samples (10·14%) for IgM and 36 samples (12·2%) for IgG. The difference between patients and blood donors was statistically significant for IgM (P = 0·006) and for IgG (P = 0·0007) antibodies. A statistically significant result was obtained for IgG (P = 0·02) but not for IgM between patients and children with aseptic meningitis. Immunoblot using three specific antigens provides novel information about the positivity of antibodies to A. phagocytophilum in children with other tick-borne infections. Taking into account clinical and laboratory findings of children despite antibody positivity, no case of human granulocytic anaplasmosis was demonstrated.

Intrathecally produced IgG and IgM antibodies to recombinant VlsE, VlsE peptide, recombinant OspC and whole cell extracts in the diagnosis of Lyme neuroborreliosis.

Detection of intrathecally produced specific antibodies (AI) is essential in the diagnosis of Lyme neuroborreliosis (LNB); however, the performance of various newer AI detection methods has not been systematically assessed. Here we assessed and compared advanced test systems for detecting borrelia IgG-AI and IgM-AI. Serum and cerebrospinal fluid (CSF) samples from well-defined LNB and tick-borne encephalitis (TBE) patients, 25 each, were tested with three antibody detection systems, one based on chemiluminescence (CLA) and two based on enzyme-linked immunosorbent assays (ELISA), employing different antigens for detection of IgG and IgM antibodies. In samples from patients with LNB, IgG-AI was detected in 20 samples by CLA, 19 by ELISA1, and 22 by ELISA2, and IgM-AI was detected in 16 samples by CLA, six by ELISA1, and 11 by ELISA2. In samples from TBE patients, IgG-AI was positive in one case by CLA and ELISA2, and in 7 cases by ELISA1, whereas IgM-AI was positive in one case by CLA and in none by ELISA. IgG-AI and IgM-AI were not detected within the first week of disease. Duration of disease correlated with IgG-AI while IgM-AI results were heterogeneous for each test assay. Moreover, the levels of IgG-AI, but not IgM-AI, correlated with protein concentration in CSF. IgG is the relevant immunoglobulin isotype for detecting intrathecal synthesis of borrelia antibodies. The highest sensitivity and specificity were achieved by the antibody detection assay using VlsE IR6 peptide. Detection of IgM-AI yielded heterogenous results and did not support the laboratory diagnosis of LNB.

[Human granulocytic anaplasmosis and its concurrence with other tick-borne infections in the Republic of Altai].

Human granulocytic anaplasmosis (HGA) cases occur everywhere in the Republic of Altai. Anaplasma phagocytophilum is transmitted to humans by at least three genera of ticks: Ixodes, Dermacentor, and Haemaphysalis. The risk of the infection is higher in the northern part of the republic. Most patients with HGA show serologic evidence of coinfection with North Asian tick typhus and other tick-borne diseases. The high probability of HGA concurrent with other tick-borne infections requires an integrated approach to preventing these diseases in the Republic of Altai.

[Coexistence of two occupational infectious diseases in one patient--borreliosis and tick-borne encephalitis--a case report]. / Borelioza i kleszczowe zapalenie mózgu jako wspólistniejace choroby zawodowe--opis przypadku klinicznego.

Recently, infectious diseases have been found to be the most frequent among occupational diseases. Borreliosis, the most common among them, as well as tick-borne encephalitis is transmitted by ticks. Recognition of occupational etiology of such diseases is possible only when the relationship between the infection, occupational exposure and performed work is proved. A case report of a forest worker with borreliosis coexisting with tick-borne encephalitis is presented. Despite nonconcurrent recognition of both diseases it was highly possible that contagion took place at the same time or at a very short time interval. Despite high prevalence of tick-borne diseases, occupational etiology of these two infectious diseases in one patient is very rarely recognized.

Experimental infection of lambs with tick-borne encephalitis virus and co-infection with Anaplasma phagocytophilum.

Tick-borne encephalitis virus (TBEV) is a zoonotic pathogen which may cause tick-borne encephalitis (TBE) in humans and animals. More than 10,000 cases of TBE are reported annually in Europe and Asia. However, the knowledge on TBE in animals is limited. Co-infection with Anaplasma phagocytophilum and louping ill virus (LIV), a close relative to TBEV, in sheep has been found to cause more severe disease than single LIV or A. phagocytophilum infection. The aim of this study was to investigate TBEV infection and co-infection of TBEV and A. phagocytophilum in lambs. A total of 30 lambs, aged five to six months, were used. The experiment was divided into two. In part one, pre- and post-infection of TBEV and A. phagocytophilum was investigated (group 1 to 4), while in part two, co-infection of TBEV and A. phagocytophilum was investigated (group 5 and 6). Blood samples were drawn, and rectal temperature was measured daily. Lambs inoculated with TBEV displayed no clinical symptoms, but had a short or non-detectable viremia by reverse transcription real-time PCR. All lambs inoculated with TBEV developed neutralizing TBEV antibodies. Our study is in accordance with previous studies, and indicates that TBEV rarely causes symptomatic disease in ruminants. All lambs inoculated with A. phagocytophilum developed fever and clinical symptoms of tick-borne fever, and A. phagocytophilum was present in the blood samples of all infected lambs, shown by qPCR. Significantly higher mean TBEV titer was detected in the group co-infected with TBEV and A. phagocytophilum, compared to the groups pre- or post-infected with A. phagocytophilum. These results indicate that co-infection with TBEV and A. phagocytophilum in sheep stimulates an increased TBEV antibody response.

Prevalence of Rickettsia spp. in Ticks and Serological and Clinical Outcomes in Tick-Bitten Individuals in Sweden and on the Åland Islands.

Tick-transmitted diseases are an emerging health problem, and the hard tick Ixodes ricinus is the main vector for Borrelia spp., tick-borne encephalitis virus and most of the spotted fever Rickettsiae in Europe. The aim of the present study was to examine the incidence of rickettsial infection in the southernmost and south central parts of Sweden and the Åland Islands in Finland, the risk of infection in humans and its correlation with a bite of a Rickettsia-infected tick, the self-reported symptoms of rickettsial disease, and the prevalence of co-infection between Rickettsia spp. and Borrelia spp. Persons with a recent tick bite were enrolled through public media and asked to answer a questionnaire, provide a blood sample and bring detached ticks at enlistment and at follow-up three months later. Blood samples were previously analysed for Borrelia spp. antibodies and, for this report, analysed for antibodies to Rickettsia spp. by immunofluorescence and in 16 cases also using Western Blot. Ninety-six (44.0%) of the 218 participants were seropositive for IgG antibodies to Rickettsia spp. Forty (18.3%) of the seropositive participants had increased titres at the follow-up, indicating recent/current infection, while four (1.8%) had titres indicating probable recent/current infection (≥1:256). Of 472 ticks, 39 (8.3%) were Rickettsia sp. positive. Five (31.3%) of 16 participants bitten by a Rickettsia-infected tick seroconverted. Experience of the self-reported symptoms nausea (p = 0.006) and radiating pain (p = 0.041) was more common among those with recent, current or probable infection compared to those who did not seroconvert. Participants who showed seroreactivity or seroconversion to Rickettsia spp. had more symptoms than those who were seronegative. Seven (3.2%) participants showed seroconversion to Borrelia spp., and three (1.4%) of these showed seroconversion to both Rickettsia spp. and Borrelia spp., in accordance with previous studies in Sweden. Symptoms of rickettsial disease were in most of the cases vague and general that were difficult to differentiate from other tick-borne diseases.

The importance of tick-borne encephalitis virus RNA detection for early differential diagnosis of tick-borne encephalitis.

BACKGROUND: Tick-borne encephalitis virus (TBEV) is one of the most important causes of human viral infections of the central nervous system in Europe. Currently, the diagnosis of TBE is based on the demonstration of specific antibodies in patient's serum, which appear only several weeks after the infection. OBJECTIVE: To determine how successfully can viral RNA be detected by RT-PCR in the samples of body fluids of patients with TBE prior to and after the appearance of antibodies. STUDY DESIGN: Serum, whole blood and CSF samples from 34 patients with a serologically confirmed TBE were collected. Samples were tested for the presence of TBEV RNA by using RT-PCR method. RESULTS: Viral RNA was detected in all blood and serum samples collected before the development of antibodies. After the appearance of IgM antibodies, the number of positive samples dropped by at least one third. After the development of IgG antibodies, only 3% of serum and 16% of blood samples tested positive for viral RNA. Samples of cerebrospinal fluid were shown to be inappropriate for the molecular diagnosis of TBE using this assay, since only one sample (10%) that was collected in the sero-negative phase of disease was found positive by the PCR assay. CONCLUSIONS: RT-PCR is an efficient method for an early detection of TBEV in blood and serum samples collected prior to the appearance of antibodies. This method can be of valuable use for a differential diagnosis of TBEV infection in patients with febrile illness after a tick bite, particularly in regions where more than one tick-transmitted diseases are endemic.

Occurrence of antibodies to Anaplasma phagocytophilum in patients with suspected tick-borne encephalitis.

INTRODUCTION AND OBJECTIVES: Human granulocytic anaplasmosis (HGA) is an emerging tick-borne infectious disease caused by Anaplasma phagocytophilum. In Europe, the first serological evidence of HGA was described in 1995 in Switzerland, and the first clinical case was confirmed in 1997 in Slovenia. Since then, many European countries, including Slovakia, have reported the occurrence of HGA. The aim of this study was to examine the occurrence of IgG antibodies against A. phagocytophilum in blood sera of humans with suspected tick-borne encephalitis. MATERIAL AND METHODS: 181 people were examined for the presence of anti-A. phagocytophilum IgG antibodies; 113 were patients with suspected TBE (65 males, 48 females), and 68 from the control group (18 males, 50 females). Respondents were aged 2-80 years (mean age: 31.39; STD: 17.1). Anti-A. phagocytophilum IgG antibodies were detected by the IFA IgG test. Relative risk (RR) and their 95% confidence intervals (95% CI) were estimated for the occurrence of IgG A. phagocytophilum antibodies. RESULTS: Of the total number of 181 people examined, 32 (17.7%) showed positive for IgG antibodies against A. phagocytophilum, 22 of whom were patients with suspected TBE (19.5%) and 10 people from control group (14.7%). The RR of occurrence of IgG A. phagocytophilum was 1.3-times higher in the patients with suspected TBE than in the control group. CONCLUSION: None of the examined patients with suspected TBE had the disease confirmed. Hoever, as shown by the results, the relative risk of occurrence of anaplasmosis is higher in people examined for some another vector-borne disease (in this case TBE). Therefore, the performance of screening examinations in patients suspected of having any tick-borne disease is very important.

Deep Sequencing Analysis of the Ixodes ricinus Haemocytome.

BACKGROUND: Ixodes ricinus is the main tick vector of the microbes that cause Lyme disease and tick-borne encephalitis in Europe. Pathogens transmitted by ticks have to overcome innate immunity barriers present in tick tissues, including midgut, salivary glands epithelia and the hemocoel. Molecularly, invertebrate immunity is initiated when pathogen recognition molecules trigger serum or cellular signalling cascades leading to the production of antimicrobials, pathogen opsonization and phagocytosis. We presently aimed at identifying hemocyte transcripts from semi-engorged female I. ricinus ticks by mass sequencing a hemocyte cDNA library and annotating immune-related transcripts based on their hemocyte abundance as well as their ubiquitous distribution. METHODOLOGY/PRINCIPAL FINDINGS: De novo assembly of 926,596 pyrosequence reads plus 49,328,982 Illumina reads (148 nt length) from a hemocyte library, together with over 189 million Illumina reads from salivary gland and midgut libraries, generated 15,716 extracted coding sequences (CDS); these are displayed in an annotated hyperlinked spreadsheet format. Read mapping allowed the identification and annotation of tissue-enriched transcripts. A total of 327 transcripts were found significantly over expressed in the hemocyte libraries, including those coding for scavenger receptors, antimicrobial peptides, pathogen recognition proteins, proteases and protease inhibitors. Vitellogenin and lipid metabolism transcription enrichment suggests fat body components. We additionally annotated ubiquitously distributed transcripts associated with immune function, including immune-associated signal transduction proteins and transcription factors, including the STAT transcription factor. CONCLUSIONS/SIGNIFICANCE: This is the first systems biology approach to describe the genes expressed in the haemocytes of this neglected disease vector. A total of 2,860 coding sequences were deposited to GenBank, increasing to 27,547 the number so far deposited by our previous transcriptome studies that serves as a discovery platform for studies with I. ricinus biochemistry and physiology.

Variabilities in the antigenic structure of persisting tick-borne encephalitis virus strains.

Antigenic variants in the E protein from persisting tick-borne encephalitis (TBE) virus strains were analyzed using monoclonal antibodies (MAbs) to an analogous protein of the reference Sofyin strain. MAbs to sequential epitopes demonstrated their ability to differentiate persisting TBE virus strains from Sofyin and from each other. Two MAbs (2H3 and 13D6) showed a higher neutralizing activity in the interaction with persisting TBE virus variants as compared to the Sofyin strain. Based on the obtained data, a comparison was made of topologically identical epitopes from the E protein of reference and persisting virus strains. The possibility of increasing the neutralizing activity of MAbs through alterations in the primary structure of sequential antigenic sites is discussed.

Classification of a new member of the TBE flavivirus subgroup by its immunological, pathogenetic and molecular characteristics: identification of subgroup-specific pentapeptides.

The antigenic, pathogenic and molecular characteristics of Turkish sheep encephalitis (TSE) virus, strain TTE80, were compared with other members of the tick-borne encephalitis (TBE) virus complex. Monoclonal antibodies with defined specificity for the flavivirus envelope glycoprotein distinguished TSE virus from louping ill (LI), western or far eastern TBE, Langat and Powassan virus in indirect immunofluorescence, haemagglutination-inhibition and neutralization tests. On the other hand, TSE virus, which produces an LI-like disease in sheep, resembled LI virus in mouse neurovirulence tests. Molecular homology data of all the structural genes of TSE virus compared with other tick-borne flaviviruses demonstrated that TSE virus is a distinct member in the TBE virus subgroup. The data are consistent with the conclusion that TSE virus has evolved by a separate evolutionary pathway as compared with the close antigenic relatives, western European, far eastern TBE viruses and LI virus. By aligning the encoded amino acids in the viral envelope glycoprotein of mosquito- and tick-borne flaviviruses, we have also identified subgroup-specific pentapeptide motifs for the tick-borne encephalitis, Japanese encephalitis and dengue subgroup viruses of the genus Flavivirus. These pentapeptides have important implications for the evolution, classification and diagnosis of flaviviruses.

The molecular biology of tick-borne encephalitis virus. Review article.

Tick-borne encephalitis (TBE) virus is a member of the flavivirus genus and the family Flaviviridae. Like other flaviviruses such as yellow fever, Japanese encephalitis or the dengue viruses, it is an important human pathogen, endemic in many European countries, Russia and China. The disease can be effectively prevented by vaccination with a formalin-inactivated whole virus vaccine. In recent years major advances have been made in the understanding of the molecular biology of TBE virus, including the complete sequence analysis of the genomic RNA of the European and Far Eastern strains. As shown in these studies, the virion RNA contains a single long open reading frame that codes for the structural proteins at the 5' end and the nonstructural proteins at the 3' end. Co- and posttranslational cleavages by a viral and cellular proteases lead to the formation of individual viral proteins. The mature virion is composed of an isometric capsid surrounded by a lipid envelope with two membrane-associated proteins. One of these, protein E, is of paramount importance for several important viral functions, especially during the entry phase of the viral life cycle. Protein E is also responsible for the induction of a protective immune response. A detailed map of antigenic sites has been established and the structure of an anchor-free form of E is currently being investigated by X-ray diffraction analysis. Understanding the molecular basis of the functions of this protein together with the knowledge of its three-dimensional structure may provide clues for developing specific antiviral agents. Protein E has also been shown to be an important determinant of virulence, with single amino acid substitutions at selected sites leading to attenuation. Engineering of such mutations into cDNA clones to produce new recombinant viruses may open up new avenues for the development of live vaccines.

Viraemia and antibody response of the mallard (Anas platyrhynchos) to infection with tick-borne encephalitis virus.

The course and intensity of viraemia after experimental infection with a TBE virus was studied in mallards of varying age and weight. Although virus titres in the blood can range from log102.65 to log104.85, in general these titres are believed to be of sufficient magnitude to infect ticks with the virus. Neutralizing antibodies in the serum of ducks are long-lasting and of sufficient titre to prevent a second viraemia. That mallards can play a role in the epidemiology of TBE virus is likely. Although some ducklings died in unexplained circumstances, none of the infected mallards showed overt disease.

Response of sheep to experimental concurrent infection with tick-borne fever (Cytoecetes phagocytophila) and louping-ill virus.

The pathogenesis of concurrent Cytoecetes phagocytophila and louping-ill virus infection was studied in two experiments. In the first experiment 18 four- to seven-year-old rams were used. Ten were infected with C phagocytophila and five days later eight of these animals and the remaining eight sheep were infected with louping-ill virus. The two rams infected with C phagocytophila alone developed no clinical signs apart from a transient pyrexia, while only three of the eight rams infected with louping-ill virus alone showed mild clinical signs. In marked contrast, all eight dually infected sheep developed severe clinical signs with pronounced depression and dysentery and three died and five were killed in extremis. They developed higher titres of viraemia and the antibody response was depressed while necrotising lesions affecting a variety of organs were detected at post mortem examination. Rhizomucor pucillus was recovered from these lesions in seven of the eight sheep. A second experiment using 10 sheep, five aged seven months and five aged two to three years, confirmed the findings of the first experiment indicating that the age of the animal had not significantly influenced the initial result. It was concluded that C phagocytophila infection could enhance the pathogenicity of louping-ill virus and that, operating together, the two pathogens facilitated fungal invasion. It is postulated that sudden deaths in sheep recently transferred to tick-infested pastures may be due to this newly described syndrome.

Experimental infection of ticks Ixodes ricinus with tick-borne encephalitis virus under different microclimatic conditions.

The influence of environmental temperature and relative air humidity on the development of ticks Ixodes ricinus and their infection rate with tick-borne encephalitis virus was studied. It was found that the temperature influenced significantly the development of ticks, but it did not influence their infection rate. To the contrary, the relative air humidity influenced the infection rate. It is assumed that the intrinsic factors play a primary role in the infection of ticks, while extrinsic factors have a secondary role; they are probably important at border values only.

Powassan virus infection in snowshoe hares (Lepus americanus).

Sera from snowshoe hares (Lepus americanus) trapped near Rochester, Alberta, Canada were tested for Powassan virus antibody by the constant virus/serum dilution neutralization test. Of 1264 serum samples tested, 137 had an antibody titer of at least 1:4 for Powassan virus. Ten hares were inoculated with Powassan virus in the laboratory. Viremia lasted 4-5 days and ceased with the appearance of Powassan antibody in the serum. Neutralizing antibody reached a peak titer of 1:119 on day 15 post-inoculation and was still detectable 13 months post-inoculation.

Double infection with tick borne encephalitis virus and Borrelia burgdorferi sensu lato.

The limited information on co-infection with Borrelia burgdorferi sensu lato and tick-borne encephalitis (TBE) virus was a stimulus for presentation of two patients with well-defined double infection of the central nervous system. TBE virus and B. burgdorferi sensu lato infections are searched for in all patients with lymphocytic meningitis and/or meningoencephalitis admitted to our department. During the last ten years we identified two patients who had ELISA IgM and IgG antibodies to TBE virus in serum and a positive PCR result for TBE virus in cerebrospinal fluid as well as B. burgdorferi sensu lato isolated from cerebrospinal fluid. Intrathecal production of borrelial antibodies was not proven in either of the two patients. These findings show that in patients with acute lymphocytic meningitis originating in regions endemic for Lyme borreliosis and TBE, the possibility of concomitant infection should be considered.

[Ecology of indigenious arbovirus in Alsace. Tick Central European Encephalitis. I.--Complex Ixodes ricinus--bank voles. II.--Study of bank voles population immunity. III.--Virologic results in bank voles population (author's transl)]. / Ecologie d'un arbovirus indigène en Alsace. L'encéphalite à tiques. I.--Complexe Ixodes ricinus--campagnol roussâtre. II.--Etude de l'immunité d'une population de campagnols roussâtres. III.--Résultats virologiques observés dans une population de campagnols roussâtres.

I.--After showing that bank voles are parasited only by Ixodes ricinus larvae, the authors attempt to found different factors (demographic, biometric, and sexual) who favor individual parasitism. The authors conclude to absent of anti tick immunity for this rodent specie. II.--The search for anti-central european encephalitis antibodies (I.H.A.) are shown that 2 p. cent animals were immuns. Yearly and monthly chronologies of antibodies apparition are shown, factors favoring the growth of specific Central european encephalitis antibodies are discussed. III.--The Central european encephalitis tick viral infection of bank vole is studied according to the number of viral strains isolated from different viscera. The monthly chronology of this infection is shown.

Persistence of tick-borne encephalitis virus IV. Virus localization after intracerebral inoculation.

Tick-borne encephalitis (TBE) virus was isolated from the brains and spinal cords, blood, livers, lymph nodes and kidneys from Macaca rhesus monkeys showing acute and subacute fatal encephalitis. In subacute encephalitis, virus titres in the CNS were lower than in acute disease (3.0--6.2 against 3.8--8.3 log LD50/ml). TBE virus localization in chronic encephalitis was largely the same as in acute and subacute disease. In monkeys with a chronic course and stable paralysis of the upper extremity, infectious TBE virus was isolated on day 383 from subcortical ganglia and spinal cord. In lymph nodes and spleen, it could be detected only by a combination of methods (co-cultivation in association with fluorescent antibody technique and complement-fixation test, explantation of organ fragments) more sensitive than is the inoculation of mice with organ homogenates. TBE virus was detected by the same methods on day 90 in the CNS and internal organs of a monkey with chronic encephalitis in the stage of remission.

Persistence of tick-borne encephalitis virus in monkeys. V. Virus localization after subcutaneous inoculation.

In 28 Macaca rhesus monkeys inoculated subcutaneously (s.c.) with different strains and mutants of tick-borne encephalitis (TBE) virus and developing asymptomatic infection, TBE virus and the virus-specific antigen were found at different intervals up to 302 days post inoculation (p.i.) in the CNS and internal organs (liver, spleen, lymph nodes, kidneys). When cyclophosphane (CP) was used as an immunosuppressor, no significant changes in virus distribution were observed with the exception of more frequent isolations at early intervals from kidneys and at late intervals from spleen. With or without CP administration virus was found in the spinal cord at 3--6 days, in the cerebellum and subcortical ganglia at 11--14 days, in the cerebral cortex at 19 days p.i. Early after inoculation the virus was more frequently isolated from the CNS than from the internal organs; later (93--302 days p.i.) the persisting virus or virus-specific antigen were predominantly found in internal organs. The set of sensitive methods used facilitated to detect the persistence of TBE virus in noninfectious form in monkeys with asymptomatic infection.