Serological Detection of Borrelia burgdorferi among Horses in Korea

Lyme disease is a tick-borne zoonotic infectious disease caused by Borrelia burgdorferi. The present study assessed the infection status of B. burgdorferi among horses reared in Korea using ELISA and PCR. Between 2009 and 2013, blood samples were collected from 727 horses throughout Korea. Data for each animal including age, gender, breed, and region of sample collection were used for epidemiological analysis. Overall, 38 (5.2%; true prevalence: 5.5%) of 727 horses were seropositive by ELISA. There were statistically significant differences according to breed and region (P<0.001) whose differences might be attributed to the ecology of vector ticks and climate conditions. Using 2 nested PCR, none of the samples tested positive for B. burgdorferi. Thus, a positive ELISA result can indicate only that the tested horse was previously exposed to B. burgdorferi, with no certainty over the time of exposure. Since global warming is likely to increase the abundance of ticks in Korea, continuous monitoring of tick-borne diseases in Korean horses is needed.     

Anaplasma phagocytophilum-infected neutrophils enhance transmigration of Borrelia burgdorferi across the human blood brain barrier in vitro

The manifestations of Lyme disease, caused by Ixodes spp. tick-transmitted Borrelia burgdorferi, range from skin infection to bloodstream invasion into the heart, joints and nervous system. The febrile infection human granulocytic anaplasmosis is caused by a neutrophilic rickettsia called Anaplasma phagocytophilum, also transmitted by Ixodes ticks. Previous studies suggest that co-infection with A. phagocytophilum contributes to increased spirochetal loads and severity of Lyme disease. However, a common link between these tick-transmitted pathogens is dissemination into blood or tissues through blood vessels. Preliminary studies show that B. burgdorferi binds and passes through endothelial barriers in part mediated by host matrix metalloproteases. Since neutrophils infected by A. phagocytophilum are activated to release bioactive metalloproteases and chemokines, we examined the enhanced B. burgdorferi transmigration through vascular barriers with co-infection in vitro. To test whether endothelial transmigration is enhanced with co-infection, B. burgdorferi and A. phagocytophilum-infected neutrophils were co-incubated with EA.hy926 cells (HUVEC-derived) and human brain microvascular endothelial cells in Transwell cultures. Transmigration of B. burgdorferi through endothelial cell barriers was determined and endothelial barrier integrity was measured by transendothelial electrical resistivity. More B. burgdorferi crossed both human BMEC and EA.hy926 cells in the presence of A. phagocytophilum-infected neutrophils than with uninfected neutrophils without affecting endothelial cell integrity. Such a mechanism may contribute to increased blood and tissue spirochete loads.     

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.     

Adaptation of Borrelia burgdorferi in the tick and the mammalian host

Borrelia burgdorferi, the causative agent of Lyme disease, shows a great ability to adapt to different environments, including the arthropod vector, and the mammalian host. The success of these microorganisms to survive in nature and complete their enzootic cycle depends on the regulation of genes that are essential to their survival in the different environments. This review describes the current knowledge of gene expression by B. burgdorferi in the tick and the mammalian host. The functions of the differentially regulated gene products as well as the factors that influence their expression are discussed. A thorough understanding of the changes in gene expression and the function of the differentially expressed antigens during the life cycle of the spirochete will allow a better control of this prevalent infection and the design of new, second generation vaccines to prevent infection with the spirochete.     

Invasion of human neuronal and glial cells by an infectious strain of Borrelia burgdorferi

Human infection by Borrelia burgdorferi, the etiological agent for Lyme disease, can result in serious acute and late-term disorders including neuroborreliosis, a degenerative condition of the peripheral and central nervous systems. To examine the mechanisms involved in the cellular pathogenesis of neuroborreliosis, we investigated the ability of B. burgdorferi to attach to and/or invade a panel of human neuroglial and cortical neuronal cells. In all neural cells tested, we observed B. burgdorferi in association with the cell by confocal microscopy. Further analysis by differential immunofluorescent staining of external and internal organisms, and a gentamicin protection assay demonstrated an intracellular localization of B. burgdorferi. A non-infectious strain of B. burgdorferi was attenuated in its ability to associate with these neural cells, suggesting that a specific borrelial factor related to cellular infectivity was responsible for the association. Cytopathic effects were not observed following infection of these cell lines with B. burgdorferi, and internalized spirochetes were found to be viable. Invasion of neural cells by B. burgdorferi provides a putative mechanism for the organism to avoid the host's immune response while potentially causing functional damage to neural cells during infection of the CNS.     

Borrelia burgdorferi sensu lato in the female cement plug of Ixodes persulcatus ticks (Acari,Ixodidae)

Borrelia burgdorferi sensu lato was detected in one out of five cement plugs of female Ixodes persulcatus ticks. The spirochetes were found by dark field microscopy as early as 18 h after attachment of the ticks to the skin of a white mouse. The relevance of this finding is discussed in relation to the epidemiology of Lyme Borreliosis.     

Detection of glycoproteins in Borrelia burgdorferi

The presence of carbohydrates on proteins of Borrelia burgdorferi, the causative agent of Lyme disease, was investigated by using a digoxigenin labeling method together with Schiff staining and N-glycosidase F assay. The two major outer surface exposed proteins of 31 kDa and 34 kDa showed to be glycosylated and gel filtration high pressure liquid chromatography (HPLC) of proteins of B. burgdorferi metabolically labeled with ¹⁴C-N-acetylglucosamine revealed the incorporation of the carbohydrate into the glycosyl residue of these proteins.Abbreviations N-glycosidase F peptide-N-glycosidase F (EC 3.5.1.52) - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - WB Western blotting - HPLC high pressure liquid chromatography - SDS sodium dodecyl sulphate - mAb monoclonal antibody - MIAF mouse immune ascitic fluid - SS Schiff staining - Osp Outer surface protein     

What Ticks Do Under Your Skin: Two-Photon Intravital Imaging of Ixodes Scapularis Feeding in the Presence of the Lyme Disease Spirochete

Lyme disease, due to infection with the Ixodes-tick transmitted spirochete Borrelia burgdorferi, is the most common tick-transmitted disease in the northern hemisphere. Our understanding of the tick-pathogen-vertebrate host interactions that sustain an enzootic cycle for B. burgdorferi is incomplete. In this article, we describe a method for imaging the feeding of Ixodes scapularis nymphs in real-time using two-photon intravital microscopy and show how this technology can be applied to view the response of Lyme borrelia in the skin of an infected host to tick feeding.     

The Lon-2 protease of Borrelia burgdorferi is critical for infection in the mammalian host

Borrelia burgdorferi encodes a functional homolog of canonical Lon protease termed Lon-2. To date, the contribution of Lon-2 to B. burgdorferi fitness and infection remains unexplored. Herein, we showed that expression of lon-2 was highly induced during animal infection, suggesting that Lon-2 is important for B. burgdorferi infection. We further generated a lon-2 deletion mutant. Compared with that of wild-type (WT) strain, the infectivity of the mutant was severely attenuated in a murine infection model. Although no growth defect was observed for the mutant in normal BSK-II medium, resistance of the lon-2 mutant to osmotic stress was markedly reduced. In addition, when exposed to tert-Butyl hydroperoxide, survival of the lon-2 mutant was impaired. In addition, we found that the protein levels of RpoS and RpoS-dependent OspC were decreased in the mutant. All these phenotypes were restored to WT or near-WT levels when lon-2 mutation was complemented in cis. Taken together, these results demonstrate that Lon-2 is critical for B. burgdorferi to establish infection and to cope with environmental stresses. This study provides a foundation for further uncovering the direct link between the dual roles of Lon-2 in protein quality control and bacterial pathogenesis.     

Nucleotide sequence and analysis of the gene in Borrelia burgdorferi encoding the immunogenic P39 antigen

Abstract The P39 antigen is a specific, highly conserved, and immunogenic protein of Lyne disease spirochetes, Borrelia burgdorferi sensu lato. The nucleotide sequence of the gene encoding this protein was determined and found to be the first of two tandemly arranged open reading frames located on the spirochete's chromosome. These two open reading frames were designated bmpA for the gene encoding P39 and bmpB for the gene encoding the putative protein ORF2 encoded by the second open reading frame. The nucleic acid sequence identity for the two open reading frames was 62% while their deduced amino acid sequences were 52% identical. Comparison to sequence data bases demonstrated that the deduced amino acid sequences of both P39 and ORF2 were homologous to TmpC, a putative outer or cytoplasmic membrane lipoprotein of the syphilis spirochete, Treponema pallidum .     

Conspicuous impacts of inconspicuous hosts on the Lyme disease epidemic

Emerging zoonotic pathogens are a constant threat to human health throughout the world. Control strategies to protect public health regularly fail, due in part to the tendency to focus on a single host species assumed to be the primary reservoir for a pathogen. Here, we present evidence that a diverse set of species can play an important role in determining disease risk to humans using Lyme disease as a model. Host-targeted public health strategies to control the Lyme disease epidemic in North America have focused on interrupting Borrelia burgdorferi sensu stricto (ss) transmission between blacklegged ticks and the putative dominant reservoir species, white-footed mice. However, B. burgdorferi ss infects more than a dozen vertebrate species, any of which could transmit the pathogen to feeding ticks and increase the density of infected ticks and Lyme disease risk. Using genetic and ecological data, we demonstrate that mice are neither the primary host for ticks nor the primary reservoir for B. burgdorferi ss, feeding 10% of all ticks and 25% of B. burgdorferi-infected ticks. Inconspicuous shrews feed 35% of all ticks and 55% of infected ticks. Because several important host species influence Lyme disease risk, interventions directed at a multiple host species will be required to control this epidemic.     

Borrelia burgdorferi sensu lato in ticks (Acari: Ixodidae) from two different foci in Spain

The prevalence of Borrelia burgdorferi sensu lato in several tick species was studied over a 2 year period in two ecologically different areas in Spain. One area was an endemic area for Lyme disease, with a number of autochthonous human cases and supported large populations of Ixodes ricinus on cattle and birds; the second area was characterized by the absence of I. ricinus together with the presence of foxes and their associated tick species. While I. ricinus was the main vector of B. burgdoreri in the endemic area (with a mean prevalence of 14% in adults and 51% in nymphs), adults of both Ixodes canisuga and Ixodes hexagonus had high rates of B. burgdorferi prevalence (30 and 28%, respectively) in the zone where I. ricinus was absent. Immatures of Ixodes frontalis were found to be carriers of the spirochete only in those zones where I. ricinus is present, suggesting evidence for reservoir competence in a tick-bird cycle.     

Vector competence of Ixodes angustus (Acari: Ixodidae) for Borrelia burgdorferi sensu stricto

The vector competence of Ixodes angustus for Borrelia burgdorferi sensu stricto (s.s.) was investigated in the laboratory. The larval progeny of female ticks from Washington State were placed on Swiss-Webster mice that had been inoculated intravenously with 10⁸ spirochetes each of a Californian isolate of B. burgdorferi. Spirochetes were detected in 6 (12%) of 50 nymphs derived from larvae that had fed on these animals. Ten nymphs from the same cohort of experimentally infected ticks were placed on each of 4 naive deer mice (Peromyscus maniculatus). One of the mice seroconverted to B. burgdorferi and spirochetes were isolated from its ear tissues 4 weeks after exposure to ticks. Further vector competence trials were conducted with I. angustus ticks from California. Larvae were fed on deer mice that had been inoculated intradermally with B. burgdorferi along with larvae of I. spinipalpis as a comparison group. There was no significant difference in the prevalence of infection in nymphs of I. angustus (8.2%) versus those of I. spinipalpis (12.1%). We conclude that I. angustus is a competent experimental vector of B. burgdorferi s.s. and its efficiency for acquiring and transstadially passing such spirochetes is similar to that of I. spinipalpis.     

Saliva,Salivary Gland,and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) ^1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick ^3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick''s enzootic cycle ^14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva ^9,16-19.As a tick feeds the host typically responds with a strong hemostatic and innate immune response ^11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding ^{3,11,20,21,23}. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens ^3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.     

The role of fat dormouse (Glis glis L.) as reservoir host for spirochete Borrelia burgdorferi sensu lato in the region of Gorski Kotar, Croatia

To determine whether some of the B. burgdorferi sensu lato genospecies associate with fat dormouse as a reservoir host, we investigated the prevalence of infection in questing animals. A total of 45 adult fat dormice (30 female and 15 male) were captured by hunters during their hunting season in the region of Gorski Kotar, Croatia. Dead animals were aseptically dissected, and the urinary bladder tissue was used for isolation attempt and for deoxyribonucleic acid (DNA) extraction. Out of 45 DNA samples extracted from urine bladder tissue, we found four (8.88%) to be polymerase chain reaction (PCR) positive. The RFLP analysis of the PCR product after cleavage with DraI and MseI distinguished between the three major genospecies: B. burgdorferi sensu stricto, B. garinii and B. afzelii. All positive samples were typed as B. afzelii with a unique DraI or MseI pattern. The results of the analysis of urinary bladder tissue samples culture for the presence of Borrelia were negative. Results showed that a prevalence of the Borrelia infection among population of fat dormice indicated their epizootiological involvement as a reservoir of Borrelia spirochetes. Furthermore, this work is an initial step in the investigation of the molecular epidemiology/epizootiology of Lyme borreliosis in Croatia.     

Ixodes (Pholeoixodes) hexagonus, an efficient vector of Borrelia burgdorferi in the laboratory

Borrelia burgdorferi Johnson et al. was first isolated from the midgut of Ixodes dammini Spielman et al. in the U.S.A. and from the midgut of I.ricinus (L.) in Europe. I.ricinus was considered to be the only tick vector of this borrelia, in Europe, until I.hexagonus Leach, the hedgehog tick, was found to harbour spirochaetes. This paper reports an evaluation of the vector competence of I.hexagonus for the spirochaete B.burgdorferi. Transovarial and trans-stadial survival were demonstrated and the spirochaete was transmitted to laboratory mice via the bites of trans-stadially infected I.hexagonus females.     

Metabolic pathway analysis and molecular docking analysis for identification of putative drug targets in Toxoplasma gondii: novel approach

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that can infect a wide range of warm-blooded animals including humans. In humans and other intermediate hosts, toxoplasma develops into chronic infection that cannot be eliminated by host's immune response or by currently used drugs. In most cases, chronic infections are largely asymptomatic unless the host becomes immune compromised. Thus, toxoplasma is a global health problem and the situation has become more precarious due to the advent of HIV infections and poor toleration of drugs used to treat toxoplasma infection, having severe side effects and also resistance have been developed to the current generation of drugs. The emergence of these drug resistant varieties of T. gondii has led to a search for novel drug targets. We have performed a comparative analysis of metabolic pathways of the host Homo sapiens and the pathogen T. gondii. The enzymes in the unique pathways of T. gondii, which do not show similarity to any protein from the host, represent attractive potential drug targets. We have listed out 11 such potential drug targets which are playing some important work in more than one pathway. Out of these, one important target is Glutamate dehydrogenase enzyme; it plays crucial part in oxidation reduction, metabolic process and amino acid metabolic process. As this is also present in the targets of tropical diseases of TDR (Tropical disease related Drug) target database and no PDB and MODBASE 3D structural model is available, homology models for Glutamate dehydrogenase enzyme were generated using MODELLER9v6. The model was further explored for the molecular dynamics simulation study with GROMACS, virtual screening and docking studies with suitable inhibitors against the NCI diversity subset molecules from ZINC database, by using AutoDock-Vina. The best ten docking solutions were selected (ZINC01690699, ZINC17465979, ZINC17465983, ZINC18141294_03, ZINC05462670, ZINC01572309, ZINC18055497_01, ZINC18141294, ZINC05462674 and ZINC13152284_01). Further the Complexes were analyzed through LIGPLOT. On the basis of Complex scoring and binding ability it is deciphered that these NCI diversity set II compounds, specifically ZINC01690699 (as it has minimum energy score and one of the highest number of interactions with the active site residue), could be promising inhibitors for T. gondii using Glutamate dehydrogenase as Drug target.