Selection of a Borrelia burgdorferi antigenic variant by cultivation in the presence of increasing amounts of homologous immune serum

This investigation was undertaken to select antigenic variants of a Borrelia burgdorferi strain in vitro. The original strain BITS was cultivated in BSK medium supplemented with increasing concentrations of homologous hyperimmune serum raised in rabbits. After a few serial passages starting from a subinhibitory serum dilution of 1:800 in BSK up to 1:200, a variant named BITSv was obtained; it grew abundantly like the control culture in the presence of hyperimmune serum. Analysis of the antigenic pattern of the original and derived variants by Western blotting revealed that BITSv, compared to the original strain BITS, had lost the reactivity with the immune serum at the level of the oligosaccharide moiety. These experiments, designed to mimic the possible action of antibodies that arise during a Borrelia infection, suggest that lipopolysaccharides are surface located and that they play a role in the integrity of the outer membrane during the multiplication of Borrelia burgdorferi.     

Mapping of genes on the linear chromosome of the bacterium Borrelia burgdorferi: Possible locations for its origin of replication

Molecular clones of Borrelia burgdorferi, aetiologic agent of Lyme borreliosis, were isolated and analysed by DNA sequence determination. This procedure yielded B. burgdorferi homologues of gidA, gyrB, gyrA, ftsA and ftsZ. The genes were located on the physical map of the B. burgdorferi linear chromosome. Also mapped were the genes fla and p60 while dnaA was mapped using a heterologous probe. gyrA and gyrB were found to be in tandem and were mapped, along with dnaA at the centre of the chromosome. gidA was located close to the left hand extremity of the chromosome. Because gyrB, dnaA and gidA are normally located within 50 kb of the origin of replication (oriC), we propose two possible sites for oriC in the B. burgdorferi linear chromosome.     

Identification of four genomic groups ofBorrelia burgdorferi sensu lato inIxodes ricinus ticks collected in a Lyme borreliosis endemic region of northern Croatia

We investigated the presence ofBorrelia burgdorferi sensu lato inIxodes ricinus ticks collected in a Lyme borreliosis (LB) endemic region of northern Croatia. Ticks (n=124) were collected at five locations and analysed by the polymerase chain reaction (PCR). A DNA fragment from the internal transcribed spacer (ITS2) ofI. ricinus was detected in all tick lysates, indicating that PCR inhibitors were not present.Borrelia burgdorferi sensu lato DNA was detected in 56 out of 124 ticks (45%). Four genomic groups were identified:Borrelia afzelii (n=26),Borrelia garinii (n=5), group VS116 (n=5) andB. burgdorferi sensu stricto (n=1). Mixed infections ofB. afzelii with group VS116 (n=10) andB. afzelii withB. burgdorferi sensu stricto (n=1) were also detected. Eight ticks containedB. burgdorferi sensu lato, which could not be typed. The detection ofB. afzelii andB. garinii in ticks was in agreement with manifestations of LB found locally. The occurrence of group VS116 in northern Croatia and in an earlier study in The Netherlands, infers that this genomic group may be well established in EuropeanI. ricinus.     

Species of Borrelia distinguished by restriction site polymorphisms in 16S rRNA genes

Abstract Three phyletic groups of Borrelia associated with Lyme disease, B. burgdorferi, B. garinii and group VS461 can be distinguished from each other and other species of Borrelia by Bfa I restriction site polymorphisms in PCR amplified 16S rRNA genes. One strain isolated from an Ixodes pacificus tick in California that was previously unclassifiable was distinguishable from B. burgdorferi by an Mnl I restriction site polymorphism.     

The flagellin gene of Borrelia miyamotoi sp. nov. and its phylogenetic relationship among Borrelia species

Abstract We have cloned and sequenced the flagellin gene from Borrelia miyamotoi strain HT31 and compared it with previously published flagellin sequences. Sequence similarity analysis demonstrated that strain HT31 is phylogenetically distant from the three species of Lyme disease borreliae and is deeply branched into the relapsing fever borrelia cluster. The result was in full agreement with the classification of Borrelia strains using 16S rRNA sequences. This finding indicates that a phylogenetic analysis using flagellin gene sequences might be useful for classification of Borrelia strains.     

Schistosoma mansoni and Fasciola hepatica: Cross-resistance in mice with single-sex schistosome infections

Primary Schistosoma mansoni single-sex infections in mice, i.e., either male only or female only, did not stimulate any detectable level of heterologous resistance to challenge with Fasciola hepatica after 22 to 76 days, while statistically significant resistance to a challenge with F. hepatica was demonstrated in the presence of patent mixed-sex S. mansoni infections. Simultaneous infections with S. mansoni and F. hepatica induced a statistically significant reduction in the number of schisto-some worms established, i.e., the burden being reduced by 40.1 and 43.9%, respectively. There was no reduction of the F. hepatica worm burden. Similar features could be observed with a time interval of 48 hr between the S. mansoni infection and the F. hepatica challenge, i.e., the schistosome burden being reduced by 34.2 and 45.6%, respectively. Furthermore, simultaneous infections with S. mansoni and F. hepatica induced a statistically significant reduction of the egg production capacity per paired female schistosome worm as compared with that of the S. mansoni control group. Tissue egg counts of the various intestinal sections were reduced by 92.8–99.6%.     

Activity of the tick Ixodes ricinus monitored in a suburban park in Brno,Czech Republic,in association with the evaluation of selected repellents

The ever‐increasing number of Lyme borreliosis patients led us to consider more effective procedures for disease prevention. The aim of our study was to monitor the annual activity and infectivity of Ixodes ricinus ticks in the Pisárky region, City of Brno, CR, and to test the responses of the locally‐captured ticks to selected repellents. The result of regular one‐hour‐perweek monitoring in 2011 was the collection of ticks that directly reflected the highest number of Lyme disease patients (4,835) detected throughout the period of recording in the Czech Republic. The ticks were examined for spirochaetes by dark field microscopy. The positive samples were identified by PCR analysis, confirming that 76% of these were infected with Borrelia burgdorferi sensu lato. Ticks were most abundant in May and June, with August having the highest risk for spirochaetal infection. Tick activity was statistically correlated with temperature. The moving‐object‐bioassay was used to study repellent efficiency on the Ixodes ricinus nymphs captured in the above‐mentioned suburban park. Five selected commercial repellents based on DEET (N, N‐diethyl3methylbenzamide) showed statistically different effects on the non‐repellent control group.     

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.     

Structural Basis for Complement Evasion by Lyme Disease Pathogen Borrelia burgdorferi

Borrelia burgdorferi spirochetes that cause Lyme borreliosis survive for a long time in human serum because they successfully evade the complement system, an important arm of innate immunity. The outer surface protein E (OspE) of B. burgdorferi is needed for this because it recruits complement regulator factor H (FH) onto the bacterial surface to evade complement-mediated cell lysis. To understand this process at the molecular level, we used a structural approach. First, we solved the solution structure of OspE by NMR, revealing a fold that has not been seen before in proteins involved in complement regulation. Next, we solved the x-ray structure of the complex between OspE and the FH C-terminal domains 19 and 20 (FH19-20) at 2.83 Å resolution. The structure shows that OspE binds FH19-20 in a way similar to, but not identical with, that used by endothelial cells to bind FH via glycosaminoglycans. The observed interaction of OspE with FH19-20 allows the full function of FH in down-regulation of complement activation on the bacteria. This reveals the molecular basis for how B. burgdorferi evades innate immunity and suggests how OspE could be used as a potential vaccine antigen.