Control of Listeria species and other bacteria in crawfish and crab processing facilities, utilizing copper drains, coatings and concrete, containing copper ions

The antibacterial properties of copper ions against Listeria spp., Pseudomonas spp., Escherichia coli and other coliforms, and total aerobic bacteria have been investigated in seafood processing environments. Our hypothesis was that drains fabricated from copper and coatings and concrete containing copper ions that have been used for sealing floors and walls could be utilized in crawfish and crab processing facilities as an effective means of controlling Listeria spp. and other unwanted bacteria. Copper in the form of copper sulfate pentahydrate was incorporated into coatings and concrete at the salt concentration of 25 ppm (6.36 ppm Cu++). Sampling sites were selected in multiple areas of the processing facilities. Sampling was performed in the course of two-month seafood production period. Bacterial counts were determined by using microbiological selective media. In addition, PCR analysis was applied to detect the presence of Listeria monocytogenes in the environmental samples. Copper drains were found to be quite effective against some of the tested bacteria. The counts of Listeria spp. and total aerobic bacteria were over one log CFU/cm2 lower on the copper drains than those detected on the control sites. Neither copper coatings nor copper concrete exerted antimicrobial activity against any microorganisms tested. Additionally, almost all PCR samples were negative for the presence of Listeria monocytogenes, suggesting that black colonies grown on Oxford media represented the other species of Listeria family. Based on the overall results, the copper-fabricated drains could be used in the seafood processing facilities as one of other approaches for reducing environmental contamination by foodborne pathogens. Regarding the application of copper coatings and concrete at the seafood production plants, future research should be conducted to find the most effective bactericidal copper forms and, subsequently, their effective minimal inhibitory concentrations

Antibody Response to Lyme Disease Spirochetes in the Context of VlsE-Mediated Immune Evasion

Lyme disease (LD), the most prevalent tick-borne illness in North America, is caused by Borrelia burgdorferi. The long-term survival of B. burgdorferi spirochetes in the mammalian host is achieved though VlsE-mediated antigenic variation. It is mathematically predicted that a highly variable surface antigen prolongs bacterial infection sufficiently to exhaust the immune response directed toward invariant surface antigens. If the prediction is correct, it is expected that the antibody response to B. burgdorferi invariant antigens will become nonprotective as B. burgdorferi infection progresses. To test this assumption, changes in the protective efficacy of the immune response to B. burgdorferi surface antigens were monitored via a superinfection model over the course of 70 days. B. burgdorferi-infected mice were subjected to secondary challenge by heterologous B. burgdorferi at different time points postinfection (p.i.). When the infected mice were superinfected with a VlsE-deficient clone (ΔVlsE) at day 28 p.i., the active anti-B. burgdorferi immune response did not prevent ΔVlsE-induced spirochetemia. In contrast, most mice blocked culture-detectable spirochetemia induced by wild-type B. burgdorferi (WT), indicating that VlsE was likely the primary target of the antibody response. As the B. burgdorferi infection further progressed, however, reversed outcomes were observed. At day 70 p.i. the host immune response to non-VlsE antigens became sufficiently potent to clear spirochetemia induced by ΔVlsE and yet failed to prevent WT-induced spirochetemia. To test if any significant changes in the anti-B. burgdorferi antibody repertoire accounted for the observed outcomes, global profiles of antibody specificities were determined. However, comparison of mimotopes revealed no major difference between day 28 and day 70 antibody repertoires

DECIPHERING MECHANISMS OF VLSE-MEDIATED IMMUNE AVOIDANCE BY BORRELIA BURGDORFERI

The current work has focused on antigenic variation of the VlsE surface lipoprotein, a key mechanism for immune evasion and persistent infection by the Lyme disease spirochete, Borrelia burgdorferi. The fact that only VlsE exhibits ongoing variation of its surface epitopes despite substantial numbers of other proteins expressed on the Borrelia burgdorferi surface suggests that spirochetes may utilize a VlsE-mediated system for immune avoidance of Borrelia burgdorferi surface antigens. The studies described in this dissertation have attempted to decipher whether such a system exists during murine infection by the Lyme pathogen, and the mechanism(s) behind this process. The data suggest that VlsE prevents recognition of Borrelia burgdorferi surface antigens from host antibodies. The work also explored VlsE involvement during reinfection and superinfection, and the potential immune barriers to secondary infection by Borrelia burgdorferi. As a result, innate immunity of the reservoir mouse host has been proposed to be a driving force for Borrelia burgdorferi heterogeneity during the enzootic cycle. The significance of this research is also reflected by the fact that findings, obtained from studies utilizing in vitro-grown or host-adapted Borrelia burgdorferi clones and laboratory strains of mice, were retested in the enzootic cycle model. This involved examination of the effects of vls mutation on persistent Borrelia burgdorferi infection of the natural reservoir, Peromyscus maniculatus, and acquisition and transmission by the tick vector, Ixodes scapularis. The findings highlight the significance of the vls system for long-term infection of the mammalian reservoir host and show that VlsE antigenic variability is advantageous for efficient tick acquisition of Borrelia burgdorferi from the infected murine hosts. Thus, the current work provides the most direct evidence of the importance of VlsE for the enzootic cycle of Lyme spirochetes, and underscores the significance of VlsE antigenic variation for maintaining the pathogen in nature. Finally, this work has also attempted to test a requirement of murine IgM for VlsE-mediated immune evasion by Borrelia burgdorferi during infection of mice. Overall, the present study represents a significant advance in our knowledge of immune evasion by Borrelia burgdorferi, and provides insight into the possible mechanisms involved in VlsE-mediated immune avoidance

Role of VIsE in host reinfection by the lyme disease spirochete, borretia burgdorferi

A key mechanism for immune evasion and persistent infection by the causative agent of Lyme borreliosis, Borrelia burgdorferi (Bb), is antigenic variation of the immunodominant VIsE surface protein. Although a multitude of other surface proteins exist that are immunogenic, VIsE is the only know Bb antigen that exhibits variation of its surface epitopes. A long-standing question regarding Bb immune escape has been how such a feat is accomplished through sequence variation of this single lipoprotein, despite the presence of a substantial number of additional antigens residing on the bacterial surface. In other bacterial pathogen systems, the dominant presence of surface-exposed variable proteins had been associated with the ability to reinfect a host. In the current study, we investigated whether host reinfection by Bb requires VIsE, and the likely mechanism involved. Host-adapted wild-type and VIsE mutant spirochetes were used to reinfect immunocompetent mice that had naturally cleared an infection with a VIsE-deficient clone. Our results demonstrated that VIsE is necessary for reinfection by Bb, and this ability is directly related to evasion of the host antibody response. Moreover, the data presented here raise the possibility that escape of Bb surface antigens from immune surveillance may involve epitope shielding by the VIsE protein

Comparison of motif-based and whole-unique-sequence-based analyses of phage display library datasets generated by biopanning of anti-Borrelia burgdorferi immune sera.

Detection of protection-associated epitopes via reverse vaccinology is the first step for development of subunit vaccines against microbial pathogens. Mapping subunit vaccine targets requires high throughput methods, which would allow delineation of epitopes recognized by protective antibodies on a large scale. Phage displayed random peptide library coupled to Next Generation Sequencing (PDRPL/NGS) is the universal platform that enables high-yield identification of peptides that mimic epitopes (mimotopes). Despite being unsurpassed as a tool for discovery of polyclonal serum mimotopes, the PDRPL/NGS is far inferior as a quantitative method of immune response. Difficult-to-control fluctuations in amounts of antibody-bound phages after rounds of selection and amplification diminish the quantitative capacity of the PDRPL/NGS. In an attempt to improve the accuracy of the PDRPL/NGS method, we compared the discriminating capacity of two approaches for PDRPL/NGS data analysis. The whole-unique-sequence-based analysis (WUSA) involved generation of 7-mer peptide profiles and comparison of the numbers of sequencing reads for unique peptide sequences between serum samples. The motif-based analysis (MA) included identification of 4-mer consensus motifs unifying unique 7-mer sequences and comparison of motifs between serum samples. The motif comparison was based not on the numbers of sequencing reads, but on the numbers of distinct 7-mers constituting the motifs. Our PDRPL/NGS datasets generated from biopanning of protective and non-protective anti-Borrelia burgdorferi sera of New Zealand rabbits were used to contrast the two approaches. As a result, the principle component analyses (PCA) showed that the discriminating powers of the WUSA and MA were similar. In contrast, the unsupervised hierarchical clustering obtained via the MA classified the preimmune, non-protective, and protective sera better than the WUSA-based clustering. Also, a total number of discriminating motifs was higher than that of discriminating 7-mers. In sum, our results indicate that MA approach improves the accuracy and quantitative capacity of the PDRPL/NGS method

Treatment of infected SCID mice with ΔVlsE-specific immune sera.

a<p>Values listed correspond to numbers of cultures positive/numbers tested.</p>b<p>ha denotes host-adapted clone.</p

Variable VlsE Is Critical for Host Reinfection by the Lyme Disease Spirochete

<div><p>Many pathogens make use of antigenic variation as a way to evade the host immune response. A key mechanism for immune evasion and persistent infection by the Lyme disease spirochete, <i>Borrelia burgdorferi</i>, is antigenic variation of the VlsE surface protein. Recombination results in changes in the VlsE surface protein that prevent recognition by VlsE-specific antibodies in the infected host. Despite the presence of a substantial number of additional proteins residing on the bacterial surface, VlsE is the only known antigen that exhibits ongoing variation of its surface epitopes. This suggests that <i>B. burgdorferi</i> may utilize a VlsE-mediated system for immune avoidance of its surface antigens. To address this, the requirement of VlsE for host reinfection by the Lyme disease pathogen was investigated. Host-adapted wild type and VlsE mutant spirochetes were used to reinfect immunocompetent mice that had naturally cleared an infection with a VlsE-deficient clone. Our results demonstrate that variable VlsE is necessary for reinfection by <i>B. burgdorferi</i>, and this ability is directly related to evasion of the host antibody response. Moreover, the data presented here raise the possibility that VlsE prevents recognition of <i>B. burgdorferi</i> surface antigens from host antibodies. Overall, our findings represent a significant advance in our knowledge of immune evasion by <i>B. burgdorferi</i>, and provide insight to the possible mechanisms involved in VlsE-mediated immune avoidance.</p> </div

Infectivity of <i>in vitro</i>-grown VlsE mutants in naïve SCID and C3H mice.

a<p>Values listed correspond to numbers of cultures positive/numbers tested.</p

Infectivity of host-adapted VlsE mutants in naïve C3H mice.

a<p>Values listed correspond to numbers of cultures positive/numbers tested.</p>b<p>ha denotes host-adapted clone.</p

<i>Borrelia burgdorferi</i> clones used in the study.

a<p><i>vls2-16</i> denotes silent cassettes of the <i>vls</i> locus.</p