Özlem Büyüktanir

Detection of the tdh and trh genes in Vibrio parahaemolyticus isolates in fish and mussels from Middle Black Sea Coast of Turkey.

Aim:  The aim of this study was to demonstrate the occurrence of potential pathogenic Vibrio parahaemolyticus in seafoods using DNA‐based techniques in comparison with bacteriological methods.

A Borrelia burgdorferi Surface-Exposed Transmembrane Protein Lacking Detectable Immune Responses Supports Pathogen Persistence and Constitutes a Vaccine Target

Borrelia burgdorferi harbors a limited set of transmembrane surface proteins, most of which constitute key targets of humoral immune responses. Here we show that BB0405, a conserved membrane-spanning protein of unknown function, fails to evoke detectable antibody responses despite its extracellular exposure. bb0405 is a member of an operon and ubiquitously expressed throughout the rodent-tick infection cycle. The gene product serves an essential function in vivo, as bb0405-deletion mutants are unable to transmit from ticks and establish infection in mammalian hosts. Despite the lack of BB0405-specific immunoglobulin M or immunoglobulin G antibodies during natural infection, mice immunized with a recombinant version of the protein elicited high-titer and remarkably long-lasting antibody responses, conferring significant host protection against tick-borne infection. Taken together, these studies highlight the essential role of an apparently immune-invisible borrelial transmembrane protein in facilitating infection and its usefulness as a target of protective host immunity blocking the transmission of B. burgdorferi.

BB0323 and Novel Virulence Determinant BB0238: Borrelia burgdorferi Proteins That Interact With and Stabilize Each Other and Are Critical for Infectivity

We have shown that Borrelia burgdorferi gene product BB0323 is essential for cell fission and pathogen persistence in vivo. Here we describe characterization of a conserved hypothetical protein annotated as BB0238, which specifically interacts with the N-terminal region of BB0323. We show that BB0238 is a subsurface protein, and similar to BB0323, exists in the periplasm and as a membrane-bound protein. Deletion of bb0238 in infectious B. burgdorferi did not affect microbial growth in vitro or survival in ticks, but the mutant was unable to persist in mice or transmit from ticks--defects that are restored on genetic complementation. Remarkably, BB0238 and BB0323 contribute to mutual posttranslational stability, because deletion of one causes dramatic reduction in the protein level of the other partner. Interference with the function of BB0238 or BB0323 and their interaction may provide novel strategies to combat B. burgdorferi infection.

Middle region of the Borrelia burgdorferi surface‐located protein 1 (Lmp1) interacts with host chondroitin‐6‐sulfate and independently facilitates infection

Borrelia burgdorferi surface‐located membrane protein 1, also known as Lmp1, has been shown to play critical roles in pathogen evasion of host‐acquired immune defences, thereby facilitating persistent infection. Lmp1 possesses three regions representing potentially discrete domains: Lmp1N, Lmp1M and Lmp1C. Because of its insignificant homology to known proteins, how Lmp1 or its specific regions contribute to microbial biology and infection remains enigmatic. Here, we show that distinct from Lmp1N and Lmp1C, Lmp1M is composed of at least 70% alpha helices and completely lacks recognizable beta sheets. The region binds to host glycosaminoglycan chondroitin‐6‐sulfate molecules and facilitates mammalian cell attachment, suggesting an adhesin function of Lmp1M. Phenotypic analysis of the Lmp1‐deficient mutant engineered to produce Lmp1M on the microbial surface suggests that Lmp1M can independently support B. burgdorferi infectivity in murine hosts. Further exploration of functions of Lmp1 distinct regions will shed new light on the intriguing biology and infectivity of spirochetes and help develop novel interventions to combat Lyme disease.

A recombinant PvpA protein-based diagnostic prototype for rapid screening of chicken Mycoplasma gallisepticum infections.

Mycoplasma gallisepticum is the primary agent of chronic respiratory disease causing important economic losses in the poultry industry. Serological monitoring is essential to maintain mycoplasma-free breeder flocks and often complicated by the cross-reactions between different mycoplasma species. To overcome serological cross-reactions, a large fragment of the M. gallisepticum PvpA cytadhesin, species-specific surface-exposed protein, was produced in E. coli as a recombinant protein (rPvpA336) and used as a potential diagnostic antigen. The rPvpA336 protein possesses 336 mycoplasma-specific amino acids with relative molecular weight of 44 kDa. A deletion region of 37 amino acids was identified when compared to the wild-type PvpA protein. Immunoreactivity of the rPvpA336 protein has been demonstrated by Western blot analysis with M. gallisepticum-positive and -negative chicken sera. Furthermore, an enzymatic rapid immunofiltration assay (ERIFA) prototype based on the rPvpA336 protein has been developed and its species-specific detection capability has been demonstrated by using M. gallisepticum and/or M. synoviae-positive and -negative chicken sera. In addition to its species-specificity, the ERIFA prototype presents certain advantages such as rapidity, field-applicability and cost-effectiveness. Therefore, these advantages would make the prototype a species-specific rapid diagnostic tool of choice in the field and limited laboratory conditions for screening M. gallisepticum infections.

Efficient Detection of Pathogenic Leptospires Using 16S Ribosomal RNA.

Pathogenic Leptospira species cause a prevalent yet neglected zoonotic disease with mild to life-threatening complications in a variety of susceptible animals and humans. Diagnosis of leptospirosis, which primarily relies on antiquated serotyping methods, is particularly challenging due to presentation of non-specific symptoms shared by other febrile illnesses, often leading to misdiagnosis. Initiation of antimicrobial therapy during early infection to prevent more serious complications of disseminated infection is often not performed because of a lack of efficient diagnostic tests. Here we report that specific regions of leptospiral 16S ribosomal RNA molecules constitute a novel and efficient diagnostic target for PCR-based detection of pathogenic Leptospira serovars. Our diagnostic test using spiked human blood was at least 100-fold more sensitive than corresponding leptospiral DNA-based quantitative PCR assays, targeting the same 16S nucleotide sequence in the RNA and DNA molecules. The sensitivity and specificity of our RNA assay against laboratory-confirmed human leptospirosis clinical samples were 64% and 100%, respectively, which was superior then an established parallel DNA detection assay. Remarkably, we discovered that 16S transcripts remain appreciably stable ex vivo, including untreated and stored human blood samples, further highlighting their use for clinical detection of L. interrogans. Together, these studies underscore a novel utility of RNA targets, specifically 16S rRNA, for development of PCR-based modalities for diagnosis of human leptospirosis, and also may serve as paradigm for detection of additional bacterial pathogens for which early diagnosis is warranted.

Development of an individual rapid test based on enzymatic immunofiltration assay for detection of anti-Brucella abortus antibody in bovine sera

To detect bovine antibody directed to smooth lipopolysaccharide (LPS), cell lysate (LYS), O-polysaccharide (OPS), and LPS-deprived chromatographic fractions (ChF) of Brucella abortus, 2 bi-antigenic diagnostic models based on the enzymatic rapid immunofiltration assay (ERIFA), ERIFALPS/LYS and ERIFAOPS/ChF, were developed. Their diagnostic performance was compared with complement fixation test (CFT), Rose Bengal test (RBT), indirect in-house and commercial enzyme-linked immunosorbent assays (iELISA and com-ELISA, respectively), based on the smooth LPS antigen, by using a total of 420 cattle sera collected from aborted-unvaccinated, aborted-unvaccinated and culture-positive, healthy-unvaccinated, and healthy-vaccinated cattle. The results demonstrated excellent agreement and no statistical difference between iELISAs and LPS-, LYS-, OPS-based ERIFA models. However, diagnostic performance of CFT, RBT, and ChF-based ERIFA was less significant than that of LPS-, LYS-, and OPS-based ERIFA models, and iELISAs. The results demonstrated a successful adaptation of the multi-antigenic ERIFA model to anti–B. abortus antibody in bovine sera and suggest that the ERIFA model can be considered as an “individual rapid ELISA” due to its similarity with ELISA, individual applicability, and rapidity in determining reactor animals within 5 minutes. In conclusion, the potential of multi-antigenic applications can make the rapid ERIFA model not only an alternative screening method but also a confirmatory test for bovine brucellosis diagnosis.

Rapid immunofiltration assay based on colloidal gold-protein G conjugate as an alternative screening test for bovine and ovine brucellosis.

A non-enzymatic rapid immunofiltration assay (NERIFA) was developed as an alternative field test for rapid detection of anti-Brucella antibody in bovine and ovine sera. The assay was based on Brucella abortus lipopolysaccharide as diagnostic antigen and colloidal gold particle–protein G conjugate as detection reagent. Its diagnostic performance was evaluated using undiluted well-defined positive and negative serum samples in comparison with Rose Bengal test (RBT), complement fixation test (CFT) and a commercial and an in-house indirect enzyme-linked immunosorbent assay (ELISA). A perfect test agreement was found between NERIFA and ELISAs by kappa statistics. In addition, McNemar’s analysis of the results showed that the RBT for bovine sera and the CFT for ovine sera were found significantly less performant than indirect ELISAs and NERIFA. The results of the present study indicated that the NERIFA could be considered as a simple, rapid, and accurate field test for screening of ovine and bovine brucellosis. Therefore, this test constitutes a high potential to be used as an alternative model particularly in brucellosis prevalent tropical and subtropical geographical areas.

RAPID IMMUNOFILTRATION ASSAY AS A FIELD DIAGNOSTIC TOOL FOR OVINE BRUCELLOSIS

This work describes the development of two rapid immunofiltration assays, enzymatic (ERIFA) and non-enzymatic (NERIFA), for the rapid detection of ovine anti-Brucella antibodies. Brucella abortus lipopolysaccharide and total bacterial extract were dotted separately as diagnostic antigens on a nitrocellulose filter-membrane of the individual assay unit along with a third dot of purified sheep IgG as an internal control. The assays diagnostic performance was evaluated in comparison with a modified rose bengal test (mRBT) and an indirect enzyme-linked immunosorbent assay (ELISA) through usage of 590 serum samples from healthy, vaccinated, or infected sheep. The ERIFA and indirect ELISA were found to be significantly more sensitive than NERIFA, while mRBT was determined to be statistically equivalent to NERIFA. A perfect agreement (κ = 0.984) and a statistical equivalence to indirect ELISA suggest that the bi-antigenic ERIFA can be used as an “individual rapid ELISA” for screening ovine anti-Brucella antibody both in the field and in limited laboratory conditions.

Borrelia burgdorferi BBI39 Paralogs, Targets of Protective Immunity, Reduce Pathogen Persistence Either in Hosts or in the Vector

Borrelia burgdorferi genome harbors several paralogous gene families (pgf) that can encode immunogenic proteins of unknown function. Protein-protein interaction assays using a transmission-blocking vaccine candidate, BBA52, as bait identified an interacting partner in spirochetes-a member of pgf 54, annotated as BBI39. We show that BBI39 is a surface-exposed membrane antigen that is immunogenic during spirochete infection, despite the gene being primarily transcribed in the vector with a transient expression in the host only at tick-bite sites. Immunization of rodents with BBI39, or a diverse paralog, BBI36, or their combination impaired pathogen acquisition by the vector, transmission from ticks to hosts, or induction of disease. High-titer BBI39 immunoglobulin G antibodies, which have borreliacidal properties, could be generated through routine subcutaneous or oral immunization, further highlighting use of BBI39 proteins as novel Lyme disease vaccines that can target pathogens in the host or in ticks.Borrelia burgdorferi genome harbors several paralogous gene families (pgf) that can encode immunogenic proteins of unknown function. Protein-protein interaction assays using a transmission-blocking vaccine candidate, BBA52, as bait identified an interacting partner in spirochetes-a member of pgf 54, annotated as BBI39. We show that BBI39 is a surface-exposed membrane antigen that is immunogenic during spirochete infection, despite the gene being primarily transcribed in the vector with a transient expression in the host only at tick-bite sites. Immunization of rodents with BBI39, or a diverse paralog, BBI36, or their combination impaired pathogen acquisition by the vector, transmission from ticks to hosts, or induction of disease. High-titer BBI39 immunoglobulin G antibodies, which have borreliacidal properties, could be generated through routine subcutaneous or oral immunization, further highlighting use of BBI39 proteins as novel Lyme disease vaccines that can target pathogens in the host or in ticks.