Nevzat Yurdusev

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 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.

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.

Bi-antigenic immunoassay models based on the recombinant PvpA proteins for Mycoplasma gallisepticum diagnosis in chickens

The present study aimed to produce the relatively conserved central fragment of the Mycoplasma gallisepticum PvpA cytadhesin as recombinant antigen and to determine its species-specific diagnostic potential in comparison with the full-length recombinant rPvpA336 protein. For this purpose, a recombinant protein (rPvpA134) consisting of 134 amino acids with apparent molecular mass of 27 kD was produced and highly purified. The rPvpA134 protein was composed of the amino acid residues at positions 133–265 with respect to the wild-type PvpA. Two bi-antigenic diagnostic models based on Western blot and enzymatic rapid immunofiltration assay (ERIFA) were developed to compare simultaneously the diagnostic potential of the recombinant antigens rPvpA134 and rPvpA336. Although 40% of the confirmed rPvpA336-positive chicken sera were detected as reactive with rPvpA134, this protein would be a useful secondary diagnostic antigen with which to confirm species-specific antibody response for monitoring M. gallisepticum infections. It can be concluded from the present study that 2 bi-antigenic models were successfully adapted to the specific diagnosis of chicken M. gallisepticum. Furthermore, by virtue of its simplicity and rapidity, the ERIFA model has multi-antigenic application potential, making it an alternative field test that is widely applicable in the veterinary diagnostic field.