Detection of Antibodies Against Three Zoonotic Bartonella spp. and Cross-reactivity Among Species and Coxiella burnetii in Dogs and Cats From Central Thailand

Bartonella henselae is a recently recognized pathogenic bacterium associated with cat-scratch disease, bacillary angiomatosis and bacillary peliosis. A recombinant clone expressing an immunoreactive antigen of B. henselae was isolated by screening a genomic DNA cosmid library by Western blotting with sera pooled from patients positive for B. henselae IgG antibodies by indirect immunofluorescence (IFA). The deduced amino acid sequence of the 43.7 kDa encoded protein was found to be 76.3 % identical to the dihydrolipoamide succinyltransferase enzyme (SucB) of Brucella melitensis. SucB has been shown to be an immunogenic protein during infections by Brucella melitensis, Coxiella burnetii and Bartonella vinsonii. The agreement between reactivity with a recombinant SucB fusion protein on immunoblot analysis and the results obtained by IFA was 55 % for IFA-positive sera and 88 % for IFA-negative sera. Cross-reactivity was observed with sera from patients with antibodies against Brucella melitensis, Mycoplasma pneumoniae, Francisella tularensis, Coxiella burnetii and Rickettsia typhi.

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Candidate Antigens for Q Fever Serodiagnosis Revealed by Immunoscreening of a Coxiella burnetii Protein Microarray

Clinical and Vaccine Immunology ◽

10.1128/cvi.00300-08 ◽

2008 ◽

Vol 15 (12) ◽

pp. 1771-1779 ◽

Cited By ~ 66

Author(s):

Paul A. Beare ◽

Chen Chen ◽

Timo Bouman ◽

Jozelyn Pablo ◽

Berkay Unal ◽

...

Keyword(s):

Coxiella Burnetii ◽

Q Fever ◽

Protein Microarray ◽

Protein S ◽

In Vitro Transcription ◽

Cross Reactivity ◽

Open Reading Frames ◽

Enzyme Linked Immunosorbent Assay ◽

Translation System

ABSTRACT Q fever is a widespread zoonosis caused by Coxiella burnetii. Diagnosis of Q fever is usually based on serological testing of patient serum. The diagnostic antigen of test kits is formalin-fixed phase I and phase II organisms of the Nine Mile reference strain. Deficiencies of this antigen include (i) potential for cross-reactivity with other pathogens; (ii) an inability to distinguish between C. burnetii strains; and (iii) a need to propagate and purify C. burnetii, a difficult and potentially hazardous process. Consequently, there is a need for sensitive and specific serodiagnostic tests utilizing defined antigens, such as recombinant C. burnetii protein(s). Here we describe the use of a C. burnetii protein microarray to comprehensively identify immunodominant antigens recognized by antibody in the context of human C. burnetii infection or vaccination. Transcriptionally active PCR products corresponding to 1,988 C. burnetii open reading frames (ORFs) were generated. Full-length proteins were successfully synthesized from 75% of the ORFs by using an Escherichia coli-based in vitro transcription and translation system (IVTT). Nitrocellulose microarrays were spotted with crude IVTT lysates and probed with sera from acute Q fever patients and individuals vaccinated with Q-Vax. Immune sera strongly reacted with approximately 50 C. burnetii proteins, including previously identified immunogens, an ankyrin repeat-domain containing protein, and multiple hypothetical proteins. Recombinant protein corresponding to selected array-reactive antigens was generated, and the immunoreactivity was confirmed by enzyme-linked immunosorbent assay. This sensitive and high-throughput method for identifying immunoreactive C. burnetii proteins will aid in the development of Q fever serodiagnostic tests based on recombinant antigen.

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A comparison of three cryotechniques (freeze-drying, cryosubstitution, cryosectioning) of specimen preparation for immunolabelling of bacterial antigens of Coxiella burnetii

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100168438 ◽

1994 ◽

Vol 52 ◽

pp. 140-141

Author(s):

T. F. McCaul ◽

R. J. Gould

Keyword(s):

Coxiella Burnetii ◽

Freeze Drying ◽

Stress Proteins ◽

Cultured Cells ◽

High Specificity ◽

Specimen Preparation ◽

Bacterial Antigens ◽

Structural Preservation ◽

Nuclear Ribonucleoprotein ◽

Cellular Components

Immuno-electron microscopy has allowed the selective localisation of molecules with high resolution and high specificity. Cryopreparatory methods have provided better retention of antigenicity suitable for precise immunolabelling together with optimal structural preservation of cellular components. Cryosubstitution and cryoultramicrotomy have widely been exploited for immunolabelling. Molecular Distillation Dryer (MDD), a form of freeze-drying technique, has recently been used for immunolabelling of Plasmodium falciparum stress proteins and nuclear ribonucleoprotein particles in cultured cells. In the present study, we report the comparison of all three cryotechniques in the immunolabelling of bacterial antigens of Coxiella burnetii.The highly infectious C. burnetii was prefixed in 3% glutaraldehyde (66 mM cacodylate buffer, pH 6.8 ). The cells were then pre-embedded in 2% low-temperature agarose on Durapore hydrophilic membrane prior to cryofixation using a LifeCell CF100 metal-mirror system. A 1% glutaraldehyde in 100% methanol was used as a medium for cryosubstitution in a Reichert CS Auto Cryosubstitution apparatus.

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Concomitant Clinical Sensitivity (CCS) and Cross-Reactivity

Allergy & Clinical Immunology International - Journal of the World Allergy Organization ◽

10.1027/0838-1925.15.1.18 ◽

2003 ◽

Vol 15 (01) ◽

pp. 018-029 ◽

Cited By ~ 1

Author(s):

Harris Steinman ◽

Steve Lee

Keyword(s):

Cross Reactivity ◽

Clinical Sensitivity

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Cow fever – High seroprevalence of Coxiella burnetii antibodies in veterinarians associated with obstetric activity on cattle, Germany, 2009

Das Gesundheitswesen ◽

10.1055/s-0030-1266357 ◽

2010 ◽

Vol 72 (08/09) ◽

Author(s):

H Bernard ◽

S Brockmann ◽

N Kleinkauf ◽

C Klinc ◽

C Wagner-Wiening ◽

...

Keyword(s):

Coxiella Burnetii ◽

High Seroprevalence

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Human Prothrombin Activation: Determination of Plasma and Serum Levels of Prothrombin Fragment 3 by Radioimmunoassay

10.1055/s-0038-1665668 ◽

1979 ◽

Author(s):

Daniel Walz ◽

Thomas Brown

Keyword(s):

Blood Clotting ◽

Factor Xa ◽

Heart Association ◽

Serum Levels ◽

Cross Reactivity ◽

Assay Procedure ◽

A Chain ◽

Prothrombin Activation ◽

Theoretical Amount

Human prothrombin activation is unique in that, in addition to the release of fragment 1.2 (FI.2) from the NH-terminus of prothrombin by factor Xa during the generation of thrombin, an additional 13 residue polypeptide, fragment 3 (F3), is autocatalytically removed from the amino-terminus of the thrombin A chain. We have developed a rapid radioimmunoassay for human F3 which incorporates short incubation times and the use of a preprecipitated second antibody; the assay can be performed in three hours. Specificity studies in buffer systems show prothrombin and prethrombin 1 cross-reacting at a level of 0.001; purified thrombin does not cross-react. In the presence of 5% BSA, prothrombin displays considerably less cross-reactivity. No immunoreactive material to F3 antibodies could be detected in 400 μL of plasma. Serum, obtained from whole blood clotting, contained measurable quantities of F3 (40-100 ng/mL). This amount in serum represents only 5-10% of the theoretical amount available should all of the fragment be hydrolytically cleaved during the conversion of prothrombin to thrombin. This assay procedure is currently being utilized to monitor the activation of purified human prothrombin in the absence and presence of selected plasma inhibitors. (Supported in part by NIH 05384-17 and the Michigan Heart Association).

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