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 crossreactivity 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.Coxiella burnetii is a gram-negative obligate intracellular bacterium and the etiological agent of the zoonosis Q ("query") fever. Human populations most at risk for infection are those routinely exposed to infected animals and their products. The organism has a diverse animal reservoir that includes domestic livestock such as dairy cattle, goats, and sheep. Chronically infected dairy cattle shed C. burnetii in milk and other secretions, and the products of livestock parturition can deposit tremendous numbers of the organisms into the environment. The insidious nature of C. burnetii is further exacerbated by the organism's aerosol route of infection, low infectious dose, and pronounced extracellular stability. Q fever most commonly manifests as a self-limiting but debilitating influenza-like illness that includes signs and/or symptoms of prolonged high fever, headache, and malaise. Chronic infection can occur, normally in predisposed individuals, that typically presents as a life-threatening endocarditis (reviewed in reference 17).Two advancements that would aid in control of Q fever are (i) a specific and sensitive serodiagnostic test based on recombinant antigen and (ii) an efficacious and safe vaccine that d...
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