BACKGROUND
coxiella burnetii is a gram-negative bacterium that causes the worldwide zoonotic disease "q fever".
in humans, the disease generally arises from inhalation of the aerosolized coxiella organisms produced by infected livestock.
acute q fever usually presents as an influenza-like illness with various degrees of pneumonia  <cit> ,which may be self limiting or effectively treated with antibiotics.
however, chronic q fever is typically manifested as endocarditis, osteomyelitis or infected aortic aneurysms  <cit> , and is difficult to treat.
the clinical diagnosis of q fever is mainly based on serological tests including indirect immunofluorescence assay , enzyme-linked immunosorbent assay  and complement fixation   <cit> .
these tests have several limitations: large sample/reagent volume requirements, complex protocols, and differing sensitivities and specificities  <cit> .
furthermore, they all need purified coxiella organisms which are difficult and hazardous to culture and purify  <cit> .
identifying novel seroreactive proteins could be a step towards the development of a fast, specific and safe molecular diagnostic assay instead of traditional serological tests.
immunoproteomic methods have been successfully applied in identifying seroreactive proteins of other pathogens  <cit> .
several immunoproteomic studies on c. burnetii have also been reported with various seroreactive proteins identified  <cit> .
in this study, the proteins of c. burnetii xinqiao, a phase i strain isolated in china  <cit> , were analyzed with sera from experimentally infected balb/c mice and q fever patients using immunoproteomic analysis.
RESULTS
c. burnetii infection in balb/c mice
five days post infection , mice showed clinical symptoms: gathered together, reduced movement, ruffled fur, but no deaths occurred.
the dna samples extracted from tissues of the c. burnetii-infected mice were detected by qpcr.
high levels of coxiella dna were found in liver and spleen tissues  and the highest level was found in tissues obtained on day  <dig> pi.
the coxiella load in spleen tissues was significantly higher than that in liver or lung tissues and significantly decreased by day  <dig> pi .
seroreactive proteins recognized with specific sera
the lysates of purified coxiella organisms was separated by 2d-page and a proteome map of c. burnetii was obtained .
more than  <dig> distinct protein spots with isoelectric points  ranging from  <dig> to  <dig> and molecular mass ranging from  <dig> to  <dig> kda were visualized by coomassie blue stain.
following the immunoblot assay,  <dig>   <dig>   <dig>  and  <dig> of the coxiella proteins were recognized by the mice sera obtained at  <dig>   <dig>   <dig>  and  <dig> days pi, respectively .
among these recognized proteins,  <dig> proteins, chaperonin groel , peptidyl-prolyl cis-trans isomerase  and putative outer membrane chaperone protein , were strongly recognized by sera obtained at days  <dig>   <dig>  and  <dig> days pi, and the  <dig> kda outer membrane protein  was recognized by sera obtained at day  <dig> and strongly recognized by sera obtained on days  <dig> and  <dig> pi .
in addition,  <dig> of the coxiella proteins were recognized by sera from two patients during the acute phase of q fever.
however,  <dig> of the  <dig> proteins, including  <dig> kda chaperone protein , lsu ribosomal protein l12p , 3-oxoacyl- synthase  <dig> , s-adenosylmethionine synthetase , acute disease antigen a , glutamine synthetase , were not recognized by the mouse sera .
serological analysis of the recombinant seroreactive proteins with q fever patient sera
twenty genes encoding the seroreactive proteins were amplified  and cloned into the pet32a/pqe <dig> plasmid.
except for the rpsb-recombinant plasmid, the rest were successfully expressed in e. coli cells.
the  <dig> recombinant proteins were purified by ni-nta agarose and analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis .
then they were used to fabricate a protein microarray.
the protein microarray was probed with  <dig> sera from patients with acute q fever and  <dig> sera from healthy persons .
the average fi value of the proteins probed with acute early, late or convalescent q fever patient sera were significantly higher compared with that probed with the normal sera  the average fi values of the proteins probed with acute late q fever patient sera were significantly higher than acute early or convalescent q fever patient sera .
the protein was considered to be seroreactive if its average fi probed with the patient sera were higher than the mean fi plus twice the standard deviation probed with normal sera .
seven recombinant proteins  were selected as major seroreactive proteins with sensitivities that ranged from 48% to 88% when probed with acute late q fever sera.
the recombinant groel gave the highest sensitivity at 88% .
asensitivity was calculated as the percentage
specificity analysis of the major seroreactive proteins
a small microarray fabricated with groel, ybgf, rpll, mip, com <dig>  omph, and dnak was probed with rickettsial spotted fever, legionella pneumonia or streptococcal pneumonia patient sera.
the average fi value of each protein probed with acute late q fever patient sera were significantly higher compared with that probed with the sera from the other three groups of patients .
a reaction was considered positive if the average fi of one protein probed with one of the tested sera were higher than the mean fi plus  <dig> times the standard deviation probed with the sera of healthy person sera .
as a result, ybgf and dnak displayed no reaction with any of the tested sera, and com <dig> and mip cross-reacted with one or two of the rickettsial spotted fever patient sera .
omph cross-reacted with one of the legionella pneumonia or streptococcal pneumonia patient sera; groel cross-reacted with one of the legionella pneumonia and two of the rickettsial spotted fever patient sera; rpll cross-reacted with two of the legionella pneumonia and three of the streptococcal pneumonia patient sera .
discussion
c. burnetii xinqiao was isolated from ticks in china and its phase i phenotype was demonstrated in a previous study  <cit> .
in this current study, c. burnetii xinqiao was used to infect balb/c mice and a large amount of c. burnetii was found in the spleens and livers of the infected mice by qpcr analysis.
the coxiella load in spleens was significantly higher compared with that in the other organs of the infected mice, indicating that the mouse spleen is the most important organ for c. burnetii propagation and its coxiella load may reflect the severity of c. burnetii infection.
the highest level of coxiella in spleens of the infected mice was found on day  <dig> pi and then gradually decreased, indicating that the infected mice recovered gradually from the severe infection.
these results also indicate that the combination of the sublethal challenge mouse model and the qpcr assay may be a useful and sensitive way to evaluate severity of the infection caused by different c. burnetii strains and evaluate efficiency of drugs or vaccines against this pathogen.
in order to identify the seroreactive proteins of c. burnetii xinqiao, the whole cell lysates of the organism was separated by 2-d electrophoresis.
immunoblot analysis using the sera of mice obtained at days  <dig>   <dig>  and  <dig> pi, indentified  <dig>   <dig>  and  <dig> of the separated proteins, respectively.
this indicated that the specific immune responses to c. burnetii developed progressively in the infected mice with additional antigens of c. burentii recognized as the immune response grew further.
in addition,  <dig> of the proteins were recognized by sera from two patients with acute q fever.
among these seroreactive proteins,  <dig> proteins were recognized by both the mouse and human sera, indicating that these proteins are able to elicit similar humoral immune responses to c. burnetii infection in both species.
a total of  <dig> seroreactive proteins were recognized by the positive mouse or human sera by mass spectra of maldi-tof-ms. groel, a conserved heat shock protein   <cit> , has been reported as a major immunodominant antigen of c. burnetii  <cit> .
ybgf, a tol-pal system protein that involved in bacterial outer membrane stability  <cit> , was found in both phases of c. burnetii  <cit> .
groel and ybgf were both recognized by the sera of c. burnetii-infected mice and the q fever patient sera in this study and have been previously documented as seroreactive antigens using a proteomic approach  <cit> .
while com <dig>  mip, and omph were recognized by the sera of c. burnetii-infected mice but were not recognized by q fever patient sera.
this difference might be due to the fact that mouse and human sera were from different infection stages or there were differences in humoral immune responses to c. burnetii infection between mice and humans.
com <dig> was first identified as an outer membrane-associated seroreactive protein of c. burnetii by hendrix and colleagues  <cit> .
mip is a cell-surface associated peptidylprolyl-isomerase related to macrophage infectivity potentiator protein  <cit>  and plays a role in enhancing clearance of bacteria from spleens of infected mice  <cit> .
omph is a putative outer membrane chaperone protein required for efficient release of translocated proteins from the plasma membrane  <cit> .
the  <dig> proteins had also been recognized as immunodominant antigens in other studies  <cit> .
dnak, a surface-associated protein playing a role in assisting with folding of nascent polypeptide chains  <cit> , and rpll, a ribosomal protein involved in translation, were previously recognized as seroreactive  <cit> .
in this study, dnak and rpll were most seroreactive when probed with the sera of patients with acute q fever but were nonreactive when probed with the sera of c. burnetii-infected mice.
additionally, another  <dig> seroreactive proteins identified in this study were housekeeping enzymes, including fbaa, atpd, and tuf <dig> which are involved in metabolism and biosynthesis.
eight of these proteins were previously identified as seroreactive antigens  <cit> .
this indicated that metabolic enzymes released from c. burnetii organisms were exposed to the host immune system and induced a specific antibodies response.
nineteen of the  <dig> seroreactive proteins identified in this immunoproteomics study were successfully expressed in e. coli cells and the resultant recombinant proteins were used to fabricate a protein microarray.
to evaluate their serodiagnostic potential, the protein microarray was probed with q fever patient sera.
as a result,  <dig> of the  <dig> proteins  gave a modest sensitivity of more than 48% when probed with acute late q fever patient sera.
we noted that inconsistency existed between immunoproteomic and microarray data: the reaction of com <dig> was stronger than that of mip, omph or ygbf in immunoblot assay, whereas fi value of mip, omph or ygbf was higher than that of com <dig> in microarray assay with q fever sera.
the inconsistency might be caused by the fact that the q fever sera recognized linear epitopes of coxiella proteins in immunoblot assay whereas they recognized conformational epitopes of recombinant proteins in protein microarray assay.
our results also showed that the average fi value of the  <dig> major seroreactive proteins probed with acute late sera were significantly higher than those probed with acute early or normal sera, which is generally in accordance with igg titers determined in ifa.
this result firmly suggests that the  <dig> major seroreactive proteins are immunodominant antigens of c. burnetii and they have capability to evoke strong humoral immune responses in c. burnetii infection.
however, compared to ifa, the lower sensitivity of some individual proteins in microarray assay with q fever patient sera, especially sera in acute early stage, was observed, which might be due to the fact that there were remarkable variation in immune recognition patterns for q fever and differences between the two assays in calculating positive values.
when the seroreactive proteins were analyzed in combination, 98% of antibody responders to one or more of the  <dig> major seroreactive proteins could be found among the q fever patients.
the remarkable variation in immune recognition patterns for q fever requires multi-antigen combination to cover the different antibody responses and thus achieve the highest possible test sensitivity.
ybgf, rpll, mip, com <dig>  and omph were considered as potential antigens for diagnosis of q fever by other investigators using in vitro transcription and translation -based microarray of c. burnetii nine mile strain, indicated that xinqiao strain isolated in china shares these major seroreactive antigens with nine mile strain  <cit> .
two heat shock proteins groel and dnak were also recognized as major seroreactive antigens in this study.
the positive frequencies of groel probed with acute early and acute late, and convalescent q fever patient sera were 84%, 88%, and 83%, respectively, higher than the other major seroreactive proteins, suggesting that groel is an excellent molecular marker for q fever.
additionally, the positive frequencies of ybgf with these q fever patient sera were 44%, 62%, and 77%, lower than groel but higher than the other  <dig> major seroreactive proteins, indicating that it is a better protein antigen for q fever diagnosis.
rickettsial spotted fever caused by tick-borne infection may share similar clinical feature with q fever.
legionella pneumonia is caused by legionella pneumophila which is the bacterium closely related to c. burnetii with genomic homology and similar clinical presentations.
pneumonia is the major clinical presentation of acute q fever and most bacterial pneumonia is caused by s. pneumoniae.
these bacterial infections must be distinguished from q fever using serological or molecular tests.
therefore, the  <dig> coxiella proteins were used to fabricate a small microarray for further analysis of specificity with the sera of patients with other infectious diseases.
the average fi value of each protein probed with acute late q fever patient sera was significantly higher than that probed with the sera of patients with one of the three other infectious diseases, which indicated that the major seroreactive proteins of coxiella can be distinguished from other bacteria in general.
ybgf and dnak displayed no cross-reaction with any of the tested sera, and com <dig>  mip, omph and groel cross-reacted with one or two of the sera of patients with rickettsial spotted fever, legionella pneumonia or bacterial pneumonia.
rpll cross-reacted with two of the legionella pneumonia patient sera and three of the streptococcal pneumonia patient sera.
in this analysis, these coxiella proteins gave a modest specificity for recognizing of q fever patient sera, suggesting that they are potential serodiagnostic markers for q fever.
notably, groel had the highest sensitivity and modest specificity for recognizing of q fever, which may be the most important antigen used for the diagnosis of q fever.
the antigen combination, groel, ybgf and com <dig>  may give a more authentic specificity.
refinement of antigen combination and the production of fusion molecules comprised of the major seroreactive antigens described herein may lead to improved sensitivity and specificity for the development of a rapid, accurate, and convenient seorodiagnostic test of q fever.
CONCLUSIONS
in summary, the combination of 2d-page, immunoblot and maldi-tof-ms permitted the identification of  <dig> seroreactive proteins of c. burnetii.
a protein microarray fabricated with recombinant proteins was probed with q fever patient sera.
seven proteins  were recognized as major seroreactive antigens.
the major seroreactive proteins fabricated in a small array were analyzed with the sera of patients with q fever, rickettsial spotted fever, legionella pneumonia or streptococcal pneumonia and they gave a moderate specificity for recognizing of q fever patient sera, suggesting these proteins are potential serodiagnostic markers for q fever.

 coxiella burnetii is a gram-negative bacterium that causes the worldwide zoonotic disease "q fever". in humans, the disease generally arises from inhalation of the aerosolized coxiella organisms produced by infected livestock. acute q fever usually presents as an influenza-like illness with various degrees of pneumonia,which may be self limiting or effectively treated with antibiotics. however, chronic q fever is typically manifested as endocarditis, osteomyelitis or infected aortic aneurysms, and is difficult to treat. the clinical diagnosis of q fever is mainly based on serological tests including indirect immunofluorescence assay, enzyme-linked immunosorbent assay and complement fixation. these tests have several limitations: large sample/reagent volume requirements, complex protocols, and differing sensitivities and specificities. furthermore, they all need purified coxiella organisms which are difficult and hazardous to culture and purify. identifying novel seroreactive proteins could be a step towards the development of a fast, specific and safe molecular diagnostic assay instead of traditional serological tests. immunoproteomic methods have been successfully applied in identifying seroreactive proteins of other pathogens. several immunoproteomic studies on c. burnetii have also been reported with various seroreactive proteins identified. in this study, the proteins of c. burnetii xinqiao, a phase i strain isolated in china, were analyzed with sera from experimentally infected balb/c mice and q fever patients using immunoproteomic analysis.  c. burnetii infection in balb/c mice five days post infection, mice showed clinical symptoms: gathered together, reduced movement, ruffled fur, but no deaths occurred. the dna samples extracted from tissues of the c. burnetii-infected mice were detected by qpcr. high levels of coxiella dna were found in liver and spleen tissues and the highest level was found in tissues obtained on day pi. the coxiella load in spleen tissues was significantly higher than that in liver or lung tissues and significantly decreased by day pi. seroreactive proteins recognized with specific sera the lysates of purified coxiella organisms was separated by 2d-page and a proteome map of c. burnetii was obtained. more than distinct protein spots with isoelectric points ranging from to and molecular mass ranging from to kda were visualized by coomassie blue stain. following the immunoblot assay, and of the coxiella proteins were recognized by the mice sera obtained at and days pi, respectively. among these recognized proteins, proteins, chaperonin groel, peptidyl-prolyl cis-trans isomerase and putative outer membrane chaperone protein, were strongly recognized by sera obtained at days and days pi, and the kda outer membrane protein was recognized by sera obtained at day and strongly recognized by sera obtained on days and pi. in addition, of the coxiella proteins were recognized by sera from two patients during the acute phase of q fever. however, of the proteins, including kda chaperone protein, lsu ribosomal protein l12p, 3-oxoacyl- synthase, s-adenosylmethionine synthetase, acute disease antigen a, glutamine synthetase, were not recognized by the mouse sera. serological analysis of the recombinant seroreactive proteins with q fever patient sera twenty genes encoding the seroreactive proteins were amplified and cloned into the pet32a/pqe plasmid. except for the rpsb-recombinant plasmid, the rest were successfully expressed in e. coli cells. the recombinant proteins were purified by ni-nta agarose and analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis. then they were used to fabricate a protein microarray. the protein microarray was probed with sera from patients with acute q fever and sera from healthy persons. the average fi value of the proteins probed with acute early, late or convalescent q fever patient sera were significantly higher compared with that probed with the normal sera the average fi values of the proteins probed with acute late q fever patient sera were significantly higher than acute early or convalescent q fever patient sera. the protein was considered to be seroreactive if its average fi probed with the patient sera were higher than the mean fi plus twice the standard deviation probed with normal sera. seven recombinant proteins were selected as major seroreactive proteins with sensitivities that ranged from 48% to 88% when probed with acute late q fever sera. the recombinant groel gave the highest sensitivity at 88%. asensitivity was calculated as the percentage specificity analysis of the major seroreactive proteins a small microarray fabricated with groel, ybgf, rpll, mip, com omph, and dnak was probed with rickettsial spotted fever, legionella pneumonia or streptococcal pneumonia patient sera. the average fi value of each protein probed with acute late q fever patient sera were significantly higher compared with that probed with the sera from the other three groups of patients. a reaction was considered positive if the average fi of one protein probed with one of the tested sera were higher than the mean fi plus times the standard deviation probed with the sera of healthy person sera. as a result, ybgf and dnak displayed no reaction with any of the tested sera, and com and mip cross-reacted with one or two of the rickettsial spotted fever patient sera. omph cross-reacted with one of the legionella pneumonia or streptococcal pneumonia patient sera; groel cross-reacted with one of the legionella pneumonia and two of the rickettsial spotted fever patient sera; rpll cross-reacted with two of the legionella pneumonia and three of the streptococcal pneumonia patient sera. discussion c. burnetii xinqiao was isolated from ticks in china and its phase i phenotype was demonstrated in a previous study. in this current study, c. burnetii xinqiao was used to infect balb/c mice and a large amount of c. burnetii was found in the spleens and livers of the infected mice by qpcr analysis. the coxiella load in spleens was significantly higher compared with that in the other organs of the infected mice, indicating that the mouse spleen is the most important organ for c. burnetii propagation and its coxiella load may reflect the severity of c. burnetii infection. the highest level of coxiella in spleens of the infected mice was found on day pi and then gradually decreased, indicating that the infected mice recovered gradually from the severe infection. these results also indicate that the combination of the sublethal challenge mouse model and the qpcr assay may be a useful and sensitive way to evaluate severity of the infection caused by different c. burnetii strains and evaluate efficiency of drugs or vaccines against this pathogen. in order to identify the seroreactive proteins of c. burnetii xinqiao, the whole cell lysates of the organism was separated by 2-d electrophoresis. immunoblot analysis using the sera of mice obtained at days and pi, indentified and of the separated proteins, respectively. this indicated that the specific immune responses to c. burnetii developed progressively in the infected mice with additional antigens of c. burentii recognized as the immune response grew further. in addition, of the proteins were recognized by sera from two patients with acute q fever. among these seroreactive proteins, proteins were recognized by both the mouse and human sera, indicating that these proteins are able to elicit similar humoral immune responses to c. burnetii infection in both species. a total of seroreactive proteins were recognized by the positive mouse or human sera by mass spectra of maldi-tof-ms. groel, a conserved heat shock protein, has been reported as a major immunodominant antigen of c. burnetii. ybgf, a tol-pal system protein that involved in bacterial outer membrane stability, was found in both phases of c. burnetii. groel and ybgf were both recognized by the sera of c. burnetii-infected mice and the q fever patient sera in this study and have been previously documented as seroreactive antigens using a proteomic approach. while com mip, and omph were recognized by the sera of c. burnetii-infected mice but were not recognized by q fever patient sera. this difference might be due to the fact that mouse and human sera were from different infection stages or there were differences in humoral immune responses to c. burnetii infection between mice and humans. com was first identified as an outer membrane-associated seroreactive protein of c. burnetii by hendrix and colleagues. mip is a cell-surface associated peptidylprolyl-isomerase related to macrophage infectivity potentiator protein and plays a role in enhancing clearance of bacteria from spleens of infected mice. omph is a putative outer membrane chaperone protein required for efficient release of translocated proteins from the plasma membrane. the proteins had also been recognized as immunodominant antigens in other studies. dnak, a surface-associated protein playing a role in assisting with folding of nascent polypeptide chains, and rpll, a ribosomal protein involved in translation, were previously recognized as seroreactive. in this study, dnak and rpll were most seroreactive when probed with the sera of patients with acute q fever but were nonreactive when probed with the sera of c. burnetii-infected mice. additionally, another seroreactive proteins identified in this study were housekeeping enzymes, including fbaa, atpd, and tuf which are involved in metabolism and biosynthesis. eight of these proteins were previously identified as seroreactive antigens. this indicated that metabolic enzymes released from c. burnetii organisms were exposed to the host immune system and induced a specific antibodies response. nineteen of the seroreactive proteins identified in this immunoproteomics study were successfully expressed in e. coli cells and the resultant recombinant proteins were used to fabricate a protein microarray. to evaluate their serodiagnostic potential, the protein microarray was probed with q fever patient sera. as a result, of the proteins gave a modest sensitivity of more than 48% when probed with acute late q fever patient sera. we noted that inconsistency existed between immunoproteomic and microarray data: the reaction of com was stronger than that of mip, omph or ygbf in immunoblot assay, whereas fi value of mip, omph or ygbf was higher than that of com in microarray assay with q fever sera. the inconsistency might be caused by the fact that the q fever sera recognized linear epitopes of coxiella proteins in immunoblot assay whereas they recognized conformational epitopes of recombinant proteins in protein microarray assay. our results also showed that the average fi value of the major seroreactive proteins probed with acute late sera were significantly higher than those probed with acute early or normal sera, which is generally in accordance with igg titers determined in ifa. this result firmly suggests that the major seroreactive proteins are immunodominant antigens of c. burnetii and they have capability to evoke strong humoral immune responses in c. burnetii infection. however, compared to ifa, the lower sensitivity of some individual proteins in microarray assay with q fever patient sera, especially sera in acute early stage, was observed, which might be due to the fact that there were remarkable variation in immune recognition patterns for q fever and differences between the two assays in calculating positive values. when the seroreactive proteins were analyzed in combination, 98% of antibody responders to one or more of the major seroreactive proteins could be found among the q fever patients. the remarkable variation in immune recognition patterns for q fever requires multi-antigen combination to cover the different antibody responses and thus achieve the highest possible test sensitivity. ybgf, rpll, mip, com and omph were considered as potential antigens for diagnosis of q fever by other investigators using in vitro transcription and translation -based microarray of c. burnetii nine mile strain, indicated that xinqiao strain isolated in china shares these major seroreactive antigens with nine mile strain. two heat shock proteins groel and dnak were also recognized as major seroreactive antigens in this study. the positive frequencies of groel probed with acute early and acute late, and convalescent q fever patient sera were 84%, 88%, and 83%, respectively, higher than the other major seroreactive proteins, suggesting that groel is an excellent molecular marker for q fever. additionally, the positive frequencies of ybgf with these q fever patient sera were 44%, 62%, and 77%, lower than groel but higher than the other major seroreactive proteins, indicating that it is a better protein antigen for q fever diagnosis. rickettsial spotted fever caused by tick-borne infection may share similar clinical feature with q fever. legionella pneumonia is caused by legionella pneumophila which is the bacterium closely related to c. burnetii with genomic homology and similar clinical presentations. pneumonia is the major clinical presentation of acute q fever and most bacterial pneumonia is caused by s. pneumoniae. these bacterial infections must be distinguished from q fever using serological or molecular tests. therefore, the coxiella proteins were used to fabricate a small microarray for further analysis of specificity with the sera of patients with other infectious diseases. the average fi value of each protein probed with acute late q fever patient sera was significantly higher than that probed with the sera of patients with one of the three other infectious diseases, which indicated that the major seroreactive proteins of coxiella can be distinguished from other bacteria in general. ybgf and dnak displayed no cross-reaction with any of the tested sera, and com mip, omph and groel cross-reacted with one or two of the sera of patients with rickettsial spotted fever, legionella pneumonia or bacterial pneumonia. rpll cross-reacted with two of the legionella pneumonia patient sera and three of the streptococcal pneumonia patient sera. in this analysis, these coxiella proteins gave a modest specificity for recognizing of q fever patient sera, suggesting that they are potential serodiagnostic markers for q fever. notably, groel had the highest sensitivity and modest specificity for recognizing of q fever, which may be the most important antigen used for the diagnosis of q fever. the antigen combination, groel, ybgf and com may give a more authentic specificity. refinement of antigen combination and the production of fusion molecules comprised of the major seroreactive antigens described herein may lead to improved sensitivity and specificity for the development of a rapid, accurate, and convenient seorodiagnostic test of q fever.  in summary, the combination of 2d-page, immunoblot and maldi-tof-ms permitted the identification of seroreactive proteins of c. burnetii. a protein microarray fabricated with recombinant proteins was probed with q fever patient sera. seven proteins were recognized as major seroreactive antigens. the major seroreactive proteins fabricated in a small array were analyzed with the sera of patients with q fever, rickettsial spotted fever, legionella pneumonia or streptococcal pneumonia and they gave a moderate specificity for recognizing of q fever patient sera, suggesting these proteins are potential serodiagnostic markers for q fever. 
the major seroreactive proteins were fabricated in a small microarray and further analyzed with the sera of patients with rickettsial spotted fever, legionella pneumonia or streptococcal pneumonia. an immunoproteomic study of c. burnetii xinqiao strain isolated in china was conducted with the sera from experimentally infected balb/c mice and q fever patients. coxiella burnetii is the etiological agent of q fever. the clinical diagnosis of q fever is mainly based on several serological tests. nineteen of the proteins were successfully expressed in escherichia coli and used to fabricate a microarray which was probed with q fever patient sera. our results demonstrate that these coxiella proteins gave a modest sensitivity and specificity for recognizing of q fever patient sera, suggesting that they are potential serodiagnostic markers for q fever. as a result, groel, ybgf, rpll, mip, omph, com and dnak were recognized as major seroreactive antigens.
 coxiella burnetii is the etiological agent of q fever. the clinical diagnosis of q fever is mainly based on several serological tests. these tests all need coxiella organisms which are difficult and hazardous to culture and purify.  an immunoproteomic study of c. burnetii xinqiao strain isolated in china was conducted with the sera from experimentally infected balb/c mice and q fever patients. twenty of whole proteins of xinqiao recognized by the infection sera were identified by mass spectrometry. nineteen of the proteins were successfully expressed in escherichia coli and used to fabricate a microarray which was probed with q fever patient sera. as a result, groel, ybgf, rpll, mip, omph, com and dnak were recognized as major seroreactive antigens. the major seroreactive proteins were fabricated in a small microarray and further analyzed with the sera of patients with rickettsial spotted fever, legionella pneumonia or streptococcal pneumonia. in this analysis, these proteins showed fewer cross-reactions with the tested sera.  our results demonstrate that these coxiella proteins gave a modest sensitivity and specificity for recognizing of q fever patient sera, suggesting that they are potential serodiagnostic markers for q fever. 