during 2015 in south korea , a total of 10,332,000 pigs were raised , of which 2,338,521 ( 22.6% ) were raised on 1,134 farms in gyeongsang province ( 6 ) . for this study , we collected 1,030 blood and 97 tissue samples from pigs ( 645 breeding and 479 fattening pigs ) reared on 209 pig farms in gyeongsang province during 20142015 . sample size was determined using a formula with an expected disease prevalence of 50% , accepted absolute error of 5% , and ci of 99% in a simple random sampling design ( technical appendix ) ; a minimum of 664 samples were required . samples were collected by practicing veterinarians during treatment or regular medical checkups ; ethical approval was not required . the number of samplings was based on the number of pigs and farms within each of the province s administrative districts ( figure 1 ) . number of pig farms in the provincial administrative districts and number of farms on which pigs were sampled for the detection and genotyping of coxiella burnetii , gyeongsang province , south korea , 20142015 . the number of samplings was based on the number of pigs and farms within each of the province s administrative districts . lung , lymph node , liver , spleen , and kidney samples were collected for differential diagnosis of diseases in pigs that aborted or had a stillbirth , respiratory symptoms , or weakness . to detect c. burnetii  positive samples , we used 2 different assays and nested pcr . we used an indirect multispecies elisa ( i d screen q fever indirect multi - species kit ; idvet , montpellier , france ) according to the manufacturer s instructions to detect c. burnetii antibodies in samples ; a sample optical density to positive - control optical density value of > 50% was considered positive . we also performed an indirect immunofluorescence assay ( ifa ) , using the coxiella burnetii ( q fever ) fa substrate slide ( vmrd , pullman , wa , usa ) , as recommended by the manufacturer ; titers > 64 to phase-1 or phase-2 antigens were considered seropositive . we used the dneasy blood and tissue kit ( qiagen , hilden , germany ) according to the manufacturer s instructions to extract dna from whole blood and tissue samples . the coxiella 16s rrna gene in extracted dna was then amplified using nested pcr and sequencing primers ( technical appendix ) . we sequenced amplification products with macrogen ( seoul , south korea ) and analyzed results using sequence alignment programs and statistical methods ( technical appendix ) . of the 1,030 sampled pigs , 70 ( 6.8% ) were positive for c. burnetii by elisa ( table ) ; these pigs were from 32 ( 15.3% ) of the 209 sampled farms . two of the 32 farms had 8 positive pigs each ; the other 30 had 13 positive pigs each . fifty - three ( 5.2% ) sampled pigs had samples identified as phase-1 or phase-2 antigen seropositive by ifa ; these samples were also seropositive by elisa . an additional 17 samples seropositive by elisa were seronegative by ifa . c. burnetii seroprevalence was significantly higher ( p<0.0001 ) in breeding than in fattening pigs by elisa and ifa . elisa and ifa results were in agreement for 1,013 ( 98.4% ) of the 1,030 samples ; 53 ( 5.2% ) samples were positive , and 960 ( 93.2% ) were negative . the cohen  coefficient was 0.85 ( i.e. , very good agreement ; 95% ci 0.790.92 ) . three ( 0.3% ) pigs were positive for c. burnetii by pcr ; all were breeding pigs and seronegative for c. burnetii . one positive sample was lung tissue from a pig that appeared to have respiratory signs ; other respiratory pathogens were also detected in the sample . however , the infection status of seropositive pigs can not be determined on the basis of a single titer . c. burnetii seronegative pigs can , however , shed the organism and , thus , might serve as a reservoir for transmission of the bacterium to humans . 16s rrna gene sequences for the 3 c. burnetii pcr - positive samples ( genbank accession nos . kt94501416 ; figure 2 ) showed 100% identity with each other ; nucleotide sequences showed high ( 96.6%96.9% ) identity with those of other c. burnetii strains . phylogenetic analysis showed that the 3 isolates belong to clade a , clustering with previously published c. burnetii sequences ( figure 2 ) . phylogenetic tree constructed using the maximum - likelihood method from coxiella burnetii 16s rrna sequences . arrows indicate c. burnetii sequences from study to detect and genotype c. burnetii in pigs in gyeongsang province , south korea , 20142015 . we found that 6.8% , 5.2% , and 0.3% of tested pig samples in gyeongsang province were positive for c. burnetii by elisa , ifa , and pcr , respectively . these rates of seropositivity are relatively low compared with the rate found in a study in uruguay , in which 18.4% ( 83/479 ) of the blood samples were seropositive by layer microagglutination ( 7 ) . in that study , the innate susceptibility of pigs to c. burnetii was confirmed during a q fever epidemic . seropositivity in our study was , however , higher than that reported in blood tested by ifa in japan ( 0/396 samples ) ( 8) and by complement fixation in bulgaria ( 0.05% ; 1/1,809 samples ) ( 9 ) .   in c. burnetii we did not test such tissues ; however , the positivity rate in our study was similar to that ( 0/16 ) in a previous examination of pig placentas by real - time pcr in the netherlands ( 10 ) . in our study , similar to results from a previous study ( 11 ) , our results showed that ifa was less sensitive than elisa at detecting c. burnetii in serum . animals can maintain seropositivity after acute infection has cleared , and they can seroconvert without shedding ( 12 ) ; thus , serologic methods are not useful for determining which animals currently pose a risk for transmission . in our study , in addition , only breeding pigs were positive for c. burnetii by pcr . because of pregnancy stress , breeding pigs probably experienced a recrudescent infection , making them more likely to shed the organism . a study on the epidemiology of q fever suggested that breeding pigs can cause infection in humans ( 13 ) . the genus coxiella is divided into 4 highly divergent genetic clades ( a  d ) ; c. burnetii belongs to clade a ( 14 ) . phylogenetic analysis showed that the 3 c. burnetii isolates in our study were closely related to clade a strains from the united states , japan , and greenland , indicating a close epidemiologic link . although the number of c. burnetii  positive pigs was low in our study , a previous study identified contact with pigs as a risk factor for c. burnetii seropositivity in humans ( 15 ) . therefore , pigs may serve as potential reservoirs for c. burnetii . however , several questions remain unanswered regarding the epidemiology of c. burnetii infection in pigs and possible transmission to humans . additional investigations of the infection prevalence in other animals are necessary to understand the epidemiology of c. burnetii .
we assessed coxiella burnetii prevalence and genotypes in pigs in south korea during 20142015 . 
 prevalence was low among 1,030 samples tested by elisa and immunofluorescent assay and 1,124 samples tested by pcr . despite this finding , possible transmission of c. burnetii from pigs to humans 
 can not be excluded .