PCR analysis included 14 isolates, 10 of which had previously been characterized as members of genetic groupings I through V [Nine Mile phase II (9Mi/II), Nine Mile RSA 514 (RSA 514), Ohio, Australian QD, Q195, Henzerling RSA 343 (Henz I), Henzerling, RSA 331 (Henz II), M44, KAV, PAV, Q238 and WAV], and 4 [Nine Mile Baca (9Mi/Baca), Scottish, Q238 and WAV] which had not been genotyped [4]. Our repository lacks the group VI Dugway isolates, thus these were not included. The analysis was completed for each isolate for each of the 20 IS elements present in 9Mi/I using the consensus primer IS1111-1 paired with a unique primer upstream of each of the IS elements. The presence or absence of certain IS elements was generally consistent among members of a particular genogroup, but allowed their differentiation from members of other genogroups. The results from each of these 14 isolates are provided (see Additional file 1) along with the element's orientation and nucleotide position in 9Mi/I [GenBank: AE016828], and GenBank accession numbers for the sequences determined in this study.
Genomic group I isolates
9Mi/II, RSA 514, Ohio, and Australian QD appeared to have all 20 IS elements and their products were indistinguishable from each other and 9Mi/I by this method. The isolates 9Mi/Baca and Scottish, which have not been genotyped previously, also had all 20 IS elements and were placed within this group.
Genomic group II isolates
PCR products amplified using DNA from isolates Henz I, Henz II, and M44 were identical in size to 9Mi/I for 15 of the 20 IS elements (2, 3, 5, 6, 7, 8, 9, 11, 12, 13, 15, 16, 17, 18, and 19). IS elements 14 and 20 were confirmed absent in these isolates by sequencing, though our M44 isolate also possessed a subpopulation which contained IS 14 (Fig 1). In regards to IS 14, the deletion totaled 1,374 bp, which corresponds exactly to the predicted length of the IS element [17]. Also within this region, Henz I and Henz II, but not M44, contain a point mutation. The IS 20 deletion in these three isolates totaled 2,246 bp.
Amplicons to IS elements 1 and 4 were present but of larger size than 9Mi/I. In all three group II isolates, indels consisted of small deletions and slightly larger insertions. The IS 10 region, however, was quite unique in that PCR products using primer pairs IS 10 and IS1111-1 produced a product of incorrect size. Upon amplification with primer pairs 1269R and 1273R, which correspond to genes flanking the IS 10 element in 9Mi/I, products were obtained. Sequence data indicated that the site where the IS 10 primer anneals is missing due to a deletion of 1,650 bp in these isolates, but that the IS element is indeed present.
Genomic group III isolate
A single isolate from genomic group III, Q195, was available in our repositories for analysis. There were no noticeable differences in amplicon sizes between 9Mi/I and Q195 for 15 of the 20 IS elements (2, 3, 5, 6, 7, 8, 9, 10, 12, 13, 15, 16, 17, 18, and 19). The only IS element which Q195 lacked was IS 14. Sequencing of this region identified a genomic deletion of 1,374 bp.
Amplicons of larger size were produced to IS elements 1, 4 and 20. Upon sequencing of these PCR products, each was missing 3 nucleotides and an identical sequence of 26 nucleotides was inserted in its place in all cases. The PCR product for IS 11 was smaller for Q195 in comparison to 9Mi/I and was found to be lacking 8 bp.
Genomic group IV isolates
Two isolates from chronic Q fever patients exhibiting endocarditis, KAV and PAV, have previously been placed into group IV [4]. Q238 is believed to be an isolate derived from the same patient as PAV, though it has not been genotyped previously. These three isolates were examined by PCR using the primer pairs to each of the 20 IS elements described above. No amplicon size differences were seen for 10 of the IS elements (2, 5, 6, 8, 11, 12, 13, 17, 19 and 20) when compared to 9Mi/I.
Analysis of IS 7 and IS 9 in the KAV, PAV and Q238 isolates showed that both of these IS elements were missing. Sequencing revealed the absence of 1,458 bp and 1,341 bp, respectively, compared to the IS 7 and IS 9 elements in the 9Mi/I genome. Similarly, IS elements 14, 15, and 16 were missing in each of the genomic group IV isolates because of identical deletions of 1,374 bp.
No products of the correct size were obtained from isolates KAV, PAV and Q238 with primers for IS 10. A long PCR product obtained using primers 1269R and 1273R was sequenced and an IS element was found in this region, although the annealing site for the IS 10 primer was eliminated. The sequence of DNA to which the IS element is connected corresponds to 9Mi/I nucleotide positions 1222860–1222964. The sequence is then interrupted by 133 bp of sequence that does not match sequence in 9Mi/I, but does match sequence found in the Dugway 7E9-12 strain [GenBank: AAQI00000000].
Products of IS 18-specific PCR in each of the three genomic group IV isolates appeared to be slightly larger than that of 9Mi/I. Upon sequencing of the KAV product, however, this appeared to be a consequence of primer mismatch to another IS element in the genome. The sequence data obtained [GenBank: DQ882594] corresponded to an IS element and a novel sequence not present in the 9Mi/I genome. However, this sequence was found in the Dugway 7E9-12 strain. Using long PCR to flanking genes and DNA from the three isolates, no products were obtained, though PCR of each of the flanking genes (data not shown) indicated they are present somewhere in the genome, suggesting that in these isolates, this region of the genome has undergone rearrangement compared to 9Mi/I.
PCR to IS elements 1, 3, and 4 produced products from KAV, PAV and Q238 differing in size relative to 9Mi/I. These variations were due to numerous indels ranging from 1 bp to 32 bp. The indels in both IS 1 and IS 3 resulted in frame shifts in the predicted IS1111 encoded protein.
Genomic group V isolate
WAV had not been characterized previously but was hypothesized to be a member of genomic group V, as it could be distinguished from each of the other isolates at a variety of IS elements. It was indistinguishable from 9Mi/I for 12 of the 20 IS elements (2, 3, 6, 8, 9, 10, 11, 12, 13, 16, 17, and 18). Six nucleotides were missing in WAV in the products amplified from IS elements 1, 4, and 20 when compared to 9Mi/I and an identical 21-bp sequence was present in its place in each case.
No amplicon was generated using primers to IS elements 5, 7, 14, 15 and 19. Sequencing confirmed the absence of IS 5, 14 and 15 due to deletions of 1,374 bp, and of IS 7 due to a 1,458 bp deletion. Lastly, PCR to IS 19 using DNA from WAV did not result in an amplicon. Long PCR using primers 1960R and 1693R resulted in a product larger than that from 9Mi/I (data not shown). Sequencing elucidated that a rearrangement had occurred in this area of the WAV genome, but was not pursued further as it was evident that an IS element was no longer present in this portion of the WAV genome.
Creation and testing of an algorithm to differentiate C. burnetii strains
Using the information gained above, an algorithm was derived which discriminated each of the five genomic groups of isolates tested (Fig. 1). Because only group IV heart valve isolates (KAV, PAV and Q238) lacked products to IS 9, Henz I, Henz II, and M44 (group II) lacked IS 20, and WAV (group V) lacked IS 5, PCR to these isolates could be used to discriminate these groups. Isolates positive to each of these IS elements could then be further tested using PCR to IS 14, which separates Q195 (group III) isolates from group I isolates. PCR to IS 14 also serves to confirm that group II, IV and V isolates, which also lack this IS element, are grouped appropriately.
Further, each of the primer pairs used in the algorithm was tested using a two-step PCR program, and all products were successfully amplified (Fig. 1). The algorithm and the two-step PCR was then tested with additional isolates of C. burnetii and veterinary samples previously shown to be PCR positive for the agent. This included two isolates previously characterized, Priscilla Q177 (group IV) and Q229 (group V), four isolates of unknown genomic group (Idaho Q, Qiyi, Poker Cat and Q172), and nine veterinary samples from a single outbreak in 2005 on a goat ranch in Colorado. As expected, Priscilla Q177 and Q229 could be differentiated appropriately due to the lack of PCR products when using primers to IS 9 and IS 5, respectively. Q172 could also be placed into group V based on its lack of product to IS 5. PCR to IS 14 was also negative in these isolates, confirming that Priscilla Q177, Q229, and Q172 lacked this IS element as predicted. The remaining isolates (Idaho Q, Qiyi and Poker Cat) produced PCR products with all primer pairs and therefore were identical to 9Mi/I in our assay and placed within group I (data not shown). Six of the veterinary samples lacked PCR products to IS 9 and IS 14, placing them in genomic group IV. Three of the veterinary samples, one from placenta and two from feces, did not give PCR products using the above conditions. Since these three samples were positive for C. burnetii when tested with a real time-PCR assay that amplifies the IS1111 element, it was suspected that an inhibitor was present. By using a master mix that has demonstrated increased sensitivity in our hands (PuReTaq Ready-To-Go PCR Beads, GE Healthcare, Fairfield, CT) in lieu of Qiagen Taq Master Mix, PCR products were successfully obtained such that these samples were also shown to be members of genomic group IV.