High prevalence and two dominant host-specific genotypes of Coxiella burnetii in U.S. milk

Background Coxiella burnetii causes Q fever in humans and Coxiellosis in animals; symptoms range from general malaise to fever, pneumonia, endocarditis and death. Livestock are a significant source of human infection as they shed C. burnetii cells in birth tissues, milk, urine and feces. Although prevalence of C. burnetii is high, few Q fever cases are reported in the U.S. and we have a limited understanding of their connectedness due to difficulties in genotyping. Here, we develop canonical SNP genotyping assays to evaluate spatial and temporal relationships among C. burnetii environmental samples and compare them across studies. Given the genotypic diversity of historical collections, we hypothesized that the current enzootic of Coxiellosis is caused by multiple circulating genotypes. We collected A) 23 milk samples from a single bovine herd, B) 134 commercial bovine and caprine milk samples from across the U.S., and C) 400 bovine and caprine samples from six milk processing plants over three years. Results We detected C. burnetii DNA in 96% of samples with no variance over time. We genotyped 88.5% of positive samples; bovine milk contained only a single genotype (ST20) and caprine milk was dominated by a second type (mostly ST8). Conclusions The high prevalence and lack of genotypic diversity is consistent with a model of rapid spread and persistence. The segregation of genotypes between host species is indicative of species-specific adaptations or dissemination barriers and may offer insights into the relative lack of human cases and characterizing genotypes.

Each TaqMan® assay we designed targets a polymorphic locus in the C. burnetii genome that was found to be specific to either a clade of C. burnetii multi locus spacer typing (MST) genotypes or a single MST genotype (Glazunova et al., 2005;Hornstra et al., 2011). Supplemental Figure 1 shows the phylogenetic placement of each SNP locus including the nucleotide for the ancestral and derived alleles for each locus for the assays presented here. In addition, for some of our assays we also designed a self-reporting synthetic positive control (see Section IV and Supplemental Table 2).

III. Genotyping
Our TaqMan® dual-probe assays were used to genotype DNA extractions from milk that were determined to be free of PCR inhibitors via a generalized 16S rRNA assay (Liu et al., 2012) and also positive for C. burnetii DNA via the IS1111 assay (Loftis et al., 2006). For each assay (Supplemental Table 1), 1 µL of DNA was used in a total reaction volume of 10 µL that contained 5 µL of 2x TaqMan® Universal PCR Master Mix (Life Technologies, CA, USA; p/n 4304437), 0.45 µL of each 20 µM primer, 0.10 µL of each 20 µM MGB probe, and 2.9 µL of sterile, molecular grade water. If synthetic positive controls were used (see section IV) than additionally, 0.10 µL of a 20 µM universal NED MGB probe (NED-CGACTGTGTTAGGTCAC) were added and the amount of water was adjusted to 2.8 µL.
Thermal cycling conditions were: 50°C for 2 min., 95°C for 10 min., followed by 45 cycles of 95°C for 15 sec., and 60°C for 1 min. and were performed on an Applied Biosystems 7900HT Fast real-time PCR system with SDS v2.4 software. Allele calls were used in conjunction with Supplemental Figure 1 to determine a sample's placement into a clade of genotypes or a specific genotype.

IV. Self-reporting Synthetic Positive Control
Real-time PCR TaqMan® dual-probe assays require the inclusion of two positive controls, one per allele SNP state, with every experiment. With this, there is always a risk of reporting false positive results from samples due to possible cross contamination with positive controls. When assays are being used for detection or genotyping of low-levels of target DNA it can be particularly difficult to differentiate cross contamination from true signal as cross contamination from positive controls often presents as a weak signal. To effectively identify false signals, we present a generic molecular approach for generating a synthetic positive control designed to self-report its synthetic status during real-time PCR.
Design. Two synthetic positive controls, one per allele SNP state, must be generated for every unique assay. Each allele-specific synthetic positive control is constructed by two separate, hierarchically ordered, PCR processes (Supplemental Figure 2) that produces a final synthetic template which contains the sequences to the following targets per unique assay: forward primer sequence, universal synthetic sequence (CGACTGTGTTAGGTCAC), allele-specific SNP containing probe sequence, and reverse primer sequence. The purpose of the first PCR is to generate an intermediate template that contains the following targets: allele-specific SNP containing probe sequence, synthetic sequence, and reverse primer sequence. Genomic DNA (gDNA) is used as the template in the first PCR (Supplemental Figure 2A). The product amplification is generated by a reverse primer and a customized forward primer where the 3'end targets the SNP allele sequence on the gDNA and the 5'end carries the synthetic sequence. The final PCR product from this reaction serves as the template for the second PCR (Supplemental Figure 2B). The second PCR (Supplemental Figure 2B) incorporates the forward primer sequence of the assay onto the intermediate template. This is accomplished by amplifying a product with a reverse primer and a customized forward primer where the 3'end targets the synthetic sequence on the intermediate templates and the 5'end carries the assay's forward primer sequence. This final PCR amplicon from the second PCR is used as the synthetic positive control template for their unique assay. The internal synthetic sequence is targeted by a NEDlabeled TaqMan® probe containing the corresponding sequence. Increased NED fluorescence over the course of the real-time PCR run confirms the success of the PCR reaction while simultaneously revealing the synthetic status of the positive control.
Allele-specific synthetic positive controls. The allele SNP-state of the synthetic positive control is determined by the base identity of the 3' end of the forward primer used in the first PCR process. The single 3'end mismatch between a forward allele-specific primer and its non-allelic genomic template does not measurably impede the PCR amplification of product (Supplemental Figure 2A). As a consequence, the same starting genomic DNA template can generate synthetic positive controls possessing either SNP states.
PCR conditions. Each PCR process was performed using conventional PCR master mix composed of 300 nM each of a forward allele-specific primer and a common reverse primer (IDT, CA, USA), 1x PCR buffer without MgCl2 (Life Technologies, CA, USA), 2 mM MgCl2 (Life Technologies, CA, USA), 200 µM of each dNTPs (Life Technologies, CA, USA), 0.8 units of Platinum Taq DNA polymerase (Life Technologies, CA, USA), 1 µl of template at ~1ng/µl or 1/100,000 dilution of intermediate PCR product, and molecular grade water to a final volume of 10 µl. PCR amplifications were conducted on an MJ Research DNA engines 96 well block thermal cycler equipped with hot bonnets. PCRs were raised to 94°C for 5 min to denature the DNA and activate the Taq DNA polymerase, then cycled (total of 35 times) at 94°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec, with a final extension at 72°C for 5 min.
Utility. To construct our synthetic positive template controls (synPTCs) for this study, we used genomic material from C. burnetii strain 9 Mi phase II c4, the primer sets in Supplemental Table 2, and the PCR conditions listed above. While this method of constructing synPTCs worked well for many of our assays we found that for one assay, Cox56bp10, the synPTCs degraded over time such that the NED signal was no longer present in our amplification reactions. Unfortunately, this negated their utility as a self-reporting positive control. We found that one solution to this issue was to purchase two oligos (see Supplemental  Table 1) that contained the exact sequence that theoretically our synPTCs for Cox56bp10 would have if we had constructed them in house. While ordering two longer oligos was more expensive than ordering four shorter ones, we have found the longer ones to be more stable and to more consistently produce a suitable NED signal. Despite this, synthetic PTCs constructed for other assays in this study and for use in similar work with Francisella tularensis have not degraded over time and consistently produce good results (D. Birdsell, unpub.) which suggests that the stability of constructed synPTCs may be dependent on the exact sequence of the nucleotides being targeted.