Evaluation of randomly amplified polymorphic DNA (RAPD) for discrimination of Coxiella burnetii ruminant strains isolated in France

Abstract

Coxiella burnetii is the causative agent of Q fever, a worldwide zoonosis. Ruminant species represent the main reservoir of the bacterium, as high levels of the bacteria are shed in birth products and other excreta. Human infection generally occurs after inhalation of contaminated aerosols [1]. The availability of methods to study the distribution and the spread of a given C. burnetii strain, from different geographical areas or hosts, would promote a better understanding of the epidemiology of this pathogen. Several genetic typing methods for C. burnetii have been evaluated and appeared to be useful tools for epidemiological and phylogenetic purposes. However, RAPD [2,3] has never been evaluated for C. burnetii typing. In this work, a RAPD protocol was used to evaluate the genetic diversity among C. burnetii ruminant strains in comparison to published MLVA data [4]. Ten French C. burnetii isolates obtained from goats, sheep, cows and the reference strain Nine Mile, were used for RAPD typing (Table 1). Whole DNA was extracted using the Qiagen Dneasy kit (Courtaboeuf, France), following the manufacturer’s protocol. To assess optimal RAPD conditions several parameters were tested, for instance using a range of MgCl2 concentrations (from 1.5 to 5.5 mM), or different brands of Taq polymerase. RAPD PCRs were performed using a set of four 10-mer primers (P4M, 5¢-AAGACGCCGT-3¢; PR5, 5¢-AGTCGTCCCC-3¢; PR10A, 5¢AGGGCCGTCT3¢; and PR12A, 5¢-CAGCTCACGA-3¢). Unless specified, the optimised PCR conditions were, for a final volume of 25 lL in dH2O: 1X Taq polymerase buffer (without MgCl2); 3 mM MgCl2; 0.2 mM each dNTPs; 1U of Taq polymerase; 0.5 lM 10-mer primer; and 10 ng of DNA (all the reagents were purchased from Invitrogen, Cergy Pontoise, France). Negative controls consisting of dH2O only were included in each run. Amplifications were performed in an Eppendorf Mastercycler (Le Pecq, France), programmed for an initial denaturation step of 5 min at 94 C, 45 cycles of 96 C for 30 s, 37 C for 30 s, 72 C for 90 s and a final extension step at 72 C for 5 min. The PCR products were separated on 0.8% agarose gel containing ethidium bromide, visualised and photographed under UV light. Gel analysis was performed using the Quantity One 1-D Analysis software from Bio-Rad (Marnes la Coquette, France). An RAPD type was defined after the combination of the patterns obtained with the four primers used in this study (Table 1). The reproducibility of the method was evaluated by repeating the same assay several times, by different manipulators, as well as by using independent DNA preparations. Briefly, RAPD profiles were stable when varying MgCl2 concentration. However, a higher yield of amplification, estimated by the intensity of the bands observed on the gels, was observed using 2.5–3.5 mM MgCl2. This yield decreased when using higher concentrations of MgCl2 (data not shown). A MgCl2 concentration of 3 mM was therefore chosen for subsequent analyses. Different RAPD patterns were observed when different brands of Taq polymerases were used (data not shown). The Hot start platinum Taq polymerase (Invitrogen) produced the best patterns in our hands and was chosen for all the experiments. After optimisation of our RAPD protocol, the use of each primer generated distinct polymorphisms allowing differentiation of the studied strains. Strains from neighbouring flocks, that were indistinguishable using MLVA analysis, could be discriminated using RAPD typing (Table 1). For instance, CbB2 and CbB5 are two strains obtained in 2001 from neighbouring flocks, but were isolated from cows displaying different clinical signs (Table 1). CbB2 was isolated from a case of metritis and CbB5 from an aborted cow. Corresponding author and reprint requests: Dr V. Duquesne, AFSSA, Sophia Antipolis, Unite de Pathologie des Petits Ruminants, 105 Route des Chappes, BP111, 06902 SophiaAntipolis, France E-mail. v.duquesne@afssa.fr