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Evaluation of Two PCR Tests for Coxiella burnetii Detection in Dairy Cattle Farms Using Latent Class Analysis.

ABSTRACT: Different tests performed on bulk tank milk samples (BTM) are available to determine the C. burnetii status of herds. However, these tests, which are based on the detection of either antibodies directed against C. burnetii (ELISA) or bacterial DNA (PCR), have limitations. A currently disease-free herd infected in the past may continue to test positive with ELISA due to the persistence of antibodies in animals that were infected and that subsequently cleared the infection. Infectious herds can also be misclassified using PCR because of the absence of bacteria in the BTM when the test is performed. Recently, PCR has been used for bacterial DNA detection in the farm environment, which constitutes the main reservoir of C. burnetii. The objectives of this study were to assess and compare the sensitivities and specificities of one commonly used PCR test in BTM (PCR BTM) and of a PCR applied to environmental samples (PCR DUST) in dairy cattle farms. BTM and dust samples were collected (using environmental swabs) in 95 herds. The evaluation of the performance of the 2 tests was conducted using latent class models accounting for within herd disease dynamics. Parameter estimation was carried out using MCMC, within a Bayesian framework. Two types of priors were used for the specificity of PCR DUST. A model with a uniform prior on 0-1 fitted the data better than a model with a uniform prior on 0.95-1. With the best model PCR DUST had a lower sensitivity than PCR BTM (0.75 versus 0.83) and a specificity of 0.72. The moderately low value for the specificity of PCR DUST suggests that the presence of bacteria on farm is not always associated with persistent infections and shedding of bacteria in milk.

SUBMITTER: Nusinovici S

PROVIDER: S-EPMC4683090 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Evaluation of Two PCR Tests for Coxiella burnetii Detection in Dairy Cattle Farms Using Latent Class Analysis.

Nusinovici Simon S Madouasse Aurélien A Hoch Thierry T Guatteo Raphaël R Beaudeau François F

PloS one 20151216 12

Different tests performed on bulk tank milk samples (BTM) are available to determine the C. burnetii status of herds. However, these tests, which are based on the detection of either antibodies directed against C. burnetii (ELISA) or bacterial DNA (PCR), have limitations. A currently disease-free herd infected in the past may continue to test positive with ELISA due to the persistence of antibodies in animals that were infected and that subsequently cleared the infection. Infectious herds can al ...[more]

PMID: 26673419

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Project description:BACKGROUND: Coxiella burnetii, the bacterium causing Q fever, is an obligate intracellular biosafety level 3 agent. Detection and quantification of these bacteria with conventional methods is time consuming and dangerous. During the last years, several PCR based diagnostic assays were developed to detect C. burnetii DNA in cell cultures and clinical samples. We developed and evaluated TaqMan-based real-time PCR assays that targeted the singular icd (isocitrate dehydrogenase) gene and the transposase of the IS1111a element present in multiple copies in the C. burnetii genome. RESULTS: To evaluate the precision of the icd and IS1111 real-time PCR assays, we performed different PCR runs with independent DNA dilutions of the C. burnetii Nine Mile RSA493 strain. The results showed very low variability, indicating efficient reproducibility of both assays. Using probit analysis, we determined that the minimal number of genome equivalents per reaction that could be detected with a 95% probability was 10 for the icd marker and 6.5 for the IS marker. Plasmid standards with cloned icd and IS1111 fragments were used to establish standard curves which were linear over a range from 10 to 10(7) starting plasmid copy numbers. We were able to quantify cell numbers of a diluted, heat-inactivated Coxiella isolate with a detection limit of 17 C. burnetii particles per reaction. Real-time PCR targeting both markers was performed with DNA of 75 different C. burnetii isolates originating from all over the world. Using this approach, the number of IS1111 elements in the genome of the Nine Mile strain was determined to be 23, close to 20, the number revealed by genome sequencing. In other isolates, the number of IS1111 elements varied widely (between seven and 110) and seemed to be very high in some isolates. CONCLUSION: We validated TaqMan-based real-time PCR assays targeting the icd and IS1111 markers of C. burnetii. The assays were shown to be specific, highly sensitive and efficiently reproducible. Cell numbers in dilutions of a C. burnetii isolate were reliably quantified. PCR quantification suggested a high variability of the number of IS1111 elements in different C. burnetii isolates, which may be useful for further phylogenetic studies.

Project description:BackgroundQ fever, a zoonosis caused by Coxiella burnetii, has adverse effects on public health. Ticks are vectors of C. burnetii and they contribute to the transmission of the pathogen. A tool for rapid, sensitive, and accurate detection of C. burnetii from ticks is important for the prevention of Q fever.MethodsUltra-rapid real-time PCR (UR-qPCR) as a chip-based real-time PCR system was developed for the detection of C. burnetii from ticks. The UR-qPCR system was established and evaluated for the rapidity, sensitivity, and specificity of C. burnetii detection.ResultsC. burnetii was detected using UR-qPCR from 5644 larval, nymphal, and adult ticks from 408 pools collected from livestock and epidemiologically linked environments in two provinces, Gangwon and Jeju, in Korea. Ticks from three species were identified; Haemaphysalis longicornis accounted for the highest number, present in 333 of 408 pools (81.62%), followed by Haemaphysalis flava in 62 pools (15.19%) and Ixodes nipponensis in 13 pools (3.19%). The rapidity and sensitivity of PCR detection was demonstrated with the sufficient amplification and detection of approximately 56 copies of C. burnetii DNA with only 20 min of PCR amplification. The kappa value for the diagnostic agreement between UR-qPCR and stationary qPCR was in perfect agreement (κ = 1). PCR detection and sequencing indicated that C. burnetii was present in 5 of the 408 pools (1.23%), in which four pools contained H. longicornis and one pool contained H. flava. The infection rates of C. burnetii in the tick pools collected from Gangwon and Jeju Provinces were 1.70% and 0.58%, respectively. Phylogenetic analysis indicated a close relationship between the detected C. burnetii and those originating from goats, humans, and ticks in different countries, such as the USA, France, Germany, and Serbia.ConclusionsThe methods described in this study could be important for the prevention and control of Q fever in the two provinces. The UR-qPCR, with its features of mobility, sensitivity, and rapidity, is helpful for constructing early alert systems in the field for C. burnetii in ticks and could help alleviate the transmission of and economic damage due to Q fever.

Dataset Information

Evaluation of Two PCR Tests for Coxiella burnetii Detection in Dairy Cattle Farms Using Latent Class Analysis.

Publications

Evaluation of Two PCR Tests for Coxiella burnetii Detection in Dairy Cattle Farms Using Latent Class Analysis.

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